pneumonia

pneumoniajourn

Vol7SpecialIssueforweb

pneumonia 2014 Aug xx;x:xx–xx

pneumonia

Special Issue

12th European Meeting on the Molecular

Biology of the Pneumococcus

7–10 July 2015, Oxford, UK

Volume 7 (2015)

pneumonia 2014 Volume x 1


EuroPneumo Special Issue / pneumonia 2015 Oct 21;7:I–72

pneumonia

pneumonia EDITORIAL TEAM

Professor Allan Cripps, Editor-in-Chief, Australia

Amanda Cox, Guest Editor, Australia

Dr Diana Otczyk, Senior Editor, Australia

Penny Chapman, Managing Editor, Australia

Kelly Poynter, Editorial Assistant, Australia

Disclaimer

This special issue of pneumonia has been produced from abstracts that were reviewed and forwarded to the journal

by the organisers of the 12th European Meeting on the Molecular Biology of the Pneumococcus (EUROPNEUMO 2015).

The abstracts have been ordered in the sections as received from the organisers of the meeting. Abstracts have been

reproduced largely as received from the authors. Minor editorial changes have been made for consistency and to be

more compliant with pneumonia’s style guidelines.

Suggested citation

Abstracts should be cited as follows:

Hackenbeck R. Penicillin resistance in Streptococcus pneumoniae: evolution by genetic communication. [Abstract

EuroPneumo - P1.01]. pneumonia 2015;7:3

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Commonly used Abbreviations and Acronyms

BALF

BLAST

CFU

CI

COPD

CPS

CSF

CST

EMSA

ICP-MS

ICU

IPD

KC

MALDI-TOF

MALDI-TOF/MS

MDM

MIC

MLSA

MLST

MOI

MST

PCR

PCV

PCV7

PCV10

PCV13

qPCR

qRT-PCR

ROS

RT-PCR

SMC

SNP

ST

TCS

VT

WGS

WHO

WT

Bronchoalveolar lavage fluid

Basic Local Alignment Search Tool

Colony forming units

Confidence interval

Chronic obstructive pulmonary disease

Capsule polysaccharide synthesis

Cerebrospinal fluid

Capsule sequence typing

Electrophoretic mobility shift assay

Inductively coupled plasma mass spectroscopy

Intensive care unit

Invasive pneumococcal disease

Killer cells

Matrix assisted laser desorption ionisation – time-of-flight

Matrix assisted laser desorption ionisation – time-of-flight/mass spectrometry

Monocyte derived macrophages

Minimum inhibitory concentrations

Multi-locus sequence analysis

Multi-locus sequence typing

Multiplicity of infection

Microscale thermophoresis

Polymerase chain reaction

Pneumococcal conjugate vaccine

7-valent pneumococcal conjugate vaccine

10-valent pneumococcal conjugate vaccine

13-valent pneumococcal conjugate vaccine

Quantitative polymerase chain reaction

Quantitative real-time polymerase chain reaction

Reductive oxygen species

Real-time polymerase chain reaction

Small marker chromosome

Single nucleotide polymorphisms

Sequence type

Two-component regulatory systems

Vaccine type

Whole genome sequence

World Health Organization

Wild type

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Contents

Editorial 1–2

Plenary abstracts 3–4

Poster abstracts 4–47

Oral abstracts 48–72

Editorial

Progress towards understanding the pathology of the pneumococcus 1

Amanda J Cox, Angela B Brueggemann, Timothy Mitchell, Jeremy S Brown

Plenary abstracts

PL.01

Penicillin resistance in Streptococcus pneumoniae: evolution by genetic communication 3

Regine Hakenbeck

PL.02

Structure and assembly of the cell wall in Streptococcus pneumoniae 3

Waldemar Vollmer

PL.03

Immuno-regulatory control of host susceptibility to pneumococcal carriage and invasive disease 3

Aras Kadioglu

PL.04

An ecological perspective on symbiosis between Streptococcus pneumoniae and the host 4

Debby Bogaert

Poster abstracts

P1.01

Growth phase regulates the localisation and activity of the pneumococcal autolysin LytA 4

Julie Bonnet, Maxime Jacq, Justine Cartannaz, Cécile Morlot, Thierry Vernet, Claire Durmort, Anne Marie

Di Guilmi

P1.02

DiiA is a newly identified cell wall protein of Streptococcus pneumoniae involved in invasive disease 5

María de la Soledad Escolano Martínez, Arnau Domenech, José Yuste, María I. Cercenado, Carmen Ardanuy,

Josefina Liñares, Adela G. de la Campa, Antonio J. Martin-Galiano

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P1.03

Induction of meningitis by a non-passaged clinical pneumococcal isolate in an infant rat model depends on

the presence of capsule 5

Lucy Hathaway, Denis Grandgirard, Belinda Ries, Suzanne Aebi, Luca Valente, Stephen Leib

P1.04

An investigation of the impact of air pollutants on Streptococcus pneumoniae 6

Shane Hussey, Peter Andrew, Paul Monks, Julian Ketley, Julie Morrissey

P1.05

Studies on pneumococcal esterases 6

Hasan Kahya, Peter Andrew, Hasan Yesilkaya

P1.06

Anti-capsular polysaccharide IgG-mediated protection on experimental human pneumococcal carriage 7

Elena Mitsi, Jesus Reine, Andrea Collins, Angela Wright, Jenna Gritzfeld, Jessica Owugha, Stephen Gordon,

Daniela Ferreira

P1.07

P4 peptide enhances phagocytic activity of neutrophils acquired from patients admitted to critical care with

severe sepsis 7

Elena Mitsi, Ben Morton, Shaun Pennington, Jesus Reine, Angela Wright, Gowrisankar Rajam, Edwin Ades,

Daniela Ferreira, Aras Kadioglu, Stephen Gordon

P1.08

Evaluation of Streptococcus pneumoniae respiratory infection in mice with different susceptibility for acute

inflammatory responses 8

Rúbia Isler Mancuso, Alessandra Soares-Schanoski, Eliane Namie Miyaji, Paulo Lee Ho, Orlando Garcia Ribeiro,

Maria Leonor Oliveira

P1.09

Stable niche adaptation within clonal lineages of Streptococcus pneumoniae 8

Zarina Amin, Claudia Trappetti, Bart Eijkelkamp, Catherine Hughes, Richard Harvey, Christopher McDevitt,

Adrienne Paton, James Paton

P1.10

The inflammatory response to Streptococcus pneumoniae is exaggerated by the polysaccharide capsule 9

Jimstan Periselneris, Suneeta Chimalapati, Gillian Tomlinson, Catherine Hyams, Alex Dyson, Mervyn Singer,

Mahdad Noursadeghi, Jeremy Brown

P1.11

Survival of Streptococcus pneumoniae and Staphylococcus aureus in sputum from intensive care unit patients 9

Jolien Seinen, Willem Dieperink, Anne Marie G.A. de Smet, Sven Hammerschmidt, Jan Maarten van Dijl

P1.12

Characterisation of a low molecular weight protein tyrosine phosphatase in Streptococcus pneumoniae 10

Zuleeza Ahmad, Renato Morona, Alistair Standish

P1.13

Quantification of the agglutinating effect of anti-pneumococcal antibodies analysed by flow cytometry 10

Saskia van Selm, Marrit Habets, Dimitri Diavatopoulos, Marien de Jonge

P1.14

Binding activities and anti-phagocytic properties of PclA, the Pneumococcal collagen-like protein A 11

Sandra Koch, Elena Fuchs, Tim J. Mitchell, Andrea M. Mitchell, Marcus Fulde, Michael Steinert, Simone

Bergmann

P1.15

Delineating the interaction of pneumococcal surface proteins with the human adhesive glycoprotein

fibronectin 11

Sajida Kanwal, Inga Jensch, Thomas P. Kohler, Roland Frank, Werner Tegge, Mark Brönstrup, Sven

Hammerschmidt

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P1.17

Pneumolysin-induced nitric oxide contributes to lysosomal membrane permeabilisation in Streptococcus

pneumoniae-infected macrophages 12

Joseph Ford, Martin Bewley, Simon Johnston, David Dockrell

P1.18

Structure of the pneumococcal L,D-carboxypeptidase DacB and impact of DacB on pathophysiological

processes 12

Mohammed R. Abdullah, Javier Gutiérrez-Fernández, Thomas Pribyl, Nicolas Gisch, Malek Saleh, Manfred

Rohde, Lothar Petruschka, Gerhard Burchhardt, Dominik Schwudke, Juan Hermoso, Sven Hammerschmidt

P1.19

Blockade of Streptococcus pneumoniae adhesion receptors of the blood-brain barrier endothelium: a new

therapeutic approach for the prevention and cure of pneumococcal meningitis 13

Federico Iovino, Birgitta Henriques-Normark

P1.20

Pneumococcal nasopharyngeal carriage in Nepalese children prior to pneumococcal conjugate vaccine

introduction 13

Rama Kandasamy, Meeru Gurung, Stephen Thorson, Shrijana Shrestha, Imran Ansari, Katherine L. O’Brien,

Sarah Kelly, Dominic F. Kelly, Andrew J. Pollard

P1.21

Phosphoenol-pyruvate phospho-transferase A interactions with putative host target receptors and their

derived peptides during Streptococcus pneumoniae adhesion 14

Tatyana Kushnir, Marilous Shagan, Anat Shahar, Raz Zarivach, Natali Elia, Ron Dagan, Yaffa Mizrachi Nebenzahl

P1.22

Contribution of a temperate bacteriophage to the virulence of a Streptococcus pneumoniae serotype 1 strain 14

Martin Norman, Anna Syk, Sarah Browall, Birgitta Henriques-Normark

P1.23

Streptococcus pneumoniae cardiac microlesions: pneumolysin-mediated immune evasion and biofilm growth 15

Ryan Gilley, Anukul Shenoy, Norberto Gonzalex-Juarbe, Carlos Orihuela

P1.24

Polyamine transport in the pneumococcus is essential for evading early innate immune responses in

pneumococcal pneumonia 15

Aswathy Rai, John Stokes, Justin Thornton, Edwin Swiatlo, Imran Sunesara, Bindu Nanduri

P1.25

IL-22 contributes to recovery from pneumococcal pneumonia by reducing collagen deposition in lung 16

Neil Ritchie, Tom Evans

P1.26

Pneumococcal colonisation and invasive disease studied in the porcine model 16

Astrid de Greeff, Saskia van Selm, Herma Buys, Jose Harders-Westerveen, Rahajeng Tunjungputri, Quirijn de

Mast, Andre van der Ven, Norbert Stockhofe-Zurwieden, Marien de Jonge, Hilde Smith

P1.27

Variome of the pneumococcal surface-exposed proteins and other virulence factors: a bioinformatics analysis 17

Andres Castro, Alejandro Gomez, Mauricio Gallego, Alejandro Bedoya, Sven Hammerschmidt, Gustavo Gamez

P1.28

Global serotypes and genotypes for Streptococcus pneumoniae: a whole genome resource for assessing

the impact of global pneumococcal immunisation 17

Rebecca Gladstone, Jukka Corander, Maaike Alaerts, Brenda Kwambana-adams, Mignon Du plessis, Paulina

Hawkins, Dean Everett, Martin Antonio, Anne von Gottberg, Keith Klugman, Lesley McGee, Stephen Bentley,

Robert Breiman

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P1.29

Investigating the pneumococcal phageome using a diverse genome dataset 18

Caroline L Harrold, Andries J van Tonder, Angus McDonnell, Ben A Edwards, Angela B Brueggemann

P1.31

Molecular pneumococcal capsular typing using whole genome sequencing: moving the Streptococcus

pneumoniae reference service into the genomic era 18

Georgia Kapatai, Carmen Sheppard, Ali Al-Shahib, David Litt, Norman Fry, Anthony Underwood, Timothy

Harrison

P1.32

Streptococcus pneumoniae shows no overall systematic genetic adaptation over the course of infections

leading to meningitis 19

John Lees, Diederik van der Beek, Julian Parkhill, Stephen Bentley

P1.33

Genomic and virulence comparisons of non-human isolates of Streptococcus pneumoniae 19

Andrea Mitchell, Ashleigh Holmes, Patricia Romero, Jenny Herbert, Mark van der Linden, Andrew Waller,

Tim Mitchell

P1.34

Analysis into the recombination of the phase variable Type I restriction modification system spnD39III locus 20

Megan De Ste Croix, Ana Manso, Shehzan Dada, Richard Haigh, Marco Oggioni

P1.35

Site matters: alternative insertion sites of integrative conjugative element Tn5253 can influence conjugation

frequency in Streptococcus pneumoniae 20

Francesco Santoro, Alessandra Romeo, Gianni Pozzi, Francesco Iannelli

P1.36

Mutagenesis of one Streptococcus pneumoniae serotype 1 strain 21

Vanessa Terra, Charles Plumptre, Emily Kay, Brendan Wren

P1.37

Improving molecular detection, identification and serotyping of Streptococcus pneumoniae in complex

samples 21

Laura Boelsen, Eileen Dunne, Katherine Gould, Fiona Russell, Kim Mulholland, Jason Hinds, Catherine Satzke

P1.38

Genome-wide assessment of pneumococcal antigenic diversity 22

Nicholas Croucher, Lisa Kagedan, Claudette Thompson, Julian Parkhill, Stephen Bentley, Jonathan Finkelstein,

Marc Lipsitch, William Hanage

P1.39

An increase in negative supercoiling in Streptococcus pneumoniae induces a global transcriptomic response

that regulates the DNA topoisomerase I gene 22

Maria-José Ferrándiz, Antonio-Javier Martín-Galiano, Isabel Camacho-Soguero, Cristina Arnanz,

Adela G. de la Campa

P1.41

Systematic nomenclature for the bacterial two-component regulatory systems, based on genomic,

structural and functional analysis of the pan-genome of Streptococcus pneumoniae 23

Gustavo Gamez, Diego Sanchez, Frank Mona, Yully Betancur, Andres Castro, Alejandro Gomez, Jose Mediavilla,

Mauricio Corredor, Sven Hammerschmidt

P1.42

Optimisation of the expression of fluorescent proteins in Streptococcus pneumoniae 23

Maria Catalão, Joana Figueiredo, Mafalda Henriques, João Gomes, Sergio Filipe

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P1.43

The single transmembrane segment of pneumococcal WalK is required for the perception of an

intramembrane or extracellular signal 24

Gro Stamsås, Daniel Straume, Zhian Salehian, Leiv Sigve Håvarstein

P2.01

Regulatory responses of Streptococcus pneumoniae D39 to ascorbic acid 24

Muhammad Afzal, Sulman Shafeeq, Birgitta Henriques-Normark, Oscar Kuipers

P2.02

Altered penicillin-binding protein 2x of Streptococcus pneumoniae: a new target of the serine protease HtrA 25

Katharina Peters, Inga Schweizer, Regine Hakenbeck, Dalia Denapaite

P2.03

Hydroxyl radicals contribute to the bactericidal effects of the fluoroquinolone moxifloxacin in Streptococcus

pneumoniae 25

Maria-José Ferrándiz, Antonio-Javier Martín-Galiano, Cristina Arnanz, Tahl Zimmerman, Adela G. de la Campa

P2.04

Regulation of PavB by TCS08 in Streptococcus pneumoniae 26

A Gómez, L. Petruschka, G. Gámez, S. Böhm1, V. Kluger, A. Klein, S. Hammerschmidt

P2.05

TpxD serves as a global regulator for pneumococcal response to H 2

O 2

, and is regulated by CodY 26

Barak Hajaj, Hasan Yesilkaya, Sulman Shafeeq, Xiangyun Zhi, Rachel Benisty, Oscar Kuipers, Nurith Porat

P2.06

Co2+-dependent transcriptional regulation in Streptococcus pneumoniae: Opposite effect of Mn2+ and

Co2+ on the expression of the virulence genes psaBCA, pcpA and prtA 27

Irfan Manzoor, Sulman Shafeeq, Tomas Kloosterman, Oscar Kuipers

P2.07

Differential complement sensitivity of Streptococcus pneumoniae and Streptococcus mitis 27

Helina E Marshall, Fernanda C Petersen, Jeremy S Brown

P2.08

Using dual RNA-Seq to characterise gene expression in an animal model of Streptococcus pneumoniae 28

Neil Ritchie, Tom Evans

P2.09

Emergence of amoxicillin-resistant variants of Spain 9V -ST156 pneumococci expressing serotype 11A correlates

with their ability to evade the host immune response 28

Leire Aguinagalde, Bruno Corsini, Arnau Domenech, Mirian Domenech, Jordi Cámara, Carmen Ardanuy, Ernesto

García, Josefina Liñares, Asunción Fenoll, Jose Yuste

P2.10

Streptococci of Mitis group, misidentified as Streptococcus pneumoniae 29

Agnieszka Bojarska, Alicja Kuch, Elzbieta Stefaniuk, Anna Skoczyńska, Waleria Hryniewicz, Ewa Sadowy

P2.11

Clonal relationship with the invasive disease potential of pneumococcal serotypes 29

Eva del Amo, Cristina Esteva, Susanna Hernandez-Bou, Carmen Galles, Marian Navarro, Goretti Sauca, Miriam

Triviño, Jordi de Batlle, Marina Cunquero, Carmina Marti, Pilar Ciruela, Carmen Muñoz-Almagro, Invasive

Disease Catalan Study Group

P2.12

Pneumococcal carriage in infants and young children in Fiji before and after PCV10 introduction 30

Eileen Dunne, Catherine Satzke, Tupou Ratu, Eric Rafai, Mike Kama, Rachel Devi, Kathryn Bright, Lisi Tikoduadua,

Joseph Kado, Casey Pell, Monica Nation, Jenna Smyth, Maha Habib, Belinda Ortika, Kate Gould, Jason Hinds, Kylie

Jenkins, Kim Mulholland, Fiona Russell

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P2.13

First description of Streptococcus pneumoniae serotype 6E in Spain 30

María Ercibengoa, Marta Alonso, Jose Maria Marimón, Emilio Pérez-Trallero

P2.14

Utility of a reverse-hybridisation strip assay for Streptococcus pneumoniae serotyping 31

María Ercibengoa, Susana Gamen, Ana Manrique, Jose María Marimón, Emilio Pérez-Trallero

P2.15

Nasopharyngeal colonisation of Streptococcus pneumoniae in Colombian children under five years of age 31

Jessica Morales, Carlos Eugenio Delgado, Beatriz Salazar, Leidy Acevedo, Johan Bolivar, Sara Saldarriaga, Milena

Cardona, Diego Florez, Steven Rivera, Alejandro Gomez, Lorena Molina, Juan David Rodriguez, Laura Sanchez,

Ana Cecilia Diez, Sandra Castro, Sven Hammerschmidt, Doracelly Hincapie, Gustavo Gamez

P2.16

Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae clinical isolates from invasive

pneumococcal diseases in Antioquia, Colombia 32

Carlos Eugenio Delgado, Jessica Morales, Beatriz Salazar, Leidy Acevedo, Johan Bolivar, Sara Saldarriaga, Milena

Cardona, Diego Florez, Steven Rivera, Alejandro Gomez, Adriana Gonzalez, Marleny Gallego, Marcela Arrubla,

Blanca Restrepo, Sven Hammerschmidt, Doracelly Hincapie, Gustavo Gamez

P2.17

Vaccination drives changes in metabolic and virulence profiles of Streptococcus pneumoniae 32

Eleanor Watkins, Caroline Buckee, Bridget Penman, Martin Maiden, Sunetra Gupta

P2.18

The relevance of a novel rapid quantitative assay to detect up to 40 major Streptococcus pneumoniae

serotypes directly in clinical nasopharyngeal and blood specimens 33

Melina Messaoudi, Milen Milenkov, Werner Albrich, Mark Van Der Linden, Monidarin Chou, Mariam Sylla,

Nathalie Richard, Patricia Barreto Costa, Keith P. Klugman, Hubert Endtz, Glaucia Paranhos Baccala, Jean

Noel Telles

P2.19

Streptococcus pneumoniae serotype identification using a rapid, low-cost, automated sequence specific

oligonucleotide typing system 33

Stephen Middle, Andrew Canterbury, Ian Crosby, Peter Maguire

P2.20

A novel multiplex real-time PCR assay for identifying and typing of bacterial meningoencephalitis pathogens:

development and validation on clinical samples 34

Milen Milenkov, Mala Rakoto Andrianarivelo, Mickael Harioly Nirina, Lalaina Rahajamanana, Angelot

Rakotomalala, Liliane Raboba, Annick Robinson, Mélina Messaoudi, Jean-Noël Telles, Hubert Endtz,

Bénédicte Contamin, Glaucia Paranhos-Baccalà

P2.21

Evolution of Streptococcus pneumoniae serotype 14 in Catalonia, Spain, after the introduction of 13-valent

pneumococcal conjugate vaccine (PCV13) (2010–2014) 34

Cristina Esteva, Eva del Amo, Paula Gassiot, Xavier Raga, Carmina Sanjose, Mar O Perez, Pilar Ciruela, Marina

Cunquero, Irene Latorre, Carmen Muñoz-Almagro, Catalan Study Group Invasive Pneumococcal Disease

P2.22

Conjugate vaccine introduction in Portugal was followed by a decrease of serotypes 6A and 6E but not of

serotypes 6B and 6C 35

Jorge Diamantino-Miranda, Sandra Isabel Aguiar, João André Carriço, José Melo-Cristino, Mário Ramirez

P2.23

PCV13 serotypes were the major constituents of the major clones causing adult IPD in Portugal in the

conjugate vaccine era 35

Andreia Neves Horácio, Catarina Silva-Costa, Jorge Diamantino-Miranda, Joana Pimento Lopes, Mário Ramirez,

José Melo-Cristino

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P2.24

The identification of pneumococci by lytA-based identification methods may retrieve false results 36

Débora A. Tavares, Alexandra S. Simões, Hermínia de Lencastre, Raquel Sá-Leão

P2.25

Characterisation of the blp locus of co-colonising pneumococci and its impact on co-colonisation 36

Carina Valente, Suzanne Dawid, Hermínia de Lencastre, Raquel Sá-Leão

P2.26

Molecular surveillance on Streptococcus pneumoniae carriage in the Netherlands: does the pneumococcus

change its niche preference with increasing host age? 37

Anne L. Wyllie, Nynke Y. Rots, Jody van Engelsdorp Gastelaars, Lidewij W. Rümke, Jacob P. Bruin, Elisabeth A.M.

Sanders, Krzysztof Trzciński

P2.27

Specificity and sensitivity of molecular methods used for detection of Streptococcus pneumoniae and

pneumococcal serotypes 38

Anne L. Wyllie, Arie van der Ende, Yvonne Pannekoek, Karin Elberse, Jody van Engelsdorp Gastelaars, James A.

Groot, Kayleigh Arp, Debby Bogaert, Elisabeth A.M. Sanders, Krzysztof Trzciński

P2.28

Molecular surveillance on nasopharyngeal carriage of Streptococcus pneumoniae in children vaccinated

with conjugated polysaccharide pneumococcal vaccines 38

Anne L. Wyllie, Alienke J. Wijmenga-Monsuur, Marlies van Houten, Astrid A.T.M. Bosch, James A. Groot, Jody

van Engelsdorp Gastelaars, Jacob P. Bruin, Debby Bogaert, Nynke Y. Rots, Elisabeth A.M. Sanders, Krzysztof

Trzciński

P2.29

PatA/PatB: a multidrug ABC transporter from Streptococcus pneumoniae energised by GTP hydrolysis 39

Benjamin Duchene, Emilie Boncoeur, Sandrine Aros-Calt, François Fenaille, Thierry Vernet, Jean-Michel Jault,

Claire Durmort

P2.30

Penicillin and erythromycin resistance among pneumococci carried by young children in Portugal: evolution

over a 15-year period 39

Sofia Félix, Sónia Nunes, Carina Valente, Ana C. Paulo, Alexandra S. Simões, Sónia T. Almeida, Débora A. Tavares,

António Brito-Avô, Hermínia de Lencastre, Raquel Sá-Leão

P2.31

Auranofin-PLGA nanoparticles as an alternative therapeutic tool against pneumococcal infections 40

Esther García-Fernández, Roberto Díez-Martínez, Miguel Manzano, Ángel M. Martínez, María Vallet-Regí,

Pedro García

P2.32

P4 immunotherapy augments neutrophil bacterial killing in patients admitted to critical care with severe

community acquired pneumonia 40

Suzanna Gore, Ben Morton, Mathieu Bangert, Emma Dearing, Robert Parker, Ingeborg Welters, Angie Wright,

Gowrisankar Rajam, Edwin Ades, Stephen Gordon, Aras Kadioglu

P2.33

Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli 41

Emily Kay, Laura Yates, Vanessa Terra, Jon Cuccui, Brendan Wren

P2.34

A novel cross protective whole cell inactivated pneumococcal vaccine 41

Rachelle Babb, Austen Chen, Abiodun D Ogunniyi, Tim Hirst, Mohammed Alsharifi, James Paton

P2.35

Development of a novel, heat shock protein enriched pneumococcal vaccine 42

Win Yan Chan, Paola Cecchini, Claire Entwistle, Christopher Bailey, Jun Wheeler, Jeremy Brown

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P2.36

Use of Δ6PLY as an immunomodulator when fused to antigens of unrelated species Mannheimia haemolytica 42

Ricardo Corona-Torres, Andrea Mitchell, Jenny Herbert, Tim Mitchell

P2.37

Pneumolysin toxoid as a vaccine adjuvant 43

Cassandra L Krone, Jenny Herbert, Andrea Mitchell, David R Withers, Tim J Mitchell

P2.38

Clonal expansion of non-susceptible non-vaccine pneumococci in non-invasive isolates following conjugate

vaccine introduction results in similar rates of antimicrobial non-susceptibility to the pre conjugate vaccine era 43

Martha McElligott, Imelda Vickers, Mary Meehan, Mary Cafferkey, Robert Cunney, Hilary Humphreys

P2.39

Salmonella outer membrane vesicles displaying high densities of pneumococcal antigen at the surface offer

protection against colonisation 44

Kirsten Kuipers, Maria Daleke-Schermerhorn, Wouter Jong, Corinne ten Hagen-Jongman, Fred van Opzeeland,

Elles Simonetti, Christa van der Gaast, Aldert Zomer, Joen Luirink, Marien de Jonge

P2.40

Calibration of an individual-based transmission model to pre-and post-PCV pneumococcal carriage 44

Marc Lipsitch, Thomas Fussell, Sarah Cobey, Daniel Weinberger

P2.41

Evaluation of the efficacy of the immunisation with PspA in a co-colonisation model in mice 45

Rafaella Tostes, Maria Leonor Oliveira, Paulo Ho, Eliane Miyaji

P2.42

New protein vaccines against pneumococcal pneumonia using experimentally induced immunity 45

Jessica Owugha, Angela Wright, Andrea Collins, Cintia Vadesilho, Eliane Miyaji, Kondwani Jambo, Stephen

Gordon, Daniela Ferreira

P2.43

Novel recombinant glycoconjugate vaccines for prevention of pneumococcal meningitis 46

Charlie Plumptre, Emily Kay, James Paton, Brendan Wren, Jeremy Brown

P2.44

Broadly cross-reactive antibodies recognising the proline-rich region of pneumococcal surface protein A

variants show cross-reactivity with skeletal muscle 46

Zoltan Magyarics, Harald Rouha, Adriana Badarau, Nels Nielson, Marisa Caccamo, Susanne Weber, Barbara

Maierhofer, Katharina Havlicek, Ivana Dolezilkova1, Karin Gross, Eszter Nagy

P2.45

Structural analysis of the choline-binding sites in CbpL from Streptococcus pneumoniae 47

Javier Gutiérrez-Fernández, Martín Alcorlo Pagés, Malek Saleh, Sergio G. Bartual, Thomas Pribyl, Sven

Hammerschmidt, Juan A. Hermoso

P2.46

Assessment of the specific role of the phosphorylcholine esterase Pce in the modification of pneumococcal

teichoic acids 47

Franziska Waldow, Thomas Kohler, Dominik Schwudke, Sven Hammerschmidt, Nicolas Gisch

Oral abstracts

O1.1

Identification of pneumococcal colonisation determinants in the stringent response pathway, facilitated by

genomic diversity 48

Yuan Li, Nicholas J. Croucher, Claudette M. Thompson, Krzysztof Trzcinski, William P. Hanage, Marc Lipsitch

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O1.2

Genomics reveals the worldwide distribution of multidrug-resistant serotype 6E pneumococci 48

Andries van Tonder, James Bray, Lucy Roalfe, Rebecca White, Marta Zancoli, Sigríður Quirk, Gunnstein

Haraldsson, Keith Jolley, Martin Maiden, Stephen Bentley, Ásgeir Haraldsson, Helga Erlendsdóttir, Karl

Kristinsson, David Goldblatt, Angela Brueggemann

O1.3

The post-vaccine microevolution of invasive pneumococcal disease 49

Fredrick M. Mobegi, Amelieke J.H. Cremers, Marien I. de Jonge, Sacha A.F.T. van Hijum, Jacques F. Meis,

Peter W.M Hermans, Gerben Ferwerda, Stephen D. Bentley, Aldert Zomer

O1.4

Mechanisms of pneumococcal evolution over short and long timescales 49

Nicholas Croucher, Paul Coupland, Abbie Stevenson, Allanna Callendrello, Stephen Bentley, William Hanage

O1.5

Genetic stabilisation of the pandemic and drug resistant PMEN1 pneumococcus lineage by its distinctive

DpnIII restriction-modification system 50

Rory Eutsey, Evan Powell, Janina Dordel, Tyson Clark, Jonas Korlach, Garth Ehrlich, N. Luisa Hiller

O2.1

MapZ beacons the division sites and positions FtsZ-rings in Streptococcus pneumoniae 50

Aurore Fleurie, Christian Lesterlin, Sylvie Manuse, Chao Zhao, Caroline Cluzel, Jean-Pierre Lavergne, Mirita

Franz-Wachtel, Boris Macek, Christophe Combet, Erkin Kuru, Michael Van Nieuwenhze, Yves Brun, David

Sherratt, Christophe Grangeasse

O2.2

Competence for genetic transformation in Streptococcus pneumoniae: Primary sigma factor mutations

enhance transcription of late genes in comW mutants 51

Yanina Tovpeko, Donald A. Morrison

O2.3

A new role for Autoinducer-2 in Streptococcus pneumoniae 51

Claudia Trappetti, Bart Eijkelkamp, Zarina Amin, Adrienne Paton, Christopher McDevitt, Marco Oggioni, James

Paton

O2.4

Spatial and dynamic organisation of the chromosome and DNA replication machinery in Streptococcus

pneumoniae 52

Morten Kjos, Renske van Raaphorst, Jan-Willem Veening

O2.5

Repressor of iron transport regulator (RitR) is a novel cysteine-activated redox sensor in Streptococcus

pneumoniae 52

David Wright, Andrew Maule, Lanlan Han, Nicholas Silvaggi, Nicholas Croucher, Stephen Bentley, Thomas Clarke,

Andrew Ulijasz

O3.1

Cpl-711, a powerful enzyme against pneumococci 53

Roberto Díez-Martínez, Héctor D. de Paz, Esther García Fernández, Noemí Bustamante, Chad W. Euler, Vincent

A. Fischetti, Margarita Menéndez, Pedro García

O3.2

Esters of bicyclic amines: a new generation of antimicrobials against pneumococcus 53

M. de Gracia Retamosa, Roberto Diez-Martinez, Beatriz Maestro, Esther Garcia-Fernandez, Bas de Waal, E. W.

Meijer, Pedro Garcia, Jesus Sanz

O3.3

Identification of new cell wall biogenesis factors in Streptococcus pneumoniae using Tn-Seq 54

Andrew Fenton, Thomas Bernhardt, David Rudner

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O3.4

Engineered liposomes sequester pneumolysin and protect from severe invasive pneumococcal disease in mice 54

Daniel Neill, Suzanna Gore, Laura Bricio Moreno, Eduard Babiychuk, Aras Kadioglu

O4.1

DNA-release by Streptococcus pneumoniae autolysin LytA induced Krueppel-like factor 4 expression

controlling pneumococci-related innate immune response in macrophages 55

Janine Zahlten, Toni Herta, Christin Kabus, Jan-Moritz Doehn, Pedro García, Norbert Suttorp, Stefan Hippenstiel

O4.2

The pneumococcal whole cell vaccine reduces influenza-induced pneumococcal disease in the ears and

lungs of co-infected infant mice 55

Jayne Manning, Eileen Dunne, Kim Mulholland, Roy Robins-Browne, Richard Malley, Odilia Wijburg, Catherine

Satzke

O4.4

The effect of macrophage polarisation and delayed apoptosis in the innate immune response to

Streptococcus pneumoniae infection 56

Lucy Morris, Helen Marriott, David Dockrell

O4.5

Murine respiratory tract microbiome: important interactions with IL-17 and pneumococcal colonisation 56

Neil Ritchie, Tom Evans

O5.1

Zinc homeostasis in Streptococcus pneumoniae during disease 57

Bart Eijkelkamp, Charles Plumptre, Jacqueline Morey, Stephanie Begg, James Paton, Christopher McDevitt

O5.2

The pneumococcal MgaSpn virulence transcriptional regulator 57

Virtu Solano-Collado1, Rudi Lurz2, Manuel Espinosa1, Alicia Bravo

O5.3

Genomic analyses of pneumococci reveal a wide diversity of bacteriocins, including pneumocyclicin—a

novel circular bacteriocin 58

Carlijn Bogaardt, Andries van Tonder, Angela Brueggemann

O5.4

Antibiotic-induced bacteriocin expression—regulatory interplay between the blp and com systems in

Streptococcus pneumoniae 58

Morten Kjos, Eric Miller, Frank Lake, Daniel E. Rozen, Jan-Willem Veening

O5.5

Conserved Streptococcus pneumoniae spirosomes point toward a single type of transformation pilus in

competence 59

Petya V. Krasteva, Raphael Laurenceau, Amy Diallo, Sahra Ouarti, Magalie Duchateau, Christian Malosse, Julia

Chamot-Rooke, Remi Fronzes

O6.1

Pathogenesis of nonencapsulated Streptococcus pneumoniae in experimental otitis media 59

Larry McDaniel

O6.2

Modulation of nasopharyngeal innate defences by viral co-infection predisposes individuals to

experimental pneumococcal carriage 60

Sarah Glennie, Jenna F Gritzfeld, Shaun H Pennington, Michael Garner-Jones, Nicholas Coombes, Mark J

Hopkins, Cintia F Vadesilho, Eliane N Miyaji, Duolao Wang, Angela D Wright, Andrea M Collins, Stephen

B Gordon, Daniela M Ferreira

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O6.3

Circulating pneumolysin is a potent inducer of cardiac injury during pneumococcal infection 60

Daniel Neill, Yasir Alhamdi, Simon Abrams, Hesham Malak, Richard Barrett-Jolley, Gouzheng Wang,

Cheng-Hock Toh, Aras Kadioglu

O6.4

Streptococcus pneumoniae invades the heart during severe pneumonia in a non-human primate model 61

Luis Reyes, Nilam Soni, Cecilia Hinojosa, Jessica Perry, Robert Shade, Melissa de la Garza, Luis Giavedoni, Marcos

Restrepo, Carlos Orihuela

O6.5

Inflammation dampening effects of pneumolysin 61

Jimstan Periselneris, Tracey Pollard, Thomas James, Mahdad Noursadeghi, Jeremy Brown

O6.6

A phase-variable genetic switch regulates pneumococcal virulence via epigenetic changes 62

Ana Manso, Chai Melissa, John Atack, Leonardo Furi, Megan De Ste Croix, Richard Haigh, Claudia Trappetti,

David Ogunniyi, Lucy Shewell, Mathew Boitano, Tyson Calrke, Jonas Korlach, Matt Blades, Evgeny Mirkes,

Alexander Gorban, James Paton, Mike Jennings, Marco Oggioni

O7.1

Pneumococcal conjugate vaccine reduces the rate, density, and duration of experimental human

pneumococcal colonisation: first human challenge testing of a pneumococcal vaccine—a double blind

randomised controlled trial 62

Jenna Gritzfeld, Andrea Collins, Angela Wright, Elena Mitsi, Carole Hancock, Shaun Pennington, Duolao Wang,

Ben Morton, Daniela Ferreira, Stephen Gordon

O7.2

Heterologous protection against Streptococcus pneumoniae colonisation by the mucosal adjuvant cholera

toxin subunit B 63

Kirsten Kuipers, Dimitri Diavatopoulos, Corné van den Kieboom, Fred van Opzeeland, Elles Simonetti, Dorette

van Ingen-Schenau, Mariska Kerstholt, Malgorzata Borczyk, Mihai Netea, Marien de Jonge

O7.3

In vivo efficacy of recombinant protein polysaccharide conjugate pneumococcal vaccines 63

Jenny Herbert, Emily Kay, Jon Cuccui, Vanessa Terra, Brendan Wren, Timothy Mitchell

O7.4

Immune responses to pneumococcal vaccination in HIV-infected adults in the UK 64

Sian Faustini, James Hodson, Sindiso Masuko, Mebie Singo, Joyful Chigiga, Jenny Herbert, Timothy Mitchell,

Tim Plant, Mark Drayson, Kaveh Manavi, Calman MacLennan, Alex Richter

O7.5

The agglutinating effects of anti-capsular antibody contribute to pneumococcal clearance 64

Aoife Roche, Jeffrey Weiser

O8.1

PBP2b, MreD and DivIVA constitute a functional unit in the peripheral peptidoglycan synthesis machinery

of Streptococcus pneumoniae 65

Daniel Straume, Kari Helene Berg, Gro Stamsås, Leiv Sigve Håvarstein

O8.2

Conformational plasticity of choline-binding modules: β-hairpin to α-helix transition of choline-binding

repeats triggered by detergent micelles and membrane vesicles 66

Hector Zamora-Carreras, Beatriz Maestro, Erik Strandberg, Anne Ulrich3, Jesus Sanz, M. Angeles Jimenez

O8.3

On the interaction between the pneumococcal cell wall and the choline-binding modules (CBMs): evaluation

of binding affinities and effect of externally added CBMs to bacterial cultures 66

Manuel Sanchez, Beatriz Maestro, Francisco Garcia-Asencio, Jesus Sanz

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O8.4

Conditional lethal mutants reveal that FtsA is needed at early and late stages of cell division in Streptococcus

pneumoniae 67

Andrea Mura, Daniela Musu, Ana Isabel Rico, Marcin Krupka, Dalia Denapaite, Pavel Branny, Miguel Vicente,

Orietta Massidda

O9.1

Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae 67

Sergio G. Bartual, Daniel Straume, Gro Anita Stamsås, Inés Muñoz, Carlos Alfonso, Martín Martínez-Ripoll,

Leiv Sigve Håvarstein, Juan A. Hermoso

O9.2

Single molecule force spectroscopy reveals interaction strength between Streptococcus pneumoniae TIGR4

pilus-1 tip protein RrgA and human fibronectin 68

Tanja Becke, Stefan Ness, Raimund Gürster, Stefanie Sudhop, Arndt F. Schilling, Anne Marie di Guilmi, Hauke

Clausen-Schaumann, Markus Hilleringmann

O9.3

Structural basis for selective recognition of microbial and endogenous polysaccharides by SIGN-R1 receptor 68

Iván Acebrón, Noella Silva-Martín, Sergio G. Bartual, Erney Ramírez-Aportela, Pablo Chacón, Chae Gyu Park,

Juan A. Hermoso

O9.4

New insights into structure, biosynthesis and pro-inflammatory potential of pneumococcal teichoic acids 69

Nicolas Gisch, Thomas Kohler, Franziska Waldow, Dominik Schwudke, Holger Heine, Regine Hakenbeck, Dalia

Denapaite, Sven Hammerschmidt

O9.5

LocZ is a new cell division protein that directs septum placement in Streptococcus pneumoniae 69

Nela Holecková, Linda Doubravová, Orietta Massidda, Virginie Molle, Karolína Buriánková, Oldrich Benada,

Olga Kofronová, Aleš Ulrych, Pavel Branny

O10.1

Streptococcus pneumoniae interaction with brain derived neural cells and alterations in functional state of

the cells 70

Reuven Afriat, Marilous Shagan, Ron Dagan, Esther Priel, Yaffa Mizrachi Nebenzahl

O10.2

PSGL-1 receptor on leukocytes is a critical component of the host immune response against invasive

pneumococcal disease 70

Elisa Ramos-Sevillano, Ana Urzainqui, Miriam Domenech, Rafael González-Tajuelo, Fernando González-Camacho,

Francisco Sánchez-Madrid, Jeremy S Brown, Ernesto García, José Yuste

O10.3

Pneumococcal adhesins PavB and PspC are important for the interplay with human thrombospondin-1 71

Ulrike Binsker, Thomas P. Kohler, Krystin Krauel, Sylvia Kohler, Hansjörg Schwertz, Sven Hammerschmidt

O10.5

Mcl–1 regulates mitochondrial ROS-mediated bacterial killing of Streptococcus pneumoniae in macrophages

and defines susceptibility to pulmonary infection in COPD 71

Martin Bewley, Julie Preston, Richard Budd, Dave Singh, Peter Barnes, Louise Donnelly, Steve Shapiro, Moira

Whyte, David Dockrell

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Editorial

Progress towards understanding the pathology of the

pneumococcus

Amanda J Cox a,b , Angela B Brueggemann c , Timothy Mitchell d , Jeremy S Brown e

a

Menzies Health Institute Queensland, Southport, QLD, Australia; b School of Medical Science, Griffith University, Southport, QLD, Australia; c Nuffield

Department of Medicine, Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, United Kingdom; d School of Immunology

and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom; e Centre for Respiratory Research,

Department of Medicine, University College London, London, United Kingdom

Corresponding author: Amanda Cox, Microbiology and Immunology Research Group, Menzies Health Institute Queensland, Parklands Drive,

Southport, Queensland 4222, Australia. Phone: +61 7 5678 0898. Email: a.cox@griffith.edu.au

Author contributions: AC wrote the first draft of the manuscript. JB, AB, TM critically reviewed the manuscript for important intellectual content. All

authors agree with the manuscript results and conclusions. All authors approved the final version of the manuscript.

Received 21 July 2015; Accepted 13 August 2015; Published 21 October 2015

Citation: Cox AJ, Brueggemann AB, Mitchell T, Brown JS. Progress towards understanding the pathology of the pneumococcus. pneumonia

2015;7:1–2

Keywords: Streptococcus pneumoniae, pneumonia, molecular biology, pathogenesis, respiratory disease

Despite the introduction of the childhood pneumococcal

vaccines in 2000 and their next-generation successors

in 2010, pneumonia remains the leading infectious

cause of death in children less than 5 years of age.

Similarly, pneumococcal infections in the elderly and

immunocompromised continue to represent a significant

healthcare burden; abatement is unlikely given expanding

elderly populations, particularly in Western countries.

The 12th European Meeting on the Molecular Biology of the

Pneumococcus (EuroPneumo) held in July 2015 in Oxford

was attended by 174 scientists from 20 countries and

provided an excellent forum for consideration of

these and other issues relating to pneumococcal

disease. The conference covered the whole range

of Streptococcus pneumoniae biology, ranging

through basic bacterial processes (e.g. cell wall

synthesis), structural biology, antibiotic resistance

and novel treatment approaches, understanding

host–pathogen interactions during carriage and disease,

the use of “–omics” technologies to characterise

pneumococcal diversity, molecular epidemiology,

immunology and novel vaccine development. The

conference abstracts published in this issue support the

continued research focus around S. pneumoniae to inform

improved treatment and management of pneumococcal

disease in at risk populations.

As an encapsulated diplococcus, the nearly 100 identified

S. pneumoniae serotypes represent a considerable source

of antigen diversity that remains a challenge in vaccine

development. While pneumococcal disease has been

attributed to a limited number of dominant serotypes,

patterns of carriage and serotype replacement postvaccination

continue to be documented, particularly

in developing nations and geographically isolated

populations. Ongoing characterisation of serotype

switching remains important for further vaccine evolution

and presented data revealed options to pursue further in

this context. Of particular interest were the data relating

to serotype-independent whole-cell vaccines [1,2] and the

development of an experimental pneumococcal human

colonisation model for assessing vaccine efficacy [3], which

also offers promise in the assessment of host–pathogen

interaction.

The role of the polysaccharide capsule as a major virulence

factor of S. pneumoniae underpins ongoing efforts to better

understand regulation of capsule synthesis. However,

non-encapsulated pneumococcal infection can cause

mucosal infection, and data presented at the conference

suggested the virulence of these strains is dependent on

protein adhesins [4]. Other determinants of streptococcal

virulence also continue to receive attention, including an

elegant characterisation of the molecular interactions

between microbial pili and the host extracellular matrix

using atomic force microscopy [5]. The biological

relevance of the substantial amount of genome variation

between S. pneumoniae strains remains unclear; hence

characterisation of phage-mediated genomic alterations in

S. pneumoniae [6,7] was of particular interest and perhaps

an important component driving S. pneumoniae evolution

over time and associated variation in pathogenicity

between strains.

Further insights into pneumococcal virulence

may also be garnered from consideration of the

host–pathogen interaction in both stable carriage and

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overt pneumococcal disease. Increasing data demonstrate

an important role of the respiratory microbiome in

shaping local immune responses to S. pneumoniae at the

mucosal surfaces [8], and these effects are likely to have

wide-reaching consequences during carriage and disease

development. The importance of the interplay between

both bacterial and host factors in determining virulence

was nicely demonstrated through the reported use of

the less pathogenic S. mitis to assess host response in

comparison to S. pneumoniae [9]. Likewise, the potential

for the S. pneumoniae toxin pneumolysin to subvert the

early host inflammatory response [10,11] and cause direct

cardiac damage [12] confirms that bacterial-mediated

disruption of host cells can also be a key determinant in

the pathogenesis of infection.

After a conference with such varied content, what can

we conclude about future directions for research into S.

pneumoniae? Firstly, S. pneumoniae biology remains a

highly energetic and innovative field with an increasing rate

of novel and exciting discoveries. Secondly, and perhaps

paradoxically, the more we learn about S. pneumoniae

the more apparent it becomes how much more we

need to know. For example, genome sequencing has

identified much larger genetic variation between strains

than expected [13], and the effects of the microbiome

has added an additional layer of complexity to assessing

host immunological responses to S. pneumoniae [8].

Considerably more research will be required if we are

to state with confidence why S. pneumoniae is a highly

successful pathogen and before we learn how to prevent it

from causing such a huge level of morbidity and mortality

across the globe.

FFunding: AB is supported by funding from the Wellcome Trust

Biomedical Research Fund (Grant 04992/Z/14/Z), Wellcome Trust Career

Development Fellowship (Grant 83511/Z/07/Z), GlaxoSmithKline (GSK)

Biologicals (Grant HBRYVH00) and the University of Oxford John Fell

Fund (Grant 123/734). TM is supported by funding from the Wellcome

Trust Medical Research Council (Grant WT094762MA) and Department

of Health, UK (Grant MR/K012053/1). JB is supported by funding from

the Medical Research Council UK, Biotechnology and Biological Sciences

Research Council, National Health and Medical Research Council (Grant

1034029), Rosetrees Trust, Research Council of Norway, Meningitis Now

and the Department of Health’s National Institute for Health Research

(NIHR) Biomedical Research Centre. The funders had no role in study

design, collection and analysis of data, decision to publish, or writing of

the manuscript.

Competing interests: JB has received consultancy fees from

ImmunoBiology Ltd and Pfizer. AB has received grant support from GSK.

All other authors declare no competing interests.

JB, AB, TM were organisers of the EuroPneumo Conference.

Provenance and peer review: Commissioned; internally peer

reviewed.

Copyright: This is an open-access article distributed under the terms of

the Creative Commons Attribution License, which permits unrestricted

use, distribution, and reproduction in any medium, provided the original

author and source are credited.

References

1. Manning J, Dunne E, Mulholland K, Robins-Browne R,

Malley R, Wijburg O, et al. The pneumococcal whole cell

vaccine reduces influenza-induced pneumococcal disease

in the ears and lungs of co-infected infant mice [Abstract

EuroPneumo - O4.2]. pneumonia. 2015;7:55

2. Babb R, Chen A, Ogunniyi AD, Hirst T, Alsharifi M, Paton J. A

novel cross protective whole cell inactivated pneumococcal

vaccine [Abstract EuroPneumo - P2.34]. pneumonia.

2015;7:41

3. Gritzfeld J, Collins A, Wright A, Mitsi E, Hancock C,

Pennington S, et al. Pneumococcal Conjugate Vaccine

Reduces the Rate, Density and Duration of Experimental

Human Pneumococcal Colonisation: First Human Challenge

Testing of a Pneumococcal Vaccine - A double blind

randomised controlled trial [Abstract EuroPneumo - O7.1].

pneumonia. 2015;7:62

4. McDaniel L. Pathogenesis of nonencapsulated

Streptococcus pneumoniae in experimental otitis media

[Abstract EuroPneumo - O6.1]. pneumonia. 2015;7:59

5. Becke T, Ness S, Gurster R, Sudhop S, Schilling AF, di Guilmi

AM, et al. Single molecule force spectroscopy reveals

interaction strength between Streptococcus pneumoniae

TIGR4 pilus-1 tip protein RrgA and human fibronectin

[Abstract EuroPneumo - O9.2]. pneumonia. 2015;7:68

6. Norman M, Syk A, Browall S, Henriques-Normark B.

Contribution of a temperate bacteriophage to the virulence

of a Streptococcus pneumoniae serotype 1 strain [Abstract

EuroPneumo - P1.22]. pneumonia. 2015;7:14

7. Harold CL van Tonder AJ, McDonnel A, Edwards BA,

Brueggemann AB. Investigating the pneumococcal

phageome using a diverse genome dataset [Abstract

EuroPneumo - P1.29]. pneumonia. 2015;7:18

8. Bogaert D. An ecological perspective on symbiosis

between Streptococcus pneumoniae and the host [Abstract

EuroPneumo - PL0.4]. pneumonia. 2015;7:4

9. Marshall HE, Petersen FC, Brown JS. Differential

complement sensitivity of Streptococcus pneumoniae

and Streptococcus mitis [Abstract EuroPneumo - P2.07].

pneumonia. 2015;7:27

10. Periselneris J, Pollar T, James T, Noursadeghi M, Brown J.

Inflammation dampening effects of pneumolysin [Abstract

EuroPneumo - O6.5]. pneumonia. 2015;7:61

11. Gilley R, Shenoy A, Gonzalex-Juarbe, Orihuela C.

Streptococcus pneumoniae cardiac microlesions:

pneumolysin-mediated immune evasion and biofilm growth

[Abstract EuroPneumo - P1.23]. pneumonia. 2015;7:15

12. Neill D, Alhamdi Y, Abrams S, Malak H, Barrett-Jolley R,

Wang G, et al. Circulating pneumolysin is a potent inducer

of cardiac injury during pneumococcal infection [Abstract

EuroPneumo - O6.3]. pneumonia. 2015;7:60

13. Bogaardt C, van Tonder A, Brueggemann A. Genomic

analyses of pneumococci reveal a wide diversity of

bacteriocins, including pneumocyclicin—a novel circular

bacteriocin [Abstract EuroPneumo - O5.3]. pneumonia.

2015;7:58

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Plenary abstracts

PL.01

Penicillin resistance in Streptococcus pneumoniae: evolution by genetic

communication

Regine Hakenbeck

Department of Microbiology, University of Kaiserslautern, Germany

In the early 80s it was discovered that penicillin-binding proteins (PBPs) are encoded by mosaic genes in penicillin

resistant clinical isolates of Streptococcus pneumoniae as a result of gene transfer followed by recombination. It was

soon recognised that commensal species are part of the game and might serve as potential donors for the mosaic

sequences. In contrast, laboratory mutants resistant to beta-lactams contain point mutations in PBP genes, but also in

other non-PBP genes. Where are we now? The astounding speed of rapidly evolving sequencing technologies has paved

the way to extend our view from mosaic genes to interspecies gene transfer events affecting the entire genomes of

the streptococcal world. I will also discuss molecular mechanisms contributing to beta-lactam resistance, based on the

interplay of altered PBPs and other enzymes affecting the biosynthetic machinery of the bacterial cell wall.

PL.02

Structure and assembly of the cell wall in Streptococcus pneumoniae

Waldemar Vollmer

The Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, UK

The cell wall protects the bacterial cell from bursting due to its turgor, and maintains cell shape. The pneumococcal

cell wall has a remarkably complex structure. The main component, peptidoglycan, carries several covalently attached

macromolecules, including wall teichoic acid, capsular polysaccharide and cell surface proteins. Secondary structural

modifications in the peptidoglycan, like N-deacetylation and O-acetylation of the glycan chains, and the D-alanylation

of teichoic acids contribute to the resistance of pneumococci against lysozyme and cationic antimicrobial peptides.

Peptidoglycan and wall teichoic acid are also linked together by strong ionic interactions via divalent cations. So far we

have gained only limited insights into the molecular mechanisms of pneumococcal cell wall synthesis, and the processes

regulating cell wall synthesis during growth and division are largely unknown. Cell wall polymers are synthesised at

the bacterial membrane and inserted into the existing wall. In the last steps phosphotransferases of the LytR-Cps2A-

Psr family of enzymes (LCP enzymes) ligate the wall teichoic acid and capsular polysaccharides to the glycan chains of

peptidoglycan to assemble the final cell wall structure. Cell wall growth is temporarily and spatially regulated to maintain

the characteristic ovoid cell shape and to reliably divide the cell into two daughters. Lactic acid bacteria including

pneumococci lack the cell elongation protein MreB which is present in many rod-shaped bacteria. The insertion of

new material occurs at or near mid-cell where two modes of cell wall synthesis, one for elongation and another for cell

division, take place simultaneously to produce the elongated, ovococcal cell shape.

PL.03

Immuno-regulatory control of host susceptibility to pneumococcal

carriage and invasive disease

Aras Kadioglu

University of Liverpool, Liverpool, UK

Pneumococcal nasopharyngeal carriage is considered to be a prerequisite for invasive disease, but the majority of carriage

episodes are asymptomatic and self-resolving. Interactions determining the development of carriage versus invasive

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disease, and in particular the immunological mechanisms underpinning nasopharyngeal carriage, are poorly understood

but are crucial to deciphering why and how carriage may lead to invasive disease as well as the effectiveness of vaccines

or therapeutics. I will describe the mechanisms by which Streptococcus pneumoniae colonise the nasopharynx without

inducing damaging host inflammation and provide insight into the role of bacterial and host constituents that allow

and maintain stable carriage, while also describing the factors whose perturbation lead to invasive disease. Similarly, I

will discuss our current understanding of the role of immune regulation during invasive disease and how these factors

contribute to host susceptibility. Overall, I will highlight the delicate balance between the requirements of the host

to prevent inflammatory tissue damage with the need to combat potentially dangerous pathogen colonisation. This

equilibrium needs to be considered carefully in the future design of anti-pneumococcal therapeutics and vaccines which

may alter the density of pneumococcal carriage and hence affect upper airway immune responses.

PL.04

An ecological perspective on symbiosis between Streptococcus

pneumoniae and the host

Debby Bogaert

UMCU-WKZ, The Netherlands

Respiratory tract infections are a major global health concern, accounting for high morbidity and mortality, especially in

young children and elderly individuals. Traditionally, it is thought that bacterial respiratory tract infections, including otitis

media and pneumonia, are caused by a limited number of pathogens like Streptococcus pneumoniae, and Haemophilus

influenzae. The ecological niche for these potential pathogens is the upper respiratory tract of humans. They commonly

reside here asymptomatically, and at equilibrium with their environment and the host. It is still unclear why in one

individual an infection may develop, and in the other one not. However, over the last years the importance of the upper

respiratory tract (URT) microbiota in maintaining respiratory health has become more apparent. Analogous to the gut

microbiome, the respiratory microbiome is thought to be beneficial to the host by priming the immune system and

providing colonisation resistance. In contrast, an imbalanced ecosystem lacking keystone bacteria might predispose to

bacterial overgrowth, dissemination and consecutive respiratory infections. Recently, we have characterised the upper

respiratory microbiota of a large group of individuals, young and old, during health and disease. We obtained evidence for

the existence of different respiratory microbiota profiles, related to environmental drivers, to stability of the ecosystem

and susceptibility to respiratory infections. Several of these profiles are positively or negatively related to streptococcal

colonisation in general, and S. pneumoniae colonisation in particular. In this lecture I will present the current body of

evidence regarding the position of S. pneumoniae within the nasopharyngeal and oropharyngeal microbial ecosystem,

and discuss the current ecological theory regarding pathogenesis of respiratory infections.

Poster abstracts

P1.01

Growth phase regulates the localisation and activity of the

pneumococcal autolysin LytA

Julie Bonnet, Maxime Jacq, Justine Cartannaz, Cécile Morlot, Thierry Vernet, Claire Durmort, Anne

Marie Di Guilmi

Institut de Biologie Structurale, Grenoble, France

In Streptococcus pneumoniae, the choline-binding proteins (CBPs) are exposed at the cell surface through association to

phosphocholine residues (PCho), which decorate the cell wall teichoic acids. Among them, LytA is an N-acetylmuramoyl-

L-alanine amidase responsible for the pneumococcal autolysis in late stationary phase. Many aspects of LytA mechanism

remain unknown, such as the molecular mechanism of release, the localisation pattern as well as the regulation of

amidase activity. We have developed a version of the superfolder Green Fluorescent Protein (sfGFPop) appropriate

to investigate the localisation of secreted proteins at the surface of S. pneumoniae. The fusion protein LytA–sfGFPop

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has been ectopically expressed in the pneumococcus and live fluorescence microscopy was realised to determine the

spatio-temporal localisation of LytA. The results showed that LytA shifts from a homogeneous cytoplasmic localisation

during the exponential growth phase to a heterogeneous cytoplasmic localisation when bacterial cell population enters

the stationary growth phase. Finally, we observed the extracellular presence of LytA at the septum site along the lytic

phase. Respective roles of the choline-binding and amidase domains of LytA were analysed in this relocalisation process.

We determined that both domains are required for efficient cell lysis and surface localisation of LytA, which is also

linked to the presence of CbpE that regulates the amount of cell wall-associated PCho. In stationary growth phase,

cell death occurs before lysis, which suggests that LytA could be released by membrane disruption. Moreover, LytA

specifically cleaves the peptidoglycan at the septal sites only when cells enter the stationary growth phase. Studying cell

physiology at the early stationary phase will allow deciphering the mechanisms underlying these processes. Preliminary

data suggest that the regulation of LytA-mediated cell lysis occurs through the substrate availability. Characterisation of

such peptidoglycan modification is currently under investigation.

P1.02

DiiA is a newly identified cell wall protein of Streptococcus pneumoniae

involved in invasive disease

María de la Soledad Escolano Martínez 1, 2 , Arnau Domenech 2, 3 , José Yuste 1, 2 , María I. Cercenado 1 ,

Carmen Ardanuy 2, 3 , Josefina Liñares 2, 3 , Adela G. de la Campa 1, 2 , Antonio J. Martin-Galiano 1, 2

1

Instituto de Salud Carlos III, Centro Nacional de Microbiología, Majadahonda, Madrid, Spain; 2 CIBER de Enfermedades Respiratorias (CIBERES),

Madrid, Spain; 3 Servicio de Microbiología, Hospital Universitari de Bellvitge, Universitat de Barcelona, Barcelona, Spain

Many outer multi-domain proteins play fundamental roles in the virulence of bacterial pathogens in an allele-dependent

manner. The hypothetical protein SP1992 of Streptococcus pneumoniae TIGR4 contains (from N- to C-terminus): two

imperfect repeats (R1 and R2), an unstructured region, and a cell-wall anchor domain. In this study we have investigated

560 clinical isolates, demonstrating that the gene coding for DiiA is part of the core genome and may carry either one

(R2) or two (R1R2) repeats. Clonal complexes carrying R1R2 were associated with invasive disease, while those carrying

R2 preferentially affected patients with underlying risk factors. Isogenic strains carrying step-wise deletions of diiA were

constructed to investigate the contribution of the different modules to the infective process. The mutants constructed

were: diiA-R2 that lacks R1 and mimics the natural short allele; diiA-NR that lacks both repeats; and the ΔdiiA null mutant.

Our results show that R1 and R2 are involved in the interaction with lung epithelial cells, and in systemic dissemination

using a mice model of pneumonia infection. Moreover, the ΔdiiA defective strain was severely impaired in its ability

to proliferate in blood in a sepsis model. This strain was also less able to avoid complement-mediated immunity and

phagocytosis. The latter phenotype may be mediated by the binding of the unstructured region to lactoferrin, for which

the DiiA-NR protein had a high affinity for, with a K D

of 2.5 nM. Based on our results, we termed SP1992 DiiA, after

Dimorphic invasion-involved A protein.

P1.03

Induction of meningitis by a non-passaged clinical pneumococcal

isolate in an infant rat model depends on the presence of capsule

Lucy Hathaway, Denis Grandgirard, Belinda Ries, Suzanne Aebi, Luca Valente, Stephen Leib

Institute for Infectious Diseases, University of Bern, Bern, Switzerland

This study aimed to use an established infant rat model of experimental pneumococcal meningitis to study the potential

of a non-passaged clinical pneumococcal isolate to induce disease. A Swiss clinical pneumococcal isolate (106.66) of

serotype 6B and its non-encapsulated mutant (106.66 Janus) were administered intracisternally in an established infant

rat model without prior passage of the bacteria in animals. Bacterial titres were determined in CSF samples to optimise

the model for study of a non-passaged strain and to determine the appropriate time to administer ceftriaxone antibiotic

therapy. Clinical parameters were recorded and CSF sampled to determine bacterial titre and cytokine concentrations.

Brain damage was quantified by histomorphological analysis. A non-passaged clinical pneumococcal isolate was able

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to establish a productive infection and cause meningitis in the model, resulting in significant mortality. Brain damage

(cortical damage, hippocampal apoptosis) and leukocyte pleocytosis were evident from histomorphological analysis. The

non-encapsulated mutant failed to establish an infection or cause any detectable pathology. The presence of capsule

is essential for establishing meningitis in this model. The modified model is appropriate for studying the meningitisinducing

potential of non-passaged clinical isolates.

P1.04

An investigation of the impact of air pollutants on Streptococcus

pneumoniae

Shane Hussey 1, 2 , Peter Andrew 2 , Paul Monks 3 , Julian Ketley 1 , Julie Morrissey 1

1

Department of Genetics, University of Leicester, Leicester, UK; 2 Department of Infection, Immunity and Inflammation, University of Leicester,

Leicester, UK; 3 Department of Chemistry, University of Leicester, Leicester, UK

Despite legislation to reduce air pollution, it is the largest global environmental and health problem we are faced with

in current times and was responsible for 1 in 8 deaths worldwide in 2012. One of its most toxic components, particulate

matter (PM) is responsible for increases in cardiovascular and respiratory diseases and infections. To date this has been

attributed to deleterious and immunomodulatory effects on the human host, including tissue damage, inflammatory

responses, and oxidative stress. However, there has been a major oversight in this field as no studies have investigated the

direct impact of air pollution on bacteria of the human respiratory tract. This may be a serious omission because bacteria

play an essential role in maintaining the health of a host. We have investigated the effect of a specific component of PM

and black carbon (BC), which is a by-product of fossil fuel combustion, on the behaviour of Streptococcus pneumoniae.

S. pneumoniae was chosen as it asymptomatically colonises much of the population whilst retaining the ability to cause

severe disease. Our data show, for the first time, that there is an interaction between air pollutants and bacteria, with

BC inducing species-specific alterations in biofilm formation and structure. Biofilms are vital in both host colonisation

and infection, providing protection from the environment and the host immune system. Biofilm alterations will have

a significant impact on bacterial survival in vivo as well as on the potential for disease progression and transmission.

Therefore our data suggests a major effect of air pollution has been overlooked.

P1.05

Studies on pneumococcal esterases

Hasan Kahya, Peter Andrew, Hasan Yesilkaya

Infection, Immunity and Inflammation/University of Leicester, Leicester, UK

The genome of pneumococcal strain contains 4 putative esterase genes (SPD_0534 (estA), SPD_0932, SPD_1239, and

SPD_1506 (axe); however, their importance in pneumococcal biology is very sparse. In this study, we determined

the contribution of each esterase gene to total esterase activity, evaluated the substrate specificity of pneumococcal

esterases, and investigated their role in potentiation of neuraminidase activity and glycoprotein utilisation. The results

showed that all mutants displayed significantly less esterase activity than the parental strain EstA, which is responsible

for the main esterase activity, and the pneumococcal esterases are specific for short acyl chains. In addition, EstA, but not

Axe, was found to catalyse tributyrin, and both EstA and Axe could use acetylated xylan as substrate. It was also found

that EstA and Axe use bovine submaxillary mucin (BSM), which is highly acetylated, as substrate to release acetate, and

pre-treatment of BSM by either EstA or Axe increases sialic acid release by neuraminidase. Esterases’ role in potentiation

of neuraminidase activity was further supported by decrease the ability of sialic acid release and growth of esteraseneuraminidase

mutants (ΔestAnanA and ΔaxenanA) in medium containing BSM as sole carbon source compared to

each of the respective single mutants. The replacement of S 121 in EstA and S 181 in Axe to alanine abolished the catalytic

activity of esterases. In addition, the mutation of estA alone or in combination with nanA reduced the pneumococcal

colonisation and virulence significantly after intranasal infection. qRT-PCR results showed that the expression level of

estA and axe were significantly up-regulated when exposed to BSM.

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P1.06

Anti-capsular polysaccharide IgG-mediated protection on experimental

human pneumococcal carriage

Elena Mitsi, Jesus Reine, Andrea Collins, Angela Wright, Jenna Gritzfeld, Jessica Owugha, Stephen

Gordon, Daniela Ferreira

Liverpool School of Tropical Medicine, Liverpool, UK

Recent studies have highlighted the role of mucosal antibodies in blocking acquisition of pneumococcal colonisation

through agglutination. We used samples from adults vaccinated with 13-PCV prior to experimental human pneumococcal

carriage (EHPC) to study the role of capsule polysaccharide (PS) IgG in mediating protection from carriage after

pneumococcal inoculation. Ninety-six healthy adults were intranasally inoculated with live 6B pneumococcus after

vaccination (10% carriage in PCV versus 48% in control). We assessed changes in IgG levels to 6B and 23F PS in serum

and nasal wash (NW) samples before and after vaccination and before and after inoculation. In the control group, for

those colonised, IgG levels against 6B PS but not 23F PS were elevated in both serum and NW at 21 days after challenge

when compared to baseline, indicating that carriage boosts IgG levels of existing PS immunity. In PCV-vaccinated

subjects, IgG levels to both PS types were increased in serum and NW after vaccination. While IgG levels to both PS

types remained increased in sera after challenge, levels of IgG specific to the PS of inoculated type strain dropped

at day 2 after challenge, an effect more pronounced in those protected from carriage. No decrease was observed in

IgG levels to 23F PS. We hypothesise that PCV protection against carriage is mediated by bacterial agglutination by

PS-specific antibodies. Mucosal antibodies are sequestered by the pneumococcal inoculum, blocking bacterial adherence

and therefore mediating protection against colonisation. We are evaluating the capacity of NW antibodies from

PCV-vaccinated subjects in mediating agglutination of pneumococcus.

P1.07

P4 peptide enhances phagocytic activity of neutrophils acquired from

patients admitted to critical care with severe sepsis

Elena Mitsi 1 , Ben Morton 1 , Shaun Pennington 1 , Jesus Reine 1 , Angela Wright 1 , Gowrisankar Rajam 3 ,

Edwin Ades 3 , Daniela Ferreira 1 , Aras Kadioglu 2 , Stephen Gordon 1

1

Liverpool School of Tropical Medicine, Liverpool, UK; 2 University of Liverpool, Liverpool, UK; 3 Centers for Disease Control and Prevention, Atlanta,

USA

P4 is a 28-aa peptide fragment of pneumococcal surface adhesion A, which shows phagocyte- activating effects in vitro

and in vivo. Passive immunotherapy augmented using P4 is a novel potential therapeutic strategy to enhance phagocytes

bacterial killing activity and we have previously shown improved ex vivo neutrophil activity in patients with severe

community acquired pneumonia. In this study, patients with severe sepsis (respiratory, abdominal, and urogenital)

admitted to critical care were recruited and blood samples obtained. We cultured neutrophils ex vivo with P4 or control

vehicle and assessed phagocyte function, bacterial killing activity and ROS. We took sequential samples at different phases

of disease (early, latent, and convalescent) from each subject to perform two biological assays: 1) opsonophagocytosis

assay with isolated neutrophils and 2) flow cytometric whole blood phagocytosis assay using fluorophore labelled

intraphagosomal reporter (oxidative burst) beads. Our preliminary data demonstrate that P4 peptide significantly

increased neutrophil bacterial killing in early and latent phases of disease. In addition, the whole blood phagocytosis

assay demonstrated both increased uptake and oxidation of intraphagosomal reporter beads in the early and latent

phases. Samples from the convalescent phase are currently being collected. The stimulation of neutrophils with P4

peptide ex vivo augments their phagocytic activity in the majority of septic patients. Its administration, in conjunction

with intravenous immunoglobulin (IVIG), could be considered as a novel treatment to enhance host immune function

and to facilitate the combat against multiple infections.

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P1.08

Evaluation of Streptococcus pneumoniae respiratory infection in mice

with different susceptibility for acute inflammatory responses

Rúbia Isler Mancuso 1 , Alessandra Soares-Schanoski 1 , Eliane Namie Miyaji 1 , Paulo Lee Ho 1, 2 ,

Orlando Garcia Ribeiro 3 , Maria Leonor Oliveira 1

1

Centro de Biotecnologia - Instituto Butantan, São Paulo, Brazil; 2 Divisão de Desenvolvimento Industrial e Produção - Instituto Butantan, São Paulo,

Brazil; 3 Laboratório de Imunogenetica - Instituto Butantan, São Paulo, Brazil

Acute inflammation is an important response against bacterial infections. Here, an invasive respiratory challenge model

with a Streptococcus pneumoniae serotype 3 strain was studied in two outbred mice lineages, genetically selected for

exacerbated (AIRmax) and low (AIRmin) acute inflammatory responses. A dose of 10 4 bacteria per animal induced the

death of 100% of AIRmin mice a few days after the challenge. In contrast, only 36.4% of AIRmax mice died after the

infection. Bacterial numbers in the airways of AIRmax mice at 48 h post-challenge were significantly lower than the

observed in AIRmin mice. Analysis of cytokines in bronchoalveolar lavage fluids, showed peaks of IFN-γ and IL-6 at 12 h

post-challenge for AIRmax mice, and significant increases in IL-4, IL-5 and IL-10, at the same time point, for AIRmin mice.

No differences were observed for IL-1β, KC or IL-17, when comparing the 2 mice strains. The profile of TNF-α response

after the challenge was similar in the 2 mice strains; however, the levels of this cytokine remained high for AIRmin mice

at later time points. Preliminary analysis showed no differences in the percentage of alveolar macrophages expressing

the CD206 mannose receptor (F4/80+ CD11c+, CD206+), for both mice strains, before or 6 hours post-challenge.

However, a significant increase in the expression of CD206 by these cells (measured by the mean fluorescence intensity)

was observed in AIRmin mice at 6 hours post-challenge. Further analysis on the role of these cells in the outcome of

pneumococcal infection in this model is underway.

P1.09

Stable niche adaptation within clonal lineages of Streptococcus

pneumoniae

Zarina Amin, Claudia Trappetti, Bart Eijkelkamp, Catherine Hughes, Richard Harvey, Christopher

McDevitt, Adrienne Paton, James Paton

University of Adelaide, Adelaide, Australia

Individual Streptococcus pneumoniae strains differ markedly in their virulence phenotypes, but genetic heterogeneity

has complicated attempts to identify any association between a given clonal lineage and propensity to cause a particular

disease type. Previously we reported that serotype 3 pneumococci of the same ST-type exhibit distinct in vitro and in

vivo phenotypes in accordance with clinical site of isolation. In this study, serotype 14 blood and ear isolates of the same

ST-type (ST15) were investigated for pathogenic phenotype. In a murine intranasal challenge model, the three blood

and two ear isolates colonised the nasopharynx to similar extents. However, they exhibited significant differences in

bacterial loads in other host niches. At 24 and 2 hours post-challenge, neither of the ear isolates was detectable in the

lungs of any of the mice, but the blood isolates were present in the lungs of the majority of mice at both time points.

None of the three ST15 blood isolates were detected in the brains of any mice at either time point. However, the ear

isolates were detected in the brains and ear compartment of most of the mice at both 24 hours and at 72 hours. Thus,

stable niche adaptation within a clonal lineage appears to be a general property of pneumococci. Interestingly, both

blood and ear isolates were present in the lungs at similar levels at 6h post-infection, suggesting early immune responses

may underpin the distinct virulence phenotypes. Transcriptional analysis of lung tissue from mice infected for 6 hours

with blood versus ear isolates using RT-PCR arrays of key immune response genes revealed 8 differentially expressed

genes. Two of these were exclusively expressed in response to infection with the ear isolate. These results suggest a link

between differential capacity to elicit early innate immune response and the distinct virulence phenotypes of clonally

related S. pneumoniae strains.

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P1.10

The inflammatory response to Streptococcus pneumoniae is

exaggerated by the polysaccharide capsule

Jimstan Periselneris, Suneeta Chimalapati, Gillian Tomlinson, Catherine Hyams, Alex Dyson,

Mervyn Singer, Mahdad Noursadeghi, Jeremy Brown

University College London, London, UK

The inflammatory response to bacteria requires the interaction of pattern recognition receptors (PRR) with bacterial

surface constituents. Streptococcus pneumoniae expresses a polysaccharide capsule (cps); this essential virulence

factor’s main function is to inhibit opsonophagocytosis, and would be expected to inhibit interactions with host PRRs, so

reducing inflammatory responses. We tested this hypothesis by characterising the effect of S. pneumoniae capsule on

the inflammatory response using the TIGR4 strain and its unencapsulated derivative Δcps. Despite being more sensitive

to phagocytosis by human monocyte derived macrophages (MDM) than TIGR4, RNA transcripts and supernatant levels

of TNF, IL1β, and IL6 were reduced in response to Δcps. Furthermore, TIGR4 generated pro-inflammatory cytokine

responses and greater neutrophilic infiltrate in a mouse pneumonia model than Δcps, as well as a trend towards

greater physiological disturbance in a rat model of septic shock. Whole genome transcriptome analysis demonstrated a

generally reduced pro-inflammatory response to the TIGR4cps strain. Notably, preventing phagocytosis preserved these

differences. In vitro experiments excluded differences in TLR2 signalling, NFκB translocation, antibody recognition, the

inflammasome, and scavenger receptor binding as mechanisms driving differences in inflammatory responses between

TIGR4 and Δcps. However, a transcription factor array suggested that Δcps activated more transcription factors than TIGR4.

These data demonstrate that S. pneumoniae capsule causes increased pro-inflammatory responses that are relevant

during infection, perhaps by restricting macrophage cell signalling responses. Identifying mechanisms responsible for

capsule-dependent inflammation may offer opportunities for adjuvant treatment of S. pneumoniae infections.

P1.11

Survival of Streptococcus pneumoniae and Staphylococcus aureus in

sputum from intensive care unit patients

Jolien Seinen 1 , Willem Dieperink 1 , Anne Marie G.A. de Smet 1 , Sven Hammerschmidt 2 , Jan Maarten

van Dijl 1

1

University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; 2 Interfaculty Institute for Genetics and Functional

Genomics, Ernst Moritz Arndt University of Greifswald, Greifswald, Germany

Pneumonia related to mechanical ventilation in hospitals is referred to as ventilator-associated pneumonia (VAP). VAP

results in increased duration of mechanical ventilation and prolonged intensive care unit (ICU) and hospital stay. Both

Streptococcus pneumoniae and Staphylococcus aureus have been associated with VAP. To date, it is not very well known

to which stresses these bacteria are exposed in the airways of ventilated patients. The present studies were therefore

aimed at establishing an ex vivo assay that mimics the in vivo situation through the incubation of bacteria in sputum

samples collected from different ICU patients. S. pneumoniae strains D39 and TIGR4 and S. aureus strain HG001 were

pre-cultured in different media and, subsequently, incubated for different periods of time in sputum samples from

mechanically ventilated ICU patients from the University Medical Center Groningen. Next, bacterial survival in the

sputum samples was tested by plating and microscopic inspection. The tested S. pneumoniae and S. aureus strains

showed different rates of survival in sputa from different patients. This did not relate to the way in which sputum

samples were pre-treated. Our ongoing research is aimed at assessing both patient parameters that affect bacterial

survival in sputum as well as the bacterial gene repertoire that is needed for survival. Results of these analyses will be

presented at the meeting.

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P1.12

Characterisation of a low molecular weight protein tyrosine

phosphatase in Streptococcus pneumoniae

Zuleeza Ahmad, Renato Morona, Alistair Standish

University of Adelaide, Adelaide, South Australia, Australia

Tyrosine phosphorylation is widely recognised as a critical regulator of bacterial virulence, with the associated protein

tyrosine phosphatases (PTPs) and bacterial tyrosine kinases (BY-kinases) major virulence factors in a range of bacterial

pathogens including Streptococcus pneumoniae. We have previously studied a phospho-regulatory system in the

pneumococcus comprising CpsB (a PTP) and CpsC and CpsD, which together form an active BY-kinase. This system plays a

crucial role in the regulation of capsule production. While homology searches did not uncover additional BY-kinases, the

protein encoded by one open reading frame (designated Sp-PTP) showed homology (46%) to the low molecular weight

protein family of tyrosine phosphatase which play a role in capsule regulation in other bacteria, as well as diverse roles

that include stress responses and biofilm formation. To determine PTPs role in the pneumococcus, His 6

-Sp-PTP purified

from Escherichia coli enabled us to verify that Sp-PTP is indeed a phosphatase, with specificity against tyrosine. We also

found that Sp-PTP’s phosphatase activity is inhibited by hydrogen peroxide. We have constructed ptp mutations on the

pneumococcal chromosome and found that PTP does not affect capsule production. We suggest that the phosphatase

may have other roles in the pneumococcus, and predict that tyrosine phosphorylation events in the pneumococcus have

wider effects outside the capsule system.

P1.13

Quantification of the agglutinating effect of anti-pneumococcal

antibodies analysed by flow cytometry

Saskia van Selm, Marrit Habets, Dimitri Diavatopoulos, Marien de Jonge

Radboud University Medical Center, Nijmegen, The Netherlands

Following entry into the airways, pneumococci must first overcome the natural barriers in the nasopharynx before it

can establish stable colonisation. The presence of antibodies at mucosal surfaces is thought to inhibit colonisation via

multiple mechanisms, including opsonisation-mediated phagocytosis and killing and neutralisation by binding to factors

such as bacterial adherence. Recently, a role for antibody-mediated agglutination in the prevention of colonisation was

identified in mice. Passive immunisation studies with anti-capsular IgG demonstrated that optimal mucosal protection

was independent of Fc-mediated signalling and complement-binding, but occurred through F(ab)2-mediated formation

of bacterial aggregates. Agglutination is thought to enhance excretion of bacteria via mucociliary flow or other ways

of mechanical clearance. At present, there is no gold standard yet to measure and quantify agglutinating antibodies,

although microscopy is frequently used. We therefore developed a novel, fast, standardised, high-throughput flow

cytometry-based assay to quantify the agglutinating capacity of antibodies, based on size (FSC) and granularity (SSC)

of pneumococcal aggregates. We demonstrate that antibodies against PspA, an antigen that is less abundantly present

on the pneumococcal surface than capsular polysaccharides, are able to induce agglutination. Flow cytometry analysis

offers the possibility to quantify the agglutinating effect of antibodies on a panel of clinical isolates, thus enabling studies

on the correlation with protection.

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P1.14

Binding activities and anti-phagocytic properties of PclA, the

pneumococcal collagen-like protein A

Sandra Koch 1 , Elena Fuchs 1 , Tim J. Mitchell 2 , Andrea M. Mitchell 2 , Marcus Fulde 3 , Michael

Steinert 1 , Simone Bergmann 1

1

Technische Universität Braunschweig, Braunschweig, Germany; 2 University of Birmingham, Birmingham, UK, 3 University of Veterinary Medicine

Hannover, Hannover, Germany

The pneumococcal collagen-like protein A (PclA) is covalently linked to the surface of Streptococcus pneumoniae via

an LPxTG motif and harbours a variable number of repetitive amino acid sequences with high homologies to human

collagen. PclA is expressed in up to 45% of tested pneumococcal isolates and mediates pneumococcal adherence to

nasopharyngeal cells [1]. Molecular analyses indicated that the collagen-like sequence repeats are clustered in up to

3 collagen-like domains spanning a maximum of 6 repeated amino acid sequences per domain. The total number of

collagen-like repeats of PclA ranges from 2 repeats in a serotype 19F isolate, to up to 10 repeats in total in serotype 2

isolates, e.g. R6. Consequently, the molecular weight of PclA varies between 187 kDa and 289 kDa depending on the

numbers of collagen-like repeats. The effect of PclA on phagocytic neutralisation was analysed in phagocytic killing

assays with macrophage-like U937 cells and with neutrophils prepared from blood of two different human donors.

Phagocytosis of a pclA-deficient mutant by U937 cells was similar to the corresponding wild type strain. In contrast,

depending on the donor, the prepared neutrophils were less efficient in phagocytosis of pclA-deficient pneumococcal

mutants. Furthermore, we demonstrate for the first time specific binding activity of the collagen-like domain 1 of S.

pneumoniae strain D39 to human plasma fibronectin and von Willebrand factor. The interaction of the PclA domain with

host proteins indicates an important contribution of PclA to pneumococcal colonisation and virulence.

1. Paterson GK, Nieminen L, Jefferies JM, Mitchell TJ. PclA, a pneumococcal collagen-like protein with selected strain distribution,

contributes to adherence and invasion of host cells. FEMS Microbiol Lett 2008;285:170–6. PMID:18557785 http://dx.doi.

org/10.1111/j.1574-6968.2008.01217.x

P1.15

Delineating the interaction of pneumococcal surface proteins with the

human adhesive glycoprotein fibronectin

Sajida Kanwal 1 , Inga Jensch 1 , Thomas P. Kohler 1 , Roland Frank 2 , Werner Tegge 2 , Mark Brönstrup 2 ,

Sven Hammerschmidt 1

1

Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, Ernst Moritz Arndt University of Greifswald,

Greifswald, Germany; 2 Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany

The virulence potential of Streptococcus pneumoniae is closely associated with its expression and exposure of a

diverse repertoire of colonising and virulence factors. Several of these factors belong to microbial surface components

recognising adhesive matrix molecules (MSCRAMMs), which exploit eukaryotic host matricellular proteins such as

fibronectin, vitronectin, thrombospondin, collagen, or plasminogen as a molecular mediator to interact with host

cells. Pneumococcal adherence and virulence factor A (PavA) and B (PavB) are fibronectin-binding proteins (FnBPs),

representing a sub-class of MSCRAMMs. PavB is covalently anchored to the pneumococcal cell wall by sortase A,

whereas the PavA protein is a member of the so-called non-classical surface proteins (NCSPs) with an ambiguous

unknown mechanism of secretion and surface-exposure. In this study direct protein–protein interaction approaches have

been used to delineate pneumococcal PavA and PavB interactions with the human glycoprotein fibronectin (Fn). Flow

cytometric analysis showed that S. pneumoniae recruits fibronectin from human plasma in a dose-dependent manner.

Pneumococci interact with the C-terminal part of Fn, while other Gram-positive bacteria bind to the N-terminal Fn type

I repeats. In order to assess the underlying molecular mechanism of these PavA/PavB–fibronectin interactions, different

Fn fragments and type repeats were used in far western blots and surface plasmon resonance (SPR) studies. Far western

blots demonstrated binding of PavA and PavB to C-terminally located type III repeats. The interaction between type III

repeats and PavA or PavB was confirmed in our SPR analysis. Among the Fn type III repeats, Fn type III12-14 (heparin

binding domain) bound with highest affinity to PavA and PavB. In addition, peptide arrays are used to identify crucial

binding motifs in Fn type III repeats. This study shows that FNBPs preferentially bind to type III repeats of Fn molecules

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and provides insights into the molecular mechanisms of Fn engagement during pneumococcal-host interactions.

P1.17

Pneumolysin-induced nitric oxide contributes to lysosomal membrane

permeabilisation in Streptococcus pneumoniae-infected macrophages

Joseph Ford, Martin Bewley, Simon Johnston, David Dockrell

University of Sheffield, Sheffield, UK

A key phase in the macrophage response to Streptococcus pneumoniae is a program of host-mediated apoptosis. This

occurs after the macrophage has engulfed bacteria, but becomes overwhelmed. This apoptotic response increases

late stage killing of bacteria when conventional phagolysosomal killing has become exhausted. The apical event in

this pathway is lysosomal membrane permeabilisation (LMP). This is caused by a currently unknown mechanism that

requires the pneumococcal toxin pneumolysin (PLY). PLY is a pore-forming toxin but pore-formation is not necessary

to induce LMP, indicating other aspects of PLY activity may be responsible. PLY is known to contribute to nitric oxide

(NO) production in macrophages. We hypothesised that PLY-induced NO production by the macrophage contributes

to LMP and that this is integral to the host response to pneumococcus. To study this system we employed the use of

primary human monocyte derived macrophages (MDMs). MDMs were infected at an MOI of 10 using opsonised D39

S. pneumoniae, a PLY negative mutant (Stop), or latex beads. MDMs were assayed for LMP using flow cytometery and

western blotting. In some experiments MDMs were treated with inhibitors of ROS (Trolox, DPI) and NO (1400W), NO

donors SNAP or NOC-13 and/or TLR agonists lipoteichoic acid (LTA) and lipopolysaccharide (LPS). D39 infected MDM

displayed significantly higher levels of LMP than Stop infected cells. Incubation with 1400W and Trolox significantly

reduced LMP in D39 infected cells, but DPI had no effect. Conversely, incubation with SNAP and NOC-13 was able to

restore LMP in Stop infected cells. Preliminary data suggests that NO is necessary but not sufficient for LMP, with TLR

activation and phagocytosis also required for NO to exert its effect; however, further verification is required. These data

identify a novel role for NO in LMP and host–pneumococcus interactions.

P1.18

Structure of the pneumococcal L,D-carboxypeptidase DacB and impact

of DacB on pathophysiological processes

Mohammed R. Abdullah 1 , Javier Gutiérrez-Fernández 2 , Thomas Pribyl 1 , Nicolas Gisch 3 , Malek

Saleh 1 , Manfred Rohde 4 , Lothar Petruschka 1 , Gerhard Burchhardt 1 , Dominik Schwudke 3 , Juan

Hermoso 2 , Sven Hammerschmidt 1

1

Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, Ernst Moritz Arndt University of Greifswald,

Greifswald, Germany; 2 Department of Crystallography and Structural Biology, Institute of Physical-Chemistry “Rocasolano”, CSIC, Madrid, Spain;

3

Division of Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Bioscience, Borstel, Germany; 4 Department of

Molecular Infection Biology, Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany

The peptidoglycan (PGN) is a complex macromolecule and composed of 2 of the alternating sugars residues,

N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which form oligo-(GlcNAc-MurNAc) glycan

strands. These heteropolymers are cross-linked by short peptides to form a complex three-dimensional scaffold

(murein). The PGN forms the essential exoskeleton needed to maintain the shape and osmotic stability of bacteria.

Pneumococcal cell wall hydrolases such as L,D- and D,D-carboxypeptidases DacB and DacA respectively, have

been shown to be important for cell division and shape. Pneumococcal ΔdacA and ΔdacB single and double

mutants were generated and characterised by immunoblot analysis, growth behaviour, and flow cytometry.

The impact of DacB on virulence was tested by phagocytosis assays by applying acute pneumonia mouse

infection model in conjunction with real-time bioimaging. To assess the muropeptide species a PGN analysis

has been carried out. Importantly, the crystal structure of DacB was solved successfully at high resolution.

The L,D-carobxypeptidase DacB is a surface-exposed lipoprotein and its structure is characterised at the atomic level

showing radically different structure, regulation and catalytic machinery than the pneumococcal D,D-carboxypeptidase

DacA. Importantly, the morphological changes observed in dac-mutants are associated with an altered PGN composition

and hence lower bacterial fitness under infection-related conditions. The in vivo mouse infections and cell cultured-based

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adherence and invasion assays indicated loss of function of DacA and/or DacB impaired full-virulence of pneumococci and

accelerated uptake by professional phagocytes, while adherence to epithelial cells is decreased. In this study, we further

characterised the crucial role of the pneumococcal carboxypeptidases DacA and DacB for PGN architecture, bacterial

shape and pathogenesis. By applying in vivo and in vitro approaches, a close relationship between PGN metabolism and

impaired bacterial pathogenesis was discovered.

P1.19

Blockade of Streptococcus pneumoniae adhesion receptors of the

blood–brain barrier endothelium: a new therapeutic approach for the

prevention and cure of pneumococcal meningitis

Federico Iovino, Birgitta Henriques-Normark

Karolinska Institutet, Stockholm, Sweden

Meningitis is thought to occur as the result of pneumococci crossing the blood–brain barrier and invading the central

nervous system (CNS). The main route taken by Streptococcus pneumoniae to invade the CNS is via the bloodstream by

translocating across the vascular endothelium of the blood–brain barrier. Bacteria have the capability to bind to receptors

expressed on the plasma membrane of epithelial and endothelial cells and, through this binding, they can invade and

cross human cell layers. Platelet endothelial adhesion molecule (PECAM-1) and poly immunoglobulin receptor (pIgR)

expressed by brain endothelial cells are cell membrane-receptors which have been recently described to be involved in

the adhesion of S. pneumoniae to the blood–brain barrier endothelium. It has been shown that pneumococci co-localise

with PECAM-1 and pIgR in the brain of mice intravenously infected with S. pneumoniae. Furthermore, blockade of pIgR

and PECAM-1 with specific antibodies reduced pneumococcal adhesion to human endothelial cells in vitro. Firstly, a

bacteraemia-derived meningitis model with PECAM-1 -/- , pIgR -/- and wild-type mice will be used as in vivo system to

unequivocally demonstrate the role of PECAM-1 and pIgR in pneumococcal meningitis pathogenesis. In the case of a

direct role of these 2 receptors, the prediction is that fewer bacteria would translocate across the blood–brain barrier

in the knock-out mice and, in addition, the typical symptoms of bacteraemia progressing towards meningitis should be

less pronounced. Lastly, antibodies against PECAM-1 and pIgR will be administered systemically in wild-type mice prior

to intravenous challenge with S. pneumoniae to determine whether the treatment with receptor-specific antibodies

counteracts meningitis onset. In the case of a significantly reduced progression towards meningitis, the systemic

administration of antibodies to block S. pneumoniae adhesion receptors has the realistic potential to become a new

therapeutic approach for the prevention and cure of pneumococcal meningitis.

P1.20

Pneumococcal nasopharyngeal carriage in Nepalese children prior to

pneumococcal conjugate vaccine introduction

Rama Kandasamy 1 , Meeru Gurung 2 , Stephen Thorson 2 , Shrijana Shrestha 2 , Imran Ansari 2 ,

Katherine L. O’Brien 3 , Sarah Kelly 1 , Dominic F. Kelly 1 , Andrew J. Pollard 1

1

University of Oxford, Oxford, UK; 2 Patan Academy of Health Sciences, Kathmandu, Nepal; 3 Johns Hopkins Bloomberg School of Public Health,

Baltimore, USA

As part of an impact evaluation of 10-valent pneumococcal conjugate vaccine (PCV10) in Nepal, we undertook a

serotype-specific community carriage study among community-based children from Kathmandu in the year prior to

PCV10 introduction. Children 6–60 months and 0–8 weeks of age were recruited for the nasopharyngeal (NP) study from

immunisation and outpatient clinics, and relatives attending inpatients at Patan Hospital, Kathmandu. Flocked NP swabs

were collected according to the WHO guidelines. Swabs were placed into skim-milk-tryptone-glucose-glycerin medium,

plated within 8 hours on Columbia blood agar, and incubated overnight at 35 –37°C in 5% carbon dioxide. Pneumococcal

colonies were identified by morphology and optochin sensitivity. Serotyping was performed by Quellung reaction using

sera from Serum Staten Institute, Denmark. Between April and December 2014 we enrolled 1,905 children (1,305 aged

6–60 months, and 600 aged 0–8 weeks). Pneumococcal carriage prevalence was 69.7% (910/1,305) and 18.8% (113/600)

in those 6–60 months and 0–8 weeks of age, respectively (p < 0.0001). Among those colonised, 32.2% (137/425 [95% CI

27.8–36.9%]) and 22.5% (22/98 [95% CI 14.6–32%]) had PCV10 serotypes in the 6–60 month and 0–8 week age groups,

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respectively (p = 0.07). Colonised 0–8-week-old children were more likely to have a nontypeable pneumococcus than

colonised 6–60-month-old children, with 33% (32/98 [95% CI 23.5–42.9%]) versus 14.1% (60/425 [95% CI 11–17.8%];

p < 0.0001). Infants in the pre-immunisation age group have a significantly lower prevalence of pneumococcal carriage

than vaccine age-eligible children; the youngest colonised children were more likely to have a nontypeable strain than

the older children. This may be due to factors such as inter-serotype competition for the nasopharyngeal niche, passively

transmitted serotype-specific maternal immunity, or differential exposure.

Funding: This study is funded by Gavi, The Vaccine Alliance

P1.21

Phosphoenol-pyruvate phospho-transferase A interactions with putative

host target receptors and their derived peptides during Streptococcus

pneumoniae adhesion

Tatyana Kushnir, Marilous Shagan, Anat Shahar, Raz Zarivach, Natali Elia, Ron Dagan, Yaffa

Mizrachi Nebenzahl

Ben Gurion University of the Negev, Beer Sheva, Israel

Streptococcus pneumoniae is a commensal pathogen and the major cause of life threatening diseases, including otitis

media, pneumonia, bacteraemia and meningitis. Adhesion of S. pneumoniae to the host mucosal cells is prerequisite

for disease development. In previous studies several proteins with known enzymatic functions were localised also

to the cell wall, and were found to function as adhesins. Current study focuses on one such adhesin, Phosphoenolpyruvate

phospho-transferase A (PtsA), which is the first enzyme of the PTS systems. Putative PtsA target receptors

were identified using combinatorial peptide library expressed in filamentous phage followed by a homology based

search of the human genome. Immunostaining proved 5 out of 6 PtsA putative target receptors to reside in the lung

derived epithelial cells. Microscale thermophoresis (MST) assay confirmed the specificity of rPtsA interaction with

each of the 5 putative target receptors derived peptides, yielding affinities in the micro-molar range, in accordance

with their inhibitory concentration range in adhesion. To decipher the precise mechanism of PtsA interactions with its

putative target receptors and their derived peptides, the 3D structure of PtsA is being resolved using X-ray diffraction.

In vivo experiments also exhibited lower bacterial load in mice infected with S. pneumoniae preincubated with putative

receptor derived peptides, indicating the essential role of PtsA in bacterial adhesion and infection. With respect to the

increasing antibiotic resistance of S. pneumoniae, the putative target receptor derived peptides are currently considered

as candidates for alternative effective therapeutics.

P1.22

Contribution of a temperate bacteriophage to the virulence of a

Streptococcus pneumoniae serotype 1 strain

Martin Norman 1, 2 , Anna Syk 1, 2 , Sarah Browall 1, 2 , Birgitta Henriques-Normark 1, 2

1

Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; 2 Dep. of Clinical Microbiology, Karolinska University

Hospital, Stockholm, Sweden

Temperate bacteriophages are frequently found in the genomes of clinical isolates of Streptococcus pneumoniae.

Currently there is a lack of knowledge on how, if at all, these genetic elements affect the virulence of pneumococcal

strains. In our study we investigated the effect on virulence of a temperate phage in an invasive clinical isolate of a

highly virulent serotype 1 strain of sequence type (ST) 217 by using a mouse model of invasive disease. We found

that the presence of the phage was associated with increased virulence and the effect was linked to a gene encoding

a phage tail protein, which in S. mitis has been described as pblB. In S. mitis the pblB platelet binding locus has been

shown to mediate binding to platelets and increase virulence in an endocarditis model. We found that in S. pneumoniae

pblB is required for sustained bacteraemia during invasive disease. Further studies are needed to elucidate the exact

mechanisms by which bacteriophages contribute to pathogenesis.

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P1.23

Streptococcus pneumoniae cardiac microlesions: pneumolysinmediated

immune evasion and biofilm growth

Ryan Gilley, Anukul Shenoy, Norberto Gonzalex-Juarbe, Carlos Orihuela

The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

Approximately 20% of adults hospitalised for pneumococcal pneumonia experience an adverse cardiac event. We have

recently reported on the formation of pneumococci-filled, non-purulent microscopic lesions (i.e. microlesions) within

the myocardium of mice with invasive pneumococcal disease (IPD). Microlesion formation was concomitant with adverse

cardiac electrophysiology thus offering one possible explanation for the occurrence of adverse cardiac events following

IPD. Using a non-anesthetised intraperitoneal mouse challenge model we have characterised the formation of cardiac

microlesions in a more physiologically relevant method and in greater detail than our first report. We report the entry

of individual pneumococci into the heart as early as 12 hours post-challenge. We show that immune cells are recruited

to the site of infection as early as 18 hours, but that these cells are no longer present or limited to the periphery

of the microlesion after 24 hours. Fluorescent and electron microscopic analysis of hearts from mice infected with a

pneumolysin-deficient mutant showed robust immune cell infiltration of both mononuclear and polymorphonuclear

cells, indicating pneumolysin plays a key role in the clearance of immune cells during the early stages of microlesion

formation. Pneumococci within microlesions were predominantly found to be the transparent phenotype and consisted

of a mixture of live and dead bacteria. Immunohistochemical analysis of the microlesions revealed elevated levels of

N-acetylglucosamine and the presence of extracellular DNA from the host. All of which suggest the formation of a

biofilm within the cardiac microlesion. Cardiomyocte death was shown to be dependent on pneumolysin and hydrogen

peroxide, together inciting activation of caspase and necroptosis signalling cell death pathways.

P1.24

Polyamine transport in the pneumococcus is essential for evading early

innate immune responses in pneumococcal pneumonia

Aswathy Rai 1 , John Stokes 1 , Justin Thornton 2 , Edwin Swiatlo 3 , Imran Sunesara 3 , Bindu Nanduri 1

1

Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA; 2 Department of Biological

Sciences, Mississippi State University, Mississippi State, MS, USA; 3 Department of Infectious Diseases, University of Mississippi Medical Center,

Jackson, MS, USA

Polyamines are ubiquitous small cationic molecules that are important for growth and virulence of human pathogens,

including pneumococcus. An isogenic deletion of polyamine transport operon (ΔpotABCD) in Streptococcus pneumoniae

TIGR4 led to attenuation in a mouse model of pneumonia. The aim of this study is to identify specific host innate immune

mechanisms that contribute to enhanced resistance in mice challenged with polyamine deficient pneumococci. We

inoculated C57BL/6 mice (n = 5) intranasally with 1 x 10 7 CFU of wild-type (WT) TIGR4, ∆potABCD. Mice were euthanised

4 hours, 12 hours, and 24 hours post-infection (p.i.) and lungs were harvested. Bacterial CFU enumeration, evaluation of

immune cell infiltration, measurement of cytokines/chemokines and mass spectrometry based expression proteomics

were carried out with lung homogenates. As early as 4 hours p.i., there was a significant difference in the in vivo bacterial

burden with WT and ∆potABCD. While the WT strain persisted in the lung 24 hours p.i., ∆potABCD was cleared from the

animal. Concentration of G-CSF, LIF, IP-10, KC, GM-CSF, IL-5, MCP-1 and MIP-1a were higher 4 hours p.i. in mice infected

with ∆potABCD. Consistent with this observation, we found a significant increase in the infiltration of neutrophils in

the lung tissue. Modelling of proteomics data using Ingenuity pathways analysis predicted activation of MyD88 and

Interferon gamma 4 hours p.i. in response to ∆potABCD and at 12 hours p.i with WT. These predictions indicate a delay

in the activation of early innate immune responses involved in bacterial clearance with the WT. In summary, impaired

polyamine transport in S. pneumoniae ∆potABCD resulted in its transition from nasopharynx to lung as early as 4 hours

p.i., and its subsequent clearance from the lung and blood by 24 hours p.i. Therefore, polyamine transport in the

pneumococcus is essential to evade early innate immune responses.

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P1.25

IL-22 contributes to recovery from pneumococcal pneumonia by

reducing collagen deposition in lung

Neil Ritchie, Tom Evans

University of Glasgow, Glasgow, UK

Pneumococcal pneumonia can cause dense pulmonary consolidation, which usually resolves within a few weeks of

treatment. IL-22 has been implicated in contributing to the resolution of pulmonary inflammation without fibrosis. We

explored the role of IL-22 in resolution of treated pneumococcal pneumonia in a murine model. IL22KO and wild-type

mice were infected intra-nasally with 3 x 10 6 cfu of serotype 3 Streptococcus pneumoniae. Infection was terminated

by administration of ceftriaxone once mice showed clinical signs of infection and again the following day. Lungs were

examined histologically and using a soluble collagen assay. Untreated mice developed severe pulmonary consolidation

with lung abscess formation and development of empyema. There was no significant difference in outcome between

IL-22KO mice and wild-type in outcome of untreated infection with similar histopathological changes and bacterial

burden. Treated mice resolved clinical illness and regained lost weight. Histological examination revealed improvement

in alveolar infiltration in both mouse types but IL-22KO mice had increased areas of sub-pleural collagen deposition

and lungs from IL-22KO mice had increased total collagen content. Treatment of pneumococcal pneumonia leads to

resolution of consolidation with relatively little pulmonary fibrosis. IL-22 was required for complete resolution and could

represent a novel therapeutic target to augment antibiotic treatment in severe pneumonia.

P1.26

Pneumococcal colonisation and invasive disease studied in the porcine

model

Astrid de Greeff 1 , Saskia van Selm 2, 3 , Herma Buys 1 , Jose Harders-Westerveen 1 , Rahajeng

Tunjungputri 4 , Quirijn de Mast 4 , Andre van der Ven 4 , Norbert Stockhofe-Zurwieden 1 , Marien de

Jonge 2, 3 , Hilde Smith 1

1

Central Veterinary Institute, part of Wageningen UR, Lelystad, The Netherlands; 2 Laboratory of Pediatric Infectious Diseases, Department of

Pediatrics and Laboratory of Medical Immunology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The

Netherlands; 3 Raboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands, 4 Department of Internal Medicine, Nijmegen, The

Netherlands

Streptococcus pneumoniae is carried in the nasopharynx and spread by human-to-human transmission causing mild

diseases such as otitis media and sinusitis as well as severe diseases including pneumonia, meningitis, and sepsis.

Furthermore, S. pneumoniae is an important cause of septic arthritis, and endocarditis. Asymptomatic colonisation of

the nasopharynx is an essential precursor for pneumococcal disease. Piglets are the natural host for Streptococcus suis

infections. There are striking similarities between S. suis pathogenesis in piglets and S. pneumoniae pathogenesis in

humans. Piglets develop severe disease like meningitis, sepsis, arthritis, endocarditis or pneumonia upon infection with

S. suis. S. suis is carried in the oropharynx, the bacterium colonises the tonsil of piglets, similar to S. pneumoniae in

children. Furthermore, genetically S. suis and S. pneumoniae are closely related. Because pigs are very similar to humans

in terms of anatomy, genetics and physiology, we investigated the use of piglets as a model for human colonisation

and for the induction of pneumococcal invasive disease. Piglets inoculated intravenously with S. pneumoniae showed

persistent bacteraemia, frequently followed by arthritis. Moreover, in piglets inoculated intra-nasally the oropharynx

was colonised with S. pneumoniae for at least 7 consecutive days. This demonstrates that central aspects of human

pneumococcal infections also occur in the porcine model, and advocates the use of piglets in future colonisation,

transmission, and intervention studies. There is growing evidence that S. pneumoniae infections are associated with an

increased risk for cardiovascular events and stroke. Since the porcine and human cardiovascular systems are very similar,

the porcine pneumococcal model can be used to gain insight into the pathogenesis of this serious health problem.

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P1.27

Variome of the pneumococcal surface-exposed proteins and other

virulence factors: a bioinformatics analysis

Andres Castro 1, 2 , Alejandro Gomez 1, 3 , Mauricio Gallego 1, 2 , Alejandro Bedoya 1, 2 , Sven

Hammerschmidt 3 , Gustavo Gamez 1, 2

1

Basic and Applied Microbiology (MICROBA) Research Group, School of Microbiology, Universidad de Antioquia, UdeA, Calle 70 No. 52 - 21, 050010,

Medellín, Colombia; 2 Genetics, Regeneration and Cancer (GRC) Research Group, University Research Center (SIU), Universidad de Antioquia, UdeA,

Calle 70 No. 52 - 21, 050010, Medellín, Colombia; 3 Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional

Genomics, University of Greifswald, Friedrich-Ludwig-Jahn Strasse 15a D-17487 Greifswald, Germany

The surface of the pneumococcus is decorated by a great amount of proteins, which have been associated with its

pathogenic functions such as adhesion, colonisation, transmigration and immune evasion. To date, few studies have

reported the genetic variability among the different pneumococcal strains from a global perspective. Therefore, there is

still a need to fully understand the conservation and distribution of all the virulence factors this human pathogen employs

to cause diseases. Here, the construction of the first variome for Streptococcus pneumoniae is reported. Twenty-five

pneumococcal strains with fully sequenced and annotated genomes were analysed, estimating the distribution (presence/

absence) of the pneumococcal virulence factors and identifying all their allelic and protein variants and mutations. The 25

S. pneumoniae strains were distributed in 37 phylogenetic groups with a variable number of represented genomes and

9 strains with a unique representation. A total of 61 different genes and proteins were identified, classified and analysed

for the construction of the variome. The genes of the pneumococcal virulence factors are distributed in the genome and

located in a co-oriented way in relation with the region were the chromosomal replication begins. The analysis of the

gene distribution showed that 24 of them belong to the core genome and 37 to the accessory genome. The estimation

of the variability for each of the studied virulence factors allowed us to establish that the virulence factors with the

highest conservation in the pneumococcus are Ply, Eno and Usp45; the virulence factors with the highest variability are

RrgB, PhtD and NanA. Finally, all these results allow us to identify potential pharmaceutical targets for new antimicrobial

therapies, and to confirm highly conserved and well-distributed protein candidates for vaccine development, supporting

the idea of a new generation of vaccines based on proteins (adhesins) to fight against S. pneumoniae and its associated

diseases.

P1.28

Global serotypes and genotypes for Streptococcus pneumoniae:

a whole genome resource for assessing the impact of global

pneumococcal immunisation*

Rebecca Gladstone 1 , Jukka Corander 2 , Maaike Alaerts 3 , Brenda Kwambana-adams 4 , Mignon Du

plessis 5 , Paulina Hawkins 6 , Dean Everett 3 , Martin Antonio 4 , Anne von Gottberg 5 , Keith Klugman 6 ,

Lesley McGee 7 , Stephen Bentley 1 , Robert Breiman 8

1

Pathogen Genomics, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK; 2 University of Helsinki, Helsinki, Finland; 3 Malawi-Liverpool-

Wellcome-Trust, Blantyre, Malawi; 4 Medical Research Council, Serrekunda, Gambia; 5 National institute for communicable diseases, Johannesburg,

South Africa; 6 Rollins School Public Health, Emory University, Atlanta, USA; 7 Centers for disease control and prevention, Atlanta, USA; 8 The Emory

Global Health Institute, Atlanta, USA

Pneumococcal serotype and genotype prevalence are known to vary geographically. However, there are substantial gaps

in data on global diversity, especially from low and middle-income countries. The Global Pneumococcal Sequencing

project (GPS) aims to sequence the genomes of 20,000 pneumococcal isolates with sampling priority for invasive disease

isolates from infants in developing countries. A major project goal is to describe geographical differences in global

pneumococcal genetic diversity in the context of pneumococcal vaccine implementation. At time of writing, over 8,500

pneumococci, representing >60 serotypes, approximately 90% from disease, have been Illumina sequenced. Isolates

originated from >20 different countries with a current focus on the African continent, from between 1991 to 2015, both

pre and post PCV, collected predominantly during the last decade. Serotype/genotype designations were derived from

whole genome data. A Bayesian clustering program (hierBAPS) was used to define population structure, designating

sequence types (STs) in the Multi Locus Sequence Typing database to sequence clusters. These cluster designations were

then applied to the GPS dataset using their derived ST. The top ten serotypes were all pneumococcal conjugate vaccine

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types, except 15B/C. Over 1000 STs were observed; the most prevalent (n = 275) was the highly clonal ST217, expressing

serotype 1, exclusively from the African continent. Eight of the 13 major lineages defined by hierBAPS, were present in all

nine countries for which >100 isolates had been sequenced, whilst the remaining 5 lineages were present in ≥7 of these

countries. All 13 lineages exhibited multiple serotypes but ≤3 serotypes accounted for >50% of isolates in each lineage.

Although most lineages were widespread, serotype/genotype profiles varied between countries, potentially overstated

by any sampling biases. As PCV use expands globally, characterising lineage and serotype distribution is valuable

for assessing changes in pneumococcal diversity, which could potentially impact the effectiveness of pneumococcal

prevention and control measures.

*On behalf of all Global Pneumococcal Sequencing project partners: www.pneumogen.net/gps

P1.29

Investigating the pneumococcal phageome using a diverse genome

dataset

Caroline L Harrold, Andries J van Tonder, Angus McDonnell, Ben A Edwards, Angela B

Brueggemann

University of Oxford, Oxford, UK

Bacteriophages are viruses that infect bacteria: many bacterial species are infected by bacteriophages and the

bacteriophages often confer benefits to their host by providing genes that encode toxins or other virulence factors.

Pneumococcal bacteriophages were first identified in 1975. Bacteriophages may play an important role in pneumococcal

biology and evolution; however, prevalence data has been limited and relatively little is known about their effects on the

pneumococcal host. We have a large and diverse global dataset of 336 pneumococcal whole genome sequences (WGS)

obtained from 31 different countries between 1916 and 2008. Preliminary analysis of this dataset used bioinformatics

tools to interrogate the pneumococcal genomes for sequence-based evidence of bacteriophages. With the view to

build a pneumococcal phageome reference database of pneumococcal bacteriophage sequences, the dataset was

screened for evidence of bacteriophage DNA using an in-house pipeline and a reference database of previously

characterised streptococcal bacteriophage genomes. Initial analyses revealed that 86% of the pneumococcal genomes

had bacteriophage DNA integrated within them, with 48 different bacteriophage types identified. In many cases the

pneumococcal genomes contained DNA from >1 bacteriophage type: 22 genomes contained 2 full length bacteriophages,

and 13 genomes contained ≥5 full length and partial bacteriophages. The results of the screen led to the identification of

10 novel bacteriophages. Four of the novel bacteriophages showed considerable sequence similarity to each other, but

were unlike the other 6 novel bacteriophages, and were widespread throughout our collection of South African isolates,

suggesting that they are a common feature of the South African pneumococcal population. These results suggest that

bacteriophage sequences in the pneumococcus are much more prevalent and diverse than previously recognised, and

that they are likely to be contributing to pneumococcal genome evolution.

P1.31

Molecular pneumococcal capsular typing using whole genome

sequencing: moving the Streptococcus pneumoniae reference service

into the genomic era

Georgia Kapatai, Carmen Sheppard, Ali Al-Shahib, David Litt, Norman Fry, Anthony Underwood,

Timothy Harrison

Public Health England, London, UK

We describe a novel bioinformatics method for accurate prediction of 88/92 (95.7%) Streptococcus pneumoniae

serotypes using WGS data. Our project is part of a large modernisation strategy within Public Health England aiming

to replace the current phenotypic pneumococcal species confirmation and serotyping reference service with a unified

WGS pipeline. Our bioinformatics pipeline integrating several components provides a comprehensive S. pneumoniae

workflow delivering species identification, MLST typing and capsular typing for each isolate. The “capsular typing” tool

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uses mapping and mutation detection to assign capsular type to bacterial genomic sequence data. The software utilises

a two-step approach; firstly, reads are mapped to capsular operon sequences for all known capsular types (n = 92).

This step can predict 36/92 (39.1%) serotypes to type level and the remaining 56/92 (60.9%) to “genogroup” (group of

capsular types with high genetic identity; often serogroups) level. If assigned to a genogroup, reads are fed into step 2

and the software uses genogroup-specific algorithms to determine the serotype. Algorithms have been developed for

18/20 genogroups, and the different approaches include presence/absence of CPS genes, detection of early stop codons,

frameshift mutations and other inactivating mutations, detection of differing alleles of genes, and SNP differences.

Using this two-step approach we can predict 88/92 serotypes to type level and 4 (32A, 32F, 24B, 24F) to serogroup

level. From the relative proportions of isolates of each serotype submitted to RVPBRU during 2014, we predict that the

serotyping pipeline will be able to type approximately 96% of circulating strains. The remaining 4% will be identified to

genogroup level and will require an additional manual serotyping step. To date, we have sequenced 1344 isolates and

have observed 90% concordance with the standard serotyping method. Following side-by-side validation of WGS and

standard methods, we anticipate implementation into the reference service at the beginning of 2016.

P1.32

Streptococcus pneumoniae shows no overall systematic genetic

adaptation over the course of infections leading to meningitis

John Lees 1 , Diederik van der Beek 2 , Julian Parkhill 1 , Stephen Bentley 1

1

Wellcome Trust Sanger Institute, Cambridge, UK; 2 Academic Medical Centre Amsterdam, Amsterdam, The Netherlands

In meningitis caused by Streptococcus pneumoniae, the bacteria will typically invade the blood stream of the patient

causing bacteraemia, then cross the blood–brain barrier into the cerebrospinal fluid (CSF) causing inflammation leading

to meningitis. We sequenced separate pneumococcal isolates from both the blood and the CSF of 675 patients with

pneumococcal meningitis. Using the resulting 1,350 whole genome sequences we robustly characterised SNP and indel

differences between pairs of strains and found no overall genetic difference between bacteria in the blood and CSF of

a single patient. In addition, we obtain equivalent results for 267 patients with meningitis caused by 3 other bacterial

species. A Bayesian model was also developed for analysis of sequence data for a phase variable inverting locus affecting

virulence. Results for observed average allele frequencies across all samples were consistent with existing mouse

infection studies, though no difference was seen between blood and CSF isolates. This work conclusively answers the

standing question of whether adaptation of S. pneumoniae occurs during an infection: no genetic elements are solely

responsible for survival in the blood compared to survival in the CSF. This result is recapitulated in 3 other invasive

bacterial species.

P1.33

Genomic and virulence comparisons of non-human isolates of

Streptococcus pneumoniae

Andrea Mitchell 1 , Ashleigh Holmes 2 , Patricia Romero 3 , Jenny Herbert 1 , Mark van der Linden 4 ,

Andrew Waller 5 , Tim Mitchell 1

1

University of Birmingham, Birmingham, UK; 2 The James Hutton Institute, Invergowrie, Dundee, UK; 3 University of Bern, Berne, Switzerland; 4 German

National Reference Center for Streptococci, Aachen, Germany; 5 Animal Health Trust, Newmarket, UK

Streptococcus pneumoniae or the pneumococcus has historically been noted as a human pathogen and commensal.

However the pneumococcus has also been isolated from non-human hosts from both carriage and disease [1]. The draft

genome sequence of an equine pneumococcal isolate A45 has been compared with other German equine isolates. A

novel multi-locus sequence type (MLST) ST6934 and putative clonal complex specific to equine isolates of serotype 3

has been identified. A previously described chromosomal deletion spanning 7Kb from pneumolysin to autolysin, and

disrupting both genes [2] appears characteristic and equine strains also appear to harbour a lysogenic prophage which

is fully inducible in the A45 strain. The representative pneumolysin-autolysin equine deletion has been transferred

into a serotype 4 genetic background. Complemented mutants, with WT Ply on plasmid, have also been generated to

compare virulence with parent strains in a murine model of pneumococcal infection. A number of genome sequences

of pneumococci from other host species including guinea pig, have also been generated. Comparison of gene content

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across these different isolates allows discussion of host restriction within the evolution of S. pneumoniae as well as

interesting insights into the mechanisms of pathogenesis employed by the pneumococcus.

1. van der Linden M, Al-Lahham A, Nicklas W, Reinert RR. Molecular characterization of pneumococcal isolates from pets and

laboratory animals. PLoS ONE 2009;4:e8286. PMID:20011527 http://dx.doi.org/10.1371/journal.pone.0008286

2. Whatmore AM, King SJ, Doherty NC, Sturgeon D, Chanter N, Dowson CG. Molecular characterization of equine isolates of

Streptococcus pneumoniae: natural disruption of genes encoding the virulence factors pneumolysin and autolysin. Infect Immun

1999;67:2776–82. PMID:10338480

P1.34

Analysis into the recombination of the phase variable Type I restriction

modification system SpnD39III locus

Megan De Ste Croix, Ana Manso, Shehzan Dada, Richard Haigh, Marco Oggioni

University of Leicester, Leicester, UK

We have identified a novel phase variable epigenetic control mechanism in Streptococcus pneumoniae with an impact on

gene expression and important phenotypes such as virulence in experimental models of infection. This genetic system is

based on the rapid inversion of the specificity determinants of the type I restriction modification (RM) system SpnD39III

which results in a change in methylation of the bacterial genome. Similarly organised RM loci are present in multiple

other species. The objective of this work was to investigate the contribution of the CreX recombinase encoded by this

locus to the phase variable rearrangement of epigenetic control. Recombination within the SpnD39III locus reverses

the orientation of a creX gene encoded in the locus. Deletion of the creX gene resulted in an almost complete block of

recombination on the sort 15 bp inverted repeat but no significant changes in recombination on the two larger 85 and

333 bp inverted repeats. RNAseq data show that the creX gene is expressed two-fold more when co-directional to the

hsdRMS genes. Somewhat surprisingly, the strains with increased creX expression recombined significantly less than

strains with lower creX expression. Our data indicate a direct but not exclusive involvement of creX in the recombination

of the hsdS genes of the SpnD39III locus responsible for the epigenetic control of pneumococcal virulence.

P1.35

Site matters: alternative insertion sites of integrative conjugative

element Tn5253 can influence conjugation frequency in Streptococcus

pneumoniae

Francesco Santoro, Alessandra Romeo, Gianni Pozzi, Francesco Iannelli

LAMMB, Department of Medical Biotechnologies, University of Siena, Siena, Italy

The integrative and conjugative element (ICE) Tn5253 of Streptococcus pneumoniae confers chloramphenicol and

tetracycline resistance, and can be transferred by conjugation. Tn5253 is found integrated at an 83-bp specific target

site (attB) located between spr1042 (iga) and spr1043 (rbgA) of the R6 chromosome. A recombinant pneumococcal

conjugation recipient was produced using a mutagenic construct containing a Kanamycin resistance cassette joined

to an engineered attB where the first 63 nucleotides (nts) were deleted and 5 nucleotide changes introduced in the

remaining 20 nts. Conjugation frequency of Tn5253 in the attB mutant recipient was considerably lower when compared

to a standard recipient (4.8 x 10 -7 versus 1.7 x 10 -5 transconjugants/donor cells). PCR analysis of transconjugants revealed

that: (i) 40% of transconjugants had Tn5253 integrated into the mutated attB; (ii) 45% had Tn5253 integrated into the

original attB with loss of the mutagenic construct; and (iii) 15% had Tn5253 integrated elsewhere in the chromosome.

Inverse PCR and sequencing disclosed 5 Tn5253 alternative insertion sites: spr1713, coding for an alpha-galactosidase,

spr0540 coding for a cell wall synthesis enzyme, spr1534 coding for the substrate binding protein of an ABC sugar

transporter, spr1983 coding for a MFS protein, and spr0546 (nrd) coding for a putative nitroreductase. Transfer of Tn5253

from transconjugants harbouring Tn5253 in alternative sites occurred at lower frequencies than the wild-type donor

(from 2 x 10 -7 to < 3.6 x 10 -8 ). One transconjugant harbouring 3 copies of Tn5253 was able to transfer the element at

a frequency 100-fold higher than the wild-type donor. Our results indicate that Tn5253 has a strong preference for its

primary insertion site, even when it is mutated, but that it can also integrate at different sites. The insertion site of

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Tn5253 affects the transposition rate, which is decreased or abolished when it is integrated in an alternative attB.

P1.36

Mutagenesis of one Streptococcus pneumoniae serotype 1 strain

Vanessa Terra 1 , Charles Plumptre 2 , Emily Kay 1 , Brendan Wren 1

1

London School of Hygiene and Tropical Medicine, London, UK; 2 University College London, London, UK

Streptococcus pneumoniae causes pneumonia, bacteraemia, meningitis and otitis media. Despite vaccination, there

are still around 1 million deaths each year worldwide. Serotype 1 is amongst the most invasive of serotypes and is

extremely important in Africa. It is also a significant cause of meningitis, is unusual in that it is rarely found colonising

the nasopharynx, and is frequently associated with outbreaks. Although included in the pneumococcal conjugate

vaccine, PCV13, the efficacy against serotype 1 has been questioned. Therefore there is a continuous need to study and

understand this particular serotype. In this study, a new method was developed to mutagenise a serotype 1 strain. The

method relies on: the known natural competence of S. pneumoniae; double recombination; and a delivery plasmid. The

delivery plasmid contains a PCR fragment where a portion of the gene of interest has been deleted and substituted by

a suitable antibiotic cassette. This is followed by natural transformation. In order to test the method, 3 genes known to

be possible to mutate and 1 putative gene were targeted and the mutations achieved. The loss of function mutants will

be firstly studied in the insect larvae model Galleria mellonella, for decreased virulence, followed by virulence studies in

mice. The ability to mutagenise this strain will help pave the way for a better and more extensive understanding of such

an important serotype, allowing for improved therapies and better vaccines.

P1.37

Improving molecular detection, identification and serotyping of

Streptococcus pneumoniae in complex samples

Laura Boelsen 1, 2 , Eileen Dunne 1 , Katherine Gould 3 , Fiona Russell 1, 4 , Kim Mulholland 1, 5 , Jason

Hinds 3 , Catherine Satzke 1, 6

1

Pneumococcal Research, Murdoch Childrens Research Institute, Parkville, VIC, Australia; 2 Department of Paediatrics, The University of Melbourne,

Parkville, VIC, Australia; 3 Bacterial Microarray Group, St. George’s University of London, London, UK; 4 Centre for International Child Health,

Department of Paediatrics, The University of Melbourne, Parkville, VIC, Australia; 5 London School of Hygiene and Tropical Medicine, London, UK;

6

Department of Microbiology and Immunology, Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, VIC, Australia

Molecular methods (lytA qPCR and serotyping by microarray) were used to examine pneumococcal carriage in adult

caregivers using nasopharyngeal and oropharyngeal (OP) samples collected as part of a PCV10 impact study in Fiji. Results

for the OP samples were more difficult to interpret, as microarray analysis indicated many samples contained partial or

divergent pneumococcal capsule homologues but also lacked pneumococci, including several lytA positive samples. False

positives in OP samples due to the presence of capsule and lytA gene homologues in non-pneumococcal species have

been reported by other groups. Culture of OP samples on gentamicin horse blood agar (gHBA) also revealed a complex

mix of bacteria present. Here we dissect the complexity of a subset of these OP samples to better understand the

component species present and to improve the sample screening approach by investigating alternative pneumococcalspecific

qPCR targets. A representative of each colony morphology on gHBA was subcultured from 11 samples resulting

in 92 isolates. Each isolate was tested by standard culture-based pneumococcal identification tests and MALDI-TOF/

MS for species identification. A subset were analysed by microarray and serotyped by latex agglutination. Preliminary

results have found 3 serotypeable pneumococcal isolates, and 9 non-pneumococcal isolates with latex agglutination

serotyping results, most commonly 19B (n = 6). MALDI-TOF/MS (n = 13) identified Streptococcus mitis/Streptococcus

oralis (n = 7), Streptococcus salivarius (n = 2), Streptococcus parasanguinis (n = 2) and Streptococcus anginosus (n = 1),

with 1 unidentified isolate. The MALDI-TOF/MS results were concordant with microarray analysis, which also found

non-pneumococcal isolates containing pneumococcal capsule gene homologues. Some non-pneumococcal isolates

were weakly positive for lytA and likely contributing to the weak positivity of some OP samples. Our results thus far

highlight the complexity of OP samples and the potential to misidentify and incorrectly serotype pneumococci using

existing conventional and molecular methods. Further work examining the performance of alternative pneumococcal

identification qPCR screening targets is underway.

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P1.38

Genome-wide assessment of pneumococcal antigenic diversity

Nicholas Croucher 1 , Lisa Kagedan 2 , Claudette Thompson 2 , Julian Parkhill 3 , Stephen Bentley 3 ,

Jonathan Finkelstein 4 , Marc Lipsitch 2 , William Hanage 2

1

Imperial College, London, UK; 2 Harvard School of Public Health, Boston, USA, 3 Wellcome Trust Sanger Institute, Cambridge, UK, 4 Harvard Medical

School, Boston, USA

Extensive variation in both polysaccharide and protein antigens is evident across pneumococcal populations. The

variation in these surface structures can be quantified through whole genome sequencing of systematic samples of

pneumococci isolated from carriage. Data from 616 isolates collected in Massachusetts between 2001 and 2007 allowed

characterisation of 20 serotype switching events. These revealed a highly significant (p < 0.0001) enrichment for withinserogroup

switching, based on the observed serotype distribution. However, this pattern could not be fully explained by

more frequent, shorter recombinations preferentially driving the smaller changes required to switch between serotypes

of the same serogroup. Although a separate dataset indicated vaccine escape sometimes associated with changes in

β–lactam resistance, the pattern of switching in this collection was not detectably affected by linkage between pbp

genes and the capsule polysaccharide synthesis (cps) locus. The absence of an explanation based on the properties

of transformation suggested the observed pattern was a consequence of selection for preserving serogroup. Hence

multiple exchanges of serotypes were experimentally constructed in common genetic backgrounds to test for epistatic

interactions between the cps locus and other pneumococcal genes. However, these data were not consistent with

epistatic interactions between cps loci of a particular serogroup and the rest of the genome, meaning they were unlikely

to account for the observed distribution of capsule types. These data suggested future work should focus on alternative

mechanisms, such as host immunity spanning multiple serotypes within the same serogroups, which might explain this

significant trend. Characterisation of proteinaceous antigens found them to be highly diverse across the population.

However, individual alleles were stably associated with particular lineages, with only pspA and pspC undergoing

diversification over short timescales. Together, these data show how individual lineages are likely to be restricted in how

they diversify under host immune selection pressure.

P1.39

An increase in negative supercoiling in Streptococcus pneumoniae

induces a global transcriptomic response that regulates the DNA

topoisomerase I gene

Maria-José Ferrándiz 1 , Antonio-Javier Martín-Galiano 1 , Isabel Camacho-Soguero 1 , Cristina

Arnanz 1 , Adela G. de la Campa 1, 2

1

Centro Nacional de Microbiología, ISCIII and CIBERES, Majadahonda/ Madrid, Spain; 2 Presidencia. CSIC, Madrid, Spain

The most basic level of transcription regulation in Streptococcus pneumoniae results from the organisation of its

chromosome into topological domains. We have previously observed a global transcriptional response to DNArelaxation.

In this study, to increase DNA supercoiling, we used seconeolitsine (SCN), an inhibitor of topoisomerase I.

Supercoiling density (σ) varied as a function of time as a result of treatment with SCN at 0.5 × MIC: 48%, 74%, and 46%

at 5 min, 15 min and 30 min of treatment, respectively. A global transcriptomic response was observed. The number

of responsive genes decreased from a high 395 at 5 min to 285 at 15 min, to 150 at 30 min (a drop of more than half).

More than one-third of the responsive genes at 15 min were found to be contiguous in the chromosome, forming

clusters that showed coordinated regulation. At least 10 clusters were discerned. Clusters were evident at 5 and 15

min. The only DNA topoisomerase gene significantly affected was topA, which was 3-fold down-regulated at 15 min.

After 30 min, topA transcripts were restored to baseline levels, coincident with partial recovery of the supercoiling and

reduction of the transcriptomic response. For the first time here, we have identified in bacteria, the existence of a global

transcriptomic response triggered by an increase in DNA supercoiling. This response is similar to the one observed

during DNA relaxation. This indicates that they are handled globally as a common type of topological stress.

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P1.41

Systematic nomenclature for the bacterial two-component regulatory

systems, based on genomic, structural and functional analysis of the

pan-genome of Streptococcus pneumoniae

Gustavo Gamez 1, 2 , Diego Sanchez 1, 2 , Frank Mona 1, 2 , Yully Betancur 1, 2 , Andres Castro 1, 2 , Alejandro

Gomez 1, 3 , Jose Mediavilla 4 , Mauricio Corredor 2 , Sven Hammerschmidt 3

1

Basic and Applied Microbiology (MICROBA) Research Group, School of Microbiology, Universidad de Antioquia, Medellin, Colombia; 2 Genetics,

Regeneration and Cancer (GRC) Research Group, University Research Center (SIU), Universidad de Antioquia, Medellin, Colombia; 3 Department

Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany; 4 Public

Health Research Institute, International Center for Public Health, Rutgers University, Newark, USA

Two-component systems (TCSs) represent the single largest paralogous gene family encoding signalling proteins in

Archaea and Eukarya. However, despite the importance of these systems for the regulation of different cellular and

physiological processes, a clear and uniform nomenclature system allowing for their systematic study does not exist.

Here, a thorough bioinformatics search and analysis was performed to identify conserved and distinctive features at the

genomic, structural and functional level, with the aim of devising a systematic and expandable model of nomenclature

for the operons, genes and proteins comprising the TCSs of the pan-genome of Streptococcus pneumoniae. The DNA

and protein sequences of 25 pneumococcal strains, whose genomes are completely sequenced and annotated, were

analysed employing different bioinformatics tools and databases. Twenty-nine different TCS-proteins (14-HKs, 15-RRs)

were identified, indicating a total pan-genomic complement of 15-TCSs, to which this species has access at a population

level. However, the modal complement was 13-TCSs and One-Orphan-RR. The TCS-variome estimation (genetic and

protein variability) confirmed the high-level of conservation among these pneumococcal regulators. Additionally,

through the structural/functional analysis of the TCS-proteins, it was possible to establish the presence of conserved and

distinctive features in terms of presence, absence, number, type, organisation and localisation of the different functional

domains. Moreover, the common names and diversity of functions under the control of the pneumococcal TCSs was

defined by a systematic research of the specialised literature. The identification of both conserved and distinctive

features at the genomic, structural and functional level allowed for the establishment of a systematic and uniform model

of nomenclature for the operons and proteins comprising the TCSs of the pneumococcal pan-genome. This model has

been already extrapolated to other bacterial species and it has the potential to serve as a reference standard to improve

research and understanding of these regulatory systems in prokaryotes.

P1.42

Optimisation of the expression of fluorescent proteins in Streptococcus

pneumoniae

Maria Catalão 1 , Joana Figueiredo 1 , Mafalda Henriques 1 , João Gomes 2 , Sergio Filipe 1

1

Laboratory of Bacterial Cell Surfaces and Pathogenesis, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal;

2

National Institute of Health, Department of Infectious Diseases, Lisbon, Portugal

Streptococcus pneumoniae is a Gram-positive bacterium usually found in association with a range of different types of

infections. The understanding of how these bacteria divide or perform specific tasks important for their survival is a

requirement for the design of efficient strategies to fight bacterial infections. This implies a detailed knowledge not only

of the function of proteins required for the infection process, but also of their localisation and role in complex molecular

machineries. In order to determine the correct subcellular localisation of fluorescent proteins in S. pneumoniae, we

have previously described tools to express derivatives of 4 fluorescent proteins—mCherry, Citrine, CFP and GFP—to

levels that allow visualisation by fluorescence microscopy, by fusing the first 10 amino acids of the S. pneumoniae

protein Wze (the i-tag), upstream of the fluorescent protein. We had proposed that this i-tag extension might facilitate

ribosome accessibility to the ribosome-binding site, thus enhancing protein translation. These tools, which we have now

confirmed that can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus, and

Bacillus subtilis, have been optimised by changing the nucleotide sequence of the i-tag and testing the effect of the first

10 amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found

that manipulating the structure and stability of the 5’ end of the mRNA molecule, which may influence the accessibility

of the ribosome, is determinant to ensure the expression of a strong fluorescent signal. We therefore propose that the

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determination of minimum free energy calculated for the 5’ end of the mRNAs molecules originated by these tools may

predict whether the expression of a specific fluorescent protein will be successful and may be used to set up a regulated

constitutive protein expression.

P1.43

The single transmembrane segment of pneumococcal WalK is required

for the perception of an intramembrane or extracellular signal

Gro Stamsås, Daniel Straume, Zhian Salehian, Leiv Sigve Håvarstein

Norwegian University of Life Sciences, Aas, Norway

The Streptococcus pneumoniae genome encodes 13 two-component regulatory systems plus an orphan response

regulator. Only one of these systems, WalRK, is essential for viability under laboratory conditions. Despite its importance,

the biological role of the WalRK system is not well understood. However, previous studies have shown that it regulates

expression of the cross wall splitting enzyme PcsB, and consequently has a crucial role in pneumococcal cell division.

Considerable efforts have been made to understand how the system is regulated, but no signal(s) sensed by the WalK

histidine kinase has been identified so far. WalK orthologs in most low-GC Gram-positive bacteria are attached to the

cytoplasmic membrane via two transmembrane segments separated by a large extracellular loop believed to function

as a sensor domain. In contrast, members of the genus Streptococcus have WalK histidine kinases that are anchored

to the cytoplasmic membrane by a single transmembrane segment. It has been a long-standing question whether this

transmembrane segment still serves as a signal-sensing domain, or if it only functions as a membrane anchor. Here, we

present data that strongly suggest that the transmembrane segment senses or relays an extracellular or intramembrane

signal that regulates the activity of WalK. Moreover, in contrast to what was previously believed, we provide evidence

suggesting that the serine/threonine protein kinase StkP upregulates PcsB expression by stimulating the kinase activity

or inhibiting the phosphatase activity of WalK rather than by direct phosphorylation of the WalR response regulator.

P2.01

Regulatory responses of Streptococcus pneumoniae D39 to ascorbic

acid

Muhammad Afzal 1 , Sulman Shafeeq 1, 2 , Birgitta Henriques-Normark 2 , Oscar Kuipers 1

1

University of Groningen, Groningen, The Netherlands; 2 Karolinska Institutet, Stockholm, Sweden

We have explored the impact of ascorbic acid on the transcriptome of Streptococcus pneumoniae D39. The expression

of several genes and operons, including the ula (utilisation of L-ascorbic acid) operon, the AdcR regulon (which has been

previously shown to be involved in zinc transport and virulence) and a PTS operon (which we denote here as the ula2

operon) were altered in the presence of ascorbic acid. We have explored the regulatory mechanism of the ula and ula2

operons. Our β-galactosidase assay and microarray data demonstrate that transcriptional regulators UlaR and UlaR2

act as transcriptional activators of the ula and ula2 operons, respectively, in the presence of ascorbic acid. We predict

putative regulatory sites for UlaR and UlaR2 binding in PulaA and Pula2, respectively. Furthermore, we have explored the

effect of ascorbic acid on the expression of the AdcR regulon in more detail. Our ICP-MS analysis showed that addition of

ascorbic acid to the medium causes zinc starvation in the cell that leads to the activation of the AdcR regulon.

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P2.02

Altered penicillin-binding protein 2x of Streptococcus pneumoniae: a

new target of the serine protease HtrA

Katharina Peters, Inga Schweizer, Regine Hakenbeck, Dalia Denapaite

Department of Microbiology, University of Kaiserslautern, D-67663 Kaiserslautern, Germany

The two cefotaxime resistant laboratory Streptococcus pneumoniae mutants C405 and C606 derived independently

from the laboratory strain R6 [1] contain less essential penicillin-binding protein 2x compared to the parental strain

[2]. In transpeptidase domain of PBP2x each of these mutants have 2 or 4 mutations, respectively. In these mutants no

effect on the transcription of pbp2x gene was detected, and thus the reduced amount of PBP2x was likely to be due

to degradation of the protein. Furthermore, both mutants carry mutation in ciaH, which encodes the histidine protein

kinase of the two-component system CiaRH [3], resulting in hyperactivation of CiaR [4]. One gene product of the CiaRH

regulon is the serine protease/chaperon HtrA [5], suggesting that HtrA is responsible for the reduced amount of PBP2x.

Analysis of HtrA deletion or active site inactivation of HtrA in C405 and C606 showed that the proteolytic activity of

HtrA is responsible for degradation of PBP2x in both mutants. Integration of an ectopic copy of htrA in the HtrA deletion

mutants restored the phenotype. Introduction of pbp2x alleles into the sensitive R6 strain, in which the CiaRH system is

not as active as in the mutants, leads to a slight reduction of PBP2x amount, but not as pronounced as in the mutants.

This suggests the contribution of other factors to this phenotype. Depletion studies of the PBP2x C405

variant in different

genetic backgrounds and in presence or absence of HtrA confirmed that HtrA degrades PBP2x in C405. A GFP-PBP2x C405

fusion protein still localises at the septum in the absence of HtrA; in the presence of HtrA high cytoplasmatic signals are

in agreement with the presence of GFP-PBP2x C405

degradation products.

1. Laible G and Hakenbeck R. Penicillin-binding proteins in beta-lactam-resistant laboratory mutants of

Streptococcus pneumoniae. Mol Microbiol. 1987;1(3):355-63

2. Maurer P, Koch B, Zerfass I, Krauss J, van der Linden M, Frère JM, Contreras-Martel C, Hakenbeck R. Penicillinbinding

protein 2x of Streptococcus pneumoniae: three new mutational pathways for remodelling an

essential enzyme into a resistance determinant. J Mol Biol. 2008;376(5):1403-16.

3. Guenzi E, Gasc AM, Sicard MA, Hakenbeck R. A two-component signal-transducing system is involved

in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae. Mol

Microbiol. 1994;12(3):505-15.

4. Müller M1, Marx P, Hakenbeck R, Brückner R. Effect of new alleles of the histidine kinase gene ciaH on the

activity of the response regulator CiaR in Streptococcus pneumoniae R6. Microbiology. 2011;157(Pt 11):

3104-12.

5. Halfmann A, Kovács M, Hakenbeck R, Brückner R. Identification of the genes directly controlled by the

response regulator CiaR in Streptococcus pneumoniae: five out of 15 promoters drive expression of small

non-coding RNAs. Mol Microbiol. 2007;66(1):110-26.

P2.03

Hydroxyl radicals contribute to the bactericidal effects of the

fluoroquinolone moxifloxacin in Streptococcus pneumoniae

Maria-José Ferrándiz 1 , Antonio-Javier Martín-Galiano 1 , Cristina Arnanz 1 , Tahl Zimmerman 1 , Adela

G. de la Campa 1, 2

1

Centro Nacional de Microbiología, ISCIII and CIBERES, Majadahonda/ Madrid, Spain; 2 Presidencia. CSIC, Madrid, Spain

We studied the transcriptomic response of Streptococcus pneumoniae to moxifloxacin, an inhibitor of DNA gyrase. At 10

× MIC, 30 and 140 responsive genes were detected at 15 and 30 minutes, respectively. This response included the upregulation

of 2 genes converting 6P-sugars to fructose 6P, together with 3 out of 7 genes of the glycolysis pathway, which

converts fructose-6P to pyruvate. In addition, an increase in acetyl-coA would be expected from the down-regulation

of the acetyl-coA carboxylase. Acetyl-coA would be converted to pyruvate by the action of formate acetyltransferase,

which gene was also up-regulated. Since pyruvate is converted to hydrogen peroxide (H 2

O 2

) by pyruvate oxidase (SpxB),

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pyruvate increase would lead to an increase in the intracellular H 2

O 2

, and in turn, in the amounts of reactive oxygen

species (ROS) resulting from the Fenton reaction (Fe 2+ + H 2

O 2

→ Fe 3+ + OH - + OH . ). ROS damage DNA, lipids and proteins.

With moxifloxacin, we observed similar increases in the production of H 2

O 2

and ROS, which contributed to the lethality

of the drug. In accordance, the lethality of moxifloxacin was attenuated in a strain lacking the spxB gene. These results

support the model of a universal mechanism of action for bactericidal antibiotics, including fluoroquinolones. These

observations are also in agreement with our previous findings that levofloxacin, an inhibitor of topoisomerase IV, triggers

the transcriptional activation of iron transport genes. Both drugs stimulate the Fenton reaction in their mechanism of

action, by causing an increase in the concentration of either iron or H 2

O 2

, respectively.

P2.04

Regulation of PavB by TCS08 in Streptococcus pneumoniae

A Gómez 1, 2 , L. Petruschka 1 , G. Gámez 2, 3 , S. Böhm 1 , V. Kluger 1 , A. Klein 1 , S. Hammerschmidt 1

1

Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, Ernst Moritz Arndt University of Greifswald,

D-17487, Greifswald, Germany; 2 Basic and Applied Microbiology (MICROBA) Research Group, School of Microbiology, Universidad de Antioquia,

UdeA, Calle 70 No. 52 - 21, 050010, Medellin, Antioquia, Colombia; 3 Genetics, Regeneration and Cancer (GRC) Research Group, University Research

Center (SIU), Universidad de Antioquia, UdeA, Calle 70 No. 52 - 21, 050010, Medellin, Antioquia, Colombia

The human pathogen Streptococcus pneumoniae (pneumococci) possess 13 two-component regulatory systems (TCSs),

which are crucial for bacterial fitness and virulence. Traditionally, these systems consist of a sensor histidine kinase

(HK) and an output, the response regulator (RR). For TCS08, its encoding genes are placed downstream of the coding

sequence of the adhesin PavB (pneumoccal adherence and virulence factor b), which has been associated with virulence

in the pneumococci [1]. Hence, the interaction of the TCS08 proteins and its effect on the regulation of PavB and other

pneumococcal surface proteins was evaluated in this work. This study aimed to assess the interaction and regulation of

TCS08 proteins and PavB. Maltose binding protein and affinity chromatography were used for TCS08 and PavB proteins

purification. Phosphotransfer profiling and EMSA were established to assess the interaction between the recombinant

HK08 and RR08, and the RR08 and the promoter region of pavB, respectively. The purity of recombinant MBP-HK08

and MBP-RR08 were determined by SDS-PAGE and immunoblot analysis. The HK08 and its cognate RR08 displayed a

phosphorylation interaction, suggesting a phosphatase activity and autophosphorylation, respectively. The interaction

between the pavB promoter fragment and the non-phosphorylated RR08 showed a shift in the electrophoretic mobility

of pavB. The expression of PavB increased dramatically for the hk08-mutant but no significant differences were

found for the rr08- or tcs08-mutants when compared with the wild-type, suggesting a repressor function of the nonphosphorylated

RR08 for the expression of PavB. The results revealed no direct relation of the TCS08 with virulence and

pathogenicity, but it is involved in the expression of PavB in S. pneumoniae.

1. Jensch I, Gámez G, Rothe M, Ebert S, Fulde M, Somplatzki D et al. PavB is a surface-exposed adhesin of

Streptococcus pneumoniae contributing to nasopharyngeal colonization and airways infections. Mol Microbiol

2010;77:22–43. http://dx.doi.org/10.1111/j.1365-2958.2010.07189.x. PMID:20444103

P2.05

TpxD serves as a global regulator for pneumococcal response to H 2

O 2

,

and is regulated by CodY

Barak Hajaj 1 , Hasan Yesilkaya 2 , Sulman Shafeeq 3 , Xiangyun Zhi 2 , Rachel Benisty 1 , Oscar Kuipers 3 ,

Nurith Porat 1

1

Pediatric Infectious Disease Unit, Soroka University Medical Center, Faculty of Health Sciences, Department of Microbiology and Immunology,

Ben-Gurion University of the Negev, Beer Sheva, Israel; 2 Department of Infection, Immunity & Inflammation, University of Leicester, Leicester,

UK; 3 Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The

Netherlands

Streptococcus pneumoniae is a facultative anaerobic pathogen. Although it maintains fermentative metabolism, during

aerobic growth pneumococci produce high levels of H 2

O 2

, which can have adverse effects on cell viability and DNA, and

influence pneumococcal interaction with its host. The pneumococcus is unusual in its dealing with toxic reactive oxygen

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species, in that it neither has catalase nor the global regulators of peroxide stress resistance such as OxyR and PerR. In

this study we investigated TpxD’s function as a sensor and global regulator in pneumococcal response to H 2

O 2

. Microarray

analysis revealed that TpxD mediates pneumococcal response to H 2

O 2

at the transcriptional level, as mutation of tpxD

abolished H 2

O 2

mediated gene expression. The mechanism underlying TpxD regulatory function was further elucidated

by replacing the catalytic cysteine 58 with alanine. This mutation prevented tpxD up-regulation upon addition of H 2

O 2

,

and eliminated pneumococcal response to H 2

O 2

, signifying that cysteine 58 is crucial for TpxD signalling activity. We then

aimed to identify the transcription factor governing tpxD expression under H 2

O 2

stress. Bioinformatic analysis discovered

a putative 15-bp consensus CodY binding site in the upstream region of tpxD-coding sequence. The functionality of this

CodY-box was confirmed by mutations and EMSA analysis. Moreover, no up-regulation of TpxD could be detected in

DcodY challenged with H 2

O 2

. These data identify CodY as the transcription factor modulating tpxD expression under H 2

O 2

stress. We propose a model in which TpxD functions as a mediator, transferring H 2

O 2

signal to down-stream effectors,

thereby controlling other targets involved in oxidative stress response.

P2.06

Co2+-dependent transcriptional regulation in Streptococcus

pneumoniae: opposite effect of Mn2+ and Co2+ on the expression of the

virulence genes psaBCA, pcpA and prtA

Irfan Manzoor 1 , Sulman Shafeeq 1, 2 , Tomas Kloosterman 1 , Oscar Kuipers 1

1

University of Groningen, Groningen, The Netherlands; 2 Karolinska Institutet, Dtockhlom, Sweden

Manganese (Mn2+)-, zinc (Zn2+)- and copper (Cu2+)-dependent gene regulation in Streptococcus pneumoniae have

already been studied extensively. However, the effect of the important transition metal ion cobalt (Co2+) on gene

expression of S. pneumoniae has not yet been explored. Here, we study the impact of Co2+-stress on the transcriptome

of S. pneumoniae strain D39. BLAST searches revealed that the genome of S. pneumoniae encodes a putative Co2+transport

operon (cbi operon), which we show here to be upregulated under Co2+-stress. By means of transcriptional

lacZ-reporter studies, we confirm that the expression of the cbi operon increases with increasing concentrations of

Co2+. Furthermore, we show that Co2+, as has been shown previously for Zn2+, can induce derepression of the genes

of the PsaR regulon, encoding the Mn2+-uptake system (PsaBCA), the choline-binding protein (PcpA) and the cell-wall

associated serine protease (PrtA). The data also corroborate a role for CzcD as a Co2+-efflux pump.

P2.07

Differential complement sensitivity of Streptococcus pneumoniae and

Streptococcus mitis

Helina E Marshall 1 , Fernanda C Petersen 2 , Jeremy S Brown 1

1

University College London, London, UK; 2 University of Oslo, Oslo, Norway

Streptococcus pneumoniae and Streptococcus mitis are naso-oropharyngeal commensals that are genetically similar.

However, S. pneumoniae is highly pathogenic and a common cause of pneumonia and septicaemia, whereas S. mitis

rarely causes disease. We hypothesise that differences in sensitivity to innate immunity may underlie these differences

in virulence phenotype. We compared sensitivity of S. pneumoniae and S. mitis to neutrophil killing. After opsonisation

with serum, but not with heat-treated serum or PBS, S. mitis was markedly more sensitive to neutrophil killing compared

to S. pneumoniae. These differences suggested S. mitis was relatively complement sensitive, and flow cytometry assays of

C3b/iC3b deposition confirmed there was increased complement opsonisation of S. mitis compared to S. pneumoniae. S.

pneumoniae resistance to complement is partially dependent on binding of the immune regulator factor H by the surface

protein PspC. We investigated factor H binding to S. mitis using flow cytometry. The results demonstrated that there

was no significant factor H binding to S. mitis. By inserting pspC of S. pneumoniae into S. mitis, we demonstrated that

expression of PspC enabled S. mitis to then bind factor H. Investigation of C3b/iC3b confirmed a decrease in opsonisation.

Furthermore, survival in whole human blood of this modified strain showed an increase, when compared to the wildtype

strain. These results suggest that an inability to bind factor H might underpin S. mitis sensitivity to opsonisation with

complement and neutrophil killing compared to S. pneumoniae, and therefore contribute to the differences in virulence

between these 2 commensal species.

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P2.08

Using dual RNA-Seq to characterise gene expression in an animal

model of Streptococcus pneumoniae

Neil Ritchie, Tom Evans

University of Glasgow, Glasgow, UK

RNA-Seq is a useful tool for characterising RNA transcription. The ability to generate large numbers of reads allows

accurate quantification of the transcriptome of both bacterial pathogens and the host response. We characterised the

transcriptome of the serotype 3 strain SRL1 during growth in broth and in vivo during a mouse model of infection.

RNA was extracted using a bead beating method from broth cultures as well as bronchoalveolar and pleural lavage.

Sequencing was conducted using the Illumina Hi-Seq platform. Samples from 3 mice were combined for the final analysis.

Ninety-eight million reads from broth culture were successfully mapped to the reference genome while 6.8 million

and 4.2 million from BALF and pleura respectively were mapped. Infection samples clustered separately from broth

samples on principle component analysis. Three genomic regions were identified as being up-regulated during infection

relative to broth: an operon coding for genes involved in purine metabolism and the VanZ glycopeptide resistance gene,

the Blp bacteriocin locus, and the PsaA locus. All 3 genomic regions were identified as being upregulated in BALF and

pleura. Analysis of the host response showed a significant up-regulation of type I interferon-regulated genes in the

pleural space compared to the lung. RNA-Seq is a useful tool to characterise the transcriptome during infection in vivo.

Consistent gene regulation changes were identified in both infection sites with upregulation of operons associated with

metabolism, intra-species competition and virulence. Further work will characterise the role of these genes in infection.

P2.09

Emergence of amoxicillin-resistant variants of Spain 9V -ST156

pneumococci expressing serotype 11A correlates with their ability to

evade the host immune response

Leire Aguinagalde 1 , Bruno Corsini 1 , Arnau Domenech 2, 3 , Mirian Domenech 3, 4 , Jordi Cámara 2, 3 ,

Carmen Ardanuy 2, 3 , Ernesto García 3, 4 , Josefina Liñares 2, 3 , Asunción Fenoll 1 , Jose Yuste 1, 3

1

Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Madrid, Spain; 2 Microbiology Department, Hospital Universitari de Bellvitge-IDIBELL-

Barcelona University, Barcelona, Spain; 3 CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain; 4 Centro de Investigaciones Biológicas-CSIC,

Madrid, Spain

Capsular switching in Streptococcus pneumoniae allows pre-existing clones expressing vaccine serotypes to escape

vaccine-induced immunity by acquisition of capsular genes from pneumococci of a non-vaccine serotype. The emergence

of penicillin-resistant serotype 11A pneumococci has been identified at the Spanish Pneumococcal Reference Laboratory,

resulting in a significant concern as this serotype is not included in the current conjugate vaccines. For this reason, we

have analysed the clonal composition of 492 clinical isolates of serotype 11A causing invasive disease in Spain (2000-

2012), and their ability to evade the host immune response. Antibiograms, molecular typing and restriction profiles of

pbp2x, pbp1a and pbp2b genes were analysed. Interaction with complement components C1q, C3b, C4BP, and factor

H was explored. Moreover, opsonophagocytosis assays were performed using HL-60 cells differentiated to neutrophils.

Biofilm formation and polymorphisms of the major autolysin LytA were also evaluated. The main genotypes of the 11A

pneumococci throughout the period 2000-2012 were ST62 (>65%), followed by ST6521, and ST838. Genotypes ST838

and ST6521 emerged from 2005 displaying penicillin and amoxicillin resistance by harbouring a different pbp2b gene.

Both genotypes were single (ST838) or double (ST6521) locus variants of the Spain 9V -ST156 clone. A different pattern

of evasion of complement immunity and phagocytosis was observed between genotypes. Emergence of ST6521 11A , a

vaccine escape variant of the S. pneumoniae Spain 9V -ST156 showing a high potential to avoid the host immune response,

was observed. Our findings anticipate the possible spread of ST6521 11A clone among pneumococcal isolates causing

invasive disease.

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P2.10

Streptococci of Mitis group, misidentified as Streptococcus pneumoniae

Agnieszka Bojarska 1 , Alicja Kuch 2 , Elzbieta Stefaniuk 2 , Anna Skoczyńska 2 , Waleria Hryniewicz 2 ,

Ewa Sadowy 1

1

Department of Molecular Microbiology, National Medicines Institute, Warsaw, Poland; 2 Department of Epidemiology and Clinical Microbiology,

National Medicines Institute, Warsaw, Poland

We aimed to perform identification and molecular analysis group of isolates initially identified as Streptococcus

pneumoniae. Sixty-six Streptococcus spp. isolates from upper and lower respiratory tract (mostly from sputum) collected

during 1995–2013, were examined for colony morphology on sheep blood agar, optochin sensitivity, bile salts solubility,

in mass spectrometry identification systems (MALDI-TOF), by biochemical test API 20 Strep, Pneumotest-Latex, as well

as molecular methods such as detection of pneumolysin gene (ply), partial sequencing of lytA and multi-locus sequence

analysis (MLSA). Colony morphology and haemolysis of all isolates resembled one of S. pneumoniae strains. Ninety per

cent of isolates were optochin sensitive and 70% of isolates were soluble in bile salts. MALDI-TOF identified collected

isolates mostly as S. pneumoniae. Agglutination was observed with sera from Pneumotest-latex for some isolates. Weak

enzymatic activity and fermentation of sugar exhibited by tested isolates prevented unequivocal species identification

by API 20 Strep. The ply gene was frequent in the analysed group (80%). All studied isolates harboured the 6-bp deletion

in the 3’ part of the lytA gene that excluded their identification as S. pneumoniae. MLSA classified isolates as S. mitis,

S. pseudopneumoniae and single representatives of S. sanguinis and S. oralis. Species identification of certain strains of

alpha-haemolytic streptococci, especially those isolated from respiratory tract, may be problematic and unreliable when

limited to the classical microbiological methods.

P2.11

Clonal relationship with the invasive disease potential of pneumococcal

serotypes

Eva del Amo 1 , Cristina Esteva 1 , Susanna Hernandez-Bou 2 , Carmen Galles 3 , Marian Navarro 4 ,

Goretti Sauca 5 , Miriam Triviño 2 , Jordi de Batlle 6 , Marina Cunquero 1 , Carmina Marti 7 , Pilar Ciruela 8 ,

Carmen Muñoz-Almagro 1 , Invasive Disease Catalan Study Group 8

1

Molecular Microbiology Department, Universitary Hospital Sant Joan de Déu, Esplugues de Llobregat, Barcelona, Spain; 2 Paediatric Department,

Universitary Hospital Sant Joan de Déu, Esplugues de Llobregat, Barcelona, Spain; 3 Microbiology Department, Calella Hospital, Calella, Barcelona,

Spain; 4 Microbiology Department, Consorci Hospitalari de Vic, Vic, Barcelona, Spain; 5 Microbiology Department, Hospital de Mataró, Mataró,

Barcelona, Spain; 6 Microbiology Department, Universitary Hospital Dr Josep Trueta, Girona, Spain; 7 Microbiology Department, Hospital General de

Granollers, Granollers, Barcelona, Spain; 8 Public Health Agency, Government of Catalonia, Barcelona, Spain

Estimation of the invasive disease potential (IDP) of pneumococcal serotypes is an important tool to identify the serotypes

with high risk to cause disease. Nowadays, the invasiveness index calculation is labour intensive because it needs to

compare the serotype distribution in a wide carrier collection versus the distribution found in patients with invasive

pneumococcal disease (IPD). The aim of this study is to determine the IDP of Streptococcus pneumoniae serotypes

causing IPD in Catalonia without using a carrier collection. We calculate the IDP using the index of clonal diversity of each

invasive serotype. All pneumococcal invasive isolates (adults and children) received between 2010 and 2013 from 26

health centres in Catalonia and characterised by the Molecular Microbiology Department at Hospital Sant Joan de Déu,

Barcelona, Spain, were included. Capsular typing and clonal analysis were performed by fluorescent Multiplex PCR and

MLST, respectively. Clonal diversity was estimated by Simpson’s numerical index of discrimination. We received 1,576

pneumococcal invasive isolates. Serotype and clonal analysis were available in 1,551 strains (98.4%). Fifty-two serotypes

and 249 clonal types were identified. We classified the invasiveness of the serotypes that were detected in at least 10

isolates. The serotypes with a clonal diversity under the percentile 50 were 10A, 23B, 7F, 12F, 22F, 1, 31,38, 5, 11A, 14,

23A, 8, 3, and 15A. The serotypes with a clonal diversity higher than the percentile 50 were 24F, 18C, 9V, 33F, 23F, 9N,

6C, 6B, 16F, 4, 19A, 15C, 6A, 15B, and 19F. Our results confirmed that serotypes highly associated with invasive disease

such as serotype 1 or 7F presented some of the lowest levels of diversity, while serotypes such as 19F and 6C that are

associated with carriage showed the highest levels of diversity, suggesting an alternative methodology based only in the

invasive strains collection using molecular methods.

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P2.12

Pneumococcal carriage in infants and young children in Fiji before and

after PCV10 introduction

Eileen Dunne 1 , Catherine Satzke 1, 2 , Tupou Ratu 3 , Eric Rafai 3 , Mike Kama 3 , Rachel Devi 3 , Kathryn

Bright 1 , Lisi Tikoduadua 3 , Joseph Kado 3 , Casey Pell 1 , Monica Nation 1 , Jenna Smyth 1 , Maha Habib 1 ,

Belinda Ortika 1 , Kate Gould 4 , Jason Hinds 4 , Kylie Jenkins 6 , Kim Mulholland 1, 5 , Fiona Russell 1, 2

1

Murdoch Childrens Research Institute, Parkville, VIC, Australia; 2 The University of Melbourne, Parkville, VIC, Australia; 3 Ministry of Health, Suva, Fiji;

4

University of London, London, UK; 5 London School of Hygiene and Tropical Medicine, London, UK; 6 Fiji Health Sector Support Program, Suva, Fiji

Fiji introduced the 10-valent pneumococcal conjugate vaccine (PCV10) in 2012 with no catch up. Annual cross-sectional

nasopharyngeal carriage surveys are being conducted to evaluate direct and indirect effects of PCV10. Nasopharyngeal

swabs were collected from healthy children aged 12–23 months and infants aged 5–8 weeks before, and for two years

after, PCV10 introduction (n ≈ 500 per cohort per year). Swabs were stored in STGG and frozen at -80°C until analysis.

Pneumococci were detected by lytA qPCR and molecular serotyping performed by microarray. All data are preliminary.

For 12- to 23- month-old children, the overall pneumococcal carriage rate was 50% in 2012 (pre-PCV10) compared to

48% in 2013 and 32% in 2014. Of pneumococci identified, 38% were PCV10 serotypes in 2012 compared to 32% in 2013.

Post-PCV10, carriage of non-typeable pneumococci has increased, and a higher proportion of pneumococcal-positive

samples contain multiple serotypes. For 5- to 8-week-old infants, the overall pneumococcal carriage rate was 34% in

2012 (pre-PCV10) compared to 23% in 2013 and 13% in 2014. Pre-PCV10, 30% of pneumococci identified were PCV10

serotypes. Serotyping is underway for post-PCV10 carriage samples. Overall, pneumococcal carriage rates are decreasing

in both age groups following PCV10 introduction. One year post-PCV10, we observed a non-significant decline in PCV10

serotypes, and an increase in multiple serotype and non-typeable pneumococcal carriage in young children. Changes in

pneumococcal carriage will be more pronounced in subsequent years, and annual carriage surveys are ongoing.

P2.13

First description of Streptococcus pneumoniae serotype 6E in Spain

María Ercibengoa 2, 3 , Marta Alonso 1 , Jose Maria Marimón 1, 2 , Emilio Pérez-Trallero 1, 3

1

University Hospital Donostia, Donostia/Gipuzkoa, Spain; 2 CIBERES, Donostia/Gipuzkoa, Spain; 3 University of Basque Country UPV/EHU, Donostia/

Gipuzkoa, Spain

The detection of Streptococcus pneumoniae serotype 6E isolates is relatively recent and it might be thought it is a result

of recent evolution, perhaps obtained by the introduction of the pneumococcal conjugated vaccines (PCV). The presence

of serotype 6E isolates before and after PCV was studied in a sample of 169 randomly selected ones (26 from invasive

disease) among the 736 serogroup 6 isolates collected at Donostia University Hospital, northern Spain, between 1981

and 2012 and in 5 serogroup 6B reference strains. Serotype 6E investigation was performed by detecting orf 1 to 4

specific of serotype 6E. Antimicrobial susceptibility testing was performed by broth microdilution. Overall, 48 serotype

6E (8 invasive) of 165 serogroup 6 isolates were identified: 39/71 (55%) before 2002 and 9/98 (9%) after. Of them, 6 had

been previously classified as serotype 6A and 42 as serotype 6B. None of the 29 serotype 6C isolates studied was 6E.

Thirty-eight were penicillin non-susceptible (MIC ≥ 0.12 µg/mL), 19 erythromycin-resistant and 4 ciprofloxacin-resistant

(MIC ≥ 4 µg/mL). Surprisingly, reference strain of the 6B-2 multidrug-resistant clones Streptococcus pneumoniae ATCC

700670 also showed orf 1 to 4 described for serotype 6E. Serotype 6E has been circulating since at least 1981, being

unlikely that the vaccines have influenced the emergence of its strains. Conversely, PVCs seems to have a protective

effect. The S. pneumoniae ATCC 700670 should be renamed. Serotype 6E were mainly found among previously identified

serotype 6B isolates and frequently showed antimicrobial resistance.

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P2.14

Utility of a reverse-hybridisation strip assay for Streptococcus

pneumoniae serotyping

María Ercibengoa 2, 3 , Susana Gamen 4 , Ana Manrique 4 , Jose María Marimón 1, 2 , Emilio Pérez-

Trallero 1, 3

1

Donostia University Hospital, Donostia, Gipuzkoa, Spain; 2 CIBERES, Donostia, Gipuzkoa, Spain; 3 EHU-UPV, Donostia, Gipuzkoa, Spain; 4 OPERON,

Zaragoza, Aragón, Spain

Surveillance of pneumococcal serotypes causing disease is essential to evaluate pneumococcal vaccination programs.

The Microbiology Department of Donostia University Hospital (San Sebastián, Spain) and the biotechnology company

Operon (Zaragoza, Spain) are developing a reverse-hybridisation strip assay to easily determine pneumococcal serotypes.

The first prototype developed includes the 13 serotypes of the PCV13 and another 4 related serotypes (6C, 6D and 18A,

18F). This first prototype was initially validated against reference isolates of the 94 pneumococcal serotypes described

up to date showing 100% specificity. Besides, it was tested with 66 clinical isolates from the Microbiology Department

collection previously serotyped with the Quellung reaction: 59 strains belonging to the 17 serotypes included in the

strip and another 7 strains of serotypes not included. All the 66 strains were correctly characterised with the reversehybridisation

strip assay. However, because of similarity of capsular sequences, serotypes 7A/7F, 9A/9V and 18B/18C

could not be differentiated. The reverse-hybridisation strip assay proved to be a good tool for pneumococcal serotyping.

An advantage of this technique as compared with the Quellung is the possibility of working in batches of samples. Other

advantages are that no experienced staff is required to obtain and interpret results, and that records of the results

(hybridised strips) can be stored. With this technique, some related serotypes could not be discriminated because of

sequences similarity.

P2.15

Nasopharyngeal colonisation of Streptococcus pneumoniae in

Colombian children under five years of age, Medellin 2014

Jessica Morales 1, 3 , Carlos Eugenio Delgado 1, 3 , Beatriz Salazar 2, 4 , Leidy Acevedo 1, 2 , Johan

Bolivar 1, 2 , Sara Saldarriaga 1, 2 , Milena Cardona 1, 2 , Diego Florez 1, 2 , Steven Rivera 1, 2 , Alejandro

Gomez 1, 6 , Lorena Molina 5 , Juan David Rodriguez 5 , Laura Sanchez 5 , Ana Cecilia Diez 5 , Sandra

Castro 5 , Sven Hammerschmidt 6 , Doracelly Hincapie 3 , Gustavo Gamez 1, 2

1

Basic and Applied Microbiology (MICROBA) Research Group, School of Microbiology, Universidad de Antioquia, Medellín, Colombia; 2 Genetics,

Regeneration and Cancer (GRC) Research Group, University Research Center (SIU), Universidad de Antioquia, UdeA, Medellín, Colombia;

3

Epidemiology Research Group, National Faculty of Public Health, Universidad de Antioquia, Medellín, Colombia; 4 Bacteria and Cancer Research

Group, School of Medicine, Universidad de Antioquia, Medellín, Colombia; 5 Programa Buen Comienzo, Secretaría de Educación de Medellín,

Medellín, Colombia; 6 Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald,

Greifswald, Germany

Human colonisation by Streptococcus pneumoniae is the main requirement for pneumococcal diseases. In Medellín,

Colombia, no studies have documented the behaviour of the pneumococcus and the public health problems it can cause

in childhood. Here, the prevalence of nasopharyngeal colonisation by S. pneumoniae and its associated factors in children


EuroPneumo Special Issue / pneumonia 2015 Oct 21;7:I–72

(18.2%). Higher colonisation rates were also associated with the presence of respiratory signs, symptoms and diseases,

as well as with the consumption of steroidal medicaments and no affiliation to the health systems (social security).

Serotyping, antimicrobial resistance, genotyping and pulsed-field gel electrophoresis and multilocus sequence typing

are currently under evaluation. This molecular epidemiology study in Medellín contributes to a better understanding of

the scenario of circulating pneumococcal strains and the factors favouring colonisation in children.

P2.16

Serotype distribution and antimicrobial resistance of Streptococcus

pneumoniae clinical isolates from invasive pneumococcal diseases in

Antioquia, Colombia

Carlos Eugenio Delgado 1, 3 , Jessica Morales 1, 3 , Beatriz Salazar 2, 4 , Leidy Acevedo 1, 2 , Johan

Bolivar 1, 2 , Sara Saldarriaga 1, 2 , Milena Cardona 1, 2 , Diego Florez 1, 2 , Steven Rivera 1, 2 , Alejandro

Gomez 1, 6 , Adriana Gonzalez 5 , Marleny Gallego 5 , Marcela Arrubla 5 , Blanca Restrepo 5 , Sven

Hammerschmidt 6 , Doracelly Hincapie 3 , Gustavo Gamez 1, 2

1

Basic and Applied Microbiology (MICROBA) Research Group, School of Microbiology, Universidad de Antioquia, Medellín, Colombia; 2 Genetics,

Regeneration and Cancer (GRC) Research Group, University Research Center (SIU), Universidad de Antioquia, Medellín, Colombia; 3 Epidemiology

Research Group, National Faculty of Public Health, Universidad de Antioquia, Medellín, Colombia; 4 Bacteria and Cancer Research Group, School of

Medicine, Universidad de Antioquia, Medellín, Colombia; 5 Laboratorio Departamental de Salud Pública, Secretaría Seccional de Salud de Antioquia,

Medellín, Colombia; 6 Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald,

Greifswald, Germany

Invasive pneumococcal diseases (IPDs) are a leading cause of morbidity and mortality among children 60% of all IPD cases in the region. According to the Agar diffusion test,

the highest rates of antimicrobial resistance were observed with penicillin (NS-45%), tetracycline (R-38%), trimethoprim/

sulfamethoxazole (R-35%), erithromycin (R-23%), and clindamycin (R-19%). Traditionally, serotype 14 is the most

prevalent serotype in Colombia; however, its incidence has decreased in the last decade. Conversely, the serotype 19A

prevalence is increasing in Colombia (emerging serotype) after the PCV introduction. Both serotypes are non-sensible

to penicillin and trimethoprim/sulfamethoxazole resistants. The reduced number of IPD cases in children, in comparison

with non-PCV-immunised adults, and the shifts in the distribution of IPD serotypes, suggest the potential impact of PCVs

on IPDs in Antioquia, Colombia.

P2.17

Vaccination drives changes in metabolic and virulence profiles of

Streptococcus pneumoniae

Eleanor Watkins 1 , Caroline Buckee 2 , Bridget Penman 1 , Martin Maiden 1 , Sunetra Gupta 1

1

University of Oxford, Oxford, UK; 2 Harvard School of Public Health, Boston, USA

Since the introduction of pneumococcal conjugate vaccines, increases in non-vaccine serotypes have been recorded in

several countries: a phenomenon termed vaccine-induced serotype replacement (VISR). Here, using a combination of

mathematical modelling and whole genome analysis, we show that targeting particular serotypes through vaccination

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also can cause their metabolic and virulence-associated components to transfer through recombination to non-vaccine

serotypes: a phenomenon we term vaccine-induced metabolic shift (VIMS). Our results provide a novel explanation

for changes observed in the population structure of the pneumococcus following vaccination, and have important

implications for strain-targeted vaccination in a range of infectious disease systems.

P2.18

The relevance of a novel rapid quantitative assay to detect up to

40 major Streptococcus pneumoniae serotypes directly in clinical

nasopharyngeal and blood specimens

Melina Messaoudi 1 , Milen Milenkov 1 , Werner Albrich 3, 4 , Mark Van Der Linden 5 , Monidarin Chou 7 ,

Mariam Sylla 8 , Nathalie Richard 9 , Patricia Barreto Costa 10 , Keith P. Klugman 6, 4 , Hubert Endtz 1, 2 ,

Glaucia Paranhos Baccala 1 , Jean Noel Telles 1

1

Fondation Merieux, Emerging Pathogens Laboratory - Centre International de Recherche en Infectiologie (CIRI), Lyon, France; 2 Departement

of Medical Microbiology & Infectious Diseases Erasmus MC, Rotterdam, The Netherlands; 3 Department of Infectious Diseases and Hospital

Epidemiology, Kantonsspital St. Gallen, St. Gallen, Switzerland; 4 Medical Research Council Respiratory and Meningeal Pathogens Research Unit,

University of the Witwatersrand, Johannesburg, South Africa; 5 National Reference Center for Streptococci, Institute of Medical Microbiology,

University Hospital RWTH Aachen, Aachen, Germany; 6 Hubert Department of Global Health and Division of Infectious Diseases, Emory University,

Atlanta, USA; 7 Faculty of Pharmacy, University of Health Sciences, Phnom Penh, Cambodia; 8 CHU Gabriel Touré, Bamako, Mali; 9 Service de

Réanimation Pédiatrique Médico-Chirurgicale, HFME, Groupement Hospitalier Est, Bron, France; 10 Laboratório de vírus respiratórios e do sarampo,

Instituto Oswaldo Cruz/Fiocruz, Rio de Janairo, Brazil

Prior to and following pneumococcal vaccine introduction it is crucial to monitor the distribution and dynamics

of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative

information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype

compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification

and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimen

from emerging and developing countries. Two multiplex PCR to specifically detect 29 and 11 serotypes, respectively,

were optimised. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance

of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from

adult and paediatric patients hospitalised with pneumonia (n = 664) from 5 different countries. Serotype 6AB was widely

represented in nasopharyngeal specimens from all 5 cohorts. The most frequent serotypes in the French, South African,

and Brazilian cohorts were 1 and 7F, 3 and 19F, and 14, respectively. When both samples were available, the serotype

in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype

to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the

serotypes that moved to the blood was 2 log-fold higher than the ones only found in the nasopharynx. This novel, rapid,

quantitative assay may potentially enable prediction of the S. pneumoniae serotypes invasiveness and assessment of

pneumococcal serotype distribution pre- and post-introduction of vaccines, and the likely serotype replacement.

P2.19

Streptococcus pneumoniae serotype identification using a rapid,

low-cost, automated sequence specific oligonucleotide typing system

Stephen Middle, Andrew Canterbury, Ian Crosby, Peter Maguire

MC Diagnostics, St. Asaph, UK

Currently used pneumococcal serotyping techniques are labour intensive, costly and restricted to a limited number of

specialist laboratories. A fast, low-cost test that utilises sequence specific oligonucleotides (SSOs) to identify pneumococcal

serotypes in an automated procedure has been designed which requires minimal user training. Primers and SSOs were

designed from genes located in the capsular polysaccharide synthesis region of the pneumococcal genome. Testing was

performed with DNA from pure cultures of pneumococcal, human, and non-pneumococcal bacterial species to ensure

no cross-amplification or non-specific SSO signals were detected. We aimed to identify serotypes that are responsible for

the majority of invasive pneumococcal disease cases. With our test it is possible to identify a pneumococcal sample to at

least the serogroup level, with those protected against by the 23 polyvalent vaccine to the serotype level. However, it is

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not possible to differentiate between serotypes that that are caused by protein truncation due the testing method (e.g.

9A and 9V). In addition to pure bacterial culture, identification of serotype direct from extracted DNA of nasopharyngeal

and ear swabs as well as serum samples is our next target to ensure rapid diagnosis can be achieved. Within 24 hours of

sample acquirement, up to 96 samples can be tested simultaneously before analysis and interpretation with a software

program. Furthermore, we aim to be able to identify multiple pneumococcal serotypes from a single sample to reflect

multi-serotype pneumococcal carriage. Overall, our system is suitable for high throughput analysis of pneumococcal

samples for both the research and medical communities.

P2.20

A novel multiplex real-time PCR assay for identifying and typing of

bacterial meningoencephalitis pathogens: development and validation

on clinical samples

Milen Milenkov 1 , Mala Rakoto Andrianarivelo 2 , Mickael Harioly Nirina 2 , Lalaina Rahajamanana 3 ,

Angelot Rakotomalala 2 , Liliane Raboba 3 , Annick Robinson 3 , Mélina Messaoudi 1 , Jean-Noël Telles 1 ,

Hubert Endtz 1 , Bénédicte Contamin 1 , Glaucia Paranhos-Baccalà 1

1

Fondation Mérieux, Lyon, France; 2 Centre d’Infectiologie Charles Mérieux, Antananarivo, Madagascar; 3 CHU Mère-Enfants Tsaralalàna,

Antananarivo, Madagascar

Bacterial meningoencephalitis is a major public health concern especially in developing countries. Common identification

techniques are time consuming as they require samples culture and often lack sensitivity. Here, we describe a rapid,

two-step multiplex real-time PCR method for identifying and typing Neisseria meningitidis, Streptococcus pneumoniae,

and Haemophilus influenzae type b. The first step of the test is a quadruplex, real-time PCR, targeting highly conserved

and species specific genes for S. pneumoniae, N. meningitidis, H. influenzae type b, and Streptococcus zooepidemicus

(internal control). The second step consists of 2 multiplex PCR assays for the identification of the 42 most prevalent S.

pneumoniae serotypes (1, 2, 3, 4, 5, 6A/B, 6C, 7F, 8, 9N/L, 9V, 10A, 10F, 11A, 12F, 13, 14, 15B/C, 16F, 18A/B/C /F, 19A,

19F, 21, 22F, 23A, 23B, 23F, 24F, 31, 33F, 34, 35A, 35B, 35F, 38, 39) and for meningococcal serogroups A, B, C, W135, X

and Y. The analytical specificity of all primers/probe pairs has been validated on laboratory strains, representative of

each serotype/serogroup targeted by the assay, as well as on closely related members of the genera Streptococcus,

Haemophilus, and Neisseria. The test was then used to analyse 252 samples of cerebrospinal fluid from patients with

suspected meningoencephalitis in Antananarivo, Madagascar. Forty-two (17%) were positive for S. pneumoniae and we

were able to determine the serotype of 35 of them (83%). N. meningitidis serogroup W135 was found in 1 sample and

another was positive for N. meningitidis serogroup C. The major advantage of the proposed molecular assay resides in

the possibility to perform a direct analysis of a clinical sample, thus providing results within a couple of hours. It can be

also used for monitoring epidemiological trends and serotype replacement upon vaccine introduction.

P2.21

Evolution of Streptococcus pneumoniae serotype 14 in Catalonia,

Spain, after the introduction of 13-valent pneumococcal conjugate

vaccine (PCV13) (2010–2014)

Cristina Esteva 1 , Eva del Amo 1 , Paula Gassiot 2 , Xavier Raga 3 , Carmina Sanjose 4 , Mar O Perez 5 ,

Pilar Ciruela 6 , Marina Cunquero 1 , Irene Latorre 1 , Carmen Muñoz-Almagro 1 , Catalan Study Group

Invasive Pneumococcal Disease 6

1

Molecular Microbiology Department, Universitary Hospital Sant Joan de Déu, Esplugues de Llobregat, Barcelona, Spain; 2 Microbiology Department,

Figueres Hospital, Figueres, Girona, Spain; 3 Microbiology Department, Sant Pau y Santa Tecla Hospital, Tarragona, Spain; 4 Microbiology Department,

Alt Penedes Hospital, Vilafranca del Penedes, Spain; 5 Microbiology Department, Verge de la Cinta Hospital, Tortosa, Spain, 6 Public Health Agency,

Government of Catalonia, Barcelona, Spain

Serotype 14 (S14) has been considered one of the main serotypes causing invasive pneumococcal disease (IPD). Since

2001, conjugate vaccines are available to prevent IPD caused by different serotypes included S14. Since 2010, PCV13

is the vaccine recommended in children in our region, but it is estimated only 50% are vaccinated. Our aim was to

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study S14 evolution in paediatric and adult IPD patients in Catalonia after the introduction of PCV13. All pneumococcal

invasive isolates received in the period 2010–2014 from 23 hospitals in Catalonia and characterised by the Microbiology

Department at Hospital Sant Joan de Déu, Barcelona, were included. Capsular typing and clonal analysis were performed

by fluorescent multiplex PCR and MLST, respectively. One thousand seven hundred and sixty-two IPD episodes were

diagnosed during the study period and 1,730 (98.2%) were available for serotyping and clonal study. The IPD rate

decreased from 15.4/100,000 habitants in 2010 to 12.9/100,000 in 2014 (p < 0.001). A total of 921 (53.2%) isolates

were PCV13 serotypes, 126 (7.3%) were S14 (30 from children 65 years (7.4% vs. 13.1%) (p =

0.032). ST156 was the main clonal type identified along the study (n = 86; 68.3%). This ST was detected in 63% of S14

isolates during 2010–2013 vs. 82.4% in 2014 (p = 0.05). Despite the progressive decrease of IPD rate in Catalonia due to

conjugate vaccines, S14 is still a major cause of IPD. Further studies are needed to determine the reasons of this.

P2.22

Conjugate vaccine introduction in Portugal was followed by a decrease

of serotypes 6A and 6E but not of serotypes 6B and 6C

Jorge Diamantino-Miranda 1 , Sandra Isabel Aguiar 1, 2 , João André Carriço 1 , José Melo-Cristino 1 ,

Mário Ramirez 1

1

Instituto de Medicina Molecular, Faculty of Medicine, University of Lisbon, Lisbon, Portugal; 2 Faculty of Veterinary Medicine, University of Lisbon,

Lisbon, Portugal

The pneumococcal vaccine PCV13 has been available since 2010 in Portugal. Two serotypes of serogroup 6 (6A and 6B) are

included in the vaccine but six others (6C, 6D, 6E, 6F, 6G and 6H) were recently proposed. Their capsule polysaccharides

are similar and one recombination or mutation event in the capsular locus may switch serotype. Particularly, a single

amino acid change in WciP is sufficient to alter its specificity resulting in a different serotype. Transformation, the main

mechanism of horizontal genetic exchange in pneumococci, is regulated by a quorum sensing system controlled by the

competence stimulating peptide (CSP). There are 3 variants of CSP and each defines a pherotype with a given strain

being able to respond to just 1 variant. The goals of this work were to determine the prevalence of invasive isolates

of serogroup 6, to evaluate the potential effect of the conjugate vaccines in this serogroup, and to assess their genetic

diversity and determine associations with pherotypes. Out of 239 isolates expressing serogroup 6 recovered between

1999 and 2012, n = 80 were 6A, n = 26 were 6B, n = 82 were 6C, and n = 51 were 6E. Serotypes 6E and 6A declined

after the introduction of PCV7 in 2001 and PCV13 in 2010, respectively. Serotype 6B remained constant with a low

prevalence. After 2010, 6C became the most frequent serotype and there is no evidence of cross-protection conferred

by the vaccine. A total of 65 sequence types were identified, reflecting the high genetic diversity of this serogroup.

However, the majority belonged to 5 major clonal complexes. Serotypes 6A and 6C were associated with pherotype CSP2

and 6B with CSP1. In conclusion, the majority of previously identified serotype 6B isolates were in fact serotype 6E and

these declined after PCV7 introduction. In spite of the presence of serotypes 6A and 6B in PCV13, this did not result in

a decline of serotype 6C isolates.

P2.23

PCV13 serotypes were the major constituents of the major clones

causing adult IPD in Portugal in the conjugate vaccine era

Andreia Neves Horácio, Catarina Silva-Costa, Jorge Diamantino-Miranda, Joana Pimento Lopes,

Mário Ramirez, José Melo-Cristino

Instituto de Medicina Molecular, Instituto de Microbiologia, Lisboa, Portugal

We have previously shown that major changes in serotype frequency occurred in the isolates causing adult (>18yrs)

invasive pneumococcal disease (IPD) between 2008 and 2011 and in this study we aimed to look at the clonal composition

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of this collection. From a total of 1661 isolates, a random sample of at least 50% of the isolates from each serotype was

chosen for multilocus sequence typing (MLST) and goeBURST analysis (n = 872). We detected a diverse clonal composition

with 205 different sequence types (STs) (SID = 0.972 [95% CI 0.967–0.976]) organised in 82 different clonal complexes

(CCs) (SID = 0.948 [95% CI 0.942–0.953). Although there was high genetic diversity half of the isolates arranged in only

6 CCs: CC156 (11.6%), CC191 (10.1%), CC180 (8.0%), CC306 (7.8%), CC62 (7.7%), CC230 (5.4%). The major clone was

mostly composed of PCV7 serotypes (89%) although these only accounted for 18.1% of the isolates analysed by MLST.

The additional serotypes found in PCV13 (42.0% of the genotyped isolates) were the major constituents of 4 of these

6 major clones (CC191, CC180, CC306, and CC230) while PPV23 additional serotypes (20.0%) were the most frequent

in CC62. The clonal frequency was greatly influenced by serotype fluctuations, especially of the CC306, represented by

serotype 1, which significantly declined from 12.8% (2008) to 2.8% (2011). A few possible capsular switches between

vaccine and non-vaccine types were identified. For instance, isolates of ST1201 presented serotypes 19A and 7C, ST230

had serotypes 19A and 24F, ST42 was from serotypes 23A or 6A, and ST241 had both 19A and 18A capsular types.

Antimicrobial resistance was more strongly associated with ST than with serotype, with different STs of the resistant

serotypes 19A and 14 behaving differently. Since the most important clones causing adult IPD are mainly composed of

PCV13 serotypes, major changes in the clonal composition of pneumococci are expected with continued vaccine use.

P2.24

The identification of pneumococci by lytA-based identification methods

may retrieve false results

Débora A. Tavares 1 , Alexandra S. Simões 1 , Hermínia de Lencastre 2, 3 , Raquel Sá-Leão 1

1

Laboratory of Molecular Microbiology of Human Pathogens, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de

Lisboa, Oeiras, Portugal; 2 Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa,

Oeiras, Portugal; 3 Laboratory of Microbiology and Infectious Diseases, The Rockefeller University, New York, NY, USA

In recent years, molecular strategies targeting lytA (coding for the major pneumococcal autolysin), have been proposed

to identify pneumococcus. In the clinical setting, the identification of pneumococci based on the amplification of lytA by

real-time PCR is increasingly used particularly when doing direct detection in clinical samples. In research laboratories,

the distinction between atypical pneumococci and closely related species has often been based on the assignment of

specific lytA-BsaAI-RFLP signatures and/or MLST and multilocus sequence analysis (MLSA) strategies. In our collection,

we detected 11 strains displaying conflicting or novel results when identified by lytA-BsaAI-RFLP and MLST. Here we

report further characterisation of these strains and discuss the utility of using lytA-real-time PCR in these cases. MLSA

for viridans streptococci divided the 11 strains in 4 pneumococci 2 lineages), 5 Streptococcus mitis (5 lineages), and 2

S. pseudopneumoniae (1 lineage). Three novel lytA-BsaAI-RFLP signatures were found. In addition, 1 pneumococcus

displayed the atypical lytA-BsaAI-RFLP signature characteristic of non-pneumococci and 2 S. pseudopneumoniae

displayed the typical lytA-BsaAI-RFLP pattern characteristic of pneumococci. lytA-real-time PCR misidentified these 3

isolates. DNA sequencing of lytA confirmed these observations. Although rare, lytA-based identification methods may

lead to false results.

P2.25

Characterisation of the blp locus of co-colonising pneumococci and its

impact on co-colonisation

Carina Valente 1 , Suzanne Dawid 2, 3 , Hermínia de Lencastre 4, 5 , Raquel Sá-Leão 1

1

Laboratory of Molecular Microbiology of Human Pathogens, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras,

Portugal; 2 Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA; 3 Department of Microbiology

and Immunology, University of Michigan, Ann Arbor, Michigan, USA; 4 Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica,

Universidade Nova de Lisboa, Oeiras, Portugal; 5 Laboratory of Microbiology, The Rockefeller University, New York, New York, USA

Pneumococcal co-colonisation of the nasopharynx is frequent and is important for pneumococcal biology by promoting

both evolution and competition. The bacteriocin-like peptides (blp) locus has been implicated in intra-species competition

but the effect of bacteriocin secretion on co-colonisation remains to be assessed. We aimed to evaluate the impact

of the blp locus and bacteriocin production on pneumococcal co-existence in the nasopharynx. A collection of 135

co-colonised nasopharyngeal samples from healthy children was used. The inhibitory activity of all strains (n = 285)

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was assessed through overlay competition assays and their blp locus was characterised regarding BlpC type (signalling

peptide), integrity of the secretion system, and bacteriocin/immunity content. Approximately one-third of co-colonising

strains displayed inhibitory activity (29%) and more than half were cheaters (54%) (expression of immunity without

cost of bacteriocin secretion). Cheater strains co-existed with other cheaters (28%) and with inhibitory strains (25%)

at a comparable frequency. The 5 known BlpC types were not equally distributed in the population—BlpCT4 was the

most prevalent (37%) and BlpCP155 was rare (2%). The proportion of co-colonisation events involving matched (41%) or

unmatched (59%) BlpC types was not significantly different (p = 0.32). Also, the observed proportions of co-colonisation

events predicted to result in inhibition of one strain (41%) or no-inhibition (59%) were not different from the estimated

proportions based on the observed frequency in the population (p = 0.81). The results show that bacteriocin producers

often co-colonise with susceptible strains, suggesting that phenotypes of bacteriocin secretion alone do not explain the

co-existence of pneumococci in the nasopharynx.

P2.26

Molecular surveillance on Streptococcus pneumoniae carriage in the

Netherlands: does the pneumococcus change its niche preference with

increasing host age?

Anne L. Wyllie 1 , Nynke Y. Rots 2 , Jody van Engelsdorp Gastelaars 1 , Lidewij W. Rümke 1 , Jacob P.

Bruin 3 , Elisabeth A.M. Sanders 1, 2 , Krzysztof Trzciński 1

1

Paediatric Immunology and Infectious Diseases, Wilhelmina’s Children Hospital, University Medical Center Utrecht, Utrecht, The Netherlands;

2

Centre for Infectious Disease Control Netherlands, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands;

3

Regional Laboratory of Public Health, Haarlem, The Netherlands

Pneumococcal colonisation of the upper airways is a disease prerequisite, which disproportionally affects infants and

elderly. Unlike in children, carriage of Streptococcus pneumoniae is rarely detected in elderly by the gold standard

method of conventional diagnostic culture. We suspected contemporary carriage rates to be underestimated and that

molecular-based analysis of samples from additional niches would enhance pneumococcal carriage detection. Here,

we compared the sensitivity of conventional and molecular methods in upper-airway samples from infants, adults and

elderly. Nasopharyngeal and saliva samples were collected from asymptomatic 24-month-old children (n = 289), parents

(n = 298) and 135 elderly aged 60–89 (2 time points, n = 270). Oropharyngeal samples were also obtained from all

adults. Following conventional diagnostic culture on pneumococci-selective media, DNA extracted from all plate growth

was tested by qPCR targeting 2 species-specific genes. For all age groups, molecular methods significantly increased

the number of carriers detected. There was no significant difference in the number of culture-positive nasopharyngeal

samples and qPCR-positive saliva samples from infants, while in both parents and elderly, qPCR-detection of pneumococci

in saliva samples was the most sensitive method. Accurate detection of S. pneumoniae is essential for monitoring

direct and indirect effects of infant pneumococcal vaccination. We demonstrate that no single method of detection is

optimal for all age groups, which is an important consideration for future surveillances. Our findings suggest a change

in niche preference with age and indicate that carriage rates in adults and elderly are underestimated when based on

nasopharyngeal samples alone. We propose that for sensitive pneumococcal carriage detection, nasopharyngeal and

saliva samples should be collected from children aged 0–6 years and that oropharyngeal and saliva samples should be

collected from all individuals aged 6–80 years.

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P2.27

Specificity and sensitivity of molecular methods used for detection of

Streptococcus pneumoniae and pneumococcal serotypes

Anne L. Wyllie 1 , Arie van der Ende 2 , Yvonne Pannekoek 2 , Karin Elberse 3 , Jody van Engelsdorp

Gastelaars 1 , James A. Groot 1 , Kayleigh Arp 1 , Debby Bogaert 1 , Elisabeth A.M. Sanders 1, 3 , Krzysztof

Trzciński 1

1

Department of Paediatric Immunology and Infectious Diseases, Wilhelmina’s Children Hospital, University Medical Centre Utrecht, Utrecht, The

Netherlands; 2 Department of Medical Microbiology and the Netherlands Reference Laboratory for Bacterial Meningitis, Academic Medical Center,

Amsterdam, The Netherlands; 3 Centre for Infectious Disease Control Netherlands, National Institute for Public Health and the Environment (RIVM),

Bilthoven, The Netherlands

The gold standard method for the detection of upper respiratory tract commensal Streptococcus pneumoniae relies

on conventional culture. Recent studies investigating culture-independent methods uniformly demonstrate the higher

sensitivity of molecular detection. Concerns regarding the specificity of molecular methods have been expressed however,

particularly when applied to oropharyngeal samples, representing high microbial diversity including other streptococci

that may carry homologues of pneumococcal genes. Here, we investigated the sensitivity and specificity of molecular

assays for pneumococcal detection with particular focus on non-pneumococcal streptococci. DNA of streptococcal strains

isolated from invasive disease (103 non-pneumococcal, 6 pneumococcal isolates) and asymptomatic carriage (64 infants,

58 elderly; 101 non-pneumococci, 19 pneumococci including 18 non-typeable isolates) was tested for the pneumococcal

genes lytA, piaA, cps, ply, and spn9802. In addition, pneumococcal serotyping was performed using serotype-specific

qPCR assays, as well as capsule sequence typing (CST). Species identification was determined by S2 sequencing. The

molecular assay targeting lytA was 100% specific and sensitive. Molecular detection of piaA was 100% specific but

less sensitive for S. pneumoniae, while assays targeting cps, ply, and spn9802 showed a lack of specificity. While CST

was highly specific, some streptococcal isolates generated false positive signals in serotype-specific qPCR assays (5,

9A/V, 18B/C, 19F). False positive signals were predominantly generated by S. pseudopneumoniae and S. mitis strains.

Accurate detection and classification of pneumococci is essential for improved understanding of pneumococcal carriage

and disease aetiology. We demonstrate that molecular identification of S. pneumoniae through lytA and piaA is highly

sensitive and specific. Furthermore, we identified species of bacteria that may confound molecular methods, used for

pneumococcal detection and serotype determination when applied to polymicrobial samples from the upper airways.

P2.28

Molecular surveillance on nasopharyngeal carriage of Streptococcus

pneumoniae in children vaccinated with conjugated polysaccharide

pneumococcal vaccines

Anne L. Wyllie 1 , Alienke J. Wijmenga-Monsuur 2 , Marlies van Houten 3 , Astrid A.T.M. Bosch 1 , James

A. Groot 1 , Jody van Engelsdorp Gastelaars 1 , Jacob P. Bruin 4 , Debby Bogaert 1 , Nynke Y. Rots 2 , Elisabeth

A.M. Sanders 1, 3 , Krzysztof Trzciński 1

1

Paediatric Immunology and Infectious Diseases, Wilhelmina’s Children Hospital, University Medical Center Utrecht, Utrecht, The Netherlands;

2

Centre for Immunology of Infectious Diseases and Vaccines, National Institute for Public Health and the Environment (RIVM), Bilthoven, The

Netherlands; 3 Linnaeus Institute, Spaarne Hospital, Hoofddorp, The Netherlands; 4 Regional Laboratory of Public Health, Haarlem, The Netherlands

As a commensal of the upper respiratory tract, Streptococcus pneumoniae is a potential pathogen causing respiratory

and invasive diseases. Following implementation of pneumococcal conjugate vaccination (PCV) for infants, surveillance

studies have proven essential for monitoring direct (carriage of serotypes targeted by vaccine, VTs) and indirect effects

(changes in carriage of non-vaccine serotypes, NVTs). We compared the detection of pneumococcal carriage and

serotypes in a unique study setting using both conventional culture and molecular methods, in nasopharyngeal samples

from healthy PCV-vaccinated infants in 2 large, cross-sectional surveillance studies on PCV effects in the Netherlands.

Nasopharyngeal samples were collected from 1,182 11- and 24-month-old children (n = 591 each) during autumn/winter

2010/11 (n = 584) and 2012/2013 (n = 598). Following conventional culture on plates selective for S. pneumoniae, DNA

extracted from all bacterial growth was tested by quantitative-PCR (qPCR) for the presence of pneumococci and a panel

of serotypes, including serotypes targeted by the 13-valent PCV (PCV13). Molecular diagnostic methods significantly

increased both the overall pneumococcal carriage rate and number of serotype carriage events detected as compared

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to culture. There was a correlation (Spearman’s rho = 0.98; p < 0.001) between the frequency of serotypes detected

using qPCR and prevalence according to conventional culture. We found no evidence of a hidden circulation of serotypes

rarely detected by culture or those targeted by vaccination. Our findings suggest that surveillances based on the culture

method alone do not underestimate carriage of VTs in immunised children.

P2.29

PatA/PatB: a multidrug ABC transporter from Streptococcus

pneumoniae energised by GTP hydrolysis

Benjamin Duchene 1 , Emilie Boncoeur 1 , Sandrine Aros-Calt 2 , François Fenaille 2 , Thierry Vernet 1 ,

Jean-Michel Jault 3 , Claire Durmort 1

1

IBS, Grenoble, France; 2 CEA DSV/iBiTec -S/SPI, Saclay, France; 3 IBCP, Lyon, France

The superfamily of ABC transporters (ATP binding cassette) is composed of membrane complexes dedicated to transport

a wide variety of compounds through the cell membrane. Transport is carried out by 2 transmembrane domains referred

to as permeases and 2 cytoplasmic nucleotide binding domain (NBD) that energise the transporter by hydrolysing ATP.

More than 60 ABC transporters have been identified in Streptococcus pneumoniae, about 20 of which are carriers

putatively involved in the efflux of xenobiotics. PatA/PatB is one of these export systems driving pneumococcal multidrug

resistance and a homologue to a multidrug ABC exporter of Bacillus subtilis, BmrC/BmrD. It is frequent to face pathogenic

strains resistant to antibiotic treatment, which poses problems in patients. To better understand these mechanisms, we

performed a functional and structural study of PatA/PatB transporter. PatA/PatB plays a role in the efflux of antibiotic

fluoroquinolone and oxazolidinones (linezolid). Single or simultaneous deletion of patA and patB in the pneumococcal

genome confers increased susceptibility to ciprofloxacin and norfloxacin; 2 antibiotics belonging to the fluoroquinolone

family. Only the heterodimer PatA/PatB has noticeable ATPase and drug transport activities in vitro when overexpressed

in Escherichia coli and using inside-out membrane vesicles; homodimers of PatA or of PatB are unstable and/or inactive.

More surprisingly, by incorporating purified PatA/PatB into nanodisc made of phospholipids bilayer, we found that this

ABC transporter preferentially hydrolyses GTP as compared to ATP. As a result, the export of drug is increased 6–8-fold

in the presence of GTP. Analyses of the intracellular concentrations of ATP and GTP performed by mass spectrometry

revealed that these 2 nucleotides are present in similar amounts, indicating that this preference for GTP over ATP has a

physiological relevance. Thus, we show for the first time that an ABC transporter has evolved and adapted to hydrolyse

GTP. This unexpected adaptation warrants further investigations.

P2.30

Penicillin and erythromycin resistance among pneumococci carried by

young children in Portugal: evolution over a 15-year period

Sofia Félix 1, 3 , Sónia Nunes 1, 3 , Carina Valente 1, 3 , Ana C. Paulo 1, 3 , Alexandra S. Simões 1, 3 , Sónia T.

Almeida 1, 3 , Débora A. Tavares 1, 3 , António Brito-Avô 2 , Hermínia de Lencastre 4 , Raquel Sá-Leão 1, 3

1

Laboratory of Molecular Microbiology of Human Pathogens, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de

Lisboa, Oeiras, Portugal; 2 Private Pediatric Clinic, Lisboa, Portugal; 3 Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica

António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal; 4 Laboratory of Microbiology and Infectious Diseases, The Rockefeller University, New

York, USA

In Portugal, PCV7 was not introduced in the national immunisation plan but was commercially available between 2001

and 2010. We aimed to analyse trends in penicillin and erythromycin resistance among colonising pneumococci over a

15-year period (1996–2010). Nasopharyngeal samples were obtained from children attending day care centres in the

urban area of Lisbon and Oeiras. Pneumococci were isolated, serotyped, and antibiotyped by standard procedures. A

total of 7,561 children were sampled. Antimicrobial use in the month preceding sampling varied between 13.5%-29.0%

and showed a decreasing trend over the years (p < 0.001). Although use of PCV7 among children younger than 2 years in

2002 and 2003 was less than 50%, from 2006 onwards PCV7 use was high (range 77.8%–85.2%). Pneumococcal carriage

remained high throughout the study (average of 61.2%). High-level resistance to penicillin ranged between 0.7%–8.0%,

with a decreasing trend over the years (p < 0.001). No sustained changes in the prevalence of low-level resistance to

penicillin (range 12.0%-26.5%) or resistance to erythromycin (range 16.4%–27.6%) were observed over time. However,

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non-susceptibility to both antimicrobials increased over the years (p < 0.001) and was detected in 15–20% of all

isolates obtained since 2006. Also, after 2006, most penicillin and/or erythromycin non-susceptible isolates belonged

to serotypes not included in PCV7. Our data on antimicrobial resistance among colonising pneumococci suggest that

antibiotic pressure remains high in the population. Although a decrease in antibiotic consumption was noted after 2002,

it remains high when compared to other countries. These results suggest that additional efforts to contain antibiotic use

must be implemented in Portugal.

P2.31

Auranofin-PLGA nanoparticles as an alternative therapeutic tool against

pneumococcal infections

Esther García-Fernández 1, 2 , Roberto Díez-Martínez 1, 2 , Miguel Manzano 3, 4 , Ángel M. Martínez 3, 4 ,

María Vallet-Regí 3, 4 , Pedro García 1, 2

1

Centro de Investigaciones Biológicas (CSIC), Madrid, Spain; 2 CIBER de Enfermedades Respiratorias, Madrid, Spain; 3 Facultad de Farmacia,

Universidad Complutense de Madrid, Madrid, Spain; 4 Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN),

Madrid, Spain

In recent years the effectiveness of the antibiotics for treatment of pneumococcal and other bacterial infections has

been compromised by the increasing prevalence of multiresistant strains, which has pushed the development of

new drugs with antimicrobial activity. Auranofin, a gold-containing compound approved in 1985 for the treatment of

rheumatoid arthritis, has been recently re-profiled by its demonstrated activity against several Gram-positive pathogens.

However, the potential pharmacological applications of this hydrophobic compound may be limited by its low solubility

in water and biological fluids. One of the most promising strategies for overcoming drug solubility and stability is the

use of nanoparticles as drug delivery vehicles. In this work we have explored the use of poly(lactic-co-glycolic acid)

(PLGA) nanoparticles loaded with auranofin as antibacterial agent against 2 important streptococcal pathogens i.e.

Streptococcus pneumoniae and S. pyogenes. In vitro experiments have shown that cell viability of cultures of several

pneumococcal strains, including multiresistant ones, decreased about 3 log units after 6 hours of treatment with 0.5 μM

of auranofin, whereas the comparative cultures treated with auranofin-PLGA nanoparticles reached the practical sterility

even at lower concentrations (0.25 μM). The different bactericidal effect of auranofin alone or loaded in nanoparticles

was more noticeable on the sessile bacterial community (biofilm) of S. pyogenes, since auranofin-nanoparticles killed

about 4 logs of the bacterial population at 0.25 μM, whereas auranofin alone was virtually ineffective at the same

concentration. These promising results have been validated in vivo using a zebrafish embryo S. pneumoniae infection

model. Experiments revealed that treatment with auranofin-nanoparticles resulted in about 15% greater protection

compared with the drug alone, in the range of 0.1-0.5 μM.

P2.32

P4 immunotherapy augments neutrophil bacterial killing in patients

admitted to critical care with severe community acquired pneumonia

Suzanna Gore 1 , Ben Morton 2 , Mathieu Bangert 1, 2 , Emma Dearing 1 , Robert Parker 4 , Ingeborg

Welters 5 , Angie Wright 2 , Gowrisankar Rajam 3 , Edwin Ades 3 , Stephen Gordon 2 , Aras Kadioglu 1

1

Clinical Infection, Microbiology and Immunology, Institute of Infection & Global Health, University of Liverpool, Liverpool, UK; 2 Respiratory Infection

Group, Liverpool School of Tropical Medicine, Liverpool, UK; 3 Division of Bacterial Diseases, Centers for Disease Control and PreventionCenters for

Disease Control and Prevention, Atlanta, USA; 4 Critical Care Department, Aintree University Hospital NHS Foundation Trust, Liverpool, UK; 5 Critical

Care Department, Royal Liverpool University Hospital, Liverpool, UK

New treatments are needed to improve outcome in severe pneumonia and sepsis. Immunotherapy using P4 peptide—a

28 amino acid fragment of the pneumococcal protein PsaA—has considerable potential as a novel therapeutic strategy

aimed at augmenting phagocytic responses to bacterial infection. To assess the potential clinical efficacy of P4 treatment,

we compared neutrophil bacterial killing activity with and without P4 stimulation, using peripheral blood neutrophils

obtained from patients admitted to critical care with severe community-acquired pneumonia. Patients admitted to critical

care with severe community-acquired pneumonia were recruited to a cross-sectional observational study of outcome

and ex vivo cellular responses. We assessed neutrophil bacterial killing, cell surface marker, and cytokine expression and

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observed clinical course until hospital discharge. Twenty-five patients were recruited and 23 had blood samples tested.

Treatment with P4 peptide resulted in significantly increased bacterial killing by neutrophils in 15/23 (65%) of patients

(mean killing index 31.4% vs. 20.3%, p = 0.0024). Clinical measures of disease severity (SOFA and APACHE II) did not

correlate with P4 peptide activity. Serum levels of IL-10 (GM 12.4 vs. 1.9, p = 0.0061) and IL-8 (GM 59.1 vs. 8.5, p = 0.015)

were significantly higher in blood samples that did not respond to P4 stimulation. Treatment with P4 peptide significantly

enhanced neutrophil phagocytic activity in the majority of patients with severe community-acquired pneumonia. This

study supports the rationale for P4 immunotherapy as a therapeutic strategy in severe community-acquired pneumonia.

P2.33

Recombinant expression of Streptococcus pneumoniae capsular

polysaccharides in Escherichia coli

Emily Kay 1 , Laura Yates 1, 2 , Vanessa Terra 1 , Jon Cuccui 1 , Brendan Wren 1

1

London School of Hygiene & Tropical Medicine, London, UK; 2 University of Alberta, Edmonton, Canada

Currently, Streptococcus pneumoniae is responsible for over 14 million cases of pneumonia worldwide annually, and

more than 1 million deaths, the majority of them children. In the UK 2 vaccines are recommended: the pneumococcal

polysaccharide vaccine (PPV23), containing purified capsular polysaccharide from 23 serotypes; and the pneumococcal

conjugate vaccine (PCV13), containing 13 common serotypes conjugated to CRM197 (mutant diphtheria toxin). Vaccine

production costs of pneumococcal conjugate vaccines can be prohibitively high, limiting accessibility of the vaccine in lowincome

countries. Protein glycan coupling technology (PGCT) can be used to produce recombinant vaccines by expressing

the glycan of interest, acceptor protein. and glycosyltransferase enzyme (PglB) within Escherichia coli. This approach is

flexible and can be exploited to produce an unlimited and purified supply of vaccine at low cost. In order to optimise

glycoconjugate yield each of the components must be optimised individually and in concert. Recombinantly expressing

a polysaccharide-encoding locus from a Gram-positive bacterium in a Gram-negative bacterium is challenging, not least

because transcriptional levels may not be maintained, all the necessary substrates and precursors may not be available,

and the resultant components may not be trafficked and assembled in the same way. Our aims were to recombinantly

express S. pneumoniae capsular polysaccharides, from several different capsule types, within E. coli; to find out the

minimum set of genes necessary to reliably and efficiently express these polysaccharides heterologously; and finally, to

assess the suitability of these polysaccharides to be used with PGCT, coupling them to a variety of immunogenic carrier

proteins, to produce an inexpensive and flexible conjugate vaccine. To date, 7 polysaccharides have been recombinantly

expressed and detected using a serotype-specific antiserum; of these, 4 have been coupled to a protein using PGCT.

P2.34

A novel cross-protective whole-cell inactivated pneumococcal vaccine

Rachelle Babb 1 , Austen Chen 1 , Abiodun D Ogunniyi 1 , Tim Hirst 2 , Mohammed Alsharifi 1 , James

Paton 1

1

School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia; 2 Gamma Vaccines Pty Ltd, Canberra, Australian Capital

Territory, Australia

Generating a pneumococcal vaccine that is serotype-independent and cost effective remains a global challenge. Gammairradiation

has recently been employed as an inactivation technique for the generation of whole cell bacterial and viral vaccines

due to its ability to conserve pathogen structure without disruption of antigenic determinants. In the present study, we

utilised gamma irradiation to inactivate an unencapsulated Streptococcus pneumoniae strain Rx1 with an unmarked deletion

of the autolysin gene lytA, and with the pneumolysin gene ply replaced with an allele encoding a non-toxic pneumolysoid

(PdT) (designated γ-PN vaccine). Intranasal γ-PN vaccination of C57BL/6 mice was shown to be protective in lethal challenge

models of pneumococcal bacteraemia, pneumonia and meningitis. Vaccine efficacy was shown to be reliant on B cells,

IFN-γ and IL-17A responses. These data are the first to demonstrate the use of gamma-irradiation as a means of generating

an effective serotype-independent pneumococcal vaccine that is dependent on both humoral and cellular immunity.

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P2.35

Development of a novel, heat shock protein enriched pneumococcal

vaccine

Win Yan Chan 1 , Paola Cecchini 2 , Claire Entwistle 2 , Christopher Bailey 2 , Jun Wheeler 3 , Jeremy

Brown 1

1

CITR, Division of Medicine, UCL, London, UK; 2 ImmunoBiology Ltd., Babraham, Cambridge, UK; 3 NIBSC, MHRA, Potters Bar, Herts, UK

Current vaccination against Streptococcus pneumoniae, using vaccines based on capsular polysaccharides from a limited

number of serotypes, has led to replacement by non-vaccine serotypes. An alternative approach based on multiple

protein antigens enriched with and bound to highly conserved heat-shock proteins (Hsps) will overcome shortcomings

of these existing strategies and provide multivalent protection. Hsps are chaperones produced in response to cell

stress and bind S. pneumoniae proteins to form Hsp-peptide complexes, which improve innate and adaptive immune

responses. S. pneumoniae cultures were enriched with Hsps by subjecting to heat stress, lysed and purified to form a

vaccine preparation. Proteomic analysis was used to confirm enrichment of heat shock proteins and to identify potential

protective antigens and their relative expression compared to an untreated homologous lysate. Serum for western

blotting, surface IgG-binding, and ELISA analysis of antibody responses were obtained from vaccinated rabbits and mice.

Protective efficacy was assessed in mouse models of S. pneumoniae infection. The Hsp-enriched vaccine contained several

known important antigens and induced robust antibody responses. Vaccinated rabbit sera contained cross-reactive

antibodies against multiple serotypes, including non-vaccine serotypes, and sera from vaccinated mice opsonised S.

pneumoniae with IgG. Active vaccination significantly protected in mouse models of pneumonia infection, whilst passive

vaccination of rabbit serum significantly protected against both homologous and heterologous infection mouse models

of sepsis. This novel vaccine induced cross-reactive antibodies against multiple serotypes of S. pneumoniae, protected

against infection and has the potential to provide serotype-independent protection against S. pneumoniae.

P2.36

Use of Δ6PLY as an immunomodulator when fused to antigens of

unrelated species Mannheimia haemolytica

Ricardo Corona-Torres, Andrea Mitchell, Jenny Herbert, Tim Mitchell

University of Birmingham, Birmingham, UK

Pneumolysin (PLY), the cholesterol-dependant cytolysin produced by Streptococcus pneumoniae, causes direct tissue

damage in the different presentations of pneumococcal infections by pore formation in host cells membranes. The

deletion of two amino acids in the PLY sequence produces a non-toxic version known as Δ6PLY, which is non-toxic but

retains immunogenicity and is therefore useful for vaccine development. Our research has demonstrated that the fusion

of Δ6PLY to other pneumococcal virulence factor such as choline-binding proteins increases their immunogenicity when

given mucosally. We hypothesise that the adjuvant properties of PLY may be useful for construction of other vaccines.

Therefore we are investigating the use of protein fusions of Δ6PLY with proteins from the ruminant pathogen Mannheimia

haemolytica, responsible for causing bovine respiratory complex. Current commercial vaccines are produced by the

chemical inactivation of the RTX toxin, leukotoxin (Lkt), produced by M. haemolytica. Four protein chimeras were

designed, 2 of them based on LKT: LktAΔ6PLY, which contains the inactive structural protein LktA, and LktAepΔ6PLY,

which is formed by a neutralising epitope of Lkt. The other 2 fusions were designed with a different virulence factor of M.

haemolytica, the neuraminidase NanH: NanHG63-H585Δ6PLY, which lacks the autotransporter domain, and NanHG63-

H435Δ6PLY, which only contains the catalytic domain of NanH. These fusion proteins were expressed in Escherichia coli

and evaluated for their ability to generate systemic immune responses when administered to mucosal surfaces. We

additionally propose the vaccination with crude E. coli lysates as a cheaper alternative for veterinary medicine so the

proteins will be assessed in a crude and in a purified version.

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P2.37

Pneumolysin toxoid as a vaccine adjuvant

Cassandra L Krone 1, 2 , Jenny Herbert 1, 2 , Andrea Mitchell 1, 2 , David R Withers 1 , Tim J Mitchell 1, 2

1

School of Immunity and Infection, University of Birmingham, Birmingham, UK; 2 Institute of Microbiology and Infection, University of Birmingham,

Birmingham, UK

Streptococcus pneumoniae (the pneumococcus) is a major human pathobiont and can cause a range of diseases including

pneumonia and meningitis. Currently, available vaccines against the pneumococcus are based on the polysaccharide

capsule and as such are serogroup specific. Therefore, there is much interest in developing protein-based vaccines. The

pneumococcal pore-forming toxin pneumolysin (PLY) is an important virulence factor, but also a potential protective

immunogen. We previously made a non-toxic version of PLY (Δ6PLY) that does not form pores. Δ6PLY is immunogenic

in mice and also acts as a mucosal adjuvant for proteins fused to the toxoid. In this study we use a mouse model to

demonstrate that mucosal vaccination with protein fusion vaccines can protect against colonisation. The mechanism

of protection is IL-17 dependant, as protection against colonisation was abrogated in IL-17 knock-out mice. We will

also describe the use of in vitro stimulation assays to investigate the possible mechanisms of the adjuvant activity of

Δ6PLY. Furthermore, use of IL17A-Cre R26R-tdRFP fate mapping reporter mice will enable us to determine which IL-17A

producing cells are involved in the mucosal immune response to Δ6PLY.

P2.38

Clonal expansion of non-susceptible non-vaccine pneumococci in

non-invasive isolates following conjugate vaccine introduction results

in similar rates of antimicrobial non-susceptibility to the

pre-conjugate-vaccine era

Martha McElligott 1, 2 , Imelda Vickers 1, 2 , Mary Meehan 1 , Mary Cafferkey 1, 2 , Robert Cunney 1, 3 ,

Hilary Humphreys 2, 4

1

Temple Street Children’s University Hospital, Dublin, Ireland; 2 Royal College of Surgeons in Ireland, Dublin, Ireland; 3 Health Protection Surveillance

Centre, Dublin, Ireland, 4 Beaumont Hospital, Dublin, Ireland

The 7- and 13- valent pneumococcal conjugate vaccines (PCV7/13) were introduced to the Irish childhood immunisation

schedules in 2008 and 2010, respectively. We collected 506 paediatric non-invasive pneumococci from 2009 to 2014 at

Temple St Children’s University Hospital and Our Lady’s Children’s Hospital Crumlin and AMNCH Tallaght during 2013 to

2014. Serotyping and susceptibility testing to 6 different antimicrobials was performed on isolates. Multilocus sequence

typing further characterised 169 isolates non-susceptible to at least 1 antimicrobial. Notable changes occurred in nonsusceptible

clones and serotypes in non-invasive isolates after conjugate vaccine introduction compared to before

conjugate vaccine introduction. In 2007, England 14 -9 and Spain 9V -3 accounted for 12% and 32% of non-susceptible clones,

respectively, but in 2009-2014 their prevalence was just 0% and 2.4%. Both England 14 -9 and Spain 9V -3 were associated

with PCV7 serotypes 14 and 9V. Furthermore, there was a significant decline in non-susceptible PCV7 serotype 6B

belonging to Spain 6B -2 and its variants from 2009 to 2014 (p = 0.0024). Fluctuations occurred in clonal complex 320

associated with non-susceptible PCV13 serotype 19A. A significant increase occurred in non-susceptible non-vaccine

type (NVT) pneumococci from 12% (3/25) in 2007 to 73% (51/69) in 2014 (p < 0.0001). Serotypes 15A (n = 21) and 35B (n

= 22) were the most frequent NVT which were non-susceptible from 2009 onwards. The increase of NVT serotypes 15A

and 35B was largely mediated by clonal expansion of Sweden 15A -25 and its variants (n = 15) and sequence type 558 (n =

21), respectively. Overall, non-susceptibility rates among non-invasive isolates did not change notably during the study

at 23% (2007), 30% (2009), 31% (2010), 21% (2011), 34% (2012), and 41% (2013-2014). This suggests that the increase

in clones expressing NVT has resulted in a similar rate of non-susceptibility among non-invasive pneumococci. Continued

surveillance will determine if non-susceptible NVT pneumococci increase in circulation with sustained conjugate vaccine

use.

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P2.39

Salmonella outer membrane vesicles displaying high densities

of pneumococcal antigen at the surface offer protection against

colonisation

Kirsten Kuipers 1 , Maria Daleke-Schermerhorn 2, 3 , Wouter Jong 2, 3 , Corinne ten

Hagen-Jongman 2, 3 , Fred van Opzeeland 1 , Elles Simonetti 1 , Christa van der Gaast 1 , Aldert Zomer 4 ,

Joen Luirink 2, 3 , Marien de Jonge 1

1

Radboud University Medical Center, Nijmegen, The Netherlands; 2 VU University, Amsterdam, The Netherlands; 3 Aberia Bioscience AB, Stockholm,

Sweden, 4 Utrecht University, Utrecht, The Netherlands

Bacterial outer membrane vesicles (OMVs) are attractive vaccine formulations because of their intrinsic immunostimulatory

properties and non-living nature. In principle, heterologous antigens incorporated into OMVs will elicit specific immune

responses, especially if presented at the surface. In this study, we explored the feasibility of the protein expression

Hbp platform for vaccine development and present an approach for a broadly protective pneumococcal vaccine. We

show that intranasally administered Salmonella OMVs displaying high antigen levels at the surface induced strong

protection in a murine model of pneumococcal colonisation, without the need for a mucosal adjuvant. Reduction in

bacterial recovery from the nasal cavity correlated with local production of antigen-specific IL-17A. Furthermore, the

protective efficacy, the production of antigen-specific IL-17A, and local and systemic IgGs, were all improved at a higher

concentration of the displayed antigen. As only the α1α2 part of PspA elicited strong protection, we investigated which

region(s) in α1α2 could mediate cross-protection between pneumococcal strains using a pneumococcal strain collection

of 350 clinical isolates. IgG cross-reactivity between pneumococcal strains appears to be mediated by 2 specific regions

of α1α2, which, remarkably, are variants present in almost all clinical isolates. Currently, experiments are ongoing to

assess cellular cross-reactivity. As PspA is highly variable among serotypes, the percentage coverage of pneumococcal

strains could potentially be increased by combining the cross-reactive regions from different sequences for surface

display on Salmonella OMVs. Here we demonstrate that intranasally administered OMVs decorated with pneumococcal

antigens induce strong protection. This discovery highlights the importance of an efficient antigen expression system for

development of recombinant OMV-based vaccines. In conclusion, our findings demonstrate the suitability of the Hbp

platform for development of a new generation of OMV vaccines, and illustrate the potential of using this approach to

develop a broadly protective mucosal pneumococcal vaccine.

P2.40

Calibration of an individual-based transmission model to pre- and

post-PCV pneumococcal carriage

Marc Lipsitch 1 , Thomas Fussell 1 , Sarah Cobey 2 , Daniel Weinberger 3

1

Harvard TH Chan School of Public Health, Boston, MA, USA; 2 University of Chicago, Chicago, IL, USA; 3 Yale School of Public Health, New Haven, CT,

USA

Predicting the effects of pneumococcal vaccination on carriage of and disease from specific pneumococcal serotypes

requires a sufficiently realistic model of the factors underlying serotype-specific incidence and prevalence before and

after vaccination. We adapted a previously-published model of pneumococcal population dynamics, which includes

serotype-specific variability in duration and within-host competitive ability, as well as serotype-specific and non-specific

acquired immunity, to fit the observed prevalence of pneumococcal serotypes, using carriage data from Massachusetts

before PCV7, during the PCV7 era, and during the PCV13 era. By varying a single parameter for each serotype that

governed both its specific duration and its within-host competitive ability, and varying another parameter governing

overall transmission intensity, the model was able to reproduce observed patterns of serotype-specific prevalence

before vaccine introduction. Following introduction of vaccine, based on fitting to vaccine-type prevalence, a direct

effect of at least a 73% reduction in the acquisition rate of carriage of vaccine types was required to reproduce

observed carriage prevalences, post-vaccine introduction. Serotypes that were not in the vaccine but are part of vaccine

serogroups expanded post-PCV7 more in observed data than in model predictions. We hypothesise that partial crossimmunity

within serogroups may have lowered prevaccine prevalence of these vaccine-related types, and that their

disproportionate expansion may be evidence for such partial cross-immunity. We conclude that calibrated, detailed

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individual-based modelling of pneumococcal vaccine effects is possible for applications in public health.

P2.41

Evaluation of the efficacy of the immunisation with PspA in a

co-colonisation model in mice

Rafaella Tostes, Maria Leonor Oliveira, Paulo Ho, Eliane Miyaji

Instituto Butantan, São Paulo/SP, Brazil

Pneumococcal surface protein A (PspA) is one of the leading candidates for a protein vaccine against pneumococcal

infections. PspA shows variability and the great majority of strains express PspA from family 1 (clades 1 and 2) or family

2 (clades 3, 4, and 5). The aim of this study is to evaluate the protection elicited by nasal immunisation with recombinant

PspAs from different clades in a mouse model of co-colonisation of the nasopharynx with two strains, one expressing PspA

from family 1 and another expressing PspA from family 2. BALB/c mice were immunised intranasally with recombinant

PspAs from clades 1 to 4 (rPspA1, rPspA2, rPspA3 and rPspA4) using the whole-cell pertussis vaccine as adjuvant.

Immunisation with rPspA1 elicited an increase in anti-rPspA1 and anti-rPspA4 IgG titres measured by ELISA, whereas

immunisation with rPspA4 elicited an increase only in anti-rPspA4 IgG titres. Mice were then challenged with a mixture

of the strains 491/00 (serotype 6B, PspA1) and 472/96 (serotype 6B, PspA4, trimethoprim resistant) and bacteria were

recovered from nasal washes 5 days after challenge. Only mice immunised with rPspA1 showed statistically significant

reduction in colonisation with both strains when compared to the control saline group. Animals immunised with rPspA3

and rPspA4 showed statistically significant reduction only in the PspA4 expressing strain when compared to saline. Our

preliminary results indicate that rPspA1 and possibly a mixture of rPspA1 and rPspA4 would be suitable for attaining

protection against colonisation with strains expressing different PspAs.

Supported by FAPESP, Fundação Butantan, CAPES and CNPq (Brazil).

P2.42

New protein vaccines against pneumococcal pneumonia using

experimentally induced immunity

Jessica Owugha 1 , Angela Wright 1 , Andrea Collins 1 , Cintia Vadesilho 2 , Eliane Miyaji 2 , Kondwani

Jambo 3 , Stephen Gordon 1 , Daniela Ferreira 1

1

Liverpool School of Tropical Medicine, Liverpool, UK; 2 Biotechnology Centre, Instituto Butantan, São Paulo, Brazil; 3 Malawi-Liverpool Wellcome Trust

Clinical Research Programme, Blantyre, Malawi

Our experimental human pneumococcal carriage (EHPC) model provides an ethical measurement of vaccine efficacy

where colonisation is a surrogate for disease. Factors associated with prevention of colonisation include T-helper 17

cell (Th-17) and humoral immune responses. The conserved yet heterogeneous virulence factor pneumococcal surface

protein (PspA) is a promising vaccine candidate, immunogenic and protective in murine disease models. Identification

of a region eliciting beneficial Th-17 and humoral responses may inform development of a vaccine conferring protection

against pneumonia. Four recombinant PspA fragments (approximately 100 amino acids) were expressed in Escherichia

coli BL21 and used in ex vivo stimulation. Volunteers were inoculated with pneumococcus and mononuclear cells isolated

from peripheral blood (PBMCs) and bronchoalveolar lavage (BAL). Th-17 cells were stained for surface phenotyping and

intracellular cytokine production. Assay optimisation revealed Th-17 responses were best detected 5 days post-antigen

stimulation. Preliminary analysis of PBMC stimulations demonstrates an increase in Th-17 response post-pneumococcal

inoculation in all antigens, with elevated responses in one of four fragments. PBMCs of volunteers inoculated with

pneumococcus demonstrate an increased Th-17 response on stimulation with all candidate antigens. The PspA

fragment inducing the highest Th-17 response in blood and BAL will be used to purify antibodies from plasma of healthy

adults susceptible to or protected from colonisation. Resultant antibodies will be employed in functional binding and

complement deposition assays to compare ability to bind and potentially clear pneumococci of differing strains.

Acknowledgements Study volunteers, LSTM PhD Studentship body, MRC/FAPESP, NIHR clinical staff

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P2.43

Novel recombinant glycoconjugate vaccines for prevention of

pneumococcal meningitis

Charlie Plumptre 1 , Emily Kay 2 , James Paton 3 , Brendan Wren 2 , Jeremy Brown 1

1

Division of Medicine, University College London, UK; 2 Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine,

UK; 3 Research Centre for Infectious Diseases, University of Adelaide, Australia

The pneumococcus is a major cause of meningitis around the world. Current vaccination strategies suffer from serious

limitations in terms of cost and serotype coverage, and novel approaches are required to overcome these challenges.

In this project, we are aiming to produce vaccines comprised of conserved pneumococcal protein antigens conjugated

to capsular polysaccharide from serotype 4 pneumococci by making use of an unusual oligosaccharyltransferase from

Campylobacter jejuni named PglB. This enzyme is able to conjugate glycans containing an acetamido group in the C2

position of their reducing end sugar to proteins containing a specific amino acid sequence (D/E-Y-N-X-S/T). We have

introduced this ‘glycotag’ sequence into 4 pneumococcal protein vaccine candidates: α-glycerophosphate oxidase

(GlpO), neuraminidase A (NanA), and the ATP-binding cassette transporters PiuA and Sp0148. All 4 of these proteins

have previously shown efficacy as vaccines in animal models of colonisation or meningitis caused by Streptococcus

pneumoniae. Previous work has allowed the expression of pneumococcal serotype 4 capsule in Escherichia coli, and

by co-expressing PglB and the target protein in the same strain, we have been able to demonstrate production and

small scale purification of protein antigens conjugated to the polysaccharide. Our current efforts are directed towards

optimisation of the efficiency of the conjugation process through modifying parameters such as the leader sequence

of the protein, the plasmid used to encode its gene, the background strain of E. coli and the growth conditions used for

expression.

P2.44

Broadly cross-reactive antibodies recognising the proline-rich region

of pneumococcal surface protein A variants show cross-reactivity with

skeletal muscle

Zoltan Magyarics 1 , Harald Rouha 1 , Adriana Badarau 1 , Nels Nielson 2 , Marisa Caccamo 1 , Susanne

Weber 1 , Barbara Maierhofer 1 , Katharina Havlicek 1 , Ivana Dolezilkova 1 , Karin Gross 1 , Eszter Nagy 1

1

Arsanis Biosciences, Vienna, Austria, 2 Adimab LLC, Lebanon, NH, USA

Pneumococcal surface protein A (PspA) is an important surface-expressed virulence factor of Streptococcus pneumoniae.

PspA displays high level of sequence variability in clinical isolates but contains a conserved proline-rich region at the

C-terminus. PspA was shown to be protective in animal models and has been considered as an attractive vaccine

antigen. One of the concerns with PspA is the anecdotal detection of serum antibodies with potential cross-reactivity to

human muscle in vaccines. The antigen responsible for inducing tissue cross-reactive antibodies was assumed to be the

N-terminal coiled coil region of PspA. We selected 4 human IgG antibodies from a yeast-based antibody library utilising

all 5 clade variants of PspA as baits. The resulting mAbs were tested for binding to native PspA by flow cytometry-based

surface staining of 61 S. pneumoniae clinical isolates. Poly-reactivity of the mAbs was tested against cell membrane

extract of Chinese hamster ovary (CHO) cells as well as against porcine myosin. We identified monoclonal antibodies

binding to the majority of tested S. pneumoniae clinical isolates, expressing clades 1 to 5 of PspA. Confirming the

specificity of the mAbs, they do not bind to S. pneumoniae D39 ΔpspA/ΔpspC strain. These antibodies were determined

to react with a PAPAPKP consensus peptide motif of the proline-rich region of PspA. However, we observed significant

binding to CHO cell extract and myosin in ELISA, suggesting that breadth of binding to PspA variants correlated with nonspecific

reactivity towards mammalian cells and specifically, skeletal muscle. Amino acid homology search identified the

presence of the PAPAPKP motif and its variants in several human proteins, including myosin, cell adhesion, and nuclear

proteins. In conclusion, broadly cross-reactive PspA monoclonal antibodies targeting the proline-rich region of PspA can

bind live bacteria, but they raise concern about off-target tissue cross-reactivity related to this region of PspA.

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P2.45

Structural analysis of the choline-binding sites in CbpL from

Streptococcus pneumoniae

Javier Gutiérrez-Fernández 1 , Martín Alcorlo Pagés 1 , Malek Saleh 2 , Sergio G. Bartual 1 , Thomas

Pribyl 2 , Sven Hammerschmidt 2 , Juan A. Hermoso 1

1

Spanish National Research Council, Madrid, Spain; 2 Ernst Moritz Arndt University of Greifswald, Greifswald, Germany

Four families of surface proteins decorate the cell surface of the human pathogen Streptococcus pneumoniae. Besides

lipoproteins and LPXTG proteins, also present in other Gram-positive bacteria, the pneumococcus presents the nonclassical

surface proteins and the choline-binding protein family. Choline binding proteins (CBPs) show a modular

organisation including, at least, the choline-binding domain and a domain exerting a biological function. The cholinebinding

domain interacts with choline molecules from teichoic and lipoteichoic acids, attaching the whole protein to the

peptidoglycan layer. Here, we show the three-dimensional structure of the choline-binding domain of CbpL displaying

8 choline-binding sites. Four of them follow the canonical sequence while the other 4 are different. The alternate

configuration of canonical and non-canonical sites is a unique property of CbpL among CBP family. Structural analysis

and specific features of this module will be provided.

P2.46

Assessment of the specific role of the phosphorylcholine esterase Pce

in the modification of pneumococcal teichoic acids

Franziska Waldow 1 , Thomas Kohler 2 , Dominik Schwudke 1, 3 , Sven Hammerschmidt 2 , Nicolas

Gisch 1

1

Division of Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany; 2 Department Genetics

of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany; 3 Airway Research

Center North (ARCN), Member of the German Center for Lung Research (DZL), Germany

Pneumococcal diseases are a global burden and Streptococcus pneumoniae is one of the most important pathogens

in bacterial meningitis, otitis media, and sinusitis. Unlike other Gram-positive bacteria, the lipoteichoic acid (LTA) and

wall teichoic acid (WTA) of S. pneumoniae possess the same structure within their repeating units. Pneumococci have

a unique nutritional requirement for choline for growth to be able to attach phosphorylcholine (P-Cho) residues to the

N-acetylgalactosamine moieties of their teichoic acids (TAs). This structural feature is of physiological importance for the

anchoring of surface-localised choline-binding proteins (CBPs) to the pneumococcal cell wall. CBPs like the pneumococcal

surface protein C (PspC) and the phosphorylcholine esterase (Pce) have been shown to be involved in pneumococcal

adhesion to host cells. Interestingly, Pce hydrolyses about 15–30% of the total P-Cho residues attached to pneumococcal

TAs. In the study presented here, we investigate which P-Cho residues are specifically removed from the TAs by the Pce.

LTAs of different pneumococcal strains were isolated by citric buffer/butan-1-ol extraction and purified by hydrophobic

interaction chromatography. These LTA preparations were further treated with anhydrous hydrazine to remove fatty

acids and D-alanine residues. This procedure has two major advantages: less complex mass spectrometry data can

be obtained and 1 H as well as 31 P NMR spectra show significantly higher resolutions. Importantly, in these spectra of

O-deacylated and therefore non-aggregated LTA molecules specific moieties can be quantified. We have shown recently

that the terminus of the pneumococcal LTA can vary in the P-Cho substitution pattern in dependency on the strain (D39

vs. TIGR4) and the culturing conditions. Here, the role of Pce in the modification of pneumococcal TAs will be elucidated

by detailed structural investigation of LTA isolated from TIGR4∆cps∆pce as well as by treatment of fully P-Cho-decorated

pneumococcal LTA with heterologously expressed Pce.

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Oral abstracts

O1.1

Identification of pneumococcal colonisation determinants in the

stringent response pathway, facilitated by genomic diversity

Yuan Li 1 , Nicholas J. Croucher 1 , Claudette M. Thompson 1 , Krzysztof Trzcinski 2 , William P.

Hanage 1 , Marc Lipsitch 1

1

Harvard T.H. Chan School of Public Health, Boston, MA, USA, 2 UMC Utrecht, Utrecht, The Netherlands

Understanding genetic determinants of a microbial phenotype generally involves creating and comparing isogenic strains

differing at the locus of interest, but the naturally existing genomic and phenotypic diversity of microbial populations has

rarely been exploited. Here we report use of a diverse collection of 616 carriage isolates and their genome sequences to

help identify a novel determinant of pneumococcal colonisation. A spontaneously arising laboratory variant (SpnYL101)

of a capsule-switched TIGR4 strain (TIGR4:19F) showed reduced ability to establish mouse nasal colonisation and lower

resistance to non-opsonic neutrophil-mediated killing in vitro, a phenotype correlated with in vivo success. Whole

genome sequencing revealed 5 single nucleotide polymorphisms (SNPs) affecting 4 genes in SpnYL101 relative to its

ancestor. To evaluate the effect of variation in each gene, we performed an in silico screen of 616 previously published

genome sequences to identify pairs of closely related, serotype-matched isolates that differ at the gene of interest, and

compared their resistance to neutrophil killing. This method allowed rapid examination of multiple candidate genes and

found phenotypic differences apparently associated with variation in SP_1645, a RelA/ SpoT homolog (RSH) involved

in the stringent response. To establish causality, the alleles corresponding to SP_1645 were switched between the

TIGR4:19F and SpnYL101. The wild-type SP_1645 conferred higher resistance to neutrophil killing and competitiveness

in mouse colonisation. Using a similar strategy, variation in another RSH gene (TIGR4 locus tag SP_1097) was found to

alter resistance to neutrophil killing.

O1.2

Genomics reveals the worldwide distribution of multidrug-resistant

serotype 6E pneumococci

Andries van Tonder 1 , James Bray 1 , Lucy Roalfe 2 , Rebecca White 2 , Marta Zancoli 2 , Sigríður Quirk 3 ,

Gunnstein Haraldsson 3 , Keith Jolley 1 , Martin Maiden 1 , Stephen Bentley 5 , Ásgeir Haraldsson 3, 4 ,

Helga Erlendsdóttir 3, 4 , Karl Kristinsson 3, 4 , David Goldblatt 2 , Angela Brueggemann 1

1

University of Oxford, Oxford, UK, 2 University College London, London, UK, 3 University of Iceland, Reykjavik, UK, 4 Landspitali University Hospital,

Reykjavik, UK, 5 Wellcome Trust Sanger Institute, Hinxton, UK

The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist and they work

by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however,

current vaccines are limited by the fact that there are nearly 100 antigenically distinct serotypes. Within the serotypes,

serogroup 6 pneumococci are a frequent cause of serious disease and a common coloniser of the nasopharynx in

children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected

serotype 6E among recent pneumococcal collections. Therefore, we analysed a diverse dataset of approximately 1000

serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analysed the genetic diversity among

serogroup 6 pneumococci and investigated whether pneumococcal conjugate vaccine (PCV)-induced serotype 6A and

6B antibodies inhibited serotype 6E pneumococci. We found that 43% of all genomes were serotype 6E and they were

recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages,

3 of which were multidrug-resistant, described approximately 90% of the serotype 6E pneumococci. Serological assays

demonstrated that vaccine-induced serotype 6B antibodies were able to kill serotype 6E pneumococci. We also revealed

3 major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified

44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic

variants may reduce vaccine efficacy in the longer term due to the emergence of serotypes that can evade vaccine-

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induced immunity.

O1.3

The post-vaccine microevolution of invasive pneumococcal disease

Fredrick M. Mobegi 1 , Amelieke J.H. Cremers 1 , Marien I. de Jonge 1 , Sacha A.F.T. van Hijum 1 ,

Jacques F. Meis 3 , Peter .W.M Hermans 1 , Gerben Ferwerda 1 , Stephen D. Bentley 2 , Aldert Zomer 1

1

Radboud University Medical Center, Nijmegen, The Netherlands; 2 Wellcome Trust Sanger Institute, Cambridge, UK; 3 Canisius-Wilhelmina Hospital,

Nijmegen, The Netherlands

After the introduction of pediatric pneumococcal vaccination in the US, rates of invasive pneumococcal disease (IPD)

decreased. However, the genetic background of the pediatric pneumococcal carriage population did not change

dramatically. We investigated whether vaccination has influenced population genomics in IPD in the Netherlands.

We serotyped and sequenced 350 strains of Streptococcus pneumoniae isolated between 2001 and 2011 from adult

IPD patients in Nijmegen. After genome assembly, mapping, annotation and orthologous group (OG) assignment, a

core genome was established. A phylogenetic tree deduced from the core genome’s super alignment revealed tight

clustering of isolates per serotype. Capsular switches were inferred from phylogeny in the core genome, and diversity

was determined by phylogenetic distances between the accessory genomes. Upon the introduction of the 7-valent

pneumococcal conjugated vaccine (PCV7) in 2006 a gradual decrease in vaccine serotypes and serotype replacement

were observed, although capsular switches remained rare. The diversity of the accessory genome dropped shortly after

the introduction of PCV7 (p < 0.0001). Genes that contributed to a re-expansion of diversity afterwards were comparable

to those pre PCV7, although few genes dispersed from their prevalence in the original gene pool. Despite serotype

replacement in pneumococcal disease after paediatric vaccination with PCV7, we observed a temporary bottleneck in

gene diversity, which re-expanded mainly by genes already present in the original gene pool. These observations show

similar dynamics in pneumococcal population restructuring, both in asymptomatic carriage and IPD. We suggest the use

of whole genome sequencing for surveillance of pneumococcal population dynamics that could give a projectile on the

course of disease, facilitating effective prevention and management of IPD.

O1.4

Mechanisms of pneumococcal evolution over short and long timescales

Nicholas Croucher 1 , Paul Coupland 2 , Abbie Stevenson 3 , Allanna Callendrello 3 , Stephen Bentley 2 ,

William Hanage 3

1

Imperial College, London, UK; 2 Wellcome Trust Sanger Institute, Cambridge, UK; 3 Harvard School of Public Health, Boston, USA

Phylogenetic analyses of core genomes suggest pneumococcal populations comprise multiple co-circulating lineages.

Using data from 616 Streptococcus pneumoniae samples collected in Massachusetts between 2001 and 2007, it was

possible to demonstrate isolates from the same lineage also shared very similar accessory genomes. By contrast, gene

content diverged considerably between lineages. This was found not to be the consequence of each lineage having

extensive unique genome content, but instead represented the cumulative effect of multiple common loci having stable

patterns of presence and absence across closely related bacteria. Detailed classification of the different components

of the pneumococcal genome found these stable loci to correspond to genomic islands moving primarily through

transformation, along with integrative and conjugative elements and phage-related chromosomal islands. However,

prophage were found to be highly diverse both within and between lineages, with strong evidence that they transmitted

rapidly though the population. Their frequency appears to be limited by the deleterious effects they have on their hosts,

indicating a strong selection pressure on pneumococci for defence mechanisms against phage infection. Correspondingly,

2-phase variable genetic loci encoding Type I restriction-modification systems were found to be conserved across all

isolates. Methylation-sensitive sequencing demonstrated the high frequency rearrangements at these loci altered the

specificity of the restriction modification systems over short timescales. This is likely to make them an effective means

of preventing the spread of phage through a clonally related population of cells. Therefore, within-lineage evolution

is substantially affected by the movement of viral sequence and intragenomic rearrangements, whereas differences

between lineages are the result of the infrequent movement of stable genomic islands.

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O1.5

Genetic stabilisation of the pandemic and drug resistant PMEN1

pneumococcus lineage by its distinctive DpnIII restriction-modification

system

Rory Eutsey 2 , Evan Powell 2 , Janina Dordel 3 , Tyson Clark 4 , Jonas Korlach 4 , Garth Ehrlich 5 , N. Luisa

Hiller 1, 2

1

Carnegie Mellon University, Pittsburgh, PA, USA; 2 Allegheny Health Network, Pittsburgh, PA, USA; 3 The Wellcome Trust Sanger Institute, Cambridge,

UK; 4 Pacific Biosciences, Menlo Park, CA, USA; 5 Drexel University College of Medicine, Philadelphia,PA, USA

The human pathogen Streptococcus pneumoniae (pneumococcus) exhibits a high degree of genomic diversity and

plasticity. Isolates with high genomic similarity are grouped into lineages that undergo homologous recombination at

variable rates. The PMEN1 is a pandemic, multi-drug resistant lineage. Heterologous gene exchange between PMEN1

and non-PMEN1 isolates is directional, with extensive gene transfer from PMEN1 and only modest transfer into PMEN1.

Restriction–modification (R–M) systems can restrict horizontal gene transfer, yet most pneumococcal strains code for

either the DpnI or DpnII R–M system and neither limits homologous recombination. Our comparative genomics analysis

revealed that PMEN1 isolates code for DpnIII, a third R-M system syntenic to the other Dpn systems. Characterisation

of DpnIII demonstrated that the endonuclease cleaves unmethylated double stranded DNA at the tetramer sequence

5’-GATC-3’, and the cognate methylase is a C-5 cytosine-specific DNA methylase. We show that DpnIII decreases the

frequency of recombination under in vitro conditions, such that the number of transformants is lower for strains

transformed with unmethylated DNA relative to cognately methylated DNA. Furthermore, we have identified two

PMEN1 isolates where the DpnIII endonuclease is disrupted, and phylogenic work by Croucher and colleagues suggests

that these strains have accumulated genomic differences at a faster rate than other PMEN1 strains. We propose that

the R–M locus is a major determinant of genetic acquisition; the resident R–M system governs the extent of genome

plasticity.

O2.1

MapZ beacons the division sites and positions FtsZ-rings in

Streptococcus pneumoniae

Aurore Fleurie 1 , Christian Lesterlin 2 , Sylvie Manuse 1 , Chao Zhao 1 , Caroline Cluzel 1 , Jean-Pierre

Lavergne 1 , Mirita Franz-Wachtel 3 , Boris Macek 3 , Christophe Combet 1 , Erkin Kuru 4 , Michael Van-

Nieuwenhze 4 , Yves Brun 4 , David Sherratt 2 , Christophe Grangeasse 1

1

CNRS - University of Lyon, Lyon, France; 2 University of Oxford, Oxford, UK; 3 University of Tuebingen, Tuebingen, Germany; 4 Indiana University,

Bloomington, USA

The Min system has been shown to prevent aberrant cell division close to the cell poles, while nucleoid occlusion (NO)

prevents cell division from occurring over the nucleoids. Interestingly, Streptococcus pneumoniae, like most streptococci,

lacks Min and NO systems. This raises the question of which alternative mechanism(s) ensure the positioning of the

Z-ring at mid-cell in streptococci. In this work, we describe that Z-ring positioning at mid-cell in S. pneumoniae relies on

a new landmark protein that functions as a molecular beacon. This membrane protein of previously unknown function,

that we named MapZ for Mid-cell associated Protein Z, is widespread in streptococci. We show that it localises at the

future cell division site before FtsZ and that it is required for the correct recruitment of FtsZ at mid-cell. We further show

that MapZ directly interacts with FtsZ. Our analyses also show that MapZ auto-positions at mid-cell as a result of cell

growth and peripheral peptidoglycan synthesis responsible for cell elongation. Cells lacking MapZ display growth defects

and an aberrant cell shape. In addition, FtsZ positioning, assembly, and constriction are altered resulting in aberrant nonparallel

cell division septa and chromosome pinching. Three-dimensional structured illumination microscopy (3D-SIM)

in live cells revealed that modification of MapZ phosphorylation leads to deregulation of Z-ring dynamics and impacts

cell constriction. This work uncovers an unprecedented mechanism that regulates Z-ring placement and constriction and

illustrates that bacteria have evolved alternative mechanisms at odds with model regulatory systems of cell division.

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O2.2

Competence for genetic transformation in Streptococcus pneumoniae:

primary sigma factor mutations enhance transcription of late genes in

comW mutants

Yanina Tovpeko, Donald A. Morrison

Molecular, Cell, and Developmental Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA

Streptococcus pneumoniae is naturally transformable, but the mechanism governing transformation is not fully

understood. Streptococcal transformation depends on transient accumulation of high levels of the alternative sigma

factor σ X , coordinated via peptide pheromones. σ X recognises a non-canonical promoter upstream of late competence

genes, including those required for DNA uptake. However, full competence also requires a second competence-specific

protein, ComW, regulated by the same pheromone circuit as σ X . We mapped 27 independent comW bypass mutations to

10 single-base transitions, all within rpoD, encoding the primary sigma, σ A . σ A* mutants transformed at an elevated rate

(1% to 20% of WT), far above the comW rate (0.01%). Eight σ A* proteins carried altered residues in RpoD region, 4 that

are implicated in interaction of σ A with the core β subunit. To test for a role for comW in RNAP function, we measured

the effect of the suppressor mutations on late gene expression. Late gene expression was restored to 30% to 80% of

WT levels in the σ A* mutant strains, compared to only 10% in the comW mutant, consistent with the idea that ComW

increases σ X access to core RNAP, and with the location of the mutations in the protein structure. While σ X activity was

restored to 80% of the WT level in one σ A* mutant, the amount of transformants reached only 10% of the WT level,

indicating an additional role of ComW in the development of competence distinct from its role in transcription of late

genes.

Supported by NSF Grant. (MCB-1020863)

O2.3

A new role for Autoinducer-2 in Streptococcus pneumoniae

Claudia Trappetti 1 , Bart Eijkelkamp 1 , Zarina Amin 1 , Adrienne Paton 1 , Christopher McDevitt 1 ,

Marco Oggioni 2 , James Paton 1

1

University of Adelaide, Adelaide, Australia; 2 University of Leicester, Leicester, UK

Bacteria are able to survive in their environment by developing complex multicellular communities, rather than

functioning as individual cells. Communication between cells is essential and is achieved by the secretion and

detection of small signalling molecules called autoinducers. To date, the only signalling molecule recognised by

both Gram-positive and Gram-negative bacteria is the Autoinducer-2 (AI-2). AI-2 is synthesised by the metabolic

enzyme LuxS (S–ribosyl–homocysteine lyase) as a by-product of the conversion of S–ribosyl–homocysteine into

homocysteine. Homologues of LuxS have been found in all bacterial species, suggesting a key role in interspecies

communication. The data presented here indicate that in Streptococcus pneumoniae, AI-2 accelerates and modulates

the progression of disease in mice. In this study, we determined whether the known reduced virulence of a luxS

mutant relative to its wild-type parent D39, could be restored by administrating purified AI-2 in a murine model.

Mice were challenged intranasally with either wild-type or luxS mutant strains and AI-2 was administrated at time zero

and 24 hours post-infection. Treatment with AI-2 significantly increased the levels of bacteraemia and lung invasion in

mice challenged with the luxS mutant. Moreover, the rate of survival was dramatically reduced and the virulence of the

luxS mutant could be reconstituted to the same level of the wild-type strain. To further investigate whether AI-2 could

modulate innate immune responses in the host, we performed a RNA-cytokine array. Surprisingly, we found that only

one cytokine receptor was differentially expressed between groups of mice challenged with either wild-type or luxS

mutant, and that the expression level of this cytokine could be restored if AI-2 is administrated. These data provide

evidence that AI-2 is critical for S. pneumoniae to modulate host immune responses in order to induce systemic disease.

Thus, an inhibitor of the universal signal molecule could have therapeutic potential.

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O2.4

Spatial and dynamic organisation of the chromosome and DNA

replication machinery in Streptococcus pneumoniae

Morten Kjos, Renske van Raaphorst, Jan-Willem Veening

University of Groningen, Groningen, The Netherlands

Chromosome segregation in oval-shaped Streptococcus pneumoniae cells has been shown to depend on other

mechanisms than those known for various rod-shaped model organisms. Since most antimicrobials target the bacterial

cell cycle, it is of great importance to understand the fundamental steps of the cell cycle in different pathogens. We

are therefore studying how chromosome segregation is mediated in S. pneumoniae and how this process is connected

to DNA replication and cell division. We have constructed new tools for visualising different chromosomal locations

during the cell cycle. The tools are based on (i), a fluorescence repressor-operator system (FROS); and (ii), a ParB-parS

plasmid segregation system. Using these novel techniques we were able to follow the position and movement of several

chromosomal positions in S. pneumoniae simultaneously using (time-lapse) fluorescence microscopy. Furthermore,

functional fluorescent fusions to different replisome proteins were constructed to study how chromosome organisation

is coordinated with DNA replication. We show that the S. pneumoniae chromosome has a longitudinal organisation with

the origin region located closest to the old cell pole and the terminus region located closest to the new cell pole. The

origins split early after initiation of DNA replication and move to the quarter positions of the cells. Following replication,

the left and right arms of the chromosome move concomitantly but do not colocalise, suggesting that they are spatially

distinct entities. The terminus region stays at mid-cell until replication is finished and the septum is formed. High

temporal resolution imaging shows that the replication machinery is highly dynamic, but mainly localises in the mid-cell

area. Using these new tools, we show that the highly conserved SMC complex is pivotal for origin segregation. Together,

our data suggest that S. pneumoniae may be using DNA spooling by the centrally located replisome as a driving force for

chromosome segregation.

O2.5

Repressor of iron transport regulator (RitR) is a novel cysteine-activated

redox sensor in Streptococcus pneumoniae

David Wright 1 , Andrew Maule 2 , Lanlan Han 3 , Nicholas Silvaggi 3 , Nicholas Croucher 1 , Stephen

Bentley 4, 5 , Thomas Clarke 1 , Andrew Ulijasz 1

1

Imperial College London, London, UK; 2 University of Wisconsin, Madison, Wisconsin, USA; 3 University of Wisconsin, Milwaukee, Wisconsin, USA;

4

Wellcome Trust Sanger Institute, Hinxton, UK; 5 University of Cambridge, Cambridge, UK

Redox sensing is key to the survival of bacterial pathogens within their host. These sensors often take on the form of

a transcription factor that directly binds DNA and modulates RNA synthesis in response to environmental oxygen and

associated reactive oxygen species (ROS). During colonisation and infection, Streptococcus pneumoniae must sense and

navigate phagocyte-produced ROS, as well as withstand its own millimolar quantities of hydrogen peroxide it produces as

a metabolic by-product. One of the mysteries of this pathogen is how it is able to withstand such atrocities in the absence

of catalase and any of the typical known redox-sensing transcription factors (e.g. PerR and OxyR). Here we describe the

orphan two-component-like response regulator RitR as a bona fide redox-sensing transcription factor in this pathogen.

RitR has lost its canonical phosphorylatable aspartate and is instead, in part, controlled through phosphorylation by the

pneumococcal Penicillin Binding and Serine-Threonine Kinase Associated (PASTA)-containing serine-threonine kinase

StkP. StkP-dependent phosphorylation of a single and highly conserved RitR residue, Ser184 located with the DNA binding

domain, was shown to be necessary for resistance to both neutrophil attack and penicillin treatment. In addition, RitR

was found to possess a lone cysteine residue that responds specifically to hydrogen peroxide both in vitro and in vivo to

control iron transporter synthesis. Biochemical, genetic and atomic analyses point to RitR as being the archetype for a

new class of ROS-sensing proteins in the Streptococci, which in the pneumococcus maintains redox control in response

to hydrogen peroxide through novel inter-promoter cysteine-mediated dimerisation. We hypothesise that, through RitR,

the pneumococcus uses an integrated signalling system to control its redox state during nasopharyngeal colonisation

and infection in response to oxygen levels.

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O3.1

Cpl-711, a powerful enzyme against pneumococci

Roberto Díez-Martínez 1, 2 , Héctor D. de Paz 1, 6 , Esther García Fernández 1 , Noemí Bustamante 4, 3 ,

Chad W. Euler 2, 5 , Vincent A. Fischetti 2 , Margarita Menéndez 4, 3 , Pedro García 1, 3

1

Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain; 2 Laboratory of

Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, USA; 3 CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain;

4

Departamento de Química-Física Biológica, Instituto Química-Física Rocasolano, CSIC, Madrid, Spain; 5 Department of Medical Laboratory Sciences,

Hunter College, CUNY, New York, USA; 6 Infectious Diseases Research Unit, Department of Molecular Microbiology, Hospital Sant Joan de Deu, Barcelona,

Spain

The increase of multi-resistant pneumococcal strains has led to the proposal of using phage-encoded murein hydrolases as

a highly effective treatment against Streptococcus pneumoniae. Cpl-1 and Cpl-7 are lysozymes encoded by pneumococcal

lytic phages; Cpl-1 only degrades choline-containing pneumococcal cell walls, whereas Cpl-7 can recognise a broader

range of bacteria. It has also been demonstrated that a Cpl-7 variant (Cpl-7S), with a lower negative charge, has a

greater bactericidal activity than its parental enzyme. To construct new enzymes with improved bactericidal activity, we

have synthesised genes encoding novel chimeric enzymes from the combination of three structural elements (catalytic

module, linker and cell wall binding module) from Cpl-1 and Cpl-7S lysozymes. The enzymatic activity of these novel

proteins was analysed in vitro and validated in vivo using a bacteraemia mouse model. Cpl-711, a lysozyme harbouring

the catalytic module from Cpl-7S and linker and cell wall binding module from Cpl-1, substantially improved the killing

capacity of parental enzymes against pneumococci, including multi-resistant strains. Specifically, 5 µg/ml of Cpl-711 killed

≥7.5 logs of pneumococcal R6 strain, and substantially reduced biofilm formation. Mice injected intraperitoneally with

D39_IU strain were protected with a single injection of Cpl-711 administered 60 min later, with 50% greater protection

than with Cpl-1. Among the new chimeric enzymes constructed, Cpl-711 was the most powerful murein hydrolase to

kill pneumococcal strains. This result supports the possibility of synthesising improved lytic enzymes as a promising

therapeutic perspective for the treatment of multi-resistant pneumococcal infections.

O3.2

Esters of bicyclic amines: a new generation of antimicrobials against

pneumococcus

M. de Gracia Retamosa 1 , Roberto Diez-Martinez 2 , Beatriz Maestro 1 , Esther Garcia-Fernandez 2 , Bas

de Waal 3 , E. W. Meijer 3 , Pedro Garcia 2 , Jesus Sanz 1

1

University Miguel Hernandez, Elche, Spain; 2 Consejo Superior de Investigaciones Cientificas, Madrid, Spain; 3 Eindhoven University of Technology,

Eindhoven, The Netherlands

We had previously shown that certain esters of bicyclic amines (EBAs), such as atropine and ipratropium, are capable

of arresting pneumococcal growth in vitro [1]. These compounds behave as choline analogues that compete with the

phosphorylcholine residues in the cell wall for the binding of the choline-binding proteins (CBPs). However, too high

(tens of millimolar) concentrations are needed for these compounds, preventing their therapeutic use. To improve the

druggability of these compounds, we have followed a double approach. First, we used the information of previous

CbpF-atropine and CbpF-ipratropium crystal complexes [2] to rationally design a library of 48 EBA derivatives that were

screened by fluorescence spectroscopy methods for their interaction with the choline-binding module of the LytA

amidase (C-LytA protein). Best binders were then selected and assayed on pneumococcal in vitro cultures, monitoring

the bacterial growth, morphology and viability. The most effective EBA derivatives containing 2 or 3 aromatic rings

induced the lysis of the cells and showed a minimal inhibitory concentration in the micromolar range. On the other

hand, we have synthetised a dendrimeric nanoparticle (g2-dendropine) containing 8 copies of the otherwise poor binder

atropine. As a result of multivalency effects, g2-dendropine increases the affinity by 45,000-fold compared to monomeric

atropine, and shows CBP inhibition in nanomolar concentrations. To sum up, we have identified and synthetised a new

family of compounds (EBAs) that are effective in vitro against S. pneumoniae at therapeutically acceptable doses, and

demonstrated that multivalent display of candidate molecules on nanoparticles may highly increase its antimicrobial

capacity.

1. Maestro B, González A, García P, Sanz JM. Inhibition of pneumococcal choline-binding proteins and cell growth by esters of

bicyclic amines. FEBS J. 2007; 274, 364-376

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2. Silva-Martín N, Retamosa MG, Maestro B, Bartual SG, Rodes MJ, García P, Sanz JM, Hermoso JA. Crystal structures of CbpF

complexed with atropine and ipratropium reveal clues for the design of novel antimicrobials against Streptococcus pneumoniae.

Biochim. Biophys. Acta 2014; 1840, 129-135

O3.3

Identification of new cell wall biogenesis factors in Streptococcus

pneumoniae using Tn-Seq

Andrew Fenton, Thomas Bernhardt, David Rudner

Harvard Medical School, Boston, Massachusetts, USA

The bacterial cell wall represents the best clinically validated target for antimicrobials against Streptococcus pneumoniae.

The main enzymes that build the cell wall are synthases belonging to the family of factors called penicillin-binding

proteins (PBPs), which are the targets of penicillin and related beta-lactam drugs. Resistance to the beta-lactams is on

the rise in S. pneumoniae, necessitating the discovery of new antibiotics to treat pneumococcal infections. One attractive

avenue towards this goal is to identify new weak points in the cell wall assembly pathway that can be exploited for the

development of novel therapeutics. Identification of these vulnerabilities will require a more thorough understanding

of cell wall biogenesis, especially with regard to how the process is regulated. Importantly, although the structure and

activity of the PBPs have been studied for decades, surprisingly little is known about how these enzymes are controlled

in vivo. To address this issue, we have used Tn-Seq to identify genetic interactions with a subset of the major synthetic

PBPs in S. pneumoniae. Our analysis has uncovered a novel regulatory factor which plays an essential role in controlling

the cell wall synthetic machinery required for cell division in pneumococci. The discovery of this factor and an analysis

of its activity will be discussed along with the implications for drug discovery.

O3.4

Engineered liposomes sequester pneumolysin and protect from severe

invasive pneumococcal disease in mice

Daniel Neill 1 , Suzanna Gore 1 , Laura Bricio Moreno 1 , Eduard Babiychuk 2 , Aras Kadioglu 1

1

University of Liverpool, Liverpool, UK, 2 University of Bern, Bern, Switzerland

Pneumolysin is a cholesterol-dependent cytolysin which forms pores in the membrane of eukaryotic cells leading to

cellular death. Artificially created liposomes with high concentrations of lipids can be used as decoy targets to sequester

bacterial membrane-damaging toxins, such as pneumolysin. It was observed that in the presence of cholesterol-containing

liposomes, epithelial and endothelial cells were protected against pneumolysin-induced cell lysis. Liposomes were also

able to reduce pneumolysin-induced CXCL8 in HUVEC endothelial cells. In an in vivo model of invasive pneumococcal

disease liposomes were able to increase the survival of mice infected with D39 after a single dose of liposome, which

also reduced the bacterial load in lungs and blood at 24 hours post-infection. During a sepsis in vivo model where

mice infected with D39 typically die 33 hours post-infection, liposomes were able to protect against bacteraemia when

administered 6 or 10 hours post-infection, but not when administered 16 hours post-infection. Liposomes were also

able to reduce bacterial load and levels of TNF-α and pro-inflammatory recruitment of polymorphonuclear leukocytes

in blood. Antibiotic treatment against pneumococci can lead to release of pneumolysin due to bacterial lysis, with

substantial pro-inflammatory and pathogenic consequences to the host. A combination of antibiotic and liposomal toxinsequestration

treatment increased the survival of mice infected with D39 when compared to antibiotic only treated

groups, suggesting that adjunct therapy with liposomes could be a highly effective treatment against infections caused

by pathogens producing cholesterol-dependent cytotoxins.

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O4.1

DNA release by Streptococcus pneumoniae autolysin LytA induced

Krueppel-like factor 4 expression controlling pneumococci-related

innate immune response in macrophages

Janine Zahlten 1 , Toni Herta 1 , Christin Kabus 1 , Jan-Moritz Doehn 1 , Pedro García 2, 3 , Norbert Suttorp

1 , Stefan Hippenstiel 1

1

Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité, Universitätsmedizin Berlin, Berlin, Germany; 2 Departamento

de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; 3 CIBER de

Enfermedades Respiratorias, Madrid, Spain

In this study we investigated Streptococcus pneumoniae-dependent KLF4 induction in bone marrow derived macrophages

(BMMs). Since the S. pneumoniae-LytA mutant did not cause KLF4 expression, exogenous supplementation of S.

pneumoniae DNA restored KLF4 induction, and addition of DNAses blocked KLF4 induction, we considered S. pneumoniae

DNA to be an important trigger for KLF4 expression in BMMs. Experiments using TLR9-/- and MyD88-/- revealed that

these molecules were only partially involved in pneumococci-related KLF4 expression. Simultaneous incubation of BMMs

with S. pneumoniae-LytA mutant and DNA of other bacterial species, as well as with eukaryotic foreign (human) or self

(mouse) DNA, induced KLF4 expression. BMMs missing putative DNA receptors/adaptor molecules (ASC-/- and STING-/-)

did not reduce the S. pneumoniae-related KLF4 expression. Application of inactivated S. pneumoniae, separation of the

bacteria from the cells, and the inhibition of phagocytosis revealed that direct contact of viable bacteria to the host

cells—independent of phagocytosis—is essential for the induction of KLF4 in BMMs. These results lead us to conclude

that the KLF4 expression is—in addition to TLR9—mediated by a hitherto unknown DNA receptor. Since DNA alone

was not sufficient to induce KLF4 expression, we hypothesise that a second signal is probably needed (direct contact of

living bacteria). To investigate the function of KLF4 in macrophages we used BMMs from in vivo tamoxifen (txf) induced

Txf-Cre+/klf4loxP/loxP mice. The loss of KLF4 reduced S. pneumoniae-dependent KC and enhanced IL-10 secretion.

Therefore KLF4 expression displays a pro-inflammatory phenotype in macrophages and may therefore contribute to an

enhanced bacterial clearance in the lung.

O4.2

The pneumococcal whole cell vaccine reduces influenza-induced

pneumococcal disease in the ears and lungs of co-infected infant mice

Jayne Manning 1, 2 , Eileen Dunne 1 , Kim Mulholland 1, 3 , Roy Robins-Browne 1, 2 , Richard Malley 4 ,

Odilia Wijburg 2 , Catherine Satzke 1, 2

1

Murdoch Childrens Research Institute, Parkville, Australia; 2 University of Melbourne, Parkville, Australia; 3 London School of Hygiene and Tropical

Medicine, London, UK; 4 Boston Children’s Hospital, Boston, USA

The pneumococcal whole cell vaccine (WCV) was developed to provide serotype-independent protection and is currently

undergoing phase II clinical trials. In mice, WCV reduces pneumococcal density in the nasopharynx via production of IL-

17A by CD4 + Th17 cells and protects against invasive disease in an antibody-dependent manner. To investigate the effect

of WCV on mucosal pneumococcal disease we used an infant mouse model of influenza-induced pneumococcal otitis

media and pneumonia. Six-day-old C57BL/6 mice were administered either WCV (killed unencapsulated pneumococcal

strain RM200 absorbed to aluminium hydroxide adjuvant) or adjuvant alone via subcutaneous injection. Mice were

subsequently intranasally challenged with pneumococci (19F or 16F at 12 days old) before infection with influenza A

virus (Udorn/72 at 18 days old). Pneumococcal density in the nose, ears and lungs was measured 6 days later (24 days

old). WCV protection varied depending on the serotype tested: 19F density was significantly reduced in the ears, and

16F density was significantly reduced in the lungs of mice administered WCV compared to mice receiving adjuvant

alone. Preliminary data suggest that WCV-induced protection against 19F in the ears may involve peripheral CD4 + T

cells, since no difference between 19F density was observed between WCV and adjuvant-vaccinated C57BL/6.GK1.5

mice, who lack peripheral CD4 + T cells. This work provides evidence that vaccination with WCV can elicit immunemediated

protection against pneumococcal otitis media and pneumonia in a clinically relevant experimental model and

these data will inform future vaccine trials. We continue to investigate the immunological mechanisms of WCV-induced

protection by correlating levels of protection with levels of antigen-specific antibodies and cytokine responses, as well

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as characterising levels of inflammation in the ears and lungs.

O4.4

The effect of macrophage polarisation and delayed apoptosis in the

innate immune response to Streptococcus pneumoniae infection

Lucy Morris, Helen Marriott, David Dockrell

University of Sheffield, Sheffield, UK

Shifts in macrophage polarisation are associated with COPD pathogenesis. Macrophages undergo apoptosis when infected

with Streptococcus pneumoniae, a common cause of COPD exacerbations, as part of the innate immune response. This

apoptosis is reduced in alveolar macrophages from COPD patients associated with increased expression of the antiapoptotic

protein Mcl-1. We hypothesise that macrophages from transgenic mice that over-express Mcl-1 (CD-68 Mcl-

1) will demonstrate altered polarisation resulting in reduced host defence to pneumococcal infection. Our objectives

were to determine any differences in basal polarisation of wild-type and CD68 Mcl-1 BMDMs; explore phagocytosis,

bacterial killing and apoptosis in polarised wild type and CD68 Mcl-1 BMDMs and MDMs after S. pneumoniae infection;

and understand how cytokine and chemokine repertoire changes in response to S. pneumoniae infection in polarised

wild-type and CD68 Mcl-1 BMDMs and MDMs. Bone marrow derived macrophages (BMDM) from Mcl-1-transgenic

and wild-type littermates were stimulated for 24 hours with cytokines; IL-4 (M2a), IL-10 (M2c) or IFNγ+LPS (M1) and

polarisation determined by western blotting, ELISA and RT-PCR. Polarised macrophages were infected with S. pneumoniae

for defined times and bacterial internalisation and survival assessed. Nuclear fragmentation of mock-infected versus

infected macrophages was quantified by staining with DAPI. Supernatants were collected and assessed for cytokine

expression by ELISA. Cell viability and hypodiploid DNA were assessed by MTT assays and PI staining, respectively. M1

polarisation caused increased levels of hyplodiploid DNA and decreased cell viability compared to other polarisation

states. Assessment of S. pneumoniae internalisation and intracellular survival over a 2–4 hour time course showed

increased bacterial clearance by M1 wild-type and transgenic macrophages. There was also increased apoptosis of M1

macrophages after S. pneumoniae infection 16 hours onwards. M1 polarisation increased proinflammatory cytokine

repertoire in BMDMs after S. pneumoniae infection, however this effect was not replicated in MDMs.

O4.5

Murine respiratory tract microbiome: important interactions with IL-17

and pneumococcal colonisation

Neil Ritchie, Tom Evans

University of Glasgow, Glasgow, UK

IL-17 is known to be important in the control of nasal colonisation with Streptococcus pneumoniae. The nasal microbiome

is a rich environment with a diverse range of bacterial species; the effect of IL-17 on the respiratory tract microbiome

is undefined. We characterised the respiratory tract microbiome of wild type and IL-17RAKO mice before and after

induction of colonisation with S. pneumoniae. Mice were inoculated with 5 x 10 5 cfu of serotype 3 S. pneumoniae.

Nasal and bronchoalveolar lavages were taken and S. pneumoniae quantified. DNA was extracted and V 1

-V 2

region

of the 16S rRNA gene was amplified using barcoded primers. Sequencing was carried out using the Illumina Mi-Seq

platform. On culture, S. pneumoniae achieved higher nasal colonisation density in IL-17RAKO mice. IL-17RAKO mice

had a lower abundance of neutrophils within nasal wash. The resting microbiome of IL-17RAKO mice was significantly

different from wild-type, with decreased diversity and increased abundance of proteobacteria. There was no difference

in the overall abundance of firmicutes (such as S. pneumoniae), although the diversity of firmicutes was significantly

lower in IL-17RAKO mice. The lower airways had much lower bacterial diversity than the nose and the microbiome

between IL-17RAKO and wild-type was similar. Nasal colonisation with S. pneumoniae was associated with a marked

loss of diversity in wild-type mice but little change in diversity among IL-17RAKO. This loss of diversity occurred even in

mice that did not have a high abundance of S. pneumoniae. Microbiome sequencing confirmed the higher abundance

of pneumococcus in nasal samples among IL17RAKO mice and showed a strong negative correlation with Ochrobactrum

anthropi, a nasopharyngeal commensal, suggesting intra-species competition for the same ecological niche. IL-17 is an

important cytokine in defining the resting nasal microbiome and contributes to control of pneumococcal colonisation.

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O5.1

Zinc homeostasis in Streptococcus pneumoniae during disease

Bart Eijkelkamp, Charles Plumptre, Jacqueline Morey, Stephanie Begg, James Paton, Christopher

McDevitt

University of Adelaide, Adelaide, SA, Australia

Acquisition of zinc by Streptococcus pneumoniae is essential for colonisation and virulence. Zinc uptake in S. pneumoniae

occurs via the ATP-binding cassette transporter AdcCB, and two zinc-binding proteins, AdcA and AdcAII. We have

previously shown that in vivo, AdcA and AdcAII act in a complementary manner during host colonisation to facilitate

a more efficient infection. We have also identified that AdcAII is reliant upon the pneumococcal histidine triad (Pht)

proteins to aid in zinc recruitment. Although zinc is scarce during early stages of infection, the host appears to induce

the release of zinc during disease progression, which has detrimental effects on the pneumococcus. Therefore, the

pneumococcus utilises the zinc efflux protein CzcD to efficiently reduce intracellular zinc concentrations under zinc

stress. Here we show that the pneumococcus utilises glutathione as an additional zinc homeostasis mechanism. Zinc

is unlikely to be present as a free metal ion in the cell and thus buffering is required. Our data shows zinc buffering is

achieved using reduced thiol groups on the peptide glutathione. Either under high extracellular zinc pressure, or high

intracellular zinc concentrations achieved by deletion of czcD, we show that the pneumococcus requires glutathione. We

are currently examining the role of the concerted mode of action of these three distinct zinc homeostasis mechanisms

using a zinc-deficiency animal model. Data collected thus far has revealed which host niches are affected by the dietary

intervention and what consequences this has on pneumococcal disease progression.

O5.2

The pneumococcal MgaSpn virulence transcriptional regulator

Virtu Solano-Collado 1 , Rudi Lurz 2 , Manuel Espinosa 1 , Alicia Bravo 1

1

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; 2 Max-Planck-Institut für molekulare Genetik,

Berlin, Germany

In Gram-positive pathogenic bacteria, regulation of virulence gene expression often rely on global response regulators

that are not associated to a membrane-bound histidine kinase. To this class of regulators belong the Mga and AtxA proteins

from Streptococcus pyogenes and Bacillus anthracis, respectively. The activity of both regulators seems to be controlled

by the phosphoenolpyruvate:carbohydrate phosphotransferase system. The MgaSpn protein from S. pneumoniae is likely

a member of the Mga/AtxA family of global regulators. It plays a significant role in both nasopharyngeal colonisation and

development of pneumonia in murine infection models. We have demonstrated that MgaSpn acts directly as a positive

transcriptional regulator. It activates the expression of a 4-gene operon from the P1623B promoter. This activation

requires a region (PB activation region) that is located upstream of the promoter. We have constructed transcriptional

fusions based on the P1623B promoter and the green fluorescence protein (gfp) reporter gene. These gene fusions are

suitable to assess the activity of MgaSpn when pneumococcal cells grow in the presence of particular carbon sources.

Using an untagged form of the MgaSpn protein, we have identified two MgaSpn binding sites (sites I and II). Site I is

located within the PB activation region, whereas site II overlaps the promoter of the mgaSpn gene. These sites have a low

sequence identity and contain a potential intrinsic curvature. On binding to the primary site, MgaSpn is able to spread

along the adjacent DNA regions generating multimeric protein-DNA complexes, as it is the case of the H-NS nucleoidassociated

protein from enteric bacteria. Our results suggest that a preference for particular DNA conformations might

contribute to the capacity of MgaSpn to control the expression of a wide range of genes.

Funded by Spanish MINECO (Grants CSD-2008-00013 and BIO2013-49148-C2-2-R)

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O5.3

Genomic analyses of pneumococci reveal a wide diversity of

bacteriocins, including pneumocyclicin—a novel circular bacteriocin

Carlijn Bogaardt, Andries van Tonder, Angela Brueggemann

University of Oxford, Oxford, UK

Pneumococci reside in the paediatric nasopharynx, where they compete for space and resources. One competition

strategy is to produce a bacteriocin (antimicrobial peptide or protein) to attack other bacteria and an immunity protein to

protect against self-destruction. Understanding the dynamics of competition is important in the context of understanding

how perturbations such as vaccine introduction affect the pneumococcal population structure and thus the competition

in the nasopharynx. The aims of this study were to: i) provide a detailed characterisation and comparison of the blp

bacteriocin cassettes from a large and diverse set of historical and modern pneumococcal genomes; ii) investigate

cassette diversity in the context of the pneumococcal population structure; and iii) investigate the genetic stability of

blp cassettes over time. We analysed a collection of 336 diverse pneumococcal genomes dating from 1916 onwards,

identified bacteriocin cassettes, detailed their genetic composition and sequence diversity, and evaluated the data in

the context of the pneumococcal population structure. We found that all genomes maintained a blp bacteriocin cassette

and we identified several novel blp cassettes and genes. The composition of the ‘bacteriocin/immunity region’ of the blp

cassette was highly variable: 1 cassette possessed 6 bacteriocin genes and 8 putative immunity genes, whereas another

cassette had only 1 of each. Both widely distributed and highly clonal blp cassettes were identified. Most surprisingly,

one-third of pneumococcal genomes also possessed a cassette encoding a novel circular bacteriocin that we called

pneumocyclicin, which shared a similar genetic organisation to well-characterised circular bacteriocin cassettes in

other bacterial species. Pneumocyclicin cassettes were mainly of 1 genetic cluster and largely found among 7 major

pneumococcal clonal complexes. These detailed genomic analyses revealed a novel pneumocyclicin cassette and a wide

variety of blp bacteriocin cassettes, suggesting that competition in the nasopharynx is a complex biological phenomenon.

O5.4

Antibiotic-induced bacteriocin expression—regulatory interplay

between the blp and com systems in Streptococcus pneumoniae

Morten Kjos 1 , Eric Miller 2, 3 , Frank Lake 1 , Daniel E. Rozen 2 , Jan-Willem Veening 1

1

University of Groningen, Groningen, The Netherlands; 2 Leiden University, Leiden, The Netherlands; 3 The University of Manchester, Manchester, UK

Exposure to antibiotics changes global gene expression and can induce the competent state in Streptococcus pneumoniae.

Using RNA-sequencing, we found that exposure to competence-inducing antibiotics caused upregulation of the

ubiquitous bacteriocin blp gene-cluster in S. pneumoniae D39. This strain contains a blp-cluster with 5 transcriptional units

controlled by the promoters P blpS

(controlling the regulatory system blpSRH), P blpA

(ABC-transporter genes and inducer

peptide BlpC), Pblp U1

(possible bacteriocin and immunity genes) and 2 operons of unknown function (P blpT

and P SPD_0046

).

blp-promoter activity is regulated by a quorum sensing mechanism via accumulation of BlpC. In order to understand

how antibiotics control blp expression, we made constructs in which all 5 blp promoters were fused to a novel tripartite

reporter cassette containing luciferase (luc), gfp and lacZ. Real-time gene expression measurements showed that while

P blpS

was constitutive, expression from the remaining blp-promoters was induced in the late exponential phase after

competence has initiated, and occurred in all cells. Induction of blp-expression was also pH-dependent and strictly

dependent on the presence of the blpSRHC regulatory genes. Since both blp-expression and competence are induced

by the same antibiotics, are pH-dependent, and are regulated by similar quorum sensing mechanisms, we studied the

interplay between these two systems. Exposure to external competence stimulating peptide (CSP) resulted in a dual

response; immediately upon addition of CSP, the blp-promoters were weakly upregulated, and after a delay the system

was highly induced. Similar to the majority of S. pneumoniae strains, the putative exporter for BlpC, blpAB is not intact

in the D39 strain, but the strain does contain an intact ABC transport system for CSP, comAB. Strikingly, we show that

blp-activation requires comAB, explaining why natural blp-induction is dependent on competence and thus induced by

several antibiotics. We discuss the potential ecological and evolutionary ramifications for this intertwined regulatory

system.

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O5.5

Conserved Streptococcus pneumoniae spirosomes point toward a

single type of transformation pilus in competence

Petya V. Krasteva 1, 2 , Raphael Laurenceau 1, 2 , Amy Diallo 1, 2 , Sahra Ouarti 1, 2 , Magalie Duchateau 1, 2 ,

Christian Malosse 1, 2 , Julia Chamot-Rooke 1, 2 , Remi Fronzes 1, 2

1

Institut Pasteur, Paris, France; 2 CNRS UMR3528, Paris, France

The success of Streptococcus pneumoniae as a major human pathogen is largely due to its remarkable genomic plasticity,

allowing efficient escape from antimicrobials action and host immune response. Natural transformation, or the active

uptake and chromosomal integration of exogenous DNA during the transitory differentiated state competence, is the

main mechanism for horizontal gene transfer and genomic makeover in pneumococci. DNA uptake requires expression

of a transformation pilus but 2 markedly different models for pilus assembly and function have been proposed. We

previously reported a long, Type 4 pilus-like appendage on the surface of competent pneumococci that likely binds

extracellular DNA as initial receptor, while a separate study proposed that secreted short, ‘plaited’ transformation pili

act simply as peptidoglycan drills to open DNA gateways. Here we show that the ‘plaited’ structures are not competencespecific

or related to transformation. We further demonstrate that these are macromolecular assemblies of the metabolic

enzyme acetaldehyde-alcohol dehydrogenase—or spirosomes—broadly conserved across the bacterial kingdom.

O6.1

Pathogenesis of non-encapsulated Streptococcus pneumoniae in

experimental otitis media

Larry McDaniel

University of Mississippi Medical Center, Jackson, MS, USA

Otitis media (OM) is the most common reason for paediatrician visits and can result in hearing loss. Streptococcus

pneumoniae (pneumococcus) remains an important cause of OM. The diverse pneumococcal polysaccharide capsule

types have long been thought to be necessary for colonisation and disease. With the increase use of pneumococcal

conjugate vaccines (PCVs), there have been increased reports of pneumococcal disease caused by nonencapsulated

S. pneumoniae (NESp). We have previously demonstrated that pneumococcal surface protein K (PspK) replaces the

capsule locus of some NESp isolates and plays a role in virulence. Additional NESp have been identified in which the

cps genes have been replaced by genes encoding AliC and AliD. Understanding how NESp are able to cause infections

is important because current pneumococcal vaccines are only effective against encapsulated strains. Thus, the purpose

of this study was to further evaluate the role of these NESp proteins in virulence. We used a murine colonisation model

and a chinchilla model of OM for in vivo studies. In addition to pathology, pneumococcal burden was determined in

each model. Human epithelial cell lines were utilised to assess NESp adhesion and invasion. In vitro assays examining

biofilm production and viability were also performed. Significant differences in epithelial cell adhesion and invasion were

strain dependent. An increase in in vitro biofilm viability and production was observed in deletion mutants. We also

demonstrated that PspK, AliC, and AliD enhanced murine nasopharyngeal colonisation and were required for OM in the

chinchilla. Historically, NESp have been considered to be comparatively avirulent. We have demonstrated that virulence

of NESp is increased as a result of PspK, AliC, and AliD. Further study of this disease causing pneumococcal population

is essential.

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O6.2

Modulation of nasopharyngeal innate defences by viral co-infection

predisposes individuals to experimental pneumococcal carriage

Sarah Glennie 1, 6 , Jenna F Gritzfeld 1 , Shaun H Pennington 1 , Michael Garner-Jones 1 , Nicholas

Coombes 1 , Mark J Hopkins 2 , Cintia F Vadesilho 3 , Eliane N Miyaji 3 , Duolao Wang 4 , Angela D

Wright 5 , Andrea M Collins 5 , Stephen B Gordon 1 , Daniela M Ferreira 1

1

Respiratory Infection Group, Liverpool School of Tropical Medicine, Liverpool, UK; 2 Liverpool Specialist Virology Centre, Royal Liverpool and Broadgreen

University Hospital, Liverpool, UK; 3 Centro de Biotecnologia, Instituto Butantan, Sao Paulo, Brazil; 4 Tropical Clinical Trial Unit, Liverpool School

of Tropical Medicine, Liverpool, UK; 5 NIHR Royal Liverpool and Broadgreen University Hospital NHS Trust, Liverpool, UK; 6 School of Cellular and

Molecular Medicine, University of Bristol, Bristol, UK

Secondary pneumococcal pneumonia is a major cause of mortality during pandemic influenza. Increased nasopharyngeal

colonisation density has been associated with pneumonia. We used an experimental human pneumococcal carriage

model to investigate whether upper respiratory tract viral infection predisposes individuals to carriage. One hundred

and one healthy subjects were screened for respiratory virus prior to pneumococcal intranasal challenge. Virus was

associated with increased likelihood of colonisation (75% virus positive became colonised versus 46% virus negative

subjects; p = 0.02). Nasal Factor H (FH) levels were increased in virus positive subjects and were associated with increased

colonisation density. Using an in vitro epithelial model we explored the impact of increased mucosal FH in the context of

co-infection. Epithelial inflammation and FH binding resulted in increased pneumococcal adherence to the epithelium.

Binding was partially blocked by antibodies targeting the FH-binding protein pneumococcal surface protein C (PspC).

PspC epitope mapping revealed individuals lacked antibodies against the FH binding region. We propose that FH binding

to PspC in vivo masks this binding site enabling FH to facilitate pneumococcal/epithelial attachment during viral infection

despite the presence of anti-PspC antibodies. We propose that a PspC-based vaccine lacking binding to FH could reduce

pneumococcal colonisation especially in those with underlying viral infection. We will now evaluate the effect of the live

attenuated influenza vaccine on pneumococcal carriage susceptibility and carriage density as well as the immunological

mechanisms involved.

Acknowledgments: Bill and Melinda Gates Foundation, National Institute for Health Research (NIHR) Local Comprehensive

Research Network, and the Medical Research Council/Sao Paulo Research Foundation (FAPESP).

O6.3

Circulating pneumolysin is a potent inducer of cardiac injury during

pneumococcal infection

Daniel Neill, Yasir Alhamdi, Simon Abrams, Hesham Malak, Richard Barrett-Jolley, Gouzheng

Wang, Cheng-Hock Toh, Aras Kadioglu

University of Liverpool, Liverpool, UK

Streptococcus pneumoniae accounts for more deaths worldwide than any other single pathogen through diverse disease

manifestations including pneumonia, sepsis, and meningitis. Life-threatening acute cardiac complications are more

common in pneumococcal infection compared to other bacterial infections. Distinctively, these arise despite effective

antibiotic therapy. Here, we describe a novel mechanism of myocardial injury, which is triggered and sustained by

circulating pneumolysin (PLY). Using a mouse model of invasive pneumococcal disease (IPD), we demonstrate that wildtype

PLY-expressing pneumococci but not PLY-deficient mutants induced elevation of circulating cardiac troponins (cTns),

which are well-recognized biomarkers of cardiac injury, which was significantly attenuated by PLY-sequestering liposomes.

Furthermore, elevated cTn levels linearly correlated with pneumococcal blood counts and levels were significantly

higher in non-surviving than in surviving mice. Intravenous injection of purified PLY, but not a non-pore forming mutant

(PdB), induced substantial increase in cardiac troponins suggesting that the membrane-binding and pore-forming

activities of circulating PLY are essential for myocardial injury in vivo. Purified PLY and PLY-expressing pneumococci

also caused myocardial inflammatory changes but apoptosis was not detected. Exposure of cultured cardiomyocytes

to PLY-expressing pneumococci caused dose-dependent cardiomyocyte contractile dysfunction and death, which was

exacerbated by further PLY release following antibiotic treatment. We found that high PLY doses induced extensive

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cardiomyocyte lysis, but more interestingly, sub-lytic PLY concentrations triggered profound calcium influx and overload

with subsequent membrane depolarisation and progressive reduction in intracellular calcium transient amplitude, a

key determinant of contractile force. This was coupled to activation of signalling pathways commonly associated with

cardiac dysfunction in clinical and experimental sepsis and ultimately resulted in depressed cardiomyocyte contractile

performance along with rhythm disturbance. Our study proposes a detailed molecular mechanism of pneumococcal

toxin-induced cardiac injury and highlights the major translational potential of targeting circulating PLY to protect against

cardiac complications during pneumococcal infections.

O6.4

Streptococcus pneumoniae invades the heart during severe pneumonia

in a non-human primate model

Luis Reyes 1, 4 , Nilam Soni 1, 2 , Cecilia Hinojosa 1 , Jessica Perry 4 , Robert Shade 4 , Melissa de la Garza 4 ,

Luis Giavedoni 4 , Marcos Restrepo 1, 2 , Carlos Orihuela 1

1

The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA; 2 South Texas Veteran Health Care System, San Antonio,

Texas, USA; 3 Universidad de la Sabana, Bogota, Colombia; 4 Southwest National Primate Research Center, Texas Biomedical Research Institute, San

Antonio, Texas, USA

Approximately 20% of adults hospitalised for pneumococcal pneumonia experience an adverse cardiac event. We have

recently made the observation that Streptococcus pneumoniae translocates into the hearts of mice during experimental

invasive pneumococcal disease; this was concomitant with development of altered cardiac electrophysiology. The extent

to which this occurs in humans and its impact on heart function remains unclear. The goal of this project was to determine

whether S. pneumoniae invades the heart of non-human primates during pneumonia. Male 12–13-year-old baboons (n

= 3) were tethered to allow continuous electrocardiogram, heart rate, temperature monitoring, and blood sampling.

Anesthetised baboons were infected intratracheally with S. pneumoniae serotype 4 strain TIGR4 (3.3-3.5 x 10 8 CFU). Daily

follow-up included full blood exams, quantitation of bacterial burden in the blood, and serum cytokine levels. Baboons

were sacrificed when they developed a moribund state or after 10 days. Fluorescent microscopy using antibody against

capsular polysaccharide was used to detect pneumococci within the heart. Cardiac damage was assessed by histological

examination of cardiac sections. Two baboons developed pneumonia with a sustained high-grade fever, tachycardia and

bacteraemia. Infection was characterised by initial leukocytosis followed by a severe leukopenia after day 3. One animal

developed severe disease and succumbed to the infection. Cytokine analysis showed severe inflammatory reactions

proportional to disease severity. Cardiomyopathy in particular, characterised by vacuolisation within cardiomyocytes,

was observed in heart sections from baboons with pneumonia. Pneumococci alone and in clusters were detected within

the myocardium. Unspecific ischemic alterations in the ECG and in the pre-mortem echocardiogram were observed.

During pneumonia in non-human primates, the pneumococcus is able to invade the heart. The extent of bacteria

detected in the heart and degree of cardiomyopathy was positively correlated with severity of disease. These findings

suggest similar events may occur in humans.

O6.5

Inflammation dampening effects of pneumolysin

Jimstan Periselneris, Tracey Pollard, Thomas James, Mahdad Noursadeghi, Jeremy Brown

University College London, London, UK

Streptococcus pneumoniae interactions with alveolar macrophages are important for protective inflammatory responses

during early lung infection. Pneumolysin is a well-recognised virulence factor for S. pneumoniae that has multiple effects

on the host immune response that are primarily thought to be pro-inflammatory. We have investigated the effects

of pneumolysin on the early macrophage response to S. pneumoniae using in vitro culture and an animal model of

early pneumonia. Wild-type and non-haemolytic purified pneumolysin induced dose dependent inflammatory cytokine

release from human monocyte derived macrophages (MDMs), as expected. However, in contrast, when MDMs were

incubated with TIGR4 S. pneumoniae, higher levels of TNF and IL6 mRNA and protein were induced in response to

pneumolysin deficient bacteria than wild-type. Transcriptome analysis of MDMs after S. pneumoniae infection confirmed

increased expression of a range of pro-inflammatory genes in response to the pneumolysin mutant. Despite differential

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cell death in response to wild-type and pneumolysin deficient TIGR4, inhibition of apoptosis or the inflammasome also

had no effect on the early MDM cytokine response. Instead, the increase in transcription of pro-inflammatory genes

and TNF and IL6 supernatant levels in response to the pneumolysin deficient strain were abrogated by inhibition of S.

pneumoniae phagocytosis with cytochalasin D. In a murine model of early pneumonia despite more rapid clearance

of the pneumolysin mutant from BALF and lung compared to wild-type TIGR4 at 4 and 24 hours, BALF leukocyte

recruitment was increased at 4 hours. These data indicate an unexpected role for pneumolysin as an initial suppressor of

macrophage inflammatory responses, which is dependent on phagocytosis. The early inflammation dampening effects

of pneumolysin released within the phagolysosome may be an important contribution to S. pneumoniae virulence,

allowing increased bacterial replication early during the course of infection.

O6.6

A phase-variable genetic switch regulates pneumococcal virulence via

epigenetic changes

Ana Manso 1 , Chai Melissa 3 , John Atack 4 , Leonardo Furi 1, 2 , Megan De Ste Croix 1 , Richard Haigh 1 ,

Claudia Trappetti 3 , David Ogunniyi 3 , Lucy Shewell 4 , Mathew Boitano 5 , Tyson Calrke 5 , Jonas Korlach

5 , Matt Blades 1 , Evgeny Mirkes 1 , Alexander Gorban 1 , James Paton 3 , Mike Jennings 4 , Marco

Oggioni 1, 2

1

University of Leicester, Leicester, UK; 2 Università di Siena, Siena, Italy; 3 University of Adelaide, Adelaide, Australia; 4 Griffith University, Southport,

Australia; 5 Pacific Biosciences, Menlo Park, USA

Switching between phenotypic forms (or ‘phases’) that favour asymptomatic carriage or invasive disease has long been

recognised in pneumococci, but the molecular mechanisms associated to this phase variation had been not elucidated.

Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I

restriction-modification (RM) system SpnD39III. The rearrangements in the target recognition domains of the specificity

subunit generate 6 different RM enzymes with 6 alternative specificities and distinct methylation patterns, as defined

by single-molecule real-time methylomics. We constructed a series of mutants which express only a single SpnD39III

variant and have shown that these strains show distinct gene expression profiles. We demonstrate distinct virulence

in experimental infection using both locked mutant strains and WT strains and identified clear in vivo selection for

switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology.

Investigation of other strains confirmed that any pneumococcal strain is composed of subpopulations with distinct

epigenetic methylation profile of their chromosome. Future studies will have to recognise the potential for switching

between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo.

O7.1

Pneumococcal conjugate vaccine reduces the rate, density, and

duration of experimental human pneumococcal colonisation: first

human challenge testing of a pneumococcal vaccine—a double blind

randomised controlled trial

Jenna Gritzfeld 1 , Andrea Collins 1, 2 , Angela Wright 1, 3 , Elena Mitsi 1 , Carole Hancock 2 , Shaun Pennington

1 , Duolao Wang 1 , Ben Morton 1 , Daniela Ferreira 1 , Stephen Gordon 1

1

Liverpool School of Tropical Medicine, Liverpool, UK; 2 Royal Liverpool and Broadgreen University Hospital Trust, Liverpool, UK; 3 Local

Comprehensive Research Network, Liverpool, UK

New vaccines are urgently needed to protect the vulnerable from bacterial pneumonia. Studies of pneumococcal vaccine

efficacy against colonisation have been proposed as an efficient and cost-effective method to down select between

vaccine candidates. We have developed a safe and reproducible experimental human pneumococcal colonisation

(EHPC) model; here we assess the impact of the 13-valent pneumococcal conjugate vaccine (PCV) Prevenar 13 ® on

experimental pneumococcal colonisation to validate whether the model can be used to test novel vaccines. One

hundred healthy participants aged 18–50 years were recruited to a double blind randomised placebo-controlled trial.

They were randomly assigned to PCV (n = 49) or Hepatitis A (control, n = 50) vaccination and then inoculated with

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80,000 CFU/100µl of Streptococcus pneumoniae (6B) per naris. Participants were followed for 21 days to determine

pneumococcal colonisation by culture of nasal wash. The PCV group had a significantly reduced 6B colonisation rate

(10% [5/48]) compared to the control group (48% [23/48]) ([Risk Ratio 0.22 [CI 0.09–0.52]; p < 0.001). Both density

and duration of colonisation were reduced in the PCV compared to the control group following inoculation. The area

under the curve (density versus day) was significantly reduced in the PCV compared to control group (geometric mean

259 versus 11,183, p = 0.017). PCV reduced pneumococcal colonisation rate, density, and duration in healthy adults.

The EHPC model is a safe and efficient method to determine the protective efficacy of new vaccines on pneumococcal

colonisation; PCV provides a gold standard against which to test these novel vaccines.

O7.2

Heterologous protection against Streptococcus pneumoniae

colonisation by the mucosal adjuvant cholera toxin subunit B

Kirsten Kuipers, Dimitri Diavatopoulos, Corné van den Kieboom, Fred van Opzeeland, Elles Simonetti,

Dorette van Ingen-Schenau, Mariska Kerstholt, Malgorzata Borczyk, Mihai Netea, Marien

de Jonge

Radboud University Medical Center, Nijmegen, The Netherlands

For decades, cholera toxin subunit B (CTB) has been used as the benchmark mucosal adjuvant in experimental vaccine

research and is the most effective and most widely applied mucosal adjuvant to date. Recent studies showed that some

vaccines induce an innate memory response. While there is some indirect evidence that CTB alone is able to protect

against mucosal pathogens, this has never been systematically studied in vaccination experiments. Here, we show that

intranasal CTB immunisation resulted in protection against Streptococcus pneumoniae, measured as a transient reduction

in bacterial load. CTB immunisation increased local expression of CD3, IFN-γ and IL-10 in nasal tissue, suggestive of both

Th1 and Th2/Treg responses. Remarkably, a single CTB immunisation caused a strong influx of T cells and neutrophils and

a minor increase in macrophages towards the nasal cavity. To establish non-specific protection, CTB required the GM-1

ganglioside receptor, as protective effects were abrogated upon treatment with anti GM-1 antibodies. Additionally,

protection was absent in SCID -/- and CCR2 -/- mice, pointing to a complementary role for T cells and macrophages in this

process. Strikingly, the CTB-mediated reduction of bacterial load appeared directly regulated by resident macrophages,

as local depletion of resident macrophages, but not neutrophils, diminished protection against S. pneumoniae carriage.

To our knowledge, we report for the first time that CTB alone recruits immune cells to the nasopharyngeal niche and

heterologously protects against S. pneumoniae colonisation. In vivo experiments are ongoing to assess the role of

the inflammasome. Insight into the in vivo effects of intranasally delivered CTB on bacterial carriage may accelerate

development of effective mucosal adjuvants.

O7.3

In vivo efficacy of recombinant protein polysaccharide conjugate

pneumococcal vaccines

Jenny Herbert 1 , Emily Kay 2 , Jon Cuccui 2 , Vanessa Terra 2 , Brendan Wren 2 , Timothy Mitchell 1

1

University of Birmingham, Birmingham, UK; 2 London School of Hygiene and Tropical Medicine, London, UK

Pneumonia is the largest killer of children under the age of 5, more than AIDS, malaria and tuberculosis combined. The

pneumococcus is the leading cause of bacterial pneumonia in children, but can also cause disease in the elderly and

immunocompromised. Prevnar 13 ® , a protein polysaccharide conjugate vaccine, is currently used in children to prevent

invasive pneumococcal disease. This vaccine protects against 13 of the pneumococcal capsular types found most

prevalent in disease. Limitations of this vaccine are the high cost, limiting the availability of the vaccine in the developing

world. Further phenomena such as serotype replacement and capsular switching limit the use of this vaccine in the long

term. Recent advances in understanding protein glycosylation in bacteria have paved the way for development of new

protein glycan coupling technology [1]. This technology is based on a well defined glycosylation system in Campylobacter

jejuni in which PglB an oligosaccharyltransferase natively couples an oligosaccharide to a number of campylobacter

proteins. PglB, due to its relaxed glycan specificity, can be used to couple a number of polysaccharides to any carrier

protein that contains a glycotag sequence. Use of this technology has enabled the coupling of a chosen carrier protein

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to pneumococcal type 4 polysaccharide to be done in Escherichia coli, rather than the traditional chemical coupling.

This technology could potentially revolutionise the way protein polysaccharide conjugate vaccines are manufactured.

Our current research aims to evaluate the efficacy of these new recombinant pneumococcal protein polysaccharide

conjugate vaccines against that of the currently licensed vaccine (Prevnar 13 ® ) using a mouse model of pneumonia. Data

will be presented showing that a glycoconjugate vaccine produced in E.coli can elicit protection in a mouse model of

pneumonia.

1. Terra VS, Mills DC, Yates LE, Abouelhadid S, Cuccui J, Wren BW. Recent developments in bacterial protein

glycan coupling technology and glycoconjugate vaccine design. J Med Microbiol 2012;61:919–26.

PMID:22516134 http://dx.doi.org/10.1099/jmm.0.039438-0

O7.4

Immune responses to pneumococcal vaccination in HIV-infected adults

in the UK

Sian Faustini 1, 2 , James Hodson 2 , Sindiso Masuko 2 , Mebie Singo 2 , Joyful Chigiga 2 , Jenny Herbert 3 ,

Timothy Mitchell 3 , Tim Plant 1 , Mark Drayson 1 , Kaveh Manavi 2 , Calman MacLennan 1 , Alex

Richter 1

1

Clinical Immunology Service, School of Immunity and Infection, University of Birmingham, Edgbaston, UK; 2 HIV Service, Queen Elizabeth Hospital

Birmingham, Edgbaston, UK; 3 Institute of Microbiology and Infection, School of Immunity and Infection, Biosciences, University of Birmingham,

Edgbaston, UK

Streptococcus pneumoniae is a major cause of disease in HIV-1 infected individuals. Pneumovax (PPV-23), a pure

polysaccharide vaccine, is currently recommended for routine vaccination of HIV-infected adults (BHIVA, 2008) but its

efficacy in this cohort is yet to be determined. AIR (assessment of immune responses to routine immunisations) is a

collaboration between the HIV services at the Queen Elizabeth Hospital, Birmingham and Clinical Immunology Services,

University of Birmingham, that has studied the immune response to UK recommended vaccinations in adults with HIV.

The AIR study found that approximately 50% of patients respond to PPV-23, as assessed by pre- and post- vaccine IgG

antibodies against 12 pneumococcal serotypes (Pn 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) at the WHO protective

threshold 0.35 µg/ml in ≥ 8/12 serotypes threshold using a 19-plex luminex-based assay (manuscript submitted). AIR

went on to examine whether Prevenar 13 ® (PCV-13), a polysaccharide-conjugate vaccine, could increase the percentage

of protected patients: 148 HIV-infected adults were vaccinated with PPV-23 and compared with 197 vaccinated with

PCV-13. PCV-13 IgG responses were at least equivalent to PPV-23 and increased for Pn 3 (p = 0.05), 9V (p = 0.01), 19A

(p = 0.003) and 19F (p = 0.01), also assessed by a multiplexed opsonophagocytic killing assay. Response rates could be

further improved by a booster dose of PCV-13. Patients who had first received PPV-23 and were not protected were

eligible to receive PCV-13 which generated a response that was lower than when PCV-13 was given alone, consistent

with published findings of hyporesponsiveness to polysaccharide vaccines. However, the hyporesponsiveness could be

overcome with a booster dose of PCV-13. PCV-13 and PPV produce similar IgG responses in HIV-infected patients but

the PCV-13 can be boosted, unlike that of PPV. This prospective study finding requires confirmation in a randomised

controlled trial.

O7.5

The agglutinating effects of anti-capsular antibody contribute to

pneumococcal clearance

Aoife Roche, Jeffrey Weiser

University of Pennsylvania, Philadelphia, PA, USA

A major benefit of the pneumococcal conjugate vaccine has been attributed to herd immunity, resulting from its ability

to decrease transmission by blocking the acquisition of colonisation. This mucosal protection against colonisation

correlates with increased serum anti-capsular antibody titres generated by vaccine. We have used a murine model

of Streptococcus pneumoniae colonisation to study the mechanism of mucosal protection by antibody. We previously

reported that mice passively immunised with anti-capsular antibody were protected from acquisition of colonisation,

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and IgG detected on the mucosa at the time of nasal challenge was sufficient to block acquisition. Protection correlated

with the agglutinating effect of antibody, as mice immunised with IgG fragments enzymatically digested or reduced to

remove di-valent binding were no longer protected. IgA1 is the major immunoglobulin on the nasal mucosa of humans,

but it is cleaved in the hinge region by a pneumococcal protease eliminating its ability to agglutinate. We also showed

that immunisation with serotype-specific hIgA1 mAb blocked colonisation of an IgA1-protease mutant (agglutinated),

but not the protease-producing wild-type parent (not agglutinated). We have now adapted a flow cytometry-based assay

to quantify antibody-mediated agglutination to further characterise this response. Using this assay we demonstrated

that antisera generated against unencapsulated whole-cell pneumococci was poorly agglutinating. Furthermore, passive

immunisation with this antiserum did not protect against the acquisition of colonisation. In addition, antiserum raised

against the abundant surface antigen PspA was unable to agglutinate. This suggests that capsule may be the only

vaccine target that can elicit agglutinating antibodies, leading to herd immunity. This data highlights the importance of

agglutinating antibodies in mucosal defence and reveal how successful pathogens evade this effect. However, high levels

of protease resistant anti-capsular IgG change the dynamics of this host-pneumococcal interaction and allow the host to

overcome this pathogen.

O8.1

PBP2b, MreD and DivIVA constitute a functional unit in the peripheral

peptidoglycan synthesis machinery of Streptococcus pneumoniae

Daniel Straume, Kari Helene Berg, Gro Stamsås, Leiv Sigve Håvarstein

Norwegian University of Life Sciences, Aas, Norway

A number of proteins are involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae. The elongasome is

believed to encompass PBP2b, MreC, MreD, RodA, DivIVA, PBP1a and presumably additional proteins. We have previously

shown that depletion of the essential penicillin-binding protein PBP2b results in extremely long chains of lentil shaped

cells that are deficient in peripheral peptidoglycan synthesis [1]. A similar phenotype was reported for pneumococci in

which the DivIVA protein had been deleted [2], suggesting that PBP2b and DivIVA have a close functional relationship.

Many biochemical and biophysical technologies have been developed to study membrane associated protein complexes.

Most of these technologies provide information on subcellular localisation or physical contact between proteins.

However, physical proximity does not necessarily imply a direct functional relationship between proteins. In the present

study we use a different approach to identify proteins that are functionally linked to PBP2b. Pneumococci that are

competent for natural transformation secrete a murein hydrolase, CbpD, which lyses susceptible pneumococci present

in the same environment. The integral membrane protein ComM protects competent cells from committing suicide by

a mechanism that is not yet fully understood. We discovered that competence-induced cells depleted in PBP2b were no

longer able to protect themselves even though they were ComM + . The same phenotype was observed in pneumococci

in which DivIVA and MreD had been deleted, demonstrating that PBP2b, DivIVA and MreD constitute a functional unit

required to establish immunity against CbpD in competent cells expressing the ComM immunity protein. Deletion of

MreC, on the other hand, did not influence immunity development. These findings demonstrate that DivIVA and MreD

are required for the activation of PBP2b and/or its correct positioning relative to other proteins in the elongasome.

1. Berg KH, Stamsås GA, Straume D, Håvarstein LS. Effects of low PBP2b levels on cell morphology and

peptidoglycan composition in Streptococcus pneumoniae R6. J Bacteriol 2013;195:4342–54. PMID:23873916

http://dx.doi.org/10.1128/JB.00184-13

2. Fleurie A, Manuse S, Zhao C, Campo N, Cluzel C, Lavergne JP et al. Interplay of the serine/threonine-kinase

StkP and the paralogs DivIVA and GpsB in pneumococcal cell elongation and division. PLoS Genet

2014;10:e1004275. PMID:24722178 http://dx.doi.org/10.1371/journal.pgen.1004275

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O8.2

Conformational plasticity of choline-binding modules: β-hairpin to

α-helix transition of choline-binding repeats triggered by detergent

micelles and membrane vesicles

Hector Zamora-Carreras 1 , Beatriz Maestro 2 , Erik Strandberg 3 , Anne Ulrich 3 , Jesus Sanz 2 , M.

Angeles Jimenez 1

1

Consejo Superior de Investigaciones Cientificas, Madrid, Spain; 2 University Miguel Hernandez, Elche, Spain; 3 Karlsruhe Institute of Technology,

Karlsruhe, Germany

Choline-binding modules have a ββ-solenoid structure composed of choline-binding repeats (CBRs), which in turn consist

of a β-hairpin followed by a short linker. To find minimal peptides able to maintain the CBR native structure and to evaluate

their remaining choline-binding ability, we have analysed the structure of the isolated 238-252 sequence from the LytA

autolysin (LytA 238-252

). Circular dichroism and nuclear magnetic resonance data demonstrate that this 14-aa peptide

forms a highly stable native-like β-hairpin in aqueous solution and displays a residual ability to bind choline. Remarkably,

the peptide acquires a stable, amphipathic α-helix conformation in both zwitterionic (dodecylphosphocholine) and

anionic (sodium dodecylsulphate) detergent micelles, as well as in small lamellar phospholipid vesicles. This β-hairpinto-α-helix

conversion is reversible upon removal of the external reagent. Furthermore, the full-length choline-binding

module of LytA (C–LytA protein, comprising residues 188–318), similarly to the isolated CBR, could also get inserted

into detergent micelles acquiring a highly helical conformation. To our knowledge, this chameleonic behaviour is the

only case described so far accounting for a micelle-induced structural transition between two highly ordered peptide

structures, and suggests that LytA and possibly other CBPs have the intrinsic ability to reversibly interact with the cell

membrane in physiologically relevant processes such as membrane translocation from the cytoplasm to the cell wall.

O8.3

On the interaction between the pneumococcal cell wall and the cholinebinding

modules (CBMs): evaluation of binding affinities and effect of

externally added CBMs to bacterial cultures

Manuel Sanchez, Beatriz Maestro, Francisco Garcia-Asencio, Jesus Sanz

University Miguel Hernandez, Elche, Spain

Pneumococcal choline-binding proteins (CBPs) make use of the so-called choline-binding modules (CBMs) to specifically

adsorb onto the bacterial cell wall through binding to the phosphorylcholine residues in the teichoic acids. CBMs are,

in turn, built from short sequences named choline-binding repeats (CBRs). It had been previously shown that addition

of the CBMs from the LytA amidase and the phagic CPL1 lysozyme (C–LytA and C–CPL1 respectively) in vitro inhibits

the lytic action of their parental cell-wall hydrolases, probably by competition for the binding to choline. To evaluate

whether this finding might constitute a novel anti-pneumococcal line, we first estimated the binding affinities of C–LytA

and C–CPL1 to DEAE-functionalised magnetic nanoparticles as bio-inspired mimics of the cell wall. Despite being built

from the same number of CBRs (6), both modules greatly differ in their binding mode and affinity to both free choline

and DEAE nanoparticles. Moreover, small amounts of choline (below 5 mM) not only did not hamper binding to the

nanoparticles, but appreciably enhanced its strength, probably by inducing the dimerisation of both modules. When the

CBMs were externally added to exponential cultures of Streptococcus pneumoniae R6, binding to the bacterial surface

was immediate and caused the inhibition of cell separation, leading to long bacterial chains and sedimenting aggregates.

This effect, which correlates with the in vitro binding assays, suggests that the use of CBMs and more stable chemical

bioconjugate derivatives might represent promising candidates for a streamlined action against pneumococcus based on

bacterial aggregation and subsequent phagocytosis.

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O8.4

Conditional lethal mutants reveal that FtsA is needed at early and late

stages of cell division in Streptococcus pneumoniae

Andrea Mura 1 , Daniela Musu 1 , Ana Isabel Rico 2 , Marcin Krupka 2 , Dalia Denapaite 3 , Pavel Branny 4 ,

Miguel Vicente 2 , Orietta Massidda 1

1

University of Cagliari, Cagliari, Italy; 2 Centro Nacional de Biotecnología, Madrid, Spain; 3 University of Kaiserslautern, Kaiserslautern, Germany;

4

Academy of Sciences of the Czech Republic, Prague, Czech Republic

The early stage of bacterial cell division requires assembly of 2 essential proteins, FtsZ and FtsA, at mid-cell. Here we

present the characterisation of 3 Streptococcus pneumoniae ftsA thermosensitive (TS) mutants, named A19 TS

, A20 TS

and

A21 TS

, obtained by error-prone random PCR, allelic replacement and screening for their ability to grow at 28 °C but not

at 40 °C. Mutations mapped in different domains of the FtsA structure, thought to be involved in the interactions with

itself, FtsZ and/or late cell division proteins. Temperature shifting experiments showed that, in contrast to the Rx1 WT

,

the A TS

mutants stopped growing and started lysing upon shifting to 40°C. Protein-protein interaction assays showed

that the FtsA TS

proteins lost the ability to self-interact but retained the ability to interact with FtsA WT

and FtsZ. GFP-

FtsA WT

, expressed from the P Zn

inducible promoter, fully complemented the A20 TS

and A21 TS

phenotype at 40 °C, but

only partially that of the A19 TS

mutant. While different cell division proteins localised in the Rx1 WT

at both temperatures,

in the A TS

mutants their localisation was retained or lost upon shifting to 40 °C, depending on the specific mutant. The

results are consistent with what was observed in a zinc dependent S. pneumoniae ftsA conditional lethal mutant, where

the only source of FtsA was GFP-FtsA expressed from the P Zn

promoter. Localisation studies showed that FtsA is required

for efficient mid-cell localisation of FtsZ, supporting previous observations that in Gram-positives, which lack ZipA, FtsA

is required for anchoring FtsZ to the membrane. Overall, the results confirm that FtsA is essential in S. pneumoniae and

validate it as a target for searching new antibiotics. Moreover, these data suggest that an early block in pneumococcal

cell division impairs growth and division, supporting a model in which a single machinery directed by FtsZ and FtsA

orchestrates both peripheral and septal cell wall synthesis.

O9.1

Structural basis of PcsB-mediated cell separation in Streptococcus

pneumoniae

Sergio G. Bartual 1 , Daniel Straume 2 , Gro Anita Stamsås 2 , Inés Muñoz 3 , Carlos Alfonso 4 , Martín

Martínez-Ripoll 1 , Leiv Sigve Håvarstein 2 , Juan A. Hermoso 1

1

Department of Crystallography and Structural Biology, Instituto de Química-Física Rocasolano, CSIC, Madrid, Spain; 2 Department of Chemistry,

Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway; 3 Macromolecular Crystallography Group. Structural Biology

and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; 4 Centro de Investigaciones Biológicas (CIB), Madrid,

Spain

In order to divide, bacteria must synthesise a new septal cell wall, which must be split down the middle by 1 or more

murein hydrolases to separate the resulting daughter cells. The 2-component regulatory system WalKR controls

peptidoglycan metabolism in low G+C Gram-positive bacteria. The product of one of these genes, PcsB (from protein

required for cell wall separation of group B streptococcus), has been shown to be essential for viability in Streptococcus

pneumoniae strains D39 and R6. Recently we have reported the crystal structure of full-length PcsB [1]. The structure

of PcsB revealed a homodimer with an unusual subunit arrangement: the catalytic domain of 1 subunit is sandwiched

between 2 arms of an elongated regulatory domain coming from the other subunit. The structure implies membraneattached

FtsEX complex activates PcsB by liberating the catalytic domain from the regulatory domain. In support of this

notion, we found that full-length PcsB had no detectable PG hydrolase activity but a truncation derivative lacking the

regulatory domain digested PG in a zymogram. We propose a model in which dimeric PcsB must interact simultaneously

with FtsEX complexes on both sides of the division septum (i.e. a PcsB dimer bridges the division site). It would mean the

catalytic activity of PcsB is only turned in a mature division septum, providing an elegant means of ensuring the enzyme

digests the cell wall only in the right place and at the right time.

1. Bartual SG, Straume D, Stamsås GA, Muñoz IG, Alfonso C, Martínez-Ripoll M et al. Structural basis of PcsBmediated

cell separation in Streptococcus pneumoniae. Nat Commun 2014;5:3842. PMID:24804636

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O9.2

Single molecule force spectroscopy reveals interaction strength

between Streptococcus pneumoniae TIGR4 pilus-1 tip protein RrgA and

human fibronectin

Tanja Becke 1, 2 , Stefan Ness 1 , Raimund Gürster 1 , Stefanie Sudhop 2 , Arndt F. Schilling 4 , Anne Marie

di Guilmi 3 , Hauke Clausen-Schaumann 2 , Markus Hilleringmann 1

1

FG Protein Biochemistry and Cellular Microbiology, Department of Applied Sciences and Mechatronics, Munich University of Applied Sciences,

Munich, Germany; 2 Center for Applied Tissue Engineering and Regenerative Medicine, Munich University of Applied Sciences, Munich, Germany;

3

Pneumococcus Group, Institut de Biologie Structurale, CEA-CNRS-Université Joseph Fourier, Grenoble, France; 4 Department for Plastic Surgery and

Hand Surgery, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany

Gram-positive Streptococcus pneumoniae represents a major human pathogen causing serious diseases including

pneumonia, meningitis, and febrile bacteraemia, with high mortality rates worldwide. Among other virulence factors,

recently discovered surface appendages (pili) are involved in pneumococcal host colonisation and invasion. Native S.

pneumoniae TIGR4 pilus-1 is composed of an RrgC cell wall anchor protein, multiple covalently linked RrgB backbone

subunits and a terminal RrgA adhesion molecule. The pilus tip protein RrgA was found to interact with specific host

components like extracellular matrix molecules (ECMs) and elements of the innate immune system. However the precise

role of RrgA and the fundamental molecular mechanisms of respective individual RrgA domains during host factor

interplay are not understood in detail. In particular, nothing is known about the specific thermodynamics and underlying

interaction forces between RrgA mediated associations and potential consequences regarding their respective role

during pneumococcal infection. In this study, we use single molecule force spectroscopy, a widely used operating mode

of the atomic force microscope to directly probe protein to protein linking, to quantify the interaction forces between

the pilus subunits and different ECMs. In a first attempt we could show specific binding forces between the tip protein

RrgA and human fibronectin, whereas the pilus backbone protein RrgB shows no specific binding towards the respective

molecule. We plan further experiments studying thermodynamics of the RrgA–fibronectin association process applying

isothermal titration calorimetry. We anticipate these studies to be a starting point for the detailed analysis of the

molecular interplay between the pneumococcal type-1 pilus subunits and various host ECMs like fibronectin, laminin,

collagen I, as well as elements of the host innate immune system and potentially whole cells.

O9.3

Structural basis for selective recognition of microbial and endogenous

polysaccharides by SIGN-R1 receptor

Iván Acebrón 1 , Noella Silva-Martín 1 , Sergio G. Bartual 1 , Erney Ramírez-Aportela 2 , Pablo Chacón 2 ,

Chae Gyu Park 3, 4 , Juan A. Hermoso 1

1

Department of Crystallography and Structural Biology, Instituto de Química Física “Rocasolano”, CSIC, Madrid, Spain; 2 Department of Biological

Chemical Physics, Instituto de Química Física “Rocasolano”, CSIC, Madrid, Spain; 3 Laboratory of Cellular Physiology and Immunology, Chris Browne

Center for Immunology and Immune Diseases, The Rockefeller University, New York, NY, USA; 4 Laboratory of Immunology, Severance Biomedical

Science Institute, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seodaemun-gu, Seoul, Korea, Republic of

Korea

SIGN-R1 is a C-type-lectin receptor involved in the recognition of exogenous polysaccharides, which facilitates the uptake

of microbes and activates C3 fixation via an unusual complement pathway on splenic marginal zone macrophages. In

addition to this, SIGN-R1 is also responsible for an anti-inflammatory activity of intravenous immunoglobulin by direct

interaction with sialylated Fcs. The crystal structures of SIGN-R1 in complex with dextran sulfate, sialic acid and the

sialylated Fc antibody at high resolution have provided insights into the selective binding of α-2,6-sialylated glycoproteins.

The exhaustive analysis of these structures has also revealed a secondary novel carbohydrate recognition domain

(CRD) found at the opposite face of the canonical binding site. This secondary binding site could bind molecules made

up of repetitive units, such as the ones observed in microbial polysaccharides. This additional binding site is calcium

independent and structurally not related to the previous identified carbohydrate recognition domain. As a result, these

two binding sites may help SIGN-R1 simultaneously bind both immune glycoproteins and microbial polysaccharide

components, accommodating SIGN-R1’s ability to combine the recognition of microbes with the activation of the

classical complement pathway.

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O9.4

New insights into structure, biosynthesis and pro-inflammatory

potential of pneumococcal teichoic acids

Nicolas Gisch 1 , Thomas Kohler 2 , Franziska Waldow 1 , Dominik Schwudke 1, 5 , Holger Heine 3, 5 ,

Regine Hakenbeck 4 , Dalia Denapaite 4 , Sven Hammerschmidt 2

1

Division of Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany; 2 Department Genetics

of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany; 3 Division of Innate

Immunity, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany; 4 Department of Microbiology, University of

Kaiserslautern, Kaiserslautern, Germany; 5 Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Germany

The members of the mitis group of streptococci are known to possess teichoic acids (LTA and WTA) that are unique

among Gram-positive bacteria. They contain phosphorylcholine residues, are of remarkable high complexity, and their

precursor are synthesised via a common biosynthetic pathway, which has been bioinformatically deduced recently. Until

now, only teichoic acids of Streptococcus pneumoniae have been analysed on the molecular level and the structural

model of the pneumococcal LTA has recently been revised by us. Here, we present the elucidation of the so far unknown

linkage of the pneumococcal WTA to the peptidoglycan as well as the structural investigation of the teichoic acids isolated

from S. oralis. The knowledge and comparison of these structures provides a significant step forward to understand the

biosynthesis of teichoic acids in these bacteria, especially with regard to the enzymes involved in the attachment of TA

precursor chains to the peptidoglycan or the glycolipid anchor. For detailed structural analysis of LTA samples, targeted

chemical degradations like hydrazine or hydrofluoric acid treatment have been performed, followed by high-resolution

mass spectrometry and NMR spectroscopy. WTA bound to small peptidoglycan fragments has been generated by

enzymatic digestion of cell walls using LytA and lysozyme-like enzymes, the specific linkage was investigated by different

two-dimensional NMR experiments. Our study provides evidence that protein(s) other than the members of the LCP

protein family (LytR, Cps2A, and Psr) have to be responsible for the transfer of TA precursor chains to the glycolipid anchor

to form the LTA. Furthermore, we examined the immunostimulatory properties of pnTAs in human mononuclear cells

(hMNCs) independently of those caused by Toll-like receptor 2 activation induced by contaminating lipoproteins. Based

on these results, we suggest that the immunostimulating activity of pnTAs is restricted to the complement activation.

O9.5

LocZ is a new cell division protein that directs septum placement in

Streptococcus pneumoniae

Nela Holecková 1 , Linda Doubravová 1 , Orietta Massidda 2 , Virginie Molle 3 , Karolína Buriánková 1 ,

Oldrich Benada 1 , Olga Kofronová 1 , Aleš Ulrych 1 , Pavel Branny 1

1

Cell and Molecular Microbiology Division, Institute of Microgiology, v.v.i., Academy of Science of the Czech Republic, Prague, Czech Republic; 2 Dipartimento

di Scienze Chirurgiche, Sez. di Anatomia Patologica e Microbiologia, Università di Cagliari, Cagliari, Italy; 3 Laboratoire de Dynamique des

Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS, Montpellier, France

Bacterial cell division is a highly ordered process regulated in time and space. How Streptococcus pneumoniae

controls proper septum placement at mid-cell to guarantee the generation of identical daughter cells is still largely

unknown. Here we report identification of new cell division protein, named LocZ (Spr0334), which is involved in proper

septum placement in S. pneumoniae. LocZ is a substrate of Ser/Thr protein kinase StkP and is conserved only among

streptococci, lactococci and enterococci, which lack homologues of the Min and nucleoid occlusion effectors. We

showed that LocZ is not essential but that its deletion results in cell division defects and shape deformation, causing

cells to divide asymmetrically and generate unequally sized, occasionally anucleated, daughter cells. LocZ has a unique

localisation profile: it arrives at mid-cell before FtsZ and FtsA and leaves the septum early, apparently moving along with

the equatorial rings that mark the future division sites. Consistently, cells lacking LocZ show misplacement of the Z-ring

suggesting that it could act as a positive regulator to determine septum placement. All these data indicate that ovoid

bacteria adapted a unique mechanism to find their middle, reflecting their specific shape and symmetry.

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O10.1

Streptococcus pneumoniae interaction with brain-derived neural cells

and alterations in functional state of the cells

Reuven Afriat, Marilous Shagan, Ron Dagan, Esther Priel, Yaffa Mizrachi Nebenzahl

Ben Gurion University of the Negev, Beer Sheva, Israel

Streptococcus pneumoniae causes meningitis with 50% mortality rate and survivors present long time sequelae including

learning impairment, deafness, mental retardation, and hydrocephalus. We have previously identified several putative

bacterial adhesin and their respective receptors on epithelial cells in the host that are proteins known to function in brain

development or affect neural cell function. We hypothesise that the very same adhesins are involved in the interaction

of the bacteria with neural cells and may affect neural cell function and survival. Currently we demonstrate a significant

reduction in pneumococcal adhesion to the neural cells (U251, NSC-34) in either presence of the recombinant adhesins

(rNOX and rGtS) or using bacteria lacking the putative adhesins compared to the wild-type (WT) strain. Infection of cells

with S. pneumoniae inhibited Topoisomerase I (Topo I) activity, significantly and to a significantly lesser extent with

the mutated bacteria. Topo I is an essential nuclear enzyme that participates in all DNA transaction processes and is

important for gene expression. The reduced Topo I activity resulted from increased ERK phosphorylation, followed by

increased ADP ribosylation of Topo I by PARP. Inhibition of both enzymes with specific inhibitors prevented the reduction

of Topo I activity in response to S. pneumoniae. Thus, pneumococci infection of neural cells inhibit Topo I activity via ERK

phosphorylation and increased ADP ribosylation by PARP. The recombinant adhesins or bacteria lacking them prevent

the inhibition of Topo I activity in the neural cells in response to the pneumococcus.

O10.2

PSGL-1 receptor on leukocytes is a critical component of the host

immune response against invasive pneumococcal disease

Elisa Ramos-Sevillano 1, 3 , Ana Urzainqui 4 , Miriam Domenech 1, 2 , Rafael González-Tajuelo 4 ,

Fernando González-Camacho 3 , Francisco Sánchez-Madrid 4 , Jeremy S Brown 5 , Ernesto García 1, 2 ,

José Yuste 2, 3

1

Centro de Investigaciones Biológicas, CSIC, Madrid, Spain; 2 CIBERES, Madrid, Spain; 3 Instituto de Salud Carlos III, Madrid, Spain; 4 Hospital de la

Princesa, Madrid, Spain; 5 Centre for Inflammation and Tissue Repair. UCL, London, UK

Phagocytic cells play a key role in the clearance of invading pathogens such as Streptococcus pneumoniae. Among

them, leukocytes play an important role in inflammatory and immune responses against disseminated infection, and

bacterial clearance depends on the efficacy of different receptors on phagocytic cells to recognise, internalise and

kill the pathogen. In this study we report the leukocyte P-selectin glycoprotein ligand-1 receptor (PSGL-1) as a novel

receptor for the interaction with S. pneumoniae during the invasive process. Co-localisation of different clinical isolates

of S. pneumoniae with PSGL-1 was demonstrated, observing a rapid and active phagocytosis when PSGL-1 was present.

Moreover, the capsular polysaccharide and the main autolytic enzyme of the bacterium, the amidase LytA, were both

identified as pneumococcal ligands recognised by PSGL-1. Experiments using wild-type mice and mice deficient in PSGL-1

demonstrated that PSGL-1 on leukocytes plays a critical role against invasive pneumococcal disease. Histological analysis

demonstrated that PSGL-1 -/- mice have lower levels of granulocytes migrating to the lung than the correspondent wildtype

mice, confirming that PSGL-1 is important for leukocyte extravasation. Bacterial levels were higher in the respiratory

tract of PSGL-1 -/- deficient mice after pneumococcal infection, which is consistent with the impaired recruitment of

granulocytes to the lung of these mice. Bacterial levels were also markedly increased in the blood of PSGL-1 -/- mice,

which confirmed the importance of this receptor in the recognition and clearance of S. pneumoniae from the systemic

circulation. This study demonstrates that PSGL-1 contributes to protection against lethal pneumococcal infection.

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O10.3

Pneumococcal adhesins PavB and PspC are important for the interplay

with human thrombospondin-1

Ulrike Binsker 1 , Thomas P. Kohler 1 , Krystin Krauel 2 , Sylvia Kohler 1 , Hansjörg Schwertz 2 , Sven

Hammerschmidt 1

1

Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany,

2 Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany

Streptococcus pneumoniae is a widespread Gram-positive commensal of the human nasopharynx but can also cause

severe infections. The adherence of Gram-positive bacteria to host cells is the obligatory step in an infection process

and is facilitated by surface-exposed structures, which especially target components of the extracellular matrix. The

human matricellular glycoprotein thrombospondin-1 (hTSP-1) is released by activated platelets and mediates adhesion

of Gram-positive bacteria to various host cells. Here we demonstrate for the first time that pneumococcal adherence and

virulence factor B (PavB) and pneumococcal surface protein C (PspC) are essential for the interaction of S. pneumoniae

(pneumococci) with hTSP-1. The contribution of PavB and PspC to contribute to hTSP-1 acquisition was analysed by

flow cytometry using deletion mutants of S. pneumoniae D39Δcps and serotype 35A. Various interaction experiments

using surface plasmon resonance (SPR) and ELISA with heterologously expressed protein domains of PavB and PspC

were performed. Furthermore, differences in adherence of S. pneumoniae D39∆cps and the isogenic double mutant

deficient for PavB and PspC to hTSP-1 pre-treated human epithelial cells were examined using fluorescence microscopy.

The impact of PavB and PspC in the activation of purified human platelets was also investigated. The deficiency in PavB

and PspC reduces hTSP-1 recruitment by pneumococci and decreases hTSP-1-mediated pneumococcal adherence to

epithelial cells. Binding studies with recombinant fragments of PavB and PspC containing repetitive structures exhibit

hTSP-1-binding activity as shown by ELISA and SPR. Furthermore, platelet assays suggested that PavB and PspC are not

involved in platelet activation by pneumococci. This study indicates a pivotal role of PavB and PspC for pneumococcal

sequestration of soluble hTSP-1 to the bacterial surface. The data further emphasises that host cell-bound hTSP-1

facilitates pneumococcal adhesion. The numbers of repeating domains within these adhesins play a crucial role for the

interaction with hTSP-1.

O10.5

Mcl–1 regulates mitochondrial ROS-mediated bacterial killing of

Streptococcus pneumoniae in macrophages and defines susceptibility

to pulmonary infection in COPD

Martin Bewley 1 , Julie Preston 1 , Richard Budd 1, 5 , Dave Singh 2 , Peter Barnes 3 , Louise Donnelly 3 ,

Steve Shapiro 4 , Moira Whyte 1 , David Dockrell 1, 5

1

University of Sheffield, Sheffield, UK; 2 Medicines Evaluation Unit, University Hospital of South Manchester, Manchester, UK; 3 National Heart and

Lung Laboratory, Imperial College London, London, UK; 4 University of Pittsburgh, Pittsburgh, USA; 5 Sheffield Teaching Hospitals, Sheffield, UK

Apoptosis-associated killing (AAK) facilitates late-stage killing of bacteria in Streptococcus pneumoniae (Spn) infected

macrophages when canonical phagolysosomal killing has become exhausted. This process is governed by levels of the

anti-apoptotic protein Mcl–1, however the exact mechanism remains unknown. Chronic obstructive pulmonary disease

(COPD) patients are at increased risk from bacterial infections, which cause acute exacerbations and pneumonia. We

hypothesised that defects in AAK result in a failure to clear bacteria in COPD and examined potential mechanisms. A

novel CD68.hMcl–1 transgenic mouse, with macrophage-specific expression Mcl–1, was created, and human alveolar

macrophages (AM) from COPD patients collected by broncho-alveolar lavage. Transgenic (Tg) BMDM had normal initial

microbial killing but failed to clear residual bacteria by AAK and reduced AM apoptosis led to pulmonary persistence

of Spn. In wild-type BMDM AAK was associated with a caspase-dependent increase in mROS, which was blocked in

Tg BMDM. COPD macrophages had elevated levels of Mcl–1 and lower levels of apoptosis. Basal levels of mROS were

higher compared to healthy controls, but there was no induction with Spn infection. COPD AM also had higher levels of

mROS scavenger superoxide dismutase (SOD)2. This lower apoptosis and increased anti-oxidant capacity translated to

increased bacterial survival in Spn challenged COPD macrophages. These data suggest elevated levels of Mcl–1 in COPD

macrophages cause a defect in apoptosis and a failure to induce mROS relative to SOD2, reducing bacterial clearance and

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contributing to bacterial colonisation in the COPD lung. Thus through its governance of AAK, Mcl–1 levels are identified

as an important determinant of pulmonary infection.

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