CLOSPATH 7 - Conference Planning and Management - Iowa State ...

7 TH INTERNATIONAL CONFERENCE ON THE
MOLECULAR BIOLOGY AND PATHOGENESIS
OF THE CLOSTRIDIA
CLOSPATH 7
PROGRAM & ABSTRACT BOOK
October 25 to 29, 2011
Iowa State Center
Ames, Iowa USA

7 th International Conference on the Molecular Biology and Pathogenesis of the Clostridia
Abstract Book
Organizing Committee:
Jimmy Ballard (USA)
Neil Fairweather (UK)
Miia Lindström (Finland)
Paola Mastrantonio (Italy)
Bruce A. McClane (USA)
Nigel Minton (UK)
Tom Riley (Australia)
Julian Rood (Australia)
Maja Rupnik (Slovenia)
Bo-Moon Shin (South Korea)
Linc Sonenshein (USA)
J.Glenn Songer (USA)
Rick Titball (UK)
Rod Tweten (USA)
Gayatri Vedantam (USA)
Vince Young (USA)
Scientific Advisory Board:
Klaus Aktories (Germany)
Frederic Barbut (France)
Marietta Flores Diaz (Costa Rica)
Bruno Dupy (France)
Dale Gerding (USA)
Gerhard Gottschalk (Germany)
Eric Johnson (USA)
Haru Kato (Japan)
Cesare Montecucco (Italy)
Peter Mullany (UK)
Shinichi Nakamura (Japan)
Michael R. Popoff (France)
Ian Poxton (UK)
Tohru Shimizu (Japan)
Francisco A. Uzal (USA)

The 7 th International Conference on the Molecular Biology of the
Clostridia would like to acknowledge the contributions of
Funding for this conference was made possible [in part] by R13 AI098104-01 from
NIH. The views expressed in written conference materials or publications and by
speakers and moderators do not necessarily reflect the official policies of the
Department of Health and Human Services, nor does mention of trade names,
commercial practices, or organizations imply endorsement by the U.S.
Government.

IOWA STATE UNIVERSITY
OF SCIENCE AND TECHNOLOGY
Dear Colleague and Fellow Delegate,
Many of you may have thought you‘d never have a good reason to come to Iowa, but here you are!
Iowa is not, on the whole, great for nightlife, although I suspect that many of you would be surprised by
the variety and extent of after hours entertainment. I‘ve lived in or been in some way connected to
Iowa for more than 50 years, and have yet to observe real, live cow-tipping.
Iowa IS a great agricultural state, leading the US most years in production of corn and hogs, and
ranking high in cattle production, as well. Disease caused by members of the genus Clostridium in
humans is as common here as anywhere, and the plethora of domestic animals have their share of
infections, as well. So, maybe Iowa is a good place for the 7 th International Conference on the
Molecular Biology and Pathogenesis of the Clostridia. In any case, it‘s here.
And all of you are absolutely welcome! Members of the Organizing Committee have done our best to
assemble a program that will be stimulating to everyone. We hope that each of you will have the
opportunity to forge new relationships, make friends, find new collaborators, and above all to learn a
lot about the current state of clostridial research – from basic to translational to applied. In addition to
the science, I think you‘ll be pleased with the meals and breaks – we should have great volumes of
food and drink, with enough variety to satisfy everyone‘s tastes.
I owe a great debt of gratitude to the Organizing Committee, and especially to the ―executive
committee,‖ consisting of Julian Rood, Bruce McClane, and Linc Sonenshein. Finally, my ―right hand
woman‖ has been Patti Thrasher. She has dealt with matters of registration, housing, and all local
arrangements at the venue single-handedly. I cannot possibly thank her enough for all she‘s done.
Without her, I would have been, long sense, a jabbering mass of coffee-stained blue jeans.
If you have trouble while here, seek help at the registration desk, where Patti and a plethora of helpers
from my laboratory will be around to assist you. As a last resort, you may come to me, but I‘m likely to
be of less value than the others. Enjoy yourselves, and let us know if there‘s anything we can do to
make your stay more fulfilling.
All the best,
Dr. J. Glenn Songer, Fellow AAM, Diplomate ACVP
Boehringer Ingelheim Vetmedica Chair in Food Animal Infectious Disease
Department of Veterinary Microbiology and Preventive Medicine
College of Veterinary Medicine
Iowa State University
Ames, IA 50011

CLOSPATH 7 CONFERENCE PROGRAM

Tuesday, October 25, 2011 — Scheman Building (1 st Floor)
5:30 pm — Open for Registration (Lobby Registration Desk )
6:00 pm — Welcome: Wine and Cheese Reception (Lobby)
7:00 pm — Keynote Address (Benton Auditorium): Rodney K. Tweten, University of
Oklahoma, USA. ―Cholesterol-Dependent Cytolysins.‖
Wednesday, October 26, 2011 — Fisher Theater
7:00 am — Open for Registration (Lobby)
— Continental Breakfast (Lobby)
Session I (Fisher Theater): Epidemiology and Laboratory Diagnosis (Chair: Nigel
Minton):
8:30 – 9:00 am — Maja Rupnik, University of Maribor, Slovenia. ―Epidemiology of
Clostridium difficile – new environments and new types.‖
9:00 – 9:20 am — David Aronoff, University of Michigan, USA. ―Lack of association
between ribotype and severe Clostridium difficile infection.‖
9:20 – 9:40 am — Cornelis Knetsch, Leiden University, The Netherlands. ―Unique genetic
markers for hypervirulent Clostridium difficile.”
9:40 – 10:00 am — Ying Cheng, Chinese Center for Disease Control and Prevention,
China. ―Genomic characteristics of a toxin A-negative, toxin B-positive C. difficile
strain from Beijing, China.‖
10:00 am — Break (Lobby)
Session II (Fisher Theater): Large Clostridial Glycosylating Toxins (Chair: Klaus
Aktories):
10:15 – 10:45 am — Jimmy Ballard, University of Oklahoma, USA. ―Critical differences
between historical and hypervirulent TcdB.‖
10:45 – 11:05 am — Borden Lacy, Vanderbilt University, USA. ―Structural determinants of
the Clostridium difficile toxin A glucosyltranserase activity.‖
11:05 – 11:25 am — Aimee Shen, University of Vermont, USA. ―Chemically interrogating
Clostridium difficile glucosylating toxin activation.‖
11:25 – 11:45 am — Shan Li, Tufts University, USA. ―A neutralizing intrabody to study
autocleavage of Clostridium difficile toxin B.‖
11:45 am – 12:05 pm — Wensheng Wei, Peking University, China. ―Identification of novel
endocytosis pathway for TcdB of Clostridium difficile through shRNAmir library
screening and high-throughput sequencing analysis.‖

12:05 pm — Buffet Lunch (Scheman Bldg., Rooms 167-179)
Session III (Fisher Theater): Genetics, including genomes and plasmids (Chair: Miia
Lindström):
1:20 – 1:50 pm — Marian Wachtel, Program Officer, NIH. ―NIAD Support of Clostridial
Research and Resources for the Research Community‖
1:50 –2:20 pm — Julian Rood, Monash University, Australia. ―The mechanism of
conjugation in C. perfringens.”
2:20 – 2:50 pm — Bruce McClane, University of Pittsburgh, USA. ―Characterization of the
toxin plasmids of Clostridium perfringens.”
2:50 – 3:20 pm — Nigel Minton, University of Nottingham, UK. ―The development of
clostridial gene systems and their exploitation.‖
3:20 – 3:40 pm — Anne Collignon, Université Paris-Sud, France. ―Analysis of the
genome-wide temporal expression of a Clostridium difficile 027 hypervirulent strain
in monoxenic mice.‖
3:40 – 4:00 pm — Eric Johnson, University of Wisconsin, USA. ―Phylogenomic analysis of
Clostridium botulinum strains.‖
4:00 – 6:30 pm — POSTERS I (Scheman Bldg., Lounge 182)
4:30 pm — Registration Closes (Fisher Theater, Lobby)
6:30 pm — Buffet Dinner (Scheman Bldg., Rooms 167-179)
Thursday, October 27, 2011—Fisher Theater
7:00 am — Continental Breakfast (Lobby)
Session IV (Fisher Theater): Membrane active toxins, binary toxins and enzymes
(Chair: Joe Barbieri):
8:30 – 9:00 am — Klaus Aktories, Alert-Ludwigs Universität, Freiburg, Germany.
―Clostridium difficile CDT and Clostridium perfringens Iota Toxin: Identification of
their membrane receptors and actions on the cytoskeleton of target cells.‖
9:00 – 9:20 am — Mark McClain, Vanderbilt University, USA. ―Identification of host factors
contributing to Clostridium perfringens epsilon-toxin-induced cell death.‖
9:20 – 9:40 am — Sérgio Fernandes da Costa, University of Exeter, UK. ―Expression and
functional characterization of Clostridium perfringens NetB Toxin.‖
9:40 – 10:00 am — Ajit Basak, Birkbeck College, UK. ―Structural Relationships in
clostridial beta-pore-forming toxins.‖
10:00 am — Break (Lobby)

Session V (Fisher Theater): Regulation of virulence genes, including transcriptomics
(Chair: Linc Sonenshein):
10:20 – 10:50 am — Bruno Dupuy, Institut Pasteur, Paris, France. ―Role of carbon
catabolite repression (CCR) in the pathogenicity of Clostridium difficile.”
10:50 – 11:10 am — Revathi Govind, Kansas State University, USA. ―A new role for C.
difficile glutamate dehydrogenase.‖
11:10 – 11:30 am — Kaori Ohtani, Kanazawa University, Japan. ―Novel regulatory
mechanism for spore formation and enterotoxin production in Clostridium
perfringens.”
11:30 – 11:50 am — Wiep Klaas Smits, Leiden University Medical Center, The
Netherlands. ―Clostridium difficile Spo0A regulates sporulation, but not toxin
production, by direct binding to target DNA.‖
11:50 am – 12:10 pm — Kate Mackin, Monash University, Australia. ―Regulator-mediated
modulation of toxin production and sporulation in an epidemic clinical isolate of C.
difficile.”
12:10 pm — Buffet Lunch (Scheman Bldg., Rooms 167-179)
Session VI (Fisher Theater): Food animal diseases; animal models (Chair: Neil
Fairweather):
1:30 – 2:00 pm — Francisco Uzal, University of California, Davis, USA. ―Animal models to
study C. perfringens diseases.‖
2:00 – 2:20 pm — Kevin Chen, Tufts University, USA. ―Systemic dissemination of C.
difficile toxins A and B is associated with severe fatal disease in the piglet and
mouse models.‖
2:20 – 2:40 pm — Anthony Buckley, University of Glasgow, UK. ―Aspects of the hamster
model of infection.‖
2:40 – 3:00 pm — Xingmin Sun, Tufts University, USA. ―A mouse relapse model of C.
difficile infection and its application in evaluating immunotherapies against
disease.‖
3:00 – 3:20 pm — Horst Posthaus, University of Berne, Switzerland. ―Lessons from
natural disease in pigs: pathogenesis of C. perfringens type C enteritis.‖
3:20 – 3:40 pm — Duncan MacCannell, CDC, USA. ―The molecular epidemiology of
Clostridium difficile in the United States‖
3:40 – 6:30 pm — POSTERS II (Scheman Bldg., Lounge 182)
6:30 pm — Buffet Dinner (Scheman Bldg., Rooms 167-179)

Friday, October 28, 2011—Scheman Building (1 st Floor)
7:00 am — Continental Breakfast (1 st Floor Lobby)
Session VII (Benton Auditorium): Physiology, including proteomics/metabolomics and
sporulation/germination (Chair: Gayatri Vedantam):
8:30 – 9:00 am — William Self, University of Central Florida, USA. ―Elucidating the role
and requirement for selenoenzymes in growth of Clostridium difficile.”
9:00 – 9:30 am — Linc Sonenshein, Tufts University, USA. ―Regulation of C. difficile
metabolism.‖
9:30 – 9:50 am — Robert Fagan, Imperial College, UK. ―Clostridium difficile has two
parallel and essential Sec secretion systems.‖
9:50 – 10:10 am — Mahfuzur Sarker, Oregon State University, USA. ―The molecular
mechanism of Clostridium perfringens spore germination.‖
10:10 – 10:30 am — Laurel Saujet, Institut Pasteur, France. ―The key sigma factor of
transition phase, SigH, controls sporulation, metabolism, and virulence factor
expression in Clostridum difficile.‖
10:30 am — Break (1 st Floor Lobby)
Session VIII (Benton Auditorium): Control and treatment (including therapeutics,
vaccines/immune responses and exploitation) (Chair: Anne Collignon):
10:50 – 11:20 am — Hanping Feng, Tufts University, USA. ―Novel C. difficile vaccines.‖
11:20 – 11:40 am — Anjana Chakravorty, Monash University, Australia. ―Opioid-based
analgesics block the progression and development of C. perfringens mediated
myonecrosis.‖
11:40 – 12:00 am — Louis Charles Fortier, Universite de Sherbrooke, Canada. ―Subinhibitory
concentrations of tigecycline inhibit sporulation of Clostridium difficile in
vitro.‖
12:00 am – 12:20 pm — Simon Cutting, Imperial College, UK. ―Immunization with Bacillus
spores expressing toxin A peptide repeats protects against infection with
Clostridium difficile strains producing toxins A and B.‖
12:20 – 12:40 pm — Shonna McBride, Tufts University, USA. ―The C. difficile CPR locus
mediates resistance to antimicrobial peptides through molecular mimicry.‖
12:40 – 1:00 pm — Rebecca McQuade, University of Arizona, USA. ―Differential
sensitivity of Clostridium difficile clinical isolates to mammalian cationic
antimicrobial peptides.‖
1:00 pm — Buffet Lunch (Rooms 167-179)

Session IX (Benton Auditorium): Neurotoxins and toxin applications (Chair: Rod
Tweten):
2:15 – 2:45 pm — Joe Barbieri, Medical College of Wisconsin, USA. ―Botulinum toxin
structure/function relationships.‖
2:45 – 3:05 pm — Miia Lindström, University of Helsinki, Finland. ―A two-component
system negatively regulates botulinum neurotoxin expression.‖
3:05 – 3:25 pm — David Kirk, University of Helsinki, Finland. ―Disruption of sigK in C.
botulinum ATCC 3502 prevents sporulation.‖
3:25 – 6:00 pm — POSTERS III (Lounge 182)
6:30 pm — Conference Banquet (Rooms 167-179)
Saturday, October 29, 2011—Scheman Building(1 st Floor)
7:00 am — Continental Breakfast (Lobby)
Session X (Benton Auditorium): Host-pathogen interactions involving toxins (Chair:
Bruce McClane):
8:30 – 9:00 am — Dena Lyras, Monash University, Australia. ―Roles of Toxins in C.
difficile pathogenesis.‖
9:00 – 9:20 am — Sarah Kuehne, University of Nottingham, UK. ―C. difficile toxins and
pathogenesis.‖
9:20 – 9:40 am — Jianming Chen, University of Pittsburgh, USA. ―The AGR quorum
sensing system is a global regulator of Clostridium perfringens toxin production
and virulence.‖
9:40 – 10:00 am — Kevin D‘Auria, University of Virginia, USA. ―Global gene expression of
epithelial cells from an in vivo model of C. difficile toxin A and B intoxication.‖
10:00 am — Break (Lobby)
Session XI (Benton Auditorium): Host-pathogen interactions involving nontoxic
virulence factors (Chair: Dena Lyras):
10:20 – 10:50 am — Gayatri Vedantam, University of Arizona, USA. ―Sporulation and
other non-toxin virulence factors.‖
10:50 – 11:20 am — Steve Melville, Virginia Tech, USA. ―Motility and adherence to
myoblasts.‖
11:20 – 11:50 am — Neil Fairweather, Imperial College, UK. ―Antigenic and phase
variation in cell wall proteins of Clostridium difficile.

11:50 am – 12:10 pm — Jihong Li, University of Pittsburgh, USA. ―Sialidases affect the
host cell adherence and epsilon toxin-induced cytotoxicity of C. perfringens type D
strain CN3718.‖
12:10 – 12:30 pm — Craig Ellermeier, ―University of Iowa, USA. ―Extra-cytoplasmic
function sigma factor CSFV regulates lysozyme resistance of Clostridium difficile”.
12:30 – 12:45 pm Meeting Closing
12:45 pm — Buffet Lunch (Rooms 167-179)
DEPART

ABSTRACTS OF ORAL PRESENTATIONS
Keynote Address
O1 to O52

Keynote
THE STRUCTURAL BIOLOGY OF CLOSTRIDIUM PERFRINGENS PERFRINGOLYSIN O:
NEW PARADIGMS, VACCINE DEVELOPMENT AND EVOLUTION
Rodney K. Tweten, Department of Microbiology and Immunology, University of Oklahoma
Health Sciences Center, Oklahoma City, OK 73104 USA,
The cholesterol-dependent cytolysins (CDCs) are a family of pore forming toxins produced
by many species of Gram-positive pathogenic and commensal bacteria. Insights into the
structural biology of pore formation of the CDCs have been largely accomplished by the
study of the archetype CDC, Clostridium perfringens perfringolysin O (PFO). The study of
PFO has revealed many new paradigms of how the CDCs recognize a cell and form a pore
in its membrane. These studies, however, have impacted a much broader expanse of
science than could have been predicted. The structure/function studies of PFO were key to
(1) revealing that the eukaryotic membrane attack complex/perforin (MACPF) proteins may
utilize a pore-forming mechanism that exhibits features of the CDC mechanism and may be
ancient ancestors of the CDCs, (2) the presence of CDCs in Gram-negative bacteria and (3)
the rational design of a pneumolysin toxoid as a component for a protein-based vaccine for
Streptococcus pneumoniae. Furthermore, methods developed to trap PFO at different stages
of its pore forming mechanism have been used to show that formation of the pore is not the
only way that CDCs can contribute to pathogenesis, which suggests that CDCs are
multifunctional proteins. Hence, the investigation of the PFO structure and pore forming
mechanism has impacted many fields of study and its continued study will likely reveal new
paradigms for how the CDCs function and contribute to bacterial survival and pathogenesis.

O1
EPIDEMIOLOGY OF CLOSTRIDIUM DIFFICILE – NEW ENVIRONMENTS AND NEW
TYPES
Maja Rupnik. Institute of Public Health Maribor, Centre for Microbiology, Maribor; University
of Maribor, Faculty of Medicine, Maribor; Centre of Excellence CIPKeBIP, Ljubljana,
Slovenia.
Clostridium difficile is currently still regarded as a nosocomial pathogen, but its importance in
the community is also rising. The hospital, as well as the non-hospital, environment can be
the source of infection that occurs either in outbreaks or as isolated cases. Even within
hospitals new modes of exposure, such as airborne dissemination, are taken into account.
Isolation of C. difficile from substantial numbers of normal or diseased food animals or
companion animals was reported relatively recently. Species infected most commonly
include piglets, calves, poultry, cats and dogs. Genotypes present in humans are much more
diverse ( > 300 ribotypes) than in animals or food (approx. 30 ribotypes) and the prevalent
genotype in human and animal reservoirs is still different. But there is also a large overlap of
genotypes from food, food animals, and humans, suggesting a contribution of food and
contact with animals to the infection process. The presence of most common PCR ribotypes
in many different sources (humans, animals, food, soil, and water) suggests that the ability to
survive in different environments plays a role in successful distribution and high prevalence
of a given genotype. Understanding of the broader epidemiology (e.g. comparison of
ribotypes in different countries, and spread of more virulent genotypes) is still limited by the
use of non-exchangeable typing techniques. Interestingly, there seems to be a large change
in circulating toxin production types. A few decades ago, C. difficile strains were distributed
into two main groups, toxinogenic and nontoxinogenic. Toxinogenic strains always produced
both toxins A and B, and nontoxigenic strains produced neither. Later discovery of A-B+
strains has dramatically changed laboratory diagnostics. In the non-outbreak situation,
approximately 25% of strains will be A-B+ or variant forms of A+B+. So far, existence of A+Bstrains
has not been confirmed. There are some interesting new A-B+ strains that in genetic
background resemble nontoxinogenic strains, but have an entire functional tcdB as well,
suggesting an association with mobile genetic elements.

O2
LACK OF ASSOCIATION BETWEEN RIBOTYPE AND SEVERE CLOSTRIDIUM DIFFICILE
INFECTION
S. T. Walk 1,2 , D. Micic 1 , R. Jain 1,2 , I. Trivedi 1 , E. W. Liu 1 , L. M. Almassalha 1 , S.A. Ewing 1 , D.
W. Newton 3,4 , A. T. Galecki 1,5,6 , C. Ring 1,2 , L. Washer 1 , V. B. Young 1,2,5 , and D. M.
Aronoff* 1,2,7 . 1 Department of Internal Medicine; 2 Division of Infectious Diseases; 3 Clinical
Microbiology Laboratories; 4 Department of Pathology; 5 Institute of Gerontology; 6 Department
of Biostatistics, and 7 Department of Microbiology, University of Michigan Health System, Ann
Arbor, MI 48109 USA.
Epidemiologic studies suggested that closely related strains of C. difficile caused more
severe clinical disease than other strains. Although new studies have challenged this
hypothesis, they were limited by heterogeneous clinical criteria defining severe disease. We
readdressed this question using recommended clinical definitions of severity and isolates
from 150 independent cases of C. difficile infection (CDI) from a single institution. Toxigenic
isolates were selectively cultured from stool samples, characterized using PCR virulence
factor screening, and typed using high-throughput, fluorescent PCR ribotyping, yielding 47
distinct ribotypes. Ribotype 014-020 was the most prevalent (21% of all isolates). Sixteen
cases met the definition of severe CDI and were caused by 9 ribotypes. CDI caused by
isolates previously implicated with more severe disease (ribotypes 027 and 078) was not
more severe than CDI caused by other ribotypes. Similarly, severe CDI cases caused by
isolates of ribotypes commonly associated with outbreaks were not more prevalent than
cases caused by non-outbreak isolates. Six epidemiologic characteristics were significantly
associated with severe CDI using univariate logistic regression but on multivariate analysis
only albumin level remained significantly (and inversely) associated with disease severity.
However, ribotype did not predict hypoalbuminemia (

O3
UNIQUE GENETIC MARKERS FOR HYPERVIRULENT CLOSTRIDIUM DIFFICILE
C.W. Knetsch* 1 , M.P.M. Hensgens 1 , C. Harmanus 1 , M.W. van der Bijl 2 , P.H.M. Savelkoul 2 ,
E.J. Kuijper 1 , J. Corver 1 * and H.C. van Leeuwen 1 . 1 Department of Medical Microbiology,
Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands;
2 Department of Medical Microbiology and Infection Control, VU University Medical Center,
Amsterdam, The Netherlands.
Clostridium difficile is an anaerobic bacillus that resides in the gut and has rapidly emerged
as a leading cause of antibiotic associated diarrheal disease in humans. Recently, the
incidence, complications and mortality of a Clostridium difficile infection have increased
dramatically due to the emergence of hypervirulent strains PCR ribotype 027 (NAP01) and
PCR ribotype 078 (NAP7/8). In this study we identified unique loci in the PCR ribotypes 027
and 078 and used these markers to detect specifically hypervirulent C. difficile strains. Whole
genome sequences of C. difficile PCR ribotypes 001, 012, 017, 027 and 078 were compared
using a bioinformatics approach. This resulted in a list of candidate marker genes unique for
hypervirulent types 027 and 078. Of these we selected the most promising candidates (i.e.
genes that are stably integrated or not abundantly present in genomes). Next, a large
collection of C. difficile strains (N=68) was screened for the presence of the selected
markers. Surprisingly, we found that the markers were also present in a few other strains.
Therefore, we analyzed if strains with the insert were genetically more related to each other
compared to strains without the insert. The strains with the insert were indeed more closely
related as evidenced by AFLP, MLST, similarity in PCR ribotype pattern and toxin gene
profile (tcdA, tcdB, tcdC, binary toxin). We have identified six PCR ribotypes that resemble
the hypervirulent type 078 and eleven PCR ribotypes that resemble the hypervirulent type
027. These markers enable us to categorize these strains into two clades of genetically
related strains. Analysis of the 078 and 027 loci revealed several interesting open reading
frames. The 078-region introduces an enzyme that is involved in the biosynthesis of
antibiotics. The 027-insert disrupts the thymidylate synthetase (thyX) gene (involved in DNA
replication) and replaces it by an equivalent, catalytically more efficient, thyA gene. Both
inserts may confer a selective advantage to the pathogen. The identified markers enable
rapid and specific recognition of hypervirulent strains and other C. difficile ribotypes with
similar characteristic features.

O4
GENOMIC CHARACTERISTICS OF A TOXIN A-NEGATIVE, TOXIN B- POSITIVE C.
DIFFICILE STRAIN FROM BEIJING, CHINA
Y. Cheng* 1 , P. Du 1 , C. Chen 1 , Q. Yan 1 , J. Lu 1 . 1 Hospital Acquired Infection Department,
State Key Laboratory for Infectious Disease Prevention and Control (SKLID), National
Institute for Communicable Disease Control and Prevention, Chinese Center for Disease
Control and Prevention, Beijing, 102206 China.
Recently, TcdA-negative, TcdB-positive (A-/B+) C. difficile strains have been reported
frequently in C. difficile infection (CDI) worldwide. However, its published isolation rate
ranged drastically from 3% to 75% in different regions, and the Asians and Latin Americans
seemed more prone to suffer from infection by A-/B+ strains. To further understand this
distinct variation in isolation rate between different regions, and to investigate the genomic
characteristics of A-/B+ C. difficile from Asia, we performed sequence comparison with seven
other published C. difficile sequences and bioinformatic analysis. First, we compared widelyused
primers NK1-NK2, NK2- NK3, NK104-NK105, A1C-A2N, and others (used to conduct
epidemic surveys in Japan, France, Argentina, and other countries). Second, we performed
bioinformatic and comparative genome analysis on the primer regions of 7 published C.
difficile sequences, looking for genomic sequence variations. Third, we selected an A-/B+ C.
difficile strain (BJ08) from a Beijing hospital. Then, a shotgun method was used to acquire its
genome sequence. Two DNA libraries, 500bp and 2kb DNA fragments for high throughput
genome sequencing with SOLEXA, were constructed. Our results show that the PCR primers
used in different countries for toxin detection do not provide uniform results. The intergroup
of toxin A+B+, and the intra group between toxin A+B+ and Toxin A-B+, shows more
diversity in the Paloc region. These diversities or single nucleotide polymorphisms (SNPs)
that occur in primers may weaken our signal when we use PCR methods to detect TcdA/B
regions. The genome of BJ08 was sequenced and assembled into 19 scaffolds, with N50 at
460,923 bp. Compared with sequenced genome of C. difficile 630, CD196 and
R20291(ribotype 017), our sequence shows a large degree of conservation in size, number
of open reading frames (ORF), GC content, gene length, and density. Thus, the genomic
information of BJ08 will provide us new primer positions for toxin detection for the coming
China CDI survey. Furthermore, our results may shed new light on the distinctly higher A-B+
incidence rate in Asia and Latin America.

O5
CRITICAL DIFFERENCES BETWEEN HISTORICAL AND HYPERVIRULENT TcdB.
Jimmy D. Ballard. The University of Oklahoma Health Sciences Center, Department of
Microbiology and Immunology, Oklahoma City, OK USA.
Infection with hypervirulent strains of Clostridium difficile is associated with a higher morbidity
and mortality compared to infection with historical strains of this pathogen. The underlying
reasons for the increased lethality of hypervirulent C. difficile strain are unknown. A major
virulence factor of C. difficile is the cytotoxin TcdB, which is responsible, in part, for the
pathology of the intestines as well as systemic effects. Unlike other proteins encoded within
the PaLoc of hypervirulent C. difficile, the sequence of TcdB has diverged from that of
historical strains. Our work has focused on determining how these sequence differences
impact the function and antigenic make-up of TcdB. Antibodies raised against the antigenic
carboxy-terminus of historical TcdB (TcdB HIST ) were found to not cross-neutralize
hypervirulent TcdB (TcdB HV ), suggesting the neutralizing epitopes differ between the two
forms of TcdB. Fine specificity mapping of epitopes in TcdB HIST and TcdB HV revealed clear
differences in the antigenic profiles of these proteins. The two forms of TcdB were found to
differ in autoprocessing, a critical step in cell entry. TcdB HV underwent more efficient
autoprocessing and was activated by previously unreported factors, including UDP and GDP.
Further studies using an activity based fluorescent probe found that TcdB HV engaged
intramolecular substrate more efficiently than TcdB HIST , providing an underlying mechanistic
explanation for differences in autoprocessing between the two forms of TcdB. These data
provide insight into a critical change in TcdB activity and indicate that variations in the
sequence of TcdB have altered the toxin‘s antigenic characteristics and efficiency of
autoprocessing.

O6
STRUCTURAL DETERMINANTS OF THE CLOSTRIDIUM DIFFICILE TOXIN A
GLUCOSYLTRANSFERASE ACTIVITY
R. N. Pruitt 1 , N. M. Chumbler 1 , M. A. Farrow 1 , S. A. Seeback 1 , D. B. Friedman 2 , B. W.
Spiller 1,3 , and D. B. Lacy* 1,2 . Departments of 1 Pathology, Microbiology and Immunology;
2 Biochemistry; Pharmacology; Vanderbilt University School of Medicine, Nashville TN 37232
USA.
The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB,
homologous glucosyltransferases capable of inactivating small GTPases within the host cell.
We present crystal structures of the TcdA glucosyltransferase domain (GTD) in the presence
and absence of the co-substrate UDP-glucose. While the enzymatic core is similar to that of
TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is
comparable to TcdB in its modification of Rho-family substrates and that, unlike TcdB, TcdA
is also capable of modifying Ras-family GTPases both in vitro and in cells. The
glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but
can be restored with autoproteolytic activation and GTD release. We propose a model
wherein the receptor-binding domain occludes the binding of GTPase substrates and discuss
the importance of cellular activation in determining the array of substrates available to the
toxins once delivered into the cell.

O7
CHEMICALLY INTERROGATING CLOSTRIDIUM DIFFICILE GLUCOSYLATING TOXIN
ACTIVATION
A. Shen* 2 , P.J. Lupardus 3 , A.W. Puri 4 , M.M. Gersch 4,5 , V.E. Albrow 1,3,6 , K.C. Garcia 3,6 , M.
Bogyo 1,2,4 . 1 Department of Pathology, Stanford University, Stanford, CA 94305 USA;
2 Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT,
05405 USA; 3 Department of Molecular and Cellular Physiology, Stanford University,
Stanford, CA, 94305 USA; 4 Department of Chemical and Systems Biology, Stanford
University, Stanford, CA, 94305 USA; 5 Department of Chemistry, Center for Integrated
Protein Science Munich (CIPSM), Technische Universitat Munchen, Garching, Germany;
6 Howard Hughes Institute, Stanford School of Medicine, Stanford, California, 94305 USA.
Multidomain toxins TcdA and TcdB are the primary mediators of Clostridium difficileassociated
disease. These toxins contain a cysteine protease domain (CPD) that
autoproteolytically releases a cytotoxic effector domain upon binding the eukaryotic-specific
small molecule inositol hexakisphosphate (InsP6) in target cells. Although CPD-mediated
processing is necessary for optimal toxin function, the mechanism by which the InsP6 signal
is sensed by the CPD remains poorly characterized. In order to study the regulation and
function of CPD-mediated processing of glucosylating toxins, we rationally designed covalent
small molecule inhibitors of TcdB CPD that inactivate TcdB holotoxin function in cells. By
converting the inhibitors into activity-based probes, we have developed tools that sensitively
detect InsP6-induced activation of TcdB CPD. Using this probe, we demonstrate that,
although InsP6 dramatically shifts the conformational equilibrium of the CPD towards an
active conformer, apo-CPD unexpectedly adopts an active conformer at low frequency.
Structural analyses combined with disulfide bond engineering studies identify residues that
functionally and mechanically couple the InsP6 binding site to the active site. Collectively, our
results define an allosteric circuit that allows the glucosylating toxins to sense and respond to
the eukaryotic environment. This work highlights the value of using chemical tools to study
mechanisms of enzyme regulation, validate the CPD as a druggable target, and provides
general insight into the nature of protein allostery. These tools may additionally facilitate
analyses of glucosylating toxins in other Clostridium spp.

O8
A NEUTRALIZING INTRABODY TO STUDY AUTOCLEAVAGE OF CLOSTRIDIUM
DIFFICILE TOXIN B
S. Li*, L. Shi, D. Schmidt, J. M. Tremblay, X. Sun, S. Tzipori, K. Chen, Y. Zhang, C. B.
Shoemaker, H. Feng. Infectious Disease Division, Department of Biomedical Sciences, Tufts
Cummings School of Veterinary Medicine, N. Grafton MA 01536, USA.
Clostridium difficile induces a serious and potentially fatal inflammatory disease of the colon
and is the most prevalent cause of antibiotic-associated diarrhea and colitis in hospitals. The
disease is mainly caused by two exotoxins, TcdA and TcdB. TcdB is a 269 KD protein
containing at least 2 enzymatic domains, the glucosyltransferase domain (GTD) and the
cysteine protease domain (CPD). After internalization, the GTD is released into the cytosol
and enzymatically inactivates Rho GTPases, leading to the intoxication of host cells. The
cytosolic liberation of GTD is thought to be mediated through auto-cleavage by CPD, a
notion that is primarily based on observations from in vitro studies. Direct evidence
generated using intact cells is necessary to prove this concept. In this study, we identified a
VHH antibody, 7F, which is capable of neutralizing the cytotoxicity of TcdB on cultured cells.
In vitro studies demonstrate that 7F blocks CPD-mediated auto-cleavage and release of
GTD. Epitope mapping found that 7F binds to the GTD sequence immediately adjacent to
the auto-catalytic site, so its binding likely blocks access to the protease cleavage site. To
study whether blocking CPD-mediated auto-cleavage in TcdB-exposed intact cells inhibits
the intoxication process, 7F was expressed as an intrabody in the mammalian cell cytosol.
The 7F VHH extracted from transiently transfected mammalian cells bound to TcdB and
neutralized the toxin in cell assays. Cells expressing 7F intrabody were resistant to
intoxication of TcdB, but not those expressing C6, a non-neutralizing VHH antibody that also
recognizes GTD of TcdB. Our data thus provide direct evidence that CPD-mediated autocleavage
is crucial for the cellular activity of TcdB.

09
IDENTIFICATION OF NOVEL ENDOCYTOSIS PATHWAY FOR TcdB OF CLOSTRIDIUM
DIFFICILE THROUGH shRNAmir LIBRARY SCREENING AND HIGH-THROUGHPUT
SEQUENCING ANALYSIS
C. Li, C. Cai, R. He, X. Wu, Y. Wang, W. Wei*. College of Life Sciences, Peking University,
Beijing China 100871.
As the leading cause of healthcare associated diarrhea in North America and Europe,
Clostridium difficile infection has drawn great attention in research. TcdA and TcdB are two
essential virulence determinants. By employing human shRNAmir library screening, in
combination with deep sequencing analysis, we have identified a number of novel host
pathways that are important for both toxins‘ endocytosis and damaging mechanism. These
screenings have been carried out in three cell lines: HeLa, HT29 and KBM7 (a human
myeloid leukemia cell), by using TcdB, TcdA, or chimeric DTA-TcdB toxins. Bioinformatic
analysis of these data reveals that TcdB employs a novel mechanism to gain its access to
host cells. In addition, a few cell surface membrane proteins have been identified to play
important roles in TcdB toxicity. More interestingly, two of these receptor/co-receptor
candidates belong to the same family, and their normal functions include the activation of
signaling molecule RhoA. Further characterization of these cell surface proteins as well as
other essential host targets are still in process. Details of this work will be presented.

O10
THE MECHANISM OF CONJUGATION IN CLOSTRIDIUM PERFRINGENS
J. I. Rood* 1 , T. L. Bannam 1 , R. Bantwal 1 , J. Wisniewski 1 , C. J Porter 2 and J. C Whisstock 2 .
ARC Centre of Excellence in Structural and Functional Microbial Genomics, 1 Department of
Microbiology and 2 Department of Biochemistry and Molecular Biology, Monash University,
Clayton, Victoria 3800, Australia.
In Clostridium perfringens antibiotic resistance genes and genes encoding extracellular
toxins are often carried on conjugative plasmids that have the tcp conjugation locus. We
have mutated each of the eleven genes in this locus and examined their role in the
conjugation process. Five tcp genes are essential for conjugative transfer; they encode a
putative FtsK-like coupling protein (TcpA), two integral membrane proteins of unknown
function (TcpD and TcpE), a potential hexameric ATPase (TcpF) and TcpH, which appears
to be the major structural component of the transfer complex. Three proteins are required for
maximal transfer efficiency, since mutation of their structural genes leads to greatly reduced
transfer frequencies. These proteins include IntP, which appears to be an unusual type of
DNA relaxase, TcpC, a transmembrane protein that localises to the cell membrane
independently of the other Tcp proteins, and TcpG, a peptidoglycan hydrolase with two
functional hydrolase domains. The remaining genes are not required for conjugative transfer.
The crystal structure of the soluble C-terminal component of TcpC (TcpC99-354) has been
determined to 1.8-Å resolution. It crystallised as a homotrimer, with each monomer
comprising two independent domains that have very similar structures, even though they
only have 16% sequence identity. Comparative structural analysis showed that TcpC was
member of the NTF-2 superfamily and that each of the closely related structural domains
was related to VirB8-like conjugation proteins, which are important components of type IV
secretion systems from Gram-negative bacteria. Both of the C-terminal domains of TcpC
were required for its function, with the second domain (aa 239-354) having a major role in
protein-protein interactions. Based on these studies, detailed site-directed mutagenesis
experiments, bacterial two-hybrid analysis and protein-protein interaction experiments we
have developed a model that provides insight into the conjugation process in C. perfringens.

O11
CHARACTERIZATION OF THE TOXIN PLASMIDS OF CLOSTRIDIUM PERFRINGENS
Bruce McClane* 1 , Jihong Li 1 , Kazuaki Miyamoto 2 , Sameera Sayeed 1 , Derek Fisher 1 , Abhijit
Gurjar 1 , Meredith Hughes 3 , Rachel Poon 3 , Vicki Adams 3 and Julian Rood 3 . 1 Department of
Microbiology and Molecular Biology, University of Pittsburgh School of Medicine, Pittsburgh,
USA, 2 Department of Microbiology, Wakayama Medical University, Wakayama, Japan and
3 Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Clostridium perfringens is a major pathogen of humans and livestock due to its prolific toxinproducing
ability. The ability to produce alpha, beta, epsilon and iota toxins is used to classify
C. perfringens isolates into types A-E. With the exception of alpha toxin and perfringolysin O,
all other C. perfringens toxins can be encoded by genes on large plasmids that range in size
from ~40 kb to >150 kb. Molecular Koch‘s postulates analyses have demonstrated that
some of these plasmid-encoded toxins are essential when C. perfringens causes diseases
originating in the intestines. There is considerable diversity amongst C. perfringens isolates
with respect to toxin plasmid carriage; a single strain can carry up to three different plasmids
and a single plasmid can encode up to three different toxin genes. Some toxin plasmids are
related and share a common backbone. Some, if not all, C. perfringens toxin plasmids can
conjugatively transfer, probably due to their carriage of a tcp locus, which could convert
normal intestinal flora C. perfringens strains to virulence, thereby enhancing disease. In
addition, most C. perfringens plasmid-borne toxin genes are flanked by IS elements that can
apparently mediate excision of these toxin genes from their plasmids. The association of
many toxin genes with both conjugative plasmids and IS elements allows toxin gene mobility
that likely impacts C. perfringens toxin plasmid diversity, evolution and virulence. Recently,
some C. perfringens isolates were identified that carry variant plasmid-borne enterotoxin and
iota toxin genes. These isolates initially genotyped as type A because their variant iota toxin
gene did not amplify with current PCR toxin genotyping assays, indicating that some PCRbased
diagnoses of C. perfringens infections could be incorrectly attributed to type A strains.
Lastly, PFGE Southern blot studies identified only limited diversity of beta toxin and epsilon
toxin plasmids in type B isolates. This observation could suggest that only certain toxin
plasmid combinations are compatibly maintained in a single C. perfringens cell. If true, this
may help to explain the limited number of C. perfringens types found in nature.

O12
THE DEVELOPMENT OF CLOSTRIDIAL GENE SYSTEMS AND THEIR EXPLOITATION
N.P. Minton*, S.T. Cartman, S.A. Kuehne, J. T. Heap, R. Ng, D. Walker, M. Ehsaan, M.L.
Kelly, S. Baban, A. Kubiak, K. Winzer and A. Cockayne. School of Molecular Medical
Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park,
Nottingham NG7 2RD, United Kingdom.
In recent years, tools for genetic modification of Clostridium difficile have been considerably
expanded by the development of both directed (Cambell-like plasmid integration and the
ClosTron) and random (transposon mariner) insertional systems for ‗knock-out‘ and ‗knockin‘.
However, as insertional mutagens, their deployment can cause polar effects. Moreover,
they are limited in the amount of DNA that can be inserted into the genome. We have now
developed a new series of systems, based on allelic exchange, with which it is possible both
to routinely make in-frame deletions of target genes, using negative selection markers, and
to insert DNA fragments of any size or complexity into the genome, a prerequisite for
synthetic biology. The former may also be used to make subtle changes (ie., the introduction
of SNPs) to specific genes by allelic exchange. The latter system, termed Allele-Coupled
Exchange (ACE) technology, provides the facility to develop in-frame deletion/ allelic
exchange methodology for virtually any clostridial species. It may additionally be deployed to
introduce wildtype alleles into the chromosome, dispensing with the need to use autonomous
plasmids for complementation studies. Moreover, it has provided a simple assay to confirm
the essentiality of genes suggested to be essential on the basis of under-representation in
transposon-directed insertion site sequencing (TraDIS) of mariner transposon mutant pools.
The basis of these systems will be discussed, together with examples of their deployment.
This work was supported by the UK Medical Research Council (G0601176) and the
European Union (HEALTH-F3-2008-223585).

O13
ANALYSIS OF THE GENOME-WIDE TEMPORAL EXPRESSION OF A CLOSTRIDIUM
DIFFICILE 027 HYPERVIRULENT STRAIN IN MONOXENIC MICE
A. Barketi-Klai 1 , M. Monot 2 , S. Hoys 1 , S. Lambert 1 , B. Dupuy 2 , I. Kansau 1 , and A.
Collignon* 1 . 1 EA 4043, USC INRA Faculté de Pharmacie, Université Paris-Sud 11, 92296
Châtenay-Malabry, France; 2 Institut Pasteur, 28, rue du Docteur Roux, 75015, Paris, France.
Clostridium difficile is a frequent cause of recurrent post-antibiotic diarrhea and
pseudomembranous colitis. Recently, new hypervirulent epidemic strains, such as the NAP1
027 strain, have emerged and have been responsible for several outbreaks worldwide. The
hypervirulence of the Clostridium difficile 027 strains seems related to several factors
including high-toxin A and B production, presence of the binary toxin and high sporulation
rate. However, other factors could be involved in its hypervirulence. In this work, we focused
our interest on the colonization mechanism of a C. difficile 027 strain by an in vivo
transcriptomic approach. We analyzed the C. difficile 027 transcriptome during the early
stages of intestinal colonization in our monoxenic mouse model. Bacterial mRNA was
extracted from the caeca at 4, 6, 8, 14 and 38 h post-infection, then transcribed into cDNA by
RT-PCR and finally labeled for hybridization to DNA microarrays (Agilent ). The overall
analysis of data (by software Ma2HTLM) showed a differential expression of 707 genes in
the early stage (4-6 h) and late stage (14-38h) of colonization. 285 genes were significantly
up-regulated, while 456 were down-regulated. Genes encoding known virulence factors such
as the toxins were up-regulated in the late phase (38 h), whereas genes encoding surface
proteins likely involved in the colonization process showed little or no variation. In addition,
genes involved in sporulation, membrane transport, metabolism and fermentation, underwent
significant changes during the infection process suggesting a potential role for the encoded
proteins in the early stages of C. difficile intestinal colonization. This analysis will be
completed by RT-qPCR on the regulated genes during the in vivo kinetics of colonization.
These results will help to elucidate the mechanisms of intestinal colonization by C. difficile
027 strain and to better understand the hypervirulence of this strain.

O14
PHYLOGENOMIC ANALYSIS OF CLOSTRIDIUM BOTULINUM STRAINS
E. A. Johnson* 1 , L. Papazisi 2 , M. J. Jacobson 1 , G. Lin 1 , and S. N. Peterson 2 . 1 Department of
Bacteriology, Food Research Institute, University of Wisconsin-Madison, Madison, WI USA;
2 The J. Craig Venter Institute (JCVI), Rockville, MD USA.
Clostridium botulinum is a heterogenous group of bacteria that have the common property of
producting botulinum neurotoxin (BoNT). There is a growing body of evidence to suggest that
genomic diversity within this taxon is relatively large and that exchange of genetic elements
among members of this group is commonplace. There is considerable genetic variation even
among the seven major seven BoNT serotypes, as demonstrated by the recognition of at
least many BoNT subtypes. The most studied serotype, BoNT/A, has been found in a large
and diverse group of clostridia, most of which express the subtype BoNT/A1. Nearly 50% of
C. botulinum strains producing BoNT/A1 have been shown to also encode unexpressed
variants of BoNT/B with a distinct cluster arrangement. The flow of genetic information within
the C. botulinum group motivated us to identify novel genes for the purpose of creating a
―species‖ DNA microarray to better understand the ancestral relationships among its
members. Based on preliminary genotyping (MLST, and CGH using a single-genome-based
array), 20 diverse C. botulinum strains were selected for targeted sequencing. We developed
a strategy we refer to as Gene Discovery (GD) that enables the identification and rapid
characterization of strain-specific sequences present in microbial genomes. Sequence
information obtained from this project, and from other publicly available sources, led to the
development of a comprehensive species microarray containing ca. 40,000 genomic markers
The availability of the C. botulinum species DNA microarray has allowed us to carry out a
CGH genotyping project to validate this microarray as well as understand the phylogenomic
relationships among members of this very diverse group. The putative role of horizontal
gene transfer in C. botulinum phylogeny is also discussed.
Acknowledgments: This work was supported by the NIAID contract No. N01-AI-15447 to Pathogen Functional
Genomics Resource Center at JCVI. Studies in EAJ‘s laboratory was partly sponsored by the NIH/NIAID Regional
Center of Excellence for Bio-defense and Emerging Infectious Diseases Research (RCE) Program. The authors
wish to acknowledge membership within and support from the Pacific Southwest Regional Center of Excellence
grant U54 AI065359 and from the Region V ‗Great Lakes‘ RCE (NIH award 1-U54-AI-057153).

O15
CLOSTRIDIUM DIFFICILE CDT AND CLOSTRIDIUM PERFRINGENS IOTA TOXIN:
IDENTIFICATION OF THEIR MEMBRANE RECEPTORS AND ACTIONS ON THE
CYTOSKELETON OF TARGET CELLS
Klaus Aktories. Institute of Experimental and Clinical Pharmacology and Toxicology,
University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany.
Clostridium difficile infection (CDI) causes antibiotic-associated diarrhea and
pseudomembranous colitis. Hypervirulent strains of the pathogen, which are responsible for
increased morbidity and mortality of CDI, produce the actin-ADP-ribosylating toxin C. difficile
transferase (CDT) in addition to the Rho glucosylating toxins A and B. CDT is a binary actin-
ADP-ribosylating toxin, which consists of an enzymatic component and a separated
binding/translocation component. The toxin ADP-ribosylates G-actin at Arg-177. Thereby, the
actin cytoskeleton is depolymerized and formation of microtubule protrusions is induced. The
protrusions form a network on the cell surface of host target cells and increase adherence
and colonization of clostridia, indicating a role as virulence factors.
We have identified the cell surface receptor for CDT by gene trapping using a haploid genetic
screen. We found that C. perfringens iota toxin, a related binary actin-ADP-ribosylating toxin,
enters target cells via the same membrane receptor. Nature and properties of the receptor
will be discussed.

O16
IDENTIFICATION OF HOST FACTORS CONTRIBUTING TO CLOSTRIDIUM
PERFRINGENS EPSILON-TOXIN-INDUCED CELL DEATH
C.M. Fennessey 1 , S.E. Ivie 1 , J.Sheng 1 , D.H. Rubin 1,2,3 , and M.S. McClain* 1 . 1 Division of
Infectious Disease, Department of Medicine, Vanderbilt University School of Medicine,
Nashville, Tennessee, USA; 2 Department of Microbiology and Immunology, Vanderbilt
University School of Medicine, Nashville, Tennessee, USA; 3 Research Medicine, VA
Tennessee Valley Healthcare System, Nashville, Tennessee, USA.
Results of recent studies indicate that the mechanism by which pore-forming toxins mediate
cell death is more complicated than previously thought and that inhibition of cellular
responses can protect from cell death induced by pore-forming toxins. Thus, identifying host
factors contributing to the activity of pore-forming toxins may identify candidates for new
therapeutic approaches. We have used gene trap selection to identify mammalian factors
contributing to Clostridium perfringens epsilon-toxin-induced cytotoxicity. One gene identified
encodes caveolin 2 (CAV2), a membrane protein involved in endocytosis, signal
transduction, lipid metabolism, cellular growth control and apoptosis. RNA interference was
used to disrupt CAV2 expression and decreased CAV2 correlated with decreased toxin
sensitivity. Immunoprecipitation of CAV2 from toxin-treated cells resulted in co-purification of
epsilon-toxin. Similarly, epsilon-toxin was co-purified when caveolin 1 (CAV1, a protein
known to interact with CAV2) was immunoprecipitated from toxin treated cells. Together,
these results suggest that interaction between epsilon-toxin and the caveolins is an important
step in the process by which the toxin induces cell death, and a new target for possible
therapeutic intervention.

O17
EXPRESSION AND FUNCTIONAL CHARACTERISATION OF CLOSTRIDIUM
PERFRINGENS NETB TOXIN
S. P. Fernandes da Costa* 1 , C. G. Savva 2 , M. Bokori-Brown 1 , C. E. Naylor 2 , A. K. Basak 2 , D.
S. Moss 2 , R. W. Titball 1 . 1 College of Life and Environmental Sciences, University of Exeter,
Exeter, United Kingdom; 2 Institute of Structural and Molecular Biology, Birkbeck College,
London, United Kingdom.
Clostridium perfringens is able to cause several diseases as a consequence of the
differential production of a variety of toxins, some of them known to be the most powerful
naturally occurring poisons. NetB (Necrotic enteritis toxin B) is a novel β-pore-forming toxin
produced by C. perfringens toxinotype A, and to a lesser extent by strains of type C. NetB
has been associated with avian necrotic enteritis, a severe gastro-intestinal disease that
manifests in gross lesions within the intestines of poultry. However, the molecular basis of
toxicity is still little understood and remains to be revealed. In this study, we present an
expression system for netB in E. coli and show that the recombinant protein is able to form
oligomeric complexes. Cytotoxicity assays on LMH cells (chicken hepatocellular carcinoma
cell line) showed that the toxin is capable of forming functional pores and to cause cell lysis.
Moreover, cryo-electron microscopy of NetB in the presence of liposomes confirm that the
NetB is able to form multimeric ring-like structures. Based on sequence similarities with
related pore-forming toxins a monomeric/heptameric model of NetB has been designed and
been used to identify potentially important amino acids for oligomerisation, cell binding, and
pore formation. Several site specific mutants have been created so far and their role on NetB
functionality is currently being investigated. Non-toxic NetB variants might have the potential
to be used as a toxoid to protect chicken against necrotic enteritis.

O18
STRUCTURAL RELATIONSHIPS IN CLOSTRIDIAL BETA-PORE-FORMING TOXINS
A. K. Basak*, C. E. Naylor, C. Savva, J. Huyet, and T. Yelland. Department of Biological
Sciences, Birkbeck College, Malet St, London, WC1E 7HX, UK.
Clostridium perfringens is a facultative anaerobe that secretes a large panel of toxins and is
associated with a broad range of diseases in man and animals. A number of these toxins,
including the epsilon, delta and beta-toxins, enterotoxin and the recently discovered NetB
have been identified as pore-forming toxins. We have determined the 3D-structures of a
number of these pore-forming toxins by X-ray crystallography, and are investigating others
by Electron Microscopy, Biophysical techniques and Crystallography. Our work has
uncovered interesting, and often unexpected, relationships between these toxins and also
with toxins from other organisms. Our recently published enterotoxin (CPE) structure is a
causative agent of type-A food poisoning and is linked to hospital- and community-acquired
antibiotic-associated and sporadic diarrhoea. We will discuss the structural homology to the
epsilon-toxin and other members of the aerolysin-like beta-pore-forming family. This
relationship has enabled us to develop several models for oligomeric pore-formation. In
addition, we have recently determined the structure of the soluble form of the delta-toxin to
2.4 Å. This toxin is implicated in haemolysis in animals. The structure is part of the alphahaemolysin
like family of beta-pore-forming toxins, and is closely related to the recently
discovered NetB toxin, which is responsible for enteric disease in farmed poultry. Our
structure has enabled us to build models for the pore-inserted forms of both delta-toxin and
NetB, which we have validated by electron microscopy and biophysical studies. The
mechanisms of pore-formation of these proteins are different, as the receptors they
recognize are diverse and the regions of the proteins associated with these functions also
differ markedly. These aspects of the proteins will be presented.

O19
ROLE OF CARBON CATABOLITE REPRESSION (CCR) IN THE PATHOGENICITY OF
CLOSTRIDIUM DIFFICILE
A. Antunes, E. Camiade, M. Monot, I. Martin-Verstraete and B. Dupuy*. Laboratoire
Pathogenèse des Bactéries Anaérobies, Institut Pasteur, 25 rue du Dr Roux, 75724, Paris,
France.
Many bacterial pathogens produce virulence factors to overcome the drastic changes in
environment that they encounter during infection. Consequently, global regulation of
virulence genes must be considered as an important step of the pathogenicity process. In
Clostridium difficile, it is now well establish that toxin production is regulated in response to
various environmental signals. Thus, in the presence of glucose or other rapidly
metabolizable sugars, C. difficile toxin synthesis is repressed involving the Carbon Catabolite
Repression (CCR) mechanism. CcpA is the global transcriptional regulator of the CCR,
which generally binds a cis-acting catabolite response element (cre) in the promoter region of
the controlled genes. Furthermore its DNA binding activity is enhanced by its interaction with
HPr-Ser-P, a component of the phosphotransferase system (PTS). We have shown by in
vivo and in vitro approaches that CCR is implicated in the glucose-dependent repression of
the toxin gene. CcpA binds to regulatory regions of tcdA and tcdB genes but not through the
cre motif. Moreover, only FBP enhances CcpA binding, establishing a novel mode of action
of CcpA. In order to understand the impact of glucose and the regulatory role of CcpA in C.
difficile physiology, we performed a transcriptome analysis during the growth of JIR8094
strain and its isogenic ccpA mutant, in the presence or absence of glucose. 18% of C. difficile
genes are regulated in response to glucose availability and half of them are controlled by
glucose in a CcpA-dependent manner. CcpA plays a central role in several metabolic
pathways and cellular processes like stress response and sporulation. We have recently
identified more than 60 CcpA direct targets by ChIP to chip analysis and we proposed a
CcpA binding motif. Finally, we showed in the hamster model that virulence is delayed in the
ccpA mutant indicating that CcpA controls the infectious process of C. difficile. To look for
processes potentially related to the CDI, co-ordinately regulated by CcpA, we used a axenic
mouse model to determine the CcpA regulon in vivo. 183 genes are specifically controlled by
CcpA in vivo including genes involved in ethanolamine utilization, an abundant compound of
the intestinal tract that can be used as carbon and nitrogen sources during the C. difficile
gastrointestinal lifestyle.

O20
A NEW ROLE FOR C. DIFFICILE GLUTAMATE DEHYDROGENASE
R. Govind*. Division of Biology, Kansas State University, Manhattan, KS, 66506 USA.
Clostridium difficile produces an NAD-specific glutamate dehydrogenase (GDH), which
converts L- glutamate into alpha-ketoglutarate through an irreversible reaction. The enzyme
GDH is detected in the stool samples of the patients with C. difficile-associated disease and
serves as one of the diagnostic tools to detect C. difficile infection. Initial experiments
detected GDH in the supernatant fluids of C. difficile cultures. To understand the role of this
extracellular GDH, a mutation was introduced into the GDH encoding gene, gdhA. The
presence of the gdhA mutation in the C. difficile chromosome was confirmed by PCR and the
absence of GDH in the mutant was documented by Western blot analysis. Various
phenotypic assays were performed to understand the effect of GDH on C. difficile growth.
Higher sensitivity of the mutant to H 2 O 2 , as compared to the parent strain, led me to
hypothesize that GDH might play an important role in fighting against reactive oxygen
species (ROS) released as a part of host defense against C. difficile infection. Results that
suggest the role of extracellular GDH in C. difficile defense against ROS will be discussed.

O21
NOVEL REGULATORY MECHANISM FOR SPORE FORMATION AND ENTEROTOXIN
PRODUCTION IN CLOSTRIDIUM PERFRINGENS
K. Ohtani* 1 , H. Hirakawa 2 , D. Pareds-Sabja 3 , K. Tashiro 4 , S. Kuhara 4 , M. Sarker 5 and T.
Shimizu 1 . 1 Kanazawa Univ., Japan; 2 Kazusa DNA Research Institute, Japan; 3 Universidad
Andres Bello, Chile, 4 Kyushu Univ., Japan; 5 Oregon State University, USA.
C. perfringens causes clostridial myonecrosis (gas gangrene) and gastrointestinal (GI)
diseases in humans. The most common cause of C. perfringens associated food poisoning is
the consumption of C. perfringens vegetative cells followed by sporulation and production of
enterotoxin in the gut. However, C. perfringens-associated gas gangrene and other nonfoodborne
GI illnesses occur when C. perfringens spores come in contact with the host,
where spores undergo germination followed by cell proliferation and toxin secretion. Despite
the importance of spore formation in C. perfringens pathogenesis, the detailed regulation of
sporulation has not yet been defined. Although C. perfringens lacks some sporulation-related
genes compared to Bacillus subtilis, the most important regulatory genes encoding the
master regulator of sporulation (Spo0A) and sigma factors are highly conserved between
these two species. Despite the identification of regulators for sporulation that are common to
both Clostridium and Bacillus species, no unique regulators for this process have been
identified in C. perfringens. In this context, our microarray and bioinformatic analyses
identified a novel candidate gene (virX) that might be involved in repression of genes
encoding positive regulators (i.e., Spo0A and sigma factors) of C. perfringens sporulation.
When a virX mutant was constructed in food poisoning strain SM101 and the sporulation
phenotype of the isogenic strains was compared, the virX mutant had a higher sporulation
efficiency compared to wild-type. Northern blot analyses demonstrated that the transcription
of sigE, sigF and sigK was strongly induced in a 2.5h - culture of the virX mutant. Moreover
the transcription of the enterotoxin gene (cpe) was also strongly induced in a virX mutant.
Western blotting analyses confirmed much higher levels of enterotoxin production in the virX
mutant compared to wild-type. Importantly, the virX mutant‘s phenotype could be restored by
complementing the mutant with wild-type virX, indicating that the higher levels of sporulation
and enterotoxin production in the virX mutant are specifically due to inactivation of the virX
gene. Collectively, our current study, for the first time, identified a novel negative regulator for
sporulation and enterotoxin production in C. perfringens, which might explain why two
different morphotypes of C. perfringens cells are required to cause gas gangrene
(vegetative) and food poisoning (sporulation).

O22
CLOSTRIDIUM DIFFICILE Spo0A REGULATES SPORULATION, BUT NOT TOXIN
PRODUCTION, BY DIRECT BINDING TO TARGET DNA
K.E. Rosenbusch 1 , D. Bakker 1 , E.J. Kuijper 1 , W.K. Smits* 1 . 1 Department of Medical
Microbiology, Leiden University Medical Center, Leiden, the Netherlands
Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant
endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the
symptoms can range from a mild diarrhea to potentially fatal toxic megacolon and
pseudomembranous colitis. Endospore formation in Firmicutes, including C. difficile, is
governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription
factor is also directly or indirectly involved in various other cellular processes. Here, we
report that C. difficile Spo0A shows a high degree of similarity to the well characterized B.
subtilis protein and recognizes a similar binding sequence, with two guanine residues that
are crucial for specific binding. We find that our laboratory strain C. difficile 630Deltaerm
contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain
630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile
Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early
sporulation genes, and several other putative targets. In silico analyses and in vitro binding
assays suggest that the genes encoding the major clostridial toxins TcdA and TcdB, the
binary toxin locus CdtAB, and their regulators TcdR and CdtR are not direct targets of
Spo0A. These results identify, for the first time, putative targets of the Spo0A protein in C.
difficile and make a direct effect of Spo0A on toxin production in this organism highly unlikely.

O23
REGULATOR-MEDIATED MODULATION OF TOXIN PRODUCTION AND SPORULATION
IN AN EPIDEMIC CLINICAL ISOLATE OF CLOSTRIDIUM DIFFICILE
K. Mackin* 1 , G. Carter 1 , P. Howarth 1 , R. Govind 2 , B. Dupuy 2 , G. Douce 3 , J. I. Rood 1 , and D.
Lyras 1 . 1 Department of Microbiology, Monash University, Victoria, Australia; 2 Unité de
Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France; 3 Division of Infection and
Immunity, FBLS Glasgow Biomedical Research Centre, University of Glasgow, UK.
Epidemic ribotype 027 isolates of Clostridium difficile have emerged throughout the world
over the last decade. These strains are reported to produce significantly more toxin than
other clinical isolates; this is thought to be due to a nonsense mutation within tcdC, which
encodes a negative regulator of toxin production. The emergence of ribotype 078 isolates in
the human population is also of concern as these strains also are associated with more
severe disease. Various hypotheses have been proposed for the emergence of these strains,
and for their persistence and increased virulence, but supportive experimental data are
lacking. To study these isolates at the molecular level, we have developed a new and more
efficient plasmid transfer system which has facilitated the genetic manipulation of both
ribotype 027 and 078 strains. We have carried out two separate series of proof-of-principle
experiments. Firstly, we have used it to inactivate the gene encoding the global regulator
Spo0A in different strain backgrounds, including ribotypes 027 and 078. Spo0A controls the
initiation of sporulation, and the mutants constructed in this study were unable to sporulate;
complementation restored this phenotype. In other bacteria Spo0A is also involved in the
regulation of other cell behaviour such as competence, motility, biofilm formation and toxin
production. Interestingly, our results show that Spo0A appears to be involved in TcdA and
TcdB production in 027 strains, but not in the other strain backgrounds tested. Secondly, we
have used the new plasmid transfer system to complement an 027 epidemic isolate with an
intact copy of tcdC. This complementation resulted in a significant repression of toxin
production. Most importantly, using the hamster infection model we showed that
complementation of the defective tcdC with an intact copy of the gene attenuated the
hypervirulent 027 phenotype. These data provide definitive evidence that TcdC is a negative
regulator of toxin production in C. difficile and, for the first time, evidence that mutation of
tcdC in epidemic 027 isolates is an important factor in the development of hypervirulence.

O24
ANIMAL MODELS TO STUDY CLOSTRIDIUM PERFRINGENS DISEASES
F.A. Uzal *1 , B.A. McClane 2 , J. Rood 3 , J. Saputo 1 , J.P. Garcia 1 , J. Caserta 2 , S. Robertson 2 , M.
Ma 2 , J.E. Vidal 2 , A. Shrestha 2 , M. Hughes 3 , R. Poon 3 and V. Adams 3 . 1 University of
California Davis, USA; 2 University of Pittsburgh, USA; 3 Monash University, Australia.
Several animal models have been developed recently to study Clostridium perfringens
diseases of animals and humans. Models in rabbits, mice, sheep, and goats have allowed us
to conduct molecular Koch‘s postulates analyses for several toxins and to study the
pathogenicity of several C. perfringens diseases. Examples of these models include a mouse
ligated intestinal loop model to study systemic effects and lethality of enterotoxin (CPE).
Lethality was observed after inoculation of CPE into ligated intestinal loops, and a correlation
was noted between intestinal histological damage and lethality. A dose-dependent increase
in serum CPE and serum potassium levels was observed, as was CPE binding to the liver
and kidney. These data suggest that CPE can be absorbed from the intestine into the
circulation, followed by binding of the toxin to internal organs to induce potassium leakage,
which can cause death. Rabbit small intestinal loops were used to study the use of a soluble
claudin-4 receptor decoy as a defense against CPE. Intestinal loops incubated with claudin-4
and then with CPE for 6 hours did not show the typical fluid accumulation and histological
damage seen in loops incubated with CPE alone. These results suggest that claudin receptor
decoys could have application in preventing or treating CPE-associated disease. Rabbit
intestinal loops and mouse intraduodenal models can be used to demonstrate that the
VirS/VirR two-component system regulates intestinal pathogenicity and lethality of C.
perfringens type C. An isogenic virR null mutant lost the ability to cause necrotic enteritis in
rabbit small intestinal loops and showed strongly attenuated lethality in a mouse
intraduodenal model. Mouse, sheep, and goat models are useful in demonstrating that
epsilon toxin (ETX) is essential for disease in sheep, goats, and mice. Cultures of a wild-type
D isolate inoculated intraduodenally caused acute clinical disease and death in sheep, goats,
and mice, while no disease was observed after inoculation of an isogenic ETX toxin null
mutant. These results provide evidence that ETX has a key role in type D disease
pathogenesis. Overall, our studies indicate the importance of combining genetic studies and
the use of animal disease models to study the pathogenesis of diseases caused by C.
perfringens.

025
SYSTEMIC DISSEMINATION OF CLOSTRIDIUM DIFFICILE TOXINS A AND B IS
ASSOCIATED WITH SEVERE FATAL DISEASE IN THE PIGLET AND MOUSE MODELS
J. Steele 1 , K. Chen* 1 , X. Sun 1 , Y. Zhang 1,2 , H. Wang 1,3 , S. Tzipori 1 , and H. Feng 1 . 1 Tufts
Cummings School of Veterinary Medicine, North Grafton, MA USA; 2 School of
Bioengineering, East China University of Science and Technology, Shanghai, China; 3 School
of Bioscience and Biotechnology, South China University of Technology, Guangzhou, China.
Clostridium difficile infection (CDI), the most common cause of antibiotic-associated diarrhea
and colitis in developed countries, can cause a wide range of disease, from mild diarrhea to
fulminant systemic disease. Disease is mainly caused by two exotoxins, TcdA and TcdB.
Although the incidence of systemic CDI and fatality has increased rapidly in recent years, the
causes for the development of systemic disease are not well understood but may be due to
the toxins liberated into the circulation, despite a current lack of such evidence. Using an
ultrasensitive cytotoxicity assay developed by our laboratory, we measured TcdA and TcdB
in serum and body fluids of piglets and mice exposed to CDI to investigate the relationship
between presence of toxins in body fluids and systemic manifestations of CDI. We found that
TcdA and TcdB are frequently liberated into the blood and body fluids in animals with
systemic CDI. The presence of toxins in the circulation caused significant systemic
manifestations and was also associated with fatal consequences. Both piglets and mice may
develop lesions of the respiratory tract and ascites, and in the piglet model, cardiac lesions
and pancreatitis were also noted in a few cases. Pro-inflammatory cytokines IL-1β and IL-6
were elevated in the serum of mice and piglets with systemic CDI, highlighting the potential
for inflammation induced tissue damage systemically. Interestingly, IL-4, an antiinflammatory
cytokine was elevated in piglets with non-systemic CDI, indicating a potential
protective effect when anti-inflammatory mediators are induced. Expectedly, systemic
administration of neutralizing antibodies against both toxins to mice blocked the development
of systemic disease. Our study demonstrates the existence of a strong correlation between
toxemia and the occurrence of systemic disease, supporting the hypothesis that systemic
CDI is most likely due to the toxicity of TcdA and TcdB and the induction of pro-inflammatory
cytokines by the toxins.

O26
ASPECTS OF THE HAMSTER MODEL OF INFECTION.
A.M. Buckley* 1 , J. Spencer 1 , L.F. Dawson 2 , E.J. Beards 3 , J.M. Ketley 3 , B.W. Wren 2 , and
G.R. Douce 1 . 1 Dept of Microbiology, University of Glasgow, Glasgow, UK G12 8TA;
2 Pathogen molecular biology unit, LSHTM, London, UK WC1E 7HT; 3 Department of
Genetics, University of Leicester, Leicester, UK LE1 7RH.
Clostridium difficile is an important cause of antibiotic associated disease, which is of high
socio-economical importance that has recently been compounded by the emergence of
epidemiological strains of different toxinotypes. C. difficile cases attributed to these strains
show increased disease severity and high recurrence rates (up to 36%), which is increasing
the financial burden faced by hospitals. The hamster model of infection is widely accepted as
an appropriate model for studying aspects of C. difficile host-pathogen interactions. Infection
of hamsters with toxigenic C. difficile manifests as hemorrhagic caecitis (wet tail) followed by
death. Using this model we characterised the virulence of naturally occurring isolates which
vary in toxin expression (A+B+, 630 & R20291; A-B+, M68 & CF5; A-B-, CD1342). 100% of
hamsters challenged with A+B+ strains succumbed to the infection by ~47 hours, whilst
those challenged with A-B+ strains showed ~60% survival rate. Hamsters challenged with
the A-B- strain remained well. Bacterial colonisation, in vivo toxin production and caecal
histology with these strains will be discussed. As with humans, the majority of clinical
symptoms observed in the hamster are thought to be due to the action of TcdA and TcdB;
making these proteins potential vaccine targets.
Vaccination using recombinant proteins encoding the repeat regions from the binding domain
of each toxin protected animals against severe disease. Vaccination with either antigen
individually could not prevent death in the hamster model, however, in combination these
antigens could protect animals challenged either 630 (100%), B1 (83%) or R20291 (62%).
Surviving animals still shed C. difficile spores up to 14 days post challenge, were colonised
at low levels, and showed adaptations to gut morphology. Ribosomal 16S sequencing of
faecal samples taken throughout the infection process showed gross microbiota changes
post clindamycin and C. difficile challenge, which recovered in the vaccinated model to pretreatment
levels from 6 days post challenge onwards. Taken together these data give
valuable insights into the infection profiles of different C. difficile strains and the use of toxin
fragment vaccines as a potential prophylactic intervention.

O27
A MOUSE RELAPSE MODEL OF CLOSTRIDIUM DIFFICILE INFECTION AND ITS
APPLICATION IN EVALUATING IMMUNOTHERAPIES AGAINST THE DISEASE
X. Sun*, H. Wang, Y. Zhang, K. , B. Davis, H. Feng. Tufts University, Cummings School of
Veterinary Medicine, North Grafton, MA USA.
Clostridium difficile is the causative agent of primary and recurrent antibiotic-associated
diarrhea and colitis in hospitalized patients. The disease is mainly caused by two exotoxins
TcdA and TcdB produced by the bacteria. Recurrent C. difficile infection (CDI) constitutes
one of the most significant clinical issues in this disease, and occurs in more than 20% of
patients after the first episode, and may be increasing in frequency. We recently established
a mouse model of CDI relapse (Sun et al, 2011, IAI, 79:2856-64) enabling us to evaluate
immunotherapies against recurrent CDI. In this study we found that the primary episode of
CDI induced little or no protective antibody responses against the two toxins and mice
continued shedding C. difficile spores. Antibiotic treatment of the surviving mice induced a
second episode of diarrhea while a simultaneous re-exposure of animals with C. difficile
bacteria or spores elicited a full spectrum of CDI symptom similar to that of the primary
infection. Moreover, mice treated with immunosuppressive agents were prone to more
severe and fulminant recurrent disease, indicating a protective role of the host innate
immune response against CDI recurrence. We have used this model to test both prophylactic
and therapeutic effects of neutralizing antitoxin polysera on recurrent CDI. We found that
vancomycin treatment only delayed disease recurrence, whereas, polysera against both
TcdA and TcdB completely protected mice against CDI relapse. Furthermore, we evaluated
protection of a novel vaccine against recurrent CDI in the mouse disease model. We
demonstrated that prophylactic vaccination induced potent neutralizing antitoxin antibodies
and conferred a complete protection against C. difficile spore-induced recurrent disease
activities, including diarrhea, weight loss and death. Our data indicate that antitoxin
neutralizing antibodies may have great therapeutic potential against recurrent CDI.

O28
LESSONS FROM NATURAL DISEASE IN PIGS: PATHOGENESIS OF CLOSTRIDIUM
PERFRINGENS TYPE C ENTERITIS
H. Posthaus* 1 , M. Wyder 1 , D. Authemann 2 , S. Christen 2 , M. Popoff 3 , F. Van Immerseel 4 .
1 Institute of Animal Pathology, Vetsuisse Faculty, University of Berne, Switzerland; 2 Institute
of Infectious diseases, Medical School, University of Bern, Switzerland; 3 Institut Pasteur,
Paris, France; 4 Department of Pathology, Bacteriology and Avian Medicine, Gent University,
Belgium.
Clostridium perfringens is an important enteric pathogen in veterinary and human medicine.
Toxinotypes of the organism are responsible for several distinct diseases in various host
species. The pathogenesis of most enteric diseases associated with these pathogens is
incompletely understood. C. perfringens type C strains induce a rapidly fatal necrotizing
enteritis in pigs, humans, and other mammalian species. One essential virulence factor of
these strains is beta toxin (CPB), a beta-barrel pore-forming toxin. Its cellular and molecular
mode of action on natural target cells are not known in detail. We studied the effects of
purified CPB and C. perfringens type C culture supernatant fluids on porcine small intestinal
mucosa and intestinal epithelial and endothelial cells using different in vitro and in vivo
approaches. Type C strains rapidly induced necrohemorrhagic lesions in ligated neonatal
porcine small intestinal loops. Purified CPB was highly toxic to primary porcine and human
endothelial cells, inducing rapid programmed necrosis by membrane pore-formation.
Moreover, it bound to endothelial cells in small intestinal mucosal explants. Porcine small
intestinal epithelial cells were not affected by CPB. Nevertheless, culture supernatants
induced morphological damage in intestinal epithelial cells. In conclusion, our results suggest
that endothelial cells are the primary target of beta toxin. Epithelial damage, required for
penetration of the toxin into the tissue, is most likely induced by additional virulence factors.
Identification of these factors will be important to understand the pathogenesis of enteric
diseases caused by C. perfringens strains.

029
THE MOLECULAR EPIDEMIOLOGY OF CLOSTRIDIUM DIFFICILE IN THE UNITED
STATES
D. MacCannell, L. Anderson, J. Cohen, F. Lessa, and B. Limbago. CDC‘s Emerging
Infections Program. CDC, Atlanta, GA USA..
Abstract unavailable.

O30
ELUCIDATING THE ROLE AND REQUIREMENT FOR SELENOENZYMES IN GROWTH
OF CLOSTRIDIUM DIFFICILE
K. Cobaugh 1 , M. Srivastava 1 , L. Bouillaut 2 , A. L. Sonenshein 2 , and W. T. Self* 1 . 1 Department
of Molecular Biology and Microbiology, University of Central Florida; 2 Department of
Molecular Biology and Microbiology, Tufts University.
The incorporation of the metalloid selenium into proteins requires the activation of selenium
into selenophosphate prior to translational insertion of the amino acid selenocysteine into the
polypeptide chain. We have previously shown that C. difficile expresses at least two
selenoenzymes, glycine reductase and D-proline reductase, when cultured in rich media. In
addition, the presence of selenium and the substrates for these enzymes (proline and
glycine) significantly improves growth. We also have shown that exposure of cells to
auranofin, a drug that blocks the metabolism of selenium, inhibits growth of C. difficile,
suggesting that these enzymes are required for growth. To test the hypothesis that C.
difficile requires one or both of these selenoenzymes for growth, we isolated mutant strains
that carry deletions in genes encoding one subunit of glycine reductase (grdA), D-proline
reductase (prdB) or selenophosphate synthetase (selD). All three mutant strains are viable,
although the selD mutant grows at a slower rate. The level of D-proline reductase enzyme is
significantly increased in the grdA mutant. Likewise the level of glycine reductase is
increased in the prdB mutant. These results indicate that the cell balances its need for ATP
synthesis through concerted regulation of these two enzymes. To our surprise the selD
mutant is viable, and radiolabeling studies confirm that selenium is not incorporated into
either enzyme in the mutant strain, but growth is noticeably slower than with the wild type.
Our ongoing studies aim to elucidate the role that these selenoenzymes play in the
physiology and bioenergetics of C. difficile, and to determine whether strains that lack one or
both of these enzymes can trigger infection in animal models of disease.

O31
INTEGRATION OF METABOLISM AND VIRULENCE IN CLOSTRIDIUM DIFFICILE.
A. L. Sonenshein* 1 , L. Bouillaut 1 , S. S. Dineen 1 , S. M. McBride 1 and W. Self 2 . 1 Department of
Molecular Biology and Microbiology, Tufts University School of Medicine and 2 Department of
Molecular Biology and Microbiology, University of Central Florida.
A broad linkage between nutrient availability and toxin production in C. difficile has been
documented through the contributions of many laboratories. When cells are grown in rich
medium, toxin gene expression and toxin production are repressed during rapid exponential
growth and induced in stationary phase. Glucose, proline, glycine, branched-chain amino
acids, cysteine and biotin have all been found to affect toxin production. The mechanistic
bases for some of these effects are now becoming known. Two global regulators have been
shown to repress toxin gene expression. CodY, a protein found in virtually all low G+C Grampositive
bacteria, is activated by the accumulation of branched-chain amino acids (BCAAs;
Ile, Leu, Val) and GTP, controls dozens of metabolic genes and also represses transcription
of tcdR, the gene that encodes the alternative RNA polymerase sigma factor that recognizes
the promoters of the toxin genes (tcdA and tcdB). CcpA links toxin gene expression to
metabolism by regulating a vast array of carbon metabolism genes and directly repressing
tcdA and tcdB when glucose is in excess. The role of proline can now be attributed at least in
part to its role as an activator of PrdR. C. difficile, like many Clostridium spp., uses the
Stickland reaction (co-fermentation of pairs of amino acids) to generate ATP. The key
enzymes, D-proline reductase (Prd) and glycine reductase (Grd), are encoded in multi-gene
loci whose transcription responds to the presence of proline and glycine. The prdR gene
located upstream of the prd genes was shown to encode a positive regulator of the prd
genes and a negative regulator of the grd genes. The effect of PrdR on prd gene
transcription appeared to be direct, while the effect of a prdR null mutation on grd
transcription was shown to be due to lack of Prd activity and the consequent failure to
produce an inhibitor of Grd gene expression. The inhibitor is postulated to be 5-
aminovalerate. Moreover, a null mutation in prdB, which led to high proline accumulation,
greatly decreased toxin gene expression, suggesting that the effect of proline on toxin
production is mediated at least in part through activation of PrdR. The BCAAs provide a
second potential interaction between the Stickland pathways and toxin synthesis. Since the
BCAAs serve as electron donors for the Stickland reactions, increased grd expression in a
prdB mutant may cause a sufficient reduction in the BCAA pool that the ability of CodY to
repress the tcdR gene is compromised.

O32
CLOSTRIDIUM DIFFICILE HAS TWO PARALLEL AND ESSENTIAL SEC SECRETION
SYSTEMS
R. P. Fagan* and N. F. Fairweather. Division of Cell and Molecular Biology, Centre for
Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, United
Kingdom.
Protein translocation across the cytoplasmic membrane is an essential process in all
bacteria. The Sec system, comprising at its core an ATPase, SecA, and a membrane
channel, SecYEG, is responsible for the majority of this protein transport. Recently, a second
parallel Sec system has been described in a number of Gram-positive species. This
accessory Sec system is characterized by the presence of a second copy of the energizing
ATPase, SecA2; where it has been studied, SecA2 is responsible for the translocation of a
subset of Sec substrates. In common with many pathogenic Gram-positive species,
Clostridium difficile possesses two copies of SecA. Here we describe the first
characterization of the Clostridium difficile accessory Sec system and the identification of its
major substrates. Using inducible antisense RNA expression and dominant negative alleles
of SecA1 and SecA2, we demonstrate that export of the S-layer proteins (SLPs) and an
additional cell wall protein (CwpV) is dependent on SecA2. Accumulation of the cytoplasmic
precursor of the S-layer protein SlpA and other cell wall proteins was observed in cells
expressing dominant negative SecA1 or SecA2 alleles, concomitant with a decrease in the
levels of mature SLP proteins in the cell wall. Furthermore, expression of either dominant–
negative allele or antisense RNA knock-down of SecA1 or SecA2 dramatically impaired
growth, indicating that both Sec systems are essential in C. difficile.

O33
THE MOLECULAR MECHANISM of CLOSTRIDIUM PERFRINGENS SPORE
GERMINATION
M. R. Sarker* 1 , D. Paredes-Sabja 2 , and P. Setlow 3 . 1 Department of Biomedical Sciences,
Oregon State University, Corvallis, OR97331, USA; 2 Departmento de Ciencias Biológicas,
Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile; 3 Department of
Molecular, Microbial and Structural Biology, University of Connecticut Health Center,
Farmington, CT06030, USA.
Clostridium perfringens is a Gram-positive, spore-forming, anaerobic pathogen that causes
diseases in animals and humans. The virulence of this bacterium is largely attributed to its
capability to produce a large number of toxins. However, in addition to toxin production, C.
perfringens has the ability to form metabolically dormant spores that are extremely resistant
to heat and other environmental stresses. To cause deleterious effects, C. perfringens
dormant spores must first go through germination and then outgrowth to be converted to
vegetative cells capable of producing toxins. In Bacillus subtilis, spore germination is initiated
when nutrient germinants bind to germinant receptors (GRs) in the spore's inner membrane
triggering the release of the spore core's depot of dipicolinic acid as a 1:1 chelate with Ca2+
(Ca-DPA), and replacement of Ca-DPA by water. These events trigger the hydrolysis of the
spore's peptidoglycan cortex by cortex-lytic enzymes, and subsequent germ cell wall
expansion allowing full spore core hydration, resumption of metabolism and macromolecular
synthesis. Although spore germination in Clostridium species is less well studied than in
Bacillus species, significant progress on the molecular mechanism of C. perfringens spore
germination has been made recently in our laboratory. First, C. perfringens spores can
germinate with the nutrient germinants L-asparagine, KCl, and inorganic phosphate (pH 6.0),
and with non-nutrients such as dodecylamine and Ca-DPA. GerKA and/or GerKC are the
main GR for these germinants, while GerAA and GerKB play auxiliary roles in germination.
Second, release of Ca-DPA is not required for the initiation of hydrolysis of the spore‘s
peptidoglycan cortex. Third, among two cortex-lytic enzymes (CLEs) (i.e., SleC and SleM),
SleC is the single essential CLE for spore‘s peptidoglycan (PG) cortex hydrolysis during C.
perfringens spore germination, with SleM playing only an auxiliary role. Finally, the serine
protease CspB is essential to generate active SleC and allow DPA release and PG cortex
hydrolysis early in C. perfringens spore germination. The ongoing research towards
determining the mechanism of activation of CspB should help in understanding further the
molecular mechanism of spore germination in C. perfringens.

O34
THE KEY SIGMA FACTOR OF TRANSITION PHASE, SigH, CONTROLS SPORULATION,
METABOLISM, AND VIRULENCE FACTOR EXPRESSION IN CLOSTRIDIUM DIFFICILE
L. Saujet*, M. Monot, B. Dupuy, O. Soutourina, and I. Martin-Verstraete. Laboratoire
Pathogenèse des Bactéries Anaérobies, Institut Pasteur, 25 rue du Dr Roux 75724 Paris
Cedex, 15 and Université Paris 7-Denis Diderot, 75205 Paris, France.
Toxin synthesis in Clostridium difficile is subject to multiple forms of environmental regulation
and increases as cells enter stationary phase. The regulatory network controlling postexponential
events in C. difficile is still poorly characterized. However, among the regulators
that trigger this transition, CodY and Spo0A were shown to modulate toxin gene expression.
So, we decided to test the role of SigH, an alternative sigma factor involved in the transition
to post-exponential phase in Bacillus subtilis. We inactivated sigH in C. difficile and
compared the expression profiles of strain 630E after 4 h and 10 h of growth as well as the
630E and the sigH mutant at the onset of stationary phase (10h). About 60% of the genes
differentially expressed between exponential and stationary phases including genes involved
in motility, sporulation, cellular division, and metabolism were regulated by SigH appearing
as a key regulator of transition phase in C. difficile. SigH positively controls genes required
for sporulation. Accordingly, sigH inactivation results in an asporogeneous phenotype. The
spo0A and CD2492 genes encoding the master regulator of sporulation and one of its
associated kinases and the spoIIA operon were transcribed from a SigH-dependent
promoter. The expression of tcdA and tcdB encoding the toxins and of tcdR encoding the
sigma factor required for toxin production increased in a sigH mutant. So, the sigH mutant is
unable to sporulate but still produces toxins demonstrating that toxin synthesis is a stationary
phase event. Finally, SigH regulated the expression of genes encoding surface-associated
proteins like the Cwp66 adhesin, the S-layer precursor and the flagellum components.
Among the 286 genes positively regulated by SigH, about 40 transcriptional units presenting
a SigH consensus in their promoter regions are good candidates for direct SigH targets. We
have recently performed a genome wide determination of transcriptional start sites of the
630E strain using RNA-seq. Among the 40 predicted SigH direct targets, we confirmed the
existence of a SigH promoter for 22 of them and proposed a SigH-dependent promoter
consensus.

O35
A CHIMERIC TOXIN VACCINE PREVENTS PRIMARY AND RECURRENT CLOSTRIDIUM
DIFFICILE INFECTION
H. Wang *1, 2 , X. Sun *1 , Y. Zhang 1, 3 , S. Li 1 , K. Chen 1 , G.P. Cordon 1 , L.Shi 1 , W. Nie 1 , R.
Kumar 4 , S. Tzipori 1 , J. Wang 2 , T. Savidge 5 , and H. Feng. 1 1 Tufts University, Cummings
School of Veterinary Medicine, North Grafton, Massachusetts 01536, US. 2 School of
Bioscience and Biotechnology, South China University of Technology, Guangzhou, China.
3 School of Bioengineering, East China University of Science and Technology, Shanghai,
China. 4 Department of Basic Sciences, The Commonwealth Medical College, Scranton, US.
5 Department of Gastroenterology & Hepatology, The University of Texas Medical Branch,
Galveston, Texas 77555, US.
*Authors contributed equally to this work.
The global emergence of hypervirulent Clostridium difficile infection (CDI) has contributed to
the recent surge in severe antibiotic-associated colonic disease. C. difficile produces two
homologous glucosylating exotoxins, TcdA and TcdB, both of which require neutralization to
prevent disease. Antitoxin immunization is widely recognized to be the preferred vaccination
strategy against CDI. However, because of their large size and complex multifunctional
domain structure, it has been a challenge to produce recombinant toxin vaccines. As a
consequence, clinical immunization has been limited to toxoids that show suboptimal
efficacy. Here we describe a novel chimeric toxin vaccine that confers complete disease
protection in animal models of CDI and characterize the underlying antitoxin protective
mechanism. Using a non-pathogenic Bacillus megaterium expression system, we generated
glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. These
holotoxins induced markedly greater antitoxin neutralizing responses than toxoid, but showed
little cross-protection. To facilitate simultaneous protection against both toxins, we generated
an active clostridial toxin chimera by replacing the receptor binding domain of TcdB with
TcdA, thus conserving immunodominant, conformation-dependent epitopes. Parenteral
immunization with the avirulent toxin chimera, cTxAB, induced rapid and complete disease
protection against oral challenge with laboratory or epidemic C. difficile strains. Moreover,
prophylactic cTxAB-vaccination prevented spore-induced disease relapse, which constitutes
one of the most significant clinical issues in CDI. Systemic and local mucosa-associated IgG
immunity conferred this disease protection. Thus, the rational design of recombinant chimeric
toxins provides a novel approach for rapidly protecting individuals at high risk of developing
infectious disease such as CDI.

O36
OPIOID-BASED ANALGESICS BLOCK THE PROGRESSION AND DEVELOPMENT OF C.
PERFRINGENS MEDIATED MYONECROSIS
A. Chakravorty*, M.M. Awad, T.J. Hiscox, J.K. Cheung, J.M. Choo, D. Lyras and J.I. Rood.
Monash University, Department of Microbiology, Melbourne, Australia, 3800.
Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that is the
causative agent of traumatic gas gangrene. The key pathological characteristic of this
disease is a paucity of polymorphonuclear leukocytes (PMNs) within sites of infection, which
allows the infection to spread unhindered. This process involves the formation of plateletplatelet
and platelet-leukocyte aggregates within the blood vessels, which in turn blocks
blood flow into the sites of infection, the resultant development of an ischemic environment.
The end result is rapid bacterial growth and widespread necrosis of the infected tissues.
Current treatment of gas gangrene involves the use of antibiotics, primarily penicillin, and
debridement of necrotic tissues. However, the disease develops rapidly and the amputation
of infected limbs is often necessary to stop the spread of infection. We have recently shown
that mice pre-treated with buprenorphine, a morphine-based opioid analgesic, do not
succumb to experimental C. perfringens-mediated myonecrosis. Independent of their role in
the neuroendorcrine system, previous studies have shown that opioids can repress the
immune response and increase the host‘s susceptibility to infection. Our study shows the
reverse effect. Infected mice that have been pre-treated with buprenorphine do not develop
the characteristic pathology associated with gas gangrene, especially blackening of the thigh
and footpad. A similar effect was observed with morphine, suggesting that it has a similar
mechanism of action. These data provide evidence that treatment with opiates retards the
progression of clostridial myonecrosis and provides valuable insights into the mechanisms by
which these effects are mediated.

O37
SUB-INHIBITORY CONCENTRATIONS OF TIGECYCLINE INHIBIT SPORULATION OF
CLOSTRIDIUM DIFFICILE IN VITRO
J. R. Garneau, L. Valiquette and L.C. Fortier*. Department of Microbiology and Infectious
Diseases, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th
Ave North, Sherbrooke, QC, Canada, J1H 5N4.
Sub-inhibitory concentrations of certain antibiotics were previously shown to affect the
expression of several genes in Clostridium difficile, but how antibiotics affect sporulation is
not well understood. Spores are insensitive to antibiotics and sporulation of C. difficile in the
colon during infection could possibly explain treatment failures and relapses reported with
metronidazole and vancomycin. The objective of our study was to evaluate the impact of
sub-inhibitory concentrations of metronidazole (MTZ), vancomycin (VAN), ciprofloxacin (CIP),
and tigecycline (TIGE) on the in vitro sporulation of C. difficile. The aim was to determine
whether these antibiotics could promote or inhibit sporulation. The reference strains ATCC
9689, 630 and VPI 10463, as well as seven other clinical isolates of C. difficile were
characterized by PCR ribotyping, tandem repeat sequence typing (TRST), binary toxin
detection, and sequencing of the tcdC gene. Three clinical isolates were PCR ribotype 027
(R027) and had common characteristics of the epidemic NAP1/027 strain. Minimum
inhibitory concentrations (MIC) were determined for MTZ, VAN, CIP and TIGE and all strains
were sensitive, except the R027 isolates that were resistant to CIP (MIC=128 µg/mL).
Sporulation was assessed on TY agar in the absence or presence of 0.5X MIC of each
antibiotic. Colonies were picked after 24, 48, and 96 h of growth and Gram stains were
observed under the microscope. The number of spores and vegetative cells from 10 different
fields was used to calculate the sporulation rate. We found that MTZ and VAN did not
significantly modify the sporulation rate of C. difficile, but CIP inhibited sporulation up to 18-
fold, but only for R027 strains and VPI 10463. On the other hand, TIGE systematically
inhibited sporulation of all strains tested by a factor of up to 254-fold. In concentration range
assays with a subset of 3 strains, we observed that the inhibition of sporulation with TIGE
and CIP was greater as the concentration of antibiotic approached the MIC. In summary,
TIGE consistently inhibited sporulation of C. difficile, and this inhibition may in part explain
the recent success of TIGE in treating recurrent CDI cases.

O38
IMMUNIZATION WITH BACILLUS SPORES EXPRESSING TOXIN A PEPTIDE REPEATS
PROTECTS AGAINST INFECTION WITH CLOSTRIDIUM DIFFICILE STRAINS
PRODUCING TOXIN A AND B
P. Permpoonpattana* 1 , H. A. Hong 1 , J. Phetcharaburanin 1 , J. Huang 1 , J. Cook 1 , N.F.
Fairweather 2 , and S. M. Cutting* 1 . 1 School of Biological Sciences, Royal Holloway,
University of London, Egham, Surrey, TW20 0EX United Kingdom; 2 Department of Life
Sciences, Imperial College London, London, SW7 2AZ United Kingdom.
Clostridium difficile is a leading cause of nosocomial infection in the developed world. Two
toxins, A and B, produced by most strains of C. difficile are implicated as virulence factors,
yet only recently has the requirement of these for infection been investigated by genetic
manipulation. Current vaccine strategies are focused mostly on parenteral delivery of
toxoids. In this work, we have used bacterial spores (Bacillus subtilis) as a delivery vehicle to
evaluate the carboxy-terminal repeat domains of toxins A and B as protective antigens. Our
findings are important and show that oral immunization of the repeat domain of toxin A is
sufficient to confer protection in a hamster model of infection designed to closely mimic the
human course of infection. Importantly, neutralizing antibodies to the toxin A repeat domain
were shown to be cross-reactive with the analogous domain of toxin B and, being of high
avidity, provided protection against challenge with a C. difficile strain producing toxins A and
B (A+B+). Thus, although many strains produce both toxins, antibodies to only toxin A can
mediate protection. Animals vaccinated with recombinant spores were fully able to survive
reinfection, a property that is particularly important for a disease with which patients are
prone to relapse. We show that mucosal immunization, not parenteral delivery, is required to
generate secretory IgA and that production of these neutralizing polymeric antibodies
correlates with protection. This work demonstrates that an effective vaccine against C.
difficile can be designed around two attributes, mucosal delivery and the repeat domain of
toxin A.

O39
THE C. DIFFICILE CPR LOCUS MEDIATES RESISTANCE TO ANTIMICROBIAL
PEPTIDES THROUGH MOLECULAR MIMICRY
S.M. McBride and A.L. Sonenshein. Department of Molecular Biology and Microbiology,
Tufts University School of Medicine, Boston, MA 02111 USA.
Clostridium difficile is the major causative agent of antibiotic-associated diarrhea. To persist
in the intestine the bacteria must cope with innate defenses within the host, such as cationic
antimicrobial peptides (CAMPs) made by the host and indigenous flora. We previously
showed that C. difficile responds to CAMPs by inducing expression of genes that lead to
CAMP resistance. The first such C. difficile gene cluster identified (cprABCK) encodes an
ABC-type transporter and a sensor kinase typical of bacterial two-component systems (TCS).
These genes are highly related to genes that encode immunity proteins in lantibiotic producer
species. Using qRT-PCR and directed mutagenesis, we showed that the transporter and
kinase genes are directly involved in resistance to CAMPs. However, no apparent response
regulator is encoded in the vicinity of cprABCK. Here, we identify an orphan response
regulator, CD3320, which encodes the response regulator that controls the cprABCK genes
in response to antimicrobial peptides. We cloned the CD3320 gene and upstream region in
a vector that replicates in C. difficile and demonstrated that cells adapted faster to CAMPs
and expression of the cprABCK operon increased. By expressing these regulators and a
Pcpr::lacZ reporter fusion in a heterologous host, Bacillus subtilis, we were able to show how
CprK and CD3320 (renamed CprR) interact to activate expression of the cpr operon. In
addition, we were able to identify putative residues of both the lantibiotics and of CprK that
are involved in sensing and activation of this system. These results demonstrate how the cpr
ABC-transporter and regulators allow for substrate recognition that is broader than the typical
narrow-substrate recognition of lantibiotic producer systems, resulting in resistance to
multiple bacterial CAMPs through a single mechanism. The cpr lantibiotic immunity system
represents a novel antimicrobial peptide resistance mechanism that has not been described
in any other species.

O40
DIFFERENTIAL SENSITIVITY OF CLOSTRIDIUM DIFFICILE CLINICAL ISOLATES TO
MAMMALIAN CATIONIC ANTIMICROBIAL PEPTIDES
R. McQuade* 1 , M. Mallozzi 1 , B. Roxas 1 , G. Vedantam 1,2,3 , and V.K. Viswanathan 1,2 . 1 Dept.
of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ; 2 The BIO5
Institute, University of Arizona, Tucson, AZ, USA; 3 The Southern Arizona VA Healthcare
System, Tucson, AZ, USA.
Background and Rationale: Clostridium difficile is a leading cause of hospital-acquired
diarrhea. Recent outbreaks of C.difficile infection (with increased morbidity and mortality)
have involved ―hypervirulent‖ (HV) strains, including those belonging to the 027 and 078
ribotypes. Cationic antimicrobial peptides (CAMPs; e.g. human LL37 & sheep SMAP29)
contribute to gut innate immunity by interacting with, and disrupting the negatively-charged
bacterial cell-membrane. Some pathogens have evolved specific defense mechanisms to
evade CAMP-mediated killing. We hypothesized that increased resistance to CAMPs
contributes to the more persistent infections associated with HV C. difficile strains.
Methods: Minimum inhibitory concentrations (MIC) of LL37 and SMAP29 were determined
for various HV and non-HV C. difficile strains using a standard broth microdilution. To
visualize the action of CAMPs on C. difficile, LL37-exposed bacteria were imaged by electron
microscopy. The kinetics of CAMP killing was evaluated by determining the number of
surviving C. difficile throughout an 8h exposure to various concentrations of LL37. To
determine whether C. difficile has inducible CAMP-resistance mechanisms, the sensitivity of
strain 630 to LL37 was assessed with and without pre-exposure to a sub-inhibitory
concentration of the peptide. Changes in the proteome of LL37-exposed C. difficile were
determined using mass spectrometry.
Results: Compared to non-epidemic strains, HV C. difficile isolates display a consistent
increase in resistance to both LL37 and SMAP29. Electron microscopy of LL37-exposed
bacteria revealed surface distortion characteristic of CAMP-mediated killing. LL37 inhibited
C. difficile growth in a dose-dependent manner, with bacterial death evident as early as 0.5h
post-exposure. Exposure of bacteria to a sub-inhibitory concentration of LL37 resulted in
increased resistance to the CAMP. Mass-spectrometry analysis revealed proteome
alterations, including the increased expression of putative ABC transporters, in bacteria
exposed to sub-inhibitory concentrations of LL37. Discussion: C. difficile strains display
variable sensitivity to CAMPs, and the increased resistance of HV strains likely contributes to
their persistence in the gut. CAMPs appear to act on C. difficile by disrupting the bacterial
membrane. Pre-exposure of C. difficile to LL37 results in altered protein expression, as well
as increased resistance to LL37, suggesting adaptability of the bacteria to CAMPs.

O41
BOTULINUM TOXIN STRUCTURE/FUNCTION RELATIONSHIPS
J.T. Barbieri, Department of Microbiology and Molecular Genetics, Medical College of
Wisconsin, Milwaukee, WI 53226 USA.
The botulinum neurotoxins (BoNT) are the most potent protein toxins for humans. There are
seven serotypes of BoNTs termed (A-G) which are defined by a lack of cross anti-sera
neutralization. Several of the BoNT serotypes (A, B, E, F, and G) utilize dual receptors, a
ganglioside and protein, to enter neurons. Initially, these serotypes of BoNT bind ganglioside
on resting neurons which supports cell association until a synaptic vesicle fuses to the cell
membrane, exposing the protein receptor and facilitating entry into the neuron via synaptic
vesicle internalization. In contrast, tetanus toxin utilizes dual-gangliosides as functional
receptors and enters through an endosome-based mechanism. BoNT serotypes C and D
include mosaic toxins that are organized as D-C and C-D toxins. One BoNT D-C mosaic
toxin, BoNT-D(South Africa), was not fully neutralized by immunization with vaccines derived
from either BoNT serotype C or D, which prompted characterization of their biological and
cellular properties. Structural studies showed that the receptor binding domains of BoNT-C,
BoNT-D, and BoNT-D(SA) possessed overall structural similarity with other BoNTs
serotypes. However, a structural alignment of the primary amino acid sequence showed that
BoNT-C, BoNT-D, and BoNT-D(SA) lacked residues that were components of the
ganglioside binding domain (GBP) and that was conserved in BoNT serotypes A,B,E, F, and
G. This alignment also identified a unique loop that was present in BoNT-C, BoNT-D, and
BoNT-D(SA) that was distanced from the conserved GBP of the other BoNT serotypes
termed the ganglioside binding loop (GBL). The GBL included a tryptophan for BoNT-C and
BoNT-D(SA) and a phenylalanine for BoNT-D. Mutation of this aromatic amino acid
eliminated the ability to bind ganglioside without perturbing protein structure. These studies
showed that BoNT-C, BoNT-D, and BoNT-D(SA) recognized and bound gangliosides
differently than the other serotypes of BoNTs, implicating a unique mechanism for entry and
intoxication of neurons. Continued studies may provide new information on the action of
these neurotoxins that may extend their clinical utility.

O42
A TWO-COMPONENT SYSTEM NEGATIVELY REGULATES BOTULINUM NEUROTOXIN
EXPRESSION
Z. Zhang 1 , H. Korkeala 1 , E. Dahlsten 1 , J. T. Heap 2 , N. P. Minton 2 , M. Lindström* 1 .
1 Department of Food Hygiene and Environmental Health, Centre of Excellence in Microbial
Food Safety Research, Faculty of Veterinary Medicine, University of Helsinki, Finland;
2 School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of
Nottingham, University Park, Nottingham NG7 2RD, United Kingdom.
Two-component signal transduction systems (TCSs) play an important regulatory role in
virulence in many pathogenic bacteria. However, little is known about the role of TCSs in
Clostridium botulinum. We identified a TCS involved in neurotoxin regulation in C. botulinum
type A strain ATCC3502. Using the ClosTron mutagenesis system 1 , we inactivated tcsR,
encoding a response regulator, and tcsK, encoding a sensor histidine kinase. Inactivation of
tcsR and tcsK resulted in a significantly higher level of neurotoxin gene (botA) transcripts and
neurotoxin production, as measured with ELISA. Complementation of tcsR mutant cells with
a plasmid 2 expressing tcsKR restored neurotoxin production to the wild-type level. Further
experiments suggested that TcsKR also down-regulates the transcription of botR, the
alternative sigma factor which activates botA transcription 3 . The regulatory role of TcsKR was
confirmed by protein-DNA binding assays. To our knowledge, this is the first report on
negative regulation of botulinum neurotoxin production and a role of TCS in C. botulinum.
Profound understanding of the negative regulation of the neurotoxin production may provide
tools to control the risk of botulism in foods.
1. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. 2007. The ClosTron: a universal gene knock-out system for
the genus Clostridium. J Microbiol Methods 70: 452-464
2. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, Minton NP. 2010. The ClosTron: mutagenesis in
Clostridium refined and streamlined. J Microbiol Methods 80: 49-55
3. Marvaud JC, Gibert M, Inoue K, Fujinaga Y, Oguma K, and Popoff MR. 1998. botR/A is a positive regulator of botulinum
neurotoxin and associated non-toxin protein genes in Clostridium botulinum A. Mol Microbiol 29: 1009-1018

O43
DISRUPTION OF sigK IN CLOSTRIDIUM BOTULINUM ATCC3502 PREVENTS
SPORULATION.
D. Kirk*, H. Korkeala, Z. Zhang, E. Dahlsten, and M. Lindström. Department of Food
Hygiene and Environmental Health, Centre of Excellence in Microbial Food Safety Research,
Faculty of Veterinary Medicine, University of Helsinki, 00014 Finland.
Clostridium botulinum is a spore-forming obligate anaerobe. The Spo0A protein is thought to
be responsible for initiating sporulation in clostridia, triggering the sporulation pathway which
involves different sigma factors at each stage. C. botulinum also produces potent
neurotoxins, the causative agents of botulism. Spore contamination is a key factor in cases
of infant and wound botulism as spores germinate and neurotoxin is produced in vivo. It is
unknown how the activation of Spo0A is regulated in C. botulinum, as those mechanisms
found in Bacillus are not present. The subsequent stages of sporulation have homologues in
most Bacillus species, though there may be differences in target genes and their expression.
Using the ClosTron tool developed by Heap et al. (2007), which utilizes a group II intron
insertion to disrupt gene function, we created a mutation in the late-stage sporulation sigma
factor gene sigK. This mutant failed to produce spores after seven days‘ incubation at 37°C
in a standard nutrient medium. Upon spore staining and observation with light microscopy,
no spores were visible. In addition, the individual mutant cells‘ morphology appeared more
elongated than that of the wild type, occasionally forming long strings of growth. By creating
a plasmid containing sigK, we were able to complement spore formation. The wildtype strain,
with and without the empty plasmid vector, formed spores as expected. The results suggest
that successful sporulation in C. botulinum ATCC 3502, a Group I type A toxin producing
strain, is dependent upon the late-stage sporulation sigma factor SigK. Expression analysis
of spo0A and other sporulation related genes is ongoing in the sigK mutant and with type
ATCC 3502.
References: Heap et al. (2007). The ClosTron: A universal gene knock-out system for the genus Clostridium. Journal of
Microbiological Methods 70: 452–464.

O44
THE ROLE OF TOXINS IN CLOSTRIDIUM DIFFICILE PATHOGENESIS
G. Carter 1 , A. Chakravorty 1 , K. Mackin 1 , M. Kelly 1 , G. Douce 2 , R. Govind 3 , B. Dupuy 4 , S.
Johnson 5 , D. Gerding 5 , J. Rood 1 , T. Lawley 6 and D. Lyras* 1 . 1 Department of Microbiology,
Monash University, Victoria, Australia; 2 Division of Infection and Immunity, FBLS Glasgow
Biomedical Research Centre, University of Glasgow, UK; 3 Division of Biology, Kansas State
University, Manhattan, KS, USA; 4 Unité de Génétique Moléculaire Bactérienne, Institut
Pasteur, Paris, France; 5 Hines Veterans Affairs Hospital, Hines, IL, USA; 6 Pathogen
Genomics, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
Cambridge, UK.
Clostridium difficile is an important cause of nosocomial disease worldwide. Most diseasecausing
C. difficile isolates produce two large clostridial cytotoxins, A and B, encoded by
tcdA and tcdB, respectively. Studies using derivatives of the historical strain 630 have shown
that toxin B plays a major role in virulence while the role of toxin A is not as clear. Recently,
―hypervirulent‖ NAP1/027 isolates have been associated with more severe disease and
higher rates of mortality. These strains appear to produce more toxin than other clinical
isolates, which is thought to be the result of a nonsense mutation within a gene encoding a
putative negative regulator of toxin production, tcdC. This hypothesis is contentious and
there are conflicting reports on the role of TcdC. To study NAP1/027 epidemic strains at the
molecular level, and to clarify the role of toxins in disease, we developed a new and more
efficient plasmid transfer system which facilitated the construction of toxin gene mutants and
the complementation of a NAP1/027 epidemic isolate with an intact copy of tcdC. We have
constructed isogenic toxin gene mutants of a virulent C. difficile NAP1/027 strain and
assessed these mutants for their virulence properties. The data obtained by studying these
strains provide new insights into the role of toxins in disease and the contribution of TcdC to
the development of hypervirulence in C. difficile.

O45
THE INDIVIDUAL ROLES OF TOXIN A AND TOXIN B IN CLOSTRIDIUM DIFFICILE
INFECTION
S.A. Kuehne*, M.M. Collery, M.L. Kelly, S.T. Cartman, A. Cockayne, and N.P. Minton.
Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular
Sciences, University of Nottingham, Nottingham, NG7 2RD, UK.
Clostridium difficile is the major cause of antibiotic associated diarrhoea in Europe and North
America. It leads to higher mortality rates than MRSA and imposes a significant financial
burden on our healthcare systems. The two large toxins, TcdA and TcdB, are the main
virulence factors in Clostridium difficile infection (CDI). Despite their undoubted importance
there has been much controversy over the years with regards to their individual contributions
to the disease. In recent years, two papers have been published, one by Lyras et al, 2009
indicating that in strain 630 only toxin B is essential for CDI, and the other one by our
laboratory (Kuehne et al, 2010) showing that the same strain producing either toxin A or toxin
B alone can cause fulminant disease. The reason for the different outcome of the two studies
is not clear, but may be a consequence of the presence of ancillary mutations in the isolates
used in the two laboratories. This is currently being investigated through genome resequencing.
In the meantime, we have extended our analysis to another strain, R20291.
This is a PCR ribotype 027/NAP1/B1 (‗hypervirulent‘) strain. Hypervirulent strains are linked
to more severe CDI, and produce a third toxin, which is a binary toxin called Cdt. We have
created stable toxin mutants in strain R20291 using ClosTron technology, to allow
examination of each individual toxin and combinations of the three toxins during the disease
process. In common with our early study using strain 630 (Kuehne et al, 2010), experiments
have shown that R20291 derivatives producing either of the two main toxins (A or B) alone
can cause fatal disease in the hamster infection model. These findings re-establish the
importance of TcdA and TcdB in CDI and emphasize a need to consider both when
developing effective countermeasures.

O46
THE AGR QUORUM SENSING SYSTEM IS A GLOBAL REGULATOR OF CLOSTRIDIUM
PERFRINGENS TOXIN PRODUCTION AND VIRULENCE
J. Chen* 1 , M. Ma 1 , J. Li 1 , J. Vidal 1 , J. Garcia 3 , J. Saputo 3 , J. Rood 2 , F. Uzal 3 , B. McClane 1 .
1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of
Medicine, Pittsburgh, PA, USA; 2 Department of Microbiology, Monash University, Clayton,
Australia; 3 California Animal Health and Food Safety Laboratory, University of California
Davis, San Bernardino, CA, USA.
Clostridium perfringens, an important pathogen of humans and livestock, causes wound
infections (e.g. gas gangrene) and diseases originating in the intestines (e.g. food poisoning,
necrotic enteritis and enterotoxemia). C. perfringens virulence is largely dependent upon
prolific toxin production, with this bacterium capable of producing at least 17 different toxins.
The agr system is a well-characterized regulator, in a quorum-sensing (QS) manner, of toxin
genes in Staphylococcus aureus. Recently an Agr-like system was shown to regulate the
production of alpha toxin and, particularly, perfringolysin O, by C. perfringens type A strain
13. We later demonstrated that the Agr-like locus also regulates sporulation and enterotoxin
production by C. perfringens type A strain F5603. C. perfringens epsilon toxin (ETX) is
considered the third most potent of all clostridial toxins and thus has been classified by the
CDC as a class B select agent toxin. ETX is also a major virulence factor in several
important, and often fatal, natural veterinary enterotoxemias caused by type B and D strains.
Similarly, when C. perfringens type C isolates cause necrotic enteritis in humans or livestock,
beta toxin (CPB) is considered the most important toxin. There has been limited information
available regarding the regulation of ETX production by type D strains or CPB production by
type C strains. To obtain a better understanding of how toxin production is regulated in these
strains, agrB null mutants were generated by Targetron insertional mutagenesis technology
in C. perfringens type C strain CN3685 and type D strain CN3718. We found that the
CN3685::agrB mutant exhibits strongly reduced CPB production and the CN3718::agrB
mutant makes much less ETX toxin compared to the wild-type parents. Initial animal testing
has shown that the CN3685::agrB mutant is attenuated in its ability to cause hemorrhagic
necrotic enteritis in rabbits or fatal enterotoxemia in mice. Mouse lethality experiments
demonstrated that the CN3718::agrB mutant also exhibits less lethality compared with wildtype
CN3718. The findings support the Agr-like QS system‘s global importance for regulating
C. perfringens toxin production and pathogenicity.

O47
GLOBAL GENE EXPRESSION OF EPITHELIAL CELLS FROM AN IN VIVO MODEL OF C.
DIFFICILE TOXIN A AND B INTOXICATION
K.M. D'Auria* 1 , G.L. Kolling 2 , G.M. Donato 2 , C.A. Warren 2 , M.C. Gray 2 , J.A. Papin †1 , E.L.
Hewlett †2 . 1 Department of Biomedical Engineering, 2 Division of Infectious Diseases and
International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia,
22908 USA.
† Equal contribution
Clostridium difficile Toxins A (TcdA) and B (TcdB) cause differing degrees of inflammation in
the same mammalian host, and the potency or cytotoxicity of each individual toxin differs
across many hosts and cell types. In our previous analysis of gene expression in human
ileocecal cells (HCT-8), the toxins elicited very similar transcriptional responses, with TcdB
inducing greater changes at earlier times. Unexpectedly, very few genes that contribute to
inflammation had altered expression, suggesting that signaling involved in pathology is due
to the interplay of multiple cell types rather than simply an epithelial cell response. To
investigate these effects in vivo, we injected 20 μg of one or both toxins directly into the
cecum of C57 black mice and harvested ceca after 2, 6 and 16 h. Pathology scores were
significantly higher for TcdA-treated compared to sham mice, and this difference increased
with time. Myeloperoxidase staining revealed increased infiltration of granulocytes 6 and 16
hours after TcdA and/or TcdB injection. Hence, TcdB elicits pathology though not to the
extent of TcdA; TcdA plus TcdB did not cause a significantly greater change than TcdA alone
in any measurement. Cecal epithelial cells were dissociated with EDTA and gene expression
quantified using transcriptome-wide microarrays. The number of differentially expressed
genes is more than 5-fold higher in TcdA- vs. TcdB-treated mice. The expression profiles of
TcdA-treated, TcdB-treated, and sham mice are all distinct from one another and expression
in TcdA/TcdB-treated mice is highly correlated with TcdA-treated mice. Computational
analyses revealed increased expression of many transcription factors and many of the genes
with altered expression at 2h are associated with intracellular organelles, such as the
endosome, mitochondria, or Golgi apparatus. At 6 hours, the most significantly affected
functions in TcdA-treated mice are inflammation, defense response, and apoptosis. TcdBtreated
mice have fewer changes in inflammation-associated genes, but affected genes are
also associated with intracellular organelles (nuclear lumen and Golgi apparatus).
Contrasting TcdA and TcdB treatments, several differentially expressed genes include small
GTPases, the targets of toxin-mediated glucosylation. At 16 hours, several TcdA-affected
genes are linked to mitochondrial membrane, peroxisome, and apoptosis. Overall, TcdA
induces greater changes in gene expression than TcdB, especially regarding the
inflammatory response, but TcdB does alter physiology and distinctly change gene
expression. A more in-depth analysis of the factors and affected pathways is currently
leading us to studies clarifying the role of epithelial cells in the development of C. difficileassociated
diarrhea and colitis.

O48
COMPARATIVE PROTEOMIC ANALYSES OF EPIDEMIC-ASSOCIATED CLOSTRIDIUM
DIFFICILE STRAINS: IDENTIFICATION OF PROTEINS WITH LIKELY ROLES IN
INTESTINAL COLONIZATION AND DISEASE
G. Vedantam. Deptartment of Veterinary Science and Microbiology, University of Arizona,
Tucson, AZ USA; and Southern Arizona VA Healthcare System, Tucson, AZ USA.
Clostridium difficile (CD) causes diarrheic disease in humans and non-human mammals.The
past decade has seen the emergence of CD ―variant‖ strains that are associated with
outbreaks/epidemics, severe disease, increased recurrence rate(s) and community onset of
disease. There is a renewed sense of urgency to define specific virulence traits underlying
the robustness of CD epidemic-associated strains. CD clinical isolates can be resistant to
genetic manipulation; therefore, we employed proteomic analyses to rapidly identify
molecules altered in epidemic-associated CD strains. A mass spectrometry-based
quantitative approach was used to identify differentially expressed proteins in BI-17, an
epidemic-associated CD strain, as compared with BI-1, a "historic" non-epidemic-associated
CD strain, phylogenetically related to BI-17. Once quantitation parameters were optimized,
similar experiments were performed with 13 different epidemic- and non-epidemic associated
CD clinical isolates. For all strains, both secreted and total cellular proteomes were analyzed
at three growth phases. 2D LC MALDI-TOF/TOF analyses revealed that approximately 550
proteins (~15% of the genome; consistent with other bacteria) were expressed by BI-1 and
BI-17 in each growth phase. Further analyses, using stringent fold-change cut-off
computations, identified a total of 121 proteins that were differentially abundant between BI-1
and BI-17. Consistent with our published studies, epidemic-associated strains showed only
marginal increases, if at all, in toxin (A & B) levels. In contrast, functional classification
analyses revealed that non-toxin CD proteins constituted the most differentially abundant
molecules; these included surface proteins, transporters and host innate immunity
modulators. Similar, and extremely consistent results, were obtained for the other epidemicassociated
CD strains tested. Alteration of phenotypes known to be dependent on some of
the specifically dysregulated molecules was also experimentally confirmed. Epidemicassociated
CD strains showed increased resistance to host innate immune system
molecules, increased host-cell adherence, altered surface morphology, and altered
sporulation/germination profiles compared with the genetically-related non-epidemicassociated
strain. Our proteomic-based approach may thus be useful in identifying attractive
targets for CDI intervention focused on preventing GI-tract establishment of C. difficile.

O49
MOTILITY AND ADHERENCE TO MYOBLASTS
Andrea Hartman, Brittany Gianetti, and Stephen Melville*. Department of Biological
Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
The anaerobic pathogen Clostridium perfringens possesses a unique type IV pilus (TFP)-
mediated gliding motility. We have recently shown that TFP are necessary for adherence of
C. perfringens to rodent myoblasts (muscle cells), which is likely an important feature of this
bacterium‘s ability to cause rapidly spreading gas gangrene. On the surface of agar plates,
elongated C. perfringens move in a group, aligned end-to-end and-side to-side, pushing
themselves away from the mother colony as they grow. The filaments must remain intact for
motility to occur. We used immunofluorescence to identify the two major pilins as colocalizing
on the surface at the poles of the cells. We also used fluorescently tagged proteins
responsible for TFP assembly to track their movement and organization in the cells as they
grow and divide. C. perfringens possesses two PilA (the pilin monomer), two PilB (the
assembly ATPase), and two PilC (an essential assembly protein) homologs, but has just one
PilT (a pilus retraction ATPase) homolog. We created translational fusions of yellow
fluorescent protein (YFP) or cyan fluorescent protein (CFP) to both PilB proteins and PilT,
expressed pairs of these fusions in C. perfringens and viewed them with fluorescent
microscopy in time-lapse anaerobic videos. We found the two PilB proteins co-localized at
the poles of cells and at points which later develop into division septa. PilT frequently
localized to the same locations as the PilB proteins, but also moved independently and
rapidly throughout the cells in a helical pattern, localizing to sites that correspond with CFPtagged
FtsA, a septal division protein. We have shown previously that the pilT gene is found
in an operon with the ftsA and ftsZ genes in C. perfringens. These findings were used to
develop a comprehensive model for the roles and locations of the TFP proteins as the cells
grow and divide, in which the division septa proteins FtsA and FtsZ provide the localization
signal for TFP proteins to assemble so they can synthesize pili, which keep the filaments
intact and anchor them to the surface the cells are gliding on.

O50
ANTIGENIC AND PHASE VARIATION IN CELL WALL PROTEINS OF CLOSTRIDIUM
DIFFICILE
N. F. Fairweather*. Department of Life Sciences and Centre for Molecular Microbiology and
Infection, Imperial College London, London SW7 2AZ, UK.
Clostridium difficile, a Gram-positive spore-forming anaerobe, is the main cause of
nosocomial antibiotic-associated diarrhea in Europe and the US. Two toxins, TcdA and
TcdB, are produced by most strains and mediate damage to host cells through glucosylation
of host GTPases. The epidemiology of C. difficile has changed over the last 10 years with
the emergence of several new endemic strains. Spores of C. difficile are highly infectious
and are difficult to eradicate from the environment. Spores germinate within the intestine and
the vegetative cells proliferate producing toxins and other virulence factors. However, little is
known of the mechanisms employed by C. difficile to colonize the enteric system.
Colonization would seem to be essential for longevity of infection and for efficient production
of toxins. C. difficile expresses a proteinaceous S-layer array on its cell surface consisting
primarily of the major S-layer protein, SlpA. A family of SlpA paralogs is also found,
collectively known as cell wall proteins (CWPs). We have investigated the function of several
CWPs and have thrown light on their putative roles in cell wall biogenesis and virulence.
One example is the cysteine protease Cwp84; this cleaves the SlpA precursor to yield the
mature S-layer proteins that then form a complex on the cell surface. Mutants in cwp84 are
viable but produce an altered cell wall that is defective in retention of some CWPs. CwpV is
the largest member of the CWP family and is expressed in a phase variable manner;
approximately 5% of cells in culture express CwpV. Phase variation is mediated by DNA
inversion that requires the recombinase RecV. We have shown that CwpV promotes C.
difficile aggregation, and that this activity is mediated by the C-terminal repetitive domain.
This domain varies markedly between strains; five distinct repeat types have been identified
and are antigenically distinct. Thus, CwpV function, regulation, and processing are highly
conserved across C. difficile strains, whilst the functional domain exists in at least five
antigenically distinct forms. Conservation of CwpV function and its phase-variable
expression hint at a role for this protein in pathogenesis.

O51
SIALIDASES AFFECT THE HOST CELL ADHERENCE AND EPSILON TOXIN-INDUCED
CYTOTOXICITY OF CLOSTRIDIUM PERFRINGENS TYPE D STRAIN CN3718
J. Li* 1 , S. Sayeed 2 , S. Robertson 1 , J. Chen 1 , and B. A. McClane 1 . 1 Department of
Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA; 2 Department of Environmental and Occupational Health, University of
Pittsburgh, PA, USA.
Clostridium perfringens type B or D isolates, which cause natural enterotoxemias in livestock,
produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class
B select toxin. These C. perfringens strains also express sialidases, although the possible
contributions of those enzymes to type D pathogenesis remain unclear. Type D isolate
CN3718 carries two genes (nanI and nanJ) encoding secreted sialidases and one gene
(nanH) encoding a cytoplasmic sialidase. Construction of single nanI, nanJ and nanH null
mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, in
CN3718 identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells
with NanI sialidase, or with C. perfringens culture supernatants containing NanI, enhanced
the binding and cytotoxic effects of ETX. Contact of CN3718 with Caco-2 cells induced more
rapid sialidase production. Inactivation of nanI reduced ETX production by CN3718; this
effect was fully reversible by complementation with the wildtype nanI. It was also partially
reversible by supplementation of cultures with sialic acid, suggesting that sialic acid could
signal upregulation of ETX production. NanI production also substantially increased CN3718
adherence to enterocyte-like Caco-2 cells. Finally, the sialidase activity of NanI (but not NanJ
or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that,
during type B/D diseases originating in the intestines, trypsin may activate NanI, which (in
turn) could contribute to intestinal colonization by C. perfringens type D isolates and also
increase ETX production and action.

O52
EXTRA-CYTOPLASMIC FUNCTION SIGMA FACTOR CSFV REGULATES LYSOZYME
RESISTANCE OF CLOSTRIDIUM DIFFICILE
T. D. Ho and C.D. Ellermeier*. University of Iowa, Department of Microbiology, Iowa City, IA
USA.
Clostridium difficile is an anaerobic, Gram-positive, spore-forming opportunistic pathogen
which is the most common cause of hospital-acquired infectious diarrhea. In numerous
pathogens, stress response mechanisms are required for survival within the host. Extra-
Cytoplasmic Function sigma (σ) factors (ECF σ factors) are a major family of signal
transduction systems which sense and respond to extracellular stresses. Activity of C.
difficile ECF σ CsfV is induced in response to lysozyme, a substantial component of innate
immunity. To further characterize the role of CsfV in responding to lysozyme, we generated
a C. difficile mutant in csfV. The csfV mutant exhibited a marked decrease in lysozyme
resistance when compared to the wild type C. difficile parent. Interestingly, transmission
electron micrographs revealed that csfV mutant cells were irregularly shaped with
impaired/damaged cell envelope only in the presence of sub-inhibitory levels of lysozyme. In
in vivo hamster experiments, the C. difficile csfV mutant was significantly attenuated
compared to the wild type, indicating that CsfV is critical during C. difficile infection. Using
quantitative RT-PCR we established that CsfV regulates expression of a putative
polysaccharide deacetylase (pdaV) and a presumed post-translational secreted protein
chaperone (prsA2). When pdaV was over-expressed in Bacillus subtilis, PdaV conferred an
increase in lysozyme resistance. These data suggest that, in C. difficile, PdaV may play a
role in lysozyme resistance which is regulated by the CsfV signal transduction system.
Taken together, our data imply that the CsfV signal transduction system is important during
C. difficile infection, likely as a means to control mechanisms for surviving host immune
system assaults such as lysozyme.

POSTER OVERVIEW

Last Name
Title
Auchtung, Jennifer P1 HIGH THROUGHPUT ANALYSIS OF INTERACTIONS
BETWEEN THE HUMAN FECAL MICROBIOME AND
CLOSTRIDIUM DIFFICILE
Babakhani, Farah P2 FIDAXOMICIN INHIBITS PRODUCTION OF TOXIN A
AND TOXIN B IN CLOSTRIDIUM DIFFICILE
Babakhani, Farah P3 FIDAXOMICIN INHIBITS SPORE PRODUCTION IN
CLOSTRIDIUM DIFFICILE
Bakker, Dennis P4 MUTATION OF tcdC IN C. DIFFICILE 630∆ERM DOES
NOT INFLUENCE TOXIN EXPRESSION
Bouillaut, Lauren P5 REGULATION OF AMINO ACID FERMENTATION IN
CLOSTRIDIUM DIFFICILE
Carlson, Paul P6 PHENOTYPIC CHARACTERIZATION OF
CLOSTRIDIUM DIFFICILE CLINICAL ISOLATES
Cartman, Stephen P7 PRECISE MANIPULATION OF THE CLOSTRIDIUM
DIFFICILE CHROMOSOME TO EXPLORE THE ROLE
OF TCDC IN TOXIN PRODUCTION
Collignon, Anne P8 FLAGELLIN OF CLOSTRIDIUM DIFFICILE 027
HYPERVIRULENT STRAIN ACTIVATES ERK1/2
MAPK IN AN EPITHELIAL TLR-5-EXPRESSING CELL
LINE
Collignon, Anne P9 ANALYSIS OF THE GENOME-WIDE EXPRESSION
OF A CLOSTRIDIUM DIFFICILE FLIC MUTANT IN
MONOXENIC MICE
Collignon, Anne P10 PROTEOMIC ANALYSIS OF THE CLOSTRIDIUM
DIFFICILE 630 STRAIN SECRETOME ACCORDING
TO GROWTH KINETICS
Corver, Jeroen P11 ANALYSIS OF A CLOSTRIDIUM DIFFICILE PCR
RIBOTYPE 078 100 KILOBASE ISLAND REVEALS
THE PRESENCE OF A NOVEL TRANSPOSON
Curry, Scott P12 A PILOT STUDY OF CLOSTRIDIUM DIFFICILE IN
RAW RETAIL MEATS FROM WESTERN
PENNSYLVANIA

Figueroa, Iris P13 RELAPSE VERSUS REINFECTION: TIMING AND
INFLUENCE OF THE INFECTING STRAIN ON
RECURRENT CLOSTRIDIUM DIFFICILE INFECTION
Heeg, Daniela P14 SPORULATION RATES AND THE
'HYPERVIRULENCE' OF CLOSTRIDIUM DIFFICILE:
STANDARDISING EXPERIMENTAL PROCEDURES
FOR ACCURATE STRAIN COMPARISON
Ho, Theresa P15 EXTRA-CYTOPLASMIC FUNCTION SIGMA FACTOR
CSFV REGULATES LYSOZYME RESISTANCE OF
CLOSTRIDIUM DIFFICILE
Janezic, Sandra P16 CLOSTRIDIUM DIFFICILE 16S-23S rRNA
INTERGENIC SPACER REGION (ISR) AS A MARKER
FOR PHYLOGENETIC STUDIES
Kõljalg, Siiri P17 SPORULATION LEVEL OF CLOSTRIDIUM DIFFICILE
INTEGRN POSITIVE AND NEGATIVE STRAINS IN
THE PRESENCE OF LACTOBACILLI
Lei, Xiang P18 USING PHENOTYPE MICROARRAYS TO
DETERMINE CULTURE CONDITIONS THAT INDUCE
OR REPRESS TOXIN PRODUCTION BY
CLOSTRIDIUM DIFFICILE
Liu, Mingyu P19 INCREASED BILE SALTS IN THE CECA OF
ANTIBIOTIC-TREATED MICE BY FLAGELLIN
ADMINISTRATION INHIBIT VEGETATIVE
REPLICATION OF CLOSTRIDIUM DIFFICILE
Mallozzi, Michael P20 SURVEILLANCE AND MOLECULAR
CHARACTERIZATION OF CLOSTRIDIUM DIFFICILE
INFECTIONS IN TUCSON
Marsh, Jane P21 CHARACTERIZATION OF RECURRENT
CLOSTRIDIUM DIFFICILE DISEASE ISOLATES BY
MULTI-LOCUS VARIABLE NUMBER TANDEM
REPEAT ANALYSIS AND TOXIN SEQUENCING
Marsh, Jane P22 GENETIC DIVERSITY OF NON-TOXIGENIC
CLOSTRIDIUM DIFFICILE DETERMINED BY MULTI-
LOCUS VARIABLE NUMBER TANDEM REPEAT
ANALYSIS AND MULTI-LOCUS SEQUENCE TYPING

Miezeiewski,
Matthew
P23 AN IN VITRO MODEL TO STUDY COLONIC
MICROBIOTA DISRUPTION IN RELATION TO
INFECTION WITH CLOSTRIDIUM DIFFICILE IN
SYRIAN GOLDEN HAMSTERS
Moura, Hercules P24 PROTEOMIC ANALYSIS OF THE Clostridium difficile
LARGE TOXINS
Naaber, Paul P25 STRAIN-SPECIFIC SUSCEPTIBILITY OF
CLOSTRIDIUM DIFFICILE TO INHIBITORY ACTIVITY
OF LACTOBACILLUS PLANTARUM IN VITRO
Olling, Alexandra P26 ROLE OF THE CROP DOMAIN OF CLOSTRIDIUM
DIFFICILE TOXINS A AND B IN BINDING AND
UPTAKE
Paredes-Sabja,
Daniel
Quesada-Gómez,
Carlos
P27
P28
ADHERENCE OF CLOSTRIDIUM DIFFICILE SPORES
TO HUMAN COLONIC ENTEROCYTE-LIKE CACO-2
CELLS
FLUOROQUINOLONE RESISTANCE, BUT NOT
TOXIN HYPERPRODUCTION, CORRELATES WITH
THE INCREASED FINDING OF A NOVEL
PULSOTYPE OF VIRULENT CLOSTRIDIUM
DIFFICILE STRAINS
Reeves, Angela P29 ROLE OF MEMBERS OF THE RESIDENT GUT
MICROBIOTA IN LIMITING CLOSTRIDIUM DIFFICILE
GROWTH AND TOXIN PRODUCTION
Robinson, Cathy P30 IDENTIFICATION AND CHARACTERIZATION OF
GENES SPECIFIC TO HYPERVIRULENT NAP1
CLOSTRIDIUM DIFFICILE STRAINS
Scholl, Dean P31 PHAGE TAIL-LIKE BACTERIOCINS OF
CLOSTRIDIUM DIFFICILE
Schoster, Angelika P32 EPIDEMIOLOGIC INVESTIGATION OF
CLOSTRIDIUM DIFFICILE AND CLOSTRIDIUM
PERFRINGENS IN HEALTHY HORSES
Shin, Bo-Moon P33 THE PREVALENCE OF Clostridium difficile AND
Clostridium perfringens AS PATHOGENS OF ACUTE
DIARRHEA IN PATIENTS VISITING A TERTIARY
CARE HOSPITAL IN KOREA

Shin, Bo-Moon P34 COMPARISON OF TWO PCR ASSAYS FOR TOXIN B
FOR DIRECT DETECTION OF TOXIN PRODUCING
Clostridium difficile IN FECAL SPECIMENS
Sorg, Joseph P35 EFFECT OF INCREASED FECAL
CHENODEOXYCHOLIC ACID LEVELS ON C.
DIFFICILE VIRULENCE
Squire, Michele P36 DETECTION OF CLOSTRIDIUM DIFFICILE IN
PIGGERY EFFLUENT AFTER TREATMENT IN A 2-
STAGE POND SYSTEM
Theriot, Casey P37 COMPARATIVE PATHOGENICITY OF CLOSTRIDIUM
DIFFICILE STRAINS IN CEFOPERAZONE-TREATED
MICE
von Eichel-Streiber,
Christoph
P38
MONOCLONAL ANTIBODIES TO SPECIFICALLY
IDENTIFY TOXIN B OF CLOSTRIDIUM DIFFICILE
RIBOTYPE 027
Walk, Seth P39 A PHYLOGENETIC ANALYSIS OF THE NEGATIVE
TOXIN REGULATOR (TcdC) OF CLOSTRIDIUM
DIFFICILE
Wright, Lorinda P40 EFFECT OF PROTON PUMP INHIBITORS WITH AND
WITHOUT ANTIBIOTICS ON CLOSTRIDIUM
DIFFICILE INFECTION IN HAMSTERS
Blasi, Juan P41 HIGH AFFINITY BINDING OF CLOSTRIDIUM
PERFRINGENS EPSILON TOXIN TO THE RENAL
SYSTEM
Butel, Marie-José P42 CLOSTRIDIAL GUT COLONIZATION IN PRETERM
NEONATES
Derman, Yagmur P43 csps PLAY A ROLE IN NaCl AND pH STRESS
RESPONSE OF CLOSTRIDIUM BOTULINUM ATCC
3502
Farias, Luana P44 MOLECULAR DIAGNOSIS OF BLACKLEG FROM
COMMON FILTER PAPER
Garcia, Jorge P45 THE EFFECT OF CLOSTRIDIUM PERFRINGENS
TYPE C AND ITS BETA TOXIN MUTANT IN GOATS
Goossens, Evy P46 CLOSTRIDIUM PERFRINGENS STRAINS OF
VARIOUS ORIGIN CAN CAUSE HEMORRHAGIC
ENTERITIS IN A CALF INTESTINAL LOOP MODEL.

Govoni, Gregory P47 A FUNCTIONAL, HIGH MOLECULAR WEIGHT
BACTERIOCIN (―DIFFOCIN‖) FROM CLOSTRIDIUM
DIFFICILE CLONED AND EXPRESSED IN BACILLUS
SUBTILIS
Kelly, Michelle P48 IMPROVING THE REPRODUCIBILITY OF INFECTION
OF THE NAP1/B1/027 HYPERVIRULENT STRAIN
R20291 IN THE HAMSTER MODEL OF INFECTION.
Keto-Timonen,
Riikka
P49
AMPLIFIED FRAGMENT LENGTH POLYMORPHISM
ANALYSIS IN STRAIN TYPING AND
IDENTIFICATION OF CLOSTRIDIUM SPECIES
Lahti, Päivi P50 COMPARATIVE GENOMIC HYBRIDIZATION
ANALYSIS SHOWS DIFFERENT EPIDEMIOLOGY OF
CHROMOSOMAL AND PLASMID-BORNE cpe-
CARRYING C. PERFRINGENS TYPE A STRAINS
Lambert, Dominic P51 CHARACTERIZATION OF SURFACE-LAYER
PROTEINS OF CLOSTRIDIUM BOTULINUM GROUP I
AND II
Lepp, Dion P52 IDENTIFICATION OF C. PERFRINGENS GENES
ASSOCIATED WITH AVIAN NECROTIC ENTERITIS
BY MICROARRAY COMPARATIVE GENOMIC
HYBRIDIZATION
Moura, Hercules P53 APPLYING TOXIN PROTEOMICS TO MEASURE
RELATIVE QUANTITIES OF PROTEINS WITHIN THE
BOTULINUM NEUROTOXIN COMPLEX
Nowell, Victoria P54 GENOMIC AND PROTEOMIC ANALYSIS OF A
BOVINE HEMORRHAGIC ABOMASITIS TYPE A
CLOSTRIDIUM PERFRINGENS ISOLATE
Shi, Lianfa P55 A FRAGMENT OF 97 AMINO ACIDS (D97) WITHIN
THE TRANSMEMBRANE DOMAIN IS ESSENTIAL
FOR THE CELLULAR ACTIVITY OF CLOSTRIDIUM
DIFFICILE TOXIN B
Vargas, Agueda P56 NECROTIZING ENTERITIS ASSOCIATED WITH
CLOSTRIDIUM PERFRINGENS TYPE B IN
CHINCHILLAS (CHINCHILLA LANIGERA)
Yan, Xuxia P57 DETERMINATION OF FUNCTIONAL RESIDUES ON
NETB TOXIN FROM CLOSTRIDIUM PERFRINGENS

Yumine, Natsuko P58 SEAFOOD AS A RESERVOIR AND A SUPPLIER OF
THE PROTOTYPE PLASMID ENCODING
CLOSTRIDIUM PERFRINGENS ENTEROTOXIN
Rupnik, Maja P59 COMPARISON OF CHANGES IN HUMAN AND
POULTRY INTESTINAL MICROBIOTA DURING
COLONISATION WITH CLOSTRIDIUM DIFFICILE
Ardis, Tara C. P60 DEVELOPMENT OF A QUANTITATIVE IMMUNO-PCR
ASSAY FOR THE DETECTION OF GROUP III
CLOSTRIDIUM BOTULINUM NEUROTOXINS IN
CATTLE
Sadighi Akha, Amir P61 THE LOCAL AND SYSTEMIC IMMUNE RESPONSE IN
A MOUSE MODEL OF ACUTE CLOSTRIDIUM
DIFFICILE INFECTION
Zemljič, Majeta P62 PORCELLIO SCABER – A NEW NONVERTEBRATE
MODEL FOR C. DIFFICILE COLONIZATION

ABSTRACTS OF POSTER PRESENTATIONS
SESSION I: P1 TO P20
WEDNESDAY, OCTOBER 26, 2011

P1
HIGH THROUGHPUT ANALYSIS OF INTERACTIONS BETWEEN THE HUMAN FECAL
MICROBIOME AND CLOSTRIDIUM DIFFICILE
J. Auchtung* 1 , C. Robinson 1 , R. Stedtfeld 2 , S. Hashsham 2 , and R. Britton 1 . 1 Department of
Microbiology and Molecular Genetics and 2 Department of Civil and Environmental
Engineering, Michigan State University, East Lansing, MI 48824, USA.
We have developed continuous-flow microbioreactors that allow us to study the interaction
between human fecal microbial communities and Clostridium difficile in vitro. The small size
of these reactors (15 ml culture volume) allows up to 36 individual reactors to operate in
parallel, providing more flexibility to examine multiple conditions simultaneously. Preliminary
experiments have shown that when a pool of human fecal samples is used to inoculate
microbioreactors containing complex media, stable microbial communities composed of
species typically found in the human gut microbiome are established. Furthermore, these
communities can inhibit the growth of C. difficile. Ongoing and future experiments will
examine the how the perturbation of the microbiota with antibiotics makes them susceptible
to C. difficle invasion. Specifically, we plan to analyze how changes in microbial community
structure (as assayed through 16S rDNA and metagenomic sequencing) and function (as
assayed through metatranscriptomics and metabolomics) correlate with the ability of C.
difficile to invade and express virulence functions.

P2
FIDAXOMICIN INHIBITS PRODUCTION OF TOXIN A AND TOXIN B IN CLOSTRIDIUM
DIFFICILE
L. Bouillaut 1 , C. Sims 2 , A. Gomez 2 , P. Sears 2 , J. Seddon 2 , A.L. Sonenshein 1 , and F.
Babakhani* 2 . 1 Tufts University School of Medicine, Boston, MA 021111, USA; 2 Optimer
Pharmaceuticals, Inc., San Diego, CA 921212, USA.
Fidaxomicin, recently approved for the treatment of C. difficile associated diarrhea,
possesses bactericidal activity against C. difficile and was superior in clinical trials to
vancomycin (VAN) in its sustained clinical response. In this study, we showed its mechanism
of antimicrobial activity to be via inhibition of RNA polymerase (RNAP). Since this
mechanism may also lead to inhibition of virulence factors, we examined toxin levels and
toxin gene expressions following exposure to FDX. Transcription inhibition studies were
performed with purified C. difficile RNAP in presence of FDX. Impact of FDX on toxin
production was determined by adding drugs to C.difficile ATCC 43255 (a high toxin
producing strain) and UK-14 (NAP1/BI/027 epidemic strain) at early stationary phase of
growth followed by incubation ~7 days. Culture supernatants were tested by commercial
ELISA-tgcBiomics for the presence of toxins A and B. Toxin gene expression was assessed
in presence of drugs (qRT-PCR) in C. difficile UK1 (NAP1/BI/027 type epidemic strain) and in
its close non-epidemic relative, CD196. Toxin gene transcript levels were normalized to
levels of 16s rRNA and rpoC mRNA. Toxin production in the absence of drugs peaked
between 3 and 5 days in C. difficile ATCC 43255, however, in the presence of FDX and its
major metabolite OP-1118 (both at 1/4xMIC), production of TcdB and TcdA levels were
strongly suppressed. In contrast, VAN did not inhibit toxin production. Similar inhibitory
effects on TcdA and TcdB expression by FDX and OP-1118 but not VAN, or metronidazole
(MTZ) were seen in the UK-14 strain. Addition of FDX (2xMIC) or OP-1118 (2.5xMIC) at the
end of exponential growth phase led to nearly complete inhibition of subsequent
accumulation of transcripts from the pathogenicity locus, i.e. tcdR, tcdA, tcdB and tcdC. By
contrast, addition of vancomycin ( 2.5xMIC) at the end of exponential growth phase had little
or no significant effect on subsequent toxin production. Transcription inhibition studies with
C. difficile
in toxin production may be mediated via the inhibition of RNAP.
Results demonstrate that both FDX and OP-1118, but not VAN or MTZ, block C. difficile toxin
synthesis even when added to stationary phase cells.

P3
FIDAXOMICIN INHIBITS SPORE PRODUCTION IN CLOSTRIDIUM DIFFICILE
A. Gomez 1 , L. Bouillaut 2 , A.L. Sonenshein 2 , P. Sears 1 , L. Nguyen 1 , and F. Babakhani* 1 .
1 Optimer Pharmaceuticals, Inc., San Diego, CA 921211, USA; 2 Tufts University School of
Medicine, Boston, MA 021112, USA.
Introduction: Fidaxomicin was recently approved for the treatment of Clostridium difficile
associated diarrhea. This is a novel narrow spectrum antibacterial agent that demonstrates
potent bactericidal activity against C. difficile. In recent clinical trials with over 1100 CDI
subjects, FDX was shown to be superior to vancomycin in providing sustained clinical
response (response without recurrence of disease) at 25 days following therapy. A possible
mechanism for reduced recurrences is that FDX may exert an inhibitory effect on sporulation.
Methods: FDX and its major metabolite, OP-1118, were evaluated with comparator drugs
vancomycin, metronidazole, and rifaximin for their effects on kinetics of C. difficile (UK-14, an
epidemic NAP1/BI/027 strain and ATCC 43255, a high toxin producer strain) growth and
sporulation. Drugs were added to cells at early stationary phase of growth followed by
collection of cells, at various time intervals, for quantitation of total viable cell count and spore
counts (by heating to kill the vegetative cells) on media containing taurocholate. The effect of
drugs on sporulation gene expression in the C. difficile NAP1/BI/027 type epidemic strain
UK1 was also compared via qRT-PCR using Roche LightCycler 480 and SYBR Green
fluorescence. Transcript levels for test genes were normalized to levels of 16s rRNA and
rpoC mRNA. Results: Both FDX and OP-1118 at sub-MIC concentrations (1/4xMIC) inhibited
sporulation when added to early stationary phase cells in C. difficile strains UK-14 and ATCC
432455. In contrast, vancomycin, metronidazole and rifaximin (at similar sub-MIC levels) did
not inhibit sporulation. The number of spores following treatment with comparator drugs
increased to the same level as the no-drug control treatment. Expression of mother cellspecific
(spoIIID) and forespore-specific (spoIIR) sporulation genes was also inhibited by
fidaxomicin and OP-1118, but not by vancomycin. Conclusion: Both FDX and OP-1118,
unlike vancomycin, rifaximin, or metronidazole, effectively inhibited sporulation by C. difficile.
Fidaxomicin‘s inhibitory effect on C. difficile sporulation may contribute to its superior
performance in sustaining clinical response and reducing recurrences and may also be
beneficial in decreasing shedding and transmission of this pathogen.

P4
MUTATION OF tcdC IN C. DIFFICILE 630∆ERM DOES NOT INFLUENCE TOXIN
EXPRESSION
D. Bakker*, W.K. Smits, E.J. Kuijper and J. Corver. Department of Medical Microbiology,
Center of Infectious Diseases, Leiden University Medical Center, the Netherlands
The main virulence factors of the enteropathogen Clostridium difficile are toxins A and B. The
pathogenicity locus (PaLoc) contains the genes coding for toxin A (tcdA) and toxin B (tcdB),
flanked by the genes for the positive regulator TcdR and the negative regulator TcdC.
Currently, there is a debate about the role of TcdC as a regulator of toxin expression. Our
aim is to clarify the role of tcdC. For this purpose we generated a Clostron-based mutant of
tcdC in Clostridium difficile strain 630∆Erm. Clostridium difficile 630∆Erm (wt) and the C.
difficile CT::tcdC (∆tcdC) mutant were grown under anaerobic conditions in pre-reduced
Brain Heart Infusion broth supplemented with Yeast extract and L-cysteine. Samples for RNA
preparation and toxin detection were taken 1, 2, 5, 7, 24, and 48 h post inoculation.
Synthesized cDNA was used in a real-time PCR for detecting transcription levels of the
PaLoc genes and compared to the levels of toxin expression in a cytotoxicity/MTS assay
measuring the viability of the cells. We find that transcription levels of the PaLoc genes are
growth dependent. The early logarithmic phase is associated with high transcription levels of
tcdC and low level expression of transcription levels of tcdA, tcdB and tcdR. The late
logarithmic and stationary growth phases are associated with low transcription levels of tcdC
and high transcription levels of tcdA, tcdB, and tcdR. The transcription levels of the PaLoc
genes in the ∆tcdC strain are similar to the transcription levels in the wt strain. Furthermore,
we detected increasing levels of toxin expression in time and the toxin expression levels
were similar in both strains. We demonstrated an inverse correlation between tcdC and the
other PaLoc genes. The increasing transcription levels of tcdA, tcdB, and tcdR correlates
with increased toxin expression. These data are consistent with prevailing models. However,
the similar transcription levels of the PaLoc genes and toxin expression levels in both wt and
tcdC strains indicate that TcdC is not a major (negative) regulator of toxin expression under
the conditions tested.

P5
REGULATION OF AMINO ACID FERMENTATION IN CLOSTRIDIUM DIFFICILE
L. Bouillaut* 1 , W. Self 2 , and A.L. Sonenshein 1 . Molecular Biology and Microbiology, Tufts
University; 2 Molecular Biology and Microbiology, University of Central Florida.
Stickland fermentation reactions are key to growth of C. difficile. In Stickland reactions, pairs
of amino acids donate and accept electrons, generating ATP and reducing power in the
process. Reduction of the electron acceptors proline and glycine requires the proline
reductase (Prd) and the glycine reductase (Grd) enzyme complexes, respectively. Addition of
proline to the medium increases the level of Prd protein but decreases the level of Grd
protein and synthesis of toxins. Upstream of the prd operon, we identified a putative
regulatory gene, prdR. To investigate PrdR function, we constructed a C. difficile prdR
mutant using Targetron technology. Our analysis revealed that PrdR is responsible for
activation of the transcription of the Prd-encoding genes in the presence of proline. We
conclude that PrdR is a proline-activated activator. Furthermore, transcripts of Grd genes
were more abundant in the prdR mutant strain compared to the wild type, suggesting a role
for PrdR in the glycine utilization pathway as well. The results suggest that PrdR is a central
metabolism regulator that controls preferential utilization and fermentation of proline and
glycine to produce energy via the Stickland reaction. Given the important role of proline in
toxin synthesis, we are investigating the role of PrdR in toxin gene expression.

P6
PHENOTYPIC CHARACTERIZATION OF CLOSTRIDIUM DIFFICILE CLINICAL ISOLATES
P. E. Carlson Jr*, and P. C. Hanna. Department of Microbiology and Immunology, University
of Michigan, Ann Arbor, MI 48104 USA.
Clostridium difficile (Cd) causes between 1 and 3 million cases of diarrhea and colitis in the
United States each year at an annual cost of $1.1B dollars for nosocomial infections alone. It
is likely that Cd spores, and not the vegetative bacilli, are the direct contagion, similar to
other pathogenic Clostridium sp. Spores are hardy, not easily cleared from the body, and
resistant to antibiotics. The robust physical resistance properties of spores necessitate
prolonged periods of antibiotic treatment and a high risk of developing serious drug
intolerances, and residual spores may cause infection to recur after initial treatment is
stopped. We hypothesize that there is a correlation between Cd germination/ sporulation and
human pathogenesis. Cd was isolated from sick patients at multiple U.S. hospitals. To date,
more than 40 isolates have been examined. Spore stocks were produced by growing each
isolate on BHIS plates for seven days at 37°C under anaerobic conditions. Cultures were
exposed to oxygen and washed repeatedly to purify spores. Germination assays were
performed by incubating spores with germinant and monitoring loss of heat resistance over
time. Sporulation was measured by observing increase in heat resistant CFU in a culture
over 3 days. Finally, spore viability was measured by examining the ratio between spores
present and those able to outgrow and produce colonies. Cd clinical isolates exhibited a
range of phenotypes. These isolates exhibited a wide range of spore viability, with some
spore preps containing fewer than 25% viable spores. Interestingly, for nearly all isolates
tested, the spores that were viable were capable of rapid germination, with 100%
germination observed within 10 minutes. Sporulation rates were even more variable. While
most isolates were capable of sporulation to some level, the total number of spores observed
for each isolate was significantly different. Clinical isolates of Cd exhibit clear differences in
sporulation and spore viability. While this fact alone is interesting, the effect of these
differences on clinical outcome remains to be elucidated. Patient information will be
examined to determine if any of these bacterial phenotypes lead to an altered disease course
in humans.

P7
PRECISE MANIPULATION OF THE CLOSTRIDIUM DIFFICILE CHROMOSOME TO
EXPLORE THE ROLE OF TCDC IN TOXIN PRODUCTION
S. T. Cartman* 1 , M. L. Kelly 1 , D. Heeg 1 , D. A. Burns 1 , S. A. Kuehne 1 , J. T. Heap 1 and N. P.
Minton 1 . 1 Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7
2RD, United Kingdom.
TcdC is widely considered to be a negative regulator of toxin production. However, this
notion has never been tested rigorously using isogenic strains of Clostridium difficile. We
have developed a new 2-step allele exchange procedure which permits precise manipulation
of the C. difficile chromosome. Using this approach we have constructed recombinant strains
of C. difficile with different tcdC genotypes. Here we will describe the construction of these
strains and the subsequent analysis, which suggests that tcdC genotype does not affect toxin
production.

P8
FLAGELLIN OF CLOSTRIDIUM DIFFICILE 027 HYPERVIRULENT STRAIN ACTIVATES
ERK1/2 MAPK IN AN EPITHELIAL TLR-5-EXPRESSING CELL LINE
P-A. Jolivot 1 , A. Collignon* 1 , and I. Kansau 1 . 1 EA 4043, USC INRA Faculté de Pharmacie,
Université Paris-Sud 11, 92296 Châtenay-Malabry, France.
Clostridium difficile is a Gram-positive bacillus responsible for antibiotic-associated diarrhea
and pseudomembranous colitis. Recently, emerging hypervirulent ribotype 027 strain was
associated with a higher mortality rate. The pathogenesis of C. difficile is essentially based
on the production of two toxins: tcdA and tcdB that disrupt cellular cytoskeleton and cause
severe tissue damage. To date, the role of flagella of C. difficile in its pathogenesis is not
understood. The aim of this study was to characterize the signaling pathways elicited in
epithelial cells by the flagella of this opportunistic pathogen through Toll-Like Receptor-5
(TLR-5), which recognizes flagellin monomers. We focused our work on a potential activation
of Mitogen-Activated Protein Kinases (MAPK) and more specifically the activation of
Extracellular signal-Regulated kinases 1 and 2 (ERK1/2). To reach this aim, first the
epithelial polarized MDCK (Madin-Darby Canine Kidney) cell line were transfected with TLR-
5 so that it expresses this receptor of the innate immune response in a homogenous and
stable way. Then, the TLR-5-expressing MDCK cells (MDCK-TLR5) and the control MDCK
cell line were infected during different times (from 5 to 60 minutes) by two C. difficile strains:
the wild-type hypervirulent 027 strain (R20291, WT) and the isogenic strain mutated in the
fliC gene that did not express flagellin (ΔFliC). Furthermore, a recombinant His-tagged FliC
flagellin from C. difficile was purified by immobilized metal ion affinity chromatography. This
recombinant FliC was incubated during various times (30 and 60 min) and at different
concentrations (from 1 to 10 µg/ml) with MDCK-TLR5 and MDCK cells. The ERK1/2
activation was analyzed on cell lysates by Western blot. The MDCK-TLR5 infection by the
WT hypervirulent strain triggered a time-dependent activation of ERK1/2 whereas no
activation was observed with the control cell line. On both MDCK-TLR5 and MDCK cell lines,
the ΔfliC strain did not activate ERK1/2. The recombinant flagellin, regardless of
concentration and during 30 and 60 min incubation, also activated ERK1/2 in MDCK-TLR5
cells but not in MDCK cells. The ERK1/2 activation by a flagellated C. difficile strain and
purified flagellin suggests that the C. difficile flagellin might play a role in the initiation of an
innate immune response and contribute to the pathogenesis of this bacterium.

P9
ANALYSIS OF THE GENOME-WIDE EXPRESSION OF A CLOSTRIDIUM DIFFICILE FLIC
MUTANT IN MONOXENIC MICE
A. Barketi-Klai 1 , M. Monot 2 , S. Hoys 1 , S. Lambert 1 , B. Dupuy 2 , A. Collignon* 1 , I. Kansau 1 . 1 EA
4043, USC INRA, Faculté de Pharmacie, Université Paris-Sud 11, 92296 Châtenay-Malabry,
France; 2 Institut Pasteur, 28, rue du Docteur Roux, 75015, Paris, France.
The hypervirulence of the Clostridium difficile 027 strains seems to be due to several factors
including high-toxin production, presence of binary toxin, and high sporulation rate. However,
other factors such as colonization factors could also be involved. We are therefore interested
in the flagellar proteins and in particular the structural flagellin protein FliC.
We previously constructed a fliC mutant in a C. difficile 027 strain by the Clostron technique.
The mutant showed: 1) inability to synthesize FliC and flagella, 2) a greater adherence on
human colonic cell line Caco-2 as compared to the parental strain, and 3) ability to colonize
the mouse intestine either in a monoxenic or a dixenic model in competition with the wild type
strain. Growth competition between the wild type and the fliC mutant strains showed the
dominance of the wild type. Surprisingly, the fliC mutant was more virulent than the wild type
strain. Actually, all monoxenic mice challenged with the fliC mutant died 48 h post-infection in
contrast to mice colonized with the wild type strain, which all survived. Thus, the absence of
FliC increases the virulence of the C. difficile 027 strain suggesting that FliC could play a role
in virulence. We then analyzed and compared the bacterial transcriptomes of the fliC mutant
and the parental strains at the early stage of intestinal colonization in the monoxenic mouse
model. The global analysis of the fliC mutant data (by Ma2HTLM) showed a differential
expression of 310 genes. High regulations were particularly observed for genes involved in
motility, membrane transport systems (PTS, ABC transporters), carbon metabolism,
regulation (Agr-2, s54, PadR system) and sporulation. Significant regulation was also
observed for genes involved in the synthesis of toxins (TcdC down-regulated), the cell wall
(cwp66 up-regulated), cell growth, fermentation, metabolism (AA, nucleic acids, lipids), stress
(antibiotic resistance) and anaerobic respiration. This in vivo transcriptomic analysis will
provide important data to better understand the hypervirulence of C. difficile.

P10
PROTEOMIC ANALYSIS OF THE CLOSTRIDIUM DIFFICILE 630 STRAIN SECRETOME
ACCORDING TO GROWTH KINETICS
C. Viala 1 , C. Boursier 2 , A. Collignon* 1 , and, S. Péchiné 1 . 1 EA 4043, USC INRA, Faculté de
Pharmacie, Université Paris-Sud 11, Châtenay-Malabry, France; 2 Plate-forme TransProt,
IFR141-IPSIT, Faculté de Pharmacie, Université Paris-Sud 11, Châtenay-Malabry, France.
Clostridium difficile, a Gram-positive spore forming anaerobe, is a major cause of antibioticassociated
diarrhea and pseudomembranous colitis. Toxins TcdA and TcdB are the primary
virulence factors and are secreted late in the exponential phase and during the stationary
phase. However, other aspects of C. difficile virulence are poorly understood. In particular,
little is known about the mechanisms of gastrointestinal colonization, an undoubtedly
essential step in pathogenesis. Different adhesins and probably also some hydrolytic and
proteolytic enzymes are implicated. They could be considered as secondary virulence
factors. Indeed, bacterial pathogens generally secrete a subset of proteins mediating
essential functions during pathogenesis such as adhesion to host tissues and modulation of
the host immune response. The identification of such proteins and the mechanisms of
secretion employed by C. difficile will give new clues regarding the pathogenic process. By a
proteomic approach that combines bidimensional electrophoresis with nano-liquid
chromatography-tandem mass spectrometry (nanoLC-MS/MS), we studied the secretome of
the C. difficile 630 strain. This work was based on an in vitro growth kinetic at five different
times. Secreted proteins were collected and precipitated from the culture supernatant fluids
at T=0, T=4h, T=6h, T=8h and T=10h. Culture and extraction was done for each point in
triplicate. Then, they were analyzed by two-dimensional gel electrophoresis. The gels were
compared two by two: T=0 was compared to T=4h, T=4h to T=6h, T=6h to T=8h and T=8h to
T=10h. Appearance or disappearance of spots, as well as significant variation in spot
intensity was noted (software ImageMaster 2-D Platinum®). 202 differential protein spots
were of interest. Some of these spots have been analyzed and identified by nanoLC-MS/MS.

P11
ANALYSIS OF A CLOSTRIDIUM DIFFICILE PCR RIBOTYPE 078 100 KILOBASE ISLAND
REVEALS THE PRESENCE OF A NOVEL TRANSPOSON
J. Corver* 1 , D. Bakker 1 , M. Brouwer 2 , C. Harmanus 1 , M. P. Hensgens 1 , A.P. Roberts 2 , E. J.
Kuijper 1 and H. C. van Leeuwen 1 . 1 Department of Medical Microbiology, Leiden University
Medical Center, Leiden, The Netherlands; 2 UCL Eastman Dental Institute, Division of
Microbial Diseases, London, United Kingdom
A collection of 229 Clostridium difficile PCR ribotype 078 isolates from human and porcine
origin was tested for the presence of a 100 kb insert, previously described to be unique for
PCR ribotype 078. The insert contained over 90 open reading frames, encoding mainly
proteins from transposons, phages and plasmids. Indeed, through PCR, the presence of a
circular transposon DNA, containing the 5‘and 3‘ends of the whole insert, could be shown.
The transposon was named Tn6164 and was present in 9 human isolates. All 9 isolates were
resistant to tetracycline and they originated from various countries in Europe. Moreover, 9
isolates, all from humans, contained only half of the transposon, suggesting multiple insertion
steps yielding the full Tn6164. MLVA analysis clearly revealed genetic relatedness between
transposon- containing isolates, but not all isolates. The presence of Tn6164 did not result in
a readily identifiable phenotype but clinical data suggest a possible increase in virulence
linked to presence of the transposon.

P12
A PILOT STUDY OF CLOSTRIDIUM DIFFICILE IN RAW RETAIL MEATS FROM WESTERN
PENNSYLVANIA
S.R. Curry * 1 , J.W. Marsh 1 , M.M. Tulenko 1 , L.H. Harrison 1 . 1 Infectious Diseaess
Epidemiology Research Unit, University of Pittsburgh, Pittsburgh, PA, 15261, USA.
Background: The prevalence of C. difficile in retail meat samples has varied widely, with
North American studies reporting 12-45% prevalence in contrast to European studies
reporting 0-4.5%.The food supply may be a source for C. difficile infections. Methods: In
phase I, 102 raw ground meat and sausage samples (n=20 beef, 2 buffalo, 22 chicken, 2
lamb, 40 pork, 10 turkey, and 5 veal) were purchased from 3 grocers in Pittsburgh, PA
between 2/28/2011 and 4/28/2011. Where available, the USDA establishment number was
recorded. All products were processed in batches of no more than 30 samples. Samples
were broth amplified using 10g sample in 100 mL cycloserine cefoxitin mannitol broth with
taurocholate and lysozyme and incubated anaerobically for five days. A negative broth
control was carried through food sample processing. Meat spiked with C. difficile of known
genotype was included as positive control. Fermenting samples were sub-cultured to 5%
sheep blood agar and cycloserine cefoxitin fructose agar with horse blood and taurocholate.
Colonies with morphologies consistent with C. difficile were confirmed by L-proline
aminopeptidase and typed using tcdC genotyping and multilocus variable number of tandem
repeats analysis (MLVA). For phase II, an additional 12 samples of brand A pork sausages
were purchased 5/14/2011 and sampled as above. Results: 2/102 (2.0%) raw meat products
sampled were positive for C. difficile. Both were from brand A pork sausage products. The
products were purchased on separate dates and cultured in different batches. Both products
were from the same processing facility (facility A). Another brand A sample from a different
processing facility was negative for C. difficile. Of the additional 12 samples of brand A, 4/5
samples from processing facility A were positive for C. difficile. None of the 7 samples from
facilities B, C, and D were positive for C. difficile. The isolates in phase I were both tcdC
genotype A (inferred ribotype 078) but were distinct by MLVA with a summed tandem repeat
difference of 21. The positive control strain was tcdC genotype 2. Conclusions: The
prevalence of C. difficile in retail meats in western Pennsylvania is 2%. These data suggest
that the prevalence of C. difficile in retail meat may not be as high as previously reported in
North America.

P13
RELAPSE VERSUS REINFECTION: TIMING AND INFLUENCE OF THE INFECTING
STRAIN ON RECURRENT CLOSTRIDIUM DIFFICILE INFECTION
I. Figueroa* 1 , S.P. Sambol 1,2 , E.J.C. Goldstein 3,4 , S. Johnson 1,2 , and D.N. Gerding 1,2 . 1 Hines
VA Hospital, Hines, IL, USA; 2 Loyola University Medical Center, Maywood, IL, USA; 3 RM
Alden Research Laboratory, Culver City, CA, USA; 4 David Geffen School of Medicine at
UCLA, Los Angeles, CA, USA.
Recurrences of Clostridium difficile infection (CDI) following successful treatment are
common and may be due to the same strain that caused initial infection (relapse) or a
different strain (reinfection). The frequency of relapse and reinfection and the timing and
influence of the initial infecting strain on these events are not well defined. Therefore, we
identified and compared CD isolates from initial and recurrent episodes of CDI in 93 patients
from a large clinical treatment trial comparing vancomycin and fidaxomicin. Culture of stool
specimens was performed anaerobically on selective media for CD and strain typing was
performed by HindIII Restriction Endonuclease Analysis (REA). Early recurrences after
successful treatment (within 14 days of treatment completion) were relapses in 87.3% and
reinfections in 12.7%. Late recurrences (15-30 days after treatment) were relapses in 76.7%
and reinfections in 23.3%. The average time to recurrence was 11.9 days for relapses and
14.9 days for reinfections (positive trend, but P=NS). The most common subtype of the
current epidemic strain in North America, REA type BI 6/8/17, was the initial infecting strain
in 26 patients of whom 25 had relapses and one who was reinfected with a different strain.
Reinfection with new strains were more common following initial infection by strains other
than BI 6/8/17. Reinfection with new strains may occur early or late after treatment
completion, but tend to occur somewhat later than relapses with the same strain. Most
recurrences following infection with the epidemic BI 6/8/17 strain were relapses of the same
strain. The influence of the treatment agent on relapse and reinfection is currently being
analyzed.

P14
SPORULATION RATES AND THE 'HYPERVIRULENCE' OF CLOSTRIDIUM DIFFICILE:
STANDARDISING EXPERIMENTAL PROCEDURES FOR ACCURATE STRAIN
COMPARISON
D. A. Burns, D. Heeg*, S. T. Cartman, and N. P.Minton. School of Molecular Medical
Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park,
Nottingham, NG7 2RD UK.
Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and a major
burden to healthcare services worldwide. In recent years, C. difficile strains belonging to the
BI/NAP1/027 type have become highly represented among clinical isolates. These so-called
‗hypervirulent‘ strains are associated with outbreaks of increased disease severity, higher
relapse rates and an expanded repertoire of antibiotic resistance. Spores, formed during
sporulation, play a pivotal role in disease transmission and it has been suggested that
BI/NAP1/027 strains are more prolific in terms of sporulation in vitro than ‗non-epidemic‘ C.
difficile types. Work in our laboratory has since provided credible evidence to the contrary
suggesting that the strain-to-strain variation in C. difficile sporulation characteristics is not
type-associated. However, the BI/NAP1/027 type is still widely stated to have an increased
rate of sporulation. On analysis of the other studies currently in the literature, it is apparent
that sample sizes have remained small and, perhaps most importantly, the methods used to
quantify sporulation have severe limitations. In this study, we analysed the sporulation rates
of 53 C. difficile strains, the largest sample size used to-date in such a study, including 28
BI/NAP1/027 isolates. Our data confirm that significant variation exists in the rate at which
different C. difficile strains form spores. However, we clearly show that the sporulation rate of
the BI/NAP1/027 type is no higher than that of non-BI/NAP1/027 strains. In addition, we
observed substantial variation in sporulation characteristics within the BI/NAP1/027 type.
Predictably, given the clinical importance of C. difficile BI/NAP1/027 a considerable amount
of time and money is now being utilised to investigate the molecular basis of virulence in this
type. This work highlights the danger of assuming that all strains of one type behave similarly
without studying adequate sample sizes. Furthermore, we stress the need for a standard set
of experimental procedures in order to quantify C. difficile sporulation more accurately in the
future.

P15
DETECTION OF CELL ENVELOPE STRESS IN C. DIFFICILE
T.D. Ho and C. Ellermeier. Department of Microbiology, University of Iowa, Iowa City, IA
52242 USA.
Extra-Cytoplasmic Function sigma (σ) factors (ECF σ factors) are a major family of signal
transduction systems which sense and respond to extracellular stresses. We have identified
three C. difficile ECF σ factors. These ECF σ factors, CsfT, CsfU, and CsfV, induce their own
expression and are negatively regulated by their cognate anti-σ factors RsiT, RsiU and RsiV,
respectively. The expression of csfT and csfU are induced following exposure to the
antimicrobial peptide bacitracin and/or lysozyme. In contrast, csfV is specifically induced by
lysozyme stress. The activity of many ECF σ factors is controlled by site-1 and site-2
proteases which cleave anti-σ factors. The C. difficile prsW mutant exhibited decreased
expression of CsfT and CsfU but not CsfV. When expressed in a heterologous host, C.
difficile PrsW induces degradation of RsiT but not RsiU. When the prsW mutant was tested in
competition assays against its isogenic parent in the hamster model of C. difficile infection,
we found that the prsW mutant was 30-fold less virulent than the wild type. The prsW mutant
was also significantly more sensitive to bacitracin and lysozyme than the wild type in in vitro
competition assays. Taken together, these data suggest that PrsW likely regulates activation
of ECF σ factor CsfT in C. difficile and controls resistance of C. difficile to antimicrobial
peptides which are important for survival in the host.

P16
CLOSTRIDIUM DIFFICILE 16S-23S rRNA INTERGENIC SPACER REGION (ISR) AS A
MARKER FOR PHYLOGENETIC STUDIES
S. Janezic* 1 , T. Weinmaier 2 , T. Rattei 2 , A. Indra 3 and M. Rupnik 1,4,5 . 1 Institute of Public Health
Maribor, Centre for Microbiology, Maribor, Slovenia; 2 University of Vienna, Faculty of Life
Sciences, Vienna, Austria; 3 Austrian Agency for Health and Food Safety (AGES), Vienna,
Austria; 4 University of Maribor, Faculty of Medicine, Maribor, Slovenia; 5 Centre of Excellence
CIPKeBIP, Ljubljana, Slovenia.
For molecular epidemiological investigation of Clostridium difficile infections, a PCR
ribotyping method based upon size variation in 16S-23S rRNA intergenic spacer region
(ISR), is widely used in European reference laboratories. The size variability seen in ISR is
due to the presence or absence of tRNA genes and spacers of different lengths separated
with direct repeats which, as suggested could result from slipped-strand mispairing and/or
homologous recombinations (Indra et al., 2010). To investigate the extent of diversity of ISRs
in C. difficile, and to see if ISRs could be a marker for phylogenetic studies, ISRs of a total of
31 strains of 26 different PCR ribotypes were amplified using primers described by Bidet et
al., (1999). PCR products or fragments excised from the gel were then cloned and
sequenced. More than 300 sequences were obtained (ISR sequences of published C.
difficile genomes; strains 630, R20291 and CD196, were also included in the analysis). To
remove the sequence variability that could have resulted from sequencing errors and to
reduce the number of sequences and facilitate analysis, sequence clustering with 97%
identity was performed. The representatives of non-redundant sets were then aligned using
M-Locarna software which first calculates the secondary structure for all ISRs using the
Vienna RNA package and then generates multiple alignment that keeps as many structural
features as possible. It has been demonstrated previously that ISRs are organized in mosaiclike
structure with sequence blocks that are present or absent in different rDNA copies with
alignment done only on the basis of sequence similarity. Sequences aligned on the basis of
the secondary structure also show many conserved blocks that could be for example used
for phylogeny calculations and would help us to better understand the evolution of the
virulent strains. Moreover, our results show that ISR sequences of different ribotypes that are
of the same or comparable sizes are very similar and hence the PCR ribotyping reflects this
relatedness.

P17
SPORULATION LEVEL OF CLOSTRIDIUM DIFFICILE INTEGRN POSITIVE AND
NEGATIVE STRAINS IN THE PRESENCE OF LACTOBACILLI
S. Kõljalg* 1 , M. Rätsep 1 , J. Stsepetova 1 , I. Smidt 1 , E. Shkut 1 , P. Naaber 1,2 , E. Sepp 1 .
1 Department of Microbiology, University of Tartu, Tartu, Estonia; 2 Department of Medical
Microbiology, Stavanger University Hospital, Stavanger, Norway.
Clostridium difficile (CD) is the leading cause of nosocomial diarrheas in adults and also the
most important agent of antibiotic-associated diarrhea. Sporulation is important for disease
transmission, but also for surviving in the intestinal tract in the presence of indigenous
bacteria such as lactobacilli. The aim of this work was to clarify sporulation level (SL) and
integrons in CD strains and the possible influence on SL of Lactobacillus plantarum (LP)
strains. Materials and methods. Eighty-two CD isolates were used, including 64 from Estonia
[22 from 1994 (EE1) and 42 from 2008 (EE2)] and 18 from Norway in 2008 (NO)]. Five LP
strains were used. The presence of class I integrons was studied by PCR. SL at room
temperature (initial conc 8x10 8 CFU/ml) was estimated. SL at body temperature was studied
at 0, 10, 24, and 48 hours of growth of CD alone or with LP. Results. Class I integrons were
found in 29/82 (35%) strains. Five sizes of integrons were detected (200 to 600bp). The most
common size was 300 bp (14/29). The majority of integron-containing strains (n = 27) were
isolates from EE2 and it differed from data of EE1 (27/42 vs 1/21, p < 0.001) and NO (27/42
vs 1/17, p < 0.001). SL at room temperature varied from 6.1 to 10.7 log 10 CFU/ml. EE2
strains exhibited more spores than NO strains (median 10 vs 8 log 10 CFU/ml, p < 0.001).
Integron-containing strains had higher SL than integron-negative ones (median 9.7 vs 8.3
log 10 CFU/ml, p = 0.02) at room temperature, but differences were not found at body
temperature. Compared to CD alone, co-growth with LP caused a decrease in SL at 24h
(median 3.0 vs 0 log 10 CFU/ml, p = 0.016) and 48h (median 4.8 vs 1.3 log 10 CFU/ml, p <
0.001). The influence of LP was more expressed to integron-positive than negative strains
(median at 10 h: 0 vs 1.6 log 10 CFU/ml, p = 0.014; at 24h: 0 vs 1.5 log 10 CFU/ml, p < 0.001;
at 48h: 0 vs 1.9 log 10 CFU/ml, p < 0.001). Conclusions. We revealed the putative influence of
probiotic LP strains on CD sporulation level. The presumed linkage between sporulation
capacity and integrons needs to be clarified.

P18
USING PHENOTYPE MICROARRAYS TO DETERMINE CULTURE CONDITIONS THAT
INDUCE OR REPRESS TOXIN PRODUCTION BY CLOSTRIDIUM DIFFICILE
X.H. Lei*, B.R. Bochner. Biolog, Inc., Hayward, CA, USA.
Background: Toxins A and B are important determinants of C. difficile virulence. In this study
we developed a simple but reliable and efficient cytotoxicity assay for C. difficile toxin to
systematically examine the effects of culture conditions on toxin production in Phenotype
MicroArray (PM) panels.
Methods: C. difficile type strain ATCC 9689 was cultured in an anaerobic chamber (5% H2,
5% CO2, 90% N2) at 36ºC for 24 hours and then inoculated into PM panels and incubated
for 72 hours. Toxins in the microplate well supernatants were harvested by filtration with a
96 well Filter Plate. Mammalian cells CHO-k1 and Vero from ATCC were grown in flasks at
37ºC with 5% CO 2 in RPMI 1640 plus 10% FBS and Pen/Strep and then plated into 96-well
tissue-culture treated microplates in an assay medium. Five to 10ul aliquots of filtered
supernatant containing toxins were transferred into each well of the plated cells. Cell
morphologies were observed and photos were taken after 18-20 hours of exposure. Then
viability of the cells was determined using a dye reduction assay with Biolog Dye Mix MB and
OmniLog instrument. The plates were also read at 590 nm with a microplate reader.
Results: Strong effects on C. difficile toxin production were seen with certain nitrogen
sources. Dipeptides and a few tripeptides had stronger stimulatory effects on toxin
production than single amino acids. Arginine peptides gave among the highest levels of
toxin production. Some carbon sources also had strong inducing effects. D-threonine gave
highest toxin production followed by L-serine, L-alanine, D-serine, and a few other amino
acids. Acetyl amino sugars or amino sugars as carbon or nitrogen sources (e.g., N-acetyl-Dglucosamine)
also showed a strong or above average stimulation effect. Fumaric acid and
bromosuccinic acid gave highest toxin production among the carboxylic acid carbon sources.
Conclusions: We demonstrated an effective approach for studying toxin production by C.
difficile under hundreds of different culture conditions. Our results provide valuable insights
into metabolic regulation of toxin production by C. difficile. The combination of Phenotype
MicroArray technology with a cytotoxicity assay, a toxin neutralization assay employing a
specific antibody, and a cell viability assay based on cellular dye reduction, makes it possible
to reliably, quantitatively, effectively, and efficiently study regulation of toxin production by C.
difficile and other bacteria.

P19
INCREASED BILE SALTS IN THE CECA OF ANTIBIOTIC-TREATED MICE BY FLAGELLIN
ADMINISTRATION INHIBIT VEGETATIVE REPLICATION OF CLOSTRIDIUM DIFFICILE
M. Liu* 1 , I. Jarchum 1 , and E.G. Pamer 1 . 1 Immunology Program, Sloan-Kettering Institute,
Department of Medicine (Division of Infectious Disease), Memorial Sloan-Kettering Cancer
Center, New York, NY 10065, USA.
Clostridium difficile (C. difficile), a spore-forming enteric bacterium, is often acquired by oral
ingestion of spores and can cause severe antibiotic-associated diarrhea in humans. To
cause disease, oral acquired spores must germinate and undergo vegetative growth in host.
Antibiotic treatment of mice increases the concentrations of small intestinal bile salts that can
stimulate germination of C. difficile. However, a few bile salt compounds inhibit vegetative
growth of C. difficile in vitro. Administration of Salmonella-derived purified flagellin, a Toll-like
receptor 5 (TLR5) ligand, delays the vegetative replication of C. difficile in mice and protects
mice from acute C. difficile colitis. We found that small intestinal extracts from mice without
antibiotic treatment, but administered with flagellin, can enhance germination of C. difficile
spores. Cholestyramine, a bile salt sequesterer, abolishes the enhanced germination,
suggesting that bile salts are involved in the process. Further analyses of bile salts in
intestinal extracts from either antibiotic-treated or untreated mice show that flagellin
administration increases the concentrations of bile salts in small intestinal extracts from
untreated mice but not antibiotic-treated mice. By contrast, the concentrations of bile salts in
cecal extracts from antibiotic-treated mice but not untreated mice are elevated by flagellin.
The effects of flagellin administration on bile salts are dependent on TLR5. Compared with
mice only treated with antibiotics, cecal extracts from mice treated with both flagellin and
antibiotics support vegetative replication of C. difficile much worse. Treatment of cecal
extracts with cholestyramine augments the vegetative growth of C. difficile in extracts and
abolishes the difference between extracts from mice with and without flagellin administration.
Addition of deoxycholate, a bile salt compound, to cecal extracts from mice only treated with
antibiotics, restores the suppression of vegetative replication of C. difficile to the extent
observed in the extracts from mice treated with both flagellin and antibiotics. Our results
support the idea that flagellin administration causes an elevated level of bile salts in the ceca
of antibiotic-treated mice, which is associated with enhanced germination, but more likely
suppressing vegetative replication of C. difficile.

P20
SURVEILLANCE AND MOLECULAR CHARACTERIZATION OF CLOSTRIDIUM DIFFICILE
INFECTIONS IN TUCSON
F. Anwar 1 , M.J. Mallozzi* 1,6 , D. Wolk 3,5 , N. Ampel 5 , A. Noon 4 , V.K. Viswanathan 1,5 , and G.
Vedantam 1,2,5,6 . 1 Dept. of Veterinary Science and Microbiology, 2 Dept. of Immunobiology,
3 Dept. of Pathology, 4 Dept. of Medicine, 5 BIO5 Research Institute, University of Arizona,
Tucson AZ, USA; 6 Southern Arizona VA Healthcare System, Tucson AZ, USA.
BACKGROUND: Clostridium difficile (CD) is a leading bacterial cause of nosocomial
infections, with over 400,000 cases reported annually in the US. Risk factors for C. difficile
infection (CDI) include age, immune-suppression and the use of antibiotics. Since the year
2000, new ―hypervirulent‖ CD strains have emerged, that are associated with CDI
epidemics/outbreaks, severe disease, increased mortality, and increased risk of recurrent
infection. Thus CDI surveillance studies are urgently required to document the various
virulent types of CD. The last CDI surveillance study in Arizona was approximately
seventeen years ago. METHODS AND RESULTS: 83 stool specimens were collected from
three Tucson-area hospitals. CD isolates were recovered through the use of selective media.
The molecular types of these isolates were determined by PCR-based ribotyping. Semiquantitative
toxin testing was done on a subset of these isolates. We identified multiple CD
ribotypes, including several which are epidemic-associated (ribotypes 001, 027, 078 and
106). Low toxin-producing CD was also recovered from multiple specimens. Epidemicassociated
CD accounted for up to 50% of CD-positive samples at one facility (private
hospital). Two facilities had one predominant ribotype: 027 (private hospital) and 001 (VA
Medical Center) respectively, while a third (University Medical Center) had a mix of various
ribotypes. Toxin analysis revealed that the samples from the various hospitals produced
varying amounts of toxin, and high toxin levels
did not necessarily correlate with epidemic-associated types. CONCLUSIONS: Epidemicassociated
molecular types of CD are prevalent and frequently isolated from Southern
Arizona hospitals. Interestingly, we found a preponderance of ribotype 001 strains at a
hospital previously associated with a CDI outbreak (17 years ago) that was also dominated
by ribotype 001 strains. Further, our toxin data indicate that the toxin-producing capability
does not vary drastically among the isolated ribotypes. Thus, other mechanisms (besides
toxin-production) likely play equally important roles in pathogenesis in vivo. These results
underscore the importance of surveillance for this important pathogen, and substantiate the
use of highly-sensitive molecular methods to identify CD.

ABSTRACTS OF POSTER PRESENTATIONS
SESSION II: P21 TO P40
THURSDAY, OCTOBER 27, 2011

P21
CHARACTERIZATION OF RECURRENT CLOSTRIDIUM DIFFICILE DISEASE ISOLATES
BY MULTI-LOCUS VARIABLE NUMBER TANDEM REPEAT ANALYSIS AND TOXIN
SEQUENCING
J. Marsh*, J. Gee, K. Shutt, S. Curry, L. Harrison. Epidemiology Research Unit, University of
Pittsburgh, Pittsburgh, PA, 15261, USA.
Background. Recurrent Clostridium difficile infection (CDI) occurs in ~25% of CDI cases.
Host immune responses against C. difficile are protective and vaccines that target toxin
peptides are under development for prevention of CDI. Discriminatory genotyping tools are
required to distinguish relapse from re-infection in patients with recurrence. In this study,
multi-locus variable number tandem repeat analysis (MLVA) was used to classify recurrent
CDI as either relapse or re-infection. C-terminal antigenic variation of toxins A and B was
examined in relapsing isolates of individual patients. Methods. MLVA and tcdC genotyping
were performed on 355 C. difficile isolates from 148 in-patients with recurrent CDI at UPMC
from 2001-2009. The summed tandem repeat difference (STRD) among isolates from
individual patients was determined from MLVA data. Serial patient isolates with STRD < 2
were defined as relapse while those with STRD > 3 were defined as reinfection. Sequence
analysis and peptide alignments of the C-terminal domains of toxins A and B were performed
on 242 isolates using SeqMan and MegAlign software in LaserGene DNAstar v.8. Results.
Among 108 patients with a single recurrence, 69 (64%) were classified as relapse with a
median interval between episodes of 36 days. Of relapsing isolates, 62% had the tcdC-1
allele characteristic of BI/NAP1. The 39 patients with a single recurrence due to re-infection
had a median interval between episodes of 92 days. Among 40 patients with >1 recurrence,
14 (35%) were relapse, 5 (13%) were re-infections and 21 (53%) were a combination of
relapses and re-infections. Among 242 selected isolates, there were 16 different TcdA and
10 different TcdB C-terminal peptides identified generating 20 different TcdA/TcdB peptide
combinations. No antigenic variation among toxin peptides was observed in relapsing CDI
cases. Of 104 relapsing cases, 60 (58%) were attributable to BI/NAP1. Conclusions. Based
on MLVA genotyping, the majority of recurrent CDI was due to relapse and many of these
were attributable to the BI/NAP1 hypervirulent clone. C-terminal toxin variation among serial
isolates from patients with relapsing disease was not observed. Further investigation of
tandem repeat mutation rates combined with tcdC and toxin genotypes will help define useful
metrics of genetic relatedness based on MLVA.

P22
GENETIC DIVERSITY OF NON-TOXIGENIC CLOSTRIDIUM DIFFICILE DETERMINED BY
MULTI-LOCUS VARIABLE NUMBER TANDEM REPEAT ANALYSIS AND MULTI-LOCUS
SEQUENCE TYPING
J. W. Marsh* 1 , T. K. Henderson 1 , S. P. Sambol 2 , D. N. Gerding 2,3 , L.H. Harrison 1 .
1 Infectious Diseases Epidemiology Research Unit, University of Pittsburgh, Pittsburgh, PA
15261, USA; 2Hines Veterans Affairs Hospital, Hines, IL, USA; 3 Loyola University Chicago
Stritch School of Medicine, Maywood, IL, USA.
Background. Colonization by non-toxigenic Clostridium difficile (NTCD) prevents CD infection
in hamsters. Since asymptomatic carriage of NTCD in humans is common, these strains may
be of therapeutic benefit. Multi-locus variable number tandem repeat analysis (MLVA) and
multi-locus sequence typing (MLST) were performed to determine the genetic diversity of
NTCD strains. A parallel serial passage experiment (PSPE) was performed to examine the
stability of MLVA loci for discrimination of NTCD. Methods. MLVA and MLST were performed
on 121 NTCD isolates from 6 different restriction enzyme analysis (REA) groups
representing 71 REA types. Minimum spanning tree (MST) was generated using the
summed tandem repeat difference (STRD) based on the MLVA data. The PSPE was
performed on a NTCD representing the most common REA type by inoculating 96 agar
plates from a single colony and performing 10 serial passages on each lineage. MLVA was
performed on the 10th passage of each lineage and the rate of mutation at each tandem
repeat locus was calculated. Results. Overall, NTCD isolates clustered on the MST
according to REA groups and sequence type. There were 115 MLVA types identified. REA
type M3, the most common NTCD in this collection could be discriminated by a 6 locus
MLVA typing scheme. Tandem repeat loci CDR4 and CDR6 had the highest mutation rates
while CDR5 and CDR48 were invariant in the PSPE. Nine sequence types (ST) were
identified. All of the REA group M strains belonged to ST-15 and the majority of REA group
T were ST-3. Conclusions. Asymptomatically carried NTCD are relatively clonal by MLST.
MLVA identifies NTCD strains according to REA groups but is substantially more
discriminatory than REA.

P23
AN IN VITRO MODEL TO STUDY COLONIC MICROBIOTA DISRUPTION IN RELATION
TO INFECTION WITH CLOSTRIDIUM DIFFICILE IN SYRIAN GOLDEN HAMSTERS
M. Miezeiewski* 1 , S. Wang 1 , T. Schnaufer 2 , M. Muravsky 2 , T. Mitchell 2 , J. Zorman 1 , K.
Soring 1 , A. Xie 1 , J. Heinrichs 1 , J. ter Meulen 1 , J. Shiver 1 , and J.L. Bodmer 1 . 1 Merck Research
Laboratories, Vaccine Basic Research, West Point, PA, USA; 2 Merck Research Laboratories,
Bioanalytics & Pathology, West Point, PA, USA.
Clostridium difficile is the pathogen commonly associated with antibiotic-associated diarrhea
in humans. Clostridium difficile infections (CDI) are caused by the outgrowth of toxigenic
strains of C. difficile in the colon of individuals whose microbiota has previously been
perturbed by antimicrobial therapy. The lethal enterocolitis model in Syrian golden hamsters
(Mesocricetus auratus) is the most commonly accepted animal model mimicking CDI in
humans. The critical components of this model are the disruption of the hamster‘s GI flora by
antibiotic treatment and the challenge with C. difficile spores. The relationship between
colonization, the status of the gut microbiota and ultimately toxin production and disease is
an ongoing area of research. Here we present an in vitro method to determine the in vivo
susceptibility of Syrian hamsters to colitis and allow for the quantitative measurement of C.
difficile growth and toxin production in a convenient format. In brief, fecal samples were
collected from Syrian hamster prior and post administration of a single dose of clindamycin.
C. difficile spores of strain VPI 10463 were then cultured in broth in the presence or absence
of the filtered soluble fraction of the fecal material for 72hr at 37C under anaerobic
conditions. Cultures were analyzed by qPCR to determine C. difficile genome copy numbers
and toxin production levels by EIA. The data demonstrated that (a) normal fecal extracts
inhibit C. difficile growth, (b) fecal extracts from animals treated with clindamycin are
permissive to C. difficile growth and toxin production, and. (c) the restoration of clindamycin
treated microbiota is gradual and closely matches recovery kinetics observed in the
challenge itself. Overall, the timing of disruption and subsequent recovery in this in vitro
system is highly suggestive of the proposed pathogenesis in humans. It is, therefore, a
central tool in the understanding of the hamster model and its applications for
prevention/treatment of CDI in humans.

P24
PROTEOMIC ANALYSIS OF THE CLOSTRIDIUM DIFFICILE LARGE TOXINS
H. Moura 1 *, R.R. Terilli 1,2 , A.R. Woolfitt 1 , Y.M. Williamson 1 , T.A. Blake 1 , M.I. Solano 1 , and
J.R. Barr 1 . 1 Division of Laboratory Sciences, National Center for Environmental Health;
Centers for Disease Control and Prevention (CDC), Atlanta, GA USA; 2 Oak Ridge Institute
for Scientific Education, Oak Ridge, TN USA.
Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitals
worldwide, caused by hypervirulent epidemic strains which have the ability to produce
greater quantities of the toxins TcdA and TcdB. The expression level of these toxins from
different toxinotypes is not completely known. Additionally, accurate quantification of TcdA
and TcdB using small samples has not yet been reported. Proteomics strategies can be used
for accurate quantification of TcdA and TcdB and the information obtained can be applied to
new toxin detection methods, investigating toxic mechanisms, and developing new
therapeutics. In the present study, we describe the analyses of both purified TcdA and TcdB
and a standard culture filtrate, using two different proteomics platforms. Three biological
samples of each analyte were analyzed in triplicate using gel-based and gel-free
approaches. The proteins were separated by SDS-PAGE and the gel bands were digested
and analyzed using a mass spectrometer (MS) instrument. In addition, purified TcdA and
TcdB were digested using five different enzymes followed by analysis on two different MS
instruments. Discovery-type proteomics experiments were applied to the culture filtrate. The
toxins were further quantified using label-free MS and the method was applied to measure
TcdA and TcdB in the standard culture filtrate. The use of two different proteomics platforms
provided unique results. First, the gel-based approach revealed basic information on toxin
integrity and provided sequence confirmation. Secondly, gel-free in-solution digestion of both
toxins generated broad amino acid sequence coverage with 84% for TcdA and 90% for
TcdB. This data coupled to in silico sequencing analysis suggested that the method has the
potential to distinguish different C. difficile toxinotypes based on protein sequence.
Proteomics analysis of the culture filtrate uncovered a number of proteins, among them
TcdA, TcdB, and S-layer protein. Lastly, MS-based label-free proteomics analysis was used
to estimate the amounts of TcdA and TcdB in small (

P25
STRAIN-SPECIFIC SUSCEPTIBILITY OF CLOSTRIDIUM DIFFICILE TO INHIBITORY
ACTIVITY OF LACTOBACILLUS PLANTARUM IN VITRO
P. Naaber* 1;2 , I. Smidt 1 , M. Rätsep 1 , S. Kõljalg 1 , E. Shkut 1 and E. Sepp 1 . 1 Department of
Microbiology, University of Tartu, Tartu, Estonia; 2 Department of Medical Microbiology,
Stavanger University Hospital, Stavanger, Norway.
Clostridium difficile (CD) causes diarrhea and colitis in patients with disrupted intestinal
microbiota. Our previous studies have shown the possible role of L. plantarum (LP) in
resistance to colonization with CD. The aim of our study was to evaluate the in vitro
antagonistic effect of indigenous LP strains on the growth of CD strains. Materials and
methods. In total, 11 LP strains (different genotypes) isolated from antibiotic-associated
diarrhea patients‘ fecal samples were initially screened for antagonistic properties against
two CD strains. Five LP strains potentially active against CD were co-cultivated with 12 CD
strains of different ribotypes. The presence of integrons and resistance patterns, counts of
CD, and spore production after 10, 24, and 48 hours of co-cultivation were measured.
Results. The highest inhibition of CD strains by lactobacilli as compared to controls was
found after 24 h co-cultivation (median inhibition 4.1 log 10 CFU/mL vs. 1.0 after 10 h, p <
0.0001; and vs. 2.85 after 48 h, p = 0.26). The CD strain-related variations in inhibition were
high (range 0 – 8.7 log 10 at 24h), while no significant differences in inhibition activity between
different LP strains were found. CD strains showed different degrees of susceptibility to LP
inhibition: highly-sensitive (inhibition > 5 log 10 by all LP strains) – 3 strains (medians 6.7; 6.9
and 8.3), moderately sensitive (inhibition 2.5 - 5) – 2 strains (medians 4.1 and 4.3) and weak
(inhibition < 2.5) – 2 strains (medians 1.3 and 1.4). In the case of 5 CD strains, high variation
of sensitivity (from highly-sensitive to insensitive; range 0-6.0) was found, depending on the
particular LP strain. There was no correlation between CD strain sensitivity to inhibition and
their PCR-ribotype, presence of integrons, spore production, or quinolone resistance.
However, these 3 CD strains that were highly-sensitive to LP were resistant to clindamycin,
whereas the other 9 strains were clindamycin sensitive. Conclusions. Our in vitro results
suggest that antagonistic activity of L. plantarum against CD is CD strain-specific, rather than
LP strain-specific. High sensitivity of CD to inhibition could be related to clindamycin
resistance. This strain-specific sensitivity to indigenous intestinal bacteria can partly explain
the clinical variation in signs and symptoms of CD infection and contradictory results of effect
of probiotics against CD infection.

P26
ROLE OF THE CROP DOMAIN OF CLOSTRIDIUM DIFFICILE TOXINS A AND B IN
BINDING AND UPTAKE
A. Olling*, S. Goy, E. Frenzel, H. Tatge, I. Just and R. Gerhard. Institute of Toxicology,
Hannover Medical School, 30625 Hannover, Germany.
The Clostridium difficile toxins TcdA and TcdB are intracellular acting toxins that inhibit small
GTP-binding proteins of the Rho subfamily after entering host cells via receptor-mediated
endocytosis. The C-terminally combined repetitive oligopeptides (CROPs) function as
receptor binding domain. Antibodies directed against the CROPs are able to neutralize TcdA
and TcdB. Based on previous findings we hypothesized that the CROPs are not essential for
function of TcdA and TcdB. To re-evaluate the role of the CROPs we investigated binding
properties and potency towards different cells of full length and CROP-truncated TcdA
(TcdA1-1874) and TcdB (TcdB1-1852). TcdA1 1874 and TcdB1-1852 induced time and
concentration dependent cell rounding and Rac1-glucosylation; however, depending on the
cell line, with up to 10-fold reduced potency compared to the respective full length toxin.
FACS and Western blot analyses revealed a correlation between CROP-binding to the cell
surface and toxin potency. This was supported by the observation of a faster internalization
process of full length TcdA compared to CROP- truncated TcdA1 1874. These findings refute
the accepted opinion of a solely CROP- mediated toxin uptake and led to the assumption of
an additional alternative uptake process at least for TcdA. This process might be based
either on the recognition of different receptor structures or on various endocytotic pathways.
To substantiate this hypothesis, we analyzed toxin potency following apical or basolateral
uptake into the polarized intestinal epithelial cell line CaCo-2 as model for different receptor
structures. In fact, cells were hardly susceptible towards apically applied TcdA1-1874
whereas basolateral treatment caused a pronounced cytopathic effect. In contrast, potency
of full length TcdA was almost identical and independent on the side of application.
Furthermore, competition experiments indicated that TcdA and TcdA1 1874 predominantly
use different receptor structures corroborating the notion of alternative toxin internalization
processes. Thus, different routes for cellular uptake might enable the toxins to enter a
broader repertoire of cell types leading to the observed multifarious pathogenesis of C.
difficile. The characterization of specific receptor structures and alternative endocytotic
pathways used by the C. difficile toxins might therefore be the basis to investigate the
opportunity of toxin uptake inhibition as a therapeutic option.

P27
ADHERENCE OF CLOSTRIDIUM DIFFICILE SPORES TO HUMAN COLONIC
ENTEROCYTE-LIKE CACO-2 CELLS
D. Paredes-Sabja 1 *, M.R. Sarker 2,3 . 1 Universidad Andrés Bello, Chile;
2 Oregon State University, USA.
Clostridium difficile is a Gram positive, anaerobic, spore-forming, entero-pathogenic
bacterium, and is the main causative agent of antibiotic-associated diarrhea. There is general
consent that C. difficile spores are the morphotype of transmission, infection and persistence
of Clostridium difficile infections (CDI). Antibiotic treatments disrupt the normal colonic
microbiota, allowing C. difficile spores to germinate, proliferate, secrete toxins and cause
CDI. During the course of CDI, the appearance of high level C. difficile spores in the patient‘s
stools suggest that C. difficile vegetative cells sporulate inside the host leading to persistence
of C. difficile spores in the intestinal tract. The persistence of C. difficile spores in the colon of
CDI-patients complicates effective treatments, as C. difficile spores exhibit resistance to all
currently available treatments and can, therefore, survive in the colon until suppression of
CDI treatments. The latter observations suggest that C. difficile spores might have unique
adherence properties to the host‘s epithelial surfaces. In this context, our current study
shows that, despite the similarities in spore-surface hydrophobicity between spores of C.
difficile and C. perfringens (another enteric pathogen that also sporulates in the gut), spores
of C. difficile adhere better to Caco-2 cells. The adherence of C. difficile spores to Caco-2
cells was localized to the apical microvilli, and adherence was significantly reduced when C.
difficile spores were trypsin-treated. A competitive inhibition assay between fluorescently
labeled and unlabeled C. difficile spores revealed that adherence of C. difficile spores to
Caco-2 cells is mediated by unknown spore-specific receptors on Caco-2 cells. Sonication of
C. difficile spores altered the ultrastructure of the outermost exosporium-like structure,
released two protein species of ~ 40-kDa, significantly reduced spore-hydrophobicity and
adherence to Caco-2 cells. Using a trifunctional crosslinker, we were able to coimmunoprecipitate
four protein species from the surface of Caco-2 cells. In conclusion, this
study provides, for the first time, evidence that C. difficile spores are well suited to adhere to
human colonic enterocyte-like cells and that the mechanism of adherence might be mediated
through specific spore-surface and enterocytic-surface receptors(s) and/or ligand(s).

P28
FLUOROQUINOLONE RESISTANCE, BUT NOT TOXIN HYPERPRODUCTION,
CORRELATES WITH THE INCREASED FINDING OF A NOVEL PULSOTYPE OF
VIRULENT CLOSTRIDIUM DIFFICILE STRAINS
C. Quesada-Gómez 1 , C. Rodríguez 1 , M. del Mar Gamboa-Coronado 1 , C. Guzmán-Verri 2 , T.
Du 3 , M.R. Mulvey 3 , E.Chaves-Olarte 1,2 *, E. Rodríguez-Cavallini* 1 . 1 Centro de Investigación
en Enfermedades Tropicales and Facultad de Microbiología, Universidad de Costa Rica, San
José, Costa Rica; 2 Programa de Investigación en Enfermedades Tropicales, Escuela de
Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica; 3 National Microbiology
Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada. *Contact:
esteban.chaves@ucr.ac.cr, evelyn.rodriguez@ucr.ac.cr
During a recent outbreak of C. difficile infection, we found 35 PFGE macrorestriction patterns
among 105 isolates. While nearly one third of these isolates were classified as the
hypervirulent NAP1 (33%), a similar proportion of isolates (34%) were grouped in a novel
pulsotype designated as NAP-CR1. The remaining isolates corresponded to NAP4 (6.7%),
NAP9 (2.8%), NAP6 (1.9%), NAP2 (1.9%) and other 21 PFGE patterns without NAP
designation (19.7%). To gain insight into the high abundance of the NAP-CR1 strains, we
compared the pulsotypes to their toxin genotype (PCR), toxin expression (Western Blot, real
time PCR, and cytotoxicity), and resistance to several antibiotics (E-test). The genes tcdA,
tcdB, and tcdC were detected in all the pulsotypes. By contrast, cdtB was only found among
the NAP1 strains. The NAP-CR1 strains contained the 18 bp deletion in tcdC previously
described in toxin hyperproducers like NAP1, but lacked the 1 bp deletion in the upstream
region of this gene. Accordingly, the hyperproduction of TcdA and TcdB was confirmed for
the NAP1 strains but not for the NAP-CR1 strains. The NAP1 and NAP-CR1 strains were
highly resistant to the fluoroquinolones (ciprofloxacin and moxifloxacin), whereas almost all
the other strains were highly sensitive to these antibiotics. The resistance to clindamycin of
the NAP-CR1 strains was distinctive because they exhibited elevated MIC for this
antimicrobial (MIC50 >256 µg ml-1) and were positive for ermB. All the other pulsotyopes,
including NAP1, had a lower MIC for clindamycin (MIC 50 = 8 µg ml-1) and were negative for
the aforementioned gene.
Altogether, our results indicate that the higher incidence of NAP1 and NAP-CR1 strains in
this outbreak correlates more with the antibiotic resistance profile than with the level of
production of TcdA and TcdB. Thus, we postulate that resistance to antibiotics plays a major
role in the intranosocomial dissemination of C. difficile strains.

P29
ROLE OF MEMBERS OF THE RESIDENT GUT MICROBIOTA IN LIMITING CLOSTRIDIUM
DIFFICILE GROWTH AND TOXIN PRODUCTION
A. Reeves* 1 , and V. Young 1,2 . 1 Department of Microbiology and Immunology; 2 Department of
Internal Medicine/Division of Infectious Diseases, University of Michigan, Ann Arbor, MI
48109, USA.
Clostridium difficile is a pathogen that causes nosocomial antibiotic-associated diarrhea and
colitis. The indigenous gut microbiota has an important role in protecting the host against
infection with C. difficile. Administration of antibiotics disrupts the gut microbiota, thus
allowing C. difficile to cause disease. In this study we employed in vivo and in vitro systems
to examine the role of members of the normal gut microbiota in limiting C. difficile growth and
toxin production. WT C57BL/6 mice were treated for 3 days with 5 antibiotics (kanamycin,
gentamicin, vancomycin, colistin and metronidazole) in drinking water. Following a two-day
recovery period without antibiotics, animals received a single dose of clindamycin and were
then challenged with C. difficile (VPI 10463) one day later. Control animals received no
treatment. Clinical signs of disease were monitored, and at necropsy, tissue was harvested
for histopathologic and culture-independent analysis of the gut community. Specific members
of the gut community were isolated by selective culture and spent culture supernatants from
each isolate were tested for the ability to inhibit C. difficile growth and toxin production in
vitro. Antibiotic-treated mice challenged with C. difficile either developed rapidly lethal CDI or
were stably colonized with mild disease. The gut community of animals with mild disease
was dominated by Lachnospiraceae, resembling the control community. Moribund animals
had high levels of Enterobacteriaceae. Treatment with only antibiotics resulted in a
predominance of Lactobacillus. Based on this information, we isolated E.coli, Lactobacillus
spp, and Lachnospiraceae spp. We also obtained a short-chain fatty acid producing
Lachnospiraceae, Butyrivibrio fibrisolvens (ATCC 19171). Spent culture supernatants from B.
fibrisolvens and the Lachnospiraceae isolate significantly decreased levels of C. difficile
growth. Correspondingly, a cytotoxin assay using Vero cells also showed decreased toxin
activity. Lactobacillus spp and E. coli showed lower levels of C. difficile growth inhibition.
Lachnospiraceae members had the greatest inhibitory effect on C. difficile growth and toxin
activity. It is likely that these organisms secrete inhibitory substances such as short-chain
fatty acids that limit C. difficile growth and toxin production. Understanding how members of
the indigenous microbiota interfere with C. difficile could lead to new modalities for
prevention and treatment of this important infection.

P30
IDENTIFICATION AND CHARACTERIZATION OF GENES SPECIFIC TO
HYPERVIRULENT NAP1 CLOSTRIDIUM DIFFICILE STRAINS
C. Robinson*, J. P. van Pijkeren and R. Britton. Department of Microbiology and Molecular
Genetics, Michigan State University, East Lansing, MI 48824, USA.
Clostridium difficile, the leading cause of antibiotic-associated diarrhea, is a prevalent
nosocomial pathogen. Growth of this enteric pathogen is normally inhibited by mechanisms
of colonization resistance by the intestinal microbiota; however, growth can ensue following a
perturbation of the microbiota, usually initiated by administration of antibiotics. Many
aspects of the physiology of this pathogen and its interactions with the microbiota are poorly
understood. In the past decade a hypervirulent strain of C. difficile has emerged
(NAP1/ribotype 027), generating an even greater need to understand this organism and its
pathology. Using Phylogenetic Profiler we compared sequenced C. difficile genomes and
identified fifty genes unique to NAP1 strains. A cluster of four genes was found that
interrupts the thymidylate synthase gene (thyX), including an alternative thymidylate
synthase (thyA), dihydrofolate reductase (dhfR), a hypothetical protein, and thiamine
synthase (thiC), respectively. This is of interest because it has been demonstrated in recent
literature that ThyA is catalytically more active than ThyX, promoting up to 10-fold faster DNA
replication rates, which has physiological implications including faster growth rates and
maintenance of larger genome sizes. Current experiments are being conducted to
investigate growth differences by competing NAP1 and non-NAP1 strains in continuous
culture competitions. A cluster of CRISPR (clustered regularly interspersed palindromic
repeats)-associated proteins was also found to be unique to NAP1 strains. CRISPR genes,
which play a role in phage resistance, may confer a competitive advantage to hypervirulent
strains over historical strains that are susceptible to phage infection. PCR detection of the
thyA and CRISPR loci in over 80 recent clinical isolates of various NAP lineages from
Michigan verified that these gene clusters are unique to, and conserved among, NAP1
hypervirulent strains. In addition to the genetic investigations, we have also conducted
phenotypic screens of several NAP1 and non-NAP1 strains using Phenotypic MicroArrays
(Biolog Inc.); these are 96-well plates used to screen preselected compounds for their effects
on growth, sporulation, and germination. Using this technology, we have identified several
compounds that increase growth yield of C. difficile, including ones that are selectively
efficacious to just the NAP1 strains screened. We expect that these investigations will
elucidate differences between hypervirulent and non-hypervirulent strains on the
physiological and ecological levels.

P31
PHAGE TAIL-LIKE BACTERIOCINS OF CLOSTRIDIUM DIFFICILE
D. Gebhart, S. Williams, L. Fortier, G. Govoni, and D. Scholl*. AvidBiotics Corp., South San
Francisco, CA 94080 USA.
Some strains of C. difficile produce high molecular weight phage tail-like particles, ―diffocins,‖
upon induction of the SOS response. We have purified these particles and showed for the
first time that they have bactericidal activity against other C. difficile strains, and can be
classified as bacteriocins. The gene cluster that encodes diffocin structural and regulatory
proteins has been identified. Diffocins produced from different strains possess unique
bactericidal spectra. One of the most interesting features of these particles is a very large
protein of ~200 kDa, encoded by Orf 1374. This large protein determines the killing spectrum
of the particles and is likely the receptor-binding protein. We plan to develop diffocins as
antimicrobial agents for both therapeutic and prophylactic use.

P32
EPIDEMIOLOGIC INVESTIGATION OF CLOSTRIDIUM DIFFICILE AND CLOSTRIDIUM
PERFRINGENS IN HEALTHY HORSES.
A. Schoster* 1 , L.G. Arroyo 1 , H.R. Staempfli 1 , P.E. Shewen 1 , J.S. Weese 1 . 1 Ontario Veterinary
College, University of Guelph, Guelph, ON Canada.
Clostridium difficile and Clostridium perfringens are important causes of equine colitis but can
also be found in healthy individuals. Epidemiologic information is restricted to cross-sectional
studies of fecal shedding with little information on prevalence in gastrointestinal
compartments other than feces and variability in shedding over time. The objectives were to
investigate the presence of C. difficile and C. perfringens in healthy horses over time and
assess prevalence in different gastrointestinal compartments. Feces were collected monthly
from 25 horses for one year. Ingesta were collected from nine GI compartments of a
separate group of 15 euthanized horses. Selective enrichment culture was performed,
followed by toxin gene detection and ribotyping (C. difficile) and multiplex PCR (C.
perfringens). Toxigenic C. difficile was isolated from 15/275 (5.5%) samples from 10/25
(40%) horses over one year. Three horses were positive in consecutive months, but different
ribotypes were found in 2/3. Ribotypes included 078 (n=6), 001 (n=5) and an A+B+CDT+
ribotype previously identified as toxinotype IX in this laboratory (n=4). C. perfringens was not
recovered despite a detection threshold of 9cfu/g feces. Toxigenic C. difficile was isolated
from 14/135 (10.3%) ingesta samples from 8/15 (50.3%) horses, from both the small and
large intestine. Multiple compartments were positive in four horses but more than one
ribotype was present in all. In 3/8 colonized horses (38%) the rectal sample was negative but
a proximal site was positive. Ribotypes included 078 (n=5), 001 (n=4) and three A+B+CDTribotypes
not previously identified in this laboratory. C. perfringens type A (CPE- and β2-)
was recovered from the colon of 1/15 horses (6.6%). In the longitudinal study only one horse
shed the same strain for more than one month, suggesting horses are short-term carriers or
that C. difficile spores are passively moving through the intestinal tract. The presence of
multiple ribotypes in different gastrointestinal compartments in the same horse further
supports transient passage. Agreement between feces and proximal compartments was
moderate to good (κ=0.61), suggesting limitations with studies using feces. The
predominance of ribotype 078, in contrast to earlier reports, suggests recent emergence in
this horse population, similar to reports from humans. The low prevalence of C. perfringens
supports results of previous studies that indicate this organism is rare in healthy horses.

P33
THE PREVALENCE OF CLOSTRIDIUM DIFFICILE AND CLOSTRIDIUM PERFRINGENS
AS PATHOGENS OF ACUTE DIARRHEA IN PATIENTS VISITING A TERTIARY CARE
HOSPITAL IN KOREA
B. Shin*, S. Mun, E. Lee, E. Kuak. Department of Laboratory Medicine, Sanggye Paik
Hospital, Inje University, Seoul, Korea.
Object: Clostridium perfringens is one of the most common pathogens associated with acute
diarrhea. Clostridium difficile is a common pathogen of health care associated infection, and
community associated CDI (CA-CDI) seems to be increasing as well. Both of these
pathogens are difficult to culture in routine microbiology laboratories and the prevalence
rates are not exactly known in Korea. Therefore, we need to investigate the prevalence rate
of both C. difficile and C. perfrignens as community associated diarrheic pathogens. Method:
We performed muliplex-PCR (Seeplex Diarrhea ACE Detection, Seegene, Seoul, South
Korea) to detect C. difficile, tcdB and C. perfringens genes with 262 stool specimens from
adult diarrheic patients visiting an emergency department in a tertiary hospital in Korea from
July 2008 to June 2011. We also perfomed C. difficile culture and tcdA and tcdB gene PCR
assay with C. difficile isolated from culture. Results: The number of C. difficile and C.
perfringens were 34 (13.0%) and 58 (22.1%), respectively. Among 34 C. difficile isolates,
tcdA+tcdB+, tcdA-tcdB+ and tcdA-tcdB- strains were 31 (91.2%), 2 (5.9%) and 1 (2.9%).
Salmonella spp, Shigella spp, Campylobacter spp and Vibrio spp were also detected in 11,
12, 32, and 9 specimens with multiplex PCR. Conclusion: C. perfringens was the most
prevalent pathogen as etiologic agent causing acute diarrhea in patients visiting the
emergency department in a tertiary care hospital in Korea. C. difficile was the second most
prevalent pathogen causing acute diarrhea in patients visiting the ED, which means C.
difficile is an emerging community associated diarrheic pathogen. Most C. difficile strains
isolated from patients visiting the ED were tcdA+tcdB+, but tcdA-tcdB+ strains were also
found. The isolation and identification of C. difficile and C. perfrignens are important,
because the treatment regimens of C. difficile and C. perfringens are different, and more
intensive infection control is needed for C. difficile.

P34
COMPARISON OF TWO PCR ASSAYS FOR TOXIN B FOR DIRECT DETECTION OF
TOXIN PRODUCING CLOSTRIDIUM DIFFICILE IN FECAL SPECIMENS
B. Shin*, S. Mun, E. Lee, E. Kuak. Department of Laboratory Medicine, Sanggye Paik
Hospital, Inje University, Seoul, Korea.
Objectives: Clostridium difficile (C. difficile) is a spore-forming, gram-positive anaerobe,
responsible for virtually all cases of pseudomembranous colitis (PMC), and for 15-25% of
cases of antibiotic-associated diarrhea (AAD). Toxigenic C. difficile strains usually produce
two toxins, enterotoxin (TcdA) and cytotoxin (TcdB), both involved in the pathogenic
characteristics of the microorganism. The aim of this study was to evaluate the performance
of the two new PCR assays (BD GeneOhm Cdiff assay and Seegene Diarrhea ACE ) and
compared the results with bacterial culture and in house PCR assay for tcdA and tcdB from
C. difficile isolates for the detection of toxigenic C. difficile. Materials and Methods: Freshly
collected samples (n = 215) were cultured on a Clostridium difficile selective media (BD) and
isolates were tested using in-house PCR assays for tcdA and tcdB. In this study, culture and
PCR assays were considered as the reference method. Each sample was analyzed using
two commercial PCR assays (BD GeneOhm and Seegene ACE B ). BD GeneOhm
(SanDiego, CA, US) Cdiff assay is a real-time PCR assay that amplifies tcdB and Seegene
ACE B is a dual priming oligo multiplex PCR assay that contains primer to detect tcdB.
Results: C. difficile was identified in 56 samples (26.0%), and 159 samples (74.0%) showed
culture-negative results. PCR assays for tcdA and tcdB genes were conducted on the 56 C.
difficile isolates. The number of tcdA+/tcdB+, tcdA-/tcdB+, and tcdA-/tcdB- strains are 36 , 5 ,
15, respectively. The concordance rate between BD GeneOhm and Seegene ACE toxin B
was 96.7% (208/215). The sensitivity and specificity and positive and negative predictability
of BD GeneOhm and Seegene ACE Toxin B for tcdB gene was 95.1%/96.0%/ 84.8%/98.8%
and 90.2%/96.6%/86.0%/97.7%, respectively. Conclusion: Both PCR assays (BD GeneOhm
and Seegene ACE Toxin B for tcdB gene) represented good performances compared to
toxigenic culture for the detection of toxigenic C. difficile directly in stool specimens.

P35
EFFECT OF INCREASED FECAL CHENODEOXYCHOLIC ACID LEVELS ON C. DIFFICILE
VIRULENCE
C. Allen and J. Sorg*. Department of Biology, Texas A&M University, College Station, TX
77843 USA.
Clostridium difficile infections remain a significant burden to the hospital and long-term
healthcare settings. C. difficile is spread in the environment in the form of a dormant spore.
Upon inoculation into susceptible hosts, C. difficile spores germinate in response to certain
bile acids and glycine. Bile acids are steroid-based compounds synthesized in the liver as
cholic acid or chenodeoxycholic acid derivatives and help emulsify fat and cholesterol in the
intestinal tract. Previously, we found that C. difficile spore germination is initiated upon
exposure to cholic acid derivatives (taurocholic acid, glycocholic acid, cholic acid and
deoxycholic acid). Chenodeoxycholic acid competitively inhibits cholic acid-mediated
germination and is toxic for growth. Inhibiting in vivo spore germination would be a powerful
way in overcoming new or relapsing C. difficile infections. Thus, to test this hypothesis, we
will feed chenodeoxycholic acid to Syrian hamsters and monitor the levels of fecal
chenodeoxycholic acid by High Performance Liquid Chromatography. Hamsters will be
treated with clindamycin and inoculated with C. difficile spores. Animals will be monitored for
signs of disease and compared to animals fed chow without the compound. These
experiments may highlight the importance of anti-germination therapy in preventing C.
difficile infections.

P36
DETECTION OF CLOSTRIDIUM DIFFICILE IN PIGGERY EFFLUENT AFTER TREATMENT
IN A 2-STAGE POND SYSTEM
M.M. Squire* 1 , S.C. Lim 1 , N.F. Foster 1 , and T.V. Riley 1,2 . 1 Microbiology and Immunology,
School of Biomedical, Biomolecular and Chemical Sciences, The University of Western
Australia, Perth, 6009, Australia; 2 Microbiology and Infectious Diseases, PathWest
Laboratory Medicine, Perth, 6009, Australia.
Clostridium difficile is associated with enteric disease in man and animals. It has recently
emerged as an agent of neonatal enteritis in piglets, with high asymptomatic carriage rates
(up to 75%). C. difficile may be part of a zoonosis; there is increasing overlap between
molecular types of C. difficile causing human and animal disease. Transmission may be via
environmental contamination or food-borne spores as C. difficile is found in retail meat and
ready-to-eat salads. The association with enteric disease is poorly understood, but it is clear
that C. difficile is prevalent in Australian piggeries. Piggery effluent is treated in anaerobic
ponds to remove pathogens and re-used to wash sheds or applied to agricultural land. We
hypothesized that C. difficile would also survive the effluent treatment process due to the
resistant nature of its spores. We conducted a study to ascertain the presence, number and
molecular type of C. difficile at various stages of piggery effluent treatment and storage. Ten
samples of effluent before and after treatment in a 2-stage pond system were collected,
cultured, and C. difficile enumerated by direct spread plate counting. C. difficile was isolated
from all 10 samples. Isolates were confirmed as PCR ribotype UK 237 which predominates
in piglets at this farm. C. difficile (8.0 x 10 1 cfu/ml) was isolated from raw effluent prior to
treatment. As expected, C. difficile numbers increased during anaerobic and facultative
treatment phases (1.3 x 10 2 cfu/ ml and 2.3 x 10 2 cfu/ ml respectively), decreasing to 9.3 x
10 1 cfu/ ml in the aerobic evaporative pond. Low numbers of C. difficile (3.5 x 10 1 cfu/ ml on
average) also remained in treated effluent storage tanks, although these samples were taken
from the surface of full tanks and may have been affected by spore settling. These results
confirm our hypothesis that C. difficile survives effluent treatment. This is the first study to
detect and count C. difficile in treated piggery effluent. Further work is planned to confirm
these findings in other piggeries and investigate the risk that re-use of treated effluent poses
to public health.

P37
COMPARATIVE PATHOGENICITY OF CLOSTRIDIUM DIFFICILE STRAINS IN
CEFOPERAZONE-TREATED MICE
C.M. Theriot*, V.B. Young. Department of Internal Medicine, Division of Infectious Diseases,
Department of Microbiology and Immunology, The University of Michigan, Ann Arbor, MI
48109 USA.
INTRODUCTION: The toxin-producing bacterium C. difficile is the leading cause of antibioticassociated
colitis, with an estimated 1-3 million cases of C. difficile infection (CDI) each year
in the U.S. Despite the significance of CDI, little is known about the pathogenesis of this
infection. The recent development of tractable murine models of CDI that utilize antibiotic
treatment prior to C. difficile infection will allow us to study determinants of C. difficile
pathogenesis in vivo. We wished to determine if challenge of cefoperazone-treated mice
could distinguish the relative pathogenicity of C. difficile strains. METHODS: 5-8 week old
C57BL/6 WT mice were pretreated with a 10 day course of cefoperazone administered in the
drinking water. Following a 2-day recovery period with plain water, the animals were orally
challenged with C. difficile strains VPI 10463, NAP1/BI/027, 630 and a non-toxigenic strain.
Animals were monitored for loss of weight and clinical signs of colitis. At the time of harvest,
C. difficile strains were isolated from cecal contents and the severity of colitis was
determined by histopathologic examination of the cecum and colon. RESULTS:
Cefoperazone-treated mice challenged with the more historically virulent C. difficile strains
(known to produce more toxin) VPI 10463 and NAP1/BI/027, developed clinically more
severe disease than those infected with 630 and the non-toxigenic strain. This increased
clinical severity was correlated with more severe microscopic lesions of disease, with
significantly more severe edema, inflammation, and epithelial damage in animals infected
with the more virulent strains. More severe histopathologic disease in cefoperazone-treated
mice also correlated with increased cytotoxicity and elevated white blood cell counts in vivo.
CONCLUSIONS: Cefoperazone-treated mice exhibit varying degrees of colitis when
challenged with different C. difficile strains. This murine model, along with clinically-relevant
strains of C. difficile, will help us better understand the pathogenesis and virulence of CDI. It
will also allow us to explore the host-pathogen relationship along with the role that the gut
microbiota plays in colonization resistance against C. difficile.

P38
MONOCLONAL ANTIBODIES TO SPECIFICALLY IDENTIFY TOXIN B OF CLOSTRIDIUM
DIFFICILE RIBOTYPE 027
C. von Eichel-Streiber* 1 , K. Gisch 1 , A. Lange 1 , C. Oester 1 . R. Murillio 2 , M. Villacampa 2 ,
T.Toribio 2 . 1 tgcBIOMICS GmbH, Carl Zeiss. Str. 51, D-55129 Mainz, Germany; 2 Oporon
S.A., Camino del Plano 19, 50410 Cuarte de Huerva (Zaragoza), Spain.
Toxin B genes of Clostridium difficile are known to be genetically diverse. tgcBIOMICS has
developed monoclonal antibodies that differentially recognize TcdB molecules of different
isolates. Partial characterization of such antibodies will be presented. Some of the antibodies
specifically recognize TcdB-027 of C. difficile ribotype 027. The antibodies were used to
establish a CE-certified lateral flow test and an ELISA for the identification of C. difficile
infections with hypervirulent isolates.

P39
A PHYLOGENETIC ANALYSIS OF THE NEGATIVE TOXIN REGULATOR (TcdC) OF
CLOSTRIDIUM DIFFICILE
S. Walk* 1 , V. Young 1 , and D. Aronoff 1 . 1 Department of Internal Medicine, Division of
Infectious Diseases, and the Department of Microbiology, University of Michigan, Ann Arbor,
MI USA.
Epidemiologic observations suggest that the prevalence of severe cases of Clostridium
difficile infection (CDI) is increasing. One hypothesis to explain this trend is that strains
associated with severe disease produce higher levels of the C. difficile toxins TcdA and
TcdB. As a result, C. difficile toxin regulation has received much attention and a negative
regulator of toxin expression, called TcdC, has been identified. In support of the above
hypothesis, certain C. difficile isolates possess an allele of tcdC that encodes a premature
stop codon, resulting in a truncated and presumably non-functional protein. However,
multiple clinical studies have not found a link between specific tcdC alleles and severe CDI.
To better examine the genetic diversity of tcdC and the abundance and distribution of
different alleles among C. difficile pathogens, we sequenced this gene in 250 ribotyped
isolates isolated from patients with C. difficile infection at the University of Michigan Health
System. Alignment of these tcdC sequences with 44 previously identified alleles from online
databases revealed that previously published alignments could be improved. An improved
alignment based on all available alleles revealed 44 variable nucleotide sites and 19 variable
amino acid sites throughout tcdC. A total of 19 unique alleles were represented among the
clinical isolates in this study and 6 of them were novel. The most abundant allele, tcdC-3,
was identical to a previously identified allele and was shared by 31% of all isolates. A
phylogenetic analysis was conducted on the updated alignment and a model for the evolution
of tcdC diversity was developed. We found evidence for intragenic recombination using
break-point analysis as well as evidence for at least 4 different homologous recombination
events (shared alleles between C. difficle ribotypes). Based on our results, we propose a
standardized alignment and naming convention for tcdC alleles that can be used in future
surveillance of C. difficile pathogens, thereby aiding the quantification of association between
tcdC alleles and CDI severity.

P40
EFFECT OF PROTON PUMP INHIBITORS WITH AND WITHOUT ANTIBIOTICS ON
CLOSTRIDIUM DIFFICILE INFECTION IN HAMSTERS
L. Wright* 1 , K. Aleksoniene 2 , S. Sambol 2 , L. Petrella 1 , D.N. Gerding 1 , S. Johnson 1,2 . 1 Hines
VA Hospital, Hines, IL USA; 2 Loyola University Medical Center, Maywood, IL USA.
The most important risk factor for Clostridium difficile infection (CDI) is exposure to
antibiotics. Controversy exists over whether proton pump inhibitors (PPIs) increase the risk of
CDI. We sought to determine whether PPI administration to hamsters renders these animals
susceptable to colonization and/or disease following challenge with toxigenic CD.
Esomeprazole in bicarbonate buffer was administered orally to groups of 10 hamsters
starting on day 1 and oral challenge with CD spores occurred on day 5. Pilot studies were
conducted to optimize the PPI dose, duration, and CD challenge inoculum. Initial
experiments did not include clindamycin treatment, but in subsequent experiments,
clindamycin was given to hamsters on day 0. In order to determine the effect of PPIs alone
(no antibiotic) on CDI, we used a high inoculum (1 million spores) of a highly virulent CD
strain (BI17) to challenge hamsters given continuous PPI (50 mg/kg esomeprazole daily by
oral gavage for 7 days). 10/10 hamsters had fecal pellet cultures positive for CD up to 48
hours after spore challenge vs. 2/10 up to 24 hours in controls given bicarbonate diluent but
no PPI (P

ABSTRACTS OF POSTER PRESENTATIONS
Session III: P41 to P61
Friday, October 28, 2011

P41
HIGH AFFINITY BINDING OF CLOSTRIDIUM PERFRINGENS EPSILON TOXIN TO THE
RENAL SYSTEM
J. Dorca-Arévalo 1,2 , M. Martín-Satué 1,2 , and J. Blasi* 1,2 . 1 Department of Pathology and
Experimental Therapeutics. School of Medicine. Campus of Bellvitge. Health Universitat de
Barcelona Campus (HUBc). University of Barcelona. c/ Feixa Llarga s/n 08907. L‘Hospitalet
de Llobregat. Spain; 2 Institut d‘Investigació Biomèdica de Bellvitge (IDIBELL).
Epsilon toxin (ε-toxin), produced by Clostridium perfringens types B and D, causes fatal
enterotoxaemia in livestock. In the renal system, the toxin binds to target cells before
oligomerization, pore formation, and cell death. Still, there is little information about the
cellular and molecular mechanism involved in the initial steps of the cytotoxic action of ε-
toxin, including the specific binding to the target sensitive cells. In the present work, the
binding step of ε-toxin to the MDCK cell line, a very sensitive cellular model for ε-toxin
cytotoxic effect, is characterized by means of an ELISA based assay and using ε-toxin-green
fluorescence protein (ε-toxin-GFP) and ε-prototoxin-GFP. In addition, different treatments
with Pronase E, detergents, N-glycosidase F and beta-elimination on MDCK cells and renal
tissue cryosections have been performed to further characterize the ε-toxin binding. ELISA
assays revealed a single binding site with a similar dissociation constant (Kd) for ε-toxin-GFP
and ε-prototoxin-GFP, but a three-fold increase in Bmax levels in the case of ε-toxin-GFP.
Double staining on kidney cryoslices with lectins and ε-prototoxin-GFP revealed specific
binding to distal and collecting tubule cells. Moreover, experiments on kidney and bladder
cryoslices demonstrated the specific binding to distal tubules and urothelium of a range of
mammalian renal systems, including mouse, cow, goat, sheep and human kidneys and
mouse, cow, goat and sheep bladders. Pronase E and beta-elimination treatments on kidney
cryoslices and MDCK cells showed that the binding of epsilon-toxin in the renal system is
mediated by an O-glycoprotein. In addition, detergent treatments revealed that the integrity of
the plasma membrane is required for the binding of ε-toxin to its receptor. This work has
been supported by the grant SAF2008/00732 and FIS 00305 from the Ministerio de Ciencia e
Innovación of the Spanish Government.

P42
CLOSTRIDIAL GUT COLONIZATION IN PRETERM NEONATES
L. Ferraris 1 , M. Butel* 1 , F. Campeotto 1 , I. Nicolis 2 , M. Vodovar 3 , J.C. Rozé 4 , and J. Aires 1 .
1 EA4065 and 2 EA2498, Sorbonne Paris Cité, Université Paris Descartes, Paris, France;
3 Néonatologie, Institut de Puériculture, Paris, France; 4 Médecine Néonatale, INSERM
CIC004, Nantes, France.
Clostridia are part of the normal gut microbiota in humans and have been associated with
endogenous infections. Several reports have suggested their role in necrotizing enterocolitis
(NEC), a devastating gastrointestinal disease in preterm infants. However, little data are
available on the gut colonization at birth by this genus. The aim of our study was to report the
distribution of clostridial species colonizing the gut of preterm neonates, and to investigate
the colonization factors. Moreover, since preterm infants are often treated with broad
spectrum antibiotics, antibiotic susceptibility patterns of the clostridial isolates have been
studied. Clostridial colonization has been investigated in the feces of 76 preterm neonates
(median gestational age 32.8 wk [range 24-35.9 wk]) collected each week throughout their
hospital stay (median 4 wk [range 2-11 wk]). The incidence of colonization was high: 78.9%
at hospital discharge (mean level of 7.4 CFU/g of feces [range 3.3-9.2 CFU/g]). This
incidence increased throughout the hospitalization from 27.5% at wk 1 to 100% at the end of
the follow-up. The most frequently isolated species were C perfringens, C butyricum, C
difficile, and C paraputrificum. No differences in the microbiota between neonates either
colonized or not by clostridia were observed. The main factor of colonization was antibiotic
courses. Ante natal antibiotic therapy decreased the level of colonization (p=0.006). Neonatal
antibiotic therapy decreased both the incidence (p=0.002) and the level (p= 0.001) of
colonization only when antibiotic treatment lasted more than 10 days. Overall, clostridia
strains were found susceptible to the usual anti-anaerobe agents. However, as already
known, all C. difficile isolates were resistant to ertapemen, cefoxitin and cefotaxim and 97%
to clindamycin. Resistance to amoxicillin and piperacillin was observed for C. butyricum
strains (12.5%). This study showed clostridial colonization as part of the microbial community
of preterm neonates; this was in contrast to other anaerobes, for which gut colonization is
sharply delayed. The increase in the incidence throughout the hospital stay suggests
colonization from the hospital environment by this spore-forming genus. The study gives new
insights on the factors of clostridial colonization in preterm infants and may participate to
better understanding of neonatal infections involving clostridia such as NEC.

P43
csps PLAY A ROLE IN NACL AND PH STRESS RESPONSE OF CLOSTRIDIUM
BOTULINUM ATCC 3502
Y. Derman* 1 , M. Lindström 1 , H. Söderholm 1 , and H. Korkeala 1 .
1 Department of Food Hygiene and Environmental Health, the Centre of Excellence in
Microbial Food Safety Research, Faculty of Veterinary Medicine, University of Helsinki,
Finland.
Growth and toxin production of Clostridium botulinum in foods may lead to life-threatening
food poisoning called botulism. Some C. botulinum strains can adapt to and survive in stress
conditions such as low pH and presence of salt and therefore they pose a risk for food
safety. Mutations in the cold shock protein coding genes (csps) cspB or cspC, but not cspA,
resulted in a cold-sensitive phenotype in C. botulinum ATCC 3502 (Söderholm H., M.
Lindström, P. Somervuo, J. Heap, N. Minton, J. Lindén, and H. Korkeala. 2011. cspB
encodes a major cold shock protein in Clostridium botulinum ATCC 3502. Int. J. Food
Microbiol. 146:23-30). In this study, the growth of csp mutants was compared to wild type
strain in different concentrations of added NaCl and various pH conditions. The mutant and
wild type strains were anaerobically grown in tryptone-peptone-glucose-yeast extract broth
with up to 4% NaCl and pH of 4 to 8 in the Bioscreen turbidity reader (BCDE Group, Helsinki,
Finland) placed in anaerobic conditions at 37°C. No growth differences between the csp
mutants and the wild type strain were observed in native broth, in the presence of 1% NaCl,
or at pH 5.5 to 8.0. In the presence of 2% to 4% NaCl, the cspB mutant showed an increased
lag phase compared to the wild type strain, with the difference being greatest at 4% NaCl.
None of the strains grew in broth with pH of 4.0 or 4.5. At pH 5.0, only the wild type strain,
but none of the csp mutants, grew. The data suggest that cspB is involved in NaCl stress,
whereas all csps seem to play a role in pH stress at 37°C. Further studies on the role of the
csp genes in the ethanol and hydrogen peroxide stress response are ongoing.

P44
MOLECULAR DIAGNOSIS OF BLACKLEG FROM COMMON FILTER PAPER
L. Farias* 1 , S. Botton, A.C. Vargas 1 . 1 Laboratory of Bacteriology, UFSM, CEP 97105-900
Santa Maria/RS, Brazil.
The absence of conclusive diagnosis of blackleg usually happens for practical and
economical infeasibility of sending sample for laboratory diagnosis. Seeking to facilitate the
collection, storage and material shipment to the laboratory, the objective of this work was to
verify the viability of Clostridium chauvoei in common filter paper for subsequent molecular
identification by using PCR (Polymerase chain reaction). For this we tested in vitro the
sensitivity, specificity and feasibility of this method over different storage periods at room
temperature. For the viability test, we used twelve bovine liver tissues which ten samples
were impregnated with suspension containing C. chauvoei inoculums and the tests were
performed at 24h, 48h, 72h, and one week after impregnation on common filter paper. The
sensitivity of the technique was not affected with the use of common filter paper. There were
no cross-reaction and no amplification from the negative control samples. Direct PCR by
using common filter paper showed amplification in 50% of the samples in 24 hours, 100% in
48h, 70% in 72h and 90% within one week of storage at room temperature. Therefore, the
common filter paper can be used as practical and economical alternative for collection,
storage and material shipment for molecular diagnosis of blackleg.

P45
THE EFFECT OF CLOSTRIDIUM PERFRINGENS TYPE C AND ITS BETA TOXIN
MUTANT IN GOATS
J. P. Garcia* 1 , J. Saputo 1 , D. J. Fisher 2,3 , S. Sayeed 2 , B. A. McClane 2,3 , H. Posthaus 4 , and F.
A. Uzal 1 . 1 California Animal Health and Food Safety Laboratory System, School of
Veterinary Medicine, University of California Davis, San Bernardino, CA, USA; 2 Department
of Microbiology and Molecular Genetics; 3 Molecular Virology and Microbiology Graduate
Program, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 4 Institute of
Animal Pathology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Clostridium perfringens type C is an important cause of enteritis and enterocolitis in several
animal species including pigs, sheep, goats, horses, and humans. The disease is a classic
enterotoxemia and the enteric lesions and associated systemic effects are thought to be
caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens
type C. This has been demonstrated recently by fulfilling molecular Koch‘s postulates in
rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a
natural host of type C disease. Nine healthy male or female Anglo Nubian goat kids were
inoculated with a virulent C. perfringens type C wild-type strain, an isogenic CPB null mutant,
or a strain where the CPB mutation had been reversed (complement strain). Three goats
inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea,
necrotizing enterocolitis, pulmonary edema, hydropericardium, and death within 24 h of
inoculation. Two goats inoculated with the CPB null mutants and two goats inoculated with
sterile culture media (negative controls) remained clinically healthy during 24 h after
inoculation and no gross or histological abnormalities were observed in any of their tissues.
Reversal of the null mutation restored CPB production and virulence; 2 goats inoculated with
this reversed mutant presented clinical and pathological changes similar to those observed in
goats inoculated with the wildtype, except that spontaneous death was not observed. These
results indicate that CPB is both required and sufficient for C. perfringens type C to induce
disease in goats, supporting a key role for this toxin in natural type C disease pathogenesis.

P46
CLOSTRIDIUM PERFRINGENS STRAINS OF VARIOUS ORIGIN CAN CAUSE
HEMORRHAGIC ENTERITIS IN A CALF INTESTINAL LOOP MODEL
B. Valgaeren 1 , E. Goossens* 2 , S. Verherstraeten 2 , B. Pardon 1 , L. Timbermont 2 , S.
Schauvlieghe 3 , R. Ducatelle 2 , F. Van Immerseel 2 , P. Deprez 1 . 1 Department of Large Animal
Internal Medicine, Ghent University, B-9820 Merelbeke, Belgium; 2 Department of Pathology,
Bacteriology and Avian Diseases, Ghent University, B-9820 Merelbeke, Belgium;
3 Department of Surgery and anaesthesiology of domestic animals, Ghent University, B-9820
Merelbeke, Belgium.
Hemorrhagic enteritis is one of numerous pathologies caused by Clostridium perfringens in
cattle. It is an acute syndrome with a case fatality rate close to 100%, that affects mainly
suckling and veal calves in good to excellent body condition up to four months of age. In
Belgian Blue cattle, losses due to hemorrhagic enteritis may be responsible for up to 20% of
total mortality. The pathogenesis and the toxins involved in bovine hemorrhagic enteritis
remain to be elucidated. Therefore, an intestinal loop model was developed to test a
collection of Clostridium perfringens strains for their ability to reproduce the pathology. Loops
were injected with logarithmic C. perfringens cultures in combination or not with commercial
milk replacer for veal calves. The tested set of strains consisted of bovine strains isolated
from both hemorrhagic enteritis cases and healthy animals, as well as human, NetB-positive
and -negative chicken strains, and beta toxin positive porcine isolates. Also a VirR, α toxin,
and a θ toxin mutant were tested. All tested strains were capable of eliciting hemorrhagic
enteritis in this experimental setup when inoculated together with commercial milk replacer. A
time course experiment showed that the pathogenesis is characterized by early capillary
congestion, followed by hemorrhages and necrosis of the lamina propria, originating from the
tips of the villi. No edema was present. C. perfringens bacteria were often attached to cellular
debris in the lumen. These observations indicate that any Clostridium perfringens strain,
independent of the isolation source, may be capable of eliciting hemorrhagic enteritis, and
that this syndrome is not caused by host-specific strains or toxins. In addition, the
pathological process seems to be independent of α- or θ-toxin or VirR-regulated
mechanisms.

P47
A FUNCTIONAL, HIGH MOLECULAR WEIGHT BACTERIOCIN (―DIFFOCIN‖) FROM
CLOSTRIDIUM DIFFICILE CLONED AND EXPRESSED IN BACILLUS SUBTILIS
G. Govoni*, D. Gebhart, S. Williams, and D. Scholl. AvidBiotics Corp., South San Francisco,
CA 94080 USA.
Several C. difficile isolates have been observed to produce phage tail-like particles with no
capsid structure. Recently, it was shown that these particles can act as high molecular
weight bacteriocins (termed ―diffocins‖) and kill other C.difficile isolates. We have cloned the
22kb diffocin gene cluster, which contains 25 ORFs and multiple predicted transcripts, and
integrated it into the amyE gene in the B. subtilis chromosome via homologous
recombination. As in C. difficile, expression of diffocin can be induced by addition of
mitomycin C during exponential phase. Preliminary studies suggest we can also express
diffocins in B. subtilis by inducing expression of a constitutively active form of E. coli RecA.
Using a bioassay on sensitive C. difficile isolates, we were able to confirm that the diffocins
produced by these B. subtilis strains were functionally active. Interestingly, the spectrum of
killing C. difficile isolates could be altered by exchanging orthologues of the putative receptor
binding protein encoded by ORF CD1374. This platform provides the opportunity to engineer
the spectrum of killing and to produce diffocins for clinical applications.

P48
IMPROVING THE REPRODUCIBILITY OF INFECTION OF THE NAP1/B1/027
HYPERVIRULENT STRAIN R20291 IN THE HAMSTER MODEL OF INFECTION
M. Kelly*, R. Ng, S. Cartman, A. Cockayne and N.P. Minton.
Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular
Science, University Park, University of Nottingham, Nottingham, NG7 2RD U.K.
Clostridium difficile has become the leading cause of diarrhoea in hospitalized patients over
the past three decades in North America and Europe, placing a substantial financial burden
on healthcare systems. The situation has been exacerbated by the emergence of so-called
‗hypervirulent‘ strains (NAP1/BI/027). As its genome sequence is available, we have been
using the strain R20291 as a model of hypervirulence. This strain was isolated from the
2003-2004 outbreak at the Stoke Mandeville Hospital in the UK, where 334 cases of C.
difficile associated disease (CDAD) and 38 mortalities were recorded. The golden Syrian
hamster has been the model of choice for in vivo investigations of CDAD for the past three
decades as it mimics the progression of the disease in humans. CDAD in the hamster is
brought about by oral infection following administration of the antibiotic clindamycin. Studies
using the 630 wild type and 630Δerm strains of C. difficile have shown that the onset of
disease is observed routinely between 48 and 72 hours post infection. Investigation of strain
R20291 using the hamster model of infection is, however, hampered by the strains‘
sensitivity to clindamycin (MIC= 16µg/ml) which leads to irreproducible infection (between 52
and 240 hours post infection). This complicates experiments being undertaken to assess the
comparative virulence of directed mutants, which, in contrast to the parental control, have
become resistant to clindamycin as a consequence of the ClosTron-mediated insertion of
ermB. We have, therefore, created an R20291 strain that is erythromycin resistant through
the insertion of an equivalent copy of the ClosTron-derived ermB gene into the genome using
Allele Coupled Exchange (ACE) technology. This new strain shows an increased resistance
to clindamycin that is comparable to the 630 wild type strain. Data on the reproducibility of
infection in the hamster model will be presented.

P49
AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS IN STRAIN TYPING AND
IDENTIFICATION OF CLOSTRIDIUM SPECIES
R. Keto-Timonen* and H. Korkeala. Department of Food Hygiene and Environmental Health,
Faculty of Veterinary Medicine, FI-00014 University of Helsinki, Finland.
Amplified fragment length polymorphism (AFLP) is a PCR-based DNA fingerprinting method,
which inspects the entire genome for polymorphism and can be used both for strain
characterization and species identification. The AFLP technique was applied to 129 strains
representing 24 different Clostridium species to assess the potential of AFLP for identification
of clostridia. The ability of the same AFLP protocol to type Clostridium perfringens at the
strain level was also assessed. All Clostridium strains were typeable by AFLP and, thus, the
method seemed to overcome the problem of extracellular DNase production. AFLP
differentiated all Clostridium spp tested, except for C. ramosum and C. limosum, which
clustered together with a 45% similarity level. Other Clostridium spp were divided into
species-specific clusters or occupied separate positions. Wide genetic diversity was
observed among C. botulinum strains, which were divided into seven species-specific
clusters. With a 93% cut-off value, a total of 29 different AFLP types were identified for 37
strains of C. perfringens. AFLP analysis of unrelated C. perfringens strains resulted in
divergent fingerprints, whereas identical banding patterns were observed for strains
originating from the same isolate or from the same food poisoning outbreak. AFLP showed
potential to subtype C. perfringens strains, and due to the high throughput of samples, the
AFLP approach is especially well-suited for screening large numbers of isolates. In addition,
AFLP may be used as a valuable additional tool in identification of Clostridium spp if an
expandable identification library with several AFLP profiles of well-defined strains for each
species is established.

P50
COMPARATIVE GENOMIC HYBRIDIZATION ANALYSIS SHOWS DIFFERENT
EPIDEMIOLOGY OF CHROMOSOMAL AND PLASMID-BORNE cpe-CARRYING C.
PERFRINGENS TYPE A STRAINS
P. Lahti* 1 , M. Lindström 1 , P. Somervuo 1 , A. Heikinheimo 1 , and H. Korkeala 1 . 1 Department of
Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of
Helsinki, Helsinki, Finland.
The reservoirs and the routes of contamination of a common food poisoning pathogen,
enterotoxin-producing C. perfringens, remain unknown, which complicates the prevention of
C. perfringens food poisonings. cpe, encoding the enterotoxin, can be chromosomal or
plasmid-borne. The chromosomal cpe-carrying strains are a more common cause of food
poisonings than the plasmid-borne cpe-genotypes, perhaps due to their better heat
tolerance. However, the plasmid-borne cpe-positive genotypes are more commonly found in
human feces and in environmental samples than are chromosomal cpe-positive genotypes.
The different prevalence of the cpe-positive genotypes in environmental niches and the
differences in heat tolerance raise a question, whether the epidemiology of C. perfringens
type A food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains is
different. A DNA microarray was designed for analysis of genetic relatedness between the
different cpe-positive and cpe-negative genotypes of C. perfringens strains isolated from
human, animal, environmental, and food samples. The DNA microarray contained two
probes for all protein coding sequences in the three genome sequenced strains (C.
perfringens type A strains 13, ATCC13124, and SM101). The analysis of the variable gene
pool was complemented with the growth studies and the growth of the cpe-positive C.
perfringens strains were examined in the minimal medium using myo-inositol, cellobiose or
ethanolamine as the sole source of energy. The chromosomal and plasmid-borne C.
perfringens genotypes were grouped into two distinct clusters, one consisting of the
chromosomal cpe-genotypes and the other consisting of plasmid-borne cpe-genotypes. The
plasmid-borne cpe-carrying strains carried operons encoding for utilization of myo-inositol
and ethanolamine, and were able to utilize these as a sole source of energy. Most
chromosomal strains carried a gene cluster encoding the utilization of cellobiose and were
able to utilize cellobiose as a sole source of energy. The epidemiology of chromosomal and
plasmid-borne cpe-carrying C. perfringens type A food poisonings seem to be different. The
plasmid-borne cpe-carrying strains are suggested to contaminate food mainly by humans,
whereas the epidemiology of the chromosomal cpe-carrying strains is suggested to be
connected to decomposing plant material. Further research should be done to elucidate the
habitat of these strains.

P51
CHARACTERIZATION OF SURFACE-LAYER PROTEINS OF CLOSTRIDIUM BOTULINUM
GROUP I AND II
D. Lambert* 1 , S. M. Twine 2 and J. W. Austin 1 . 1 Microbiology Research Division, Bureau of
Microbial Hazards, HPFB, Health Canada, Ottawa, Ontario, Canada; 2 Institute for Biological
Sciences, National Research Council, Ottawa, Ontario, Canada.
Interactions between bacteria and their environments, including their hosts in the case of
pathogens, regularly involve surface proteins. For Gram-positive bacteria like Clostridium
botulinum, such surface proteins are putative virulence factors involved in the colonization of
wounds or gastrointestinal tracts of adults or infants. These infections lead to bacterial
growth and the subsequent production of botulism neurotoxin (BoNT) in vivo, causing rare
forms of the disease, viz. wound botulism, adult gut colonization botulism, or infant botulism.
The surface proteins of C. botulinum can also potentially be used in detection assays as
surrogates for the presence of BoNT in contaminated foods during an outbreak or possible
bio-terrorist attacks. Other C. botulinum surface proteins have now been characterized, but
little is known about its surface (S-) layer. It comprises a planar array of regularly ordered
often self-assembling proteinaceous subunits, representing some of the most abundant
cellular proteins. We thus performed an initial characterization of the S-layer proteins from C.
botulinum groups I and II. We isolated putative S-layer proteins using various mechanical
and chemical extraction techniques, followed by electrophoresis. Protein identities were
confirmed using mass spectrometry analysis using MASCOT. We observed signs of posttranslational
glycosylation using glycan-specific dyes, but not with commercially available
lectins. Using colony and genomic DNA PCR, followed by amplicon sequencing and
sequence alignments (Blast), we determined various levels of homology between S-layer
protein genes of selected group I and II strains. We are now exploring the possibility of using
these findings to develop new detection methods for C. botulinum contamination.

P52
IDENTIFICATION OF C. PERFRINGENS GENES ASSOCIATED WITH AVIAN NECROTIC
ENTERITIS BY MICROARRAY COMPARATIVE GENOMIC HYBRIDIZATION
D. Lepp* 1,2 , V. Parreira 1 , J. G. Songer 3 , A.Kropinski 4 , P. Boerlin 1 , J. Gong 2 , J. Prescott 1 .
1 Department of Pathobiology, University of Guelph; 2 Guelph Food Research Centre,
Agriculture and Agri-Food Canada; 3 College of Veterinary Medicine, Iowa State University;
4 Laboratory for Foodborne Zoonoses, Public Health Agency of Canada.
Clostridium perfringens type A causes poultry necrotic enteritis (NE), an enteric disease of
considerable economic importance. A recently-identified novel pore-forming toxin, NetB, is
critical to NE pathogenesis and closely associated with virulent strains. More recently, we
demonstrated that netB resides on a large, plasmid-borne pathogenicity locus (NELoc-1)
that, in conjunction with two other loci (NELoc-2 and 3), is highly conserved in poultry-virulent
Cp strains. To determine the prevalence of these loci in a larger group of poultry isolates and
to identify other genes associated with NE-causing strains, we have developed a C.
perfringens microarray for comparative genomic hybridization analyses (CGH). Based on the
genome sequences of two NE-causing strains, a microarray consisting of 3,335
oligonucleotide probes, also representing 85% - 92% of the three publicly available C.
perfringens genome sequences (ATCC13124, SM101 and Str13) was constructed.
Hybridization of the array with two sequenced strains (ATCC13124 and Str13) and
comparison with the predicted gene content based on sequence analysis revealed it to be
accurate and sensitive (sensitivity >99%, specificity >95%). A set of 57 C. perfringens
strains, 28 from birds with NE and 29 from healthy birds, were chosen for analysis by array
CGH. Pulsed field gel electrophoresis (PFGE) of the strains confirmed that they were
genetically distinct. The three NE loci were more prevalent in isolates from diseased than
from healthy birds. In particular, NELoc-2 was present in 85.7% of isolates from diseased
and only 41.3% of isolates from healthy birds. Several other smaller loci were also
significantly associated with isolates from diseased birds, which include an alternative sigma
factor, a two-component signal transduction system, and a ferric iron siderophore transport
system. These findings suggest that the three NE loci are important for pathogenesis, in
particular NELoc-2. Additionally, several other fitness-related chromosomal genes may
contribute to a strain‘s ability to cause disease.

P53
APPLYING TOXIN PROTEOMICS TO MEASURE RELATIVE QUANTITIES OF PROTEINS
WITHIN THE BOTULINUM NEUROTOXIN COMPLEX
H. Moura*, A.R Woolfitt, R.R. Terilli, M.I. Solano & J.R. Barr. Division of Laboratory Sciences,
National Center for Environmental Health; Centers for Disease Control and Prevention
(CDC), Atlanta, GA USA.
Botulinum neurotoxins (BoNT) are specific endopeptidases that block the neurotransmitter
release in peripheral nerve endings, causing botulism. There are seven known types of
BoNTs (A through G). BoNTs are large proteins (~150 kDa) and exist as non-covalent
complexes with neurotoxin-associated proteins (NAPs). Accurate quantification of BoNT and
NAPs (composed of NTNH and hemagglutinin) within complexes has not been
accomplished. Accurate quantification of BoNT and NAPs within the BoNT complex would
reveal key information with applications on toxin detection, toxic mechanisms, and
therapeutics. To determine the ratio of BoNT and NAPs present in different lots of
commercially available BoNT complexes, we applied a label-free mass spectrometry
quantification method based on nanoscale ultra-performance liquid chromatography coupled
to electrospray-ionization mass spectrometry with data-independent acquisition (UPLC-ESI-
MSE). A rapid (3-minute) digestion method was used to obtain tryptic peptides from each lot
of BoNT complex. The digests were run in triplicate and analyzed by UPLC-ESI-MSE; the
proteomics data sets were further analyzed using commercial software (PLGS, IdentityE)
and custom software written in-house. All BoNTs and NAPs were detected with high protein
sequence coverage in the commercial complexes along with other proteins such as flagellin
and ORFs. The relative quantity of proteins in the BoNT complexes was accurately
determined. The BoNT:NAPs ratios were higher for BoNT /E and /F complexes (1:1) than for
BoNT /A, /B, /C, and /D (1:5 – 1:7); hemagglutinins were the most abundant proteins
detected in the complexes in the latter. The label-free method brings accurate quantification
to the study of BoNT, and will contribute to a better understanding of BoNT complexes, their
effects and applications.

P54
GENOMIC AND PROTEOMIC ANALYSIS OF A BOVINE HEMORRHAGIC ABOMASITIS
TYPE A CLOSTRIDIUM PERFRINGENS ISOLATE
V. J. Nowell* 1 , J. F. Prescott 1 , J. G. Songer 2 , A. M. Kropinski 3 , J. I. MacInnes 1 and V. R.
Parreira 1 . 1 Department of Pathobiology, University of Guelph, Guelph, ON, Canada. 2 Iowa
State University, Ames, IA, USA. 3 Public Health Agency of Canada, Laboratory for
Foodborne Zoonoses, Guelph, ON, Canada.
Type A Clostridium perfringens is a cause of hemorrhagic abomasitis in calves, a serious
and often fatal infection in milk-fed animals. The disease has been reproduced
experimentally, but the basis of its virulence is not known. To understand the characteristics
of C. perfringens associated with hemorrhagic abomasitis, an isolate of type A C. perfringens
obtained from a small outbreak in calves was analyzed using whole genome sequencing.
The outbreak strain sequenced was shown by PFGE to be identical to that isolated from
another calf that died of hemorrhagic abomasitis, supporting evidence that the correct strain
was sequenced. The genome was sequenced using the Roche 454 GS FLX Titanium
massively parallel shotgun sequencing technology with ~69X coverage total. The genome
was annotated and compared to other C. perfringens sequences to identify genes unique to
the isolate. A hypothesized novel toxin gene was not identified, but interesting findings
included a chromosomally-integrated plasmid fragment (~25 kb), three circular plasmids (~5
kb, ~9 kb and ~55 kb) and a large plasmid fragment (50 kb) of unknown location.
Approximately 300 kb of DNA and 261 ORFs were unique to this strain, including a number
of recombination-associated elements, phage sequences and CRISPR elements. The most
surprising finding was a frameshift in the virS gene that would result in the truncation of ~100
amino acids at the C-terminal end. Given this, it is likely that the entire VirR/VirS system
would be non-functional and therefore that VirR would be unable to activate transcription of
many virulence genes. Despite this, the strain expressed perfringolysin O, alpha-toxin and
beta2-toxin. We were unable to identify any novel toxin genes by genome analysis or using
mass spectrometry of the supernatant. It is possible that hemorrhagic abomasitis caused by
type A C. perfringens is the result of a combination of factors unrelated to the presence of
novel toxins or that novel virulence factors remain hidden in the 60% of hypothetical proteins
found among the unique ORFs.

P55
A FRAGMENT OF 97 AMINO ACIDS (D97) WITHIN THE TRANSMEMBRANE DOMAIN IS
ESSENTIAL FOR THE CELLULAR ACTIVITY OF CLOSTRIDIUM DIFFICILE TOXIN B
Y. Zhang 1,2 , L.Shi* 1 , X. Sun 1 , R. Kumar 3 , T. Savidge 3 , R. Zhuge 4 , Z. Yang 2 , X. Wang 2 , and H.
Feng 1 . 1 Tufts Cummings School of Veterinary Medicine, North Grafton, MA, 01536, USA;
2 School of Bioengineering, East China University of Science and Technology, Shanghai
200237, China; 3 The University of Texas Medical Branch, Galveston, TX, 77555, USA;
4 University of Massachusetts School of Medicine, Worcester.
Clostridium difficile toxin B (TcdB) is one of the major virulence factors associated with C.
difficile-associated disease, which intoxicates target cells by glucosylating Rho GTPases.
TcdB is a 269 kDa protein containing at least 4 functional domains including a
glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a transmembrane
domain (TD), and a receptor binding domain (RBD). In this study, we have found that a 97-
amino acid fragment (D97) located in the C-terminus of the TD is essential for the cellular
activity of TcdB. We deleted this fragment and expressed a truncated protein designated as
TxB-D97, and found that this deletion did not significantly alter the conformation of the toxin
or the functions of the other domains. The toxin cell binding and uptake were similar between
the mutant and wild type TcdB. Both wildtype and mutant toxins released their GTDs
similarly in the presence of inositol hexakisphosphate (InsP6), and showed similar activity in
a cell-free glucosylating assay. Despite these similarities, the cytotoxic activities of TxB-D97
were reduced by more than 5 logs compared to wild type toxin. Cells exposed to TxB-D97
also had an undetectable level of glucosylated Rac1. Unlike wild type TcdB, the mutant toxin
failed to induce macrophages to produce tumor necrosis factor alpha (TNF-α), an outcome
dependent on the GT activity of the toxin. Cellular fractionation of toxin-exposed cells
demonstrated that the TxB-D97 was unable to release its GT domain efficiently into cytosol.
Thus, the D97 fragment, located in the C-terminus of the TD, adjacent to the RBD, appears
to play an essential role in the delivery of the GT domain into cell cytosol.

P56
NECROTIZING ENTERITIS ASSOCIATED WITH CLOSTRIDIUM PERFRINGENS TYPE B
IN CHINCHILLAS (CHINCHILLA LANIGERA)
R. Lucena 1 , L. Farias 2 , F. Libardoni 2 , A.C. Vargas* 2 , P. Giaretta 1 , C.S. L. Barros 1 .
1 Laboratory of Veterinary Pathology, UFSM,CEP 97105-900 Santa Maria/RS, BRAZIL;
2 Laboratory of Bacteriology, UFSM,CEP 97105-900 Santa Maria/RS, BRAZIL.
Four 3-4 month-old chinchillas (Chinchilla lanigera) from a commercial flock of 395
chinchillas, were found dead with evidence of previous diarrhea and prolapsed rectum. A fifth
8 month-old chinchilla died 8 hours after being found recumbent, apathetic, diarrheic, and
with a prolapsed rectum. Two chinchillas were necropsied and gross lesions consisted of
extensive hemorrhagic enteritis, mild pulmonary edema, and enlarged and yellow liver; this
latter finding was particularly prominent in the chinchilla presenting with the longer clinical
course. Histologically, there was necrotizing enteritis associated with abundant aggregates of
bacterial rods on the intestinal epithelial surface and within the lamina propria. In the lungs,
there were small amounts of pink proteinaceous material (edema) in the interstitium and
marked vacuolar hepatocellullar degeneration (lipidosis) in the liver. Anaerobic cultures from
the intestinal contents of one of the affected chinchillas yielded Clostridium perfringens.
Genotyping of this C. perfringens isolate was achieved by multiplex polymerase chain
reaction (mPCR), which revealed that the isolates was genotype B, due to detection of alpha,
beta, and epsilon-toxin genes. These findings suggest C. perfringens type B as a cause of
sudden or acute death in chinchillas.

P57
DETERMINATION OF FUNCTIONAL RESIDUES ON NETB TOXIN FROM CLOSTRIDIUM
PERFRINGENS
X. Yan* 1 , C. J. Porter 2 , A. L. Keyburn 1,3 , D. Steer 2 , A. I. Smith 2 , N. Quinsey 2 , V. Hughes 2 , J.
K. Cheung 1 , J. C. Whisstock 2 , R. J. Moore 1,3 , T. L. Bannam 1 and J. I. Rood 1 . Australia
Research Council Centre of Excellence in Structural and Functional Microbial Genomics,
Departments of 1 Microbiology and 2 Biochemistry and Molecular Biology, Monash University,
VIC, Australia; 3 CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong,
VIC, Australia.
The novel toxin NetB is a putative β-barrel pore-forming toxin with 31% amino acid sequence
identity to α-hemolysin (Hla) from Staphylococcus aureus. NetB is almost exclusively found
in avian isolates of Clostridium perfringens type A and is associated with necrotic enteritis in
chickens. The objective of this study was to determine NetB residues essential for its
function. Both site-directed and random mutagenesis were used to generate netB point
mutations that led to single amino acid substitutions in NetB. Based on the similarity of NetB
to Hla, two residues that were equivalent to amino acids known to be important for Hla
function were substituted by site-directed mutagenesis. One of the resultant NetB
derivatives, R230Q, was no longer functional, whereas the other, D186N, had wild-type
activity. Subsequently, a non-haemolytic C. perfringens strain was used as the host to screen
for netB mutants after passage of a complementation plasmid through the mutator strain XL-
1 Red. A library of over 200 mutants was obtained; subsequent analysis led to the
determination of 14 unique NetB amino acid substitutions that resulted in a non-haemolytic
phenotype on horse blood agar. Seven of these substituted NetB proteins were overexpressed
and purified. Analysis of their CD spectra showed that they all adopted a
predominant β-sheet fold that was similar to wild-type NetB. Functional analysis of these
proteins led to the identification three residues that were essential for the lysis of chicken and
duck red blood cells. These results represent the first structure-function data generated for
NetB and will have significant implications for our understanding of the mode of action of this
novel toxin.

P58
SEAFOOD AS A RESERVOIR AND A SUPPLIER OF THE PROTOTYPE PLASMID
ENCODING CLOSTRIDIUM PERFRINGENS ENTEROTOXIN
N. Yumine*, K. Mimura, K. Miyamoto. Wakayama Medical University, Department of
Microbiology, Wakayama, Japan.
Clostridium perfringens is a ubiquitous bacterium in the environment and in the
gastrointestinal (GI) tract of human and domestic animals. This bacterium can produce up to
16 different toxins. Of these toxins, C. perfringens enterotoxin (CPE) is the most important for
human GI diseases. C. perfringens isolates carrying the CPE gene (cpe) are rarely found in
retail seafood, although freshwater sediments contain many cpe-positive isolates. Therefore,
the current study investigated the frequency of cpe-positive C. perfringens in raw retail
seafood and then performed a genetic analysis of those isolates. PCR survey using DNA
prepared from enrichment culture of seafood samples revealed that 80% of 70 surveyed
samples showed a positive reaction in a cpe-PCR assay. For the investigated seafood
samples, 49 of 50 retail oyster samples without shells contained cpe-positive C. perfringens.
In estimating the amount of iron-reducing Clostridia present in oyster samples with a three
tube MPN method, iron-reducing Clostridia including C. perfringens were 1,000 colonies/gram. Moreover, only 28 of 908 lecito-vetelline-positive colonies
(mostly C. perfringens) showed a positive reaction with cpe-PCR assay. From these findings,
seafood might be a reservoir for cpe-positive C. perfringens. However, retail seafood should
not be a food vehicle for food poisoning, if prepared promptly. To further characterize
seafood isolates, genetic assays were performed. Toxin genotype PCR assay indicated that
all 34 cpe-positive and 11 cpe-negative isolates classify as type A C. perfringens. To identify
the cpe location by cpe-genotyping PCR assay, 33 of 34 cpe-positive isolates carry their cpe
gene on plasmid; 27 have IS1151 near their cpe gene, 6 have IS1470-like near their cpe
gene, 1 has an unknown cpe locus. The genetic relationship among cpe-positive seafood
and human clinical isolates using groEL sequence typing revealed that those cpe-positive
isolates might be closely related. Collectively, these results suggest that retail seafood might
be a major reservoir of plasmid borne cpe-positive C. perfringens.

P59
COMPARISON OF CHANGES IN HUMAN AND POULTRY INTESTINAL MICROBIOTA
DURING COLONISATION WITH CLOSTRIDIUM DIFFICILE
J. Škraban 1 , M. Medved 2 , M. Rupnik* 1,2,3 . 1 University of Maribor, Faculty of Medicine, 2000
Maribor, Slovenia; 2 Institute of Public Health Maribor, Centre for Microbiology, 2000 Maribor,
Slovenia; 3 Centre of Excellence CIPKeBiP, 1000 Ljubljana, Slovenia.
Although the changes in intestinal microbiota during colonization of C. difficile have become
an important part of research in humans, the data are still very limited. Few published studies
describe changes of the bacterial microbiota in humans only and there are virtually no data
on changes/effects of gut microbiota during colonisation with C. difficile in animals or on
changes in non-bacterial microbial groups. The aim of our study was to analyse the changes
in bacterial, fungal and archaeal gut microbiota in C. difficile colonised and non – colonised
individuals. We used a new molecular high-throughput method for DNA fragment analysis
called denaturing high performance liquid chromatography (DHPLC; Wave system,
Transgenomic). The level of the analysis is comparable to gradient gels (DGGE), with the
advantage of high automatisation and repeatability. In the study we have analysed 200
human and 143 poultry faecal samples from individuals colonised (n (humans) = 105, n
(poultry) = 86) and non-colonised (n (humans) = 95, n (poultry) = 57) with C. difficile. In
humans the main differences were observed in the species diversity. The results show that
C. difficile colonised subjects have a simpler bacterial and archaeal microbiota and more
complex fungal microbiota. The colonisation status correlates to the presence/absence of
certain bacterial groups like clostridia, Bacteroides spp, and bifidobacteria. In poultry, we
have followed also the development of the gut microbiota over time. As the bacterial
microbiota complexity increased with age, the C. difficile colonisation rates dropped. The
colonisation status was associated to the presence/absence of certain enterococci. While the
archaeal microbiota developed between 2 and 4 months of age, the fungal microbiota
seemed to be relatively diverse from the beginning. Our results show a change in diversity
and a shift in the composition of the bacterial, but also in the fungal and archaeal, gut
microbiota in correlation to C. difficile colonisation.

P60
DEVELOPMENT OF A QUANTITATIVE IMMUNO-PCR ASSAY FOR THE DETECTION OF
GROUP III CLOSTRIDIUM BOTULINUM NEUROTOXINS IN CATTLE
T.C. Ardis* 1 , C.E. Brooks 1 , D. Fairley 2 , and H.J. Ball 1 . 1 Agri-food & Bioscience Institute,
Veterinary Science Division, Stoney Road, Belfast, BT4 3SD UK; 2 Regional Virus
Laboratory, Belfast Health & Social Care Trust, Kelvin Building, Grosvenor Road, Belfast,
BT12 6BA UK.
Introduction: Cattle botulism typically involves Group III neurotoxins, types C and D,
produced by Clostridium botulinum. The ‗gold standard‘ diagnostic method for detection of
botulinum neurotoxin (BoNT) is the mouse bioassay. This assay has greater sensitivity than
any in vitro assay available, but it also has certain disadvantages. The quantitative Immuno-
PCR (qIPCR) technique, which combines PCR with the standard ELISA method, can
reportedly lead to a 10- to 1000-fold increase in the limit of detection as compared to the
original ELISA. The aim of this work was to increase the sensitivity of a previously
developed monoclonal antibody-based sELISA that detects both types C and D BoNTs, by
developing a qIPCR assay which could provide a possible alternative to the standard mouse
bioassay. Method: A sequential qIPCR method was employed using the same capturedetection
MAbs as the original sELISA. Recombinant streptavidin was used to bridge the
biotinylated detection antibody with a biotinylated 438 bp DNA marker. Quantitation of BoNT
type D was achieved by real-time PCR amplification of the DNA marker. Results: The
sequential qIPCR protocol allowed for approximately a 40-fold (98.4 pg/ml) increase in
sensitivity as compared to the conventional sELISA. Detection of BoNT type D was linear
over a range of 10 – 0.078 ng/ml (R 2 = 0.986). Conclusions: While a considerable
improvement on the sensitivity of the original sELISA, this level of sensitivity is not yet
sufficient to provide an in vitro replacement for the mouse bioassay, which detects BoNT at
approximately 20 pg/ml. Alternative qIPCR methods, direct and modular, are currently being
investigated. These methods are reported to be more sensitive than the sequential method.

P61
THE LOCAL AND SYSTEMIC IMMUNE RESPONSE IN A MOUSE MODEL OF ACUTE
CLOSTRIDIUM DIFFICILE INFECTION
A.A. Sadighi Akha *1 , C.M. Theriot 2 , A.J. McDermott 3 , A.E. Reeves 3 , V.B. Young 2,3 and G.B.
Huffnagle 1,3 . 1 Divisions of Pulmonary and Critical Care Medicine and 2 Infectious Diseases,
3 Department of Internal Medicine, and Department of Microbiology and Immunology, The
University of Michigan Medical School, Ann Arbor, MI 48109 USA.
Clostridium difficile infection (CDI) is a significant cause of morbidity and mortality in
hospitals and hospices. While the higher susceptibility of immuno-compromised individuals
and the elderly to CDI underscore the protective role of the immune response to this
organism, the topography, kinetics, and mechanisms of the host response that mediate this
protection remain largely unknown. Here we report our initial findings in an acute mouse
model of CDI. 5-8 week old male C57BL/6 mice were either left untreated or made
susceptible to C. difficile infection and colitis by administering a cocktail of 5 antibiotics in
drinking water for 3 days followed by a single intra-peritoneal dose of clindamycin. The
antibiotic-treated mice were then infected with 10 5 CFUs of C. difficile strain VPI 10463, and
sacrificed 42 hours after the infection. Flow-cytometric study of the spleens, mesenteric
lymph nodes (MLN), colons and cecums of C. difficile-infected mice showed a significant
increase in the number of CD45 + cells in their colons and ceca, but not their spleens or MLN,
in comparison to non-antibiotic-treated, uninfected controls. This in turn was due to a
significant across-the-board increase in the numbers of B cells, CD4 and CD8 T cells,
FOXP3 + CD4 T cells, NK cells, neutrophils, macrophages, and dendritic cells in the colons
and ceca of the infected mice. The spleens and MLN of the infected mice did not display any
such increase. A fraction of the recruited neutrophils in the colons and ceca of the infected
mice displayed up-regulated levels of CD11b, an indication of their activated state. By
contrast, a similar fraction of CD4 and CD8 T cells in the colons and ceca of uninfected and
infected mice showed an up-regulated level of the activation marker CD69. These data
demonstrate the diversity of the hematopoetic cells recruited to the colons and ceca of mice
acutely infected with C. difficile, provide no indication of overwhelming activation in the
recruited lymphocytes to these organs, and underscore the local nature of the response.

P62
PORCELLIO SCABER – A NEW NONVERTEBRATE MODEL FOR C. DIFFICILE
COLONIZATION
M. Zemljič* 1,2 , M. Rupnik 1,2,3 , and D.Drobne 4 . 1 University of Maribor, Faculty of Medicine,
Maribor, Slovenia; 2 Centre of Excellence for Integrated Approaches in Chemistry and Biology
of Proteins, Ljubljana, Slovenia; 3 Institute of Public Health Maribor, Centre for Microbiology,
Department for Microbiology Research, Maribor, Slovenia; 4 University of Ljubljana,
Biotechnical Faculty, Department of Biology,Večna pot 111, Ljubljana, Slovenia.
The terrestrial isopod Porcellio scaber (Crustacea) is widely used as a model for toxicity
testing of pollutants. The aim of this study was to test P. scaber as a possible model for
Clostridium difficile colonization. P. scaber is a decomposer that preferentially feeds on
decayed plant material colonised by microorganisms, which are also utilized as a source of
nutrients and enzymes. Whereas some undigested microorganisms are passive transients,
others proliferate in the gut. The gut‘s simple tube-like anatomy, the rapid passage of the
food and frequent renewal of the gut cuticle appeared to be unsuitable for development of
resident and anaerobic microbiota. However, the reports on isolation of strictly anaerobic
bacteria from P. scaber confirmed the presence of anaerobic microniches. As in a standard
P. scaber experiment, animals were divided into groups of ten and were fed with hazelnut
tree leaves (Corylus avellana) for 30 days. Test groups were exposed to leaves with C.
difficile spores, and the control group to leaves without C. difficile. The selected endpoints
were the following: i) feeding activity between control and tested animals, ii) colonization with
C. difficile and iii) survival of the animals. In the first set of experiments, we compared two
control groups exposed to C. difficile 630, one of them previously exposed also to
clindamycin. Strain 630 caused death of animals in a very broad time interval from 3 to 21
days after exposure. Use of antibiotic did not affect the survival rates. In the second set of
experiments, animals were exposed to a combination of two different strains. Strain pairs
included a) toxigenic and nontoxigenic strains, b) toxigenic strain with high and toxigenic
strain with low virulence potential, and c) two strains with high virulence potential. All
combinations resulted in 50 to 100% colonization and in 30 to 100% mortality. Survival was
lowest in the group exposed to two strains with high virulence. These results suggest that P.
scaber could be a potentially useful model for assessing C. difficile virulence or to compare
fitness among strains under in vivo conditions.

CLOSPATH 7 PARTICIPANTS

ClosPath
Institution
Participants Country E-mail Abstract
Aktories, Klaus Germany University of Freiburg O15
klaus.aktories@pharmakol.uni-freiburg.de
Ardis, Tara United Kingdom Agri-Food and Biosciences Institute P60
jenna.donaldson@afbini.gov.uk
Aronoff, David United States The University of Michigan O2
daronoff@umich.edu
Aubry, Annie Canada National Research Council
annie.aubry@nrc-cnrc.gc.ca
Auchtung, Jennifer United States Michigan State University P1
auchtun3@msu.edu
Babakhani Farah Optimer Pharmaceuticals, Inc. P2, P3
fbabakhani@optimerpharma.com
Bakker, Dennis Netherlands Leiden University Medical Center P4
d.bakker@lumc.nl
Ballard, Jimmy United States OUHSC O5
jimmy-ballard@ouhsc.edu
Barbieri, Joseph United States Medical College of Wisconsin O41
jtb01@mcw.edu
Basak, Ajit United Kingdom Birkbeck College O18
a.basak@mail.cryst.bbk.ac.uk
Blasi, Juan Spain University of Barcelona P41
blasi@ub.edu
Bordeleau, Eric Canada University de Sherbrooke
eric.bordeleau@usherbrooke.ca
Bouillaut, Laurent United States Tufts University P5
laurent.bouillaut@tufts.edu
Bradshaw, Marite United States University of Wisconsin-Madison
mbradsha@wisc.edu

ClosPath
Institution
Participants Country E-mail Abstract
Buckley, Anthony United Kingdom University of Glasgow O26
Anthony.Buckley@Glasgow.ac.uk
Burrus, Vincent Canada University de Shebrooke
Vincent.burrus@usherbrooke.ca
Buss, Janice USA TechLab, Inc.
jebuss2@gmail.com
Butel, Marie-José France University Paris Descartes/Faculty of
Pharmacy
marie-jose.butel@parisdescartes.fr
P42
Carlson, Paul United States University of Michigan P6
pecarl@umich.edu
Cartman, Stephen United Kingdom University of Nottingham P7
stephen.cartman@nottingham.ac.uk
Chakravorty, Anjana Australia Monash University O36
anjana.chakravorty@monash.edu
Chen, Jianming United States University of Pittsburgh O46
jic40@pitt.edu
Chen, Kevin United States Tufts University O25
Chun-Nian.Chen@tufts.edu
Cheng, Ying China National Institute for Communicable Disease
Control and Prevention
chengying@icdc.cn
Collignon, Anne France University Paris Sud 11 O13, P8,
anne.collignon@u-psud.fr
P9, P10
Coppe, Philippe Belgium Bio-X Diagnostics
p.coppe@biox.com
Corver, Jeroen Netherlands Leiden University Medical Center P11
j.corver@lumc.nl
Curry, Scott United States University of Pittsburgh P12
currysr@upmc.edu
O4

ClosPath
Institution
Participants Country E-mail Abstract
Cuttingm, Simon United Kingdom Royal Holloway, University of London O38
S.Cutting@rhul.ac.uk
D'Auria, Kevin United States University of Virginia O47
kd3jd@virginia.edu
Derman, Yagmur Finland University of Helsinki, Faculty of Veterinary
Medicine
yagmur.derman@helsinki.fi
Di Paolo, Emmanuel Beligum GlaxoSmithKline Biologicals R & D
Emmanuel.di-paolo@gskbio.com
Downing, Mariann United States ViroPharma, Inc.
mariann.downing@viropharma.com
Dupuy, Bruno France Institut Pasteur O19
bdupuy@pasteur.fr
Dyson, Walter United States Merck
walter.dyson@merck.com
Ellermeier, Craig United States University of Iowa O52
craig-ellermeier@uiowa.edu
Etzel, Lisa United States Iowa State University
iaponygirl@aol.com
Fagan, Robert United Kingdom Imperial College London O32
r.fagan@imperial.ac.uk
Fairweather, Neil United Kingdom Imperial College London O50
n.fairweather@imperial.ac.uk
Farias, Luana Brazil UFSM P44
luana.vett@gmail.com
Feng, Hanping United States Tufts University O35
hanping.feng@tufts.edu
Fernandes Da Costa, United Kingdom University of Exeter O17
Sérgio
s.p.fernandes-da-costa@exeter.ac.uk
Figueroa, Iris United States Hines VA Hospital P13
iristfig@gmail.com

ClosPath
Institution
Participants Country E-mail Abstract
Fortier, Louis-Charles Canada Universite de Sherbrooke O37
Louis-Charles.Fortier@USherbrooke.ca
Garcia, Jorge United States University of California, Davis P45
jorgepampa@yahoo.con.ar
Gaskin, Duncan United Kingdom Institute of Food Research
duncan.gaskin@ifr.ac.uk
Gergen, Linda United States Merck Animal Health
linda.gergen@merck.com
Gibert, Xavier Spain Laboratorios Hipra, S.A.
xgp@hipra.com
Ginter, Annita Belgium Bio-X Diagnostics
a.ginter@biox.com
Goossens, Evy Belgium Ghent University P46
evy.goossens@UGent.be
Govind, Revathi United States Kansas State University O20
rgovind@ksu.edu
Govoni, Gregory United States AvidBiotics Corporation P47
greg.govoni@avidbiotics.com
Hanna, Philip United States University of Michigan Medical School
pchanna@umich.edu
Heeg, Daniela United Kingdom University of Nottingham P14
daniela.heeg@nottingham.ac.uk
Heinrichs, Jon United States Merck & Co., Inc.
jon_heinrichs@merck.com
Henderson, Abigail United States Hipra Scinetific USA, LLC
xgp@hipra.com
Ho, Theresa United States University of Iowa P15
theresa-ho@uiowa.edu
Huang, Haihui China Fudan University Hospital Huashan
huanghaihui@fudan.edu.cn

ClosPath
Institution
Participants Country E-mail Abstract
Huynh, Hong United Kingdom Royal Holloway University
hong.huynh@rhul.ac.uk
Jabbari, Sara United Kingdom University of Nottingham
sara.jabbari@nottingham.ac.uk
Janezic, Sandra Slovenia Institute of Public Health Maribor P16
sandra.janezic@zzv-mb.si
Johnson, Eric United States University of Wisconsin O14
eajohnso@wisc.edu
Johnson, Stuart United States Hines VA Hospital
sjohnson@lumc.edu
Kelly, Michelle United Kingdom University of Nottingham P48
michelle.kelly@nottingham.ac.uk
Kempker, Jennifer United States Boehringer Ingelheim Vetmedica
jennifer.kempker@boehringeringelheim.com
Keto-Timonen, Riikka Finland University of Helsinki P49
riikka.keto-timonen@helsinki.fi
Kirk, David Finland University of Helsinki O43
david.kirk@helsinki.fi
Knetsch, Wilco Netherlands Leiden University Medical Center O3
c.w.knetsch@lumc.nl
Kõljalg, Siiri Estonia University of Tartu P17
siiri.koljalg@ut.ee
Korkeala, Hannu Finland University of Helsinki
hannu.korkeala@helsinki.fi
Kuehne, Sarah United Kingdom University of Nottingham O45
sarah.kuehne@nottingham.ac.uk
Lacy, Dana United States Vanderbilt University School of Medicine O6
borden.lacy@vanderbilt.edu

ClosPath
Institution
Participants Country E-mail Abstract
Lahti, Päivi Finland University of Helsinki P50
paivi.lahti@helsinki.fi
Lambert, Dominic
Canada
Health Canada, Health Product and Food
Branch
dominic.lambert@hc-sc.gc.ca
Lei, Xiang United States Biolog, Inc. P18
xlei@biolog.com
Leite, Fernando USA Iowa State University
fleite@iastate.edu
Lepp, Dion Canada University of Guelph P52
dlepp@uoguelph.ca
Li, Jihong United States University of Pittsburgh O51
jihongli@pitt.edu
Li, Shan United States Tufts University O8
lishan.li@tufts.edu
Lindström, Miia Finland University of Helsinki O42
miia.lindstrom@helsinki.fi
Liu, Mingyu United States Memorial Sloan-Kettering Cancer Center P19
mymingyuliu@gmail.com
Liu, Shie-Chau United States Stanford University
liusc@stanford.edu
Lu, Jinxing China National Institute for Communicable
Disease Control and Prevention
lujinxing@icdc.cn
Lu, Shan United States University of Massachusetts
Cindi.callaghan@umassmed.edu
Lyras, Dena Australia Monash University O44
dena.lyras@monash.edu
MacCannell, Duncan United States Centers for Disease Control and Prevention O29
fms2@cdc.gov
P51

ClosPath
Institution
Participants Country E-mail Abstract
Mackin, Kate Australia Monash University O23
kate.mackin@monash.edu
Mallozzi, Michael United States University of Arizona P20
mallozzi@email.arizona.edu
Marsh, Jane United States University of Pittsburgh P21, P22
jwmarsh@pitt.edu
McBride, Shonna United States Tufts University School of Medicine O39
shonna.mcbride@tufts.edu
McClain, Mark United States Vanderbilt University O16
mark.mcclain@vanderbilt.edu
McClane, Bruce United States University of Pittsburgh O11
bamcc@pitt.edu
McQuade, Rebecca United States University of Arizona O40
rmcquade@email.arizona.edu
Melnyk, Roman Canada University of Toronto
roman.melnyk@sickkids.ca
Melville, Stephen United States Virginia Tech O49
melville@vt.edu
Miezeiewski, Matthew United States Merck P23
matthew_miezeiewski@merck.com
Minton, Nigel United Kingdom University of Nottingham O12
nigel.minton@nottingham.ac.uk
Miyamoto, Kazuaki Japan Wakayama Medical University
kazuaki@wakayama-med.ac.jp
Moura, Hercules United States Centers for Disease Control and Prevention P24, P53
HMoura@cdc.gov
Naaber, Paul Estonia University of Tartu P25
paul.naaber@gmail.com
Nowell, Vicki Canada University of Guelph P54
vnowell@uoguelph.ca

ClosPath
Institution
Participants Country E-mail
Ohtani, Kaori Japan Kanazawa University O21
ohtanik@med.kanazawa-u.ac.jp
Olling, Alexandra Germany Hannover Medical School P26
olling.alexandra@mh-hannover.de
Paredes-Sabja, Chile Universidad Andres Bello P27
Daniel
daniel.paredes.sabja@gmail.com
Parreira, Valeria Canada University of Guelph
vparreir@uoguelph.ca
Posthaus, Horst Switzerland University of Bern O28
horst.posthaus@vetsuisse.unibe.ch
Quesada-Gómez, Costa Rica Rica. San José, Costa Rica P28
Carlos
carlos.quesada@ucr.ac.cr
Reeves, Angela United States University of Michigan P29
angelhop@umich.edu
Riley, Thomas Australia University of Western Australia
Thomas.riley@uwa.edu.au
Robinson, Catherine United States Michigan State University P30
pohlcath@msu.edu
Rood, Julian Australia Monash University O10
julian.rood@monash.edu
Abstract
Rupnik, Maja Slovenia Institute of Public Health Maribor O1, P59
maja.rupnik@zzv-mb.si
Sadighi Akha, Amir United States University of Michigan Medical School P61
asadighi@umich.edu
Sarker, Mahfuzur United States Oregon State University O33
sarkerm@oregonstate.edu
Saujet, Laure France Institut Pasteur O34
laure.saujet@pasteur.fr
Schmidt, Diane United States Tufts University
diane.schmidt@tufts.edu

ClosPath
Institution
Participants Country E-mail
Scholl, Dean United States AvidBiotics Corporation P31
dean@avidbiotics.com
Schoster, Angelika Canada University of Guelph P32
angelika_schoster@hotmail.com
Secore, Susan United States Merck & Co., Inc.
susan_secore@merck.com
Seibel, Janice United States Iowa State University
jseibel@iastate.edu
Self, William United States University of Central Florida O30
wself@mail.ucf.edu
Sepp, Epp Estonia University Tartu
epp.sepp@ut.ee
Shen, Aimee United States University of Vermont O7
aimee.shen@uvm.edu
Shi, Lianfa United States Tufts University P55
lianfa.shi@tufts.edu
Shimizu, Tohru Japan Kanazawa University
tshimizu@med.kanazawa-u.ac.jp
Abstract
Shin, Bo-Moon Korea Sanggye Paik Hospital, Inje University P33, P34
bmshin@unitel.co.kr
Smits, Wiep Klaas Netherlands Leiden University Medical Center O22
w.k.smits@lumc.nl
Sonenshein, Abraham United States Tufts University School of Medicine O31
linc.sonenshein@tufts.edu
Songer, Glenn United States Iowa State University
jgsonger@iastate.edu
Sorg, Joseph United States Texas A&M University P35
jsorg@bio.tamu.edu
Springer, Sven Germany IDT Biologika GmbH
sven.springer@idt-biologika.de

ClosPath
Institution
Participants Country E-mail
Squire, Michele Australia University of Western Australia P36
mspb@bigpond.net.au
Staempfli, Henry Canada University of Guelph
hstaempf@uoguelph.ca
Stringer, Sandra United Kingdom Institute of Food Research
sandra.stringer@ifr.ac.uk
Strong, Philippa Canada NRC
philippa.strong@nrc.ca
Sun, Xingmin United States Tufts University O27
xingmin.sun@tufts.edu
Tadepalli, United States Novartis Animal Health US, Inc.
Sambasivarao
sambasivarao.tadepalli@novartis.com
Tamayo, Rita United States University of North Carolina at Chapel Hill
rita_tamayo@med.unc.edu
Tangudu, Chandra United States Iowa State University
ctangudu@iastate.edu
Tatarowicz, Walter United States ViroPharma Incorporated
walter.tatarowicz@viropharma.com
Theile, Teri United States USDA
teri.l.theile@aphis.usda.gov
Therien, Alex United States Merck & Co., Ltd.
alex_therien@merck.com
Theriot, Casey United States University of Michigan P37
caseythe@med.umich.edu
Abstract
Tweten, Rodney K. United States University of Oklahoma Keynote
rod_tweten@ouhsc.edu
Uzal, Francisco United States University of California, Davis O24
fuzal@cahfs.ucdavis.edu
Vargas, Agueda Brazil UFSM P56
agueda.vargas@gmail.com

ClosPath
Institution
Participants Country E-mail
Vedantam, Gayatri United States University of Arizona O48
vkv@email.arizona.edu
Viswanathan, Vish United States University of Arizona
vkv@email.arizona.edu
von Eichel-Streiber, Germany tgcBIOMICS GmbH P38
Christoph
chv.eichel@tgcbiomics.de
Wachtel, Marian United States NIH
waschtelm@niaid.nih.gov
Walk, Seth United States University of Michigan P39
sethwalk@umich.edu
Weeramantri, Lakmini Australia Monash University
lakmini.weeramantri@monash.edu
Wei, Wensheng China Peking University O9
wswei@pku.edu.cn
Wilson, Janet M. United States USDA
Janet.M.Wilson@aphis.usda.gov
Wright, Lorinda United States Edward Hines Jr. VA Hospital P40
lorinda.wright2@va.gov
Yan, Xuxia Australia Monash University P57
xuxia.yan@monash.edu
Yu, Brian United States Loyola University Medical Center
bryu@lumc.edu
Yumine, Natsuko Japan Wakayama Medical University P58
yu-mie.n-i0o.o0j.@docomo.ne.jp
Zemljic, Mateja Slovenia University of Maribor P62
mateja.zemljic@zzv-mb.si
Zhang, Zhen Finland University of Helsinki
zhen.zhang@helsinki.fi
Zhu, Jun United States University of Pennsylvania
junzhu@upenn.edu
Abstract

AUTHOR INDEX

Adams, V. – O11, O24
Monash University, Australia
Aktories, K. – O15
University of Freiburg, Germany
Albrow, V.E. – O7
Standford University, USA
Technische Universitat Munchen, Germany
Aleksoniene, K. – P40
Hines VA Hospital, USA
Allen, C. – P35
Texas A & M University, USA
Almassalha, L.M. – O2
University of Michigan, USA
Aires, J. – P42
Université Paris Descartes, France
Ampel, N. – P20
University of Arizona, USA
Anderson, L. – O29
Centers for Disease Control and Prevention, USA
Antunes, A. – O19
Institut Pasteur, France
Anwar, F. – P20
University of Arizona, USA
Ardis, T.C. – P60
Agri-food & Bioscience Institute, UK
Aronoff, D.M. – O2, P39
University of Michigan, USA
Arroyo, L.G. – P32
University of Guelph, Canada
Auchtung. J. – P1
Michigan State University, USA
Austin, J.W. – P51
Institute for Biological Sciences, Canada
Authemann, D. – O28
University of Berne, Switzerland
Awad, M.M. – O36
Monash University, Australia
Babakhani, F. – P2, P3
Optimer Pharmaceuticals, Inc., USA
Baban, S. – O12
University of Nottingham, UK
Bakker, D. – O22, P4, P11
Leiden University, Netherlands
Ball, H.J. – P60
Agri-food and Bioscience Institute, UK
Ballard, J. – O5
University of Oklahoma, USA
Bannam, T.L. – O10, P57
Monash University, Australia
Bantwal, R. – O10
Monash University, Australia
Barbieri, J. T. – O41
Medical College of Wisconsin, USA
Barketi-Klai, A. – O13, P9
Université Paris-Sud, France
Barr, J.R. – P24, P53
Centers for Disease Control and Prevention, USA
Barros, C.S.L. – P56
UFSM, Brazil
Basak, A.K. – O17, O18
Birkbeck College, UK
Beards, E.J. – O26
University of Leicester, UK
Blake, T.A. – P24
Centers for Disease Control and Prevention, USA
Blasi, J. – P41
University of Barcelona, Spain
Bochner, B.R. – P18
Biolog, Inc., USA
Bodmer, J.L. – P23
Merck Research Laboratories, USA
Boerlin, P. – P52
University of Guelph, Canada
Bogyo, M. – O7
Stanford University, USA
University of Vermont, USA
Bokori-Brown, M. – O17
University of Exeter, UK
Botton, S. – P44
UFSM, Brazil
Bouillaut, L. – O30, O31, P2, P3, P5
Tufts University, USA
Boursier, C. – P10
Université Paris-Sud, France
Britton, R. – P1, P30
Michigan State University, USA
Brooks, C.E. – P60
Agri-food & Bioscience Institute, UK
Brouwer, M. – P11
UCL Eastman Dental Institute, UK
Buckley, A.M. – O26
University of Glasgow, UK
Burns, D.A. – P7, P14
University of Nottingham, UK
Butel, M. – P42
Université Paris Descartes, France
Cai, C. – O9
Peking University, China
Camiade, E. – O19
Institut Pasteur, France
Campeotto, F. – P42
Université Paris Descartes, France

Carlson Jr., P.E. – P6
University of Michigan, USA
Carter, G. – O23, O44
Monash University, Australia
Cartman, S.T. – O12, O45, P7, P14, P48
University of Nottingham, UK
Caserta, J. – O24
University of Pittsburgh, USA
Chakravorty, A. – O36, O44
Monash University, Australia
Chaves-Olarte, E. – P28
Universidad de Costa Rica, Costa Rica
Universidad Nacional, Costa Rica
Chen, C. – O4
Chinese CDC & Prevention, China
Chen, J. – O46, O51
University of Pittsburgh, USA
Chen, K. – O8, O25, O35
Tufts University, USA
Cheng, Y. – O4
Chinese CDC & Prevention, China
Cheung, J.K. – O36, P57
Monash University, Australia
Choo, J.M. – O36
Monash University, Australia
Christen, S. – O28
University of Berne, Switzerland
Chumbler, N.M. O6
Vanderbilt University, USA
Cobaugh, K. – O30
University of Central Florida, USA
Cockayne, A. – O12, O45, P48
University of Nottingham, UK
Cohen, J. – O29
Centers for Disease Control and Prevention, USA
Collery, M.M. – O45
University of Nottingham, UK
Collignon, A. – O13, P8, P9, P10
Université Paris-Sud, France
Cook, J. – O38
University of London, UK
Cordon, G.P. – O35
Tufts University, USA
Corver, J. – O3, P4, P11
Leiden University, UKCurry, S.R. – P12, P21
University of Pittsburgh, USA
Cutting, S.M. – O38
Imperial College, UK
Dahlsten, E. – O42, O43
University of Helsinki, Finland
D‘Auria, K.M. – O47
University of Virginia, USA
Davis, B. – O27
Tufts University, USA
Dawson, L.F. – O26
University of Leicester, UK
del Mar Gamboa-Coronado, M. – P28
Universidad de Costa Rica, Costa Rica
Deprez, P. – P46
Ghent University, Belgium
Derman, Y. – P43
University of Helsinki, Finland
Dineen, S. – O31
Tufts University, USA
Donato, G.M. – O47
University of Virginia, USA
Dorca-Arévalo. J. – P41
University of Barcelona, Spain
Douce, G. – O23, O26, O44
University of Glasgow, UK
Drobne, D. – P62
University of Ljubljana, Slovenia
Du, P. – O4
Chinese CDC & Prevention, China
Du, T. – P28
Natinal Microbiology Laboratory, Canada
Ducatelle, R. P46
Ghent University, Belgium
Dupuy, B. – O13, O19, O23, O34, O44, P9
Institut Pasteur, France
Ehsaan, M. – O12
University of Nottingham, UK
Ellermeier, C.D. – O52, P15
University of Iowa, USA
Ewing, S.A. – O2
University of Michigan, USA
Fagan, R.P. – O32
Imperial College, UK
Fairley, D. – P60
Belfast Health & Social Care Trust, UK
Fairweather, N. – O32, 038, O50
Imperial College, UK
Farias, L. – P44, P56
UFSM, Brazil
Farrow, M.A. O6
Vanderbilt University, USA
Feng, H. – O8, O25, O27, O35, P55
Tufts University, USA
Fennessey, C.M. – O16
Vanderbilt University, USA
Fernandes da Costa, S. – O17
University of Exeter, UK
Ferraris, L. – P42
Université Paris Descartes, France

Figueroa, I. – P13
Hines VA Hospital, USA
Fisher, D. J.– O11, P45
University of Pittsburgh
Fortier, L.C. – O37, P31
Universite de Sherbrooke, Canada
Foster, N.F. – P36
University of Western Australia, Australia
Frenzel, E. – P26
Hanover Medical School, Germany
Friedman, D.B. – O6
Vanderbilt University, USA
Galecki, A.T. – O2
University of Michigan, USA
Garcia, K.C. – O7
Stanford University, USA
Garcia, J.P. – O24, O46, P45
University of California Davis, USA
Garneau, J.R. – O37
Université de Sherbrooke, Canada
Gebhart, D. – P31, P47
AvidBiotics Corp., USA
Gee, J. – P21
University of Pittsburgh, USA
Gerding, D. – O44, P13, P22, P40
Hines Veterans Affairs Hospital, USA
Gerhard, R. – P26
Hanover Medical School, Germany
Gersch, M.M. – O7
Standford University, USA
Gianetti, B. – O49
Virginia Tech, USA
Giaretta, P. – P56
UFSM, Brazil
Gisch, K. – P38
tgcBIOMICS GmbH, Germany
Goldstein, E.J.C. – P13
RM Aiden Research Laboratory, USA
UCLA, USA
Gomez, A. – P2, P3
Optimer Pharmaceuticals, Inc., USA
Gong, J. – P52
Guelph Food Research Centre, Canada
Goossens, E. – P46
Ghent University, Belgium
Govind, R. – O20, O23, O44
Kansas State University, USA
Govoni, G. – P31, P47
AvidBiotics Corp., USA
Goy, S. – P26
Hanover Medical School, Germany
Gray, M.C. – O47
University of Virginia, USA
Gurjar, A. – O11
University of Pittsburgh
Guzmán-Verri, C. – P28
Universidad Nacional, Costa Rica
Hanna, P.C. – P6
University of Michigan, USA
Harmanus, C. – O3, P11
Leiden University Medical Center, Netherlands
Harrison, L.H. – P12, P21, P22
University of Pittsburgh, USA
Hartman, A. – O49
Virginia Tech, USA
Hashsham, S. – P1
Michigan State University, USA
He, R. – O9
Peking University, China
Heap, J.T. – O12, O42, P7
University of Nottingham
Heeg, D. – P7, P14
University of Nottingham, UK
Henderson, T.K. – P22
University of Pittsburgh, USA
Heikinheimo, A. – P50
University of Helsinki, Finland
Heinrichs, J. – P23
Merck Research Laboratories, USA
Hensgens, M.P.M. – O3, P11
Leiden University Medical Center, Netherlands
Hewlett, E.L. – O47
University of Virginia, USA
Hirakawa, H. – O21
Kazusa DNA Research Institute, Japan
Hiscox, T.J. – O36
Monash University, Australia
Ho, T.D. – O52, P15
University of Iowa, USA
Hong, H.A. – O38
University of London, UK
Howarth, P. – O23
Monash University, Australia
Hoys, S. – O13, P9
Université Paris-Sud, France
Huang, J. – O38
University of London, UK
Huffnagle, G.B.
University of Michigan, USA
Hughes, M. – O11, O24
Monash University, Australia
Hughes, V. – P57
Monash University, Australia

Huyet, J. – O18
Birkbeck College, UK
Indra, A. – P16
Austrian Agency for Health & Food Safety, Vienna
Ivie, S.E. – O16
Vanderbilt University, USA
Jacobson, M.J. – O14
University of Wisconsin-Madison, USA
Jain, R. – O2
University of Michigan, USA
Janezic, S. – P16
Institute of Public Health Maribor, Slovenia
Jarchum, I. – P19
Sloan-Kettering Institute, USA
Johnson, E. – O14
University of Wisconsin, USA
Johnson, S. – O44, P13, P40
Hines Veterans Affairs Hospital, USA
Jolivot, P-A. – P8
Université Paris-Sud, France
Just, I. – P26
Hanover Medical School, Germany
Kansau, I. – O13, P8, P9
Université Paris-Sud, France
Kelly, M.L. – O12, O44, O45, P7, P48
University of Nottingham, UK
Keto-Timonen, R. – P49
University of Helsinki, Finland
Keyburn, A.L. – P57
Monash University, Australia
CSIRO Livestock Industries, Australia
Kirk, D. – O43
University of Helsinki, Finland
Knetsch, C.W. – O3
Leiden University, Netherlands
Kõljalg, S. – P17, P25
University of Tartu, Estonia
Kolling, G.L. – O47
University of Virginia, USA
Korkeala, H. – O42, O43, P43, P49, P50
University of Helsinki, Finland
Kropinski, A. – P52, P54
Public Health Agency of Canada, Canada
Kuak, E. – P33, P34
Inje Univesity, South Korea
Kubiak, A. – O12
University of Nottingham, UK
Kuehne, S.A. – O12, O45, P7
University of Nottingham, UK
Kuhara, S. – O21
Kyushu University, Japan
Kuijper, E.J. – O3, O22, P4, P11
Leiden University Medical Center, Netherlands
Kumar, R. – O35, P55
The Commonwealth Medical College, USA
Lacy, D.B. – O6
Vanderbilt University, USA
Lahti, P. – P50
University of Helsinki, Finland
Lambert, D. – P9, P51
Health Canada, Canada
Lange, A. – P38
tgcBIOMICS GmbH, Germany
Lapp, D. – P52
University of Guelph, Canada
Lawley, T. – O44
The Wellcome Trust Sanger Institute, UK
Lee, E. – P33, P34
Inje Univesity, South Korea
Lei, X. – P18
Biolog, Inc., USA
Lepp, D.
University of Guelph, Canada
Lessa, F. – O29
Centers for Disease Control and Prevention, USA
Li, C. – O9
Peking University, China
Li, J. – O11, O46, O51
University of Pittsburgh, USA
Li, S. – O8, O35
Tufts University, USA
Libardoni, F. – P56
UFSM, Brazil
Lim, S.C. – P36
University of Western Australia, Australia
Limbago, B. – O29
Centers for Disease Control and Prevention, USA
Lindström, M. – O42, O43, P43, P50
University of Helsinki, Finland
Liu, E.W. – O2
University of Michigan, USA
Liu, M. – P19
Sloan-Kettering Institute, USA
Lu, J. – O4
Chinese CDC & Prevention, China
Lucena, R. – P56
UFSM, Brazil
Lupardus, P.J. – O7
Stanford University, USA
Lyras, D. – O23, O36, O44
Monash University, Australia
Ma, M. – O24, O46
University of Pittsburgh, USA

MacCannell, D. – O29
Centers for Disease Control and Prevention, USA
MacInnes, J.I. – P54
University of Guelph, Canada
Mackin, K. – O23, O44
Monash University, Australia
Mallozzi, M. – O40, P20
University of Arizona, USA
Marsh, J.W. – P12, P21, P22
University of Pittsburgh, USA
Martín-Satué, M. – P41
University of Barcelona, Spain
Martin-Verstraete, I. – O19
Institut Pasteur, France
McBride, S.M. – O31, O39
Tufts University, USA
McClane, B. – O11, O24, O46, O51, P45
University of Pittsburgh, USA
McClain, M.S. – O16
Vanderbilt University, USA
McDermott, A.J. – P61
University of Michigan, USA
McQuade, R. – O40
University of Arizona, USA
Medved, M. – P59
Institute of Public Health Maribor, Slovenia
Melville, S. – O49
Virginia Tech, USA
Micic, D. – O2
University of Michigan, USA
Miezeiewski, M. – P23
Merck Research Laboratories, USA
Mimura, K. – P58
Wakayama Medical University, Japan
Minton, N.P. – O12, O42, O45, P14, P48
University of Nottingham, UK
Mitchell, T. – P23
Merck Research Laboratories, USA
Miyamoto, K. – O11, P58
Wakayama Medical University, Japan
Monot, M. – O13, O19, O34, P9
Institut Pasteur, France
Moore, R.J. – P57
Monash University, Australia
CSIRO Livestock Industries, Australia
Moss, D.S. – O17
Birkbeck College, UK
Moura, H. – P24, P53
Centers for Disease Control and Prevention, USA
Mulvey, M.R. – P28
National Microbiology Laboratory, Canada
Mun, S. – P33, P34
Inje Univesity, South Korea
Muravsky, M. – P23
Merck Research Laboratories, USA
Murillio, R. – P38
Oporon S.A., Spain
Naaber, P. – P17, P25
Stavanger University Hospital, Norway
Naylor, C.E. – O17, O18
Birbeck College, UK
Newton, D.W. – O2
University of Michigan, USA
Ng, R. – O12, P48
University of Nottingham, UK
Nguyen, L. – P3
Optimer Pharmaceuticals, Inc., USA
Nicolis, I. – P42
Université Paris Descartes, France
Nie, W.
Tufts University, USA
Noon, A. – P20
University of Arizona, USA
Nowell, V.J. – P54
University of Guelph, Canada
Oester, C. – P38
tgcBIOMICS GmbH, Germany
Ohtani, K. – O21
Kanazawa University, Japan
Olling, A. – P26
Hanover Medical School, Germany
Pamer, E.G.– P19
Sloan-Kettering Institute, USA
Papazisi, L. – O14
The J. Craig Venter Institute, USA
Papin, J.A. – O47
University of Virginia, USA
Pardon, B. P46
Ghent University, Belgium
Paredes-Ssabja, D. – O21, O33, P27
Universidad Andrés Bello, Chile
Parreira, V. – P52, P54
University of Guelph, Canada
Péchiné, S. – P10
Université Paris-Sud, France
Permpoonpattana, P. – O38
University of London, UK
Petrella, L. – P40
Hines VA Hospital, USA
Peterson, S.N. – O14
The J. Craig Venter Institute, USA
Phetcharaburanin, J. – O38
University of London, UK

Poon, R. – O11, O24
Monash University, Australia
Popoff, M. – O28
Institut Pasteur, France
Porter, C.J. – O10, P57
Monash University, Australia
Posthaus, H. – O28, P45
University of Berne, Switzerland
Prescott, J.F. – P52, P54
University of Guelph, Canada
Pruitt, R.N. – O6
Vanderbilt University, USA
Puri, A.W. – O7
Standford University, USA
Quesada-Gómez, C. – P28
Universidad de Costga Rica, Costa Rica
Quinsey, N. – P57
Monash University, Australia
Rätsep, M. – P17, P25
University of Tartu, Estonia
Rattei, T. – P16
University of Vienna, Austria
Reeves, A.E. – P29, P61
University of Michigan, USA
Riley, T.V. – P36
University of Western Australia, Australia
PathWest Laboratory Medicine, Australia
Ring, C. – O2
University of Michigan, USA
Roberts, A.P. – P11
UCL Eastman Dental Institute, UK
Robertson, S. – O24, O51
University of Pittsburgh, USA
Robinson, C. – P1, P30
Michigan State University, USA
Rodríguez, C. – P28
Universidad de Costa Rica, Costa Rica
Rodríguez-Cavallini, E. – P28
Universidad de Costa Rica, Costa Rica
Rood, J. – O10, O11, O23, O24, O36, O44, O46,
P57
Monash University, Australia
Rosenbusch, K.E. – O22
Leiden University, Netherlands
Roxas, B. – O40
University of Arizona, USA
Rozé, J.C. – P42
Médecine Néonatale, France
Rubin, D.H. – O16
Vanderbilt University, USA
Rupnik, M. – O1, P16, P59, P62
University of Maribor, Slovenia
Sadighi Akha, A. – P61
University of Michigan, USA
Sambol, S.P. – P13, P22 P40
Hines VA Hospital, USA
Loyola Univesity Medical Center, USA
Saputo, J. – O24, O46, P45
University of California Davis, USA
Sarker, M.R. – O21, O33, P27
Oregon State University, USA
Saujet, L. – O34
Institut Pasteur, France
Savelkoul, P.H.M. – O3
VU University Medical Center, Netherlands
Savidge, T. – O35, P55
University of Texas, USA
Savva, C.G. – O17, O18
Birkbeck College, UK
Sayeed, S. – O11, O51, P45
University of Pittsburgh, USA
Schauvlieghe, S. P46
Ghent University, Belgium
Schmidt, D. – O8
Tufts University, USA
Schnaufer, T. – P23
Merck Research Laboratories, USA
Scholl, D. – P31, P47
AvidBiotics Corp., USA
Schoster, A. – P32
University of Guelph, Canada
Sears, P. – P2, P3
Optimer Pharmaceuticals, Inc., USA
Seeback, S.A. O6
Vanderbilt University, USA
Self, W.T. – O30, O31, P5
University of Central Florida, USA
Sepp, E. – P17, P25
University of Tartu, Estonia
Setlow, P. – O33
University of Connecticut, USA
Shen, A. – O7
University of Vermont, USA
Sheng, J. – O16
Vanderbilt University, USA
Shewen, P.E. – P32
University of Guelph, Canada
Shi, L. – O8, P55
Tufts University, USA
Shimizu, T. – O21
Kanazawa University, Japan
Shin, B. – P33, P34
Inje University, South Korea

Shiver, J. – P23
Merck Research Laboratories, USA
Shkut, E. – P17, P25
University of Tartu, Estonia
Shoemaker, C.B. – O8
Tufts University, USA
Shrestha, A. – O24
University of Pittsburgh, USA
Shutt, K. – P21
University of Pittsburgh, USA
Sims, C. – P2
Optimer Pharmaceuticals, Inc., USA
Škraban, J. – P59
University of Maribor, Slovenia
Smidt, I. – P17, P25
University of Tartu, Estonia
Smith, A.I. – P57
Monash University, Australia
Smits, W.K. – O22, P4
Leiden University Medical Center, Netherlands
Solano, M.I. – P24, P53
Centers for Disease Control and Prevention, USA
Somervuo, P. – P50
University of Helsinki, Finland
Sonenshein, A. L. – O30, O31, O39, P2, P3, P5
Tufts University, USA
Songer, J.G. – P52, P54
Iowa State University, USA
Sorg, J. – P35
Texas A & M University, USA
Soring, K. – P23
Merck Research Laboratories, USA
Söderholm, H. – P43
University of Helsinki, Finland
Soutourina, O. – O34
Institut Pasteur, France
Spencer, J. – O26
University of Glasgow, UK
Spiller, B.W. O6
Vanderbilt University, USA
Squire, M.M. – P36
University of Western Australia, Australia
Srivastava, M. – O30
University of Central Florida, USA
Staempfli, H.R. – P32
University of Guelph, Canada
Stedtfield, R. – P1
Michigan State University, USA
Steele, J. – O25
Tufts University, USA
Steer, D. – P57
Monash University, Australia
Stsepetova, J. – P17
University of Tartu, Estonia
Sun, X. – O8, O25, O27, O35, P55
Tufts University, USA
Tashiro, K. – O21
Kyushu University, Japan
Tatge, H. – P26
Hanover Medical School, Germany
ter Meulen, J. – P23
Merck Research Laboratories, USA
Terilli, R.R. – P24, P53
Centers for Disease Control and Prevention, USA
Oak Ridge Institute for Scientific Education, USA
Theriot, C.M. – P37, P61
University of Michigan, USA
Timbermont, L. P46
Ghent University, Belgium
Titball, R.W. – O17
University of Exeter, UK
Toribio, T. – P38
Oporon S.A., Spain
Tremblay, J.M. – O8
Tufts University, USA
Tulenko, M.M. – P12
University of Pittsburgh, USA
Tweten, R.K. – Keynote
University of Oklahoma, USA
Twine, S.M. – P51
HPFB Health Canada, Canada
Tzipori, S. – O8, O25, O35
Tufts University, USA
Uzal, F.A. – O24, O46, P45
University of California, Davis, USA
Valgaeren, B. – P46
Ghent University, Belgium
Valiquette, L. – O37
Université de Sherbrooke, Canada
van der Bijl, M.W. – O3
VU University Medical Center, Netherlands
Van Immerseel, F. – O28, P46
Gent University, Belgium
van Leeuwen, H.C. – O3, P11
Leiden University Medical Center, Netherlands
van Pijkeren, J.P. – P30
Michigan State University, USA
Vargas, A.C. – P44, P56
Universidade Federal de Santa Maria, Brazil
Vedantam, G. – O40, O48, P20
University of Arizona, USA
Verherstraeten, S. – P46
Ghent University, Belgium

Viala, C. – P10
Université Paris-Sud, France
Vidal, J.E. – O24, O46
University of Pittsburgh, USA
Villacampa, M. – P38
Oporon S.A., Spain
Viswanathan, V.K. – O40, P20
University of Arizona, USA
Vodovar, M. – P42
Institut de Puériculture, France
von Eichel-Streiber, C. – P38
tgcBIOMICS GmbH, Germany
Walk, S.T. – O2, P39
University of Michigan, USA
Walker, D. – O12
University of Nottingham, UK
Wang, H. – O25, O27, O35
Tufts University, USA
South China University of Science and
Technology, China
Wang, J. – O35
South China University of Technology, China
Wang, S. – P23
Merck Research Laboratories, USA
Wang, X. – P55
East China University of Science and Technology,
China
Wang, Y. – O9
Peking University, China
Washer, L. – O2
University of Michigan, USA
Wei, W. – O9
Peking University, China
Weese, J.S. – P32
University of Guelph, Canada
Weinmaier, T. – P16
University of Vienna, Austria
Whisstock, J.C. – O10, P57
Monash University, Australia
Williams, S. – P31, P47
AvidBiotics Corp., USA
Williamson, Y.M. – P24
Centers for Disease Control and Prevention, USA
Winzer, K. – O12
University of Nottingham, UK
Wisniewski, J. – O10
Monash University, Australia
Wolk, D. – P20
University of Arizona, USA
Woolfitt, A.R.– P24, P53
Centers for Disease Control and Prevention, USA
Wren, B.W. – O26
University of Leicester, UK
Wright, L. – P40
Hines VA Hospital, USA
Wu, X. – O9
Peking University, China
Wyder, M. – O28
University of Berne, Switzerland
Xie, A. – P23
Merck Research Laboratories, USA
Yan, Q. – O4
Chinese CDC & Prevention, China
Yan, X. – P57
Monash University, Australia
Yang, Z. – P55
East China University of Science and Technology,
China
Yelland, T. – O18
Birkbeck College, UK
Young, V.B. – O2, P29, P37, P39, P61
University of Michigan, USA
Yumine, N. – P58
Wakayama Medical University, Japan
Zemljič, M. – P62
University of Maribor, Slovenia
Zhang, Y. – O8, O25, O27, O35, O42, O43, P55
Tufts University, USA
Zhuge, R. – P55
University of Massachusetts, USA
Zorman, J. – P23
Merck Research Laboratories, USA