ABMM/ABMLI Professional Recognition Award

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ABMM/ABMLI Professional Recognition Award

ABMM/ABMLI Professional

Recognition Award

Patrick R. Murray, PhD

May 22, 2011


Clinical Microbiology:

The Future is in Good Hands

• My mentors and their influence

• Contributions of fellows working in

my NIH lab

• The future of clinical microbiology


John Washington

1936-2010

Henry Isenberg

1922-2006

My Mentors

Alex Sonnenwirth

1923-1984

Al Balows

1921-2006


NIH Microbiology Fellows

• 2001-2003 Pattarachai Kiratisin, MD, PhD

• 2003-2006 Adrian Zelazny, PhD

• 2004-2006 Susan Harrington, PhD

• 2006-2009 Clemente Montero, PhD

• 2007-2009 Lindsay Stevenson, PhD

• 2008-present Lilia Mijares, PhD

• 2011-present Anna Lau, PhD

• 2011-present Stella Antonara, PhD


• Bangkok, Thailand

• MD, Mahidol University

Pattarachai Kiratisin, MD, PhD

• PhD, University of Rochester Medical

Center, New York

• NIH Microbiology Fellow (2001-03)

• Recipient of ASM Dade MicroScan

Young Investigator Award (2004)

• Chairman of Clinical Microbiology

Society of Thailand

• Professor, Mahidol University and

Deputy Chairman, Faculty of Medicine,

Siriraj Hospital, Bangkok Thailand


Bacterial Identification by Gene Sequencing

• Initiated the program for identification of bacteria by Sanger

sequencing of 16S rRNA gene.

• March 2002 – May 2003: 5% of all bacterial isolates identified

by sequencing including –

Abiotrophia defectiva Capnocytophaga sputigena Helicobacter cinaedi

Achromobacter

xylosoxidans

Cardiobacterium hominis Herbaspirillum huttiense

Brevundimonas diminuta Corynebacterium accolens Kingella denitrificans

Campylobacter fetus Dysgonomonas capnocytophagoides Kytococcus schroeteri

Campylobacter upsaliensis Haemophilus aphrophilus Tsukamurella pulmonis

• Following year, determined that sequencing the housekeeping

genes for glucose-6-phosphate dehydrogenase and glucose

kinase could be used to identify members of the

Streptococcus mitis group.


• Born Buenos Aires, Argentina

• PhD, Weizmann Institute, Israel

Adrian Zelazny, PhD

• NIH Microbiology Fellow (2003-06)

• Recipient of NIH Fellows Award for

Research Excellence (2005)

• Fellow, ABMM (2006)

• NIH Director’s Award for Research

(2007)

• Recipient of ASM Dade MicroScan

Young Investigator Award (2008)

• Staff Scientist, NIH Clinical

Microbiology Laboratory


Identification of Mycobacterium

and Nocardia Species by Gene Sequencing

• A number of gene targets had been used to identify

mycobacteria and nocardia with variable success including

16S rRNA, 16S-23S internal transcribed spacer, heat shock

protein, recA, rpoB, and gyrB genes.

• Speculated that since SecA1 protein is essential for protein

export, secA1 gene would be a good target for identification

of these genera.


Identification of Mycobacterium Species

• Identified the 700-bp gene sequence that codes the substrate

specificity domain and protein translocation domain of the

secA1 gene.

• 47 reference strains representing 30 species and 59 clinical

isolates representing 9 species were evaluated.

• Variability of the target sequence allowed differentiation of all

species except members of the M. tuberculosis complex

which had identical sequences.

• Strains belonging to the same species had high intraspecies

similarity and all strains were correctly identified.


Identification of Nocardia Species

• A similar study was performed with reference strains and

clinical isolates of Nocardia.

• 30 reference strains (30 species) and 40 clinical isolates

representing 12 species were evaluated.

• All 30 species could be differentiated by sequence analysis of

the SecA1 gene target. Additionally, all clinical isolates were

identified accurately using this gene target.

• Subsequent studies have demonstrated that multiple gene

targets may be necessary for the identification of some

uncommonly isolated species.


Direct Detection and Identification

of Mycobacteria and Nocardia in Clinical Specimens

• All acid-fast organisms in smear-positive clinical

specimens were identified directly by sequencing the

SecA1 gene.

• This approach was used in the last 5 years to identify

mycobacteria and nocardia in clinical specimens from

more than patients.

• 17 species of mycobacteria (including M. tuberculosis,

M. bovis, M. leprae) and 3 species of Nocardia were

identified in many different specimens (e.g., respiratory,

CSF, synovial fluid, duodenal fluid, skin lesions, sinus

aspirates, urine, and tissues).


Susan Harrington, PhD

• PhD, Department of Microbiology,

University of Maryland School of

Medicine

• NIH Microbiology Fellow (2004-06)

• Fellow, ABMM (2007)

• Former Associate Director,

Microbiology Laboratory, Albany

Medical Center, New York

• Current position: Cleveland Clinic -

Associate Medical Director,

Mycobacteriology & Specimen

Processing


Genotypic Analysis of Invasive S. pneumoniae

Strains from Mali Africa

• Previous work demonstrated that 78% of the S. pneumoniae

strains responsible for IPD in Mali were not in PCV7.

• Used PFGE and repPCR to determine if clonal dissemination

of predominant serotypes had occurred.

• The two methods were equally discriminatory within a

specific pneumococcal serotype.

• Using both methods, isolates within serotypes 2, 5 and 7

(the most common serotypes) formed 3 large clusters

containing 1 genotype each, which is consistent with clonal

dissemination of these serotypes.


• Born Venezuela

Clemente Montero, PhD

• PhD, Department of Chemical

and Biomolecular Engineering,

North Carolina State University

• NIH Microbiology Fellow (2006-

09)

• Fellow, ABMM (2008)

• Current position: Research

Associate, Weill Medical College

of Cornell University


Evaluation of Pyrosequencing for Yeast Identification


Evaluation of Pyrosequencing for Yeast Identification

• 133 isolates of clinically relevant yeasts, representing 43

species, were analyzed by gene sequencing.

• Sanger cycle sequencing of the ITS1-5.8S-ITS2 region

identified 79% of the isolates at the species level.

• Pyrosequencing of the hyper-variable ITS region identified

69% of the isolates at the species level.

• Although most species of Candida could be identified

accurately by pyrosequencing, Trichosporon species and

some Cryptococcus species could not be differentiated.


Lindsay Stevenson, PhD

• PhD, Department of Microbiology

and Molecular Genetics, Emory

University

• NIH Microbiology Fellow (2007-

09)

• Fellow, ABMM (2010)

• Current position: CPT, US Army

Assistant Chief, Infectious Disease

Laboratory, Walter Reed Army

Medical Center and Bethesda

Naval Hospital, Washington, DC


Microbial Identification by Matrix Assisted Laser

Desorption/Ionization Time of Flight (MALDI-TOF)

Mass Spectrometry

Intens.

[a.u.]

500

0

400

0

300

0

200

0

100

0

4364

5096

5380

6254

6315

6410

7157

7273

0

4000 5000 6000 7000 8000

7870

8368

m/z


MALDI-TOF Mass Spectrometry

Technical Considerations

• Most biomarkers detected in MALDI spectra are intracellular

basic proteins (2000 to 25,000 Da).

– Bacteria – ribosomal proteins

– Fungi – ribosomal and mitochondrial proteins

• Spectral profiles are influenced by culture conditions (e.g.,

media, age of colonies), pre-analytic processing, and assay

conditions.

• Although reproducibility of spectra can be poor, unique

profiles of conserved, discriminatory peaks are obtained and

are sufficient to identify a wide spectrum of organisms.


MALDI-TOF Mass Spectrometry

Yeast Identification

• 194 clinical isolates were tested (6 genera, 23 species)

• 192 (99.0%) were identified correctly; 2 isolates (Candida

rugosa and Cryptococcus neoformans) had spectral scores


MALDI-TOF Mass Spectrometry

Identification of Isolates in Positive Blood Cultures

• 212 positive blood cultures were analyzed:

– 20% with no identification

– 95% of the remaining organisms were identified

correctly at the species level

– 8 organisms were misidentified (all Streptococcus mitis

isolates were misidentified as S. pneumoniae)

• The most common organisms with no ID were

Propionibacterium and coagulase-negative

staphylococci


• PhD, Yale University School of

Medicine

Lilia Mijares, PhD

• NIH Fellow in Clinical Microbiology

Laboratory and National Human

Genome Institute, Julie Segre lab

(2008-present)


MALDI-TOF Mass Spectrometry

Nocardia and Mycobacteria Identification

• Techniques used to bacteria and yeasts failed to produce

acceptable spectra for nocardia and mycobacteria.

• A modified protein extraction procedure was developed

using heat inactivation and mechanical steps to disperse

bacteria and fragment their cell walls.

• Databases were developed for 37 species of mycobacteria

and 43 species of nocardia, and then challenged with >300

strains of mycobacteria and nocardia.

• M. tuberculosis complex strains were identified accurately

at the complex level but could not be identified at the

species level. All other strains were identified accurately at

the species level.


MALDI-TOF Mass Spectrometry

• Routinely used in the NIH lab for the

identification:

– Aerobic and anaerobic bacteria

– Yeasts and limited selection of molds

– Bacteria and yeasts recovered in blood culture broths

– Nocardia and mycobacteria

• Virtually all organisms identified at the species

level unless:

– Inoculum insufficient

– Organism not adequately represented in database


Reflections

• My career is rooted in mentors and colleagues who taught me

the clinical significance of this discipline, the skills to critically

analyze data and write my thoughts coherently, and the

intricacies of managing a clinical lab; they gave me the

opportunities and support to function at a national level;

• I have benefitted most recently by being surrounded by

fellows and colleagues who are willing to explore new

approaches for improving diagnostic testing.

• I think the future of clinical microbiology is bright because we

have a new generation of microbiologists who have the

training and intellectual skills to discover novel solutions to

clinical problems.

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