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.