Transcript Staphylococcus aureus CC398: Host Adaption and Emergence of

Staphylococcus aureus CC398:
Host Adaption and Emergence
of Methicillin Resistance in
Livestock
Price et al., 2012
Jourdan Meltzer
November 4, 2013
S
Background: S. aureus
S bacteria frequently found in human respiratory tract & skin.
S not always pathogenic: 20% long-term carriers
S disease-associated strains: potent protein toxins & cell-surface proteins
that inactivate antibodies
S Methicillin Resistant Staphylococcus aureus: strain of S. aureus
bacteria resistant to most β-lactam antibiotics
S not always so: 1943-- antibacterial activity of Penicillium fungus
demonstrated against S. aureus
S Rates of resistance rising
Background: MRSA and MSSA
S Types based on sources:
S health care-associated
S community-associated
S health care-associated w/ community onset
S livestock-associated
S Differentiating from methicillin-susceptible S. aureus
(MSSA): MSSA unable to resist β-lactam antibiotics
S  MRSA not more virulent, simply harder to treat
Background: Risks, Diagnosis,
& Treatment
S Hospital patients, prisoners, homeless, athletes, people in
contact w/ live food-producing animals, & children
S Grow tissue sample in presence of antibiotic & check for
bacterial growth  developing DNA methods
S Draining of superficial abscesses in certain cases; bactrim &
vancomycin most common antibiotics
S Exciting research: bacteriophage
Clonal Complex 398
S new strain of MRSA discovered June 2011
S responsible for livestock-associated MRSA (LA-MRSA)
S found primarily in food-producing animals (e.g. pig, bovine,
and turkey)
S dangerous to humans; often asymptomatic in animals
Question
What are the origins and evolution of
MRSA CC398?
Methods: Sample Selection
S 48 MRSA & 40 MSSA CC398 isolates
S 25 human strains & 63 livestock strains (live animals, meat
samples, & environmental contamination)
S referenced against ST398 strain, SO385 (2,872,582 bps)
Methods Overview
S whole-genome sequence typing (WGST)
S + multilocus sequence typing (MLST)
S + spa typing
S Identifying SNPs & building phylogenetic tree using maximum
parsimony
S SCCmec typing
S Detection of antimicrobial resistance & host adaption genes
Methods Background:
Next Generation Sequencing
S Sequencing avg. of 2,651,848 bases: previously major endeavor
Methods: Building Tree from
Data
S w/ Burrows-Wheeler Aligner
software, aligned data sequences
against ST398 reference genome
S analyzed w/ SolSNP: excluding
SNP loci
w/ coverage <10X
S w/ variants present in <90% of
base calls for given position
S in duplicated regions on
reference genome
S
S Tree built w/ maximum
parsimony method in PAUP
Methods: “Enriching” the Tree
S MLST: sequence-based genotyping of 7 housekeeping genes
S allelic profiles (sequence type: ST) associated with known clonal
complexes (CCs) via computer program eBURST
S spa typing: sequence-based analysis of VNTRs in gene encoding
Protein A (spa)
S use primers targeting specific locus
S enter sequence results into BioNumerics software
S SCCmec typing: genomic island containing antibiotic resistance
gene mecA
S Detection of antimicrobial resistance genes
Results
S In building tree took major care: making adjustments post initial
analysis
S Rooted first using ST36 as out-group  revealed 4 isolates of t899
spa type 1st to diverge
S
Similarity between cluster & ST36 single 123,000bp region, which
comparative genetic analysis revealed was acquired horizontally
S SNP’s from region excluded: clade of human MSSA isolates from
France, French Guiana, & U.S. 1st to diverge
S 4,238 SNPs ID’d, 1,102 parsimony informative w/ CI=0.951
S Lack of homeoplasy demonstrated w/out need for addt’l
measured
Results: Most Ancestral Lineage
Results: spa types & SCCmec
S limited # of spa types: 15 ID’d among 89 isolates
S two most common represented 67%
S spa types inconsistent w/ overall CC398 phylogeny
S 30/49 CC398 MRSA isolates had cadmium zinc resistance
gene
Key Conclusions
S CC398 lineage originated in humans as MSSA  then
spread to livestock where acquired SCCmec cassette &
methicillin resistance
S Potential power of WGST for epidemiological
investigations: revealing common ancestors & avoiding
homeoplasy w/in spa gene
Implications
S Potential public health risk in using broad-spectrum
cephalosporins in food animal production selects  select
for MRSA
S Presence of zinc in feed  select for MRSA CC398 strains
with zinc r gene
S WGST as most reliable method
Further Directions
S Categorizing bi-directional nature: jump from humans to
animals truly assoc. w/  capacity for human colonization,
transmission, & virulence?
S Further examining genetic changes in shift
S Surveillance to predict public health impact
References
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http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/phage/phage-virulence.html
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http://en.wikipedia.org/wiki/Maximum_parsimony_(phylogenetics)
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http://www.mayoclinic.org/mrsa/index.html
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http://en.wikipedia.org/wiki/Methicillin-resistant_Staphylococcus_aureus#Strains
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http://www.webmd.com/skin-problems-and-treatments/understanding-mrsa-detection-treatment
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http://www.st398.com
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http://res.illumina.com/documents/products/illumina_sequencing_introduction.pdf
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http://www.premierbiosoft.com/tech_notes/multiplex-pcr.html
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http://en.wikipedia.org/wiki/Dna_sequencing#Next-generation_methods
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http://phylo.bio.ku.edu/slides/BIOL848-lec9-Distances.pdf
Papers Consulted
S Kondo Y, Ito T, Ma XX, Watanabe S, Kreiswirth BN, Etienne J, Hiramatsu
K. Combination of multiplex PCRs for staphylococcal cassette
chromosome mec type assignment: rapid identification system for mec, ccr,
and major differences in junkyard regions. Antimicrob Agents Chemother.
2007 Jan;51(1):264-74. Epub 2006 Oct 16.
S J. Ross Fitzgerald. Livestock-associated Staphylococcus aureus: origin,
evolution and public health threat. Trends in Microbiology. 2012
April;20(4):192–98.
S B. Strommenger, C. Kettlitz, Weniger,D. Harmsen, W. Friedrich, and W.
Witte1. Assignment of Staphylococcus Isolates to Groups by spa Typing,
SmaI Macrorestriction Analysis, and Multilocus Sequence Typing. J Clin
Microbiol. 2006 July; 44(7): 2533–2540.
S Chambers HF and DeLeo FR. Waves of resistance: Staphylococcus aureus
in the antibiotic era. Nature. 2009 Sep; 7:629-641.
Thank You!