Evolution of MRSA During Hospital Transmission and Intercontinental Spread   (Harris  et al , 2010) Mariam Reyad Rizkallah...
<ul><li>Current methods  for differentiating isolates  (MLST)   </li></ul><ul><li>Do not define  variation  within a   ver...
<ul><li>A  high-throughput genomics approach </li></ul><ul><li>Epidemiology  &  microevolution  of a dominant strain of (M...
Prospects <ul><li>Applicable to a range of  infectious diseases  &  microbial ecology . </li></ul>
Why  characterization  of pathogenic microorganisms? Characterization Local epidemiology: (hospital outbreaks)   Same or d...
What are the  current methods  used for the characterization of pathogenic microorganisms? <ul><li>Traditional and molecul...
Methods   The clone <ul><li>HA-MRSA sequence type 239  (ST239) </li></ul><ul><li>90% of HA-MRSA throughout China, Thailand...
<ul><li>Global isolates & isolates from a hospital in northern Thailand. </li></ul><ul><li>Mapping against TW20. </li></ul...
Methods Genome-wide analysis, Genomic fingerprinting  & DNA comparison 63 isolates  2 samples 1 st  Sample 43 isolates Glo...
Methods Genome-wide analysis, Genomic fingerprinting  & DNA comparison <ul><li>Rapid diagnostic tools (e.g. bioterrorism a...
Genome Analyzers <ul><li>Brands:  </li></ul><ul><li>Illumina </li></ul><ul><li>Solexa </li></ul><ul><li>Genomatix </li></u...
Genome Analyzer Applications http :// www . geneworks . com . au / library / MultipleApp_image . jpg
Genome Analyzer Applications <ul><li>Applications: </li></ul><ul><li>DNA Sequencing,  </li></ul><ul><li>Gene Regulation An...
Results 6714 SNPs With marked  uneven distribution   across the genome Core  ( 4310) Chromosomal Vertical gene transfer No...
Results <ul><li>Understanding of the phylogenetic relation between clones:  How similar/different ?? </li></ul><ul><li>Det...
Results   –  Phylogenetic analysis & geographical relation
<ul><li>Asia </li></ul><ul><li>North America </li></ul><ul><li>South America </li></ul><ul><li>Europe </li></ul><ul><li>Au...
Source:  Pathogen Microevolution in High Resolution. (Aziz  et al , 2010,   Science Translational Medicine) A map showing ...
<ul><li>Asia </li></ul><ul><li>North America </li></ul><ul><li>South America </li></ul><ul><li>Europe </li></ul><ul><li>Au...
<ul><li>Asia </li></ul><ul><li>North America </li></ul><ul><li>South America </li></ul><ul><li>Europe </li></ul><ul><li>Au...
What did they find? Results <ul><li>Understood the phylogenetic relation between clones:  How similar/different ?? </li></...
Results  -  Mutation rate   <ul><li>Estimated mutation rate per site per year:  </li></ul><ul><li>3.3 × 10 −6  [95% from 2...
Results  –   SNPs analysis   from  homoplasies in the core regions   SNP position Region/gene Isolate SNP (amino acid repl...
Results  –   SNPs analysis   from  homoplasies in the core regions
Results  -  Fine - scale transmission <ul><li>5  out of 20 isolates differ in only  14 SNPs :  </li></ul><ul><li>4 isolate...
spa , PFGE vs. phylogenetic analysis
 
Prospects/Future applications <ul><li>Whole-genome & phylogenetic analysis may help in: </li></ul><ul><li>Gaining insight ...
Comments & Questions
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MRR_Harris_JC_20100217

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This IS my first power-point presentation, presenting at the Journal Club Harris et al's paper from Science entitled: "Evolution of MRSA During Hospital Transmission and Intercontinental Spread" Many thanks to Dr. Ramy A. for the choice and the fulltext and Dr. Tamer I. for the opportunity, encouragement and great help.

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MRR_Harris_JC_20100217

  1. 1. Evolution of MRSA During Hospital Transmission and Intercontinental Spread (Harris et al , 2010) Mariam Reyad Rizkallah 5 th year student, Faculty of Pharmacy, Cairo University
  2. 2. <ul><li>Current methods for differentiating isolates (MLST) </li></ul><ul><li>Do not define variation within a very small sample of the genome ( 2 Mb) . </li></ul><ul><li>Cannot distinguish between closely-related isolates, as it measures variation in housekeeping genes not virulence-related or antibiotic-resistant ones . </li></ul>What is the problem? (Can’t depend on a snapshot) Genes that are transcribed at a relatively constant level, code for products for maintaining the basic cell processes or structure.
  3. 3. <ul><li>A high-throughput genomics approach </li></ul><ul><li>Epidemiology & microevolution of a dominant strain of (MRSA) </li></ul><ul><li>1) Global geographic structure. </li></ul><ul><li>2) Intercontinental transmission through four decades. </li></ul><ul><li>3) Potential trace of person-to-person transmission within a hospital environment. </li></ul>What is the solution provided by the research?
  4. 4. Prospects <ul><li>Applicable to a range of infectious diseases & microbial ecology . </li></ul>
  5. 5. Why characterization of pathogenic microorganisms? Characterization Local epidemiology: (hospital outbreaks) Same or different strain? Global epidemiology: How geographically-related?
  6. 6. What are the current methods used for the characterization of pathogenic microorganisms? <ul><li>Traditional and molecular typing </li></ul><ul><li>Phage typing </li></ul><ul><li>Multilocus sequence typing ( MLST ) </li></ul><ul><li>Full-genome sequencing </li></ul><ul><li>Illumina Genome Analyzer </li></ul>
  7. 7. Methods The clone <ul><li>HA-MRSA sequence type 239 (ST239) </li></ul><ul><li>90% of HA-MRSA throughout China, Thailand, Turkey, and much of Asia. </li></ul><ul><li>Detected in South America </li></ul><ul><li>Currently circulating in Eastern Europe </li></ul><ul><li>Variations in: </li></ul><ul><li>large type III SCCmec element, </li></ul><ul><li>spa data, </li></ul><ul><li>pulsed-field gel electrophoresis (PFGE), </li></ul><ul><li>Little discrimination using MLST. </li></ul>
  8. 8. <ul><li>Global isolates & isolates from a hospital in northern Thailand. </li></ul><ul><li>Mapping against TW20. </li></ul>Methods Genome-wide analysis, Genomic fingerprinting & DNA comparison
  9. 9. Methods Genome-wide analysis, Genomic fingerprinting & DNA comparison 63 isolates 2 samples 1 st Sample 43 isolates Global collection (1982-2003) Intercontinental transmission 2 nd Sample 20 isolates Northeast Thailand (7-month period) Fine-scale transmission
  10. 10. Methods Genome-wide analysis, Genomic fingerprinting & DNA comparison <ul><li>Rapid diagnostic tools (e.g. bioterrorism and SARS) </li></ul><ul><li>Microbial forensics </li></ul><ul><li>Reverse vaccinology </li></ul><ul><li>Understanding the relatedness of microbial </li></ul><ul><li>strains </li></ul><ul><li>Study the emergence and geographical spread of virulent or drug-resistant clones </li></ul>
  11. 11. Genome Analyzers <ul><li>Brands: </li></ul><ul><li>Illumina </li></ul><ul><li>Solexa </li></ul><ul><li>Genomatix </li></ul>Genome Analyzer IIe http://www.illumina.com/systems/genome_analyzer.ilmn
  12. 12. Genome Analyzer Applications http :// www . geneworks . com . au / library / MultipleApp_image . jpg
  13. 13. Genome Analyzer Applications <ul><li>Applications: </li></ul><ul><li>DNA Sequencing, </li></ul><ul><li>Gene Regulation Analysis, </li></ul><ul><li>Sequencing-Based Transcriptome Analysis, </li></ul><ul><li>SNP Discovery and Structural Variation Analysis , </li></ul><ul><li>Cytogenetic Analysis, </li></ul><ul><li>DNA-Protein Interaction Analysis (ChIP-Seq), </li></ul><ul><li>Sequencing-Based Methylation Analysis, </li></ul><ul><li>Small RNA Discovery and Analysis </li></ul>
  14. 14. Results 6714 SNPs With marked uneven distribution across the genome Core ( 4310) Chromosomal Vertical gene transfer Non-core MGEs Horizontal gene transfer (Major source) 38 homoplasic genes involved in drug resistance “ similar in form & structure, but not in origin”
  15. 15. Results <ul><li>Understanding of the phylogenetic relation between clones: How similar/different ?? </li></ul><ul><li>Determine the source of SNPs: core or non-core ?? </li></ul><ul><li>Explain of the geographical relation between clones: Who did it ?? </li></ul><ul><li>Outbreaks studied: </li></ul><ul><li>Explaining the Portugal outbreak in the 1990s </li></ul><ul><li>Explaining the London outbreak in 2001-2003 </li></ul><ul><li>Estimation of the mutation rate and linked it back to first emergence of MRSA: Playback the mutation tape ! </li></ul>
  16. 16. Results – Phylogenetic analysis & geographical relation
  17. 17. <ul><li>Asia </li></ul><ul><li>North America </li></ul><ul><li>South America </li></ul><ul><li>Europe </li></ul><ul><li>Australasia </li></ul>Recent expansion of a single variant Single, but more diverse clade more diverse, most positioned basally , consistent Europe is the possible origin for ST239 with geographical clustering
  18. 18. Source: Pathogen Microevolution in High Resolution. (Aziz et al , 2010, Science Translational Medicine) A map showing the geographical spread of MRSA ST239 as deduced by Harris et al .
  19. 19. <ul><li>Asia </li></ul><ul><li>North America </li></ul><ul><li>South America </li></ul><ul><li>Europe </li></ul><ul><li>Australasia </li></ul>Portuguese clone,1990-1993 together Explaining the relationship between ST239 clones in Portugal in the 1990s: Portuguese clone,1990s Brazilian clone, 1997 Brazilian clone, 1997 clustered with South America isolates Second wave in Portugal resulted from the introduction of a South American variant
  20. 20. <ul><li>Asia </li></ul><ul><li>North America </li></ul><ul><li>South America </li></ul><ul><li>Europe </li></ul><ul><li>Australasia </li></ul>phiSPb-like (TW20) prophage characteristic of the Asian clade. Single intercontinental transmission event from southeast Asia. Explaining the origin of London outbreak 2001-2003: DEN907, isolate from Denmark, and TW20, from London among an Asian clade ??
  21. 21. What did they find? Results <ul><li>Understood the phylogenetic relation between clones: How similar/different ?? </li></ul><ul><li>Determined the source of SNPs: core or non-core ?? </li></ul><ul><li>Explained the geographical relation between clones: Who did it ?? </li></ul><ul><li>Outbreaks studied: </li></ul><ul><li>Explaining the Portugal outbreak in the 1990s </li></ul><ul><li>Explaining the London outbreak in 2001-2003 </li></ul><ul><li>Estimated the mutation rate and linked it back to first emergence of MRSA: Playback the mutation tape ! </li></ul>
  22. 22. Results - Mutation rate <ul><li>Estimated mutation rate per site per year: </li></ul><ul><li>3.3 × 10 −6 [95% from 2.5 × 10 -6 to 4.0 × 10 -6 ] </li></ul><ul><li>The most recent common ancestor </li></ul><ul><li>mid-late 1960s (emergence of MRSA in Europe). </li></ul><ul><li>Suggested explanation: </li></ul><ul><li>effective population size, accumulation of mutations </li></ul><ul><li>Some core SNPs transferred by recombination.. rare </li></ul><ul><li>Rate of mutation in the population before selection, purifying selection acts on all mutations, including intergenic, synonymous & nonsynonymous sites. </li></ul>
  23. 23. Results – SNPs analysis from homoplasies in the core regions SNP position Region/gene Isolate SNP (amino acid replaced, syn, intergenic) Antibiotic resistance Nucleotide replaced
  24. 24. Results – SNPs analysis from homoplasies in the core regions
  25. 25. Results - Fine - scale transmission <ul><li>5 out of 20 isolates differ in only 14 SNPs : </li></ul><ul><li>4 isolates within 16-day period & 1 after 11 weeks </li></ul><ul><li>Estimated mutation rate </li></ul><ul><li>one core SNP every 6 weeks </li></ul>
  26. 26. spa , PFGE vs. phylogenetic analysis
  27. 28. Prospects/Future applications <ul><li>Whole-genome & phylogenetic analysis may help in: </li></ul><ul><li>Gaining insight into evolution/virulence of a clone . </li></ul><ul><li>Development of global surveillance strategies for prevention of intercontinental transmission (e.g. bioterrorism and SARS ) </li></ul><ul><li>Epidemiological analysis ( geographical origin & intra-hospital spread) due to absolute discrimination of isolates & detection of minor differences between clones. </li></ul><ul><li>Estimation of mutation rate, which will assist contact tracing in endemics & outbreaks. </li></ul><ul><li>Reduction of the cost of measures taken to control MRSA, by targeting of diagnostics & interventions according to need. </li></ul><ul><li>Design of tailored-typing schemes for specific clones and locales based on non-core regions. </li></ul>
  28. 29. Comments & Questions
  29. 30. Thank you

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