20091201 Transfer Seminar Final

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MPhil->PhD transfer seminar to Infection and Immunity Department, 1st December, 2009, Windeyer Building, UCL, London, UK

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20091201 Transfer Seminar Final

  1. 1. Tipping the supragenome iceberg: Phenotypic and genotypic diversity of Streptococcus pneumoniae Marcus Leung Divison of Infection and Immunity Centre for Medical Microbiology UCL Medical School, Royal Free Hampstead Campus 1 st December 2009
  2. 2. Streptococcus pneumoniae <ul><li>Aerotolerant anaerobe </li></ul><ul><li>Gram-positive, encapsulated </li></ul><ul><li>In pairs, may also exist as </li></ul><ul><li>singlets and chains </li></ul><ul><li>α – haemolytic </li></ul><ul><li>Optochin-sensitive </li></ul><ul><li>Adapts rapidly (highly transformable) </li></ul>CDC, Janice Carr
  3. 3. Epidemiology and Pathogenesis <ul><li>More than 1.6 million deaths (WHO, 2007) </li></ul><ul><ul><li>Infants < 5, elderly, immuno-compromised vulnerable </li></ul></ul><ul><ul><li>Deaths in infants/yr > Malaria + TB + HIV/AIDS combined </li></ul></ul><ul><li>As high as 18 million cases of pneumococcal infections reported per year (Lancet, 2009, 374: 854) </li></ul><ul><li>Secondary infections </li></ul><ul><ul><li>Influenza </li></ul></ul><ul><ul><li>Compromised immune system: pneumococcal infection rises by 100x </li></ul></ul>
  4. 4. Epidemiology and Pathogenesis <ul><li>Disease burden in children </li></ul><ul><ul><li>Pneumonia (740,000+ deaths) </li></ul></ul><ul><ul><li>Meningitis (60,000+ deaths) </li></ul></ul><ul><ul><li>Others (25,000+ deaths) </li></ul></ul><ul><ul><li>Sinusitis </li></ul></ul><ul><ul><li>Otitis media (20+ million clinician </li></ul></ul><ul><ul><li>visits in US alone, 60% caused </li></ul></ul><ul><ul><li>by pneumococcus) </li></ul></ul>Lancet, 2004, 4 :144
  5. 5. Colonisation and Adaptation to Environment <ul><li>Opportunistic pathogen </li></ul><ul><li>Normal microbiota of nasopharynx </li></ul><ul><li>Human: sole habitat </li></ul><ul><ul><li>- With related Streptococci </li></ul></ul><ul><li>Up to 70% in children, 15% adults, rises again in elderly </li></ul><ul><li>Pre-requisite for invasive diseases </li></ul><ul><li>Transmission / dissemination </li></ul>
  6. 6. Colonisation and Adaptation to Environment <ul><li>Sessile form of growth, biofilm-forming </li></ul><ul><ul><li>Contribute to enhanced horizontal gene transfer </li></ul></ul><ul><ul><ul><li>Transformation efficiency ↑ 100x </li></ul></ul></ul><ul><ul><ul><li>Architecture of biofilm made up of DNA </li></ul></ul></ul><ul><ul><ul><li>Pathway for biofilm formation coupled with transformation (Com signal transduction system) </li></ul></ul></ul>Mol Microbiol, 2006, 61 : 1196 Int Microbiol, 2009, 12 : 77 Red = high Green = low
  7. 7. Capsular Polysaccharide <ul><li>Major colonisation factor -> evades complement-mediated opsonophagocytosis </li></ul><ul><li>90+ types (serotypes) -> evades immune response </li></ul><ul><ul><li>Differences in capsulation operon </li></ul></ul><ul><ul><li>20-30 involved in most colonisation and invasion </li></ul></ul><ul><ul><li>Common types: 6B, 14, 19F ( colonisation + invasion ) </li></ul></ul><ul><ul><li>Rarely captured from carriage but invasive: 1, 5, 46 </li></ul></ul><ul><ul><li>7 serotypes covered in polyvalent conjugated vaccine (PCV7) -> 4, 6B, 9V, 14, 18C, 19F, 23F </li></ul></ul>
  8. 8. Colonisation and Adaptation to Environment <ul><li>Serotype switching </li></ul><ul><ul><li>Horizontal gene transfer of capsulation operon </li></ul></ul><ul><ul><li>Serotypes not covered in vaccine increase in prevalence (19A) </li></ul></ul><ul><ul><li>Age‐specific incidence of invasive pneumococcal disease caused by serotype 19A in United States, 1998–2005 </li></ul></ul>Introduction of PCV7 Modified from J Infect Dis, 2008, 197 : 1016 < 5 > 80
  9. 9. Colonisation and Adaptation to Environment <ul><li>Antibiotic-resistant genes acquisition </li></ul><ul><li>- Intra-species genetic exchange </li></ul><ul><ul><ul><li>Also inter-species ( S. oralis, S. mitis ) </li></ul></ul></ul><ul><ul><li>Antibiotic-resistant strains: exchanges of genes fragments in antibiotic-resistance determinants </li></ul></ul><ul><ul><li>Sensitive strains : conserved sequences </li></ul></ul><ul><ul><li>Resistance strains: 20% sequence variation </li></ul></ul><ul><ul><ul><li>Increased genetic rearrangements to escape selective pressure </li></ul></ul></ul>
  10. 10. Colonisation and Adaptation to Environment
  11. 11. Colonisation and Adaptation to Environment
  12. 12. Multiple Colonisation <ul><li>Greater gene pool, greater ability to adapt </li></ul><ul><li>Simultaneous colonisation by more than one strain </li></ul><ul><li>Distributed genome hypothesis: pan-genome of all pneumococcal strains colonising together </li></ul>
  13. 13. Supragenome <ul><li>Distributed genome hypothesis </li></ul><ul><ul><li>Full genome of species > genome of a single strain </li></ul></ul><ul><li>total clusters: 3,170 </li></ul><ul><li>S. pneumoniae : only </li></ul><ul><li>46% of gene clusters </li></ul><ul><li>found in all strains </li></ul><ul><li>Each strain contain at </li></ul><ul><li>least one cluster not </li></ul><ul><li>found in any other </li></ul><ul><li>genome </li></ul>J Bacteriol, 2007, 189 : 8186
  14. 14. Aim To determine the diversity and genetic relatedness of pneumococcal strains within a single colonisation Objectives <ul><li>Determine phenotypic diversity of pneumococci colonising together in Tanzanian children </li></ul><ul><ul><li>- Serotype/group </li></ul></ul><ul><ul><li>Antibiotype (Penicillin/Co-trimoxazole minimum inhibitory concentrations) </li></ul></ul><ul><li>2. Determine genotypic diversity in colonisations with 2 or more phenotypes </li></ul><ul><li>3. Investigate genetic relatedness of strains within a colonisation </li></ul><ul><li>4. Investigate genetic relatedness of strains circulating within children </li></ul>
  15. 15. Methodology for Isolating and Phenotyping + gentamycin 10 colonies <ul><li>Serotyping </li></ul><ul><ul><li>Pooled antisera </li></ul></ul><ul><ul><li>Differentiates 23 </li></ul></ul><ul><ul><li>prevalent serotypes/groups </li></ul></ul><ul><ul><li>Non-typeables (NT) put </li></ul></ul><ul><ul><li>into single serogroup </li></ul></ul><ul><li>Antibiotic Susceptibility </li></ul><ul><ul><li>Disc diffusion </li></ul></ul><ul><ul><li>E-test (MIC) </li></ul></ul>
  16. 16. 21 children sampled over 12 months, 61 pneumococcal colonisations observed 13 (21%) colonisations with multiple phenotypes 8 colonisations 1 colonisation 4 colonisations <ul><li>Phenotypic diversity of pneumococci colonising together </li></ul>
  17. 17. Diversity Within a Colonisation by Serotype/group <ul><li>Five colonisations (8%) </li></ul><ul><li>had multiple serotypes/groups </li></ul><ul><li>Up to 5 serotypes/groups </li></ul><ul><li>colonising together </li></ul><ul><ul><ul><li>- 1, 5, 6, 18, NT </li></ul></ul></ul>
  18. 18. <ul><li>Twelve colonisations </li></ul><ul><li>(20%) with multiple </li></ul><ul><li>antibiotypes </li></ul><ul><li>Highest number of </li></ul><ul><li>antibiotypes = 5 </li></ul>Diversity Within a Colonisation by Antibiotype
  19. 19. Diversity in a Colonisation by Serotype/group and Antibiotype Serotype/group Antibiotype (MIC in μ g/mL) Pen Sxt 1 S 0.38 5 0.125 S 6 0.125 4 3 0.5 18 S 0.5 NT S S
  20. 20. Summary 1 (Phenotype Diversity) <ul><li>Eight percent of colonisations contained multiple serotypes/groups </li></ul><ul><li>Twenty percent of colonisations contained multiple antibiotypes </li></ul><ul><li>When combining serotype/group and antibiotype diversity, six </li></ul><ul><li>phenotypes were observed </li></ul>
  21. 21. 2. Genotypic Diversity in Colonisations with Multiple Phenotypes <ul><li>Multilocus Sequencing Typing (MLST): seven loci distributed around genome </li></ul><ul><li>Each strain given a sequence type (ST) </li></ul><ul><ul><li>- ST of a strain represents the strain’s genotype </li></ul></ul>
  22. 22. Diversity within a Colonisation by Genotype <ul><li>11 colonisations (85%) </li></ul><ul><li>contained multiple STs </li></ul><ul><li>Up to 5 genotypes </li></ul><ul><li>colonising together </li></ul>Number of MLST-defined genotypes Number of colonisations with multiple phenotypes (n = 13) 1 2 2 5 3 3 4 2 5 1
  23. 23. Diversity within a Colonisation by Phenotype and Genotype <ul><li>An additional colonisation had 6 different strains colonising together </li></ul><ul><li>Suggestive of </li></ul><ul><li>capsular switching event </li></ul>4164 NT Serotype/group MLST-defined genotype (ST) 6 4433 6 4157 10 4163 10 4162 19 4162
  24. 24. In this study of multiple colonisation, we made two unique discoveries!
  25. 25. <ul><li>1) Multiple genotypes expressing the same serotype in the same colonisation </li></ul><ul><li>ST4370 </li></ul><ul><li>ST4371 </li></ul><ul><li>ST4367 </li></ul><ul><li>ST1145 </li></ul><ul><li>ST4430 </li></ul>Two Unique Discoveries! Serotype 13 Serotype 21
  26. 26. Two Unique Discoveries! 2) The same genotype expressing multiple serotypes/ groups in the same colonisation ST852 ST217 6 10 1 5 18 NT
  27. 27. Sequence Types with Multiple Antibiotypes <ul><li>Same STs may also have </li></ul><ul><li>multiple antibiotypes </li></ul><ul><li>Confirmed previous </li></ul><ul><li>results </li></ul>Sequence Type Child, Month Number of Antibiotypes MICs of Antibiotype ( μ g/mL) Pen Sxt 4432 11, April 5 0.19 8 0.19 32 1.5 1 2 32 6 32 4432 11, June 2 0.19 0.75 0.25 4
  28. 28. 3. Genetic Relatedness of Strains Within a Colonisation eBURST diagram - STs = dots - Single-Locus Variants (SLVs) - Identical sequences in 6/7 loci - Connected by lines - Serotypes/groups indicated Red circle = found in same colonisation 7/13 (54%) colonisations: presence of genetically related strains colonising together Suggesting gene arrangements occurring in colonisations (less likely to acquire SLVs from different sources) Suggestion strengthened by observation that SLVs have same serotypes/groups, except for one pair, which may be due to serogroup switching
  29. 29. Summary 2 (Genotype Diversity) <ul><li>Eleven colonisations (85%) had multiple genotypes </li></ul><ul><li>Strains expressing same serotypes/groups with different genotypes </li></ul><ul><li>Same genotypes expressing different serotypes/groups and antibiotypes </li></ul><ul><li>Genetically-related strains colonising together </li></ul>
  30. 30. 4. Genetic Relatedness of Strains Circulating Within Children <ul><li>Clusters of genetically related strains in children </li></ul><ul><ul><li>Clonal complexes </li></ul></ul><ul><ul><li>CC4429: survival advantage, expansion of </li></ul></ul><ul><ul><li>clones expressing a common colonising </li></ul></ul><ul><ul><li>serogroup (6) </li></ul></ul>
  31. 31. Conclusion <ul><li>Gene pool of 6 different strains could be found in a single </li></ul><ul><li>colonisation </li></ul><ul><li>According to finite supragenome model, potentially </li></ul><ul><li>increases the supragenome by 35% </li></ul>
  32. 32. Current/Future Projects <ul><li>Determine the size of supragenome </li></ul><ul><ul><li>Whole-genome sequencing of strains colonising together </li></ul></ul><ul><li>Whether successful clones is associated with good biofilm formation </li></ul><ul><li>- Compare biofilm formation of STs in CC4429 and other clusters </li></ul><ul><li>Common serotypes in carriage show greater gene variation </li></ul><ul><ul><li>Aim: Intra-Serotype Polymorphisms in Capsulation Gene cpsB </li></ul></ul><ul><li>- Determine extent of sequence variation within different serotypes </li></ul><ul><li>Allelic variants of the quorum sensing peptide (CSP) have an effect in biofilm formation </li></ul><ul><ul><li>- study CSP allelic variants in carriage and invasive strains and compare biofilm development </li></ul></ul>
  33. 33. Acknowledgements Royal Free Hospital, London Dr. Bambos Charalambous Prof Stephen Gillespie Kathrin Freystätter Ashley York Bisi Obamakin Dhriti Dosani Kilimanjaro Christian Medical Centre, Moshi, Tanzania Dr. Harry Mwerinde (Clinical Director, Tanganyika Plantation Hospital) Prof N. Sam Ndekya Oriyo
  34. 34. Finite Supragenome Model <ul><li>Supragenome = core genes, distributed genes, unique genes </li></ul><ul><li>Answers question: how many genomes are required to calculate the entire supragenome in question? </li></ul><ul><li>Developed by Hoggs et al. 2007 </li></ul><ul><li>Takes into account unequal probability in retrieving distributed genes -> accuracy </li></ul><ul><li>Based on complex mathematical derivations </li></ul><ul><li>Predict size of supragenome for a given number of genomes analysed </li></ul>
  35. 35. Classes of Streptococci α - haemolytic S. pneumoniae, S. mutans, S. mitis, S. sanguinis β – haemolytic Group A: S. pyogenes Group B: S. agalactiae Group C: S. zooepidemicus Group D (enterococci): S. faecalis
  36. 36. Strains with 6 Different Genotypes and Phenotypes Child 35, January Serotypes/groups (STs): 6 (4433/4157) 10 (4163/4162) 19 (4162) NT (4164) Child 3, September Five serotypes/groups, two antibiotypes in serogroup 6

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