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20100311 M Sc Lecture Final
- 1. Genomic Plasticity and the Supragenome of Streptococcus pneumoniae Marcus Leung 11 th March 2010 [email_address]
- 2. Outline Intro Colonisation and Biofilm/Competence Bacterial Pleurality Supragenome: Hiller et al paper Research
- 3. Streptococcus pneumoniae Aerotolerant anaerobe Gram-positive, encapsulated In pairs, may also exist as singlets and chains α – haemolytic Optochin-sensitive Adapts rapidly (highly transformable) CDC, Janice Carr
- 4. Colonisation and Adaptation to Environment Sessile form of growth, biofilm-forming Contribute to enhanced horizontal gene transfer Transformation efficiency ↑ 100x Architecture of biofilm made up of DNA Pathway for biofilm formation coupled with transformation (Com signal transduction system) Mol Microbiol, 2006, 61 : 1196 Int Microbiol, 2009, 12 : 77 Red = high Green = low
- 5. Multiple Colonisation Simultaneous colonisation by more than one strain Greater gene pool, greater ability to adapt Distributed genome hypothesis: pan-genome of all pneumococcal strains colonising together
- 6. Bacterial Pleurality Phenotypic Diversity Serotype Antibiotic susceptibility Environmental factors (identical strains) Differential protein expression (different strains) Genetic Diversity
- 7. Bacterial Pleurality Genetic Heterogeneity –different alleles - aka genetic differences Genomic Plasticity – unique genes - aka genic differences Genetic Diversity
- 8. Supragenome Distributed genome hypothesis Full genome of species > genome of a single isolate
- 9. Supragenome Distributed genome hypothesis Full genome of species > genome of a single isolate Terminology: Gene cluster : group of genes where all genes share > 70% sequence similarity - 3 groups
- 10. Supragenome Gene clusters Distributed - in some but not all Conserved/Core - in all 17 strains Unique - in only 1 of 17
- 11. Supragenome Distributed genome hypothesis Full genome of species > genome of a single isolate total clusters: 3,170 S. pneumoniae : only 46% of gene clusters found in all strains Each isolate contain at least one cluster not found in any other genome J Bacteriol, 2007, 189 : 8186 Core: clusters found in all genomes Unique: only found in one genome
- 12. J Bacteriol, 2007, 189 : 8186 Dendogram based on gene content differences Strains found in patients in same location could be very diverse in gene content (CGSS).
- 13. Number of conserved clusters as new strain genome added to database Number of total clusters as new strain genome added to database Number of unique clusters as new strain genome added to database
- 14. Finite Supragenome Model Answers question: how many genomes are required to calculate the entire supragenome in question? Complex mathematical derivations Predict size of supragenome for a given number of genomes analysed Number of total clusters plateaus, and number of core clusters stabilises as number of genomes sequenced increases.
- 15. Finite Supragenome Model As number of genomes increases, 1) Number of total clusters increases and plateaus 2) Number of core clusters decreases and stabilises 3) Number of new clusters decreases and stabilises 1 2 3
- 16. Within close proximity, multiple strains may be present that are genetically and “genically” diverse. This increases the size of the supragenome present in a population. With the ability to undergo horizontal gene transfer, strains present may acquire new alleles and genes from other strains -> adaptation for survival as a population.
- 17. Aim To determine the diversity and genetic relatedness of strains within a single colonisation Objectives 1. Determine phenotypic diversity of pneumococci colonising together in Tanzanian children - Serotype Antibiotype (Penicillin/Co-trimoxazole minimum inhibitory concentrations) 2. Determine genotypic diversity in colonisations with 2 or more phenotypes
- 18. 21 children sampled over 12 months, 61 pneumococcal colonisations observed 12 (19.7%) colonisations with multiple phenotypes 8 colonisations 0 colonisation 4 colonisations Phenotypic diversity of pneumococci colonising together
- 19. Diversity Within a Colonisation by Serotype Four colonisations (6.5%) had multiple serotypes Up to 4 serotypes colonising together 1, 5, 6B, NT 6B, 10A, 19A/B/C, NT
- 20. Twelve colonisations (20%) with multiple antibiotypes Highest number of antibiotypes = 5 Diversity Within a Colonisation by Antibiotype
- 21. Diversity in a Colonisation by Serotype and Antibiotype 4 serotypes + 2 antibiotypes within 6B = 5 PHENOTYPES Serotype Antibiotype (MIC in μ g/mL) Pen Sxt 1 S 0.38 5 0.125 S 6B 0.125 4 3 0.5 NT S S
- 22. Summary 1 (Phenotype Diversity) Over 6% of colonisations contained multiple serotypes/groups Twenty percent of colonisations contained multiple antibiotypes When combining serotype and antibiotype diversity, number of phenotypes present could be increased
- 23. 2. Genotypic Diversity in Colonisations with Multiple Phenotypes Multilocus Sequencing Typing: seven loci distributed around genome Each strain given a multilocus sequence type (MLST) - MLST of a strain represents the strain’s genotype spi gki gdh aroE ddl xpt recP S. pneumoniae G54 2.08Mbp
- 24. Diversity within a Colonisation by Genotype 11 colonisations (92%) contained multiple MLSTs Up to 4 genotypes colonising together Number of MLST-defined genotypes Number of colonisations with multiple phenotypes (n = 12) 1 1 2 6 3 3 4 2
- 25. Diversity within a Colonisation by Genotype and Phenotype Increased number of colonisations with multiple strains, however did not increase maximum of different strains found Maximum number of strains found colonising simultaneously = 5
- 26. In this study of multiple colonisation, we made two unique discoveries!
- 27. 1) Multiple genotypes expressing the same serotype in the same colonisation ST4370 ST4371 ST4367 ST1145 ST4430 Two Unique Discoveries! Serotype 13 Serotype 21
- 28. Two Unique Discoveries! 2) The same genotype expressing multiple serotypes/ groups in the same colonisation MLST 4162 MLST 217 19A/B/C 10A 1 5 NT
- 29. Sequence Types with Multiple Antibiotypes Same MLSTs may also have multiple antibiotypes Confirmed previous results 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
- 30. Summary 2 (Genotype Diversity) Eleven colonisations (92%) had multiple genotypes Strains expressing same serotypes with different genotypes Same genotypes expressing different serotypes and antibiotypes
- 31. Review Gene pool of 5 different strains could be found in a single colonisation According to finite supragenome model, potentially increases the supragenome by 25% with genome = 2 x 10 6 nucleotides, an extra 5 x 10 5 is present for adaptation 5 2500 clusters 2000 clusters = 25% increase in supragenome size
- 32. 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
Editor's Notes
- #3: Homologous recombination
- #4: Homologous recombination
- #5: - EXPLAIN RED GREEN AND EXPRESSION PROFILES,
- #6: G reater the gene pool present in NP, greater ability to adapt, and this is facilitated by the presence of multiple strains colonising together in the nasopharynx. The presence of multiple strains colonising together contributes to the distributed genome hypothesis, which describe the pan-genome of all pneumococcal strains colonising together
- #7: G reater the gene pool present in NP, greater ability to adapt, and this is facilitated by the presence of multiple strains colonising together in the nasopharynx. The presence of multiple strains colonising together contributes to the distributed genome hypothesis, which describe the pan-genome of all pneumococcal strains colonising together
- #8: G reater the gene pool present in NP, greater ability to adapt, and this is facilitated by the presence of multiple strains colonising together in the nasopharynx. The presence of multiple strains colonising together contributes to the distributed genome hypothesis, which describe the pan-genome of all pneumococcal strains colonising together
- #9: Led to investigation in determining dissimilarities in pneumococcal strains, whole sequencing of prevalence disease strains (show from paper, the size of one genome, number of clusters) This observation was followed by A pneumococcous has on average Emphasize unique genes
- #10: Led to investigation in determining dissimilarities in pneumococcal strains, whole sequencing of prevalence disease strains (show from paper, the size of one genome, number of clusters) This observation was followed by A pneumococcous has on average Emphasize unique genes
- #11: Led to investigation in determining dissimilarities in pneumococcal strains, whole sequencing of prevalence disease strains (show from paper, the size of one genome, number of clusters) This observation was followed by A pneumococcous has on average Emphasize unique genes
- #12: Led to investigation in determining dissimilarities in pneumococcal strains, whole sequencing of prevalence disease strains (show from paper, the size of one genome, number of clusters) This observation was followed by A pneumococcous has on average Emphasize unique genes
- #18: Fixed semi closed population, pneumococcal population dynamics within this population Critical for colonisation and disease in pneumococci Evolution in semi-closed community, evolution within nasopharynx
- #19: Venn-diagram!!!!!
- #20: Remove “results” change to Diversity within a Colonisation by Serotype/Group
- #21: Remove “results” change to Diversity within a Colonisation by Antibiotype
- #22: Big bright yellow blocks between S and 4!!!
- #23: So in summary, 8% of our colonisations contained multiple serotypes/groups, and a higher number of them contained multiple antibiotypes. When we combined the two properties together, the extent of diversity is greater, which was to be expected.
- #24: Genotyped strains by MLST, involved sequencing of 7 housekeeping genes sequencing of 7 loci provides an ST number
- #25: percentage
- #26: percentage
- #29: Remove eBURST diagram, put table with same sequence types with different serotypes COLONMISING TOGETHER!
- #30: Remove eBURST, only show table with 4432, bigger font!
- #31: Conclusion: distributed genome hypothesis. Provide evidence that up to 6 strains may share a distributed gene pool which based on finite model, it increases the pan-genome by 35%, major finding!!!!!!