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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

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

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  • Homologous recombination
  • Pneumococcous is truly one of the most deadly bacterial pathogens today. They are responsible for an annual of over 1.6 million deaths each year. Most of these fatalities are in infants, the elderly and anyone with compromised immune responses. Children under the age of 5 make up a majority of the deaths, and it’s the leading bacterial cause of death in this age group, causing over 10% of death in general in these children. The bacterium is held accountable for more than 18 million recorded diseases annually. And given the recent hype of swine flu, the fact that secondary pneumococcus infection as a cause of death in patients with flu make pneumococcal research very relevant.
  • Given all the diseases listed it is interesting to note that the pneumococcus is also a common coloniser of the nasopharynx, along with other bacteria including related Streptococci species. Prevalence is especially high in children, where 95% of children would have encountered a pneumococcus by the age of 2. Probably due to maturity of the immune system, prevalence drops in adults, only to rise again later on, mainly due to increase contacts with grandchildren. The colonisation stage is a pre-requisite for most invasive diseases, and it acts as a reservoir for transmission between hosts. An obvious indication of that is the increased prevalence in pneumococcal colonisation in crowded areas such as DCCs, prisons, and mines. Therefore, understanding physiology of colonisation may be a big step towards understanding the biology behind pneumococcal diseases.
  • Vaccine introduced, not made in 2000 Decrease in prevalence of carriage and invasion of vaccine serotypes, but since 2000 acted as a selective pressure, resulting in the increase in prevalence of serotypes not included in the vaccine 3 doses: 3 months, 6 months, 18 months! Protects from disease, and elimination of carriage groups
  • Stress selective pressure behind sequence variation! Survival!!
  • This recent paper compared nucleotide sequences from a few species of the Streptococcus genus on pubmed, and have divided strains into clusters based on nucleotide sequence similarities. What they have found is that there is a cluster within Streptococcus pneumoniae (cluster 4) which in each strain has gene segments indicative of origins from other clusters. They came up with a figure depicting that a sizable portion of the genes found in this cluster comes from other clusters, including clusters representing the non-pneumo clusters, possibly acquired from horizontal gene transfer. What they have further showed is that strains in cluster 4 have a higher odds ratio of being antibiotic-resistant. Clearly there is a corrolation between high frequency of horizontal gene transfer and antibiotic resistance.
  • It’s interesting to note that a capsular switch and antibiotic resistance can be gained from a single re-combinational event. Brueggemann’s group in Oxford identified a 39kb recombinational event that have swapped a serotype 19A capsule into a strain with a genotype only to be found in serotype 4. recombination also involved two flanking penicillin-binding protein genes, resulting in penicillin non-susceptibility.
  • 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
  • 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
  • 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
  • Resuspending in 1ml media -> 10uL aliquots onto blood agar Made selective for strep pneumo -> gram negative Draftsman-like colonies Many serotypes have shared epitopes and put into same serogroup
  • Venn-diagram!!!!!
  • Remove “results” change to Diversity within a Colonisation by Serotype/Group
  • Remove “results” change to Diversity within a Colonisation by Antibiotype
  • Big bright yellow blocks between S and 4!!!
  • 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.
  • Genotyped strains by MLST, involved sequencing of 7 housekeeping genes sequencing of 7 loci provides an ST number
  • percentage
  • percentage
  • Remove eBURST diagram, put table with same sequence types with different serotypes COLONMISING TOGETHER!
  • Remove eBURST, only show table with 4432, bigger font!
  • Add eburst diagram, explain presence of related STs colonising together!
  • 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!!!!!!
  • Greater clusters of related genotypes found in the cohort of children.
  • Conclusion! Gene pool of 6 strains could be found in a single colonisation This according to finite supragenome model, increases the supragenome by 35%
  • Transcript

    • 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. 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
    • 3. Epidemiology and Pathogenesis
      • More than 1.6 million deaths (WHO, 2007)
        • Infants < 5, elderly, immuno-compromised vulnerable
        • Deaths in infants/yr > Malaria + TB + HIV/AIDS combined
      • As high as 18 million cases of pneumococcal infections reported per year (Lancet, 2009, 374: 854)
      • Secondary infections
        • Influenza
        • Compromised immune system: pneumococcal infection rises by 100x
    • 4. Epidemiology and Pathogenesis
      • Disease burden in children
        • Pneumonia (740,000+ deaths)
        • Meningitis (60,000+ deaths)
        • Others (25,000+ deaths)
        • Sinusitis
        • Otitis media (20+ million clinician
        • visits in US alone, 60% caused
        • by pneumococcus)
      Lancet, 2004, 4 :144
    • 5. Colonisation and Adaptation to Environment
      • Opportunistic pathogen
      • Normal microbiota of nasopharynx
      • Human: sole habitat
        • - With related Streptococci
      • Up to 70% in children, 15% adults, rises again in elderly
      • Pre-requisite for invasive diseases
      • Transmission / dissemination
    • 6. 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
    • 7. Capsular Polysaccharide
      • Major colonisation factor -> evades complement-mediated opsonophagocytosis
      • 90+ types (serotypes) -> evades immune response
        • Differences in capsulation operon
        • 20-30 involved in most colonisation and invasion
        • Common types: 6B, 14, 19F ( colonisation + invasion )
        • Rarely captured from carriage but invasive: 1, 5, 46
        • 7 serotypes covered in polyvalent conjugated vaccine (PCV7) -> 4, 6B, 9V, 14, 18C, 19F, 23F
    • 8. Colonisation and Adaptation to Environment
      • Serotype switching
        • Horizontal gene transfer of capsulation operon
        • Serotypes not covered in vaccine increase in prevalence (19A)
        • Age‐specific incidence of invasive pneumococcal disease caused by serotype 19A in United States, 1998–2005
      Introduction of PCV7 Modified from J Infect Dis, 2008, 197 : 1016 < 5 > 80
    • 9. Colonisation and Adaptation to Environment
      • Antibiotic-resistant genes acquisition
      • - Intra-species genetic exchange
          • Also inter-species ( S. oralis, S. mitis )
        • Antibiotic-resistant strains: exchanges of genes fragments in antibiotic-resistance determinants
        • Sensitive strains : conserved sequences
        • Resistance strains: 20% sequence variation
          • Increased genetic rearrangements to escape selective pressure
    • 10. Colonisation and Adaptation to Environment
    • 11. Colonisation and Adaptation to Environment
    • 12. Multiple Colonisation
      • Greater gene pool, greater ability to adapt
      • Simultaneous colonisation by more than one strain
      • Distributed genome hypothesis: pan-genome of all pneumococcal strains colonising together
    • 13. Supragenome
      • Distributed genome hypothesis
        • Full genome of species > genome of a single strain
      • total clusters: 3,170
      • S. pneumoniae : only
      • 46% of gene clusters
      • found in all strains
      • Each strain contain at
      • least one cluster not
      • found in any other
      • genome
      J Bacteriol, 2007, 189 : 8186
    • 14. Aim To determine the diversity and genetic relatedness of pneumococcal strains within a single colonisation Objectives
      • Determine phenotypic diversity of pneumococci colonising together in Tanzanian children
        • - Serotype/group
        • Antibiotype (Penicillin/Co-trimoxazole minimum inhibitory concentrations)
      • 2. Determine genotypic diversity in colonisations with 2 or more phenotypes
      • 3. Investigate genetic relatedness of strains within a colonisation
      • 4. Investigate genetic relatedness of strains circulating within children
    • 15. Methodology for Isolating and Phenotyping + gentamycin 10 colonies
      • Serotyping
        • Pooled antisera
        • Differentiates 23
        • prevalent serotypes/groups
        • Non-typeables (NT) put
        • into single serogroup
      • Antibiotic Susceptibility
        • Disc diffusion
        • E-test (MIC)
    • 16. 21 children sampled over 12 months, 61 pneumococcal colonisations observed 13 (21%) colonisations with multiple phenotypes 8 colonisations 1 colonisation 4 colonisations
      • Phenotypic diversity of pneumococci colonising together
    • 17. Diversity Within a Colonisation by Serotype/group
      • Five colonisations (8%)
      • had multiple serotypes/groups
      • Up to 5 serotypes/groups
      • colonising together
          • - 1, 5, 6, 18, NT
    • 18.
      • Twelve colonisations
      • (20%) with multiple
      • antibiotypes
      • Highest number of
      • antibiotypes = 5
      Diversity Within a Colonisation by Antibiotype
    • 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. Summary 1 (Phenotype Diversity)
      • Eight percent of colonisations contained multiple serotypes/groups
      • Twenty percent of colonisations contained multiple antibiotypes
      • When combining serotype/group and antibiotype diversity, six
      • phenotypes were observed
    • 21. 2. Genotypic Diversity in Colonisations with Multiple Phenotypes
      • Multilocus Sequencing Typing (MLST): seven loci distributed around genome
      • Each strain given a sequence type (ST)
        • - ST of a strain represents the strain’s genotype
    • 22. Diversity within a Colonisation by Genotype
      • 11 colonisations (85%)
      • contained multiple STs
      • Up to 5 genotypes
      • colonising together
      Number of MLST-defined genotypes Number of colonisations with multiple phenotypes (n = 13) 1 2 2 5 3 3 4 2 5 1
    • 23. Diversity within a Colonisation by Phenotype and Genotype
      • An additional colonisation had 6 different strains colonising together
      • Suggestive of
      • capsular switching event
      4164 NT Serotype/group MLST-defined genotype (ST) 6 4433 6 4157 10 4163 10 4162 19 4162
    • 24. In this study of multiple colonisation, we made two unique discoveries!
    • 25.
      • 1) Multiple genotypes expressing the same serotype in the same colonisation
      • ST4370
      • ST4371
      • ST4367
      • ST1145
      • ST4430
      Two Unique Discoveries! Serotype 13 Serotype 21
    • 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. Sequence Types with Multiple Antibiotypes
      • Same STs 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
    • 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. Summary 2 (Genotype Diversity)
      • Eleven colonisations (85%) had multiple genotypes
      • Strains expressing same serotypes/groups with different genotypes
      • Same genotypes expressing different serotypes/groups and antibiotypes
      • Genetically-related strains colonising together
    • 30. 4. Genetic Relatedness of Strains Circulating Within Children
      • Clusters of genetically related strains in children
        • Clonal complexes
        • CC4429: survival advantage, expansion of
        • clones expressing a common colonising
        • serogroup (6)
    • 31. Conclusion
      • Gene pool of 6 different strains could be found in a single
      • colonisation
      • According to finite supragenome model, potentially
      • increases the supragenome by 35%
    • 32. Current/Future Projects
      • Determine the size of supragenome
        • Whole-genome sequencing of strains colonising together
      • Whether successful clones is associated with good biofilm formation
      • - Compare biofilm formation of STs in CC4429 and other clusters
      • Common serotypes in carriage show greater gene variation
        • Aim: Intra-Serotype Polymorphisms in Capsulation Gene cpsB
      • - Determine extent of sequence variation within different serotypes
      • Allelic variants of the quorum sensing peptide (CSP) have an effect in biofilm formation
        • - study CSP allelic variants in carriage and invasive strains and compare biofilm development
    • 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. Finite Supragenome Model
      • Supragenome = core genes, distributed genes, unique genes
      • Answers question: how many genomes are required to calculate the entire supragenome in question?
      • Developed by Hoggs et al. 2007
      • Takes into account unequal probability in retrieving distributed genes -> accuracy
      • Based on complex mathematical derivations
      • Predict size of supragenome for a given number of genomes analysed
    • 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. 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