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
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
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24. In this study of multiple colonisation, we made two unique discoveries!
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26. Two Unique Discoveries! 2) The same genotype expressing multiple serotypes/ groups in the same colonisation ST852 ST217 6 10 1 5 18 NT
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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
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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
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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
Editor's Notes
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.
- EXPLAIN RED GREEN AND EXPRESSION PROFILES,
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
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%