Genomic Plasticity and the Supragenome of Streptococcus pneumoniae
Marcus Leung
11th March [email protected]
Outline
- Intro
- Colonisation and Biofilm/Competence
- Bacterial Pleurality
- Supragenome: Hiller et al paper
- Research
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
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 = highGreen = low
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
Bacterial Pleurality
Phenotypic Diversity- Serotype- Antibiotic susceptibility
1) Environmental factors (identical strains)2) Differential protein expression (different strains)
Genetic Diversity
Bacterial Pleurality
1) Genetic Heterogeneity –different alleles- aka genetic differences
2) Genomic Plasticity – unique genes- aka genic differences
Genetic Diversity
Supragenome
• Distributed genome hypothesis– Full genome of species > genome of a single isolate
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
SupragenomeGene clusters
Distributed - in some but not allDistributed - in some but not all
Conserved/Core - in all 17 strainsConserved/Core - in all 17 strains
Unique - in only 1 of 17Unique - in only 1 of 17
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 genomesUnique: only found in one genome
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).
• 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
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.
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
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.
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
21 children sampled over 12 months, 61 pneumococcal
colonisations observed
12 (19.7%) colonisations with multiple phenotypes
8 colonisations
0 colonisation
4 colonisations
1. Phenotypic diversity of pneumococci colonising together
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
• Twelve colonisations (20%) with multiple antibiotypes
• Highest number of antibiotypes = 5
Diversity Within a Colonisation by Antibiotype
Serotype
Antibiotype (MIC in μg/mL)
Pen Sxt
1 S 0.38
5 0.125 S
6B0.125 4
3 0.5
NT S S
Diversity in a Colonisation by Serotype and Antibiotype
4 serotypes + 2 antibiotypes within 6B = 5 PHENOTYPES
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
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
gdharoE
ddl
xpt
recP
S. pneumoniae G542.08Mbp
Diversity within a Colonisation by Genotype
Number of MLST-defined
genotypes
Number of colonisations with multiple phenotypes
(n = 12)
1 1
2 6
3 3
4 2
• 11 colonisations (92%) contained multiple MLSTs
• Up to 4 genotypes colonising together
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
In this study of multiple colonisation, we made two unique discoveries!
1) Multiple genotypes expressing the same serotype in the same colonisation
ST4370ST4371
ST4367ST1145ST4430
Two Unique Discoveries!
Serotype 13
Serotype 21
2) The same genotype expressing multiple serotypes/ groups in the same colonisation
MLST 4162
MLST 217
Two Unique Discoveries!
19A/B/C
10A
1
5
NT
• Same MLSTs may also have multiple antibiotypes
• Confirmed previous results
Sequence Types with Multiple Antibiotypes
Sequence Type
Child, Month
Number of Antibiotypes
MICs of Antibiotype (μg/mL)
Pen Sxt
4432 11, April 5 0.19 8
0.19 32
1.5 1
2 32
6 32
443211,
June2 0.19 0.75
0.25 4
Summary 2 (Genotype Diversity)
• Eleven colonisations (92%) had multiple genotypes
• Strains expressing same serotypes with different genotypes
• Same genotypes expressing different serotypes and antibiotypes
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 106
nucleotides, an extra 5 x 105 is present for adaptation
5
2500 clusters
2000 clusters
= 25% increasein supragenome size
Acknowledgements
Royal Free Hospital, London
Dr. Bambos CharalambousProf Stephen GillespieKathrin FreystätterAshley YorkBisi ObamakinDhriti Dosani
Kilimanjaro Christian Medical Centre, Moshi, Tanzania
Dr. Harry Mwerinde (Clinical Director, Tanganyika Plantation Hospital)Prof N. SamNdekya Oriyo
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