Genomic Plasticity and the Supragenome of Streptococcus pneumoniae

Marcus Leung

11th March 2010m.leung@ucl.ac.uk

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