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

1st December 2009

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

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

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

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

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

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

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 fromJ Infect Dis, 2008, 197: 1016

< 5

> 80

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

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

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

Aim

To determine the diversity and genetic relatedness of pneumococcal strains within a single colonisation

Objectives

1. 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

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)

21 children sampled over 12 months, 61 pneumococcal

colonisations observed

13 (21%) colonisations with multiple phenotypes

8 colonisations

1 colonisation

4 colonisations

1. Phenotypic diversity of pneumococci colonising together

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

• Twelve colonisations (20%) with multiple antibiotypes

• Highest number of antibiotypes = 5

Diversity Within a Colonisation by Antibiotype

Serotype/group

Antibiotype (MIC in μg/mL)

Pen Sxt

1 S 0.38

5 0.125 S

60.125 4

3 0.5

18 S 0.5

NT S S

Diversity in a Colonisation by Serotype/group and Antibiotype

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

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

Diversity within a Colonisation by Genotype

Number of MLST-defined

genotypes

Number of colonisations with multiple phenotypes

(n = 13)

1 2

2 5

3 3

4 2

5 1

• 11 colonisations (85%) contained multiple STs

• Up to 5 genotypes colonising together

Diversity within a Colonisation by Phenotype and Genotype

Serotype/group MLST-defined genotype (ST)

6 4433

6 4157

10 4163

10 4162

19 4162

NT 4164

• An additional colonisation had 6 different strains colonising together

• Suggestive of capsular switching event

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

ST852

ST217

Two Unique Discoveries!

6

10

1

5

18

NT

• Same STs 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

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)

3. Genetic Relatedness of Strains Within a Colonisation

Suggestion strengthened by observation that SLVs have same serotypes/groups, except for one pair, which may be due to serogroup switching

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

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)

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%

Current/Future ProjectsDetermine 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 variationAim: 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

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