This document summarizes the results of whole genome sequencing performed on 19 Streptococcus pneumoniae serotype 6C strains isolated from carriage and invasive disease in the UK. Key findings include: - Two distinct genetic clusters of 6C strains were identified based on gene content differences. - MLST sequence type 1692 strains showed less genetic diversity than strains of the second cluster. - Novel antibiotic resistance genes were identified in some strains. - Single nucleotide polymorphisms and gene content variability were analyzed to explore diversity within and between strains.

1. Diversity of the emerging
pneumococcal serotype 6C
in the UK
SGM Autumn Conference - University of Nottingham
6-9 September 2010
Rebecca Gladstone
06 September 2010

2. Overview
• Introduction –Why is pneumococcal serotype 6C
diversity important?
• Collection of samples
• Strain selection - How were the strains chosen for
further analysis?
• Whole genome sequencing
• Summary and future work

3. Introduction
Streptococcus pneumoniae is common coloniser of the
nasopharynx but also a major cause of morbidity and mortality
in the UK
S. pneumoniae serotype 6C is a recently recognised serotype that
arose from 6A
Prevalence of 6C has significantly increased in our carriage study
Driven by expansion of Multi-locus sequence type 1692 (MLST-
genotyping tool based on the sequence of seven house keeping genes)
Capacity to cause invasive disease
Not included in any conjugate vaccine
Carvalho et al., 2009, Cooper et al., 2010, Park et al., 2007a, Park et al., 2007b, Tocheva et al., 2010

4. Sample collection and strain selection
• Nasopharyngeal specimens obtained from healthy children
<4years during the winter months of 2006-9
301 S. pneumoniae strains isolated
32 serotype 6C
17 sequence type 1692
15 strains selected with representatives from each of the three
winters covering each of the 9 observed MLST
• IPD isolates obtained from HPA SE regional microbiology
laboratory, Southampton in 2004-2010
– 315 S. pneumoniae IPD strains
– 6 serotype 6C
– 4 most recent strains selected for analysis
– 3 ST1692
Carvalho et al., 2009, Tocheva et al., 2010

5. Whole genome sequencing (WGS)
• Objectives of WGS study:
– the diversity of the serotype 6C
– the diversity of ST1692
– clinical relevance of any diversity
• Preliminary analysis:
– Confirmation of MLST
– Gene content
– SNPs
Hiller et al., 2007, Silva et al., 2006

6. Whole genome sequencing (WGS)
•454 Genome Sequencer FLX System
•Whole genome shotgun methodology
• Analysis was perfomed via xBASE-NG (http://ng.xbase.ac.uk) and
(http://xbase.ac.uk/annotation), utilising the following software
– De novo assembly (Newbler 2.5, Roche)
– Annotation (xBASE annotation pipeline)
– Mapping (Newbler gsMapper, Roche)
• Sequencing averages- 113,178 reads, 39,434,823 bases, and 18.55
coverage

7. A plot of the
gene-
distance
matrix
The number
of genes not
shared
between any
given pair
Image produced by Dr Nick Loman

8. SNPs –Isolates vs 2073(ST1692)
Strain Sequence Type Total SNP CDS Non-Synonymous
2074 1692 81 4 3
3022 1692 233 51 29
2105 1692 217 49 30
802M 1692 202 77 45
954Q 1692 251 70 45
1058S 1692 237 77 46
0081 1692 189 80 48
3055 1692 304 102 70
2371 395 367 123 81
3074 1714 353 123 82
0237 1714 334 122 83
0113 65 16772 2042 636
2029 3460 18835 10360 2977
3060 1150 17824 11935 3466
3050 1600 16534 11551 3494
3088 398 17735 12275 3721
1060N 1150 19298 13403 3874
2300 1862 24959 17209 5054
Key: IPD strains, Carriage strains

9. Novel genes
• Novel genes were detected by orthologue comparison of
study strains and removal of any genes found in other
completed S. pneumoniae genomes in GenBank.
– ermB and a gene encoding a tetracycline-resistance
protein were unique to 2029 (ST3460). Erythromycin
and tetracycline resistance were confirmed as functional.
– Two novel putative beta-lactamase genes in 2300
(ST1862) homologous to coding sequences in two plant
pathogens (Ralstonia solanacearum and Dickeya
dadantii).
– 6C group 1 strains contain a region encoding for a
lantibiotic biosynthesis protein and lantibiotic efflux
protein
Ding et al., 2009

10. Summary
• Two distinct 6C genetic clusters could be observed
by gene-distance analysis
• The cluster that included ST1692 was more
homogenous than the second cluster
• MLST is a good indicator of genotype
• MLST can be determined from WGS however there
is diversity within the STs of 6C

11. Future work
• Analysis of insertions and deletions including previously
reported regions of diversity
• Comparison with other streptococcal genomes
• Analysis of gene content/diversity for known virulence
factors
• Significance of identified SNPs and novel genes
• Further comparison of disease and carriage isolates
Hava & Camilli, 2002; Hiller et al., 2007; Obert et al., 2006; Silva et al., 2006.

12. Acknowledgements
University of Southampton
Dr Stuart Clarke
Dr Saul Faust
Dr Jo Jefferies
Anna Tocheva Centre for Systems Biology
University of Birmingham
Professor Mark Pallen
Dr Nick Loman - Bioinformatics
Dr Chrystala Constantinidou - Sequencing
Mala Patel - Sequencing

13. Thank you for your
attention
SGM Autumn Conference - University of Nottingham, 6-9 September 2010
Rebecca Gladstone
(R.A.Gladstone@soton.ac.uk)
06 September 2010

14. References
•xBASE-NG website http://ng.xbase.ac.uk
•Carvalho, M., Pimenta, F. C., Gertz, R. E., Jr., Joshi, H. H., Trujillo, A. A., Keys, L. E., Findley, J., Moura, I. S., Park, I. H. &
other authors (2009). PCR-Based Quantitation and Clonal Diversity of the Current Prevalent Invasive Serogroup 6
Pneumococcal Serotype, 6C, in the United States in 1999 and 2006 to 2007. J Clin Microbiol 47, 554-559.
•Cooper, D., Yu, X., Sidhu, M., Nahm, M. H., Fernsten, P. & Jansen, K. U. (2010). Development of an opsonophagocytic assay
to Streptococcus pneumoniae serotype 6C: Demonstration of cross-functional responses to 6C in Prevenar-13 immune sera.
In ESPID. Nice.
•Ding, F., Tang, P., Hsu, M.-H., Cui, P., Hu, S., Yu, J. & Chiu, C.-H. (2009). Genome evolution driven by host adaptations
results in a more virulent and antimicrobial-resistant Streptococcus pneumoniae serotype 14. BMC Genomics 10, 158.
•Hava, D. L. & Camilli, A. (2002). Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol
Microbiol 45, 1389-1406.
•Hiller, N. L., Janto, B., Hogg, J. S., Boissy, R., Yu, S., Powell, E., Keefe, R., Ehrlich, N. E., Shen, K. & other authors (2007).
Comparative genomic analyses of seventeen Streptococcus pneumoniae strains: insights into the pneumococcal
supragenome. J Bacteriol 189, 8186-8195.
•Obert, C., Sublett, J., Kaushal, D., Hinojosa, E., Barton, T., Tuomanen, E. I. & Orihuela, C. J. (2006). Identification of a
Candidate Streptococcus pneumoniae core genome and regions of diversity correlated with invasive pneumococcal disease.
Infect Immun 74, 4766-4777.
•Park, I. H., Park, S., Hollingshead, S. K. & Nahm, M. H. (2007a). Genetic basis for the new pneumococcal serotype, 6C.
Infect Immun 75, 4482-4489.
•Park, I. H., Pritchard, D. G., Cartee, R., Brandao, A., Brandileone, M. C. C. & Nahm, M. H. (2007b). Discovery of a New
Capsular Serotype (6C) within Serogroup 6 of Streptococcus pneumoniae. J Clin Microbiol 45, 1225-1233.
•Park, I. H., Moore, M. R., Treanor, J. J., Pelton, S. I., Pilishvili, T., Beall, B., Shelly, M. A., Mahon, B. E. & Nahm, M. H. (2008).
Differential effects of pneumococcal vaccines against serotypes 6A and 6C. J Infect Dis 198, 1818-1822.
•Silva, N. A., McCluskey, J., Jefferies, J. M. C., Hinds, J., Smith, A., Clarke, S. C., Mitchell, T. J. & Paterson, G. K. (2006).
Genomic Diversity between Strains of the Same Serotype and Multilocus Sequence Type among Pneumococcal Clinical
Isolates. Infect Immun 74, 3513-3518.
•Tocheva, A. S., Jefferies, J. M., Christodoulides, M., Faust, S. N. & Clarke, S. C. (2010). Increase in serotype 6C
pneumococcal carriage, United Kingdom. Emerg Infect Dis 16, 154-155.

15. Sequencing results
Strain Number of reads Number of bases Coverage
0081 91237 28733390 13.58
0113 110623 38489470 17.02
0237 116400 38374563 17.87
2029 107703 35591041 15.76
2073 122684 41618252 19.66
2074 226938 74015324 34.98
2105 96988 32480840 15.37
2300 78683 23267452 10.55
2371 117005 35530396 16.64
3022 127553 39499836 18.68
3050 133283 40967776 18.87
3055 94317 31276976 14.90
3060 74967 25060487 12.20
3074 136821 45703125 21.42
3088 107105 36219264 17.44
0802M 117813 41369459 20.37
0954Q 167622 58368746 27.67
1058S 122634 43260411 20.91
1060N 124573 44306824 21.79
Average 113178 39434823 18.55
Key Lowest Highest

16. Structural difference between serotype 6A
and 6C CH2OH
O
CH2OH
O
HO H OH H H OH
OH H OH H
H H HO H
H OH H OH
galactose glucose
• There is one structural difference between serotype 6A and serotype 6C
• There are two structural differences between serotype 6B and serotype 6C
6A [P 2) – galactose – (1 3) – glucose – (1 3) – rhamnose – (1 3) – ribitol – (5 P ]
6B [P 2) – galactose – (1 3) – glucose – (1 3) – rhamnose – (1 4) – ribitol – (5 P ]
6C [P 2) – glucose – (1 3) – glucose – (1 3) – rhamnose – (1 3) – ribitol – (5 P ]

17. MLST tree
ST1150
ST398
ST395
ST1692
ST1714
ST398
ST65
ST1862
ST3460

18. MLST: WGS vs Qiagen
Gene and allele number
Isolate aroe gdh gki recP spi xpt ddl ST WGS ST Qiagen Agreement
SOT0081 1 5 7 12 17 158 14 ST1692 ST1692 Y
SOT0113 2 7 4 10 10 1 27 ST65 ST65 Y
SOT0237 1 5 7 12 17 148 14 ST1714 ST1714 Y
SOT2029 2 34 1 5 42 28 75 ST3460 ST3460 Y
SOT2073 1 5 7 12 17 158 14 ST1692 ST1692 Y
SOT2074 1 5 7 12 17 158 14 ST1692 ST1692 Y
SOT2105 1 5 7 12 17 158 14 ST1692 ST1692 Y
SOT2300 79 1 40 13 6 1 6 ST1862 ST1862 Y
SOT2371 1 5 7 12 17 1 14 ST395 ST395 Y
SOT3022 1 5 7 12 17 158 14 ST1692 ST1692 Y
SOT3050 1 5 9 12 94 1 20 ST1600 ST1600 Y
SOT3055 1 5 7 12 17 158 14 ST1692 ST1692 Y
SOT3060 7 25 8 6 25 6 8 ST1150 ST1150 Y
SOT3074 1 5 7 12 17 148 14 ST1714 ST1714 Y
SOT3088 37 25 4 4 15 20 28 ST398 ST398 Y
SOT0802M 1 5 7 12 17 158 14 ST1692 ST1692 Y
SOT0954Q 1 5 7 12 17 158 14 ST1692 ST1692 Y
SOT1058S 1 5 7 12 17 158 14 ST1692 ST1692 Y
SOT1060N 7 25 8 6 25 6 8 ST1150 N/A N/A

19. Wheel comparison of 9 S.
pneumoniae strains with R6 genome

20. Clinical data for IPD strains
,' 67 <HDU 6H[ $JH 6SHFLPHQ 'LDJQRVLV 2XWFRPH
7SH
0 )HPDOH %ORRG 23' 5HFRYHUHG
H[DFHUEDWLRQ
4 0DOH %ORRG 3QHXPRQLD 'HFHDVHG
6 )HPDOH 6) 0HQLQJLWLV 'HFHDVHG
1 )HPDOH %ORRG 6HSVLV 'HFHDVHG