Streptococcus pneumoniae is a major cause of sepsis, meningitis and respiratory
disease worldwide. Pneumococcal conjugate vaccines (PCVs) have now been implemented in
many countries worldwide, including Singapore. To evaluate the effectiveness of these
vaccines, pneumococcal surveillance studies are required. Detailed and unified
pneumococcal epidemiology data are currently scarce in South East Asia. Thus, we present
data on invasive pneumococcal (IPD) isolates from Singapore that could assist in
evaluating the effectiveness of pneumococcal vaccine in Singapore. One hundred and
fifty-nine invasive pneumococcal disease isolates were received by the National Public
Health Laboratory in Singapore between June 2009 and August 2010. Isolates were
characterized using serotyping and multilocus sequence typing. Twenty-four different
serotypes were found, the most common of which were 19A, 3, 7F, 23F, 6B, 14, 8 and 19F (in
rank order). One hundred and two sequence types were observed, of which 38 were novel due
to new alleles or new combinations of already existing alleles. Based on the
Simpson's Index of Diversity, serotypes 3, 6B and 19A were the most genetically
diverse. Novel sequence types were more prevalent among conjugate vaccine serotypes 3, 19F
and 23F and non-conjugate vaccine serotype 8, serogroup 15 and in non-typable isolates. We
have demonstrated considerable genetic diversity among invasive pneumococci before and
during the widespread use of conjugate vaccines in Singapore. Approximately half of all
novel IPD clones identified in this study were non-conjugate vaccine serotypes. Although
PCVs would target the most common serotypes, the high genetic diversity in non-vaccine
serotypes would require further surveillance studies.
Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and noninvasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole-genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole-genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51 to 264 bp, one insertion resulting in a coding frameshift, and seven nonsense single-nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae and therefore may have important implications for colonization, carriage, and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development.
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