Carbon Source-Dependent Changes of the Structure of Streptococcus pneumoniae Capsular Polysaccharide with Serotype 6F.

The structure of the exopolysaccharide capsule of Streptococcus pneumoniae is defined by the genetic arrangement of the capsule operon allowing the unequivocal identification of the pneumococcal serotype. Here, we investigated the environment-dependent composition of the polysaccharide structure of S. pneumoniae serotype 6F. When grown in a chemically defined medium (CDM) with glucose versus galactose, the exopolysaccharide capsule of the serotype 6F strains reveals a ratio of 1/0.6 or 1/0.3 for galactose/glucose in the capsule by 1H-NMR analyses, respectively. Increased production of the capsule precursor UDP-glucose has been identified by 31P-NMR in CDM with glucose. Flow cytometric experiments using monoclonal antibodies showed decreased labelling of Hyp6AG4 (specific for serotype 6A) antibodies when 6F is grown in glucose as compared to galactose, which mirrors the 1H-NMR results. Whole-genome sequencing analyses of serotype 6F isolates suggested that the isolates evolved during two different events from serotype 6A during the time when the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced. In conclusion, this study shows differences in the capsular structure of serotype 6F strains using glucose as compared to galactose as the carbon source. Therefore, 6F strains may show slightly different polysaccharide composition while colonizing the human nasopharynx (galactose rich) as compared to invasive locations such as the blood (glucose rich).

Phenotypic and genotypic characterization of serogroup 6 Streptococcus pneumoniae isolates collected during 10-valent pneumococcal conjugate vaccine era in Bulgaria.

Serogroup 6 remains common in the pneumococcal-conjugated vaccine era in Bulgaria; therefore, we investigated its clonal and serotype dynamics. The antibiotic susceptibilities were assessed by broth microdilution. Strains identified as serogroup 6 with latex agglutination method were subjected to serotype-specific PCRs. Erythromycin-resistant strains were analyzed by PCR for presence of ermB and mefE genes. MLST was performed to define clonal composition of the sequence types (STs). Serogroup 6 was represented by 40 (13.3%) from 301 invasive and non-invasive Streptococcus pneumoniae isolates. Molecular serotyping revealed new emerging serotype 6C (6.6%), not detected in pre-vaccine era. Among unvaccinated patients, mostly we observed serotypes 6А (57.1%) and 6В (28.6%). Serotype 6C was distinctive for vaccinated children (64%), followed by 6A (24%). Penicillin and ceftriaxone non-susceptible serogroup 6 strains were 65% and 5%, respectively; erythromycin- and clindamycin-resistant were 70.0% and 52.5%, respectively. Multidrug-resistant strains were 57.5%. Prevalent genetic determinant for macrolide resistance was ermB gene (75%). MLST revealed 17 STs into 5 clonal complexes and 7 singletons. Predominant genetic lineage was CC386, represented by MDR-6C non-invasive strains. Serotype 6B, principally responsible for invasive diseases in the pre-vaccine era, retreated this position to serotype 6A.

Capsular polysaccharide gene diversity of pneumococcal serotypes 6A, 6B, 6C, and 6D.

This study was performed to better understand the genetic diversity and evolutionary relatedness of pneumococcal serotypes 6A, 6B, 6C, and 6D. Multi-locus sequence typing (MLST) was performed for 160 serogroup 6 isolates from clinical specimens collected from children between 1991 and 2010. We identified 38 sequence types (STs) comprising five clonal complexes with 12 singletons. Although most STs were confined to a single serotype, some STs were shared by two serotypes, and one ST was shared by three serotypes. Many STs of serotype 6A showed genetic relatedness with those of serotype 6C or 6D in eBURST analysis. Five capsular polysaccharide (cps) genes - wchA, wciO, wciP, wzy, and wzx - were analysed in 74 isolates from our clinical samples and in 36 isolates from GenBank. There were several profiles and clades in each serotype on the analysis of the concatenated sequences of the five cps genes. Small genetic distances between serotypes 6A and 6B and between serotypes 6C and 6D were observed while serotype 6B with an indel sequence formed a distinct clade. When comparing the individual cps genes between the serotypes, there was also a high level of similarity in the wchA and wciO gene sequences between serotype 6C and serotype 6D. On the other hand, serotypes 6A and 6D had the most highly similar wzy and wzx gene sequences. The wzy sequences of serotype 6C were nearly identical (99.6%) to those of serotype 6A clade II strains. In conclusion, we revealed the diversity of the genetic background and cps sequences in each pneumococcal serotype of serogroup 6. Pneumococcal serotype diversity might be attributable to complex serial mutation and recombination events.