A novel genetic variant of Streptococcus pneumoniae serotype 11A discovered in Fiji

Abstract

Objectives: As part of annual cross-sectional Streptococcus pneumoniae carriage surveys in Fiji (2012-2015), we detected pneumococci in over 100 nasopharyngeal swabs that serotyped as '11F-like' by microarray. We examined the genetic basis of this divergence in the 11F-like capsular polysaccharide (cps) locus compared to the reference 11F cps sequence. The impact of this diversity on capsule phenotype, and serotype results using genetic and serologic methods were determined.

Methods: Genomic DNA from representative 11F-like S. pneumoniae isolates obtained from the nasopharynx of Fijian children was extracted and subject to whole genome sequencing. Genetic and phylogenetic analyses were used to identify genetic changes in the cps locus. Capsular phenotypes were evaluated using the Quellung reaction and latex agglutination.

Results: Compared to published 11F sequences, the wcwC and wcrL genes of the 11F-like cps locus are phylogenetically divergent, and the gct gene contains a single nucleotide insertion within a homopolymeric region. These changes within the DNA sequence of the 11F-like cps locus have modified the antigenic properties of the capsule, such that 11F-like isolates serotype as 11A by Quellung reaction and latex agglutination.

Conclusions: This study demonstrates the ability of molecular serotyping by microarray to identify genetic variants of S. pneumoniae and highlights the potential for discrepant results between phenotypic and genotypic serotyping methods. We propose that 11F-like isolates are not a new serotype but rather are a novel genetic variant of serotype 11A. These findings have implications for invasive pneumococcal disease surveillance as well as studies investigating vaccine impact.

Keywords: Atypical pneumococcal serotype; Genetic variant; Pneumococcal capsule; Pneumococcal serotyping; Pneumococcus; Streptococcus pneumoniae.

Fig. 1

Phylogenetic analysis of serogroup 11 capsular polysaccharide genes from 11F-like isolates (PMP1342 and PMP1343) with serogroup 11 strains MNZ272 (11A), 8087/40 (11B), Eddy no. 53 (11C), 70/86 (11D), MNZ264 (11E) and 34356 (11F), (GenBank accession nos. GU074952, CR931654, CR931655, CR931656, GU074953, CR931657 respectively). Genes specific to serogroup 11 include; wchA (A), wchJ (B), wchK (C), wcyK (D), wcwC (E), wcrL (F), wzy (G), wcwT (H), wcwU (I), wzx (J), gct (K) and wcjE (L). Trees for wcwC and wcjE do not include sequences from 11B and 11C as these serotypes lack these genes. Using MEGA 6 package , DNA sequences were aligned using MUSCLE; and maximum likelihood of phylogeny trees were generated based on Tamura-Nei model. Scale bars represent number of substitutions per site. Statistical support for branches was determined by bootstrapping (1000 replicates) and are displayed as percentages.

Fig. 2

Alignment of 11F-like WcrL amino acid sequence with 11A and 11F sequences generated by Clustal Omega. Identical residues are indicated with an asterisk; conserved residents, a colon; and semiconserved residues by a period respectively. Highlighted is residue 112 (A, alanine; N, asparagine).

Fig. 3

Alignment of 11F-like gct DNA sequences with 11A and 11F sequences generated using Clustal Omega. Identical residues are indicated with an asterisk. Box represents homopolymeric region with mutation site.

Fig. 4

Representative latex agglutination reactions of Statens Serum Institut (SSI) 11F and 11A reference strains, and an 11F-like isolate from this study. Latex reagents were prepared using SSI antisera (11b, 11c, 11f, 11g) as Quellung as previously described .