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We characterized Streptococcus pneumoniae serotype 6D from among previously identified S. pneumoniae serotype 6B strains from Jewish and Bedouin children in southern Israel during a decade before vaccination. S. pneumoniae serotype 6D isolates constituted 6.7% of the presumed S. pneumoniae serotype 6B isolates. S. pneumoniae serotype 6D strains belonged to 20 sequence types that were differentially distributed between the two ethnic groups.
The discovery of a new Streptococcus pneumoniae serotype, serotype 6C, led to the logical suggestion that recombination might also produce a new member of the serogroup 6 family which would have the wciP serotype 6B (wciP6B) allele that differentiates S. pneumoniae serotype 6B from S. pneumoniae serotype 6A and the wciN6C allele (1, 2). This serotype, designated S. pneumoniae serotype 6D, has been detected in both nasal carriage and invasive isolates (2–11).
The 7- and 13-valent pneumococcal conjugate vaccines (PCV7 and PCV13, respectively) were introduced into the Israeli national immunization program in 2009 and 2010, respectively. Preliminary in vitro data suggest that the S. pneumoniae serotype 6B component of these vaccines elicits antibodies that are cross-opsonic to S. pneumoniae serotype 6D (12). However, clinical data are not yet available to confirm these findings. Evaluation of the prevalence of S. pneumoniae serotype 6D before and after PCV implementation would indirectly determine whether these vaccines provide protection against S. pneumoniae serotype 6D. The aim of this study was therefore to identify S. pneumoniae serotype 6D strains from among previously characterized S. pneumoniae serotype 6B isolates, explore their molecular epidemiology, and determine the circulation of S. pneumoniae serotype 6D clones among the members of two distinct ethnic subpopulations.
This retrospective study was conducted in southern Israel during the decade (1999 to 2008) preceding PCV7 implementation. The study population included children <5 years of age: Jewish children with lifestyles resembling those of populations in developed countries and Muslim Bedouin children with standards of living resembling those of populations in developing countries (13). Contact between children of the two populations is rare. S. pneumoniae was cultured from four isolation sites: blood/cerebrospinal fluid (CSF), middle ear fluid (MEF), conjunctiva, and nasopharynx (14). Identification, antimicrobial susceptibility testing, and serotyping were performed as previously described (13). A PCR system targeting both wciN6C (wciNβ) and wciP6B (wciP584a) was used to segregate S. pneumoniae serotype 6D from S. pneumoniae serotype 6B (2).
Of 1,628 presumed S. pneumoniae serotype 6B isolates, 1,593 (97.9%) pneumococci, isolated from 1,201 children, were available for S. pneumoniae serotype 6D typing by PCR. S. pneumoniae serotype 6D was identified in 98/1,201 (8.2%) individuals, of which 41 individuals also presented S. pneumoniae serotype 6B during the study period. Overall, S. pneumoniae serotype 6D constituted 6.7% (107/1593) of the presumed S. pneumoniae serotype 6B isolates: 1/34 (2.9%) from blood/CSF cultures, 24/337 (7.1%) from MEF cultures, 0/21 (0%) from conjunctival swab cultures, and 82/1,094 (7.5%) from nasopharyngeal swab cultures. S. pneumoniae serotype 6D constituted 7.9% (48/610) of the presumed S. pneumoniae serotype 6B isolates from Jewish children and 6.0% (59/983) from Bedouin children (P = 0.148).
Most isolates, 90.7% (97/107), were nonsusceptible to at least one antibiotic, and 73.8% (79/107) were nonsusceptible to ≥3 antimicrobial agents (Table 1). Pulsed-field gel electrophoresis (PFGE) (15) revealed the presence of 6 major clusters (with each cluster having ≥6 isolates) and 7 minor clusters, which constituted 72.9% (78/107) and 20.6% (22/107) of the S. pneumoniae serotype 6D strains, respectively. Seven isolates exhibited unique PFGE patterns. Representative strains from each PFGE cluster and all the strains with unique PFGE patterns (total, 44/107; 41.1%) were further characterized by multilocus sequence typing (MLST) (16) (www.mlst.net queried on 5 November 2012), which yielded 12 known and 8 novel sequence types (STs) (Table 1).
Comparison of the clonal distribution between Jewish and Bedouin children was done using the χ2 method; a P value of <0.05 was considered significant. The distribution of 5/6 major STs was significantly different between the two ethnic groups (P < 0.05) (Fig. 1). ST553 and ST6984 were isolated only from Bedouin children, and ST90, ST315, ST8597, and ST8599 were highly frequent among Jewish rather than Bedouin children. Of note is the finding that all S. pneumoniae serotype 6D STs found in this study which were already published in the MLST database were associated with serogroup 6. In addition, the 8 novel STs were single- or double-locus variants (SLVs and DLVs, respectively) of serogroup 6 as well. eBURST analysis (http://spneumoniae.mlst.net/eburst), based on the minimal identity of 6/7 loci, revealed that 14/20 S. pneumoniae serotype 6D clones were grouped into 4 clonal clusters (CCs; numbered by the ST of the predicted founder) (Table 1). Interestingly, the distribution of 3/4 CCs was significantly different between the two ethnic groups (P < 0.001): CC4220 was more frequently isolated from Bedouin children, while CC156 and CC315 were more frequently isolated from Jewish children. The fourth CC, CC2779, was also more common among Bedouin children but its differential distribution did not reach significance due to the small number of strains. ST8594, ST8595, and ST8598 are SLVs of each other and thus form a CC. This predicted CC, isolated only from Bedouin children, is not yet displayed in the MLST website.
In this study, we used a specific PCR method (2) to identify S. pneumoniae serotype 6D from presumed S. pneumoniae serotype 6B isolates. Identification by the classical Quellung reaction did not give conclusive results, since PCR-positive S. pneumoniae serotype 6D isolates reacted poorly or not at all with the 6D factor serum. Overall, of all isolates that were formerly typed as S. pneumoniae serotype 6B by the Quellung reaction, 6.7% were identified as S. pneumoniae serotype 6D by PCR.
Our study describes a large collection of S. pneumoniae serotype 6D strains that have been causing mucosal and invasive diseases for more than a decade. Almost three-quarters of the S. pneumoniae serotype 6D strains were multidrug resistant. Characterization by PFGE and MLST revealed the presence of 20 genotypes, 8 of which were first described in this study. One cannot rule out the possibility that more STs were present than detected, given that not all isolates were evaluated with MLST. eBURST analysis assigned 14 STs in our collection to 4 CCs. The founders of three CCs (ST315, ST2779, ST4220) belong to S. pneumoniae serotype 6A and S. pneumoniae serotype 6B, while the founder of the fourth CC (ST156) is the international serotype 9V/14 clone, suggesting that S. pneumoniae serotype 6D arose mainly from S. pneumoniae serotypes 6A and 6B but also had other evolutionary pathways (17). Five STs (ST8594, ST8595, ST8596, ST8598, ST8601) were newly identified in this study and have not yet been assigned to a CC.
A striking clonal segregation was found between the two study populations; e.g., ST6984, the major S. pneumoniae serotype 6D clone, which constituted 31% of the strains, was found only among Bedouin children, whereas ST8599, which constituted 16% of the strains, was found mostly (16/17, 94%) among Jewish children. The differential clonal distribution between the two study populations was already shown by us for additional serotypes (13, 18–20), suggesting that lifestyle and microenvironment are major determinants in child-to-child transmission of pneumococci.
This study provides broad baseline information regarding the molecular epidemiology of S. pneumoniae serotype 6D in southern Israel, prior to PCV implementation. Further surveillance is needed to assess the effect of vaccination on this highly resistant, vaccine-related serotype.
This work was partially supported by a grant from Wyeth (0887X1-4463).
Published ahead of print 27 February 2013