Pherotypes of Co-Colonizing Pneumococci Among Portuguese Children

Introduction

S treptococcus pneumoniae (the pneumococcus) remains a major cause of morbidity and mortality worldwide. Nasopharyngeal colonization, which is asymptomatic, is particularly frequent among young children. ¹⁷

Pneumococci are naturally competent for transformation, and horizontal gene transfer (HGT) by homologous recombination is considered the main mechanism of evolution of this species. ²⁵ The nasopharynx, where co-colonization may occur, is the privileged niche for such events. ²⁶ Evidence for in vivo intra- and interspecies HGT involving colonizing strains has been described.^2,13

In pneumococci, competence is triggered by the competence-stimulating peptide (CSP), a pheromone that is prone to allelic variation.^10,28 Two dominant pherotypes, CSP1 and CSP2, have been identified.^19,20,28 Each pherotype is only recognized by its specific membrane-associated histidine kinase ComD receptor that, upon binding of CSP, initiates competence.^7,11

In recent years, it has been shown that pneumococci can compete in cocultivation through a mechanism called fratricide, in which competent cells can kill their noncompetent sisters. ⁹ During competence, an immunity membrane protein, ComM, which protects competent cells from their own hydrolases, is produced. ¹² Bacteria not responding to the external CSP will not express ComM and will be killed by their competent siblings. ¹⁴ It has been proposed that fratricide could provide access to nutrients, stabilize the relationship with the host through an immune response, or drive genetic evolution through increased gene exchange. ¹²

The role of pherotypes in competition and genetic differentiation remains a matter of debate. Specifically, whether or not it restricts recombination in the species is not totally clear. Some authors have proposed that the CSP/ComD specificity should imply a limited interpherotype recombination, resulting in two genetic populations associated with the two dominant pherotypes.^3,4 Others argued that the CSP polymorphisms are determinant in driving pneumococcal genetic differentiation by acting as a starting point for genetic diversity. ⁶

A recent study by Vestrheim et al. has looked at pairs of cocolonizing strains to evaluate the impact of pherotype on the coexistence of pneumococci in the nasopharynx. ²⁷ The authors showed that pneumococcal pherotypes often coexist in the nasopharynx and suggested that the impact of competence-induced fratricide on competition is limited.

Considering this seminal and very recent observation, we aimed to determine the pherotype of pneumococcal strains from Portuguese cocolonized samples.

Methods

Results

Of the 368 pneumococcal isolates, all but one isolate were of either CSP1 or CSP2 corroborating previous studies on the dominance of these pherotypes on the population.^3,6,20 The single exception was obtained in a serotype 38 strain that probably expresses a different pherotype. For this reason, one cocolonized sample was excluded from further analyses. Of the 366 isolates remaining under study, 32 serotypes were represented. Nineteen capsular types contained representatives of both pherotypes (Fig. 1). Serotypes 5, 7F, 9V, 11A, 14, 15B/C, 16F, 17F, 21, and 35F contained only representatives of CSP1; serotypes 9L/9N and 24F contained only representatives of CSP2 (Fig. 1). Of all isolates, 63.7% were of pherotype CSP1 (n=233) and 36.3% were of pherotype CSP2 (n=133) reflecting the pherotype distribution and the predominance of CSP1 described in the literature for clinical isolates.^3,6,20

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FIG. 1.

Pherotype and serotype distributions. Serotypes with an absolute frequency of 1 (i.e., serotypes 8, 9A, 31, 33A, 36, and 38) were grouped as others. CSP, competence-stimulating peptide; NT, nontypeable.

Regarding antibiotic resistance, significant associations between pherotype CSP2 and resistance to at least one class of antibiotics, multidrug resistance, and individually with each of the antibiotics tested (with the exception of chloramphenicol) were observed (Table 1). As a substantial proportion (41%) of the CSP2 strains were nontypeable (NT) pneumococci, which are frequently drug-resistant, ²³ the analysis was repeated excluding all NT pneumococci. Again, the strength of the association between CSP2 and resistance remained significantly higher compared to the strength of CSP1: multidrug resistance was 27.9% among CSP1 versus 42.3% among CSP2, p=0.02, χ² test; resistance to at least one antibiotic was 15.3% among CSP1 versus 38.5% among CSP2, p<0.0001, χ² test. The exclusion of NT pneumococci also did not abolish the significant association of resistance to each antibiotic and pherotype CSP2.

Among the cocolonized samples, 39.9% contained two strains of CSP1, 12.6% contained two strains of CSP2, and 47.5% had one strain of each pherotype. When the observed and estimated proportions of concordant and discordant pherotypes in the cocolonized samples were compared no significant differences were observed (p=0.9, χ² test) (Table 2), suggesting that co-colonization of pherotypes CSP1 and CSP2 occurs independently.

As the serotype distribution in our samples was characterized by a predominance of NT pneumococci (18.0%) and strains of serotype 3 (13.7%) (Fig. 1), which could potentially bias the results, we repeated the analysis after exclusion of these strains. The results indicated that, again, the observed and estimated proportions of concordant (55.6% and 56.6%, respectively), and discordant (44.4% and 43.4%, respectively), pherotypes were not significantly different (p=0.8, χ² test). This confirmed the results obtained with all samples regarding the independent coexistence of pneumococcal pherotypes in the nasopharynx.

Discussion

The results obtained in our study replicate the observations by Vestrheim et al. regarding the independent coexistence of different pherotypes in co-colonization.

In addition, we report a significant association of pherotype CSP2 with antimicrobial resistance. As no information on the genetic backgrounds of the strains was obtained, the possibility that these findings might result from high clonality between the resistant isolates could be raised. In fact, Carrolo et al. observed that pherotype is a clonal property and is not randomly dispersed within the pneumococcal population. ³ Nevertheless, given the high genetic diversity of the pneumococcal population, the high assortment of serotypes in our collection, and the fact that the samples were collected in a wide temporal range (2001–2010), clonal diversity within each pherotype is expected.

Of interest, the general association between CSP2 and antibiotic resistance among Portuguese isolates from carriage contrasts with the observation that among Portuguese isolates from invasive disease, an association was described between penicillin resistance and CSP1. ³ However, this latter collection was obtained in a shorter time period (1999–2002) and before widespread use of conjugate vaccines occurred in the country^1,21,24 that have resulted in major changes among circulating pneumococci. In addition, an asymmetric distribution of circulating lineages among carriage and disease is also expected ²² and may also have accounted for these contrasting observations.

Our study (as the one from Vestrheim et al. ²⁷ ) has some limitations. First, samples originated from cross-sectional studies and, therefore, duration of carriage was not taken into account. ⁸ Whether detection of co-colonization results, in general, from a true coexistence event or reflects mostly a transitional state remains to be ascertained. Second, the relative abundance of the cocolonizing strains was not quantified. One can imagine that if the proportion of two strains of different pherotypes in the host is very different, competition may be occurring despite their coexistence. Third, we used a convenience sample based on the detection of samples where pneumococcal colonies exhibited different morphologies. Hence, it is anticipated that we underestimated co-colonization events in our collections and that only a subset of it was analyzed. Fourth, we did not attempt to measure whether the isolates had the capacity to become competent and induce fratricide. Such approach, could, however, be still inconclusive, as lack of capacity to induce competence in vitro does not necessarily imply the strain is unable to undergo competence in vivo.^19,20,28

Still, it is of particular interest that in both studies—ours and the one from Vestrheim et al. ²⁷ —using collections from different years and geographical origins, the same conclusions were reached, suggesting a low impact of CSP-mediated competition on pneumococcal coexistence in the nasopharynx. Johnsborg et al. ¹⁴ have previously reported the existence of ComD promiscuity between S. pneumoniae and closely related species, which might allow target bacteria to sense a noncognate CSP, resulting in the expression of the ComM immunity protein. To our best knowledge, this cross talk has not been found to occur between pneumococcal isolates producing CSP1 or CSP2 and their corresponding ComD1 and ComD2 proteins. This finding would, however, support the hypothesis that CSP-mediated competition has little impact on pneumococcal coexistence.

In summary, our data support the hypothesis that pherotype-mediated fratricide does not seem to be an important mechanism of within-host competition. However, further studies of longitudinal design with systematic detection of co-colonization and assessment of colonization density are needed to provide further insights on the nature of pneumococcal co-colonization.