Site of Isolation Determines Biofilm Formation and Virulence Phenotypes of Streptococcus pneumoniae Serotype 3 Clinical Isolates

Abstract

Streptococcus pneumoniae is a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. In the present study, S. pneumoniae serotype 3 blood (n ‫؍‬ 12) or ear (n ‫؍‬ 13) isolates were multilocus sequence typed (MLST) and assessed for biofilm formation and virulence phenotype. Blood and ear isolates exhibited similar MLST distributions but differed markedly in phenotype. Blood isolates formed robust biofilms only at pH 7.4, which were enhanced in Fe(III)-supplemented medium. Conversely, ear isolates formed biofilms only at pH 6.8, and Fe(III) was inhibitory. Biofilm formation paralleled luxS expression and genetic competence. In a mouse intranasal challenge model, blood isolates did not stably colonize the nasopharynx but spread to the blood; none spread to the ear. Ear isolates colonized the nasopharynx at higher levels and also spread to the ear compartment in a significant proportion of animals; none caused bacteremia. Thus, pneumococci of the same serotype and MLST exhibit distinct phenotypes in accordance with clinical site of isolation, indicative of stable niche adaptation within a clonal lineage.

Isolation Site Influences Virulence Phenotype of Serotype 14 Streptococcus pneumoniae Strains Belonging to Multilocus Sequence Type 15

“…However, in the present study, we not only observed highly significant differences in virulence and pathogenic profiles between isolates belonging to the same serotype and clonal lineage but also found a close correlation between pathogenic profiles in mice and the original sites of isolation from human cases of pneumococcal disease. This extends an observation that we have previously reported for serotype 3 strains belonging to ST180, ST232, and ST233 (8). Thus, stable niche adaptation within a clonal lineage appears to be a general phenomenon.…”

“…The plethora of capsular serotypes and clonal lineages (MLST types) no doubt contribute to their adaptability in different host microenvironments, but they have also complicated previous attempts to unequivocally associate particular serotypes or clonal groups with specific virulence attributes (6, 7). However, we have previously described multiple examples of serotype 3 strains belonging to the same ST that consistently and reproducibly exhibit distinct virulence phenotypes in mice that directly correlate with the original site of isolation from human patients (i.e., ear versus blood) (8). This suggested that, at least in serotype 3, pneumococci exhibit stable niche adaptation within a clonal lineage.…”

“…The challenge dose was confirmed retrospectively by serial dilution and plating on BA. Groups of five mice were euthanized by CO 2 asphyxiation at either 24 or 72 h. Blood and tissue samples (lungs, nasopharynx, brain, and ear) were harvested and pneumococci enumerated in blood or tissue homogenates as described previously (8,16). For in vivo competition experiments, groups of mice were challenged intranasally with a mixed inoculum comprising approximately 5 ϫ 10 7 CFU of each strain, as previously described (17).…”

“…S. pneumoniae strains used in this study are listed in Table 1. Cells were grown in a casein-based, semi-synthetic liquid medium (CϩY) or in serum broth (SB), as required (8), or on Columbia agar supplemented with 5% (vol/vol) horse blood (BA) at 37°C in a CO 2 -enriched atmosphere. Bacterial stocks were prepared from mid-log-phase cultures and stored at Ϫ80°C in 20% glycerol.…”

“…In the murine intranasal challenge model, only blood isolates caused sepsis, while only otitis media isolates spread to the ear. These findings were indicative of stable niche adaptation within a clonal lineage (8). To further examine the nexus between the clinical source of isolates and their respective virulence-related phenotypes, we have now conducted similar studies in S. pneumoniae serotype 14 strains.…”