Characterization of In Vitro Biofilm-Associated Pneumococcal Phase Variants of a Clinically Relevant Serotype 3 Clone

Abstract

An increasing proportion of children with acute otitis media due to Streptococcus pneumoniae have serotype 3 infections since licensure of the seven-valent pneumococcal conjugate vaccine. These serotype 3 strains are genetically related by molecular subtyping. During otitis media with effusion and recurrent otitis media, biofilms commonly develop. Pneumococcal in vitro biofilms are comprised of phase variants that differ in colony morphology. By using a representative strain of the mucoid serotype 3 clone, rough phase variants with a diverse array of mutations were detected in biofilms formed in vitro. Most phase variants had mutations in the cps3D gene, the first gene of the capsular operon. Eleven had single nucleotide polymorphisms (SNPs) in the cps3D gene, one had an SNP in the ؊10 promoter, and three had large deletions in the cps3D gene. Reversion to the mucoid phenotype was associated with reversion of the mutation in the cps3D gene. Unlike the phase variants detected in the nasopharynx, which have at least 20% of the parental amount of capsule, the in vitro biofilm-associated phase variants had <12% of the parental amount of capsule, as determined by capsule enzyme-linked immunosorbent assays. Using real-time reverse transcription-PCR, we determined that capsule expression in the phase variants was likely regulated at multiple levels. These in vitro phase variation data, which underscore the plasticity of the pneumococcus, need to be confirmed with in vivo analyses of the middle ear mucosa during otitis media.

Isolation of Streptococcus pneumoniae Biofilm Mutants and Their Characterization during Nasopharyngeal Colonization

“…Thus, a limitation of our screen is that it did not allow the identification of factors that contribute to this aspect of biofilm formation in vitro. Interestingly, biofilm formation by S. pneumoniae serotype 3 in vitro results in the enrichment of acapsular phase variants (5,82), which express less than 10% of wild-type capsule levels (57). Thus, the capsule polysaccharide may not constitute an important component of the S. pneumoniae biofilm.…”

“…Interestingly, it seems that once the bacteria cross the mucus layer, the capsule contributes less to the ability of S. pneumoniae to persist. Moreover, clinical isolates from the nasopharynx often express low levels of capsule compared to those of wild-type laboratory strains (53), and isolates from otitis media patients have even lower levels of capsule expression (57). In addition, it has long been known that acapsular strains of S. pneumoniae or low-capsule-expressing variants adhere to epithelial layers much more efficiently than wild-type isogenic strains (77) and that colonization of the nasopharynx favors S. pneumoniae transparent variants which express lower capsule levels than their opaque counterparts (47,85).…”

“…The experiment was done once. (35,47,77,86) and controlled at both the transcriptional and posttranscriptional levels (57,63,90). We thus decided to perform a second screen, this time using an acapsular serotype 4 TIGR4 strain.…”

Signal diffusion and the mitigation of social exploitation in pneumococcal competence signalling

“…Second, cells in vivo persist in conditions that deviate markedly from the assumptions of a mass action environment. Rather, growth is more likely to be found on mucosal surfaces as biofilms, within highly viscous mucous itself or within cells of the mucosal lining than in a highly diffusive liquid (Hall-Stoodley et al 2006;Allegrucci & Sauer 2007;McEllistrem et al 2007;Reid et al 2009). These differences, together with our results, imply that even at low densities there may be opportunities for competence induction at rates that will have been underestimated from laboratory studies.…”

“…In the laboratory, transformation is typically studied in batch culture that approximates a highly diffusive mass action environment. By contrast, pneumococci in vivo persist on or within cells of the mucosal surface within biofilms or as microcolonies (Hall-Stoodley et al 2006;Allegrucci & Sauer 2007;McEllistrem et al 2007;Reid et al 2009), conditions that violate the assumptions of efficient mass transfer.…”