Streptococcus pneumoniae is a main cause of child mortality worldwide, but strains also asymptomatically colonize the upper airways of most children and form biofilms. Recent studies have demonstrated that ∼50% of colonized children carry at least two different serotypes (i.e., strains) in the nasopharynx; however, studies of how strains coexist are limited. In this work, we investigated the physiological, genetic, and ecological requirements for the relative distribution of densities, and spatial localization, of pneumococcal strains within biofilm consortia. Biofilm consortia were prepared with vaccine type strains (i.e., serotype 6B [S6B], S19F, or S23F) and strain TIGR4 (S4). Experiments first revealed that the relative densities of S6B and S23F were similar in biofilm consortia. The density of S19F strains, however, was reduced to ∼10% in biofilm consortia, including either S6B, S23F, or TIGR4, in comparison to S19F monostrain biofilms. Reduction of S19F density within biofilm consortia was also observed in a simulated nasopharyngeal environment. Reduction of relative density was not related to growth rates, since the Malthusian parameter demonstrated similar rates of change of density for most strains. To investigate whether quorum sensing (QS) regulates relative densities in biofilm consortia, two different mutants were prepared: a TIGR4ΔluxS mutant and a TIGR4ΔcomC mutant. The density of S19F strains, however, was similarly reduced when consortia included TIGR4, TIGR4ΔluxS, or TIGR4ΔcomC Moreover, production of a different competence-stimulating peptide (CSP), CSP1 or CSP2, was not a factor that affected dominance. Finally, a mathematical model, confocal experiments, and experiments using Transwell devices demonstrated physical contact-mediated control of pneumococcal density within biofilm consortia.IMPORTANCEStreptococcus pneumoniae kills nearly half a million children every year, but it also produces nasopharyngeal biofilm consortia in a proportion of asymptomatic children, and these biofilms often contain two strains (i.e., serotypes). In our study, we investigated how strains coexist within pneumococcal consortia produced by vaccine serotypes S4, S6B, S19F, and S23F. Whereas S6B and S23F shared the biofilm consortium, our studies demonstrated reduction of the relative density of S19F strains, to ∼10% of what it would otherwise be if alone, in consortial biofilms formed with S4, S6B, or S23F. This dominance was not related to increased fitness when competing for nutrients, nor was it regulated by quorum-sensing LuxS/AI-2 or Com systems. It was demonstrated, however, to be enhanced by physical contact rather than by a product(s) secreted into the supernatant, as would naturally occur in the semidry nasopharyngeal environment. Competitive interactions within pneumococcal biofilm consortia regulate nasopharyngeal density, a risk factor for pneumococcal disease.

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