Competitive Dominance within Biofilm Consortia Regulates the Relative Distribution of Pneumococcal Nasopharyngeal Density

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 similar to 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 similar 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 TIGR4 Delta luxS mutant and a TIGR4 Delta comC mutant. The density of S19F strains, however, was similarly reduced when consortia included TIGR4, TIGR4 Delta luxS, or TIGR4 Delta comC. 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 Trans-well devices demonstrated physical contact-mediated control of pneumococcal density within biofilm consortia.