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

Abstract

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 ϳ50% 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 ϳ10% 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 competencestimulating 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.IMPORTANCE Streptococcus 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 ϳ10% 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 quorumsensing 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 in-

Pneumococcal carriage, density, and co-colonization dynamics: A longitudinal study in Indonesian infants

“…Within-host serotype replacement was observed; this may be due to an immune-mediated reduction in density of the original colonizing serotype that facilitates expansion of a more recently acquired serotype and/or direct competition between pneumococcal strains, which has been observed in experimental models (Dawid et al, 2007;Wu et al, 2017).…”

A Mechanism of Unidirectional Transformation, Leading to Antibiotic Resistance, Occurs within Nasopharyngeal Pneumococcal Biofilm Consortia

“…In the above-mentioned study, inoculation of strains at a 1:1 ratio was enough to completely eradicate susceptible pneumococci by a BlpC-producing strain ( 42 ). A decrease in density, but by no means eradication of a pneumococcal strain, was observed in recent studies in which two strains were inoculated at a similar density and cultured under static conditions ( 44 ). Moreover, when inoculated at similar densities in the bioreactor the density of strains was similar throughout the incubation period.…”

“…We, and others, have clearly demonstrated that under static culture conditions irreversible autolysis occurs in monostrain pneumococcal biofilms ( 40 , 43 ). Heterolysis also happened in consortial biofilms inoculated with two different strains incubated under static conditions ( 42 , 44 ). All of this evidence correlates with the finding that release of DNA in the supernatants is highest in stationary cultures because it comes from lysis of pneumococci ( 32 ).…”

“…So far, we have not been able to obtain recombinants within 8 h of incubation in a static plate model, whether or not it contains human pharyngeal cells ( 40 , 43 ). The absence of recombination may be because of the heterolysis phenotype reported using static incubation conditions ( 42 , 44 ) or the accumulation of DNases in static cultures, which may degrade available eDNA for transformation ( 32 ). In summary, we have demonstrated in this study that unidirectional transformation occurred within pneumococcal biofilm consortia and that unidirectional transformation is mediated by inhibition of transformation within pneumococcal nasopharyngeal biofilms.…”

Clinical Relevance and Molecular Pathogenesis of the Emerging Serotypes 22F and 33F of Streptococcus pneumoniae in Spain

“…For instance, serotypes 11A and 35B, which are serotypes frequently associated to multidrug resistance, are good biofilm formers (Domenech et al, 2015). Biofilm formation is a critical aspect from the pathogenesis perspective because S. pneumoniae has been reported to form biofilms in the upper respiratory tract (Wu et al, 2017;Iverson et al, 2019;Silva and Sillankorva, 2019) and nasopharyngeal colonization is the initial step in this process and the prerequisite for developing IPD (Bogaert et al, 2004). Interestingly, active infection is also associated to pneumococcal biofilms in meningitis by binding to brain microvascular endothelial cells and in persistent respiratory infection affecting patients with cystic fibrosis or chronic obstructive pulmonary disease (COPD) (Orihuela et al, 2009;Vidal et al, 2013;Vandevelde et al, 2014;Dennis et al, 2018).…”