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Pseudomonas aeruginosa dominates the complex polymicrobial cystic fibrosis (CF) airway and is a leading cause of death in persons with CF. Interestingly, oral streptococcal colonization has been associated with stable CF lung function. The most abundant streptococcal species found in stable patients, Streptococcus salivarius, has been shown to downregulate pro-inflammatory cytokines in multiple colonization models. However, no studies have demonstrated how S. salivarius potentially improves lung function. Our lab previously demonstrated that the P. aeruginosa exopolysaccharide Psl promotes S. salivarius biofilm formation in vitro, suggesting a possible mechanism by which S. salivarius is incorporated into the CF airway microbial community. In this study, we demonstrate that co-infection of rats leads to enhanced S. salivarius colonization and reduced P. aeruginosa colonization. Histological scores for tissue inflammation and damage are lower in dual-infected rats compared to P. aeruginosa infected rats. Additionally, pro-inflammatory cytokines IL-1β, IL-6, CXCL2, and TNF-α are downregulated during co-infection compared to P. aeruginosa single-infection. Lastly, RNA sequencing of cultures grown in synthetic CF sputum revealed that P. aeruginosa glucose metabolism genes are downregulated in the presence of S. salivarius, suggesting a potential alteration in P. aeruginosa fitness during co-culture. Overall, our data support a model in which S. salivarius colonization is promoted during co-infection with P. aeruginosa, whereas P. aeruginosa airway bacterial burden is reduced, leading to an attenuated host inflammatory response.

Chronic infections in the cystic fibrosis (CF) airway are composed of both pathogenic and commensal bacteria. However, chronic Pseudomonas aeruginosa infections are the leading cause of lung deterioration in individuals with CF. Interestingly, oral commensals can translocate to the CF lung and their presence is associated with improved lung function, presumably due to their ability to antagonize P. aeruginosa. We have previously shown that one commensal, Streptococcus parasanguinis, produces hydrogen peroxide that reacts with nitrite to generate reactive nitrogen intermediates (RNI) which inhibit P. aeruginosa growth. In this study, we sought to understand the global impact of commensal-mediated RNI on the P. aeruginosa transcriptome. RNA sequencing analysis revealed that S. parasanguinis and nitrite-mediated RNI dysregulated expression of denitrification genes in a CF isolate of P. aeruginosa compared to when this isolate was only exposed to S. parasanguinis. Further, loss of a nitric oxide reductase subunit (norB) rendered an acute P. aeruginosa isolate more susceptible to S. parasanguinis-mediated RNI. Additionally, S. parasanguinis-mediated RNI inactivated P. aeruginosa aconitase activity. Lastly, we report that P. aeruginosa isolates recovered from CF individuals are uniquely hypersensitive to S. parasanguinis-mediated RNI compared to acute infection or environmental P. aeruginosa isolates. These findings illustrate that S. parasanguinis hinders the ability of P. aeruginosa to respond to RNI, which potentially prevents P. aeruginosa CF isolates from resisting commensal and host-induced RNI in the CF airway.

Commensal streptococci regulate health and homeostasis within oral polymicrobial communities. Remarkably, high salivary nitrite concentrations have also been associated with improved health in the oral cavity. We previously demonstrated that nitrite assists hydrogen peroxide-producing oral commensal streptococci in regulating homeostasis via the generation of reactive nitrogen species (RNS), which have antimicrobial activity on oral pathogens. However, it is unknown how nitrite and commensal streptococci work in concert to influence the metabolome of oral polymicrobial communities. In this study, we report that nitrite aids commensal streptococci in the inhibition of multi-kingdom pathogens that reside in distinct oral niches, which supports commensal dominance. More importantly, we show that commensal streptococci utilize nitrite to drive the metabolic signature of multispecies biofilms in a manner that supports commensal metabolism and resistance to RNS, and restricts metabolic processes that are required for pathogen virulence. Taken together, our study provides insight into how commensal streptococci use nitrite to trigger shifts in the oral polymicrobial metabolome to support health and homeostasis.

Early childhood caries (ECC) is a highly prevalent and costly chronic oral infectious disease in preschool children. Candida albicans has been frequently detected in children and has demonstrated cariogenic traits. However, since ECC is a multifactorial infectious disease with many predisposing non-microbial factors, it remains to be elucidated whether the presence and accumulation of C. albicans in ECC is merely a consequence of the adaptation of C. albicans to a cariogenic oral environment, or it plays an active role in the initiation and progression of dental caries. This review aims to summarize the current knowledge on C. albicans and the risk of ECC, with a focus on its synergistic relationship with the cariogenic pathogen Streptococcus mutans. We also highlight recent advances in the development of approaches to disrupt C. albicans-S. mutans cross-kingdom biofilms in ECC prevention and treatment. Longitudinal clinical studies, including interventional clinical trials targeting C. albicans, are necessary to ascertain if C. albicans indeed contributes in a significant manner to the initiation and progression of ECC. In addition, further work is needed to understand the influence of other bacteria and fungi of oral microbiota on C. albicans-S. mutans interactions in ECC.