The oral cavity harbors a diverse community of microbes that are physiologically unique. Oral microbes that exist in this polymicrobial environment can be pathogenic or beneficial to the host. Numerous oral microbes contribute to the formation of dental caries and periodontitis; however, there is little understanding of the role these microbes play in systemic infections. There is mounting evidence that suggests that oral commensal streptococci are cocolonized with Pseudomonas aeruginosa during cystic fibrosis pulmonary infections and that the presence of these oral streptococci contributes to improved lung function. The goal of this study was to examine the underlying mechanism by which Streptococcus parasanguinis antagonizes pathogenic P. aeruginosa. In this study, we discovered that oral commensal streptococci, including Streptococcus parasanguinis, Streptococcus sanguinis, and Streptococcus gordonii, inhibit the growth of P. aeruginosa and that this inhibition is mediated by the presence of nitrite and the production of hydrogen peroxide (H 2 O 2 ) by oral streptococci. The requirement of both H 2 O 2 and nitrite for the inhibition of P. aeruginosa is due to the generation of reactive nitrogenous intermediates (RNI), including peroxynitrite. Transposon mutagenesis showed that a P. aeruginosa mutant defective in a putative ABC transporter permease is resistant to both streptococcus/nitrite-and peroxynitrite-mediated killing. Furthermore, S. parasanguinis protects Drosophila melanogaster from killing by P. aeruginosa in a nitrite-dependent manner. Our findings suggest that the combination of nitrite and H 2 O 2 may represent a unique anti-infection strategy by oral streptococci during polymicrobial infections.T he human oral cavity provides residence to more than 700 diverse bacterial species (1). A fraction of the microbial communities that colonize the oral cavity are responsible for localized pathogenic infections, such as dental caries and aggressive periodontitis (1, 2). In contrast, other oral microbes are classified as commensal bacteria (Streptococcus parasanguinis, Streptococcus sanguinis, Streptococcus gordonii, and Streptococcus salivarius) and are involved in facilitating the attachment of pathogenic bacteria to the tooth surface (3) and modulating bacterial dysbiosis (4). Microorganisms unique to the oral cavity have been considered to be mainly involved in localized oral infections; however, there is increasing evidence that suggests that oral microbes may play a more prominent role in systemic infections (5). Oral pathogens and commensals have the ability to disseminate through the bloodstream and cause infective endocarditis (6). Moreover, periodontal pathogens have been associated with cases of atherosclerosis and respiratory tract infections (5, 7).Current evidence suggests that some oral streptococci reside in locations of the body that are distant to the oral cavity, including the respiratory tracts of cystic fibrosis (CF) patients (8). Historically, Pseudomonas aeruginosa was considered to...

Pseudomonas aeruginosa causes devastating chronic pulmonary infections in cystic fibrosis (CF) patients. Although the CF airway is inhabited by diverse species of microorganisms interlaced within a biofilm, many studies focus on the sole contribution of P. aeruginosa pathogenesis in CF morbidity. More recently, oral commensal streptococci have been identified as cohabitants of the CF lung, but few studies have explored the role these bacteria play within the CF biofilm. We examined the interaction between P. aeruginosa and oral commensal streptococci within a dual species biofilm. Here we report that the CF P. aeruginosa isolate, FRD1, enhances biofilm formation and colonization of Drosophila melanogaster by the oral commensal Streptococcus parasanguinis. Moreover, production of the P. aeruginosa exopolysaccharide, alginate, is required for the promotion of S. parasanguinis biofilm formation and colonization. However, P. aeruginosa is not promoted in the dual species biofilm. Furthermore, we show that the streptococcal adhesin, BapA1, mediates alginate-dependent enhancement of the S. parasanguinis biofilm in vitro, and BapA1 along with another adhesin, Fap1, are required for the in vivo colonization of S. parasanguinis in the presence of FRD1. Taken together, our study highlights a new association between streptococcal adhesins and P. aeruginosa alginate, and reveals a mechanism by which S. parasanguinis potentially colonizes the CF lung and interferes with the pathogenesis of P. aeruginosa.

Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in cystic fibrosis (CF) patients. However, recent evidence suggests that the polymicrobial community of the CF lung may also harbour oral streptococci, and colonization by these micro-organisms may have a negative impact on P. aeruginosa within the CF lung. Our previous studies demonstrated that nitrite abundance plays an important role in P. aeruginosa survival during co-infection with oral streptococci. Nitrite reductase is a key enzyme involved in nitrite metabolism. Therefore, the objective of this study was to examine the role nitrite reductase (gene nirS) plays in P. aeruginosa survival during co-infection with an oral streptococcus, Streptococcus parasanguinis. Inactivation of nirS in both the chronic CF isolate FRD1 and acute wound isolate PAO1 reduced the survival rate of P. aeruginosa when co-cultured with S. parasanguinis. Growth of both mutants was restored when co-cultured with S. parasanguinis that was defective for H 2 O 2 production. Furthermore, the nitrite reductase mutant was unable to kill Drosophila melanogaster during co-infection with S. parasanguinis. Taken together, these results suggest that nitrite reductase plays an important role for survival of P. aeruginosa during co-infection with S. parasanguinis.

Streptococcus mutans and Candida albicans are frequently co-isolated from dental plaque of children with early childhood caries (ECC) and are only rarely found in children without ECC, suggesting that these species interact in a manner that contributes to the pathogenesis of ECC. Previous studies have demonstrated that glucans produced by S. mutans are crucial for promoting the formation of biofilm and cariogenicity with C. albicans; however, it is unclear how non-glucan S. mutans biofilm factors contribute to increased biofilm formation in the presence of C. albicans. In this study we examined the role of S. mutans antigen I/II in two-species biofilms with C. albicans, and determined that antigen I/II is important for the incorporation of C. albicans into the two-species biofilm and is also required for increased acid production. The interaction is independent of the proteins Als1 and Als3, which are known streptococcal receptors of C. albicans. Moreover, antigen I/II is required for the colonization of both S. mutans and C. albicans during co-infection of Drosophila melanogaster in vivo. Taken together, these results demonstrate that antigen I/II mediates the increase of C. albicans numbers and acid production in the two-species biofilm, representing new activities associated with this known S. mutans adhesin.

Summary Balanced bacterial biofilm communities help to maintain host health. Disturbance of such communities can lead to bacterial dysbiosis and pathogenesis. However, complex and dynamic bacterial interactions within the biofilm communities are poorly understood. In this study, we used a dual-species biofilm consisting of the periodontal pathogen Aggregatibacter actinomycetemcomitans, and a commensal Streptococcus parasanguinis to investigate bacterial interactions since the two organisms have been found to coexist during the development of localized aggressive periodontal disease. We report that A. actinomycetemcomitans promoted biofilm formation of S. parasanguinis in vitro and in vivo. Protein profiling of S. parasanguinis co-cultured with A. actinomycetemcomitans revealed a significant decrease in the protein level of pyruvate oxidase(PoxL), an enzyme required for the generation of hydrogen peroxide (H2O2). Consistently, the H2O2 concentration was concurrently decreased. However, the complete removal of H2O2 impaired the biofilm formation. H2O2 at a low concentration range regulated by A. actinomycetemcomitans enhanced the biofilm formation. These results demonstrate that A. actinomycetemcomitans promotes the S. parasanguinis biofilm formation through modulating the production of H2O2 by fine-tuning the expression of poxL, indicating that H2O2 functions as a signaling molecule. Taken together, this report revealed a previously unknown bacteria-bacteria interaction mechanism.

Oral commensal streptococci are primary colonizers of the oral cavity. These streptococci produce many adhesins, metabolites, and antimicrobials that modulate microbial succession and diversity within the oral cavity.

Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in patients with cystic fibrosis (CF). The metabolic pathways utilized by P. aeruginosa during these infections, which can persist for decades, are poorly understood. Several lines of evidence suggest that the glyoxylate pathway, which utilizes acetate or fatty acids to replenish intermediates of the tricarboxylic acid cycle, is an important metabolic pathway for P. aeruginosa adapted to the CF lung. Isocitrate lyase (ICL) is one of two major enzymes of the glyoxylate pathway. In a previous study, we determined that P. aeruginosa is dependent upon aceA, which encodes ICL, to cause disease on alfalfa seedlings and in rat lungs. Expression of aceA in PAO1, a P. aeruginosa isolate associated with acute infection, is regulated by carbon sources that utilize the glyoxyate pathway. In contrast, expression of aceA in FRD1, a CF isolate, is constitutively upregulated. Moreover, this deregulation of aceA occurs in other P. aeruginosa isolates associated with chronic infection, suggesting that high ICL activity facilitates adaptation of P. aeruginosa to the CF lung. Complementation of FRD1 with a PAO1 clone bank identified that rpoN negatively regulates aceA. However, the deregulation of aceA in FRD1 was not due to a knockout mutation of rpoN. Regulation of the glyoxylate pathway by RpoN is likely to be indirect, and represents a unique regulatory role for this sigma factor in bacterial metabolism. INTRODUCTIONBronchopulmonary infections caused by Pseudomonas aeruginosa are the leading cause of mortality for cystic fibrosis (CF) patients. These infections resist eradication by antibiotic therapy and the host immune system, and indicate a need for novel therapeutic strategies. The ability of P. aeruginosa to maintain decade-long infections within the CF lung is attributed in part to virulence mechanisms that evolve as the bacterium adapts to this environment (Lindsey et al., 2008;Nguyen & Singh, 2006). Although P. aeruginosa is nutritionally versatile, within human niches it must adapt to the availability of host-derived nutrients. Within the lungs of CF patients, these nutrients are probably contained in sputum.The composition of CF sputum is complex. It contains host and bacterial cells, as well as various host-and bacterialderived compounds (Hoiby, 1998). Transcriptome studies have indicated that PAO1, a wound isolate of P. aeruginosa, primarily uses amino acids as a carbon source when grown in CF sputum (Palmer et al., 2005;Son et al., 2007). In contrast, a CF isolate of P. aeruginosa uses amino acids and lipids (Palmer et al., 2005;Son et al., 2007). The different carbon utilization patterns by these isolates suggest that P. aeruginosa alters its metabolic pathways during chronic infection of the CF lung. This is supported by the observation that regulatory control of several central metabolic enzymes is altered in FRD1, a CF isolate, compared with PAO1 (Lindsey et al., 2008;Silo-Suh et al., 2005).In a previous study, we exploited t...

Pseudomonas aeruginosa causes persistent infections in the airways of cystic fibrosis (CF) patients. Airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphotidylcholine, a major component of host cell membranes. Phosphotidylcholine can be degraded by P. aeruginosa to glycerol and fatty acids to increase the availability of glycerol in the CF lung. In this study, we explored the role that glycerol metabolism plays in biofilm formation by P. aeruginosa. We report that glycerol metabolism promotes biofilm formation by both a chronic CF isolate (FRD1) and a wound isolate (PAO1) of P. aeruginosa. Moreover, loss of the GlpR regulator, which represses the expression of genes involved in glycerol metabolism, enhances biofilm formation in FRD1 through the upregulation of Pel polysaccharide. Taken together, our results suggest that glycerol metabolism may be a key factor that contributes to P. aeruginosa persistence by promoting biofilm formation.