Commensal Streptococcus Research Articles

Live and let die: Hydrogen peroxide production by the commensal flora and its role in maintaining a symbiotic microbiome.

The majority of commensal oral streptococci are able to generate hydrogen peroxide (H2 O2 ) during aerobic growth, which can diffuse through the cell membrane and inhibit competing species in close proximity. Competing H2 O2 production is mainly dependent upon the pyruvate oxidase SpxB, and to a lesser extent the lactate oxidase LctO, both of which are important for energy generation in aerobic environments. Several studies point to a broad impact of H2 O2 production in the oral environment, including a potential role in biofilm homeostasis, signaling, and interspecies interactions. Here, we summarize the current research regarding oral streptococcal H2 O2 generation, resistance mechanisms, and the ecological impact of H2 O2 production. We also discuss the potential therapeutic utility of H2 O2 for the prevention/treatment of dysbiotic diseases as well as its potential role as a biomarker of oral health.

Thymus essential oil extraction, characterization and incorporation in phospholipid vesicles for the antioxidant/antibacterial treatment of oral cavity diseases.

The aim of the work was to extract, characterize, and formulate Thymus capitatus (Tymbra capitata) essential oil in phospholipid vesicles: liposomes, glycerosomes and Penetration Enhancer-containing Vesicles (PEVs). The steam-distilled essential oil was mainly composed of carvacrol. The oil was mixed with lecithin and water to produce liposomes, or different ratios of water/glycerol or water/propylene glycol (PG) to produce glycerosomes and PG-PEVs, respectively. Cryo-TEM showed the formation of unilamellar, spherical vesicles, and light scattering disclosed that their size increased in the presence of glycerol or PG, which improved long-term stability. The formulations were highly biocompatible, and capable of counteracting oxidative stress and favouring wound repair in keratinocytes, thanks to enhanced uptake. The antibacterial activity of the oil was demonstrated against cariogenic Streptococcus mutans, Lactobacillus acidophilus, and commensal Streptococcus sanguinis. The combination of antioxidant and antibacterial activities of Thymus essential oil formulations may be useful for the treatment of oral cavity diseases.

Sublingual administration of 2-hydroxyethyl methacrylate enhances antibody responses to co-administered ovalbumin and Streptococcus mutans

Objective: The oral mucosa of patients undergoing dental procedures is often exposed to residual monomers leaking from incompletely cured acrylic resins. We investigated whether 2-hydroxyethyl methacrylate (HEMA) monomers applied to the sublingual mucosa in mice modulate the antibody responses towards co-administered ovalbumin (OVA) or live oral bacteria.Material and methods: OVA, live mouse oral commensal Lactobacillus murinus or live human oral commensal Streptococcus mutans were administered sublingually with or without HEMA to BALB/c mice on four weekly occasions. One week after the last administration, the experiment was terminated and serum antibody levels were analyzed using ELISA.Results: Significantly increased IgG and IgE anti-OVA antibody activity was found in the sera from mice immunized with OVA together with HEMA, as compared to mice immunized with OVA alone. Likewise, S. mutans together with HEMA induced an IgG anti-S. mutans antibody response that was significantly higher than the antibody response detected after application of S. mutans alone. No IgG anti-L. murinus antibody response was detected in mice immunized with L. murinus together with HEMA, as compared to the background activity.Conclusions: We report that HEMA monomers have adjuvant properties when sublingually administered in combination with OVA or S. mutans.

Interplay between Porphyromonas gingivalis and EGF signalling in the regulation of CXCL14.

Porphyromonas gingivalis is a keystone pathogen in chronic periodontitis. Its expression of gingipain proteases (Kgp and RgpA/B) is central to the stimulation of chronic inflammation. In this study, we investigated the inflammatory response of oral epithelial cells to P.gingivalis. The cells responded by upregulating the expression of the orphan chemokine CXCL14. The stimulation of CXCL14 expression was largely triggered by the gingipain proteases and was dependent on the host protease-activated receptor PAR-3. Significantly, CXCL14 expression was transcriptionally repressed in response to epidermal growth factor (EGF)-induced activation of the MEK-ERK1/2 pathway. P.gingivalis overcomes the repression of CXCL14 via the gingipain protease-mediated degradation of EGF. Therefore, P.gingivalis not only directly stimulates CXCL14 expression via PAR-3 but also promotes its expression by antagonising EGF signalling. In addition to chemotactic activity, some chemokines also have antimicrobial activities. CXCL14 was demonstrated to have bactericidal activity, against commensal oral streptococci associated with health. Notably though, P.gingivalis was not susceptible to killing by CXCL14, potentially because the gingipain proteases can degrade CXCL14. This suggests that the stimulation of dysregulated CXCL14 expression by P.gingivalis may help promote dysbiosis and the development of chronic periodontitis.

Macrophage Polarization Alters Postphagocytosis Survivability of the Commensal Streptococcus gordonii.

Oral streptococci are generally considered commensal organisms; however, they are becoming recognized as important associate pathogens during the development of periodontal disease as well as being associated with several systemic diseases, including as a causative agent of infective endocarditis. An important virulence determinant of these bacteria is an ability to evade destruction by phagocytic cells, yet how this subversion occurs is mostly unknown. Using Streptococcus gordonii as a model commensal oral streptococcus that is also associated with disease, we find that resistance to reactive oxygen species (ROS) with an active ability to damage phagosomes allows the bacterium to avoid destruction within macrophages. This ability to survive relies not only on the ROS resistance capabilities of the bacterium but also on ROS production by macrophages, with both being required for maximal survival of internalized bacteria. Importantly, we also show that this dependence on ROS production by macrophages for resistance has functional significance: S. gordonii intracellular survival increases when macrophages are polarized toward an activated (M1) profile, which is known to result in prolonged phagosomal ROS production compared to that of alternatively (M2) polarized macrophages. We additionally find evidence of the bacterium being capable of both delaying the maturation of and damaging phagosomes. Taken together, these results provide essential insights regarding the mechanisms through which normally commensal oral bacteria can contribute to both local and systemic inflammatory disease.

Circuitry Rewiring Directly Couples Competence to Predation in the Gut Dweller Streptococcus salivarius

Small distortions in transcriptional networks might lead to drastic phenotypical changes, especially in cellular developmental programs such as competence for natural transformation. Here, we report a pervasive circuitry rewiring for competence and predation interplay in commensal streptococci. Canonically, in streptococci paradigms such as Streptococcus pneumoniae and Streptococcus mutans, the pheromone-based two-component system BlpRH is a central node that orchestrates the production of antimicrobial compounds (bacteriocins) and incorporates signal from the competence activation cascade. However, the human commensal Streptococcus salivarius does not contain a functional BlpRH pair, while the competence signaling system ComRS directly couples bacteriocin production and competence commitment. This network shortcut might underlie an optimal adaptation against microbial competitors and explain the high prevalence of S.salivarius in the human digestive tract. Moreover, the broad spectrum of bacteriocin activity against pathogenic bacteria showcases the commensal and genetically tractable S.salivarius species as a user-friendly model for competence and bacterial predation.

Beneficial Oral Biofilms as Smart Bioactive Interfaces

Periodontitis is a very common health problem caused by formation of pathogenic bacterial biofilm that triggers inflammation resulting in either reversible gingivitis or irreversible periodontal hard and soft tissue damages, leading to loss of teeth when left untreated. Commensal bacteria play an important role in oral health in many aspects. Mainly by colonizing oral tissues, they (i) contribute to maturation of immune response, and (ii) foreclose attachment of pathobiont and, therefore, prevent from infection. The main goal of the study was to investigate if blocking of receptors on a commensal biofilm can prevent or reduce the attachment of pathogenic strains. To do so, biofilm produced by commensal Streptococcus sanguinis was treated with whole cell lysate of pathobionts Fusobacterium nucleatum or Porphyromonas gingivalis, followed by incubation with respective strain(s). The study revealed significant reduction in pathobiont adhesion to lysate-treated commensal biofilm. Therefore, adhesion of pathobionts onto the lysate-blocked biofilm was hindered; however, not completely eliminated supporting the idea that such approach in the oral cavity would benefit the production of a well-balanced and healthy bioactive interface.

Getting to Know "The Known Unknowns": Heterogeneity in the Oral Microbiome.

Technological advances in DNA sequencing have provided unprecedented insights into the composition of the oral microbiome in health and disease, and RNA-sequencing and metabolomics-related technologies are beginning to yield information on the activities of these organisms. Importantly, progress in this area has brought the scientific community closer to an understanding of what constitutes a health-associated microbiome and is supporting the notion that the microbiota in healthy sites assumes an active role in promoting health and suppressing the acquisition, persistence, and activities of overt and opportunistic pathogens. It is also becoming clear that a significant impediment to developing a conclusive body of evidence that defines a healthy microbiome and the mechanisms by which beneficial bacteria promote health is that an inherent characteristic of the most abundant members of the oral flora, those that potentially play the greatest roles in health and disease, is intraspecies genomic diversity. In particular, individual isolates of abundant commensal and pathogenic streptococci show tremendous variability in gene content, and this variability manifests in tremendous phenotypic heterogeneity. Analysis of the consequences of this diversity has been complicated by the exquisite sensitivity these bacteria have evolved to environmental inputs, inducing rapid and substantial fluctuations in behaviors, and often only within subpopulations of the organisms. Thus, the conditions under which the oral microbiota is studied can produce widely different results within and between species. Fortunately, continually diminishing costs and ongoing refinements in sequencing and metabolomics are making it practical to study the oral microbiome at a level that will create a sufficiently robust understanding of the functions of individual organisms and reveal the complex interrelationships of these microbes ("the known unknowns") in a way that researchers will be able to engage in the rational design of reliable and economical risk assessments and preventive therapies.

Streptococcus gordonii pheromone s.g.cAM373 may influence the reservoir of antibiotic resistance determinants of Enterococcus faecalis origin in the oral metagenome.

Streptococcus gordonii produces a pheromone heptapeptide, s.g.cAM373, which induces a conjugative mating response in Enterococcus faecalis cells carrying the responsive plasmid, pAM373. We investigated the extent of this intergeneric signaling on DNA acquisition by streptococcal species likely to cohabit oral biofilms. E. faecalis/pAM373/pAMS470 cells were incubated with synthetic s.g.cAM373, reverse peptide s.g.cAM373-R, or peptide-free medium and examined for their abilities to transfer plasmid DNA to streptococcal species in the presence of DNase. Preinduction of E. faecalis donors with s.g.cAM373 resulted in transconjugation frequencies in non-pheromone producing strains of Streptococcus mutans, Streptococcus sanguinis, Streptococcus anginosus, and Streptococcus suis that were significantly higher than frequencies when donors were preincubated with s.g.cAM373-R or medium alone. Peptide-mediated communication between commensal streptococci and E. faecalis carrying pheromone-responsive plasmids may facilitate conjugative DNA transfer to bystander species, and influence the reservoir of antibiotic resistance determinants of enterococcal origin in the oral metagenome.

Real-Time Metabolic Interactions between Two Bacterial Species Using a Carbon-Based pH Microsensor as a Scanning Electrochemical Microscopy Probe.

We have developed a carbon-based, fast-response potentiometric pH microsensor for use as a scanning electrochemical microscopy (SECM) chemical probe to quantitatively map the microbial metabolic exchange between two bacterial species, commensal Streptococcus gordonii and pathogenic Streptococcus mutans. The 25 μm diameter H+ ion-selective microelectrode or pH microprobe showed a Nernstian slope of 59 mV/pH and high selectivity against major ions such Na+, K+, Ca2+, and Mg2+. In addition, the unique conductive membrane composition aided us in performing an amperometric approach curve to position the probe and obtain a high-resolution pH map of the microenvironment produced by the lactate-producing S. mutans biofilm. The x-directional pH scan over S. mutans also showed the influence of the pH profile on the metabolic activity of another species, H2O2-producing S. gordonii. When these bacterial species were placed in close spatial proximity, we observed an initial increase in the local H2O2 concentration of approximately 12 ± 5 μM above S. gordonii, followed by a gradual decrease in H2O2 concentration (>30 min) to almost zero as lactate was produced, and a subsequent decrease in pH with a more pronounced metabolic output of S. mutans. These results were supported by gene expression and confocal fluorescence microscopic studies. Our findings illustrate that H2O2-producing S. gordonii is dominant while the buffering capacity of saliva is valid (∼pH 6.0) but is gradually taken over by S. mutans as the latter species slowly starts decreasing the local pH to 5.0 or less by producing lactic acid. Our observations demonstrate the unique capability of our SECM chemical probes for studying real-time metabolic interactions between two bacterial species, which would not otherwise be achievable in traditional assays.

AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis.

Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (ΔatlA) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an ΔatlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the ΔatlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in ΔatlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans, which contributes to biofilm formation in infective endocarditis.

Impact of a 7-Day Field Training on Oral Health Condition in Japan Ground Self-Defense Force Personnel.

In the Japan Ground Self-Defense Force (JGSDF), personnel periodically perform intensive training that mimics the conditions seen in battle and during natural disasters. Military training involves intensive, stressful conditions, and changes in immune responses have been found in personnel following training. Good oral condition is important for military personnel to fulfill their duties; however, they have difficulty performing daily oral care under training conditions. In this study, we investigated the impact of a 7-day field training on the oral health status of JGSDF personnel by comparing their oral condition before and just after training. The participants were 59 male and 3 female JGSDF personnel undergoing a 7-day field training. All personnel provided informed written consent to participate, and this study was approved by the ethics committee of the Kagoshima University Graduate School of Medical and Dental Sciences. Oral health behaviors before and during the training period were surveyed using a self-administered questionnaire. Dental caries was assessed before training in terms of decayed, missing and filled teeth (DMFT), and periodontal condition was examined before and immediately after training using the community periodontal index (CPI). The presence of eight species of bacteria in dental plaque, including commensal streptococci that are early colonizers on the tooth surface, cariogenic bacteria, and periodontopathic bacteria, was determined using real-time polymerase chain reaction. We also assessed antibacterial factors and a stress marker in saliva samples. Sample collection was performed before and just after training. In addition to difference analysis between groups, logistic regression analysis was performed to examine the association between each health behavior and periodontal deterioration. The frequency of toothbrushing decreased, and snacking increased during the training period. Thirty-five personnel (56.5%) showed an increase in individual CPI code, and 57 personnel (91.9%) showed deterioration in the CPI code in 1 or more sextants after training (Figure 1). Toothbrushing frequency was significantly associated with CPI deterioration; the odds ratio in subjects who did not brush their teeth was 7.51 compared to those who brushed at least once during the training period. Severe periodontal deterioration was observed in the high-DMFT group (Figure 2), and toothbrushing frequency during the training period decreased more in this group compared to the low-DMFT group. The percentages of Streptococcus sanguinis and Streptococcus gordonii increased significantly after the training period suggesting dental plaque maturation, and an increase in S. sanguinis was associated with toothbrushing frequency. The lactoferrin concentration in saliva increased significantly after training. We demonstrated periodontal deterioration in JGSDF personnel after a 7-day training. Behavioral changes, especially discontinuation of regular toothbrushing, fostered dental plaque maturation, resulting in inflammatory changes in participants' periodontal condition. The results indicate the importance of performing toothbrushing at least once over a 7-day training period for prevention of periodontal deterioration. The regimen could be applicable to evacuees from disasters because they are under conditions of stress that may limit oral hygiene activity.

Long Persistence of a Streptococcus pneumoniae 23F Clone in a Cystic Fibrosis Patient.

Streptococcus pneumoniae isolates of serotype 23F with intermediate penicillin resistance were recovered on seven occasions over a period of 37months from a cystic fibrosis patient in Berlin. All isolates expressed the same multilocus sequence type (ST), ST10523. The genome sequences of the first and last isolates, D122 and D141, revealed the absence of two phage-related gene clusters compared to the genome of another ST10523 strain, D219, isolated earlier at a different place in Germany. Genomes of all three strains carried the same novel mosaic penicillin-binding protein (PBP) genes, pbp2x, pbp2b, and pbp1a; these genes were distinct from those of other penicillin-resistant S.pneumoniae strains except for pbp1a of a Romanian S.pneumoniae isolate. All PBPs contained mutations that have been associated with the penicillin resistance phenotype. Most interestingly, a mosaic block identical to an internal pbp2x sequence of ST10523 was present in pbp2x of Streptococcus mitis strain B93-4, which was isolated from the same patient. This suggests interspecies gene transfer from S.pneumoniae to S.mitis within the host. Nearly all genes expressing surface proteins, which represent major virulence factors of S.pneumoniae and are typical for this species, were present in the genome of ST10523. One exception was the hyaluronidase gene hlyA, which contained a 12-nucleotide deletion within the promoter region and an internal stop codon. The lack of a functional hyaluronidase might contribute to the ability to persist in the host for an unusually long period of time. IMPORTANCEStreptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S.pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S.mitis as donor species. Sequences identical to one such mosaic gene were identified in an S.mitis strain from the same patient, suggesting that in this case S.pneumoniae played the role of donor.

A commensal streptococcus hijacks a Pseudomonas aeruginosa exopolysaccharide to promote biofilm formation.

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.

Insight into the Diversity of Penicillin-Binding Protein 2x Alleles and Mutations in Viridans Streptococci.

ABSTRACTThe identification of commensal streptococci species is an everlasting problem due to their ability to genetically transform. A new challenge in this respect is the recent description of Streptococcus pseudopneumoniae as a new species, which was distinguished from closely related pathogenic S. pneumoniae and commensal S. mitis by a variety of physiological and molecular biological tests. Forty-one atypical S. pneumoniae isolates have been collected at the German National Reference Center for Streptococci (GNRCS). Multilocus sequence typing (MLST) confirmed 35 isolates as the species S. pseudopneumoniae. A comparison with the pbp2x sequences from 120 commensal streptococci isolated from different continents revealed that pbp2x is distinct among penicillin-susceptible S. pseudopneumoniae isolates. Four penicillin-binding protein x (PBPx) alleles of penicillin-sensitive S. mitis account for most of the diverse sequence blocks in resistant S. pseudopneumoniae, S. pneumoniae, and S. mitis, and S. infantis and S. oralis sequences were found in S. pneumoniae from Japan. PBP2x genes of the family of mosaic genes related to pbp2x in the S. pneumoniae clone Spain23F-1 were observed in S. oralis and S. infantis as well, confirming its global distribution. Thirty-eight sites were altered within the PBP2x transpeptidase domains of penicillin-resistant strains, excluding another 37 sites present in the reference genes of sensitive strains. Specific mutational patterns were detected depending on the parental sequence blocks, in agreement with distinct mutational pathways during the development of beta-lactam resistance. The majority of the mutations clustered around the active site, whereas others are likely to affect stability or interactions with the C-terminal domain or partner proteins.

Streptococcal adhesin SspA/B analogue peptide inhibits adherence and impacts biofilm formation of Streptococcus mutans.

Streptococcus mutans, the major causative agent of dental caries, adheres to tooth surfaces via the host salivary glycoprotein-340 (gp340). This adherence can be competitively inhibited by peptides derived from the SspA/B adhesins of Streptococcus gordonii, a human commensal microbe that competes for the same binding sites. Ssp(A4K-A11K), a double-lysine substituted SspA/B peptide analogue, has been shown to exhibit superior in vitro binding affinity for a gp340-derived peptide (SRCRP2), suggesting that Ssp(A4K-A11K) may be of clinical interest. In the present work, we tested the inhibitory effects of Ssp(A4K-A11K) on adherence and biofilm formation of S. mutans by reconstructing an artificial oral environment using saliva-coated polystyrene plates and hydroxyapatite disks. Bacterial adherence (adherence period: 1 h) was assessed by an enzyme-linked immunosorbent assay using biotinylated bacterial cells. Biofilm formation (periods: 8, 11, or 14 h) was assessed by staining and imaging of the sessile cells, or by recovering biofilm cells and plating for cell counts. The pH values of the culture media were measured as a biofilm acidogenicity indicator. Bactericidality was measured by loss of optical density during culturing in the presence of the peptide. We observed that 650 μM Ssp(A4K-A11K) significantly inhibited adherence of S. mutans to saliva-coated polystyrene; a similar effect was seen on bacterial affinity for SRCRP2. Ssp(A4K-A11K) had lesser effects on the adherence of commensal streptococci. Pretreatment of polystyrene and hydroxyapatite with 650 μM Ssp(A4K-A11K) significantly attenuated biofilm formation, whether tested with glucose- or sucrose-containing media. The SspA/B peptide’s activity did not reflect bactericidality. Strikingly, pH in Ssp-treated 8-h (6.8 ± 0.06) and 11-h (5.5 ± 0.06) biofilms showed higher values than the critical pH. Thus, Ssp(A4K-A11K) acts by inhibiting bacterial adherence and cariogrnic biofilm formation. We further consider these results in the context of the safety, specificity, and stability properties of the Ssp(A4K-A11K) peptide.

Modulation of behaviour and virulence of a high alginate expressing Pseudomonas aeruginosa strain from cystic fibrosis by oral commensal bacterium Streptococcus anginosus.

Cystic fibrosis (CF) airways harbour complex and dynamic polymicrobial communities that include many oral bacteria. Despite increased knowledge of CF airway microbiomes the interaction between established CF pathogens and other resident microbes and resulting impact on disease progression is poorly understood. Previous studies have demonstrated that oral commensal streptococci of the Anginosus group (AGS) can establish chronic pulmonary infections and become numerically dominant in CF sputa indicating that they play an important role in CF microbiome dynamics. In this study a strain of Pseudomonas aeruginosa (DWW2) of the mucoid alginate overproducing phenotype associated with chronic CF airway infection and a strain of the oral commensal AGS species Streptococcus anginosus (3a) from CF sputum were investigated for their ability to co-exist and their responses to biofilm co-culture. Bacteria in biofilms were quantified, pyocyanin expression by DWW2 was measured and the effect of AGS strain 3a on reversion of DWW2 to a non-mucoidal phenotype investigated. The virulence of DWW2, 3a and colony variant phenotypes of DWW2 in mono- and co-culture were compared in a Galleria mellonella infection model. Co-culture biofilms were formed in normoxic, hypercapnic (10% CO2) and anoxic atmospheres with the streptococcus increasing in number in co-culture, indicating that these bacteria would be able to co-exist and thrive within the heterogeneous microenvironments of the CF airway. The streptococcus caused increased pyocyanin expression by DWW2 and colony variants by stimulating reversion of the mucoid phenotype to the high pyocyanin expressing non-mucoid phenotype. The latter was highly virulent in the infection model with greater virulence when in co-culture with the streptococcus. The results of this study demonstrate that the oral commensal S. anginosus benefits from interaction with P. aeruginosa of the CF associated mucoid phenotype and modulates the behaviour of the pseudomonad in ways that may be clinically relevant.

Microbial Community Composition Impacts Pathogen Iron Availability during Polymicrobial Infection.

Iron is an essential nutrient for bacterial pathogenesis, but in the host, iron is tightly sequestered, limiting its availability for bacterial growth. Although this is an important arm of host immunity, most studies examine how bacteria respond to iron restriction in laboratory rather than host settings, where the microbiome can potentially alter pathogen strategies for acquiring iron. One of the most important transcriptional regulators controlling bacterial iron homeostasis is Fur. Here we used a combination of RNA-seq and chromatin immunoprecipitation (ChIP)-seq to characterize the iron-restricted and Fur regulons of the biofilm-forming opportunistic pathogen Aggregatibacter actinomycetemcomitans. We discovered that iron restriction and Fur regulate 4% and 3.5% of the genome, respectively. While most genes in these regulons were related to iron uptake and metabolism, we found that Fur also directly regulates the biofilm-dispersing enzyme Dispersin B, allowing A. actinomycetemcomitans to escape from iron-scarce environments. We then leveraged these datasets to assess the availability of iron to A. actinomycetemcomitans in its primary infection sites, abscesses and the oral cavity. We found that A. actinomycetemcomitans is not restricted for iron in a murine abscess mono-infection, but becomes restricted for iron upon co-infection with the oral commensal Streptococcus gordonii. Furthermore, in the transition from health to disease in human gum infection, A. actinomycetemcomitans also becomes restricted for iron. These results suggest that host iron availability is heterogeneous and dependent on the infecting bacterial community.

Methods to Study Antagonistic Activities Among Oral Bacteria.

Most bacteria in nature exist in multispecies communities known as biofilms. In the natural habitat where resources (nutrient, space, etc.) are usually limited, individual species must compete or collaborate with other neighboring species in order to perpetuate in the multispecies community. The human oral cavity is colonized by >700 microbial species known as the indigenous microbiota. This indigenous flora normally maintains an ecological balance through antagonistic as well as mutualistic interspecies interactions. However, environmental perturbation may disrupt this balance, leading to overgrowth of pathogenic species which could in turn initiate diseases such as dental caries (tooth decay) and periodontitis (gum disease). Understanding the mechanisms of diversity maintenance may help developing novel approaches to manage these "polymicrobial diseases." In this chapter, we focus on a well-characterized form of biochemical warfare: bacteriocins produced by Streptococcus mutans, a primary dental caries pathogen, and hydrogen peroxide (H2O2) produced by several oral commensal streptococci. We will describe detailed methodologies on the competition assay, isolation, purification, and characterization of bacteriocins.

Capsular Polysaccharide Expression in Commensal Streptococcus Species: Genetic and Antigenic Similarities to Streptococcus pneumoniae.

ABSTRACTExpression of a capsular polysaccharide is considered a hallmark of most invasive species of bacteria, including Streptococcus pneumoniae, in which the capsule is among the principal virulence factors and is the basis for successful vaccines. Consequently, it was previously assumed that capsule production distinguishes S. pneumoniae from closely related commensals of the mitis group streptococci. Based on antigenic and genetic analyses of 187 mitis group streptococci, including 90 recognized serotypes of S. pneumoniae, we demonstrated capsule production by the Wzy/Wzx pathway in 74% of 66 S. mitis strains and in virtually all tested strains of S. oralis (subspecies oralis, dentisani, and tigurinus) and S. infantis. Additional analyses of genomes of S. cristatus, S. parasanguinis, S. australis, S. sanguinis, S. gordonii, S. anginosus, S. intermedius, and S. constellatus revealed complete capsular biosynthesis (cps) loci in all strains tested. Truncated cps loci were detected in three strains of S. pseudopneumoniae, in 26% of S. mitis strains, and in a single S. oralis strain. The level of sequence identities of cps locus genes confirmed that the structural polymorphism of capsular polysaccharides in S. pneumoniae evolved by import of cps fragments from commensal Streptococcus species, resulting in a mosaic of genes of different origins. The demonstrated antigenic identity of at least eight of the numerous capsular polysaccharide structures expressed by commensal streptococci with recognized serotypes of S. pneumoniae raises concerns about potential misidentifications in addition to important questions concerning the consequences for vaccination and host-parasite relationships both for the commensals and for the pathogen.