Candida–streptococcal mucosal biofilms display distinct structural and virulence characteristics depending on growth conditions and hyphal morphotypes

Candida albicans and streptococci of the mitis group form communities in multiple oral sites, where moisture and nutrient availability can change spatially or temporally. This study evaluated structural and virulence characteristics of Candida–streptococcal biofilms formed on moist or semidry mucosal surfaces, and tested the effects of nutrient availability and hyphal morphotype on dual‐species biofilms. Three‐dimensional models of the oral mucosa formed by immortalized keratinocytes on a fibroblast‐embedded collagenous matrix were used. Infections were carried out using Streptococcus oralis strain 34, in combination with a C. albicans wild‐type strain, or pseudohyphal‐forming mutant strains. Increased moisture promoted a homogeneous surface biofilm by C. albicans. Dual biofilms had a stratified structure, with streptococci growing in close contact with the mucosa and fungi growing on the bacterial surface. Under semidry conditions, Candida formed localized foci of dense growth, which promoted focal growth of streptococci in mixed biofilms. Candida biofilm biovolume was greater under moist conditions, albeit with minimal tissue invasion, compared with semidry conditions. Supplementing the infection medium with nutrients under semidry conditions intensified growth, biofilm biovolume and tissue invasion/damage, without changing biofilm structure. Under these conditions, the pseudohyphal mutants and S. oralis formed defective superficial biofilms, with most bacteria in contact with the epithelial surface, below a pseudohyphal mass, resembling biofilms growing in a moist environment. The presence of S. oralis promoted fungal invasion and tissue damage under all conditions. We conclude that moisture, nutrient availability, hyphal morphotype and the presence of commensal bacteria influence the architecture and virulence characteristics of mucosal fungal biofilms.     

Characterization of a stress tolerance‐defective mutant of Lactobacillus rhamnosus LRB

Lactobacillus rhamnosus is a lactic acid bacterium that survives diverse ecological niches, including the human oral cavity and gastrointestinal tract. L. rhamnosus is an acidogenic bacterium that produces copious amounts of lactic acid. The organism is also considered as aciduric, since it can survive prolonged exposure to an acidic environment. For a probiotic bacterium such as L. rhamnosus, it is necessary to understand how this organism survives acid stress. In this study we used L. rhamnosus LRB to isolate one spontaneous mutant that was sensitive to acid stress. The mutant, which we named RBM1, also displayed sensitivity to a wide range of stresses including osmotic, thermal, and others. Using whole genome sequencing, we mapped the putative mutations in the mutant strain. It appears that three single nucleotide substitutions occurred in the mutant as compared to the wild‐type LRB strain. Among those, the most relevant mutation occurred in the ftsH gene that created a single amino acid change in the protein. We performed a comparative proteomic study to understand the molecular basis for stress sensitivity and found that ~15% of the proteome is altered in the mutant strain. Our study suggests that generation of spontaneous mutants during L. rhamnosus colonization could drastically affect bacterial physiology and survival under stress conditions.     

Porphyromonas gingivalis gingipain is involved in the detachment and aggregation of Aggregatibacter actinomycetemcomitans biofilm

Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are major periodontal pathogens that cause several types of periodontal disease. Our previous study suggested that P. gingivalis gingipains secreted in the subgingival environment are related to the detachment of A.actinomycetemcomitans biofilms. However, it remains unclear whether arginine‐specific cysteine proteinase (Rgp) and lysine‐specific proteinase (Kgp) play different roles in the detachment of A. actinomycetemcomitans biofilm. The aim of this study was to investigate possible disruptive roles of Kgp and Rgp in the aggregation and attachment of A. actinomycetemcomitans. While P. gingivalis ATCC33277 culture supernatant has an ability to decrease autoaggregation and coaggregation of A. actinomycetemcomitans cells, neither the boiled culture supernatant of ATCC33277 nor the culture supernatant of KDP136 showed this ability. The addition of KYT‐1 and KYT‐36, specific inhibitors of Rgp and Kgp, respectively, showed no influence on the ability of P. gingivalis culture supernatant. The result of gelatin zymography suggested that other proteases processed by gingipains mediated the decrease of A. actinomycetemcomitans aggregations. We also examined the biofilm‐destructive effect of gingipains by assessing the detachment of A. actinomycetemcomitans from polystyrene surfaces. Scanning electron microscope analysis indicated that A. actinomycetemcomitans cells were detached by P. gingivalis Kgp. The quantity of A. actinomycetemcomitans in biofilm was decreased in co‐culture with P. gingivalis. However, this was not found after the addition of KYT‐36. These findings suggest that Kgp is a critical component for the detachment and decrease of A. actinomycetemcomitans biofilms.     

Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics

Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co‐aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that whereas antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity.     

Streptococcus oralis maintains homeostasis in oral biofilms by antagonizing the cariogenic pathogen Streptococcus mutans

Bacteria residing in oral biofilms live in a state of dynamic equilibrium with one another. The intricate synergistic or antagonistic interactions between them are crucial for determining this balance. Using the six‐species Zürich “supragingival” biofilm model, this study aimed to investigate interactions regarding growth and localization of the constituent species. As control, an inoculum containing all six strains was used, whereas in each of the further five inocula one of the bacterial species was alternately absent, and in the last, both streptococci were absent. Biofilms were grown anaerobically on hydroxyapatite disks, and after 64 h they were harvested and quantified by culture analyses. For visualization, fluorescence in situ hybridization and confocal laser scanning microscopy were used. Compared with the control, no statistically significant difference of total colony‐forming units was observed in the absence of any of the biofilm species, except for Fusobacterium nucleatum, whose absence caused a significant decrease in total bacterial numbers. Absence of Streptococcus oralis resulted in a significant decrease in Actinomyces oris, and increase in Streptococcus mutans (P < .001). Absence of A. oris, Veillonella dispar or S. mutans did not cause any changes. The structure of the biofilm with regards to the localization of the species did not result in observable changes. In summary, the most striking observation of the present study was that absence of S. oralis resulted in limited growth of commensal A. oris and overgrowth of S. mutans. These data establish highlight S. oralis as commensal keeper of homeostasis in the biofilm by antagonizing S. mutans, so preventing a caries‐favoring dysbiotic state.     

Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms

As a member of subgingival multispecies biofilms, Tannerella forsythia is commonly associated with periodontitis. The bacterium has a characteristic cell surface (S‐) layer modified with a unique O‐glycan. Both the S‐layer and the O‐glycan were analyzed in this study for their role in biofilm formation by employing an in vitro multispecies biofilm model mimicking the situation in the oral cavity. Different T. forsythia strains and mutants with characterized defects in cell surface composition were incorporated into the model, together with nine species of select oral bacteria. The influence of the T. forsythia S‐layer and attached glycan on the bacterial composition of the biofilms was analyzed quantitatively using colony‐forming unit counts and quantitative real‐time polymerase chain reaction, as well as qualitatively by fluorescence in situ hybridization and confocal laser scanning microscopy. This revealed that changes in the T. forsythia cell surface did not affect the quantitative composition of the multispecies consortium, with the exception of Campylobacter rectus cell numbers. The localization of T. forsythia within the bacterial agglomeration varied depending on changes in the S‐layer glycan, and this also affected its aggregation with Porphyromonas gingivalis. This suggests a selective role for the glycosylated T. forsythia S‐layer in the positioning of this species within the biofilm, its co‐localization with P. gingivalis, and the prevalence of C. rectus. These findings might translate into a potential role of T. forsythia cell surface structures in the virulence of this species when interacting with host tissues and the immune system, from within or beyond the biofilm.     

Alpha‐amylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa.LPS) is a major virulence factor associated with aggressive periodontitis. Although the recognition of Aa.LPS is potentially initiated by salivary proteins in the oral cavity, Aa.LPS‐binding proteins (Aa.LPS‐BPs) in saliva are poorly characterized. The purpose of this study was to capture and identify Aa.LPS‐BPs in human saliva using a LTQ‐Orbitrap hybrid Fourier transform mass spectrometry. Aa.LPS conjugated onto N‐hydroxysuccinimidyl‐Sepharose^® 4 Fast Flow beads (Aa.LPS‐beads) activated Toll‐like receptor 4 and produced nitric oxide and Interferon gamma‐inducible protein‐10, implying that the conjugation process did not alter the biological properties of Aa.LPS. Aa.LPS‐BPs were subsequently isolated from the nine human saliva samples from healthy individuals with the Aa.LPS‐beads followed by identification with the mass spectrometry. Aa.LPS‐BPs include α‐amylase, serum albumin, cystatin, lysozyme C, submaxillary gland androgen‐regulated protein 3B, immunoglobulin subunits, polymeric immunoglobulin receptor, deleted in malignant brain tumors 1, prolactin‐inducible protein, lipocalin‐1, and basic salivary proline‐rich protein 2. Specific binding was validated using a pull‐down assay with α‐amylase which was captured at the highest frequency. Alpha‐amylase demonstrated to interfere with the adherence and biofilm formation of A. actinomycetemcomitans. Even heat‐inactivated α‐amylase showed the interference to the same extent. Conclusively, we identified unique Aa.LPS‐BPs that provide useful information to understand bacterial pathogenesis and host innate immunity in the oral cavity.     

Lactobacillus rhamnosus LRB mediated inhibition of oral streptococci

Lactobacillus rhamnosus is a lactic acid bacterium with a diverse ecological habitat. We recently isolated a L. rhamnosus strain (LRB) from a healthy baby‐tooth that had naturally fallen out. We determined the whole genome sequence of LRB and found that the isolate is closely genetically related to an intestinal isolate, L. rhamnosus GG (ATCC 53103). However, the LRB genome had lost about a 75‐kb segment and undergone a genomic rearrangement. We assessed LRB's capacity to survive in the gut environment, at least temporarily. We found that LRB, like the intestinal isolate ATCC 53103, showed resistance to low pH but sensitive to bile salt. Surprisingly, we found that this oral isolate LRB showed strong antimicrobial activity against a variety of oral streptococci including Streptococcus mutans. The production of antimicrobial activity is dependent on media composition since some media supported the production while others did not. The production of antimicrobial activity is also dependent on growth temperature, with optimal production at 37°C. The antimicrobial activity was not restricted to streptococci, but effective against a variety of organisms, including ESKAPE pathogens.     

The pga gene cluster in Aggregatibacter actinomycetemcomitans is necessary for the development of natural competence in Ca2+‐promoted biofilms

Natural competence is the ability of bacteria to incorporate extracellular DNA into their genomes. This competence is affected by a number of factors, including Ca²⁺ utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca²⁺, the additive effects of Ca²⁺‐promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca²⁺‐promoted biofilms, and examined whether the pga gene cluster, required for bacterial cell aggregation, is necessary for competence development. The A. actinomycetemcomitans cells grown in the presence of 1 mm CaCl₂ exhibited enhanced cell aggregation and increased levels of cell‐associated Ca²⁺. Biofilm‐derived cells grown in the presence of Ca²⁺ exhibited the highest levels of natural transformation frequency and enhanced expression of the competence regulator gene, tfoX. Natural competence was enhanced by the additive effects of Ca²⁺‐promoted biofilms, in which high levels of pga gene expression were also detected. Mutation of the pga gene cluster disrupted biofilm formation and competence development, suggesting that these genes play a critical role in the ability of A. actinomycetemcomitans to adapt to its natural environment. The Ca²⁺‐promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.     

Antigen I/II mediates interactions between Streptococcus mutans and Candida albicans

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.     

Fermentative 2‐carbon metabolism produces carcinogenic levels of acetaldehyde in Candida albicans

Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast acetaldehyde is a by‐product of the pyruvate bypass that converts pyruvate into acetyl‐Coenzyme A (CoA) during fermentation. The aims of our study were: (i) to determine the levels of acetaldehyde produced by Candida albicans in the presence of glucose in low oxygen tension in vitro; (ii) to analyse the expression levels of genes involved in the pyruvate‐bypass and acetaldehyde production; and (iii) to analyse whether any correlations exist between acetaldehyde levels, alcohol dehydrogenase enzyme activity or expression of the genes involved in the pyruvate‐bypass. Candida albicans strains were isolated from patients with oral squamous cell carcinoma (n = 5), autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) patients with chronic oral candidosis (n = 5), and control patients (n = 5). The acetaldehyde and ethanol production by these isolates grown under low oxygen tension in the presence of glucose was determined, and the expression of alcohol dehydrogenase (ADH1 and ADH2), pyruvate decarboxylase (PDC11), aldehyde dehydrogenase (ALD6) and acetyl‐CoA synthetase (ACS1 and ACS2) and Adh enzyme activity were analysed. The C. albicans isolates produced high levels of acetaldehyde from glucose under low oxygen tension. The acetaldehyde levels did not correlate with the expression of ADH1, ADH2 or PDC11 but correlated with the expression of down‐stream genes ALD6 and ACS1. Significant differences in the gene expressions were measured between strains isolated from different patient groups. Under low oxygen tension ALD6 and ACS1, instead of ADH1 or ADH2, appear the most reliable indicators of candidal acetaldehyde production from glucose.     

Deficiency of PdxR in Streptococcus mutans affects vitamin B6 metabolism,acid tolerance response and biofilm formation

Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA‐binding N‐terminal domain and a C‐terminal domain highly homologous to the pyridoxal phosphate‐dependent aspartate aminotransferases. A pdxR‐deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR‐deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real‐time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B₆ biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR‐deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B₆. Further studies revealed that although S. mutans is known to require vitamin B₆ to grow in defined medium, B₆ vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B₆ metabolism, acid tolerance response and biofilm formation.     

SMU.940 regulates dextran‐dependent aggregation and biofilm formation in Streptococcus mutans

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence‐stimulating peptide. Eight competence‐stimulating peptide‐dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran‐dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild‐type. GbpC is known to be involved in the dextran‐dependent aggregation of S. mutans. An SMU.940‐gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran‐dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran‐dependent aggregation and biofilm formation.     

Bifidobacteria inhibit the growth of Porphyromonas gingivalis but not of Streptococcus mutans in an in vitro biofilm model

There is growing interest in the use of probiotic bifidobacteria for enhancement of the therapy, and in the prevention, of oral microbial diseases. However, the results of clinical studies assessing the effects of bifidobacteria on the oral microbiota are controversial, and the mechanisms of actions of probiotics in the oral cavity remain largely unknown. In addition, very little is known about the role of commensal bifidobacteria in oral health. Our aim was to study the integration of the probiotic Bifidobacterium animalis subsp. lactis Bb12 and of oral Bifidobacterium dentium and Bifidobacterium longum isolates in supragingival and subgingival biofilm models and their effects on other bacteria in biofilms in vitro using two different in vitro biofilms and agar‐overlay assays. All bifidobacteria integrated well into the subgingival biofilms composed of Porphyromonas gingivalis, Actinomyces naeslundii, and Fusobacterium nucleatum and decreased significantly only the number of P. gingivalis in the biofilms. The integration of bifidobacteria into the supragingival biofilms containing Streptococcus mutans and A. naeslundii was less efficient, and bifidobacteria did not affect the number of S. mutans in biofilms. Therefore, our results suggest that bifidobacteria may have a positive effect on subgingival biofilm and thereby potential in enhancing gingival health; however, their effect on supragingival biofilm may be limited.