Interactions between Streptococcus oralis,Actinomyces oris,and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual‐species biofilms, or three‐species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non‐fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.     

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.     

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.     

Genetic basis of coaggregation receptor polysaccharide biosynthesis in Streptococcus sanguinis and related species

Interbacterial adhesion between streptococci and actinomyces promotes early dental plaque biofilm development. Recognition of coaggregation receptor polysaccharides (RPS) on strains of Streptococcus sanguinis, Streptococcus gordonii and Streptococcus oralis by Actinomyces spp. type 2 fimbriae is the principal mechanism of these interactions. Previous studies of genetic loci for synthesis of RPS (rps) and RPS precursors (rml, galE1 and galE2) in S. gordonii 38 and S. oralis 34 revealed differences between these strains. To determine whether these differences are strain‐specific or species‐specific, we identified and compared loci for polysaccharide biosynthesis in additional strains of these species and in several strains of the previously unstudied species, S. sanguinis. Genes for synthesis of RPS precursors distinguished the rps loci of different streptococci. Hence, rml genes for synthesis of TDP‐L‐Rha were in rps loci of S. oralis strains but at other loci in S. gordonii and S. sanguinis. Genes for two distinct galactose epimerases were also distributed differently. Hence, galE1 for epimerization of UDP‐Glc and UDP‐Gal was in galactose operons of S. gordonii and S. sanguinis strains but surprisingly, this gene was not present in S. oralis. Moreover, galE2 for epimerization of both UDP‐Glc and UDP‐Gal and UDP‐GlcNAc and UDP‐GalNAc was at a different locus in each species, including rps operons of S. sanguinis. The findings provide insight into cell surface properties that distinguish different RPS‐producing streptococci and open an approach for identifying these bacteria based on the arrangement of genes for synthesis of polysaccharide precursors.     

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.     

Microbial dynamics during conversion from supragingival to subgingival biofilms in an in vitro model

The development of dental caries and periodontal diseases result from distinct shifts in the microbiota of the tooth‐associated biofilm. This in vitro study aimed to investigate changes in biofilm composition and structure, during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile. Biofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans and Veillonella dispar were aerobically grown in saliva‐containing medium on hydroxyapatite disks. After 64 h, Campylobacter rectus, Prevotella intermedia and Streptococcus anginosus were further added along with human serum, while culture conditions were shifted to microaerophilic. After 96 h, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola were finally added and the biofilm was grown anaerobically for another 64 h. At the end of each phase, biofilms were harvested for species‐specific quantification and localization. Apart from C. albicans, all other species gradually increased during aerobic and microaerophilic conditions, but remained steady during anaerobic conditions. Biofilm thickness was doubled during the microaerophilic phase, but remained steady throughout the anaerobic phase. Extracellular polysaccharide presence was gradually reduced throughout the growth period. Biofilm viability was reduced during the microaerophilic conversion, but was recovered during the anaerobic phase. This in vitro study has characterized the dynamic structural shifts occurring in an oral biofilm model during the switch from aerobic to anaerobic conditions, potentially modeling the conversion of supragingival to subgingival biofilms. Within the limitations of this experimental model, the findings may provide novel insights into the ecology of oral biofilms.     

In vitro study of biofilm formation and effectiveness of antimicrobial treatment on various dental material surfaces

Elevated proportions of Candida albicans in biofilms formed on dentures are associated with stomatitis whereas Streptococcus mutans accumulation on restorative materials can cause secondary caries. Candida albicans, S. mutans, saliva‐derived and C. albicans/saliva‐derived mixed biofilms were grown on different materials including acrylic denture, porcelain, hydroxyapatite (HA), and polystyrene. The resulting biomass was analysed by three‐dimensional image quantification and assessment of colony‐forming units. The efficacy of biofilm treatment with a dissolved denture cleansing tablet (Polident^®) was also evaluated by colony counting. Biofilms formed on HA exhibited the most striking differences in biomass accumulation: biofilms comprising salivary bacteria accrued the highest total biomass whereas C. albicans biofilm formation was greatly reduced on the HA surface compared with other materials, including the acrylic denture surface. These results substantiate clinical findings that acrylic dentures can comprise a reservoir for C. albicans, which renders patients more susceptible to C. albicans infections and stomatitis. Additionally, treatment efficacy of the same type of biofilms varied significantly depending on the surface. Although single‐species biofilms formed on polystyrene surfaces exhibited the highest susceptibility to the treatment, the most surviving cells were recovered from HA surfaces for all types of biofilms tested. This study demonstrates that the nature of a surface influences biofilm characteristics including biomass accumulation and susceptibility to antimicrobial treatments. Such treatments should therefore be evaluated on the surfaces colonized by the target pathogen(s).     

Effect of the quorum‐sensing luxS gene on biofilm formation by Enterococcus faecalis

Enterococcus faecalis is the species of bacterium most frequently isolated from the root canals of teeth that exhibit chronic apical periodontitis refractory to endodontic treatment. In this study, we evaluated the effect of the S‐ribosylhomocysteine lyase (luxS) quorum‐sensing gene on E. faecalis biofilm formation by constructing a knockout mutant. The biofilms formed by both E. faecalis and its luxS mutant strain were evaluated using the MTT method. Important parameters that influence biofilm formation, including cell‐surface hydrophobicity and the nutrient content of the growth medium, were also studied. Biofilm structures were observed using confocal laser scanning microscopy (CLSM), and expression of biofilm‐related genes was investigated using RT‐PCR. The results showed that the luxS gene can affect biofilm formation, whereas it does not affect the bacterial growth rate. Deletion of the luxS gene also increased cell‐surface hydrophobicity. Biofilm formation was accelerated by the addition of increasing concentrations of glucose. The CLSM images revealed that the luxS mutant strain tends to aggregate into distinct clusters and relatively dense structures, whereas the wild‐type strain appears confluent and more evenly distributed. All genes examined were up‐regulated in the biofilms formed by the luxS mutant strain. The quorum‐sensing luxS gene can affect E. faecalis biofilm formation.     

Architectural analysis, viability assessment and growth kinetics of Candida albicans and Candida glabrata biofilms

The human fungal pathogen Candida is able to form biofilms in almost all the medical devices in current use. Indeed, biofilm formation is a major virulence attribute of microorganisms and account for a majority of human infections. Therefore, understanding processes appertaining to biofilm development is an important prerequisite for devising new strategies to prevent or eradicate biofilm-related infections. In the present study we used an array of both conventional and novel analytical tools to obtain a comprehensive view of Candida biofilm development. Enumeration of colony forming units, colorimetric (XTT) assay, Scanning Electron Microscopy (SEM) and novel Confocal Laser Scanning Microscopy (CLSM) coupled with COMSTAT software analyses were utilised to evaluate growth kinetics; architecture and viability of biofilms of a reference (ATCC) and a clinical strain each of two Candida species, C. albicans and C. glabrata. Biofilm growth kinetics on a polystyrene substrate was evaluated from the initial adhesion step (1.5 h) up to 72 h. These analyses revealed substantial inter- and intra-species differences in temporal organisation of Candida biofilm architecture, spatiality and cellular viability, while reaching maturity within a period of 48 h, on a polystyrene substrate. There were substantial differences in the growth kinetics upon methodology, although general trend seemed to be the same. Detailed architectural analysis provided by COMSTAT software corroborated the SEM and CSLM views. These analyses may provide a strong foundation for down stream molecular work of fungal biofilms.     

Adherence of Candida albicans to silicone is promoted by the human salivary protein SPLUNC2/PSP/BPIFA2

Interactions between Candida albicans, saliva and saliva‐coated oral surfaces are initial events in the colonization of the oral cavity by this commensal yeast, which can cause oral diseases such as candidiasis and denture stomatitis. Candida albicans also colonizes silicone voice prostheses, and the microbial biofilm formed can impair valve function, necessitating frequent prosthesis replacement. We have previously shown that saliva promoted binding of C. albicans cells to silicone in vitro, and that the selective binding of specific salivary proteins to voice prosthesis silicone mediated attachment of C. albicans cells. The C. albicans cells adhered to a polypeptide (or polypeptides) of ~36 kDa eluted from saliva‐treated silicone. We show here that a protein of similar size was identified in replicate blots of the eluate from saliva‐treated silicone when the blots were probed with antibodies to human SPLUNC2, a salivary protein with reported microbial agglutination properties. In addition, SPLUNC2 was depleted from saliva that had been incubated with silicone coupons. To determine whether SPLUNC2 is a yeast‐binding protein, SPLUNC2 cDNA was expressed in Escherichia coli. Purified recombinant His‐tagged protein (SPLUNC2r) bound to silicone as demonstrated by immunoblot analysis of an eluate from SPLUNC2r‐treated silicone coupons and ³⁵S‐radiolabelled C. albicans cells adhered in a dose‐dependent manner to SPLUNC2r‐coated silicone. We conclude that SPLUNC2 binds to silicone and acts as a receptor for C. albicans adherence to, and subsequent colonization of, voice prosthesis silicone.     

Extracellular proteinases of Candida species pathogenic yeasts

The increased incidence of severe disseminated infections caused by the opportunistic yeast‐like fungi Candida spp. highlights the urgent need for research into the major virulence factors of these pathogens—extracellular aspartic proteinases of the candidapepsin and yapsin families. Classically, these enzymes were considered to be generally destructive factors that damage host tissues and provide nutrients for pathogen propagation. However, in recent decades, novel and more specific functions have been suggested for extracellular candidal proteinases. These include contributions to cell wall maintenance and remodeling, the formation of polymicrobial biofilms, adhesion to external protective barriers of the host, the deregulation of host proteolytic cascades (such as the complement system, blood coagulation and the kallikrein–kinin system), a dysregulated host proteinase–inhibitor balance, the inactivation of host antimicrobial peptides, evasion of immune responses and the induction of inflammatory mediator release from host cells. Only a few of these activities recognized in Candida albicans candidapepsins have been also confirmed in other Candida species, and characterization of Candida glabrata yapsins remains limited.     

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.     

In vitro characterization of biofilms formed by Kingella kingae

The Gram‐negative bacterium Kingella kingae is part of the normal oropharyngeal mucosal flora of children <4 years old. K. kingae can enter the submucosa and cause infections of the skeletal system in children, including septic arthritis and osteomyelitis. The organism is also associated with infective endocarditis in children and adults. Although biofilm formation has been coupled with pharyngeal colonization, osteoarticular infections, and infective endocarditis, no studies have investigated biofilm formation in K. kingae. In this study we measured biofilm formation by 79 K. kingae clinical isolates using a 96‐well microtiter plate crystal violet binding assay. We found that 37 of 79 strains (47%) formed biofilms. All strains that formed biofilms produced corroding colonies on agar. Biofilm formation was inhibited by proteinase K and DNase I. DNase I also caused the detachment of pre‐formed K. kingae biofilm colonies. A mutant strain carrying a deletion of the pilus gene cluster pilA1pilA2fimB did not produce corroding colonies on agar, autoaggregate in broth, or form biofilms. Biofilm forming strains have higher levels of pilA1 expression. The extracellular components of biofilms contained 490 μg cm^?2 of protein, 0.68 μg cm^?2 of DNA, and 0.4 μg cm^?2 of total carbohydrates. We concluded that biofilm formation is common among K. kingae clinical isolates, and that biofilm formation is dependent on the production of proteinaceous pili and extracellular DNA. Biofilm development may have relevance to the colonization, transmission, and pathogenesis of this bacterium. Extracellular DNA production by K. kingae may facilitate horizontal gene transfer within the oral microbial community.     

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.     

Innocent until proven guilty: mechanisms and roles of Streptococcus–Candida interactions in oral health and disease

Candida albicans and streptococci of the mitis group colonize the oral cavities of the majority of healthy humans. While C. albicans is considered an opportunistic pathogen, streptococci of this group are broadly considered avirulent or even beneficial organisms. However, recent evidence suggests that multi‐species biofilms with these organisms may play detrimental roles in host homeostasis and may promote infection. In this review we summarize the literature on molecular interactions between members of this streptococcal group and C. albicans, with emphasis on their potential role in the pathogenesis of opportunistic oral mucosal infections.     

Salivary pellicles equalise surfaces’ charges and modulate the virulence of Candida albicans biofilm

IntroductionNumerous environmental factors influence the pathogenesis of Candida biofilms and an understanding of these is necessary for appropriate clinical management.AimsTo investigate the role of material type, pellicle and stage of biofilm development on the viability, bioactivity, virulence and structure of C. albicans biofilms.MethodsThe surface roughness (SR) and surface free energy (SFE) of acrylic and titanium discs was measured. Pellicles of saliva, or saliva supplemented with plasma, were formed on acrylic and titanium discs. Candida albicans biofilms were then generated for 1.5 h, 24 h, 48 h and 72 h. The cell viability in biofilms was analysed by culture, whilst DNA concentration and the expression of Candida virulence genes (ALS1, ALS3 and HWP1) were evaluated using qPCR. Biofilm metabolic activity was determined using XTT reduction assay, and biofilm structure analysed by Scanning Electron Microscopy (SEM).ResultsWhilst the SR of acrylic and titanium did not significantly differ, the saliva with plasma pellicle increased significantly the total SFE of both surface. The number of viable microorganisms and DNA concentration increased with biofilm development, not differing within materials and pellicles. Biofilms developed on saliva with plasma pellicle surfaces had significantly higher activity after 24 h and this was accompanied with higher expression of virulence genes at all periods.ConclusionInduction of C. albicans virulence occurs with the presence of plasma proteins in pellicles, throughout biofilm growth. To mitigate such effects, reduction of increased plasmatic exudate, related to chronic inflammatory response, could aid the management of candidal biofilm-related infections.