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.     

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.     

Candida albicans induces early apoptosis followed by secondary necrosis in oral epithelial cells

The capacity of Candida albicans to invade and damage oral epithelial cells is critical for its ability to establish and maintain symptomatic oropharyngeal infection. Although oral epithelial cells are reported dead after 18 h of candidal infection, activation of specific epithelial cell‐death pathways in response to C. albicans infection has not yet been demonstrated. Considering the key role of oral epithelial cell damage in the pathogenesis of oropharyngeal candidiasis, the aim of this study was to characterize this event during infection. Using an oral epithelial–C. albicans co‐culture system, we examined the ability of C. albicans to induce classic necrotic, pyroptotic and apoptotic cellular alterations in oral epithelial cells such as osmotic lysis, exposure of phosphatidylserine on the epithelial cell plasma membrane and internucleosomal DNA fragmentation. It was found that the ability of C. albicans to kill oral epithelial cells depends on its capacity to physically interact with and invade these cells. Caspase‐dependent apoptotic pathways were activated early during C. albicans infection and contributed to C. albicans‐induced oral epithelial cell death. Earlier apoptotic events were followed by necrotic death of infected oral epithelial cells. Hence, C. albicans stimulates oral epithelial signaling pathways that promote early apoptotic cell death through the activation of cellular caspases, followed by late necrosis.     

Induction of apoptosis in oral epithelial cells by Candida albicans

During infection, interactions between Candida albicans and oral epithelial cells result in oral epithelial cell death. This is clinically manifested by the development of oral mucosal ulcerations generally associated with discomfort. In vitro studies have shown that C. albicans induces early apoptotic alterations in oral epithelial cells; however, these studies have also shown that treatment of infected cells with caspase inhibitors does not prevent their death. The reasons for these contradictory results are unknown and it is still not clear if C. albicans stimulates oral epithelial signaling pathways that promote apoptotic cell death. Activation of specific death pathways in response to microbial organisms plays an essential role in modulating the pathogenesis of a variety of infectious diseases. The aim of this study was to (i) characterize C. albicans‐induced apoptotic morphological alterations in oral epithelial cells, and (ii) investigate the activation of apoptotic signaling pathways and expression of apoptotic genes during infection. Candida albicans induced early apoptotic changes in over 50% of oral epithelial cells. However, only 15% of those showed mid‐late apoptotic alterations. At the molecular level, C. albicans caused a loss of the mitochondrial transmembrane potential and translocation of mitochondrial cytochrome c. Caspase‐3/9 activities increased only during the first hours of infection. Moreover, poly[ADP ribose] polymerase 1 was cleaved into apoptotic and necrotic‐like fragments. Finally, five anti‐apoptotic genes were significantly upregulated and two pro‐apoptotic genes were downregulated during infection. Altogether, these findings indicate that epithelial apoptotic pathways are activated in response to C. albicans, but fail to progress and promote apoptotic cell death.     

Granulocyte‐macrophage colony‐stimulating factor responses of oral epithelial cells to Candida albicans

Candida albicans is the principal fungal species responsible for oropharyngeal candidiasis, the most frequent opportunistic infection associated with immune deficiencies. Cytokines, such as granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), are important in the generation of effective immunity to C. albicans. The purposes of this investigation were to determine whether C. albicans triggers secretion of GM‐CSF by oral epithelial cells in vitro and to investigate mechanisms of host cell–fungal interactions that trigger such responses. Oral epithelial cell lines as well as primary oral mucosal epithelial cells were challenged with stationary phase viable C. albicans, added to human cell cultures at varying yeast:oral cell ratios. Yeast were allowed to germinate for up to 48 h and supernatants were analyzed for GM‐CSF by ELISA. Fixed organisms, germination‐deficient mutants and separation of yeast from epithelial cells using cell culture inserts were used to assess the effects of viability, germination and physical contact, respectively, on the GM‐CSF responses of these cells. Two out of three cell lines and three out of six primary cultures responded to C. albicans with an increase in GM‐CSF secretion. GM‐CSF responses were contact‐dependent, strain‐dependent, required yeast viability and were optimal when the yeast germinated into hyphae.     

Increase in detectable opportunistic bacteria in the oral cavity of orthodontic patients

Abstract: Objectives: This study was performed to detect the opportunistic bacteria and fungi from the oral cavities of orthodontic patients and examine the ability of the organisms to adhere to saliva‐coated metallic brackets. Methods: Opportunistic bacteria and fungi were isolated from 58 patients (orthodontic group: 42; non‐orthodontic group: 16) using culture methods and were identified based on their biochemical and enzymatic profiles. Seven opportunistic and four streptococcal strains were tested for their ability to adhere to saliva‐coated metallic brackets. Results: More opportunistic bacteria and fungi were detected in the orthodontic group than in the non‐orthodontic group (P < 0.05). Opportunistic bacteria adhered to saliva‐coated metallic brackets to the same degree as oral streptococci. Conclusions: The isolation frequencies of opportunistic bacteria and fungi increase during orthodontic treatment, suggesting the importance of paying special attention to oral hygiene in orthodontic patients to prevent periodontal disease and the aggravation of systemic disease in immunocompromised conditions.     

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.     

Oral candidosis in relation to oral immunity

Symptomatic oral infection with Candida albicans is characterized by invasion of the oral epithelium by virulent hyphae that cause tissue damage releasing the inflammatory mediators that initiate and sustain local inflammation. Candida albicans triggers pattern‐recognition receptors of keratinocytes, macrophages, monocytes and dendritic cells, stimulating the production of IL‐1β, IL‐6 and IL‐23. These cytokines induce the differentiation of Th17 cells and the generation of IL‐17‐ and/or IL‐22‐mediated antifungal protective immuno‐inflammatory responses in infected mucosa. Some immune cells including NKT cells, γδ T cells and lymphoid cells that are innate to the oral mucosa have the capacity to produce large quantities of IL‐17 in response to C. albicans, sufficient to mediate effective protective immunity against C. albicans. On the other hand, molecular structures of commensal C. albicans blastoconidia, although detected by pattern‐recognition receptors, are avirulent, do not invade the oral epithelium, do not elicit inflammatory responses in a healthy host, but induce regulatory immune responses that maintain tissue tolerance to the commensal fungi. The type, specificity and sensitivity of the protective immune response towards C. albicans is determined by the outcome of the integrated interactions between the intracellular signalling pathways of specific combinations of activated pattern‐recognition receptors (TLR2, TLR4, Dectin‐1 and Dectin‐2). IL‐17‐mediated protective immune response is essential for oral mucosal immunity to C. albicans infection.     

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.     

Antifungal drug susceptibility of oral Candida albicans isolates may be associated with apoptotic responses to Amphotericin B

J Oral Pathol Med (2010) 39 182–187 Background: Candida albicans is the important opportunistic fungal pathogens which can cause oral Candidiasis and even more seriously systemic infection. Apoptosis of C. albicans induced by environmental factor such as weak acid and antifungal drugs were studied recently. Illustrating the phenomenon of apoptosis in C. albicans may help us to discover new antifungal therapy by activating the fungal cells to suicide. Methods: Two oral C. albians clinical isolates which isolated respectively from healthy host [Strain 23C: minimal inhibition concentration (MIC) is 0.125 μg/ml for Amphotericin B (AmB)] and advanced cancer patient (Strain 28A: MIC is 2 μg/ml for AmB), were induced by 1 μg/ml AmB in vitro for 200 min, and then studied the apoptosis markers using terminal deoxynucletidyltransferase‐mediated dUTP nick end labeling (TUNEL) (shown by diaminobenzidine and fluorescent isothiocyanate), and the ultrastructure of cell nuclear using transmission electron microscope (TEM), quantitative analysis using flow cytometry for the rapid exposure of phosphatidylserine at the outer membrane and propodium iodide (PI) double staining. C. albicans conference strain YEM30 was used as the control strain. Results: With TUNEL assay and TEM, we detected the typical characteristics of apoptosis. Strain 23C (with low MIC) showed significantly higher percentage of apoptosis (19.92%) compared with Strain 28A (with high MIC) which was isolated from the cancer patient (7.29%) (P < 0.01). In addition, 7.3% of early apoptosis cells of Strain 23C can form colonies on the plates, while 15% for Strain 28A. None of the PI (+) cells can form colony. Conclusions: Apoptosis of oral C. albicans isolates can be induced by AmB. The feature of antifungal drug susceptibility of the oral C. albicans clinical isolates may associate with the response of apoptosis inducing.     

Differences between salivary and blood neutrophils from elderly and young denture wearers

Summary We previously showed that neutrophils from patients with Candida‐related denture stomatitis exhibited damaged function, and the advance in age intensified this condition. Because such alterations had been determined in elderly people that were not denture wearers, the purpose of this study was to clarify functional and phenotypic characteristics of neutrophils from elderly denture wearers (EDW) and young denture wearers (YDW) without oral lesion. We enrolled 20 denture wearers (12 EDW and 8 YDW) and determined the positivity of Candida species on maxillary prosthesis and palate. Additionally, blood and salivary neutrophils were evaluated. Furthermore, cytokines and chemokines salivary levels were detected. YDW presented higher positivity of Candida albicans than elderly ones. However, blood neutrophils from EDW expressed less CXCR1, CD62L and CD11b and had lower C. albicans phagocytosis than YDW. Although myeloperoxidase and elastase activity was significantly higher in C. albicans‐stimulated blood neutrophils from elderly, they produced high levels of IL‐10 and low levels of Granulocyte macrophage‐colony stimulating factor (GM‐CSF). Despite apoptosis rate of salivary neutrophils was enhanced, these cells were at a high number in YDW. GM‐CSF and IL10 were lower in saliva from elderly group. These data confirmed that ageing affects blood and salivary neutrophils and could predispose elderly to persistent oral infections.     

Correlation between the relative abundance of oral bacteria and Candida albicans in denture and dental plaques

ObjectivesThe opportunistic fungus Candida albicans is a component of denture plaque and is associated with denture-related stomatitis. Inter-kingdom interactions between C. albicans and bacteria exist in such multi-species biofilms, which may affect the microbial composition of the plaque. This study was performed to investigate the bacterial composition of denture plaques, and the correlation between the relative abundance of these bacteria and C. albicans.MethodsThirty denture plaque and 16 dental plaque samples were collected from 18 denture wearers (mean age, 80.3 years). After DNA extraction, a meta 16S rDNA amplicon library was constructed using PCR primers targeting the V3–V4 hypervariable region of bacteria. The amplicon was evaluated by high-throughput sequencing, followed by bacterial population analysis. The concentrations of both C. albicans DNA and total bacterial DNA were determined by real-time PCR. The correlation between the relative abundance of each bacterial genus and C. albicans was analyzed through Spearman's rank correlation.ResultsThe genera Streptococcus, Lactobacillus, Rothia, and Corynebacterium were found to be more abundant in dentures than in dental plaques. The predominant bacteria in healthcare-associated pneumonia also inhabited denture surfaces. C. albicans was positively correlated with three acidogenic bacteria and negatively correlated with Leptotrichia and pathogens associated with periodontitis and endocarditis.ConclusionsDentures may be significant reservoirs of pathogens causing aspiration pneumonia. Bacteria showing negative correlation with C. albicans, such as Leptotrichia, may be useful for controlling the growth of C. albicans in antifungal therapies.     

Oral epithelium–Candida glabrata interactions in vitro

Background: Oropharyngeal candidiasis is a common opportunistic infection and Candida glabrata is the second or third most frequently isolated species from oropharyngeal candidiasis lesions, after Candida albicans. The aim of this study was to study the cytokine‐inducing and cell‐damaging potential of C. glabrata in oral epithelial cells and compare this to C. albicans. Methods: Oral epithelial cell lines and primary gingival epithelial cells were cocultured with C. glabrata strains GDH2269 and 94‐11 or C. albicans strains SC5314 and ATCC28366. Supernatants were analysed for the presence of interleukin‐1α (IL‐1α), IL‐8 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) by enzyme‐linked immunosorbent assay. The cytotoxity of different strains was determined using the CytoTox‐96 assay. Results: Compared to C. albicans, C. glabrata induced different proinflammatory cytokine responses in oral epithelial cells; a high level of GM‐CSF induction was only detected in C. glabrata‐infected cells and not in C. albicans‐infected cells, regardless of the origin of these cells (cell lines or primary cells) or the strain used. Like C. albicans, C. glabrata induced an IL‐1α response by oral epithelial cells, but this response was both strain‐dependent and epithelial cell origin‐dependent. Unlike C. albicans, C. glabrata failed to induce a strong IL‐8 response in any of the cell systems studied. Finally, in these studies C. glabrata showed lower cytotoxicity than C. albicans. Conclusions: C. glabrata is less cytotoxic than C. albicans and induces different proinflammatory cytokine responses in oral epithelial cells.     

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.     

Absence of capsule reveals glycan‐mediated binding and recognition of salivary mucin MUC7 by Streptococcus pneumoniae

Salivary proteins modulate bacterial colonization in the oral cavity and interact with systemic pathogens that pass through the oropharynx. An interesting example is the opportunistic respiratory pathogen Streptococcus pneumoniae that normally resides in the nasopharynx, but belongs to the greater Mitis group of streptococci, most of which colonize the oral cavity. Streptococcus pneumoniae also expresses a serine‐rich repeat (SRR) adhesin, PsrP, which is a homologue to oral Mitis group SRR adhesins, such as Hsa of Streptococcus gordonii and SrpA of Streptococcus sanguinis. As the latter bind to salivary glycoproteins through recognition of terminal sialic acids, we wanted to determine whether S. pneumoniae also binds to salivary proteins through possibly the same mechanism. We found that only a capsule‐free mutant of S. pneumoniae TIGR4 binds to salivary proteins, most prominently to mucin MUC7, but that this binding was not mediated through PsrP or recognition of sialic acid. We also found, however, that PsrP is involved in agglutination of human red blood cells (RBCs). After removal of PsrP, an additional previously masked lectin‐like adhesin activity mediating agglutination of sialidase‐treated RBCs becomes revealed. Using a custom‐spotted glycoprotein and neoglycoprotein dot blot array, we identify candidate glycan motifs recognized by PsrP and by the putative S. pneumoniae adhesin that could perhaps be responsible for pneumococcal binding to salivary MUC7 and glycoproteins on RBCs.     

Polymicrobial interactions of Candida albicans and its role in oral carcinogenesis

The oral microbiome is composed of microorganisms residing in the oral cavity, which are critical components of health and disease. Disruption of the oral microbiome has been proven to influence the course of oral diseases, especially among immunocompromised patients. Oral microbiome is comprised of inter‐kingdom microorganisms, including yeasts such as Candida albicans, bacteria, archaea and viruses. These microorganisms can interact synergistically, mutualistically and antagonistically, wherein the sum of these interactions dictates the composition of the oral microbiome. For instance, polymicrobial interactions can improve the ability of C albicans to form biofilm, which subsequently increases the colonisation of oral mucosa by the yeast. Polymicrobial interactions of C albicans with other members of the oral microbiome have been reported to enhance the malignant phenotype of oral cancer cells, such as the attachment to extracellular matrix molecules (ECM) and epithelial‐mesenchymal transition (EMT). Polymicrobial interactions may also exacerbate an inflammatory response in oral epithelial cells, which may play a role in carcinogenesis. This review focuses on the role of polymicrobial interactions between C albicans and other oral microorganisms, including its role in promoting oral carcinogenesis.