Although the mouse and human oral commensal microbiota share very few similarities in health [ 48 ], there is increasing evidence that in states of immunosuppression lower abundance oral commensals such asare selectively enriched in both mice and humans [ 12 39 ]. Further systematic metagenomics studies are needed to identify global mucosal bacterial community differences in different mucosal sites and different immunosuppression states and mechanistically link these differences to the antimicrobial effector or regulatory functions of immune or mucosal epithelial cells affected by immunosuppression.

The type of immunosuppression can play a decisive role in modifying the local microbial environment whereinfections occur, since different types of immune deficiencies may drive qualitatively different bacterial community shifts. For example, mice given cortisone have an increased number of functionally competent infiltrating neutrophils, whereas mice given cytotoxic chemotherapy are severely neutropenic [ 12 46 ]. It is thus possible that bacteria that are primarily cleared by neutrophils overgrow during cytotoxic chemotherapy treatment, whereas neutrophil-resistant bacteria are more likely to survive during chronic use of corticosteroids. In support of this hypothesis, we recently showed that both cortisone and cytotoxic chemotherapy treatment with 5-FU cause a significant increase in the total bacterial biomass on the mouse oral mucosa [ 12 ]. However, the effects of these two immunosuppressive treatments on certain mucosal bacteria were different. Whereas in cortisone-treated mice the oral enterococcal biomass decreased, in 5-FU-treated mice the enterococcal biomass increased compared to untreated control mice [ 12 ]. This could be explained by the central role of neutrophils in the control of endogenous enterococcal overgrowth in mice [ 47 ].

A recent prospective study in a cancer cohort showed that the salivary bacterial microbiome was significantly altered during chemotherapy with enrichment of several Gram-negative species [ 41 ]. Chemotherapy disrupted the oral microbiome profoundly, with salivary bacterial communities showing a decrease in diversity correlating with the dose of the cytotoxic drug 5-fluorouracil (5-FU). Oral microbiome shifts could not be explained by antibiotic intake or by a selective antibacterial action of 5-FU [ 41 ]. Interestingly, salivary fungal communities were not disturbed by chemotherapy while antibiotic use was not correlated with the risk for developing oropharyngeal candidiasis [ 22 41 ]. A chemotherapy-induced increase in oral commensal bacterial burdens, particularly aciduric bacteria such asandhas also been reported in a chemotherapy treated breast cancer cohort [ 42 ]. Moreover, a recent systematic review focusing on evaluation of the impact of chemotherapeutic treatment on the oral microbiota in patients with cancer showed that during chemotherapy, there is an increase in bacteria of thefamily and inspecies, potentially contributing to local oral manifestations, such as oral mucositis, or even systemic infections such as septicemia [ 43 ]. Finally, HIV infection was recently associated with lower richness and diversity estimates in the oral bacterial microbiome, with several taxa having increased abundance in this host background. However, as in the cancer chemotherapy cohort above, in this cohort there were no major oral fungal taxonomic shifts associated with HIV infection [ 44 ].

All aspects ofvirulence and pathogenesis must be examined in the context of the specific host immune status, since most mucosal infections with this organism afflict immunocompromised hosts. Three well-recognized human immunodeficient states that are risk factors for candidiasis are chronic use of corticosteroids, intensive cancer chemotherapy, and advanced AIDS [ 3 37 ]. In addition to allowingovergrowth leading to infection, immunosuppression may change the overall microbial equilibrium by allowing certain bacterial species to overgrow as well. We conducted the first comprehensive evaluation of the effect of long- term immunosuppression on the oral bacterial microbiome in solid organ transplant recipients using high throughput sequencing of salivary 16S rRNA gene amplicons [ 38 ]. Ninety percent of these patients were on chronic corticosteroid use. Compared to a control group, transplant status significantly influenced salivary bacterial community membership with the corticosteroid dose showing significant correlation with bacterial richness and the relative abundance of several bacteria genera.frequency and abundance was significantly increased in transplant patients, together with, andspecies. Network correlation analysis also showed abundance of mitis groupto be positively associated with transplant status and with opportunistic pathogens such as 38 ]. In a similar study of the colon microbiome in transplant patients, major shifts were also reported with a predominant increase in the proportion ofand a decrease in other Firmicutes, evident as early as the immediate post-transplant period when these patients are extremely susceptible to gastrointestinal candidiasis [ 39 ]. Finally, a study conducted in lung transplant recipients showed distinct shifts in the oropharyngeal bacterial communities associated with immunosuppression, with the vast majority of patients being co-colonized with an increased abundance ofandspecies [ 40 ].

Prior to our work, the role of indigenous bacteria in alimentary tract colonization was studied using combinations of broad-spectrum antibiotics in healthy mice and monitoring the growth of both bacteria andin the post-antibiotic period. Enhanced oropharyngeal and intestinal colonization ofwas noted with most broad-spectrum antibiotics in mice [ 30 33 ]. However, none of these studies examined the parallel growth of indigenous bacterial communities in the oropharyngeal region post-antibiotics. These studies revealed that a rise inspecies in the stomach and small and large intestine [ 30 33 ], andspecies in the colon [ 32 ] in the post-antibiotic period were associated with increasedcolonization. On the other hand, abundance ofwas inversely associated withcolonization in the lower GI-tract mucosa [ 30 32 ]. Importantly, using innovative predictive statistical models, Shankar and colleagues [ 32 ] showed greater dependence ofcolonization in the murine ileum and colon on certain bacterial genera such as streptococci than on the mucosal cytokine response in both sites. It is also well-established that commensal anaerobic bacteria are critical in limitingintestinal colonization in mice, and colonization levels are generally proportional to the level of antibiotic depletion of anaerobic bacteria [ 34 ]. However, such an antagonistic relationship between oral anaerobic bacteria andcolonization has not been established in the healthy murine oral mucosa.

We conducted the first experimental study that examined the influence ofcolonization on mucosal resident bacteria of mice with unperturbed indigenous microbiota [ 12 ]. Mice are not naturally colonized with, although they may harbor other indigenous species [ 29 ]. Our studies showed that daily oral inoculation of C57B/L6J mice with differentstrains induced a decrease in bacterial diversity in the oral mucosa. Surprisingly daily inoculation withhad an impact on oral biodiversity even though fungal colonization was below the sensitivity limit of the CFU assay in most healthy mice. Lower diversity was associated with an increase in the relative abundance of the genusand a decrease in. This positive effect ofinoculation onspecies was evident even with filamentation- defective strains associated with lower colonization. In contrast in the jejunum of the same mice,colonization caused an increase in bacterial diversity, suggesting that the effects ofcolonization on the local bacterial communities are site-specific [ 12 ]. Our findings in the small intestinal mucosa were in agreement with older studies showing that low level colonization of the murine cecum of healthy C57B/L6J mice byleads to shifts in the community structure such that bacterial communities in colonized mice are distinct from and more diverse than naive mice [ 30 ].

The oral cavity is home to hundreds of bacterial species and close to one hundred fungal species [ 19 20 ].is the most abundant fungal species in the oral microbiome [ 20 22 ], colonizes the alimentary tract of 30–70% of healthy individuals within the first few weeks of life, and persists without causing disease [ 23 ]. The frequency of colonization, number of species, and strains of oral. vary with age [ 24 ]. Interestingly, aging individuals with higherload have a lower bacterial diversity in their salivary microbiome and a distinct bacterial composition dominated by streptococci [ 25 ]. This positive relationship betweenand streptococcal mucosal colonization is corroborated by multiple studies reporting that in women of reproductive age vaginal carriage ofis an independent risk factor for vaginal colonization by group B 28 ].

It is widely accepted that in a healthy host, unperturbed resident commensal bacterial communities play an important role in limitingcolonization in mucosal sites. This may be accomplished by direct fungal–bacterial cell interactions involving secreted bacterial products, metabolic interactions, or indirectly by influencing the host response [ 11 ]. However, epidemiologic evidence for a significant effect of prolonged treatment with antibiotics on the incidence of oropharyngeal candidiasis is not strong and weakens the notion that homeostasis can only be maintained by unperturbed local bacterial communities. In fact, experimental evidence on the use of antibiotics in murine models shows that although antibiotics significantly increase oral fungal burdens, infection as evidenced by pathologic changes in the oral mucosa, requires some form of immunodeficiency [ 12 ]. On the other hand, several recent studies using murine models of oral infection have challenged the traditional thinking that most ubiquitous commensal bacteria have antagonistic relationships with. Indeed, these studies showed that certain oral streptococci have pathogenic synergy withleading to more severe oral opportunistic infections [ 13 18 ]. Most of these studies focused on individual bacterial species and very few investigated the interactions betweenand resident mucosal bacteriomes in health and disease. In this perspective we briefly highlight our recent work in a mouse model which focused on interactions ofwith resident oral mucosal bacteria and review recent evidence from human studies linking oropharyngeal candidiasis (OPC) to a dysbiotic shift of the oral bacterial microbiota.

Oropharyngeal candidiasis is the most prevalent fungal infection in patients with weakened or immature immune systems, such as HIV+ children, neonates, and patients with malignancies [ 1 3 ]. Persistent oropharyngeal and esophageal thrush is refractory to most antifungals and a significant clinical problem [ 4 5 ]. In states of severe immunosuppression these infections are associated with high morbidity and may lead to systemic disease with mortality ranging from 25–30% [ 6 ]. In patients treated with high dose cancer chemotherapy regimens cytotoxic damage to the oropharyngeal, esophageal, and gastrointestinal mucosae combined with myelosuppression, are thought to promote invasion of the mucosal barriers byhyphae and ultimately dissemination via the hepatic portal vein to the liver [ 7 8 ]. In most immunosuppressed states the main portal of entry ofinto the blood vessels is through the esophageal and gastrointestinal epithelium, while the oropharyngeal mucosa is thought to be a fungal reservoir seeding the lower gastrointestinal tract and alveolar mucosa [ 9 ]. Because the majority of such systemicinfections are acquired across the alimentary tract [ 9 10 ], it is of paramount importance to discover new approaches to treat or prevent these infections.

Although strong positive associations between C. albicans and certain oral bacterial species can be shown in human cohorts with candidiasis, causality is almost impossible to prove. Thus, the question whether certain indigenous bacteria influence C. albicans infection or whether changes in the bacterial microbiota are a result of C. albicans infection is extremely difficult to answer in human studies, even with prospective study designs and longitudinal repeated sampling of the same individuals. This limitation of human studies strengthens the rationale for asking these questions experimentally using mouse models where C. albicans can be introduced de novo and the effects on the local bacterial microbiota assessed. In addition, by conducting bacterial add-back experiments in antibiotics-depleted C. albicans -infected mice the effects of different bacterial species on the course of fungal infection can be elucidated.

In healthy patients with denture candidiasis an enrichment of the tongue mucosa withandwas recently reported [ 54 ]. This finding supports the concept that candidiasis occurs in a polymicrobial environment enriched with these bacteria consistent with our mouse tongue infection models [ 13 49 ]. We also conducted the first prospective study of oral microbiome changes in chemotherapy-treated cancer patients who develop oropharyngeal candidiasis [ 22 ]. In this cohort, development of oropharyngeal candidiasis was not associated with mycobiome structure shifts but was the result of increasedload, withbeing the most abundant species [ 22 ]. Similar to our mouse 5-FU model, in this human cohort consisting of nine patients who developed oral candidiasis, infection was associated with an increase in oral bacterial burdens, albeit not statistically significant. Although the identification of distinct dysbiotic shifts during the development of candidiasis was not possible in this small patient cohort, we were able to link a lower baseline bacterial diversity with a significantly increased risk for infection. In addition, subjects with increased risk were more abundantly colonized by aciduric bacteria including certainspecies. Our findings thus suggested that increased abundance of aciduric bacteria may be a risk factor underlying susceptibility to oropharyngeal candidiasis in chemotherapy. It is also worth noting that the effect of certain bacteriome members on infection risk was greater than the effect of the baseline proportions ofin the oral microbiome. Finally, in a recent study oral bacterial dysbiosis was implicated in oropharyngeal candidiasis in humans with hyper-IgE inflammatory syndrome, withidentified as the top abundant bacterial species during infection [ 55 ].

To explore a role of endogenousin fungal mucosal invasion in the 5-FU model,isolates from mice with oropharyngeal candidiasis were co-inoculated within organotypic mucosal constructs. These experiments showed increased invasion of mixed biofilms into the submucosal compartment.isolates synthesized gelatinase E and degraded recombinant E-cadherin increasing the permeability of oral epithelial cells in a transwell in vitro assay. Importantly, depletion of these organisms with antibiotics in vivo attenuated oral mucosal E- cadherin degradation andinvasion without affecting fungal burdens, indicating that bacterial community changes contribute to pathogenesis and represent overt dysbiosis [ 12 ].

To test whether these findings are immunosuppression type-specific we performed similar analyses in the cortisone-associated oropharyngeal candidiasis mouse model. Consistent with the 5- FU model, infection within cortisone-immunosuppressed mice caused a further increase in total oral bacterial burdens. However, in cortisone-treated mice with candidiasis the mucosal enterococcal biomass, as assessed by qPCR, was similar to healthy control mice. FISH staining of oral biofilms in these mice showed that the majority of endogenous bacteria forming biofilms withwere 53 ]. This finding together with evidence from others that cortisone-immunosuppressed mice inoculated with bothandshow increased oral pathology [ 51 ], implicatesas accessory pathogens in cortisone-associated oropharyngeal candidiasis.

Time-dependent analysis of beta diversity changes in the microbiomes of these mice showed thatinfection caused a profound disruption of the tongue and small intestinal community structure after 6 days of chemotherapy.infection was associated with reduction in mucosal bacterial diversity in both sites, with indigenousandspecies dominating the small intestinal, andrepresenting >90% of the oral mucosal communities. Endogenouswere identified in mixed tongue biofilms withusing genus and species ()-specific FISH probes, and their increase in these biofilms was validated by species-specific qPCR. In these mixed biofilmswas noted invading into the submucosal tongue compartment [ 12 ].

In immunocompromised hosts indigenous bacterial species that form mutualistic relationships withmay increase in abundance leading to a well-coordinated dysbiosis which amplifies mucosal damage. To support this concept, we conducted experiments using a cytotoxic cancer chemotherapy model, which recapitulates oral mucosal and bone marrow toxicity in cancer patients receiving 5-FU [ 45 ]. Whenis orally inoculated indigenous oral bacterial burdens rise in parallel with fungal burdens in mice receiving 5-FU [ 12 ], making the model ideal for the study of the role of resident oral bacteria in fungal pathogenesis. 5-FU-treated mice orally inoculated withgradually develop severe oroesophageal and intestinal candidiasis over the course of 8 days [ 12 52 ]. This prompted the longitudinal examination of site-specific indigenous bacterial changes associated with infection.infection led to a significant further increase in the oral mucosal bacterial biomass compared to 5-FU alone, with a strong positive correlation between fungal and bacterial loads in the same samples. This was contrasted by findings in the jejunum where bacterial loads decreased in response toinfection.

There is also mounting evidence for pathogenic synergy betweenandin murine models.andsynergy was shown in a cortisone oral infection model [ 51 ]. In a peritonitis model disease progression and microbial loads in mice infected with bothandwere significantly higher compared to those with monomicrobial infections [ 52 ]. In our cortisone oropharyngeal model and the peritonitis model developed by the Noverr lab, pathogenic synergy between bacteria andwas shown to be host-response mediated via induction of a significantly higher proinflammatory response [ 15 52 ]. In the peritonitis model, pathogenic synergy was primarily eicosanoid-mediated [ 52 ], whereas in our oropharyngeal model, an exaggerated TLR-2-dependent chemokine and neutrophil response was involved in pathogenesis [ 15 ].

Mounting experimental evidence in murine immunosuppression models supports a synergistic role of oralwith. Using a cortisone immunosuppression model, we revealed mutualistic relationships betweenand the mitis group member. We showed that whenis co-inoculated with, there is an increase in oral mucosal biofilms andvirulence as evidenced by increased mucosal fungal invasion [ 13 49 ]. Our work further showed thatsynergizes withto augment virulence directly by transcriptional activation of the Efg1 filamentation pathway. In particular,promoted- mediated filamentous growth ofand increased-dependentgene and protein expression on the surface of hyphae, enhancing interspecies co-aggregation and streptococcal colonization of the oral mucosa [ 17 ]. We also found that in orally co-inoculated cortisone-treated mice expression ofsecreted aspartyl protease genes (2, 4, 5, and 6) was increased relative to mono-infection. To examine the requirement of these proteases in mucosal invasion during co-infection, we used a Δ2456 deficient mutant which has strongly attenuated virulence in oral models. Surprisingly, in both organotypic and mouse streptococcal co-infection models the Δ2456 mutant partially regained the ability to form biofilms, invade, and disseminate to distant organs [ 16 ]. These studies suggest that virulence factors ofthat have been well established in other models, may become redundant in the polymicrobial environment of the alimentary tract, as was also shown recently by Noble and colleagues [ 50 ]. To investigate- independent factors that enhance fungal mucosal invasion we examined host enzymatic pathways that may play a role in mucosal barrier breach. We discovered thatanddecreased epithelial E-cadherin levels and increased mucosal invasion by synergistically increasing μ-calpain, a proteolytic enzyme that targets E-cadherin and occludin from epithelial junctions [ 16 ].

Based on the studies above it is evident that oral and other mucosal sites along the alimentary tract provide a shared ecological niche for members of the bacterial genera, and, with. Since immunosuppression can enrich the abundance of these bacteria on mucosal surfaces [ 38 40 ] as well as increase the risk of oropharyngeal candidiasis, this may have important implications in fungal pathogenesis.

3. Conclusions and Future Directions

C. albicans overgrowth results in bacterial dysbiosis with dominant species that have the ability to act as synergistic or accessory pathobionts ( Streptococci , or with low abundance transient species not considered part of the healthy oral microbiota, such as Staphylococci and Enterococci [ Enterococci [ Based on the human and experimental evidence presented above we propose a pathogenesis model in oropharyngeal candidiasis whereby immunosuppression coupled withovergrowth results in bacterial dysbiosis with dominant species that have the ability to act as synergistic or accessory pathobionts ( Figure 1 ). According to this pathogenesis model immunosuppression may lead to further enrichment with bacterial species which are ubiquitous members of the oral microbiota such as, or with low abundance transient species not considered part of the healthy oral microbiota, such asand 38 ]. Given the different effect of cortisone and 5-FU-induced immunosuppression on oral 12 ] we propose that the type of immunosuppression influences the type of bacterial dysbiosis associated with mucosal candidiasis. Whether oral bacterial community changes are affected by the type of immunosuppression in humans or mice requires more investigation. More prospective metagenomics studies are needed in humans with elevated risk for oropharyngeal candidiasis assessing both bacterial and fungal genomic components in the same oral samples longitudinally.

C. albicans virulence. In these studies, we identified endogenous Enterococci as synergistic pathobionts that augment C. albicans mucosal invasion [ C. albicans , Enterococcus species are a major concern in patient critical care due to resistance to multiple antibiotics [ Enterococci are considered transient commensals and carriage rates are low [ Enterococcus species (predominantly E. faecalis ) in chemotherapy patients or HIV positive patients rises to 82% [59, Candida and E. faecalis abundance increase over time and may place individuals at higher risk for mucosal pathology [ Enterococci and C. albicans in this host background. An older study using live Enterococcus organisms in a mouse intraperitoneal infection model showed that C. albicans promoted microbial tissue burdens and worsened infection outcomes [ Our studies in the cancer chemotherapy murine model underpin the hypothesis that bacterial community changes during infection represent a dysbiotic shift promotingvirulence. In these studies, we identified endogenousas synergistic pathobionts that augmentmucosal invasion [ 12 ]. Likespecies are a major concern in patient critical care due to resistance to multiple antibiotics [ 56 ]. In the oral cavity of healthy humansare considered transient commensals and carriage rates are low [ 57 ]. However, the oral carriage rate ofspecies (predominantly) in chemotherapy patients or HIV positive patients rises to 82% [ 58 60 ]. In particular, in chemotherapy patients bothandabundance increase over time and may place individuals at higher risk for mucosal pathology [ 61 ]. These are also some of the most high-risk populations for oropharyngeal candidiasis. Thus, our findings of a mutualistic relationship between these organisms in the murine chemotherapy model are relevant to the human condition and may have serious clinical implications for cancer chemotherapy patients [ 62 63 ]. However, more studies are needed to mechanistically dissect the synergistic interactions ofandin this host background. An older study using liveorganisms in a mouse intraperitoneal infection model showed thatpromoted microbial tissue burdens and worsened infection outcomes [ 64 ]. There are currently no infection models studying the interaction of these organisms in the oral mucosa and such models are urgently needed.

C. albicans breach of mucosal barriers may be exacerbated by mucosal bacteria, in a well-coordinated dysbiosis. There is currently limited information on how interactions of C. albicans with the resident bacterial microbiota can affect the oral mucosal barrier. Recent combined genomics and culture approaches have identified 76 culturable bacterial species in the murine lower GI tract [ As shown in our murine models,breach of mucosal barriers may be exacerbated by mucosal bacteria, in a well-coordinated dysbiosis. There is currently limited information on how interactions ofwith the resident bacterial microbiota can affect the oral mucosal barrier. Recent combined genomics and culture approaches have identified 76 culturable bacterial species in the murine lower GI tract [ 65 ]. A similar large-scale cultivation study in the murine oral cavity, which would allow more precise taxonomic and functional classification of bacterial sequences is not yet available. Without a comprehensive genomic database of sequences corresponding to murine culturable and uncultured bacterial species the information we glean from murine oral metagenomics studies will be of limited value.

Candida species [ Lactobacilli may reduce the oral colonization of C. albicans . In vitro studies have shown the ability of different Lactobacillus species to have variable effects on the macrophage cytokine response and pattern recognition receptor expression in response to C. albicans [ Lactobacillus or S. salivarius strains with a confirmed protective effect in preclinical murine immunosuppression models. Pathogenic synergy between C. albicans and oral dysbiotic bacteria also raises the possibility of exploring a convergent immunity approach to develop novel vaccine strategies, as has been reported for C. albicans and S. aureus [ C. albicans to promote pathogenesis may contribute to overall protective immunity against C. albicans . The limited efficacy and increased toxicity of available antifungal drugs, in addition to the emergence of drug-resistance inspecies [ 66 ], bring urgency to exploring alternative therapy or preventative strategies against fungal infections. US and international guidelines for the management of oropharyngeal candidiasis in high risk patients include local antifungal treatments as first line treatment, such as nystatin or amphotericin B mouthwashes, or miconazole mucoadhesive tablets. Second line antifungal treatment is usually with an oral systemic azole such as fluconazole [ 67 ]. We propose that in addition to addressing the fungal pathogen with such treatments, correcting the underlying bacterial dysbiosis will restrict fungal breach of mucosal barriers leading to bloodstream infections in hosts with weakened immune systems. Animal [ 68 69 ] and human studies [ 70 71 ] have shown that the daily consumption of probioticmay reduce the oral colonization of. In vitro studies have shown the ability of differentspecies to have variable effects on the macrophage cytokine response and pattern recognition receptor expression in response to 72 ]. Clinical trials are needed with probioticstrains with a confirmed protective effect in preclinical murine immunosuppression models. Pathogenic synergy betweenand oral dysbiotic bacteria also raises the possibility of exploring a convergent immunity approach to develop novel vaccine strategies, as has been reported forand 73 ]. Targeting an epitope from a bacterial species that occupies the same mucosal niche and interacts withto promote pathogenesis may contribute to overall protective immunity against