ABSTRACT
The clinical significance of polymicrobial interactions, particularly those between commensal species with high pathogenic potential, remains largely understudied. Although the dimorphic fungal species Candida albicans and the bacterium Staphylococcus aureus are common cocolonizers of humans, they are considered leading opportunistic pathogens. Oral candidiasis specifically, characterized by hyphal invasion of oral mucosal tissue, is the most common opportunistic infection in HIV+ and immunocompromised individuals. In this study, building on our previous findings, a mouse model was developed to investigate whether the onset of oral candidiasis predisposes the host to secondary staphylococcal infection. The findings demonstrated that in mice with oral candidiasis, subsequent exposure to S. aureus resulted in systemic bacterial infection with high morbidity and mortality. Histopathology and scanning electron microscopy of tongue tissue from moribund animals revealed massive C. albicans hyphal invasion coupled with S. aureus deep tissue infiltration. The crucial role of hyphae in the process was demonstrated using a non-hypha-producing and a noninvasive hypha-producing mutant strains of C. albicans. Further, in contrast to previous findings, S. aureus dissemination was aided but not contingent upon the presence of the Als3p hypha-specific adhesion. Importantly, impeding development of mucosal C. albicans infection by administering antifungal fluconazole therapy protected the animals from systemic bacterial disease. The combined findings from this study demonstrate that oral candidiasis may constitute a risk factor for disseminated bacterial disease warranting awareness in terms of therapeutic management of immunocompromised individuals.
INTRODUCTION
A considerable number of infectious diseases involve multiple microbial species coexisting and interacting in a host (1, 2). In these infections, the presence of one microorganism predisposes the host to colonization by others and in additive polymicrobial infections two or more nonpathogenic microorganisms together can cause disease (1, 3). Adherence of organisms to surfaces and to each other is a prerequisite for establishing concurrent infections, which often stem from the formation of polymicrobial biofilms that form on biotic or abiotic surfaces (2, 4–6). In the oral cavity, the oral mucosa serves as an important barrier to the myriad of microbial species present in this complex environment. However, the interactions between the various species can be synergistic in that the presence of one microorganism generates a niche for other microorganisms (1, 5, 7–11).
Candida albicans is an opportunistic fungal species commonly colonizing human mucosal surfaces as a component of the normal microflora (12–14). However, when host defenses are weakened, C. albicans can proliferate causing an array of infections ranging from mucosal such as oral candidiasis (OC) to systemic infections that are often life-threatening (12, 15–17). Therefore, candidal infections are unsurprisingly often endogenous occurring when there is a disruption in host environment (13). Specifically, OC is considered an AIDS-defining opportunistic infection with up to 80% of HIV+ individuals suffering recurrent episodes during the course of their HIV disease progression (15, 18).
The ability of this highly adaptable fungal pathogen to transition from commensal to pathogen is primarily the result of its ability to morphologically switch between yeast and hyphal forms (14, 19, 20). In fact, the majority of C. albicans infections are associated with its ability to form biofilms where adhesion of yeast cells to the substrate is followed by proliferation and hypha formation resulting in a network of cells embedded in extracellular polymeric matrix (21–23). In the oral cavity, C. albicans coexists and forms tight associations with various commensal bacterial species forming complex mucosal biofilms, a phenomenon known to play an important factor in C. albicans colonization (2, 5, 10, 11, 24–26). Thus, when Candida infections arise, they often occur in association with bacteria (27). Among the bacterial species, Staphylococcus aureus specifically is a precarious opportunistic pathogen implicated in a variety of diseases, ranging from minor skin infections to more serious invasive diseases (28–30). However, it primarily exists as a commensal organism with approximately 30% of healthy humans colonized in their nasopharynx or on their skin. Although colonization itself is a harmless state, colonized individuals are at risk of endogenous infection when S. aureus enters otherwise sterile sites via wounds or indwelling medical devices. With the emergence of methicillin-resistant S. aureus (MRSA), this ubiquitous pathogen is becoming an even greater therapeutic challenge (31–33).
Our in vitro studies have demonstrated that S. aureus exhibits high affinity to the C. albicans hyphae as these species coexist in biofilm and identified the C. albicans hyphal specific adhesin Als3p to be involved in the coadherence process (34, 35). Further, our previous in vivo studies have indicated that the interaction of these species may carry important clinical implications (36, 37). In fact, several studies have reported the coisolation of these diverse species from a multitude of diseases such as periodontitis, denture stomatitis, cystic fibrosis, keratitis, ventilator-associated pneumonia, urinary tract catheters and burn wound infections (38–42).
Given the prevalence of OC as an opportunistic infection in immunocompromised populations and the established clinical similarity of OC in mice to that in the human host, we developed a mouse model to assess whether OC predisposes to secondary bacterial infection. Further, hypha-deficient mutant strains of C. albicans were used to demonstrate the crucial role of hyphae in the disease process and antifungal therapy was implemented as a strategy to impede the development of bacterial dissemination.
MATERIALS AND METHODS
Ethics statement.All animal experiments were conducted at the AAALAAC accredited Animal Facility of the University of Maryland in accordance with the USA Animal Welfare Act as regulated by the U.S. Department of Agriculture. Animal studies were approved by the University of Maryland Animal Care and Use Committee (IACUC protocol 0814012). This institution has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare, National Institutes of Health. The assurance number is A-3199-01.
Strains and growth conditions.The C. albicans wild-type strain SC5314 (43), the non-hypha-producing double mutant cph1Δ/efg1Δ strain (HLC54) (44), the tup1Δ mutant strain (BCa2-10) lacking the transcriptional repressor of filamentous growth and its parent strain (SN250) (45, 46), the als3Δ strain lacking the hyphal specific adhesin Als3p (47, 48) and an MRSA strain (MRSA-M2) recently sequenced by us (GenBank accession number AMTC00000000) (49) were used in the present study. C. albicans strains were grown in yeast peptone dextrose broth (Difco Laboratories) overnight at 30°C, and cells were equilibrated in fresh medium to an optical density at 600 nm of 1.0. S. aureus cultures were grown overnight in Trypticase soy broth (TSB; Difco) at 37°C and then grown in fresh TSB to mid-log phase. The cells were harvested, washed with phosphate-buffered saline (PBS), and then suspended in Hanks' balanced salt solution (Fisher Scientific), and suspensions were maintained at 30°C. C. albicans was used at a final cell density of 2 × 107 cells/ml, and S. aureus was used at a final cell density of 1 × 106 cells/ml.
Animals.Three-month-old pathogen-free female C57BL/6 mice (Charles River Laboratories) were used in these studies. Mice were housed at a maximum of five animals per cage. To suppress bacterial flora and allow C. albicans colonization, mice received ampicillin at a dose of 0.5 mg/ml in their drinking water 2 days prior to infection and throughout the study.
Infection model.The coinfection mouse model was performed in accordance with an established model of OC (50) with modification. Time-line of infection and treatment is illustrated in Fig. 1. Mice were rendered susceptible to candidiasis by subcutaneous administration (0.2 ml) of cortisone acetate (225 mg/kg of body weight) in the dorsum of the neck every other day starting 1 day before infection (total of three injections). Animals were divided into groups, with five mice in each group: mono- or coinfected groups with WT or mutant strains and groups with or without therapy. On the day of infection, mice were anesthetized by intraperitoneal injections (0.5 ml) of Tribromoethanol (Sigma-Aldrich; 250 mg/kg of body weight). While under anesthesia, the animals were placed on heating pads (Braintree Scientific) maintained at 37°C. Anesthetized animals were weighed and then orally inoculated by placing calcium alginate swabs (Fisher Scientific) saturated for 5 min with C. albicans yeast cell suspension sublingually for 50 min (Fig. 2A). Animals were placed in a supine position on isothermal pads and monitored until they recovered from anesthesia. Two days after C. albicans infection, the animals were similarly infected with S. aureus cell suspension; however, in addition, S. aureus was also added to drinking water at a final cell density of 106 cells/ml for the duration of experiments. Animals were monitored daily for any developing clinical signs of distress, and those showing signs of severe disease or loss of significant body weight were euthanized. Animals were euthanized 5 days postinfection with C. albicans by CO2 inhalation, followed by cervical dislocation, and then weighed. The tongues and kidneys were harvested, weighed, homogenized, and cultured in triplicate on bacterial and yeast chromogenic media CHROMagar (DRG International, Inc.). The plates were incubated for 48 h at 37°C, and viable counts were enumerated and expressed as the log CFU/g of tissue.
Infection timeline. A timeline for infection and antifungal therapy model beginning with immunosuppression the day prior to infection with C. albicans until animals are euthanized 5 days after C. albicans infection is shown.
Mouse infection model. (A) Anesthetized mice were infected sublingually using swabs saturated with C. albicans cell suspensions. (B) At 3 days postinfection with the wild-type strains, white lesions can be seen on the surface of the tongue, and at the day animals are euthanized, advanced oral candidiasis characterized by white plaques is seen covering the tongue and other oral surfaces (red arrows). (C) A similar clinical picture is seen in animals infected with the als3 strain (red arrows). (D) No lesions, however, were visible in mice infected with C. albicans receiving fluconazole therapy.
Antifungal therapy.In order to demonstrate that S. aureus does not disseminate in the absence of OC, some animals were given daily intraperitoneal injections (0.2 ml) of fluconazole (West-Ward Pharmaceutical Corp.; 16 mg/kg of body weight) beginning the day of C. albicans infection until they were euthanized, and then the tissue was harvested and processed as described. Control animals were treated with sterile PBS. Experiments were performed to demonstrate lack of any effect for fluconazole on S. aureus colonization.
Histopathology of infected tongue tissue.One half of harvested tongues were fixed and processed for histopathology. In addition, kidneys from mice with severe disease were also subjected to histopathology. The tissue was embedded in paraffin and sectioned, the sections were deparaffinized with xylene and stained with periodic acid-Schiff (PAS) or Gram stain for better visualization of bacteria, and the slides were examined using light microscopy. In order to rule out the gastrointestinal tract as a portal of entry for S. aureus dissemination, sections from the small and large intestines were also harvested and processed.
SEM of infected tongue tissue.For scanning electron microscopy (SEM) analysis, tongue tissue was fixed in 2% paraformaldehyde–2.5% glutaraldehyde and, after washing steps with PBS, postfixed with 1% osmium tetroxide, rinsed with PBS, and dehydrated using a series of washes with ethyl alcohol (30 to 100%). Samples were dried by critical point drying using an Autosamdri-810 (Tousimi), mounted on aluminum stubs, sputter coated with 10 to 20 nm of platinum/palladium, and imaged with a Quanta 200 scanning electron microscope (FEI Co., Hillsboro, OR).
Data analysis.The database for statistical analysis was built up by incremental addition of experimental data. In order to determine the optimal variables and conditions (infectious doses, endpoint of infection, etc.) and standardize the model, initial experiments were performed using small groups of animals. For the main experiments, a total of 11 experimental groups were tested, each including five randomly selected mice for each strain and experimental condition analyzed. All experiments were performed on at least three separate occasions, and averages were used to present the data. Since the experiments were dynamic (systemic infection, testing mutant strains, fluconazole therapy, etc.), they were conducted sequentially in phases, and several experimental sessions were performed to complete the database. Since some groups served as controls for subsequent experimental sets (such as coinfected), the number of animals in these groups was substantially higher than a total of 15 mice. Therefore, more data points for these groups were included in the analysis.
All statistical analysis was performed using GraphPad Prism 5.0 software. A Kruskal-Wallis one-way analysis of variance test was used to compare differences between multiple groups, and Dunn's multiple-comparison test was used to determine whether the difference between two samples was statistically significant. A Student unpaired t test was used to compare differences between two samples (Fig. 3D). P values of <0.05 were considered significant.
Tissue microbial recovery from mono- and coinfected mice with or without fluconazole therapy. At 5 days postinfection with C. albicans, mice were euthanized, and tissue samples were harvested and assessed for microbial burden. (A) Tongue fungal burden. A high level of C. albicans colonization was seen in mice infected with the wild-type alone (WT) or in combination with S. aureus (WT+SA). In contrast, no C. albicans was recovered from mice infected with the efg1/cph1 strain with some recovery from mice coinfected with S. aureus. On the other hand, mice infected with the asl3 strain with or without S. aureus recovery of als3 was comparable to that for the wild type. (B) Recovery of S. aureus from tongues. S. aureus was recovered from the majority of mice infected with S. aureus alone (SA) or in combination with the efg1/cph1 strain. However, significantly higher level of colonization was noted in mice coinfected with wild-type C. albicans (WT+SA) compared to infection with the efg1/cph1 strain. (C) C. albicans and S. aureus recovery from kidneys of coinfected animals. The microbial presence in kidneys was assessed as an indication of systemic disease. S. aureus was consistently recovered from the kidneys of all mice coinfected with wild-type strain (WT+SA) concomitant with clinical signs of disease. Similarly, although not consistent, S. aureus was recovered from kidneys of mice coinfected with the als3 strain (als3+SA). No organisms were recovered from the kidneys of all other groups, i.e., animals monoinfected or coinfected with C. albicans mutants. (D) C. albicans and S. aureus recovery from the tongues of fluconazole-treated and untreated animals. No C. albicans was recovered from any of the mice receiving fluconazole therapy with (CA/SA/F) or without (CA/F) S. aureus. However, S. aureus was recovered from coinfected fluconazole treated (SA/CA/F) mice although at significantly lower levels than the coinfected group not treated with fluconazole (SA/CA). Five animals were included in each experimental and control group, and all experiments were performed on at least three separate occasions. Since some groups served as controls for subsequent experiments, higher number of animals were tested, and therefore more data points were available for some experimental groups. Error bars represent standard error of the means; P values of <0.05 (*) were considered significant. NS, not significant.
RESULTS
Tissue microbial burden.To determine whether OC constitutes a risk factor for the development of secondary bacterial infection, 5 days following infection with C. albicans, mice were euthanized, and tissue samples were harvested for assessment of oral colonization and systemic infection. Findings demonstrated that all mice infected with the wild-type C. albicans strains (SC5314 and SN250) developed clinical OC where, 3 days postinfection, white lesions could be seen which rapidly progressed into overt candidiasis (white plaques) covering the tongue surface (Fig. 2B) with high level of C. albicans recovered. In the group coinfected with S. aureus and wild-type strains, in addition to OC, animals exhibited symptoms indicative of severe morbidity, including weight loss, hunch back, ruffled fur, and lethargy with a mortality rate of up to 20% and extensive recovery of both species from homogenized tongues (see Fig. 3A and B). More significantly, S. aureus was recovered from the kidneys of all moribund animals, and in some (30%) C. albicans was also recovered from kidneys. In contrast, mice infected with S. aureus or C. albicans individually, although organisms were recovered from the tongues (Fig. 3B), animals did not show any signs of systemic disease with no microbial presence in kidneys. Interestingly, significantly higher level of S. aureus colonization was seen when C. albicans was present (Fig. 3B). Although otherwise healthy, mice infected with C. albicans alone exhibited some weight loss, likely due to interference of lesions on ability to eat; however, in animals coinfected with both species, up to 25% of loss in body weight was seen concomitant with high morbidity and S. aureus presence in the kidneys.
In contrast to the wild-type C. albicans strains, mice infected with the non-hypha-producing mutant strain (efg1/cph1) did not develop any clinical disease, and no C. albicans was recovered from the tongues (Fig. 3A). Importantly, no organisms were recovered from the kidneys from any of the mice in this group.
To provide more insight into the mechanism leading to S. aureus dissemination, two additional C. albicans mutant strains were tested in the animal model in order to assess the importance of hyphal tissue invasiveness for disseminated disease (tup1 mutant strain) and to determine whether the staphylococcal dissemination is contingent upon the presence of the C. albicans hypha-specific Als3 adhesin (als3 mutant strain). As expected, the findings demonstrated that the tup1 did not colonize the oral cavity, affirming the importance of hyphae not only in causing disease but also in mediating colonization (data not shown). On the other hand, animals infected with the als3 mutant developed OC (Fig. 2C), and the recovery of both species from the tongues was comparable to that observed with the parent strain (Fig. 3A and B). However, in contrast to the parent strain, S. aureus was recovered from the kidneys of 50% of mice coinfected with S. aureus and the als3 mutant.
Fluconazole treatment.Of the mice receiving antifungal therapy, those infected with the wild-type strain and administered fluconazole did not develop OC (Fig. 2D), and no C. albicans was recovered from tongues with or without S. aureus coinfection (Fig. 3D). Similarly, significantly less S. aureus was recovered from the tongues of the coinfected group treated with fluconazole compared to those not treated (Fig. 3D). Notably, no organisms were recovered from the kidneys of any of the animals receiving fluconazole.
Histopathology of infected tissue.In addition to assessing microbial burden, tissue harvested from animals was also processed for histopathology in order to visualize the tissue invasion process. Microscopic analysis of PAS-stained (Fig. 4A and B) and Gram-stained (Fig. 4C) tissue sections revealed extensive fungal adherence and hyphal penetration of the epithelial tissue with a massive influx of inflammatory cells. Significantly, extensive and deep tongue tissue infiltration of the typically noninvasive S. aureus was seen (see Fig. S1 in the supplemental material) and, more importantly, in mice with severe disease S. aureus was also seen in stained kidney sections (Fig. 4D). Similarly, based on histopathology analysis of tongue tissue, the als3 strain exhibited invasive ability comparable to that of the wild-type strain (see Fig. S2A in the supplemental material). Although S. aureus could be seen within the damaged epithelium, its association with the invasive hyphae was to a lesser extent than that with the wild-type strain (see Fig. S2B, C, and D in the supplemental material). Histopathology analysis of gastrointestinal tissue sections indicated no signs of hyphal invasion or pathology to the mucosal tissue (data not shown).
Histopathology images of tissue sections of tongues from coinfected animals. Tongues were also subjected for histopathology and examined by light microscopy. (A) Representative image from PAS-stained section of tongues from mice coinfected with S. aureus and wild-type C. albicans demonstrating extensive presence and penetration of epithelial tissue by the invasive C. albicans hyphae. (B) Upon magnification, massive amounts of S. aureus could be seen on the outer epithelial layer intertwined with the invading hyphae with marked presence of host inflammatory cells. (C) Tissue sections were also stained with Gram stain for better visualization of the bacteria. An image of tissue from within the epithelium shows bacterial cells adhering to penetrated hyphae. (D) Image of Gram-stained kidney tissue sections from coinfected mice with systemic disease showing the presence of S. aureus cells. Arrows: white, hyphae; black, S. aureus. Bar, 20 μm.
SEM of infected tongue tissue.In addition, infected tongues were also subjected to SEM analysis. Consistent with histopathology, SEM analysis revealed a thick matrix covering the whole surface of the tongue consisting of C. albicans hyphae, S. aureus, and host cells (Fig. 5A and B). Upon higher magnification, C. albicans hyphae could be seen penetrating the outer epithelial layer of the tongue from the sublingual area where it was inoculated (Fig. 5C), leaving gaps in the tissue at sites of emergence (Fig. 5D). In addition to the outer surface, tongues were also sectioned prior to processing in order to visualize the subepithelial tissue (Fig. 6A, red box). Analysis of the deep tissue area revealed extensive presence of both hyphae and S. aureus (Fig. 6B). Importantly, upon higher magnification, significant amount of S. aureus was seen within the tissue in some cases associated with the hyphae (Fig. 6C and D). Figure 7 is a false-colored image for better visualization of the deep copenetration of C. albicans hyphae and S. aureus in coinfected mice.
Scanning electron micrographs of outer tongue epithelium from mice with oral candidiasis and systemic infection. (A) Low-magnification (×100) overview image of an excised tongue with advanced OC showing the thick biofilm formed on the surface. (B) Magnified image (×2,000) of the surface of tongue showing the massive matrix consisting of C. albicans hyphae invading the tissue with S. aureus intertwined with the hyphae. (C) Higher-magnification (×5,000) image of the outer surface of the tongue showing the spiny layer of the tongue surface with hyphae penetrating the surface from the sublingual area where C. albicans was inoculated. (D) Magnified image (×10,000) showing a significant gap in the tissue caused by hyphae invading from the sublingual area as it emerges through the tongue surface.
Representative SEM images from within deep tissue of mice with oral candidiasis and systemic infection. (A) Lower-magnification (×200) image demonstrating the massive biofilm matrix formed on the surface of the tongue with sloughing of tissue. (B) Image (×5,000) from within the tongue tissue (red box) showing deep hyphal invasion and the associated presence of S. aureus. (C and D) High-magnification images (×8,000 and ×10,000, respectively) showing hyphae penetrating crevices within the tissue, with an overwhelming amount of S. aureus adhering to tissue within the crevices.
False-colored SEM image. In order to better visualize the coinvasion of C. albicans hyphae and S. aureus into the subepithelial tissue, a captured image was false colored using Adobe Photoshop. The micrograph shows the invasive hyphae (purple) within the tissue, with infiltrated S. aureus adhering to the hyphae and the surrounding tissue.
DISCUSSION
The human mouth with its diverse niches and ample supply of nutrients is undoubtedly conducive for the unrestricted formation of natural microbial biofilms, such as those found on the oral mucosal tissue, where a multitude of microbial species coexist (10, 51). Mucosal biofilms have been implicated in a wide variety of infections, and it is therefore imperative that we understand the implications of the interactions between colonizing microbial species as they coexist on host tissue surfaces (7).
Oral candidiasis is perhaps the most common oral mucosal infection particularly in immunocompromised individuals. Although C. albicans is commonly encountered as a commensal in healthy individuals, this opportunistic pathogen is capable of invading virtually any site of the human host, from superficial sites to deep tissues and organs. C. albicans and S. aureus are frequent colonizers of human mucosal surfaces; using confocal fluorescent and atomic force microscopy, our previous in vitro studies revealed a unique mixed biofilm architecture wherein S. aureus associated closely with the hyphae (34, 52). However, in order to investigate the clinical implications of this interaction during cocolonization of host mucosal tissue, a suitable animal model is needed.
In the present study, at 3 days after oral infection with C. albicans, localized lesions consistent with OC were readily seen on the tongues rapidly progressing into overt candidiasis. Given the massive hyphal invasion and deep infiltration of S. aureus observed upon microscopic analysis of tongues, it was not surprising that the mice with OC became moribund within 2 days of exposure to S. aureus, indicating that S. aureus rapidly gained entry into the circulatory system.
The ability of C. albicans to switch morphology between a yeast and hyphal form is central to its pathogenesis (19). Where the yeast form is associated with systemic disease the hyphae are adept at causing mucosal infections such as OC and Candida vaginitis, and therefore mutant strains of C. albicans that do not produce hyphae are not capable of causing mucosal infections (44, 46, 48). The importance of the hypha-mediated tissue damage manifested as OC in the development of secondary bacterial infection was clearly established when a mutant strain of C. albicans unable to form hyphae (efg1/cph1) and a strain producing noninvasive hyphae (tup1) failed to induce staphylococcal infection. Interestingly, however, although the mutant strain was not efficient at colonizing the oral cavity by itself, some recovery was noted in coinfected mice, indicating that the presence of S. aureus aided the mutant in colonization. Along these lines, it was also interesting that although the C. albicans was not recovered from the kidneys of any of the monoinfected mice, in some cases, C. albicans disseminated in some coinfected mice. Although it is not clear whether S. aureus contributed to C. albicans dissemination, it is noteworthy that a study by Xu et al. (53) demonstrated that mucosal commensal bacteria, namely, Streptococcus oralis, modified C. albicans virulence, suggesting pathogenic synergy. Whether a similar process occurs between C. albicans and S. aureus, however, remains to be investigated.
The dissemination of S. aureus was clearly via the extensive tissue damage caused by the invading hyphae. In fact, in some of our SEM images red blood cells could be seen around the hyphae and S. aureus as they coinvaded the tissue (data not shown), indicating that the process of invasion caused injury to the vascular system, thereby allowing S. aureus to gain entry. Adherence of S. aureus to the C. albicans biofilm matrix also likely played a key role in the process by enhancing its colonization and persistence on the tissue within the biofilm hyphal matrix as indicated by the significantly higher level of S. aureus recovery in the presence of C. albicans. In fact, in some images of infected tissue, infiltrating bacterial cells could be seen adhered to the hyphae. Since we have previously shown that the Als3p hyphal adhesin plays a role in S. aureus adherence to the hyphae, the als3 mutant strain was also used to coinfect a group of mice. Although our in vitro adherence studies demonstrated diminished S. aureus adherence to the hyphae, in the animal model the level of S. aureus recovery from tongues was comparable to that with the wild type. However, S. aureus was only recovered from the kidneys of 50% of the mice coinfected with the als3 mutant strain. Interestingly, in our previous study using a different model, animals were only directly inoculated once with S. aureus simultaneously with C. albicans to investigate whether Als3p is crucial during the early stages of colonization prior to development of OC (37). In contrast to the findings from the present study, Als3p was found to be necessary for S. aureus to disseminate (37). Combined, these findings are in line with the hypothesis that the process of C. albicans-mediated staphylococcal dissemination is multifactorial and OC development prior to bacterial exposure is sufficient to mediate systemic disease, and although not contingent upon, the process is likely augmented by the ability of S. aureus to adhere to the invasive hyphae via Als3p.
The azole fluconazole is the most commonly used antifungal agent for the prevention and treatment of OC (22, 54). Therefore, to further affirm the significant clinical implications of OC, a group of animals were administered daily fluconazole treatments. In contrast to the untreated group, those receiving therapy did not develop OC or systemic disease. Notably, significantly less S. aureus was recovered from the treated group, highlighting the importance of enhanced colonization and persistence of biofilm-associated microbial communities. Our findings are in line with those from a recent study by Roux et al. (55), who, using a rat model, showed that C. albicans airway colonization favors the development of bacterial pneumonia. Importantly, and similar to our findings, antifungal treatment decreased airway fungal colonization and in turn the risk of bacterial pneumonia. These findings are significant since they not only solidify the importance of implementing antifungal therapy upon the indication of OC but also suggest that antibiotic therapy may be similarly warranted in immunocompromised individuals, including HIV+ patients, who are prone to recurrent episodes of OC.
It is important to note, however, that although individuals with immune suppression are the most vulnerable, other mundane factors are also considered risk factors for candidiasis; these factors include steroid and antibiotic usage, hormonal imbalances, nutritional deficiency, age, diabetes, metabolic disorders, and denture usage, among others (13, 56, 57). In fact, denture stomatitis stemming from the adherence of C. albicans to denture material, followed by hyphal infiltration of the denture-associated palatal tissue, is prevalent in approximately 70% of denture wearers (58). Therefore, although our model is an immunocompromised model, it is conceivable to speculate that under certain conditions, potentially other individuals with OC could also be predisposed to secondary bacterial infections.
In the case of S. aureus, nasal carriage is a well-defined risk factor of infection with this bacterium, supported by the fact that in most of the cases strains isolated from colonization and infection sites are indistinguishable (59). Therefore, given the demonstrated propensity of staphylococci to disseminate in our animal model, it is feasible to speculate that a similar phenomenon may take place in a susceptible human host as these species cocolonize the oral and nasal mucosa. It is important to note, however, that although we focused here on C. albicans and S. aureus, C. albicans coexists in the oral cavity with more than 700 different bacterial species, so it is also conceivable that similar interactions with clinical relevance may occur between C. albicans and other species.
As our knowledge of interspecies interactions on mucosal niches expands, the role of C. albicans in pathogenesis might prove to be more intricate than currently recognized. The identification of oral candidiasis as a risk factor for disseminated bacterial disease carries serious clinical implications warranting awareness in terms of therapeutic management, particularly in immunocompromised individuals who experience recurrent episodes of oral candidiasis.
ACKNOWLEDGMENTS
This study was supported by the National Institutes of Health grant DE14424 to M.A.J.-R., by the Interuniversity Attraction Poles Programme initiated by the Belgian Science Policy Office (P.V.D. and M.A.J.-R.), by Flemish Science Foundation (FWO) grant WO.026.11N (P.V.D. and M.A.J.-R.), and by FWO postdoctoral fellowships awarded to S.K.
We thank Scott Filler for his input and assistance. We also thank Ru-Ching Hsia and the University of Maryland Core Imaging Facility for assistance with electron microscopy, Shariq Khan and Christina Tsui for their contributions, and Vincent Bruno for providing C. albicans strains.
FOOTNOTES
- Received 28 October 2014.
- Returned for modification 11 November 2014.
- Accepted 13 November 2014.
- Accepted manuscript posted online 24 November 2014.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.02843-14.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.
