Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans

Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model.


Introduction
The oral cavity is colonized with different microbial species that are usually organized in biofilms adhered to a solid surface such as dental enamel, root surface, or dental implants. An interesting characteristic of biofilms is the presence of a wide variety of microbial species and the interactions between these microorganisms [1][2][3]. Despite the abundant interactions between fungi and bacteria in the oral cavity, our knowledge of the mechanisms involved in these interactions is still limited. The elucidation of the interaction mechanisms between different microbial species is extremely important for the understanding of the pathogenesis of human diseases. Furthermore, knowledge of the natural mechanisms whereby microorganisms compete with each other and establish antagonistic interactions may contribute to the discovery of new therapeutic strategies for human infections [4].
Candida albicans is a human pathogen that, in addition to oral candidiasis, can cause various polymicrobial diseases due to its ability to form multispecies biofilms. In this respect, the ecological interactions between yeast of the genus Candida and different bacterial species found in the oral cavity, such as Streptococcus mutans, have become the subject of interest in scientific studies. Pereira-Cenci et al. [5] observed that S. mutans (UA159) stimulated the growth of C. albicans in in vitro biofilms, but suppressed the formation of hyphae by this yeast. On the basis of these results, Jarosz et al. [1] evaluated the interaction between S. mutans UA159 and C. albicans based on production of quorum sensing molecules. Filter sterilized spent medium of S. mutans inhibited germ tube formation by C. albicans indicating that S. mutans secretes one or more diffusible molecules that affect hypha formation by C. albicans. Next, Joyner et al. [6] attributed the inhibitory effects on C. albicans morphogenesis to a natural peptide produced by S. mutans, which was called mutanobactin A.
All of these studies on the interaction between S. mutans and C. albicans cited earlier have used in vitro biofilm models. In vivo studies are becoming increasingly limited due to ethical issues to the use of rats or mice in scientific research. More recently, invertebrate models of infection, such as Galleria mellonella, are being developed because of their numerous advantages compared to mammalian models, including their low cost, easy handling and the possibility of large-scale studies. Additionally, these models have no ethical restrictions and can be used as a screening tool for studies using vertebrate models, thus reducing the number of rats or mice necessary [7][8][9][10][11]. G. mellonella has been successfully used in the medical field as a model for the study of Candida pathogenesis, since it permits the injection of a standardized inoculum of C. albicans and contains different types of hemocytes and antimicrobial peptides that play an important role in the defense against pathogens [7,[12][13][14][15][16].
Since the previous studies showed that S. mutans produces signaling molecule capable to inhibit C. albicans cultured in vitro and no study was conducted to investigate the activity of S. mutans on the candidiasis development, the objective of the present study was to evaluate the effects of S. mutans on biofilm formation and morphogenesis of C. albicans in vitro and expand these findings for in vivo studies using G. mellonella as a model of experimental candidiasis.

Materials and Methods
Inoculum preparation of C. albicans and S. mutans In this study, we used reference strains of Candida albicans (ATCC 18804) and Streptococcus mutans (UA159) maintained in a freezer at -80°C in the Laboratory of Microbiology and Immunology, Institute of Science and Technology, UNESP, Campus of São José dos Campos, Brazil. C. albicans was cultured for 18 h at 37°C in yeast nitrogen base broth (YNB; Difco, Detroit, USA) supplemented with 100 mM glucose. S. mutans was grown in brain-heart infusion broth (BHI; Difco, Detroit, USA) supplemented with 5% sucrose for 4 h at 37°C in a bacteriological incubator under a partial pressure of 5% CO 2 . Cells were collected by centrifugation and washed three times with phosphate-buffered saline (PBS). Suspensions of each microorganism were standardized in PBS at a concentration of 10 7 cells/mL using a spectrophotometer (B582, Micronal, São Paulo, Brazil). Cells densities of the inoculum were confirmed by CFU/ mL counting after plating in Sabouraud dextrose agar for C. albicans and BHI agar for S. mutans.
Preparation of the S. mutans culture filtrate Firstly, the standardized suspension of S. mutans containing 10 7 cells/mL was prepared as described above. A volume of 1 mL of the standardized suspension was transferred to a Falcon tube containing 6 mL of brain-heart infusion broth supplemented with 5% sucrose and the mixture was incubated for 4 h at 37ºC under a partial pressure of 5% CO 2 . After this period, the culture was centrifuged and the supernatant was filtered through a membrane with a pore diameter of 0.22 μm (MFS, Dublin, USA).

In vitro biofilm formation
The method of biofilm formation on the bottom of a 96-well microtiter plate (Costar Corning, New York, USA) was used according to described by Thein et al. [17]. Firstly, 100 μL of the standardized suspension of C. albicans was added to each well of the plate. The plate was incubated for 90 min at 37°C under shaking at 75 rpm (Quimis, Diadema, São Paulo, Brazil) to promote initial adhesion of the microorganisms. The suspension was then aspirated and each well was washed two times with PBS to remove weakly adhered cells. Next, 100 μL fetal bovine serum was added to each well and the plates were incubated again for 2 h under shaking. The wells were washed two times with PBS and 50 μL of the standardized S. mutans suspension or 50 μL of the S. mutans culture filtrate was added to each well. In the control groups, 50 μL of PBS (Control PBS) or 50 μL of BHI + 5% sucrose were performed (Control BHI). In addition, a control group formed only by 50 μL of the standardized S. mutans suspension (without C. albicans) was added.
For biofilm growth and maintenance, 140 μL yeast nitrogen base (YNB) supplemented with 100 mM glucose and 60 μL BHI broth supplemented with 5% sucrose were added in each well. The media were changed at intervals of 24 h and the plates were incubated at 37°C under shaking at 75 rpm for 48 h.

Analysis of biofilm formation by CFU/mL count
After 48 h of incubation, the wells were washed two times with PBS. Next, 250 μL PBS was added to each well and the biofilm adhered to the bottom of the plate was disrupted by homogenization for 30 s in an ultrasonic homogenizer (Sonics Vibra Cell) at an amplitude of 25%. Serial dilutions were prepared from the solution obtained and 100-μL aliquots were seeded onto Sabouraud dextrose agar and incubated at 37°C. In the groups containing S. mutans cells, the serial dilutions were seeded onto Mitis Salivarius Bacitracin Sucrose (MSBS) agar and incubated at 37°C under a partial pressure of 5% CO 2 . After 48 h, the number of colony-forming units (CFU) was determined. The study was supported by two experiments at different times with ten biofilms per group.

Analysis of biofilm formation by total biomass quantification
After biofilm formation as described above, the biofilm biomass was quantified based on the crystal violet (CV) assay described by Peeters et al. [18] with modifications. For fixation of the biofilms, 100 μl of 99% methanol was added (Sigma-Aldrich, São Paulo, Brasil). After 15 min, supernatants were removed and the plates were air-dried. Then, 100 μl of a 1% CV solution was added to all wells. After 20 min, the excess CV was removed by washing with PBS. Finally, bound CV was released by adding 150 μl of 33% acetic acid (Sigma-Aldrich, São Paulo, Brasil). The absorbance was measured at 540 nm. All steps were carried out at room temperature. The CV assay was performed in two independent experiments with six biofilms per group.

Analysis of biofilm formation by scanning electron microscopy (SEM)
Acrylic resin discs measuring 8 mm in diameter were placed on a 24-well plate for biofilm formation as previously cited. After biofilm formation, the specimens were fixed in 1 mL 2.5% glutaraldehyde for 1 h. The specimens were then dehydrated in an increasing ethanol series (10,25,50,75 and 90%) for 20 min each, followed by immersion in 100% alcohol for 1 h. The plates were kept in an oven at 37°C for 24 h to permit complete drying of the specimens.
After drying, the specimens were transferred to aluminum stubs and sputtered with gold for 160 s at 40 mA (Denton Vacuum Desk II). The specimens were examined and photographed under a JEOL JSM5600 scanning electron microscope at the National Institute for Space Research (Instituto Nacional de Pesquisas Espaciais-INPE). These experiments were performed at two different times with three biofilms per group.

Induction of in vitro filamentation by C. albicans
For the study of in vitro filamentation, the following groups were evaluated: PBS control (C. albicans + PBS), BHI control (C. albicans + BHI broth), cell interaction (C. albicans + S. mutans cells), and supernatant interaction (C. albicans + S. mutans supernatant). In a 24-well culture plate (Costar Corning, New York, USA), 1 mL of distilled water was mixed with 10% fetal bovine serum and 100 μL of the standardized C. albicans suspension.
According to the experimental group, 50 μL standardized S. mutans suspension or 50 μL S. mutans culture supernatant were also added. In the control groups, 50 μL PBS or BHI broth were added to each well. The plates were incubated at 37°C under a partial pressure of 5% CO 2 . Five assays were used per group and the experiment was performed independently in duplicate.
After 24 h of incubation, 50 μL of the inoculum were transferred to glass slides with 10 previously demarcated fields on the back of the slide and observed under a light microscope at 400x magnification. The images were analyzed regarding C. albicans morphology and 10 microscopic fields per slide were chosen for the quantification of hyphae. According to Vilela et al. [19] the following scores were attributed for the number of hyphae present in each microscopic field: 0 (no hyphae), 1 (1-3 hyphae), 2 (4-10 hyphae), 3 (11-20 hyphae), and 4 (more than 20 hyphae).

Experimental candidiasis in the Galleria mellonella model
The methodology described by Mylonakis et al. [20] and Fuchs et al. [7] was used in this study. Galleria mellonella (Embrapa Gado de Leite, Juiz de Fora, MG, Brazil) in the final stage of the larval phase and weighing approximately 250 mg were stored in the dark and used within 7 days from shipment. The larvae were kept without food throughout the experiment.
Before the study of the interaction between C. albicans and S. mutans, the susceptibility of G. mellonella to infection with S. mutans was analyzed to determine the sublethal concentration of this microorganism in these animals. Standardized suspensions containing different concentrations (10 4 to 10 7 cells/larva) of S. mutans were injected into G. mellonella and the survival curve was determined. The suspensions were standardized in a spectrophotometer as described above and a group of 16 larvae was used per concentration. These experiments were performed independently in duplicate.
For the study of C. albicans and S. mutans interaction, standardized suspensions of C. albicans (10 6 cells/larva) were injected into the hemolymph of each larva through the last left proleg using a Hamilton syringe (Hamilton, Inc., Reno, USA). Next, standardized suspensions of S. mutans containing 10 5 cells/larva (sublethal concentration for G. mellonella as defined in the previous test) or S. mutans supernatant were inoculated into the right proleg. A group inoculated only with PBS was used to show that death was not due to needle trauma. Another non-injected group was included to monitor the health status of the G. mellonella larvae throughout the experiment.

G. mellonella survival assays
After microbial injections, the larvae were incubated at 37°C in a bacteriological oven and observed 18, 24, 48, 72, 96, 120, 144 and 168 hours after infection. The number of dead G. mellonella was recorded daily. The larvae were considered to be dead when they showed no sign of movement after touch. Sixteen larvae were used per experimental group. These experiments were performed independently in duplicate.
CFU/mL count of C. albicans in the hemolymph of G. mellonella To quantify the presence of C. albicans in G. mellonella infection, samples of larval hemolymph were removed at 0, 8 and 12 h after injections. Hemolymph was collected from a pool of three larvae per time point and experimental group, which is sufficient to prepare serial dilutions. The experiment was carried out in duplicate using 9 larvae per group.
The larvae were cut with a scalpel blade in the ventral part and gently squeezed to remove the hemolymph. Serial dilutions were prepared from 10 μL of collected hemolymph and seeded onto Sabouraud dextrose agar containing 100 mg/L chloramphenicol for the growth of C. albicans. The plates were incubated for 48 h at 37°C and colonies were counted for the calculation of CFU/mL.

G. mellonella histological analysis
Histological analysis was performed to determine the effects of S. mutans on C. albicans filamentation in G. mellonella. Eighteen hours after inoculation, the fat body and other internal structures of G. mellonella was removed with a scalpel blade, stored in 10% formalin, and sent for histological processing. Histological sections (5 μm) were mounted on glass slides and stained with periodic acid Schiff (PAS). The yeast and hyphal forms of C. albicans present in the internal tissues of G. mellonella were observed under a light microscope. For analysis of filamentation, all areas stained with PAS, indicating the presence of yeast cells and hyphae, were photographed with a Cyber-Shot DSC-585 digital camera (Sony Corporation) coupled to a Zeiss Axiophot 2 light microscope (Carl Zeiss, Oberkochen, Germany) at 100x magnification. The area (μm) occupied by yeast cells and hyphae was determined in each image using the Image J program (version 1.32 for Windows, National Institutes of Health/NIH, Bethesda, USA) according to Vilela et al. [19]. In each histological section, the delimited areas were summed and Log 10 transformed. Five larvae were used per group.

Statistical analysis
Analysis of variance (ANOVA) and the Tukey test were used for the CFU/mL and biomass quantification of the in vitro biofilm formation tests, to analyze the presence of C. albicans in G. mellonella hemolymph, and for histological analysis of G. mellonella. The scores obtained by the analysis of in vitro filamentation were compared by the Kruskal-Wallis and Dunn's test. A survival curve was constructed to analyze the survival of G. mellonella and differences were estimated by the log-rank method (Mantel-Cox). All analyses were performed using the Graph Pad Prism 5 Program and a level of significance of 5% was adopted.

Effects of S. mutans on biofilm formation by C. albicans (in vitro study)
Analysis of the interaction between S. mutans and C. albicans in the in vitro model of biofilm formation showed a higher C. albicans count (CFU/mL) in the mixed biofilms formed by C. albicans and S. mutans cells compared to the single biofilms formed by C. albicans and PBS (Control group). However, a reduction in C. albicans counts was observed when the biofilms of C. albicans were associated with the S. mutans culture supernatant ( Fig 1A). These data indicate that the number of C. albicans in the biofilms were stimulated by the S. mutans cells and inhibited by the S. mutans supernatant.
In the group with mixed biofilms formed by C. albicans and S. mutans cells, the number of S. mutans was also determined (Fig 1B). The mixed biofilms showed similar results of CFU/ mL compared to the single biofilm formed only by S. mutans (without C. albicans).
In the crystal violet assay, the biofilms formed by C. albicans and S. mutans cells (Interaction group) exhibited a significant increase of the total biomass compared to the control groups formed only by C. albicans or S. mutans. In relation to the indirect effects of S. mutans on C. albicans, the presence of S. mutans supernatant was not able to reduce the total biofilm formed by C. albicans (Fig 2). These results show that S. mutans supernatant can cause reduction of C. albicans viable cells (CFU/mL), but it is not enough to reduce the total biomass of C. albicans biofilm.
The biofilms formed in vitro were also analyzed by scanning electron microscopy (SEM), in which we observed a mature biofilm formation on acrylic resin discs after 48 hours of incubation. The biofilms showed C. albicans cells, an extracellular polymeric matrix and water channels responsible for biofilm nutrition. In the mixed biofilms, the presence of S. mutans cells was also confirmed (Fig 3). The C. albicans cells observed in the biofilms showed morphological variations according to the experimental group. In the biofilms formed by C. albicans and PBS (Control group), we verified a large number of hyphae in contrast with a few yeast cells (Fig 4A and 4B). However, we found a predominance of yeast cells with a reduction in the hyphae formation in the biofilms formed by C. albicans and S. mutans cells (Fig 4C and 4D) and a total absence of hyphae in the biofilms formed by C. albicans and S. mutans supernatant (Fig 4E and 4F), indicating that the S. mutans inhibited the hyphae formation by C. albicans in the biofilms.
Effects of S. mutans on C. albicans filamentation (in vitro study) After we verified that S. mutans inhibited the hyphae formation by C. albicans in biofilms formed in vitro, another experiment focusing in the C. albicans filamentation was conducted. In this experiment, C. albicans was placed in a 24-well culture plate containing fetal bovine serum to induce the hyphae formation. The plates were incubated for 24 h and transferred to glass slides for morphological analysis. We observed a large number of C. albicans hyphae in the following groups: Control with PBS; Control with BHI; and Interaction with S. mutans cells. The presence of S. mutans cells was not capable to alter hyphae formation by C. albicans. However, we verified a significant inhibition of the hyphae formation when C. albicans was incubated in the presence of the S. mutans supernatant compared to the control groups (PBS or BHI broth) (Figs 5 and 6).
Besides bovine serum, another factor that plays an important role in filamentation is the pH of the medium. Therefore, the pH of the culture medium was measured. The pH of the S. mutans supernatant was 7.0 and the BHI broth supplemented with 5% sucrose was 7.3. These data show that the inhibition of C. albicans filamentation by S. mutans supernatant cannot be attributed to pH variations in the culture medium. Taken together, the in vitro results obtained in this study suggest that S. mutans secretes subproducts into the culture medium, which exert inhibitory effects on C. albicans, interfering with the number of viable cells in the biofilm formation and filamentation capacity. In order to determine whether S. mutans also exerts inhibitory effects on the pathogenesis of C. albicans, we extended this study to an in vivo host model.

Effects of S. mutans on experimental candidiasis: G. mellonella survival curve
First, we evaluated the susceptibility of G. mellonella to infection with different concentrations of S. mutans (10 4 to 10 7 cells/larva) to determine the sublethal concentration of this microorganism in these animals. The results showed that S. mutans pathogenicity was dose-dependent in G. mellonella; concentrations of 10 6 and 10 7 S. mutans cells/larva resulted in mortality rates of 20 and 30% in the end of the experiment, whereas concentrations of 10 4 and 10 5 cells/larva did not cause lethal infection in these animals (S1 Fig). Thus, the sublethal concentration of 10 5 cells/larva was adopted for the subsequent assays.
After that, we moved on to the study of the effects of S. mutans on C. albicans pathogenicity in the G. mellonella larvae. Analysis of the survival curve of G. mellonella showed that the pathogenicity of C. albicans in G. mellonella varied according to the group studied. The groups infected by C. albicans and inoculated only with PBS or BHI broth (Control groups) resulted in death of 100% of the larvae within 24 h after infection. However, the larval survival rate increased significantly when the infection by C. albicans in G. mellonella was followed by the injection of S. mutans cells or supernatant (Interaction groups) (Fig 7). These findings indicate Fig 2. Mean and standard deviation of the biofilm mass obtained in the crystal violet assay for the following groups: biofilms formed by C. albicans + PBS (Control group); biofilms formed by S. mutans + PBS (Control group); mixed biofilms formed by C. albicans + S. mutans cells (Interaction group); biofilms formed by C. albicans + BHI (Control group); and biofilms formed by C. albicans and S. mutans supernatant filtrate (Interaction group). Tukey test, P 0.05. that S. mutans was able to attenuate the infection caused by C. albicans in G. mellonella model. Then, we investigated the effects of S. mutans on the number of C. albicans present in the hemolymph and in the internal tissues of G. mellonella using, respectively, assays of CFU/mL count and histological analysis.
Effects of S. mutans on experimental candidiasis: CFU/mL count of C. albicans in the hemolymph of G. mellonella The study of G. mellonella hemolymph culture revealed a similar growth pattern of C. albicans in all groups at the different time points studied. Immediately after infection with C. albicans  In this experiment, statistically significant differences were not observed between the interaction and control groups.

Effects of S. mutans on experimental candidiasis: G. mellonella histological analysis
Histological analysis of the fat body and other internal structures of G. mellonella showed the presence of extensive aggregations of yeast and hyphae in the tissues of animals infected with C. albicans (Fig 8). The number of C. albicans was quantified in all groups. Significant reduction in Candida count was only observed in the supernatant interaction group when compared to the control group (Fig 9). Therefore, as observed in the in vitro tests, the S. mutans culture supernatant was able to inhibit the morphological transition of C. albicans.

Discussion
It has been estimated that 65% of human infections are associated with the formation of biofilms on the surfaces of host tissues or medical devices [21]. Studies have shown that bacteria and fungi present in biofilms can influence each other through the secretion of extracellular signaling molecules or physical interactions of cell-cell contact or aggregation [4]. Since C. albicans and S. mutans are found together in the oral biofilms on dental surfaces [22,23], in this study we investigate the influence of S. mutans on the growth and pathogencity of C. albicans.
In an attempt to understand the effect of S. mutans on the growth of C. albicans in mixed biofilms, we used three different assays to evaluate the biofilms formed in vitro: CFU/mL count, crystal violet assay, and scanning electron microscopy (SEM).
In the CFU/mL analyses, the results showed a higher C. albicans count in the mixed biofilms formed by C. albicans and S. mutans cells compared to the single biofilms formed only by C. albicans, indicating that S. mutans cells were capable to stimulate the growth of C. albicans. It has been described that S. mutans can improve the growth of C. albicans cells in mixed biofilms [5,23]. According to Sztajer et al. [23], C. albicans can be more efficient than S. mutans in taking up sucrose. The sucrose has been considered an important factor in the interaction of C. albicans and S. mutans in biofilms formed in vitro. When sucrose is present in the culture medium, as in our study, the adhesive interaction between these two microorganisms is enhanced [24,25]. Thein et al. [17] studied mixed biofilms of S. mutans and C. albicans in the presence of glucose and no significant effect of S. mutans on the viability of C. albicans was found.
Novel assays for quantification of bacteria and fungi in biofilms have been used rather than assays based on the quantification of viable cells (CFU/mL count). Crystal violet assay allows to quantifying the biofilm biomass in the entire well of microtiter plates. This dye binds to negatively charged molecules and polysaccharides, staining both living and dead cells, as well as extracellular matrix [18]. In this study, the mixed biofilms formed by C. albicans and S. mutans cells had a significant increase of the total biomass compared to the control groups formed only by C. albicans or S. mutans. Although in this study, we focused on the influence of the S. mutans on C. albicans, this assay demonstrated that C. albicans also provides benefits to S. mutans in mixed biofilms. Falsetta et al. [25] reported that the association between C. albicans and S. mutans can be mediated by a physical interaction that relies on the production of glucans, which are produced by bacterial exoenzymes (glucosyltransferases) on yeast and hyphal  cell surface. These interactions are essential for the assembly of an exopolysaccharides-rich matrix and the development of cospecies biofilms. The synergism between C. albicans and S. mutans can enhance the virulence of mixed biofilms formed on tooth surfaces and contribute for the severity of early childhood caries and other polymicrobial biofilm infections [23,25].
On the other hand, in the scanning electron microscopy analyses, we noticed that the presence of S. mutans cells caused a reduction in the hyphae formation in the mixed biofilms composed by C. albicans and S. mutans. Similar results have been reported in the study of Pereira-Cenci et al. [5], in which the presence of S. mutans cells in the biofilm increased C. albicans growth. However, using confocal laser scanning microscopy, these authors observed that S. mutans suppressed hyphae formation by C. albicans and emphasized that the effects of S. mutans on C. albicans filamentation should be considered in studies on the prevention of oral candidiasis.
In order to investigate how S. mutans affects C. albicans, we also tested the effects of S. mutans culture filtrate (without S. mutans cells) on C. albicans biofilms. S. mutans culture filtrate was able to inhibit the CFU/mL number of C. albicans and block the hyphae formation, with total absence of filamentation in the biofilms analyzed by SEM. After that, we performed another experiment focused in the C. albicans filamentation. The hyphae formation was induced in vitro by incubation of C. albicans with fetal bovine serum. The presence of S. mutans cells was not capable to inhibit hyphae formation by C. albicans. Possibly, S. mutans cells did not affect C. albicans morphogenesis because S. mutans was placed in the plates with only bovine serum (without culture medium), in which these microorganisms were unable to grow and produce acids or bacterial secreted molecules that could inhibit C. albicans. On the other hand, S. mutans culture filtrate, that probably contained acids or other metabolites, was capable to inhibit C. albicans filamentation.
Then, the presence of acids in the S. mutans supernatant was evaluated by pH measurement. It has been reported that a neutral to alkaline pH favors the growth of hyphae, while an acidic pH has a inhibitory effect on filamentation [26]. The S. mutans supernatant and the BHI broth (Control group) showed similar pH values, represented respectively by 7.0 and 7.3. These findings indicate that C. albicans filamentation was not affected by pH in the culture medium. Jarosz et al. [1] investigated the interaction between C. albicans and S. mutans based on production of secreted molecules. They tested the effect of spent medium of S. mutans on C. albicans germ-tube formation at different phases of S. mutans growth (4, 6, 8 and 24 h cultures). Only S. mutans spent medium of 4 h culture was capable to inhibit germ-tube formation, indicating that S. mutans secretes quorum sensing molecule during the early stages of growth that inhibits the C. albicans morphological transition.
These results suggest that, when cultured in vitro, S. mutans can produce signaling molecules with antifungal activity that inhibit the growth of C. albicans cells and mainly block its filamentation capacity. Then, the in vitro results were investigated in more detail through in vivo studies, using G. mellonella as a model host of Candida infection. This is the first study that attempts to investigate the effects of S. mutans on the pathogenicity of C. albicans and development of experimental candidiasis in animal models. Initially, we performed an experiment to elucidate the host response of G. mellonella to mono-infection by S. mutans. We found that S. mutans cells were capable of infecting and killing G. mellonella larvae. The increasing concentrations of the S. mutans cell number resulted in gradually decreasing survival of the infected larvae and 10 5 cells/larva was defined as a sublethal concentration. Abranches et al. [27] also injected S. mutans in G. mellonella larvae and demonstrated that G. mellonella can be a suitable model to study the virulence potential of S. mutans strains.
Once the sublethal concentration of S. mutans was defined, the microbial interaction between C. albicans and S. mutans was investigated in the G. mellonella model. The results from survival analysis showed that the injection with S. mutans cells or S. mutans filtrate culture prolonged the survival rate of the larvae infected by C. albicans when compared to the control groups, suggesting that S. mutans reduced the pathogenicity of C. albicans in these animals.
Since in this study, G. mellonella larvae were inoculated with a known quantity of C. albicans by injecting the fungal cells directly into the hemocoel, we also evaluated the effects of S. mutans on C. albicans cells present in the hemolymph of G. mellonella at different times of infection. The results showed that S. mutans cells and S. mutans culture filtrate did not affect the number of C. albicans in the hemolymph. The changes in the C. albicans CFU/mL occurred only by the action of immune system. The number of C. albicans cells recovered immediately after inoculation (0 h) did not differ from the fungal load inoculated; however, a reduction in the number of recovered cells was observed at the subsequent time after inoculation (8 h), suggesting that the larval immune system was able to combat infection with C. albicans, reducing the number of Candida CFU/mL at these times of infection. After this period, C. albicans was able to overcome the immune system of G. mellonella, reaching its peak proliferation (12 h) and a fungal load similar to that inoculated at the beginning of the test.
In addition to CFU/mL count of C. albicans in the hemolymph of G. mellonella, the experimental candidiasis can be evaluated by histological analysis. It has been described that C. albicans is capable to form hyphae in the G. mellonella tissues during the infection process. Therefore, analysis of internal structures can be used to observe the fungal state and identify forms of yeast and hyphae [7]. In this study, histological analysis of the fat body and other internal structures of G. mellonella infected with C. albicans showed a significant reduction in the presence of hyphae when C. albicans was associated with the S. mutans supernatant, confirming the results of the in vitro tests. These results suggest that S. mutans may secrete subproducts into the culture medium in vitro that are able to inhibit the morphological transition of C. albicans in vivo.
Although there is an abundance of published reports illustrating the extent to which bacteria and fungi are capable of interacting with other microorganisms within their vicinity, the majority of biomolecules responsible for influencing these biological processes remain unknown [28]. Recently, some studies were developed to characterize biomolecules excreted by S. mutans into the extracellular environment. Joyner et al. [6] described the structural features of the major putative hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) derived metabolite from S. mutans UA159. This biomolecule, which was dubbed the mutanobactin, was capable to suppress the morphological transition of C. albicans from yeast to hyphae. Vílchez et al. [29] identified another compound secreted by S. mutans, trans-2-decenoic acid, which suppressed morphogenesis at concentrations that do not affect fungal growth. Zvanych et al. [30] tested whether mutanobactins display any immunomodulatory activity using in vitro models of macrophage cell line. Interestingly, mutanobactin B caused a significant increase in pro-inflammatory cytokines, such as IL-6 and Il-12, suggesting that mutanobactins may play an important role in modulating immune response.
In summary, the results obtained showed that the S. mutans culture filtrate exerted inhibitory effects on the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in animal models. These results will certainly contribute to the development of new therapeutic strategies for human candidiasis. However, the exact mechanism whereby S. mutans interferes with morphogenesis and pathogenicity of C. albicans still needs to be elucidated. Therefore, comprehensive studies involving molecular and immunological assays are needed to better understand how oral bacteria S. mutans can modulate the pathogenicity of C. albicans.