Role of Candida albicans on enamel demineralization and on acidogenic potential of Streptococcus mutans in vitro biofilms

Abstract There is growing evidence that C. albicans is associated with dental caries, but its role on caries development needs to be better clarified. Objective: To evaluate at the hard tissue level the effect of C. albicans on the cariogenic potential of S. mutans biofilms focusing on the mineral profile of induced carious lesions. This study also aimed to evaluate the effect of C. albicans on the acidogenic potential of S. mutans biofilms. Methodology: Dual-species (CA+SM) and single-species biofilms (CA or SM) were grown on the surface of enamel slabs in the presence of glucose/sucrose supplemented culture medium for 24, 48 and 72 hours. Demineralization was evaluated through percentage of surface microhardness change (%SMC) and transversal microradiography analysis (ILM and LD) and pH of the spent medium was recorded daily. Data were analyzed by two-way ANOVA followed by Bonferroni correction. Results: %SMC was statistically different among the biofilms at each time point being the highest for SM biofilms and the lowest for CA biofilms which also differed from CA+SM biofilms [SM (24 h: 47.0±7.3; 48 h: 66.3±8.3; 72 h: 75.4±3.9); CA (24 h: 7.3±3.3; 48 h: 7.1±6.4; 72 h: 6.6±3.6); CA+SM (24 h: 35.9±7.39.1; 48 h: 47.2±9.5; 72 h: 47.6±9.5)]. pH of spent medium was statistically lower for SM biofilms compared to the other biofilms at each time point and remained constant over time while pH values increased from 24 to 72 h for both CA and CA+SM biofilms [SM (24 h: 4.4±0.1; 48 h: 4.4±0.1; 72 h: 4.5±0.1); CA (24 h: 6.9±0.3; 48 h: 7.2±0.2; 72 h: 7.5±0.2); CA+MS (24 h: 4.7±0.2; 48 h: 5.1±0.1; 72 h: 6.1±0.6)]. IML and LD for SM biofilms increased over time while no difference was observed from 24 to 72 h for the other biofilms. Conclusions: The present data suggest that C. albicans has low enamel demineralization potential and the presence of C. albicans can reduce both the cariogenic and acidogenic potentials of S. mutans biofilms.


Introduction
Acid production, through fermentation of carbohydrates by a selective group of microorganisms, results in pH reduction within biofilms.This acidification is directly related to the dissolution of tooth tissues that might occur if the balance between demineralization and remineralization process is disturbed over an extended period. 1 So, in dental caries, frequent sugar intake results in a dysbiotic change in dental plaque where the acid-tolerant microorganisms prevail over the less acid-tolerant ones in response to biofilm acidification. 2 Candida albicans has a high acid tolerance and is capable of producing acids even under low pH conditions.These characteristics may favor the fungus in the microbial shifts associated with dental caries. 3,4[7][8][9] Furthermore, one of the microorganisms most frequently associated with dental caries is the bacterial species S. mutans.Despite being not alone in this task, S. mutans has a high acid production ability.
In addition, it has the unique ability of converting sucrose into extracellular insoluble glucans (EPS) that constitute the main component of the biofilm matrix.
Insoluble glucans alter the diffusion properties of biofilm matrix by acting as a barrier to the buffering effects of saliva.[12] Literature has extensively provided evidences that biofilms formed by C. albicans and S. mutans present increased cariogenic potential compared to single-species biofilms.4][15][16][17][18] Rodent models have also been used to show that carious lesions produced by the association between C. albicans and S. mutans are more severe. 13[21] However, the role played by this microbial association on dental caries onset is still controversial since other studies have indicated opposite findings. 22,23t, the association between surrogate outcomes, such as the carriage of streptococci and C. albicans on dental plaque and saliva, and caries onset 21 is questioned. 24ditionally, to the best of our knowledge, no other study has objectively assessed the profile of enamel mineral loss in the presence of that microbial association.

Enamel slabs preparation
Enamel slabs were cut from sound bovine anterior teeth, which were previously disinfected in 10% formaldehyde solution, 25  diamond grit hole saw (DeWALT, Baltimore, MD, USA).The resulting 6.4 mm diameter enamel slabs were then flattened and polished with a grinding and polishing machine (Arotec S/A, Cotia, SP, Brazil).A 1 mm nail varnish strip was made on the side of each slab to create an unexposed (control) area.Slabs with cracks, scratches, exposed dentin or with mean enamel baseline hardness out of the range from 320 to 360 Vickers microhardness (VMH) were discarded.
Baseline surface hardness determination is described below.Slabs were then randomized and balanced into experimental groups by mean VMH values.Finally, the slabs were mounted on individual nylon cylinders attached to the lid of 24-well plates 26 and they were subjected to hydrogen peroxide gas sterilization. 27

Inoculum preparation
Candida albicans (ATCC 90028) and Streptococcus mutans (UA 159) were cultivated from frozen stocks on Brain Heart Infusion Agar (BHI; Kasvi, São José dos Pinhais, PR, Brazil).Colony-forming units (CFU) were then inoculated into tubes containing Tryptic Soy Broth (TSB; HiMedia, Mumbai, MH, India baseline pH from 7.1 to 7.3; 14 mM glucose basal concentration) supplemented with 12.5 mM sucrose.After 24 h of incubation, aliquots of each suspension were transferred into fresh medium and were further incubated for 24 h at 37°C.Optical density was adjusted independently for each microorganism to 0.5±0.1 at 550 nm with a spectrophotometer (Milton Roy Co., Ivyland, PA, USA), corresponding to approximately 10 7 CFU/mL of C. albicans and 10 8 CFU/mL of S. mutans.Counts of viable cells were confirmed at each experiment by plating the inoculum on BHI agar plates and incubating them under the conditions described above.Inoculum presented a mixture of hyphae and yeasts forms (qualitatively determined by microscopic analysis).Dual-species consortium was obtained by combining equal amounts of the density-adjusted cultures 26 .

In vitro biofilm growth
The modified lids with the slabs were put onto 24well plates with 0.4 mL inoculum and 1 mL TSB (pH 7; supplemented with 6 mM glucose and 3 mM sucrose; TSBGS; 20 mM glucose final concentration) on each well and kept at 37°C.After 8 h of incubation, the slabs were transferred to new plates containing 1.4 mL of TSBGS medium and incubated at 37°C overnight.
Biofilms were then transferred every 24 hours to new 24-well plates containing fresh TSBGS medium and kept at 37°C 26 .The pH values of the spent medium (at 8, 24, 48 and 72 h) were individually measured for each well using a pH meter (Digimed, São Paulo, SP, Brazil) previously calibrated with pH 7.0 and pH 4.0 standards.

Biofilm harvesting and viable cell counts
At the end of each experimental period (after 24, 48 and 72 h of biofilm growth), enamel slabs (n=8) were aseptically removed from the lids and individually transferred to tubes containing 1 mL of sterile 0.9% NaCl and 4 sterile glass beads (4-5 mm diameter).
Tubes were vortexed for 30 s to disperse the biofilms.
Aliquots of the microbial suspensions were serially diluted and plated on BHI agar.Plates were incubated at 37°C under microaerophilic conditions for 24 h.CFU were then counted under a stereomicroscope and the results expressed as CFU/mL.Enamel slabs were gently cleaned and stored in pre-codified vials at 4°C under humid environment until further assessment of caries lesion development.(Softex Co. Ltd., Ebina, Japan) on a high precision glass plate (Konica Minolta Inc., Tokyo, Japan) at a distance of 42 cm using 20 kV and 20 mA for 13 minutes.Plates were developed for 5 min, rinsed with deionized water and fixed for 8 min in a dark room at 20°C.All plates were then washed in running water for 10 min and air-dried.Microradiographs were examined using a microscope (Carl Zeiss Microscopy GmbH, Jena, TH, Germany) in conjunction with a camera (Canon, Tokyo, Japan) and a computer running data-acquisition and calculation software programs (Inspektor Research Inc., Amsterdam, NH, Netherlands).The lesion depth (LD) was calculated using a threshold of 95% of the mineral content of sound enamel.Integrated mineral loss (IML; vol% mineral x µm) was also calculated. 28l analyses were performed by a blinded examiner.

Statistical analysis
The mean and standard deviation (pH of spent medium, % SMC, LD and IML) were calculated for each tested condition.The assumptions of homogeneity of variances and normality of the distribution were checked.Counts of viable cells were log transformed and were descriptively reported as median and quartiles.Two-way analysis of variance (ANOVA) was used to determine the effects and interactions of the tested conditions (biofilm microbial composition and biofilm age) on response variables.Bonferroni adjusted comparisons were used when two-way ANOVA indicated statistically significant effects.Correlation between pH and counts of fungal viable cells on dual-species biofilms was tested using Pearson's coefficient, while correlation between %SMC and

Results
Regarding counts of viable cells, only changes lower than 1 log 10 (10-fold) were found on median of CFU among the different tested conditions for both fungal and bacterial cells.Overall, CFU remained constant among different periods of biofilm growth irrespective to the biofilm composition (Table 1).For all the other outcomes (pH, %SMC, IML and LD) statistical analysis indicated an interaction effect between microbial composition and biofilm age (p<0.01).In relation to carious lesion development, higher %SMC was found in the presence of S. mutans singlespecies biofilms than for other biofilms at each time point.In addition, S. mutans related demineralization increased with time, being statistically different among the different time points.On the other hand, carious lesion development in the presence of C. albicans single-species biofilms was statistically lower than in the presence of other biofilms and it remained constant over time.Additionally, the % SMC in the presence of dual-species biofilms at 48 h of biofilm formation was statistically higher than at 24 h, but it was not statistically different compared to enamel demineralization after 72 h of biofilm growth (Table 2).

The mean of pH values
At 24 h of biofilm growth, no statistical difference was found on IML and LD among the different biofilms, but enamel slabs exposed to S. mutans single-species biofilms presented IML and LD higher than slabs exposed to C. albicans single-species biofilms at 48 and 72 h and higher than dual-species biofilms at 72 h of biofilm growth.Furthermore, IML of enamel slabs exposed to S. mutans biofilms increased over time, being statistically different at each time point, whereas LD at 72 h was statistically higher than that found at 24 h of biofilm growth.Moreover, IML of enamel slabs exposed to C. albicans and to dual-species biofilms remained constant over time.This same behaviour was observed in relation to LD (Table 2).
Figure 1 shows representative transversal microradiographs of enamel slabs according to the experimental conditions where it can be easily seen that all carious lesions were sub-superficial presenting a well-mineralized surface layer.Lesions formed in the presence of S. mutans biofilms were deeper and had lower mineral content compared to the other biofilms.

Discussion
As the presence of C. albicans has been increasingly related to dental caries, 21 the present study used an in vitro biofilm model for the assessment of enamel carious lesion development at the hard tissue level by using surface hardness and transversal microradiography aiming to investigate the effect of C. albicans on the cariogenic potential of S. mutans biofilms as well as on their acidogenic potential.
It is well known that S. mutans, in addition to its acid-tolerance and acidogenic potential, can produce exoenzymes, known as glucosyltransferase B (GtfB), that synthesize EPS from sucrose, an essential factor associated to the cariogenic potential of biofilms. 10,12,29, as expected, enamel slabs exposed to S. mutans single-species biofilms presented an increased mineral loss over time, taking both superficial microhardness and transversal microradiography outcomes into consideration (Table 2, Figure 1).On the other hand, less acidification was found in the presence of C. albicans single-species biofilms which lead to very little enamel demineralization (Table 2, Figure 1).This low acidogenic potential found in C. albicans single-species biofilms could be related to the fact that this fungus may present a more complex acid production profile than lactic acid-producing bacteria. 3ruvate and acetate may be produced by this fungus in the presence of glucose alone, but almost no spent medium acidification has been found for C. albicans single-species biofilms grown in the presence of glucose and sucrose. 23Chemical characterization of C. albicans single-species related spent medium suggests minimum carbohydrate utilization and a poor ability of the fungus in metabolizing sucrose when growing alone. 17Additionally, it has been also suggested that in the presence of culture medium supplemented with sucrose, C. albicans may produce ethanol that does not influence the pH of the medium. 23Important to mention that the less acidified environment found in the presence of C. albicans could not be attributed to any colonization impairment or lack of fungi viability, since, although slightly changes had been found over time, CFU counts remained constant and similar to those of S. mutans throughout the experiments (Table 1).
It is important to acknowledge though that the low cariogenicity of C. albicans single-species biofilms found in this study might be strain-related and might also be a result of different biofilm growing conditions and/or experimental conditions compared to other studies since there are evidences showing that biofilms of C. albicans strains isolated from HIV+ children can cause enamel demineralization after 5 days of biofilm growth 30,31 and that carious lesions are developed in rats infected with C. albicans. 13,22Breakdown of exogenous amino acids can lead to extrusion of ammonia, a highly basic compound.

Carbon source has a direct influence on biofilm
Consequently, in a glucose-limited milieu, C. albicans can actively modulate extracellular pH by alkalinisation of acidic environments. 36ditionally, C. albicans consumption of organic acids as carbon sources, such as pyruvate and lactate, is also responsible for the neutralization of acidic environments.It is likely that both mechanisms are acting together leading to the reduced acidification of dual-species biofilms over time.This way, in the presence of dual-species biofilms enamel slabs showed an initial caries lesion development, but no additional mineral loss could be observed after 48 hours (Figure 1).By providing data related to surface hardness and mineral profile of demineralized enamel specimens, our results corroborate with the results of Willems, et al. 23 (2016) reinforcing that C. albicans reduce the cariogenic potential of S. mutans biofilms.It is important to highlight that Willems's study and ours used similar biofilm growth conditions (up to 72 hours in a medium with high protein and low carbohydrate contents).Thus, this reduced cariogenic potential may only occur in environments with high nutritional content but low carbohydrate concentrations.
One may argue that the reduced cariogenicity found on dual-species biofilms was a result of the less carbohydrate availability for biofilm growth, since the same sugar concentration was used for both singleand dual-species biofilms.It is important to emphasize though that the use of higher sugar concentration would lead to enamel surface softening compromising the assessment of enamel demineralization by means of surface hardness.Even having plenty of available sugars, C. albicans as single-species biofilm was not able to induce considerable enamel demineralization suggesting that the available sugar concentration was not the reason related to the reduced cariogenicity of dual-species biofilms.Moreover, it could also be argued that the increase on pH of spent medium of dual-species biofilms would be the result of a reduction on viable cells counts over time.It is important to emphasize that changes on counts of viable cells were too small (less than 1log 10 -fold) and do not present any relevance for caries development.
However, the data of the present study differ from previous studies.A more aggressive onset of carious lesions was found in the presence of dual-species biofilms. 13It has been shown that GtfB secreted by S. mutans adheres to C. albicans yeast cell surface being kept under an enzymatically active form promoting the formation of EPS-rich biofilm matrix and enhancing accumulation of S. mutans on biofilms. 37t, C. albicans-bound GtfB produces more EPS than S. mutans-bound exoenzyme, leading to an enhanced EPS accumulation on biofilm matrix. 14On the other hand, downregulation of bacterial exoenzymes can occur in the presence of C. albicans leading to lack of EPS production by S. mutans 38 which could also reduce the cariogenic potential of S. mutans biofilms. 22It is unclear though at this time the effect on three-dimensional structure of biofilms imposed by the present dualspecies biofilm model.Further studies are necessary to assess the impact of the tested model and of the tested growth conditions on biofilm matrix production and its relation to carious lesion development.

Conclusion
Overall, the data of the present study suggest that the presence of C. albicans can reduce the cariogenic potential of S. mutans biofilms when there is a competition for nutrients.Under this condition, C. albicans also modulates spent medium pH decreasing the acidogenic potential of S. mutans biofilms.
, the aim of this study was to evaluate at the hard tissue level the effect of Candida albicans on the cariogenic potential of Streptococcus mutans biofilms focusing on the mineral profile of induced carious lesions.Additionally, this study aimed to evaluate the effect of C. albicans on the acidogenic potential of Streptococcus mutans biofilms.To this end, an in vitro biofilm model was used to induce enamel carious lesions that were assessed via surface hardness and whose mineral profile was evaluated by transversal microradiography.The null hypothesis was that C. albicans do not affect the cariogenic and acidogenic potentials of S. mutans biofilms.Methodology Experimental design Dual-species (MIX; C. albicans + S. mutans; n=24) and single-species (C.albicans; n=24 or S. mutans; n=24) biofilms were grown on the surface of sound bovine enamel slabs in the presence of culture medium supplemented with 6 mM glucose and 3 mM sucrose for 24, 48 and 72 hours (n=8 for each biofilm experimental period).The medium was refreshed daily and the pH of the spent medium was recorded.Biofilms were harvested and viable cell counts were performed at the end of each experimental period.Enamel demineralization and mineral profile were evaluated through the determination of percentage of surface hardness change, via hardness analysis, and integrated mineral loss and lesion depth, via transversal microradiography.The experiments were done in triplicate.
using a drill bench (Schulz S.A., Joinville, SC, Brazil) coupled with a ¼ inch Role of Candida albicans on enamel demineralization and on acidogenic potential of Streptococcus mutans in vitro biofilms Baseline enamel surface hardness was determined by a microhardness tester (ISH-TDV2000; Insize Co. Ltd, Suzhou, JS, China) by making five Vickers indentations, spaced 100 µm from each other, with a load of 200 gf for 10 seconds.After each experimental period of biofilm growth, five indentations were placed 100 µm at the right side of the baseline indentations following the same parameters described above.The mean Vickers microhardness (VMH) values of baseline (B) and post-biofilm (P) VMH were averaged and the percentage of surface microhardness change (% SMC) was calculated as 26 % SMC=[(P-B)÷B]×100.Transversal microradiography (TMR)After post-biofilm hardness analysis, half of the specimens were sectioned through the center with a low-speed diamond saw (Isomer; Buehler Ltd., Lake Bluff, IL, USA), perpendicularly to the surface and transversally to the varnish strip, in order to obtain slices approximately 150 µm thick representing both exposed and unexposed (sound) enamel surfaces.Slices were then hand polished plane-parallel from both cut sides with 600 and 1200 grit sandpapers to a thickness of approximately 100 µm.Specimens were mounted in a custom-made sample-holder with an aluminium calibration step wedge with 14 steps.Microradiographs were taken with an X-ray generator EIDT G, ANDRADE CG, NEGRINI TC, ARTHUR RA after 8 hours of biofilm growth was 6.7±0.2,4.8±0.1 and 4.4±0.1 for C. albicans, dual-species and S. mutans biofilms respectively.Spent medium pH related to S. mutans biofilms remained between 4.4 -4.5 under all the tested experimental conditions being significantly lower than for other biofilms at each time point.The pH of dual-species biofilms increased over time being below the critical pH for enamel demineralization for up to 48 h of biofilm growth.Under all the tested conditions, pH of the spent medium of C. albicans biofilms slightly increased over time but remained higher than the critical pH needed to induce enamel carious lesions (Table2).A moderate correlation between fungi CFU and spent medium pH (r=0.559;p<0.01) was found in dual-species biofilms.

Figure 1 -
Figure 1-Representative microradiographs and mineral profile after 24, 48 and 72 hours of biofilm growth.Different coloured lines represent mean values of the percentage of mineral volume (vol %) according to lesion depth (LD) for each biofilm microbial composition

Table 1 -
Viable cell counts [Log CFU/mL; median (25 th /75 th quartiles)] according to biofilm age and biofilm microbial composition Different uppercase letters show a significant difference between biofilm age and different lowercase letters show a significant difference between biofilm microbial compositions by two-way ANOVA followed by Bonferroni test (p<0.01)

Table 2 -
Spent medium pH, percentage of Surface Microhardness Change (%SMC), Integrated Mineral Loss (IML; Vol% x µm) and Lesion Depth (LD; µm) according to biofilm age and biofilm microbial composition Role of Candida albicans on enamel demineralization and on acidogenic potential of Streptococcus mutans in vitro biofilms J Appl Oral Sci.