Study of Microbial Interaction Formed by Candida krusei and Candida glabrata : In Vitro and In Vivo Studies

of infection and oral colonization by association of different species in a large number of immunosuppressed patients. The objective of this study was to evaluate the interactions between C. krusei and C. glabrata in biofilms formed in vitro and their ability to colonize the oral cavity of mouse model. Monospecies and mixed biofilms were developed of each strain, on 96-well microtiter plates for 48 h. These biofilms were analyzed by counting colony-forming units (CFU/mL) and by determining cell viability, using the XTT hydroxide colorimetric assay. For the in vivo study, twenty-four mice received topical applications of monospecie or mixed suspensions of each strain. After 48 h, yeasts were recovered from the mice and quantified by CFU/mL count. In the biofilm assays, the results for the CFU/mL count and the XTT assay showed that the two species studied were capable of forming high levels of in vitro monospecie biofilm. In mixed biofilm, the CFU of C. krusei increased (p=0.0001) and C. glabrata decreased (p=0.0001). The metabolic activity observed in XTT assay of mixed biofilm was significantly reduced compared with a single C. glabrata biofilm (p=0.0001). Agreeing with CFU in vitro count, C. glabrata CFU/mL values recovered from oral cavity of mice were statistically higher in the group with single infection (p=0.0001) than the group with mixed infection. We concluded that C. krusei inhibits C. glabrata and takes advantage to colonize the oral cavity and to form biofilms. Study of Microbial Interaction Formed by Candida krusei and Candida glabrata: In Vitro and In Vivo Studies


Introduction
Some decades ago, Candida albicans represented 80% of the Candida species recovered from patients with oral and systemic candidiasis.Although C. albicans continues to be the most frequently isolated species, the number of infections caused by non-albicans species has increased significantly (1).Furthermore, C. glabrata has emerged as the second most common cause of candidemia, followed by C. parapsilosis, C. tropicalis and C. krusei (2).
One of the major factors contributing to the virulence of Candida is its ability in acclimatize to a variety of different habitats for growth and formation of surface-attached microbial communities known as "biofilms".Biofilms are defined as microbial communities encased in a matrix of extracellular polymeric substance (EPS), which display phenotypic features that differ from their planktonic or free-floating counterparts (3)(4)(5).It is known that C. glabrata exhibits intrinsically low susceptibility to azoles and develops resistance after exposure to these drugs (6) and C. krusei is naturally resistant to fluconazole, but in most cases, it is sensitive to voriconazole (cross-resistance is uncommon in this specie) (7,8), which complicates the treatment of candidiasis caused by this species.Because of that, it is important to know the behavior of these species when they grow together in a biofilm.
Recently, Rossoni et al. (9) and Barros et al. (10) evaluated the interaction of C. albicans associated with C. krusei and C. glabrata in biofilm formation.Both of the studies suggest that these interactions decrease the ability of C. albicans to form biofilm.Moreover, Santos et al. (11) studied the interaction between C. albicans, C. krusei, C. glabrata and C. tropicalis.Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans.However, none of above studies evaluated the interaction between non-albicans Candida species.
There is just one study that investigated the in vitro interaction between C. krusei and C. glabrata (12) and our study is the first one that has additional in vitro data and also in vivo.In this context, the objective of this study was to evaluate the interactions between C. krusei and C. glabrata in in vitro biofilms and its ability to colonize the oral cavity of mice.Univ.Estadual Paulista, São Paulo, Brazil.The strains were stored as frozen stocks with 20% glycerol at -80 °C.The strains were routinely grown in Yeast Nitrogen Base (YNB, Himedia, Mumbai, India) liquid medium at 37 °C in a shaking incubator.

Strains and Culture Conditions
The strains were grown on Sabouraud Dextrose agar (Himedia) and incubated at 37 °C for 24 h.Next, a loopful of growth was inoculated in Yeast Nitrogen Base (YNB) broth supplemented with 100 mM glucose (Vetec, Rio de Janeiro, RJ, Brazil) and incubated at 37 °C for 18 h.The cells were collected by centrifugation (MPW Med.Instruments, Warsaw, Poland) and washed three times with phosphate buffered saline (PBS) (Laborclin, São José dos Pinhais, PR, Brazil).Yeast cells were counted using a Neubauer counting chamber (Laboroptik GmbH, Bad Homburg, Germany).The cell number was confirmed by determining CFU/mL on SDA plates (10 7 cells/mL).

Biofilm Formation In Vitro
The biofilm formation was performed as described by Seneviratne et al. (13), with some modifications.Standardized suspension containing 10 7 cells/mL was prepared for each strain of Candida.The monotypic biofilm were formed by adding 200 μL of standardized suspension of C. krusei or C. glabrata to each well of flat-bottom 96-well microtiter plates (Costar Corning, New York, NY, USA).For mixed biofilm were added 100 μL of standardized suspension of C. krusei and 100 μL of standardized suspension of C. glabrata.For the initial stage of adhesion the plates were incubated for 90 min at 37 °C at 75 rpm in an orbital shaker (Quimis, Diadema, SP, Brazil).After this step, the cell suspensions were carefully aspirated and each well was washed twice with 200 μL of phosphate buffered saline (PBS) to remove any planktonic cells.Then, 200 μL of YNB with 100 mM of glucose were added, and the plates were incubated for 48 h at 37 °C in an orbital shaker.The liquid medium was replaced daily.

Analysis of Biofilm by Counting Colony Forming Units (CFU/mL)
After the biofilms formation, each well was washed four times with 200 μL of PBS.Next, 200 μL of PBS was added into each well plate and the biofilm cells were carefully scraped using a sterile pick.Subsequently, 100 μL aliquot was transferred to a tube (J Prolab, São José dos Pinhais, PR, Brazil) containing PBS.To disaggregate the cells on biofilms an ultrasonic homogenizer was used (Sonoplus HD 2200, Bandelin Electronic, Berlin, Germany) with output power of 50 W for 30 s. From the solution obtained in the microtubes, decimal dilutions of the biofilm suspension were performed and 100 mL aliquots of the dilutions were inoculated into Petri dishes containing chromogenic HiCromeCandida medium (Himedia), followed by incubation of the plates at 37 °C for 48 h.After incubation, Candida species from the mixed biofilms were differentiated by colony color on HiChrome Candida agar (cream for C. glabrata and purple for C. krusei) and the CFU/mL values were determined.For statistical analysis and comparison among the groups, the data of CFU/mL were converted to logarithmic form.

Analysis of Biofilm By Colorimetric Assay (XTT)
The biofilms formed were subjected to XTT assay (2-methoxy-4-nitro-5-sulfophenyl-5-phenylalaninocarbonyl-2H-tetrazolium hydroxide), which assessed the presence of metabolically active yeast (14).Each well containing the adhered biofilm was rinsed twice with 200 µL phosphate-buffered saline solution (PBS) to remove weakly adhered cells.Then, each of the wells was inoculated with 158 µL PBS, 40 µL XTT and 2 µL menadione.After incubation in the dark for 3 h at 37 °C, 100 µL of solution was transferred to a new plate and the colorimetric change of the solution was measured using a microplate reader (TP Reader; Thermoplate, Sdorf, Araras, SP, Brazil) at 490 nm (14).

Candida Interactions in Mice
The Animal Research Ethics Committee from the Institute of Science and Technology at UNESP, approved this study under protocol number 014/2011 -PA/CEP.Twenty-four adult male mice (Mus musculus, albinus, Swiss), weighing between 30 and 60 g were included in this study.Animals were divided into 3 groups: C. krusei (n=8), C. glabrata (n=8), and C. krusei + C. glabrata (n=8).The design of the study of interaction between Candida species was performed according Rossoni et al. (9) and Takakura et al. (15) and is shown in Table 1.In summary, animals were immunosuppressed with 3 subcutaneous injections of prednisolone (Depo-Medrol; Pfizer Laboratories Ltd., Guarulhos, SP, Brazil) at 100 mg/kg of body weight alternated with two inoculations of Candida.Tetracycline chloride (Terramicina; Pfizer) was administered in the drinking water of the animals at a concentration of 0.83 mg/ mL, beginning one day before infection and was maintained throughout the experiment.A 50 μL intramuscular injection of chlorpromazine chloride, equivalent to 10 mg/kg of body weight (Amplictil; Sanofi Aventis, Suzano, SP, Brazil) in each thigh was used to sedate the animals.Each strain of Candida was cultured for 24 h at 37 °C on Sabouraud Dextrose Agar (Himedia), and were re-suspended in 10 mL of PBS, subsequently being centrifuged at 358 xg for 10 min.The resulting pellet was re-suspended in 10 mL PBS and adjusted to 1x10 8 cells/mL after counting in a Neubauer chamber (Laboroptik).A sterile swab (Absorve; Cral, São Paulo, SP, Brazil) soaked in the Candida suspension was used to inoculate the sedated mice by rubbing the swab for 3 min on the tongue dorsum.For the groups with mixed infections, the same procedure was performed, but the swab was soaked in a standard mixed suspension containing 1x10 8 cells/mL for each Candida species.
In order to confirm the final concentration of each Candida species in the swab before the oral inoculation in mice, we performed another experiment for counting CFU/mL of Candida cells adhered to the swab.Therefore, after the sterile swab had been soaked in the Candida suspension, it was transferred to a falcon tube containing PBS and submitted to ultrasonic homogenizer for 30 s.A series of dilutions were made and plated in chromogenic medium HiCrome Candida (37 °C for 48 h) for quantification of CFU/mL for each Candida species.

Recovery of C. krusei and C. glabrata from the Tongue Dorsum of Mice
Samples from the oral cavity were collected with a swab and placed in a tube containing 0.9 mL of PBS and shaken for one min.Considering that the swab absorbed approximately 0.1 mL of saliva from the oral cavity of mice, this solution was estimated to be a 10 -1 starting dilution of Candida from the soaked swab.
A series of dilutions were subsequently performed and 0.1 mL of each dilution was plated in duplicate onto the surface of plates containing chromogenic HiCrome Candida (Himedia) in order to differentiate the species recovered.Plates were incubated at 37 °C for 48 h and Candida colonies were counted to determine colony-forming units (CFU/mL).

Euthanasia of the Mice
The euthanasia of mice was performed within 2 days after the second inoculation with Candida, corresponding to 6 days of experiments.This procedure was performed by administration of an overdose of anesthetic.

Statistical Analysis
The CFU/mL data of in vitro biofilms of Candida and of experiments in mice were converted to logarithmic values and submitted Student t-test (p≤0.05).

Results
In biofilms formed in vitro at the bottom of 96-well plates it was observed that C. krusei and C. glabrata species were capable to form biofilms, reaching a median of 6.00 and 6.46 CFU/mL (log) in the monotypic biofilms, respectively.In the mixed biofilms, C. krusei showed significant higher number of CFU/mL (p=0.0001) and C. glabrata decreased (p=0.0001)compared to the single biofilm.These data indicate that the presence of C. krusei in the biofilm inhibited the growth of C. glabrata.The mean and standard deviation values of the CFU/mL obtained from the experiments described above are shown in Figure 1.
Regarding the colorimetric method based on XTT, which measured the metabolic activity of the biofilms formed, C. glabrata strain exhibited higher metabolic activity in relation to the C. krusei in single biofilms, with optical densities (OD490 nm) of 0.80±0.103and 0.21±0.296,respectively (Fig. 2).When the above values were compared to the mixed biofilms (OD490 nm= 0.24±0.168),a significant reduction was observed of the metabolic activity of C. glabrata (p=0.0001), while the values for C. krusei remained almost unchanged (p=0.8116).
Before the in vivo study, we did an assay to confirm the concentration of non-albicans Candida cells adhered to the swab.There was no difference on the capacity of C. krusei and C. glabrata to adhere the swab when they were soaked in both monotypic and heterotypic suspensions (p=0.5728)(Data not shown).Samples from the oral cavity were collected and plated to count CFU/mL number.In the groups with single infections, we found values ranging from 6.05±0.27CFU/ mL (Log) for C. krusei and 4.49±0.22CFU/mL (Log) for C. glabrata inoculations (Fig. 3).Both non-albicans species studied were capable to colonize the oral cavity of mice.Analyzing the C. krusei colonization profile in single and mixed infections, we observed that CFU/mL values were slightly higher in mixed infection (p=0.0913), with 6.30 CFU/mL values when compared to single infection (6.05 CFU/mL values).However, there was a significant reduction (p=0.0001) for C. glabrata in mixed infection (3.05 CFU/ mL values) compared to single infection (4.49CFU/mL values), agreeing with the in vitro results and suggesting an ecological interaction by competition between these two Candida species.

Discussion
C. glabrata is generally considered a species of low virulence but with a higher mortality rate than C. albicans and is the most common non-albicans Candida isolated species (16).The emergence of yeasts other than C. albicans and of mixed infections has suggested that the epidemiology of Candida infections is changing.Consequently, these infections may require higher doses of antifungal agents and may predispose patients to recurrent candidiasis, mainly because Candida species such as C. glabrata and C. krusei are becoming resistant to currently available antifungal treatments (17,18).
In this report, the interaction between C. krusei and C. glabrata was evaluated in in vitro biofilms and their ability to colonize the oral cavity of mice.In the in vitro study, the results obtained by counting CFU/mL show that C. krusei obtained higher growth when associated with C. glabrata compared to single species biofilm.However, C. glabrata was inhibited in the interaction compared with monotypic group.These data suggest that the growth of C. krusei was stimulated by the presence of C. glabrata.Most studies on polymicrobial biofilms have focused on the interaction between bacteria and Candida spp. or association between Candida albicans and non-albicans species.This is the first study that evaluate the interaction of C. krusei and C. glabrata in mixed biofilms.
In agreement with Kirkpatrick et al. ( 19) and Thein et al. (20), interaction of two Candida species seemed to suppress each other's growth, possibly because they competed for nutrients and/or one of the species generated toxic metabolites.Competitive inhibition may occur even in the initial step of adhesion onto a substrate during dual-species
C. glabrata is a non-dimorphic specie and its biofilm is composed of scattered chains of cells with multiple blastospores embedded in thin extracellular matrix.In contrast, C. krusei biofilm maintained its 'large American rice grain' form devoid of hyphal elements, which yielded a thick multi-layer ultrastructure enveloped by voluminous extracellular material (21).
Taff et al. (22) observed that XTT reduction activity is associated with the CFU in the biofilm.Furthermore, this method is the most accurate and reproducible assay for biofilm quantification.In this study, we selected the XTT activity method to determine biofilm forming activity of Candida spp.Single biofilms produced by C. glabrata showed the highest metabolic activity than that produced by C. krusei, although it produced less biomass.These results were similar with other reports (12,(22)(23)(24).Consequently, C. glabrata cells in the biofilms seem to be metabolically more active than cells from other species that produce biofilms with more biomass.In mixed biofilms, the metabolic activity was reduced significantly in comparison with single biofilm of C. glabrata, and it was significantly similar to the single biofilm of C. krusei, suggesting a possible competition between the species.These findings corroborate with Pathak et al. (12) that evaluated the interaction of these species by XTT assay and found C. krusei decreased the biofilm production of C. glabrata.
Considering that C. krusei was stimulated in mixed biofilm and C. glabrata had reduction in CFU counts, the present study was expanded to encompass the oral colonization of these species in mice models.Based on the CFU/mL data analysis of yeast recovered from the buccal cavity of immunosuppressed mice with single infections, the recovery observed were equivalent to 6.05 log10 for groups infected with C. krusei and 4.49 log10 for groups infected with C. glabrata, showing that C. krusei had a higher colonization ability compared to C. glabrata.The low CFU count of C. glabrata is consistent with Tati et al. (25) who tried to establish a model of oropharyngeal candidiasis with C. glabrata.The authors used inocula size of ranging 1x10 10 cells/mL and recovered 4-7x10 2 cells/mL from the tongue.Moreover, it was not observed infection with C. glabrata resulted in no clinical appearance of disease or weight loss in animals.
Regarding the quantification of C. glabrata CFU/ mL in mixed infections, we verified that the amount of yeast recovered was significantly lower (1.44 log10 of reduction) compared to single infection.The present study is pioneering in documenting of oral colonization by mixed infection of C. krusei and C. glabrata in mice models and indicates that further studies are required to investigate the interactions among Candida species using a larger number of strains in order to elucidating the mechanisms of how these species compete amongst themselves during the biofilm formation and oral colonization.
Within the parameters of this study, we concluded that C. krusei inhibits C. glabrata and takes advantage to colonize the oral cavity and to form biofilms.

For
this study, we used C. krusei (ATCC 6258) and C. glabrata (ATCC 90030) strains provided from the Microbiology and Immunology Laboratory of the Institute of Science and Technology of São José dos Campos, UNESP-R.D. Rossoni et al.

Figure 1 .
Figure 1.Quantification of colonies in biofilms formed at the bottom of 96-well plates (in vitro study).Mean values and standard deviation of CFU/mL (log10) of C. krusei and C. glabrata, organized in single and mixed biofilms.Student t-test.C. krusei counts: statistically significant difference between biofilm formed only by C. krusei and biofilm formed by C. krusei+C.glabrata (***p<0.0001).C. glabrata counts: statistically significant difference between biofilm formed only by C. glabrata and biofilm formed by C. krusei+C.glabrata (***p<0.0001).

Figure 2 .
Figure 2. Mean values and standard deviation of the metabolic activity (XTT analysis) for single biofilms formed by C. krusei and C. glabrata, and for mixed biofilms formed by the interactions of C. krusei+C.glabrata.Student t-test: statistically significant differences between C. glabrata (single biofilm) vs. C. glabrata+C.krusei (p=0.0001) and no difference between C. krusei (single biofilm) vs. C. glabrata+C.krusei (p = 0.8116).

Figure 3 .
Figure 3. Quantification of fungal cells recovered from the buccal cavity of mice.Mean and standard deviation of the CFU/mL (Log) of C. krusei and C. glabrata recovered from the buccal cavity of immunosuppressed mice with single and mixed infections.Student-t test.CFU/mL of C. krusei: comparison between single infection by C. krusei and mixed infection by C. krusei+C.glabrata (p=0.0913);CFU/mL of C. glabrata: comparison between single infection by C. glabrata and mixed infection by C. krusei+C.glabrata (***p=0.0001).

Table 1 .
Design of the study of the colonization between non-albicans Candida species in the immunosuppressed mice model 6Recovery of Candida from the tongue dorsum of mice euthanasia of the mice