Abstract The morphological transition from yeast to a hyphal form, as well as the adhesion capability to the gastrointestinal tract, are implicated virulent determinant in Candida albicans and could be potential targets for prevention of the opportunistic pathogen. Based on this rationale, two yeast strains Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC along with reference strain Saccharomyces boulardii NCDC 363 were screened for the probiotic potential. Characters like pH, temperature, bile, simulated gastrointestinal juice tolerance tests, and Caco-2 cell line adhesion assay were determined in the present study. Further, the evaluation of its impact on C. albicans morphological transition and adhesion was assessed using microtitre germ tube test. In terms of probiotic characteristics, both the strains were tolerant to pH 2.5 and the presence of bile (0.3 to 0.6%) with an optimum growth temperature of 37°C. The strain KTP was also resistant to simulated gastric and intestinal juices as compared to control (13% and 41%, respectively) and NCDC 363 (55% and 35%, respectively). In contrast, both the yeasts had reduced adhesiveness to Caco-2 monolayer. Candida virulence in in vitro systems indicated that treatment of live probiotic yeast cells (108 ml) effectively reduced the filamentation and adhesion of C. albicans. The S. cerevisiae KTP had a profound effect on the hyphal development and adhesion when compared to the ApC and NCDC 363. The strain significantly reduced (P < .05) the hyphal growth in co-cultivated (93% and 94%, respectively) and pre-existing hyphae (54% and 68%) of strains C. albicans 183 and 1151. Isolates KTP and ApC also reduced the adhesion (≈ 22% and 41%, respectively) and transition of blastoconidia at two hours of incubation in abiotic surface. This study provides knowledge on the effect of potential probiotic yeasts such as Saccharomyces and non- Saccharomyces strains against virulence characteristic of Candida albicans. probiotics, simulated gastrointestinal juices, Caco-2 cells, germ tube assay, morphological transition Introduction Fungal pathogens are well associated with morbidity and mortality, and approximately 1.5 million people die from lethal fungal infections every year.1Candida albicans is the most common opportunistic pathogen associated with various clinical manifestations ranging from superficial to life-threatening systemic infections like candidemia. The significant changes in morphology and extracellular metabolites of C. albicans promotes its pathogenesis, which includes phenotypic switching, yeast to hyphal transition, secretion of extracellular proteins that promote adhesion, and invasion to host cells leading to infection.2 Moreover, the frequent prescription of antifungal compounds for candidiasis and candidemia, resulted in incessant emergence of drug-resistant superbugs. In addition, the drug resistance could increase aneuploidy, enhancing genomic plasticity and rapid evolutionary selection during infection in C. albicans.3 The azoles are widely used antifungal drugs, and phenotypic and/or inherited azole resistance reduces direct impact on its target. The alarming situation can be assessed with Candida infections in India, with fluconazole and amphotericin B resistance being recorded to approximately 3.3% leading to mortality varying from 35 to 75%.4 This has led to the renewed interest in the search for a new medication to replace the traditional antibiotic therapy for candidiasis. Probiotics represent as an effective alternate to the use of therapeutics and are defined as ‘live microorganisms which when administered in adequate amount confer a health benefit on the host.’5 Bacteria and yeast are more commonly used as probiotics. The probiotic yeast Saccharomyces cerevisiae var. boulardii has been successfully used for the prevention and treatment of various diseases, such as antibiotic-associated diarrhoea, Helicobacter pylori infections, inflammatory bowel disease, allergy, bacterial and fungal associated urinary tract infections.6,7 Researchers had focused on fermented foods as a source of probiotic yeast, and the natural environment of fermented foods provide them the inherent property to withstand the host's natural barriers.8 Additionally, microorganisms from fermented sources gain various functional properties such as antimicrobial and antioxidant capacity, synthesis of bioactive peptide, degradation of antinutritive compounds, cholesterol reduction, and immune-modulatory effect, which may be of additional importance for the probiotic application.9–11 Yeast such as S. cerevisiae, Pichia kudriavzevii, and Kluyveromyces lactis from fermented sources had been reported for their remarkable tolerance capacity towards acidic pH, bile, gastric and pancreatic enzymes.12–15 The use of probiotics such as Lactobacillus acidophilus, L. rhamnosus GR-1, L. fermentum, Bifidobacterium animalis, and S. boulardii had been widely studied to reduce Candida pathogenesis on mucosal surfaces in different mammalian and non-mammalian animal models. Additionally, clinical trials of probiotics successfully controlled mucosal candidiasis.16Saccharomyces boulardii is a commercialised probiotic yeast, extensively applied to control the virulence factors of C. albicans.17In vitro experiments have indicated that S. boulardii and its cell-extract can significantly inhibit the adhesion, morphological transition and biofilm formation.18 Additionally, more recent evidence proved that vaginal administration of S. cerevisiae CNCM I-3856 in mice, significantly reduced the C. albicans colonisation in vulvovaginal candidiasis (VVC).19 The antagonistic effect of probiotic yeast might be due to secretion of inhibitory molecules, stimulation of immunity, competitive inhibition, detoxification of microbial toxin, which successively inhibits the pathogen and its virulent behaviour.20 The lack of understanding of mechanism as well as continuous advent in search of new probiotic yeasts opens an arena for assessing the ability of other yeast against candidiasis. With the above background, we have assessed the ability of two yeasts, Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC along with reference strain S. boulardii NCDC 363 for their probiotic attributes. Further, we studied the influence of screened probiotic yeasts on morphology and adhesion ability of C. albicans in an in vitro system with an aim to open the gateway for yeast strains apart from S. boulardii for treatment of Candida infection. Methodology Yeast strains and its culture conditions The Saccharomyces cerevisiae KTP (represented as KTP) and Issatchenkia occidentalis ApC (ApC) were isolated from toddy and fermented apple juice, respectively. The yeast strains were identified by sequencing of D1/D2 region of 28S rDNA encoding genes /ITS region. Saccharomyces boulardii NCDC 363 (NCDC 363) obtained from National Collection Centre for Dairy Cultures, Karnal, India was used as probiotic reference strain. The Candida albicans MTCC 183 (CA 183) and C. albicans MCC 1151 (CA 1151) procured from Microbial Type Culture Collection and Gene Bank, Chandigarh, India, and Microbial Culture Collection, Pune, India, respectively, were used for the antagonistic study. All the yeast strains were maintained in yeast extract-peptone-dextrose (YPD) media (pH 6.8 at 30°C). The 24 h old yeast cells were harvested by centrifugation at 8000 rpm for 5 min, washed thrice with phosphate buffered saline (PBS, pH 7.4), and cell number was adjusted as per the requirement. Heat-killed cells were prepared by exposing the cell suspension at 70°C for 60 min and washed three times with PBS. Hyphae or/and pseudohyphae development of C. albicans was induced by inoculating the 24 h old pre-grown yeast (106 cells ml) in Roswell Park Memorial Institute (RPMI) 1640 media (HiMedia Laboratories, Mumbai, India) supplemented with 20% (v/v) fetal bovine serum (FBS) (HiMedia Laboratories, Mumbai, India) at 37 °C. In vitro assessment of KTP, and ApC as potential probiotics pH, bile, temperature tolerance, and survival assays For the pH stress tolerance, initially 107 cells ml of KTP, ApC, and NCDC 363 were inoculated in YPD media –adjusted to pH 1.5, 2.5, and 6.8; pH 6.8 was considered as control.13 The survival rate of yeast in the presence of bile was examined using YPD broth supplemented with ox-bile (HiMedia Laboratories, Mumbai, India) at three different concentrations (0.3, 0.6, and 0.9% (w/v)). YPD broth without ox-bile was used as a control. The cultures were incubated at 37°C for 24 h. For temperature tolerance assay, cells were incubated at 28°C (optimum yeast growth temperature) and 37°C (host body temperature). Samples were withdrawn at 4 and 24 h of incubation.21 Growth was estimated by viable count method using a haemocytometer (Rohem Instrument Pvt. Ltd, Nasik, India) with methylene blue stain and expressed as number of cells per ml. In vitro survival assay in simulated gastric and bile juice The 24 h old cell suspension (107 ml) was inoculated into simulated gastric juice (glucose 3.5 g/l, sodium chloride 1.28 g/l, potassium phosphate monobasic 0.6 g/l, calcium chloride 0.11 g/l, potassium chloride 0.23 g/l (HiMedia Laboratories, Mumbai, India), pepsin 0.3 g/l (Sigma-Aldrich, India) with pH adjusted to 2.5)22 and bile juice media (glucose 3.5 g/l, sodium chloride 3.5 g/l, potassium phosphate monobasic 0.6 g/l, pancreatin (HiMedia Laboratories, Mumbai, India) and ox-bile 3 g/l with pH 8.00). The normal saline with pH 6.8 served as control for both the assays. After incubation at 37°C for 4 h, viability was calculated by viable count method. Adherence stability of yeast strains to Caco-2 cell monolayer Caco-2 cell line (passage number 44) was procured from National Centre for Cell Sciences (NCCS), Pune, India. The cells were grown in Minimum Essential Medium Eagle (MEM) (HiMedia Laboratories, Mumbai, India) supplemented with 20% (v/v) FBS. The 104 ml of Caco-2 cells were used to promote monolayer in 96-well tissue culture plate (SPL Life Sciences Co., Ltd, Korea) and incubated at 37°C in 5% CO2 for 23 days. For adhesion assay, 108 cells ml of yeast were seeded in each well and plates were incubated at 37°C in 5% CO2 for 90 min. After incubation, wells were washed three times with sterile PBS of pH 7.4 (HiMedia Laboratories, Mumbai, India) to remove nonadhered yeast cells. Yeast cells attached to the cell lines were harvested using trypsin-EDTA (0.25% (w/v) trypsin and 0.02% (w/v) EDTA) (HiMedia Laboratories, Mumbai, India) treatment for 5 min at 37°C.23 Cell number was monitored through viable count method. In vitro antagonistic effect of yeast strains on C. albicans filamentation and adherence Quantification of C. albicans hyphal inhibition Candida albicans CA 183 and CA 1151 (106 cells ml) were co-cultured with live and heat- killed cells (108 cells ml) of NCDC 363, KTP, and ApC in RPMI-1640 media with 20% (v/v) of FBS in 24 well plates in a shaker at 90 rpm and 37°C for 6 h. After incubation, number of hyphae developed was counted using hemocytometer and expressed as number of hyphae per ml. The trans, trans-farnesol (400 μM) (Sigma-Aldrich, India) was used as control for hyphal inhibition. To detect the effect of KTP and ApC on pre-existing hyphae, Candida strains were incubated for germ tube development at 37°C for 90 min and 108 ml cells of live and heat-killed NCDC 363, KTP, and ApC were inoculated into the wells and then incubated and measured at the same condition as mentioned above.24 Microtiter germ tube assay for adhesion The live and heat-killed yeast strains of KTP and ApC were co-cultivated with CA 183 and CA 1151 for 90 min in nucleon delta surface treated flat bottom microtiter plates (Thermo Fisher Scientific, China) on a shaker at 90 rpm, and nonadhered cells were removed by washing with PBS. Adhesion capacity was quantified by crystal violet staining method.25 Briefly, plates were washed three times with PBS to remove nonadhered cells. The adhered cells were air-dried and incubated with 50 μl of gram's crystal violet for 45 min, followed by PBS wash, and the stained cells were destained with 95% (v/v) of ethanol and absorbance was measured at 595 nm to analyze the amount of adhered cells. To understand the effect of yeast against pre-existing adhesion of Candida, the Candida cell suspension (106 ml) was grown on microtiter plates, and the plates were incubated for 90 min at 37°C in a shaker at 90 rpm to promote the adherence of yeast cells to the surface of the wells on plates. Nonadhered cells were removed by PBS washing. Adherence was followed by inoculation of 100 μl live and heat-killed yeast cell suspension (108 ml) of KTP, ApC, and NCDC 363 and incubated and measured at the same condition as mentioned earlier.26 The adhesion was also analysed under phase contrast microscope (CKX40SF, Olympus, Philippines). Statistical analysis Statistical analysis was performed using GraphPad Prism-5 software (Graph Pad Software Inc., San Diego, CA, USA). Results were expressed in mean ± standard deviation (SD). Variation in treatments were compared using One-way analysis of variance (ANOVA) followed by post hoc analysis using Tukey's t test at a significance level of P < .05. Results Effect of in vitro gastrointestinal (GI) tract survival assays on S. cerevisiae KTP and I. occidentalis ApC In the present study, the ability of the isolated yeast strains to survive in the GI conditions during the transit was assessed indirectly using in vitro assays (pH, temperature, simulated gastric and bile juice tolerance) and were compared with reference probiotic yeast S. boulardii (NCDC 363). Caco-2 intestine derived cell line was used to assess the adhesiveness of yeast. At acidic pH 1.5, both the yeast strains indicated decreased survival ability and increased mortality with increased time of incubation. The growth of NCDC 363, KTP, and ApC declined to 23, 56, and 57%, respectively, by 24 h of incubation as compared to control. However, at pH 2.5 and above, all the strains significantly (P < .05) recovered their growth (20, 47, and 50% of regained growth rate respectively as compared to pH 1.5) (Fig 1a.). Figure 1b. illustrates that incubation at 37°C slightly inhibited the growth of S. cerevisiae KTP and I. occidentalis ApC (mortality was 8 and 19% with respect to control). However, both KTP and ApC had remarkably decreased growth as compared to reference strain NCDC 363, which is naturally resistance to temperature of 37°C (34 and 40%, respectively). Survival capacity of KTP and ApC in bile was observed at 4 and 24 h of incubation (Fig 2), which indicated that the isolates could tolerate 0.3% (w/v) bile. Further, the ApC exhibited a minor inhibition in 0.6 and 0.9% (w/v) of bile (6 and 22% respectively) at 24 h of incubation when compared to control. Figure 1. View largeDownload slide Effect of acidic pH (a.) and temperature (b.) on the viability of S. cerevisiae KTP and I. occidentalis ApC incubated for 4 and 24 h. Saccharomyces boulardii NCDC 363 was used as reference strain. In acidic tolerance, two different pH levels (pH 1.5 and 2.5) were used along with control (indicated as C, pH 6.8) and for temperature tolerance 28 and 37°C were applied. Results are expressed as mean ± SD; # significantly decreased value with respect to control (#P < 0.05 and ###P < 0.001) Figure 1. View largeDownload slide Effect of acidic pH (a.) and temperature (b.) on the viability of S. cerevisiae KTP and I. occidentalis ApC incubated for 4 and 24 h. Saccharomyces boulardii NCDC 363 was used as reference strain. In acidic tolerance, two different pH levels (pH 1.5 and 2.5) were used along with control (indicated as C, pH 6.8) and for temperature tolerance 28 and 37°C were applied. Results are expressed as mean ± SD; # significantly decreased value with respect to control (#P < 0.05 and ###P < 0.001) Figure 2. View largeDownload slide Growth rate of yeast strains in the ox-bile at 37°C. Three different concentrations (0.3, 0.6 and 0.9%) of ox-bile were used for to assess stability to the tolerance. Data is expressed as mean ± SD; # significantly decreased value with respect to control; * significantly increased value with respect to control (#P < 0.05; **/##P < 0.01 and ###P < 0.001). Figure 2. View largeDownload slide Growth rate of yeast strains in the ox-bile at 37°C. Three different concentrations (0.3, 0.6 and 0.9%) of ox-bile were used for to assess stability to the tolerance. Data is expressed as mean ± SD; # significantly decreased value with respect to control; * significantly increased value with respect to control (#P < 0.05; **/##P < 0.01 and ###P < 0.001). In vitro survival assay in simulated gastric and bile juice In order to determine GI tract enzyme tolerance ability of isolates, yeast strains were exposed to simulated gastric and bile juices, which are mixtures of digestive enzymes, inorganic salts, and bile. Issatchenkia occidentalis ApC had high survival rate (P < .001; 35% higher than the control) in simulated gastric juice, on other hand, it showed significantly reduced viability in bile juice (63%). The isolate KTP exhibited remarkable resistance to synthetic gastric and bile juice as compared to reference strain NCDC 363 (Table 1). Table 1. Viable number of yeast treated with simulated gastric and intestinal juices. Control Gastric juice Bile juice (107 ml) (107 ml) (107 ml) NCDC 363 2.36 ± 0.41 1.64 ± 0.30# 2.32 ± 0.35 KTP 2.11 ± 0.33 2.42 ± 0.21 3.58 ± 0.18*** ApC 2.41 ± 0.25 3.71 ± 0.07 *** 1.53 ± 0.31### Control Gastric juice Bile juice (107 ml) (107 ml) (107 ml) NCDC 363 2.36 ± 0.41 1.64 ± 0.30# 2.32 ± 0.35 KTP 2.11 ± 0.33 2.42 ± 0.21 3.58 ± 0.18*** ApC 2.41 ± 0.25 3.71 ± 0.07 *** 1.53 ± 0.31### Abbreviation used: NCDC 363, S. boulardii NCDC 363; KTP, S. cerevisiae KTP; ApC, I occidentalis ApC. Results are expressed as mean ± SD; # significantly decreased value with respect to control. *Significantly increased value with respect to control (#P < .05 and ***/###P < .001). View Large Table 1. Viable number of yeast treated with simulated gastric and intestinal juices. Control Gastric juice Bile juice (107 ml) (107 ml) (107 ml) NCDC 363 2.36 ± 0.41 1.64 ± 0.30# 2.32 ± 0.35 KTP 2.11 ± 0.33 2.42 ± 0.21 3.58 ± 0.18*** ApC 2.41 ± 0.25 3.71 ± 0.07 *** 1.53 ± 0.31### Control Gastric juice Bile juice (107 ml) (107 ml) (107 ml) NCDC 363 2.36 ± 0.41 1.64 ± 0.30# 2.32 ± 0.35 KTP 2.11 ± 0.33 2.42 ± 0.21 3.58 ± 0.18*** ApC 2.41 ± 0.25 3.71 ± 0.07 *** 1.53 ± 0.31### Abbreviation used: NCDC 363, S. boulardii NCDC 363; KTP, S. cerevisiae KTP; ApC, I occidentalis ApC. Results are expressed as mean ± SD; # significantly decreased value with respect to control. *Significantly increased value with respect to control (#P < .05 and ***/###P < .001). View Large Adhesive ability of yeast to Caco-2 cell monolayer Human epithelial colorectal adenocarcinoma cell line Caco-2 was used to analyze adhesion ability of yeast. Candida albicans was used as the positive control. The result suggested that ApC had higher adhesion (24%) when compared to NCDC 363, which was significantly lower (P < .05; 51%) than positive control, C. albicans (Fig 3a). Saccharomyces cerevisiae KTP and NCDC 363 displayed 17% and 36% of adhesion with respect to positive control. Figure 3. View largeDownload slide Adhesion of yeast strains NCDC 363, KTP and ApC to Caco-2 cell monolayer. C. albicans (CA) was used as positive control for the study (a.); the light microscopic image of adhered yeast cells on Caco-2 cell monolayer, adhered yeast cells are indicated by arrows (b.); P < 0.05 considered as level of significance with respect to positive control C. albicans (*P < 0.05; **P < 0.01 and ***P < 0.001). Figure 3. View largeDownload slide Adhesion of yeast strains NCDC 363, KTP and ApC to Caco-2 cell monolayer. C. albicans (CA) was used as positive control for the study (a.); the light microscopic image of adhered yeast cells on Caco-2 cell monolayer, adhered yeast cells are indicated by arrows (b.); P < 0.05 considered as level of significance with respect to positive control C. albicans (*P < 0.05; **P < 0.01 and ***P < 0.001). Effect of yeast isolates on the morphology of C. albicans 183 and 1151 In order to determine, the effect of KTP and ApC against the cell morphology of Candida, the strains C. albicans 183 and 1151 were treated with KTP, ApC, and NCDC 363 cell suspension (108 cells ml). Quantification of C. albicans hyphal inhibition The experiment was designed in two conditions. In the first set, planktonic cells of C. albicans and probiotic yeasts were co-inoculated, and trans, trans- farnesol was used as control. The results revealed that KTP, ApC, and NCDC 363 significantly inhibited the filamentation of C. albicans. Saccharomyces cerevisiae KTP was most effective for inhibiting C. albicans filamentation (63 and 57% in CA 183 and CA 1151, respectively) (Fig 4a.). In the second set of experiment, C. albicans was pre-incubated for 90 min and treated with yeast cultures. The yeast treated for pre-existing hyphae/pseudohyphae of Candida indicated decreased antagonistic capacity against filamentation (Fig 4b.). Here, we noted that the probiotic yeast was most effective in early stages of filamentation, and the effect was similar to farnesol treated group. However, the isolate KTP significantly reduced the filamentation of CA 183 and CA 1151 (55 and 68%, respectively) in pre-existing stage. Both live and heat-killed cells were assayed for effectiveness against the filamentation. Heat-killed cells were not efficient against retardation of hyphal development of Candida indicating the requirement of live cells of probiotic yeast for the activity (result not included). Figure 4. View largeDownload slide The effect of co-cultured (a) and pre-incubated (b) KTP, ApC and reference probiotic strain NCDC 363 against hyphal development of C. albicans. The results are expressed in number of hyphae with respect to probiotic yeast treatment. Trans, trans-farnesol (400μM) was used as a control for hyphal inhibition. Abbreviations used: C. albicans, CA; C. albicans with S. boulardii NCDC 363, CA+NCDC 363; C. albicans with S. cerevisiae KTP, CA+ KTP; C. albicans with I. occidentalis ApC, CA+ApC; C. albicans with trans, trans-farnesol, CA+F. All the values are expressed in mean ± SD, P < 0.05 were considered as level of significance with respect to control (*P < 0.05; **P < 0.01 and ***P < 0.001). Figure 4. View largeDownload slide The effect of co-cultured (a) and pre-incubated (b) KTP, ApC and reference probiotic strain NCDC 363 against hyphal development of C. albicans. The results are expressed in number of hyphae with respect to probiotic yeast treatment. Trans, trans-farnesol (400μM) was used as a control for hyphal inhibition. Abbreviations used: C. albicans, CA; C. albicans with S. boulardii NCDC 363, CA+NCDC 363; C. albicans with S. cerevisiae KTP, CA+ KTP; C. albicans with I. occidentalis ApC, CA+ApC; C. albicans with trans, trans-farnesol, CA+F. All the values are expressed in mean ± SD, P < 0.05 were considered as level of significance with respect to control (*P < 0.05; **P < 0.01 and ***P < 0.001). Microtiter germ tube assay for adhesion Microtiter germ tube assay under mild shaking condition was employed to understand the effect of probiotic isolates against C. albicans adhesion capacity. Co-culturing of Candida with the live yeasts NCDC 363, KTP, and ApC, completely inhibited the adhesion of Candida strain (result not included). In pre-incubated set of experiment, CA 183 was found to be sensitive for probiotic yeasts and number of adhesion significantly reduced compared to the control (Fig 5). In contrast, inhibition capacity of CA 1151 was not statistically significant in pre-existing set of experiment; however, when compared to control, 10–13% of inhibition was recorded. Heat-killed cells did not exhibit any inhibition against adhesion. Figure 5. View largeDownload slide Effect of probiotic yeasts on adhesion ability of pre-incubated C. albicans. The strains KTP, ApC and NCDC 363 were treated with the concentration of 108 ml against C. albicans 183 and 1151, unadhered cells were removed and examined by phase contrast microscopy, inset images indicates effects of KTP, ApC and NCDC 363 on the morphology of C. albicans. The trans, trans-farnesol (400μM) used as a control for hyphal inhibition. Abbreviation used: C. albicans 183, CA 183 (a.); CA 183 treated with NCDC 363, CA 183-NCDC 363 (b.); CA 183 treated with KTP, CA 183-KTP (c.); CA 183 treated with ApC, CA 183-ApC (d.); C. albicans 1151, CA 1151 (e.); CA 1151 treated with NCDC 363, CA 1151-NCDC 363 (f.); CA 1151 treated with KTP, CA 1151-KTP (g.); CA 1151 treated with ApC, CA 1151-ApC (h.); CA 183 treated with farnesol, CA 183-F (i.); CA 1151 treated with farnesol, CA 1151-F (j.); Inhibition of C. albicans adhesion on microtitre plate surface. The results are expressed in absorbance ability (A595) of crystal violet stain with respect to treatment (k.); (**P < 0.01 and *** P < 0.001). Figure 5. View largeDownload slide Effect of probiotic yeasts on adhesion ability of pre-incubated C. albicans. The strains KTP, ApC and NCDC 363 were treated with the concentration of 108 ml against C. albicans 183 and 1151, unadhered cells were removed and examined by phase contrast microscopy, inset images indicates effects of KTP, ApC and NCDC 363 on the morphology of C. albicans. The trans, trans-farnesol (400μM) used as a control for hyphal inhibition. Abbreviation used: C. albicans 183, CA 183 (a.); CA 183 treated with NCDC 363, CA 183-NCDC 363 (b.); CA 183 treated with KTP, CA 183-KTP (c.); CA 183 treated with ApC, CA 183-ApC (d.); C. albicans 1151, CA 1151 (e.); CA 1151 treated with NCDC 363, CA 1151-NCDC 363 (f.); CA 1151 treated with KTP, CA 1151-KTP (g.); CA 1151 treated with ApC, CA 1151-ApC (h.); CA 183 treated with farnesol, CA 183-F (i.); CA 1151 treated with farnesol, CA 1151-F (j.); Inhibition of C. albicans adhesion on microtitre plate surface. The results are expressed in absorbance ability (A595) of crystal violet stain with respect to treatment (k.); (**P < 0.01 and *** P < 0.001). Discussion Microorganisms to be considered as effective probiotics, must be functionally active when exposed to the environmental barriers of GI tract in the host. In addition, initial screening of strains based on these GI tract stress are important, especially for nonencapsulated strains used in food and therapeutic applications.27 The pH of gastric juice varies within /different individuals and it ranges from 1.5 to 3.5.28 Therefore, pH 2.5 is considered a satisfactory limit for probiotic screening. In this study, isolates KTP and ApC had significant growth in pH 2.5 compared to pH 1.5 (40 and 39% of regained growth, respectively). When similar selection parameters were adopted in the previous studies, several strains of Saccharomyces and non-Saccharomyces species isolated from kefir grains depicted 50–90% of viability after 3 h at pH 2.5.29,30 This also indicates the survival capacity may also depends on strain specificity and source of origin. A successful probiotics should be able to tolerate the human body temperature (37°C) on the other hand; the optimum temperature for the growth of yeast is between 25–30°C. Therefore, assessment of survivability at 37°C for the probiotic potential is essential. In our previous study, we reported that the non-Saccharomyces strains Pichia kudriavzevii P1, P2, and P3 had remarkably high growth rate compared to the Saccharomyces strain at 37°C14; however in the current study, 18–20% of mortality was observed in the isolates tested. Bile tolerance is another parameter for probiotic selection. Tolerance to bile salt in the range 0.15 to 0.5% has been recommended for probiotics, which is in the range of the physiological concentrations.31 Psomas et al. (2001), revealed that non-Saccharomyces strain Kluyveromyces marxianus, I. orientalis and P. farinosa had higher survival capacity than Saccharomyces in 0.5% (w/v) bile which, further support the findings of our study.21 GI tract environment is a combination of stress factors, and their synergistic influence might be different from individual stress factor. Furthermore, simulate GI tract juices have been better represented in the in vivo conditions than the individual stress tolerance assays. There were differential responses of the strains when exposed to synthetic bile juice with a significant decrease in the growth rate of ApC compared to the reference yeast strain (NCDC 363). Earlier, studies had reported a slight inhibitory effect of gastric juice to Saccharomyces and non-Saccharomyces yeasts.31 Adherence of probiotic strain can increase colonization, which further plays an important role in mucosal immune modulation. The Caco-2 cell line was employed to evaluate the adhesion capacity of yeast, and it was found that yeast indicated poor adhesive ability to these cell lines as compared to positive control C. albicans. Previous reports had suggested that non-Saccharomyces strains such as Kluyveromyces lactis and K. lodderae had better adherence when compared to S. cerevisiae.15,32 Similar observations was recorded in the experiments. On the other hand, robust size of a yeast cell needs a larger space to adhere to the Caco-2 cell line than a bacteria, and this makes the difference between bacterial and yeast adhesion capacity. Collado et al. (2007) suggested the role of adhesion to the intestinal immune modulation,33 whereas, independent studies by Tasteyre et al. (2002) and Van der Aa kuhle et al. (2005) highlighted the importance of poor adhesive yeasts as probiotics.34,35 During GI disorder, the pathogenic organisms take over and disrupt the gut homeostasis. Depending on anatomical niche, more than 70% of population colonising the oral, GI and urogenital tracts were found to be C. albicans.36 Probiotics are the one considered as a major bio-therapeutic method, which are successfully used to treat C. albicans infections. Candida albicans exhibit polymorphism, hence it can grow as budding yeast, pseudohyphal form (cells with constricted septa) or as hyphae (elongated filamentous form).40 On the other hand, the morphological changes in Candida has its own clinical significance during infection, with its filamentous form being more virulent as compared to blastoconidial stage. Inhibition of this morphological transition can be used as therapeutic applications. In the present study, the co-cultured and pre-existed hyphal treatments with isolate KTP and ApC successfully inhibited the hyphal development of C. albicans. This is in agreement to the reports, where S. boulardii cell extract and supernatant were shown to have a similar effect on C. albicans morphology.37 In contrast, there was no effect on the viability of C. albicans, co-cultivated and/or pre-incubated with KTP, ApC and NCDC 363 cells. The similar results were observed when S. boulardii was treated for inhibiting the biofilm formation of C. albicans.37 The effect of yeast against pre-existed hyphae will provide evidence for bio-therapeutic applications of probiotics against antifungal resistant Candida. Adherence of C. albicans to biotic surfaces is considered a crucial step in the pathogenesis in Candida infections. In addition, the Candida biofilms have defined phases of development, adhesion and colonisation phases which provide a platform to biofilm formation.40 The co-cultured and pre-existing treatments remarkably reduced the adhesion to abiotic surfaces. Similar result was recorded in probiotic Lactobacillus and Bifidobacteria strains when co-cultivated with food borne pathogens like Lischeria monocytogenes, Salmonella tphimurium, Shigella boydii and Staphylococcus aureus.38 The competitive inhibition might be enhancing the inhibition of C. albicans in microtitre plates, and it replicates the same phenomenon of GI tract, where, competitive inhibition of intestinal microflora hinders the adhesion of Candida. The abiotic adhesion studies indicated that factors such as low pH, cell surface mannoprotein, cell surface hydrophobicity, surface roughness and the surface free energy of the resin strips modify the adhesion and colonisation capacity of microorganisms.39 Other than these factors, we also observed that strain-dependent adhesion by Candida, with CA 1151 having higher adhesion ability than CA 183. Yeast form of C. albicans is initially attached to the biotic and abiotic surfaces, and then, these microcolonies become interlinked through filamentation. This complex interlinked filamentation increases with time, and finally, it leads to a complex structure known as biofilm.40 The formation of biofilms on biotic and abiotic (especially in medical devices) surfaces has direct relation with human disease. In the present study, the assessed yeast were found to have the probiotic potential. It was revealed that S. cerevisiae KTP and non-Saccharomyces strain I. occidentalis ApC are capable of surviving in the GI tract. Interestingly, the study also confirms that yeast isolates could control C. albicans filamentation and adhesion property. Antagonistic effect against pre-existing hyphae and adhesion has a clinical significance to treat antifungal resistance related candidiasis. However, the beneficial effect of strains in preventing or treating candidiasis can be substantiated by well-designed in vivo and clinical trials. To our knowledge, this is the first report of a non-Saccharomyces yeast I. occidentalis to control virulence factors of C. albicans. Acknowledgments We would like to thank the Director, CSIR-CFTRI for excellent technical support. Mr. K. Lohith is indebted to INSPIRE program, Department of Science and Technology (Ministry of Science and Technology), Government of India for their financial assistance for his doctoral research. 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Medical Mycology – Oxford University Press
Published: Nov 1, 2018
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