Antifungal effects of Lactobacillus acidophilus and Lactobacillus plantarum against different oral Candida species isolated from HIV/ AIDS patients: an in vitro study

ABSTRACT Oropharyngeal Candidiasis (OPC) is an opportunistic fungal infection occurring in immunocompromised patients such as HIV/AIDS. The purpose of this study was to evaluate the antifungal properties of Lactobacillus acidophilus and Lactobacillus plantarum on different Candida species isolated from oral cavity of HIV/AIDS patients compared to Fluconazole (FLC). In this study, the antifungal effects of both cells and cell-free supernatants (CFSs) of L. acidophilus and L. plantarum were investigated against different oral Candida species by co-aggregation, agar overlay interference and broth microdilution assays, respectively. Our results showed that the highest co-aggregation ratio of the two tested Lactic acid bacteria (LAB) was observed for C. krusei. Both L. acidophilus and L. plantarum at cell concentrations 1010 to 102 cfu/ml were able to inhibit the growth of most of the oral Candida species, except for C. albicans, and to some C. krusei. In this study, MIC and MFC values for CFS of L. acidophilus ranged from 100 to 200 µl/ml and 100 to 200 µl/ml, respectively, and MIC and MFC values for CFS of L. plantarum were 50 to 200 µl/ml and 50 to 200 µl/ml, respectively. The ranges of MIC and MFC for FLC were 256–1024 µg/ml and 512–2048 µg/ml, respectively. C. albicans and C. parapsilosis displayed the highest and least susceptibility to CFSs of two LAB, respectively. Our findings showed that both cells and CFSs of L. acidophilus and L. plantarum had antifungal effects against oral Candida species.


Introduction
Probiotics are live microorganisms that, when consumed in sufficient quantities can increase the microbial balance in the host's gut and be beneficial to human health. The major probiotics include Lactobacillus spp, Bacillus spp, Bifidobacterium spp, Escherichia coli, and Saccharomyces cerevisiae [1]. Lactic acid bacteria (LAB) are known as major probiotics and are considered as a group of normal gram-positive microbiota living in the gastrointestinal tract mucosa. The colonization of these bacteria has a vital role in protection against pathogenic microorganisms [2,3].
Lactobacillus acidophilus and Lactobacillus plantarum are the most common species of Lactobacillus spp in the gut, and a number of these species are introduced as probiotics [4]. Lactobacillus species have the ability to produce several antimicrobial substances including hydrogen peroxide, acetic acid, lactic acid, bacteriocins such as small heat-stable lantibiotics (SHSL), non-lanthionine-containing membrane-active peptides (MAP), larger heat-labile proteins (LHLP), and complex bacteriocins containing one or several of chemical components. Because of the ability to produce various antimicrobial agents, these probiotics could be candidates for the control and treatment of different infections [5].
Oropharyngeal Candidiasis (OPC) is known as an opportunistic fungal infection in immunocompromised patients [6]. Candida albicans is the most common cause of OPC. Moreover, other Candida species such as C. tropicalis, C. glabrata, C. krusei, C. kefyr, C. parapsilosis, and C. dubliniensis have been isolated from infected areas in the mouth [7,8]. The different clinical signs of OPC in HIV/ADIS patients include oral thrush (pseudomembranous candidiasis), linear gingival erythema, erythematous candidiasis, perleche or angular cheilitis, salivary gland swellings, sore formation in the oral cavity, and oral hairy leukoplakia [9].
At present, development of resistant fungal strains and treatment failures following high or long-term use of antifungal drugs have increased in immunocompromised patients [10,11]. Therefore, finding an alternative bio-ecological method for better control and treatment of fungal infections has been suggested [12]. The aim of the present study was to investigate the ability of L. acidophilus and L. plantarum to inhibit the growth of different oral Candida species isolated from HIV/AIDS patients under in vitro conditions.

Materials and methods
Probiotic species and culture conditions Two lactobacillus species, L. acidophilus and L. plantarum were used in this study. These species generously provided by Dr Hamid Frootanfar from the Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. The two LAB species were initially cultured on De Man-Rogosa-Sharpe (MRS) agar (Liofilchem Company, Italy) at 37°C for 24 h in anaerobic conditions. Detached colonies of each LAB species were transferred to 5 ml MRS broth (Liofilchem Company, Italy), and then incubated in a shaker incubator at 37°C for 48 h. At the end of incubation time, two LAB species were kept in glycerol stocks at −20°C until use. For recultivation, 1 ml of L. acidophilus and L. plantarum stock were added to 5 ml MRS broth medium. Fifty microliters L-cysteine was added and microtubes placed in a shaker incubator at 37°C for 20 h (Lab companion, South Korea) for 48 h at 37°C.

Candida species and culture conditions
In this study, five different Candida species including C. albicans, C. parapsilosis, C. glabrata, C. kefyr, and C. krusei were used. These clinical Candida species isolated from oral cavity of HIV/AIDS patients and identified previously by the specific color the colony created on CHROMagar Candida media and PCR-RFLP with Msp I enzyme [9,13,14].

Co-aggregation assay
The co-aggregation was determined spectrophotometrically by UV-VIS/VIS spectrophotometer AE-S60 (AELAB Company, Guangzhou, Guangdong, China) in mixtures L. acidophilus and L. plantarum, and suspensions of each Candida species after 1, 2 and 4 h incubation and presented as the aggregation ratio (%) according to Jørgensen et al. study [7]. Briefly, the detached colonies of each 24 h culture of L. acidophilus and L. plantarum were transferred to a sterile microtube containing 5 mL MRS broth and were incubated in a shaker incubator at 84 rpm for 24 h at 37°C in an anaerobic chamber. On the other hand, different five Candida species were collected from Sabouraud Dextrose Agar (Liofilchem Company, Italy) and incubated in Sabouraud Dextrose broth (Liofilchem Company, Italy) at 37°C for 24 h. After 24 h incubation, the microtubes containing two LAB and Candida species were centrifuged separately at 855 rpm (Eppendorf Company, Hamburg, Germany) for 10 min at 25°C. Obtained pellets washed carefully thrice in phosphate-buffered saline (PBS), and suspended in 10 mmol/L PBS (pH = 7.0). The absorbance rate was set to an optical density (OD) equivalent to a McFarland standard of 600 nm (approximately equal to 10 8 cfu/ml for two LAB species and 10 6 cfu/ml for each Candida species) using a UV-VIS/VIS spectrophotometer AE-S60. 1 ml of each the LAB and 1 ml of each Candida species were completely mixed and incubated in a shaker incubator at 100 rpm at 37°C for 1, 2, and 4 h without any stimulation. Prior to each OD measurement, the microtubes containing each LAB and Candida species mixture were completely vortexed for at least 10 s. After 4 h incubation at 37°C, the OD measurement was carried out using a spectrophotometer at OD 600 nm . The experiments were performed in triplicate. Then, the co-aggregation percentage was calculated using the following formula [7,15]: where OD 0 shows the absorption amount of the complex suspension of each LAB with each Candida species at the beginning of the experiment (0 h) and OD h shows the absorption amount of the complex solutions at various times (1, 2, and 4 h).

Agar overlay interference assay
The growth inhibition of five oral Candida species by L. acidophilus and L. plantarum was done base on Keller et al. study [16]. Briefly, one distinct colony of 24 h cultured two LAB was transferred to a sterile microtube containing 5 ml MRS broth and was incubated anaerobically at 37°C for one day. The next day, the LAB species were harvested by centrifugation for 10 minutes at 855 g. The supernatants of two LAB species culture were removed. Then, the pellets were washed thrice in PBS and transferred again to the MRS broth. Cell suspensions corresponding to approximately 10 10 , 10 8 , 10 6 , 10 4 , and 10 2 cfu/ ml of L. acidophilus and L. plantarum were made. 1 ml of different cell concentrations of two LAB (10 10 cfu/ml to 10 2 cfu/ml) was added to 24 ml sterilized molten MRS agar (approximately 45°C) in petri dishes. When the medium became solid, the plates were anaerobically incubated at 37°C for 24 h. After incubation, 24 ml of sterilized molten sabouraud dextrose agar (approximately 45°C) were added to the top of the MRS agar layer containing cultured two LAB. The plates were kept at room temperature for 3 hours to solidify. 40 µl of cell suspension equivalent to 10 6 cfu/ml from each Candida species was distributed on top of sabouraud dextrose agar with a sterilized steer's replicator and was left to dry. The plates were placed at room temperature (approximately 24-25.5°C) for one hour and incubated for one day at 37°C in an anaerobic chamber.
As controls, each Candida species was distributed on top of sabouraud dextrose agar on the plate containing MRS agar layer without two LAB. All experiments were performed in triplicate. The obtained results were evaluated based on Simark-Mattsson et al. study [17]. A score of 0 = Full containment (no visible colonies), Score 1 = partial inhibition (at least one colony is visible but certainly smaller than the control plate), and Score 2 = without containment (similar growth with the control plate).

Susceptibility of different Candida species to FLC, CFSs of L. acidophilus and L. plantarum
Preparation of Cell-free supernatants (CFSs) of L. acidophilus and L. plantarum L. acidophilus and L. plantarum were grown into MRS broth and held at 37°C for 24 h. On the next day, the MRS broth containing each LAB species centrifuged for 10 min at 12,000 rpm at 4°C. Cells of L. acidophilus and L. plantarum were removed and the CFSs of two LAB species were harvested. Each CFS of LAB was filtered via a 0.22 µm sterilized syringe-driven filter (Jet Biofil, Guangdong, China) [18][19][20]. The CFSs of two LAB were kept at −20°C until use. in turbidity in comparison with the growth of control well considered as MIC value. All the tests were carried out in triplicate. Finally, average results for MICs were presented as µl/ml for two LAB species and μg/ml for FLC, respectively. The minimum fungicidal concentration (MFC) was considered as the lowest concentration for FLC, CFSs of L. acidophilus and L. plantarum, which were able to kill ≥99.9% of the five Candida species. Briefly, 10 μl of the wells with invisible growth were transferred to SDA plates. Then, the plates were incubated for 24 h at 35°C. The lowest amount of FLC, CFSs of L. acidophilus and L. plantarum, that created three colonies or less in the SDA medium was determined as MFC values [10,22].

Statistical Analysis
The results of susceptibility of different Candida species to FLC, CFSs of L. acidophilus and L. plantarum were presented as µl/ml for two LAB and μg/ml for FLC, respectively. These data analyzed by Graph Pad Prism version 8 (Graph Pad Software In, San Diego, USA) and ANOVA multiple comparison test. Data analysis on co-aggregation assay was done using student's t-test. Results of agar overlay interference assay were analyzed by the chi-square test and expressed as the median inhibition score. The significance rate for all experiments was considered p < 0.05.

Results
Co-aggregation percentage between L. acidophilus and L. plantarum with different oral Candida species The co-aggregation results after 4 h are demonstrated in percentages (%) in Figure 1. Both L. acidophilus and L. plantarum species had coaggregation ability with different oral Candida species with varying degrees. Co-aggregation percentage enhanced significantly with increase in time (p < 0.05). L. acidophilus displayed the highest coaggregation ratio for C. krusei (78%) followed by C. glabrata (70%) after 4 h incubation. The coaggregation ratio ranking of L. acidophilus with the tested five Candida species was C. krusei > C. glabrata> C. albicans > C. kefyr > C. parapsilosis. The highest co-aggregation ratio of L. plantarum was observed with C. krusei (72%), followed by C. albicans (63%) and C. glabrata Table 1 shows growth inhibition of five oral Candida spp isolated from HIV/ADIS patients with OPC at different cell concentrations of L. acidophilus and L. plantarum. At high cell concentrations (10 10 cfu/ ml and 10 8 cfu/ml), both L. acidophilus and L. plantarum inhibited the growth of all tested Candida spp. At cell concentrations 10 6 cfu/ml and 10 4 cfu/ml, the two LAB species showed slight inhibition on the five Candida spp. Also, at lower cell concentrations (10 2 cfu/ml), a slight inhibition in growth of C. glabrata, C. kefyr and C. parapsilosis by L. acidophilus and for C. glabrata, C. kefyr, C. parapsilosis, and C. krusei by L. plantarum were observed, respectively. L. acidophilus displayed no inhibition for C. albicans and C. krusei at cell concentrations 10 2 cfu/ml, and no growth inhibition was viewed only for C. albicans by L. plantarum at this concentration. Overall, at concentrations 10 10 cfu/ml to 10 2 cfu/ml, no statistically significant differences were observed between inhibitory effects of two both L. acidophilus and L. plantarum on Candida species except C. krusei. At in concentration 10 2 cfu/ml, L. plantarum displayed superiority at inhibiting C. krusei compared L. acidophilus (p < 0.05).
Susceptibility of different oral Candida species to FLC, Cell-free supernatants of L. acidophilus and L. plantarum    The CFS of L. acidophilus displayed equal inhibitory effects on C. albicans, C. krusei, C. kefyr, and C. glabrata. The susceptibility ranking of Candida spp to the CFS of L. acidophilus was: C. albicans, C. krusei, C. kefyr and C. glabrata > C. parapsilosis. The CFS of L. plantarum inhibited the growth of C. albicans significantly, followed by C. krusei and C. kefyr (p < 0.05). The susceptibility ranking of Candida spp to the CFS of L. plantarum was: C. albicans> C. krusei and C. kefyr> C. glabrata and C. parapsilosis. Generally, C. albicans and C. parapsilosis displayed the highest and least susceptibility to CFSs of two LAB, respectively. The susceptibility ranking of Candida species to FLC was: C. albicans > C. krusei and C. parapsilosis > C. kefyr and C. glabrata. Therefore, C. albicans showed the highest sensitivity to FLC among the tested Candida species. The lowest inhibitory effect of FLC was found on C. kefyr and C. glabrata. For all tested Candida spp, the antifungal effects of L. acidophilus and L. plantarum were higher than FLC among five oral Candida species (p < 0.05).
Comparison of fungicidal effects of FLC, Cell-free supernatants of L. acidophilus and L. plantarum on different oral Candida species Comparison of fungicidal effects of supernatants of L. acidophilus and L. plantarum and FLC was shown in Figure 3. The fungicidal effects ranking of Candida spp to the CFS of L. acidophilus was: C. krusei and C. kefyr > C. albicans and C. parapsilosis> C. glabrata. The CFS of L. acidophilus had highest fungicidal effects on C. krusei and C. kefyr (p < 0.05). The lowest lethal effect of CFS of L. acidophilus was found on C. glabrata. The fungicidal effects ranking of Candida spp to the CFS of L. plantarum was: C. albicans > C. krusei and C. kefyr > C. glabrata and C. parapsilosis. Generally, C. albicans and C. glabrata and C. parapsilosis displayed the highest and least lethal effects to CFS of L. plantarum, respectively. The fungicidal effects ranking of Candida species to FLC was: C. albicans, C. krusei and C. parapsilosis > C. glabrata > C. kefyr. The fungicidal effects of FLC were equal on C. albicans, C. krusei and C. parapsilosis. C. kefyr showed the lowest lethal effects to FLC among the tested Candida species. The fungicidal effects of L. acidophilus and L. plantarum were higher than FLC for five different Candida spp (p < 0.05).
Comparison of susceptibilities of different Candida species to supernatants of L. acidophilus and L. plantarum and FLC The antifungal effects of supernatants of L. acidophilus and L. plantarum on Candida species were compared to FLC. For C. albicans, the inhibitory effect of L. plantarum CFS is higher than the CFS of L. acidophilus (p < 0.0036). In addition, the inhibitory properties of the CFSs of two LAB species were greater than FLC. The difference between the growth inhibition of C. albicans by the CFSs of two LAB and FLC was significant (p < 0.001). For C. glabrata, the most intense inhibition was observed at low concentrations of L. acidophilus CFS compared to CFS of L. plantarum and FLC (p < 0.0001). In addition, significant difference was detected between the antifungal effects of the CFSs of the two LAB species (p < 0.003). CFSs of L. acidophilus and L. plantarum exhibited equal antifungal activities against C. krusei, C. parapsilosis and C. kefyr (p > 0.999). However, for these three species, the CFSs of the two LAB species had a significantly greater inhibitory effect on Candida growth than FLC (p < 0.0001).
LB21, and L. paracasei F19 exhibited week inhibition properties, and L. acidophilus La5 had no inhibitory effect. However, L. plantarum 931, L. plantarum 299v, and L. reuteri ATCC 55730 demonstrated strong inhibition. Similar to our study, at low cell concentration (10 3 cfu/ml) of LAB strains cells, except for L. plantarum strain, no growth inhibition was observed [3].
In another part of this study, we examined the antifungal effects of CFSs of L. acidophilus and L. plantarum at different concentrations on five oral Candida species. In this study, C. albicans was the most susceptible to CFSs of two LAB. Here, MIC and MFC values for CFS of L. acidophilus ranged from 100 to 200 μL/ml. These values greater than those reported by Aminnezhad et al. [32], who reported that the growth of P. aeruginosa was inhibited by CFSs of L. casei and L. rhamnosus at concentration of 62.5 μl/ml. Coman et al. showed that the most of the pathogenic yeasts and bacteria were inhibited by L. rhamnosus and L. paracase with various degrees [26].
Lower antibacterial effects for CFSs of L. acidophilus LA5 and L. casei 431 compared to our study was reported by Koohestani et al. [19]. Contrary to our results, a strong antifungal activity of L. pentosus strain LAP1 was observed versus C. tropicalis, followed by C. albicans and C. krusei [33]. CFSs of L. gasseri and L. rhamnosus inhibited the mixed biofilms of non-albicans Candida species and damaged the cells [34]. Cell-free supernatant of L. acidophilus was inhibited biofilm development and filamentation of C. albicans [24]. The differences in results of different studies may be related to differences in the examined lactobacilli strains, the experiments for evaluating antifungal effects, examined Candida species, the initial counts of LAB species, the duration of incubation, and the origin of the Candida spp isolation.
The mechanism of action of Lactobacillus strains as an effective probiotic is related to the presentation of a 29 kD collagen-binding protein on the surface and the production of biosurfactants such as surlactin that allow them to prevent the binding and decampment of harmful microorganisms into different tissues of the host's body, reduction in luminal pH, and the production of H 2 O 2 , which is toxic for harmful microorganisms. Stimulation of innate and adaptive immune responses includes the synthesis of inflammatory cytokines, producing various antimicrobial substances including hydrogen peroxide, acetic acid, lactic acid, bacteriocins such as small heat-stable lantibiotics (SHSL), non-lanthionine-containing membrane-active peptides (MAP), larger heat-labile proteins (LHLP), and complex bacteriocins containing one or several of chemical components are number of mechanisms suggested for the action of probiotics [1,35,36]. It is noteworthy that these mechanisms vary in different species of lactobacillus.
A potentially interesting and novel aspect of this study is the comparison of antifungal effects of both cells and CFSs of L. acidophilus and L. plantarum on different species using clinical isolates. These clinical species involved C. albicans, C. parapsilosis, C. glabrata, C. kefyr, and C. krusei that isolated from oral cavity of HIV/AIDS patients. During HIV infection period, the incidence of candidiasis is related to reduce the immunity level of these patients due to decreased CD 4 + cells, which is dependent on the use of antiviral therapy [37]. C. albicans, nonalbicans Candida species and Cryptococcus neoformans are the most common yeasts isolated from HIV/AIDS patients [38]. One limitation of the present study is the lack of investigation of the possible antifungal effects of L. acidophilus and L. plantarum on some species such as C. dubliniensis, C. tropicalis and C. guilliermondi.

Conclusion
Both cells and CFSs of L. acidophilus and L. plantarum showed antifungal effects against the five oral Candida species. Our finding revealed that both L. acidophilus and L. plantarum at cell concentrations 10 10 to 10 2 cfu/ml was able to inhibit the growth of most of the oral Candida species, except for C. albicans, and to some C. krusei. Here, C. albicans and C. parapsilosis displayed the highest and least susceptibility to CFSs of two LAB, respectively. Considering the obtained results and importance of candidiasis in immunocompromised hosts, treatment failures due to formation of resistant species, and the side effects of chemical drugs, further investigations for evaluating of the antifungal properties of L. acidophilus and L. plantarum and other lactobacillus species, identifying the exact mechanisms of their action, and performing antifungal studies in infected experimental animals are suggested.

Disclosure statement
There is not any conflict of interest in this study.