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Article

In Vitro Inhibitory Effects and Co-Aggregation Activity of Lactobacilli on Candida albicans

by
Lyubomira Yocheva
1,
Lilia Tserovska
1,
Antonia Danguleva-Cholakova
2,
Teodora Todorova
2,
Galina Zhelezova
1,
Elena Karaivanova
2 and
Ralitsa Georgieva
2,*
1
Department of Biology, Medical Genetics and Microbiology, Faculty of Medicine, Sofia University “St. Kliment Ohridski”, 1407 Sofia, Bulgaria
2
Lactina Ltd., 1320 Bankya, Bulgaria
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2024, 15(3), 1576-1589; https://doi.org/10.3390/microbiolres15030104
Submission received: 22 July 2024 / Revised: 14 August 2024 / Accepted: 15 August 2024 / Published: 17 August 2024
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Abstract

:
Lactobacilli are considered important probiotics for the prevention of some infections. In this study, the antifungal effect of both cells and cell-free supernatants of twenty-three strains of lactobacilli were investigated against Candida albicans by co-aggregation, agar diffusion assay, agar spot assay and co-culture assay. In all cases, a fungistatic effect was recorded. In the agar diffusion assay and agar spot assay, an effect was established primarily for heterofermentative species via the production of lactic acid. The anti-Candida effect was higher with microbial suspension than with cultural supernatants in the co-culture assay. A strain-specific reduction in the yeast growth up to 28.9% in MRS broth and up to 17.1% in BHI broth was observed. Cells of Limosilactobacillus fermentum LLF-01 and Limosilactobacillus reuteri LLR-K67 showed the highest activity in both model systems. For all strains, a lower reduction up to 9.7% was recorded with cultural supernatants. L. fermentum LLF-01 showed the highest ability of co-aggregation (64.8%) with C. albicans, followed by Lactobacillus acidophilus LLA-01, Lactobacillus gasseri LLG-V74, Lactobacillus delbrueckii subsp. bulgaricus LLB-02 and two strains of Lactobacillus delbrueckii subsp. lactis LLL-14 and LLL-F18. The present study showed the potential of several strains of lactobacilli to affect the population of C. albicans in vitro. The combination of cultures with proved anti-Candida and co-aggregation activity in a probiotic formula may have a positive effect for the prevention of yeast overgrowth in the gut and hence for the suppression of candidiasis.

1. Introduction

Lactobacilli are known as major probiotics and are considered as a group of normal Gram-positive microbiota living in the gastrointestinal (GI) and genitourinary tracts. The colonization of these bacteria has a vital role on the maintenance, stabilization and restoration of the resident microflora, especially after the development of dysbiosis as a result of use of broad-spectrum antibiotics [1,2].
The states of dysbiosis involving fungi such as Candida species and especially Candida albicans are severe and difficult to overcome. C. albicans is the most common Candida species inhabiting the skin and the mucosal surfaces (oral cavity, GI tract and vagina) both in health and disease [3]. This organism can cause superficial to deep systemic candidiasis only when there is overgrowth, impaired host immunity, hormonal imbalance or metabolic disorders [4]. Most of the systemic infections originate from the GI tract, which is the main reservoir of C. albicans [5]. These diseases are essentially caused by candidal biofilms (yeast-to-hyphae transition) attached to body surfaces, as opposed to the planktonic form of the yeast, which exists in the suspended phase [4,6]. Severe cases of candidiasis may cause serious complications to internal organs and require prolonged treatments (weeks or months) to clear up completely. However, the toxicity of available antifungals (polyenes and azoles), their adverse effects, most commonly GI discomfort, rash, hepatic and nephrotoxicity, and the emergence of resistance to these drugs put restrictions on their use as long-term therapeutic agents for candidal infections [6,7]. Incomplete courses of treatment by patients often result in recurrent symptoms.
In the search of innovative approaches for preventing and treating Candida-related dysbiosis, probiotics have been suggested as a useful alternative. Their protective effect against Candida is based on various mechanisms that act synergistically: production of metabolites inhibiting the yeast growth, such as lactic acid, H2O2, bacteriocin-like proteins, small peptides, short-chain fatty acids and biosurfactants as well as reduction in biofilm formation and adhesion by co-aggregation, competition for nutrients and binding sites of mucosal surfaces and by immunomodulation of the intestinal epithelial barrier [8,9]. Some in vitro studies and clinical trials showed positive results regarding the effectiveness of specific lactobacilli strains against C. albicans [10,11,12,13,14,15]. The results from studies testing one strain should not be extrapolated to other strains because of the specific properties and effects on Candida.
The aim of the present study was to investigate the ability of lactobacilli strains to inhibit the growth of the Candida albicans reference strain and their co-aggregation capability under different in vitro conditions. Most of the tested lactobacilli were selected at an earlier stage based on their antimicrobial activity and antibiotic susceptibility [16]. Some strains of commercial importance [17,18] and some newly isolated strains of human origin were also part of the study.

2. Materials and Methods

2.1. Lactobacilli Strains

Twenty-three strains of lactobacilli isolated from a variety of sources and previously identified at species level by API-50 CHL biochemical identification, species-specific PCR, 16S ARDRA, 16S rRNA Sequence Analysis and MALDI-TOF MS (unpublished data) were provided by Lactina Ltd. (Bankya, Bulgaria) (Table 1). Nineteen of the strains are on safe deposit in the National Bank for Microorganisms and Cell Cultures of Bulgaria. The cultures were stored in glycerol stocks at −65 °C. Before the assay, the strains were pre-cultivated twice in de Man Rogosa Sharpe (MRS) broth (HiMedia, Mumbai, India) at 37 °C for 24 h.

2.2. Test Organism

A reference strain of Candida albicans NBIMCC 74 (originally ATCC 10231) was received from the National Bank for Microorganisms and Cell Cultures of Bulgaria. It is a standard susceptible strain recommended as test culture for determining the effectiveness of antimicrobial agents. C. albicans was grown in Yeast Extract Peptone Dextrose (YPD) broth (HiMedia) and on Sabouraud Dextrose (SD) agar (HiMedia).

2.3. Agar Diffusion Assay

The inhibitory effect was tested using acid and neutralized cell-free supernatants (aCFSs, nCFSs) prepared as previously described [16]. The samples were stored as single-use aliquots at −20 °C until use.
A suspension of C. albicans (OD = 0.5, λ = 530 nm, ~106 CFU/mL) was mixed with melted and cooled SD agar to a final concentration of ~104 CFU/mL. Wells (8 mm diameter) were cut by cork-borer and 100 µL CFS was placed into each well. The plates were kept in a refrigerator for 2 h and then incubated at 32 °C for 24 h. Finally, the zones of growth inhibition were measured and their diameters (in mm) were recorded. The test was performed in triplicate.

2.4. Agar Spot Assay

The inhibitory activity of the method was analyzed as described by Schillinger and Lüke [19]. Overnight cultures of lactobacilli were spotted (2 to 3 µL) on the surface of MRS agar plates and MRS agar plates buffered with 35 mM sodium bicarbonate. The plates were dried out at room temperature and incubated for 24 h at 37 °C to develop the spots. Afterwards, 10 mL of SD agar (0.7% agar) previously inoculated with an overnight culture of C. albicans at 106 CFU/mL were poured over the plates and incubated aerobically at 32 °C for additional 24 h. The growth inhibition of C. albicans by lactobacilli was quantitatively assessed by measuring the diameter of growth inhibition zone, subtracting the diameter of the central spot (bacterial colony). The test was performed in triplicate.

2.5. Growth Inhibition of C. albicans Using Co-Culture Assay

After cultivation in an appropriate broth media, MRS for lactobacilli and YPD for C. albicans, for 18–24 h, the cultures were centrifuged at (6000× g/10 min), washed three times in sterile phosphate-buffered saline (PBS) and finally resuspended in PBS (pH 7.0). The absorbance rate was set to an optical density (OD 600 nm) approximately equal to 106–107 CFU/mL for lactobacilli species and to an optical density (OD 530 nm) approximately equal to 106 for C. albicans using a Jenway 6300 visible spectrophotometer (Cole-Parmer Ltd., St Neots, UK). The quantification of the cell number of the inoculums was optimized at an earlier stage by counting CFU/mL after plating on MRS agar for lactobacilli and on SD agar for C. albicans.
For the experiment, 250 µL of C. albicans suspension and 250 µL of lactobacilli cell suspension or acid cultural filtrate (CFS) were combined with 2 mL MRS broth (first model system, pH 6.8) or BHI broth (HiMedia) (second model system, pH 6.9). In the control groups, microbial suspensions of lactobacilli were replaced by PBS (Control 1) or CFSs were replaced by MRS (Control 2). The method was previously described by Rossoni et al. [15]. After mixing, all samples were incubated overnight at 37 °C. The number of C. albicans cells was counted on SD agar with chloramphenicol (0.05 g/L). Each cell suspension of lactobacilli and CFS was assayed in two independent experiments, each with two replicates. Percentage growth reduction was calculated using the following formula: %Growth reduction = [(log10 CFU/mL in control − log10 CFU/mL in co-culture)/log10 CFU/mL in control] × 100. The pH of the lactobacilli-C. albicans mixed cultures was measured with pH meter (Mettler-Toledo AG, Schwerzenbach, Switzerland).

2.6. Co-Aggregation Assay

To evaluate the co-aggregation ability between lactobacilli cells and C. albicans cells, two procedures were used—spectrophotometric determination and microscopic observation. For the first procedure, standardized cell suspensions of C. albicans and lactobacilli were prepared as described above. For co-aggregation abilities, 1.5 mL aliquots of both suspensions (lactobacilli and yeast) were vortexed at least 10 s and immediately transferred to a cuvette. Samples containing 3 mL aliquots of a single bacterial or yeast suspension were used as control. After 4 h incubation at 37 °C, the absorbance (OD 600 nm) of the mixture was measured. The co-aggregation percentages were finally calculated as follows [20]: %Co-aggregation = [((Ax + Ay)/2) − A(x + y)/Ax + Ay/2] × 100, where Ax and Ay are the individual aggregation properties of the lactobacilli and the yeast, and A(x + y) is the combined aggregation of the lactobacilli and the yeast after 4 h incubation. All experiments were performed in triplicate.
For visual observation of co-aggregates, an aliquot of 100 µL of each suspension was spread on a microscope slide and Gram-stained (Gram stain kit Deltalab, Barcelona, Spain). For visualization, light microscope (1000× total magnification) with Olympus SC30 Camera (Tokyo, Japan) and AnalySIS getIT5.1 image software was used. Results were expressed as co-aggregation score: 0—no visible clumps or bound micro-organisms; 1—presence of small and sparsely distributed clumps; 2—presence of large and dense visible clumps of bacteria and yeast; 3—maximum score characterized with presence of large and dense visible clumps entrapping most of the bacteria and yeast, based on the assays of Younes et al. [21].

2.7. Statistical Analysis

Statistical analyses were performed via t-test and Tukey’s test (parametric) to compare the CFU/mL results from co-culture assay. All analyses were performed using the software KyPlot 6.0 (KyensLab Inc., Tokyo, Japan) and p < 0.05 was considered significantly different.

3. Results

3.1. Agar Diffusion Assay and Agar Spot Assay

The anti-Candida effects of cells and supernatants of twenty-three strains of lactobacilli were evaluated based on different commonly accepted methods. A negligible zone of inhibition was registered for four strains only with acid supernatants (pH between 3.84 and 4.75) in an agar well diffusion assay (Table 2). Co-incubation of lactobacilli–C. albicans in agar spot assay revealed visible fungistatic zone of inhibition around the colonies of thirteen strains on MRS agar varied between 6.0 and 10.5 mm (Table 2, Figure 1). Smaller or absent zones of inhibition were found around the colonies of the same strains in the parallel analysis with buffered MRS-NaHCO3 agar plates. The majority of the strains showed activity in both tests belonging to the group of heterofermentative lactobacilli.

3.2. Growth Inhibition of C. albicans Using Co-Culture Assay

All lactobacilli strains were screened for antifungal activity against planktonic culture of C. albicans. The direct effect of the cells and the indirect effect of their cultural supernatants were quantitatively assessed in two model systems. In general, similar growth of C. albicans was achieved in the control groups (107 CFU/mL) in MRS and BHI broth (Figure 2). After 24 h of incubation, the recovered number (CFU/mL) of C. albicans in the cell and supernatant group of lactobacilli were determined and subjected to statistical analysis (Figure 2). Additionally, the mean values of log10 CFU/mL of C. albicans in the cell and supernatant group of lactobacilli in relation to the log10 CFU/mL in untreated control groups were used to calculate the percentage reduction (Table 3).
The growth of C. albicans was inhibited by most of the tested lactobacilli strains when interacting directly with their cells in both model systems (Figure 2).
In MRS broth, all lactobacilli strains gave rise to significant decrease (p < 0.001) of yeast growth compared with the control culture. Better growth of lactobacilli in MRS broth and a decrease in pH (4.02–5.06) determined higher yeast reduction of 12.1–28.9% (equivalent to 0.9–2.1 logarithms reduction) for 87% of tested lactobacilli (Table 3). Three strains, L. acidophilus LLA-01, L. gasseri LLG-V33 and LLG-V74, for which slower growth in MRS broth was characterized, showed lower yeast reduction of 5.7–6.5% (equivalent to 0.4–0.5 logarithms reduction). Weaker growth of lactobacilli in BHI broth and higher pH values (5.38–6.53) after 24 h of incubation determined lower percentage reduction in yeast growth 2.0–17.1% (equivalent to 0.1–1.2 logarithms reduction) in compare with MRS broth (Table 3). Nevertheless, significant decrease (p < 0.001) of C. albicans showed 91% of tested lactobacilli in BHI broth.
Generally, the highest percentage of reduction was calculated for L. fermentum LLF-01 and L. reuteri LLR-K67 in both model systems (Table 3). Although the relative reduction rate was higher for heterofermentative species, some homofermentative lactobacilli showed comparable levels of inhibition (L. delbrueckii subsp. bulgaricus LLB-02, L. delbrueckii subsp. lactis LLL-F18, LLL-14 and L. helveticus LLH-108) in MRS broth.
Next, the growth of C. albicans was inhibited when interacting with the acid supernatants (1:8 dilution in broth media). The percentage of reduction varied between 3.5 and 9.7% (0.25–0.7 logarithms reduction) in MRS broth and 2.2–7.9% (0.2–0.6 logarithms reduction) in BHI broth. A statistically significant difference between the control group and treatment group was determined for all CFSs in MRS broth and for 17 (74%) of CFSs in BHI broth.
To compare the anti-Candida effects between the cells of different lactobacilli from one hand, and between their acid supernatants on the other hand, an additional statistical analysis was performed (Table 3, lowercase letters in the same column). While the direct effect of cells on the yeast growth was determined as strain specific, no significant difference in the effects of investigated CFSs was calculated in both model systems. In addition, the anti-Candida effect was higher with microbial suspension than with cultural supernatants (Table 3, capital letters in the same raw). All strains in MRS broth and 11 (48%) strains in BHI broth showed a significantly (p < 0.05) higher reduction in C. albicans’ cell number when compared with their cultural supernatants.

3.3. Co-Aggregation Assay

The co-aggregation ability between cells of lactobacilli and C. albicans varied widely from one strain to another. L. fermentum LLF-01 displayed the highest co-aggregation percentage (64.8%) followed by L. delbrueckii subsp. lactis LLA-F18 and LLL-14, L. acidophilus LLA-01, L. delbrueckii subsp. bulgaricus LLB-02, L. gasseri LLG-74 and L. paracasei LLC-115 (17.2–37.7%) (Figure 3). Albeit to a lesser extent, co-aggregation was registered for L. casei LLC-4K, L. reuteri LLR-K67, L. gasseri LLG-V33 and L. paracasei LLC-J31 (8.3–10.8%).
The microscopic examination clearly demonstrated the formation of clusters between the cells of lactobacilli and C. albicans for some strains and the absence of similar clusters for other strains (Figure 4A–I). The co-aggregates were observed for all strains with a high rate of co-aggregation determined spectrophotometrically. Thus, the highest co-aggregation score was observed for L. fermentum LLF-01 (score = 3). L. delbrueckii subsp. bulgaricus LLB-02, L. delbrueckii subsp. lactis LLL-14 and LLL-F18, L. gasseri LLG-V74, L. acidophilus LLA-01 and L. paracasei LLC-115 (score = 2) formed visible clusters with different sizes. In the cases of a low rate of co-aggregation (≤10%), the formation of small clusters (L. reuteri LLR-K67, score = 1) or the absence of co-aggregates (65% of the strains, score = 0) was observed (Figure 4).

4. Discussion

The present study assessed the anti-Candida and the co-aggregation activity of lactobacilli strains previously qualified as probiotic candidates [16] and of newly isolated strains of vaginal origin. The effects of the presence of lactobacilli cells and cultural supernatants on the growth of C. albicans were determined in vitro on agar plates and in liquid cultures. The detection of activity only with acid supernatants for some heterofermentative strains by agar diffusion assay and the decrease in the activity on MRS agar plates when the media is buffered with sodium carbonate clearly indicate the role of lactic acid released in the growth media. Althought some heterofermentative species produce other organic acids such as acetic acid, the lactic acid is mainly responsible for the decrease in pH in the growth media. Besides lactic acid, the main byproduct of the sugar fermentation of lactobacilli is more acidic than acetic acid. Both organic acids are lipophilic acids that are able to dissociate directly inside a microbial cell. This induces intracellular acidification and internal accumulation of negatively charged counter-ions, leading to multiple deleterious effects for the yeast cells [22]. At low pH (<6), C. albicans remains in the less virulent budding yeast form [4]. In addition to organic acids, biosurfactants, bacteriocins, H2O2, exopolysaccharides and fatty acids produced by lactobacilli are also factors contributing to the inhibition of C. albicans growth [23,24,25,26,27,28].
In our study, the insufficient growth of some lactobacilli before collecting the supernatants or before the overlay with Candida results in inadequate production of inhibitory compounds. This could explain the lack of activity for L. gasseri strains and most of the strains in L. delbrueckii groups. In accordance with our study, 56.6% of the tested vaginal homofermentative lactobacilli (mainly L. gasseri, L. jensenii and L. johnsonii strains) showed no inhibitory effect on C. albicans by agar overlay methods, unlike most of the heterofermentative lactobacilli (L. paracasei, L. rhamnosus and L. reuteri) [29].
In the planktonic culture, most lactobacilli exerted an anti-Candida activity when the yeast was placed in contact with the cells or supernatant. The extent of anti-Candida activity in co-culture with lactobacilli cells was determined by the growth characteristic of individual strains and the media used. The higher growth rate and biomass accumulation for some strains determined a lower pH in the media for the time of incubation. A species- and strain-specific antifungal effect was registered in both model systems but it was higher in MRS broth. BHI is a highly enriched media but with a very low amount of glucose and without yeast extract, which is the preferred nitrogene source by lactobacilli. More abundant growth of lactobacilli in MRS compared to BHI determined stronger competition for nutrients and the secretion of sufficient amount of lactic acid, which may have an impact on C. albicans. The obtained results in both model systems confirm the hypothesis that only elevated levels of lactic acid efficiently inhibit fungal growth [6,30,31]. However, the ability of the selected strain to incorporate in the microbiome and its ability to produce sufficient amounts of antimicrobial metabolites in the prevailing conditions of a given habitat are of crucial importance in vivo [32].
The higher activity of heterofermentative lactobacilli might not be accidental, considering their very close association with intestinal habitats [33]. Commonly reported as inhibitors of C. albicans and non-albicans, Candida species are strains of L. fermentum, L. reuteri, L. rhamnosus, L. paracasei and L. plantarum species. Some vaginal lactobacilli such as L. acidophilus, L. gasseri and L. jonsoni are also documented to reduce the growth, biofilm formation and hyphal formation of Candida spp. [9,23,34]. In our study, aCFSs were characterized as less effective on the planktonic C. albicans culture than lactobacilli cell suspensions. In the test with supernatants, a significant reduction in C. albicans cells was registered in comparison to the control culture but showed similar effects irrespective of the lactobacilli strains within the same model system. In a study by Rossoni et al. [15], 86% of the Lactobacillus strains had a comparable inhibitory effect on C. albicans growth only when their supernatant was placed in contact with C. albicans. The comparison with other studies is difficult due to the different model systems and the ratio of dilution of the analyzed supernatants. A generally higher concentration and lower pH of the cultural supernatants determined a stronger anti-Candida effect [24,30,35].
A possible explanation for the better anti-Candida activity of lactobacilli than their CFS was given by Xu et al. [36]. The authors demonstrated that L. plantarum culture reduced to a higher level C. albicans yeast cell proliferation than CFS in the mature L. plantarum culture. In their work, the potential role of lactic acid was excluded due to the lack of a significant reduction in yeast cell proloferation in a CFS minicking acidified medium. Upon polymicrobial transcriptomics of the dual-species interaction, interesting changes were identified in both L. plantarum and C. albicans gene expression. This included contrary changes in two L. plantarum quorum-sensing (QS) systems, including upregulation of genes involved in the co-aggregation of L. plantarum and C. albicans and downregulation of genes contributing to interspecific communication. Dual-species interaction reduced cell survival-related and pathogenesis (adhesion/invasion, phenotypic switch, environmental adaption, biofilm formation, drug resistance and others) determinants in C. albicans [36].
The production and/or the activity of antifungal compounds in vitro are influenced by the cultural condition such as culture media, pH and incubation time. For example, Yang et al. [37] found high bacteriocin activity in MRS broth in comparison with no activity detected in BHI broth in the same growth conditions. In addition, considering our results, we found MRS broth to be more suitable than BHI broth for the selection of strains with antifungal activity in a planktonic culture. The importance of the method (solid vs. liquid culture) was also demonstrated. The presence of antifungal activity was observed for some cultures, such as L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis and L. helveticus in broth, but a lack of activity on agar media could underestimate the potential of some cultures in the case of using one method for selection. This was also demonstrated by Osset et al. [13], who reported good anti-Candida activity of lactobacilli in a liquid assay in contrast to no activity in a solid assay. In a study by De Gregorio et al. [29], some vaginal lactobacilli inhibited the growth of at least one Candida strain via an agar overlay and liquid media. However, such activity was not evidenced by the agar plate diffusion method. Although the methods outlined above allowed the selection of several lactobacilli strains (L. reuteri LLR-K67, L. fermentum LLF-01, L. paracasei LLC-115, L. casei LLC-4K, L. rhamnosus LLR-L1 and LLR-L2, L. plantarum LLP-4B), applying other methods for selection is recommended, taking into consideration the different mechanisms used by the strains to exercise their antifungal activity.
Another important property, linked to the beneficial effects of some lactobacilli, is their co-aggregation ability with pathogenic micro-organisms. The co-aggregation can create a microenvironment around the pathogen with a higher concentration of inhibitory substances and can prevent pathogens from binding to tissue receptors [21,38]. This leads to a reduction in the pathogenic load during infections. Here, species and strain specific co-aggregation activity were registered with C. albicans in accordance with other studies [34,39]. L. fermentum strains are reported by other authors for their high level of co-aggregation with C. albicans [34,40]. Boris et al. [41] found that L. acidophilus and L. gasseri, isolated from the vaginas of healthy premenopausal women, co-aggregated in vitro with C. albicans isolated from the same vaginal samples. Co-aggregation ability was reported for L. reuteri ATCC PTA 5289 and L. reuteri DSM 17938 with both clinical and reference Candida strains [39]. In the same study, the percentage of co-aggregation of L. reuteri DSM 17938 with both C. albicans strains was in the same range as the one registered for our L. reuteri LLR-K67. Good co-aggregation potential was reported for L. plantarum strains with C. albicans [42,43], which was in contrast to our results. Although high co-aggregation activity with C. albicans was reported for a strain of L. delbrueckii isolated from traditional Egyptian dairy products [28], the data for this species are scarce. The close association of L. delbrueckii with dairy products but not with human and animal specimens should not underestimate their potential application as probiotics with verified health effects.

5. Conclusions

The anti-Candida activity of lactobacilli is closely related with their ability to proliferate in the target habitat, where lactobacilli should produce antifungal metabolites. Hence, the in vivo production of lactic acid by lactobacilli would be a more efficient anti-Candida treatment than the use of preparations based on lactic acid only. Using different methods for the in vitro selection of strains with antifungal activity as well as a combination of lactobacilli with complementary antagonism would be a good approach in developing probiotics with anti-Candida activity. Amongst the tested strains, L. fermentum LLF-01 showed promising anti-Candida properties based on the results of the agar diffusion assay, agar spot assay, co-culture assay and co-aggregation assay. The inclusion of this strain in a probiotic blend with other lactobacilli that also displayed good antifungal and/or co-aggregation activities (L. reuteri LLR-K67, L. rhamnosus LLR-L1 and LLR-L2, L. paracasei LLC-115, L. plantarum LLP-4B, L. acidophilus LLA-01, L. gasseri LLG-V74, L. delbrueckii subsp. bulgaricus LLB-02, and L. delbrueckii subsp. lactis LLL-F18 and LLL-14) would be a sound alternative in the prevention of candidiasis. The obtained results represent a good base for further investigation of other mechanisms involved in the inhibition of C. albicans and non-albicans Candida species by these lactobacilli.

Author Contributions

Conception, design, methodology, L.Y., L.T. and R.G.; data curation, L.Y., L.T., A.D.-C., T.T. and R.G.; literature search and data analysis, L.Y. and R.G.; writing—original draft, R.G.; writing—review and editing, L.Y., L.T., G.Z. and E.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study is partially funded by the Research Fund at Sofia University “St. Kliment Ohridski” Project No. 80-10-15/09.04.2019 and Lactina Ltd. (Bankya, Bulgaria).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this published article and are available from the authors upon reasonable request.

Conflicts of Interest

The research was funded in part by Lactina Ltd. (Bankya, Bulgaria), and Ralitsa Georgieva, Antonia Danguleva-Cholakova, Teodora Todorova and Elena Karaivanova are employed by the company. The funders had no role in the design and conduct of the study, nor the decision to prepare and submit the manuscript for publication.

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Figure 1. Growth inhibition of C. albicans by agar spot assay after 24 h incubation at 32 °C.
Figure 1. Growth inhibition of C. albicans by agar spot assay after 24 h incubation at 32 °C.
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Figure 2. Effect of lactobacilli cells and their cultural supernatants on the growth of C. albicans in co-culture assay using MRS and BHI broth model systems after 24 h at 37 °C. The control groups included non-treated suspension of C. albicans (control 1—C. albicans + PBS and control 2—C. albicans + MRS). Data are means ± S.D, n = 2 of the number of C. albicans (Log10 CFU/mL). Significant differences from the control are indicated small letters: a (p < 0.001); b (p < 0.01) and c (p < 0.05).
Figure 2. Effect of lactobacilli cells and their cultural supernatants on the growth of C. albicans in co-culture assay using MRS and BHI broth model systems after 24 h at 37 °C. The control groups included non-treated suspension of C. albicans (control 1—C. albicans + PBS and control 2—C. albicans + MRS). Data are means ± S.D, n = 2 of the number of C. albicans (Log10 CFU/mL). Significant differences from the control are indicated small letters: a (p < 0.001); b (p < 0.01) and c (p < 0.05).
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Figure 3. Co-aggregation degrees (%) between lactobacilli and C. albicans after 4 h incubation. Data are means ± S.D, n = 3.
Figure 3. Co-aggregation degrees (%) between lactobacilli and C. albicans after 4 h incubation. Data are means ± S.D, n = 3.
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Figure 4. Microscope images (1000×) of co-aggregates between lactobacilli and C. albicans NBIMCC 74: (A) L. fermentum LLF-01 (score = 3); (B) L. delbrueckii subsp. bulgaricus LLB-02 (score = 2); (C) L. delbrueckii subsp. lactis LLL-14 (score = 2); (D) L. acidophilus LLA-01 (score = 2); (E) L. gasseri LLG-V74 (score = 2); (F) L. paracasei LLC-115 (score = 2); (G) L. reuteri LLR-K67 (score = 1); (H) L. rhamnosus LLR-L2 (score = 0); (I) L. delbrueckii subsp. bulgaricus LKZ200 (score = 0). Results were expressed as co-aggregation score: 0—no visible clumps or bound micro-organisms; 1—presence of small and sparsely distributed clumps; 2—presence of large and dense visible clumps of bacteria and yeast; 3—maximum score characterized with presence of large and dense visible clumps entrapping most of the bacteria and yeast, based on the assays of Younes et al. [21].
Figure 4. Microscope images (1000×) of co-aggregates between lactobacilli and C. albicans NBIMCC 74: (A) L. fermentum LLF-01 (score = 3); (B) L. delbrueckii subsp. bulgaricus LLB-02 (score = 2); (C) L. delbrueckii subsp. lactis LLL-14 (score = 2); (D) L. acidophilus LLA-01 (score = 2); (E) L. gasseri LLG-V74 (score = 2); (F) L. paracasei LLC-115 (score = 2); (G) L. reuteri LLR-K67 (score = 1); (H) L. rhamnosus LLR-L2 (score = 0); (I) L. delbrueckii subsp. bulgaricus LKZ200 (score = 0). Results were expressed as co-aggregation score: 0—no visible clumps or bound micro-organisms; 1—presence of small and sparsely distributed clumps; 2—presence of large and dense visible clumps of bacteria and yeast; 3—maximum score characterized with presence of large and dense visible clumps entrapping most of the bacteria and yeast, based on the assays of Younes et al. [21].
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Table 1. List of tested lactobacilli and sources of isolation.
Table 1. List of tested lactobacilli and sources of isolation.
Sources of IsolationSpecies/Strain
DairyHome-made yogurtLactobacillus delbrueckii subsp. bulgaricus LLB-02
L. delbrueckii subsp. bulgaricus LLB-05
L. delbrueckii subsp. bulgaricus LLB-06
Lactiplantibacillus plantarum LLP-4B
Yellow cheese wheyLactobacillus delbrueckii subsp. lactis LLL-14
CheeseLacticaseibacillus rhamnosus LLR-L2
Lacticaseibacillus casei LLC-4K
Human originInfant fecesLactobacillus acidophilus LLA-01
L. delbrueckii subsp. lactis LLL-F18
Lactobacillus helveticus LLH-108
Lacticaseibacillus paracasei LLC-J31
L. paracasei LLC-J35
L. paracasei LLC-115
L. rhamnosus LLR-L1
Limosilactobacilllus reuteri LLR-K67
VaginaL. reuteri LLR-V31
Lactobacillus gasseri LLG-V33
L. gasseri LLG-V74
L. rhamnosus LLR-V57
SalivaLimosilactobacilllus fermentum LLF-01
Ligilactobacillus salivarius LLS-23
OthersRaw-dried sausagesL. plantarum LLP-2L
PlantL. delbrueckii subsp. bulgaricus LKZ-200
Geranium sanguineum
All strains are part of the laboratory collection of Lactina Ltd. (Bankya, Bulgaria).
Table 2. Antifungal activity of lactobacilli against C. albicans by agar diffusion (ADA) and agar spot assay (ASA) after 24 h incubation at 32 °C.
Table 2. Antifungal activity of lactobacilli against C. albicans by agar diffusion (ADA) and agar spot assay (ASA) after 24 h incubation at 32 °C.
StrainsInhibition Zone (mm) ADA/ASAStrainsInhibition Zone (mm) ADA/ASA
L. bulgaricus LLB-020/0L. rhamnosus LLR-L20/8.0 (±0)
L. bulgaricus LLB-050/0L. rhamnosus LLR-V570/8.0 (±2.3)
L. bulgaricus LLB-060/0L. casei LLC-4K11.0(±1.0)/7.0 (±1.2)
L. bulgaricus LKZ-2000/0L. paracasei LLC-J310/6.0 (±0)
L. lactis LLL-140/0L. paracasei LLC-J350/6.5 (±1.0)
L. lactis LLL-F180/0L. paracasei LLC-1150/9.5 (±1.9)
L. helveticus LLH-1080/7.5 (±1.9)L. plantarum LLP-4B14.0 (±1.0)/9.0 (±1.2)
L. acidophilus LLA-010/0L. plantarum LLP-2L0/8.0 (±1.6)
L. gasseri LLG-V330/0L. fermentum LLF-0112.6 (±1.2)/10.5 (±1.7)
L. gasseri LLG-V740/0L. reuteri LLR-K670/6.0 (±0)
L. salivarius LLS-230/7.0 (±1.2)L. reuteri LLR-V310/0
L. rhamnosus LLR-L114.3 (±1.2)/7.5 (±1.9)
The table includes results obtained with acid CFS (ADA) and non-buffered MRS agar (ASA). The results with neutralized CFS and buffered MRS agar are not included due to the very low or absent of activity. Data are means ± SD, n = 3.
Table 3. Growth reduction (%) of C. albicans in the presence of lactobacilli cell suspensions and their supernatants in MRS and BHI broth model systems in comparison with untreated groups.
Table 3. Growth reduction (%) of C. albicans in the presence of lactobacilli cell suspensions and their supernatants in MRS and BHI broth model systems in comparison with untreated groups.
Lactobacilli StrainsGrowth Reduction (%) of C. albicans
MRS Broth Model SystemBHI Broth Model System
C. albcans + CellsC. albicans + SupernatantsC. albicans + CellsC. albicans + Supernatants
L. bulgaricus LLB-0221.2 (±4.9) Aac9.7 (±3.6) Ba11.4 (±2.7) Aac5.9 (±2.0) Ba
L. bulgaricus LLB-0512.1 (±5.4) Ac4.7 (±1.2) Ba2.0 (±1.7) Abcd5.4 (±0.5) Ba
L. bulgaricus LLB-0614.0 (±0.7) Aac5.1 (±1.0) Ba12.2 (±4.2) Aac6.2 (±2.7) Ba
L. bulgaricus LKZ-20014.4 (±5.1) Aac3.9 (±1.4) Ba9.5 (±2.9) Aac4.3 (±1.0) Ba
L. lactis LLL-1420.7 (±2.4) Aac5.4 (±1.3) Ba5.5 (±4.0) Abcd2.7 (±1.4) Aa
L. lactis LLL-F1824.6 (±3.5) Aa6.6 (±1.2) Ba9.6 (±2.2) Aac2.8 (±0.9) Ba
L. helveticus LLH-10822.5 (±3.9) Aac9.2 (±2.7) Ba4.2 (±2.2) Abcd2.2 (±0.8) Aa
L. acidophilus LLA-016.5 (±1.1) Abc3.5 (±0.8) Ba7.8 (±2.7) Aacd4.5 (±1.2) Ba
L. gasseri LLG-V335.7 (±1.3) Ab3.9 (±0.8) Ba6.5 (±1.6) Aabcd3.3 (±2.5) Ba
L. gasseri LLG-V746.4 (±1.8) Abc7.5 (±1.5) Ba6.5 (±2.1) Aabcd3.8 (±1.0) Ba
L. salivarius LLS-2318.6 (±1.9) Aac4.8 (±1.4) Ba8.0 (±2.9) Aacd6.4 (±3.3) Aa
L. rhamnosus LLR-L128.2 (±3.2) Aa7.3 (±4.2) Ba7.1 (±2.7) Aabcd6.5 (±3.6) Aa
L. rhamnosus LLR-L226.6 (±3.6) Aa8.6 (±5.1) Ba7.2 (±3.2) Aabcd5.1 (±2.3) Aa
L. rhamnosus LLR-V5724.7 (±4.6) Aac7.6 (±3.7) Ba9.9 (±2.9) Aac7.3 (±1.2) Aa
L. casei LLC-4K25.1 (±3.0) Aa5.0 (±2.9) Ba8.5 (±3.2) Aacd7.7 (±4.1) Aa
L. paracasei LLC-J3123.1 (±2.2) Aac6.5 (±2.2) Ba10.3 (±2.9) Aac7.9 (±4.5) Aa
L. paracasei LLC-J3520.3 (±1.6) Aac5.5 (±1.6) Ba11.8 (±4.9) Aacd6.9 (±1.6) Aa
L. paracasei LLC-11525.0 (±3.3) Aa6.6 (±1.9) Ba17.1 (±2.2) Aa4.7 (±3.2) Ba
L. plantarum LLP-4B26.1 (±5.1) Aac5.4 (±0.7) Ba8.1 (±2.8) Aacd7.1 (±2.9) Aa
L. plantarum LLP-2L21.5 (±4.2) Aac6.3 (±2.3) Ba10.2 (±5.5) Aacd7.6 (±1.6) Aa
L. fermentum LLF-0128.9 (±2.4) Aa5.1 (±3.1) Ba14.3 (±2.7) Aa4.0 (±3.3) Ba
L. reuteri LLR-K6728.3 (±3.1) Aa6.9 (±0.6) Ba14.9 (±3.4) Aa7.9 (±2.9) Ba
L. reuteri LLR-V3121.6 (±2.9) Aac5.2 (±1.2) Ba7.1 (±3.6) Aabcd5.9 (±1.3) Aa
The reduction in the growth of C. albicans was calculated using the following equation: %Growth reduction = [(log10 CFU/mL in control − log10 CFU/mL in co-culture)/log10 CFU/mL in control] × 100. Data are means ± S.D, n = 2. A, B: different superscript capital letters in the same row denote significant differences in cell number of C. albicans (p < 0.05) between cells and supernatant group for the same strain in the respective model system (MRS or BHI broth), according to the t-test. a–d: different superscript lowercase letters in the same column denote significant differences in cell number of C. albicans (p < 0.05) between different cells or supernatant group in the respective model system (MRS or BHI broth), according to Tukey’s test.
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Yocheva, L.; Tserovska, L.; Danguleva-Cholakova, A.; Todorova, T.; Zhelezova, G.; Karaivanova, E.; Georgieva, R. In Vitro Inhibitory Effects and Co-Aggregation Activity of Lactobacilli on Candida albicans. Microbiol. Res. 2024, 15, 1576-1589. https://doi.org/10.3390/microbiolres15030104

AMA Style

Yocheva L, Tserovska L, Danguleva-Cholakova A, Todorova T, Zhelezova G, Karaivanova E, Georgieva R. In Vitro Inhibitory Effects and Co-Aggregation Activity of Lactobacilli on Candida albicans. Microbiology Research. 2024; 15(3):1576-1589. https://doi.org/10.3390/microbiolres15030104

Chicago/Turabian Style

Yocheva, Lyubomira, Lilia Tserovska, Antonia Danguleva-Cholakova, Teodora Todorova, Galina Zhelezova, Elena Karaivanova, and Ralitsa Georgieva. 2024. "In Vitro Inhibitory Effects and Co-Aggregation Activity of Lactobacilli on Candida albicans" Microbiology Research 15, no. 3: 1576-1589. https://doi.org/10.3390/microbiolres15030104

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