Inhibitory effect of lactobacilli supernatants on biofilm and filamentation of Candida albicans, Candida tropicalis, and Candida parapsilosis

Abstract

Introduction: Probiotic Lactobacillus strains had been investigated for the potential to protect against infection caused by the major fungal pathogen of human, Candida albicans. Besides antifungal activity, lactobacilli demonstrated a promising inhibitory effect on biofilm formation and filamentation of C. albicans. On the other hand, two commonly isolated non-albicans Candida species, C. tropicalis and C. parapsilosis, have similar characteristics in filamentation and biofilm formation with C. albicans. However, there is scant information of the effect of lactobacilli on the two species.

Methods: In this study, biofilm inhibitory effects of L. rhamnosus ATCC 53103, L. plantarum ATCC 8014, and L. acidophilus ATCC 4356 were tested on the reference strain C. albicans SC5314 and six bloodstream isolated clinical strains, two each of C. albicans, C. tropicalis, and C. parapsilosis.

Results and discussion: Cell-free culture supernatants (CFSs) of L. rhamnosus and L. plantarum significantly inhibited in vitro biofilm growth of C. albicans and C. tropicalis. L. acidophilus, conversely, had little effect on C. albicans and C. tropicalis but was more effective on inhibiting C. parapsilosis biofilms. Neutralized L. rhamnosus CFS at pH 7 retained the inhibitory effect, suggesting that exometabolites other than lactic acid produced by the Lactobacillus strain might be accounted for the effect. Furthermore, we evaluated the inhibitory effects of L. rhamnosus and L. plantarum CFSs on the filamentation of C. albicans and C. tropicalis strains. Significantly less Candida filaments were observed after co-incubating with CFSs under hyphae-inducing conditions. Expressions of six biofilm-related genes (ALS1, ALS3, BCR1, EFG1, TEC1, and UME6 in C. albicans and corresponding orthologs in C. tropicalis) in biofilms co-incubated with CFSs were analyzed using quantitative real-time PCR. When compared to untreated control, the expressions of ALS1, ALS3, EFG1, and TEC1 genes were downregulated in C. albicans biofilm. In C. tropicalis biofilms, ALS3 and UME6 were downregulated while TEC1 was upregulated. Taken together, the L. rhamnosus and L. plantarum strains demonstrated an inhibitory effect, which is likely mediated by the metabolites secreted into culture medium, on filamentation and biofilm formation of C. albicans and C. tropicalis. Our finding suggested an alternative to antifungals for controlling Candida biofilm.

Keywords: Candida; Lactobacillus; biofilm; filamentation; gene expression; non-albicans Candida species.

Figure 1

Effects of living cell suspension and CFS of three Lactobacillus strains on biofilm formation of seven Candida strains. (A,B) L. rhamnosus GG, (C,D) L. plantarum ATCC 8014, (E,F) L. acidophilus ATCC 4356. Data are shown as percentage relative to paired control (MRS broth) and the mean ± SEM of triplicate experiments. Comparison with control was performed with Student’s t-test. *p < 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 2

Effects of acidified MRS broth (AMRS) and neutralized CFS (NCFS) on Candida biofilm. Data are shown as percentage relative to a paired control (untreated MRS) and as mean ± SEM of triplicate experiments. (A,B) Effects of AMRS on the metabolic activity and biomass of biofilms produced by three C. albicans (SC5314, A14, and A69), two C. tropicalis (T18S and T38R), and one C. parapsilosis (P152) strains. Asterisks indicate data that are significantly different from control (MRS) at p < 0.05 as determined by one-sample t-test. (C,D) Effects of untreated CFS (pH 4.0) and NCFS (pH 7.0) of LGG on the C. albicans and C. tropicalis strains. Comparison between CFS, NCFS, and control (MRS) was done with one-way ANOVA with Tukey’s test. Asterisks indicate significant difference between CFS and NCFS results at p < 0.05.

Figure 3

Light micrographs of C. albicans and C. tropicalis cells in the filamentation assay. In each row, images of one Candida strain under three conditions: Control (left), LGG CFS (middle), and LP8014 CFS (right) are shown. Scale-bar: 50 μm.

Figure 4

Quantification of filament in the filamentation inhibition assays. (A) C. albicans SC5314, (B) C. albicans A14, (C) C. albicans A69, (D) C. tropicalis T18S, and (E) C. tropicalis T38R. Each data point represents the mean ± SEM of the number of filaments in one experiment. Comparisons with the control group were performed with results of triplicate experiments and Dunnett’s test. *p < 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 5

Expression levels filamentation and biofilm-related genes in biofilms of C. albicans and C. tropicalis incubated with LGG or LP8014 CFSs. (A) ALS1, (B) ALS3, (C) BCR1, (D) EFG1, (E) TEC1, and (F) UME6. Expression levels were expressed as the normalized relative quantity (NRQ). NRQ of the control group (MRS) is constantly 1.0 and not shown on the figures. Values represent the mean ± SEM of triplicate experiments. Comparison with control group was performed with the binary logarithm of NRQ by Student’s t-test. *p < 0.05; **p ≤ 0.01.