Lactobacillus acidophilus ATCC 4356 inhibits biofilm formation by C. albicans and attenuates the experimental candidiasis in Galleria mellonella

Abstract

Probiotic strains of Lactobacillus have been studied for their inhibitory effects on Candida albicans. However, few studies have investigated the effect of these strains on biofilm formation, filamentation and C. albicans infection. The objective of this study was to evaluate the influence of Lactobacillus acidophilus ATCC 4356 on C. albicans ATCC 18804 using in vitro and in vivo models. In vitro analysis evaluated the effects of L. acidophilus on the biofilm formation and on the capacity of C. albicans filamentation. For in vivo study, Galleria mellonella was used as an infection model to evaluate the effects of L. acidophilus on candidiasis by survival analysis, quantification of C. albicans CFU/mL, and histological analysis. The direct effects of L. acidophilus cells on C. albicans, as well as the indirect effects using only a Lactobacillus culture filtrate, were evaluated in both tests. The in vitro results showed that both L. acidophilus cells and filtrate were able to inhibit C. albicans biofilm formation and filamentation. In the in vivo study, injection of L. acidophilus into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, the number of C. albicans CFU/mL recovered from the larval hemolymph was lower in the group inoculated with L. acidophilus compared to the control group. In conclusion, L. acidophilus ATCC 4356 inhibited in vitro biofilm formation by C. albicans and protected G. mellonella against experimental candidiasis in vivo.

Keywords: ATCC, American type culture collection; BHI, Brain heart infusion; CFU, colony-forming unit; Candida albicans; Galleria mellonella; HE, hematoxylin-eosin; Lactobacillus acidophilus; MRS, Man, Rogosa and Sharpe; NIH, National Institutes of Health; PAS, periodic acid-Schiff; PBS, phosphate buffered saline; SEM, Scanning electron microscopy; YNB, Yeast nitrogen base; biofilm; candidiasis; filamentation; pH, potential hydrogen ion; probiotic.

Figure 1.

Candida albicans biofilm formed in vitro: (A) Percentage of reduction, expressed as mean values (CFU/mL), in the viability of C. albicans in the groups treated with L. acidophilus cells obtained from cultures in different phases of growth (4, 6, 18 and 24 h) in relation to the control group (PBS). (B) Percentage of reduction, expressed as mean values (CFU/mL), in the viability of C. albicans in the groups treated with L. acidophilus cells or culture filtrate obtained from 24-h cultures in relation to the control groups (PBS or MRS broth). (C) Number of C. albicans CFU/mL (log) in biofilms of the control group (PBS or MRS broth) and groups treated with L. acidophilus cells or culture filtrate. *Significant difference between the control group (PBS) and C. albicans + L. acidophilus cell group (p = 0.0001). **Significant difference between the control group (MRS broth) and C. albicans + L. acidophilus culture filtrate group (p = 0.0001). Student t-test, P ≤ 0.05.

Figure 2.

Light microscopy photomicrographs of in vitro Candida albicans filamentation. (A) Control group (PBS): intense formation of hyphae. (B) C. albicans + L. acidophilus cells group: presence of some hyphae. (C) Control group (MRS broth): intense formation of hyphae. (D) C. albicans + L. acidophilus culture filtrate group: note the presence of few hyphae. Original magnification: 400×.

Figure 3.

Quantification of hyphae in the in vitro C. albicans filamentation assays. Scores were attributed to the number of C. albicans hyphae formed in each group. (A) Comparison between the control group (PBS) and C. albicans + L. acidophilus cells group. (B) Comparison between the control group (PBS) and C. albicans + L. acidophilus culture filtrate group. (C) Comparison between the PBS and MRS control groups. Mann-Whitney test, p ≤ 0.05.

Figure 4.

Effects of L. acidophilus on experimental candidiasis based on the analysis of survival curves of G. mellonella larvae (A) Therapeutic groups: significant differences were observed between the C. albicans + L. acidophilus cells group and PBS control group (p=0.0001) and between the C. albicans + L. acidophilus culture filtrate group and MRS control group (p=0.0002) (B) Prophylactic groups: significant differences were observed between the C. albicans + L. acidophilus cells group and PBS control group (p=0.0001) and between the C. albicans + L. acidophilus culture filtrate group and MRS control group (p=0.0490) Log-rank test, p ≤ 0.05.

Figure 5.

Mean and standard deviation of C. albicans counts (CFU/mL) in the hemolymph of Galleria mellonella immediately after inoculation and after 4, 8, 12 and 24 h of experimental infection. The following groups were compared at each time of infection: control group (MRS broth), C. albicans + L. acidophilus cells group, and C. albicans + L. acidophilus culture filtrate group. A significant difference between groups was only observed after 24 h of infection (ANOVA, P ≤ 0.05), with a larger number of CFU/mL in the control group compared to the C. albicans + L. acidophilus cells (p = 0.0205) and C. albicans + L. acidophilus culture filtrate groups (p = 0.0135). No significant difference was observed between the last 2 groups (p = 0.9251). Tukey test, P ≤ 0.05.

Figure 6.

Mean and standard deviation of L. acidophilus counts (CFU/mL) in the hemolymph of G. mellonella at times 0, 4, 8, 12, 18 and 24 h for the group formed by interaction of C. albicans + L. acidophilus cells. No significant difference between the times was observed (p = 0.840). ANOVA, P ≤ 0.05).

Figure 7.

Photomicrographs of a histologically normal fat body of Galleria mellonella larvae not infected with the microorganisms. (A) Note the presence of Malpighi tubules (tm), part of the intestine (int), and trophocytes (t). HE; original magnification: 100x. (B) Presence of the trachea (TR) surrounded by trophocytes of the fat body. HE; original magnification: 630×. (C) Malpighi tubule (tm) responsible for the removal of excreta from the hemolymph. HE; original magnification: 630×. (D) Presence of trophocytes (t) with irregular nuclei and oenocytes (e). HE; original magnification: 630×.

Figure 8.

Histological sections of the fat body of Galleria mellonella.(A) Normal appearance of the fat body of G. mellonella not infected with Candida albicans. (B) C. albicans and PBS control group: observe the presence of clusters of hyphae and yeast cells (arrow). PAS; original magnification: 100×. (C) C. albicans and MRS control group. (D) C. albicans + L. acidophilus culture filtrate group. (E) C. albicans and PBS control group. (F) C. albicans + L. acidophilus cells group. (G) C. albicans + L. acidophilus cells group: with demarcation taken by Image J program for obtaining occupied by hyphae and yeasts area. PAS; original magnification: 1000×.

Figure 9.

Mean and standard deviation of the area occupied by Candida albicans yeast cells and hyphae in histological sections of Galleria mellonella with experimental candidiasis. No significant difference was observed between groups: control group (PBS), C. albicans + L. acidophilus cells group, control group (MRS broth), and C. albicans + L. acidophilus culture filtrate group. ANOVA, P ≤ 0.05.