Antibiotic-induced decreases in the levels of microbial-derived short-chain fatty acids correlate with increased gastrointestinal colonization of Candida albicans

Abstract

Candida albicans is the fourth most common cause of systemic nosocomial infections, posing a significant risk in immunocompromised individuals. As the majority of systemic C. albicans infections stem from endogenous gastrointestinal (GI) colonization, understanding the mechanisms associated with GI colonization is essential in the development of novel methods to prevent C. albicans-related mortality. In this study, we investigated the role of microbial-derived short-chain fatty acids (SCFAs) including acetate, butyrate, and propionate on growth, morphogenesis, and GI colonization of C. albicans. Our results indicate that cefoperazone-treated mice susceptible to C. albicans infection had significantly decreased levels of SCFAs in the cecal contents that correlate with a higher fungal load in the feces. Further, using in vivo concentration of SCFAs, we demonstrated that SCFAs inhibit the growth, germ tube, hyphae and biofilm development of C. albicans in vitro. Collectively, results from this study suggest that antibiotic-induced decreases in the levels of SCFAs in the cecum enhances the growth and GI colonization of C. albicans.

Figure 1

Cefoperazone-treated mice susceptible to C. albicans have decreased levels of SCFAs in the cecum. C. albicans SC5314 load in fecal contents after 5 days of infection in mice receiving sterile water with or without cefoperazone. Fungal load (Log₁₀ CFU/g feces) determined by CFU count (a). SCFA levels (µmol/g) in the cecal contents from mice receiving sterile water with or without cefoperazone (b). Data is represented as means ± SEM with n = 5–6 mice in each treatment group. Statistical significance was evaluated using student’s t-test and P values (* ≤ 0.05, ** ≤ 0.01) were considered as significant.

Figure 2

SCFAs inhibit C. albicans growth in vitro. Growth of C. albicans strains SC 5314 (a) and ATCC 10231 (b) in the presence of SCFAs or in pH-adjusted RPMI media determined by spectrophotometer analysis at an optical density of 600 nm after 24 and 48 hours of incubation. Experiment was repeated three times and the three combined replicates were shown here with total n = 9 for each group. Data is represented as means ± SEM. Statistical significance was evaluated using student’s t-test and P values (* ≤ 0.05, ** ≤ 0.01) were considered as significant. Significance is shown only for data points that exhibited significant decreases in growth compared to respective controls. Significance for pH-adjusted RPMI values was assessed using pH 7.00 as the comparative data set; significance for SCFA-treated conditions was assessed using respective pH controls for each SCFA condition in statistical analyses. Significance (**) for acetic acid (50 mM), butyric acid (12.5 mM and 25 mM), and propionic acid (25 mM) indicates p ≤ 0.01 at both 24 and 48 hours in both strains.

Figure 3

SCFAs inhibit germ tube formation. Germ tube formation in C. albicans ATCC 10231 strain in the presence of SCFAs or pH-adjusted media supplemented with 30% FBS. Representative images of germ tubes formed after 2 hours in control and treatment groups determined by microscopic analysis at 40X magnification (a). Quantification of the percent C. albicans cells with germ tubes in pH-adjusted controls; pH-adjusted controls (12.5 mM and 25 mM) were normalized to the RPMI control (pH 7.00) (b). Quantification of the percent C. albicans cells with germ tubes in SCFA treatment groups (c). SCFA treatments were normalized to their respective pH controls. The germ tube experiment was repeated three times and two 40X images were taken from each replicate for each treatment group, with a minimum n = 1000 cells for each group. C. albicans (CFU/mL) viability determined after 2 hours of incubation in germ tube-inducing conditions (d). The experiment was repeated three times with n = 12 for each treatment group. Combined replicates for both experiments are shown here. Data is represented as means ± SEM. Statistical significance was evaluated using student’s t-test and P values (* ≤ 0.05, ** ≤ 0.01) were considered as significant.

Figure 4

SCFAs inhibit C. albicans hyphae formation in vitro. C. albicans ATCC 10231 was grown in the presence of SCFAs or in pH-adjusted RPMI media supplemented with 30% FBS and examined using bright field microscopy at 40× (a). Quantification of C. albicans hyphae attachment to polystyrene plates in pH-adjusted controls; pH-adjusted controls (12.5 mM and 25 mM) were normalized to the RPMI control (pH 7.00) (b). Quantification of C. albicans hyphae attachment to polystyrene plates in SCFA-treatment groups; SCFA treatment groups were normalized to their respective pH controls (c). C. albicans (CFU/mL) viability determined after 12 hours of incubation in hyphae-inducing conditions (d). The experiment was repeated three times with n = 24 for the hyphae formation and n = 12 for the CFU viability determination in each treatment group. Data is represented as means ± SEM. Statistical significance was evaluated using student’s t-test and P values (* ≤ 0.05, ** ≤ 0.01) were considered as significant.

Figure 5

SCFAs reduce the metabolic activity of fungal cells in the C. albicans biofilm. C. albicans ATCC 10231 was grown in the presence of SCFAs or in pH-adjusted RPMI and the metabolic activity of the fungal cells in the biofilm was assessed using MTS assay. Percent metabolic activity of fungal cells in the biofilm formed in pH-adjusted controls was determined; pH adjusted controls (12.5 mM and 25 mM) were normalized to the RPMI control (pH 7.00) (a). Percent metabolic activity of fungal cells in the biofilm formed in SCFA-treatment groups; SCFA treatments groups were normalized to their respective pH controls (b). C. albicans (CFU/mL) viability determined after 48 hours of incubation in biofilm-inducing conditions (c). All experiments were repeated three times, with n = 18 to determine the metabolic activity in the biofilm and n = 12 for the CFU viability analysis in each treatment group. Data is represented as means ± SEM. Statistical significance was evaluated using student’s t-test and P values (* ≤ 0.05, ** ≤ 0.01) were considered as significant.