In Vitro Antibacterial Activity of Unconjugated and Conjugated Bile Salts on Staphylococcus aureus - PubMed (original) (raw)
In Vitro Antibacterial Activity of Unconjugated and Conjugated Bile Salts on Staphylococcus aureus
Thippeswamy H Sannasiddappa et al. Front Microbiol. 2017.
Abstract
Bile salts are potent antimicrobial agents and are an important component of innate defenses in the intestine, giving protection against invasive organisms. They play an important role in determining microbial ecology of the intestine and alterations in their levels can lead to increased colonization by pathogens. We have previously demonstrated survival of the opportunistic pathogen Staphylococcus aureus in the human colonic model. Thus investigating the interaction between S. aureus and bile salts is an important factor in understanding its ability to colonize in the host intestine. Harnessing bile salts may also give a new avenue to explore in the development of therapeutic strategies to control drug resistant bacteria. Despite this importance, the antibacterial activity of bile salts on S. aureus is poorly understood. In this study, we investigated the antibacterial effects of the major unconjugated and conjugated bile salts on S. aureus. Several concentration-dependent antibacterial mechanisms were found. Unconjugated bile salts at their minimum inhibitory concentration (cholic and deoxycholic acid at 20 and 1 mM, respectively) killed S. aureus, and this was associated with increased membrane disruption and leakage of cellular contents. Unconjugated bile salts (cholic and deoxycholic acid at 8 and 0.4 mM, respectively) and conjugated bile salts (glycocholic and taurocholic acid at 20 mM) at their sub inhibitory concentrations were still able to inhibit growth through disruption of the proton motive force and increased membrane permeability. We also demonstrated that unconjugated bile salts possess more potent antibacterial action on S. aureus than conjugated bile salts.
Keywords: antibacterial; bile salts; intracellular pH; membrane permeability; viability.
Figures
FIGURE 1
Effect of bile salts on viability of S. aureus SH1000. Experiments were performed at a cell density of 108 CFU/ml following exposure to (A) CA, (B) DCA, (C) GCA, and (D) TCA. Data represents mean ± standard error of mean from three independent experiments.
FIGURE 2
Effect of bile salts on intracellular pH of S. aureus SH1000. Experiments were performed at a density of 107 CFU/ml cells preloaded with fluorescent probe (cFSE) and energized with 10 mM glucose following exposure to (A) CA, (B) DCA, (C) GCA, and (D) TCA. Nigericin at 4 μM was added to check the dissipation of intracellular pH. Data represents mean ± standard error of mean from three independent experiments.
FIGURE 3
Effect of bile salts on the transmembrane electrical potential of S. aureus SH1000. Experiments were performed at a density of 107 CFU/ml cells preloaded with the DiSC3 (5) dye and energized with 10 mM glucose following exposure to (A) CA, (B) DCA, (C) GCA, and (D) TCA. Dissipation of transmembrane electric potential was measured as the increase in the DiSC3 (5) fluorescence. VM, valinomycin at 4 μM was used as a positive control to check the dissipation of transmembrane electric potential. Data represents mean ± standard error of mean from three independent experiments.
FIGURE 4
Effect of bile salts on intracellular leakage of potassium from S. aureus SH1000. Experiments were performed at a cell density of 108 CFU/ml following exposure to (A) CA, (B) DCA, (C) GCA, and (D) TCA. NC, negative control (untreated cells); VM, valinomycin at 4 μM; LS, cells treated with 100 μg/ml lysostaphin for 30 min. Data represents mean ± standard error of mean from three independent experiments.
FIGURE 5
Measurement of cellular leakage of nucleic salts (A) and protein (B) from S. aureus SH1000 upon exposure to bile salts. Experiments were performed at a cell density of 108 CFU/ml following exposure to 20 mM CA, 1 mM DCA, 20 mM GCA, and 20 mM TCA. NC, negative control (untreated cells); LS, cells treated with 100 μg/ml lysostaphin. Data represents mean ± standard error of mean from three independent experiments.
FIGURE 6
Surface morphology of S. aureus SH1000 in the presence of bile salts. Scanning electron microscopy was used to investigate the surface morphology of (A) cells untreated or treated with (B) 20 mM CA, (C) 1 mM DCA, (D) 20 mM GCA, and (E) 20 mM TCA at a density of 108 CFU/ml for 30 min. Bar = 500 nm.
FIGURE 7
Ultra structural morphology of S. aureus SH1000 in the presence of bile salts. Transmission electron microscopy was used to investigate the interior morphological details of (A) cells untreated or treated with (B) 20 mM CA, (C) 1 mM DCA, (D) 20 mM GCA, and (E) 20 mM TCA at a density of 108 CFU/ml for 30 min. Bar = 500 nm. White arrows represent mesosome like structures. White triangles represent ghost cells.
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