The Mechanism of Antimicrobial Activity of Conjugated Bile Acids against Lactic Acid Bacilli

Abstract

The mechanism underlying antimicrobial activity of conjugated bile acids against strains of lactic acid bacilli is not well understood. The purpose of this study was to investigate two typical conjugated bile acids (glycochenodeoxycholic acid and taurochenodeoxycholic acid) for their mechanisms of antimicrobial activity against four strains of different species of lactic acid bacilli at the physiological pH of the small intestine of humans. The bacterial cell membrane integrity, transmembrane potential, and transmembrane pH gradient were examined using the fluorescence probes SYTO 9 plus propidium iodide, 3,3'-dipropylthiadicarbocyanine iodide, and 5(6)-carboxyfluorescein diacetate N-succinimidyl ester, respectively. The intracellular ATP levels were measured by the firefly luciferase-based bioluminescence method. It was found that the antimicrobial activity of conjugated bile acids against the strains of lactic acid bacilli is strain-specific, and glycochenodeoxycholic acid showed significantly greater antimicrobial activity than taurochenodeoxycholic acid against the strains of lactic acid bacilli. The conjugated bile acids inhibited the growth of strains of lactic acid bacilli by disrupting membrane integrity, dissipating transmembrane potential, reducing the transmembrane pH gradient, and depleting intracellular ATP. In conclusion, the antimicrobial activity of conjugated bile acids against lactic acid bacilli is a multifactorial phenomenon. This study will provide valuable information for developing strategies to improve the ability of lactic acid bacilli to tolerate bile in vivo.

Keywords: antimicrobial activity; conjugated bile acid; intracellular ATP; membrane integrity; transmembrane potential.

Figure 1

Antimicrobial activity of GCDCA and TCDCA against Lactiplantibacillus plantarum ATCC 14917 (a), Lacticaseibacillus casei ATCC 334 (b), Lacticaseibacillus rhamnosus ATCC 53103 (c), and Lactobacillus acidophilus ATCC 700396 (d). Each bile acid was used at a concentration of 5 mM. Data are expressed as the mean ± SD (n = 3). Means not sharing a common letter differ significantly (p < 0.05).

Figure 2

Effects of different concentrations of GCDCA and TCDCA on the cellular membrane integrity of Lactiplantibacillus plantarum ATCC 14917 (a), Lacticaseibacillus casei ATCC 334 (b), Lacticaseibacillus rhamnosus ATCC 53103 (c), and Lactobacillus acidophilus ATCC 700396 (d). Data are expressed as the mean ± SD (n = 3). Means not sharing a common letter differ significantly (p < 0.05).

Figure 3

Effects of different concentrations of GCDCA and TCDCA on the ΔΨ of Lactiplantibacillus plantarum ATCC 14917 (a), Lacticaseibacillus casei ATCC 334 (b), Lacticaseibacillus rhamnosus ATCC 53103 (c), and Lactobacillus acidophilus ATCC 700396 (d). Data are expressed as the mean ± SD (n = 3). Means not sharing a common letter differ significantly (p < 0.05).

Figure 4

Effects of different concentrations of GCDCA and TCDCA on the cellular ΔpH of Lactiplantibacillus plantarum ATCC 14917 (a), Lacticaseibacillus casei ATCC 334 (b), Lacticaseibacillus rhamnosus ATCC 53103 (c), and Lactobacillus acidophilus ATCC 700396 (d). ΔpH = intracellular pH − extracellular pH. Data are expressed as the mean ± SD (n = 3). Means not sharing a common letter differ significantly (p < 0.05).

Figure 5

Effects of different concentrations of GCDCA and TCDCA on the intracellular ATP levels of Lactiplantibacillus plantarum ATCC 14917 (a), Lacticaseibacillus casei ATCC 334 (b), Lacticaseibacillus rhamnosus ATCC 53103 (c), and Lactobacillus acidophilus ATCC 700396 (d). Data are expressed as the mean ± SD (n = 3). Means not sharing a common letter differ significantly (p < 0.05).

Figure 6

Correlation heatmap analysis between the variables. The correlation was assessed using Pearson correlation coefficients (r). Red represents a positive correlation, and blue represents a negative correlation. In subfigures (a) and (b), GCDCA and TCDCA were used at 5 mM, respectively.