Bile acids improve the antimicrobial effect of rifaximin

Abstract

Diarrhea is one of the most common infirmities affecting international travelers, occurring in 20 to 50% of persons from industrialized countries visiting developing regions. Enterotoxigenic Escherichia coli (ETEC) is the most common causative agent and is isolated from approximately half of the cases of traveler's diarrhea. Rifaximin, a largely water-insoluble, nonabsorbable (<0.4%) antibiotic that inhibits bacterial RNA synthesis, is approved for use for the treatment of traveler's diarrhea caused by diarrheagenic E. coli. However, the drug has minimal effect on the bacterial flora or the infecting E. coli strain in the aqueous environment of the colon. The purpose of the present study was to evaluate the antimicrobial effect and bioavailability of rifaximin in aqueous solution in the presence and absence of physiologic concentrations of bile acids. The methods used included growth measurement of ETEC (strain H10407), rifaximin solubility measurements, total bacterial protein determination, and assessment of the functional activity of rifaximin by monitoring inhibition of bacterial beta-galactosidase expression. Solubility studies showed rifaximin to be 70- to 120-fold more soluble in bile acids (approximately 30% in 4 mM bile acids) than in aqueous solution. Addition of both purified bile acids and human bile to rifaximin at subinhibitory and inhibitory concentrations significantly improved the drug's anti-ETEC effect by 71% and 73%, respectively, after 4 h. This observation was confirmed by showing a decrease in the overall amount of total bacterial protein expressed during incubation of rifaximin plus bile acids. Rifaximin-treated samples containing bile acids inhibited the expression of ETEC beta-galactosidase at a higher magnitude than samples that did not contain bile acids. The study provides data showing that bile acids solubilize rifaximin on a dose-response basis, increasing the drug's bioavailability and antimicrobial effect. These observations suggest that rifaximin may be more effective in the treatment of infections in the small intestine, due to the higher concentration of bile in this region of the gastrointestinal tract than in the colon. The water insolubility of rifaximin is the likely explanation for the drug's minimal effects on colonic flora and fecal pathogens, despite in vitro susceptibility.

FIG. 1.

Solubility of rifaximin (12 mg) in water and equimolar concentrations of the bile acids cholic, chenodeoxycholic, deoxycholic, glycocholic, lithocholic, and taurocholic acids in a mixture at pH 7.4. The total bile acid concentration at each reading is equal to the individual bile acid concentration multiplied by 6.

FIG. 2.

Growth of ETEC strain H10407 in the presence of 16 μg/ml rifaximin in water and 4 mM human bile at pH 7.4. Cells were grown with rifaximin in the presence and absence of human bile, and the absorbance at 600 nm was measured at 30-min intervals. Mann-Whitney two-tailed nonparametric t test analysis showed statistically significant differences between treatments, as follows: no rifaximin plus no bile acids versus 16 μg/ml rifaximin, P = 0.026 (n = 4); no rifaximin plus no bile acids versus 16 μg/ml rifaximin plus bile acids, P = 0.002 (n = 4); and 16 μg/ml rifaximin versus 16 μg/ml rifaximin plus bile acids, P = 0.012 (n = 4). The error bars represent the standard deviations between four replicate experiments.

FIG. 3.

Growth of ETEC strain H10407 in the presence of rifaximin (16 μg/ml) in water and an equimolar mixture of the synthetic bile acids cholic, deoxycholic, chenodeoxycholic, glycocholic, lithocholic, and taurocholic acids at pH 7.4. The total bile acid concentration was 4 mM. Cells were grown with rifaximin in the presence and absence of bile acids, and the absorbance at 600 nm was measured at 30-min intervals. Mann-Whitney two-tailed nonparametric t test analysis showed a statistically significant difference between treatments, as follows: no rifaximin plus no bile versus 16 μg/ml rifaximin, P = 0.026 (n = 4); no rifaximin plus no bile acids versus 16 μg/ml rifaximin plus bile acids, P = 0.002 (n = 4); and 16 μg/ml rifaximin versus 16 μg/ml rifaximin plus bile acids, P = 0.007 (n = 4). The error bars represent the standard deviations between four replicate experiments.

FIG. 4.

Growth of ETEC strain H10407 after 4 h incubation at 37°C in the presence of rifaximin (8 μg/ml, 16 μg/ml, and 32 μg/ml) in water and 4 mM total synthetic bile acids in a pooled mixture of cholic, deoxycholic, chenodeoxycholic, glycocholic, lithocholic and taurocholic acids at pH 7.4. The error bars represent the standard deviations between four replicate experiments.

FIG. 5.

Effect of bile acids on the expression of β-galactosidase enzyme. Each treatment contained 4 mM total synthetic bile acids in a mixture of cholic, deoxycholic, chenodeoxycholic, glycocholic, lithocholic, and taurocholic acids at pH 7.4. One unit is the amount of enzyme required to convert a micromole of o-nitrophenyl-β-d-galactoside to o-nitrophenol and galactose per minute at pH 7.4 at 30°C. The error bars represent the standard deviations between three replicate experiments.

FIG. 6.

Evaluation of total protein content of bacteria treated with 16 μg/ml rifaximin and bile acids. Each treatment contained 4 mM total synthetic bile acids in a mixture of cholic, deoxycholic, chenodeoxycholic, glycocholic, lithocholic, and taurocholic acids at pH 7.4. Cells were grown with rifaximin in the presence and absence of bile acids, and aliquots were taken every hour for total protein concentration determination. The total protein concentration was determined using the Bradford assay. The error bars represent the standard deviations between three replicate experiments.