The effect of physiological concentrations of bile acids on in vitro growth of Mycobacterium tuberculosis

Abstract

Intestinal tuberculosis (ITB) involves the ileocaecum in the majority of cases with upper gastrointestinal tract tuberculosis accounting for less than 3% of all cases.1 Gallbladder TB, in particular, is very rare, with few cases reported in the literature. ITB results from haematogenous spread or spread from contiguous lymph nodes or swallowed infected sputum.2 Why ITB preferentially affects the ileocaecum is unclear, but proposed mechanisms include bowel stasis in the terminal ileum, due to the ileocaecal valve, and the high density of lymphoid tissue found in the terminal ileum.Bile acids are the major constituent of bile and the primary bile acids, cholic and chenodeoxycholic acid, and secondary bile acids, lithocholic and deoxycholic acid, make up more than 95% of the bile acid pool. Bile acids have a number of biological effects, including generation of reactive oxygen species, a detergent action on lipidcontaining cell membranes and antimicrobial effects. Enterohepatic circulation of bile acids results in efficient reabsorption of bile acids, resulting in a concentration gradient between proximal small intestine and terminal ileum. Only 10% of bile acids escape reabsorption and studies have shown that the bile acid concentration in the caecum is below the micellisation concentration.3 Could the high concentration of bile acids in the gallbladder and the proximal intestine be protective and explain the anatomical distribution of ITB?We determined the effect of physiological concentrations of bile acids on the in vitro growth of Mycobacterium tuberculosis (MTB). MethodsThe 4 major bile acids, lithocolic acid, cholic acid, deoxycholic acid and chenodeoxycholic acid (all supplied by Sigma Aldrich, SA) were selected for study. Individual bile acids were added to standard Lowenstein-Jensen (LJ) media to achieve physiological concentrations, 4 as shown in Table 1. A fifth LJ-bile acid solution was prepared using all 4 bile acids. Due to difficulties in solubilising the combination of 4 bile acids at physiological concentrations, the concentration of each had to be reduced by 50% in this LJ slope.Each 'physiological' LJ-bile acid solution was subsequently doublediluted 4 times with pure LJ solution, to achieve a decreasing concentration of bile acid in the culture medium. A standard inoculum (0.5 McFarland) of MTB H37Rv was inoculated onto each LJ slope in triplicate, as well as onto LJ slopes with no bile salts added. The slopes were incubated at 37°C for 8 weeks. Mycobacterial growth was measured at 2 and 8 weeks in a semiquantitative fashion using cut-offs of >5, >10, >20, >100 colony-forming units (CFUs) per slope. ResultsThere were >100 CFUs of typical MTB colonies on the control slopes at both 2 and 8 weeks. Similarly, all lithocolic acid cultures showed growth of >100 CFUs at 2 and 8 weeks.Three of the bile acids alone (chenodeoxycholic acid, deoxycholic acid and cholic acid) and the combination of all 4 showed inhibition of growth at 2 and 8 weeks. Chenodeoxycholic acid appeared to be the most active of th...

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