Expression of bacterial β-glucuronidase in human bile: An in vitro study

Background: Bacterial β-glucuronidase causes deconjugation of bilirubin diglucuronide resulting in the precipitation of calcium bilirubinate, which contributes to biliary sludge and stone formation. This process is attributed to enzyme activity produced by the aerobic enterobacteriaceae such as Escherichia coli and Klebsiella sp. The presence of Clostridium sp. was detected in 48 of 56 intrahepatic stones by using polymerase chain reaction techniques and cultured Clostridium perfringens from 14 of 18 unblocked biliary stents. Such bacteria are reported to produce β-glucuronidase activity. The aim of this study was to determine the proportion of biliary bacteria isolated from pigment stones and stents that produce β-glucuronidase and to compare the enzyme activity expressed by the different bacteria in human bile. Methods: A total of 202 bacteria were isolated from blocked and unblocked biliary stents and pigment ductal stones recovered from patients. Of these, 61 bacteria expressed β-glucuronidase activity in brain heart infusion broth. These 61 bacteria were subsequently grown in human bile under aerobic or anaerobic conditions to the early stationary phase and assayed for β-glucuronidase activity by using p-nitrophenyl β-D glucuronide as substrate. Results were normalized and reported as units of enzyme activity per milligram protein of the bacteria. Results: C perfringens produced β-glucuronidase enzyme activity that was 34-fold higher than that for E coli, Staphylococcus, Corynebacterium sp., Bacillus sp., Enterococcus sp., Acinetobacter sp., Streptococcus sp., and Klebsiella sp. Conclusion: C perfringens with its higher enzyme activity is more important in the deconjugation of bilirubin diglucuronide than E coli and Klebsiella sp.