Increased antibiotic resistance exhibited by the biofilm of Vibrio cholerae O139

Background: Vibrio cholerae, the aetiological agent of the deadly diarrhoeal disease cholera, is known to form biofilm. The antibiotic susceptibility status of biofilm of V. cholerae O139, an important epidemic strain in India and other countries, has not previously been studied in detail. Methods: Antibiotic susceptibility status of planktonic and biofilm cultures of V. cholerae O139 was evaluated by determining MIC, MBC and minimum biofilm eradication concentration (MBEC) values of five different classes of antibiotics using established methods. Effects of antibiotic treatment on planktonic and biofilm cultures were analysed by scanning electron microscopy. The virulence of the antibiotic-surviving population (ASP) was evaluated using an infant mouse model. The frequency of spontaneous mutants and inheritability of antibiotic resistance were determined with standard methods. Results: The antibiotic resistance exhibited by biofilm of V. cholerae O139 was found to be significantly higher (P < 0.05) than its planktonic counterpart. The biofilm-associated antibiotic resistance was found to be transient and exclusive to the biofilm culture. The frequency of ASP clones among antibiotic-treated biofilm cultures occurred at a rate of 0.012%-0.95% and these clones were found to retain the virulence and antibiotic resistance of their parent strains. Conclusions: The biofilm of V. cholerae O139 was found to be resistant to different types of antibiotics tested. This unconventional biofilm resistance highlights the hidden danger of antimicrobial escape by V. cholerae, increased risk of cholera transmission and its continued persistence in the environment.