Inhibition of protein synthesis eradicates persister cells of V. cholerae.

In the present work, we studied the antibiotic-induced persister formation in Vibrio cholerae. Persisters vary with the bacterial growth phase with minimum persisters in log phase and maximum in stationary phase. Only 10% of the stationary phase cells of V. cholerae were tolerant of ampicillin and ciprofloxacin. In comparison, more than 90% of the stationary phase cells of E. coli were tolerant of ampicillin and ciprofloxacin. Frequency of ciprofloxacin-induced persisters of V. cholerae would vary with the bacteriological media used for the growth of the cells. In tryptone soy broth (TSB) and in buffered peptone water (BPW), V. cholerae could form more than 10% persisters, whereas in Luria-Bertani broth (LB) and alkaline peptone water (APW) persister fraction was less than 1%. When exposed to protein synthesis inhibitors (kanamycin, chloramphenicol, tetracycline, erythromycin and gentamicin), V. cholerae did not form persisters. Persister recovery assay, LIVE/DEAD analysis and QRDR sequence analysis showed that persister population neither included resistant mutants nor VBNC population. Starvation, anaerobic conditions and inhibition of ATP synthesis also induced persisters, but not when protein synthesis is inhibited. These observations suggest that the protein synthesis is critical for persister formation, persister maintenance, and also for dormancy maintenance in V. cholerae. Contrary to these observations, E. coli can form persisters when protein synthesis is inhibited, suggesting fundamental mechanistic differences between the two species.