Glucose sensitizes the stationary and persistent population of Vibrio cholerae to ciprofloxacin.

The subject of analysis in this report was the antibiotic susceptibility of V. cholerae under glucose supplementation since the metabolites can significantly alter the antibiotic sensitivity of bacteria. Glucose could change the antibiotic susceptibility in a growth phase-dependent manner, however, the antibiotic susceptibility of exponentially growing cells was not affected in the presence of glucose. What has been shown is that the stationary phase cells which show higher antibiotic tolerance, could be sensitized to ciprofloxacin and ampicillin by glucose supplementation (tenfold sensitive). The glucose increases the respiration which in turn increases the metabolism and cell division rate. Furthermore, the addition of glucose could increase the susceptibility of persister cells to ciprofloxacin only. In general, the bacterial susceptibility can be increased by combining the antibiotics with glucose.

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.

Comparison of Starvation-Induced Persister Cells with Antibiotic-Induced Persister Cells.

The phenotypic heterogeneity in a large population arises because of fluctuation in microenvironments and stochastic gene expressions. In this report, we isolated two types of persistent sub-populations of Vibrio cholerae, one triggered by starvation and another by antibiotics. We characterised starvation-induced (E-cells) and antibiotic-induced (P-cell) persister cells for stress tolerance, colony morphology and toxin gene expressions. Both the sub-populations differ with respect to morphology, temperature tolerance and oxidative stress tolerance. The E-cells were smaller than the P-cells and formed tiny colonies (1-2 mm). The E-cells were more sensitive to heat and oxidative stress compared with P-cells. The up-regulated genes of P-cells include, genes of antioxidant enzymes (>5 fold), cholera toxin (>26 fold) and toxin: antitoxin protein hipA (>100 fold). Upon nutrient up-shift, the E-cells recovered after lag time of 6 h. However, such lag extension was not visible during P-cell recovery, suggesting that P-cell physiology is more akin to normal cells than E-cells. This is the first comparative report on the two different persister sub-populations of V. cholerae. The E-cells and P-cells are similar regarding antibiotic tolerance. However, the sub-populations differ significantly in stress tolerance and other phenotypes studied.

The ploidy of Vibrio cholerae is variable and is influenced by growth phase and nutrient levels.

The ploidy of Vibrio cholerae was quantified under different growth conditions. The V. cholerae was found to be (mero-) oligoploid or polyploid. The ploidy levels per cell were found to be growth phase regulated. The ploidy is highest during the early stationary phase (56-72 per cell) and lowest in the long-term starved state. In addition to growth phase, an external parameter such as nutrient level influences the ploidy, i.e. ploidy reduces rapidly at the onset of the starvation. The reduction is significant with P-value < 0.05 within 2 h of starvation. Even after prolonged starvation of 10 days, the ploidy number remained above 2 per cell. Failure to obtain a monoploid V. cholerae indicates that during starvation the genome is not distributed equally to daughter cells. The activity of DNase enzyme increased during starvation that decreased the ploidy. The ploidy was restored to the pre-starvation levels with nutrient supplementation.