Inhibitory activities of Typhonium trilobatum (L.) Schott on virulence potential of multi-drug resistant toxigenic Vibrio cholerae

Abstract

Cholera is a serious epidemic disease caused by the toxigenic strains of Vibrio cholerae belonged to O1 or O139 serogroups. The emergence of antibacterial resistance in V. cholerae is an increasing concern. Natural product drug invention and Ethnopharmacology may demonstrate a considerable expectation under this circumstance. Traditionally, leaves of Typhonium trilobatum (L.) Schott (locally known as Ghatkanchu or Bengal Arum) are employed for treatment of gastrointestinal disorder in different region of India. The objective of the present study was to evaluate the antibacterial, and antibiofilm activities of methanol extract of T. trilobatum leaves (METTL) against the strains of multi-drug resistant (MDR) Vibrio cholerae (serotypes O1, O139, non-O1, and non-O139) which are responsible for watery diarrhea such as cholera. MIC, MBC and time-kill kinetic studies were used for evaluation of In vitro antibacterial activity of METTL. Microdilution method and Confocal laser scanning microscopy were used to evaluate biofilm-inhibitory activities. The gene expression was analyzed by performing Quantitative real-time PCR (qRT-PCR). METTL showed antibacterial activity with MIC and MBC at 1-32 mg/mL and 8-32 mg/mL, respectively against the clinical strains of Vibrio cholerae belonged to different serogroups. METTL showed significant (P < 0.05) inhibitory activity on the formation of biofilm by V. cholerae SG24, with 81.3, 75.8, and 69.6% of inhibition at MIC, ½ MIC and ¼ MIC, respectively. METTL showed also significant (P < 0.05) inhibitory activity on the formation of extracellular polymeric substances (EPS) formation by V. cholerae SG24, with 89.41, and 99.26% of inhibition of EPS protein and EPS carbohydrate at MIC, respectively. METTL significantly (p < 0.01) inhibited the Cholera toxin (CT) production by the V. cholerae strain SG24 evaluated by the CT - ELISA assay. The cholera toxin production was reduced by 76.26%, 48.76% and 29.93 at MIC (8 mg/mL), ½ MIC (4 mg/mL) and ¼ MIC (2 mg/mL), respectively. METTL was shown to repress ctxAB gene transcription 1.76 fold (p < 0.05) at sub-bactericidal concentration (¼ MIC). We also found that the expression of cholera toxin activator genes, toxT and tcpP was reduced by 11.56- fold (p < 0.001) and 23.52- fold (p < 0.001), respectively, at sub-bactericidal concentration (¼ MIC). Transcription of the following genes was repressed: vpsR (1.8-fold; p < 0.05), Bap1 (1.53-fold; p ≤ 0.05), and rmbA (2.89-fold) by METTL at sub-bactericidal concentration. The expression of vpsT was also repressed by 1.5-fold (p < 0.01) at sub-bactericidal concentration. The active Typhonium trilobatum (L.) leaves extract may be suggested as an substitute for the treatment of MDR V. cholerae infection and could be used as prospective source for the development of novel antimicrobial compound/s and biofilm-inhibitory drug/s useful for the treatment of cholera and diarrheal patients. The results obtained here also validate scientifically the traditional uses of Typhonium trilobatum (L.) in India employed for the treatment of gastrointestinal disorder. Further studies should be directed at purifying and characterizing these antibacterial principles against Vibrio cholerae.

Keywords: Anti-biofilm; Anti-colonization; Antibacterial; Typhonium trilobatum; Vibrio cholerae.