Kutajarista is an Ayurvedic formulation prescribed for the treatment of dysentery, piles etc. Initial characterisation of bacterial diversity of Kutajarista by the 16S rRNA gene clone library [GenBank: HQ875575-HQ875614] provided evidence about the richness of Lactobacillus spp. in the preparation of ayurvedic medicine. Therefore, the current study was aimed at characterization of probiotic and antibacterial properties of L. plantarum VR1 isolated from Kutajarista, against a known cytotoxic and virulent strain, A. veronii. Previously, it has been reported that L. delbrueckii, L. lactis and L. mesenteroides can prevent cellular damage caused by A. salmonicida, a fish pathogen [35, 36]. Here we report that VR1 possess strong probiotic properties and abrogated the cytotoxicity of A. veronii MTCC 3249, an isolate from mosquito midgut. To the best of our knowledge this is the first report of the preventive role of CFS from VR1 in cellular and epithelial damage caused by A. veronii.
Traditionally fermented products are rich source of Lactobacilli, which can be exploited for their probiotic potential. Indian fermented foods like Kallappam, koozh and Mor Kuzhambu were reported as a source of potential probiotic Lactobacillus spp. and which is useful as biopreservative . Ayurveda is traditionally practised medicinal science for many centuries and medicines are prepared from herbs. However, very little efforts have been made in utilizing these preparations as a source of probionts. There is only major study which reported the isolation and charactarisation of seventeen Lactobacillus spp. from Kanjika, an Ayurvedic formulation, for probiotic attributes . In the present study, we used Kutajarista, an Ayurvedic herbal decoction, for isolation of potential probiont. VR1 showed highest homology to L. plantarum and exhibited probiotic characteristics such as tolerance to acidic pH, bile salts and simulated gastric juice. VR1 also showed adherence to intestinal cell line HT-29, which is one of the essential prerequisites for a probiotic microorganism. All these features indicate this strain of L. plantarum as a potential probiont. A recent report by Anderson et al.  suggests that L. plantarum has better probiotic characteristics and it also reduces enteropathogenic effect of E. coli as compared to commercial strains like L. rhamnosus. Moreover, L. plantarum has been reported to inhibit pathogens in in vitro and in vivo systems [9, 13]. On the same lines, L. plantarum isolated from Kutajarista showed inhibition of the tested type strains and clinical isolates of P. aeruginosa and E. coli. Interestingly VR1 also prevented the growth of A. veronii, for which virulent attributes have already been established [[26–28]].
The pathogenicity of genus Aeromonas is multifactorial and is attributed to factors such as; cytotoxin, aerolysin, hemolysin, adhesins and secretory systems. Apart from other virulence factors which may contribute to the pathogenesis of A. veronii, here we report the presence of type three secretion system and aerolysin (additional file 2, Fig S2), putatively involved in secretion of virulence factors to the host cell and haemolytic activity respectively. Our previous studies have also demonstrated that A. veronii MTCC 3249 is multi-drug resistant, and harbours three uncharacterised plasmids and one of the plasmids codes for functional type four secretion system [[26, 28, 29]].
After establishing the fact that A. veronii was cytotoxic to mammalian cells and harbours many virulence factors, our next goal was to observe the protective or preventive role of VR1 in A. veronii infection. We used CFS of VR1 to examine its efficacy in amelioration of cytotoxicity caused by A. veronii supernatant. We observed high level of vacuole formation as an indication of cytotoxicity and morphological changes in Vero cells. Earlier, in an enterohaemorrhagic E. coli infection model, it was shown that pre-incubation with L. plantarum abolished the cytotoxicity caused by enteropathogenic strain . To test whether VR1 had similar effects, we studied the time dependent effects of CFS of A. veronii, VR1, in combination or treatment of A. veronii on VR1 pre-incubated cells. We found that pre-incubation of Vero cells with VR1 CFS delayed cytotoxicity, which was induced by A. veronii. Vacuolating cytotoxic factor from A. veronii was earlier reported to cause cell death .
Tight junction disruption is considered to be one of the indicators of morphological damage caused due to cytotoxicity. MDCK cell line infected with V. cholerae cytotoxin and S. typhimurium showed a clear indication of epithelial barrier dysfunction by disruption of tight junction [39, 40]. In fish, pre-incubation with prospective probiont L. delbrueckii sub sp. lactis could prevent epithelial damage caused by A. salmonicida . To investigate the effect of CFS derived from VR1, and A. veronii on epithelial barrier, we selected MDCK cell line over Caco2 cell line because it exhibits similar epithelial characteristics like formation of uniform columnar epithelia, tight junction, and it has an advantage of a short culture period of 5-7 days in comparison to Caco2 which has 21 days of growth period [[41–43]]. We found that A. veronii indeed caused epithelial damage by disruption of ZO-1 and F-Actin in MDCK cell line, which was prevented by pre-incubation with VR1 supernatant for 6 h, whereas co-incubation was not able to restore the epithelial integrity. ZO-1 is a cytoplasmic protein which interacts directly with F-Actin and is very important in structural and functional organisation of tight junction. In this study, microscopic observation of cellular damage is well supported by immunolocalization of ZO-1 and F-Actin, which give clear evidence of VR1 in ameliorating the epithelial damage caused by A. veronii. This finding is consistent with earlier report that, L. rhamnosus GG treatment ameliorated the redistribution of ZO-1 and claudin in MDCK cell line caused by enterohemorrhagic E. coli . In another study, incubation with CFS of B. lactis 420 has been shown to increase the intestinal epithelial integrity against enteropathogenic E. coli (EPEC) .
Cell viability assessed by MTT assay revealed that VR1 CFS treatment was not detrimental to cells and there was no loss in viability when pre-incubated with VR1 CFS. On the other hand, co-incubation could not prevent the loss in cell viability caused by A. veronii. Pre-incubation of Caco2 with p40 and p75 isolated from the soluble protein of L. rhamnosus GG, abrogated the disruptive effect of H2O2 on tight junctions of Caco2 cells . The protective effect of soluble proteins was shown to be by activation of MAP kinase and PKC dependent signalling pathways. One more study (Parassol et al., ) documented that pre-incubation of L. casei with T84 cells could abolish the invasion and adhesion of EPEC. On these lines, we speculate, pre-incubation of mammalian cells with CFS of Lactobacilli sp. initiates cellular signalling which either inhibits or upregulate tight junction proteins that may get damaged by entero pathogens.
In view of the increasing prevalence of Aeromonas spp. in food products, this study assumes significance of its application of L. plantarum as a potential probiotic microorganism. The findings also suggest that the regular usage of probiotic microorganisms in food preparations can prevent the cytotoxicity or manifestation of pathogenicity in future encounter with pathogens. Further in depth studies will be necessary to understand the preventive role of VR1 in invivo model for A. veronii infection and to identify its active component which may be used as potential preventive cure against gastro-intestinal infection.