Diarrheal disease caused by Vibrio cholerae is endemic in developing countries including India and is associated with high rate of mortality especially in children. V. cholerae is known to form biofilms on the gut epithelium, and the biofilms once formed are resistant to the action of antibiotics. Therefore agents that prevent the biofilm formation and disperse the preformed biofilms are associated with therapeutic benefits. The use of antibiotics for the treatment of cholera is associated with side effects such as gut dysbiosis due to depletion of gut microflora, and the increasing problem of antibiotic resistance. Thus search for safe alternative therapeutic agents is warranted. Herein, we screened the lactobacilli spp. isolated from the fecal samples of healthy children for their abilities to prevent biofilm formation and to disperse the preformed biofilms of V. cholerae and V. parahaemolyticus by using an in vitro assay. The results showed that the culture supernatant (CS) of all the seven isolates of Lactobacillus spp. used in the study inhibited the biofilm formation of V. cholerae by more than 90%. Neutralization of pH of CS completely abrogated their antimicrobial activities against V. cholera, but had negligible effects on their biofilm inhibitory potential. Further, CS of all the lactobacilli isolates caused the dispersion of preformed V. cholerae biofilms in the range 62–85%; however, pH neutralization of CS reduced the biofilm dispersal potential of the 4 out of 7 isolates by 19–57%. Furthermore, the studies showed that CS of none of the lactobacilii isolates had antimicrobial activity against V. parahaemolyticus, but 5 out of 7 isolates inhibited the formation of its biofilm in the range 62–82%. However, none of the CS dispersed the preformed biofilms of V. parahaemolyticus. The ability of CS to inhibit the adherence of Vibrio spp. to the epithelial cell line was also determined. Thus, we conclude that the biofilm dispersive action of CS of lactobacilli is strain-specific and pH-dependent. As Vibrio is known to form biofilms in the intestinal niche having physiological pH in the range 6–7, the probiotic strains that have dispersive action at high pH may have better therapeutic potential.
The nutritional challenge faced by the monogastric animals due to the chelation effects of phytic acid, fuel the research on bioprospecting of probiotics for phytase production. Pediococcus acidilactici SMVDUDB2 isolated from Kalarei, exhibited extracellular phytase activity of 5.583 U/mL after statistical optimization of fermentation conditions viz. peptone (1.27%); temperature (37 °C); pH (6.26) and maltose (1.43%). The phytase enzyme possessed optimum pH and temperature of 5.5 and 37 °C, respectively and was thermostable at 60 °C. The enzyme was purified 6.42 fold with a specific activity of 245.12 U/mg with hydrophobic interaction chromatography. The purified enzyme had K m and V max values of 0.385 mM and 4.965 μmol/min respectively, with sodium phytate as substrate. The strain depicted more than 80% survival rate at low pH (pH 2.0, 3.0), high bile salt concentration (0.3 and 0.5%), after gastrointestinal transit, highest hydrophobicity affinity with ethyl acetate (33.33 ± 0%), autoaggregation (77.68 ± 0.68%) as well as coaggregation (73.57 ± 0.47%) with Staphylococcus aureus (MTCC 3160). The strain exhibited antimicrobial activity against Bacillus subtilis (MTCC 121), Mycobacterium smegmatis (MTCC 994), Staphylococcus aureus (MTCC 3160), Proteus vulgaris (MTCC 426), Escherichia coli (MTCC 1652) and Lactobacillus rhamnosus (MTCC 1408). The amount of exopolysaccharide produced by the strain was 2 g/L. This strain having the capability of phytate degradation and possessing probiotic traits could find application in food and feed sectors.
Background
Increase in the number of infections caused by Gram-negative bacteria in neutropenic cancer patients has prompted the search for novel therapeutic agents having dual anticancer and antimicrobial properties. Bacteriocins are cationic proteins of prokaryotic origin that have emerged as one of the most promising alternative antimicrobial agents with applications as food preservatives and therapeutic agents. Apart from their antimicrobial activities, bacteriocins are also being explored for their anticancer potential.
Results
In this study, a broad-spectrum, cell membrane-permeabilizing enterocin with a molecular weight of 65 kDa was purified and characterized from the culture supernatant of vaginal Enterococcus faecium 12a. Enterocin 12a inhibited multidrug-resistant strains of various Gram-negative pathogens such as Salmonella enterica, Shigella flexneri, Vibrio cholerae, Escherichia coli and Gram-positive, Listeria monocytogenes, but had no activities against different strains of gut lactobacilli. The mass spectrometric analysis showed that the enterocin 12a shared partial homology with 4Fe-4S domain-containing redox protein of E. faecalis R712. Further, enterocin 12a selectively inhibited the proliferation of various human cancer cell lines in a dose-dependent manner but not that of normal human peripheral blood mononuclear cells. Enterocin 12a-treated cancer cells showed apoptosis-like morphological changes.
Conclusion
Enterocin 12a is a novel bacteriocin that has anticancer properties against human cell lines and negligible activity towards non-malignant cells. Therefore, it should be further evaluated for its anticancer potential in animal models.
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