Aims: To elucidate the antibiotic resistance and virulence genes of nisinresistant Enterococcus faecalis isolated from raw buffalo milk and to study the effect of nisin-sensitive and -resistant E. faecalis on the innate immunity of rats. Methods and Results: Slanetz-Bartley agar plates containing nisin were used to isolate nisin-resistant E. faecalis. The virulence factors were ascertained using quantitative real-time polymerase chain reaction. Cell viability, phagocytosis, intracellular survival and enzyme assays were performed to investigate the interaction of E. faecalis with rat macrophages. Nisin-resistant E. faecalis was less prone to phagocytosis and survived longer inside the macrophages, due to reduced production of reactive oxygen species and nitric oxide. The viability and activation of macrophages was also reduced in the presence of resistant E. faecalis, as observed by enhanced lactate dehydrogenase production and reduced b-galactosidase. Conclusions: Nisin-resistant E. faecalis and its virulence factors were reported in raw buffalo milk. This study shows that nisin-resistant variants exhibited cross resistance to antibiotics and suppressed the innate immune responses of rats by directly affecting macrophage activity. Significance and Impact of the Study: This study elucidated the contamination of raw buffalo milk by nisin-resistant E. faecalis, which may pose food safety risk.Enterococcus faecalis is prevalent in human and animal faeces and is a major cause of contamination in dairy products (Giraffa et al. 1997). Enterococcus faecalis collected from the milk of healthy animals and humans has also been reported as a reservoir of virulence and antibiotic-resistant genes, which exhibit resistance to clinically used drugs such as tetracycline, quinolones, vancomycin, macrolides, streptogramins and glycopeptides (Jim enez et al. 2008;Klibi et al. 2015). There are complex relationships between multidrug-resistant and virulence genes in bacteria. Schaufler et al. (2016) reported that extended spectrum b-lactamase plasmids for resistance enhance virulence in the carrier strains by affecting gene expression. Enterococcus faecalis harbours many virulence factors including aggregation substances, as-I, as-II and asa-Journal of Applied Microbiology 127, 897--910

Guava-Papaya jam was developed and evaluated for changes in its physico-chemical, microbiological and sensory parameters at monthly interval for three months storage period. Total soluble solids increased significantly, while acidity decreased significantly in guava-papaya jam with the advancement in storage period. Yeast and mold count increased significantly in blended jam during storage. The colour and appearance, flavour, taste, mouthfeel and overall acceptability of guava-papaya jam decreased significantly during storage, however, the products were found acceptable even after three months storage. Jam prepared with 40 guava: 60 papaya pulp ratio was found to be the most acceptable.

Nisin is used for food preservation due to its antibacterial activity. However, some bacteria survive under the prevailing conditions owing to the acquisition of resistance. This study aimed to characterize nisin-resistant E. faecalis isolated from raw buffalo milk and investigate their fitness cost. FE-SEM, biofilm and cytochrome-c assay were used for characterization. Growth kinetics, HPLC, qPCR, and western-blotting were performed to confer their fitness cost. Results revealed that nisin-resistant E. faecalis were morphologically different from sensitive strain and internalize more glucose. However, no significant difference was observed in the growth pattern of the resistant strain compared to that of the sensitive strain. A non-phosphotransferase glucose permease (GlcU) was found to be associated with enhanced glucose uptake. Conversely, Mpt, a major phospho-transferase system responsible for glucose uptake, did not play any role, as confirmed by gene expression studies and western blot analysis of HPr protein. The phosphorylation of His-15 residue of HPr phosphoprotein was reduced, while that of the Ser-46 residue increased with progression in nisin-resistance, indicating that it may be involved in the regulation of pathogenicity. In conclusion, resistance imposes a significant fitness cost and GlcU plays a key role in maintaining the fitness cost in nisin-resistant variants.

Chutney prepared from guava-papaya blends was analyzed for changes in chemical constituents and overall acceptability at monthly interval for three months storage period. Total sugars, reducing sugars and browning increased, while ascorbic acid, total carotenoids and total phenols decreased significantly in chutney during storage. Chutney prepared with 40 guava:60 papaya pulp ratio was most acceptable. The overall acceptability of chutney decreased significantly during three months storage period.

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