Samples were collected in triplicate (n = 15) from five locations

Samples were collected in triplicate (n = 15) from five locations situated in up-to-down-gradient fashion (Figure

1). In brief, three transects were established randomly at each site and water samples (1 L) were collected 30 cm below water surface from left, mid and right bank Doramapimod of the river along each transect. Surface water samples were stored in sterile glass bottles, labeled and transported on ice to the laboratory for analysis. Sample processing and analysis was conducted within 6 hr after sample collection. Isolation and enumeration of Enterococci Quantitative enumeration of enterococci from selected sites was performed as per APHA [40] using the Multiple Tube Fermentation Technique and reported as MPN index/100 ml surface water. Additionally, enterococci were enumerated from each sample using standard membrane filtration method and reported as CFU/100 ml surface water [41]. Presumptive enterococci recovered (n = 30) from each sample were identified by biochemical tests including catalase test and PYR test. The growth of isolates was determined in 6.5% NaCl, pH 9.6, and at 10 and 45°C, respectively. All confirmed enterococci isolates were archived in tryptic soy broth with 15% glycerol at -80°C for further analyses. Characterization of Enterococcus spp. using Polymerase Chain Reaction All isolates confirmed by biochemical tests were subjected to genotypic characterization

by Polymerase Chain Reaction (PCR) technique. The GSK690693 ic50 presence of tuf gene encoding the elongation factor EF-Tu in genus Enterococcus and the

sodA variant for E. faecalis, E. faecium, E. durans and E. hirae species were investigated by PCR as reported earlier [42, 43]. An isolate not belonging to the four species of enterococci genotypically characterized by PCR in this study was listed as “”other Enterococcus spp.”" Antimicrobial susceptibility testing A panel of thirteen antimicrobials (antimicrobial abbreviation:mcg/disc) impregnated on paper discs (Himedia Ltd., India) belonging to eight different group of antimicrobials as Fluoroquinolone: Norfloxacin (Nx:10 mcg), β-lactam: Ampicillin (A:10 mcg), Oxacillin (Ox:1 mcg), PenicillinG (P:10 units), Methicillin (M:5 mcg), Aminoglycoside: Gentamicin (G:10 mcg), Streptomycin (S:10 mcg), Tetracycline: Tetracycline (T:30 mcg), Phenicol: Etoposide mouse Chloramphenicol (C:30 mcg), Macrolide: Erythromycin (E:15 mcg), Rifamycin: Rifampicin (R:5 mcg), Glycopeptides: Vancomycin (Va:30 mcg), Teicoplanin (Te:30 mcg) were used for testing the sensitivity of isolated organisms by Kirby-Bauer disc diffusion test as described by CLSI [31, 44]. The diameter of zones showing inhibition were measured to the nearest mm and recorded. A zone size interpretive chart was used to determine sensitivity/resistance of antimicrobials as described by CLSI [44]. Determination of virulence-markers distribution in enterococci Polymerase Chain Reaction technique was used to generate a profile for virulence-markers’ distribution in enterococci.

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