Study site and Sample collection
The study was conducted along 10 km stretch of river Gomti in Lucknow city (Latitude: 25.55′N, Longitude: 80.58′E and Altitude: 123 m). River Gomti, flowing through the northern part of the country, is a major tributary of the Ganga River in India. Originating from a natural reservoir in the swampy and densely forested area near Madho-Tanda (altitude 200 m; latitude 28°34′N and E longitude 80°07′E) in the foothills of Himalayas, the river traverses a distance of about 730 km in the Indo-Gangetic alluvial region before its confluence with river Ganga. The river flowing through the Lucknow city (population: ~3.5 million), the State capital of Uttar Pradesh, meets about half of the total domestic water demand (about 550 mLd) of the town. The average flow of the river at Lucknow varies between 500 mLd in summers and 55,000 mLd during the monsoons. The river contains water throughout the year and exhibits sluggish flow except for the monsoon period. The Gomti River, identified as one of the most polluted tributary of river Ganga, receives 450 mLd of untreated domestic waste water in Lucknow city (Singh et al. 2004). In this study, five sites located in up-to-downstream on the river, namely Gau-ghat (site 1: located in upstream of river), Daliganj Bridge (site 2: bathing ghat and holy spot), Shaheed Smarak (site 3: bathing ghat and picnic spot), Lakshaman mela ground (site 4: recreational and picnic spots), Gandhi setu (site 5, most downstream location in the landscape) were selected based on various human activities on the banks of river Gomti. A cross-sectional approach was used to collect river water samples. Samples were collected in triplicate (n = 15) from five locations situated in up-to-down-stream fashion (Fig. 1). In brief, three transects were established randomly at each site and water samples (1 L) were collected 30 cm below the water surface from left, mid and right banks of the river along each transect. The samples of river water were stored in sterile glass bottles, labeled and transported on ice to the laboratory for analysis. Sample processing and analysis were conducted within 6 h after sample collection.
Isolation and enumeration of Enterococci
Quantitative enumeration of fecal streptococci or enterococci at selected sites was performed using the standard multiple tube fermentation technique (APHA 1998). In brief, tubes containing 20 mL Azide Dextrose Broth (ADB) were prepared. A series of tubes of ADB was inoculated with appropriate graduated quantities of sample (10, 1 and 0.1 mL). The inoculated tubes were incubated at 37 ± 0.5 °C and examined for turbidity after 24–48 h and calculated MPN index/100 mL surface water depending upon the number of positive tubes. Enterococci were recovered from each sample using standard membrane filtration method (APHA 1998). Briefly, tenfold dilutions of each water sample was prepared (100, 10−1, 10−2 and 10−3), and 10 mL of each dilution were filtered through 0.45-µm, 47-mm mixed cellulose ester filters (Millipore, Bedford, MA, USA) The filters were then placed on mE agar plates incubated at 41 ± 0.5 °C for 48 h for detection and enumeration of enterococci (Hi-Media, India). After 48 h, membrane filters from mE agar plates were placed on Bile Esculin Azide (BEA) agar plates and incubated at 41 ± 0.5 °C for 20 min. Colonies characteristic of enterococci, ranging from pink to dark red on mE agar and producing a brown to black precipitate on BEA agar, were considered presumptive enterococci (APHA 1998). Negative control filters and agar plates were included in each membrane filtration analysis. All colonies were counted, and concentration of enterococci per 100 mL water was determined from dilution plates containing 30–300 CFU. Presumptive enterococci recovered (n = 60) 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°s and 45 °C, respectively. All confirmed enterococci isolates were archived in tryptic soy broth with 15 % (v/v) glycerol at -80 °C for further analyses.
Multiplex PCR assay
All enterococci isolates confirmed by biochemical tests were subjected to genotypic characterization by multiplex Polymerase Chain Reaction (PCR) technique. The multiplex PCR assay format was designed comprising four genes; either ace or efaA gene with sodA (specific for detection of E. faecalis), vanB and gelE gene. All the primers used in this assay have been reported earlier in singleplex PCR format for detection of respective genes in clinical settings (Table 2; supplementary information). Genomic DNA was extracted from overnight grown cells of enterococci isolates (n = 60) using Genelute bacterial genomic DNA kit (Sigma, USA). PCR assay was performed in reaction volume of 50 µl. In brief, The mastermix consisted of 1× hot start Taq buffer, 3.0 mM MgCl2, 0.4 mM deoxynucleotide triphosphate mix, 1.5 U of Hotstart Taq polymerase (MBI Fermentas), 300 pg of template DNA, sterile molecular grade water, forward and reverse primer (Metabion, Germany) concentration optimized for this study has been described in (Table 2; supplementary information). The PCR protocol employed an initial denaturation at 95 °C for 3 min followed by 30 cycles of denaturation at 95 °C; 30 s, annealing at 51 °C; 30 s, elongation at 72 °C; 50 s and final extension at 72 °C for 8 min. The amplicons were electrophoresed on 1.5 % agarose gel in Tris–acetate-EDTA buffer supplemented with 0.5 µg/mL of ethidium bromide. Standard DNA molecular weight markers used were 100 bp DNA ladder (MBI Fermentas, USA). The presence of gene esp encoding enterococcal surface protein in different E. faecalis isolates was also examined (Table 5.2). E. faecalis ATCC 51229, E. faecium ATCC 35667, 27270, E. durans ATCC 49470, E. hirae ATCC 9790, E. coli MTCC 723, Vibrio cholerae ATCC 51394, Salmonella typhimurium ATCC 13311 and Salmonella typhimurium ATCC 14028 were used throughout the study as reference/standard strains.
Gelatinase phenotype determination
Gelatinase production was detected by spotting mid-log phase grown pure culture of individual E. faecalis isolates (n = 33) on freshly prepared tryptic soy agar plates (Himedia, India) containing 1.5 % of skimmed milk. Plates were incubated overnight at 37 °C and then cooled to ambient temperature for 2 h. The appearance of a transparent halo around the colonies was considered to be a positive indication of gelatinase production (Gilmore et al. 2002).
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:10mcg), β-lactam: Ampicillin (A:10mcg), Oxacillin (Ox:1mcg), PenicillinG (P:10 units), Methicillin (M:5mcg), Aminoglycoside: Gentamycin (G:10mcg), Streptomycin (S:10mcg), Tetracycline: Tetracycline (T:30mcg), Phenicol: Chloramphenicol (C:30mcg), Macrolide: Erythromycin (E:15mcg), Rifamycin: Rifampicin (R:5mcg), Glycopeptides: Vancomycin (Va:30mcg), Teicoplanin (Te:30mcg) were used for testing the sensitivity of isolated organisms by Kirby-Bauer disc diffusion method as described by Clinical Laboratory Standards Institute (CLSI 2002). In brief, pure E. faecalis isolate culture colonies (3–4) were transferred to tubes containing 5 mL brain heart infusion broth and incubated in rotary shaker (Innova 4230, New Brunswick, USA) at 150 rpm and 37 + 1 °C for 4–6 h to yield a suspension of 106 cells/mL. The inoculum was uniformally spread on the sterile Muller-Hinton agar plates (90 mm diameter) using the sterile cotton swab. Four to six antimicrobial discs were applied aseptically, at least 24 mm apart on Mueller–Hinton agar plates. The plates were incubated immediately at 35 + 1 °C; 16–18 h for all enlisted antimicrobials and 24 h for vancomycin. The test was performed in triplicate for each E. faecalis isolate. The diameter of zones showing inhibitions were measured to the nearest mm and recorded. A zone size interpretive chart was used to determine sensitivity/resistance of antimicrobials (CLSI 2002).
Determination of Vancomycin-resistant VanA and VanB phenotype of E. faecalis
VanA phenotyping
Antimicrobials: Vancomycin (Van) and Teicoplanin (Te) were used for this study (Sigma-aldrich, USA). After Biochemical and microbiological confirmation, E.
faecalis isolates from each sampling site were subjected to tube macrodilution method of antimicrobial susceptibility testing (Van: ≥ 64 µg/mlL, Te: ≥ 16 µg/mL) with slight modification. In brief, well characterized E. faecalis isolates were grown in tubes containing 5 mL Brain Heart Infusion broth and incubated in rotary shaker (INNOVA 4230, New Brunswick, USA) at 150 rpm and 37 + 1 °C for 4–6 h to yield a suspension of 106 cells/mL. The inoculum of 106 cells was added to sterile Muller-Hinton broth tubes containing Van: ≥ 64 µg/mL, Te: ≥ 16 µg/mL. The tubes were incubated immediately at 35 + 1 °C for 24 h. The tubes showing consistent growth were recorded as VanA positive. E. faecium ATCC 51559 was used as VanA positive and sterile water as negative control.
VanB phenotyping
Antimicrobial Vancomycin (Van) was used for this study (Sigma-aldrich, USA). The E. faecalis isolates from each sampling site were subjected to tube macrodilution method of antimicrobial susceptibility testing (Van: 4–1024 µg/mL) with slight modification. The tube macrodilution method CLSI (2005) for antimicrobial susceptibility testing (Van: 4–1024 µg/mL) was adopted with slight modification. In brief, well characterized E. faecalis isolates were grown in tubes containing 5 mL Brain Heart Infusion broth and incubated in rotary shaker (INNOVA 4230, New Brunswick, USA) at 150 rpm and 37 + 1 °C for 4–6 h to yield a suspension of 106 cells/mL. The inoculum of 106 cells was added to sterile Muller-Hinton broth tubes containing Van: 4–1024 µg/mL in twofold dilution series. The tubes were incubated immediately at 35 + 1 °C for 24 h. The tubes showing consistent growth were recorded as VanB positive. E. faecalis ATCC 51299 served as VanB positive and sterile water as negative control.
Statistical analyses
The concentrations of enterococci obtained using MPN analysis test and membrane filtration method from up-to-downstream river water samples was compared, Chi square test for trend was applied for the purpose. The prevalence and distribution of antimicrobial-resistance and virulence-markers among isolates from up-to-downstream landscape was assessed using wilcoxon rank-sum tests. All statistical tests were performed using GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com).
