*2.2. Antibacterial Susceptibility Testing and Detection of ARGs*

Coliforms were detected with the most probable number procedure [16]. A presumptive test involved three subsets of tubes containing different amounts of lactose or lauryl tryptose broth. Each subset contained five tubes with inverted Durham tubes to collect gas produced by fermentation. The three subsets were inoculated with water samples of 10, 1.0, and 0.1 mL, respectively. The tubes were then incubated for 24 h at 35–37 ◦C. A positive test for gas formation was

presumptive evidence of coliforms. A confirmatory test for coliforms was made by inoculating another broth from one of the positive tubes. The test was completed by final isolation of the coliforms on selective and differential media, Gram staining the isolates, and reconfirming gas production. Coliform isolates were then sub-cultured on *Brilliance*TM UTI agar to collect presumptive *E. coli* isolates. Following biochemical confirmation using standard tests, identified *E. coli* isolates were tested for antibiotic susceptibility using the standard Kirby Bauer disc diffusion method for: (i) amoxicillin/clavulanic acid; (ii) ceftazidime; (iii) ceftriaxone; (iv) ciprofloxacin; (v) co-trimoxazole (trimethoprim/sulfamethoxazole); (vi) fosfomycin; (vii) gentamicin; and (viii) imipenem. The selected antibiotics were commonly used in the hospitals and routinely tested in clinical laboratories. Antibiotic susceptibility test results were interpreted as resistant, intermediate, and susceptible using Clinical and Laboratory Standard Institute guidelines (CLSI M100-2013) [17]. Minimum inhibitory concentrations (MICs) were determined for ciprofloxacin and ceftazidime using E-test. Disc diffusion zone diameters were also compared with epidemiological cut-off values (ECOFF) [18].

For *E. coli* isolates resistant to third generation cephalosporins, extended-spectrum beta-lactamase (ESBL) production was tested using combined disc diffusion. Genotypic confirmation was done through polymerase chain reactions. Genes coding for beta-lactam resistance, *blaCTX-M* and *blaTEM*, were tested in ESBL-producing isolates and *qepA* gene was tested for in ciprofloxacin-resistant isolates [19,20].
