**2.1. Antibiotic susceptibility test**

Antibiotic susceptibility test for all 450 strains of *E.coli* were done using Mueller Hinton(MH) agar plate with commercially available antibiotic discs (Himedia Pvt Ltd, India) by Kirby Bauer disc diffusion method [64] according to CLSI guidelines [65]. *E.coli* ATCC 25922 was used as control.

Lawn culture of test strain (turbidity adjusted to 0.5 Mc Farland standard) was put on MH Agar plate. The antibiotic disks were put on inoculated plate with all aseptic precaution. Antibiotic susceptibility test was done for Aminoglycosides like Amikacin (AK-30μg), Gentamicin (GEN-10μg), Cephalosporins like Ceftazidime (CAZ-30μg), Cefotaxime (CTX-30μg), Fluoroquinolones like Ciprofloxacin (CIP–5μg), Monobactams like Aztreonam (AT-30μg), Carbapenems such as Imipenem (IPM-10μg), Etrapenem (ETP-10μg) etc. For urine sample an additional disk of Nitrofurantoin (NIT-300μg) and only for MBL producing strains, Colistin (CL-10μg) disk were used

All 450 *E.coli* strains were tested for newer β-lactamases e.g. Extended Spectrum β-lactamas‐ es(ESBLs), AMPC β-lactamases Metallobetalactamases(MBLs) and Klebsiella pneumoniae producing Carbapenemases (KPCs) [28, 30, 39, 58, 59, 61]. As Metallobetalactamases are also found in carbapenem susceptible organisms., we have screened carbapenem sensitive strains also for MBL production.

#### **2.2. Detection of newer β-lactamases**

### *2.2.1. Detection of ESBL production [28]*

Combined disk method as per CLSI guideline and ESBL E-test were used for ESBL detection.

In Combined disk method, lawn cultures of test strains (turbidity adjusted to McFarland 0.5 standard) were put on MH agar plates. Ceftazidime (CAZ) 30 μg disc and Ceftazidime plus Clavulanate (CAC) 30μg plus 10μg discs were put widely apart on that MH plate. After overnight incubation at 370 C increase in zone diameter of ≥5 mm with CAC disk as compared to CAZ disk alone was considered positive for ESBL detection.

In ESBL E-test, lawn culture of test strain (turbidity adjusted to McFarland 0.5 standard) was done on a MH agar plate & ESBL E-test strip (AB Biomeriux) was placed. After overnight incubation at 370 C, MIC ratio of ceftazidime/Ceftazidime Clavulanic acid (TZ/TZL) ≥ 8 or deformation of ellipse or phantom zone present was considered positive for ESBL production.
