Ertapenem

The antimicrobial susceptibility of the two investigated isolates were tested according to the Clinical Laboratory Standard Institute guidelines, CLSI (M100-S32, 2022) recommendations using Escherichiacoli ATCC 25922 as quality control strain [14 ]. The investigated isolates were tested against the following antimicrobial agents, viz., ampicillin (30 µg), cefepime (30 µg), ceftriaxone (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), aztreonam (30 µg), ertapenem (10 µg), imipenem (10 µg), meropenem (10 µg), amikacin (10 µg), gentamicin (10 µg) and ciprofloxacin (5 µg) (HiMedia, India) via Kirby-Bauer disc diffusion method. The minimal inhibitory concentrations (MICs) of ertapenem (MSD, France), imipenem (Merck, France) and meropenem (AstraZeneca, UK) were determined through agar dilution method (concentration range : 1–64 µg/ml).

Antibiotics discs containing amikacin (30 μg), amoxicillin-clavulanic acid (30 μg), aztreonam (30 μg), ampicillin (10 μg), azithromycin (30 μg), cefepime (30 μg), Cefoperazone/Sulbactam (75/30 μg), ceftriaxone (30 μg), cefotaxime (30 μg), cefuroxime (30 μg), cephalexin (30 μg), ciprofloxacin (1 μg), clindamycin (2 μg), cloxacillin (30 μg), trimethoprim/sulfamethoxazole (25 μg), ertapenem (10 μg), erythromycin (15 μg), gatifloxacin (5 μg), gentamicin (10 μg), imipenem (10 μg), levofloxacin (5 μg), linezolid (30 μg), meropenem (10 μg), netilmicin (30 μg), norfloxacin (10 μg), ofloxacin (5 μg), piperacillin-tazobactam (100/10 μg), teicoplanin (30 μg), tetracycline (30 μg), and vancomycin (30 μg) were obtained from Himedia Laboratories (Mumbai, India) and used as per manufacturer's instructions.

The selected bla_NDM harbouring Enterobacterales isolates were further tested by Kirby Bauer disc diffusion tests with CAZ (30μg), AZT (30μg), CAZ-AVI (30 μg /20 μg), imipenem (10μg), ertapenem (10μg) and meropenem (10μg) discs (HiMedia Laboratories, India). The results were interpreted as per zone diameter breakpoints described in the performance standards of antimicrobial susceptibility testing by the Clinical and Laboratory Standards Institute (CLSI) in 2022 [12 ].

To identify VRE, stool samples were inoculated onto VRE agar (HiCrome VRE Agar, HiMedia Laboratories). To identify Gram-negative bacilli, stool samples were inoculated onto eosin methylene blue agar. All isolates were identified by MALDI-TOF MS (Bruker MALDI Biotyper, Germany). Antibiotic susceptibility was assessed using the disk diffusion method using the following antibiotics: 30 µg cefepime, 5 µg cefotaxime, 10 µg ceftazidime, 30 µg ceftriaxone, 5 µg ciprofloxacin, 10 µg ertapenem, 10 µg gentamicin, 10 µg imipenem, 10 µg meropenem and 36 µg piperacillin–tazobactam (HiMedia Laboratories, India). Suspected isolates were tested for the production of ESBLs with the combination disk test.⁷ Isolates resistant to cefoxitin, and cefotaxime or ceftazidime were tested for the production of AmpC β-lactamases (AmpC) using a disk test (Mast Discs Combi, UK). All disc diffusion tests were evaluated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST).⁷ Multi-drug resistance (MDR) is defined as combined resistance to third-generation cephalosporins [cefotaxime or ceftazidime or ceftriaxone, fluoroquinolones (ciprofloxacin) and aminoglycosides (gentamycin)]. No VRE was identified.

Antimicrobial susceptibility of bla_CTX-M harbouring parent strains as well as transformants were determined by Kirby Bauer disc diffusion method and results were interpreted as per CLSI guidelines [17 ]. Following antibiotics were tested: cefotaxime (30μg), cefoxitin (30μg), ceftazidime (30μg), amikacin (30μg), gentamicin (10μg), kanamicin (30μg), ciprofloxacin (5μg), trimithoprim/sulphamethoxazole (1.25/23.75μg), imipenem (10μg), ertapenem (10μg), tigecycline (15μg) and polymyxin B (300 units) (Hi-Media, Mumbai). MIC was also determined for donor strain and transformants against cefotaxime, ceftazidime and ceftriaxone (Hi-Media, Mumbai, India) by agar dilution method.