Piperacillin tazobactam

Antibiotic resistance profiles were obtained from 166 isolates. A standardized amount was inoculated (standard 0.5 of McFarland) and antimicrobial susceptibility testing was performed by the disk diffusion method on Mueller–Hinton (MH) agar (Difco Laboratories, Detroit, MI, USA) using interpretative criteria of the Clinical and Laboratory Standard Institute (CLSI) [34 ]. The following antimicrobials were used: trimethoprim/sulfametoxazol (SXT, 23.75 μg + 1.25 μg), gentamicin (GEN, 10 μg), ciprofloxacin (CIP, 5 μg), ceftazidime (CAZ, 30 μg), cefoxitin (FOX, 30 μg), cefotaxime (CTX, 30 μg), meropenem (MEM, 10 μg), piperacillin/tazobactam (TZP, 40 μg), and cefixime (CFM, 5 μg) (Oxoid Ltd., Hampshire, United Kingdom). ESBL-positive isolates were identified by using the double-disk synergy test with third-generation cephalosporins (cefotaxime, ceftazidime, and cefixime) alone and with clavulanic acid. Quality control was carried out using standard strains of Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27953). Intermediate susceptibility to each antibiotic was considered to be resistant.
According to the definitions proposed by Magiorakos et al., resistance to three or more antibiotic classes was defined as multidrug resistant (MDR) [35 (link)].

Antimicrobial susceptibility testing (AST) was carried out using Kirby Bauer’s Disc Diffusion method and interpreted according to the Clinical and Laboratory Standard Institute (CLSI) guidelines (2018).
The following antibiotics for the Enterobacteriaceae were tested: Ceftriaxone (30μg), Sulfamethoxazole-trimethoprim (25μg), Piperacillin-tazobactam (110 μg), Ticarcillin-clavulanate (85μg), Tetracycline (30μg), Amikacin (30μg), Gentamicin (10μg), Ciprofloxacin (5μg), Meropenem (10μg), Azithromycin (15μg), Chloramphenicol (30μg), Nitrofurantoin (300μg), Nalidixic acid (30μg), Ceftazidime (30μg) and Amoxicillin-clavulanate (30μg) (Oxoid, Basingstoke, Hants, UK). Quality control was ensured by using standard strains (Klebsiella pneumoniae, ATCC 700603 and Escherichia coli, ATCC 25922) in determining susceptibility or otherwise of stool isolates. The definition of multidrug resistance (MDR) was based on the resistance to three or more classes of antimicrobial agents [27 (link)].

The Kirby–Bauer disk diffusion method was used for antibiotic susceptibility testing. The following antibiotic discs were used: penicillin (10 Unit, Oxoid), ampicillin (10 µg, Oxoid) high-level gentamicin resistance (HLGR) (120 µg, Oxoid), piperacillin/tazobactam (100/10 μg, Oxoid), teicoplanin (30 µg, Oxoid) and vancomycin (30 µg, Oxoid). All results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [24 ].

Antibiotic susceptibility testing was performed according to the Clinical Laboratory Standard Institute (CLSI) guidelines using Kirby-Bauer method (CLSI, 2014) (12 ). Antibiotics used were benzyl penicillin (10U), piperacillin tazobactam (100-10 μg), amoxicillin clavulanic acid (20-10 μg), impenem (10 μg), ceftriaxone (30 μg) chloramphenicol (30 μg), tetracycline (30 μg), moxifloxacin (5 μg), ciprofloxacin (5 μg), levofloxacin (5 μg) vancomycin (5 μg) and metronidazole (4 μg) (Oxoid, UK). Inoculated Mueller Hinton agar plates (HiMedia, India) were incubated at 37°C for 24 hours in the anaerobic jar using Anaerogen gas packs (90% N2 /10% CO2) (Oxoid, UK). E. coli ATCC 25922 was used as standard strain to check the standardization of the disks.

The isolates were initially processed using disk-diffusion antimicrobial susceptibility testing for amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, doripenem, gentamicin, imipenem, levofloxacin, meropenem, netilmicin, piperacillin-tazobactam, ticarcillin-clavulanate, and tobramycin with Oxoid (Basingstoke, United Kingdom) disks. Complementarily, an in-house broth microdilution method using cation-adjusted Mueller–Hinton Broth (Sigma-Aldrich, St. Louis, MO, USA) was performed to determine the minimum inhibitory concentration (MIC) for amikacin gentamicin, imipenem, meropenem, colistin, polymyxin B, tigecycline, and ceftazidime-avibactam, and all salts were purchased from Sigma-Aldrich (St. Louis, MO, USA), except for avibactam, which was donated by Pfizer Inc. To complete the antimicrobial susceptibility panel, novel antimicrobials/combinations were evaluated with Liofilchem (Roseto degli Abruzzi, Italy) MIC test strips for ceftolozane–tazobactam, meropenem–vaborbactam, imipenem–relebactam, cefoperazone–sulbactam, cefiderocol, plazomicin, eravacycline, and fosfomycin.

Pure colonies from each sample with E. coli and P. aeruginosa contamination were randomly selected for antibiotic susceptibility testing. Pure isolates of E. coli, and P. aeruginosa were subjected to antibiotic susceptibility testing using the Kirby–Bauer Disc Diffusion method on Mueller–Hinton agar, as recommended by Clinical Laboratory Standards Institute (CLSI) guidelines [23 ]. Zones of inhibition were measured in millimeters and recorded for each antibiotic.
Antibiotics used for E. coli isolates included amoxicillin–clavulanate (20/10 µg), aztreonam (30 µg), ertapenem (10 µg), gentamicin (10 µg), chloramphenicol (20/10 µg), ciprofloxacin (5 µg), cefuroxime (30 µg), ceftriaxone (30 µg) and trimethoprim–sulphamethoxazole (1.25/23.75 µg) (Oxoid, Hampshire, UK). For P. aeruginosa, piperacillin–tazobactam (100/10µg), aztreonam (30 µg), gentamicin (10 µg) and ciprofloxacin (5 µg) (Oxoid, Hampshire, UK) were used.