Ciprofloxacin

All isolates were examined for resistance to routine antimicrobial agents by standard disk diffusion method using Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) as control strains (17 ). The antibiotics tested were gentamicin, amikacin, ceftazidime, ceftizoxime, cefotaxime, ceftriaxone, imipenem, ciprofloxacin, co-trimoxazole, chloramphenicol, penicillin, oxacillin, ampicillin, vancomycin, rifampicin and erythromycin (Mast Co, the UK). Isolates showing intermediate levels of susceptibility were classified as nonsusceptible.

Antibiotic susceptibility of the bla_NDM-1 positive isolates was determined by the Kirby–Bauer method as recommended by the CLSI. The 11 standard antibiotic disks used include: imipenem (10 µg), meropenem (10 µg), ertapenem (10 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefepime (30 µg), gentamicin (10 µg), piperacillin/tazobactam (100/10 µg), amikacin (30 µg), ciprofloxacin (5 µg) and aztreonam (30 µg) (Mast Group Ltd, UK). The ESBL phenotype was identified using combined disk method by disks of ceftazidime (30 mg) with (10 mg) and without clavulanic acid (Mast Group Ltd, UK), applied to all bla_NDM−1 positive isolates (15). Moreover, the minimum inhibitory concentrations (MICs) of imipenem (10 µg/ml) [≤ 2 mg/L (susceptible), 4 mg/L (intermediate), and ≥ 8 mg/L (resistant)] (Liofilchem, Roseto degli Abruzzi, Italy) were applied by gradient test strips to bla_NDM−1 positive P. aeruginosa isolates [18 ].

Antimicrobial susceptibility testing (AST) of E. coli and K. pneumoniae isolates was performed by the Kirby Bauer disk diffusion method according to the CLSI guideline [19 ]. The antimicrobial disks used for testing were ampicillin (10 µg) [tested only for E. coli], Amoxicillin-clavulanic acid (20/10 µg), piperacillin-tazobactam (100/10 µg), cefazolin (30 µg), cefuroxime (30 µg). cefixime (5 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefepime (30 µg), imipenem (10 µg), ciprofloxacin (5 µg), trimethoprim sulphamethoxazole (1.25/23.75 µg), nitrofurantoin (300 µg), and amikacin (30 µg). Amoxicillin-clavulanic acid, piperacillin-tazobactam, ceftazidime, ciprofloxacin, and imipenem disks were purchased from the manufacturer Mast (Mast group Ltd, Liverpool, UK) with the remainder from HiMedia (HiMedia, India). The minimum inhibitory concentration (MIC) of ciprofloxacin was determined by E-test (0.002-32 µg/mL) (HiMedia, India), and those isolates with MIC ≥ 32 µg/mL were further tested by agar dilution method following the procedures described by CLSI [20 ]. An isolate was defined to display multidrug resistance (MDR) if non-susceptible to ≥ 1 agent in ≥ 3 antimicrobial categories [21 (link)]. Escherichia coli ATCC 25922 was used for quality control.

DST was performed using disk diffusion method (Kirby-Bauer) on Mueller-Hinton agar (Merck, Germany) plates according to the Clinical and Laboratory Standards Institute (CLSI) guideline [13 ]. The tested antibiotic disks were: imipenem (10μg), meropenem (10μg), amikacin (30μg), ciprofloxacin (5μg), ceftriaxone(30μg), ceftazidime (30μg), colistin-sulfate (10μg), pipracillin-tazobactam (100/10μg), and gentamicin (10μg) [Mast Co., UK]. P. aeruginosa ATCC 27853 was used as a control strain for DST.