Cefotaxime

The antibiotic susceptibility of all the isolates was tested by employing the Kirby-Bauer’s technique as suggested by the CLSI (10 ). The eleven antibiotic disks used include: imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), ciprofloxacin (5 μg), ceftazidime (30 μg), cefepime (30 μg), cefotaxime (30 μg), amikacin (30 μg), gentamicin (10 μg), piperacillin/tazobactam (100/10 μg), aztreonam (30 μg) (Mast Group Ltd, UK). Isolates with resistance against a minimum of three groups of antibacterial agents were considered as MDR (11 (link)). To detect ESBL phenotype combined disk method using disks of ceftazidime (30 mg) with (10 mg) and without clavulanic acid (Mast Group Ltd, UK) was applied to all positively screened isolates by modified hodgE test (MHT) (11 (link)). A growth in the area diameter of ≥5 mm around ceftazidime disc with and without clavulanic acid was expected to be a positive result for ESBL production (12 (link), 13 (link)). The MHT was performed for all isolates as recommended by CLSI (10 ). The E test (imipenem 0.002–32μg/mL) (Liofilchem, Roseto degli Abruzzi, Italy) was applied (according to the manufacturer’s instructions) to all positively screened isolates by PCR test for bla_NDM gene, to determine minimum inhibitory concentrations (MICs).

Screened-positive bacterial isolates were confirmed for ESBL production using the combined-disk method according to the CLSI guidelines [20 ]. Zones of inhibition were determined for each isolate to antibiotic disks containing 30 μg of cefotaxime, 30 μg of ceftazidime and 10 μg of cefpodozime either alone or in combination with 10μg of clavulanic acid (MAST Group Ltd.). All zones of inhibition which differed by ≥ 5 mm between at least one of the standard antibiotic disks and its corresponding clavulanic combination disks were classified as having an ESBL-producer phenotype. Escherichia coli control strain ATCC 25922 was used to monitor the performance of ESBL detection agents.

The profile of this test was investigated based on CLSI [30 (link)] and EUCAST guidelines [31 ] using VITEK 2 compact system (Biomerieux, Craponne, France) in accordance with the manufacturer’s instructions and Kirby–Bauer disk diffusion method [32 (link)] for different antibiotics, including Ampicillin, Piperacillin-Tazobactam, Amoxiclav, Ceftriaxone, Cefotaxime, Ceftazidime, Cefepime, Meropenem, Amikacin, Ciprofloxacin and Levofloxacin (Mast Group, Bootle, England).

The antibiotic susceptibility pattern of the isolates was determined by the disk agar diffusion method on Muller Hinton agar (Merck, Germany) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines²¹. The antibiotics included piperacillin (100 µg), piperacillin-tazobactam (100/10 µg), imipenem (10 µg), meropenem (10 µg), doripenem (10 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), trimethoprim-sulfamethoxazole (1.25-23.75 µg), ceftazidime (30 µg), cefotaxime (30 µg), and cefepime (30 µg) (MAST Co., England). The susceptibility pattern of the isolates against aminoglycosides including kanamycin, amikacin, spectinomycin, netilmicin, gentamicin, streptomycin, and tobramycin was determined using the micro-broth dilution method according to the CLSI guidelines²¹. For interpretation of the minimum inhibitory concentration (MIC) values, we referred to the CLSI guidelines and previous studies^1,21,22. Escherichia coli ATCC 25922 and A. baumannii ATCC 19606 were used as control strains for antibiotic susceptibility testing.