Imipenem

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.

Minimum inhibitory concentrations (MICs) of the 11 antimicrobial agents routinely prescribed in burn centers against the 55 isolates of MBL-producing P. aeruginosa were determined by the E-test method (AB BIODISK, Solna, Sweden), as recommended by the National Committee For Clinical Laboratory Standard Institute (CLSI) (19 (link)). A bacterial suspension from growth in a tryptic soy agar (TSA) plate was prepared in 2 mL of Mueller-Hinton broth (MHB), and the turbidity was adjusted so that it was equivalent to that of a 0.5 McFarland standard. The bacterial suspension was streaked onto a 150-mm-diameter plate containing Mueller-Hinton agar (MHA); the plate was later incubated at 35 to 37℃ in ambient air for 16 to 18 hours. The MIC was read on the basis of the interception of the elliptical zone of growth inhibition with the graded E-test strip according to the manufacturer’s instructions. The antibiotics (Mast Co., UK) consisted of imipenem (10 μg), meropenem (10 μg), cefepime (30 μg), ceftazidime (30 μg), piperacillin/tazobactam (110 μg), ciprofloxacin (5 μg), tobramycin (10 μg), amikacin (30 μg), gentamicin (10 μg), ampicillin (10 μg), and aztreonam (30 μg). American typing collection (ATCC 27853) of P. aeruginosa was used as a control strain to determine antibacterial susceptibility.

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.

Combination disk diffusion test (CDDT) was performed for presumptive identification of MBLs by imipenem, meropenem, doripenem, and ertapenem (Mast Group, Merseyside, UK) alone and in combination with EDTA [9 (link)]. Detection of ESBLs was tested for all the isolates by combination disk diffusion test (CDDT) containing ceftazidime (CAZ) and cefotaxime (CTX) with CAZ 30 μg + CA 10 μg and CTX 30 μg + CA 10 μg per disc (Mast Group, Merseyside, UK). The zones of inhibition were compared for the CTX, CAZ discs with that of the CAZ 30 μg + clavulanic acid (CA) 10 μg and CTX 30 μg + CA 10 μg disc. An increase in zone diameter of ≥5 mm in the presence of clavulanic acid was interpreted positive for the presence of ESBL in the test organism. Escherichia coli ATCC25922 and K. pneumoniae ATCC700603 were used as negative and positive controls for ESBL production, respectively. Presumptive identification of KPC enzyme was performed for all the K. pneumoniae isolates by modified Hodge test [7 (link)]. Amp-C was detected using the D69C AmpC Detection kit developed by Mast Group [10 (link)].