Meropenem

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)].

Eight K. pneumoniae isolates were received from the Medical Microbiology laboratories of three hospitals in Armenia between January 2019 and August 2019. All isolates were recovered from various clinical specimens (urine, sputum, throat, blood, and stool) of hospitalized patients. The isolates were identified using a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS) as described previously (59 (link)).
All isolates were tested using a disk diffusion method for susceptibility to a panel of 11 antibiotics, including ampicillin (10 mg), piperacillin-tazobactam (30/6 mg), amoxicillin-clavulanic acid (20 and 10 mg, respectively), ceftazidime (10 mg), cefepime (30 mg), norfloxacin (10 mg), levofloxacin (5 mg), amikacin (30 mg), imipenem (10 mg), meropenem (10 mg), and chloramphenicol (30 mg) (Mast Group, Merseyside, United Kingdom) according to the European Committee on Antimicrobial Susceptibility Testing protocol (EUCAST v.6.0, 2017) (60 ). The antibiotics chosen were those most frequently used in clinical settings in Armenia. Isolates resistant to three or more antibiotic classes were considered multidrug resistant.

Combined disk diffusion test (CDDT) was performed for identification of MBLs by imipenem and meropenem (Mast Group, Merseyside, UK) alone and in combination with EDTA. An increase in zone diameter of ≥7 mm around the imipenem+EDTA and meropenem+EDTA disks compared to that of imipenem and meropenem disks alone, respectively, was considered positive for MBL production [12 (link)].

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).