Meropenem

Using the Clinical and Laboratory Standards Institute (CLSI) guidelines [9 ], an antibiogram assay was performed on the isolated K. pneumoniae colonies. The antibiotic discs contained ampicillin (30 µg), cotrimoxazole (25 µg), cefixime (5 µg), cefotaxime (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), gentamicin (10 µg), amikacin (30 µg), tetracycline (30 µg), doxycycline (30 µg), minocycline (30 µg), tigecycline (15 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), ampicillin/sulbactam (100/10 µg), piperacillin/tazobactam (100/10 µg), imipenem (10 µg), meropenem (10 µg), ertapenem (10 µg), doripenem (10 µg), aztreonam (30 µg), colistin (10 µg), and fosfomycin (200 µg) (Mast Diagnostics, United Kingdom) [4 , 10 ].
The minimum inhibitory concentration (MIC) of imipenem, meropenem, and colistin for isolates resistant to carbapenems was determined by using E-test (Liofilchem, Italy) according to the 2021 CLSI guidelines [9 ].

Antimicrobial susceptibility was tested by employing disk diffusion, gradient test, and broth microdilution test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations, 2021 [43 ]. The disks impregnated with the following antimicrobial agents were tested: imipenem (10 μg), meropenem (10 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), ceftazidime (10 µg), cefepime (30 µg), amikacin (30 µg), piperacillin/tazobactam (30/6 µg), aztreonam (30 µg), ticarcillin (75 µg), ceftazidime-avibactam (10–4 µg), and ceftolozane/tazobactam (38–10 µg) (BioRad, Watford, UK). Minimum inhibitory concentrations (MICs) for colistin, imipenem, and meropenem were evaluated by ComASP Colistin (Liofilchem, Roseto, Italy) and Gradient strip test (Liofilchem, Roseto, Italy), respectively. P. aeruginosa ATCC 27853 was used as the control strain in the antibiotic susceptibility testing. MDR P. aeruginosa were defined as isolates that tested resistant to at least one antimicrobial agent of three or more different classes. Extensively drug-resistant (XDR) P. aeruginosa were defined as a subset of MDR isolates that tested non-susceptible to at least one antimicrobial agent of five different classes. P. aeruginosa was defined as pandrug-resistant (PDR) when the organism was resistant to all tested agents.

A total of 18 antibiotics were used in the susceptibility test, by disk diffusion, with amoxicillin, amoxicillin and clavulanic acid, cefoxitin, cefotaxime, ceftazidime, ceftiofur, cefepime, ceftaroline, aztreonam, meropenem, ciprofloxacin, gentamicin, sulfamethoxazole-trimethoprim, tigecycline, tetracycline, fosfomycin, chloramphenicol, and nitrofurantoin (Liofilchem, Roseto degli Abruzzi, Italy). The interpretation was performed according to CLSI [45 ]. tigecycline resistance was interpreted according to EUCAST [46 ]. E. coli ATCC 25922 was used as quality control. In accordance with EUCAST standards, the minimal inhibitory concentration (MIC) for colistin was determined by the broth microdilution method [46 ]. MDR profiles were determined according to standard criteria [26 (link)]. ESBL detection was performed using the double-disk synergy test (DDST) [45 ].

Enterobacterales isolates carrying bla_OXA-48-like genes were obtained between 2013 and 2020 from clinical samples obtained at the University Hospital Cologne. They were detected during routine diagnostics due to elevated carbapenem MICs, and bla_OXA-48 was verified via the automated PCR system Xpert Carba-R (Cepheid, Sunnyvale, CA, USA) (18 (link)). Only one isolate per species for each patient was included in the study. Antibiotic susceptibility was determined by the Micronaut-S broth microdilution panel (Merlin, Bornheim, Germany). Due to a small MIC range for carbapenems in this panel, susceptibility testing was complemented by gradient tests for ertapenem, meropenem, and imipenem (Liofilchem, Roseto degli Abruzzi, Italy).

Eleven carbapenem resistance genes (bla_IMP, bla_VIM, bla_NDM, bla_SPM, bla_AIM, bla_DIM, bla_GIM, bla_SIM, bla_KPC, bla_BIC, and bla_OXA–48) (Poirel et al., 2011 (link)) were screened using PCR detection from the presumptive carbapenemase-producing bacteria. The amplicons were sequenced (Macrogen) and identified using NCBI BLAST¹. For the screened carbapenemase-producing bacteria, MDR to 16 antibiotics was determined using Kirby-Bauer disk diffusion, and resistance to colistin was determined using broth dilution methods. For MDR, the following antibiotic disks were used: ampicillin-sulbactam (10/10 μg), cefotaxime (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg) ciprofloxacin (5 μg), colistin (2 mg/L), doripenem (10 μg), fosfomycin (200 μg), gentamicin (10 μg), levofloxacin (5 μg), meropenem (10 μg), netilmicin (10 μg), piperacillin (100 μg), tetracycline (30 μg), tobramycin (10 μg), and trimethoprim-sulfamethoxazole (1.25/23.75 μg) (Liofilchem, Roseto degli Abruzzi, Italy). Resistance to the antibiotics was determined according to the Clinical and Laboratory Standards Institute (CLSI) guideline (Clinical Laboratory Standars and Institue, 2016 ). Subsequently, MICs of 16 antibiotics for E. coli strain N7 were evaluated using the broth dilution method (Hasselmann, 2003 (link)).

The pellet was dissolved in sterile physiological saline (Polpharma) to obtain a suspension with an optical density of 0.5 McFarland. The prepared suspension was plated on Mueller Hinton Agar with 5.0% horse blood and β-NAD (MHF, Graso) and then gradient strips with antibiotics, i.e., penicillin (0.016-256 μg/ml) (Liofilchem), ampicillin (0.016-256 μg/ml) (Liofilchem), meropenem (0.002-32 μg/ml) (Liofilchem), erythromycin (0.016-256 μg/ml) (Liofilchem) and trimethoprim*/sulfamethoxazole (1/19) (co-trimoxazole) (0.002-32* μg/ml) (Liofilchem) were applied. After 20-h incubation at 35 °C MICs (based on the eclipse-shaped inhibition zone) were determined. The results were interpreted in accordance with EUCAST v. 13.0 recommendations [33 ].