Meropenem

Because the disk diffusion method allows to analysis of the impact of a single concentration of an antibiotic/disk, in this experiment E-test strips with exponentially decreasing antibiotic gradient were applied. In this investigation line, the antibiotics were selected whose application, coupled with exposure to RMF, caused the largest and the smallest differences in the zones of growth inhibition in the disc diffusion method (compared to unexposed controls). The E-tests containing cefoxitin, amoxicillin, imipenem, ceftriaxone, cefuroxime, ceftazidime and cefepime were obtained from Liofilchem (Roseto degli Abruzzi, Italy), whereas the E-test containing meropenem was purchased from BioMérieux (Craponne, France).
Similarly, as in the case of the disc diffusion method, the optimal RMF frequency and exposure time, as well as unexposed controls were used. The analyses were performed on M-H agar in accordance with the E-test manufacturer’s recommendations.

Antimicrobial susceptibilities were determined at the time of isolation by the modified Bauer–Kirby disc diffusion method, as recommended by the CLSI guidelines (CLSI, 2012 ). The antimicrobials tested were piperacillin/tazobactam (100/10 μg), imipenem (10 μg), meropenem (10 μg), amikacin (30 μg), ceftriaxone (30 μg), ceftazidime (30 μg), ticarcillin/clavulanic acid (75/10 μg), cefepime (30 μg), levofloxacin (5 μg) and co-trimoxazole (1.25/23.75 μg). Mueller–Hinton agar and antimicrobial discs were purchased from Oxoid. For colistin, meropenem, imipenem, ceftazidime and ceftriaxone, MICs were determined by E-test based on the manufacturer's recommendations (bioMérieux). The results were interpreted as resistant or sensitive according to current CLSI guidelines (CLSI, 2012 ). Escherichia coli ATCC 25922 was used as the control for these assays. For isolates that had an MIC above the range of the E-test, MICs were determined by the broth dilution method (Wiegand et al., 2008 (link)). Briefly, 5 × 10⁵ c.f.u. (ml bacteria)^− 1 were inoculated into a series of Mueller–Hinton broths containing two-fold dilutions of the antimicrobial agent. Following inoculation, the broth was incubated at 37 °C for 18–24 h. The MIC was defined as the lowest concentration of antimicrobial that inhibited the growth of bacteria.

The clinical samples reviewed in this study were blood cultues, general cultues and rectal swabs for screening received by the Clinical Microbiology Laboratory in The Chaim Sheba Medical Center in Israel during 2013. General cultures include synovial, pericardial, pleural and peritoneal fluids, peritoneal dialysate, brain abscess, lung biopsy/tissue, pericardial fluid/tissue, heart valve, bone, joint, internal organs tissues, biopsies, heart valves, electrodes, catheters and wounds. All cultures suspected to be CRE were tested by Modified Hodge test, MBL E-test and by E-tests for imipenem, meropenem and ertapenem (bioMérieux, France) following CLSI guidelines [30 ].

Clinical BFG isolates were evaluated by gradient diffusion method for susceptibility to antimicrobial agents piperacillin-tazobactam (LiofilChem), ertapenem, meropenem, and metronidazole (bioMérieux). BFG isolates cultured on PRAS Brucella blood agar for 24 to 28 h were used to make 1 McFarland standard organism suspension in Brucella broth (BD BBL™) which was then lawn-struck onto PRAS Brucella blood agar plates. Gradient diffusion strips were placed on inoculated plates and incubated at 35°C in EZ Anaerobe Gas-pack systems (BD) for up to 72 h. MICs were read at 72h. Species-specific differences in MIC were investigated using Tukey’s multiple-comparison test with ordinary one-way ANOVA (GraphPad Prism v9.0.0) to detect significant differences between the geometric mean of each species group (defined as an adjusted P value <0.05).

The antimicrobial susceptibility testing of K. pneumoniae was examined using Vitex II cards (AST-N292) (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s guidelines. The following antimicrobial agents were tested: amikacin (AK), ampicillin (AMP), amoxicillin/clavulanate (AMC), aztreonam (ATM), ceftriaxone (CRO), cefepime (CFPM), cefuroxime (CXM), ciprofloxacin (CIP), trimethoprim/sulfamethoxazole (SXT), colistin (CST), gentamicin (GN), imipenem (IPM), meropenem (MEM), piperacillin/tazobactam (TZP), and tigecycline (TGC) (bioMérieux, Marcy l’Etoile, France). Clinical and Laboratory Standards Institute (CLSI) recommendations were used to determine the minimum inhibitory concentration (MIC) and breakpoints [13 ]. MDR K. pneumoniae was defined as non-susceptibility to at least one agent in three or more antimicrobial categories [14 (link)]. K pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as quality control strains. The isolates were immediately stored in brain heart infusion broth containing 20% glycerol at −86 °C for further genetic analysis.