Imipenem

Clinical CRK isolates (SCHP191, SCHP192) were obtained from Soonchunhyang University Hospital Pathogenic Resource Bank and cultured in MacConkey broth (BD Difco, USA) at 37°C under aerobic conditions for 24 h. The cultures were streaked onto MacConkey agar (BD Difco) plates containing 10 mg/ml imipenem (Sigma-Aldrich, USA) and incubated at 30°C for 18 h. The isolated colonies were transferred to MacConkey broth and cultured at 30°C for 18 h. The bacterial culture (OD₆₀₀ = 1.0, 2 × 10⁹ CFU/ml) was stocked in sterile glycerol and kept at -80°C. Antimicrobial susceptibilities were determined using the standard broth dilution method according to the CLSI guideline. Minimum inhibitory concentrations (MICs) were >1,025 mg/l for imipenem (Sigma-Aldrich), 1,025-512.5 mg/l for vancomycin (Sigma-Aldrich), 1,025-512.5 mg/l for kanamycin (Sigma-Aldrich), and >1,025 mg/l for metronidazole (Sigma-Aldrich). In the CRK mice infection experiment, CRK (SCHP191) was used in models 1,2, and 4, and CRK (SCHP191) and CRK (SCHP192) were used in model 3.

A. baumannii included in the study were isolated from the blood of septicemic neonates admitted to the NICU of IPGMER and SSKM hospital, Kolkata, India, during 2007–2015. Strains were identified initially by the Vitek 2 compact system (bioMérieux, Marcy l’Etoile, France). Further confirmation was done by detection of bla_OXA-51. MLST was carried out using specific primers of 7 housekeeping genes (cnp60, fusA, gltA, pyrG, recA, rplB, and rpoB) and conditions described in the A. baumannii MLST Pasteur Scheme (https://pubmlst.org/abaumannii/). The allele numbers are combined to yield a specific ST using the pubMLST database.
The MIC values (mg/L) of two carbapenems (meropenem and imipenem) (Sigma-Aldrich, St. Louis, MO, USA) were determined using broth microdilution method and interpreted according to the CLSI (Clinical and Laboratory Standards Institute) breakpoints for Acinetobacter spp. (meropenem and imipenem: susceptible ≤2 mg/L; resistant ≥8 mg/L) (54 ). The MIC₉₀ values for carbapenems were also calculated. To assess whether carbapenem resistance is reversible with an efflux pump inhibitor, MIC of meropenem and imipenem was determined with and without EPI PAβN (Sigma) at a concentration of 50 mg/L. A significant inhibition was defined as a 4-fold or greater reduction of MIC in the presence of PAβN (55 (link)).

We initially treated four different groups of mice (4–5 per group) separately with either metronidazole (5 mg/mL) or a combination of metronidazole (5 mg/mL) + clindamycin (10 mg/mL), metronidazole (5 mg/mL) + imipenem (5 mg/mL), or metronidazole (5 mg/mL) + imipenem (5 mg/mL) + clindamycin (10 mg/mL) (Sigma Aldrich, USA) (Figure 1A). All mice received a daily dose of 200 µL of antibiotic preparations via the oral gavage for four weeks. The metronidazole (5 mg/mL) + clindamycin (10 mg/mL) treatment was repeated in two additional independent experiments using different sets of mice (Figure 1B,C). Fresh antibiotics were prepared at every 3-day interval.

Overnight cultures were grown in Muller Hinton broth before subculturing (1:100) and growing for 3 hours at 37 °C. Cultures were diluted (1:10) and used to inoculate 96 well microtiter plates containing 2-fold serial dilutions of; ceftazidime, co-trimoxazole, doxycycline, meropenem, imipenem or carbenicillin (Sigma Aldrich) with a final inoculum of 10⁵ bacteria per well (confirmed by CFU counts). Plates were sealed and statically incubated for 24 hours at 37 °C before the MIC results were recorded. The MIC was the lowest concentration of antibiotic for which there was no visible bacterial growth. Experiments were repeated 3 times.