Broth microdilution assay

The minimum inhibitory concentration (MIC) of ATM, CIP, CZA, and C/T were determined through broth microdilution (BMD) assay following CLSI M100 guidelines (CLSI, 2020). Cation-adjusted Mueller-Hinton broth (MHB-II) (BBL^TM BD, Sparks, MD) and 96-well round-bottom plates were used (Thermo Scientific™). All antimicrobial compounds were obtained from Merck, except CAZ and PAβN, which were obtained from Sigma-Aldrich, and avibactam, which was donated by Pfizer, Inc. Concentrations spanned the doubling dilution range of 0.125 μg/ml to 64 μg/ml for ceftazidime and ceftolozane, whereas the concentration of avibactam and tazobactam were kept at 4 μg/ml. The double dilution range analyzed for ATM and CIP concentrations were 0.25–128 μg/ml and 0.008–4 μg/ml, respectively. MIC values were determined after incubation for 18–20 h at 37°C, and three or more biological replicates were performed. As a growth control, strains were inoculated in wells containing MHB-II or MHB-II + PAβN without CZA and C/T. As sterile control, bacteria were not inoculated in wells containing MHB-II or MHB-II + PAβN without CZA and C/T. QC tests were performed using the reference strain P. aeruginosa ATCC 27853, and all QC were within the CLSI acceptable range. The phenotypes “susceptible”, “intermediate” and “resistant” were defined for CZA and C/T based on CLSI breakpoints (CLSI, 2020). In the case of C/T, the “non-susceptible” group included the “intermediate” and “resistant” categories. To evaluate the effect of RND efflux pump inhibition on ATM, CIP, CZA, and C/T susceptibility, BMD was performed as described above, but in the presence of 25 μg/ml of PAβN (Askoura et al., 2011; Blanco et al., 2018). It was considered that the RND efflux activity contributed to the baseline susceptibility to CZA and/or C/T when a two-fold (named “RND 2x”) or more (named “RND ≥4x”) decrease in MIC values were observed in the presence of PAβN (overall named “RND” isolates). On the contrary, isolates with equal or higher CZA and/or C/T MIC values in the presence of PAβN were classified as “Non-RND”. Four of the 161 Non-CP-CRPA clinical isolates were excluded due to the lack of growth observed in the presence of PAβN.