Ertapenem

Lyophilised meropenem (Ranbaxy, Haryana, India), imipenem, ertapenem and meropenem (Sigma-Aldrich, Missouri, USA) antibiotics were dissolved in sterile 0.85% saline to produce 5120 mg/L stock solutions, syringe-filtered at 0.1 µm and stored below −20 °C. FAST meropenem working stocks were made by serial 1:2 dilutions in filtered sterile Mueller-Hinton broth (MHB) to produce 1 mL aliquots ranging from 2560 mg/L to 2.5 mg/L.

Mtb H37Rv, Msm mc²-155 and Msm PM965 (ept-1 rpsL4 ΔblaS1), a mutant strain deficient for the major beta-lactamase BlaS (Raymond et al., 2005 (link)), were used in this study. Bacteria were grown in Middlebrook 7H9 medium (BD Biosciences) or in Middlebrook 7H10 medium (BD Biosciences), supplemented with 0.2% or 0.5% of glycerol, respectively. Both media were supplemented with 10% oleic acid-albumin-dextrose-catalase (BD Difco) for Mtb and 0.5% glucose for Msm. Tyloxapol (Sigma-Aldrich) was added to liquid medium to a final concentration of 0.05% to prevent clump formation. Stocks of amoxicillin (AMX), biapenem (BIA), cefotaxime (CTX), doripenem (DOR), ertapenem (ETP), ethambutol (EMB), meropenem (MEM) and vancomycin (VAN) (Sigma-Aldrich) were prepared in purified water. Potassium clavulanate (CLA) (Sigma-Aldrich) was prepared in phosphate buffer pH 6.0, 0.1 M. Isoniazid (INH) and rifampicin (RIF) (Sigma-Aldrich) were prepared in dimethyl sulfoxide or methanol, respectively.

Conjugation experiments were performed for CR-hvKP strains by a solid mating assay at 37°C, using the Escherichia coli J53 (azide-resistant) recipient (39 (link)). Transconjugants (Tc) were selected on LB agar plates supplemented with 1 mg/L ertapenem and 100 mg/L sodium azide (Sigma-Aldrich, St. Louis, USA). Putative transconjugants were screened for AMR determinants (including bla_NDM), replicon types using PCR-based replicon typing (PBRT)-kit (Diatheva, Italy), and pLVPK-associated markers.

The antimicrobial susceptibility testing was conducted using the broth microdilution test, involving meropenem (Sigma-Aldrich, Darmstadt, Germany), ertapenem (Sigma-Aldrich, Darmstadt, Germany), and imipenem (Sigma-Aldrich, Darmstadt, Germany) antibiotics, in accordance with the guidelines established by the Clinical and Laboratory Standards Institute (CLSI) [62 ]. Briefly, 100 µL of cation-adjusted Mueller–Hinton (M-H) broth 2 (Merck Millipore, Darmstadt, Germany) was added to the 96-well polystyrene microtiter sterile plate. Serially diluted concentrations of meropenem, imipenem, or ertapenem ranging from 0.25 to 128 µg/mL were established. In each plate, wells without antibiotics were included as positive controls of growth, and wells without inocula and antibiotics were used as negative control. The bacterial suspension at 0.5 McFarland (1 × 10⁸ CFU/mL) was diluted 1:20 to achieve 5 × 10⁶ CFU/mL, and 5 µL was added to the plate wells as an inoculum. The plates were placed in a plastic bag and incubated at 37 °C for 18 h. Minimum inhibitory concentrations (MICs) were expressed in µg/mL using an inverted mirror to display the visual growth. The MICs were expressed in µg/mL using an inverted mirror to display the visual growth. The interpretation of the results was also in accordance to recommendations provided by the CLSI [63 ].

The Minimal Inhibitory Concentrations (MICs) for ampicillin/sulbactam, ceftriaxone, ceftazidime, cefepime, aztreonam, ertapenem, imipenem, meropenem, ciprofloxacin, levofloxacin, amikacin, gentamicin, and minocycline (Sigma, Saint Louis, MO, USA) were determined by agar dilution, except for polymyxins and tigecycline, where the cation-adjusted broth microdilution method was performed [28 (link)]. The results were interpreted according to the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST/EUCAST) guidelines using the breakpoint Table version 10.0, published in may 20, 2020 (http://brcast.org.br/ (accessed on 1 January 2021)). E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as control strains.

The Minimum Inhibitory Concentrations (MIC) of recombinant BL21(DE3) cells harboring bla_KPC-2 gene were determined by micro dilution method for different carbapenems alone and in combination with inhibitors (clavulanic acid, sulbactam, tazobactam, ZINC01807204 and ZINC02318494) against BL21 control cells (cells with null vector). The recombinant E. coli cells were induced with IPTG prior to performing experiments. The antimicrobial agents, imipenem, meropenem, ertapenem, clavulanic acid, sulbactam and tazobactam were obtained from Sigma Aldrich (USA) and compounds ZINC01807204 and ZINC02318494 were procured from Vitas M (Netherlands). The inhibitors were taken at a fixed concentration of 4 µg/ml. The results were interpreted according to the guidelines laid by Clinical Laboratory Standards Institute (CLSI) [12] . The MIC was determined as the lowest concentration that totally inhibits visible bacterial growth.