Trimethoprim

Conjugation experiments were carried out with E. coli NalR as the recipient. The recipients were selected on Luria-Bertani (LB) medium containing streptomycin (25μg/ml), nalidixic acid (50μg/ml), trimethoprim (5μg/ml) and ampicillin (100μg/ml) and the donors were selected on LB containing only streptomycin and trimethoprim (BioMérieux, France). A total of 5 ml of fresh LB broth was inoculated with either recipient or donor bacteria and incubated for 24 h at 37°C. After overnight incubation, cultures were diluted at 1:50 (400μl in 20ml of LBB) and incubated at 37°C with strong agitation for 4 hours until they reached the logarithmic growth phase (OD 600nm = 0.6–0.8). Donor and recipient strains were then mixed at the following ratios 1:1; 1:2; 1:10 and incubated at 37°C for 3 hours. Transconjugants were selected on LB agar plates supplemented with streptomycin (25μg/ml), nalidixic acid (50μg/ml), trimethoprim (5μg/ml) and ampicillin (100μg/ml). PCR was performed on transconjugants to determine whether resistance genes had been transferred.

MICs were determined by the Etest method according to the manufacturer’s instructions. Antimicrobial agents included aztreonam, ceftazidime, ciprofloxacin, colistin, gentamicin, imipenem, nalidixic acid, tetracycline, tigecycline, tobramycin, and trimethoprim (bioMe’rieux, Marcy-l’_Etoile, France). E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. Interpretation of antimicrobial susceptibility was based on guidelines of the Clinical Laboratory Standards Institute (CLSI) [49 ].

Oxi/Ferm Pluri Test^® (Liofilchem, Italy) was used. The automatic characterization was then carried out using the VITEK^®-2 GN identification cards (bioMérieux, Marcy-l’Étoile, France). The motility of the bacterium was tested in Motility Test Agar (Liofilchem, Italy). Antimicrobial sensitivity was determined using E-test in Müller Hinton agar (Pronadisa^® Madrid, Spain) using different antibiotics for each strain. For SAICEU11^T strain we used cefepime and sulfamethoxazole and trimethoprim (bioMérieux, Marcy-l’Étoile, France); amoxicillin, amoxicillin-clavulanic acid, cefotaxime, cefpirome ciprofloxacin and nalidixic acid (Liofilchem, Italy). For SAICEU22^T strain we used piperacillin and piperacillin with tazobactam, cefepime (bioMérieux, Marcy-l’Étoile, France); ceftazidime, imipenem, imipenem with EDTA, amikacin, gentamicin, and ciprofloxacin (Liofilchem, Italy).
Mercury MBC was performed in Müller Hinton agar (Pronadisa^®, Madrid, Spain), supplemented with different concentrations of HgCl₂: 400, 350, 200, 175, 150, 100, 87.5, 75, 50, 43.75, and 25 μg mL⁻¹. MBC was determined as the lowest concentration of HgCl₂ capable of inhibiting > 99.9% of bacterial growth.