Amoxicillin

The antibiotics susceptibility pattern of the isolates was determined using the disk diffusion method on Mueller-Hinton agar (Oxoid, England). Inhibition zone diameter values were interpreted as recommended by the Antibiogram Committee of the French Society of Microbiology [11 ]. The 13 antibiotics (BioMérieux, France) used in this study are amoxicillin/clavulanic acid (20/10 μg), cefotaxime (30 μg), ceftriaxone (30 μg), amoxicillin (30 μg), imipenem (10 μg), gentamicin (10 μg), tobramycin (10 μg), amikacin (30 μg), kanamycin (30 μg), nalidixic acid (30 μg), ofloxacin (5 μg), ciprofloxacin (5 μg), and trimethoprim/sulfamethoxazole (1.25/23.75 μg).

The Kirby–Bauer technique was used (Bauer et al., 1966 (link)). Briefly, LB agar plates were spread with a suspension of approximately 10⁸ CFU/ml of wild-type P. putida DOT-T1E or DOT-T1E-18 mutant strain to produce a lawn. Once the plate surface was dried, antibiotic disks of ofloxacin (5 μg), pefloxacin (5 μg), amoxicillin (25 μg), ticarcillin (75 μg), ampicillin (10 μg), ceftazidime (30 μg), chloramphenicol (30 μg), erythromycin (15 μg) and tetracycline (30 μg) (BioMerieux, Spain) were placed on the surface of plates. After 18–20 h at 30°C, the inhibition zone was measured around each disc.

The antibiotic resistance profile of the strain was evaluated using the E‐test method and the following molecules: benzylpenecilin, amoxicillin, cefotaxime, ceftriaxone, imipenem, rifampicin, minocycline, tigecycline, amikacin, teicoplanin, vancomycin, colistin, daptomycin, and metronidazole (Biomerieux, France).

Microbial identification was confirmed by Bruker Biotyper MALDI-TOF MS (Bruker, Billerica, MA). For MRSA designation, a PBP2a SA culture colony test (Alere) was performed according to the manufacturer’s instructions using 18- to 24-h subculture growth. The presence of mecA and the homolog mecC were determined by in-house PCRs (22 (link), 23 (link)). Susceptibility testing included that for cefoxitin by disk diffusion and for oxacillin by disk diffusion and gradient diffusion. Methods followed the procedural guidelines outlined by the Clinical and Laboratory Standards Institute (documents M02 and M23) (55 , 56 ). Disk diffusion testing of cefoxitin (Hardy Diagnostics) and oxacillin (BD) was performed on conventional Mueller-Hinton agar (MHA) (Hardy Diagnostics). Gradient diffusion testing of oxacillin (bioMérieux) was performed on MHA with 2% NaCl agar (Remel). Detection of beta-lactamase production was assessed by the disk diffusion penicillin zone edge test (Hardy Diagnostics) and nitrocefin-based Cefinase disk test (Hardy Diagnostics). Beta-lactamase inhibitor rescue phenotype was determined by assessing the fold change in MIC from amoxicillin (bioMérieux) and amoxicillin-clavulanic acid (bioMérieux) gradient diffusion testing.