Nalidixic acid

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).

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 ].

Antibiotic susceptibility was determined by disc diffusion on Mueller–Hinton agar, in accordance with the guidelines of the Antibiogram Committee of the French Society for Microbiology (CA-SFM & EUCAST, 2014 ). The following 32 antimicrobial drugs (Bio-Rad) were tested: ampicillin, ticarcillin, piperacillin, piperacillin/tazobactam, cefamandole, cefoperazone, cefoxitin, cefotaxime, amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, imipenem, meropenem, ertapenem, cefepime, ceftazidime, streptomycin, spectinomycin, kanamycin, amikacin, gentamicin, netilmicin, tigecycline, isepamicin, nalidixic acid, pefloxacin, ciprofloxacin, sulfonamides, trimethoprim, sulfamethoxazole/trimethoprim, chloramphenicol, tetracycline and azithromycin. Minimal inhibitory concentration (MIC) values for nalidixic acid, ciprofloxacin and azithromycin were determined by Etests (bioMérieux). E. coli CIP 76.24 (ATCC 25922) was used as a control.

The isolated colonies were further analyzed for antibiotic susceptibility to chloramphenicol, amikacin, erythromycin, tetracycline, ciprofloxacin, nalidixic acid, and enrofloxacin (Sigma-Aldrich, St Louis, MO, USA), according to the guidelines of the Clinical & Laboratory Standards Institute [29 ]. To determine antibiotic resistance, the breakpoints suggested by CLSI [29 ], CDC [30 ], Hong et al. [31 (link)], and Kang et al. [32 (link)] were used as follows: chloramphenicol at 32 μg/mL, amikacin at 64 μg/mL, erythromycin at 32 μg/mL, tetracycline at 16 μg/mL, ciprofloxacin at 4 μg/mL, nalidixic acid at 64 μg/mL, and enrofloxacin at 4 μg/mL. The Campylobacter isolates on Colombia agar (bioMérieux, Marcy-l’Étoile, France) were suspended in Mueller-Hinton broth (MHB; Becton, Dickinson and Company, Sparks, MD, USA) to obtain a McFarland 0.5 standard, and further diluted 10-fold. Using needles, Campylobacter isolates were spotted on Mueller-Hinton agar (MHA; Becton, Dickinson and Company, Sparks, MD, USA) with 5% lysed horse blood plates (Oxoid Ltd., Basingstoke, UK), formulated at 0.5–128 μg/mL with seven antibiotics. The plates were incubated under microaerobic conditions at 37 °C for 48 h. MIC was determined by colony formation on the plates and the reference strain used was Campylobacter jejuni ATCC33560.

From the soil extract obtained in saline solution (NaCl 0.45%), it was verified that the density of viable microorganisms was >10⁸ ufc mL⁻¹ (optical density [OD] = 0.5 McFarland). The bacterial extraction was sown in Mueller–Hinton agar (Condalab^®, Madrid, Spain), and the minimum inhibitory concentration (MIC) was evaluated by the Kirby–Bauer method, using ε-test antibiotic strips, in triplicate, for the following antibiotics: cefuroxime, cefuroxime axetil, cefoxitin, cefotaxime, ceftazidime, cefepime, ertapenem, imipenem, amikacin, gentamicin, nalidixic acid, ciprofloxacin, tigecycline and trimethoprim/sulfamethoxazole (BioMérieux^®, Marcy l’Etoile, France). Plates were then incubated according to the manufacturer’s instructions. For the quantification of the MIC, the most restrictive halo was used as reference.