Gentamicin

The antibiotic susceptibility of the all isolates was determined by the disk diffusion method on Mueller–Hinton agar (Becton–Dickinson, Sparks, MD, USA) according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). The disk diffusion assay was run with 15 antibiotics: bacitracin (10 U), enrofloxacin (5 μg) (OXOID), streptomycin (10 μg), spiramycin (100 μg), sulfadiazine (25 μg), chloramphenicol (30 μg) (BD), doxycycline (30 μg), erythromycin (15 μg), gentamicin (10 μg), neomycin (30 μg), penicillin (10 U), tetracycline (30 μg), cefoxitin (30 μg), trimethoprim/sulfamethoxazole (1.25 μg/23.75 μg, respectively), and vancomycin (30 µg and 5 µg) (BIO-RAD, Hercules, CA, USA). S. aureus strain ATCC 25923 and Enterococcus faecalis strain ATCC 29212 were used as controls in the susceptibility test.

Susceptibility to 13 antibiotics was determined by the disc diffusion method and using the ADAGIO™ Automated System (Bio-Rad, Hercules, CA, USA) as described. The antibiotics tested included ampicillin (10 µg), penicillin (6 µg), ampicillin/sulbactam (20 µg), chloramphenicol (30 µg), vancomycin (5 µg), teicoplanin (30 µg), streptomycin (300 µg), gentamicin (120 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), quinupristin-dalfopristin (15 µg), linezolid (30 µg) and tigecycline (15 µg) (Bio-Rad, Hercules, CA, USA)]. Susceptibility to aminoglycosides, glycopeptides, quinolones and β-lactam antibiotics was also determined by an E-test (M.I.C. Evaluator™, OXOID, Basingstoke, UK). The methods and the interpretation of the results followed the CLSI guidelines [32 ]. Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 were used as control strains.

Isolation of E. coli was performed as follows: liquid aspirates were diluted approximately ×10⁶ fold with sterile PBS (phosphate saline buffer). Approximately 0.05 ml volume of feces were placed into 0.5 ml of sterile PBS, vortexed to homogeneity, an aliquot was diluted approximately ×10⁶ fold. Biopsy samples were vortexed in 0.2 ml of sterile PBS. For all samples, 0.1 ml of the resulting liquid was spread onto the Luria-Bertani agar plates. After overnight incubation on 37 °C, isolated colonies were identified with the Matrix Assisted Laser Desorbtion/Ionization (MALDI) Biotyper software (Bruker Daltonics, Germany) using the Microflex LT mass spectrometer (Bruker Daltonics, Germany). For DNA extraction, all E. coli strains were grown in the Luria-Bertani broth at 37 °C with shaking (200 RPM) overnight and collected by centrifugation. Samples and corresponding E. coli isolates are listed in Table 1.
The testing of susceptibility to ampicillin/sulbactam, ceftriaxone, cefotaxime, ceftazidime, cefepime, imipenem, meropenem, gentamicin, levofloxacin, and ciprofloxacin (all from Bio-Rad, USA) was performed by the disc-diffusion method using the Mueller-Hinton agar plates. The E. coli strain ATCC 25922 was used as a control. Current CLSI and EUCAST criteria were used for interpretation.

The antibiotic susceptibilities of CTX-R E. coli and their transconjugants were determined by disk diffusion method according to CLSI protocols (19 ). The following antibiotic disks were used: ampicillin (10 μg), amoxicillin (20 μg) plus clavulanic acid (10 μg), cefoxitin (30 μg), ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30 μg), aztreonam (30 μg), imipenem (10 μg), streptomycin (10 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), tetracycline (30 μg), chloramphenicol (30 μg), trimethoprim (5 μg), and sulfonamides (300 μg) (Bio-Rad, Marnes-la-Coquette, France if available or Oxoid, Dardilly, France). The susceptibility breakpoints for all antimicrobials were those recommended by CLSI (20 ).
ESBL production was detected by double-disk synergy test on Mueller-Hinton agar between clavulanic acid and ceftazidime, cefotaxime, or cefepime (20 , 21 (link)). AmpC beta-lactamases detection was based on the inhibitory effect of cloxacillin on AmpC production observed on plates supplemented with 200 mg/L cloxacillin. The control strains used were E. coli ATCC 25922, K. pneumoniae ATCC 700603, and K. pneumoniae CMY-2 from Pr R. Bonnet, France.

Antimicrobial susceptibility testing was performed on all isolates recovered from the two selective media using the disc diffusion method on Mueller–Hinton (MH) agar plates (Neogen, Lansing, Michigan) for ticarcillin (75 μg), amoxicillin/clavulanic acid (20–10 μg), cefotaxime (30 μg), ceftazidime (10 μg), temocillin (30 μg), cefoxitin (30 μg), ertapenem (10 μg), imipenem (10 μg), meropenem (10 μg), ceftazidime/avibactam (10–4 μg), aztreonam (30 μg), ciprofloxacin (5 μg), trimethoprim-sulfamethoxazole (SXT) (1.25–23.75 μg), tetracycline (30 μg), amikacin (30 μg), gentamicin (15 μg), and tobramycin (10 μg) (Bio-Rad Laboratories, Algés, Portugal), following EUCAST recommendations and breakpoint tables. Susceptibility to fosfomycin was evaluated by the disk diffusion method (50 μg) on MH agar plates supplemented with 25 μg/mL glucose-6-phosphate, according to EUCAST guidelines [24 ]. Strain E. coli ATCC 25922 was used for quality control. Multidrug resistance was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories [25 (link)].

Antibiotic susceptibility was performed by the disc diffusion method on Mueller Hinton agar (Bio-Rad, Marne la Coquette, France) according to the recommendations of the Antibiogram Committee of the French Society for Microbiology (Comité de l’Antibiogramme de la Société Française de Microbiologie) [9 ]. The following antibiotic discs (drug concentration in μg) were tested: amoxicillin (25 μg), amoxicillin/clavulanic acid (20/10 μg), ampicillin (10 μg), imipenem (10 μg), cefotaxime (30 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg), norfloxacin (5 μg), amikacin (30 μg), gentamicin (15 μg) and trimethoprim/ sulfamethoxazole (1.25/23.75 μg), all from Bio-Rad (Bio-Rad, Marne la Coquette, France).
ESBL phenotypes were detected by double-disk synergy according to the method described by Jarlier et al. [10 (link)]. Disks of cefotaxime and ceftriaxone were placed 20 mm from an amoxicillin/clavulanate disk. Enhancement of the inhibition zone of the third-generation cephalosporin toward the amoxicillin/clavulanate disk indicated the possible presence of an ESBL. Escherichia coli ATCC 25922 was used as control.