Cefotaxime

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).

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.

Using Cefoxitin-cloxacillin double disk synergy test (13 (link)), and AmpC induction tests (14 (link)). Klebsiella pneumoniae strain (M40) was used as the positive control strain for all the tests. Briefly, the Cefoxitin-cloxacillin double disk synergy test was performed on all the potentially AmpC producing isolates. These were grown on Mueller Hinton agar (Oxoid, USA). Cefoxitin/cloxacillin disks (30 μg) and Cefoxitin disk (30 μg) were used to determine the inhibition zone difference. A minimum diameter of 4 mm of inhibition of Cefoxitin/cloxacillin over Cefoxitin alone was considered a confirmation of AmpC production (13 (link)).
In this study, all patients admitted were screened for the fecal carriage of AmpC. Preliminary screening was done by inoculating stools on MacConckey agar supplemented with 2 μg/ml of Cefotaxime (bioMérieux, La Balme-les-Grottes, France). Suspect colonies growing after 24–48 h of incubation were subject to phenotypic and genotypic identification of pAmpC. We defined “Infected” any patient who had an infection in any part of the body that grew an AmpC producer regardless if an AmpC producer was isolated from the stools of this patient. We defined “Carrier” any patient from whom an AmpC producer was isolated (stools screen) without a documentation of infection in this patient.

Antimicrobial susceptibility testing was done by the Kirby-Bauer standard disk diffusion method [11] according to CLSI guidelines [12] for different antimicrobial agents like: ceftazidime (30 µg), cefotaxime (30 µg), cefpodoxime (10 µg), ceftriaxone (30 µg), cefepime (30 µg), aztreonam (30 µg), ampicillin (10 µg), piperacillin (100 µg), cefoxitin (30 µg), gentamicin (120 µg), amikacin (30 µg), ciprofloxacin (5 µg), tetracycline (30 µg), minocycline (30 µg), chloramphenicol (30 µg), trimethoprim/sulfamethoxazole (1.25 µg/23.75 µg), colistin (10 µg), ertapenem (10 µg) and meropenem (10 µg) (BD Diagnostics, Franklin Lakes, NJ, USA).
The MIC values (mg/L) of cefotaxime, ertapenem, meropenem, amikacin, gentamicin and tigecycline were determined using Etest method (AB Biodisk, Solna, Sweden) and were interpreted according to CLSI guidelines as modified in 2013. The clinical breakpoints for meropenem were as follows: susceptible (S) ≤1.0 mg/L, intermediate (I) 2.0–3.0 mg/L, and resistant (R) ≥4.0 mg/L. The same for ertapenem were as follows: S ≤0.5 mg/L, I: 1.0 mg/L, R ≥2 mg/L. MIC50 and MIC90 of meropenem were calculated as the MIC at which 50% and 90% of the isolates were inhibited.