Amoxicillin

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.

All GNB isolates which were non-naturally colistin-resistant and grown on the LBJMR medium were subjected to an AST according to the current (DD) test method (Kirby-Bauer procedure). The minimal inhibition concentration (MIC) was confirmed by CLSI and EUCAST guidelines [28 ]. AST was performed with a definite turbidity bacterial suspension in NaCl (0.5 McFarland; 1.5 × 10⁸ cells/mL). Antibiogram test included the following sixteen antibiotics: amoxicillin (AMX), amoxicillin-clavulanic acid (AMC), cefepime (FEP), piperacillin/tazobactam (TPZ), cefalotin (KF), ceftriaxone (CRO), ertapenem (ETP), imipenem (IMP), fosfomycin (FF), nitrofurantoin (F), trimethoprim-sulfamethoxazole (SXT), amikacin (AK), ciprofloxacin (CIP), doxycycline (DO), colistin (CT), and gentamicin (GT) (Bio-Rad, Marne-la-Coquette, France). Hierarchical clustering of the antibiotic resistance phenotype was performed using Multi-Experiment Viewer (MeV 4.9.0).
Strains with a narrow diameter zone of inhibition (ZOI) less than 15 mm were picked out to confirm the minimal inhibition concentration value using other complementary tests, namely the E-tests method (BioMérieux) and UMIC test (Biocentric Bandol, France) [29 (link)]. Furthermore, strains were considered to be multidrug-resistant (MDR) if bacteria were resistant to more than three different classes of antibiotics.

Antimicrobial susceptibility was determined by the disc diffusion method of Kirby-Bauer on agar Mueller-Hinton (bioMérieux, Marcy l'Etoile, France) as recommended by the Antibiogram Committee of the French Microbiology Society [22 ]. After 24 h at 37°C, inhibition zone was measured. For susceptibility to Oxacillin, inoculum of 10⁷ CFU/mL was prepared, and the plate was incubated at 37°C for 24 h on Mueller-Hinton agar + 2% NaCl. The tested antibiotics (Bio-Rad, Marne la Coquette, France) were Pristinamycin, Erythromycin, Lincomycin, Oxacillin, Amoxicillin, Ceftriaxone, Gentamicin, Tobramycin, Sisomicin, Oxytétracycline, Tetracycline, Trimethoprim/sulfonamides, Cefotaxime, Ofloxacine, Pefloxacin, Vancomycin, Rifampicin, and Imipenem.

Disk diffusion method was used to test and confirm the antimicrobial susceptibility of the Enterobacteriaceae isolates using Muller–Hinton agar (MHA, Oxoid, Milan, Italy) and an incubation time of 16–18 h at 37 °C, following the Clinical and Laboratory Standards Institute Guidelines (CLSI) [24 ]. The antimicrobial used were: ciprofloxacin (CIP, 5 µg), nalidixic acid (NA, 30 µg), amoxicillin/clavulanic acid (AMC, 20/10 µg), amoxicillin (AML, 25 µg), levofloxacin (LEV, 5 µg), cefotaxime (CTX, 30 µg), sulphonamides (SSS, 300 µg), tetracycline (TE, 30 µg), trimethoprim/sulphamethoxazole (SXT, 1,25/23,75 µg), trimethoprim (TMP, 5 µg), chloramphenicol (C, 30 µg), and neomycin (N, 30 µg) (Bio-Rad, Marnes la Coquette, France). The results were assessed following the CLSI guidelines [24 ].

Screening for ESBL-E was performed by inoculating rectal swabs on selective medium supplemented with ceftazidime (bioMérieux, Marcy l’Etoile, France). After 24 h at 37 °C, the species were identified by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionisation, time-of-flight mass spectrometry) analysis. Antibiotic susceptibility was tested using the standard agar diffusion method on Mueller-Hinton agar (Bio-Rad, Marnes-la-Coquette, France) according to CA-SFM 2012 guidelines. The following antibiotics (bioRad) were tested: amoxicillin, amoxicillin-clavulanate, ticarcillin, piperacillin, piperacillin-tazobactam, cefalotine, cefoxitine, moxalactam, cefotaxime, ceftazidime, aztreonam, cefepime, imipenem, ertapenem, meropenem, gentamicin, tobramycin, netilmicin amikacin, nalidixic acid, ofloxacin, ciprofloxacin and sulfamethoxazole. The double-disk synergy method was used to confirm ESBL production [25 (link)]. All ESBL-E − producing isolates were stored at −80 °C.

From an 18-24h bacterial culture onto trypticase soy agar, a bacterial suspension in saline was prepared at McFarland 0.5 (equivalent to ≈10⁸ cfu/ml). Antibiotic susceptibilities were determined by the disc diffusion method on Mueller-Hinton agar (Oxoid) according to the procedure described in [34 (link)]. The results were interpreted according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST: http://www.eucast.org). The antimicrobial drugs tested and their concentrations on the discs (BioRad) were the following: amoxicillin (25 μg), amoxicillin-clavulanic acid (20 μg/10 μg), cefalotin (30 μg), cefoxitin (30 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg), nalidixic acid (30 μg), trimethoprim (5 μg), sulphonamide (200 μg), tetracycline (30 UI) and ticarcillin (75 μg).

Urine analysis and strain identification were performed by conventional methods. Antimicrobial susceptibility study was determined using the standard disk diffusion method on Mueller Agar-Hinton (Oxoid) according to Clinical Laboratory Guidelines and Institute Standards [18 ]. Tested antibiotics (Bio-Rad) were as follows: amoxicillin-clavulanate, (10 μg) (AMX); amoxicillin, (20 μg/10 μg) (AMC); cefotaxime, (30 μg) (CTX); ceftazidime, (30 μg) (CAZ); cefoxitin (30 μg) (FOX); amikacin (30 μg) (AN); ciprofloxacin (10 μg) (CIP); nalidixic acid (10 μg) (NA); gentamicin (10 μg) (GEN); netilmicin (30 μg) (NET); tobramycin (10 μg) (NN); fosfomycine (10 μg) (FFL); trimethoprim + sulfamide (10 μg) (SXT); imipenem (10 μg) (IPM); and colistin (10 μg) (CL). The diameters of the zones of inhibition were interpreted according to the recommendations of the CLSI [18 ]. All strains isolated were screened for extended-spectrum β-lactamase (ESBL) production by the double-disk synergy test [19 (link)].