Ceftriaxone

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.

Escherichia coli isolates were screened for ESBL using a disc diffusion test with expanded-spectrum cephalosporins (which are hydrolysed by all TEM, SHV and CTX-M types of ESBLs) and aztreonam on Mueller-Hinton II agar (Pitout et al., 1997 (link); Pitout and Laupland, 2008 (link); Smet et al., 2008 (link); Falagas and Karageorgopoulos, 2009 (link)). The cephalosporins used were ceftazidime (30 μg), cefotaxime (30 μg), ceftriaxone (30 μg) and cefpodoxime (10 μg) (Bio-Rad, Laboratories, Hercules CA). Extended-spectrum β-lactamase phenotypic production was confirmed using the modified double-disc diffusion method or the combined-disc method (Stürenburg and Mack, 2007 (link)). cefotaxime + clavulanic acid (30 μg + 10 μg) and ceftazidime + clavulanic acid (30 μg + 10 μg) discs were used (Bio-Rad Laboratories). Extended-spectrum β-lactamase was positive when the zone diameters given by the discs with clavulanate were ≥ 5 mm larger than those without the inhibitor for at least one of the combinations. Escherichia coli ATCC 25922 (ESBL negative), E. coli ATCC 35218 (ESBL positive control), K. pneumonia ATCC 700603 (ESBL positive) and Pseudomonas aeruginosa ATCC 27853 (ESBL negative) strains were used as control strains for test performance.

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.

The stock solutions of A. fruticosa fruits EO used for further assays were prepared 1:1 in DMSO (dimethyl sulfoxide). The antimicrobial activity screening was determined by employing an adapted disk diffusion technique, using 10 μL of stock solution of EO, which contained 295 mg EO solubilized in 1 mL of DMSO. The minimum inhibitory concentrations (MICs) were measured as described previously [43 ]. Briefly, serial dilutions of the stock solutions in liquid medium (Brain heart infusion broth for bacterial strains and Sabouraud broth for yeasts) were prepared in 96 wells plates (concentrations 29.5–0.46 mg/mL). Then 10 μL of the microbial suspension with the standard density of 0.5 Mc Farland was added to each well, positive and negative controls were used for each strain. The positive control antibiotic disks used in diffusion method include oxacillin (1 µg) (Bio-Rad, Hercules, CA, USA); ticarcillin-clavulanic acid (75 + 10 µg) (Bio-Rad); ceftriaxone (30 µg) (Bio-Rad); cefalexin (30 µg) (Bio-Rad); vancomycin (30 µg) (Bio-Rad); ofloxacin (5 µg) (Bio-Rad); colistin (10 µg) (Bio-Rad); erythromycin (15 μg) (Bio-Rad). The antibiotics were chosen according to CLSI and literature data for each tested strain. The plates were incubated for 24 h at 37 °C. The absorbance was measured at 620 nm with an Apollo LB 911.

In vitro susceptibility of E. coli isolates against antibiotics was performed using Kirby Bauer Agar Diffusion Method [19 (link)]. The antibiotics tested were penicillins (ampicillin, 10 μg; ticarcillin, 75 μg; piperacillin, 75 μg; oxacillin, 1 μg; and amoxicillin, 25 μg), cephalosporins (ceftriaxone, 30 μg; cefoxitin, 30 μg; cefalotin, 30 μg; cefepime, 30 μg; cefixime, 5 μg; cefotaxime, 30 μg; and ceftazidime, 10 μg), fluoroquinolones (ciprofloxacin, 5 μg and ofloxacin, 5 μg), glycopeptides (vancomycin, 5 μg), lincosamides (clindamycin, 2 μg), aminoglycosides (amikacin, 30 μg and gentamicin, 30 μg), and trimethoprim-sulfamethoxazole (25/23.75 μg) (BIORAD, France). Each assay was performed in triplicate. Antibacterial activity of antibiotics against E. coli isolates was evaluated by measuring the clear zone of growth inhibition (diameter of zone of inhibition) on agar surface around the discs as per clinical and laboratory standards institute (CLSI) guideline [20 ]. E. coli classified as susceptible, intermediate, and resistant strains according to the criteria of the CLSI [20 ]. Escherichia coli isolates were regarded as multidrug resistant (MDR) when they were resistant to one or more antibiotics in three or more classes of antimicrobials that the isolate is expected to be susceptible.

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).