Erythromycin

The disk diffusion assay was performed in accordance with the method described by the antibiogram committee of the “French Society of Microbiology” CA-SFM/2014. All isolates were tested for their susceptibility to the following antibiotics: Amoxicillin/clavulanic acid (20/10 μg), ampicillin (10 μg), erythromycin (15 IU), tetracycline (30 IU), gentamicin (10 μg), ciprofloxacin (5 μg), and chloramphenicol (30 μg) (Bio-Rad, France).
From a pure culture of 18-24 h, the bacterial suspension was adjusted to match the 0.5 McFarland turbidity standard. A sterile swab was immersed into the adjusted suspension and then seeded by swabbing onto the entire surface of Mueller-Hinton agar supplemented with 5% defibrinated horse blood and β-NAD (MH-F). After streaking, the inoculum was dried for 5-10 min, and four antimicrobial disks were placed onto the surface of the plate. The plates were incubated at 37°C for 24 h under a microaerophilic atmosphere. Inhibition zones were measured by a caliper, and diameters were interpreted as recommended by the Antibiogram Committee of the French Society of Microbiology [8 ]. C. jejuni ATCC 33560 and C. coli ATCC 33876 were used as control strains.

The antimicrobial susceptibility was performed by the disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [9 ]. Fifteen antimicrobial agents on Mueller-Hinton agar plates (Becton Dickinson, Franklin Lakes, NJ, USA) were tested: oxacillin, cefoxitin, gentamicin, erythromycin, clindamycin, tetracycline, chloramphenicol, ciprofloxacin, trimethoprim/sulfamethoxazole, fusidic acid, linezolid, rifampicin, tigecycline and vancomycin (Bio-Rad, Marnes la Coquette, France) and penicillin G (Oxoid, Basingstoke, England). The phenotype of resistance to macrolide-lincosamide-streptogramin B was tested and interpreted according to the EUCAST. vancomycin susceptibility was determined with E-test method (bioMerieux, Marcy-l’Etoile, France). Multidrug resistance (MDR) was defined as a resistance to three or more classes of antibiotics.

Susceptibility to cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, fusidic acid, erythromycin, gentamicin, kanamycin, linezolid, mupirocin, quinupristin-dalfopristin, penicillin, rifampin, tetracycline, tobramycin and trimethoprim/sulfamethoxazole (BioRad, USA) was performed by disk diffusion method and to vancomycin and teicoplanic by Etest (bioMérieux, France) in accordance to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendation (http://www.eucast.org). Multidrug resistance (MDR) was defined as resistance of MRSA to three or more district antimicrobial classes in addition to beta-lactams.

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.

The MRSA isolates were tested for antibiotic susceptibility by disc diffusion test using the following discs: chloramphenicol (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), fusidic acid (10 μg), gentamycin (10 μg), erythromycin (15 μg), imipenem (10 μg), rifampicin (5 μg), tetracycline (30 μg), trimethoprim/sulfamethoxazole (1.25 + 23.75 μg) and vancomycin (30 μg) (BioRad, USA). The rates of susceptibility and resistance of isolated MRSA strains to the used antibiotic discs were detected.

The antibiotic susceptibility of the MRSA isolates was determined by the disk diffusion method on Mueller-Hinton agar (MHA, CONDA, Spain), according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). The disk diffusion assay was done with 14 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), and trimethoprim/sulfamethoxazole (1.25 μg/23.75 μg, respectively) (BIO-RAD). Minimum inhibitory concentrations (MIC) for cefoxitin and vancomycin was determined by using MIC Test Strip (Liofilchem) on inoculated Mueller Hinton agar plates and the results were interpreted according EUCAST breakpoints. S. aureus strain ATCC 25923 and Enterococcus faecalis strain ATCC 29212 were used as controls in the susceptibility test.