Mueller hinton agar (mha)

The antibiotic susceptibility test was performed according to the standard method recommended by the CLSI. We used 12 antibiotics such as meropenem (10 µg), ceftazidime (30 µg), ceftriaxone (30 µg), erythromycin (15 µg), amoxicillin (25 µg), rifampicin (05 µg), oxytetracycline (30 µg), gentamicin (10 µg), vancomycin (30 µg), ciprofloxacin (05 µg), levofloxacin (05 µg) and amikacin (30 µg). The opacity of the inoculums was adjusted to 0.5 McFarland standards. The isolates were well spread over Mueller-Hinton Agar (MHA) (Oxoid Limited, England) plates whereas fastidious bacterial isolates were subcultured on 5% sheep blood supplemented MHA plates. The antibiotic discs were placed at an equal distance on the bacterial inoculated plates. Finally, the plates were incubated for 24 hours at 37°C with 5% CO₂ and results were recorded (mm).16

The isolated bacteria from MacConkey agar was inoculated into 5 ml of Tryptic soy broth (TSB) (Merck, Germany) and incubated at 37°C. After 24 hours, the inoculum with turbidity equivalent to 0.5 McFarland standards were then inoculated evenly on Mueller-Hinton agar (Oxoid, England) using a sterile cotton swab. The antibiotics used were amoxicillin-clavulanate (AMC-30), aztreonam (ATM-30), cefotaxime (CTX-30), gentamicin (CN-10), ampicillin (AMP-10), tetracycline (TE-30) (all from BD BBL^TM, United States), cefpodoxime (CPD-10), ceftazidime (CAZ-30), ceftriazone (CRO-30), imipenem (IMP-10), meropenem (MEM-10), norfloxacin (NOR-10), oxacillin (OX-1), penicillin (P-1), and piperacillin (PRL-75) (all from Oxoid, England). The discs were placed on the Mueller-Hinton agar (Oxoid, England) plate. The plates were then inverted and incubated aerobically at 37°C for 16 to 18 hours. In this study, indication of the bacteria as “susceptible,” “intermediate,” or “resistant” towards the antibiotic discs was based on the Zone Diameter Interpretative Standards of the Clinical Laboratory Standard Institute (CLSI) (17 ).

Antimicrobial susceptibility testing was performed by the disk diffusion method using Mueller-Hinton agar (Oxoid Ltd.) supplemented with 5% defibrinated sheep blood, according to the French Institute of Susceptibility (Manel et al., 2018 (link)). Disks impregnated with antibiotics (bioMérieux, Marcy-l’Etoile, France) and their corresponding concentrations were as follows: ciprofloxacin (CIP: 5 μg); erythromycin (E: 30 μg); gentamicin (GM: 10 μg); ampicillin (AMP: 30 μg); amoxicillin/clavulanic acid (AMC: 30 μg); tetracycline (TE: 30 μg). Briefly, well-isolated colonies of the same morphological type were selected from an agar culture plate and transferred into 10 mL of sterile saline buffer (NaCl 0.9%). After homogenization and a 1/10 dilution, 1 mL of the mixture were flooded onto the surface of a Mueller-Hinton agar (Oxoid Ltd.) containing 5% defibrinated sheep blood. The inoculum was allowed to dry for 5 min and antibiotic disks were placed on the plate. After 48 h of microaerobic incubation at 37°C according to the European Committee for Antibiotic Susceptibility Testing (EUCAST), the inhibition zone diameters were measured with calipers. C. Jejuni ATCC 33560 was used as the reference stress. The isolates were classified as susceptible, intermediate, and resistant according to the EUCAST.