Cefoxitin

The antibiotic resistance profiles of S. aureus strains (NCCP 16830, NCCP 14567, NCCP 14754, and KVCC-BA0500624) were investigated using disc diffusion assay according to the guidelines from the Clinical and Laboratory Standards Institute [53 ]. Briefly, each of the S. aureus strains was aerobically grown at 37 °C on the BHI agar plate for 18 h, and colonies were picked and suspended in sterilized saline to meet turbidity of 0.5 McFarland standard. Each bacterial suspension was spread on the Mueller–Hinton agar plate and incubated in the presence of antibiotic discs at 35 °C for 18 h. The antibiotic discs used were cefoxitin (30 μg), oxacillin (1 μg), penicillin (10 μg), erythromycin (15 μg), clindamycin (2 μg), and chloramphenicol (30 μg) purchased from Thermo Fisher Scientific (Waltham, MA, USA). After incubation, the diameters of the inhibition zones were measured to evaluate antibiotic resistance or susceptibility of S. aureus strains.

Antimicrobial susceptibility testing was performed with the disk-diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). From an overnight culture of each S. aureus strains, a 0.5 McFarland standard bacteria solution was prepared were inoculated into a Mueller Hinton agar (MHA; Biolife, Milan, Italy) plate. The antimicrobial discs, purchased from Oxoid (Thermo Fisher Scientific, Basingstoke, UK), were penicillin G (P, 6 µg), cefoxitin (FOX, 30 µg), cefotaxim (CTX, 30 µg), kanamycin (K, 30 µg), tobramycin (TM, 10 µg), erythromycin (E, 15 µg), clindamycin (CMN, 2 µg), norfloxacine (NOR, 5 µg), ofloxacin (OFX, 5 µg), fusidic acid (FAD, 10 µg), rifampicin (RIF, 30 µg) and chloramphenicol (CHL, 30 µg). After incubation at 37 °C for 24 h, the diameters of the inhibition zones around the discs were determined.

The antimicrobial susceptibility of all the confirmed methicillin-resistant staphylococci and Mammaliicoccus was tested against a total of twenty-nine antimicrobials through the disk-diffusion technique on Mueller-Hinton agar. The following antibiotic (Oxoid) were tested: cefoxitin (30 µg), ceftaroline (30 µg), penicillin (10 UI), clindamycin (2 µg), fusidic acid (10 µg), trimethoprim (5 µg), trimethoprim-sulfamethoxazole (1.25:23.75 µg), tetracycline (30 µg), doxycycline (30 µg), minocycline (30 µg), enrofloxacin (5 µg), ciprofloxacin (5 µg), gatifloxacin (5 µg), levofloxacin (5 µg), norfloxacin (5 µg), gentamicin (10 µg), amikacin (30 µg), streptomycin (10 UI), kanamycin (30 µg), tobramycin (10 µg), sulfadiazine (300 µg), erythromycin (15 µg), tylosin (30 µg), lincomycin (15 µg), mupirocin (200 µg), chloramphenicol (30 µg), nitrofurantoin (300 µg), linezolid (30 µg), tedizolid (2 µg), rifampicin (5 µg), and vancomycin (30 µg). After incubation at 37 °C for 18 to 24 h, inhibition zones were measured and scored as resistant, susceptible, or intermediate (reduced susceptibility) in accordance with the Clinical and Laboratory Standards Institute’s guidelines [40 ].

Susceptibility testing of the GNB was examined by the Kirby–Bauer disk diffusion assay on Mueller-Hinton agar medium (Oxoid, England) against 18 antibiotic disks following the Clinical and Laboratory Standard Institute guidelines [17 ]. The following antibiotics were examined: amikacin (30 μg), amoxicillin/clavulanate (20/10 μg), aztreonam (30 μg), cefepime (30 μg), cefotaxime (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), cefuroxime (30 μg), ciprofloxacin (5 μg), colistin (10 μg), gentamicin (10 μg), imipenem (10 μg), meropenem (10 μg), nitrofurantoin (50 μg), piperacillin(100 μg), piperacillin/tazobactam (100/10 μg), tobramycin (10 μg), and trimethoprim/sulfamethoxazole (23.75 μg/1.25 μg) (Oxoid, England). In brief, standardized suspension of each isolate conforming 0.5 McFarland turbidity was inoculated onto two Mueller-Hinton agar plates. Then, nine antibiotic disks were placed onto each plate with recommended distance, followed by overnight incubation at 37°C. The strain of E. coli ATCC 25922 was used as control and was tested each time when susceptibility testing was performed.

The standardized disc diffusion method can be used commonly for susceptibility testing. The disc diffusion test was performed using Mueller Hinton agar (Himedia, Mumbai, India) with cefoxitin (30 µg) and ciprofloxacin (5 µg) discs (Oxoid Ltd.; Basingstoke, Hampshire, UK). The bacterial suspension of 1 × 10⁸ CFU/mL of S. aureus ATCC 25923 and five MRSA strains were spread on MHA plates. The drug discs were placed over the plates, followed by incubation at 37 °C for 24 h.
Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) testing was conducted. Susceptibility to nutmeg crude and essential oil extractions were determined by broth microdilution methods performed using Mueller Hinton broth (Himedia, Mumbai, India). Briefly, the extractions were diluted using twofold serial dilutions from 5000 to 0.5 µg/mL of CE and 50 to 0.0005% of EO. Then, bacterial suspension of 1 × 10⁸ CFU/mL of S. aureus ATCC 29213 and five MRSA strains were added in the wells, followed by incubation at 37 °C for 24 h. The experiments were performed according to the Clinical & Laboratory Standards Institute (CLSI) recommendations.

Antibiotic resistance profiles were obtained from 166 isolates. A standardized amount was inoculated (standard 0.5 of McFarland) and antimicrobial susceptibility testing was performed by the disk diffusion method on Mueller–Hinton (MH) agar (Difco Laboratories, Detroit, MI, USA) using interpretative criteria of the Clinical and Laboratory Standard Institute (CLSI) [34 ]. The following antimicrobials were used: trimethoprim/sulfametoxazol (SXT, 23.75 μg + 1.25 μg), gentamicin (GEN, 10 μg), ciprofloxacin (CIP, 5 μg), ceftazidime (CAZ, 30 μg), cefoxitin (FOX, 30 μg), cefotaxime (CTX, 30 μg), meropenem (MEM, 10 μg), piperacillin/tazobactam (TZP, 40 μg), and cefixime (CFM, 5 μg) (Oxoid Ltd., Hampshire, United Kingdom). ESBL-positive isolates were identified by using the double-disk synergy test with third-generation cephalosporins (cefotaxime, ceftazidime, and cefixime) alone and with clavulanic acid. Quality control was carried out using standard strains of Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27953). Intermediate susceptibility to each antibiotic was considered to be resistant.
According to the definitions proposed by Magiorakos et al., resistance to three or more antibiotic classes was defined as multidrug resistant (MDR) [35 (link)].