Cefoxitin

Antimicrobial susceptibility profiles of the isolates were carried out using the disk diffusion method [61 ]. Firstly, from an overnight pure culture, three to five colonies were suspended in 5 mL of normal saline (Fisher Scientific, Loughborough, UK). The suspension was adjusted to 0.5 McFarland (approximately 1–2 × 10⁸ CFU/mL) using a CrystalSpec nephlometer (BD Diagnostics, Baltimore, MD, USA) following the manufacturer’s protocol. The antibiotic disks included ampicillin (AMP 10 mg), piperacillin/tazobactam (TZP 110 mg), cefepime (FEP 30 mg), cefotaxime (CTX 30 mg), cefoxitin (FOX 30 mg), ceftazidime (CAZ 30 mg), imipenem (IPM 10 mg), meropenem (MEM 10 mg), amikacin (AK 30 mg), gentamicin (CN 10 mg), and ciprofloxacin (CIP 5 mg) (Liofilchem, Roseto degli Abruzzi, Italy and BioMérieux, Voie Romaine, Craponne, France). The susceptibility assay was carried out according to the CLSI standards [62 (link)], placed on the nutrient agar plates (Oxoid, Hampshire, UK), and inoculated using sterile forceps. The plates were then incubated for 18–24 h at 37 °C. E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853) were used as susceptible control strains. The readings were taken after 18–24 h, and the interpretive categories and zone diameter breakpoints are listed in Table 4.

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.

A direct PCR was performed in the KEMRI-NUITM laboratory to confirm the presence of Neisseria gonorrhoeae. The direct PCR was done using the mighty amp reagents and the simpliprep machine. Specifically designed primers, forward-CGGCAGCATTCAATTTGTTAAAGC and reverse-CGCCATTTTTGTA, which were to yield a PCR product of 162 base pairs each, were used [14 ]. The thermal cycle conditions for the simpliprep machine were: 94°C, 60 sec (denaturation), 59°C, 60 secs (annealing), 72°C, 26 sec/max 429bp (1 min. kbp), (elongation), 72°C, 10 minutes (extended elongation). When the reaction was over, the samples were loaded on gel electrophoresis, stained, and viewed under ultraviolet light (UV).
The antimicrobial susceptibility testing was done by disk diffusion using 13 antimicrobials from Liofilchem Company (Ciprofloxacin (CIP,30μg), Ceftriaxone (CRO,30μg), Cefepime (FEP,25μg), Cefotaxime (CTX,30μg), Cefuroxime (CXM,30μg), Cefazolin (KZ,30μg), Ceftazidime (CAZ,30μg), Cefoxitin (FOX,30μg), Tetracycline (TE,30μg), Ampicillin (AMP,10μg), Amikacin (AK,30μg), Gentamycin (CN,30μg) and Amoxillin (AUG,30μg).The antimicrobial discs were inoculated onto a culture of Neisseria gonorrhoeae and incubated for 24–48 hours. The zone of inhibition was measured and interpreted using the CLSI 2016 guidelines.