Penicillin g

The MIC was determined by an agar dilution method according to guidelines of the Clinical and Laboratory Standards Institute (2014[12 ]). Antimicrobial agents used in this study include penicillin G (Bio Basic Inc.), cefixime (Fluka), ceftriaxone (Sigma), ciprofloxacin (Fluka), tetracycline (Bio Basic Inc.), azithromycin (Fluka) and spectinomycin (Bio Basic Inc.) Each isolate was tested on a GC agar supplemented with 1 % of defined growth supplement containing 2-fold-increasing concentrations of antimicrobial agents. Plates were incubated in 5 % CO₂ at 37 °C for 24 h followed by recording the antimicrobial susceptibility results. The production of β-lactamase enzyme was detected using nitrocefin discs. N. gonorrhoeae strain ATCC 49226 was used as a control. The criteria of reduced susceptible to cefixime and ceftriaxone is according to the WHO document (WHO, 2012[65 ]) where N. gonorrhoeae strains displaying reduced susceptibility to cefixime exhibit MICs of ≥ 0.25 μg/ml and N. gonorrhoeae strain displaying reduced susceptibility to ceftriaxone yield MICs of ≥ 0.125 μg/ ml.

Unless otherwise stated, the chemicals were of analytical and molecular grade, and were purchased from Merck KGaA (Darmstadt, Germany). Marine broth was purchased from Condalab (Torrejón de Ardoz, Madrid, Spain). Marine agar was prepared by solidifying marine broth with 1.5% (w/v) V-agar (Condalab). Ampicillin, penicillin G, and chloramphenicol were obtained from BioBasic (Amherst, NY, USA), Amresco (Solon, OH, USA), and Sigma-Aldrich (St. Louis, MO, USA), respectively. Kanamycin sulphate and tetracycline hydrochloride were purchased from Calbiochem (San Diego, CA, USA). High-grade (≥98% purity) cellobiose, cellotriose, p-nitrophenyl-β-D-glucopyranoside (pNPG), and β-glucan from barley were obtained from Megazyme (County Wicklow, Ireland, UK).