Nitrocefin test

Determinations of antimicrobial susceptibility of N. gonorrhoeae isolates and their minimum inhibitory concentrations (MICs) of azithromycin (AZ), ceftriaxone (TXE), cefixime (IXE), tetracyclines (TCE), ciprofloxacin (CIPE), and penicillin (PC) were performed by using the GC agar base supplemented with IsoVitaleX Enrichment. The MICs were determined by the E-test method in accordance with the guidelines of the Clinical Laboratory Standards Institute (CLSI document m100-s25). The E-test strip evaluates the inhibitory potential of a single antimicrobial agent over a large range of concentrations (e.g., 0.032–256 μg/mL). The MICs were interpreted by reading the scale in terms of μg/mL where the ellipse edge intersects the strip. After the required incubation period, the MIC value is read where the edge of the inhibition ellipse intersects the side of the strip. The presence of beta-lactamase enzyme production and resistance to penicillin were detected by using the nitrocefin test (Becton, Dickinson and Company, Thailand). The penicillinase-producing N. gonorrhoeae (PPNG) was defined as isolates testing positive for beta-lactamase enzyme. The working stock of N. gonorrhoeae isolates was stored at -80°C. N. gonorrhoeae ATCC 49226 and WHO reference strains K and L were used as quality control in each assay for validation.

Resistance to methicillin was primary tested using disc diffusion method on Mueller Hinton agar (Oxoid, UK), with oxacillin and cefoxitin discs (Becton Dickinson, Germany) and confirmed by the detection of mecA gene by polymerase chain reaction (PCR) (Barski et al. 1996 (link)). The susceptibility to antimicrobial agents was determined by the disc diffusion method according to recommendations given by Hryniewicz et al. (2005 ) and the CLSI guidelines (2006 ). The antibiotics tested were the following: penicillin, erythromycin, clindamycin, ciprofloxacin, co-trimoxazole, tetracycline, gentamicin, vancomycin, teicoplanin, fusidic acid, rifampicin, linezolid, quinupristin-dalfopristin, chloramphenicol (Becton Dickinson, Germany), and mupirocin (Oxoid, UK). For all the isolates beta-lactamase production was checked by nitrocefin test (Becton Dickinson, Germany). For isolates identified as resistant to erythromycin, but susceptible to clindamycin, D-test was performed to detect inducible clindamycin resistance (Fiebelkorn et al. 2003 (link)). The susceptibility to vancomycin was confirmed by the minimal inhibitory concentration (MIC) using E-tests, as described by the manufacturer (AB Biodisc, Sweden).