Nitrocefin test

In WA, gonorrhoea is a Department of Health notifiable infectious disease and all isolates are referred to the Western Australian Gonococcal Surveillance Programme (WAGSP) laboratory for antimicrobial susceptibility testing. Antimicrobial resistance profiles of all isolates to penicillin, spectinomycin, azithromycin, ciprofloxacin, ceftriaxone and high-level resistance to tetracycline (tetracycline HLR) were assessed by the agar dilution method and interpreted using the Calibrated Dichotomous Sensitivity guidelines [20 ]. Decreased susceptibility to ceftriaxone (0.06–0.126 mg/L) is confirmed by the E-test (bioMérieux, France). Penicillinase production is detected using the nitrocefin test (Oxoid, Australia). Anatomical isolation site, geographical location and postcode are available from the Communicable Disease Control Directorate.
Fifty nine N. gonorrhoeae isolates from patients living in the remote (n = 33) and metropolitan population centers (n = 26) of WA from 2011 and 2013 were obtained from the WAGSP laboratory [16 ]. Isolates were stored in GC broth with 20% glycerol at − 80 °C and were passaged fewer than five times and were cultured under aerobic conditions with 5% CO₂ at 37 °C on GC agar (Oxoid, Australia) supplemented with 0.4% glucose, 0.01% glutamine, 0.2 mg/L of cocarboxylase and 5 mg/L of iron (III) nitrate.

The minimum inhibitory concentration (MIC; mg/L) of ceftriaxone, cefixime, spectinomycin, azithromycin, ciprofloxacin, benzylpenicillin, tetracycline, gentamicin, and kanamycin were determined using the Etest method (bioMérieux, Marcy-l’Etoile, France), according to the instructions from the manufacturer. All results were interpreted using whole MIC doubling dilutions and, where available, current clinical breakpoints for susceptibility (S) and resistance (R) according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST [48 ]]. For azithromycin, EUCAST does not recommend any clinical breakpoints, and the EUCAST azithromycin epidemiological cut-off value (ECOFF) of MIC> 1 mg/L [48 ] was used to indicate isolates with azithromycin resistance determinants (referred to as resistant hereafter). Previously published interpretative criteria were used for gentamicin [49 (link)] and kanamycin [50 (link), 51 (link)]. MIC₉₀ and MIC₅₀ values were defined as the lowest concentration of the antimicrobial at which 90% and 50% of the isolates were inhibited, respectively. β-lactamase production was identified using a Nitrocefin test (Oxoid, Basingstoke, England). The 2008 WHO N. gonorrhoeae reference strains [52] were used for quality controls of all phenotypic and molecular characterisation.

The disc diffusion method established by Kirby-Bauer was carried out for antimicrobial susceptibility testing [17 (link)]. Mueller-Hinton culture medium was supplemented with 5 % horse blood and 20 mg/L beta-nicotinamide adenine dinucleotide (BD, Heidelberg, Germany). Plates were inoculated with samples of each isolate and set to a turbidity of 0.5 McFarland. Antibiotic discs were applied to the dried surface of the inoculated culture medium and later incubated at 35 ± 1 °C for 18 ± 2 h in a 5 % CO₂ atmosphere. In cases of discrepancies or insufficient readings, the accurate determination of the MIC was executed by an E-test for particular pathogens, and the outcomes were interpreted according to the EUCAST criteria [16 ]. Intermediate isolates were grouped together with resistant isolates. Beta-lactamase production was evaluated using the nitrocefin test (Oxoid, Wesel, Germany). Gram-negative strains were described as beta-lactamase-negative strains that were resistant to ampicillin (zone diameter > 16 mm or MIC ≥ 4 μg/mL) [16 ]. Inhibition zone diameters were based on the 2014 EUCAST guidelines [16 ].