Isovitalex

From January 2014 to December 2018, a total of 986 gonococcal isolates were collected from male patients with symptomatic urethritis (urethral discharge and/or dysuria) attending the STD Clinic at the Institute of Dermatology, Chinese Academy of Medical Sciences, Nanjing, China. All men except one reported that they were heterosexual. Urethral exudates were collected with cotton swabs, immediately inoculated onto Thayer-Martin medium (Zhuhai DL Biotech, China), and cultured in candle jars at 36°C for 24 to 48 h. Gonococcal isolates were identified by colony morphology, Gram’s stain and oxidase testing, and growth on GC chocolate agar base (Difco, Detroit, MI) supplemented with 1% IsoVitaleX (Oxoid, USA). Gonococcal colonies were suspended in tryptone-based soy broth and frozen (−70°C) until used for antimicrobial testing.

The Hp 60190 strain (ATCC 49503) was grown in Brucella broth, supplemented with 0.3% β-cyclodextrin (Sigma-Aldrich), Hp selective supplement Dent (Oxoid) and Isovitalex (Oxoid), under microaerophilic conditions at 37 °C for 72 h. Bacteria were first grown to an optical density of 0.6–1.0 at 600 nm (OD₆₀₀) and subsequently diluted to a starting OD₆₀₀ of 0.05, as previously described [43 (link), 44 (link)].

All viable isolates were examined for susceptibility to azithromycin, cefixime, ceftriaxone, ciprofloxacin, spectinomycin, and gentamicin by E-test (bioMérieux) and MIC Test Strip (Liofilchem, Italy) in accordance with the manufacturer's instructions, after growth on Thayer–Martin medium (Oxoid, Ltd.), with 1% IsoVitaleX (Oxoid, Ltd.) at 37°C in a 5% CO₂ atmosphere. Chromogenic reagent nitrocefin (Oxoid, Ltd; Beta-lactamase test, Liofilchem) was used to evaluate the penicillinase production.
After the introduction of the azithromycin ECOFF value, the MIC values were interpreted by referring to both the 2018 and 2021 EUCAST clinical breakpoint criteria (version 8.1, 2018 and version 11, 2021).^{23 ,24} For testing purposes, the azithromycin ECOFF is 1 mg/L,²³ and isolates with MIC values >1 mg/L were considered resistant to azithromycin. For gentamicin, the breakpoints were not available. According to the Euro-GASP Reporting Protocol,^{7 (link)} for the isolates with cefixime MIC value ≥0.25 mg/L and azithromycin MIC value ≥256 mg/L, the MIC was repeated and the identification was confirmed. The World Health Organization (WHO) N. gonorrhoeae G, K, M, O, and P reference strains were used as controls.^{25 (link)}

Biopsy samples were vortexed vigorously for 5 min and plated on Brain Heart Infusion (BHI) agar (Oxoid Ltd, Hampshire, United Kingdom) supplemented with 7.5% sheep blood, 0.4% Isovitalex, and H. pylori Dent supplement (Oxoid, United Kingdom). Plates were incubated at 37 °C in an atmosphere of 5% O₂, 15% CO₂, and 80% N2 for 3 to 7 days. H. pylori colonies were identified based on their typical morphology, characteristic appearance on Gram staining, a positive urease test, and subsequently confirmed by MALDI_TOF (Bruker, Germany). Isolates were stored at minus 80 °C in 0.5 ml of brain heart infusion (BHI) broth with 20% glycerol.

A total of 407 gonococcal isolates were recovered from men with symptomatic urethritis (urethral discharge and/or dysuria) attending the sexually transmitted disease (STD) clinic at the Institute of Dermatology, Chinese Academy of Medical Sciences, in Nanjing, China, from January 2016 to December 2017. Urethral specimens were collected with cotton swabs and immediately streaked onto modified Thayer-Martin medium (Zhuhai DL Biotech Co. Ltd.) and cultured in candle jars at 36°C for 24 to 48 h. N. gonorrhoeae was identified by colonial morphology, Gram’s stain, and oxidase testing, which are sufficient to identify N. gonorrhoeae colonies isolated on selective medium, particularly for samples from the urethral tracts of symptomatic men (37 , 38 (link)). Isolates were subcultured onto GC chocolate agar base (Difco, Detroit, MI) supplemented with 1% IsovitaleX (Oxoid, USA); pure cultures were swabbed, suspended in tryptone-based soy broth, and frozen (−80°C) until being used for antimicrobial testing.