Colistin sulfate

The collected samples were streaked into Edward’s modified medium with colistin sulfate (5 mg/l) and oxolinic acid (2.5 mg/l) (OXOID, UK). This culture medium showed the highest sensitivity (100%) and specificity (100%) for streptococci, and it is a selective media for primary isolation [23 (link)]. The inoculated plates were incubated in 5% (v/v) CO₂ at 37ºC for 24–48 h. Typical β hemolytic streptococci dew pinpointed-like colonies developed on the inoculated plates and were identified by the characteristic colony morphology, Gram’s technique, and biochemical testing including catalase test confirmed the isolates. Recovered isolates identified as S. equi fermented sucrose and salicin but not lactose, sorbitol, or trehalose, while isolates identified as S. zooepidemicus developed the same biochemical results but fermented lactose and sorbitol [24 , 25 (link)].

The in vitro sensitivity of each E. coli and Enterococcus spp. strain to the following antibiotics (Oxoid Ltd. Basingstoke, Hampshire, UK) was tested: aztreonam (30 µg), amikacin (30 µg), amoxycillin-clavulanic acid (30 μg), ampicillin (10 µg), cephalothin (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), cephalexin (30 µg), ciprofloxacin (5 µg), colistin sulfate (10 µg), doxycycline (30 µg), erythromycin (10 µg), enrofloxacin (5 µg), gentamicin (10 µg), neomycin (30 µg), piperacillin (100 µg), rifampicin (30 µg), streptomycin (10 µg), sulphametoxazole-trimethoprim (25 µg), tetracycline (30 µg), tobramycin (10 µg). The in vitro sensitivity to the antibiotics was evaluated by Kirby-Bauer agar disk diffusion method and the results were interpreted as indicated by the National Committee for Clinical Laboratory Standards (NCCLS) [45 ].
Antibacterial activity of each EO was tested by Kirby-Bauer agar disk diffusion method following the procedures reported by Clinical and Laboratory Standards Institute (CLSI) [46 ] with some modifications. In details, each EO and mixture was diluted 1:10 in dimethyl sulfoxide (DMSO, Oxoid Ltd.) and one absorbent paper disk was impregnated with 10 µL of each dilution, respectively. A paper disk impregnated with 10 µL of DMSO was included as negative control. All tests were performed in triplicate.

Antimicrobial susceptibility test was conducted to further understand the exposure of the strain toward antibiotics by standard antibiotics disc and disc diffusion technique [20 (link)]. A suspension (100 µl) of fresh cultured of S. agalactiae <24 h on TSB, diluted to a turbidity equivalent to a MacFarland No. 0.5 standard solution, was spread onto triplicate Mueller–Hinton agar (Oxoid, England) plates, and tested against 18 chemotherapeutic agent discs namely nitrofurantoin (F - 50 µg), flumequine (UB - 30 µg), florfenicol (FFC - 30 µg), amoxylin (AML - 25 µg), doxycycline (DO - 30 µg), oleandomycin (OL - 15 µg), tetracycline (TE - 30 µg), ampicillin (AMP - 10 µg), lincomycin (MY - 15 µg), colistin sulfate (CT - 25 µg), oxolinic acid (OA - 2 µg), novobiocin (NV - 30 µg), spiramycin (SP - 100 µg), erythromycin (E - 15 µg), fosfomycin (FOS - 50 µg), neomycin (N - 10 µg), gentamicin (GM - 10 µg), and polymyxin B (PB - 30 µg) (Oxoid, England). The susceptibility test agars were incubated at 37°C for 24 h and the diameter of inhibition zones around the discs was measured and compared to the standard table for antimicrobial susceptibility provided by CLSI [21 ].

134 isolates collected from urethral swabs of patients with positive N. gonorrhoeae infections at Thai Red Cross Anonymous Clinic, Thai Red Cross AIDS Research Centre, and King Chulalongkorn Memorial Hospital, Bangkok, Thailand, from patients with gonococcal infections during 2016–2019. For culture preservation, all isolates were grown on TM medium (GC agar base supplemented with 1% haemoglobin, 1% IsoVitaleX, and vancomycin, colistin sulfate, and nystatin selective supplement (Oxoid, United Kingdom). All N. gonorrhoeae isolates were preserved at − 80 °C. N. gonorrhoeae ATCC 49226 reference strain was used for quality control of all phenotypic and molecular characterizations.

The in-vitro antimicrobial resistance patterns of the recovered isolates were determined on Mueller–Hinton agar (Oxoid, UK) using the disc diffusion method according to the procedures of (CLSI 2017 ). E.coli-ATCC 35218 was involved as a quality control strain. A blank disc impregnated with 30 μl of sterile distilled water was used as a negative control. The main concentration of bacteria was 1 × 10⁸ CFU/mL, which is equivalent to the McFarland 0.5 Turbidity Standard. Moreover, twelve antimicrobial discs were tested include ceftazidime (CAZ) (30 μg), amikacin (AK) (30 μg), cefotaxime (CTX) (30 μg), tigecycline (TCG) (15 μg), trimethoprim-sulfamethoxazole (SXT) (25 μg), amoxicillin-clavulanic acid (AMC) (30 μg), tetracycline (TE) (30 μg), levofloxacin (LEV) (5 μg), aztreonam (ATM) (30 μg), amoxicillin (AMX) (30 μg), colistin sulfate (CT) (10 μg), and meropenem (MEM) (10 μg) (ThermoFisher Scientific, USA). The tested antimicrobial agents are the most commonly used antibiotics in Egypt in both the veterinary and health sectors. The examined isolates are categorized as MDR (MDR: resistant to ≥ one antibiotic in ≥ three antimicrobial classes) as previously described by Magiorakos et al. (2012 (link)).