Oxolinic acid

Antibiotic susceptible test was performed using disk diffusion method [1 ]. The bacterial cells were prepared by propagated the bacteria using Luria Broth (Oxoid, England), followed by centrifugation at 14,500 rpm for 10 min using MiniSpin (Eppendorf, Germany). The collected bacterial cells were adjusted into 10⁹ colony forming unit (CFU) using physiological saline and monitored using Biophotometer (Eppendorf, Germany). The suspensions were swabbed on Mueller-Hinton agar (Oxoid, England) using sterile cotton bud. The inoculated media was then left for 10 min before antibiotic discs were placed onto the agar surface. 16 types of antibiotics tested were nalidixic acid (30 µg/disk), oxolinic acid (2 µg/disk), compound sulfonamides (300 µg/disk), doxycycline (30 µg/disk), tetracycline (30 µg/disk), novobiocin (30 µg/disk), chloramphenicol (30 µg/disk), kanamycin (30 µg/disk), sulfamethoxazole (25 µg/disk), flumequine (30 µg/disk), erythromycin (15 µg/disk), ampicillin (10 µg/disk), spiramycin (100 µg/disk), oxytetracycline (30 µg/disk), amoxicillin (25 µg/disk), and fosfomycin (50 µg/disk) (Oxoid, England). After 24 h of incubation period, diameter of the inhibition zone was measured in millimeter (mm) using a ruler and results were interpreted with reference to the standard provided by Clinical Laboratory Standards Institute [9 ].

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)].

Agar disc diffusion method on Mueller-Hinton Agar (Oxoid, Basingstoke, UK) was used to determine the antibiotic resistance patterns among E. coli isolates. A total of 15 antibiotics belonging to 7 classes were tested, including aminoglycosidase: gentamicin (CN, 10 μg), kanamycin (K, 30 μg), streptomycin (S, 25 μg), and neomycin (N, 30 μg); β-lactams: amoxicillin/clavulanic acid (AMC, 30μg), ampicillin (AMP, 10 μg), ceftriaxone (CRO, 30 μg), ceftiofur (EFT, 30 μg); quinolones: nalidixic acid (NA, 30 μg), oxolinic acid (OA, μg); fluoroquinolone: enrofloxacin (ENR, 5 μg), ciprofloxacin (CIP, 5 μg); tetracycline: tetracycline (TE, 30 μg); phenicols: chloramphenicol (C, 30 μg), and sulfamethoxazole/trimethoprim (SXT, 25 μg) (Oxoid Ltd.,Basingtoke, Hampshire, England). The bacteria were designated as resistant, intermediate and sensitive according to the Clinical Laboratory Standards Institute (CLSI)'s recommendations (CLSI, 2012 ). Bacteria strains that were resistant and intermediate were categorized as non-susceptible while the sensitive strains were categorized as susceptible.