Sulfamethoxazole

The antimicrobial susceptibility profile for Salmonella isolates (n = 75) was investigated against 8 different antimicrobial agents using the Kirby–Bauer disc diffusion method (Oxoid Ltd., Basingstoke, UK). The sensitivity pattern was interpreted according to the guidelines of Clinical and Laboratory Standards Institute [40 ]. The antibiotics selected were those commonly used in the poultry industry, including Cefotaxime (CTX, 30 μg), Ceftriaxone (CRO, 30 μg), Ampicillin–Sulbactam (SAM, 10 μg), Amoxicillin–clavulanic (AMC, 30 μg), Impenem (IPM, 10 μg), Sulfamethoxazole–trimethoprim (SXT, 25 μg), Ciprofloxacin (CIP, 5 μg) and (Gentamicin CN, 10 μg) (Oxoid Ltd., Basingstoke, UK). The diameter of the inhibition zone was measured compared with the zone size interpretation chart and was graded as susceptible (S), intermediate (I), and resistant (R).

All Salmonella isolates were tested for antimicrobial susceptibility after their isolation. The testing was performed in the Sanitary Veterinary Laboratory for Food Safety situated in Cluj County, Romania. The testing was made on eleven antimicrobials that are normally used in human and animal therapy. The method applied was the disk diffusion method and the protocol followed exactly the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [35 ,36 ]. The antimicrobial classes tested were ampicillin (AMP, 10 μg), cefotaxime (CTX, 30 μg), ceftazidime (CAZ, 30 μg), chloramphenicol (CHL, 30 μg), ciprofloxacin (CIP, 5 μg), gentamicin (GEN, 10 μg), nalidixic acid (NA, 30 μg), streptomycin (S, 10 μg), sulfamethoxazole (SMX, 300 μg) trimethoprim/sulfamethoxazole (SXT, 1.25/23.75 μg), and tetracycline (TET, 30 μg) (Oxoid, Basingstoke, UK). The interpretation of the results was made according to the CLSI breakpoints.

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 ].

Antibiotic resistance pattern of E. coli isolates was determined by simple disk diffusion method. The Mueller–Hinton agar (Merck, Germany) media were used for antibiotic susceptibility test. Principles of the Clinical and Laboratory Standards Institute (CLSI) were used for this purpose [19 ]. Susceptibility of E. coli isolates were tested against tetracycline (30 u/disk), ampicillin (10 u/disk), cefotaxime (30 μg/disk), gentamicin (10 μg/disk), ciprofloxacin (5 μg/disk), amikacin (30 u/disk), ceftazidime (30 μg/disk), imipenem (30 u/disk), cotrimoxazole (30 μg/disk), enrofloxacin (5 μg/disk), sulfamethoxazole (25 μg/disk), trimethoprim (5 μg/disk), streptomycin (10 μg/disk) and chloramphenicol (30 μg/disk) antibiotic agents (Oxoid, UK). All of the inoculated plates were aerobically incubated at 37 °C for 18-24 h. Results were interpreted based on the instruction provided by CLSI [19 ]. E. coli ATCC 25922 was used as positive control.

The agar disk diffusion test was performed for all the recovered P. putida isolates (n = 18) according to the method described by Ali et al. [35 ]. Susceptibility for florfenicol (10 µg), ampicillin (10 µg), erythromycin (15 µg), sulfamethoxazole-trimethoprim (25 µg), ciprofloxacin (5 µg), doxycycline (30 µg), and tylosin (15 µg), Oxoid, UK. Muller-Hinton agar plates (Oxoid, Uk) were incubated at 37 °C for 48 h due to the slow growth rate of the tested isolates. The inhibition zone (mm) was measured to the nearest mm and interpreted according to the breakpoints mentioned [36 (link)].

Mueller-Hinton (MH) agar medium was purchased from Guangzhou Dijing Microbiology Co., Ltd.. Drug sensitivity test paper (combining sulfamethoxazole, SXT; minocycline, MH; levofloxacin, LVX; ticarcillin/clavulanic acid, TIM; ceftazidime, CAZ; chloramphenicol, CL and imipenem, IPM) was purchased from OXOID (United Kingdom).