Antimicrobial susceptibility tests were conducted using antimicrobial impregnated discs (Oxoid) containing tetracycline (TET; 30 μg), erythromycin (EM; 15 μg), norfloxacin (NFLX; 10 μg), ciprofloxacin (CPFX; 10 μg), ofloxacin (OFLX; 10 μg), nalidixic acid (NA; 30 μg), ampicillin (ABPC; 10 μg), and gentamicin (GEN; 10 μg) according to the standard Kirby-Bauer disc diffusion method (5 (link)) and performed according to the recommendations of the Clinical Laboratory Standards Institute (8 ).
Antimicrobial impregnated discs
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Antimicrobial impregnated discs are a type of laboratory equipment used for antimicrobial susceptibility testing. These discs are impregnated with specific antimicrobial agents and are placed on agar plates inoculated with bacterial cultures. The antimicrobial agents diffuse from the discs into the agar, creating zones of inhibition around the discs. The size of these zones is used to determine the susceptibility or resistance of the bacteria to the tested antimicrobials.
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3 protocols using antimicrobial impregnated discs
1
Antimicrobial Susceptibility Testing Protocol
2
Antimicrobial Resistance Screening Protocol
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All of the isolates were tested for their phenotypic antimicrobial resistance by agar disc diffusion method with antimicrobial impregnated discs (Oxoid, Hampshire, England) against polymyxin-B (PB; 300 U), trimethoprim-sulfamethoxazole (STX; 1.25/23.75 μg), chloramphenicol (C; 50 μg), gentamycin (G; 10 μg), trimethoprim (W; 5 μg), neomycin (N; 10 μg) and oxytetracycline (OT; 30 μg). Antimicrobial resistance tests were performed on Mueller-Hinton agar (Oxoid) according to Bauer Kirby agar disc diffusion [23] (link) following Clinical Laboratory Standards Institute's protocol [24] . The isolates were sub-cultured onto standard-I nutrient agar (Merck) and incubated at 37 °C for 24 hrs. They were then inoculated into 3 ml of brain heart infusion broth (BHI) (Merck) and again incubated for 1 hr at 37 °C. The inoculum density was standardized to 0.5 McFarland standard; from which 0.1 ml was spread onto Mueller-Hinton agar (Oxoid). After the plates were allowed to absorb the moisture; antimicrobial impregnated discs were applied; and the plates were incubated at 35 ± 2 °C for 16-18 hr. Based on the diameter of zone inhibition for Enterobacteriaceae, results were recorded as susceptible, intermediate or resistant [24] .
3
Antimicrobial Susceptibility Profiling of Salmonella
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All Salmonella isolates were tested for susceptibility to 13 different antimicrobial drugs using Kirby Bauer disc diffusion method as described by CLSI (2014). The antimicrobial impregnated discs (Oxoid Ltd, Hampshire, England) used were streptomycin, amikacin, gentamicin, amoxicillin, cefotaxime, ceftazidime, cefuroxime, chloramphenicol, ciprofloxacin, nitrofurantoin, tetra-cycline, trimethoprim-sulfamethoxazole and norfloxacin with appropriate concentration. Two to three pure colonies were picked up and emulsified in 3 to 4 ml of sterile normal saline. Dilution of the suspended colonies was performed until the turbidity matches the 0.5 McFarland Standards and transferred to Mueller-Hinton agar (HiMedia, Pvt. Ltd., India) using a sterile cotton swab. Following air drying the plates were incubated aerobically at 37 °C for 18 to 24 hrs. The diameters of the zone of inhibitions were measured with a caliper and the susceptibility of the isolates was recorded as susceptible, intermediate, and resistant. Escherichia coli ATCC 35218 was used as quality control.
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