A cross-sectional study was carried out among the slaughter houses of Bharatpur Metropolis, Nepal and laboratory analyses were performed at the Microbiology laboratory of Birendra Multiple Campus from February to June 2018. Random sampling was done to collect 200 non-repeated single meat samples from different slaughterhouses located at different places of Bharatpur (Baseni, Dipendra chowk, Hope chowk, Junhal road, Bel chowk, Malpot Chowk and Gitanagar). The sample size was determined in accordance with the incidence rate based on the previous study [8]. Each butcher was briefed of the purpose of sample collection and verbal informed consent was taken assuring them of total confidentiality. Slaughterhouse’s sanitary and salubrious status was studied by brief interview using semi-structured questionnaire and through observations as well.
Methodology
Fresh chicken liver samples were collected separately in sterile zip-locked plastic bags with the help of sterile forceps and scissors, stored in cold box and transported aseptically to the laboratory for further processing within an hour. The samples were ground in sterile mortar and pestle to make fine particles and 1 g of them was inoculated into 9 ml of distilled water and dilutions up to 10–5 were made. From each of 10–3, 10–4 and 10–5 dilutions, 0.1 ml of inoculum was spread in nutrient agar plates in triplicate and incubated at 37℃ for 24 h to obtain viable count of the bacteria. For the isolation of Salmonella, 1 ml of the inoculum was enriched in Selenite F-broth (Himedia, M025S) and incubated at 37℃ for 24 h. A loopful of culture in Selenite F-broth was directly streaked on XLD agar (Himedia, MH031) and incubated at 37˚C for 24 h. Black-centered red colonies on XLD agar were sub-cultured on NA plates at 37˚C for 24 h to obtain pure culture of the isolates [19]. For further identification of Salmonella species, Gram staining and various biochemical tests (SIM, MR-VP, citrate, catalase, oxidase, urease and TSI) were performed. A slide agglutination test using antisera (Statens serum institute, Copenhagen) was used to detect S. Typhi O9, poly O and H antigens.
All the isolates were tested for susceptibility to antimicrobial agents on MHA by Modified Kirby-Bauer disc diffusion method as recommended by the Clinical Laboratory Standards Institute. The antibiotic discs used were amikacin (30 µg), cotrimoxazole (25 µg), ciprofloxacin (5 µg), colistin (10 µg), tetracycline (30 µg), gentamicin (10 µg) and azithromycin (15 µg). The turbidity of inoculums from a pure culture of Salmonella isolates on NA plates incubated at 37°C for 24 h were adjusted to the equivalent turbidity of 0.5 McFarland standards before spreading uniformly over the surface of Mueller-Hinton agar (MHA) (Titan Biotech Ltd. Bhiwadi–301019, Rajasthan, India) plates. Using sterile tweezers, the antibiotic discs were placed widely spaced aseptically on the surface of MHA plate. The organism was classified as resistant, intermediate or sensitive according to the interpretative chart [20]. Resistance to more than three structural classes of the antimicrobials tested was considered as MDR [21]. Salmonella Typhimurium ATCC 14028 was used as a reference strain for quality control purposes.
Primary screening test for ESBL production was done by using ceftazidime and cefotaxime discs against which the organisms showing the zone of inhibition ≤22 mm for ceftazidime (CAZ) (30 µg) and ≤27 mm for cefotaxime (CTX) (30 µg) were considered to be probable ESBL producers. The phenotypic confirmatory test was done for suspected ESBL producing isolates for which antibiotics combinations of ceftazidime + clavulanic acid (CAZ/CAC) (30/10 µg) and cefotaxime + clavulanic acid (CTX/CTC) (30/10 µg) were used according to the protocols recommended by CLSI [22]. An increase in the zone of inhibition by ≥5 mm around the discs containing cephalosporin with clavulanate over the discs containing cephalosporin alone were ESBL producers [22].
The data obtained from laboratory investigation were tabulated and presented in defined tables and p-value of the obtained results was calculated using SPSSv20 software. P-value ≤ 0.05 was considered to have a significant association.