Novobiocin

In this study, a total of 240 fecal samples, including 20 samples per month, were collected from freeranging wild red deer (Cervus elaphus) in an area of 100 hectares in Bahçeköy Deer Breeding Farm, under the body of the General Directorate of Nature Conservation and National Parks of the Ministry of Agriculture and Forestry in Istanbul Belgrad Forests between April 2019 and March 2020 for the isolation and identification of Salmonella. Fresh fecal samples collected every month regularly from different regions of a 100-hectare land were taken into sterile dry vessels containing feces and delivered to the laboratory as soon as possible within cold chain (Carroll et al., 2015) .
Among the samples, isolation of Salmonella species were performed according to the Protocol of the World Health Organization on Salmonella spp. isolation from Food and Animal feces (WHO 2010). The samples delivered to the laboratory were incubated for 18-20 hours at 37°C in Buffered Peptone Water for pre-enrichment. After the pre-enrichment process, cultures were passaged into Tetrathionate Broth (Oxoid) and Rappaport-Vassiliadis Soya Broth (Hi-Media) for selective enrichment and incubated for 18-24 hours at 37°C and 41.5°C, respectively. Hektoen Enteric Agar (Condalab) including novobiocin (15 μg/ml final concentration) and MacConkey Agar (Hi-Media) were used for the selective agar for isolation (Cakin et al., 2020) . Black colonies on Hektoen Enteric Agar and lactose-negative semitransparent yellowish colonies on MacConkey Agar were passaged to obtain pure cultures. As a result of the biochemical tests performed from the pure cultures, the isolates which were urease negative and methyl red, citrate positive, and formed H2S and gas in Triple Sugar Iron Agar, were evaluated as presumptive Salmonella spp. These isolates were examined by PCR by using Salmonella spp.-specific invA gene region primer-139 (5'GTG AAA TTA TCG CCA CGT TCG GGC AA3') and primer-141 (5'TCA TCG CAC CGT CAA AGG ACC C3') (Rahn et al., 1992) .
As a positive control, Salmonella Typhimurium ATCC 14028, from the culture collection of Department of Microbiology, Faculty of Veterinary Medicine, Istanbul University-Cerrahpaşa, was used. Salmonella spp.-specific gene region was sought in the electrophoresis that was conducted after the DNA extraction and DNA amplification procedures.

Antibiotic sensitivity of test strains was determined by the standard Disc diffusion method against Ampicillin (A10) Penicillin (P 30) Norfloxin (N30) Vancomycin (Va30) Chloramphenicol (C30) Novobiocin (Nv 30) and Carbenicillin (Cb100) Antibiotic discs were purchased from Hi-Media Pvt. Ltd. (Mumbai, India). β-Lactamase assays: Crude β-lactamase extracts were prepared from 5 mL of overnight cultures. Bacterial cells containing β-lactamase were grown to mid-log phase (optical density at 540 nm: 0.5) in LB broth containing 100 mg of ampicillin mL -1 . The cells were pelleted, washed and resuspended in 500 mL of 50 mM Tris-HCl buffer, pH 7.4. A 40 mg mL -1 stock solution of freshly prepared lysozyme in Tris-HCl buffer was added to a final concentration of 10 mg mL -1 and bacterial cells were incubated for 15 min at room temperature. EDTA was added to a final concentration of 1 mM and the mixture was gently shaken for 10 min. The cell suspension was clarified by centrifugation at 14,000 g for 15 min and the supernatant was collected. β-Lactamase activity in the supernatant was measured with the chromogenic cephalosporin nitrocefin The amount of protein in each β-lactamase preparation was determined by Lowry et al. [17] . The specific activity present in each sample was estimated by measuring the hydrolysis of 100 mM nitrocefin at 25°C in a spectrophotometer at 482 nm. Specific activity was defined as U mg -1 of protein. One unit was defined as the amount of nitrocefin hydrolyzed (micromolar) min -1 .