**2.5** *Salmonella* **serotyping and genotyping**

Frozen (-70°C) presumptive *Salmonella* cultures from the above sources were thawed and stabbed into 2ml tryptic soy agar (Difco, Becton Dickinson) deeps and shipped to the

Antimicrobial Drug Resistance and Molecular Characterization

observed at a similarity level of 80% (Figure 1).

Amoxicillin/clavulanic acid

Trimethoprim-

(N = 15)

Antibiotics Susceptible

Isolates (%)

(1/0.5–32/16) 2(3.5) 1(1.7) 55(94.8) Ampicillin (2–32) 2(5.3) - 56(94.7) Cefoxitin (0.5–32) 58(100.0) - - Cetriaxone (0.25–64) 58(100.0) - - Chloramphenicol (2–32) - 2(5.3) 56(94.7) Ciprofloxacin (0.015–4) 58(100.0) - - Gentamycin (0.25–16) 58(100.0) - - Kanamycin (6–64) 58(100.0) - - Nalidixic acid (0.5–32) 58(100.0) - - Streptomycin (32–64) NI NI 56(94.7) Sulfizoxazole (16–512) 2(5.3) - 56(94.7) Tetracycline(4–32), 2(5.3) - 56(94.7)

Amikacin (0.5–64), 58(100.0) - -

sulfamethoxazole (4-76) 58(100.0) - -

Table 3. Number (%) of *Salmonella* isolates resistant/susceptible to various antimicrobials

Intermediate Isolates (%)

Resistant Isolates (%)

of *Salmonella* Isolated from Domestic Animals, Humans and Meat Products 223

*Salmonella* Typhimurium. AMR testing showed that all isolates were resistant to more than one of the antibiotics (Table 3). All but two of the isolates were resistant to more than two of the antibiotics tested with 96.6% (56 of 58) of the isolates showing MDR antibiogram. All isolates tested were susceptible to amikacin, cefoxitin, cetriaxone, ciprofloxacin, gentamycin, gentamycin, nalidixic acid, and trimethoprim-sulfamethoxazole (Table 3). Almost all the isolates recovered from this study had a similar antimicrobial pattern. Regardless of sampling period (1, 2, or 3), 29 (3 Salmonella serovars Typhimurium and 26 Salmonella serovars Typhimurium var Copenhagen) were positive for class I integron (280 bp product) while only two of the isolates showed a 233-bp PCR product using primers *intI2* thus suggesting the presence of integron 2. These two isolates also had integron 1. Upon PFGE analysis, 9 distinguishable *Salmonella* genotypes were identified. For clarity, the genotypes were numbered I to IX with genotype V (28 of 58; 46.6%) being the most prevalent followed by type VII (15 of 58; 25.9 %) (Figure 1). Genotypes I, II, and III had the least prevalence (1 of 58; 1.7 % each). From the 58 isolates, types IV, V, VII, VIII, and IX (38 of 58; 65.5 %) isolated from the cattle at two sampling periods were observed at a similarity level of 100 %. Type V (28 of 58 isolates; 48.2 %) genotypes comprised of the most common isolates; of the 28 isolates from type V, 8 of 28 (28.6%), 18 of 28 (64.3%), and 2 of 28 (7.1%) were derived from sampling 1, 2 and 3 respectively. (Figure1). The 2 isolates which were positive for both *Int* 1 and 2 belonged to genotypes I and IV, respectively. Sampling time had a significant effect on the recovery of *Salmonella* (*P =* 0.004) while pen (*P* = 0.79) did not. All 58 *Salmonella* isolates which were grouped into two clusters (d and e) and five single isolates (a, b, c, f, and g) were

National Veterinary Service Laboratories, Animal and Plant Health Inspection Services, U.S. Department of Agriculture, Ames, Iowa, for serotyping. PFGE assays on *Salmonella* cultures to investigate their genotypic relatedness were performed at the *E. coli* Reference Centre, Pennsylvania State University, University Park. The sample preparation, restriction digestion, electrophoresis, and gel staining for PFGE were accomplished following the CDCstandardized procedure as described (CDC, 2004) (http://www.cdc.gov/pulsenet/ protocols.htm). Restriction endonuclease *XbaI* (Roche Diagnostics Corporation, Indianapolis, IN) was used for restriction digestion of genomic DNA. The size standard used for all gels was *XbaI*-digested DNA from *Salmonella* Braenderup strain H9812 (American Type Culture Collection catalogue no. BAA-664), i.e. the universal size standard used by all PulseNet laboratories. Fingerprints were analyzed using BioNumerics software version 3.5 (Applied Maths, Austin, Texas). Strain relatedness was done based on previously recommended criteria (Gebreyes et al. 2006) using 'different bands' algorithm for clustering and the unweighted pair group for arithmetic means (UPGMA) tree-building approach with optimization of 1 and 0.5% position tolerance. Visual inspection of the patterns was performed as a final step for analysis.
