Salmonella Isolation and PFGE Molecular Characterization

Feces in selenite was incubated overnight at 42°C for Salmonella enrichment (52). A 10 μl loopful of the overnight enrichment was plated onto xylose lysine deoxycholate (XLD) and brilliant green (BG) plates (Remel Inc., Lenexa, KS) and incubated overnight at 37°C as previously described (10, 53). H₂S-positive, black colonies were picked and subcultured onto blood agar plates (tryptic soy agar with 5% sheep blood). Final Salmonella confirmation was determined with the following tests on a single isolated colony: citrate, triple sugar iron (TSI), and motility-indole-ornithine media (Beckton and Dickson, Franklin Lakes, NJ); and a whole-cell agglutination test using Salmonella-specific poly A-I and Vi antiserum (Fisher Scientific, Pittsburgh, PA). Microbial identification as Salmonella was based on possessing all of the following criteria. Salmonella grows on TSI slant producing a red slant, yellow/black (H₂S-production) butt, and gas. In addition, Salmonella is motile, citrate-positive, indole and ornithine negative and agglutinates with poly A-I/Vi antiserum (54). Samples were considered culture negative if no black or pink colonies were observed on XLD or BG sections, respectively. A delayed-secondary enrichment was done for samples that were culture negative after the primary enrichment and initial plating on XLD and BG. A 10 μl loopful of the secondary enrichment in selenite overnight was plated onto XLD and BG plates. Salmonella identification of suspect colonies was confirmed as previously stated. Isolates were forwarded to the National Veterinary Service Laboratory (NVSL) at Ames, Iowa, for definitive Salmonella serotyping.

At the time of sample submission, PFGEs were still the primary method utilized by the CDC to determine genetic relatedness among Salmonella isolates by comparison with human isolates in the CDC PulseNet USA national database. Agarose plugs and PFGE conditions were performed as previously described (53, 55–57). Electrophoresis was done using the CHEF DR II electrophoresis unit (Bio-Rad; Hercules, CA), with 0.5X Tris-borate-EDTA buffer (Sigma-Aldrich; St. Louis, MO); 6 V/cm with pulse times 2.25–63.85 s at 14°C for 15.5 h. A master database of Salmonella PFGE patterns in BioNumerics (Applied Maths; Austin, TX) contains over 1,000 PFGE entries for Salmonella isolated from water and various animal species (10, 11, 21). Comparisons were made between PFGE patterns in BioNumerics using Dice coefficient and unweighted pair group method of arithmetic averages (UPGMA) clustering. Clusters were based on a 75% similarity cut-off (21). Turtle isolates were also compared to archived isolates previously acquired from animal and water samples from the Oconee River watershed (21).