Evaluation of enrichment methods for recovery of Yersinia enterocolitica O:3 and O:8 from swine feeds and its transformation with pGFPuv vector

Part II. Swine are major reservoirs of Yersinia enterocolitica and pigs are easily colonized through oral infections. The objective of this study was to evaluate five different enrichment methods to recover Y. enterocolitica from swine feeds since optimal methods for isolation of Y. enterocolitica are not available for farm samples having a heavy background microflora. Recovery of Y. enterocolitica from swine feeds using enrichment was evaluated with an inoculum level of 1000,100, and 10 CFU/g feed. Enrichment media evaluated included: Phosphate Buffered Saline (PBS) at 4°C, PBS with sorbitol (1%) and bile salts (0.15%) (PSB) at 4°C and 21°C, ITC enrichment at 22°C and tryptic soy broth with polymyxin B(5 lU/ml) and novobiocin (10 µg/ml) (TSBPN) at 18°C. Incubation periods for enrichment were evaluated from 24 h up to 6 weeks. CIN, MacConkey (MAC) and SSDC agar plates were used for differential plating. KOH treatments at concentrations of 0.25% to 0.13% were evaluated. PBS at 4°C and PSB at 4°C or 21°C were not effective for recovery of Y. enterocolitica 0:3 and 0:8 from swine feeds. KOH treatment did not generally improve recovery of Y. enterocolitica in the methods studied except for recovery of 0:8 from MAC agars after two day enrichment in TSBPN. TSBPN at 18°C with 24-h incubation was better than any other methods evaluated for recovery of both 0:3 and 0:8, which showed good recovery up to 10 and 1000 CFU/g feed for 0:3 (89%) and 0:8 (100%), respectively. ITC enrichment gave good recovery (56%) of serotype 0:3 up to 10 CFU/g feed but was not effeetive for recovery of 0:8. CIN agar plates were preferred in this study due to the distinctive colony morphology and high selectivity. Overall, optimal recovery of Y. enterocolitica from swine feeds, based on these data, was achieved using TSBPN enrichment at 18°C with incubation for 24 h followed by differential plating on CIN agar at 30°C for 24 h. Part III. Green fluorescent proteins (GFPs) producing bacteria have been increasingly used in food microbiology area because of easy identification of it. GFP producing Y. enterocolitica has never been used in this area. Y. enterocolitica was transformed with the pGFPuv plasmid vector which encodes for GFP by using electroporation. Fluorescence of Y. enterocolitica colonies on Luria-Bertani (LB) and also CIN agars was visualized under UV light. The presence of pGFPuv vectors inside the cells was confirmed on agarose gel by electrophoresis at 3.3 kb. Because the presence of green fluorescent Y. enterocolitica can be easily identified, its use is promising for many applications such as evaluation of culture media for isolation of Y. enterocolitica from naturally contaminated materials. This technique is also very useful for evaluation of spatial location or gene expression of cells.