Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 15, 2005
Publication Date: May 7, 2005
Citation: Bhaduri, S. 2005. Effect of freeze-stress on survival, injury, and virulence of plasmid-bearing virulent Yersinia enterocolitica in ground pork. American Society for Microbiology Abstract. Paper No. P-032. Technical Abstract: Swine are a reservoir for Yersinia enterocolitica, an important human pathogen. Since relatively little is known concerning the ability of this organism to adapt to freeze-stress in raw pork products, the effect of freeze-stress on survival, injury, the stability of the virulence plasmid, and expression of virulence-associated determinants of pathogenic Y. enterocolitica in ground pork was assessed. Ten-gram portions of ground pork were irradiated in an irradiator (42kGy dose of cesium137 at -30 deg C) and artificially contaminated with 107 CFU/g of plasmid-bearing Y. enterocolitica (YEP+) strain GER (serotype O:3). The samples were held at -20 deg C for 0, 2, 4, 6, 10, and 12 weeks. At the various time intervals, triplicate samples were removed from the freezer, 90 ml of 0.1% peptone water was added, and the samples were pummeled in a Stomacher Lab Blender for 2 min. Serial dilutions were surface plated onto non-selective brain heart infusion (BHI) agar (BHA) and the selective media, Cefsulodin-Irgasan-Novobiocin (CIN) agar and MacConkey (MAC) agar, to enumerate surviving YEP+ colonies. Samples were also plated onto BHA with 1.5% and 2% NaCl (BHAS) and BHA with 1% sodium pyruvate (BHAP) to enumerate injured YEP+ cells. The presence of the virulence plasmid in the freeze-stressed YEP+ cells was assessed by Congo red (CR) binding on CR BHI agarose (CR-BHO). All plates were incubated for 24 h at 37 deg C. The viable counts on BHA, CIN agar, MAC agar, BHAP, BHAS, and CR-BHO after two weeks of freeze-stress were similar to counts obtained before freezing, indicating that the YEP+ strain survived the freezing process. The initial inoculum level decreased from 7.4 to 7.1 log10 CFU/g after 12 weeks of frozen storage with no evidence of freeze-stress injury as evidenced by similar number of viable counts on BHAP and BHAS. Results of a multiplex PCR targeting the chromosomal ail gene and a plasmid-associated virF gene confirmed that YEP+ isolates from CR-BHO possessed the virulence plasmid. Moreover, in vitro virulence assays indicate that these freeze-stressed isolates are potentially capable of causing food borne illness.