Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/1/2005
Publication Date: 8/1/2005
Citation: Bhaduri, S. 2005. Survival, injury, and virulence of freeze-stressed plasmid-bearing virulent yersinia enterocolitica in ground pork. Foodborne Pathogens and Disease. 2:4. Interpretive Summary: Yersinia enterocolitica is a bacterium that is a common cause of food-borne illness in humans. Swine are the only known reservoir for disease-causing Y. enterocolitica, and this bacterium is a serious concern to the pork production and processing industries in the United States. While Y. enterocolitica is prevalent in swine, relatively little is known concerning the ability of this organism to survive on pork products when stored under freezing conditions. Therefore, the effect of freezing at -20 deg C on the survival of pathogenic Y. enterocolitica in ground pork was examined. When ground pork was inoculated with pathogenic Y. enterocolitica, most of the bacteria were able to survive for at least twelve weeks storage at -20 deg C. Therefore, since pathogenic Y. enterocolitica survive freezing, consumption of contaminated frozen pork products that are not sufficiently cooked may still cause illness. This study also indicates that freezing is not a substitute for safe handling and proper cooking of pork.
Technical Abstract: Swine are a reservoir for Yersinia enterocolitica, an important human pathogen. However, relatively little is known concerning the ability of this organism to adapt to freeze-stress in raw pork products. Thus, we evaluated the effect of freeze-stress at -20 deg C on survival, or injury of the bacterium, and on the stability of the virulence plasmid, and expression of virulence-associated determinants of pathogenic Y. enterocolitica (YEP+)in ground pork. Samples were 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 ca. 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 numbers 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. This study also indicates that freezing is not a substitute for safe handling and/or proper cooking of pork.