Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2007
Publication Date: 7/1/2007
Citation: Fratamico, P.M., Bagi, L.K. 2007. Comparison of methods for detection and isolation of cold-and freeze-stressed escherichia coli o157:h7 in raw ground beef. Journal of Food Protection. Vol.70(7):1663-1669. Interpretive Summary: E. coli O157:H7 is an important food-borne pathogen associated with cases and outbreaks of bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Foods of bovine origin, in particular, ground beef, are major vehicles of infection. Various methods have been described for detection of E. coli O157:H7 in food; however, relatively few of these have been evaluated for their ability to detect low levels of the pathogen that may have been sub-lethally injured due to conditions in the food environment and during food processing and storage, such as exposure to cold temperatures. Therefore, research was conducted to evaluate the sensitivity of cultural methods using three different growth media and four selective agar plating media for detection of cold- and freeze-stressed E. coli O157:H7 inoculated at low levels in ground beef. Our study also compared the sensitivity of the method based on enrichment and plating onto selective agars to that of a commercially-available immunoassay and a polymerase chain reaction (PCR) assay for detection of the pathogen. Analysis of the data indicated that it was 2.5 to 3 times more likely to obtain a positive result using freeze-stressed or non-stressed bacteria compared to cold-stressed bacteria, and 2.5 times more likely by plating than by using the immunoassay or the PCR. The overall performance of a commercially available growth medium known as Rapid-Check E. coli O157:H7 Enrichment Broth for enrichment of cold- and freeze-stressed E. coli O157:H7 present in low levels in ground beef was superior to that of the other media evaluated. This investigation provides information on improved methods that can be used by the food industry and regulatory agencies for detection of low levels of cold/freeze-stressed E. coli O157:H7 in ground beef, helping to ensure the microbiological safety of this food category.
Technical Abstract: A comparison was made of the relative efficiencies of three enrichment media, Rapid-Chek E. coli O157:H7 Enrichment Broth (REB), R&F Broth (RFB), and modified E. coli broth containing novobiocin (mEC+n), and four selective plating media for detection of cold- and freeze-stressed Escherichia coli O157:H7 in raw ground beef. Ground beef samples (25 g) were inoculated with less than or equal to 0.5 and less than or equal to 2 CFU of E. coli O157:H7 per gram and subjected to enrichment immediately or stored at 4 deg C for 72 h or at -20 deg C for 2 weeks prior to enrichment. After enrichment for 8 and 20 h, the cultures were diluted and plated onto BCM O157:H7(+) agar, CT-SMAC, CHROMagar O157, and Rainbow Agar O157 and also tested using the Rapid-Chek E. coli O157 lateral flow immunoassay and a multiplex PCR assay targeting the E. coli O157:H7 eae gene and the stx1 and stx2 genes. The number of E. coli O157:H7 obtained on the 4 agars were 4.0 to 7.9 log CFU/ml from the REB enrichment, 1.4 to 7.4 log CFU/ml from RFB, 1.7 to 6.7 log CFU/ml from mEC+n incubated at 42 deg C and 1.3 to 3.3 log CFU/ml from enrichment in mEC+n incubated at 35 deg C. In total, the percentage of positive results from ground beef samples containing non-stressed, cold-stressed, and freeze-stressed E. coli O157:H7 by plating, the immunoassay, and by the PCR were 97, 88, and 97%, respectively, using REB, 92, 81, and 78%, respectively, using RFB, 97, 58, and 53%, respectively, using mEC+n incubated at 42 deg C, and 22, 31, and 25%, respectively, using mEC+n incubated at 35 deg C. Analysis of the data indicated that a positive result was 1.1 times more likely to occur after 20 h of enrichment compared to 8 h, 25 times more likely using RFB and REB compared to mEC+n at 35 deg C, 3.7 times more likely using an initial inoculum of less than or equal to 2.0 CFU/g compared to less than or equal to 0.5 CFU/g, 2.5 to 3 times more likely using freeze-stressed or non-stressed bacteria compared to cold-stressed bacteria, and 2.5 times more likely by plating than by using the immunoassay or the PCR. Higher sensitivities were obtained by plating, the immunoassay, and the PCR for samples with non-stressed and freeze-stressed E. coli O157:H7 compared to samples that were cold-stressed. Although RFB was superior to enrichment in mEC+n, in particular, when the mEC+n enrichments were incubated at 35 deg C, overall,the highest percentages of positive samples by plating, the immunoassay, and the multiplex PCR were obtained with samples enriched in REB.