Submitted to: United States Japan Natural Resources Protein Panel
Publication Type: Proceedings
Publication Acceptance Date: 9/20/2003
Publication Date: 11/9/2003
Citation: Fratamico, P.M., Bagi, L.K., Briggs, C.E., Debroy, C. 2003. Detection and characterization of pathogenic escherichia coli. United States Japan Natural Resources Protein Panel. p. 92-98.
Technical Abstract: A comparison was made of the relative efficiencies of three enrichment media, Rapid-Chek E. coli O157:H7 Enrichment Broth (REB), BCM Escherichia coli O157:H7 Enrichment Broth (BCM), and modified E. coli broth containing novobiocin (mEC+n), and four selective plating media for detection/isolation of cold-stressed Escherichia coli O157:H7 in raw ground beef. Ground beef samples (25 g) were inoculated with 1 to 5 or 10 to 50 CFU of E. coli O157:H7 and stored at 4ºC for 72 h or -20ºC for 2 weeks prior to enrichment. After enrichment at 42ºC with aeration or at 35ºC with no aeration for 8 and 20 h, the cultures were diluted and directly plated onto BCM O157:H7(+) agar, CT-SMAC, CHROMagar O157, and Rainbow Agar O157 plates. The cultures were also tested with the Rapid-Chek E. coli O157 lateral flow immunoassay cassette and with a multiplex PCR assay amplifying segments of the rfbEO157:H7, fliCh7, stx1, and stx2 genes. Generally, after 8 h of enrichment at 42ºC in REB and mEC+n, E. coli O157:H7 colonies were detected on all four selective agar media, even in samples inoculated at a level of 1 to 5 CFU/25g; however, the number of CFU/ml obtained was ca. 1 to 2 log10 higher using REB compared to mEC+n. E. coli O157:H7 was not detected by plating on the selective agars when inoculated at levels of 1 to 5 CFU/g of ground beef after 8 or 20 h of enrichment in mEC+n at 35ºC without aeration. In most cases, positive results were obtained with the Rapid-Chek E. coli O157 lateral flow cassette when levels of E. coli O157:H7 reached ca. 4 to 5 log10 CFU/ml; however, the organism was detected at levels lower than 4 log10 CFU/ml by the multiplex PCR in both REB and mEC+n enrichments incubated at 42ºC for 8 or 20 h. Results of this study indicate that enrichment in REB at 42ºC with aeration is superior to enrichment in mEC+n at 35ºC without aeration for the detection/isolation of cold-stressed E. coli O157:H7 by direct plating, the PCR, and by an immunoassay after 8 h of enrichment when inoculated at levels of 1 to 5 CFU/25 g of ground beef. In a related study, PCR assays were developed to detect enterohemorrhagic E. coli O103. To accomplish this, the DNA sequence of the 12,032-bp O antigen gene cluster of Escherichia coli O103 was determined, and 12 open reading frames were identified with all having the same transcriptional direction. Analyses of results indicated that the wzx (O antigen flippase) and wzy (O antigen polymerase) genes were E. coli serogroup O103 specific, thus regions in these two genes were chosen for development of PCR assays. The PCR assays showed specificity for E. coli O103, as PCR products of the expected sizes were not produced using DNA from other E. coli serogroups and other bacterial genera tested. Thus, the PCR assay can be employed to reliably identify E. coli serogroup O103 and to potentially detect the organism in food, fecal, or environmental samples.