Detection by multiplex real-time PCR assays and isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 in ground beef

Authors: Fratamico, Pina; Bagi, Lori; Cray Jr, William; Narang, Neelam; Yan, Xianghe; Medina, Marjorie; Liu, Yanhong

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/2/2010
Publication Date: 1/1/2011
Citation: Fratamico, P.M., Bagi, L.K., Cray Jr, W.C., Narang, N., Medina, M.B., Liu, Y. 2011. Detection by multiplex real-time PCR assays and isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 in ground beef. Foodborne Pathogens and Disease. DOI: 10.1089=fpd.2010.0773.

Interpretive Summary: Food-borne pathogenic bacteria known as non-O157 Shiga toxin-producing Escherichia coli (STEC) belonging to six specific types (also known as serogroups), namely serogroups O26, O45, O103, O111, O121, and O145, are responsible for the majority of non-O157 STEC infections in the United States, representing a growing public health concern. Cattle and other ruminants are reservoirs for these pathogens, thus food of bovine origin may be a vehicle for infection with non-O157 STEC. Methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to develop intervention strategies. This study describes a method for detection of these pathogens in ground beef, which consists of polymerase chain reaction assays targeting genes that are specific for each of the STEC serogroups and a method for isolating the target pathogen for further confirmation of its identity. The pathogens were detected when present in low levels in ground beef, and were able to be readily isolated and confirmed. This work provides a method for detection and isolation in ground beef and potentially other foods of non-O157 STEC serogroups of major public health concern.

Technical Abstract: Six Shiga toxin-producing Escherichia coli (STEC) serogroups, which include serogroup O26, O45, O103, O111, O121, and O145, are responsible for the majority of non-O157 STEC infections in the United States, representing a growing public health concern. Cattle and other ruminants are reservoirs for these pathogens, thus food of bovine origin may be a vehicle for infection with non-O157 STEC. Methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to develop intervention strategies. This study describes a method for detection of non-O157 STEC in ground beef, consisting of: enrichment in mTSB at 42 deg C, followed by real-time multiplex PCR assays targeting stx1, stx2, and eae genes and the wzx gene in the O-antigen gene clusters of the six serogroups, and then immunomagnetic separation (IMS) followed by plating onto Rainbow Agar O157 and PCR assays for confirmation of isolates. All ground beef samples artificially inoculated with 1-2 and 10-20 CFU/25 g of ground beef consistently gave positive results for all of the target genes, including the internal amplification control using the multiplex real-time PCR assays after enrichment in mTSB for a total of 24 h (6 h at 37 deg C and 18 h at 42 deg C). The detection limit of the real-time multiplex PCR assays was ca. 50 CFU per PCR. IMS for O26, O103, O111, and O145 was performed with commercially available magnetic beads, and the IMS beads for O45 and O121 were prepared using polyclonal antiserum against these serogroups. A large percentage of the presumptive colonies of each serogroup picked from Rainbow Agar O157 were confirmed as the respective serogroups; however, the percent recovery of STEC O111 was lower than that of the other serogroups. This work provides a method for detection and isolation in ground beef and potentially other foods of non-O157 STEC of major public health concern.