Detection of pathogenic Escherichia coli on potentially contaminated beef carcasses using cassette PCR and conventional PCR

Abstract

Background Over a one year period, swabs of 820 beef carcasses were tested for the presence of Shiga toxin-producing Escherichia coli by performing Polymerase Chain Reaction (PCR) in a novel technology termed “cassette PCR”, in comparison to conventional liquid PCR. Cassette PCR is inexpensive and ready-to-use. The operator need only add the sample and press “go”. Cassette PCR can simultaneously test multiple samples for multiple targets. Carcass swab samples were first tested for the presence of STEC genes (O157, eae , stx1 and stx2 ). Samples were considered to be pathogenic if positive for eae plus stx1 and/or stx2 . For samples scored as pathogenic, further testing screened for 6 additional high frequency O-antigens (O26, O45, O103, O111, O121, and O145). Results Of the 820 samples, 41% were pathogenic and 30% were O157 positive. Of these, 19% of samples were positive for O157 and carried potentially pathogenic E. coli ( eae plus stx1 and/or stx2 ). Of all samples identified as carrying pathogenic E. coli , 18.9, 38.8, 41.4, 0, 36.1, and 4.1% respectively were positive for O26, O45, O103, O111, O121, and O145. To validate cassette PCR testing, conventional PCR using STEC primers was performed on each of the 820 samples. Only 148 of 3280 cassette PCR tests were discordant with conventional PCR results. However, further fractional testing showed that 110 of these 148 PCRs reflected low numbers of E. coli in the enrichment broth and could be explained as due to Poisson limiting dilution of the template, affecting both cassette PCR and conventional PCR. Of the remaining 38 discordant tests, 27 initial capillary PCRs and 10 initial conventional tests were nominally discordant between cassette and conventional PCR, perhaps reflecting human/technical error on both sides of the comparison. Conclusions Contaminated beef carcass swabs were often complex, likely harboring more than one strain of pathogenic E. coli . Cassette PCR had 98.8% concordance with parallel conventional PCR for detection of STEC genes. This indicates that cassette PCR is highly reliable for detecting multiple pathogens in beef carcass swabs from processing plants.

Shiga Toxin-Producing Escherichia coli Outbreaks in the United States, 2010–2017

“…The proportion of outbreaks associated with various food types for O157 and non-O157 were similar except for beef. It is not known why beef was the only category with significantly more O157 than non-O157 outbreaks given that non-O157 STEC are more commonly detected on beef carcasses [ 17 ]. One possibility is that detection of outbreaks might be correlated with production of Shiga toxin 2 (most STEC O157 produce only Shiga toxin 2); strains that produce only Shiga toxin 2 cause the most severe illnesses, followed by those that produce both Shiga toxins 1 and 2, then only Shiga toxin 1 [ 18 , 19 , 20 ].…”

Molecular characterization of Escherichia coli isolated from milk samples with regard to virulence factors and antibiotic resistance

“…Polymerase chain reaction (PCR) analysis has detected that all strains of E. coli possess these virulence genes. However, the procurement of one or more virulence genes does not place bacteria in a harmful grade if that strain has not harbored the proper virulence gene that initiates disease in specific species [9]. "The dispersal of antibacterial resistance genes has been noticed between E. coli isolates from human, animal, and environmental sources."…”

Quantitative Imaging of Bacteriophage Amplification for Rapid Detection of Bacteria in Model Foods

“…In addition, many molecular approaches such as PCR methods also rely on the enrichment of bacteria before detection. In many cases, these enrichment processes can range from 6 to 24 h before sample preparation steps, such as isolation and purification of nucleic acids for PCR assay (Choi et al, 2018;Manage et al, 2019). Therefore, there is a need to develop molecular-specific detection methods using low cost, stable reagent, simple sample preparation steps, detection within 6-8 h of sample collection, and reduce the need for specialized laboratory environments.…”