Multiplex Quantitative PCR Assays for the Detection and Quantification of the Six Major Non-O157 Escherichia coli Serogroups in Cattle Feces

Abstract

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, called non-O157 STEC, are important foodborne pathogens. Cattle, a major reservoir, harbor the organisms in the hindgut and shed them in the feces. Although limited data exist on fecal shedding, concentrations of non-O157 STEC in feces have not been reported. The objectives of our study were (i) to develop and validate two multiplex quantitative PCR (mqPCR) assays, targeting O-antigen genes of O26, O103, and O111 (mqPCR-1) and O45, O121, and O145 (mqPCR-2); (ii) to utilize the two assays, together with a previously developed four-plex qPCR assay (mqPCR-3) targeting the O157 antigen and three virulence genes (stx1, stx2, and eae), to quantify seven serogroups and three virulence genes in cattle feces; and (iii) to compare the three mqPCR assays to a 10-plex conventional PCR (cPCR) targeting seven serogroups and three virulence genes and culture methods to detect seven E. coli serogroups in cattle feces. The two mqPCR assays (1 and 2) were shown to be specific to the target genes, and the detection limits were 4 and 2 log CFU/g of pure culture-spiked fecal samples, before and after enrichment, respectively. A total of 576 fecal samples collected from a feedlot were enriched in E. coli broth and were subjected to quantification (before enrichment) and detection (after enrichment). Of the 576 fecal samples subjected, before enrichment, to three mqPCR assays for quantification, 175 (30.4%) were quantifiable (≥4 log CFU/g) for at least one of the seven serogroups, with O157 being the most common serogroup. The three mqPCR assays detected higher proportions of postenriched fecal samples (P > 0.01) as positive for one or more serogroups compared with cPCR and culture methods. This is the first study to assess the applicability of qPCR assays to detect and quantify six non-O157 serogroups in cattle feces and to generate data on fecal concentration of the six serogroups.

Non-O157 Shiga toxin-producing Escherichia coli—A poorly appreciated enteric pathogen: Systematic review

“…Detecting STEC strains in the feces of cattle, the source of many of these infections, is challenging; however, researchers have begun identifying non-O157 strains using qPCR (Shridhar et al, 2016). In a study comparing culture versus PCR-based methods in detecting non-O157 strains, researchers found that each method detected one or more serogroups that the other method found negative, concluding that both methods are required for accurate detection (Noll et al, 2015).…”

“…The reservoirs and vehicles of transmission of non-O157 E. coli strains resemble those seen for O157 strains. Cattle, goats, and sheep, which harbor the pathogen in their hindguts and shed them in feces (Shridhar et al, 2016), have been shown to be important reservoirs for non-O157 as well as O157 STEC. Researchers examining naturally infected beef cows and steer calves found that non-O157 STEC shedding is highest in younger animals at the stage of post-weaning and before entry into the feedlot (Ekiri et al, 2014).…”

Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays

“…Because Stx is highly potent, the presence of stx genes is tested routinely for distinguishing the virulent strains from commensal strains for epidemiological studies, outbreak investigations and food monitoring. E. coli O157 and other STEC strains are detected by microbiological procedures and multiplex PCR [ 10 , 11 , 12 , 13 ]. The process takes a long time, as the cells have to be grown for several hours before testing can be conducted.…”

Comparison of Agar Media for Detection and Quantification of Shiga Toxin–Producing Escherichia coli in Cattle Feces

“…Testing of more colonies may give a more accurate concentration value. Although the spiral plate method used here only tested 10 colonies from an agar plate, the benefit of using this approach is that it can test whether multiple genes originate from one organism, unlike current real-time PCR assays (1,7,31). Most suspensions were quantifiable when concentrations were above 10 4 CFU/g.…”

“…Concentration has been determined by use of spiral plate methods for STEC O157:H7 (3,28) or real-time PCR assays that quantify total STEC load or specific serogroups of STEC (1,7,22,31). Real-time PCR assays using multiple targets can detect genes contributed by multiple organisms.…”