Evaluation of Cattle for Naturally Colonized Shiga Toxin-Producing Escherichia coli Requires Combinatorial Strategies

Abstract

Shiga toxin-producing Escherichia coli (STEC) serogroups O157, O26, O103, O111, O121, O145, and O45 are designated as food adulterants by the U.S. Department of Agriculture-Food Safety and Inspection Service. Cattle are the primary reservoir of these human pathogens. In this study, 59 Angus crossbred heifers were tested specifically for these seven STEC serogroups using a combination of standard culture, serological, PCR, and cell cytotoxicity methods to determine if comparable results would be obtained. At the time of fecal sampling, the animals were approximately 2 years old and weighed 1000–1200 lbs. The diet comprised of 37% ground alfalfa hay, 25% ground Sudan hay, and 38% ground corn supplemented with trace minerals and rumensin with ad libitum access to water. Non-O157 STEC were isolated from 25% (15/59) of the animals tested using a combination of EC broth, CHROMagar STECTM, and Rainbow Agar O157. Interestingly, the O157 serogroup was not isolated from any of the animals. Non-O157 STEC isolates were confirmed to be one of the six adulterant serogroups by serology and/or colony PCR in 10/15 animals with the predominant viable, serogroup being O103. PCR using DNA extracted from feces verified most of the colony PCR results but also identified additional virulence and O-antigen genes from samples with no correlating culture results. Shiga toxin- (Stx-) related cytopathic effects on Vero cells with fecal extracts from 55/59 animals could only be associated with the Stx gene profiles obtained by fecal DNA PCR and not culture results. The differences between culture versus fecal DNA PCR and cytotoxicity assay results suggest that the latter two assays reflect the presence of nonviable STEC or infection with STEC not belonging to the seven adulterant serogroups. This study further supports the use of combinatorial culture, serology, and PCR methods to isolate viable STEC that pose a greater food safety threat.

Escherichia coli 0157:H7 virulence factors and the ruminant reservoir

“…This may lead to the emergence of new, potentially more pathogenic strains colonizing cattle [173]. Combinatorial strategies using culture, serology, and PCR methods to identify STEC that pose a greater food safety threat are necessary [174].…”

Comparative evaluation of antimicrobial activity of human granulysin, bovine and porcine NK-lysins against Shiga toxin-producing Escherichia coli O157:H7

“…Plates were read after overnight incubation at 37˚C and colonies that did not ferment sorbitol or utilize 4-methylumbelliferyl-β-d-glucuronide (non-fluorescent under UV light) were confirmed to be O157 via latex agglutination ( E . coli O157 latex, Oxoid Diagnostic Reagents, Oxoid Ltd., Hampshire, UK) [ 32 , 33 ].…”

“…Bacterial cultures were prepared by growing each isolate from a single colony in 5 mL LB broth (Sigma-Aldrich) per assay for 20 h at 37˚C with constant shaking at 150 rpm. Assay isolates were streaked on Difco sorbitol MacConkey agar (BD Biosciences, Franklin Lakes, NJ) containing 4-methylumbelliferyl-β-d-glucuronide (100 mg/L; Sigma-Aldrich) (SMAC-MUG) or SMAC-MUG supplemented with cefixime (50 μg/L), potassium tellurite (2.5 mg/L), and vancomycin (40 mg/L) (SMAC-CTMV) agar [32,33]. Plates were read after overnight incubation at 37˚C and colonies that did not ferment sorbitol or utilize 4-methylumbelliferyl-β-d-glucuronide (non-fluorescent under UV light) were confirmed to be O157 via latex agglutination (E. coli O157 latex, Oxoid Diagnostic Reagents, Oxoid Ltd., Hampshire, UK) [32,33].…”

Phenotypic and genomic comparison of human outbreak and cattle-associated Shiga toxin-producing Escherichia coli O157:H7

“…Fecal samples were collected six- or nine-days pre-challenge, on the day of challenge, and on days 1, 2, 3, 4, 5, 7, 9, 11, and 14 relative to day of challenge. Bacterial enumeration of fecal E. coli O157 was performed by direct and enrichment culture on selective media, as previously described (85). Briefly, non-enrichment O157 culture enumeration involved placing 10g of feces in 50ml of tryptic soy broth supplemented with cefixime (50 µg/liter), tellurite (2.5 mg/liter), and vancomycin (40 mg/liter) (TSB-CTV) with vortexing to generate a homogenous suspension.…”