Prevalence of heat-resistant bacteria in beef poses a potential problem as thermal interventions are routinely used in beef processing to control contamination. Despite extreme heat-resistant (XHR) Escherichia coli having been isolated from a ground beef processing plant, there has not been a study to assess the prevalence of XHR E. coli among types of cattle. Therefore, this study used a screening assay for XHR gram-negative bacteria and its molecular determinant, the locus of heat resistance (LHR), on feces collected from U.S. cattle. Fecal samples were collected from fed (n = 538), cull dairy (n = 425), and cull beef (n = 475) cattle at nine regional beef processing plants located across the United States. Among the 1,438 cattle sampled from northern (n = 288), southern (n = 288), eastern (n = 287), western (n = 287), and central (n = 288) regions of the United States, 91 (6.3%) cattle showed presence of XHR bacteria, as evident by growth in MacConkey broth following heat treatment of 80°C for 15 min, in their feces. Heat-resistant bacteria (n = 140) were isolated from the 91 fecal samples. Prevalence of XHR bacteria was highest (11%) in cattle from the northern region. Ninety percent of the XHR isolates were identified as E. coli. Multiplex PCR of all 1,438 fecal samples showed that the LHR was absent in 40.7% of samples and intact in 18.7% of samples. Despite the higher prevalence of intact LHR from PCR analysis, only 11 samples (0.8%) were confirmed to contain bacteria with an intact LHR. The LHR was absent in 91% of XHR bacteria, and only 7.9% of XHR bacteria had intact LHR, suggesting a novel mechanism of heat resistance. By developing and using the screening assays, we established the prevalence of XHR bacteria (6.3%) and LHR+ bacteria (0.8%) in U.S. beef cattle.

  • A screening assay for extreme heat-resistant bacteria in cattle fecal samples was developed.

  • A four-plex PCR assay can detect LHR in fecal samples and bacterial isolates.

  • Prevalence of XHR bacteria and the intact LHR in feces of U.S. cattle is low.

  • A novel mechanism(s) of heat resistance in XHR bacteria may exist.

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