Distribution of Escherichia coli O157:H7 in ground beef: Assessing the clustering intensity for an industrial-scale grinder and a low and localized initial contamination.

Undercooked ground beef is regularly implicated in food-borne outbreaks involving pathogenic Shiga toxin-producing Escherichia coli. The dispersion of bacteria during mixing processes is of major concern for quantitative microbiological risk assessment since clustering will influence the number of bacteria the consumers might get exposed to as well as the performance of sampling plans used to detect contaminated ground beef batches. In this study, batches of 25kg of ground beef were manufactured according to a process mimicking an industrial-scale grinding with three successive steps: primary grinding, mixing and final grinding. The ground beef batches were made with 100% of chilled trims or with 2/3 of chilled trims and 1/3 of frozen trims. Prior grinding, one beef trim was contaminated with approximately 106-107CFU of E. coli O157:H7 on a surface of 0.5cm2 to reach a concentration of 10-100cells/g in ground beef. The E. coli O157:H7 distribution in ground beef was characterized by enumerating 60 samples (20 samples of 5g, 20 samples of 25g and 20 samples of 100g) and fitting a Poisson-gamma model to describe the variability of bacterial counts. The shape parameter of the gamma distribution, also known as the dispersion parameter reflecting the amount of clustering, was estimated between 1.0 and 1.6. This k-value of approximately 1 expresses a moderate level of clustering of bacterial cells in the ground beef. The impact of this clustering on the performance of sampling strategies was relatively limited in comparison to the classical hypothesis of a random repartition of pathogenic cells in mixed materials (purely Poisson distribution instead of Poisson-gamma distribution).

Prevalence of carriage of Shiga toxin-producing Escherichia coli serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among slaughtered adult cattle in France.

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these "top five" STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples.O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P<0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover,simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.

Intimin gene (eae) subtype-based real-time PCR strategy for specific detection of Shiga toxin-producing Escherichia coli serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in cattle feces.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, ß1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae, and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.