James E. Keen

Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing

A survey was performed to estimate the frequency of enterohemorrhagic Escherichia coli O157:H7 or O157:nonmotile (EHEC O157) in feces and on hides within groups of fed cattle from single sources (lots) presented for slaughter at meat processing plants in the Midwestern United States, as well as frequency of carcass contamination during processing from cattle within the same lots. Of 29 lots sampled, 72% had at least one EHEC O157-positive fecal sample and 38% had positive hide samples. Overall, EHEC O157 prevalence in feces and on hides was 28% (91 of 327) and 11% (38 of 355), respectively. Carcass samples were taken at three points during processing: preevisceration, postevisceration before antimicrobial intervention, and postprocessing after carcasses entered the cooler. Of 30 lots sampled, 87% had at least one EHEC O157-positive preevisceration sample, 57% of lots were positive postevisceration, and 17% had positive postprocessing samples. Prevalence of EHEC O157 in the three postprocessing samples was 43% (148 of 341), 18% (59 of 332) and 2% (6 of 330), respectively. Reduction in carcass prevalence from preevisceration to postprocessing suggests that sanitary procedures were effective within the processing plants. Fecal and hide prevalence were significantly correlated with carcass contamination (P = 0.001), indicating a role for control of EHEC O157 in live cattle.

Prevalence of Escherichia coli O157:H7 in range beef calves at weaning.

This study was designed to determine the prevalence of Escherichia coli O157:H7 infection of beef calves at weaning, prior to arrival at the feedlot or mixing with cattle from other sources. Fifteen range cow-calf herds, which weaned calves in October and November, were sampled in Kansas, Missouri, Montana, Nebraska and South Dakota. Faecal culture for E. coli O157:H7 was performed and anti-O157 serum antibody titres were determined by blocking ELISA. Thirteen of the 15 herds (87%) were found to have at least one positive isolation of E. coli O157:H7 in faecal samples. Within positive herds, prevalence ranged from 1.7-20.0%, with an average of 7.4+/-6.2% S.D. of individual animals shedding E. coli O157:H7 in faeces. All herds had high prevalence of anti-O157 antibodies, ranging 63-100% of individuals within herds seropositive. This study indicates that E. coli O157:H7 infection before weaning, prior to entry into feedlots, is widespread. Furthermore, serologic evidence suggests that most calves (83%) and all herds (100%) have been exposed to E. coli O157.

Mutations in the csgD Promoter Associated with Variations in Curli Expression in Certain Strains of Escherichia coli O157:H7

ABSTRACT Single-base-pair csgD promoter mutations in human outbreak Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 coincided with differential Congo red dye binding from curli fiber expression. Red phenotypecsgD::lacZ promoter fusions had fourfold-greater expression than white promoter fusions. Cloning the red variant csgDEFG operon into white variants induced the red phenotype. Substrate utilization differed between red and white variants.

Longitudinal Study of Escherichia coli O157:H7 in a Beef Cattle Feedlot and Role of High-Level Shedders in Hide Contamination

ABSTRACT The objectives of the study described here were (i) to investigate the dynamics of Escherichia coli O157:H7 fecal and hide prevalence over a 9-month period in a feedlot setting and (ii) to determine how animals shedding E. coli O157:H7 at high levels affect the prevalence and levels of E. coli O157:H7 on the hides of other animals in the same pen. Cattle (n = 319) were distributed in 10 adjacent pens, and fecal and hide levels of E. coli O157:H7 were monitored. When the fecal pen prevalence exceeded 20%, the hide pen prevalence was usually (25 of 27 pens) greater than 80%. Sixteen of 19 (84.2%) supershedder (>104 CFU/g) pens had a fecal prevalence greater than 20%. Significant associations with hide and high-level hide (≥40 CFU/100 cm2) contamination were identified for (i) a fecal prevalence greater than 20%, (ii) the presence of one or more high-density shedders (≥200 CFU/g) in a pen, and (iii) the presence of one or more supershedders in a pen. The results presented here suggest that the E. coli O157:H7 fecal prevalence should be reduced below 20% and the levels of shedding should be kept below 200 CFU/g to minimize the contamination of cattle hides. Also, large and unpredictable fluctuations within and between pens in both fecal and hide prevalence of E. coli O157:H7 were detected and should be used as a guide when preharvest studies, particularly preharvest intervention studies, are designed.

Animal-to-animal variation in fecal microbial diversity among beef cattle.

ABSTRACT The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.

Variations in the csgD Promoter of Escherichia coli O157:H7 Associated with Increased Virulence in Mice and Increased Invasion of HEp-2 Cells

ABSTRACT Promoter alterations in the csgD gene of Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 are associated with variations in curli expression and the ability to bind Congo red dye. Red variants of each strain were more invasive for cultured HEp-2 cells than were white variants. An ATCC 43895 red variant was more virulent than a white variant in a mouse model. However, there were no differences in Shiga toxin production between red and white variants.

Genotypic Analyses of Escherichia coli O157:H7 and O157 Nonmotile Isolates Recovered from Beef Cattle and Carcasses at Processing Plants in the Midwestern States of the United States

ABSTRACT Escherichia coli O157:H7 and O157 nonmotile isolates (E. coli O157) previously were recovered from feces, hides, and carcasses at four large Midwestern beef processing plants (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999–3003, 2000). The study implied relationships between cattle infection and carcass contamination within single-source lots as well as between preevisceration and postprocessing carcass contamination, based on prevalence. These relationships now have been verified based on identification of isolates by genomic fingerprinting.E. coli O157 isolates from all positive samples were analyzed by pulsed-field gel electrophoresis of genomic DNA after digestion with XbaI. Seventy-seven individual subtypes (fingerprint patterns) grouping into 47 types were discerned among 343 isolates. Comparison of the fingerprint patterns revealed three clusters of isolates, two of which were closely related to each other. Remarkably, isolates carrying both Shiga toxin genes and nonmotile isolates largely fell into specific clusters. Within lots analyzed, 68.2% of the postharvest (carcass) isolates matched preharvest (animal) isolates. For individual carcasses, 65.3 and 66.7% of the isolates recovered postevisceration and in the cooler, respectively, matched those recovered preevisceration. Multiple isolates were analyzed from some carcass samples and were found to include strains with different genotypes. This study suggests that mostE. coli O157 carcass contamination originates from animals within the same lot and not from cross-contamination between lots. In addition, the data demonstrate that most carcass contamination occurs very early during processing.

Shiga-toxigenic Escherichia coli O157 in agricultural fair livestock, United States.

Organisms were common in ruminants, swine, and pest flies.

Genetic Diversity among Clonal Lineages within Escherichia coli O157:H7 Stepwise Evolutionary Model

Molecular characterization and subtyping show genetic diversities within clonal complexes.

Association of Escherichia coli O157:H7 tir polymorphisms with human infection

BackgroundEmerging molecular, animal model and epidemiologic evidence suggests that Shiga-toxigenic Escherichia coli O157:H7 (STEC O157) isolates vary in their capacity to cause human infection and disease. The translocated intimin receptor (tir) and intimin (eae) are virulence factors and bacterial receptor-ligand proteins responsible for tight STEC O157 adherence to intestinal epithelial cells. They represent logical genomic targets to investigate the role of sequence variation in STEC O157 pathogenesis and molecular epidemiology. The purposes of this study were (1) to identify tir and eae polymorphisms in diverse STEC O157 isolates derived from clinically ill humans and healthy cattle (the dominant zoonotic reservoir) and (2) to test any observed tir and eae polymorphisms for association with human (vs bovine) isolate source.ResultsFive polymorphisms were identified in a 1,627-bp segment of tir. Alleles of two tir polymorphisms, tir 255 T>A and repeat region 1-repeat unit 3 (RR1-RU3, presence or absence) had dissimilar distributions among human and bovine isolates. More than 99% of 108 human isolates possessed the tir 255 T>A T allele and lacked RR1-RU3. In contrast, the tir 255 T>A T allele and RR1-RU3 absence were found in 55% and 57%, respectively, of 77 bovine isolates. Both polymorphisms associated strongly with isolate source (p < 0.0001), but not by pulsed field gel electrophoresis type or by stx1 and stx2 status (as determined by PCR). Two eae polymorphisms were identified in a 2,755-bp segment of 44 human and bovine isolates; 42 isolates had identical eae sequences. The eae polymorphisms did not associate with isolate source.ConclusionPolymorphisms in tir but not eae predict the propensity of STEC O157 isolates to cause human clinical disease. The over-representation of the tir 255 T>A T allele in human-derived isolates vs the tir 255 T>A A allele suggests that these isolates have a higher propensity to cause disease. The high frequency of bovine isolates with the A allele suggests a possible bovine ecological niche for this STEC O157 subset.