Terrance M. Arthur

Seasonal Prevalence of Shiga Toxin–Producing Escherichia coli, Including O157:H7 and Non-O157 Serotypes, and Salmonella in Commercial Beef Processing Plants†

The seasonal prevalence of Escherichia coli O157:H7, Salmonella, non-O157 E. coli (STEC), and stx-harboring cells was monitored at three Midwestern fed-beef processing plants. Overall, E. coli O157:H7 was recovered from 5.9% of fecal samples, 60.6% of hide samples, and 26.7% of carcasses sampled before the preevisceration wash. This pathogen also was recovered from 1.2% (15 of 1,232) of carcasses sampled at chilling (postintervention) at approximate levels of <3.0 cells per 100 cm2. In one case, the E. coli O157:H7 concentration dropped from ca. 1,100 cells per 320 cm2 at the preevisceration stage to a level that was undetectable on ca. 2,500 cm2 at the postintervention stage. The prevalence of E. coli O157:H7 in feces peaked in the summer, whereas its prevalence on hide was high from the spring through the fall. Overall, Salmonella was recovered from 4.4, 71.0, and 12.7% of fecal, hide, and preevisceration carcass samples, respectively. Salmonella was recovered from one postintervention carcass (of 1,016 sampled). Salmonella prevalence peaked in feces in the summer and was highest on hide and preevisceration carcasses in the summer and the fall. Non-O157 STEC prevalence also appeared to vary by season, but the efficiency in the recovery of isolates from stx-positive samples ranged from 37.5 to 83.8% and could have influenced these results. Cells harboring stx genes were detected by PCR in 34.3, 92.0, 96.6, and 16.2% of fecal, hide, preevisceration carcass, and postintervention carcass samples, respectively. The approximate level of non-O157 STEC and stx-harboring cells on postintervention carcasses was > or = 3.0 cells per 100 cm2 for only 8 of 199 carcasses (4.0%). Overall, the prevalence of E. coli O157:H7, Salmonella, and non-O157 STEC varied by season, was higher on hides than in feces, and decreased dramatically, along with pathogen levels, during processing and during the application of antimicrobial interventions. These results demonstrate the effectiveness of the current interventions used by the industry and highlight the significance of hides as a major source of pathogens on beef carcasses.

Post-harvest interventions to reduce/eliminate pathogens in beef.

In 1999 the foodborne pathogens Salmonella, Listeria, Campylobacter, and Escherichia coli (both O157 and non-O157) were estimated to cause more than 6 million illnesses and approximately 9000 deaths each year. However, the most recent Centers for Disease Control and Prevention report on the sources and incidence of foodborne disease, released in 2004, has shown a dramatic decrease in E. coli O157:H7 infections. Since raw beef products are the most frequently foodborne sources of these pathogens, the results of this report demonstrate that the microbiological quality of raw beef has improved greatly. During the intervening years, post-harvest interventions have continually improved, with new attention to hide decontamination and innovative treatments of carcasses. In addition, a system to hold and test beef trim or ground beef for E. coli O157:H7 before its release into commerce has provided an even greater level of safety. In this paper, we review the latest information on the prevalence of E. coli O157:H7 and other pathogens on beef, the evidence identifying the hide as the primary source of pathogens on beef carcasses, the efficacy of various hide and carcass interventions, and other developments that have led or have the potential to lead to even greater improvements in the microbial quality of beef.

Prevalence and Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli on Carcasses in Commercial Beef Cattle Processing Plants

ABSTRACT Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic E. coli hlyA, eae, and at least one stx gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (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) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beef-processing industry effected a sevenfold reduction in carcass contamination by STEC.

Prevalence of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella in two geographically distant commercial beef processing plants in the United States

For two large beef processing plants, one located in the southern United States (plant A) and one located in the northern United States (plant B), prevalence of Escherichia coli O157:H7, Listeria spp., Listeria monocytogenes, and Salmonella was determined for hide, carcass, and facility environmental samples over the course of 5 months. The prevalence of E. coli O157:H7 (68.1 versus 55.9%) and Salmonella (91.8 versus 50.3%) was higher (P < 0.05), and the prevalence of Listeria spp. (37.7 versus 75.5%) and L. monocytogenes (0.8 versus 18.7%) was lower (P < 0.05) for the hides of cattle slaughtered at plant A versus plant B. Similarly, the prevalence of Salmonella (52.0 versus 25.3%) was higher (P < 0.05) and the prevalence of Listeria spp. (12.0 versus 40.0%) and L. monocytogenes (1.3 versus 14.7%) was lower (P < 0.05) for the fence panels of the holding pens of plant A versus plant B. The prevalence of E. coli O157:H7 (3.1 versus 10.9%), Listeria spp. (4.5 versus 14.6%), and L. monocytogenes (0.0 versus 1.1%) was lower (P < 0.05) for preevisceration carcasses sampled at plant A versus plant B. Salmonella (both plants), Listeria spp. (plant B), and L. monocytogenes (plant B) were detected on fabrication floor conveyor belts (product contact surfaces) late during the production day. For plant B, 21 of 148 (14.2%) late-operational fabrication floor conveyor belt samples were L. monocytogenes positive. For plant B, E. coli O157:H7 and L. monocytogenes were detected in preoperational fabrication floor conveyor belt samples. Overall results suggest that there are regional differences in the prevalence of pathogens on the hides of cattle presented for harvest at commercial beef processing plants. While hide data may reflect the regional prevalence, the carcass data is indicative of differences in harvest practices and procedures in these plants.

Transportation and lairage environment effects on prevalence, numbers, and diversity of Escherichia coli O157:H7 on hides and carcasses of beef cattle at processing.

Hide has been established as the main source of carcass contamination during cattle processing; therefore, it is crucial to minimize the amount of Escherichia coli O157:H7 on cattle hides before slaughter. Several potential sources of E. coli O157: H7 are encountered during transportation and in the lairage environment at beef-processing facilities that could increase the prevalence and numbers of E. coli O157:H7 on the hides of cattle. On three separate occasions, samples were obtained from cattle at the feedlot and again after cattle were stunned and exsanguinated at the processing plant (286 total animals). The prevalence of E. coli O157:H7 on hides increased from 50.3 to 94.4% between the time cattle were loaded onto tractor-trailers at the feedlot and the time hides were removed in the processing plant. Before transport, nine animals had E. coli O157:H7 in high numbers (> 0.4 CFU/cm2) on their hides. When sampled at the slaughter facility, the number of animals with high hide numbers had increased to 70. Overall, only 29% of the E. coli O157:H7 isolates collected postharvest (221 of 764) matched pulsed-field gel electrophoresis types collected before transport. The results of this study indicate that transport to and lairage at processing plants can lead to increases in the prevalence and degree of E. coli O157:H7 contamination on hides and the number of E. coli O157:H7 pulsed-field gel electrophoresis types associated with the animals. More study is needed to confirm the mechanism by which additional E. coli O157:H7 strains contaminate cattle hides during transport and lairage and to design interventions to prevent this contamination.

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.

Salmonella and Escherichia coli O157:H7 Contamination on Hides and Carcasses of Cull Cattle Presented for Slaughter in the United States: an Evaluation of Prevalence and Bacterial Loads by Immunomagnetic Separation and Direct Plating Methods

ABSTRACT The hide and carcass hygiene of cull cattle at slaughter in four geographically distant regions of the United States was examined from July 2005 to April 2006 by measuring the aerobic plate counts (APC) and the prevalences and loads of Salmonella and Escherichia coli O157:H7. The geometric mean log10 APC CFU/100 cm2 levels on hides and preevisceration and postintervention carcasses ranged from 6.17 to 8.19, 4.24 to 6.47, and 1.46 to 1.96, respectively, and were highest in the summer (P < 0.0001). The average prevalences of Salmonella on hides and preevisceration and postintervention carcasses were 89.6% (95% confidence interval [CI], 85.1 to 94.0), 50.2% (95% CI, 40.9 to 59.5), and 0.8% (95% CI, 0.18 to 1.42), respectively. The prevalences of E. coli O157:H7 were 46.9% (95% CI, 37.3 to 56.6) and 16.7% (95% CI, 9.8 to 23.6) on hides and preevisceration carcasses, respectively. Examination of the concomitant incidence of Salmonella and E. coli O157:H7 showed that, on average, 33.3% (95% CI, 15.9 to 69.8) of cattle hide and 4.1% (95% CI, 0.98 to 17.3) of preevisceration carcass samples were contaminated with both pathogens. The pathogen prevalence on hides and carcasses was not significantly affected by the season; however, significant differences were observed between plants with respect to the incoming pathogen load and the ability to mitigate hide-to-carcass transfer. In spite of these differences, postintervention carcass contamination was significantly reduced (P < 0.001), likely as a result of the use of one or more of the processing interventions employed at each of the four processing plants examined.

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.

Prevalence of Escherichia coli O157 and Levels of Aerobic Bacteria and Enterobacteriaceae Are Reduced When Hides Are Washed and Treated with Cetylpyridinium Chloride at a Commercial Beef Processing Plant

The objective of this experiment was to test the potential of a combined water wash and cetylpyridinium chloride (CPC) treatment as a hide intervention applied to cattle in the holding pens of a processing plant immediately before stunning. Over 2 processing days, 149 control and 139 treated cattle were tested. Control cattle were processed in the normal manner. The treatment group was prewashed with water the day before harvest. Immediately before stunning, these cattle were sprayed twice with 1% CPC, first for 3 min, then for 1 min. Hides and preevisceration carcasses were sampled to determine aerobic plate counts, Enterobacteriaceae counts (EBC), and Escherichia coli O157 prevalence. The treatment reduced the prevalence of E. coli O157 on hides from 56% to 34% and the prevalence on preevisceration carcasses from 23% to 3%. The treatment decreased aerobic plate counts from 4.9 log CFU/100 cm2 to 3.4 log CFU/100 cm2 and EBC from 3.1 log CFU/100 cm2 to 2.0 log CFU/100 cm2 on preevisceration carcasses. The treatment of hides did not result in any detectable CPC contamination of the chilled carcasses. These data indicated that a 1% CPC treatment preceded by a water wash was capable of reducing hide prevalence of E. coli O157 from as high as 80% to less than 50%, resulting in preevisceration carcass prevalence of 5% or less. We conclude that water washing followed by an antimicrobial treatment, such as CPC, has great potential as an effective hide intervention step and should be further evaluated for implementation as a processing step after stunning and before hide removal.

Development of methods for the recovery of Escherichia coli O157:H7 and Salmonella from beef carcass sponge samples and bovine fecal and hide samples

Culture methods were developed for the concurrent recovery of Escherichia coli O157:H7 and Salmonella from bovine carcass, hide, and fecal samples. Several enrichment conditions were tested for the overall growth of pure cultures; tryptic soy broth for 2 h at 25 degrees C and then for 6 h at 42 degrees C was the protocol selected for use. Immunomagnetic separation (IMS) was incorporated for sensitivity and selectivity, along with a post-IMS enrichment for the recovery of Salmonella as recommended by the manufacturer. Selective agars for plating after IMS were chosen on the basis of ease of target colony identification. Sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and Rainbow agar supplemented with novobiocin and potassium tellurite were chosen for the recovery of E. coli O157:H7. Brilliant green agar with sulfadiazine and Hektoen enteric agar supplemented with novobiocin were selected for the recovery of Salmonella. The resulting methods were evaluated along with standard or previously used methods for the recovery of E. coli O157:H7 and Salmonella from bovine hide and fecal samples and carcass sponge samples. The Meats Research Unit (MRU) methods performed at least as well as the established methods, except that a secondary enrichment in tetrathionate (TT) broth prior to IMS was required for the optimal recovery of Salmonella from feces. Thus, the MRU and MRU-TT methods are effective in the recovery of both E. coli O157:H7 and Salmonella from a single bovine carcass, hide, or fecal sample.