Proportions and Serogroups of Enterohemorrhagic Shiga Toxin-producing Escherichia coli in Feces of Fed and Cull Beef and Cull Dairy Cattle at Harvest.

Cattle are considered a primary reservoir of Shiga toxin (stx)-producing Escherichia coli that cause enterohemorrhagic disease (EHEC), and contaminated beef products are one vehicle of transmission to humans. However, animals entering the beef harvest process originate from differing production systems: feedlots, dairies, and beef breeding herds. The objective of this study was to determine if fed cattle, cull dairy, and or cull beef cattle carry differing proportions and serogroups of EHEC at harvest. Feces were collected via rectoanal mucosal swabs (RAMSs) from 1,039 fed cattle, 1,058 cull dairy cattle, and 1,018 cull beef cattle at harvest plants in seven U.S. states (CA, GA, NE, PA, TX, WA, and WI). The proportion of the stx gene in feces of fed cattle (99.04%) was not significantly different (P > 0.05) than in the feces of cull dairy (92.06%) and cull beef (91.85%) cattle. When two additional factors predictive of EHEC (intimin and ecf1 genes) were considered, EHEC was significantly greater (P < 0.05) in fed cattle (77.29%) than in cull dairy (47.54%) and cull beef (38.51%) cattle. The presence of E. coli O157:H7 and five common non-O157 EHEC of serogroups O26, O103, O111, O121, and O145 was determined using molecular analysis for single nucleotide polymorphisms (SNPs) followed by culture isolation. SNP analysis identified 23.48%, 17.67%, and 10.81% and culture isolation confirmed 2.98%, 3.31%, and 3.00% of fed, cull dairy, and cull beef cattle feces to contain one of these EHEC, respectively. The most common serogroups confirmed by culture isolation were O157, O103, and O26. Potential EHEC of fourteen other serogroups were isolated as well, from 4.86%, 2.46%, and 2.01% of fed, cull dairy, and cull beef cattle feces, respectively; with the most common being serogroups O177, O74, O98, and O84. The identification of particular EHEC serogroups in different types of cattle at harvest may offer opportunities to improve food safety risk management.

Validation of a New Method of Sampling Beef Manufacturing Trimmings for Pathogen Testing Using a Manual Sampling Mitt Approach.

The USDA Food Safety Inspection Service has declared Escherichia coli O157:H7, and six additional Shiga toxin-producing E. coli (STEC) are adulterants for nonintact raw beef products. The U. S. beef processing industry has implemented several antimicrobial intervention technologies throughout the carcass dressing process to remove or destroy foodborne pathogens present on beef carcasses. Despite these efforts, STEC have been shown to cause finished product contamination, albeit at prevalences typically <0.5%. Recent work described the development and validation of improved methods for collecting samples from raw beef trimmings. One of the methods, the Manual Sampling Device (MSD) method, uses the manual implementation of the MicroTally® Swab (MT-Swab) to vigorously scrub the surface of raw beef manufacturing trimmings for pathogen detection. The work described herein reports the data from an evaluation of a novel MSD method using the MicroTally® Mitt (MT-Mitt). The MT-Mitt provides a more user-friendly option for sample collection than the MT-Swab. A series of trials were conducted with a total of 360 matched samples comparing manual sampling of raw beef manufacturing trimmings using the MT-Swab, N60-excision, or N60-plus methods to a novel method using the MT-Mitt. The results of these trials collectively demonstrate that manual sampling of raw beef manufacturing trimmings using the MT-Mitt provides organism recovery that is not significantly different from that of the MT-Swab, N60-excision, and N60-plus methods. Thus, the MT-Mitt method provides an alternative sampling method with organism recovery that is not significantly different from previous methods for sampling beef manufacturing trimmings for pathogen detection and some implementation advantages pertaining to labor and ease of use.

Identifying a list of Salmonella serotypes of concern to target for reducing risk of salmonellosis.

There is an increasing awareness in the field of Salmonella epidemiology that focusing control efforts on those serotypes which cause severe human health outcomes, as opposed to broadly targeting all Salmonella, will likely lead to the greatest advances in decreasing the incidence of salmonellosis. Yet, little guidance exists to support validated, scientific selection of target serotypes. The goal of this perspective is to develop an approach to identifying serotypes of greater concern and present a case study using meat- and poultry-attributed outbreaks to examine challenges in developing a standardized framework for defining target serotypes.

Evaluation of Peracetic Acid Treatment on Beef Trimmings and Subprimals Against Salmonella and E. Coli O157:H7 Within Regulatory Retained Water Limitations.

The application of antimicrobial treatments to beef trimmings prior to grinding for the reduction of microbial contamination in ground beef has increased recently. However, raw single-ingredient meat products are not permitted by Food Safety and Inspection Services (FSIS) to retain more than 0.49% water resulting from postevisceration processing. The effectiveness of antimicrobials with the limited water retention is not well documented. The objective of this study was to determine the effectiveness of peracetic acid at varied concentrations against E. coli O157:H7 and Salmonella on the surface of beef trimmings and beef subprimals that was applied at industry operating parameters within the retained water requirement. One hundred and forty-four each of beef trimmings and subprimals were used to evaluate the effect of different concentrations of peracetic acid solution on reducing E. coli O157:H7 and Salmonella on surfaces of fresh beef within the FSIS requirement of ≤0.49% retained water from antimicrobial spray treatments using a conveyor system. A ten-strain cocktail mixture was inoculated on surfaces of fresh beef and subjected to water or four different concentrations of peracetic acid (130, 150, 200, and 400 ppm). Spray treatments with 130, 150, and 200 ppm peracetic acid reduced (P ≤ 0.05) E. coli O157:H7 and Salmonella at least 0.2 log on surfaces of beef trimmings and subprimals. Spray treatment with 400 ppm peracetic acid resulted in approximately 0.5 and 0.3 log reduction of E. coli O157:H7 and Salmonella, respectively. Results indicate that all concentrations (130-400 ppm) of peracetic acid significantly reduced E. coli O157:H7 and Salmonella on beef trimmings and subprimals compared to untreated controls. Thus, a range from 130 to 400 ppm of peracetic acid can be used during beef processing to improve the safety of beef trimmings and subprimals when weight gain is limited to ≤0.49% to meet regulatory requirements.

Fate of Shiga Toxin-Producing Escherichia coli (STEC) and Salmonella during Kosher Processing of Fresh Beef.

Traditional kosher meat processing involves the following steps after slaughtering: soaking with water to remove blood, salting to help draw out more blood, and rinsing to remove salt. However, the impact of the salt used on foodborne pathogens and beef quality is not well understood. The objectives of the current study were to determine the effectiveness of salt in reducing pathogens in a pure culture model, on surfaces of inoculated fresh beef during kosher processing, and the effect of salt on beef quality. The pure culture studies indicated that the reduction of E. coli O157:H7, non-O157 STEC, and Salmonella increased with increasing salt concentrations. With salt concentrations from 3 to 13%, salt reduced E. coli O157:H7, non-O157 STEC, and Salmonella ranging from 0.49 to 1.61 log CFU/mL. For kosher processing, the water-soaking step did not reduce pathogenic and other bacteria on the surface of fresh beef. Salting and rinsing steps reduced non-O157 STEC, E. coli O157:H7, and Salmonella ranging from 0.83 to 1.42 log CFU/cm2, and reduced Enterobacteriaceae, coliforms, and aerobic bacteria by 1.04, 0.95, and 0.70 log CFU/cm2, respectively. The salting process for kosher beef resulted in reducing pathogens on the surface of fresh beef, color changes, increased salt residues, and increased lipid oxidation on the final products.

Efficacy of Short Thermal Treatment Time Against Escherichia coli O157:H7 and Salmonella on the Surface of Fresh Beef.

Thermal treatment interventions consistently provide effective pathogen reductions. However, the cost of maintaining high temperature of 95°C in order to raise the surface temperature of carcasses to 82°C is very expensive. Therefore, beef processors need to identify thermal application times and temperatures that optimize the treatment effects with less maintenance cost. The objectives of this study were to determine the efficacy of hot water or steam at 71°C for 6 s and cascade e-ion plasma treatment for 2 s in reducing pathogens on the surface of fresh beef compared to the thermal treatment at 82°C for 15 s. Hot water at 71°C for 6 s reduced Escherichia coli O157:H7 and Salmonella by 2.38 and 2.48 log CFU/cm2, while steam treatment at 71°C for 6 s reduced E. coli O157:H7 and Salmonella by 2.94 and 3.06 log CFU/cm2, respectively. Cascade e-ion plasma treatment for 2 s reduced E. coli O157:H7 on surface of fresh beef by 1.89 log CFU/cm2. The findings indicate that short treatment time with appropriate temperature could serve as an effective carcass intervention to improve the safety of fresh beef.

Determination of Verification Parameters for Using the Manual Sampling Device for Fresh Raw Beef Trim.

Multifaceted food safety systems are used by the beef processing industry to minimize risk of bacterial contamination of the finished product. These systems are comprised of several parts including the conditional release of product requiring a sample to produce a negative result on a pathogen test prior to sending the product into the food supply. The methods of sample collection require verification activities that ensure the sampling protocols are performed adequately. The research described herein was done to determine the parameters for use in verifying adequate beef trim sampling for the Manual Sampling Device (MSD) method. In addition, the efficacy of repeated sampling via multiple applications of the MSD procedure on a fresh raw beef trim combo was investigated. The results show that MSD sample collection thatcoversless than the entire combo surface, but at least one-halfof the combo surface and is collected for a minimum of 90 s, is adequate for the recovery of organisms and prevalence targets from fresh raw beef trim. In addition, the evidence that MSD sample collection thatoccurs forless than the recommended time, butnot less than 30 s per side of the swab, is adequate for the recovery of organisms and prevalence targets from raw beef trim. Finally, results show that in a scenario where an in-plant MSD sample and a regulatory MSD sample are required from the same combo, two MSD samples can be collected from the same combo bin with similar test results for both samples. While the recommended MSD protocol specifications will not be changed, the data presented herein provide support for some flexibility in accepting test results when verification activities indicate that sampling did not occur as specified in the recommended procedure.

Genetic and phenotypic associations of mitochondrial DNA copy number, SNP, and haplogroups with growth and carcass traits in beef cattle.

Mitochondrial DNA copy number (mtDNA CN) is heritable and easily obtained from low-pass sequencing (LPS). This study investigated the genetic correlation of mtDNA CN with growth and carcass traits in a multi-breed and crossbred beef cattle population. Blood, leucocyte, and semen samples were obtained from 2,371 animals and subjected to LPS that resulted in nuclear DNA (nuDNA) and mtDNA sequence reads. Mitochondrial DNA CN was estimated as the ratio of mtDNA to nuDNA coverages. Variant calling was performed from mtDNA, and 11 single nucleotide polymorphisms (SNP) were identified in the population. Samples were classified in taurine haplogroups. Haplogroup and mtDNA type were further classified based on the 11 segregating SNP. Growth and carcass traits were available for between 7,249 and 60,989 individuals. Associations of mtDNA CN, mtDNA haplogroups, mtDNA types, and mtDNA SNP with growth and carcass traits were estimated with univariate animal models, and genetic correlations were estimated with a bivariate animal model based on pedigree. Mitochondrial DNA CN tended (P-value ≤0.08) to be associated with birth weight and weaning weight. There was no association (P-value >0.10) between mtDNA SNP, haplogroups, or types with growth and carcass traits. Genetic correlation estimates of mtDNA CN were -0.30 ± 0.16 with birth weight, -0.31 ± 0.16 with weaning weight, -0.15 ± 0.14 with post-weaning gain, -0.11 ± 0.19 with average daily dry-matter intake, -0.04 ± 0.22 with average daily gain, -0.29 ± 0.13 with mature cow weight, -0.11 ± 0.13 with slaughter weight, -0.14 ± 0.13 with carcass weight, -0.07 ± 0.14 with carcass backfat, 0.14 ± 0.14 with carcass marbling, and -0.06 ± 0.14 with ribeye area. In conclusion, mtDNA CN was negatively correlated with most traits investigated, and the genetic correlation was stronger with growth traits than with carcass traits.

This study investigated mitochondrial DNA copy number (mtDNA CN) as a potential genetic indicator of growth and carcass traits in a composite beef cattle population. Mitochondrial DNA CN was previously shown to be under genetic control. The current study estimated the genetic relationship of mtDNA CN with growth and carcass traits. Blood, leucocyte, and semen samples were obtained from 2,371 animals and subjected to whole-genome sequencing at a low depth that resulted in nuclear DNA and mtDNA sequence reads. Mitochondrial DNA CN was estimated as the ratio of mtDNA to nuclear DNA coverages. Growth and carcass traits were available for between 7,249 and 60,989 individuals. Genetic parameters were estimated from an animal model based on pedigree. Genetic correlation estimates of mtDNA CN with growth and carcass traits were low to moderate and mostly negative. These indicate that selection for lower mtDNA would be associated with an increase in birth weight, weaning weight, post-weaning gain, average daily dry-matter intake, mature cow weight, slaughter weight, and carcass weight. Therefore, the by-product of whole-genome sequencing at a low depth could be used as an indicator trait for growth and carcass traits in genetic evaluations, but the genetic relationships are not likely strong enough to prioritize mtDNA ahead of routinely used indicator traits.

Evaluating the Shelf Life and Sensory Properties of Beef Steaks from Cattle Raised on Different Grass Feeding Systems in the Western United States.

Consumer interest in grass-fed beef has been steadily rising due to consumer perception of its potential benefits. This interest has led to a growing demand for niche market beef, particularly in the western United States. Therefore, the objective of this study was to assess the impact of feeding systems on the change in microbial counts, color, and lipid oxidation of steaks during retail display, and on their sensory attributes. The systems included: conventional grain-fed (CON), 20 months-grass-fed (20GF), 25-months-grass-fed (25GF) and 20-months-grass-fed + 45-day-grain-fed (45GR). The results indicate that steaks in the 20GF group displayed a darker lean and fat color, and a lower oxidation state than those in the 25GF group. However, the feeding system did not have an impact on pH or objective tenderness of beef steaks. In addition, consumers and trained panelist did not detect a difference in taste or flavor between the 20GF or 25GF steaks but expressed a preference for the CON and 45GR steaks, indicating that an increased grazing period may improve the color and oxidative stability of beef, while a short supplementation with grain may improve eating quality.

Efficacy of Antimicrobial Interventions Used in Meat Processing Plants against Antimicrobial Tolerant Non-Antibiotic-Resistant and Antibiotic-Resistant Salmonella on Fresh Beef.

ABSTRACT: Salmonella is a common cause of foodborne illness in the United States, and several strains of Salmonella have been identified as resistant to antibiotics. It is not known whether strains that are antibiotic resistant (ABR) and that have some tolerance to antimicrobial compounds are also able to resist the inactivation effects of antimicrobial interventions used in fresh meat processing. Sixty-eight Salmonella isolates (non-ABR and ABR strains) were treated with half concentrations of lactic acid (LA), peracetic acid (PAA), and cetylpyridinium chloride (CPC), which are used in beef processing plants to screen for tolerant strains. Six strains each from non-ABR and ABR Salmonella that were most tolerant of LA (2%), PAA (200 ppm), and CPC (0.4%) were selected. Selected strains were inoculated on surfaces of fresh beef and subjected to spray wash treatment with 4% LA, 400 ppm PAA, or 0.8% CPC for the challenge study. Tissue samples were collected before and after each antimicrobial treatment for enumeration of survivors. Spray treatment with LA, PAA, or CPC significantly reduced non-ABR Salmonella and ABR Salmonella on surfaces of fresh beef by 1.95, 1.22, and 1.33 log CFU/cm2, and 2.14, 1.45, and 1.43 log CFU/cm2, respectively. The order of effectiveness was LA > PAA = CPC. The findings also indicated that LA, PAA, and CPC were equally (P ≤ 0.05) effective against non-ABR and ABR Salmonella on surfaces of fresh beef. These data contribute to the body of work that indicates that foodborne pathogens that have acquired both antibiotic resistance and antimicrobial tolerance are still equally susceptible to meat processing antimicrobial intervention treatments.

Differences in carcass chilling rate underlie differences in sensory traits of pork chops from pigs with heavier carcass weights.

Pork hot carcass weights (HCW) have been increasing 0.6 kg per year, and if they continue to increase at this rate, they are projected to reach an average weight of 118 kg by the year 2050. This projection in weight is a concern for pork packers and processors given the challenges in product quality from heavier carcasses of broiler chickens. However, previous work demonstrated that pork chops from heavier carcasses were more tender than those from lighter carcasses. Therefore, the objective was to determine the effects of pork hot carcass weights, ranging from 90 to 145 kg with an average of 119 kg, on slice shear force and sensory traits of Longissimus dorsi chops when cooked to 63 or 71 °C, and to assess if differences in chilling rate can explain differences in sensory traits. Carcasses were categorized retrospectively into fast, medium, or slow chilling-rates based on their chilling rate during the first 17 h postmortem. Loin chops cut from 95 boneless loins were cooked to either 63 or 71 °C and evaluated for slice shear force and trained sensory panel traits (tenderness, juiciness, and flavor) using two different research laboratories. Slopes of regression lines and coefficients of determination between HCW and sensory traits were calculated using the REG procedure in SAS and considered different from 0 at P ≤ 0.05. As hot carcass weight increased, chops became more tender as evidenced by a decrease in SSF (63 °C ß = -0.0412, P = 0.01; 71 °C ß = -0.1005, P < 0.001). Furthermore, HCW explained 25% (R2 = 0.2536) of the variation in chilling rate during the first 5 h of chilling and 32% (R2 = 0.3205) of the variation in chilling rate from 5 to 13 h postmortem. Slow- and medium-rate chilling carcasses were approximately 12 kg heavier (P < 0.05) than fast chilling carcasses. Slice shear force of chops cooked to 63 and 71 °C was reduced in slow and medium chilling compared with fast chilling carcasses. Carcass temperature at 5 h postmortem explained the greatest portion of variation (R2 = 0.071) in slice shear force of chops cooked to 63 °C. These results suggest that carcasses tend to chill slower as weight increases, which resulted in slight improvements in sensory traits of boneless pork chops regardless of final degree of doneness cooking temperature.

Pork carcass weights have increased year over year for at least the past 25 yr. The poultry industry has experienced similar increases in carcass weights in the recent past. The increases in broiler carcass weights have resulted in detrimental impacts on quality. Contrary to the poultry industry, increases in pork carcass weights have resulted in a general improvement in pork quality, including tenderness. The underlying cause of these improvements has not been explained. In the present study, chilling rate was associated with carcass weights, particularly during the first 5 h postmortem. In fact, carcass temperature measured in the Longissimus dorsi muscle at 5 h postmortem was the most predictive of instrumental tenderness values when boneless pork chops were cooked according to UDSA guidelines for whole-muscle pork products. The metabolic conversion of muscle to meat is most active during this initial chilling period. Therefore, chilling rate, which is associated with carcass weight, may be influencing the conversion of muscle to meat and provide some explanation as to why heavy carcasses result in more tender pork chops.

Resistomes and microbiome of meat trimmings and colon content from culled cows raised in conventional and organic production systems.

BACKGROUND: The potential to distribute bacteria resistant to antimicrobial drugs in the meat supply is a public health concern. Market cows make up a fifth of the U.S. beef produced but little is known about the entire population of bacteria (the microbiome) and entirety of all resistance genes (the resistome) that are found in this population. The objective of this study was to characterize and compare the resistomes and microbiome of beef, dairy, and organic dairy market cows at slaughter. METHODS: Fifty-four (N = 54) composite samples of both colon content and meat trimmings rinsate samples were collected over six visits to two harvest facilities from cows raised in three different production systems: conventional beef, conventional dairy, and organic dairy (n = 3 samples per visit per production system). Metagenomic DNA obtained from samples were analyzed using target-enriched sequencing (resistome) and 16S rRNA gene sequencing (microbiome). RESULTS: All colon content samples had at least one identifiable antimicrobial resistance gene (ARG), while 21 of the 54 meat trimmings samples harbored at least one identifiable ARGs. Tetracycline ARGs were the most abundant class in both colon content and carcass meat trimmings. The resistome found on carcass meat trimmings was not significantly different by production system (P = 0.84, R2 = 0.00) or harvest facility (P = 0.10, R2 = 0.09). However, the resistome of colon content differed (P = 0.01; R2 = 0.05) among production systems, but not among the harvest facilities (P = 0.41; R2 = 0.00). Amplicon sequencing revealed differences (P < 0.05) in microbial populations in both meat trimmings and colon content between harvest facilities but not production systems (P > 0.05). CONCLUSIONS: These data provide a baseline characterization of an important segment of the beef industry and highlight the effect that the production system where cattle are raised and the harvest facilities where an animal is processed can impact associated microbiome and resistomes.

A Farm-to-Fork Quantitative Microbial Exposure Assessment of ß-Lactam-Resistant Escherichia coli among U.S. Beef Consumers.

Integrated quantitative descriptions of the transmission of ß-lactam-resistant Escherichia coli (BR-EC) from commercial beef products to consumers are not available. Here, a quantitative microbial exposure assessment model was established to simulate the fate of BR-EC in a farm-to-fork continuum and provide an estimate of BR-EC exposure among beef consumers in the U.S. The model compared the per-serving exposures from the consumption of intact beef cuts, non-intact beef cuts, and ground beef. Additionally, scenario analysis was performed to evaluate the relative contribution of antibiotic use during beef cattle production to the level of human exposure to BR-EC. The model predicted mean numbers of BR-EC of 1.7 × 10-4, 8.7 × 10-4, and 6.9 × 10-1 CFU/serving for intact beef cuts, non-intact beef cuts, and ground beef, respectively, at the time of consumption. Sensitivity analyses using the baseline model suggested that factors related to sectors along the supply chain, i.e., feedlots, processing plants, retailers, and consumers, were all important for controlling human exposure to BR-EC. Interventions at the processing and post-processing stages are expected to be most effective. Simulation results showed that a decrease in antibiotic use among beef cattle might be associated with a reduction in exposure to BR-EC from beef consumption. However, the absolute reduction was moderate, indicating that the effectiveness of restricting antibiotic use as a standalone strategy for mitigating human exposure to BR-EC through beef consumption is still uncertain. Good cooking and hygiene practices at home and advanced safety management practices in the beef processing and post-processing continuum are more powerful approaches for reducing human exposure to antibiotic-resistant bacteria in beef products.

Meat tenderness: advances in biology, biochemistry, molecular mechanisms and new technologies.

Meat tenderness is an important quality trait critical to consumer acceptance, and determines satisfaction, repeat purchase and willingness-to-pay premium prices. Recent advances in tenderness research from a variety of perspectives are presented. Our understanding of molecular factors influencing tenderization are discussed in relation to glycolysis, calcium release, protease activation, apoptosis and heat shock proteins, the use of proteomic analysis for monitoring changes, proteomic biomarkers and oxidative/nitrosative stress. Each of these structural, metabolic and molecular determinants of meat tenderness are then discussed in greater detail in relation to animal variation, postmortem influences, and changes during cooking, with a focus on recent advances. Innovations in postmortem technologies and enzymes for meat tenderization are discussed including their potential commercial application. Continued success of the meat industry relies on ongoing advances in our understanding, and in industry innovation. The recent advances in fundamental and applied research on meat tenderness in relation to the various sectors of the supply chain will enable such innovation.

Twenty-Four-Month Longitudinal Study Suggests Little to No Horizontal Gene Transfer In Situ between Third-Generation Cephalosporin-Resistant Salmonella and Third-Generation Cephalosporin-Resistant Escherichia coli in a Beef Cattle Feedyard.

ABSTRACT: Third-generation cephalosporins (3GCs) are preferred treatments for serious human Salmonella enterica infections. Beef cattle are suspected to contribute to human 3GC-resistant Salmonella infections. Commensal 3GC-resistant Escherichia coli are thought to act as reservoirs of 3GC resistance because these strains are isolated more frequently than are 3GC-resistant Salmonella strains at beef cattle feedyards. During each of 24 consecutive months, four samples of pen surface material were obtained from five pens (N = 480) at a Nebraska feedyard to determine to the contribution of 3GC-resistant E. coli to the occurrence of 3GC-resistant Salmonella. Illumina whole genome sequencing was performed, and susceptibility to 14 antimicrobial agents was determined for 121 3GC-susceptible Salmonella, 121 3GC-resistant Salmonella, and 203 3GC-resistant E. coli isolates. 3GC-susceptible Salmonella isolates were predominantly from serotypes Muenchen (70.2%) and Montevideo clade 1 (23.1%). 3GC-resistant Salmonella isolates were predominantly from serotypes Montevideo clade 2 (84.3%). One bla gene type (blaCMY-2) and the IncC plasmid replicon were present in 100 and 97.5% of the 3GC-resistant Salmonella, respectively. Eleven bla gene types were detected in the 3GC-resistant E. coli, which were distributed across 42 multilocus sequence types. The blaCMY-2 gene and IncC plasmid replicon were present in 37.9 and 9.9% of the 3GC-resistant E. coli, respectively. These results suggest that 3GC resistance in Salmonella was primarily due the persistence of Salmonella Montevideo clade 2 with very minimal or no contribution from 3GC-resistant E. coli via horizontal gene transfer and that 3GC-resistant E. coli may not be a useful indicator for 3GC-resistant Salmonella in beef cattle production environments.

A Comparative Quantitative Assessment of Human Exposure to Various Antimicrobial-Resistant Bacteria among U.S. Ground Beef Consumers.

ABSTRACT: Consumption of animal-derived meat products is suspected as an important exposure route to antimicrobial resistance, as the presence of antimicrobial-resistant bacteria (ARB) along the beef supply chain is well documented. A retail-to-fork quantitative exposure assessment was established to compare consumers' exposure to various ARB due to the consumption of ground beef with and without "raised without antibiotics" claims and to inform potential exposure mitigation strategies related to consumer practices. The microbial agents evaluated included Escherichia coli, tetracycline-resistant (TETr) E. coli, third-generation cephalosporin-resistant E. coli,Salmonella enterica, TETrS. enterica, third-generation cephalosporin-resistant S. enterica, nalidixic acid-resistant S. enterica, Enterococcus spp., TETrEnterococcus spp., erythromycin-resistant Enterococcus spp., Staphylococcus aureus, and methicillin-resistant S. aureus. The final model outputs were the probability of exposure to at least 0 to 6 log CFU microorganisms per serving of ground beef at the time of consumption. It was estimated that tetracycline resistance was more prevalent in ground beef compared with other types of resistance, among which the predicted average probability of ingesting TETrEnterococcus was highest (6.2% of ingesting at least 0 log CFU per serving), followed by TETrE. coli (3.1%) and TETrSalmonella (0.0001%), given common product purchase preferences and preparation behaviors among beef consumers in the United States. The effectiveness of consumer-related interventions was estimated by simulating the differences in exposure as a result of changes in consumer practices in purchasing, handling, and preparing ground beef. The results indicated that proper use of recommended safe cooking and food preparation practices mitigates ARB exposure more effectively than choosing raised without antibiotics compared with conventional beef.

Validation of Additional Approaches and Applications for Using the Continuous and Manual Sampling Devices for Raw Beef Trim.

ABSTRACT: In this work, the goal was to determine the efficacy of MicroTally-based sampling in scenarios commonly encountered in the commercial beef processing industry, but outside of the parameters evaluated during the initial proof-of-concept work. The data were derived from 1,650 matched samples collected from 540 individual combo bins at six commercial beef processing plants, comparing MicroTally-based sampling (continuous and manual sampling devices [CSD and MSD]) to N60 Excision and/or N60 Plus methods. Mounting a 61-cm CSD cartridge to a 30-cm-wide conveyor provided sampling that is equivalent to N60 Excision and N60 Plus methods. Mounting a CSD to a chute instead of a conveyor was equivalent to the N60 Plus sampling method. The CSD was shown to be effective for sampling when used in conjunction with a "swinging arm trim diverter" and receiving product in batch mode as opposed to continuous flow. MSD sampling of oval combo bins with trim surface area (≈0.93 m2 [≈1,439 in2]) less than 1 m2 (1,600 in2) was shown to be equivalent to the N60 Plus sample collection method. Peracetic acid applied at the end of the trim conveyor did not negatively impact pathogen index target detection of the CSD even if the samples were shipped overnight before analysis. Pathogen index targets were demonstrated to be useful tools for validating methods designed to measure pathogen prevalence. The data presented herein support equivalency criteria of within 0.5 log CFU per sample for indicator organism counts. These data collectively support various alternative applications of MicroTally-based trim sampling and the application and interpretation of alternative methods for pathogen detection.

Antimicrobial Resistance in U.S. Retail Ground Beef with and without Label Claims Regarding Antibiotic Use.

ABSTRACT: Antibiotics used during food animal production account for approximately 77% of U.S. antimicrobial consumption by mass. Ground beef products labeled as raised without antibiotics (RWA) are perceived to harbor lower levels of antimicrobial-resistant bacteria than conventional (CONV) products with no label claims regarding antimicrobial use. Retail ground beef samples were obtained from six U.S. cities. Samples with an RWA or U.S. Department of Agriculture Organic claim (n = 299) were assigned to the RWA production system. Samples lacking these claims (n = 300) were assigned to the CONV production system. Each sample was cultured for the detection of five antimicrobial-resistant bacteria. Genomic DNA was isolated from each sample, and a quantitative PCR assay was used to determine the abundance of 10 antimicrobial resistance (AMR) genes. Prevalence of tetracycline-resistant Escherichia coli (CONV, 46.3%; RWA, 34.4%; P < 0.01) and erythromycin-resistant Enterococcus (CONV, 48.0%; RWA, 37.5%; P = 0.01) was higher in CONV ground beef. Salmonella was detected in 1.2% of samples. The AMR gene blaCTX-M (CONV, 4.1 log-normalized abundance; RWA, 3.8 log-normalized abundance; P < 0.01) was more abundant in CONV ground beef. The AMR genes mecA (CONV, 4.4 log-normalized abundance; RWA, 4.9 log-normalized abundance; P = 0.05), tet(A) (CONV, 3.9 log-normalized abundance; RWA, 4.5 log-normalized abundance; P < 0.01), tet(B) (CONV, 3.9 log-normalized abundance; RWA, 4.5 log-normalized abundance; P < 0.01), and tet(M) (CONV, 5.4 log-normalized abundance; RWA, 5.8 log-normalized abundance; P < 0.01) were more abundant in RWA ground beef. Although these results suggest that antimicrobial use during U.S. cattle production does not increase human exposure to antimicrobial-resistant bacteria via ground beef, quantitative microbiological risk assessments are required for authoritative determination of the human health impacts of the use of antimicrobial agents during beef production.

Consecutive Treatments with a Multicomponent Sanitizer Inactivate Biofilms Formed by Escherichia coli O157:H7 and Salmonella enterica and Remove Biofilm Matrix.

ABSTRACT: Many foodborne pathogens, including Escherichia coli O157:H7 and Salmonella enterica, can develop biofilms on contact surfaces at meat processing plants. Owing to the high tolerance of the biofilm cells associated with the three-dimensional biofilm structure and the well-expressed bacterial extracellular polymeric substances, it is a real challenge to completely inactivate and remove mature biofilms, as well as further prevent biofilm reoccurrence and pathogen survival. In the present study, we evaluated the effectiveness of consecutive treatments (10 to 120 min per treatment) by repeatedly applying a multicomponent sanitizer, based on a functional mechanism by synergistic combination of hydrogen peroxide and quaternary ammonia compounds, against biofilms formed by E. coli O157:H7 and S. enterica strains. Biofilms on stainless steel surfaces were treated with 2.5, 5, or 10% (recommended working concentration) of the sanitizer applied as a foam or liquid solution. Our results showed that the multicomponent sanitizer significantly (P < 0.05) reduced the amount of viable biofilm cells at all concentrations, as enumerable bacteria were only detected after low-concentration treatments (2.5 or 5%) with short exposure periods (10 or 20 min per treatment). Treatments with high concentrations (5 or 10%) of the sanitizer, multiple consecutive treatments (2 or 3 treatments), and sufficient exposure time (>60 min per treatment) effectively controlled pathogen survival postsanitization. Examination with a scanning electron microscope showed that treatment with the sanitizer at 5% strength significantly dissolved the connecting extracellular polysaccharide matrix and removed the majority of the biofilm matrix. No intact biofilm structure was detected after the 10% sanitizer treatment; instead, scattered individual bacteria with visibly altered cell morphology were observed. The treated bacteria exhibited indented and distorted shapes with shortened cell length and increased surface roughness, indicating severe cell injury and death. Our observations indicated that consecutive treatments with the multicomponent sanitizer was effective in inactivating E. coli O157:H7 and S. enterica biofilms and preventing pathogen reoccurrence.

Metagenomic Characterization of the Microbiome and Resistome of Retail Ground Beef Products.

Ground beef can be a reservoir for a variety of bacteria, including spoilage organisms, and pathogenic foodborne bacteria. These bacteria can exhibit antimicrobial resistance (AMR) which is a public health concern if resistance in pathogens leads to treatment failure in humans. Culture-dependent techniques are commonly used to study individual bacterial species, but these techniques are unable to describe the whole community of microbial species (microbiome) and the profile of AMR genes they carry (resistome), which is critical for getting a holistic perspective of AMR. The objective of this study was to characterize the microbiome and resistome of retail ground beef products labeled as coming from conventional or raised without antibiotics (RWA) production systems. Sixteen ground beef products were purchased from 6 retail grocery outlets in Fort Collins, CO, half of which were labeled as produced from cattle raised conventionally and half of products were from RWA production. Total DNA was extracted and isolated from each sample and subjected to 16S rRNA amplicon sequencing for microbiome characterization and target-enriched shotgun sequencing to characterize the resistome. Differences in the microbiome and resistome of RWA and conventional ground beef were analyzed using the R programming software. Our results suggest that the resistome and microbiome of retail ground beef products with RWA packaging labels do not differ from products that do not carry claims regarding antimicrobial drug exposures during cattle production. The resistome predominantly consisted of tetracycline resistance making up more than 90% of reads mapped to resistance gene accessions in our samples. Firmicutes and Proteobacteria predominated in the microbiome of all samples (69.6% and 29.0%, respectively), but Proteobacteria composed a higher proportion in ground beef from conventionally raised cattle. In addition, our results suggest that product management, such as packaging type, could exert a stronger influence on the microbiome than the resistome in consumer-ready products. Metagenomic analyses of ground beef is a promising tool to investigate community-wide shifts in retail ground beef. Importantly, however, results from metagenomic sequencing must be carefully considered in parallel with traditional methods to better characterize the risk of AMR in retail products.