The GEA pipeline for characterizing Escherichia coli and Salmonella genomes.

Salmonella enterica and Escherichia coli are major food-borne human pathogens, and their genomes are routinely sequenced for clinical surveillance. Computational pipelines designed for analyzing pathogen genomes should both utilize the most current information from annotation databases and increase the coverage of these databases over time. We report the development of the GEA pipeline to analyze large batches of E. coli and S. enterica genomes. The GEA pipeline takes as input paired Illumina raw reads files which are then assembled followed by annotation. Alternatively, assemblies can be provided as input and directly annotated. The pipeline provides predictive genome annotations for E. coli and S. enterica with a focus on the Center for Genomic Epidemiology tools. Annotation results are provided as a tab delimited text file. The GEA pipeline is designed for large-scale E. coli and S. enterica genome assembly and characterization using the Center for Genomic Epidemiology command-line tools and high-performance computing. Large scale annotation is demonstrated by an analysis of more than 14,000 Salmonella genome assemblies. Testing the GEA pipeline on E. coli raw reads demonstrates reproducibility across multiple compute environments and computational usage is optimized on high performance computers.

Antibiotic Resistance and Disinfectant Resistance Among Escherichia coli Isolated During Red Meat Production.

Escherichia coli commonly found in the gastrointestinal tracts of food animals include Shiga toxin-producing E. coli (STEC, stx+, eae-), Enterohemorrhagic E. coli (EHEC, stx+, eae+), Enteropathogenic E. coli (EPEC, stx-, eae+), and "nondiarrheagenic" E. coli (NDEC, stx-, eae-). EHEC, EPEC, and STEC are associated with foodborne disease outbreaks. During meat processing, disinfectants are employed to control various bacteria, including human pathogens. Concerns exist that E. coli resistant to antibiotics are less susceptible to disinfectants used during meat processing. Since EHEC, EPEC, and STEC with reduced susceptibility to disinfectants are potential public health risks, the goal of this study was to evaluate the association of antibiotic resistant (ABR) E. coli with increased tolerance to 4% lactic acid (LA) and 150 ppm quaternary ammonium compounds (QACs). A pool of 3,367 E. coli isolated from beef cattle, veal calves, swine, and sheep at various processing stages was screened to identify ABR E. coli. Resistance to ≥1 of the six antibiotics examined was identified in 27.9%, 36.1%, 54.5%, and 28.7% among the NDEC (n = 579), EHEC (n = 693), EPEC (n = 787), and STEC (n = 1308) isolates evaluated, respectively. Disinfectant tolerance did not differ (P > 0.05) between ABR and antibiotic susceptible EHEC isolates. Comparable frequencies (P > 0.05) of biofilm formation or congo red binding were observed between ABR and antibiotic susceptible strains of E. coli. Understanding the frequencies of ABR and disinfectant tolerance among E. coli present in food-animal is a critically important component of meat safety.

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.

Distribution of Extremely Heat-Resistant Escherichia coli in the Beef Production and Processing Continuum.

Understanding the dynamics of stress-resistant Escherichia coli (E. coli) across the meat production and processing continuum is important for tracking sources of such microbes and devising effective modes of control. The Locus of Heat Resistance (LHR) is a ∼14-19 Kb genetic element imparting extreme heat resistance (XHR) in Enterobacteriaceae. It has been hypothesized that thermal and antimicrobial interventions applied during meat processing may select for LHR+E. coli. Thus, our goal was to study the prevalence and molecular biology of LHR+E. coli among lots of beef cattle (n = 3) from production through processing. Two hundred thirty-two generic E. coli isolated from the same animals through seven stages of the beef processing continuum (cattle in feedyards to packaged strip loins) were examined. LHR+E. coli were rare (0.6%; 1 of 180) among the early stages of the beef continuum (feces and hides at feedlot, feces and hides at harvest, and preevisceration carcasses), whereas the prevalence of LHR+E. coli on final carcasses and strip loins was remarkably higher. Half (14 of 28) of the final carcass E. coli possessed the LHR, while 79.2% (19 of 24) of the strip loin E. coli did. Eighty-five percent (29 of 34) of the LHR+E. coli presented with the XHR phenotype. The selection or enrichment of LHR+E. coli from harvest steps to the final products appeared unlikely as the LHR+E. coli isolates were effectively controlled by antimicrobial interventions typically used during beef processing. Further, whole-genome sequencing of the isolates suggested LHR+E. coli are persisting in the chilled processing environment and that horizontal LHR transfer among E. coli isolates may take place.

Tetracycline-Resistant, Third-Generation Cephalosporin-Resistant, and Extended-Spectrum ß-Lactamase-Producing Escherichia coli in a Beef Cow-Calf Production System.

ABSTRACT: Cow-calf production plays a significant role in the beef production chain. However, bacteria in these systems are not typically monitored for antimicrobial resistance (AMR). We determined the baseline level of AMR in fecal bacteria collected from preweaned calves prior to feedlot entry and evaluated the effects of type of graze and age on AMR occurrence. Two grazing experiments (16 cow-calf pairs each) were conducted on tall fescue or wheat. Fecal samples were cultured for the detection of tetracycline-resistant (TETr), third-generation cephalosporin-resistant (3GCr), and extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. Isolates were characterized for resistance to other antibiotics and resistance mechanisms. Concentrations (P < 0.001) and prevalence (P = 0.007) of TETrE. coli isolates were significantly higher in the calves (5.1 log CFU/g and 93%, respectively) than in the cows (4.4 log CFU/g and 80%, respectively). Wheat grazing did not affect TETr isolates phenotypically; however, it significantly expanded (P = 0.005) the resistant population carrying tet(A) over that carrying tet(B). Fecal prevalence of 3GCr and ESBL-producing isolates was 31.3 and 3.4%, respectively, with no significant effects of age (P = 0.340) or wheat grazing (P = 0.597). All 3GCr and ESBL-producing isolates were multidrug resistant (resistant to at least three antimicrobial classes). 3GCr isolates were positive for blaCMY-2 (73%) or blaCTX-M (27%), and blaCTX-M-15 was the most prevalent gene (94%, n = 17) among the CTX-M-positive isolates. Wheat grazing significantly expanded (P < 0.001) the 3GCr population carrying blaCTX-M and reduced the population carrying blaCMY-2. Five of the seven ESBL-producing isolates were positive for blaCTX-M. Our study revealed age-dependent occurrence of TETrE. coli and that wheat grazing expanded the resistant population carrying certain resistance genes. Cow-calf production is a significant reservoir for antibiotic-resistant bacteria of significant public health importance such as 3GCr and CTX-M ESBL-producing E. coli.

Characterization of Escherichia coli harboring colibactin genes (clb) isolated from beef production and processing systems.

Certain strains of Escherichia coli possess and express the toxin colibactin (Clb) which induces host mutations identical to the signature mutations of colorectal cancer (CRC) that lead to tumorigenic lesions. Since cattle are a known reservoir of several Enterobacteriaceae including E. coli, this study screened for clb amongst E. coli isolated from colons of cattle-at-harvest (entering beef processing facility; n = 1430), across the beef processing continuum (feedlot to finished subprimal beef; n = 232), and in ground beef (n = 1074). Results demonstrated that clb+ E. coli were present in cattle and beef. Prevalence of clb+ E. coli from colonic contents of cattle and ground beef was 18.3% and 5.5%, respectively. clb+ E. coli were found susceptible to commonly used meat processing interventions. Whole genome sequencing of 54 bovine and beef clb+ isolates showed clb occurred in diverse genetic backgrounds, most frequently in phylogroup B1 (70.4%), MLST 1079 (42.6%), and serogroup O49 (40.7%).

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.

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.

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.

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.

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.

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.

Seasonal Prevalence of Shiga Toxin-Producing Escherichia coli on Pork Carcasses for Three Steps of the Harvest Process at Two Commercial Processing Plants in the United States.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that has a significant impact on public health, with strains possessing the attachment factor intimin referred to as enterohemorrhagic E. coli (EHEC) and associated with life-threatening illnesses. Cattle and beef are considered typical sources of STEC, but their presence in pork products is a growing concern. Therefore, carcasses (n = 1,536) at two U.S. pork processors were sampled once per season at three stages of harvest (poststunning skins, postscald carcasses, and chilled carcasses) and then examined using PCR for Shiga toxin genes (stx), intimin genes (eae), aerobic plate count (APC), and Enterobacteriaceae counts (EBC). The prevalence of stx on skins, postscald, and chilled carcasses was 85.3, 17.5, and 5.4%, respectively, with 82.3, 7.8, and 1.7% of swabs, respectively, having stx and eae present. All stx-positive samples were subjected to culture isolation that resulted in 368 STEC and 46 EHEC isolates. The most frequently identified STEC were serogroups O121, O8, and O91 (63, 6.7, and 6.0% of total STEC, respectively). The most frequently isolated EHEC was serotype O157:H7 (63% of total EHEC). Results showed that scalding significantly reduced (P < 0.05) carcass APC and EBC by 3.00- and 2.50-log10 CFU/100 cm2, respectively. A seasonal effect was observed, with STEC prevalence lower (P < 0.05) in winter. The data from this study show significant (P < 0.05) reduction in the incidence of STEC (stx) from 85.3% to 5.4% and of EHEC (stx plus eae) from 82.3% to 1.7% within the slaughter-to-chilling continuum, respectively, and that potential EHEC can be confirmed present throughout using culture isolation.IMPORTANCE Seven serogroups of STEC are responsible for most (>75%) cases of severe illnesses caused by STEC and are considered adulterants of beef. However, some STEC outbreaks have been attributed to pork products, although the same E. coli are not considered adulterants in pork because little is known of their prevalence along the pork chain. The significance of the work presented here is that it identifies disease-causing STEC, EHEC, demonstrating that these same organisms are a food safety hazard in pork as well as beef. The results show that most STEC isolated from pork are not likely to cause severe disease in humans and that processes used in pork harvest, such as scalding, offer a significant control point to reduce contamination. The results will assist the pork processing industry and regulatory agencies to optimize interventions to improve the safety of pork products.

No Change in Risk for Antibiotic-Resistant Salmonellosis from Beef, United States, 2002-2010.

Restricting antibiotic use in food production animals is a target for reducing antimicrobial drug-resistant infections in humans. We used US surveillance data to estimate the probability of antibiotic-resistant nontyphoidal salmonellosis per meal made with beef during 2002-2010. Applying data for nontyphoidal Salmonella in raised-without-antibiotics cattle, we tested the effect of removing antibiotic use from all beef cattle production. We found an average of 1.2 (95% credible interval 0.6-4.2) antibiotic-resistant nontyphoidal salmonellosis cases per 1 million beef meals made with beef initially contaminated with antibiotic-resistant nontyphoidal Salmonella at slaughter or retail and 0.031 (95% credible interval 0.00018-0.14) cases per 1 million meals irrespective of beef contamination status. Neither outcome showed sustained change except for increases in 2003 and 2009 (>98% confidence) when larger or more outbreaks occurred. Switching all beef production to a raised-without-antibiotics system may not have a significant effect on antibiotic-resistant nontyphoidal salmonellosis (94.3% confidence).

Antimicrobial Resistance at Two U.S. Cull Cow Processing Establishments.

ABSTRACT: Culled beef cows (cows that have reached the end of their productive life span in cow-calf operations) and culled dairy cows represent approximately 18% of the cattle harvested in the United States annually, but data on antimicrobial resistance (AMR) in these cull cattle are extremely limited. To address this data gap, colon contents were obtained from 180 culled conventional beef cows, 179 culled conventional dairy cows, and 176 culled organic dairy cows (produced without using antimicrobials). Sponge samples were also collected from 181 conventional beef, 173 conventional dairy, and 180 organic dairy cow carcasses. These samples were obtained on 6 days (3 days each at two beef harvest and processing establishments). At one establishment, 30 samples of beef manufacturing trimmings from conventional cows and 30 trim samples from organic dairy cows were acquired. All 1,129 samples were cultured for Escherichia coli, tetracycline-resistant (TETr) E. coli, third-generation cephalosporin-resistant (3GCr) E. coli, Salmonella, and 3GCrSalmonella. Metagenomic DNA was isolated from 535 colon content samples, and quantitative PCR assays were performed to assess the abundances of the following 10 antimicrobial resistance genes: aac(6')-Ie-aph(2″)-Ia, aadA1, blaCMY-2, blaCTX-M, blaKPC-2, erm(B), mecA, tet(A), tet(B), and tet(M). For colon contents, only TETrE. coli (P < 0.01), 3GCrE. coli (P < 0.01), and erm(B) (P = 0.03) levels were higher in conventional than in organic cows. Sampling day also significantly affected (P < 0.01) these levels. Production system did not affect the levels of any measured AMR on carcasses or trim. The human health impact of the few significant AMR differences could not be determined due to the lack of standards for normal, background, safe, or basal values. Study results provide key heretofore unavailable data that may inform quantitative microbial risk assessments to address these gaps.

Medicine: A Prescription for Medical Student Leadership Education.

This chapter proposes a medical school leadership curriculum that is context-driven, dovetails seamlessly with the medical student experience, and goes beyond managerial and professional interests by developing purposeful physician-leaders and focusing leadership knowledge on patient outcomes.

In-Feed Tylosin Phosphate Administration to Feedlot Cattle Minimally Affects Antimicrobial Resistance.

ABSTRACT: The macrolide class antimicrobial tylosin (trade name Tylan) is approved by the U.S. Food and Drug Administration for continuous inclusion in feed for liver abscess prevention. To address concerns that this antimicrobial application may threaten human health, a population of feedlot steers was split into a control treatment (n = 42) and a tylosin treatment (n = 42). Feed rations were identical except for the inclusion of tylosin at 60 to 90 mg per head per day. Fecal swab (n = 335), pen surface material (n = 256), feed (n = 56), and water trough (n = 32) samples were obtained over four sample occasions: November (1 day before the start of tylosin inclusion in feed), January (80 days of tylosin in feed), April (167 days), and June (253 days). These samples were cultured for Escherichia coli, tetracycline-resistant E. coli, third-generation cephalosporin-resistant E. coli, Enterococcus, tetracycline-resistant Enterococcus, and erythromycin-resistant Enterococcus. Metagenomic DNA was isolated from each June fecal swab and pen surface material sample. Metagenomic DNA samples were pooled by pen for 14 fecal and 14 pen surface material samples. Quantitative PCR was employed to assess the abundances of the following 10 antimicrobial resistance genes: aac(6')-Ie-aph(2″)-Ia, aadA1, blaCMY-2, blaCTX-M, blaKPC-2, erm(B), mecA, tet(A), tet(B), and tet(M). Nasal swab samples (n = 335) were obtained from each steer during each sample period and cultured for the presence of Staphylococcus aureus and methicillin-resistant S. aureus. Of these measurements, only January and June mean fecal swab erythromycin-resistant Enterococcus colony counts for tylosin-treated cattle were significantly higher (P ≤ 0.05) than the range of mean values for control treatments. These results suggest that in-feed tylosin through the end of finishing has a narrow and minimal antimicrobial resistance impact.

Food Service Pork Chops from Three U.S. Regions Harbor Similar Levels of Antimicrobial Resistance Regardless of Antibiotic Use Claims.

Pork products from animals "raised without antibiotics" (RWA) are assumed to harbor lower levels of antimicrobial resistance (AMR) than conventional (CONV) pork products with no claims regarding use of antimicrobial agents during production. A total of 372 pork chop samples from CONV (n = 190) and RWA (n = 182) production systems were collected over 13 months from three food service suppliers. The following bacteria were cultured: Escherichia coli, tetracycline-resistant (TETr) E. coli, third-generation cephalosporin-resistant (3GCr) E. coli, Salmonella enterica, TETr Salmonella, 3GCr Salmonella, nalidixic acid-resistant Salmonella, Enterococcus spp., TETr Enterococcus, erythromycin-resistant Enterococcus, Staphylococcus aureus, and methicillin-resistant S. aureus. Production system did not significantly impact the detection of cultured bacteria (P > 0.05). Metagenomic DNA was isolated from each sample, and equal amounts of metagenomic DNA were pooled by supplier, month, and production system for 75 pooled samples (38 CONV, 37 RWA). Quantitative PCR was used to assess the abundances of the following 10 AMR genes: aac(6')-Ie-aph(2″)-Ia, aadA1, blaCMY-2, blaCTX-M, blaKPC-2, erm(B), mecA, tet(A), tet(B), and tet(M). For all 10 AMR genes, abundances did not differ significantly (P > 0.05) between production systems. These results suggest that use of antimicrobial agents during swine production minimally impacts the AMR of bacteria in pork chops.

Similar Levels of Antimicrobial Resistance in U.S. Food Service Ground Beef Products with and without a "Raised without Antibiotics" Claim.

U.S. ground beef with "raised without antibiotics" (RWA) label claims are perceived as harboring fewer bacteria with antimicrobial resistance (AMR) than are found in conventional (CONV) ground beef with no such label claim. A total of 370 ground beef samples from CONV ( n = 191) and RWA ( n = 179) production systems were collected over 13 months from three food service suppliers. The following bacteria were cultured: Escherichia coli, tetracycline-resistant (TETr) E. coli, third-generation cephalosporin-resistant (3GCr) E. coli, Salmonella enterica, TETr S. enterica, 3GCr S. enterica, nalidixic acid-resistant S. enterica, Enterococcus spp., erythromycin-resistant Enterococcus spp., TETr Enterococcus spp., Staphylococcus aureus, and methicillin-resistant S. aureus. TETr E. coli was more frequently detected in CONV ground beef (CONV, 54.2%; RWA, 35.2%; P < 0.01), but supplier ( P < 0.01) and production system × suppler interaction ( P < 0.01) effects were also significant. Metagenomic DNA was isolated from each sample, and equal amounts of metagenomic DNA were pooled by supplier, month, and production system for 75 pooled samples (38 CONV, 37 RWA). The abundance of aac(6')-Ie-aph(2″)-Ia, aadA1, blaCMY-2, blaCTX-M, blaKPC-2, erm(B), mecA, tet(A), tet(B), and tet(M) genes was assessed by quantitative PCR. The tet(A) (2.9-log2-fold change, P = 0.04) and tet(B) (5.6-log2-fold change) ( P = 0.03) genes were significantly more abundant in RWA ground beef. Phylogenetic analyses revealed that ground beef microbiomes differed more by supplier than by production system. These results were consistent with prior research suggesting antimicrobial use in U.S. beef cattle has minimal impact on the AMR of bacteria found in these products. These results should spur a reevaluation of assumptions regarding the impact of antimicrobial use during U.S. beef production on the AMR of bacteria in ground beef.

Effects of In-Feed Chlortetracycline Prophylaxis in Beef Cattle on Antimicrobial Resistance Genes.

Approximately 20% of U.S. beef cattle receive prophylactic in-feed administration of chlortetracycline (CTC) to reduce bovine respiratory disease (BRD) incidence during the transition into feedlots. To determine the impact of prophylaxis on selected antimicrobial resistance genes (ARGs), 300 beef cattle were placed into 10 pens (30 head/pen). Five "CTC group" pens received in-feed CTC (10 mg/lb of body weight/d) from the fifth to ninth day after feedlot arrival, whereas the five "Control group" pens received no CTC. Fecal swabs and pen surface materials were collected for metagenomic DNA isolation on five sample occasions: arrival at the feedlot, 5 d posttreatment (dpt), and 27, 75, and 117 dpt. For each sample occasion, fecal samples and pen surface material samples were pooled by pen. Quantitative polymerase chain reaction was used to determine the abundances of 10 ARGs. Due to low detection percentages (%D) and quantification percentages (%Q), the abundances of five ARGs were not analyzed: aac(6')-Ie-aph(2'') (%D = 43%, %Q = 4%), blaCMY-2 (%D = 41%, %Q = 0%), blaCTX-M (%D = 0%, %Q = 0%), blaKPC-2 (%D = 21%, %Q = 16%), and mecA (%D = 4%, %Q = 0%). The %D and %Q for the ARGs aadA1, erm(B), tet(A), tet(B), and tet(M) were ≥98% and ≥90%, respectively. The abundances of aadA1, erm(B), tet(A), tet(B), and tet(M) resistance genes did not differ (p > 0.05) between the CTC and control groups at any sampling time for feces or pen surface material. Although only 10 ARGs were examined in this study, the results suggest that a single 5-d in-feed CTC prophylaxis of beef cattle to prevent BRD has a negligible impact on the abundances of ARGs.