MEGARes 2.0: a database for classification of antimicrobial drug, biocide and metal resistance determinants in metagenomic sequence data.

Antimicrobial resistance (AMR) is a threat to global public health and the identification of genetic determinants of AMR is a critical component to epidemiological investigations. High-throughput sequencing (HTS) provides opportunities for investigation of AMR across all microbial genomes in a sample (i.e. the metagenome). Previously, we presented MEGARes, a hand-curated AMR database and annotation structure developed to facilitate the analysis of AMR within metagenomic samples (i.e. the resistome). Along with MEGARes, we released AmrPlusPlus, a bioinformatics pipeline that interfaces with MEGARes to identify and quantify AMR gene accessions contained within a metagenomic sequence dataset. Here, we present MEGARes 2.0 (https://megares.meglab.org), which incorporates previously published resistance sequences for antimicrobial drugs, while also expanding to include published sequences for metal and biocide resistance determinants. In MEGARes 2.0, the nodes of the acyclic hierarchical ontology include four antimicrobial compound types, 57 classes, 220 mechanisms of resistance, and 1,345 gene groups that classify the 7,868 accessions. In addition, we present an updated version of AmrPlusPlus (AMR ++ version 2.0), which improves accuracy of classifications, as well as expanding scalability and usability.

Nix Pro Color Sensor provides comparable color measurements to HunterLab colorimeter for fresh beef.

The HunterLab MiniScan (HunterLab) colorimeter is used in meat quality research worldwide for measuring meat color; however, the Nix Pro Color Sensor (Nix) could be a less expensive alternative that is easier to operate. Therefore, the objective of this study was to compare the two colorimeters to objectively evaluate fresh beef color. Longissimus thoracis muscle from one side of A maturity beef carcasses (n = 200) was evaluated using both the HunterLab (3 technical replicate scans) and Nix (3, 5, 7, and 9 technical replicate scans) colorimeters. The correlation between the HunterLab and Nix for L* (lightness), a* (redness), and b* (yellowness) values ranged between r = 0.80 to 0.85 and the Bland Altman Limits of Agreement analysis indicated good agreement between the Nix and HunterLab colorimeters for all the color parameters. These results indicated that the Nix colorimeter could be a viable alternative for HunterLab colorimeters.

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).

MEGARes: an antimicrobial resistance database for high throughput sequencing.

Antimicrobial resistance has become an imminent concern for public health. As methods for detection and characterization of antimicrobial resistance move from targeted culture and polymerase chain reaction to high throughput metagenomics, appropriate resources for the analysis of large-scale data are required. Currently, antimicrobial resistance databases are tailored to smaller-scale, functional profiling of genes using highly descriptive annotations. Such characteristics do not facilitate the analysis of large-scale, ecological sequence datasets such as those produced with the use of metagenomics for surveillance. In order to overcome these limitations, we present MEGARes (https://megares.meglab.org), a hand-curated antimicrobial resistance database and annotation structure that provides a foundation for the development of high throughput acyclical classifiers and hierarchical statistical analysis of big data. MEGARes can be browsed as a stand-alone resource through the website or can be easily integrated into sequence analysis pipelines through download. Also via the website, we provide documentation for AmrPlusPlus, a user-friendly Galaxy pipeline for the analysis of high throughput sequencing data that is pre-packaged for use with the MEGARes database.

Succinct colored de Bruijn graphs.

MOTIVATION: In 2012, Iqbal et al. introduced the colored de Bruijn graph, a variant of the classic de Bruijn graph, which is aimed at 'detecting and genotyping simple and complex genetic variants in an individual or population'. Because they are intended to be applied to massive population level data, it is essential that the graphs be represented efficiently. Unfortunately, current succinct de Bruijn graph representations are not directly applicable to the colored de Bruijn graph, which requires additional information to be succinctly encoded as well as support for non-standard traversal operations. RESULTS: Our data structure dramatically reduces the amount of memory required to store and use the colored de Bruijn graph, with some penalty to runtime, allowing it to be applied in much larger and more ambitious sequence projects than was previously possible. AVAILABILITY AND IMPLEMENTATION: https://github.com/cosmo-team/cosmo/tree/VARI. CONTACT: martin.muggli@colostate.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effects of Ceftiofur and Chlortetracycline on the Resistomes of Feedlot Cattle.

Treatment of food-producing animals with antimicrobial drugs (AMD) is controversial because of concerns regarding promotion of antimicrobial resistance (AMR). To investigate this concern, resistance genes in metagenomic bovine fecal samples during a clinical trial were analyzed to assess the impacts of treatment on beef feedlot cattle resistomes. Four groups of cattle were exposed, using a 2-by-2 factorial design, to different regimens of antimicrobial treatment. Injections of ceftiofur crystalline-free acid (a third-generation cephalosporin) were used to treat all cattle in treatment pens or only a single animal, and either chlortetracycline was included in the feed of all cattle in a pen or the feed was untreated. On days 0 and 26, respectively, pre- and posttrial fecal samples were collected, and resistance genes were characterized using shotgun metagenomics. Treatment with ceftiofur was not associated with changes to ß-lactam resistance genes. However, cattle fed chlortetracycline had a significant increase in relative abundance of tetracycline resistance genes. There was also an increase of an AMR class not administered during the study, which is a possible indicator of coselection of resistance genes. Samples analyzed in this study had previously been evaluated by culture characterization (Escherichia coli and Salmonella) and quantitative PCR (qPCR) of metagenomic fecal DNA, which allowed comparison of results with this study. In the majority of samples, genes that were selectively enriched through culture and qPCR were not identified through shotgun metagenomic sequencing in this study, suggesting that changes previously documented did not reflect changes affecting the majority of bacterial genetic elements found in the predominant fecal resistome.IMPORTANCE Despite significant concerns about public health implications of AMR in relation to use of AMD in food animals, there are many unknowns about the long- and short-term impact of common uses of AMD for treatment, control, and prevention of disease. Additionally, questions commonly arise regarding how to best measure and quantify AMR genes in relation to public health risks and how to determine which genes are most important. These data provide an introductory view of the utility of using shotgun metagenomic sequencing data as an outcome for clinical trials evaluating the impact of using AMD in food animals.

Impact of "Raised without Antibiotics" Beef Cattle Production Practices on Occurrences of Antimicrobial Resistance.

The specific antimicrobial resistance (AMR) decreases that can be expected from reducing antimicrobial (AM) use in U.S. beef production have not been defined. To address this data gap, feces were recovered from 36 lots of "raised without antibiotics" (RWA) and 36 lots of "conventional" (CONV) beef cattle. Samples (n = 719) were collected during harvest and distributed over a year. AMR was assessed by (i) the culture of six AM-resistant bacteria (ARB), (ii) quantitative PCR (qPCR) for 10 AMR genes (ARGs), (iii) a qPCR array of 84 ARGs, and (iv) metagenomic sequencing. Generally, AMR levels were similar, but some were higher in CONV beef cattle. The prevalence of third-generation cephalosporin-resistant (3GCr) Escherichia coli was marginally different between production systems (CONV, 47.5%; RWA, 34.8%; P = 0.04), but the seasonal effect (summer, 92.8%; winter, 48.3%; P < 0.01) was greater. Erythromycin-resistant (ERYr) Enterococcus sp. concentrations significantly differed between production systems (CONV, 1.91 log10 CFU/g; RWA, 0.73 log10 CFU/g; P < 0.01). Levels of aadA1, ant(6)-I, bla ACI, erm(A), erm(B), erm(C), erm(F), erm(Q), tet(A), tet(B), tet(M), and tet(X) ARGs were higher (P < 0.05) in the CONV system. Aggregate abundances of all 43 ARGs detected by metagenomic sequencing and the aggregate abundances of ARGs in the aminoglycoside, ß-lactam, macrolide-lincosamide-streptogramin B (MLS), and tetracycline AM classes did not differ (log2 fold change < 1.0) between CONV and RWA systems. These results suggest that further reductions of AM use in U.S. beef cattle production may not yield significant AMR reductions beyond MLS and tetracycline resistance.IMPORTANCE The majority of antimicrobial (AM) use in the United States is for food-animal production, leading to concerns that typical AM use patterns during "conventional" (CONV) beef cattle production in the United States contribute broadly to antimicrobial resistance (AMR) occurrence. In the present study, levels of AMR were generally similar between CONV and "raised without antibiotics" (RWA) cattle. Only a limited number of modest AMR increases was observed in CONV cattle, primarily involving macrolide-lincosamide-streptogramin B (MLS) and tetracycline resistance. Macrolides (tylosin) and tetracyclines (chlortetracycline) are administered in-feed for relatively long durations to reduce liver abscesses. To ensure judicious AM use, the animal health, economic, and AMR impacts of shorter duration in-feed administration of these AMs should be examined. However, given the modest AMR reductions observed, further reductions of AM use in U.S. beef cattle production may not yield significant AMR reductions beyond MLS and tetracycline resistance.

Use of Metagenomic Shotgun Sequencing Technology To Detect Foodborne Pathogens within the Microbiome of the Beef Production Chain.

Foodborne illnesses associated with pathogenic bacteria are a global public health and economic challenge. The diversity of microorganisms (pathogenic and nonpathogenic) that exists within the food and meat industries complicates efforts to understand pathogen ecology. Further, little is known about the interaction of pathogens within the microbiome throughout the meat production chain. Here, a metagenomic approach and shotgun sequencing technology were used as tools to detect pathogenic bacteria in environmental samples collected from the same groups of cattle at different longitudinal processing steps of the beef production chain: cattle entry to feedlot, exit from feedlot, cattle transport trucks, abattoir holding pens, and the end of the fabrication system. The log read counts classified as pathogens per million reads for Salmonella enterica,Listeria monocytogenes,Escherichia coli,Staphylococcus aureus, Clostridium spp. (C. botulinum and C. perfringens), and Campylobacter spp. (C. jejuni,C. coli, and C. fetus) decreased over subsequential processing steps. Furthermore, the normalized read counts for S. enterica,E. coli, and C. botulinumwere greater in the final product than at the feedlots, indicating that the proportion of these bacteria increased (the effect on absolute numbers was unknown) within the remaining microbiome. From an ecological perspective, data indicated that shotgun metagenomics can be used to evaluate not only the microbiome but also shifts in pathogen populations during beef production. Nonetheless, there were several challenges in this analysis approach, one of the main ones being the identification of the specific pathogen from which the sequence reads originated, which makes this approach impractical for use in pathogen identification for regulatory and confirmation purposes.

National Beef Quality Audit-2022 Phase 1: face-to-face and digital interviews.

The National Beef Quality Audit (NBQA) has been conducted regularly since 1991 to assess and benchmark quality in the U.S. beef industry, with the most recent iteration conducted in 2022. The goal of NBQA Phase I is to evaluate what needs to be managed to improve beef quality and demand. Interviews (n = 130) of industry personnel were conducted with the aid of routing software. In total, packers (n = 24), retailers (n = 20), further processors (n = 26), foodservice (n = 18), and allied government agencies and trade organizations (n = 42) were interviewed. Interviews were routed in software based on interviewee involvement in either the fed steer and heifer market cow and bull sectors, or both. Interviews were structured to elicit random responses in the order of determining "must-have" criteria (quality factors that are required to make a purchase), best/worst ranking (of quality factors based on importance), how interviewees defined quality terms, a strength, weakness, opportunities, threats (SWOT) analysis, general beef industry questions, and sustainability goals (the latter four being open-ended). Quality factors were 1) visual characteristics, 2) cattle genetics, 3) food safety, 4) eating satisfaction, 5) animal well-being, 6) weight and size, and 7) lean, fat, and bone. Best/worst analysis revealed that "food safety" was the most (P < 0.05) important factor in beef purchasing decisions for all market sectors and frequently was described as "everything" and "a way of business." Culture surrounding food safety changed compared to previous NBQAs with interviewees no longer considering food safety as a purchasing criterion, but rather as a market expectation. The SWOT analysis indicated that "eating quality of U.S. beef" was the greatest strength, and cited that educating both consumers and producers on beef production would benefit the industry. Irrespective of whether companies' products were fed or market cow/bull beef, respondents said that they believed "environmental concerns" were among the major threats to the industry. Perceived image of the beef industry in the market sectors has improved since NBQA-2016 for both fed cattle and market cow/bull beef.

National Beef Quality Audit-2022: Transportation, mobility, live cattle, and hide assessments to determine producer-related defects that affect animal welfare and the value of market cows and bulls at processing facilities.

The National Beef Quality Audit (NBQA)-2022 serves as a benchmark of the current market cow and bull sectors of the U.S. beef industry and allows comparison to previous audits as a method of monitoring industry progress. From September 2021 through May 2022, livestock trailers (n = 125), live animals (n = 5,430), and post-slaughter hide-on animals (n = 6,674) were surveyed at 20 commercial beef processing facilities across the U.S. Cattle were transported in a variety of trailer types for an average distance of 490.6 km and a mean transport time of 6.3 h. During transit, cattle averaged 2.3 m2 of trailer space per animal indicating sufficient space was provided according to industry guidelines. Of all trailers surveyed, 55.3% transported cattle from an auction barn to a processing facility. When surveyed, 63.6% of all truck drivers reported to be Beef Quality Assurance certified. The majority (77.0%) of cattle were sound when evaluated for mobility. Mean body condition scores (9-point scale) for beef cows and bulls were 3.8 and 4.4, respectively, whereas mean body condition scores (5-point scale) for dairy cows and bulls were 2.3 and 2.6, respectively. Of the cattle surveyed, 45.1% had no visible live animal defects, and 37.9% had only a single defect. Of defects present in cows, 64.6% were attributed to an udder problem. Full udders were observed in 47.5% of all cows. Nearly all cattle were free of visible abscesses and knots (97.9% and 98.2%, respectively). No horns were observed in 89.4% of all cattle surveyed. Beef cattle were predominantly black-hided (68.9% and 67.4% of cows and bulls, respectively). Holstein was the predominant dairy animal observed and accounted for 85.7% of the cows and 98.0% of the bulls. Only 3.1% of all animals had no form of identification. Findings from the NBQA-2022 show improvements within the industry and identify areas that require continued education and research to improve market cow and bull welfare and beef quality.

Efficacy of Antimicrobial Spray Treatments in Reducing Salmonella enterica Populations on Chilled Pork.

Studies reporting on alternative antimicrobial interventions for pathogen control on chilled pork carcasses and cuts are limited. In this study, the antimicrobial effects of various spray treatments against Salmonella enterica inoculated on skin-on pork samples were evaluated. Chilled pork jowls were portioned (10 by 5 by 1 cm) and inoculated, on the skin side, with a mixture of six S. enterica serotype strains to target levels of 6 to 7 log CFU/cm2 (high inoculation level) or 3 to 4 log CFU/cm2 (low inoculation level). Samples were then left nontreated (control) or were treated (10 s) using a laboratory-scale spray cabinet with water, formic acid (1.5%), a proprietary blend of sulfuric acid and sodium sulfate (SSS, pH 1.2), peroxyacetic acid (PAA, 400 ppm), or PAA (400 ppm) that was pH-adjusted (acidified) with acetic acid (1.5%), formic acid (1.5%), or SSS (pH 1.2). Samples (n = 6) were analyzed for Salmonella populations after treatment application (0 h) and after 24 h of refrigerated (4°C) storage. Irrespective of inoculation level, all spray treatments effectively reduced (P < 0.05) Salmonella levels immediately following their application. Overall, pathogen reductions for the chemical treatments, compared to the respective high and low inoculation level nontreated controls, ranged from 1.2 to 1.9 log CFU/cm2 (high inoculation level) and 1.0 to 1.7 log CFU/cm2 (low inoculation level). Acidification of PAA with acetic acid, formic acid, or SSS did not (P ≥ 0.05) enhance the initial bactericidal effects of the nonacidified PAA treatment. Salmonella populations recovered from all treated samples following 24 h of storage were, in general, similar (P ≥ 0.05) or up to 0.6 log CFU/cm2 lower (P < 0.05) than those recovered from samples analyzed immediately after treatment application. The results of the study may be used by processing establishments to help identify effective decontamination interventions for reducing Salmonella contamination on pork.

More than an anthropogenic phenomenon: Antimicrobial resistance in ungulates from natural and agricultural environments.

Widely considered an anthropogenic phenomenon, antimicrobial resistance (AMR) is a naturally occurring mechanism that microorganisms use to gain competitive advantage. AMR represents a significant threat to public health and has generated criticism towards the overuse of antimicrobial drugs. Livestock have been proposed as important reservoirs for AMR accumulation. Here, we show that assemblages of AMR genes in cattle and ungulates from natural environments (Yellowstone and Rocky Mountain National Parks) are all dominated by genes conferring resistance to tetracyclines. However, cattle feces contained higher proportions of erm(A-X) genes conferring resistance to macrolide antibiotics. Medically important AMR genes differed between cattle and natural ungulates, but cumulatively were more predominant in natural soils. Our findings suggest that the commonly described predominance of tetracycline resistance in cattle feces is a natural phenomenon among multiple ungulate species and not solely a result of antimicrobial drug exposure. Yet, the virtual absence of macrolide resistance genes in natural ungulates suggests that macrolide usage in agriculture may enrich these genes in cattle. Our results show that antimicrobial use in agriculture may be promoting a potential reservoir for specific types of AMR (i.e., macrolide resistance) but that a significant proportion of the ungulate resistome appears to have natural origins.

Not All Liver Abscesses Are Created Equal: The Impact of Tylosin and Antibiotic Alternatives on Bovine Liver Abscess Microbial Communities and a First Look at Bacteroidetes-Dominated Communities.

Liver abscesses (LAs) are extremely prevalent in cattle and result in significant economic losses due to liver condemnation, decreased growth and production, and lower carcass quality. LAs are commonly attributed to the transition to diets high in rapidly fermentable starch which results in rumen epithelial inflammation that allows pathogenic bacteria to gain entry to liver through transport via the hepatic portal vein. The most common intervention for LAs is the inclusion of antibiotics in feedlot diets, under the supervision of a veterinarian; this treatment is associated with reduced occurrence of LAs in this and other studies. Here, through the largest LA 16S rRNA gene sequencing study to date, we demonstrate that the inclusion of tylosin and antibiotic alternatives (the essential oil limonene and Saccharomyces cerevisiae fermentation product) had little impact on LA microbial community composition. Importantly, members of Bacteroidetes (Bacteroides spp. and Porphyromonas spp.) were identified as the dominant taxa in conjunction with low proportions of Fusobacteria in nearly a quarter (61/259) of all LA communities analyzed in this study. The relative abundances of the phyla Fusobacteria and Bacteroidetes had a strongly negative correlation, and LA microbial communities rarely contained high abundances of both of these dominant phyla. Further, based on the presence of taxa discriminant of Bacteroidetes-dominated LAs within over 400 bovine gut communities, we provide evidence suggestive of Bacteroidetes-dominated abscess communities originating in more distal portions of the bovine gut. Together, these findings suggest that some LA microbial communities may originate from portions of the gut other than the rumen.

Bacteroidetes and Firmicutes Drive Differing Microbial Diversity and Community Composition Among Micro-Environments in the Bovine Rumen.

Ruminants are a critical human food source and have been implicated as a potentially important source of global methane emissions. Because of their unique digestive physiology, ruminants rely upon a symbiotic relationship with the complex and rich community of microorganism in the foregut to allow digestion of complex carbohydrates. This study used 16S rRNA gene sequencing to investigate the composition of microbial communities from three rumen micro-environments of cattle fed identical diets: (1) free fluid, (2) the fibrous pack, and (3) the mucosa. Community composition analysis revealed that while a phylogenetic core including the most abundant and most common ruminal taxa (members of Bacteroidetes and Firmicutes) existed across micro-environments, the abundances of these taxa differed significantly between fluid- and mucosa-associated communities, and specific lineages were discriminant of individual micro-environments. Members of Firmicutes, specifically Clostridiales, Lachnospiraceae, Mogibacteriaceae, Christenellaceae, and Erysipelotrichaceae were significantly more abundant in fluid communities, while members of Bacteroidetes, namely Muribaculaceae and Prevotellaceae were more abundant in mucosa-associated communities. Additionally, Methanobacteriaceae, a family of methanogenic Archaea, was more abundant in fluid-associated communities. A set of four more diverse lineages were discriminant of pack-associated communities that included Succinivibrionaceae, RFP12 (Verruco-5), Fibrobacteraceae, and Spirochaetaceae. Our findings indicate that different ecological niches within each micro-environment have resulted in significant differences in the diversity and community structure of microbial communities from rumen fluid, pack, and mucosa without the influence of diet that will help contextualize the influence of other environmental factors.

Evaluating the effects of antimicrobial drug use on the ecology of antimicrobial resistance and microbial community structure in beef feedlot cattle.

Introduction: Use of antimicrobial drugs (AMDs) in food producing animals has received increasing scrutiny because of concerns about antimicrobial resistance (AMR) that might affect consumers. Previously, investigations regarding AMR have focused largely on phenotypes of selected pathogens and indicator bacteria, such as Salmonella enterica or Escherichia coli. However, genes conferring AMR are known to be distributed and shared throughout microbial communities. The objectives of this study were to employ target-enriched metagenomic sequencing and 16S rRNA gene amplicon sequencing to investigate the effects of AMD use, in the context of other management and environmental factors, on the resistome and microbiome in beef feedlot cattle. Methods: This study leveraged samples collected during a previous longitudinal study of cattle at beef feedlots in Canada. This included fecal samples collected from randomly selected individual cattle, as well as composite-fecal samples from randomly selected pens of cattle. All AMD use was recorded and characterized across different drug classes using animal defined daily dose (ADD) metrics. Results: Overall, fecal resistome composition was dominated by genes conferring resistance to tetracycline and macrolide-lincosamide-streptogramin (MLS) drug classes. The diversity of bacterial phyla was greater early in the feeding period and decreased over time in the feedlot. This decrease in diversity occurred concurrently as the microbiome represented in different individuals and different pens shifted toward a similar composition dominated by Proteobacteria and Firmicutes. Some antimicrobial drug exposures in individuals and groups were associated with explaining a statistically significant proportion of the variance in the resistome, but the amount of variance explained by these important factors was very small (<0.6% variance each), and smaller than associations with other factors measured in this study such as time and feedlot ID. Time in the feedlot was associated with greater changes in the resistome for both individual animals and composite pen-floor samples, although the proportion of the variance associated with this factor was small (2.4% and 1.2%, respectively). Discussion: Results of this study are consistent with other investigations showing that, compared to other factors, AMD exposures did not have strong effects on antimicrobial resistance or the fecal microbial ecology of beef cattle.

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.

Evaluation of Immersion and Spray Applications of Antimicrobial Treatments for Reduction of Campylobacter jejuni on Chicken Wings.

The decontamination efficacy of antimicrobial treatments against Campylobacter jejuni on chicken wings was evaluated. Chicken wings surface-inoculated with C. jejuni (3.9 log colony-forming units [CFU]/mL) were left untreated (control) or were treated by immersion (5 s) or in a spray cabinet (4 s) with water, a sulfuric acid and sodium sulfate blend (SSS; pH 1.2), formic acid (1.5%), peroxyacetic acid (PAA; 550 ppm), or PAA (550 ppm) that was pH-adjusted (acidified) with SSS (pH 1.2) or formic acid (1.5%). All evaluated immersion and spray chemical treatments effectively (p < 0.05) lowered C. jejuni populations on chicken wings. Spray application of chemical treatments resulted in immediate pathogen reductions ranging from 0.5 to 1.2 log CFU/mL, whereas their application by immersion lowered initial pathogen levels by 1.7 to 2.2 log CFU/mL. The PAA and acidified PAA treatments were equally (p ≥ 0.05) effective at reducing initial C. jejuni populations, however, following a 24 h refrigerated (4 °C) storage period, wings treated with acidified PAA had lower (p < 0.05) pathogen levels than samples that had been treated with PAA that was not acidified. Findings of this study should be useful to the poultry industry in its efforts to control Campylobacter contamination on chicken parts.

Comparative Whole Genome Analysis of Escherichia coli O157:H7 Isolates From Feedlot Cattle to Identify Genotypes Associated With the Presence and Absence of stx Genes.

A comparative whole genome analysis was performed on three newly sequenced Escherichia coli O157:H7 strains with different stx profiles, previously isolated from feedlot cattle [C1-010 (stx1-, stx2c+), C1-057 (stx-), and C1-067 (stx1+, stx2a+)], as well as five foodborne outbreak strains and six stx-negative strains from NCBI. Phylogenomic analysis demonstrated that the stx2c-carrying C1-010 and stx-negative C1-057 strains were grouped with the six NCBI stx-negative E. coli O157:H7 strains in Cluster 1, whereas the stx2a-carrying C1-067 and five foodborne outbreak strains were clustered together in Cluster 2. Based on different clusters, we selected the three newly sequenced strains, one stx2a-carrying strain, and the six NCBI stx-negative strains and identify their prophages at the stx insertion sites. All stx-carrying prophages contained both the three Red recombination genes (exo, bet, gam) and their repressor cI. On the other hand, the majority of the stx-negative prophages carried only the three Red recombination genes, but their repressor cI was absent. In the absence of the repressor cI, the consistent expression of the Red recombination genes in prophages might result in more frequent gene exchanges, potentially increasing the probability of the acquisition of stx genes. We further investigated each of the 10 selected E. coli O157:H7 strains for their respective unique metabolic pathway genes. Seven unique metabolic pathway genes in the two stx2a-carrying strains and one in the single stx2c-carrying and seven stx-negative strains were found to be associated with an upstream insertion sequence 629 within a conserved region among these strains. The presence of more unique metabolic pathway genes in stx2a-carrying E. coli O157:H7 strains may potentially increase their competitiveness in complex environments, such as feedlot cattle. For the stx2c-carrying and stx-negative E. coli O157:H7 strains, the fact that they were grouped into the same phylogenomic cluster and had the same unique metabolic pathway genes suggested that they may also share closely related evolutionary pathways. As a consequence, gene exchange between them is more likely to occur. Results from this study could potentially serve as a basis to help develop strategies to reduce the prevalence of pathogenic E. coli O157:H7 in livestock and downstream food production environments.

Air versus Water Chilling of Chicken: a Pilot Study of Quality, Shelf-Life, Microbial Ecology, and Economics.

The United States' large-scale poultry meat industry is energy and water intensive, and opportunities may exist to improve sustainability during the broiler chilling process. By USDA regulation, after harvest the internal temperature of the chicken must be reduced to 40°F or less within 16 h to inhibit bacterial growth that would otherwise compromise the safety of the product. This step is accomplished most commonly by water immersion chilling in the United States, while air chilling methods dominate other global markets. A comprehensive understanding of the differences between these chilling methods is lacking. Therefore, we assessed the meat quality, shelf-life, microbial ecology, and techno-economic impacts of chilling methods on chicken broilers in a university meat laboratory setting. We discovered that air chilling methods resulted in superior chicken odor and shelf-life, especially prior to 14 days of dark storage. Moreover, we demonstrated that air chilling resulted in a more diverse microbiome that we hypothesize may delay the dominance of the spoilage organism Pseudomonas Finally, a techno-economic analysis highlighted potential economic advantages to air chilling compared to water chilling in facility locations where water costs are a more significant factor than energy costs.IMPORTANCE As the poultry industry works to become more sustainable and to reduce the volume of food waste, it is critical to consider points in the processing system that can be altered to make the process more efficient. In this study, we demonstrate that the method used during chilling (air versus water chilling) influences the final product microbial community, quality, and physiochemistry. Notably, the use of air chilling appears to delay the bloom of Pseudomonas spp. that are the primary spoilers in packaged meat products. By using air chilling to reduce carcass temperatures instead of water chilling, producers may extend the time until spoilage of the products and, depending on the cost of water in the area, may have economic and sustainability advantages. As a next step, a similar experiment should be done in an industrial setting to confirm these results generated in a small-scale university lab facility.

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.