Escherichia coli O157:H7 tir 255 T > A allele strains differ in chromosomal and plasmid composition.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains with the T allele in the translocated intimin receptor polymorphism (tir) 255 A > T gene associate with human disease more than strains with an A allele; however, the allele is not thought to be the direct cause of this difference. We sequenced a diverse set of STEC O157:H7 strains (26% A allele, 74% T allele) to identify linked differences that might underlie disease association. The average chromosome and pO157 plasmid size and gene content were significantly greater within the tir 255 A allele strains. Eighteen coding sequences were unique to tir 255 A allele chromosomes, and three were unique to tir 255 T allele chromosomes. There also were non-pO157 plasmids that were unique to each tir 255 allele variant. The overall average number of prophages did not differ between tir 255 allele strains; however, there were different types between the strains. Genomic and mobile element variation linked to the tir 255 polymorphism may account for the increased frequency of the T allele isolates in human disease.

16S rRNA gene-based assessment of common broiler chicken sampling methods: Evaluating intra-flock sample size, cecal pair similarity, and cloacal swab similarity to other alimentary tract locations.

16S rRNA gene sequencing for characterization of microbiomes has become more common in poultry research and can be used to both answer specific research questions and help inform experimental design choices. The objective of this study was to use 16S rRNA gene sequencing to examine common sampling practices in broiler chicken studies such as: the required number of birds selected from a flock to adequately capture microbiome diversity, the differences between cecal pairs within the same bird, and whether cloacal swabs are representative of other alimentary tract (AT) locations. To do this, nine market age broilers were euthanized and immediately sampled in ten AT locations: crop, gizzard, proventriculus, duodenum, jejunum, ileum, cecal samples from each pouch, colon, and cloacal swab. DNA was extracted and subjected to 16S rRNA gene amplification and sequencing. Each location within the broiler AT hosts distinct microbial communities. When each sampling location was considered, it was found that sampling after 2.8 birds (range 2-4) resulted in less than 10% new amplicon sequencing variants (ASV) being added while sampling after 7.6 birds (range 6-10) increases new observed ASVs by less than 1%. Additionally, when cecal pairs from the same bird were evaluated, it was found that cecal pair mates are an adequate replication if interested in the total cecal microbiome but may be less useful if a rare lineage is of interest. Furthermore, when compared to other AT locations, the cecal microbiome was enriched in Firmicutes and Bacteroides while several lineages, most notably Lactobacillus, were under-represented. Finally, when cloacal swabs were compared to other AT locations, community similarity exhibited a direct distance relationship, i.e., the more aborad samples were the more similar they were to the swab. These findings indicate that while cloacal swabs can approximate overall changes in microbiome composition, they are not adequate for inferring changes to specific taxa in other parts of the AT tract-even those that are highly abundant within the microbial community. These data provide new insights guiding appropriate sample size selection within flocks and add to the consensus data regarding cecal pair similarity and destructive versus non-destructive sampling methods.

Comparative Genomics of Escherichia coli Serotype O55:H7 Using Complete Closed Genomes.

Escherichia coli O55:H7 is a human foodborne pathogen and is recognized as the progenitor strain of E. coli O157:H7. While this strain is important from a food safety and genomic evolution standpoint, much of the genomic diversity of E. coli O55:H7 has been demonstrated using draft genomes. Here, we combine the four publicly available E. coli O55:H7 closed genomes with six newly sequenced closed genomes to provide context to this strain's genomic diversity. We found significant diversity within the 10 E. coli O55:H7 strains that belonged to three different sequence types. The prophage content was about 10% of the genome, with three prophages common to all strains and seven unique to one strain. Overall, there were 492 insertion sequences identified within the six new sequence strains, with each strain on average containing 75 insertions (range 55 to 114). A total of 31 plasmids were identified between all isolates (range 1 to 6), with one plasmid (pO55) having an identical phylogenetic tree as the chromosome. The release and comparison of these closed genomes provides new insight into E. coli O55:H7 diversity and its ability to cause disease in humans.

Increased Prevalence of Salmonella Infantis Isolated from Raw Chicken and Turkey Products in the United States Is Due to a Single Clonal Lineage Carrying the pESI Plasmid.

Infantis has recently become one of the most common serotypes of Salmonella isolated in the U.S. from raw meat samples collected in processing facilities and in retail stores. Investigations have determined that the majority of these isolates contain the pESI plasmid, but there has not been a large-scale investigation of the chromosome of these isolates. Here, we investigated 3276 whole-genome sequences of Salmonella Infantis with and without the pESI plasmid to understand chromosomal differences between plasmid carriage groups. S. Infantis genomes arranged into multiple clades with a single clade containing the isolates carrying the plasmid. Fifty-eight SNPs were identified in complete linkage disequilibrium between isolates that did and did not carry the plasmid. However, there were no unique genes present only in the genomes of isolates containing the plasmid. On average, isolates with the plasmid did contain more insertion sequences than those without (p < 0.05). Given that S. Infantis isolates carrying pESI form a single clade, it can be inferred that the increase in carriage of this plasmid in the U.S. is due to rapid clonal expansion of a single strain rather than as a result of multiple transfer events. As this S. Infantis clone does not contain any unique chromosomal genes, its proliferation appears to be due to pESI plasmid-encoded genes that may be advantageous in the chickens and turkeys or in their environment.

Rates of evolutionary change of resident Escherichia coli O157:H7 differ within the same ecological niche.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a pathogen known to reside in cattle feedlots. This retrospective study examined 181 STEC O157:H7 strains collected over 23 years from a closed-system feedlot. All strains were subjected to short-read sequencing, with a subset of 36 also subjected to long-read sequencing. RESULTS: Over 96% of the strains fell into four phylogenetically distinct clades. Clade membership was associated with multiple factors including stx composition and the alleles of a well-characterized polymorphism (tir 255 T > A). Small plasmids (2.7 to 40 kb) were found to be primarily clade specific. Within each clade, chromosomal rearrangements were observed along with a core phageome and clade specific phages. Across both core and mobile elements of the genome, multiple SNP alleles were in complete linkage disequilibrium across all strains within specific clades. Clade evolutionary rates varied between 0.9 and 2.8 SNP/genome/year with two tir A allele clades having the lowest evolutionary rates. Investigation into possible causes of the differing rates was not conclusive but revealed a synonymous based mutation in the DNA polymerase III of the fastest evolving clade. Phylogenetic trees generated through our bioinformatic pipeline versus the NCBI's pathogen detection project were similar, with the two tir A allele clades matching individual NCBI SNP clusters, and the two tir T allele clades assigned to multiple closely-related SNP clusters. CONCLUSIONS: In one ecological niche, a diverse STEC O157:H7 population exhibited different rates of evolution that associated with SNP alleles in linkage disequilibrium in the core genome and mobile elements, including tir 255 T > A.

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.

Considerations and best practices in animal science 16S ribosomal RNA gene sequencing microbiome studies.

Microbiome studies in animal science using 16S rRNA gene sequencing have become increasingly common in recent years as sequencing costs continue to fall and bioinformatic tools become more powerful and user-friendly. The combination of molecular biology, microbiology, microbial ecology, computer science, and bioinformatics-in addition to the traditional considerations when conducting an animal science study-makes microbiome studies sometimes intimidating due to the intersection of different fields. The objective of this review is to serve as a jumping-off point for those animal scientists less familiar with 16S rRNA gene sequencing and analyses and to bring up common issues and concerns that arise when planning an animal microbiome study from design through analysis. This review includes an overview of 16S rRNA gene sequencing, its advantages, and its limitations; experimental design considerations such as study design, sample size, sample pooling, and sample locations; wet lab considerations such as field handing, microbial cell lysis, low biomass samples, library preparation, and sequencing controls; and computational considerations such as identification of contamination, accounting for uneven sequencing depth, constructing diversity metrics, assigning taxonomy, differential abundance testing, and, finally, data availability. In addition to general considerations, we highlight some special considerations by species and sample type.

Rapid estimation of Salmonella enterica contamination level in ground beef - Application of the time-to-positivity method using a combination of molecular detection and direct plating.

Little progress has been made in decreasing the incidence rate of salmonellosis in the US over the past decade. Mitigating the contribution of contaminated raw meat to the salmonellosis incidence rate requires rapid methods for quantifying Salmonella, so that highly contaminated products can be removed before entering the food chain. Here we evaluated the use of Time-to-Positivity (TTP) as a rapid, semi-quantitative approach for estimating Salmonella contamination levels in ground beef. Growth rates of 14 Salmonella strains (inoculated at log 1 to -2 CFU/g) were characterized in lean ground beef mTSB enrichments and time-to-detection was determined using culture and molecular detection methods. Enrichments were sampled at five timepoints and results were used to construct a prediction model of estimated contamination level by TTP (superscript indicates time in hours) defined as TTP4: ≥5 CFU/g; TTP6: ≤5, ≥1 CFU/g; TTP8: ≤1, ≥0.01 CFU/g; with samples negative at 8 h estimated ≤0.01 CFU/g. Model performance measures showed high sensitivity (100%) and specificity (83% and 93% for two detection methods) for samples with a TTP4, with false negative rates of 0%.

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.

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.

A Cautionary Report for Pathogen Identification Using Shotgun Metagenomics; A Comparison to Aerobic Culture and Polymerase Chain Reaction for Salmonella enterica Identification.

This study was conducted to compare aerobic culture, polymerase chain reaction (PCR), lateral flow immunoassay (LFI), and shotgun metagenomics for identification of Salmonella enterica in feces collected from feedlot cattle. Samples were analyzed in parallel using all four tests. Results from aerobic culture and PCR were 100% concordant and indicated low S. enterica prevalence (3/60 samples positive). Although low S. enterica prevalence restricted formal statistical comparisons, LFI and deep metagenomic sequencing results were discordant with these results. Specifically, metagenomic analysis using k-mer-based classification against the RefSeq database indicated that 11/60 of samples contained sequence reads that matched to the S. enterica genome and uniquely identified this species of bacteria within the sample. However, further examination revealed that plasmid sequences were often included with bacterial genomic sequence data submitted to NCBI, which can lead to incorrect taxonomic classification. To circumvent this classification problem, we separated all plasmid sequences included in bacterial RefSeq genomes and reassigned them to a unique taxon so that they would not be uniquely associated with specific bacterial species such as S. enterica. Using this revised database and taxonomic structure, we found that only 6/60 samples contained sequences specific for S. enterica, suggesting increased relative specificity. Reads identified as S. enterica in these six samples were further evaluated using BLAST and NCBI's nr/nt database, which identified that only 2/60 samples contained reads exclusive to S. enterica chromosomal genomes. These two samples were culture- and PCR-negative, suggesting that even deep metagenomic sequencing suffers from lower sensitivity and specificity in comparison to more traditional pathogen detection methods. Additionally, no sample reads were taxonomically classified as S. enterica with two other metagenomic tools, Metagenomic Intra-species Diversity Analysis System (MIDAS) and Metagenomic Phylogenetic Analysis 2 (MetaPhlAn2). This study re-affirmed that the traditional techniques of aerobic culture and PCR provide similar results for S. enterica identification in cattle feces. On the other hand, metagenomic results are highly influenced by the classification method and reference database employed. These results highlight the nuances of computational detection of species-level sequences within short-read metagenomic sequence data, and emphasize the need for cautious interpretation of such results.

Investigation of tylosin in feed of feedlot cattle and effects on liver abscess prevalence, and fecal and soil microbiomes and resistomes1.

Liver abscesses in feedlot cattle are detrimental to animal performance and economic return. Tylosin, a macrolide antibiotic, is used to reduce prevalence of liver abscesses, though there is variable efficacy among different groups of cattle. There is an increased importance in better understanding the etiology and pathogenesis of this condition because of growing concern over antibiotic resistance and increased scrutiny regarding use of antibiotics in food animal production. The objective of this study was to compare the microbiomes and antimicrobial resistance genes (resistomes) of feces of feedlot cattle administered or not administered tylosin and in their pen soil in 3 geographical regions with differing liver abscess prevalences. Cattle (total of 2,256) from 3 geographical regions were selected for inclusion based on dietary supplementation with tylosin (yes/no). Feces and pen soil samples were collected before harvest, and liver abscesses were identified at harvest. Shotgun and 16S rRNA amplicon sequencing were used to evaluate the soil and feces. Microbiome and resistome composition of feces (as compared by UniFrac distances and Euclidian distances, respectively) did not differ (P > 0.05) among tylosin or no tylosin-administered cattle. However, feedlot location was associated with differences (P ≤ 0.05) of resistomes and microbiomes. Using LASSO, a statistical model identified both fecal and soil microbial communities as predictive of liver abscess prevalence in pens. This model explained 75% of the variation in liver abscess prevalence, though a larger sample size would be needed to increase robustness of the model. These data suggest that tylosin exposure does not have a large impact on cattle resistomes or microbiomes, but instead, location of cattle production may be a stronger driver of both the resistome and microbiome composition of feces.

Ground beef microbiome changes with antimicrobial decontamination interventions and product storage.

Ground beef makes up more than half of the beef consumed in the U.S. market. Although numerous studies have been conducted on microbial safety and shelf life of ground beef limited work has been done using a culture-independent approach. While past studies have allowed for the evaluation of a few organisms of interest, there is limited work on the microbial community associated with fresh ground beef. In order to have a more complete picture of the microbial ecology of the product, a culture-independent approach utilizing 16S rRNA gene amplicon sequencing was used. The objectives of this study were to characterize the fresh ground beef microbiome and the effect that antimicrobial interventions and antioxidants, applied to beef trim before grinding, and product storage have on community composition using 16S rRNA gene amplicon sequencing. Beef trimmings were treated with antimicrobials and an antioxidant. Samples were ground, loafed, and overwrapped before being packaged in modified-atmosphere packaging. Samples were in dark storage for 21 days followed by five days in retail display. Periodically during storage, samples were collected for microbiological analysis and DNA isolation. Due to low microbial biomass, only 52 of 210 samples were included in the final analysis. These samples represented two antimicrobial treatments (peroxyacetic acid, and a sulfuric acid and sodium sulfate blend) and a control, from day-15 of dark storage and day-5 of retail display. As sample age increased, so did the number of raw reads (P < 0.001) and aerobic plate counts (P < 0.001), which were correlated (r = 0.94, P = 0.017). Across all samples, lactic acid bacteria were most abundant followed by Enterobacteriaceae; several rare taxa were also identified (namely Geobacillus, Thermus, and Sporosarcina). Antimicrobial treatment altered the bacterial alpha (P < 0.001) and beta (P = 0.001) diversity, while storage day altered alpha (P = 0.001) diversity. Enterobacteriaceae relative abundance differed (P < 0.05) among treatments and was highest in control samples. In addition to confirming previously described dominant microbial differences in culture-dependent results, these data identified genera not typically associated with ground beef and allowed for study of shifts in the entire microbiome and not just a subset of indicator organisms.

Effects of a Saccharomyces cerevisiae fermentation product on liver abscesses, fecal microbiome, and resistome in feedlot cattle raised without antibiotics.

Liver abscesses in feedlot cattle form secondary to high concentrate feeds and rumen acidosis. Antimicrobial drugs are commonly included in cattle feed for prevention of liver abscesses, but concerns regarding antimicrobial resistance have increased the need for alternative treatments. A block randomized clinical trial was conducted to evaluate the effects of a Saccharomyces cerevisiae fermentation product (SCFP) on liver abscesses, fecal microbiomes, and resistomes in cattle raised without antibiotics in a Colorado feedlot. At enrollment, steers (n = 4,689) were sorted, by weight and source, into 2 pens comprising a block (n = 14 blocks, 28 pens); pens were randomly allocated to either the control group or the treatment group, where the diet was supplemented with SCFP. Prior to harvest, composited feces were collected for characterization of the microbiome and resistome using 16S rRNA gene and shotgun sequencing. At harvest, liver abscess severity was quantified for individual cattle. There were no statistical differences detected by treatment group in animal health, liver abscess prevalence or severity. Organisms classified to phylum, Elusimicrobia were more abundant in the feces of treated cattle, however, there were no differences in the resistome by treatment group. Both microbiome and resistome varied significantly among enrollment blocks.

Investigating Effects of Tulathromycin Metaphylaxis on the Fecal Resistome and Microbiome of Commercial Feedlot Cattle Early in the Feeding Period.

The objective was to examine effects of treating commercial beef feedlot cattle with therapeutic doses of tulathromycin, a macrolide antimicrobial drug, on changes in the fecal resistome and microbiome using shotgun metagenomic sequencing. Two pens of cattle were used, with all cattle in one pen receiving metaphylaxis treatment (800 mg subcutaneous tulathromycin) at arrival to the feedlot, and all cattle in the other pen remaining unexposed to parenteral antibiotics throughout the study period. Fecal samples were collected from 15 selected cattle in each group just prior to treatment (Day 1), and again 11 days later (Day 11). Shotgun sequencing was performed on isolated metagenomic DNA, and reads were aligned to a resistance and a taxonomic database to identify alignments to antimicrobial resistance (AMR) gene accessions and microbiome content. Overall, we identified AMR genes accessions encompassing 9 classes of AMR drugs and encoding 24 unique AMR mechanisms. Statistical analysis was used to identify differences in the resistome and microbiome between the untreated and treated groups at both timepoints, as well as over time. Based on composition and ordination analyses, the resistome and microbiome were not significantly different between the two groups on Day 1 or on Day 11. However, both the resistome and microbiome changed significantly between these two sampling dates. These results indicate that the transition into the feedlot-and associated changes in diet, geography, conspecific exposure, and environment-may exert a greater influence over the fecal resistome and microbiome of feedlot cattle than common metaphylactic antimicrobial drug treatment.

Genetics and microbiology of meat.

Detection, reduction, and public health monitoring of foodborne pathogens has advanced in precision and efficiency as technology has progressed. Here, we look back on the evolution of food safety management and public health, and attempt to provide a view of how the technology and tools have changed, and how emerging technologies and tools may impact how we manage food safety and public health in the future. With the revolution of gene editing techniques (e.g. CRISPR-Cas9, etc.), Next Generation Sequencing (NGS) and "omics"-based technologies, along with the bioinformatics tools that go with them, we now have a very new array of tools that can impact foodborne disease management. In addition to overall improvement in food safety, these tools have helped understand antibiotic resistance and virulence factors in meat to a higher degree than ever before. These technological advancements will allow food safety to move beyond strain-level characterization and control of pathogens to pinpointing genes of public health concern.

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.