Effectiveness of stabilization methods for the immediate and short-term preservation of bovine fecal and upper respiratory tract genomic DNA.

Previous research on stabilization methods for microbiome investigations has largely focused on human fecal samples. There are a few studies using feces from other species, but no published studies investigating preservation of samples collected from cattle. Given that microbial taxa are differentially impacted during storage it is warranted to study impacts of preservation methods on microbial communities found in samples outside of human fecal samples. Here we tested methods of preserving bovine fecal respiratory specimens for up to 2 weeks at four temperatures (room temperature, 4°C, -20°C, and -80°C) by comparing microbial diversity and community composition to samples extracted immediately after collection. Importantly, fecal specimens preserved and analyzed were technical replicates, providing a look at the effects of preservation method in the absence of biological variation. We found that preservation with the OMNIgene®â€¢GUT kit resulted in community structure most like that of fresh samples extracted immediately, even when stored at room temperature (~20°C). Samples that were flash-frozen without added preservation solution were the next most representative of original communities, while samples preserved with ethanol were the least representative. These results contradict previous reports that ethanol is effective in preserving fecal communities and suggest for studies investigating cattle either flash-freezing of samples without preservative or preservation with OMNIgene®â€¢GUT will yield more representative microbial communities.

Associations between early lactation intramammary infections and udder health and performance during the first 180 days in milk in first-lactation organic dairy cows.

Prior data from our group showed that first-lactation cows under organic management in United States have a high prevalence of Staphylococcus aureus, Streptococcus spp. and Staphylococcus chromogenes-IMI in early lactation. Nonetheless, the relationship between IMI, udder health and milk production in organically reared primiparous cows remains elusive. The objectives of this observational study were to investigate the relationship between presence and persistence of IMI in the first 35 DIM and SCC and milk production during the first 6 mo of lactation on first-lactation organic dairy cows. The analysis included a total of 1,348 composite milk samples collected during the first 35 DIM that were submitted for milk culture and 1,674 DHIA tests during the first 180 DIM from 333 heifers in 4 organic dairy farms, enrolled between February 2019 and January 2020. The association between IMI in the first 35 DIM and new high SCC (SCC > 200,000 cells/mL) and milk production during the first 6 mo of lactation was investigated using Cox proportional hazards regression and mixed linear regression, respectively. The association between IMI persistence (harboring the same microorganism as reported by the laboratory for 2 or more samples) in the first 35 DIM and number of DHIA tests with high SCC during the first 6 mo of lactation was modeled using negative binomial regression. The presence of IMI by Staphylococcus aureus (HR [95%CI]): 3.35 [2.64, 4.25]) or Streptococcus spp. (HR [95%CI]: 2.25 [2.12, 2.39]) during the first 35 DIM was associated with an increased risk of new high SCC during the first 6 mo of lactation. Milk production was reduced when Streptococcus spp. were identified in milk samples. However, there was no evidence of a difference in milk production in Staphylococcus aureus-IMI. Isolation of non-aureus Staphylococci and closely related Mammaliicoccal species was related to a mild increase in the hazards of high SCC (HR [95%CI]: 1.34 [0.97, 1.85]) and a decrease in milk production during 1 or more postpartum tests. Presence of gram-negative or Streptococcus-like organisms IMI was not associated with either high SCC or milk production. Presence of Bacillus-IMI was associated with a lower hazard of new high SCC (HR [95%CI]: 0.45 [0.30, 0.68]), and higher milk production during the first 180 d of lactation (overall estimate [95%CI]: 1.7 kg/day [0.3, 3.0]). The persistence of IMI in the first 35 DIM was associated to the number of tests with high SCC during the lactation for all microorganisms except for Staphylococcus chromogenes. Therefore, our results suggest that the persistence of IMI in the first 35 DIM could be an important factor to understand the association between IMI detected in early lactation and lactational SCC and milk production in organic dairy heifers. Our study described associations between IMI, udder health, and milk production in first-lactation organic dairy cows that are consistent with findings from conventional dairy farms.

Investigation of intramammary infections in primiparous cows during early lactation on organic dairy farms.

Previous studies have shown that organically raised dairy cows have an increased prevalence of Staphylococcus aureus compared with conventionally raised dairy cows. However, little information exists about the dynamics of intramammary infection (IMI) in primiparous cows during early lactation on organic dairy farms. The objective of this study was to describe the IMI dynamics of primiparous cows on certified organic farms during early lactation. This longitudinal study enrolled 503 primiparous cows from 5 organic dairy farms from February 2019 to January 2020. Quarter-level milk samples were collected aseptically on a weekly basis during the first 5 wk of lactation. Samples were pooled by cow and time point into composite samples inside a sterilized laminar hood and submitted for microbiological culture. For each of the different microorganisms identified, we estimated the prevalence in each postpartum sample, period prevalence (PP), cumulative incidence, and persistence of IMI. Logistic regression models were used to investigate whether the prevalence of IMI differed by farm or sampling time points and whether IMI persistence differed between detected microorganisms. Our findings revealed a high prevalence of Staphylococcus aureus (PP = 18.9%), non-aureus staphylococci and closely related mammaliicoccal species (PP = 52.1%), and Streptococcus spp. and Streptococcus-like organisms (PP = 32.1%) within the study population. The prevalence of these microorganisms varied significantly between farms. Staphylococcus aureus and Staphylococcus chromogenes exhibited significantly higher IMI persistence compared with other detected bacterial taxa, confirming the divergent epidemiological behavior in terms of IMI chronicity across different microorganisms. This study improves our understanding of the epidemiology of mastitis-causing pathogens in organically raised primiparous cows, which can be used to tailor mastitis control plans for this unique yet growing subpopulation of dairy cows.

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.

MEGARes and AMR++, v3.0: an updated comprehensive database of antimicrobial resistance determinants and an improved software pipeline for classification using high-throughput sequencing.

Antimicrobial resistance (AMR) is considered a critical threat to public health, and genomic/metagenomic investigations featuring high-throughput analysis of sequence data are increasingly common and important. We previously introduced MEGARes, a comprehensive AMR database with an acyclic hierarchical annotation structure that facilitates high-throughput computational analysis, as well as AMR++, a customized bioinformatic pipeline specifically designed to use MEGARes in high-throughput analysis for characterizing AMR genes (ARGs) in metagenomic sequence data. Here, we present MEGARes v3.0, a comprehensive database of published ARG sequences for antimicrobial drugs, biocides, and metals, and AMR++ v3.0, an update to our customized bioinformatic pipeline for high-throughput analysis of metagenomic data (available at MEGLab.org). Database annotations have been expanded to include information regarding specific genomic locations for single-nucleotide polymorphisms (SNPs) and insertions and/or deletions (indels) when required by specific ARGs for resistance expression, and the updated AMR++ pipeline uses this information to check for presence of resistance-conferring genetic variants in metagenomic sequenced reads. This new information encompasses 337 ARGs, whose resistance-conferring variants could not previously be confirmed in such a manner. In MEGARes 3.0, the nodes of the acyclic hierarchical ontology include 4 antimicrobial compound types, 59 resistance classes, 233 mechanisms and 1448 gene groups that classify the 8733 accessions.

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.

The impacts of viral infection and subsequent antimicrobials on the microbiome-resistome of growing pigs.

BACKGROUND: Antimicrobials are used in food-producing animals for purposes of preventing, controlling, and/or treating infections. In swine, a major driver of antimicrobial use is porcine reproductive and respiratory syndrome (PRRS), which is caused by a virus that predisposes infected animals to secondary bacterial infections. Numerous antimicrobial protocols are used to treat PRRS, but we have little insight into how these treatment schemes impact antimicrobial resistance (AMR) dynamics within the fecal microbiome of commercial swine. The aim of this study was to determine whether different PRRS-relevant antimicrobial treatment protocols were associated with differences in the fecal microbiome and resistome of growing pigs. To accomplish this, we used a metagenomics approach to characterize and compare the longitudinal wean-to-market resistome and microbiome of pigs challenged with PRRS virus and then exposed to different antimicrobial treatments, and a group of control pigs not challenged with PRRS virus and having minimal antimicrobial exposure. Genomic DNA was extracted from pen-level composite fecal samples from each treatment group and subjected to metagenomic sequencing and microbiome-resistome bioinformatic and statistical analysis. Microbiome-resistome profiles were compared over time and between treatment groups. RESULTS: Fecal microbiome and resistome compositions both changed significantly over time, with a dramatic and stereotypic shift between weaning and 9 days post-weaning (dpw). Antimicrobial resistance gene (ARG) richness and diversity were significantly higher at earlier time points, while microbiome richness and diversity were significantly lower. The post-weaning shift was characterized by transition from a Bacteroides-dominated enterotype to Lactobacillus- and Streptococcus-dominated enterotypes. Both the microbiome and resistome stabilized by 44 dpw, at which point the trajectory of microbiome-resistome maturation began to diverge slightly between the treatment groups, potentially due to physical clustering of the pigs. Challenge with PRRS virus seemed to correspond to the re-appearance of many very rare and low-abundance ARGs within the feces of challenged pigs. Despite very different antimicrobial exposures after challenge with PRRS virus, resistome composition remained largely similar between the treatment groups. Differences in ARG abundance between the groups were mostly driven by temporal changes in abundance that occurred prior to antimicrobial exposures, with the exception of ermG, which increased in the feces of treated pigs, and was significantly more abundant in the feces of these pigs compared to the pigs that did not receive post-PRRS antimicrobials. CONCLUSIONS: The fecal microbiome-resistome of growing pigs exhibited a stereotypic trajectory driven largely by weaning and physiologic aging of the pigs. Events such as viral illness, antimicrobial exposures, and physical grouping of the pigs exerted significant yet relatively minor influence over this trajectory. Therefore, the AMR profile of market-age pigs is the culmination of the life history of the individual pigs and the populations to which they belong. Disease status alone may be a significant driver of AMR in market-age pigs, and understanding the interaction between disease processes and antimicrobial exposures on the swine microbiome-resistome is crucial to developing effective, robust, and reproducible interventions to control AMR. Video Abstract.

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.

Characterization and comparison of the microbiomes and resistomes of colostrum from selectively treated dry cows.

Professionals in animal agriculture promote prudent use of antimicrobials to address public and animal health concerns, such as reduction of antimicrobial residues and antimicrobial resistance (AMR) in products. Few studies evaluate the effect of selective dry-cow therapy on preservation of the milk microbiome or the profile of AMR genes (the resistome) present at freshening. Our objectives were to characterize and compare the microbiomes and resistomes in the colostrum of cows with low somatic cell count that were treated or not treated with intramammary cephapirin benzathine at dry-off. From a larger parent study, cows on a New York dairy farm eligible for dry-off and with histories of somatic cell counts ≤200,000 cells/mL were enrolled to this study (n = 307). Cows were randomly assigned to receive an intramammary antimicrobial and external teat sealant (ABXTS) or sealant only (TS) at dry-off. Composite colostrum samples taken within 4 h of freshening, and quarter milk samples taken at 1 to 7 d in milk were subjected to aerobic culture. The DNA extraction was performed on colostrum from cows with culture-negative samples (ABXTS = 43; TS = 33). The DNA from cows of the same treatment group and parity were pooled (26 pools; ABXTS = 12; TS = 14) for 16S rRNA metagenomic sequencing. Separately, the resistome was captured using a custom RNA bait library for target-enriched sequencing. Sequencing reads were aligned to taxonomic and AMR databases to characterize the microbiome and resistome, respectively. The R statistical program was used to tabulate abundances and to analyze differences in diversity measures and in composition between treatment groups. In the microbiome, the most abundant phyla were Firmicutes (68%), Proteobacteria (23%), Actinobacteria (4%), and Bacteroidetes (3%). Shannon and richness diversity means were 0.93 and 14.7 for ABXTS and 0.94 and 13.1 for TS, respectively. Using analysis of similarities (ANOSIM), overall microbiome composition was found to be similar between treatment groups at the phylum (ANOSIM R = 0.005), class (ANOSIM R = 0.04), and order (ANOSIM R = -0.04) levels. In the resistome, we identified AMR gene accessions associated with 14 unique mechanisms of resistance across 9 different drug classes in 14 samples (TS = 9, ABXTS = 5). The majority of reads aligned to gene accessions that confer resistance to aminoglycoside (TS = ABXTS each 35% abundance), tetracycline (TS = 22%, ABXTS = 54%), and ß-lactam classes (TS = 15%, ABXTS = 12%). Shannon diversity means for AMR class and mechanism, respectively, were 0.66 and 0.69 for TS and 0.19 and 0.19 for ABXTS. Resistome richness diversity means for class and mechanism were 3.1 and 3.4 for TS and 1.4 and 1.4 for ABXTS. Finally, resistome composition was similar between groups at the class (ANOSIM R = -0.20) and mechanism levels (ANOSIM R = 0.01). Although no critical differences were found between treatment groups regarding their microbiome or resistome composition in this study, a larger sample size, deeper sequencing, and additional methodology is needed to identify more subtle differences, such as between lower-abundance features.

Characterization of the microbiota and resistome of bulk tank milk samples from Prince Edward Island dairy farms.

Bulk tank milk (BTM) is regularly used for surveillance on dairy farms for disease conditions such as mastitis and Johne's disease. In this study, we used 16S rRNA sequencing and bait-capture enrichment to characterize the microbiota and resistome of BTM, and investigate potential differences between the cream or pellet fractions. A total of 12 BTM samples were taken from 12 Prince Edward Island dairy farms, in Atlantic Canada, in duplicates. The DNA was analyzed by high-throughput sequencing of the 16S rRNA gene and a suite of antimicrobial resistance (AMR) genes. Target-capture enrichment of AMR genes was conducted before shotgun sequencing. The bioinformatics pipelines QIIME 2 and AMR++ were used for microbiota and resistome analysis, respectively. Differences between microbiotae were evaluated qualitatively with nonmetric multidimensional scaling and quantitatively with permutational ANOVA of UniFrac distances. A total of 47 phyla were present across the BTM samples. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the most abundant phyla. At the genus level, Corynebacterium, Acinetobacter, Lactobacillus, and Turicibacter were the most abundant. There was no significant difference in the Faith's phylogenetic diversity between the cream and pellet fraction. Faith's phylogenetic diversity differed marginally by stall type. There were 10,217 hits across 80 unique AMR genes, with tetracycline resistance being the most common class.

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.

Genomic diversity of class I integrons from antimicrobial resistant strains of Salmonella Typhimurium isolated from livestock, poultry and humans.

INTRODUCTION: Multidrug resistance (MDR) is a serious issue prevalent in various agriculture-related foodborne pathogens including Salmonella enterica (S. enterica) Typhimurium. Class I integrons have been detected in Salmonella spp. strains isolated from food producing animals and humans and likely play a critical role in transmitting antimicrobial resistance within and between livestock and human populations. OBJECTIVE: The main objective of our study was to characterize class I integron presence to identify possible integron diversity among and between antimicrobial resistant Salmonella Typhimurium isolates from various host species, including humans, cattle, swine, and poultry. METHODS: An association between integron presence with multidrug resistance was evaluated. One hundred and eighty-three S. Typhimurium isolates were tested for antimicrobial resistance (AMR). Class I integrons were detected and sequenced. Similarity of AMR patterns between host species was also studied within each integron type. RESULTS: One hundred seventy-four (95.1%) of 183 S.Typhimurium isolates were resistant to at least one antimicrobial and 82 (44.8%) were resistant to 5 or more antimicrobials. The majority of isolates resistant to at least one antimicrobial was from humans (45.9%), followed by swine (19.1%) and then bovine (16.9%) isolates; poultry showed the lowest number (13.1%) of resistant isolates. Our study has demonstrated high occurrence of class I integrons in S. Typhimurium across different host species. Only one integron size was detected in poultry isolates. There was a significant association between integron presence of any size and specific multidrug resistance pattern among the isolates from human, bovine and swine. CONCLUSIONS: Our study has demonstrated a high occurrence of class I integrons of different sizes in Salmonella Typhimurium across various host species and their association with multidrug resistance. This demonstration indicates that multidrug resistant Salmonella Typhimurium is of significant public health occurrence and reflects on the importance of judicious use of antimicrobials among livestock and poultry.

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.

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.

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.

Characterization of the Microbial Resistome in Conventional and "Raised Without Antibiotics" Beef and Dairy Production Systems.

Metagenomic investigations have the potential to provide unprecedented insights into microbial ecologies, such as those relating to antimicrobial resistance (AMR). We characterized the microbial resistome in livestock operations raising cattle conventionally (CONV) or without antibiotic exposures (RWA) using shotgun metagenomics. Samples of feces, wastewater from catchment basins, and soil where wastewater was applied were collected from CONV and RWA feedlot and dairy farms. After DNA extraction and sequencing, shotgun metagenomic reads were aligned to reference databases for identification of bacteria (Kraken) and antibiotic resistance genes (ARGs) accessions (MEGARes). Differences in microbial resistomes were found across farms with different production practices (CONV vs. RWA), types of cattle (beef vs. dairy), and types of sample (feces vs. wastewater vs. soil). Feces had the greatest number of ARGs per sample (mean = 118 and 79 in CONV and RWA, respectively), with tetracycline efflux pumps, macrolide phosphotransferases, and aminoglycoside nucleotidyltransferases mechanisms of resistance more abundant in CONV than in RWA feces. Tetracycline and macrolide-lincosamide-streptogramin classes of resistance were more abundant in feedlot cattle than in dairy cow feces, whereas the ß-lactam class was more abundant in dairy cow feces. Lack of congruence between ARGs and microbial communities (procrustes analysis) suggested that other factors (e.g., location of farms, cattle source, management practices, diet, horizontal ARGs transfer, and co-selection of resistance), in addition to antimicrobial use, could have impacted resistome profiles. For that reason, we could not establish a cause-effect relationship between antimicrobial use and AMR, although ARGs in feces and effluents were associated with drug classes used to treat animals according to farms' records (tetracyclines and macrolides in feedlots, ß-lactams in dairies), whereas ARGs in soil were dominated by multidrug resistance. Characterization of the "resistance potential" of animal-derived and environmental samples is the first step toward incorporating metagenomic approaches into AMR surveillance in agricultural systems. Further research is needed to assess the public-health risk associated with different microbial resistomes.

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.

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.