Whole-genome sequences of Campylobacter coli and Campylobacter jejuni isolates from rhesus macaques (Macaca mulatta) with and without intestinal disease.

Campylobacter jejuni or Campylobacter coli infection can lead to post-infectious irritable bowel syndrome in humans and may produce a similar syndrome in rhesus macaques (Macaca mulatta). We report the complete genomes of 8 C. jejuni isolates and 103 C. coli isolates obtained from rhesus macaques with and without intestinal disease.

Temporal dynamics of the fecal microbiome in female pigs from early life through estrus, parturition, and weaning of the first litter of piglets.

BACKGROUND: Age-associated changes in the gastrointestinal microbiome of young pigs have been robustly described; however, the temporal dynamics of the fecal microbiome of the female pig from early life to first parity are not well understood. Our objective was to describe microbiome and antimicrobial resistance dynamics of the fecal microbiome of breeding sows from early life through estrus, parturition and weaning of the first litter of piglets (i.e., from 3 to 53 weeks of age). RESULTS: Our analysis revealed that fecal bacterial populations in developing gilts undergo changes consistent with major maturation milestones. As the pigs progressed towards first estrus, the fecal bacteriome shifted from Rikenellaceae RC9 gut group- and UCG-002-dominated enterotypes to Treponema- and Clostridium sensu stricto 1-dominated enterotypes. After first estrus, the fecal bacteriome stabilized, with minimal changes in enterotype transition and associated microbial diversity from estrus to parturition and subsequent weaning of first litter piglets. Unlike bacterial communities, fecal fungal communities exhibited low diversity with high inter- and intra-pig variability and an increased relative abundance of certain taxa at parturition, including Candida spp. Counts of resistant fecal bacteria also fluctuated over time, and were highest in early life and subsequently abated as the pigs progressed to adulthood. CONCLUSIONS: This study provides insights into how the fecal microbial community and antimicrobial resistance in female pigs change from three weeks of age throughout their first breeding lifetime. The fecal bacteriome enterotypes and diversity are found to be age-driven and established by the time of first estrus, with minimal changes observed during subsequent physiological stages, such as parturition and lactation, when compared to the earlier age-related shifts. The use of pigs as a model for humans is well-established, however, further studies are needed to understand how our results compare to the human microbiome dynamics. Our findings suggest that the fecal microbiome exhibited consistent changes across individual pigs and became more diverse with age, which is a beneficial characteristic for an animal model system.

Two-Dose Ceftiofur Treatment Increases Cephamycinase Gene Quantities and Fecal Microbiome Diversity in Dairy Cows Diagnosed with Metritis.

Antimicrobial resistance is a significant concern worldwide; meanwhile, the impact of 3rd generation cephalosporin (3GC) antibiotics on the microbial communities of cattle and resistance within these communities is largely unknown. The objectives of this study were to determine the effects of two-dose ceftiofur crystalline-free acid (2-CCFA) treatment on the fecal microbiota and on the quantities of second-and third-generation cephalosporin, fluoroquinolone, and macrolide resistance genes in Holstein-Friesian dairy cows in the southwestern United States. Across three dairy farms, 124 matched pairs of cows were enrolled in a longitudinal study. Following the product label regimen, CCFA was administered on days 0 and 3 to cows diagnosed with postpartum metritis. Healthy cows were pair-matched based on lactation number and calving date. Fecal samples were collected on days 0, 6, and 16 and pooled in groups of 4 (n = 192) by farm, day, and treatment group for community DNA extraction. The characterization of community DNA included real-time PCR (qPCR) to quantify the following antibiotic resistance genes: blaCMY-2, blaCTX-M, mphA, qnrB19, and the highly conserved 16S rRNA back-calculated to gene copies per gram of feces. Additionally, 16S rRNA amplicon sequencing and metagenomics analyses were used to determine differences in bacterial community composition by treatment, day, and farm. Overall, blaCMY-2 gene copies per gram of feces increased significantly (p ≤ 0.05) in the treated group compared to the untreated group on day 6 and remained elevated on day 16. However, blaCTX-M, mphA, and qnrB19 gene quantities did not differ significantly (p ≥ 0.05) between treatment groups, days, or farms, suggesting a cephamycinase-specific enhancement in cows on these farms. Perhaps unexpectedly, 16S rRNA amplicon metagenomic analyses showed that the fecal bacterial communities from treated animals on day 6 had significantly greater (p ≤ 0.05) alpha and beta diversity than the untreated group. Two-dose ceftiofur treatment in dairy cows with metritis elevates cephamycinase gene quantities among all fecal bacteria while paradoxically increasing microbial diversity.

An Experimental Field Trial Investigating the Use of Bacteriophage and Manure Slurry Applications in Beef Cattle Feedlot Pens for Salmonella Mitigation.

Post-harvest Salmonella mitigation techniques are insufficient at addressing Salmonella harbored in cattle lymph nodes, necessitating the exploration of pre-harvest alternatives that reduce Salmonella prior to dissemination to the lymph nodes. A 2 × 2, unbalanced experiment was conducted to determine the effectiveness of pre-harvest treatments applied to the pen surface for Salmonella mitigation in cattle. Treatments included manure slurry intended to mimic pen run-off water (n = 4 pens), a bacteriophage cocktail (n = 4), a combination of both treatments (n = 5), and a control group (n = 5) that received no treatment. Environment samples from 18 feedlot pens and fecal grabs, hide swabs, and subiliac lymph nodes from 178 cattle were collected and selectively enriched for Salmonella, and Salmonella isolates were sequenced. The combination treatment was most effective at reducing Salmonella, and the prevalence was significantly lower compared with the control group for rump swabs on Days 14 and 21. The treatment impact on Salmonella in the lymph nodes could not be determined due to low prevalence. The reduction on cattle hides suggests that bacteriophage or water treatments applied to the feedlot pen surface may reduce Salmonella populations in cattle during the pre-harvest period, resulting in reduced contamination during slaughter and processing.

A Longitudinal Study on the Dynamics of Salmonella enterica Prevalence and Serovar Composition in Beef Cattle Feces and Lymph Nodes and Potential Contributing Sources from the Feedlot Environment.

Salmonella can persist in the feedlot pen environment, acting as a source of transmission among beef cattle. Concurrently, cattle that are colonized with Salmonella can perpetuate contamination of the pen environment through fecal shedding. To study these cyclical dynamics, pen environment and bovine samples were collected for a 7-month longitudinal comparison of Salmonella prevalence, serovar, and antimicrobial resistance profiles. These samples included composite environment, water, and feed from the feedlot pens (n = 30) and cattle (n = 282) feces and subiliac lymph nodes. Salmonella prevalence across all sample types was 57.7%, with the highest prevalence in the pen environment (76.0%) and feces (70.9%). Salmonella was identified in 42.3% of the subiliac lymph nodes. Based on a multilevel mixed-effects logistic regression model, Salmonella prevalence varied significantly (P < 0.05) by collection month for most sample types. Eight Salmonella serovars were identified, and most isolates were pansusceptible, except for a point mutation in the parC gene, associated with fluoroquinolone resistance. There was a proportional difference in serovars Montevideo, Anatum, and Lubbock comparing the environment (37.2, 15.9, and 11.0%, respectively), fecal (27.5, 22.2, and 14.6%, respectively), and lymph node (15.6, 30.2, and 17.7%, respectively) samples. This suggests that the ability of Salmonella to migrate from the pen environment to the cattle host-or vice versa-is serovar specific. The presence of certain serovars also varied by season. Our results provide evidence that Salmonella serovar dynamics differ when comparing environment and host; therefore, developing serovar-specific preharvest environmental Salmonella mitigation strategies should be considered. IMPORTANCE Salmonella contamination of beef products, specifically from the incorporation of bovine lymph nodes into ground beef, remains a food safety concern. Current postharvest Salmonella mitigation techniques do not address Salmonella bacteria that are harbored in the lymph nodes, nor is it well understood how Salmonella invades the lymph nodes. Alternatively, preharvest mitigation techniques that can be applied to the feedlot environment, such as moisture applications, probiotics, or bacteriophage, may reduce Salmonella before dissemination into cattle lymph nodes. However, previous research conducted in cattle feedlots includes study designs that are cross-sectional, are limited to point-in-time sampling, or are limited to sampling of the cattle host, making it difficult to assess the Salmonella interactions between environment and hosts. This longitudinal analysis of the cattle feedlot explores the Salmonella dynamics between the feedlot environment and beef cattle over time to determine the applicability of preharvest environmental treatments.

Age influences the temporal dynamics of microbiome and antimicrobial resistance genes among fecal bacteria in a cohort of production pigs.

BACKGROUND: The pig gastrointestinal tract hosts a diverse microbiome, which can serve to select and maintain a reservoir of antimicrobial resistance genes (ARG). Studies suggest that the types and quantities of antimicrobial resistance (AMR) in fecal bacteria change as the animal host ages, yet the temporal dynamics of AMR within communities of bacteria in pigs during a full production cycle remains largely unstudied. RESULTS: A longitudinal study was performed to evaluate the dynamics of fecal microbiome and AMR in a cohort of pigs during a production cycle; from birth to market age. Our data showed that piglet fecal microbial communities assemble rapidly after birth and become more diverse with age. Individual piglet fecal microbiomes progressed along similar trajectories with age-specific community types/enterotypes and showed a clear shift from E. coli/Shigella-, Fusobacteria-, Bacteroides-dominant enterotypes to Prevotella-, Megaspheara-, and Lactobacillus-dominated enterotypes with aging. Even when the fecal microbiome was the least diverse, the richness of ARGs, quantities of AMR gene copies, and counts of AMR fecal bacteria were highest in piglets at 2 days of age; subsequently, these declined over time, likely due to age-related competitive changes in the underlying microbiome. ARGs conferring resistance to metals and multi-compound/biocides were detected predominately at the earliest sampled ages. CONCLUSIONS: The fecal microbiome and resistome-along with evaluated descriptors of phenotypic antimicrobial susceptibility of fecal bacteria-among a cohort of pigs, demonstrated opposing trajectories in diversity primarily driven by the aging of pigs.

Long-Term Effects of Single-Dose Cephalosporin or Macrolide Use on the Prevalence of AmpC and Extended-Spectrum ß-Lactamase Producing Escherichia coli in the Feces of Beef Cattle.

Extended-spectrum-ß-lactamase (ESBL) and AmpC-lactamase-producing Enterobacteriaceae are serious public health threats. Due to an increasing number of reports of ESBL and AmpC producing Escherichia coli in agricultural settings, it is critical to understand the relationship between the use of two of the highest priority critically important human antibiotics (e.g., third generation cephalosporins [3GC] and macrolides) in food animals and their potential contribution to the selection of ESBL/AmpC E. coli. The objective of our randomized controlled feedlot trial was to measure the effects of ceftiofur crystalline-free acid and tulathromycin on 3GC resistant fecal E. coli populations in cattle before and at various time points after treatment up to and including at slaughter. Multi-level mixed-effects linear regression showed no effect of ceftiofur and tulathromycin on 3GC E. coli CFU counts at slaughter (Day 99); however, a significant (p < 0.05) population shift was observed from susceptible to 3GC resistant E. coli immediately after ceftiofur administration (Day 7). Among 799 fecal samples screened using selective media, 17.7% were ESBL/AmpC E. coli positive, which were further tested for phenotypic antibiotic susceptibility. The majority of the isolates from these plates were multidrug-resistant (94.3%) and expressed either AmpC (78.1%) or ESBL (28.1%) phenotype. A subset of isolates was whole-genome sequenced (n = 20) and identified to harbor chromosomal and/or plasmidal bla genes such as CMY-2, CTX-M, and TEM. Our findings show a time-dependent selection of antibiotics on 3GC-resistant E. coli. High prevalence of multidrug-resistant ESBL/AmpC E. coli found in cattle feces highlights the importance of prudent use of antibiotics in livestock.

A longitudinal investigation of the effects of age, dietary fiber type and level, and injectable antimicrobials on the fecal microbiome and antimicrobial resistance of finisher pigs.

Age and diet are among the factors that influence the community composition of the fecal microbiome. Additionally, antimicrobial use can alter the composition of bacterial communities. An 86-d study with finisher pigs aimed to evaluate age-related dynamics (day 98 to 177 of age), effects of types and levels of dietary fiber, and injectable antimicrobials on the fecal microbiome and antimicrobial resistance (AMR) was conducted. A total of 287 pigs, housed in 36 pens, with 7 to 8 pigs per pen, fed a corn grain and soybean meal-based basal diet, formulated to contain 8.7% neutral detergent fiber (NDF), were randomly assigned to one of three treatments: 1) basal diet with no supplement, 2) basal diet supplemented with 20% distillers dried grains with solubles (DDGS) formulated to contain 13.6% NDF, or 3) basal diet supplemented with 14.5% sugar beet pulp (SBP) formulated to contain 13.6% NDF. Five finisher pigs from each treatment group were selected randomly, and fecal samples were collected on days 98, 110, 144, and 177 of age. In addition, fecal samples were collected from pigs that were injected intramuscularly ceftiofur hydrochloride or penicillin G on days 1 and 3 along with pen-mate-untreated controls on day 1. Fecal samples were subjected to 16S rRNA amplicon-based microbiome analysis and culture methods to quantify the abundance of total AMR coliforms and enterococci populations. The alpha-diversity, such as species richness, increased with age, and the overall bacterial composition changed with age (P =0.001) and diet (P = 0.001). Diet-associated shifts in the specific bacterial taxa were observed. The richness, diversity, and evenness of bacterial taxa did not differ between pigs that were injected with ceftiofur vs. their untreated pen mates or by dietary treatments but differed in pigs that received penicillin G injection. Both antimicrobial treatments contributed to changes in the overall fecal bacterial composition at the genus level. Collectively, the data demonstrate that both age and the diet (control vs. DDGS-, control vs. SBP-, or DDGS- vs. SBP-based diets) were associated with the overall bacterial community composition, and the impact of age on variations in fecal microbiome composition was greater than the diet. Antibiotic treatment had minimal effect on bacterial diversity and relative abundance of taxa. Furthermore, diets and antimicrobial treatment had minimal impact on the overall counts of AMR coliforms and enterococci populations in feces.

Bacterial communities in the gut and the feces are strongly influenced by a number of factors, particularly the age of the animal and the diet. In addition, antibiotic administration routinely used to treat bacterial diseases can also affect the community composition. A study with finisher pigs was conducted to evaluate age-related changes, effects of types­distiller's dried grains with solubles (DGGS) or sugar beet pulp (SBP)­and levels of dietary fiber, and injectable antibiotics on the fecal bacterial composition and antibiotic resistance in fecal bacteria. Fecal samples were collected from five pigs in each of the three dietary treatment groups, control diet with no supplement or supplemented with DDGS or SBP, on days 98, 110, 144, and 177 of age and on days 1 and 3 after the first injection of antibiotics, ceftiofur or penicillin G. Samples were analyzed to identify the bacterial community composition and prevalence of antibiotic resistance in fecal bacteria. Data generated suggested that the overall bacterial composition changed with age and diet, and age appeared to have a greater impact than diet. Antibiotics had only a modest impact on the bacterial community and had minimum impact on antibiotic resistance of fecal bacteria.

Prevalence and Profiles of Antibiotic Resistance Genes mph(A) and qnrB in Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli Isolated from Dairy Calf Feces.

The use of antibiotics to treat dairy calves may result in multidrug-resistant extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. This study investigated fluoroquinolone and macrolide resistance genes among ESBL-producing E. coli isolated from dairy calves. Fresh fecal samples from 147 dairy calves across three age groups were enriched to select for ESBL-producing E. coli. Plasmid-mediated fluoroquinolone (qnrB), macrolide (mph(A)), and beta-lactam (blaCTX-M groups 1 and 9) resistance genes were identified by PCR and gel electrophoresis in ESBL-producing E. coli. Beta-lactamase variants and antibiotic resistance genes were characterized for eight isolates by whole-genome sequencing. Seventy-one (48.3%) samples were positive for ESBL-producing E. coli, with 159 (70.4%) isolates identified as blaCTX-M variant group 1 and 67 (29.6%) isolates as blaCTX-M variant group 9. Resistance gene mph(A) was more commonly associated with blaCTX-M variant group 1, while resistance gene qnrB was more commonly associated with variant group 9. E. coli growth was quantified on antibiotic media for 30 samples: 10 from each age group. Significantly higher quantities of ceftriaxone-resistant E. coli were present in the youngest calves. Results indicate the dominant blaCTX-M groups present in ESBL-producing E. coli may be associated with additional qnrB or mph(A) resistance genes and ESBL-producing E. coli is found in higher abundance in younger calves.

High-Resolution Genomic Comparisons within Salmonella enterica Serotypes Derived from Beef Feedlot Cattle: Parsing the Roles of Cattle Source, Pen, Animal, Sample Type, and Production Period.

Salmonella enterica is a major foodborne pathogen, and contaminated beef products have been identified as one of the primary sources of Salmonella-related outbreaks. Pathogenicity and antibiotic resistance of Salmonella are highly serotype and subpopulation specific, which makes it essential to understand high-resolution Salmonella population dynamics in cattle. Time of year, source of cattle, pen, and sample type (i.e., feces, hide, or lymph nodes) have previously been identified as important factors influencing the serotype distribution of Salmonella (e.g., Anatum, Lubbock, Cerro, Montevideo, Kentucky, Newport, and Norwich) that were isolated from a longitudinal sampling design in a research feedlot. In this study, we performed high-resolution genomic comparisons of Salmonella isolates within each serotype using both single-nucleotide polymorphism-based maximum-likelihood phylogeny and hierarchical clustering of core-genome multilocus sequence typing. The importance of the aforementioned features in clonal Salmonella expansion was further explored using a supervised machine learning algorithm. In addition, we identified and compared the resistance genes, plasmids, and pathogenicity island profiles of the isolates within each subpopulation. Our findings indicate that clonal expansion of Salmonella strains in cattle was mainly influenced by the randomization of block and pen, as well as the origin/source of the cattle, i.e., regardless of sampling time and sample type (i.e., feces, lymph node, or hide). Further research is needed concerning the role of the feedlot pen environment prior to cattle placement to better understand carryover contributions of existing strains of Salmonella and their bacteriophages. IMPORTANCESalmonella serotypes isolated from outbreaks in humans can also be found in beef cattle and feedlots. Virulence factors and antibiotic resistance are among the primary defense mechanisms of Salmonella, and are often associated with clonal expansion. This makes understanding the subpopulation dynamics of Salmonella in cattle critical for effective mitigation. There remains a gap in the literature concerning subpopulation dynamics within Salmonella serotypes in feedlot cattle from the beginning of feeding up until slaughter. Here, we explore Salmonella population dynamics within each serotype using core-genome phylogeny and hierarchical classifications. We used machine learning to quantitatively parse the relative importance of both hierarchical and longitudinal clustering among cattle host samples. Our results reveal that Salmonella populations in cattle are highly clonal over a 6-month study period and that clonal dissemination of Salmonella in cattle is mainly influenced spatially by experimental block and pen, as well by the geographical origin of the cattle.

Can the use of older-generation beta-lactam antibiotics in livestock production over-select for beta-lactamases of greatest consequence for human medicine? An in vitro experimental model.

Though carbapenems are not licensed for use in food animals in the U.S., carbapenem resistance among Enterobacteriaceae has been identified in farm animals and their environments. The objective of our study was to determine the extent to which older-generation ß-lactam antibiotics approved for use in food animals in the U.S. might differentially select for resistance to antibiotics of critical importance to human health, such as carbapenems. Escherichia coli (E. coli) strains from humans, food animals, or the environment bearing a single ß-lactamase gene (n = 20 each) for blaTEM-1, blaCMY-2, and blaCTX-M-* or else blaKPC/IMP/NDM (due to limited availability, often in combination with other bla genes), were identified, along with 20 E. coli strains lacking any known beta-lactamase genes. Baseline estimates of intrinsic bacterial fitness were derived from the population growth curves. Effects of ampicillin (32 µg/mL), ceftriaxone (4 µg/mL) and meropenem (4 µg/mL) on each strain and resistance-group also were assessed. Further, in vitro batch cultures were prepared by mixing equal concentrations of 10 representative E. coli strains (two from each resistance gene group), and each mixture was incubated at 37°C for 24 hours in non-antibiotic cation-adjusted Mueller-Hinton II (CAMH-2) broth, ampicillin + CAMH-2 broth (at 2, 4, 8, 16, and 32 µg/mL) and ceftiofur + CAMH-2 broth (at 0.5, 1, 2, 4, and 8µg/mL). Relative and absolute abundance of resistance-groups were estimated phenotypically. Line plots of the raw data were generated, and non-linear Gompertz models and multilevel mixed-effect linear regression models were fitted to the data. The observed strain growth rate distributions were significantly different across the groups. AmpC strains (i.e., blaCMY-2) had distinctly less robust (p < 0.05) growth in ceftriaxone (4 µg/mL) compared to extended-spectrum beta-lactamase (ESBL) producers harboring blaCTX-M-*variants. With increasing beta-lactam antibiotic concentrations, relative proportions of ESBLs and CREs were over-represented in the mixed bacterial communities; importantly, this was more pronounced with ceftiofur than with ampicillin. These results indicate that aminopenicillins and extended-spectrum cephalosporins would be expected to propagate carbapenemase-producing Enterobacteriaceae in food animals if and when Enterobacteriaceae from human health care settings enter the food animal environment.

Comprehensive phenotypic and genotypic characterization and comparison of virulence, biofilm, and antimicrobial resistance in urinary Escherichia coli isolated from canines.

Urinary tract infections (UTIs) affect nearly half of women and an estimated 14 % of the canine companion animal population at least once in their lifetime. As with humans, Escherichia coli is the most commonly isolated bacteria from canine UTIs and infections are dominated by specific phylogenetic groups with notable virulence attributes. In this study, we evaluated uropathogenic E. coli (UPEC) (n = 69) isolated from canine UTIs phenotypically and genotypically for virulence factors, biofilm formation and antimicrobial resistance profiles. Biofilm formation in UPEC strains was positively associated with common virulence factors including papG (p = 0.006), fimH (p < 0.0001), sfaS (p = 0.004), focA (p = 0.004), cnf-1 (p = 0.009) and hlyA (p = 0.006). There was a negative association between biofilm formation and phenotypic antimicrobial resistance for ampicillin (p < 0.0004), ciprofloxacin (p < 0.0001), and trimethoprim-sulfamethoxazole (p < 0.02), as well as multidrug resistance (isolates resistant to ≥ 3 classes of antimicrobials) (p < 0.0002), and the presence of extended spectrum beta-lactamase (ESBL)-producing genes (p < 0.05). In conclusion, UPECs isolated from clinical cases of canine UTIs show a broad negative association between antimicrobial resistance and biofilm formation, and this observation is supported both by phenotypic and genotypic endpoints. As the biofilm formation may result in antimicrobial tolerance, this could be a secondary evasive tactic of UPEC lacking traditional antimicrobial resistance traits. This observation is important for veterinary practitioners to consider when treating puzzling chronic intractable and/or recurrent cases of UTI that appear to be susceptible to antimicrobial therapy via traditional antimicrobial susceptibility testing (AST) methods.

Quantitative dynamics of Salmonella and E. coli in feces of feedlot cattle treated with ceftiofur and chlortetracycline.

Antibiotic use in beef cattle is a risk factor for the expansion of antimicrobial-resistant Salmonella populations. However, actual changes in the quantity of Salmonella in cattle feces following antibiotic use have not been investigated. Previously, we observed an overall reduction in Salmonella prevalence in cattle feces associated with both ceftiofur crystalline-free acid (CCFA) and chlortetracycline (CTC) use; however, during the same time frame the prevalence of multidrug-resistant Salmonella increased. The purpose of this analysis was to quantify the dynamics of Salmonella using colony counting (via a spiral-plating method) and hydrolysis probe-based qPCR (TaqMan® qPCR). Additionally, we quantified antibiotic-resistant Salmonella by plating to agar containing antibiotics at Clinical & Laboratory Standards Institute breakpoint concentrations. Cattle were randomly assigned to 4 treatment groups across 16 pens in 2 replicates consisting of 88 cattle each. Fecal samples from Days 0, 4, 8, 14, 20, and 26 were subjected to quantification assays. Duplicate qPCR assays targeting the Salmonella invA gene were performed on total community DNA for 1,040 samples. Diluted fecal samples were spiral plated on plain Brilliant Green Agar (BGA) and BGA with ceftriaxone (4 µg/ml) or tetracycline (16 µg/ml). For comparison purposes, indicator non-type-specific (NTS) E. coli were also quantified by direct spiral plating. Quantity of NTS E. coli and Salmonella significantly decreased immediately following CCFA treatment. CTC treatment further decreased the quantity of Salmonella but not NTS E. coli. Effects of antibiotics on the imputed log10 quantity of Salmonella were analyzed via a multi-level mixed linear regression model. The invA gene copies decreased with CCFA treatment by approximately 2 log10 gene copies/g feces and remained low following additional CTC treatment. The quantities of tetracycline or ceftriaxone-resistant Salmonella were approximately 4 log10 CFU/g feces; however, most of the samples were under the quantification limit. The results of this study demonstrate that antibiotic use decreases the overall quantity of Salmonella in cattle feces in the short term; however, the overall quantities of antimicrobial-resistant NTS E. coli and Salmonella tend to remain at a constant level throughout.

Population Dynamics of Salmonella enterica within Beef Cattle Cohorts Followed from Single-Dose Metaphylactic Antibiotic Treatment until Slaughter.

Antibiotic use in cattle can select for multidrug-resistant Salmonella enterica, which is considered a serious threat by the U.S. Centers for Disease Control and Prevention. A randomized controlled longitudinal field trial was designed to determine the long-term effects of a single dose of ceftiofur or tulathromycin on Salmonella population characteristics in cattle feces and peripheral lymph nodes and on hides. A total of 134 beef cattle from two sources were divided among 12 pens, with cattle in each of the 3-pen blocks receiving a single dose of either ceftiofur or tulathromycin or neither (control) on day 0. Fecal samples were collected before treatment (day 0) and repeatedly following treatment until slaughter (day 99+). Hide and lymph node samples were collected at slaughter age. Salmonella prevalence, phenotypic antimicrobial resistance, serotype, and phylogenetic relationships were examined. Multilevel mixed logistic regression models indicated no significant effects (P ≥ 0.218) of metaphylactic antibiotics on the prevalence of Salmonella across sample types. However, there was a significant time effect observed, with prevalence increasing from spring through the midsummer months (P < 0.0001) in feces. The majority of Salmonella isolates were pansusceptible to a panel of 14 antibiotics both before and after treatment. Highly prevalent Salmonella serotypes were Salmonella enterica serovar Montevideo, Salmonella enterica serovar Anatum, Salmonella enterica serovar Cerro, and Salmonella enterica serovar Lubbock across all sample types. Strong pen and cattle source serotype clustering effects were observed among Salmonella isolates originating from fecal, lymph node, and hide samples; however, the potential role of Salmonella isolates from the pen environment prior to animal placement was not assessed in this study.IMPORTANCESalmonella is a leading bacterial foodborne pathogen, causing a significant number of human infections and deaths every year in the United States. Macrolides and 3rd-generation cephalosporins play critical roles in the treatment of human salmonellosis. Use of these antibiotics in beef cattle can select for resistant bacteria that may enter the food chain or spread from the farm via manure. There is a lack of longitudinal research concerning the long-term effects of metaphylactic antibiotic administration. Here, we assessed Salmonella population dynamics during the feeding period until slaughter following single-dose antibiotic treatment. We found no long-term effects of antibiotic use early in the cattle-feeding period on Salmonella prevalence and antimicrobial resistance at slaughter. We identified the pens in which cattle were housed as the factor that contributed most to Salmonella serotypes being shared; importantly, the dominant strain in each pen changed repeatedly over the entire feeding period.

Whole-Genome Sequences of Salmonella enterica Serovar I 4,[5],12:i:- Isolates from Swine.

Salmonella enterica (non-Typhi) is one of the top five pathogens causing enteric infections worldwide. Draft whole-genome sequences of multidrug-resistant (MDR) Salmonella enterica serovar I 4,[5],12:i:- isolates from swine tissue samples collected at slaughter were evaluated for antimicrobial resistance genotypes. In recent years, Salmonella enterica serovar I 4,[5],12:i:- has increased in prevalence in humans and animals and has been linked to the consumption of contaminated pork.

Population dynamics of enteric Salmonella in response to antimicrobial use in beef feedlot cattle.

A randomized controlled longitudinal field trial was undertaken to assess the effects of injectable ceftiofur crystalline-free acid (CCFA) versus in-feed chlortetracycline on the temporal dynamics of Salmonella enterica spp. enterica in feedlot cattle. Two replicates of 8 pens (total 176 steers) received one of 4 different regimens. All, or one, out of 11 steers were treated with CCFA on day 0 in 8 pens, with half of the pens later receiving three 5-day regimens of chlortetracycline from day 4 to day 20. Salmonella was isolated from faecal samples and antimicrobial susceptibility was analysed via microbroth dilution. Serotype was determined by whole-genome sequencing. On day 0, mean Salmonella prevalence was 75.0% and the vast majority of isolates were pansusceptible. Both antimicrobials reduced overall prevalence of Salmonella; however, these treatments increased the proportion of multi-drug resistant (MDR) Salmonella from day 4 through day 26, which was the last day of faecal collection. Only six Salmonella serotypes were detected. Salmonella serotype Reading isolates were extensively MDR, suggesting a strong association between serotype and resistance. Our study demonstrates that the selection pressures of a 3rd generation cephalosporin and chlortetracycline during the feeding period contribute to dynamic population shifts between antimicrobial susceptible and resistant Salmonella.

Effects of In-Feed Copper, Chlortetracycline, and Tylosin on the Prevalence of Transferable Copper Resistance Gene, tcrB, Among Fecal Enterococci of Weaned Piglets.

Heavy metals, such as copper, are increasingly supplemented in swine diets as an alternative to antibiotics to promote growth. Enterococci, a common gut commensal, acquire plasmid-borne, transferable copper resistance (tcrB) gene-mediated resistance to copper. The plasmid also carried resistance genes to tetracyclines and macrolides. The potential genetic link between copper and antibiotic resistance suggests that copper supplementation may exert a selection pressure for antimicrobial resistance. Therefore, a longitudinal study was conducted to investigate the effects of in-feed copper, chlortetracycline, and tylosin alone or in combination on the selection and co-selection of antimicrobial-resistant enterococci. The study included 240 weaned piglets assigned randomly to 6 dietary treatment groups: control, copper, chlortetracycline, tylosin, copper and chlortetracycline, and copper and tylosin. Feces were collected before (day 0), during (days 7, 14, 21), and after (days 28 and 35) initiating treatment, and enterococcal isolates were obtained from each fecal sample and tested for genotypic and phenotypic resistance to copper and antibiotics. A total of 2592 enterococcal isolates were tested for tcrB by polymerase chain reaction. The overall prevalence of tcrB-positive enterococci was 14.3% (372/2592). Among the tcrB-positive isolates, 331 were Enterococcus faecium and 41 were E. faecalis. All tcrB-positive isolates contained both erm(B) and tet(M) genes. The median minimum inhibitory concentration of copper for tcrB-negative and tcrB-positive enterococci was 6 and 18 mM, respectively. The majority of isolates (95/100) were resistant to multiple antibiotics. In conclusion, supplementing copper or antibiotics alone did not increase copper-resistant enterococci; however, supplementing antibiotics with copper increased the prevalence of the tcrB gene among fecal enterococci of piglets.

Effects of chlortetracycline and copper supplementation on the prevalence, distribution, and quantity of antimicrobial resistance genes in the fecal metagenome of weaned pigs.

Use of in-feed antibiotics such as chlortetracycline (CTC) in food animals is fiercely debated as a cause of antimicrobial resistance in human pathogens; as a result, alternatives to antibiotics such as heavy metals have been proposed. We used a total community DNA approach to experimentally investigate the effects of CTC and copper supplementation on the presence and quantity of antimicrobial resistance elements in the gut microbial ecology of pigs. Total community DNA was extracted from 569 fecal samples collected weekly over a 6-week period from groups of 5 pigs housed in 32 pens that were randomized to receive either control, CTC, copper, or copper plus CTC regimens. Qualitative and quantitative PCR were used to detect the presence of 14 tetracycline resistance (tet) genes and to quantify gene copies of tetA, tetB, blaCMY-2 (a 3rd generation cephalosporin resistance gene), and pcoD (a copper resistance gene), respectively. The detection of tetA and tetB decreased over the subsequent sampling periods, whereas the prevalence of tetC and tetP increased. CTC and copper plus CTC supplementation increased both the prevalence and gene copy numbers of tetA, while decreasing both the prevalence and gene copies of tetB. In summary, tet gene presence was initially very diverse in the gut bacterial community of weaned pigs; thereafter, copper and CTC supplementation differentially impacted the prevalence and quantity of the various tetracycline, ceftiofur and copper resistance genes resulting in a less diverse gene population.

Nasal carriage of mecA-positive methicillin-resistant Staphylococcus aureus in pigs exhibits dose-response to zinc supplementation.

Zinc (Zn) is often supplemented at elevated concentrations in swine diets, particularly in piglets, to prevent enteric infections and promote growth. Previous studies from Denmark have suggested a genetic linkage and a phenotypic association between Zn resistance, encoded by czrC, and methicillin-resistance conferred by mecA in Staphylococcus aureus. Such an association has not been reported in the U.S. swine population. We conducted an analysis of the effects of Zn, supplemented as zinc oxide (ZnO), on the nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) in nursery (n=40) and finisher pigs (n=40) enrolled in a nutritional study. Nasal swabs, collected from nursery and finisher pigs, were inoculated onto MRSA CHROMagar and presumptive MRSA colonies were tested for the presence of mecA and czrC genes by polymerase chain reaction. Zinc susceptibility was determined by the agar dilution method. The prevalence of mecA-positive MRSA was 10% (4/40) and 20% (8/40) among nursery and finisher pigs, respectively. Of the 12 mecA-positive S. aureus isolates, 7 had the czrC gene (58.3%) compared to none among the 68 mecA-negative isolates. The presence of both mecA (p=0.002) and czrC (p=0.006) genes were positively associated with higher levels of Zn supplementation. The median minimum inhibitory concentrations of Zn for czrC-positive and czrC-negative isolates were 12 and 2 mM, respectively (p<0.0001). The link between czrC and mecA genes suggests the importance of elevated Zn supplementation in the co-selection and propagation of methicillin resistance among S. aureus in pigs.

Impact of treatment strategies on cephalosporin and tetracycline resistance gene quantities in the bovine fecal metagenome.

The study objective was to determine the effects of two treatment regimens on quantities of ceftiofur and tetracycline resistance genes in feedlot cattle. The two regimens were ceftiofur crystalline-free acid (CCFA) administered to either one or all steers within a pen and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. A 26-day randomized controlled field trial was conducted on 176 steers. Real-time PCR was used to quantify bla(CMY-2), bla(CTX-M), tet(A), tet(B), and 16S rRNA gene copies/gram of feces from community DNA. A significant increase in ceftiofur resistance and a decrease in tetracycline resistance elements were observed among the treatment groups in which all steers received CCFA treatment, expressed as gene copies/gram of feces. Subsequent chlortetracycline administration led to rapid expansion of both ceftiofur and tetracycline resistance gene copies/gram of feces. Our data suggest that chlortetracycline is contraindicated when attempting to avoid expansion of resistance to critically important third-generation cephalosporins.