Multidrug-resistant (MDR) Salmonella enterica can be spread from cattle to humans through direct contact with animals shedding Salmonella as well as through the food chain, making MDR Salmonella a serious threat to human health. The objective of this study was to use whole-genome sequencing to compare antimicrobial-resistant (AMR) Salmonella enterica serovars Typhimurium, Newport, and Dublin isolated from dairy cattle and humans in Washington State and New York State at the genotypic and phenotypic levels. A total of 90 isolates were selected for the study (37 S. Typhimurium, 32 S. Newport, and 21 S. Dublin isolates). All isolates were tested for phenotypic antibiotic resistance to 12 drugs using Kirby-Bauer disk diffusion. AMR genes were detected in the assembled genome of each isolate using nucleotide BLAST and ARG-ANNOT. Genotypic prediction of phenotypic resistance resulted in a mean sensitivity of 97.2 and specificity of 85.2. Sulfamethoxazole-trimethoprim resistance was observed only in human isolates (P Ͻ 0.05), while resistance to quinolones and fluoroquinolones was observed only in 6 S. Typhimurium isolates from humans in Washington State. S. Newport isolates showed a high degree of AMR profile similarity, regardless of source. S. Dublin isolates from New York State differed from those from Washington State based on the presence/absence of plasmid replicons, as well as phenotypic AMR susceptibility/nonsusceptibility (P Ͻ 0.05). The results of this study suggest that distinct factors may contribute to the emergence and dispersal of AMR S. enterica in humans and farm animals in different regions.IMPORTANCE The use of antibiotics in food-producing animals has been hypothesized to select for AMR Salmonella enterica and associated AMR determinants, which can be transferred to humans through different routes. Previous studies have sought to assess the degree to which AMR livestock-and human-associated Salmonella strains overlap, as well as the spatial distribution of Salmonella's associated AMR determinants, but have often been limited by the degree of resolution at which isolates can be compared. Here, a comparative genomics study of livestock-and humanassociated Salmonella strains from different regions of the United States shows that while many AMR genes and phenotypes were confined to human isolates, overlaps between the resistomes of bovine and human-associated Salmonella isolates were observed on numerous occasions, particularly for S. Newport. We have also shown that whole-genome sequencing can be used to reliably predict phenotypic resistance across Salmonella isolated from bovine sources.

Escherichia coli are believed to be associated with postpartum metritis and endometritis but their role in the pathogenesis of both diseases is still undefined. In this study, uterine swabs for E. coli isolation were collected from 374 lactating Holstein cows housed on 4 commercial farms near Ithaca, New York. A total, 125 of 374 cows (33.4%) were positive for E. coli culture. Standard multiplex PCR protocols were used to screen the isolates for the presence of 32 virulence factor genes. Cows that had twin parturition were 4.4 times more likely to have intrauterine E. coli contamination than those that gave birth to single live female calves. Stillborn parturition and birth of single live male calves also increased the odds of intrauterine contamination by E. coli (3.7- and 1.6-fold, respectively) compared with birth of live female calves. Six virulence factors, common to extraintestinal and enteroaggregative E. coli, were found to be associated with metritis and endometritis: fimH, hlyA, cdt, kpsMII, ibeA, and astA. The virulence factor gene fimH was the most prevalent and the most significant: intrauterine E. coli carrying fimH and at least 1 of the other 5 identified virulence factors were pathogenic, and phylogenetic analysis based on the nucleotide sequence of DNA gyrase from 41 such IUEC revealed 2 clades.

No abstract

Antimicrobial resistance represents a major global threat to modern medicine. In vitro studies have shown that very low concentrations of drugs, as frequently identified in the environment, and in foods and water for human and animal consumption, can select for resistant bacteria. However, limited information is currently available on the in vivo impact of ingested drug residues. The objective of our study was to evaluate the effect of feeding preweaned calves milk containing antimicrobial drug residues (below the minimum inhibitory concentration), similar to concentrations detected in milk commonly fed to dairy calves, on selection of resistant fecal E. coli in calves from birth to weaning. At birth, thirty calves were randomly assigned to a controlled feeding trial where: 15 calves were fed raw milk with no drug residues (NR), and 15 calves were fed raw milk with drug residues (DR) by adding ceftiofur, penicillin, ampicillin, and oxytetracycline at final concentrations in the milk of 0.1, 0.005, 0.01, and 0.3 µg/ml, respectively. Fecal samples were rectally collected from each calf once a week starting at birth prior to the first feeding in the trial (pre-treatment) until 6 weeks of age. A significantly greater proportion of E. coli resistant to ampicillin, cefoxitin, ceftiofur, streptomycin and tetracycline was observed in DR calves when compared to NR calves. Additionally, isolates from DR calves had a significant decrease in susceptibility to ceftriaxone and ceftiofur when compared to isolates from NR calves. A greater proportion of E. coli isolates from calves in the DR group were resistant to 3 or more antimicrobial drugs when compared to calves in the ND group. These findings highlight the role that low concentrations of antimicrobial drugs have on the evolution and selection of resistance to multiple antimicrobial drugs in vivo.

Respiratory disease and diarrhea are the 2 most common diseases that result in the use of antimicrobial drugs in preweaned calves. Because the use of drugs in food animals, including dairy calves, has the potential for generating cross-resistance to drugs used in human medicine, it is vital to propose farm practices that foster the judicious use of antimicrobials while assuring animal health and productivity. The objective of this study was to use dairy farm calf treatment records to identify antimicrobial drug treatments in calves and to evaluate their effects on the prevalence of antimicrobial-resistant Escherichia coli from rectal swabs of preweaned dairy calves. Eight farms from central New York participated in the study, 3 farms using individual pen housing management and 5 farms using group pen housing management. Eligible study farms could not add antimicrobial drugs to the milk fed to preweaned calves and were required to have farm records documenting antimicrobial drug treatment of calves from birth to weaning. Three fecal E. coli isolates per calf were tested for susceptibility to 12 antimicrobial drugs using a Kirby-Bauer disk diffusion assay. A total of 473 calves were sampled, from which 1,423 commensal E. coli isolates were tested. Of the 9 antimicrobial drugs used on study farms, only enrofloxacin was significantly associated with reduced antimicrobial susceptibility of E. coli isolates, although treatment with ceftiofur was associated with reduced susceptibility to ceftriaxone. The median numbers of days from treatment with ceftiofur and enrofloxacin to rectal swab sampling of calves were 16 d (range: 1–39) and 12 d (range: 6–44), respectively. At the isolate level, treatment with enrofloxacin resulted in odds ratios of 2 [95% confidence interval (CI): 1–4] and 3 (95% CI: 2–6), respectively, for isolation of nonsusceptible E. coli to nalidixic acid and ciprofloxacin compared with calves not treated with enrofloxacin. Treatment with ceftiofur resulted in an odds ratio of 3 (95% CI: 0.9–12) for isolation of nonsusceptible E. coli to ceftriaxone compared with calves not treated with ceftiofur. Treatment with enrofloxacin resulted in selection of isolates that presented phenotypic resistance to both ciprofloxacin and ceftriaxone. Treatment with ceftiofur resulted in a higher prevalence of isolates resistant to ≥3 antimicrobial drugs (97%) compared with no treatment with ceftiofur (73%). These findings reinforce the necessity for continued implementation of practices at the dairy farm that support the sustainable and judicious use of antimicrobial drugs in dairy calves.

Although antimicrobial drugs are central to combat disease in modern medicine, the use of these drugs can have undesired consequences for human and animal health. One consequence is the post-therapy excretion of pharmacological agents, such as the elimination of drug residues at very low concentrations in the milk of lactating mammals. Limited information is currently available on the impact from the exposure of the gut microbiota to drug residues using in vivo natural models. The objective of our study was to address this knowledge gap and evaluate the effect on the fecal microbiota composition from feeding preweaned dairy calves raw milk with residual concentrations of ampicillin, ceftiofur, penicillin, and oxytetracycline from birth to weaning. At birth, thirty calves were randomly assigned to a controlled feeding trial where: 15 calves were fed raw milk with no drug residues (NR), and 15 calves were fed raw milk with drug residues (DR) by adding ceftiofur, penicillin, ampicillin, and oxytetracycline at final concentrations in the milk of 0.1, 0.005, 0.01, and 0.3 μg/ml, respectively. Fecal samples were rectally collected from each calf once a week starting at birth, prior to the first feeding in the trial (pre-treatment), until 6 weeks of age. Sequencing of the microbial 16S rRNA genes was conducted using the Illumina MiSeq, which provides a high resolution of the microbiota down to the genus level. Discriminant analysis showed that, except for pre-treatment samples, calves fed milk with drug residues and calves fed milk without drug residues easily discriminated at the genus level on their weekly microbial profile. However, analysis comparing the abundance of taxon between NR and DR showed significant differences only at the genus levels, and not at the phylum, class, order or family levels. These results suggest that although drug residues can result in clear discriminate gut microbial communities, they do not result in disruption of taxonomic levels above the genus.

Probiotics are a promising alternative to improve food animal productivity and health. However, scientific evidence that specific microbes can be used to benefit animal health and performance is limited. The objective of this study was to evaluate the effects of administering a live culture of Faecalibacterium prausnitzii to newborn dairy calves on subsequent growth, health, and fecal microbiome. Initially, a safety trial was conducted using 30 newborn bull calves to assess potential adverse effects of the oral and rectal administration of F. prausnitzii to neonatal calves. No adverse reactions, such as increased body temperature or heart and respiratory rates, were observed after the administration of the treatments. All calves survived the experimental period, and there was no difference in fecal consistency score, attitude, appetite or dehydration between the treatment groups. The rectal route was not an efficient practice while the oral route ensures that the full dose is administered to the treated calves. Subsequently, a randomized field trial was completed in a commercial farm with preweaned calves. A total of 554 Holstein heifers were assigned to one of two treatment groups: treated calves (FPTRT) and non-treated calves (control). Treated calves received two oral doses of F. prausnitzii, one at treatment assignment (1st week) and another one week later. The FPTRT group presented significantly lower incidence of severe diarrhea (3.1%) compared with the control group (6.8%). Treated calves also had lower mortality rate associated with severe diarrhea (1.5%) compared to control calves (4.4%). Furthermore, FPTRT calves gained significantly more weight, 4.4 kg over the preweaning period, than controls calves. The relative abundance of F. prausnitzii in the fecal microbiota was significantly higher in the 3rd and 5th weeks of life of FPTRT calves than of the control calves, as revealed by sequencing of the 16S rRNA gene. Our findings showed that oral administration of F. prausnitzii improves gastrointestinal health and growth of preweaned calves, supporting its use as a potential probiotic.

Papillomatous digital dermatitis (PDD) is one of the most prevalent diseases of cattle, adversely affecting the dairy industry by its negative effect on milk production and reproductive performance. Our objective was to use culture-independent methods to determine the microbial diversity in different strata of PDD lesions of three Holstein dairy cows, analyzing whether major differences exist compared to foot skin of three non-infected cows. Both group-specific 16S rRNA gene PCR-denaturing gradient gel electrophoresis and clone library sequencing of broad-range 16S rRNA gene showed differences between the microbial composition of healthy dairy cows and the different strata of the lesion. The predominant bacterial community in the lesion, regardless of the stratum, consisted of 166 specific phylotypes belonging to seven bacterial phyla. Spirochetes (particularly, treponemes) was the most prominent group detected in PDD deep biopsies and was only found in samples from the lesion. Additionally, one phylotype phylogenetically affiliated with uncultured Euryarchaeota was detected in two strata of the lesion. Sequences from healthy foot skin samples revealed 86 specific phylotypes that were affiliated with Firmicutes and Proteobacteria. Our study corroborates the theory that treponemes are involved in PDD disease etiology and suggests, for the first time, the presence of archaeal members in this particular bovine infection.