Genomic and Metagenomic Analysis of Antibiotic Resistance in Dairy Animals

The extent to which carriage of antibiotic resistant bacteria in food animals is responsible for the burden of antibiotic resistance in human infections is currently not well known. Thus, there is a need to further evaluate the genomic diversity of multidrug resistant (MDR) bacteria and the microbial communities in which they are found within these animals. To further evaluate the prevalence of antibiotic resistance in the feces of dairy animals, we sequenced the genomes of 160 antibiotic resistant Escherichia coli and 28 fecal metagenomes isolated from adult dairy cows, veal calves, and pre-weaned calves. E. coli isolates were collected from composite fecal samples and fecal grab samples taken directly from the animal. Isolates were screened for susceptibility to a panel of 14 antibiotics, and 150 showing resistance to at least one antibiotic and 10 antibiotic-susceptible isolates were selected for whole genome shotgun sequencing using a paired-end sequencing approach on an Illumina NextSeq 500. Twenty-eight fecal grab samples collected from animals shedding variable levels of antibiotic resistant E. coli were selected for shotgun metagenomics sequencing. Phylogenetic relationships among the E. coli genomes were inferred using a maximum likelihood approach in RAxML and sequence types were determined in silico. At least four E. coli isolates from each of the major phylogroups were detected. The most frequently detected sequence types were ST88 (10% of all isolates), ST10 (9%), and ST117 (6%). Within these sequence types, isolates from different animals on the same farm and isolates from different farms were closely related indicating that some strains of antibiotic resistant E. coli may be circulating among dairy herds within the same region. Interestingly several MDR ST117 isolates were closely related to human ExPEC isolates and isolates recovered from chickens with colibacillosis. Metagenomic analyses indicated that for all samples tetracycline resistance genes were the most frequently identified resistance genes (61% of all resistance genes), followed by aminoglycoside (20%), macrolide (8%), beta-lactam (3%), sulphonamide (0.8%), and trimethoprim (0.6%) resistance genes. In veal calf fecal samples trimethoprim and floroquinolone resistance genes were more frequently detected than in pre-weaned dairy calf samples. Future work will identify genomic features that may confer persistence of high frequency antibiotic resistant E. coli strains and sequence types in the dairy cow hindgut and factors that influence the presence of antibiotic resistance in pre-weaned calves.