Methodology   

Morphologically distinct colonies on each agar plate were counted for enumeration or considered presumptive positive for prevalence. All presumptive colonies were picked and confirmed by PCR using uidA and yaiO genes for E. coli and sodA for Enterococcus. Microbial analysis of fecal samples for Salmonella was conducted similarly to that of Agga et al. (2016). Enumeration with the pre-enriched TSB-diluted fecal samples was conducted using a spiral plater onto XLD agar. Enriched fecal samples from the procedure described earlier were used to determine fecal prevalence of Salmonella. Each enriched sample was transferred to a deep-well, 96-well block preloaded with phosphate buffered saline and anti-Salmonella immunomagnetic (IMS) beads. Samples with IMS beads were mixed on a vibrating incubating micro plate shaker, and the beads were removed, washed twice in PBS-Tween and eluted. An aliquot of the bead-bacterial complex was transferred to RVS broth and enriched overnight at 42° C. A loop of the RVS secondary enrichment was plated onto XLD, XLD-tet (32 mg·L-1 tetracycline), or XLD-ctx (2 mg·L-1 cefotaxime) agar and incubated at 37° C overnight.   

Subiliac lymph nodes were aseptically denuded, weighed, and sterilized in a boiling water bath. Then, each lymph node was placed into a lateral filtered stomacher bag and pulverized with a rubber mallet. Phosphate buffered saline was added. The mixture was then homogenized using a stomacher mixer. From the homogenate, an aliquot was collected and spiral plated using onto XLD and BGA containing novobiocin (nBGA). Plates were incubated at 37°C for 24h and counted using an automated colony counter. From the lymph node homogenate, an additional aliquot was placed into a dilution of RV enrichment broth, mixed, and incubated overnight. Likewise, another 1 mL of the lymph node homogenate was placed into a dilution of Tetrathionate Broth with iodine, mixed, and incubated at 37°C overnight. After incubation, enrichments were streaked onto XLD and nBGA agars, and plates were incubated at 37°C for 24h. After incubation, the XLD plates were held at room temperature for another 24h to allow full phenotype development. Phenotypic colonies were streaked onto fresh agar and confirmed by latex agglutination.   

PCR-confirmed E. coli isolates were plated and incubated overnight at 37°C. Following incubation, colonies were picked and tested for susceptibility to 14 antimicrobials. Sensititre broth microdilution CMV4AGNF test were used to determine the minimum inhibitory concentration for each of the 14 antimicrobials according to the manufacturer’s protocol with Sensititre ARIS HiQ System. Each E. coli isolate was classified as susceptible, intermediately resistant, or resistant to antimicrobials. Any isolate exhibiting resistance to 3 or more antimicrobials was determined multi-drug resistant (MDR). Salmonella and E. coli isolates were selected based on AMR profiles as determined using the Sensititre antimicrobial sensitivity testing. A total of 94 Salmonella genomic isolates were sequenced using metagenome shotgun sequencing, and were assembled and in silico serotyped using SISTR.