- Antimicrobial resistance in feedlot cattle is not caused solely by using a metaphylactic antimicrobial on arrival, but more likely a multitude of environmental and management factors. Administering metaphylaxis after arrival with either tulathromycin or ceftiofur can decrease the morbidity rate of high-risk cattle.
- Use of metaphylactic antimicrobials affected the minimum inhibitory concentration (MIC) of several antimicrobials tested in Salmonella isolates; however, resistance was only detected sporadically for others. Lack of observed multi-drug resistance suggests that the use of a metaphylactic antimicrobial at feedlot arrival does not produce a reservoir of multi-drug resistant Salmonella in feedlot cattle.
- Multi-drug resistant E. coli strains are widely disseminated. The MIC of the antimicrobials tested for fecal strains isolated after metaphylaxis was not different from that of the strains isolated at feedlot arrival.
- A constant trend of metal resistance in all genomic isolates of Salmonella such as gold, copper, arsenic, and tellurium was observed, and resistance genes against tetracycline, macrolide, lincosamide, cephalosporin, kasugamycin, sulfonamide, chloramphenicol, florfenicol, and streptomycin were observed among the Salmonella strains sequenced.
Antimicrobial resistance (AMR) is invariably complex and is comprised of unpredictable systems such as transmission routes of resistant bacteria and genes that can alter selective pressure in humans, animals, and the environment. The use of antimicrobials in the livestock industry can directly contribute to the emergence of drug-resistant bacterial pathogens. Foodborne transmission of AMR from animals to humans may occur through human infection with Salmonella enterica
, a cattle bacterial pathogen that can harbor AMR genes. Although interventions have been employed to reduce S. enterica
entry into the food chain, its prevalence in ground beef remains constant because it can concentrate in lymph nodes. Horizontal gene transfer (HGT) allows bacteria to exchange their genetic materials with other species of bacteria, which can include AMR genes. This exchange of AMR genes can result in the bacteria being resistant to multiple drugs; more specifically, zoonotic pathogens such as S. enterica
may harbor AMR to multiple antimicrobials. In vitro studies have demonstrated that MDR E. coli
can transfer AMR to 60% of the tested strains of S. enterica
Newport. Recognizing the potential for an antimicrobial to increase MDR S. enterica
, a knowledge gap exists relative to the ability of MDR strains to develop after antimicrobial use and to longitudinally persist in beef cattle over the course of the feeding period up to slaughter. A major use of antimicrobials in the beef cattle industry is for disease prevention and treatment. Bovine respiratory disease complex (BRDC) is the most common disease in beef cattle and its control is the primary use of antimicrobials. Furthermore, marketing practices in the U.S. beef cattle industry can result in acute exposure to pathogens at auction barns where calves from multiple sources are commingled and moved through the production continuum and a longitudinal evaluation of AMR up until the day of slaughter has not specifically been studied in high-risk cattle.
249 high-risk crossbred beef steers were used in the experiment. Metaphylactic antimicrobial treatments administered on d 0 consisted of: a sterile saline negative control, florfenicol, ceftiofur, or tulathromycin. Fecal grab samples were collected on d 0, 28, 56, 112, 182, and at study the day before shipment to a commercial abattoir. Briefly, each sample was placed into a filter bag with modified TSB and mixed well by hand massage. Next, the solution was subsampled for Salmonella
and for AMR bacterial enumerations. The remaining solution was enriched for 8 h at 37°C to determine AMR bacteria and pathogen prevalence. The AMR bacterial enumerations were conducted by spiral plating serial dilutions from each subsample onto CHROMagar E. coli
, or CHROMagar Orientation. Targeted AMR populations were total, tetracycline resistant (TETR), trimethoprim-sulfamethoxazole resistant (COTR), and cefotaxime resistant (CTXR) E. coli; total, erythromycin resistant (ERYR), and highly ERYR Enterococcus
. Resistance levels were determined from the National Antimicrobial Resistance Monitoring System and the suggested concentration of each antimicrobial was added to the selective agar plates.
and E. coli
prevalence from samples not confirmed enumerable was determined by direct plating each enrichment onto individualized selective media agar plates. All plates were incubated for 8h at 37°C.
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.
Antimicrobial use at arrival as metaphylaxis did not result in detectable strains of Salmonella
developing antimicrobial resistance. Prevalence and fecal counts of total Salmonella
were greater in cattle given tulathromycin than those that did not receive metaphylaxis. Therefore, Salmonella
may be influenced by extrinsic factors with elevated concentrations resulting from the use of tulathromycin when compared to other metaphylactic antimicrobials commonly used. Multi-drug resistant E. coli
strains are widely disseminated. The MIC of trimethoprim sulfamethoxazole-resistant E. coli
was affected by the use of a metaphylactic antimicrobial in cattle at feedlot arrival. Nonetheless, with the exceptions of azithromycin, ampicillin, and chloramphenicol, the MIC of the antimicrobials tested for fecal strains isolated after metaphylaxis was not different from that of the strains isolated upon arrival. Use of metaphylactic antimicrobials affected the MIC of several antimicrobials tested in Salmonella
isolates; however, resistance was only detected sporadically for ampicillin, azithromycin, streptomycin, and tetracycline. Furthermore, lack of multi-drug resistance suggests that the use of a metaphylactic antimicrobial at feedlot arrival does not produce a reservoir of multi-drug resistant Salmonella
in feedlot cattle. Genomic isolates were sequenced using metagenome shotgun sequencing. The majority of Salmonella
isolates in feces and from the pen surface belonged to S. enterica
ser. Montevideo, followed by S. enterica
ser. Anatum, S. enterica
ser. Meleagridis, S. enterica
ser. Okerara, S. enterica
ser. Cerro, S. enterica
ser. Mbandaka, S. enterica
ser. Infantis, S. enterica
ser. Enteritidis. A constant trend of metal resistance was observed in all of the genomic isolates, including resistance to metals such as gold, copper, arsenic, and tellurium and resistance to Fosfomycin. Additionally, resistance genes against tetracycline, macrolide, lincosamide, cephalosporin, kasugamycin, sulfonamide, chloramphenicol, florfenicol, and streptomycin was observed among the Salmonella
At feedlot arrival, some cattle receive a metaphylactic antimicrobial. This practice potentially provides a reservoir for accumulation of antimicrobial-resistant Escherichia coli
, and Enterococcus
spp. in animals destined for human consumption. This study is significant because it measures AMR longitudinally throughout the receiving and finishing phase of feedlot cattle.