Novel Pre-Harvest Interventions to Protect Antimicrobials of Critical Importance in Human and Veterinary Medicine

Non-Technical Summary: Because of the potential, although contentious, for adverse outcomes associated with resistant enteric bacteria from food animal sources on public health, regulatory organizations around the world have promulgated rules to protect public health by either reducing the number and/or formulations of antimicrobial drugs available for use in food animal agriculture or by tightening the approval and monitoring processes for new antimicrobial drugs intended for food animal use in the United States. If such precautionary measures become more frequent or even universal, it will limit food animal producers' ability to control pathogens of importance to animal health. This may have implications for human health in that healthy animals harbor fewer human pathogens than animals experiencing less than optimal health. Controlling antimicrobial resistance in animal production systems and maintaining availability of efficacious antimicrobial drugs, therefore, may have benefits for both human and animal health. The premise for our proposed research is that both the relative fitness cost conferred on bacteria by carrying certain antimicrobial resistance genes (R-genes), along with a readily available source of susceptible bacteria (bacteria that do not carry the R-genes in question) are useful for promoting the rapid re-colonization of animals harboring resistant bacteria. We will exploit this principle to evaluate potential interventions to manage antimicrobial-resistant bacteria in animal agriculture. We hypothesize that the rate at which re-colonization with susceptible bacteria occurs will depend on: 1) the levels of exposure of treated cattle to susceptible bacteria from non-treated pen mates and the environment, and/or 2) bacteria resistant to another antimicrobial. Hence, we propose to use interventions that will reflect different levels of exposure of antibiotic-treated cattle to non-treated cattle. To test our overall hypothesis, we propose to conduct two studies; one in a research feedlot and one in a commercial feedlot. In the research feedlot we will expose healthy cattle to different antibiotic treatments with the aim to determine how the interventions will reduce the overall levels of resistance in treated cattle. In the commercial feedlot we will focus slightly different interventions and use cattle with naturally occurring bovine respiratory disease. The outcomes for both trials include phenotypic and genotypic resistance in E. coli to the antibiotics used to treat the feedlot cattle in this study. We hypothesize that the interventions will lead to a faster decline to the baseline levels of resistance and/or lower levels of resistance at any time during and after treatment with antibiotics. Additionally we will determine the fitness of E. coli that are resistant or susceptible to the antibiotics used to treat the feedlot cattle. We aim at determining interventions that in the long run can be used to maintain low levels of resistance to antibiotics used to treat sick feedlot cattle, and hence 1) secure their continued use and 2) decrease the potential risk to public health. <P> Approach: Objective 1 Eight treatment groups of cattle will be assessed in a complete 2x2x2 factorial design. Cattle will be treated with either short- or long-acting ceftiofur (Factor 1). The additional interventions will consist of mixing, which refers to whether all or just some animals in a group are treated with one of the ceftiofur products (Factor 2) and treatment with chlortetracycline (CTC) in the feed at the conclusion of ceftiofur treatment (Factor 3). A total of 160 steers will be enrolled in the study. In addition, to sampling feces on the day of arrival at the feedlot, all cattle will be sampled on the first day of treatment and every other day thereafter through the treatment and follow-up periods. A direct plate count of the total number of colony forming units (CFUs) of E. coli per one gram of feces will be determined using plain MacConkey agar. In addition, CFUs will be determined using MacConkey agar containing ceftiofur and tetracycline. To assess the overall burden of ceftiofur resistance in all enteric bacteria, we will use a quantitative real-time PCR for amplification of the plasmid-mediated blaCMY2 gene and the 16S rRNA gene. The latter gene represents the background bacterial content in the DNA samples to standardize the blaCMY2 quantities. Objective 2 Three groups of animals with naturally occurring bovine respiratory disease (BRD) will be used in an incomplete 2x2 factorial design conducted at a large commercial feedlot. The three treatment groups are as follow: 1) Steers that develop BRD will be treated with Excede and remain in the hospital facility during the treatment period (7 days), then returned to their home pen, 2) Steers that develop BRD will be treated with Naxcel and remain in the hospital facility during the treatment period (5 days), then returned to their home pen. 3) Steers that develop BRD will be treated with Excede and returned to their home pen immediately after treatment. A total of 60 steers with naturally occurring BRD will be needed in this study. The inclusion criteria for naturally occurring BRD will be typical clinical signs of BRD as determined by the pen-checker, and a rectal temperature at or above 39.7oC. Sampling and phenotypic and genotypic resistance will be performed as previously described for Objective 1. Objective 3 E. coli isolates for the 160 steers enrolled in Objective 1 of this proposal will be available for Objective 3. Since only two steers in each of the four antibiotic treated groups, only 88 steers will be available for determining the time-dependent return to baseline. We will use a total of 9 E. coli isolates from each of the 88 steers: 3 susceptible, 3 tetracycline resistant, and 3 ceftiofur resistant. In our proposed experiments, we will use the maximum growth rate, VMAX, as surrogates for the differences in biological fitness between strains of bacteria that carry resistance to tetracycline and ceftiofur and those that do not. The biological fitness of resistant and susceptible E. coli isolates will be determined in triplicate for each isolate using a turbidimetric method in a Bioscreen-C Automated Microbiology Growth Curve Analysis System.