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We plan to fulfill our immediate goals by pursuing the following research objectives: Objective 1: We will investigate experimental intervention strategies that can be used by feedlot managers to sustain the fitness burden, and relatively low prevalence, conferred by ceftiofur resistance in E. coli. Objective 2: We will determine group- and time-dependent variability in prevalence of phenotypic and genotypic resistance to ceftiofur in E. coli from cattle in a commercial feedlot setting in response to different and practical treatment options. These options will include: 1) varying antibiotics and formulations, 2) speeding the turnover rate in hospital pens, and 3) effectively removing the hospital pen by sending treated animals to their home pen immediately post-treatment. Objective 3: We will develop and empirically assess models useful for predicting the interactions between susceptible and single- and multi-resistant bacteria and the time-dependent return to baseline antimicrobial resistance levels post treatment. Research for Objective 1 has been completed. The objective of this trial was to study the effect of two interventions on ceftiofur resistance in host enteric microbiota as determined by genotypic quantification of the blaCMY2 gene and phenotypic quantification of ceftiofur resistance in NTS E. coli. Two interventions were applied at the pen level in a 2-by-2 factorial design where the first factor was differential mixing of ceftiofur treated and non-treated animals (8 pens where all 11 animals/pen were treated with Excede versus 8 pens where 1 of 11 animals/per pen were treated with Excede) and the second factor was treatment with chlortetracycline in the feed during three 5-day periods following Excede treatment. Preliminary examination of genotypic data using a 3-way full factorial multi-level mixed model with random intercepts for replicate, pen and animal and a random slope for ceftiofur-treatment, and fixed effects for CTC-treatment, mixing and day (period) and all 2- and 3-way interactions showed 1) a highly significant treatment by period effect, 2) that CTC treatment consistently increased blaCMY2 gene copies across other factors, and 3) mixing had a varied decreasing effect on blaCMY2 gene copies which was inconsistent across other factors. Preliminary visual and descriptive assessment of phenotypic data showed that 1) treatment of all cattle in a pen with long-acting ceftiofur resulted in an overall absolute decrease in NTS E. coli immediately after treatment 2) Absolute counts of NTS ceftiofur-resistant E. coli increased more dramatically for pens where all animals were treated with ceftiofur as compared to pen where 1 animal was treated with ceftiofur; however, in relative number (ceftiofur-resistant E. coli/all E. coli) an increase was only seen in pens where all 11 animal were treated with ceftiofur, 3) treatment with chlortetracycline appeared to result in co-selection for ceftiofur resistance determinants.
Non-Technical Summary:<br/>
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.
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Approach:<br/>
Objective 1 has been completed. In objective 1, eight treatment groups of cattle will be assessed in a complete 2x2 factorial design. Cattle will be treated with a long-acting ceftiofur. The additional interventions will consist of mixing, which refers to whether all or just some animals in a group are treated with the ceftiofur products and treatment with chlortetracycline (CTC) in the feed at the conclusion of ceftiofur treatment. A total of 176 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.
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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.
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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.
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Progress:<br/>
2012/05 TO 2013/02<br/>
OUTPUTS: During this reporting period efforts have focused on: 1) Isolation of E. coli to be used for growth curve experiments and determination of their antibiograms using sensititre plates for Gram negative bacteria. Approximately, 500 of 864 isolates have been isolated thus far. These isolates were characterized as either a) susceptible to ceftiofur and tetracycline, b) resistant to ceftiofur, but susceptible to tetracycline, c) resistant to tetracycline, but susceptible to ceftiofur, or d) resistant to ceftiofur and tetracycline; 2) Validation of our method to determine the quantity of E. coli resistant to ceftiofur, tetracycline or ceftiofur and tetracycline in a gram of feces using samples from our studies; 3) Determination of growth parameters of E. coli susceptible to or resistant to tetracycline and ceftiofur; 4) Analysis of samples for E. coli carrying extended-spectrum beta-lactamases; 5) Analysis of phenotypic and genotypic data. Results of research efforts have been presented at one international meeting and 4 national meetings. Fecal samples, bacterial isolates and bacterial community DNA have been used bycollaborators (Dr. Patrick Boerlin, Ontario Veterinary College, Canada). <br/>PARTICIPANTS: Principal investigators: Bo Norby, H. Morgan Scott, Guy Loneragan, Mindy Brashears and Roger Harvey. Genotypic determination of the blaCMY2 gene (confers ceftiofur resistance) as well as tetracycline-resistances genes was conducted by Neena Kanwar (PhD student in Dr. Scott's laboratory) and Dr. Javier Vanisco (Research Associate in Dr. Scott's Laboratory). E. coli isolations for growth curve and antibiogram determination were conducted by Neena Kanwar (Dr. Scott's laboratory) and Scott Henderson (Dr Norby's laboratory). Growth curve determination was conducted by Matthew McGowan (Dr. Scott's laboratory). PCR for detection of the blcCTX-M-32 was conducted by Dr. Jennifer Cottel (Dr. Patrick Boerlin's laboratory)
<br/>TARGET AUDIENCES: Target audiences so far have been researchers and federal agency representatives. Cattle producers, industry organizations, and federal regulators are the future target audiences for the work related to antimicrobial resistance ecology in cattle.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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IMPACT: The project has generated new knowledge about the ecology of resistant coliform bacteria recovered from cattle that were or were not treated with one or more antimicrobial drugs. Preliminary growth rate experiments suggested that multidrug resistant E. coli (including resistance to tetracycline) may exhibit better growth fitness than E. coli resistant to tetracycline alone. Minimum inhibitory concentrations (MICs) of ATCC strains and field strains of E. coli resistant to ceftiofur, tetracycline, ampicillin and ciprofloxacin on MacConkey agar were generally within one dilution as compared to Muller-Hinton agar. Muller-Hinton agar is the gold standard for agar dilution determination of MICs. Preliminarily, we concluded that adding antibiotics to MacConkey agar at the resistance breakpoint is a valid method to quantify the colony forming units of E. coli which are resistant to the four tested antibiotics. Furhtermore, analyses for the blcCTX-M-32 genes suggested that dissemination may be a result of both clonal expansion and horizontal gene transfer.