NOVEL PRE-HARVEST INTERVENTIONS TO PROTECT ANTIMICROBIALS OF CRITICAL IMPORTANCE IN HUMAN AND VETERINARY MEDICINE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0214812
• Grant No.: 2008-35201-04682
• Proposal No.: 2008-01844
• Project Start Date: Sep 1, 2008
• Project End Date: Feb 28, 2011
• Grant Year: 2008
• Program Code: [32.0B]- Food Safety and Epidemiology (B): Epidemiological Approaches for Food Safety

Project Director
Norby, B.

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
VETERINARY INTEGRATIVE BIOSCIENCES

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.

Animal Health Component

60%

Research Effort Categories

Basic

30%

Applied

60%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3399	1070	30%
712	3399	1100	40%
712	3399	1170	30%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3399 - Beef cattle, general/other;

Field Of Science
1070 - Ecology; 1100 - Bacteriology; 1170 - Epidemiology;

Keywords

antibiotic

antimicrobial drug

antimicrobial resistance

bacterial fitness

cattle

control

e. coli

enteric bacteria

feedlot

intervention

Goals / Objectives
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.

Project Methods
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.

Progress 09/01/08 to 02/28/11

Outputs
OUTPUTS: During the reporting period from September 1st 2010 through February 28th 2011, we have completed phenotypic quantification of non-type specific (NTS) Escherichia coli, as well as NTS Escherichia coli resistant to ceftiofur, tetracycline, and both ceftiofur and tetracycline. Furthermore, genotypic quantification of the blaCMY2 gene, which confers resistance to ceftiofur, was completed. Phenotypic and genotypic data were generated from 2596 fecal samples originating from 176 steers sampled 15 times in two 28 day periods. Furthermore, we have refined a method to quantify a 16S rRNA gene, which will be used as a denominator in analyses of the blaCMY2 gene data. We also have optimized quantification protocols for TetA and TetB genes which will be employed on these sample samples to compare phenotypic and phenotypic results and explain paradoxical responses where observed. PARTICIPANTS: Principal investigators: Bo Norby, Guy Loneragan, H. Morgan Scott, Mindy Brashears and Roger Harvey. Savannah Moore, master student on the project, and several other graduate students, technicians and student workers conducted sample collection at the experimental feedlot. Lacey Farrow (laboratory technician) and a student worker conducted phenotypic quantification of NTS E. coli. Genotypic determination of the blaCMY2 gene (ceftiofur resistance-determinant) was conducted by Neena Kanwar (PhD student in Dr. Scott's laboratory) and Javier Vinasco (Research Associate in Dr. Scott's Lab). TARGET AUDIENCES: The results on quantification of target resistance genes have been presented to several groups including the American Society for Microbiology (Toronto, Canada June 2010), the Conference of Workers in Animal Diseases (Chicago, December 2010), the Pew Trusts (Washington, DC January 2011). PROJECT MODIFICATIONS: The PD, Bo Norby, is moving from Texas A&M university to Michigan State University, and this grant is being transferred from Texas A&M University to Michigan State University.

Impacts
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. This project will continue to provide valuable data for determining ecological changes occurring in the intestinal microbiota following treatment with various combinations of antimicrobial drugs. The information gained from this study will enable scientists and the agricultural sector to further investigate and develop innovative methods to maintain a low level of resistance in intestinal bacteria in cattle during treatment with antibiotics.

Publications

• Kanwar, N.; Scott, H.; Vanisco, J.; Norby, B. (2010) Effects of controlled intervention strategies on the quantities of a ceftiofur resistance gen (blaCMY-2) in the feces of feedlot cattle. Conference of Research Workers in Animal Diseases, December 5-7, Chicago, Illinois.

Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: During this progress report period, efforts have focused on quantifying phenotypic resistance to ceftiofur and tetracycline in non-type-specific (NTS) Escherichia coli, as well as genotypic quantification of the blaCMY2 gene, which confers resistance to ceftiofur. Approximately two thirds of the 176 fecal samples have been analyzed. Additionally, detection of NTS E. coli by direct plating on MacConkey agar containing ceftiofur was compared to pre-enrichment in MacConkey broth plus ceftiofur followed by plating on MacConkey agar containing ceftiofur. Findings resulting from this research project have been reported at national and international conferences. PARTICIPANTS: Principal investigators: Bo Norby, Guy Loneragan, H. Morgan Scott, Mindy Brashears and Roger Harvey. Savannah Moore, master student on the project, and several other graduate students and student workers conducted sample collection at the experimental feedlot. Lacey Farrow (laboratory technician) and a student worker conducted phenotypic quantification of NTS E. coli. Genotypic determination of ceftiofur resistance-determinants was conducted by Neena Kanwar (PhD student, Dr. Scott's laboratory). TARGET AUDIENCES: The target audience has so far 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. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Preliminary results from the first replicate of the study (88 steers) suggested 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 for approximately 10 days after ceftiofur treatment, 3) treatment with chlortetracycline appeared to co-select for ceftiofur resistance determinants. In a study of samples from 88 steers collected before their treatment with ceftiofur and/or chlortetracycline (on day 0 of the study), direct plating on MacConkey broth containing ceftiofur revealed that 20.3 percent of samples contained E. coli phenotypically resistant to ceftiofur as compared to 83.5 percent if the feces had been incubated in MacConkey broth containing ceftiofur prior to plating on MacConkey agar containing ceftiofur. This suggests that the a large proportion of samples carried E. coli that were resistant to ceftiofur, but that the quantity of ceftiofur-resistant E. coli was below the detection limit for direct plating of feces on ceftiofur-containing MacConkey plates. The trial for the first objective is near completion. This project provides valuable information on the intestinal ecology of E. coli and ceftiofur and tetracycline resistance determinants in feedlot cattle during antibiotic treatment. The information gained may enable producers and scientists to develop innovative methods to maintain a low level of resistance in intestinal bacteria in cattle during treatment with antibiotics.

Publications

• N. Kanwar, H.M. Scott, B. Norby, S. Moore, J. Vinasco, G.H. Loneragan. Effects of controlled intervention strategies on the quantities of a ceftiofur resistance gene (blacmy-2) in the feces of feedlot steers. 2nd ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens in Animals, Humans and the Environment. June 8-11, 2010, Toronto, Canada.
• B. Norby, H.M. Scott, G.H. Loneragan, R.B. Harvey, M.M. Brashears, N. Kanwar, S. Moore, J. Vanisco. Effects of controlled intervention strategies on the quantities of geno- and phenotypic ceftiofur resistance in the feces of feedlot cattle. 2010 IFT Annual Meeting & Food Expo; NIFA NRI/AFRI Meeting, July 17-20, in Chicago, Illinois.
• R.M. Sanders, G.H. Loneragan, R.L. Farrow, R.M. McCarthy, H.M. Scott, B. Norby. Analysis of E. coli populations within bovine feces stored at refrigerator temperatures. 90th Annual Meeting of Research Workers in Animal Diseases, December 6-8, 2009, Chicago, Illinois.
• J.R. Graves, G.H. Loneragan, R.L. Farrow, R.M. McCarthy, H.M. Scott, B. Norby. Impact of isolation methods on estimates of prevalence of antimicrobial resistance and implications for surveillance. 90th Annual Meeting of Research Workers in Animal Diseases, December 6-8, 2009, Chicago, Illinois.

Progress 09/01/08 to 08/31/09

Outputs
OUTPUTS: OUTPUTS: During this progress report period, our efforts have focused on the experimental trial that aims at investigating 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. The trial, including 176 steers, has been completed at the experimental feedlot. Fecal samples were collected from all enrolled steers at the planned time-points and processed and frozen for phenotypic and genotypic analyses on E. coli. Our next step is to quantify phenotypic and genotypic resistance of E. coli to a third generation cephalosporin and tetracycline. PARTICIPANTS: Principal investigators: Bo Norby, Guy Loneragan, H. Morgan Scott oversaw and participated in sample collection. Savannah Moore, master student on the project, and several other graduate students and student workers conducted sample collection at the experimental feedlot. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: In July of 2009 FDA sent out a proposed order to ban the extra-label use of all cephalosporins in all food animals. This would potentially affect our proposed use of Naxcel in the experimental trial at the research feedlot. Hence, we decided to eliminate the treatment with Naxcel in the experimental trial (obj. 1). The modified design included treatment with Excede or no exceed (factor 1), mixing, which refers to whether all or just some animals in a group are treated with the ceftiofur products (Factor 2) and treatment with chlortetracycline (CTC) in the feed at the conclusion of ceftiofur treatment (Factor 3).

Impacts
We have not yet generated results that can be shared with cattle producers, veterinarians, researchers, administrators or federal regulators.

Publications

• No publications reported this period