Source: MICHIGAN STATE UNIV submitted to
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.
0227723
Grant No.
2008-35201-30235
Cumulative Award Amt.
(N/A)
Proposal No.
2011-06353
Multistate No.
(N/A)
Project Start Date
May 15, 2011
Project End Date
Feb 24, 2013
Grant Year
2012
Program Code
[32.0B]- Food Safety and Epidemiology (B): Epidemiological Approaches for Food Safety
Project Director
Norby, B.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Large Animal Clinical Sciences
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
7123399107030%
7123399110040%
7123399117030%
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;
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. 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.
Project Methods
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. 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 05/15/12 to 02/24/13

Outputs
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). 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) 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. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
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.

Publications

  • Kanwar N., Cottel J., Scott H.M., Norby B., Vinasco J., Loneragan G.H., Chengappa M.M., Bai J,, Boerlin P. Effect of Intervention Strategies on Antimicrobial Susceptibility Profiles and their Relationship with tetA, tetB, and blaCMY-2 genes in the E. coli Isolates in Cattle. Electronic conference proceedings (Paper), The 3rd ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens in Animals, Humans and the Environment, 26-29 June 2012, Aix-en-provence, France.
  • Cottel J., Kanwar N., Scott H.M., Boerlin P. Characterisation of E. coli antibiotic resistance mechanisms and plasmids recovered from US dairy cattle exposed to antibiotic agents. Electronic conference proceedings (Paper), The 3rd ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens in Animals, Humans and the Environment, 26-29 June 2012, Aix-en-provence, France.
  • Cottell J., Kanwar, Scott H.M., Norby B., Loneragan G.H., and Boerlin P. Characterization of E. coli antibiotic resistance mechanisms and plasmids recovered from US beef cattle exposed to antibiotic agents. CD ROM (Abstract), The 52nd Interscience Conference on Antimicrobial Agents and Chemotherapy, held 9-12 September 2012 in San Francisco, California.
  • McGowan M., Scott H.M., Kanwar N., Cottell J.L., Boerlin P., Norby B., Loneragan G.H. Antibiotic use versus antibiotic resistance profiles of commensal E. coli in beef cattle: explaining their association via bacterial growth parameters. Electronic conference proceedings (Abstract), The Conference of Research Workers in Animal Diseases, held 2-4 Dec 2012 in Chicago, Illinois.
  • Kanwar N., Scott H.M., Norby B., Loneragan G.H., Vinasco J., Cottell J.L., Chalmers G., Chengappa M.M., Bai J., Boerlin P. Metagenomic versus microbiological culture based approaches to evaluate the effects of interventions strategies on ceftiofur and tetracycline resistance in cattle feces. Electronic conference proceedings (Abstract), The Conference of Research Workers in Animal Diseases, held 2-4 Dec 2012 in Chicago, Illinois.
  • Kanwar N., Scott H.M. , Norby B., Loneragan G.H., Vinasco J., Cottell J.L., Chalmers G., Chengappa M.M., Bai J., Boerlin P. Approaches to evaluate the effects of interventions strategies on antibiotic resistance in cattle. Electronic conference proceedings (abstract) The Phi Zeta Day at College of Veterinary Medicine, 5 March 2013, College of Veterinary Medicine, KSU, Manhattan, Kansas
  • Journal Article: Cottell J.L., Kanwar N., Castillo-Courtade L., Chalmers G., Scott H.M., Norby B., Loneragan G.H., Boerlin P. (2013). blaCTX-M-32 on an IncN Plasmid in Escherichia coli from Beef Cattle in the United States. Antimicrob Agents Chemother.57(2):1096-1097
  • Abstracts: Kanwar N., Cottel J., Scott H.M., Norby B., Vinasco J., Loneragan G.H., Chengappa M.M., Bai J., Boerlin P. Effect of Intervention Strategies on Antimicrobial Susceptibility Profiles and their Relationship with tetA, tetB, and blaCMY-2 genes in the E. coli Isolates in Cattle Electronic conference proceedings (abstract) The Annual Meeting of the Missouri Valley Branch of the American Society of Microbiology, 12-14 April 2012, Kansas State University, Manhattan, Kansas


Progress 05/15/11 to 02/24/13

Outputs
Target Audience: Target audiences have been researchers, professional and graduate students,and technical and industry personnel participants in local, national and internationalscientific meetings,producer organizationsand federal agency representatives. Cattle producers, industry organizations, and federal regulators will be continued target audiences for the work related to antimicrobial resistance ecology in cattle. Changes/Problems: The livestock species used in objective 2 was changed from feedlot steers to dairy cattle due logistical challenges when the PI moved form Texas to Michigan. The study design and antibiotic treatment (Excede [ceftiofur crytaline free acid]) remained the same. However, the proposed naturally occurring disease was switched from bovine respiratory disease in feedlot steers (original plan) to metritis in dairy cattle. Fifty-three dairy cattle were enrolled in this study for 30 days. All cows fulfilled theinclusion criteria of being within 10 of parturition, had fetid uterine discharge and a temperature at or above 103.5F. Cow were treated with Excedeon the day of diagnosis of clinical metritis and again three days later according to label.Fifty percent of cows were randonmly assigned to be placed ina hospital pen following their firsttreatment. They stayed in the hospital pen for 16 days and then were returned to their pen of origin.Feces was collected from enrolled cows on day 0 and then every third day for 30 days The microbiological methodology did not change from what was originally proposed. What opportunities for training and professional development has the project provided? Two graduate students (one Master and one PhD degree) were trained with funds from this grant. Both students used data from this project, and their thesis/dissertation were successfully defended. Technicians and graduate students have attained better and more proficient work skills through training activities with other laboratory personnel with this grant, as well as through classroom and one-on-one interactions with their mentors. Additionally, a number of undergraduate students and veterinary students have gained knowledge about study conduct and laboratory methodologies and antimicrobial resistance epidemiology/ecology. Graduate students have gained invaluable professional development through participation and presentation at several local, national and international meetings as well as through participation in seminars. How have the results been disseminated to communities of interest? The results from this study have been disseminated to research communities via peer-reviewed publications and through 6 local, 6 national and 5 international meetings. Additionally, results from the study have been used in several invited talks at national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The two trials (objectives 1 and 2), proposed for this project, have been completed. Laboratory and data analyses directly related to objective 1 have been completed; however other studies using samples and data from trial 1 are ongoing. Two manuscripts using data from trial 1 have been published or accepted for publication, one manuscript is submitted and four are ready for submission or are in preparation. The majority of laboratory analyses for trial 2 have been concluded, and data analyses are ongoing. For objective 3, antibiograms of E. coli isolates susceptible or resistant to ceftiofur, tetracycline or both ceftiofur and tetracycline have been obtained. Data analyses are ongoing. Based on results from previous studies by our group, we hypothesized that the time it takes for the prevalence levels of ceftiofur-resistant E. coli to decline to pre-treatment levels after ceftiofur treatment will vary with the proportion of ceftiofur treated cattle in a group and the availability of sources of ceftiofur susceptible E. coli from both the environment and other untreated cattle, and whether or not cattle were treated with chlortetracycline in the feed. Chlortetracycline use did not, as hypothesized, reduce the time it took for ceftiofur-resistant E. coli to return to baseline, however ceftiofur resistance was lower in pens where only 1 of 11 steers were treated with a long-acting ceftiofur as compared to pens where all steers were treated with a long-acting ceftiofur. Further investigation of group- and time-dependent variability in prevalence of resistance to ceftiofur in E. coli form cattle treated with ceftiofur in a commercial setting was undertaken in trial 2 of the project. In a recent publication in Antimicrobial Agents and Chemotherapy (Cottell et al, 2013), we described the detection of the blaCTX-M-32 gene not previously described in US animals. The gene was found in 29 of 88 single source steers included in trial 1 of this project. Pulse-field gel electrophoresis showed 6 distinct E. coli strains and seven with identical banding patterns, indicating that dissemination of blaCTM-M-32 may be a result of both clonal and horizontal gene transfer. The plasmid carrying blaCTX-M-32 was shown to be on an IncN type 1, and was shown to be self-transmissible to an E. coli recipient. Among fecal community DNA, long-acting ceftiofur treatment significantly decreased tetracycline resistance genes copies (tet(A) and tet(B)) and chlortetracycline treatment led to an increase in gene copies of the two ceftiofur resistance genes (blaCMY-2 and blaCTX-M). Among E. coli isolates, contrary to community DNA results, an increase in proportion of isolates harboring both tetracycline resistance genes was observed upon ceftiofur treatment and there was preferential selection of isolates harboring blaCMY-2 gene over blaCTX-M gene upon chlortetracycline treatment. These differences suggest that bias may be introduced if conclusions pertaining to entire microbial populations are made solely on certain sentinel culturable organisms (e.g. E. coli). Overall, results from this study indicate that chlortetracycline use following long-acting ceftiofur treatment may exacerbate ceftiofur resistance and therefore should be avoided. For trial 2, we hypothesized that the ability to leave cattle in their pen of origin, as opposed to keeping them in a hospital pen, would produce a more rapid decline in resistant coliform bacteria. The use of a long-acting ceftiofur product allowed us to test this hypothesis, because the product does not have a milk-withdrawal period, and cows can be kept in their pen of origin. Trial 2 was conducted in dairy cows that were to be treated for metritis. A long-acting ceftiofur product labeled for treatment of metritis in dairy cows was used, and half the treated cows were moved to the hospital pen for the first 16 days after treatment while the remaining half was kept in their pen of origin. Preliminary statistical analyses including days 0 through 9, suggest that cows remaining in their pen of origin had a slightly lower prevalence level of ceftiofur-resistant E. coli as compared to cow that spent the first 16 days after treatment in the hospital pen. For objective 3, Growth rate experiments suggested that multidrug resistant E. coli (including resistance to tetracycline) may exhibit better growth fitness that E. coli resistant to tetracycline alone. Additionally, antibiograms for 688 E. coli isolated were conducted. Final analyses of those data are pending. We validated the agar dilution method utilizing MacConkey agar which was used for phenotypic quantification of total colony forming units (CFUs) per gram of feces and CFUs for ceftiofur- and tetracycline-resistant coliform bacteria. Minimum inhibitory concentrations of ATCC strains and ‘wild type’ strains of E. coli to ceftiofur and tetracycline on MacConkey agar were generally within one dilution as compared to Muller-Hinton agar (CLSI gold-standard agar). 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 ceftiofur and tetracycline antibiotics.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Cottell J. L., Kanwar N., Castillo L., Chalmers G., Scott H. M., Norby B., Loneragan G., and Boerlin P. blaCTX-M-32 on an IncN plasmid in E. coli from U.S. beef cattle, Antimicrobial Agents and Chemotherapy. 2013, 57(2): 1096-1097.
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Kanwar N., Scott H. M., Norby B., Loneragan G. H., Vinasco J., McGowan M., Cottell J. L., Chengappa M. M., Bai J., and Boerlin P. Effects of ceftiofur and chlortetracycline treatment strategies on antimicrobial susceptibility and on tet(A), tet(B), and blaCMY-2 resistance genes among E. coli isolated from the feces of feedlot cattle. PlosOne, doi 10.1371/journal.pone.0080575 (In Press, October 2013)
  • Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2013 Citation: TREATMENT STRATEGIES IMPACTING CEFTIOFUR RESISTANCE IN CATTLE by NEENA KANWAR B.V.Sc., Maharashtra Animal and Fishery Sciences University, Mumbai, India, 2005 M.V.Sc., Sardarkrushinagar Dantiwada Agricultural University, Gujarat, India, 2007 A DISSERTATION submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Diagnostic Medicine/ Pathobiology College of Veterinary Medicine KANSAS STATE UNIVERSITY Manhattan, Kansas 2013 Major Professor Harvey Morgan Scott
  • Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Kanwar N., Scott H. M., Norby B., Loneragan G. H., Vinasco J., Cottell J. L., Chalmers G., Chengappa M.M., Bai J., and Boerlin P. Impact of treatment strategies on cephalosporin and tetracycline resistance gene quantities in bovine fecal metagenome
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: McGowan M., Scott H.M., Kanwar N., Cottell J.L., Boerlin P., Norby B., Loneragan G.H. Antibiotic use versus antibiotic resistance profiles of commensal E. coli in beef cattle: explaining their association via bacterial growth parameters. Paper for the 5th Congress of European Microbiologists: FEMS 2013, held 21-25 July 2013 in Leipzig, Germany.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Kanwar N., Scott H.M. , Norby B., Loneragan G.H., Vinasco J., Cottell J.L., Chalmers G., Chengappa M.M., Bai J., Boerlin P. Approaches to evaluate the effects of interventions strategies on antibiotic resistance in cattle. Paper for the K-State Research Forum held 27 March 2013 in Kansas State University, KS
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Kanwar N., Scott H.M. , Norby B., Loneragan G.H., Vinasco J., Cottell J.L., Chalmers G., Chengappa M.M., Bai J., Boerlin P. Approaches to evaluate the effects of interventions strategies on antibiotic resistance in cattle. Paper for the Phi Zeta Day at College of Veterinary Medicine, held 5 March 2013 in College of Veterinary Medicine, KSU
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Kanwar N., Scott H.M., Norby B., Loneragan G.H., Vinasco J., Cottel J., Bai J., Chalmers G., Chengappa M., Bai J., Boerlin P. Metagenomic versus microbiological culture based approaches to evaluate the effects of interventions strategies on ceftiofur and tetracycline resistance in cattle feces. Paper for the Conference of Research Workers in Animal Diseases, held 2-4 Dec 2012 in Chicago, Illinois.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: McGowan M., Scott H.M., Kanwar N., Cottell J.L., Boerlin P., Norby B., Loneragan G.H. Antibiotic use versus antibiotic resistance profiles of commensal E. coli in beef cattle: explaining their association via bacterial growth parameters. Paper for the Conference of Research Workers in Animal Diseases, held 2-4 Dec 2012 in Chicago, Illinois.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Cottell J., Kanwar N., Scott H.M., Norby B., Loneragan G.H., and Boerlin P. Characterization of E. coli antibiotic resistance mechanisms and plasmids recovered from US beef cattle exposed to antibiotic agents. Paper for the 52nd Interscience Conference on Antimicrobial Agents and Chemotherapy, held 9-12 September 2012 in San Francisco, California.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Kanwar N., Cottel J., Scott H.M., Norby B., Vinasco J., Loneragan G.H., Chengappa M.M., Bai J., Boerlin P. Effect of Intervention Strategies on Antimicrobial Susceptibility Profiles and their Relationship with tetA, tetB, and blaCMY-2 genes in the E. coli Isolates in Cattle. Paper for the 3rd ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens in Animals, Humans and the Environment, held 26-29 June 2012 in Aix-en-provence, France.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Cottell J., Kanwar N., Scott H.M., Boerlin P. Characterisation of E. coli antibiotic resistance mechanisms and plasmids recovered from US dairy cattle exposed to antibiotic agents. Paper for the 3rd ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens in Animals, Humans and the Environment, held 26-29 June 2012 in Aix-en-provence, France.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Kanwar N., Scott H.M., Norby B., Vinasco J., Loneragan G. H. Challenges in the use of multi-level mixed linear models to evaluate factors influencing quantitative real-time PCR (qPCR) resistance gene numbers in feces Paper for the Innovations in Design, Analysis, and Dissemination: Frontiers in Biostatistical Methods Symposium, held 26-27 April 2012 in Cerner Corporations World Headquarters Campus, Kansas City, MO
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Kanwar N., Cottell J., Scott H.M., Norby B., Vinasco J., Loneragan G.H., Chengappa M.M. , Bai J., Boerlin P. Effect of Intervention Strategies on Antimicrobial Susceptibility Profiles and their Relationship with tetA, tetB, and blaCMY-2 genes in the E. coli Isolates in Cattle. Paper for the Annual Meeting of the Missouri Valley Branch of the American Society of Microbiology, held 12-14 April 2012 in Kansas State University
  • Type: Other Status: Other Year Published: 2011 Citation: 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. 2011 IAFP Annual Meeting; NIFA NRI/AFRI Meeting, July 30, in Milwaukee, Wisconsin.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Norby, B.; Scott, H.M.; Loneragan, G.; Moore, S.; Kanwar, N.; Harvey, R. (2011) The effect of two intervention strategies on ceftiofur resistance in E. coli. 4th Symposium on Antimicrobial Resistance in Animals and the Environment 27 - 29 June, Tours, Loire Valley, France
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Kanwar, N.; Scott, H.M.; Norby, B.; Moore, s.; Vinasco, J.; Loneragan, G.; Jai, J.; Chengappa, M.. (2011) Intervention strategies to control ceftiofur resistance in cattle. 4th Symposium on Antimicrobial Resistance in Animals and the Environment 27 - 29 June, Tours, Loire Valley, France
  • Type: Theses/Dissertations Status: Published Year Published: 2011 Citation: Resistance of Ceftiofur and Tetracycline among Escherichia coli in Feedlot Cattle by Savannah Holmes A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE West Texas A&M University Canyon, Texas August, 2011


Progress 05/15/11 to 05/14/12

Outputs
OUTPUTS: During most of the reporting period from May 15,2011 to May 14, 2012, the award was inactive due to transfer with the PI from Texas A&M University (TAMU) to Michigan State University (MSU). The award was terminated at TAMU on March 01, 2011. Although the start date of the award at MSU was May 15, 2011, the award agreement between USDA and MSU was dated February 21, 2012 and an advance account was set up on March 09, 2012. Consequently spending on the award was suspended for 12 months. During this reporting period, efforts have focused on 1) genotypic quantification of the blaCMY2 gene, which confers resistance to ceftiofur, 2) quantification of TetA and TetB gene which confer resistance to tetracyclines, 3) detection of plasmid replicon types in a subset of 1,050 samples and to determine associations with ceftiofur and tetracycline resistance genes, and 4) analyses of phenotypic and genotypic data. Results of research efforts have been presented at four national conferences and one international research conference. Additionally, results from this study have been used in several invited talks. Fecal samples, bacterial isolates and bacterial community DNA have been used in outreach to current and new collaborators (Dr. Patrick Boerlin, Ontario Veterinary College, Canada). PARTICIPANTS: Principal investigators: Bo Norby, Guy Loneragan, H. Morgan Scott, Mindy Brashears and Roger Harvey. Genotypic determination of the blaCMY2 gene (ceftiofur resistance-determinant) as well as Tet genes 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 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
The project has generated new knowledge about the ecology of intestinal bacteria and their resistance determinants when cattle are or are not treated with one or more antimicrobial drugs. Phenotypic and genotypic results suggested that 1) treatment of all cattle in a pen with long-acting ceftiofur resulted in an overall absolute decrease in non-type specific (NTS) E. coli immediately after treatment, 2) Absolute counts of NTS ceftiofur-resistant E. coli and the blaCMY-2 gene copies increased for approximately 10 days after ceftiofur treatment, 3) treatment with chlortetracycline appeared to co-select for ceftiofur resistance determinants. In a full factorial design the interaction with time (days), top-dressing feed with chlortetracycline increased blaCMY2 and TetA gene copies consistently across other factors. Reducing the number of cattle treated with a ceftiofur product had a significant but inconsistent sparing effect on blaCMY2 and TetA gene copies. There was a strong positive correlation between blaCMY2 and TetA genes. Furthermore, tetA was associated with higher levels of multidrug resistant phenotypes as compared to TetB. In analyses of plasmids in E. coli from a subset of fecal samples, IncA/C and IncI plasmids were identified from cattle treated with a ceftiofur product. The blaCMY2 gene was associated with plasmid replicon IncA/C but was also found in IncIα plasmids. The information made available by this project may aid scientists in developing new innovative methods to maintain a low level of resistance in intestinal bacteria in cattle during treatment with antibiotics as well as methods to further investigate the ecology of resistant bacteria and resistance determinants in the intestinal tract of animals and humans.

Publications

  • Kanwar, N.; Scott, H.M.; Norby, B.; Moore, s.; Vinasco, J.; Loneragan, G.; Jai, J.; Chengappa, M. (2011) Intervention strategies to control ceftiofur resistance in cattle. 4th Symposium on Antimicrobial Resistance in Animals and the Environment 27 - 29 June, Tours, Loire Valley, France
  • Norby, B.; Scott, H.M.; Loneragan, G.; Moore, S.; Kanwar, N.; Harvey, R. (2011) The effect of two intervention strategies on ceftiofur resistance in E. coli. 4th Symposium on Antimicrobial Resistance in Animals and the Environment 27 - 29 June, Tours, Loire Valley, France
  • B. Norby, H.M. Scott, G.H. Loneragan, R.B. Harvey, M.M. Brashears, N. Kanwar, S. Moore, J. Vanisco. (2011) Effects of controlled intervention strategies on the quantities of geno- and phenotypic ceftiofur resistance in the feces of feedlot cattle. IAFP Annual Meeting; NIFA NRI/AFRI Meeting, July 30, in Milwaukee, Wisconsin.
  • B. Norby (2011) Antimicrobial use and antimicrobial resistance in food animals. USAHA 115th Annual Meeting, October 2, Buffalo, NY
  • N. Kanwar, H.M. Scott, B. Norby, S. Moore, J. Vinasco, G.H. Loneragan, J. Bai., M. Chengappa. (2011) Effect of intervention strategies on ceftiofur resistance determinant (blaCMY-2 gene) and its relationship with TetA and TetB genes in cattle. The Conference of Research Workers in Animal Diseases (CRWAD), 4-6 December, Chicago, Illinois.
  • N. Kanwar, H.M. Scott, B. Norby, S. Moore, J. Vinasco, G.H. Loneragan (2012) Challenges in the use of multi-level mixed linear models to evaluate factors influencing quantitative real-time PCR (qPCR) resistance gene numbers in feces. Innovations in Design, Analysis, and Dissemination. Frontiers in Biostatistical Methods Symposium Cerner Corporation's World Headquarters Campus, 26-27 April, Kansas City, MO
  • N. Kanwar, J. Cottel, H.M. Scott, B. Norby, Vinasco, G.H. Loneragan, M. Chengappa, J. Bai, P. Boerlin. (2012) Effect of Intervention Strategies on Antimicrobial Susceptibility Profiles and their Relationship with tetA, tetB, and blaCMY-2 genes in the E. coli Isolates in Cattle. . Frontiers in Biostatistical Methods Symposium Cerner Corporation's World Headquarters Campus, 26-27 April, Kansas City, MO