Source: NORTH CAROLINA STATE UNIV submitted to
THE IMPACT OF DOSING SCHEDULE OF ENROFLOXACIN ON GASTRINTESTINAL TRACT DRUG CONCENTRATION AND SELECTION FOR FLUOROQUINOLONE-RESISTANT ESHERICHIA COLI
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1007631
Grant No.
(N/A)
Project No.
NCV-VMAH-0146
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 15, 2015
Project End Date
Jun 30, 2016
Grant Year
(N/A)
Project Director
Foster, DE, .
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Population Health and Pathobiology
Non Technical Summary
Prudent use of antimicrobials by veterinarians is widely considered critical to mitigate the development of AMR in cattle,1-2 and is a key component of the recently announced National Action Plan for Combating Antibiotic-Resistant Bacteria. Nowhere is this more important than the treatment of respiratory disease on feedlots as these animals are close to harvest and the predominant antimicrobials used are prescription drugs similar to those critical for human use (including enrofloxacin).3-4 Epidemiological associations between parenteral antibiotic administration in these animals and resistance in enteric organisms often only evaluate a single bacterial species or isolate.5-6 While useful in describing broad associations, these studies are inherently limited by the inability to correlate the development of AMR with antibiotic concentrations in the GI tract. The lack of a clear method of evaluating antimicrobials' risk of promoting resistance is an inherent problem in adequately mitigating the food safety risk of AMR in cattle.7Upon initial approval of enrofloxacin for treatment in cattle, the Food and Drug Administration prohibited of extra-label use of this drug in food animals to preserve the effectiveness of fluoroquinolones for treating disease in humans, yet made no distinction between the two approved dosing regimens of enrofloxacin and how that may be a factor in producing resistance. Yet, evidence suggests that it is not necessarily a specific drug that causes resistance, but the method and duration of exposure as potentially determined by the dose and dosing schedule.8-9 The approval of administration of enrofloxacin as a single, high dose or repeated lower doses provides us with a perfect opportunity to test these assumptions in cattle using novel techniques that are not possible in humans.From our studies, we expect determine the impact of dosing regimen on distribution of enrofloxacin into the GI tract. We believe that many factors, including pharmacokinetics of antibiotics, the physiochemical properties, formulations, and dosing scheme, determine the flux from systemic administration to the intestinal lumen. As enrofloxacin is partially metabolized into ciprofloxacin,10 which has a different lipophilicity and protein binding, its use in this study provides an ideal opportunity to additionally investigate the impact of these chemical differences on drug movement into the gut. Further, we believe that our methods will provide a framework to evaluate new and existing antibiotics for their potential to produce resistance in intestinal bacteria, allowing regulatory authorities and drug manufacturers to screen future antimicrobial medications for food animals. The research proposed in this application is significant because the findings are expected form the basis for rational prudent antimicrobial use guidelines for cattle, a key mechanism to reduce the food safety risk of AMR in cattle.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71133991180100%
Goals / Objectives
Development of antimicrobial resistance (AMR) in the intestinal microbial flora of cattle remains one of the most significant issues of public concern faced by the beef industry, and has led the FDA to drastically restrict the use of fluoroquinolones. Yet, the role of therapeutic use of these drugs in selecting for resistant fecal bacteria is unclear, which led us to develop a novel method to measure active antimicrobial concentrations in multiple areas of the gastrointestinal tract of steers. There remains significant unanswered questions concerning the impact of dosing regimens on the GI tract concentration of antimicrobials, the subsequent selection for AMR, and the persistence of these resistant bacteria. The work outlined in this project will fill this immediate need by directly comparing two FDA-approved dosing regimens for enrofloxacin in cattle and their impact on AMR, and the persistence of these changes in the feces.Our hypothesis is that administration of a single, high dose of enrofloxacin will produce higher drug concentrations in the GI tract and induce less AMR than repeated, lower doses.
Project Methods
Determine the active drug concentration in the GI tract after parenteral administration of enrofloxacin once at 12.5 mg/kg or for three doses at 5 mg/kg.Currently, adequate AMR risk assessment of antibiotic usage for respiratory disease in cattle is difficult as it is unclear what drug concentrations are achieved in the GI tract. Without this information, we cannot define which factors drive these concentrations. The goal of the research under Aim #1 is to measure the GI tract concentrations produced from two approved and commonly used dosing regimens of enrofloxacin in cattle to determine the impact of dosing schedule on GI tract concentrations. Our working hypothesis is that a single dose of enrofloxacin will produce brief, but high concentrations of active antibiotic in the GI tract, while repeated doses of enrofloxacin will produce repeated, smaller spikes of a short duration. We will test this hypothesis by using the experimental approach of repeatedly collecting an ultrafiltrate from the ileum and spiral colon of steers for drug measurement. The rationale for this aim is that determining how dosing schemes affect the GI tract concentrations is fundamental to developing responsible antibiotic use guidelines for enrofloxacin, and potentially other antimicrobials used in cattle. When the proposed studies for research objective #1 have been completed, we expect the overall outcome to be clear associations between enrofloxacin dosing schedule and GI tract concentrations of antibiotics. This result is expected to have a significant impact on the mitigation of AMR as it will inform drug use guidelines, and potentially drug labeling.Six 200 kg Holstein steers will be obtained from Lake Wheeler Dairy Education Unit for a crossover study. From experience, 6 animals per group are adequate for meaningful statistical analysis, while minimizing cost and animal use. Calves will receive a single subcutaneous dose of enrofloxacin (Baytril 100, Bayer Corporation) at 12.5 mg/kg or 5 mg/kg every 24 hours for 3 days and after a 7 day washout the calves will be reallocated to the other treatment group. We will have assessed all the commonly used dosing schedules for enrofloxacin between this study and our previous work.Intestinal fluid collection will be performed with an in-vivo ultrafiltration sampling kit (BAS Bioanalytical Systems, W.Lafayette, IN). The ultrafiltration probe contains 3 semi-permeable loops connected to a non-permeable tube extending to the exterior of the animal and attached to a Vacutainer. The Vacutainer provides the negative pressure for fluid collection through the small pores in the loop membrane. Each calf will be surgically fit with 3 intestinal ultrafiltration sampling devices (in the ileum, in the spiral colon, and a modified probe in the ileum). The membrane in the loop consists of pores allowing water, electrolytes, and low molecular weight molecules to pass and excludes the passage of protein, protein-bound drugs, and other large molecular weight compounds. Prior to drug administration an Intracath® jugular catheter (Becton Dickinson) will be inserted in the jugular vein to collect blood samples, and ultrafilitration probe will be placed subcutaneously to collect interstitial fluid. Plasma, interstitial fluid, and intestinal fluid samples will be collected at time 0, and at appropriate intervals for optimum pharmacokinetic modeling for at least 3 drug half-lives, accounting for 90% of drug elimination. The tubes will be centrifuged at 1,000 x g for 10 minutes to collect plasma, and samples will be stored at -70?C until assayed.Plasma and tissue fluid samples will be analyzed by reverse-phase high performance liquid chromatography (HPLC) with or fluorescence to determine the concentrations of enrofloxacin and its metabolite, ciprofloxacin. All drug concentrations will be determined from calibration curves made from fortified blank plasma and tissue fluids collected from the experimental calves. The drug concentrations will be analyzed using standard pharmacokinetic methods to determine the drug disposition for each drug in each calf. A computer program (Phoenix WinNonlin, Version 6.0 Pharsight Corporation, Mountain View, CA) will be used to determine pharmacokinetic parameters as well as deriving statistical values.Evaluate the change in proportion of fluoroquinolone-resistant E. coli in the ileum and feces of cattle treated with either a single high dose of enrofloxacin or multiple lower doses both during and after treatment. Exposure to antimicrobials inherently selects for resistance within a population of bacteria. The objective of the studies outlined in this Specific Aim is to determine the impact of the drug concentrations measured in Aim 1 on enteric bacteria. To achieve the objective of this Aim, we will test our working hypothesis that a single, high dose of enrofloxacin will induce less resistance in E. coli obtained from the ileum and feces than repeated lower doses. We plan to test our working hypothesis assessing the percentage of E. coli obtained from the ileum and feces in Aim 1 that are resistant to nalidixic acid. Our rationale for this aim is that we will demonstrate a clear association between dosing scheme, subsequent GI tract drug concentration, and the potential to select for resistance within the GI tract. At completion of these studies, we expect to make clear recommendations for use of enrofloxacin, and begin to create a model to predict a "microbiological withdrawal time."Feces and ileal fluid will be collected every 12 hours from each steer starting just prior to drug administration and continuing during treatment and for 5 days after the last dose of enrofloxacin. Samples will be weighed and serially diluted ten-fold in sterile phosphate buffered saline. Representative dilutions will be plated in triplicate onto a chromogenic selective media (HardyChrom ECC; Hardy Diagnostics) to quantify the concentration of E. coli. Ten colonies per collection time point, sample type and steer will be saved at -80°C for future analysis of flouroquinolone resistance. For susceptibility determination, colonies will be re-isolated on Columbia blood agar plates and incubated for 24 h at 35°C. A 0.5 McFarland concentration of each isolate will be obtained and used to inoculate Mueller Hinton agar. A Kirby-Bauer disk diffusion using a 30 mcg disk impregnated with nalidixic acid will be used to evaluate susceptibility to flouroquinolones. For each animal, sample type and time point, the proportion of resistant isolates will be calculated.Previous studies investigating the transfer of resistant organisms or foodborne pathogens from the feces of cattle to the carcass have suggested that fecal concentrations of less than 4log CFU/ml are unlikely to lead to significant food safety risks, making this is a reasonable target to determine if an animal is safe to go to slaughter. Therefore, time for fecal concentrations of resistant pathogens to drop below this level would be an appropriate microbiological withdrawal time analogous to the current drug withdrawal time. We suggest that the longer of these two withdrawal times should be followed to maximize food safety by preventing both drug residues and transfer of resistant bacteria through the food chain. Depending on the level of AMR E. coli in the fecal samples obtained, we may be able to calculate this directly as the concentration drops below 4 log after treatment. We will develop a mathematical model that integrates the population dynamics of the bacteria with the pharmacokinetics and pharmacodynamics effects of the drug. We developed similar models to evaluate the effects of ceftiofur on the gut microflora. The differential equation based model will be parameterized with the data collected in this study. To determine which factors influence the predicted microbiological withdrawal time, we will perform global sensitivity analysis on model parameters.

Progress 09/15/15 to 06/30/16

Outputs
Target Audience:These results are aimed at veterinary practitioners in order to help them make decisions on the appropriate dosing regimens of antimicrobials for cattle. Changes/Problems:The study was initially proposed as a crossover study, but sampling in the second half of the crossover was sparse due to failure of the intestinal probes after a long period of time. Further, the microbiological findings were clouded by the repeated administration of drug. Therefore, we ultimately repeated the project in a separate set of calves to have an adequate number of samples at each time point and unambiguous microbiological data. What opportunities for training and professional development has the project provided?A veterinary student and undergraduate student assisted with this project, and the veterinary student is currently writing the manuscript describing the results. The project also led to a collaboration with Dr. Sophia Kathariou, and has formed the basis of a PhD project for her graduate student, Sicun Fan. Fan is investing the impact of the different dosing regimens on Campylobacter and Listeria in the feces of treated animals. How have the results been disseminated to communities of interest?The results were presented at the ACVIM Forum in Denver in June 2016, and will be presented at the European College of Bovine Health Management meeting in Bilbao, Spain in September, 2017. The campylobacter results were presented at the International Association of Food Protection meeting in Athens, Greece in July, 2016. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have successfully measured the drug concentrations of enrofloxacin and its metabolite ciprofloxacin in the GI tract of steers after different dosing regimens. Changes in the fecal microbial populations suggest that the low, repeated dosing has a greater impact on the MIC of E. coli than does a single dose. Nonetheless, neither of these dosing regimens caused significant longterm changes to the microbial populations that were measured.

Publications


    Progress 09/15/15 to 09/30/15

    Outputs
    Target Audience:Cattle Industry, Veterinarians, Food Safety Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Data from this project were instrumental in obtaining a collaborative grant with the College of Agriculture and Life Sciences at NC State which will fund a graduate student. How have the results been disseminated to communities of interest?Results from this project will be presented as a portion of a research presentation at the ACVIM Forum (Impact of Gastrointestinal Antibiotic Concentrations On Antimicrobial Resistance), as an abstract at the World Buiatrics Congress (Impact of enrofloxacin dosing regimens in cattle on intestinal pharmacokinetics and the selection for resistant enteric bacteria), and at the International Association of Food Protection in Athens, Greece (Impact of Enrofloxacin Treatment on Fecal Populations of Campylobacter spp. in Calves). What do you plan to do during the next reporting period to accomplish the goals?Analysis of data. We anticipate 1-2 publications being produced describing the intestinal pharmacokinetics and the subsequent impact on the gastrointestinal flora.

    Impacts
    What was accomplished under these goals? This project sampled 12 steers in a crossover design to assess antibiotic concentrations in the gastrointestinal tract of steers when they are given either of the two FDA-approved regimens of enrofloxacin. Fecal samples were taken simultaneously to assess the change in concentration and MIC of E. coli. Drug analysis is ongoing, but it is clear that the E. coli is dramatically reduced initially after treatment, which is associated with an increase in MIC. Yet, in approximately 1 week, the E. coli count has returned to normal. In the low dose group, the change in MIC appears to persist longer. We also took advantage of these samples to assess the impact on Campylobacter. This work has demonstrated a prolonged shift in the species and an associated increase in resistance.

    Publications