Performing Department
Population Health and Pathobiology
Non Technical Summary
North Carolina State University, as a research-based land grant university, has anobligation to respond to the National Action Plan for Combating Antibiotic-Resistant Bacteria released onSeptember 18, 2014.1 Specifically, the Plan calls "for agricultural stakeholders to facilitate identificationand implementation of interventions to reduce the spread of antibiotic-resistance" and to "identifyvalidated interventions to reduce the spread of antibiotic resistance and work with public and privatesector partners to incorporate them into veterinary practice." Antibiotics administered in feed and waterare being phased out, but there will always be a need for parenteral antibiotics to treat specific diseases.Parenteral antibiotics administered to treat infections in animals diffuse into the intestine and influencethe population and susceptibility of the intestinal bacteria. However, there is little is known about theduration of this effect, particularly on bacteria that might become foodborne pathogens. Earlier attemptshave not measured drug concentrations in the animal's gastrointestinal tract (GIT), and rarely assessmicrobial changes over time. Measurement of active, unbound drug concentrations at the site of action(e.g. GIT lumen) is critical to correlate pharmacokinetic-pharmacodynamic (PK-PD) indices with microbialchanges. This PK-PD model approach is novel and will allow us to understand antimicrobial movementinto the GIT and determine the association between these concentrations with changes in gut bacteriaover time. This understanding will allow us to respond to the call from the National Action Plan and tofuture funding opportunities associated with this call by providing us with the knowledge and tools torecommend specific treatment protocols with appropriate withdrawal times and to harness themicrobiome to mitigate the emergence of resistant pathogens. These results will provide research-baseddata to drive the conversation on antimicrobial use and resistance in food animals.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The long-term goal of our research program is to develop rational, research-based recommendations for food animal veterinarians to maximize antibiotic efficacy while minimizing risk of antimicrobial resistance (AMR). The objective of this proposal is to determine the GIT concentrations of the fluoroquinolone danofloxacin and macrolide tulathromycin, their impact on the microbiota and foodborne pathogens, and the persistence of these changes. These findings would drive the development of a "microbiological withdrawal time." These twodrugs represent antibiotic classes widely used in veterinary medicine and human medicine that exhibit links to AMR in zoonotic pathogens. Based on preliminary data with enrofloxacin presented below, the central hypothesis for this proposal is that danofloxacin will cause significant, but short-lived changes to the gut microbiota, while tulathromycin will produce more long lasting changes due to its slow elimination and concentration in tissues. Measuring changes to the microbiota and fecal pathogens over time willallow us to calculate a "microbiological withdrawal time," which would be the time until the fecal concentration of AMR pathogens drops below a safe threshold. This will provide an easily implemented, scientifically-justified recommendation to mitigate emergence and spread of AMR foodborne pathogens.
Project Methods
Aim 1: Determine the active drug concentration in the GI tract of steers after parenteral administration of danofloxacin ortulathromycin. Our working hypothesis is that a single FDAapproved dose of danofloxacin will result in a brief period of highconcentrations of active drug in the GIT, while tulathromycin at the approved dose will lead to prolonged, low concentrations inthe GIT due to its long half-life and persistent tissue concentrations. Antimicrobials assessed: Twelve 200 kg Holstein steers will beused for the study returned to the farm at the conclusion. Based on our previous work, 6 animals per group are adequate formeaningful statistical analysis. Each calf will receive a single subcutaneous injection of either 8 mg/kg danofloxacin or 2.5 mg/kg tulathromcyin. Blood samples will be collected via jugular catheters at time 0, and at appropriate intervals for optimum pharmacokineticmodeling based on our previous studies. ISF will be collected through an ultrafiltration probe placed subcutaneously as previously described.Intestinal Fluid Collection: Intestinal fluid collection will be performed with an in-vivo ultrafiltration sampling kit (BAS Bioanalytical Systems, W. Lafayette, IN) as previously described by our group. A standing surgery will be performed to insert 2 ultrafiltration sampling devices in the lumen of the GIT--one in the ileum and one in the spiral colon. The intestinal fluid will be collected at time 0 and atappropriate intervals for each drug to account for a complete time course of drug disposition.Drug Analysis and PK Modeling: Plasma and tissue fluid samples will be analyzed by reverse-phase highpressure liquid chromatography (HPLC) using fluorescence detection for danofloxacin and MS fortulathromycin and a method previously validated in our laboratory. The drug concentrations will beanalyzed using PK methods to determine the drug disposition for each drug in each calf. A computerprogram (Phoenix Modeling Software, Version 6.3 Pharsight Corporation, Certara, St. Louis, MO) will beused to determine PK parameters as well as derive statistical values. Tissue fluid:plasma concentrationswill be calculated as well as PK modeling of the drug transfer (intercompartmental rates) from the plasmacompartment to each tissue fluid compartment.Aim 2: Determine the impact of danofloxacin or tulathromycin administration on fecal bacteria and foodborne pathogens. Our working hypothesis is that danofloxacin will induce a dramatic, but brief decrease in the bacteria cultured but minimal change in resistance due to the short exposure in most pathogens. Fluoroquinolone selection of Campylobacter is expected to result in fluoroquinolone-resistantbacteria that persist long after treatment. Tulathromycin will have minimal impact on concentrations of E. coli, Salmonella and Enterococcus due to their inherent resistance, while we expect a decrease in Campylobacter, which will slowly return to pretreatment level over 4 weeks. Campylobacter resistance to tulathromycin is expected to be short-lived due to the high fitness cost of this mutation.Fecal sampling: Fecal samples from each steer will be manually collected once a day for 5 days prior to surgery and for two days after surgery. After administration of danofloxacin, fecal samples will be collected every 12 hours over 3 half-lives of the drug. Following this period, fecal samples will continue to be collected every 2 days for 4 weeks after drug administration in order to sample beyond the meatwithdrawal time for each drug (4 days for danofloxacin, 18 days for tulathromycin). Isolation and enumeration of Escherichia coli, Salmonella, Campylobacter and Enterococcus and MIC determination: Fecal samples will be weighed and serially diluted ten-fold in sterile phosphate buffered saline. Representative dilutions will be plated in triplicate onto selective media (E. coli, HardyChrom ECC,Hardy Diagnostics, Santa Maria, CA; Salmonella, XLD agar, Hardy Diagnostics; Campylobacter, mCCDA, Oxoid; Enterococcus, mEnterococcus, Becton Dickinson and Co., Sparks MD) and incubated at the appropriate temperature (under microaerobic conditions in the case of Campylobacter) to quantify the concentration of the pathogens. For each pathogen, 10 colonies per collection time point and steer will be saved at -80°C for future characterizations, including analysis of resistance. For susceptibility determination, colonies will be re-cultured on non-selected media (e.g. Columbia blood agar plates for E. coli and Enterococcus, Mueller Hinton Agar plates for Campylobacter) and incubated for 24h at 35°C or other temperature as appropriate for each pathogen (e.g. 42°C for Campylobacter). Reference strains will be included for quality assurance in each MIC determination. For each archived colony, the MIC ofciprofloxacin or tulathromycin will be determined by broth microdilution according to CLSI standards. The MIC distributions will be compared across treatment groups and to reference strain distributions.Aim 3: Determine the impact of danofloxacin or tulathromycin administration on the fecal microbiota. Our working hypothesis is that both danofloxacin and tulathromycin will cause significant changes in the fecal microbiota, and mathematical modeling will determine a microbiological withdrawal time. Microbiota analysis: Fecal samples used to isolate bacterial pathogens and determine MICs from Aim 2will be used for microbiota analysis. Briefly, microbial DNA will be extracted from fecal samples using the MoBio PowerSoil DNA isolation kit and the V4 region of the 16S rRNA gene will be amplified and sequenced using the Illumina MiSeq sequencing platform. Analysis of the V4 region of the 16S rRNA gene will be done using mothur SOP. The percent relative abundance of bacterial phyla and family members ineach sample will be calculated and compared across all treatment groups. Standard packages in R will be used to create heat maps and principal component analysis (PCA) using analysis of molecular variance (AMOVA) to determine statistical differences between groups.Mathematical prediction of microbiological withdrawal time: Time for fecal concentrations of resistant bacteria or pathogens to decline below that associated with carcass contamination would be an appropriate microbiological withdrawal time analogous to the current meat withdrawal time. In order to estimate microbial withdrawal times, we will use a quantitative microbial risk assessment approach. Firstwe will predict microbial loads associated with different antimicrobial treatments, and then we will adapt the slaughter-processing plant module of a risk assessment to associate the fecal microbial load with the risk of carcass contamination. To predict microbial loads, we will develop a mathematical model that integrates the population dynamics of the bacteria with the PK-PD effects of the drug. We have previously developed similar models to evaluate the effects of ceftiofur on the gut microflora. The differential equations describing the pharmacokinetics will be based on the compartmental model from Aim 1. The bacterial growth submodel will incorporate two subpopulations (susceptible and resistance) with logistic growth and non-linear pharmacodynamics effects represented by a sigmoid function. Mutation or plasmid-mediated resistance will be incorporated depending on the modeled antimicrobial drug. Thedifferential equation based model will be parameterized with the in vivo data collected in this study using maximum likelihood methods. The slaughter and processing plant modules from the risk assessment of public health impact of Escherichia coli O157 in ground beef will be adapted to address antimicrobial resistant bacteria. To determine which factors most influence the predicted microbiological withdrawaltime, we will perform global sensitivity analysis on the model parameters.