Antimicrobial resistance (AMR) is a global threat to both human and veterinary medicine leading to recommendations to use lower tier antimicrobials such as florfenicol, a common antibiotic used in cattle. Our objective was to quantify co-selection of enteric AMR bacteria in cattle treated with two labeled dosing regimens of florfenicol. In addition, we wanted to gain insight on the gastrointestinal pharmacokinetics of florfenicol and how it correlates with AMR. We hypothesized the prevalence of AMR isolates would be higher and persist longer in the steers administered the repeated, lower dose of florfenicol. We also hypothesized that the gastrointestinal pharmacokinetics of florfenicol for the lower, repeated dose would be below a therapeutic level for a longer period of time. Twelve 6 mo old steers underwent gastrointestinal surgery to facilitate placement of an ultrafiltration probe in the ileum and colon. An interstitial probe was placed in the region of the withers. Jugular vein catheterization allowed for blood to be collected. Ultrafiltrate, interstitial fluid and blood were collected for the pharmacokinetic analysis. Twenty-four after surgery, the steers were either administered 20 mg/kg florfenicol intramuscularly (IM) (n=6) 48 hr apart or 40 mg/kg florfenicol subcutaneously (SC) (n=6) once. Feces was collected manually until day 38. To determine the pharmacokinetic parameters of florfenicol administration, HPLC with UV detection will be utilized. To assess the phenotypic resistance changes over time, two different methods were used. Feces was serially diluted and plated on selective media to ensure growth of E. coli and Enterococcus. The pure colonies were grown on blood agar plates and used on gram positive and gram negative NARMs plates to assess a wide variety of resistance to antibiotics. In addition, the serially diluted feces were plated on either plain MacConkey, plain Enterococcus or MacConkey or Enterococcus plates infused with either tetracycline (16 ug/mL), ampicillin (32 ug/mL, E. coli; 16 ug/mL, Enterococcus), or ceftiofur (8 ug/mL) in triplicate. After incubation, plates were counted and averaged to determine the concentration (CFU/g) of either E. coli or Enterococcus at each time point. To determine the prevalence AMR isolates, the log CFU/g of the resistant isolates was divided by the log CFU/g of wild type isolates at each time point. These were averaged across steers at each time to compare over time within the dosing group and between the dosing groups using student T-tests with appropriate Bonferroni correction to account for multiple testing. Pharmacokinetic analysis will be completed using Phoenix to determine pharmacokinetic parameters
There was no significant difference from baseline log growth for E. coli or Enterococcus in either dosing group. Based upon the antibiotic infused media, there was a significant increase in log growth of tetracycline resistant E. coli compared to baseline in the IM group (p= 0.007). No other comparisons were significant. We anticipate that these findings will provide guidance to veterinarians on the most appropriate dosing regimen of florfenicol in cattle in order to minimize selection of AMR enteric bacteria.microbial resistance (AMR) is a global threat to both human and veterinary medicine leading to recommendations to use lower tier antimicrobials such as florfenicol, a common antibiotic used in cattle. Our objective was to quantify co-selection of enteric AMR bacteria in cattle treated with two labeled dosing regimens of florfenicol. In addition, we wanted to gain insight on the gastrointestinal pharmacokinetics of florfenicol and how it correlates with AMR. We hypothesized the prevalence of AMR isolates would be higher and persist longer in the steers administered the repeated, lower dose of florfenicol. We also hypothesized that the gastrointestinal pharmacokinetics of florfenicol for the lower, repeated dose would be below a therapeutic level for a longer period of time. Twelve 6 mo old steers underwent gastrointestinal surgery to facilitate placement of an ultrafiltration probe in the ileum and colon. An interstitial probe was placed in the region of the withers. Jugular vein catheterization allowed for blood to be collected. Ultrafiltrate, interstitial fluid and blood were collected for the pharmacokinetic analysis. Twenty-four hours after surgery, the steers were either administered 20 mg/kg florfenicol intramuscularly (IM) (n=6) 48 hr apart or 40 mg/kg florfenicol subcutaneously (SC) (n=6) once. Feces was collected manually until day 38. To determine the pharmacokinetic parameters of florfenicol administration, HPLC with UV detection will be utilized. To assess the phenotypic resistance changes over time, two different methods were used. Feces was serially diluted and plated on selective media to ensure growth of E. coli and Enterococcus. The pure colonies were grown on blood agar plates and used on gram positive and gram negative NARMs plates to assess a wide variety of resistance to antibiotics. In addition, the serially diluted feces were plated on either plain MacConkey, plain Enterococcus or MacConkey or Enterococcus plates infused with either tetracycline (16 ug/mL), ampicillin (32 ug/mL, E. coli; 16 ug/mL, Enterococcus), or ceftiofur (8 ug/mL) in triplicate. After incubation, plates were counted and averaged to determine the concentration (CFU/g) of either E. coli or Enterococcus at each time point. To determine the prevalence AMR isolates, the log CFU/g of the resistant isolates was divided by the log CFU/g of wild type isolates at each time point. These were averaged across steers at each time to compare over time within the dosing group and between the dosing groups using student T-tests with appropriate Bonferroni correction to account for multiple testing. Pharmacokinetic analysis will be completed using Phoenix to determine pharmacokinetic parameters There was no significant difference from baseline log growth for E. coli or Enterococcus in either dosing group. Based upon the antibiotic infused media, there was a significant increase in log growth of tetracycline resistant E. coli compared to baseline in the IM group (p= 0.007). No other comparisons were significant. We anticipate that these findings will provide guidance to veterinarians on the most appropriate dosing regimen of florfenicol in cattle in order to minimize selection of AMR enteric bacteria.
