Extended period of selection for antimicrobial resistance due to recirculation of persistent antimicrobials in
Aram F Swinkels1, Bjorn J A Berendsen2, Egil A J Fischer1
1Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Objectives:
Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens.
Methods:
The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism.
Results:
Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations.
Conclusions:
Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.
Insights
Stable antimicrobials like doxycycline and enrofloxacin persist in chicken feces, driving antimicrobial resistance. Unstable amoxicillin showed minimal long-term resistance effects, highlighting the need to consider antimicrobial persistence in livestock risk assessments.
Area of Science:
- Veterinary Pharmacology
- Microbiology
- Environmental Science
Background:
- Antimicrobials are crucial in livestock but can select for resistant bacteria.
- Excreted antimicrobials can persist in the environment, potentially leading to subinhibitory concentrations.
- Coprophagic behavior in animals like chickens can facilitate antimicrobial recirculation.
Purpose of the Study:
- To determine the persistence of amoxicillin, doxycycline, and enrofloxacin in broiler chicken feces.
- To evaluate the impact of these persistent antimicrobials on antimicrobial resistance (AMR) genotypically and phenotypically.
- To inform risk assessments for antimicrobial use in livestock.
Main Methods:
- Four groups of broilers were treated with amoxicillin (unstable), doxycycline (stable), enrofloxacin (stable), or left untreated (control).
- Fecal samples were analyzed for antimicrobial concentrations using LC-MS/MS.
- Antimicrobial resistance was assessed genotypically (shotgun metagenomics) and phenotypically (using Escherichia coli as an indicator).
Main Results:
- Doxycycline and enrofloxacin persisted above minimal selective concentrations (MSC) for up to 37 days post-treatment.
- Doxycycline treatment led to a 52% increase in phenotypic resistance and significant genotypic resistance throughout the trial.
- Enrofloxacin resulted in a non-wild-type E. coli population, with resistance likely due to point mutations.
- Amoxicillin treatment showed significant genotypic resistance only immediately after administration.
Conclusions:
- Stable antimicrobials pose a significant risk for driving antimicrobial resistance in livestock due to their environmental persistence.
- Antimicrobial persistence in feces is a critical factor that should be integrated into the priority classification of antimicrobials used in animal agriculture.
- Risk assessment frameworks for veterinary antimicrobials must account for environmental fate and potential for resistance selection.
