Extended period of selection for antimicrobial resistance due to persistency of antimicrobials in broilers
Antimicrobials can select for antimicrobial resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable and very persistent, recirculation of sub-inhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens.  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 (non-persistent), doxycycline or enrofloxacin (persistent). Antimicrobials were extracted from the faecal samples and concentrations were measured  by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism.  Up to 37 days after treatment, persistent antimicrobials (doxycycline and enrofloxacin) had  concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the non-persistent (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 and a significant difference (p = 0.05 and p = 0.0001) in genotypic resistance throughout the trial. Furthermore, the enrofloxacin treatment resulted in a complete enrofloxacin-resistant E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations.   Based on our findings, we suggest that persistency of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.