Defining minimal selective concentrations of amoxicillin, doxycycline and enrofloxacin in broiler-derived cecal fermentations by phenotype, microbiome and resistome

Antimicrobial resistance (AMR) is an emerging worldwide problem. Exposure to antimicrobials selects for resistant bacteria which are a health threat for humans and animals. The concentration at which selection for resistant bacteria occurs is often lower than the minimum inhibitory concentration (MIC) and also differs between environments. Defining this minimal selective concentration (MSC) under natural conditions is essential to understand the selective window for resistant bacteria which are exposed to residual antimicrobials in humans, animals and the environment. In this study we estimated the MSCs of three antimicrobial compounds, amoxicillin, doxycycline and enrofloxacin in a complex microbial community by conducting fermentation assays with cecal material derived from broilers. We examined the phenotypic resistance of Escherichia coli, resistome and microbiome after 6 and 30 hours of fermenting in the presence of antimicrobials of interest. The concentrations are 10 to 100 times lower than the epidemiological cut-off values in E. coli for the respective antimicrobials as determined by EUCAST (https://mic.eucast.org/). In contrast to the amoxicillin and doxycycline exposure we could not determine any molecular resistance mechanism in the resistome analysis for the enrofloxacin exposure, likely because they are the result of point mutations. Our findings show at which concentrations there still is selection for AMR bacteria. This knowledge can be used to manage the risk of the emergence of AMR bacteria.