*4.2. Effects of the Development of Resistance to Antibacterial Agents in E. coli by Water Loss Simulation (Indirect Administration)*

To the best of our knowledge, using water loss simulations by spraying the water containing enrofloxacin exclusively into the litter or onto the slatted flooring in the drinking area in order to study the development of resistance to antibacterial agents has not been previously reported. In this study, it was hypothesized that excreted or metabolized enrofloxacin might alone influence the occurrence of resistance to antibacterial agents. However, in the present study, we could not verify the occurrence of enrofloxacin resistance due to spraying water with enrofloxacin directly into the animals' environment.

Earlier reports suggested that the carry-over effect of antibacterial agents like FQ as well as their active metabolites in the stable could foster the development of antibacterial resistance via oral ingestion by animals [27]. However, in the present investigation, we sprayed enrofloxacin containing water directly into the environment. In contrast, in the aforementioned study, subtherapeutic dosages (3% and 10% of the recommended dosage of 10 mg/kg body weight) were directly applied to drinking water for 21 days, which could explain the difference. The active dose may therefore have been significantly lower in our own experiments.

Chuppava et al. [25] stated from their experimental model that removing the animals from contaminated pens after antibiotic treatment might be the reason for the lower percentage of resistant *E. coli* isolates in the observed animals. Changing the environment was assumed to lead to a lower percentage of resistant *E. coli* isolates in manure. A lower exposure to resistant bacteria in manure as well as antibacterial agent residues was discussed as the cause for this observation. Additionally, in poultry, dirty or contaminated litter and other animal management parameters affect the microbial composition of the chicken gastrointestinal tract. This influence can be either directly, by providing a continuous source of bacteria, or indirectly, by influencing the physical condition and defence of the birds [37].
