*4.3. Effect of Different Types of Flooring Design on the Development of Resistant E. coli*

Up to now, little is known about reducing the development of resistance to antibacterial agents by using different flooring designs simulating varying contact intensity between animals and manure. The development of enrofloxacin and ampicillin resistance in *E. coli* was almost independent of flooring design in the present study. Differences in antibacterial susceptibility of commensal *E. coli* isolates from turkeys depending on flooring design have been previously reported [25]. Chuppava et al. [25] mentioned that flooring design had hardly any effect on the development of resistance against antibacterial agents. Nevertheless, in fully slatted flooring systems, with animals having no contact to their litter, resistance to antibacterial agents still develops in the animals.

In T1, overall, the group with floor heating (G2; average floor temperature in all trials: G1 = 27.0/G2 = 30.5/G3 = 26.5/G4 = 26.0 ◦C) showed a significantly higher number of resistant *E. coli* isolates than the other groups. Previous studies showed that the resistance to antibacterial agents in animals can change when they are kept in a heat stress environment [25,38]. A high amount of enrofloxacin resistant isolates from cloacal swabs in fattening turkeys was already reported by Chuppava et al. [25] in a group with floor heating. Also, in swine, Moro et al. [38] found a significant

increase in resistant *E. coli* isolates in the intestinal flora after the animals had been exposed to heat stress (environmental temperature: 34 ◦C).

A significantly higher prevalence of ampicillin resistance in *E. coli* isolates from excreta material from cloacal swabs and manure samples was found in the entire floor pen with litter (G1) even when no ampicillin had been administered to the animals and the pens and the stable had been tested and found to be free of *Enterobacteriaceae* at the start of the trial. Further genetic analyses were not conducted. Therefore, the reason for this difference remains unknown.

#### *4.4. Natural Resistance to Antibacterial Agents Found in Day-Old Chickens*

Turkey poults in this study had not been previously exposed to antibacterial agents. However, *E. coli* was isolated from day-old chicks' meconium in trial 1. Isolates showed resistance to enrofloxacin (48%) and ampicillin (42%). Similar results were reported in previous studies that found one-day-old chicks to be *E. coli* resistant to enrofloxacin [39] and 100% resistant to ampicillin [25]. It has to be mentioned that also other research groups observed high rates of resistance to antibacterial agents already before treatment as well as in the absence of treatment [40]. A vertical transmission of resistant isolates along the production pyramid can occur [3,41]. Also, contamination in the hatchery environment is possible [42]. Persoons et al. [43] stated that besides management, also hatchery-related factors can influence the occurrence of resistance to antibacterial agents. In newly hatched chicks, the common bacteria in the environment, whether antibacterial susceptible or resistant, colonize the intestines and become part of the intestinal normal microflora. Thus, contamination of chickens via vertical transmission could be a possible explanation for the resistance rates found in our study.

The natural enrofloxacin resistance observed in this present study increased strongly. This increase was higher than after one time treatment, as previously reported [25], despite the absence of antibacterial agent usage (T1). Chuppava et al. [25] suggested, according to their findings, that resistance could be reduced or increased, but not eliminated from the animals even with strict disinfection procedures during the experiment. From literature, it is known that a large number of animals carry resistant *E. coli*. These animals can shed huge numbers of resistant organisms. This could result in a rapid contamination of the other individuals in the same pen and in the stable environment [41]. Resistant bacteria can be ingested by birds from the environment. After entering their gut, these may cause the development of resistant *E. coli.* However, there are several possible mechanisms responsible for the development of quinolone resistance [44].

Therefore, further research is strongly recommended to analyze the genetic basis of resistance in the isolates in order to understand the resistance mechanism's origin, development and transfer.

## **5. Conclusions**

In this study, resistance to enrofloxacin was detected at a very high frequency after treatments with enrofloxacin via drinking water. Therefore, the oral administration of enrofloxacin seems to be associated with a significant increase in the frequency of resistance to enrofloxacin in commensal *E. coli* isolates from turkeys. In addition, prevalence of isolates resistant to ampicillin rose significantly. Resistance to enrofloxacin was not detected when the antibacterial agent substance was indirectly sprayed with water into the environment of fattening turkeys. Flooring structure designs did not directly affect the development of resistance to antibacterial agents, or in groups where the animals had no contact to litter. The existence of resistant *E. coli* isolates in one-day-old birds strongly suggests vertical transmission from parent flocks as one possible explanation.

Furthermore, our results can provide useful information, prompting further studies on quinolone resistance mechanisms in commensal *E. coli* depending on different housing systems. However, we cannot consider all interactions when only one isolate is taken from a sample and then, by way of example, we try to deduce the complexity of the development of resistance. Therefore, research is needed to further investigate possible explanations regarding the mechanism behind the dissemination of enrofloxacin-resistant *E. coli* in fattening turkeys.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-4601/15/9/1993/ s1, Table S1a: Means of enrofloxacin- resistant E. coli isolates from cloacal swab and manure samples from turkeys, Table S1b: Means of ampicillin-resistant E. coli isolates from cloacal swab and manure samples from turkeys.

**Author Contributions:** Conceptualization, C.V. and M.K.; Methodology, B.K., J.M., M.K. and C.V.; Validation, B.C., C.V., B.K. and J.M.; Formal Analysis, B.C., B.K., and C.V.; Investigation, B.C., C.V., A.A.E.-W. and J.M.; Resources, C.V. and M.K.; Data Curation, B.C. and C.V.; Writing-Original Draft Preparation, B.C. and C.V.; Writing-Review & Editing, B.C., C.V., B.K., A.A.E.-W., J.M. and M.K.; Visualization, B.C. and C.V.; Supervision, C.V.; Project Administration, C.V. and B.K.; Funding Acquisition, C.V. and M.K.

**Funding:** This project was supported by funds of the Federal Ministry of Food and Agriculture (BMEL, Germany) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE, Germany) under the innovation support program. This publication was supported by the Deutsche Forschungsgemeinschaft and University of Veterinary Medicine Hannover, Foundation, Germany within the funding program Open Access Publishing.

**Acknowledgments:** We would like to thank Frances Sherwood-Brock for proof-reading the manuscript to ensure correct English.

**Conflicts of Interest:** The authors declare no conflicts of interest.
