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Resistances against antibiotics represent a growing challenge for both human and veterinarymedicine around the globe. Plasmid-transmitted beta lactamases play a pivotal role in the development of resistances against beta-lactam antibiotics in Enterobacteriaceae. The bacterial synthesis of hydrolases with the capability of cleaving the beta lactam ring and thereby enzymatically inactivating beta lactam antibiotics is one of the most common bacterial antibiotic resistance mechanisms. In this context, a subgroup of beta lactameses termed 'extended spectrum beta lactamases' (ESBL) are of particular relevance, as they are capable of dramatically reducing the efficacy even of modern extended-spectrum cephalosporine und monobactame antibiotics. In recent years, increasing evidence of ESBLs have been described worldwide. Studies have reported of ESBL - producing E. coli, and other enterobacteriaceae; in healthy livestock in Europe .inter alia, in healthy broilers. In addition, resistant bacteria have been found in end products and foods such as meat, fish and raw milk. Due to the limited data available on the presence and potential distribution of 'ESBLs/AmpCs' in humans, animals and foods in Germany, the collaborative project RESET was launched, which is financed by assets from the German Ministry of Education and Research (BMBF). In this project, research groups from the fields of veterinary and human medicine have collected and are continuing to collect (during a second promotion period) substantial amounts of data on the dissemination of ESBLs in the food chain. As one aspect of the RESET collaboration, the main focus of the work presented here was the analysis of the detection frequency of ESBL/AmpC - producing E. coli in conventional broiler fattening farms in Germany throughout the entire fattening period. In particular, it was the aim to achieve new insights into the colonization dynamic of the resistant bacteria as well as their distribution in the broiler fattening farm houses. An additional focus was the analysis of a variety of transmission pathways of ESBL/AmpC- producing Enterobacteriaceae, particularly E. coli, from broiler fattening farms to the surrounding environment or vice versa. In initial investigations, the ESBL/AmpC - status of 16 broiler fattening farms was determined using boot swab samples. Of these 16 farms, seven ESBL/AmpC - positive broiler fattening farms were included in the long-term study on the prevalence and transmission of resistant microorganisms which is presented here. To determine the detection frequencies of ESBL/AmpC - producing E. coli in broiler fattening farms during the course of the fattening period, extensive measurements and sample collections from animals, their surroundings, the stable air and exhaust as well as the stable surroundings were carried out in each of the examined broiler fattening farms on three occasions during the fattening period (beginning, middle and end). Moreover, impingement was used to determine the presence of ESBLproducing E. coli and other microorganisms in the stable air and exhaust in order to identify potential emission pathways of ESBL/AmpC-producing Enterobacteriaceae, particularly E. coli, from animal farms into the environment. In doing so, the air samples from outside animal housings as well as the floor samples from the direct stable surroundings were collected in dependence of the respective wind direction. In addition, slurry samples were collected directly from the slurry pit and examined for ESBL/AmpC - E. coli. All suspected ESBL/AmpC- producing colonies isolated from MacConkey agar, containing antibiotics, gathered from these samples were identified as E. coli using MALDI-TOF and subsequently confirmed as carriers of an ESBL gene or plasmid-coded AmpC gene using PCR. In addition, selected isolates from the stable surroundings and the stable were analyzed for clonal relation using pulsed field gel electrophoresis (PFGE).
As a first result it should be emphasized that, at every sampling occasion, there was at least one sample from each broiler fattening farm where ESBL/AmpC-producing E. coli were identified and their resistance genes could be identified using PCR. Moreover, the resistant microorganisms were regularly found in the stable air, e.g. in dust or litter, as well as on the floor of the stable surroundings, the slurry and the stable air and exhaust. The detection frequencies of ESBL/AmpC-producing E. coli in animals, from each 20 swab samples from individual animals per farm varied between 0% and 100%. Nevertheless, the results of our work show that ESBL/AmpC-producing E. coli are regularly present in cloacal swabs of clinically healthy broilers as well as their stable surroundings. In addition, high prevalences of the resistant microorganisms could even be found in day-old chicks. Moreover, our results are the first report of a significant increase of the detection frequency of resistant bacteria during the course of the fattening period. This increase in prevalence was shown during the whole fattening period in samples from individual animals as well as in samples from the animal's surroundings. Therefore, apart from the possible entry via positive day-old chicks, the current results demonstrate an influence of the stable environment on the ESBL/AmpC status of a broiler flock. As a further main result, these work strengthen the hypothesis of fecal and aerogenic dissemination from broiler farms into the environment, as PFGE analyses showed clonal matching between different isolates from inside and outside the animal housings. PFGE shows results of restriction profile analyses with 86.3% to 100% clonal matching of isolates from the stables (air and slurry) in comparison to isolates from the stable surroundings. Based on these results, it can be concluded that fecal and aerogenic emission of ESBL/AmpCproducing E. coli from broiler fattening farms through, among others, distribution of contaminated slurry and stable exhaust is highly probable. However, the results of this work do not exclude that there may also be an influx of the resistant microorganisms.