Langzeituntersuchungen von Extended-Spektrum-Beta-Laktamase (ESBL)/AmpC Beta-Laktamase-bildenden Escherichia coli in Schweinemastbetrieben und deren Umgebung

Abstract

Extended-spectrum beta-lactamases (ESBL)/AmpC beta-lactamases-producing Enterobacteriaceae, especially Escherichia coli (E. coli), are dramatically limiting the therapeutic options in today’s medicine. The development and potential spread of these resistant microorganisms amongst farm animals and the potential emission from livestock to the environment are discussed critically. However, there is only little information available on the occurrence of ESBL/AmpC-producing E. coli in German pig farms and their vicinity, especially with regard to their detection over the course of a fattening period and potential paths of transmission. Therefore, the main objectives of this long-term study were to determine the potential sources of emission, the prevalence dynamics and the quantities of ESBL/AmpC-producing E. coli in seven German conventional pig fattening farms over the course of one fattening period, and to simultaneously investigate the surroundings of the pig farms for these resistant bacteria to determine faecal, airborne and other potential emission routes. Samples tested were taken at three different times within one finishing fattening period and included 20 individual faeces samples as well as various samples from the animals’ housing environment inside the barn such as pooled faeces, boot swabs, dust, environmental swabs, barn air, flies and mice faeces. Samples from the surroundings of the pig barns were taken simultaneously to the samples inside and included ground surfaces and ambient air on the up- and downwind side of the barn as well as slurry and digestate from biogas plants. One suspected E. coli colony was randomly selected for each sample, confirmed using the MALDI-TOF method and tested for antimicrobial susceptibility by the disk diffusion method. In addition, PCR and sequencing of resistance genes were performed to determine the presence and type of ESBL/AmpC beta-lactamases genes in these isolates. Moreover, selected E. coli isolates from samples from inside and outside the pig barns were typed by pulsed-field gel electrophoresis (PFGE) analysis to identify the clonal relationship of the isolates. Different detection levels of ESBL/AmpC-producing E. coli were observed at different times of investigation during the fattening period. In individual faeces average detection levels of 45% (63/140), 29% (41/140) and 36% (51/140) at the three sampling times were accompanied by decreasing faecal counts from 2.97 x 104 cfu/g at the first to 2.17 x 103 cfu/g at the third visit (p = 0.000). Moreover, detection frequencies of ESBL/AmpC-producing E. coli in individual faeces samples differed amongst the different pig farms: There were two farms with a continuous high prevalence, three farms with a low prevalence and two farms with prevalences in between. In the animals’ housing environment inside the barn pooled faeces and boot swab samples each showed a detection rate of 47.6% (10/21). 5.9% (4/68) of environmental swabs, 9.5% (6/63) of barn air samples, as well as 25% (3/12) of flies and 33% (1/3) of mice faeces samples, but none of the dust samples tested positive for ESBL/AmpC-producing E. coli. In the vicinity of the pig barns ESBL/AmpC-producing E. coli were detected in 16.1% (14/87) of the examined boot swab samples taken from various ground surfaces and in 6% (2/36) of ambient air samples. The majority of slurry samples (82.4%; 14/17) and three of four samples of digestate from biogas plants were also tested positive for these resistant bacteria. In total 274 E. coli isolates were further analysed by phenotypical and genotypical methods. Using antimicrobial susceptibility testing, 32 E. coli isolates were AmpC- and 224 E. coli isolates were ESBL-positive. By PCR analyses and subsequent sequencing, ESBL or AmpC beta-lactamases genes were detected in 215 of the 274 E. coli isolates. The dominant ESBL gene family detected was blaCTX-M with 97.2% (209/215) blaCTX-M-positive isolates. In addition, a new beta-lactamase encoding gene, blaTEM-206, was found during this study. PFGE analyses proved faecal emission of resistant E. coli as well as a possible distribution via flies. The present study provides novel information about amounts and dynamics of ESBL/AmpC-producing E. coli in the German pig production. Moreover, this is the first systematic study on a potential emission and transmission of ESBL /AmpC-producing bacteria between pig fattening farms and their surroundings. Contaminated slurry presented the major emission source for ESBL/AmpC- producing E. coli in the pig fattening farms. A spread via the airborne route or via different vectors also seems possible, but appears to play a minor role. A potential risk of colonisation for exposed animals or humans cannot be estimated at the moment and needs to be further investigated.