Antimicrobial resistance through extended-spectrum beta-lactamases (ESBLs) and transferable (plasmid-encoded) cephamycinases (pAmpCs) represents an increasing problem in human and veterinary medicine. The presence of ESBL-/ pAmpC-producing commensal enterobacteria in farm animals, such as broiler chickens, is considered one possible source of food contamination and could therefore also be relevant for human colonization. Studies on transmission routes along the broiler production chain showed that 1-day-old hatchlings are already affected. In this study, ESBL-/pAmpC-positive broiler parent flocks and their corresponding eggs, as well as various environmental and air samples from the hatchery, were analyzed. The eggs were investigated concerning ESBL-/pAmpC-producing enterobacteria on the outer eggshell surface (before/after disinfection), the inner eggshell surface, and the egg content. Isolates were analyzed concerning their species, their phylogroup in the case of Escherichia coli strains, the respective resistance genes, and the phenotypical antibiotic resistance. Of the tested eggs, 0.9% (n ϭ 560) were contaminated on their outer shell surface. Further analyses using pulsed-field gel electrophoresis showed a relationship of these strains to those isolated from the corresponding parent flocks, which demonstrates a pseudo-vertical transfer of ESBL-/pAmpC-producing enterobacteria into the hatchery. Resistant enterobacteria were also found in environmental samples from the hatchery, such as dust or surfaces which could pose as a possible contamination source for the hatchlings. All 1-day-old chicks tested negative directly after hatching. The results show a possible entry of ESBL-/pAmpC-producing enterobacteria from the parent flocks into the hatchery; however, the impact of the hatchery on colonization of the hatchlings seems to be low.IMPORTANCE ESBL-/pAmpC-producing enterobacteria occur frequently in broilerfattening farms. Recent studies investigated the prevalence and possible transmission route of these bacteria in the broiler production chain. It seemed very likely that the hatcheries play an important role in transmission and/or contamination events. There are only few data on transmission investigations from a grandparent or parent flock to their offspring. However, reliable data on direct or indirect vertical transmission events in the hatchery are not available. Therefore, we conducted our study and intensively investigated the broiler hatching eggs from ESBL-/pAmpCpositive broiler parent flocks as well as the hatchlings and the environment of the hatchery.KEYWORDS antibiotic resistance, Enterobacteriaceae, broiler chicken, hatchery, ESBL, pAmpC, AmpC

The occurrence of extended-spectrum beta-lactamase- (ESBL) and/or AmpC beta-lactamase- (AmpC) producing Enterobacteriaceae in livestock, especially in broiler fattening flocks, has been demonstrated in previous studies. Nevertheless, data on transmission routes of these resistant bacteria into the fattening farms are rare. Therefore, seven broiler fattening flocks were investigated for the occurrence of ESBL-/AmpC-producing Enterobacteriaceae during the course of the fattening period with the special focus on horizontal transmission routes. ESBL-/AmpC-producing Enterobacteriaceae from both individual animals and their housing environment were isolated at different time points and the housing environment was even sampled before the arrival of the chickens. All obtained ESBL-/AmpC-producing Enterobacteriaceae were examined for their bacterial species, Escherichia coli phylogroup, and occurrence of resistance genes. Selected isolates were further analyzed via whole-genome sequencing. All seven investigated flocks were tested positive for ESBL-/AmpC-producing Enterobacteriaceae with widely varying prevalence between the flocks. In one flock, the ESBL-/AmpC-producing Enterobacteriaceae were already detected in the housing environment before the arrival of the animals. In general, among the different types of ESBL-/AmpC-producing Enterobacteriaceae determined E. coli harboring a bla gene was the most frequent. Using whole-genome analyses we observed a horizontal transmission of ESBL-/AmpC-producing Enterobacteriaceae through contaminated housing environment as two flocks consecutively fattened in the same farm harbored closely related ESBL-producing isolates. This demonstrates the influence of a previous fattened flock on the ESBL-/AmpC-status of a following broiler flock and, therefore, the importance of hygiene measures on farm level.

SummaryHigh prevalence numbers of extended‐spectrum beta‐lactamase‐ (ESBL‐)/plasmid‐mediated AmpC beta‐lactamase‐ (pAmpC‐) producing Escherichia coli in broiler chicken and their distribution along the broiler production chain is an ongoing problem in food production. We, therefore, investigated resistant isolates along the broiler production chain to determine whether there is a constantly occurring direct vertical transmission of the ESBL‐/pAmpC‐producing E. coli from the parent flocks to their offspring or not. We, furthermore, analysed the isolates concerning the occurrence of virulence factors and their ability to form biofilms to estimate their potential to effectively colonize broiler chickens and/or persist and survive in the environment of the broiler production facilities. Using whole genome sequencing, we could show that ESBL‐/pAmpC‐producing E. coli were likely transferred in a step‐wise process along the broiler production chain but not directly from the parent flock to the fattening flock with every single batch of offspring chickens. Additionally, resistant E. coli strains showing an extraintestinal pathogenic genotype as well as high numbers of virulence‐associated genes including the production of curli fibres and cellulose have high capabilities to persist and spread in the broiler production chain.

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Applying broiler litter containing extended-spectrum beta-lactamase ( ESBL )–producing Escherichia coli ( E. coli ) to arable land poses a potential risk for humans to get colonized by contact with contaminated soil or vegetables. Therefore, an inactivation of these bacteria before land application of litter is crucial. We performed 2 short-term litter storage trials (one in summer and winter, respectively), each covering a time span of 5 D to investigate the effectiveness of this method for inactivation of ESBL-producing E. coli in chicken litter. Surface and deep litter samples were taken from a stacked, ESBL-positive chicken litter heap in triplicates in close sampling intervals at the beginning and daily for the last 3 D of the experiments. Samples were analyzed quantitatively and qualitatively for ESBL-producing E. coli , total E. coli , and enterococci. Selected isolates were further characterized by whole-genome sequencing ( WGS ). In the depth of the heap ESBL-producing E. coli were detected quantitatively until 72 h and qualitatively until the end of the trial in winter. In summer detection was possible quantitatively up to 36 h and qualitatively until 72 h. For surface litter samples a qualitative detection of ESBL-producing E. coli was possible in all samples taken in both trials. In the deep samples a significant decrease in the bacterial counts of over 2 Log 10 was observed for total E. coli in the winter and for total E. coli and enterococci in the summer. Genetic differences of the isolates analyzed by WGS did not correlate with survival advantage. In conclusion, short-term storage of chicken litter stacked in heaps is a useful tool for the reduction of bacterial counts including ESBL-producing E. coli . However, incomplete inactivation was observed at the surface of the heap and at low ambient temperatures. Therefore, an extension of the storage period in winter as well as turning of the heap to provide aerobic composting conditions should be considered if working and storage capacities are available on the farms.

ESBL-producing Klebsiella pneumoniae (K. pneumoniae) represent an increasing problem both in human and veterinary medicine. As SHV-2 - encoding K. pneumoniae were recently detected in the broiler production we were interested in investigating a possible transmission along the broiler production chain and furthermore, in evaluating their possible impact on human health. Therefore, 41 ESBL-producing K. pneumoniae originating from a parent flock, from the hatcherys' environment during the hatching of that parent flocks' chickens, and from an associated fattening flock were investigated on an Illumina Miseq. Whole genome sequences were analyzed concerning their MLST-type, cgMLST-type, genotypic and phenotypic resistance, plasmid profiles and virulence genes. Irrespective of the origin of isolation all investigated isolates were multi-drug resistant, harbored the same ESBL-gene blaSHV−2, shared the same sequence type (ST3128) and displayed 100% similarity in core genome multilocus sequence typing (cgMLST). In addition, in silico plasmid typing found several Inc/Rep types associated with ESBL-plasmids. Summarizing, identical clones of SHV-2—producing K. pneumoniae were detected in different stages of the industrial broiler production in one out of seven investigated broiler chains. This proves the possibility of pseudo-vertical transmission of multi-resistant human pathogens from parent flocks to hatcheries and fattening flocks. Furthermore, the importance of cross-contamination along the production chain was shown. Although the ESBL-producing K. pneumoniae clone detected here in the broiler production has not been associated with clinical settings so far, our findings present a potential public health threat.

The colonization of broilers with extended-spectrum β-lactamase- (ESBL-) and plasmid-mediated AmpC β-lactamase- (pAmpC-) producing Enterobacteriaceae has been extensively studied. However, only limited data on intervention strategies to reduce the colonization throughout the fattening period are available. To investigate practically relevant management measures for their potential to reduce colonization, a recently published seeder-bird colonization model was used. Groups of 90 broilers (breed Ross 308) were housed in pens under conventional conditions (stocking of 39 kg/m2, no enrichment, water and feed ad libitum). Tested measures were investigated in separate trials and included (I) an increased amount of litter in the pen, (II) the reduction of stocking density to 25 kg/m2, and (III) the use of an alternative broiler breed (Rowan x Ranger). One-fifth of ESBL- and pAmpC- negative broilers (n = 18) per group were orally co-inoculated with two E. coli strains on the third day of the trial (seeder). One CTX-M-15-positive E. coli strain (ST410) and one CMY-2 and mcr-1-positive E. coli strain (ST10) were simultaneously administered in a dosage of 102 cfu. Colonization of all seeders and 28 non-inoculated broilers (sentinel) was assessed via cloacal swabs during the trials and a final necropsy at a target weight of two kilograms (= d 36 (control, I-II), d 47 (III)). None of the applied intervention measures reduced the colonization of the broilers with both the ESBL- and the pAmpC- producing E. coli strains. A strain-dependent reduction of colonization for the ESBL- producing E. coli strain of ST410 by 2 log units was apparent by the reduction of stocking density to 25 kg/m2. Consequently, the tested management measures had a negligible effect on the ESBL- and pAmpC- colonization of broilers. Therefore, intervention strategies should focus on the prevention of ESBL- and pAmpC- colonization, rather than an attempt to reduce an already existing colonization.

ESBL-/AmpC-producing Escherichia coli from organic fertilizers were previously detected on soil surfaces of arable land and might be emitted by wind erosion. To investigate this potential environmental transmission path, we exposed ESBL-/AmpC-positive chicken litter, incorporated in agricultural soils, to different wind velocities in a wind tunnel and took air samples for microbiological analysis. No data exist concerning the airborne tenacity of ESBL-/AmpCproducing E. coli. Therefore, we explored the tenacity of two ESBL/AmpC E. coli strains and E. coli K12 in aerosol chamber experiments at different environmental conditions. In the wind tunnel, ESBL/AmpCproducing E. coli were detected in none of the air samples (n = 66). Non-resistant E. coli were qualitatively detected in 40.7% of air samples taken at wind velocities exceeding 7.3 m s −1. Significantly increased emission of total viable bacteria with increasing wind velocity was observed. In the aerosol chamber trials, recovery rates of airborne E. coli ranged from 0.003% to 2.8%, indicating a low airborne tenacity. Concluding, an emission of ESBL/ AmpC E. coli by wind erosion in relevant concentrations appears unlikely because of the low concentration in chicken litter compared with non-resistant E. coli and their low airborne tenacity, proven in the aerosol chamber trials.