Presence and distribution of extended-spectrum and AmpC beta-lactamases-producing Escherichia coli on poultry farms in Slovenia

Presence and distribution of extended-spectrum and AmpC beta-lactamases-producing Escherichia... SUMMARY The purpose of the study was to investigate the presence, distribution and dynamics of extended-spectrum beta-lactamases (ESBL) and plasmid-mediated AmpC beta-lactamases (AmpC) producing Escherichia coli (E. coli) in meat type poultry and their environment. Three broiler breeder flocks, four broiler flocks originating from these breeders, and four meat-type turkey flocks were included in a longitudinal study. A total of 349 samples were tested. All environmental samples collected before placement of birds on the farms were negative for ESBL- and AmpC-producing E. coli. However, during the observation period ESBL-producing E. coli were detected in 41 out of 186 samples (22.04%), while AmpC-producing isolates were identified in 10.75% of samples. Resistant strains belonged predominantly to E. coli phylogenetic group A1 (36.2%), followed by group D1 (24.1%), B1 (22.4%) and D2 (10.3%). The only genes detected were blaSHV-12, blaCMY-2 and blaCTX-M. No significant differences were seen in the rates of detection of resistant strains in feces (18.84%), air samples (31.42%) or organs of dead birds (20.73%), which suggested that air samples can be successfully used as an indicator for the identification of positive flocks in order to prevent or reduce cross contaminations in slaughter houses. DESCRIPTION OF THE PROBLEM Antimicrobial resistance continues to be a global concern in veterinary and human medicine. Broad-spectrum beta-lactamases-producing Enterobacteriaceae, in particular Escherichia coli (E. coli), have been detected increasingly in humans and other animals [1, 2]. The production of extended-spectrum beta-lactamases (ESBL) and plasmid-mediated AmpC beta-lactamases (AmpC) are 2 mechanisms of bacterial resistance that have recently received increasing attention. The presence of ESBL confers resistance to most beta-lactam antibiotics, including ceftazidime, ceftiofur, and aztreonam, but they can be inactivated by beta-lactamase inhibitors such as clavulanic acid. AmpC beta-lactamases hydrolyze third-generation cephalosporins and cephomycins and are not inhibited by clavulanic acid or other inhibitors. In Europe, cephalosporins are not allowed to be use in poultry, but ampicillin and amoxicillin are very often used for treating various bacterial diseases of poultry [3]. ESBL- and AmpC-producing isolates are now being found in increasing numbers in animals produced for food, in retail meats and in pets [1, 2]. Although it is not clear how ESBL/AmpC-producing bacteria colonize humans, some studies have suggested that it takes place via other animals [4]. A high prevalence of ESBL/AmpC-producing E. coli strains has been reported in almost all species of commercially reared poultry, poultry meat, and poultry house surroundings [5–9]. Slovenia is a relatively small producer of poultry meat; the number of birds slaughtered per year is approximately 35 million. A survey performed on chicken meat in 2013 showed that more than 50% of poultry meat and meat products taken at slaughter were positive for ESBL-producing E. coli, thus constituting a serious issue for public health [10]. No data on the prevalence of ESBL- or AmpC-producing E. coli in Slovenian meat-type and layer flocks are available. The purpose of this study was to investigate the presence, distribution, and dynamics of ESBL/AmpC-producing E. coli in meat-type poultry and their environment. Additionally, resistance gene families were identified, and the E. coli were assigned to phylogenetic groups in order to gain basic insights into the variability of ESBL/AmpC isolates. MATERIALS AND METHODS Flocks Included in the Study Three broiler breeder, 4 broiler and 4 meat-type turkey flocks owned by the 3 biggest poultry-producing companies were included in this study. Before placement of birds, all poultry houses were cleaned and disinfected. Broiler Breeders Three broiler breeder flocks (designated A, B, and C) from different owners were sampled. Chicks from all the flocks were imported as 1-d olds. The farms were located at various geographic regions throughout Slovenia. Two poultry houses on farms A and B and 1 house on farm C were investigated. Birds stayed on the rearing farm until they were 19 wk old. They were then moved to a production farm in such a way that all hens from a selected barn were placed in one barn on the production farm. Flock sizes ranged from 19,143 to 29,040 animals. All flocks were vaccinated using similar immunoprophylactic programs against viral diseases and Salmonella, the difference being in the protocol against coccidiosis and E. coli. Flocks A and B were vaccinated against coccidiosis with live attenuated vaccines, while birds from flock C received coccidiostats (salinomycin sodium salt) until 14 wk old. Birds from flock B were treated at the end of the first week with sulfamonomethoxine, with trimethoprim at 5 d, and at 21 wk with oxytetracycline for 3 d. Flocks A and C were not treated with antibacterial drugs during the observation period of 36 wk. Broilers Four broiler flocks derived at 1 d of age from 26-wk-old breeders were investigated. Two flocks were derived from breeder flock A (designated flocks A/1 and A/2) and 2 flocks from broiler breeder flock B (designated flocks B/1 and B/2). Flocks were placed on 4 farms at different locations. The number of birds in each flock ranged from 7,000 to 11,000 birds. All broiler flocks received coccidiostats until 5 d before slaughter. The fattening period was between 36 and 42 d. No antibiotic treatment was needed during the fattening period in any of the sampled broiler flocks. Meat-Type Turkeys Four meat-type turkey farms were investigated (designated D, E, F, and G). Locations of the farms were chosen at random throughout the country. All 1-d-old turkeys were transported and placed into clean and disinfected barns. The sizes of the flocks ranged from 4,000 to 4,100 birds and all flocks received coccidiostats until 12 wk old. Respiratory disorders and higher mortality occurred at day 112 in the flock G and birds were treated with enrofloxacin for 5 d. In flocks D, E, and F, no antibacterial treatment was needed. Sampling Procedures Environmental samples were collected on all farms before the day-old chicks arrived. For collecting environmental swabs, premoistened 3 M Enviro Swabs [11] were used. At least 10 swabs of the barn walls, floor, and equipment were taken by swabbing a surface of approximately 15 cm2. In addition, 1 sample of water (1 L), litter (1 kg), and feed (1 kg) was taken for each flock. Birds that died during transport were collected on arrival at the farms. Necropsies were performed and organs were taken aseptically for bacteriological examination. Meconium taken from the paper of transport boxes was also sampled. Dead or culled birds from broiler breeder flocks were collected at weeks 4, 12, 24, and 36. In broiler flocks, dead birds were collected at weeks 1 and 5, and in turkey flocks at weeks 1, 5, 9, and 16. At each time point, at least 4 birds were necropsied and livers were taken for bacteriological examination. Samples were pooled according to flock and time of sampling; each pool contained at least 4 liver samples. When other organs were affected (e.g., fibrinous pericarditis, oophoritis, cellulitis, inflammation of tarsometatarsal joints), each tissue sample was taken separately. At the same time, point fecal samples were collected by boot swabs after walking the whole length of the poultry house. In each broiler breeder house, 5 pairs of boot swabs were collected; in broiler and turkey houses at least 2 pairs of boot swabs were collected and pooled. Air samples were collected in broiler breeder houses at weeks 4 and 12, in broiler houses at weeks 1 and 5, and in turkey houses at weeks 1 and 9. Air samples were taken using a Coriolis Delta air sampler [12]. The sampler aspirated of 100 L of air per minute and transferred the airborne particles into a liquid medium by centrifugal vortex of air. Phosphate-buffered saline (15 mL) was used to collect the samples. The sampler was run for 10 min collecting 1000 L of air particles for each sample. A total of 349 samples were tested; 148 were obtained from broiler breeder flocks (Supplemental Table 1), 85 from broilers (Supplemental Table 2), and 116 from meat-type turkey flocks (Supplemental Table 3). Microbiological Analysis All samples were processed within 24 h of sampling. In the meantime, they were kept at 4°C. Fecal samples were first diluted 1:10 in buffered peptone water [13], all other samples were plated directly onto chromogenic medium [14] and incubated at 37°C for 24 h. Suspect colonies were identified by matrix-assisted laser desorption/ionization, time of flight (MALDI-TOF) mass spectrometry [15]. All E. coli strains were subcultured onto blood agar [16] and stored at –80°C for further testing. Phenotypic Detection of ESBL- and AmpC-Producing Isolates All samples were enriched in BPW at 37°C for 18–22 h before plating onto ESBL Chrom ID agar [17]. Based on colony morphology and appearance (presumptive ESBL/AmpC-producing E. coli colonies were colored purple/red) up to 3 colonies were subcultured by restreaking onto a fresh blood agar plate and incubated at 37°C for 18–22 h. The species of the isolates was confirmed by MALDI-TOF [18]. Presumptive ESBL/AmpC-producing E. coli isolates were tested for antimicrobial susceptibility using the microdilution method Sensititre™ [19]. All isolates were tested using the primary EUVSEC panel, containing essential first line antimicrobials. If the isolate was resistant to cefotaxime and/or ceftazidime, it was further tested using the second panel (EUVSEC2). This panel includes a cefoxitin, cefepime, and clavulanate synergy test in combination with cefotaxime and ceftazidime for detection of ESBL and AmpC production. The results were interpreted according to the protocol of the European Union Reference Laboratory for Antimicrobial Resistance based on EUCAST epidemiological cut-off values and clinical resistance breakpoints [20]. Molecular Confirmation of ESBL/AmpC-Producing Isolates and Identification of Resistance Gene Families All phenotypically confirmed ESBL/AmpC-producing isolates were further tested by PCR. DNA was released from bacterial cells by boiling [21]. All isolates were screened with multiplex PCR using primers specific for blaCTX-M groups as described by Woodford [22]. Briefly, each reaction mixture contained 2 μL of the bacterial lysate, 10 pg of each primer (blaCTX-M group 1F, blaCTX-M group 1R, blaCTX-M group 2F, blaCTX-M group 2R, blaCTX-M group 8F, blaCTX-M group 8R, blaCTX-M group 9F, blaCTX-M group 9R, blaCTX-M group 25F, blaCTX-M group 25R), 12,5 μL of DreamTaq PCR Master Mix [23], and 0.5 μL of sterile nuclease free water [23]. The cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, primer annealing at 52°C for 25 s, elongation at 72°C for 50 s, and the final elongation step for 6 min at 72°C. Genes blaTEM and blaSHV were first screened for with primer pairs blaTEM-F/blaTEM-R and blaSHV-F/blaSHV-R employing the protocol explained in detail by Dallenne et al. [24]. The blaTEM-R and blaSHV-F genes from positive isolates were subsequently multiplied for sequencing using primer pairs TEM seqF/TEM seqR and SHV seqF/SHV seqR [25] and the following cycling protocol: initial denaturation at 95°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, elongation at 72°C for 1.5 min, and the final elongation for 10 min at 72°C. For the detection of AmpC genes encoding beta-lactamases MOX, CMY, LAT, DHA, ACC, MIR, ACT, and FOX primers described by Perez-Perez and Hanson [26] were employed. The single PCR mixtures, with a total volume of 25 μL, which contained 10 pg of each primer, 12.5 μL of DreamTaq PCR Master Mix and sterile nuclease free water, were subjected to 30 cycles of denaturation at 94°C for 30 s, primer annealing at 64°C for 30 s and primer extension at 72°C for 1 min. The initial denaturation was performed at 95°C for 5 min and the final extension step for 10 min at 72°C. In positive isolates (only for CMY), the blaCMY gene was again amplified using primers CMY2seqF/CMY2seqR according Dierikx et al. [27]. Amplifications were done in a total volume of 50 μL, containing 3 μL of the bacterial lysate, 25 μL of DreamTaq PCR Master Mix, 20 pg of each primer and sterile nuclease free water. Initial denaturation was set at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, primer annealing at 58°C for 30 s, primer extension at 72°C for 1.5 min and completed with the final elongation step for 7 min at 72°C. All PCR sequencing reactions were performed at Macrogen [28] on our request. Primers, product sizes, and references are listed in Table 1. Table 1. Oligonucleotide Primers Used for PCR Amplification, PCR Product Size, and References. Primer name  Primer sequence  Size of PCR product (bp)  Reference  blaCTX-M group 1F  5΄-AAA AAT CAC TGC GCC AGT T C-3΄  415  [22]  blaCTX-M group 1R  5΄-AGC TTA TTC ATC GCC ACG TT-3΄      blaCTX-M group 2F  5΄-CGA CGC TAC CCC TGC TAT T-3΄  552  [22]  blaCTX-M group 2R  5΄-CCA GCG TCA GAT TTT TCA GG-3΄      blaCTX-M group 8F  5΄-TCG CGT TAA GCG GAT GAT GC-3΄  666  [22]  blaCTX-M group 8R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaCTX-M group 9F  5΄-CAA AGA GAG TGC AAC GGA TG-3΄  205  [22]  blaCTX-M group 9R  5΄-ATT GGA AAG CGT TCA TCA CC-3΄      blaCTX-M group 25F  5΄-GCA CGA TGA CAT TCG GG-3΄  327  [22]  blaCTX-M group 25R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaTEM-F  5΄-CAT TTC CGT GTC GCC CTT ATT C-3΄  800  [24]  blaTEM-R  5΄-CGT TCA TCC ATA GTT GCC TGA C-3΄      blaSHV-F  5΄-AGC CGC TTG AGC AAA TTA AA C-3΄  713  [24]  blaSHV-R  5΄-ATC CCG CAG ATA AAT CAC CAC-3΄      TEM seqF  5΄-ATT CTT GAA GAC GAA AGG GC-3΄  1150  [25]  TEM seqR  5΄-ACG CTC AGT GGA ACG AAA AC-3΄      SHV seqF  5΄-CAC TCA AGG ATG TAT TGT G-3΄  885  [25]  SHV seqR  5΄- TTA GCG TTG CCA GTG CTC G-3΄      MOXMF  5΄- GCT GCT CAA GGA GCA CAG GAT-3΄  520  [26]  MOXMR  5΄-CAC ATT GAC ATA GGT GTG GTG C-3΄      CITMF  5΄-TGG CCA GAA CTG ACA GGC AAA-3΄  462  [26]  CITMR  5΄-TTT CTC CTG AAC GTG GCT GGC-3΄      DHAMF  5΄-AAC TTT CAC AGG TGT GCT GGG T-3΄  405  [26]  DHAMR  5΄-CCG TAC GCA TAC TGG CTT TGC-3΄      ACCMF  5΄-AAC AGC CTC AGC AGC CGG TTA-3΄  346  [26]  ACCMR  5΄-TTC GCC GCA ATC ATC CCT AGC-3΄      EBCMF  5΄-TCG GTA AAG CCG ATG TTG CGG-3΄  302  [26]  EBCMR  5΄-CTT CCA CTG CGG CTG CCA GTT-3΄      FOXMF  5΄-AAC ATG GGG TAT CAG GGA GAT G-3΄  190  [26]  FOXMR  5΄-CAA AGC GCG TAA CCG GAT TGG-3΄      CMY2seqF  5΄-ATG ATG AAA AAA TCG TTA TGC TGC-3΄  1117  [27]  CMY2seqR  5΄-GCT TTT CAA GAA TGC GCC AGG-3΄      ChuA-F  5΄-GACGAACCAACGGTCAGGAT -3΄  279  [33]  ChuA-R  5΄-TGCCGCCAGTACCAAAGACA-3΄      YjA-F  5΄-TGAAGTGTCAGGAGACGCTG-3΄  211  [36]  YjA-R  5΄-ATGGAGAATGCGTTCCTCAAC-3΄      TspE4.C2-F  5΄-GAGTAATGTCGGGGCATTCA-3΄  152  [37]  TspE4.C2-R  5΄-CGCGCCAACAAAGTATTACG-3΄      Primer name  Primer sequence  Size of PCR product (bp)  Reference  blaCTX-M group 1F  5΄-AAA AAT CAC TGC GCC AGT T C-3΄  415  [22]  blaCTX-M group 1R  5΄-AGC TTA TTC ATC GCC ACG TT-3΄      blaCTX-M group 2F  5΄-CGA CGC TAC CCC TGC TAT T-3΄  552  [22]  blaCTX-M group 2R  5΄-CCA GCG TCA GAT TTT TCA GG-3΄      blaCTX-M group 8F  5΄-TCG CGT TAA GCG GAT GAT GC-3΄  666  [22]  blaCTX-M group 8R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaCTX-M group 9F  5΄-CAA AGA GAG TGC AAC GGA TG-3΄  205  [22]  blaCTX-M group 9R  5΄-ATT GGA AAG CGT TCA TCA CC-3΄      blaCTX-M group 25F  5΄-GCA CGA TGA CAT TCG GG-3΄  327  [22]  blaCTX-M group 25R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaTEM-F  5΄-CAT TTC CGT GTC GCC CTT ATT C-3΄  800  [24]  blaTEM-R  5΄-CGT TCA TCC ATA GTT GCC TGA C-3΄      blaSHV-F  5΄-AGC CGC TTG AGC AAA TTA AA C-3΄  713  [24]  blaSHV-R  5΄-ATC CCG CAG ATA AAT CAC CAC-3΄      TEM seqF  5΄-ATT CTT GAA GAC GAA AGG GC-3΄  1150  [25]  TEM seqR  5΄-ACG CTC AGT GGA ACG AAA AC-3΄      SHV seqF  5΄-CAC TCA AGG ATG TAT TGT G-3΄  885  [25]  SHV seqR  5΄- TTA GCG TTG CCA GTG CTC G-3΄      MOXMF  5΄- GCT GCT CAA GGA GCA CAG GAT-3΄  520  [26]  MOXMR  5΄-CAC ATT GAC ATA GGT GTG GTG C-3΄      CITMF  5΄-TGG CCA GAA CTG ACA GGC AAA-3΄  462  [26]  CITMR  5΄-TTT CTC CTG AAC GTG GCT GGC-3΄      DHAMF  5΄-AAC TTT CAC AGG TGT GCT GGG T-3΄  405  [26]  DHAMR  5΄-CCG TAC GCA TAC TGG CTT TGC-3΄      ACCMF  5΄-AAC AGC CTC AGC AGC CGG TTA-3΄  346  [26]  ACCMR  5΄-TTC GCC GCA ATC ATC CCT AGC-3΄      EBCMF  5΄-TCG GTA AAG CCG ATG TTG CGG-3΄  302  [26]  EBCMR  5΄-CTT CCA CTG CGG CTG CCA GTT-3΄      FOXMF  5΄-AAC ATG GGG TAT CAG GGA GAT G-3΄  190  [26]  FOXMR  5΄-CAA AGC GCG TAA CCG GAT TGG-3΄      CMY2seqF  5΄-ATG ATG AAA AAA TCG TTA TGC TGC-3΄  1117  [27]  CMY2seqR  5΄-GCT TTT CAA GAA TGC GCC AGG-3΄      ChuA-F  5΄-GACGAACCAACGGTCAGGAT -3΄  279  [33]  ChuA-R  5΄-TGCCGCCAGTACCAAAGACA-3΄      YjA-F  5΄-TGAAGTGTCAGGAGACGCTG-3΄  211  [36]  YjA-R  5΄-ATGGAGAATGCGTTCCTCAAC-3΄      TspE4.C2-F  5΄-GAGTAATGTCGGGGCATTCA-3΄  152  [37]  TspE4.C2-R  5΄-CGCGCCAACAAAGTATTACG-3΄      View Large Table 1. Oligonucleotide Primers Used for PCR Amplification, PCR Product Size, and References. Primer name  Primer sequence  Size of PCR product (bp)  Reference  blaCTX-M group 1F  5΄-AAA AAT CAC TGC GCC AGT T C-3΄  415  [22]  blaCTX-M group 1R  5΄-AGC TTA TTC ATC GCC ACG TT-3΄      blaCTX-M group 2F  5΄-CGA CGC TAC CCC TGC TAT T-3΄  552  [22]  blaCTX-M group 2R  5΄-CCA GCG TCA GAT TTT TCA GG-3΄      blaCTX-M group 8F  5΄-TCG CGT TAA GCG GAT GAT GC-3΄  666  [22]  blaCTX-M group 8R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaCTX-M group 9F  5΄-CAA AGA GAG TGC AAC GGA TG-3΄  205  [22]  blaCTX-M group 9R  5΄-ATT GGA AAG CGT TCA TCA CC-3΄      blaCTX-M group 25F  5΄-GCA CGA TGA CAT TCG GG-3΄  327  [22]  blaCTX-M group 25R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaTEM-F  5΄-CAT TTC CGT GTC GCC CTT ATT C-3΄  800  [24]  blaTEM-R  5΄-CGT TCA TCC ATA GTT GCC TGA C-3΄      blaSHV-F  5΄-AGC CGC TTG AGC AAA TTA AA C-3΄  713  [24]  blaSHV-R  5΄-ATC CCG CAG ATA AAT CAC CAC-3΄      TEM seqF  5΄-ATT CTT GAA GAC GAA AGG GC-3΄  1150  [25]  TEM seqR  5΄-ACG CTC AGT GGA ACG AAA AC-3΄      SHV seqF  5΄-CAC TCA AGG ATG TAT TGT G-3΄  885  [25]  SHV seqR  5΄- TTA GCG TTG CCA GTG CTC G-3΄      MOXMF  5΄- GCT GCT CAA GGA GCA CAG GAT-3΄  520  [26]  MOXMR  5΄-CAC ATT GAC ATA GGT GTG GTG C-3΄      CITMF  5΄-TGG CCA GAA CTG ACA GGC AAA-3΄  462  [26]  CITMR  5΄-TTT CTC CTG AAC GTG GCT GGC-3΄      DHAMF  5΄-AAC TTT CAC AGG TGT GCT GGG T-3΄  405  [26]  DHAMR  5΄-CCG TAC GCA TAC TGG CTT TGC-3΄      ACCMF  5΄-AAC AGC CTC AGC AGC CGG TTA-3΄  346  [26]  ACCMR  5΄-TTC GCC GCA ATC ATC CCT AGC-3΄      EBCMF  5΄-TCG GTA AAG CCG ATG TTG CGG-3΄  302  [26]  EBCMR  5΄-CTT CCA CTG CGG CTG CCA GTT-3΄      FOXMF  5΄-AAC ATG GGG TAT CAG GGA GAT G-3΄  190  [26]  FOXMR  5΄-CAA AGC GCG TAA CCG GAT TGG-3΄      CMY2seqF  5΄-ATG ATG AAA AAA TCG TTA TGC TGC-3΄  1117  [27]  CMY2seqR  5΄-GCT TTT CAA GAA TGC GCC AGG-3΄      ChuA-F  5΄-GACGAACCAACGGTCAGGAT -3΄  279  [33]  ChuA-R  5΄-TGCCGCCAGTACCAAAGACA-3΄      YjA-F  5΄-TGAAGTGTCAGGAGACGCTG-3΄  211  [36]  YjA-R  5΄-ATGGAGAATGCGTTCCTCAAC-3΄      TspE4.C2-F  5΄-GAGTAATGTCGGGGCATTCA-3΄  152  [37]  TspE4.C2-R  5΄-CGCGCCAACAAAGTATTACG-3΄      Primer name  Primer sequence  Size of PCR product (bp)  Reference  blaCTX-M group 1F  5΄-AAA AAT CAC TGC GCC AGT T C-3΄  415  [22]  blaCTX-M group 1R  5΄-AGC TTA TTC ATC GCC ACG TT-3΄      blaCTX-M group 2F  5΄-CGA CGC TAC CCC TGC TAT T-3΄  552  [22]  blaCTX-M group 2R  5΄-CCA GCG TCA GAT TTT TCA GG-3΄      blaCTX-M group 8F  5΄-TCG CGT TAA GCG GAT GAT GC-3΄  666  [22]  blaCTX-M group 8R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaCTX-M group 9F  5΄-CAA AGA GAG TGC AAC GGA TG-3΄  205  [22]  blaCTX-M group 9R  5΄-ATT GGA AAG CGT TCA TCA CC-3΄      blaCTX-M group 25F  5΄-GCA CGA TGA CAT TCG GG-3΄  327  [22]  blaCTX-M group 25R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaTEM-F  5΄-CAT TTC CGT GTC GCC CTT ATT C-3΄  800  [24]  blaTEM-R  5΄-CGT TCA TCC ATA GTT GCC TGA C-3΄      blaSHV-F  5΄-AGC CGC TTG AGC AAA TTA AA C-3΄  713  [24]  blaSHV-R  5΄-ATC CCG CAG ATA AAT CAC CAC-3΄      TEM seqF  5΄-ATT CTT GAA GAC GAA AGG GC-3΄  1150  [25]  TEM seqR  5΄-ACG CTC AGT GGA ACG AAA AC-3΄      SHV seqF  5΄-CAC TCA AGG ATG TAT TGT G-3΄  885  [25]  SHV seqR  5΄- TTA GCG TTG CCA GTG CTC G-3΄      MOXMF  5΄- GCT GCT CAA GGA GCA CAG GAT-3΄  520  [26]  MOXMR  5΄-CAC ATT GAC ATA GGT GTG GTG C-3΄      CITMF  5΄-TGG CCA GAA CTG ACA GGC AAA-3΄  462  [26]  CITMR  5΄-TTT CTC CTG AAC GTG GCT GGC-3΄      DHAMF  5΄-AAC TTT CAC AGG TGT GCT GGG T-3΄  405  [26]  DHAMR  5΄-CCG TAC GCA TAC TGG CTT TGC-3΄      ACCMF  5΄-AAC AGC CTC AGC AGC CGG TTA-3΄  346  [26]  ACCMR  5΄-TTC GCC GCA ATC ATC CCT AGC-3΄      EBCMF  5΄-TCG GTA AAG CCG ATG TTG CGG-3΄  302  [26]  EBCMR  5΄-CTT CCA CTG CGG CTG CCA GTT-3΄      FOXMF  5΄-AAC ATG GGG TAT CAG GGA GAT G-3΄  190  [26]  FOXMR  5΄-CAA AGC GCG TAA CCG GAT TGG-3΄      CMY2seqF  5΄-ATG ATG AAA AAA TCG TTA TGC TGC-3΄  1117  [27]  CMY2seqR  5΄-GCT TTT CAA GAA TGC GCC AGG-3΄      ChuA-F  5΄-GACGAACCAACGGTCAGGAT -3΄  279  [33]  ChuA-R  5΄-TGCCGCCAGTACCAAAGACA-3΄      YjA-F  5΄-TGAAGTGTCAGGAGACGCTG-3΄  211  [36]  YjA-R  5΄-ATGGAGAATGCGTTCCTCAAC-3΄      TspE4.C2-F  5΄-GAGTAATGTCGGGGCATTCA-3΄  152  [37]  TspE4.C2-R  5΄-CGCGCCAACAAAGTATTACG-3΄      View Large Phylogenetic Group Assignment of ESBL/AmpC Positive E. coli Isolates The phylogenetic group of each isolate (A0, A1, B1, B22, B23, D1, and D2) was determined by triplex PCR [29, 30]. The reaction mixture containing 2 μL of the bacterial lysate, 20 pg of each primer (primer (ChuA-F, ChuA-R, YjA-F, YjA-R, TspE4.C2-F, and TspE4.C2-R), PCR DreamTaq Master mix, and sterile nuclease free water was first denatured at 94°C for 5 min and then subjected to 30 PCR cycles as follows: denaturation at 94°C for 30 s, primer annealing at 60°C for 30 s, and elongation at 72°C for 30 s. The final elongation step was set for 7 min at 72°C. Statistical Analysis To determine the influence of the antibacterial treatment on the presence of ESBL/AmpC-producing E. coli in broiler breeder flocks, a Chi-square test and a Z test with Bonferroni's correction were used. The similarity of proportion between the number of ESBL- and AmpC-producing E. coli isolates and different types of samples was tested using Fisher's exact test. For those analyses, the Statistical Package for the Social Sciences was utilized [31]. RESULTS AND DISCUSSION Eleven poultry flocks owned by different companies and from different locations were included in our investigation. ESBL/AmpC-producing E. coli strains were isolated from all breeder and broiler flocks, as well as from 2 turkey flocks (Tables 3–5). Contamination of barns, due to insufficient cleaning and disinfection, could be one of the causes for the high incidence of ESBL/AmpC-producing isolates [6, 32]. In our study, ESBL/AmpC-producing E. coli were not detected in samples taken from the farm environment before the start of production. In addition, all samples of feed, litter, and water also tested negative for ESBL/AmpC-producing E. coli (Table 2). The proportion of these samples containing E. coli was very low (9.2%), suggesting effective implementation of good hygiene management on the poultry farms. Table 2. The Prevalence of Escherichia coli in Environmental Samples Taken Before Placement of Chicks on Farms.   Number of positive samples/number of samples tested  Sample type  Broiler breeders  Broilers  Meat-type turkeys  Total (%1)  Swabs  3/50  6/40  3/35  12/125 (9.60)  Feed  2/5  0/4  0/4  2/13 (15.38)  Litter  1/5  0/4  0/4  1/13 (11.12)  Water  0/5  0/4  0/3  0/12 (0.0)  Total (%1)  6/65 (9.23)  6/52 (11.53)  3/46 (6.52)  15/163 (9.20)    Number of positive samples/number of samples tested  Sample type  Broiler breeders  Broilers  Meat-type turkeys  Total (%1)  Swabs  3/50  6/40  3/35  12/125 (9.60)  Feed  2/5  0/4  0/4  2/13 (15.38)  Litter  1/5  0/4  0/4  1/13 (11.12)  Water  0/5  0/4  0/3  0/12 (0.0)  Total (%1)  6/65 (9.23)  6/52 (11.53)  3/46 (6.52)  15/163 (9.20)  1Percentage of positive samples. View Large Table 2. The Prevalence of Escherichia coli in Environmental Samples Taken Before Placement of Chicks on Farms.   Number of positive samples/number of samples tested  Sample type  Broiler breeders  Broilers  Meat-type turkeys  Total (%1)  Swabs  3/50  6/40  3/35  12/125 (9.60)  Feed  2/5  0/4  0/4  2/13 (15.38)  Litter  1/5  0/4  0/4  1/13 (11.12)  Water  0/5  0/4  0/3  0/12 (0.0)  Total (%1)  6/65 (9.23)  6/52 (11.53)  3/46 (6.52)  15/163 (9.20)    Number of positive samples/number of samples tested  Sample type  Broiler breeders  Broilers  Meat-type turkeys  Total (%1)  Swabs  3/50  6/40  3/35  12/125 (9.60)  Feed  2/5  0/4  0/4  2/13 (15.38)  Litter  1/5  0/4  0/4  1/13 (11.12)  Water  0/5  0/4  0/3  0/12 (0.0)  Total (%1)  6/65 (9.23)  6/52 (11.53)  3/46 (6.52)  15/163 (9.20)  1Percentage of positive samples. View Large In Slovenia, all breeder flocks and meat-type turkeys are imported as day-old chicks. As some reports indicated that vertical or pseudo-vertical transmission may play an important role in the spread of ESBL/AmpC-producing E. coli [7, 33, 34], the second step was to investigate the presence of such strains in day-old chicks before placement on farms. Except 1 broiler breeder flock that was positive for AmpC-producing E. coli, ESBL/AmpC-producing E. coli were not detected in meconium or organ samples from other broiler breeders or turkey flocks. AmpC-producing E. coli were detected in meconium and 1 pooled liver sample from flock A (Tables 3 and 5). In broilers, ESBL/AmpC-producing E. coli was not detected in day-old chicks, although they were hatched from parent flocks from which ESBL/AmpC-producing E. coli had been isolated (Tables 3 and 4). Experience elsewhere suggests that the presence of ESBL-producing E. coli in day-old chicks will always result in subsequent isolation of these E. coli from the flock, while a flock can be negative when the chicks are placed and then become positive and subsequently negative again [7]. Table 3. Frequencies, phylogenetic group, and Extended-Spectrum Beta-Lactamases (ESBL)/AmpC resistance genes of Escherichia coli isolates obtained from different samples in broiler breeder flocks. Sampling    Flock A  Flock B  Flock C  Time  Sample type  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  Before housing  Meconium1  2/2  0/2  2/2a  0/2  0/2  0/2  1/2  0/2  0/2    Organs2  4/4  0/4  1/4a (Liver)  0/2  0/2  0/2  2/2  0/2  0/2  4 wk  Air3  3/3  3/3b  0/3  2/2  0/2  2/2a  2/2  0/2  0/2    Boot swabs1  2/2  0/2  0/2  2/2  1/2c  1/2d  2/2  0/2  0/2    Organs2  2/2  2/2b (Liver)  0/2  2/2  2/2c (Liver)  0/2  1/1  0/1  0/1  12 wk  Air3  2/2  1/2e  0/2  2/2  2/2f  2/2f  2/4  0/4  1/4g    Boot swabs1  2/2  0/2  0/2  2/2  2/2h  0/2  2/2  0/2  0/2    Organs2  2/2  2/2e,i (Liver)  0/2  2/2  1/2c (Liver)  0/2  2/2  0/2  1/2g  24 wk  Boot swabs1  2/2  2/2i,j  1/2j  2/2  2/2c  0/2  2/2  0/2  0/2    Organs2  2/2  1/2a (Liver)  0/2  4/4  1/4c (Liver)  0/4  3/4  0/4  0/4  36 wk  Boot swabs1  2/2  0/2  0/2  2/2  1/2k  0/2  3/3  0/3  1/3g    Organs2  2/2  0/2  0/2  3/4  0/4  2/4g (Liver, tarsometatarsal joints)  1/2  0/2  0/2  Total (%)    27/27 (100%)  11/27x (40.07%)  4/27z (14.81%)  23/28 (28.14%)  12/28x (42.86%)  7/28z (25.00%)  23/28 (82.14%)  0/28y (0.00%)  3/28z (10.71%)  Sampling    Flock A  Flock B  Flock C  Time  Sample type  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  Before housing  Meconium1  2/2  0/2  2/2a  0/2  0/2  0/2  1/2  0/2  0/2    Organs2  4/4  0/4  1/4a (Liver)  0/2  0/2  0/2  2/2  0/2  0/2  4 wk  Air3  3/3  3/3b  0/3  2/2  0/2  2/2a  2/2  0/2  0/2    Boot swabs1  2/2  0/2  0/2  2/2  1/2c  1/2d  2/2  0/2  0/2    Organs2  2/2  2/2b (Liver)  0/2  2/2  2/2c (Liver)  0/2  1/1  0/1  0/1  12 wk  Air3  2/2  1/2e  0/2  2/2  2/2f  2/2f  2/4  0/4  1/4g    Boot swabs1  2/2  0/2  0/2  2/2  2/2h  0/2  2/2  0/2  0/2    Organs2  2/2  2/2e,i (Liver)  0/2  2/2  1/2c (Liver)  0/2  2/2  0/2  1/2g  24 wk  Boot swabs1  2/2  2/2i,j  1/2j  2/2  2/2c  0/2  2/2  0/2  0/2    Organs2  2/2  1/2a (Liver)  0/2  4/4  1/4c (Liver)  0/4  3/4  0/4  0/4  36 wk  Boot swabs1  2/2  0/2  0/2  2/2  1/2k  0/2  3/3  0/3  1/3g    Organs2  2/2  0/2  0/2  3/4  0/4  2/4g (Liver, tarsometatarsal joints)  1/2  0/2  0/2  Total (%)    27/27 (100%)  11/27x (40.07%)  4/27z (14.81%)  23/28 (28.14%)  12/28x (42.86%)  7/28z (25.00%)  23/28 (82.14%)  0/28y (0.00%)  3/28z (10.71%)  1On arrival, 5 chicken box liners per pool. During the rearing and production period, 5 pairs of boot swabs per pool. 2Four liver samples per pool; where other organs (heart, ovaries, and tarsometatarsal joints) were affected, each type of tissue was examined separately. Type of organ sample positive to ESBL/AmpC is given in brackets. 3Two to 4 air samples were taken. 4Number of positive samples/number of samples. a–kDifferent letters indicated phylogenetic group and resistance gene/s of isolate/s. Where only 1 phylogenetic group/resistance gene is listed, all isolates were of the same group and contained the same resistance gene/s. aD1/blaCMY-2; bD1/blaSHV-12; cA1/blaCTX-M; dD2/blaCMY-2; eB1/blaSHV-2; f B1/blaCTX +blaCMY-2; gB1/blaCMY-2; hA1/blaCTX-M-1; iA1/blaSHV-12; jB1/blaSHV+blaCMY-2; kB23/blaCTX-M. x,yDifferent letters indicated statistical significance in ESBL detection between flocks at the 0.05 level using the Bonferroni's correction. zSame letter indicated that flocks did not differ significantly in AmpC detection from each other at the 0.05 level using the Bonferroni's correction. View Large Table 3. Frequencies, phylogenetic group, and Extended-Spectrum Beta-Lactamases (ESBL)/AmpC resistance genes of Escherichia coli isolates obtained from different samples in broiler breeder flocks. Sampling    Flock A  Flock B  Flock C  Time  Sample type  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  Before housing  Meconium1  2/2  0/2  2/2a  0/2  0/2  0/2  1/2  0/2  0/2    Organs2  4/4  0/4  1/4a (Liver)  0/2  0/2  0/2  2/2  0/2  0/2  4 wk  Air3  3/3  3/3b  0/3  2/2  0/2  2/2a  2/2  0/2  0/2    Boot swabs1  2/2  0/2  0/2  2/2  1/2c  1/2d  2/2  0/2  0/2    Organs2  2/2  2/2b (Liver)  0/2  2/2  2/2c (Liver)  0/2  1/1  0/1  0/1  12 wk  Air3  2/2  1/2e  0/2  2/2  2/2f  2/2f  2/4  0/4  1/4g    Boot swabs1  2/2  0/2  0/2  2/2  2/2h  0/2  2/2  0/2  0/2    Organs2  2/2  2/2e,i (Liver)  0/2  2/2  1/2c (Liver)  0/2  2/2  0/2  1/2g  24 wk  Boot swabs1  2/2  2/2i,j  1/2j  2/2  2/2c  0/2  2/2  0/2  0/2    Organs2  2/2  1/2a (Liver)  0/2  4/4  1/4c (Liver)  0/4  3/4  0/4  0/4  36 wk  Boot swabs1  2/2  0/2  0/2  2/2  1/2k  0/2  3/3  0/3  1/3g    Organs2  2/2  0/2  0/2  3/4  0/4  2/4g (Liver, tarsometatarsal joints)  1/2  0/2  0/2  Total (%)    27/27 (100%)  11/27x (40.07%)  4/27z (14.81%)  23/28 (28.14%)  12/28x (42.86%)  7/28z (25.00%)  23/28 (82.14%)  0/28y (0.00%)  3/28z (10.71%)  Sampling    Flock A  Flock B  Flock C  Time  Sample type  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  Before housing  Meconium1  2/2  0/2  2/2a  0/2  0/2  0/2  1/2  0/2  0/2    Organs2  4/4  0/4  1/4a (Liver)  0/2  0/2  0/2  2/2  0/2  0/2  4 wk  Air3  3/3  3/3b  0/3  2/2  0/2  2/2a  2/2  0/2  0/2    Boot swabs1  2/2  0/2  0/2  2/2  1/2c  1/2d  2/2  0/2  0/2    Organs2  2/2  2/2b (Liver)  0/2  2/2  2/2c (Liver)  0/2  1/1  0/1  0/1  12 wk  Air3  2/2  1/2e  0/2  2/2  2/2f  2/2f  2/4  0/4  1/4g    Boot swabs1  2/2  0/2  0/2  2/2  2/2h  0/2  2/2  0/2  0/2    Organs2  2/2  2/2e,i (Liver)  0/2  2/2  1/2c (Liver)  0/2  2/2  0/2  1/2g  24 wk  Boot swabs1  2/2  2/2i,j  1/2j  2/2  2/2c  0/2  2/2  0/2  0/2    Organs2  2/2  1/2a (Liver)  0/2  4/4  1/4c (Liver)  0/4  3/4  0/4  0/4  36 wk  Boot swabs1  2/2  0/2  0/2  2/2  1/2k  0/2  3/3  0/3  1/3g    Organs2  2/2  0/2  0/2  3/4  0/4  2/4g (Liver, tarsometatarsal joints)  1/2  0/2  0/2  Total (%)    27/27 (100%)  11/27x (40.07%)  4/27z (14.81%)  23/28 (28.14%)  12/28x (42.86%)  7/28z (25.00%)  23/28 (82.14%)  0/28y (0.00%)  3/28z (10.71%)  1On arrival, 5 chicken box liners per pool. During the rearing and production period, 5 pairs of boot swabs per pool. 2Four liver samples per pool; where other organs (heart, ovaries, and tarsometatarsal joints) were affected, each type of tissue was examined separately. Type of organ sample positive to ESBL/AmpC is given in brackets. 3Two to 4 air samples were taken. 4Number of positive samples/number of samples. a–kDifferent letters indicated phylogenetic group and resistance gene/s of isolate/s. Where only 1 phylogenetic group/resistance gene is listed, all isolates were of the same group and contained the same resistance gene/s. aD1/blaCMY-2; bD1/blaSHV-12; cA1/blaCTX-M; dD2/blaCMY-2; eB1/blaSHV-2; f B1/blaCTX +blaCMY-2; gB1/blaCMY-2; hA1/blaCTX-M-1; iA1/blaSHV-12; jB1/blaSHV+blaCMY-2; kB23/blaCTX-M. x,yDifferent letters indicated statistical significance in ESBL detection between flocks at the 0.05 level using the Bonferroni's correction. zSame letter indicated that flocks did not differ significantly in AmpC detection from each other at the 0.05 level using the Bonferroni's correction. View Large Table 4. Incidences, Phylogenetic Groups, and Extended-Spectrum Beta-Lactamases (ESBL)/AmpC Resistance Genes of Escherichia coli Isolates From Broiler Flocks.   All samples  Flock A/1  Flock A/2  Flock B/1  Flock B/2  Sampling time  ESBL1  AmpC2  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Before housing  0/8  0/8  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/13  3/13  +1a  +1a  +1b  +2c  +2d  +2d  –  –  –  –  –  –  Week 5  3/12  3/12  +1e  +1e  +1e  –  +2d  –  –  +2d  –  –  +2d  –    All samples  Flock A/1  Flock A/2  Flock B/1  Flock B/2  Sampling time  ESBL1  AmpC2  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Before housing  0/8  0/8  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/13  3/13  +1a  +1a  +1b  +2c  +2d  +2d  –  –  –  –  –  –  Week 5  3/12  3/12  +1e  +1e  +1e  –  +2d  –  –  +2d  –  –  +2d  –  1ESBL-producing E. coli positive samples/number of samples tested. 2AmpC-producing E. coli positive samples/number of samples tested. 3On arrival, meconium on chicken box liners was examined. At each visit during the fattening period, 2 pairs of boot swabs were collected per flock and pooled. 4Four liver samples per pool; in flock B/1, besides pooled liver sample 1 heart sample was tested at week 1. +Positive for ESBL- or AmpC-producing E. coli. –Negative for ESBL- or AmpC-producing E. coli. Samples were not taken. a–ePhylogenetic group and resistance gene of isolate; aD1/blaSHV-12; bA1/blaCTX-M; cD1/blaCMY-2; dD2/blaCMY-2; eB1/blaSHV-12. View Large Table 4. Incidences, Phylogenetic Groups, and Extended-Spectrum Beta-Lactamases (ESBL)/AmpC Resistance Genes of Escherichia coli Isolates From Broiler Flocks.   All samples  Flock A/1  Flock A/2  Flock B/1  Flock B/2  Sampling time  ESBL1  AmpC2  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Before housing  0/8  0/8  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/13  3/13  +1a  +1a  +1b  +2c  +2d  +2d  –  –  –  –  –  –  Week 5  3/12  3/12  +1e  +1e  +1e  –  +2d  –  –  +2d  –  –  +2d  –    All samples  Flock A/1  Flock A/2  Flock B/1  Flock B/2  Sampling time  ESBL1  AmpC2  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Before housing  0/8  0/8  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/13  3/13  +1a  +1a  +1b  +2c  +2d  +2d  –  –  –  –  –  –  Week 5  3/12  3/12  +1e  +1e  +1e  –  +2d  –  –  +2d  –  –  +2d  –  1ESBL-producing E. coli positive samples/number of samples tested. 2AmpC-producing E. coli positive samples/number of samples tested. 3On arrival, meconium on chicken box liners was examined. At each visit during the fattening period, 2 pairs of boot swabs were collected per flock and pooled. 4Four liver samples per pool; in flock B/1, besides pooled liver sample 1 heart sample was tested at week 1. +Positive for ESBL- or AmpC-producing E. coli. –Negative for ESBL- or AmpC-producing E. coli. Samples were not taken. a–ePhylogenetic group and resistance gene of isolate; aD1/blaSHV-12; bA1/blaCTX-M; cD1/blaCMY-2; dD2/blaCMY-2; eB1/blaSHV-12. View Large The surveillance performed in our study showed an increase in ESBL/AmpC-producing E. coli in all 3 production types of poultry over the sampling period. In the breeder flocks, ESBL/AmpC-producing E. coli were detected in 2 flocks at week 4 and at 12 wk of age. AmpC-producing E. coli were also detected in the third flock. Two flocks were positive at the end of the observation period, at week 36 (Table 3). In broilers and turkeys, the first ESBL/AmpC-producing E. coli were detected in 3 of 8 flocks in the first week. At week 5, all 4 broiler flocks became positive (Table 4 and 5). Similar findings have been reported in studies on broiler and turkey farms in Germany, Great Britain, and the Netherlands [5, 33, 35], although those investigations were carried out on farms with a history of ESBL/AmpC-producing E. coli. Table 5. Number, Phylogenetic Group, and Extended-Spectrum Beta-Lactamases (ESBL) Resistance Genes of Escherichia coli Isolates Obtained From Different Samples in Meat-Type Turkey Flocks.   All samples  Flock D  Flock E  Flock F  Flock G  Sampling time  ESBL1  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Before housing  0/9  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/17  –  –  –  –  –  –  –  –  –  +a  +a  +b  Week 5  2/12  /  –  –  /  –  –  /  –  –  /  +a  +b  Week 9  3/17  –  –  –  +b  –  –  –  –  –  +b  –  +b  Week 16  4/15  /  –  –  /  –  –  /  –  –  /  +b  +b*    All samples  Flock D  Flock E  Flock F  Flock G  Sampling time  ESBL1  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Before housing  0/9  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/17  –  –  –  –  –  –  –  –  –  +a  +a  +b  Week 5  2/12  /  –  –  /  –  –  /  –  –  /  +a  +b  Week 9  3/17  –  –  –  +b  –  –  –  –  –  +b  –  +b  Week 16  4/15  /  –  –  /  –  –  /  –  –  /  +b  +b*  1ESBL-producing E. coli positive samples/number of samples tested. 2On arrival, meconium on chicken box liners was examined, in the fattening period 2 pairs of boot swabs per pool were tested. +Positive for ESBL-producing E. coli. –Negative for ESBL-producing E. coli. /Samples were not taken. a,bPhylogenetic group and resistance gene of isolate; aA0/blaCTX-M;bA1/blaSHV-12. *Three organ samples, e.g., pooled liver sample, tarsometatarsal joints and subcutis were positive for ESBL-producing E. coli. View Large Table 5. Number, Phylogenetic Group, and Extended-Spectrum Beta-Lactamases (ESBL) Resistance Genes of Escherichia coli Isolates Obtained From Different Samples in Meat-Type Turkey Flocks.   All samples  Flock D  Flock E  Flock F  Flock G  Sampling time  ESBL1  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Before housing  0/9  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/17  –  –  –  –  –  –  –  –  –  +a  +a  +b  Week 5  2/12  /  –  –  /  –  –  /  –  –  /  +a  +b  Week 9  3/17  –  –  –  +b  –  –  –  –  –  +b  –  +b  Week 16  4/15  /  –  –  /  –  –  /  –  –  /  +b  +b*    All samples  Flock D  Flock E  Flock F  Flock G  Sampling time  ESBL1  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Before housing  0/9  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/17  –  –  –  –  –  –  –  –  –  +a  +a  +b  Week 5  2/12  /  –  –  /  –  –  /  –  –  /  +a  +b  Week 9  3/17  –  –  –  +b  –  –  –  –  –  +b  –  +b  Week 16  4/15  /  –  –  /  –  –  /  –  –  /  +b  +b*  1ESBL-producing E. coli positive samples/number of samples tested. 2On arrival, meconium on chicken box liners was examined, in the fattening period 2 pairs of boot swabs per pool were tested. +Positive for ESBL-producing E. coli. –Negative for ESBL-producing E. coli. /Samples were not taken. a,bPhylogenetic group and resistance gene of isolate; aA0/blaCTX-M;bA1/blaSHV-12. *Three organ samples, e.g., pooled liver sample, tarsometatarsal joints and subcutis were positive for ESBL-producing E. coli. View Large The proportion of ESBL-producing E. coli among all E. coli is needed to estimate the occurrence of ESBL populations in chicken flocks and other areas of production [7]. In our study, E. coli were found in 87.09% of samples taken during the production period. ESBL-producing E. coli were detected in 22.04% of samples, while AmpC-producing isolates were detected in 10.75% of samples, although initially the number of phenotypically confirmed isolates was higher due to the presence of inhibitor-resistant beta-lactamase TEM-32. Similar detection frequencies of ESBL- and AmpC-producing E. coli were found in organs of dead or culled birds, in feces and in air samples (Table 6). The fact that all 3 types of samples were positive at the same sampling time indicated a high rate of contamination and rapid spread of these strains among the birds within one poultry house. Table 6. Frequencies of Detection of E. coli, and Extended-Spectrum Beta-Lactamases (ESBL)- and AmpC-producing E. coli in Different Types of Samples. Type of sample  Number of E. coli-positive samples/number of samples tested (%)  Number of ESBL-positive samples/number of samples (%)  Number of AmpC-positive samples/number of samples (%)  Feces (boot swabs, meconium)  62/69 (89.86)  13/69 (18.84)  9/69 (13.04)  Organs of dead or culled animals  70/82 (85.36)  17/82 (20.73)  5/82 (6.09)  Air  30/35 (85.71)  11/35 (31.42)  6/35 (17.14)  Total (%1)  162/186 (87.09)  41/186 (22.04)  20/186 (10.75)  Type of sample  Number of E. coli-positive samples/number of samples tested (%)  Number of ESBL-positive samples/number of samples (%)  Number of AmpC-positive samples/number of samples (%)  Feces (boot swabs, meconium)  62/69 (89.86)  13/69 (18.84)  9/69 (13.04)  Organs of dead or culled animals  70/82 (85.36)  17/82 (20.73)  5/82 (6.09)  Air  30/35 (85.71)  11/35 (31.42)  6/35 (17.14)  Total (%1)  162/186 (87.09)  41/186 (22.04)  20/186 (10.75)  1Percentage of positive samples. View Large Table 6. Frequencies of Detection of E. coli, and Extended-Spectrum Beta-Lactamases (ESBL)- and AmpC-producing E. coli in Different Types of Samples. Type of sample  Number of E. coli-positive samples/number of samples tested (%)  Number of ESBL-positive samples/number of samples (%)  Number of AmpC-positive samples/number of samples (%)  Feces (boot swabs, meconium)  62/69 (89.86)  13/69 (18.84)  9/69 (13.04)  Organs of dead or culled animals  70/82 (85.36)  17/82 (20.73)  5/82 (6.09)  Air  30/35 (85.71)  11/35 (31.42)  6/35 (17.14)  Total (%1)  162/186 (87.09)  41/186 (22.04)  20/186 (10.75)  Type of sample  Number of E. coli-positive samples/number of samples tested (%)  Number of ESBL-positive samples/number of samples (%)  Number of AmpC-positive samples/number of samples (%)  Feces (boot swabs, meconium)  62/69 (89.86)  13/69 (18.84)  9/69 (13.04)  Organs of dead or culled animals  70/82 (85.36)  17/82 (20.73)  5/82 (6.09)  Air  30/35 (85.71)  11/35 (31.42)  6/35 (17.14)  Total (%1)  162/186 (87.09)  41/186 (22.04)  20/186 (10.75)  1Percentage of positive samples. View Large The only genes detected in the study were blaSHV-12, blaCMY-2, and blaCTX-M, which were detected in 15.4%, 12.3%, and 9.8% of E. coli strains isolated during the breeding period, respectively (Tables 3–5). These results are in line with reports from other countries [7, 33]. However, TEM-52, which, along with SHV-12, dominates in some European countries, was not detected in our study nor in previous studies that include screening of poultry meat for ESBL-producing E. coli (unpublished results). The relative proportion of blaSHV-12, blaCMY-2, and blaCTX-M genes differed between flocks. In some flocks, only 1 ESBL and/or AmpC gene was detected throughout the whole sampling period (e.g., blaSHV-12/blaCMY-2 in flock A or blaCTX-M/blaCMY-2 in flock B). Interestingly, these genes were harbored by E. coli assigned to different phylogenetic groups, thus anticipating the entry of different E. coli strains into the flocks from one or more unknown sources or horizontal gene transfer events between E. coli strains within most flocks. Vertical transmission was presumably involved in flock G, where blaSHV-12-encoding E. coli belonging to phylogenetic group A1 could be isolated from the first week onwards (Table 5). ESBL/AmpC-producing isolates belonged predominantly to phylogenetic group A1 (36.2%), followed by group D1 (24.1%), B1 (22.4%), and D2 (10.3%). Only 3 isolates from air and boot swabs (blaCTX-M) in flock G were from phylogenetic group A0 and 1 boot swab isolate from flock B was from phylogenetic group B23, which is rarely found among poultry, indicating a possible single externally sourced contamination event. An association between particular E. coli phylogroups and ESBL or AmpC genes could not be detected in our study [36]. The relative high proportion of ESBL/AmpC-producing E. coli from human pathogenic groups D1, D2, and B23 in air and fecal samples could pose a risk for people working on the farms. In the extensive study carried out in the Netherlands, a significantly greater prevalence of ESBL/AmpC-producing E. coli was seen among people working on broiler farms compared with the general population or patients. Moreover, an increased risk of transfer has been detected among individuals with a high level of contact with live poultry [4]. Antibiotic treatment can have a substantial influence on the prevalence of ESBL/AmpC-producing E. coli [33, 37–39]. The use of antibiotics in the flocks in our study was limited. Only 1 turkey flock was treated with enrofloxacin. The treatment was needed at the end of the observation period (at 112 d old) and, therefore, had no influence on our studies of the prevalence of ESBL-producing E. coli. Among breeder flocks, only flock B received sulfamonomethoxine, with trimethoprim in the first week and oxytetracycline after placement on the production farm. The frequency of detection of ESBL/AmpC seen in this flock was comparable to the untreated flock A. The lowest detection frequency of ESBL/AmpC was in untreated flock C, in which coccidiostats were included in the feed during the rearing period (Table 3). These results could suggest that the intake of antibiotics was less important for the occurrence/selection of ESBL/AmpC E. coli strains than horizontal or vertical transmission of resistance genes between E. coli in the flocks included in our study. In an investigation of broiler farms positive for ESBL/AmpC-producing E. coli, Laube et al. [6] detected ESBL-producing E. coli on boot swabs taken from the area around poultry houses as well as in ambient air samples at a distance of 50 m. The broiler breeder houses included in our study were located on larger farms, so it is very likely that airborne emission from the houses’ surroundings could take place. However, the fact that fattening flocks were placed in houses isolated from other poultry farms does not support such an explanation. Other sources such as rodents, wild birds, waste water, contaminated slurry, or flies could also have been responsible [40]. CONCLUSIONS AND APPLICATIONS Over the fattening period an increase of ESBL/AmpC-producing E. coli can be expected within the flock, although high hygienic measurements are implemented before the placement of the chicks. The entry of different E. coli carrying ESBL/AmpC determinants and rapid spread of these strains among the birds within 1 poultry house was confirmed. ESBL/AmpC-producing E. coli from human pathogenic groups D1, D2, and B23 in air and fecal samples could pose a risk for people working on the farms. 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PLoS One  10: e0135402. Google Scholar CrossRef Search ADS PubMed  Acknowledgements This work was financially supported by Slovene Ministry of Agriculture, Forestry and Food and Slovene Research Agency. All poultry companies and poultry veterinarians are gratefully acknowledged for their contributions to this study. © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Poultry Research Oxford University Press

Presence and distribution of extended-spectrum and AmpC beta-lactamases-producing Escherichia coli on poultry farms in Slovenia

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© 2018 Poultry Science Association Inc.
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1056-6171
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1537-0437
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10.3382/japr/pfy021
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Abstract

SUMMARY The purpose of the study was to investigate the presence, distribution and dynamics of extended-spectrum beta-lactamases (ESBL) and plasmid-mediated AmpC beta-lactamases (AmpC) producing Escherichia coli (E. coli) in meat type poultry and their environment. Three broiler breeder flocks, four broiler flocks originating from these breeders, and four meat-type turkey flocks were included in a longitudinal study. A total of 349 samples were tested. All environmental samples collected before placement of birds on the farms were negative for ESBL- and AmpC-producing E. coli. However, during the observation period ESBL-producing E. coli were detected in 41 out of 186 samples (22.04%), while AmpC-producing isolates were identified in 10.75% of samples. Resistant strains belonged predominantly to E. coli phylogenetic group A1 (36.2%), followed by group D1 (24.1%), B1 (22.4%) and D2 (10.3%). The only genes detected were blaSHV-12, blaCMY-2 and blaCTX-M. No significant differences were seen in the rates of detection of resistant strains in feces (18.84%), air samples (31.42%) or organs of dead birds (20.73%), which suggested that air samples can be successfully used as an indicator for the identification of positive flocks in order to prevent or reduce cross contaminations in slaughter houses. DESCRIPTION OF THE PROBLEM Antimicrobial resistance continues to be a global concern in veterinary and human medicine. Broad-spectrum beta-lactamases-producing Enterobacteriaceae, in particular Escherichia coli (E. coli), have been detected increasingly in humans and other animals [1, 2]. The production of extended-spectrum beta-lactamases (ESBL) and plasmid-mediated AmpC beta-lactamases (AmpC) are 2 mechanisms of bacterial resistance that have recently received increasing attention. The presence of ESBL confers resistance to most beta-lactam antibiotics, including ceftazidime, ceftiofur, and aztreonam, but they can be inactivated by beta-lactamase inhibitors such as clavulanic acid. AmpC beta-lactamases hydrolyze third-generation cephalosporins and cephomycins and are not inhibited by clavulanic acid or other inhibitors. In Europe, cephalosporins are not allowed to be use in poultry, but ampicillin and amoxicillin are very often used for treating various bacterial diseases of poultry [3]. ESBL- and AmpC-producing isolates are now being found in increasing numbers in animals produced for food, in retail meats and in pets [1, 2]. Although it is not clear how ESBL/AmpC-producing bacteria colonize humans, some studies have suggested that it takes place via other animals [4]. A high prevalence of ESBL/AmpC-producing E. coli strains has been reported in almost all species of commercially reared poultry, poultry meat, and poultry house surroundings [5–9]. Slovenia is a relatively small producer of poultry meat; the number of birds slaughtered per year is approximately 35 million. A survey performed on chicken meat in 2013 showed that more than 50% of poultry meat and meat products taken at slaughter were positive for ESBL-producing E. coli, thus constituting a serious issue for public health [10]. No data on the prevalence of ESBL- or AmpC-producing E. coli in Slovenian meat-type and layer flocks are available. The purpose of this study was to investigate the presence, distribution, and dynamics of ESBL/AmpC-producing E. coli in meat-type poultry and their environment. Additionally, resistance gene families were identified, and the E. coli were assigned to phylogenetic groups in order to gain basic insights into the variability of ESBL/AmpC isolates. MATERIALS AND METHODS Flocks Included in the Study Three broiler breeder, 4 broiler and 4 meat-type turkey flocks owned by the 3 biggest poultry-producing companies were included in this study. Before placement of birds, all poultry houses were cleaned and disinfected. Broiler Breeders Three broiler breeder flocks (designated A, B, and C) from different owners were sampled. Chicks from all the flocks were imported as 1-d olds. The farms were located at various geographic regions throughout Slovenia. Two poultry houses on farms A and B and 1 house on farm C were investigated. Birds stayed on the rearing farm until they were 19 wk old. They were then moved to a production farm in such a way that all hens from a selected barn were placed in one barn on the production farm. Flock sizes ranged from 19,143 to 29,040 animals. All flocks were vaccinated using similar immunoprophylactic programs against viral diseases and Salmonella, the difference being in the protocol against coccidiosis and E. coli. Flocks A and B were vaccinated against coccidiosis with live attenuated vaccines, while birds from flock C received coccidiostats (salinomycin sodium salt) until 14 wk old. Birds from flock B were treated at the end of the first week with sulfamonomethoxine, with trimethoprim at 5 d, and at 21 wk with oxytetracycline for 3 d. Flocks A and C were not treated with antibacterial drugs during the observation period of 36 wk. Broilers Four broiler flocks derived at 1 d of age from 26-wk-old breeders were investigated. Two flocks were derived from breeder flock A (designated flocks A/1 and A/2) and 2 flocks from broiler breeder flock B (designated flocks B/1 and B/2). Flocks were placed on 4 farms at different locations. The number of birds in each flock ranged from 7,000 to 11,000 birds. All broiler flocks received coccidiostats until 5 d before slaughter. The fattening period was between 36 and 42 d. No antibiotic treatment was needed during the fattening period in any of the sampled broiler flocks. Meat-Type Turkeys Four meat-type turkey farms were investigated (designated D, E, F, and G). Locations of the farms were chosen at random throughout the country. All 1-d-old turkeys were transported and placed into clean and disinfected barns. The sizes of the flocks ranged from 4,000 to 4,100 birds and all flocks received coccidiostats until 12 wk old. Respiratory disorders and higher mortality occurred at day 112 in the flock G and birds were treated with enrofloxacin for 5 d. In flocks D, E, and F, no antibacterial treatment was needed. Sampling Procedures Environmental samples were collected on all farms before the day-old chicks arrived. For collecting environmental swabs, premoistened 3 M Enviro Swabs [11] were used. At least 10 swabs of the barn walls, floor, and equipment were taken by swabbing a surface of approximately 15 cm2. In addition, 1 sample of water (1 L), litter (1 kg), and feed (1 kg) was taken for each flock. Birds that died during transport were collected on arrival at the farms. Necropsies were performed and organs were taken aseptically for bacteriological examination. Meconium taken from the paper of transport boxes was also sampled. Dead or culled birds from broiler breeder flocks were collected at weeks 4, 12, 24, and 36. In broiler flocks, dead birds were collected at weeks 1 and 5, and in turkey flocks at weeks 1, 5, 9, and 16. At each time point, at least 4 birds were necropsied and livers were taken for bacteriological examination. Samples were pooled according to flock and time of sampling; each pool contained at least 4 liver samples. When other organs were affected (e.g., fibrinous pericarditis, oophoritis, cellulitis, inflammation of tarsometatarsal joints), each tissue sample was taken separately. At the same time, point fecal samples were collected by boot swabs after walking the whole length of the poultry house. In each broiler breeder house, 5 pairs of boot swabs were collected; in broiler and turkey houses at least 2 pairs of boot swabs were collected and pooled. Air samples were collected in broiler breeder houses at weeks 4 and 12, in broiler houses at weeks 1 and 5, and in turkey houses at weeks 1 and 9. Air samples were taken using a Coriolis Delta air sampler [12]. The sampler aspirated of 100 L of air per minute and transferred the airborne particles into a liquid medium by centrifugal vortex of air. Phosphate-buffered saline (15 mL) was used to collect the samples. The sampler was run for 10 min collecting 1000 L of air particles for each sample. A total of 349 samples were tested; 148 were obtained from broiler breeder flocks (Supplemental Table 1), 85 from broilers (Supplemental Table 2), and 116 from meat-type turkey flocks (Supplemental Table 3). Microbiological Analysis All samples were processed within 24 h of sampling. In the meantime, they were kept at 4°C. Fecal samples were first diluted 1:10 in buffered peptone water [13], all other samples were plated directly onto chromogenic medium [14] and incubated at 37°C for 24 h. Suspect colonies were identified by matrix-assisted laser desorption/ionization, time of flight (MALDI-TOF) mass spectrometry [15]. All E. coli strains were subcultured onto blood agar [16] and stored at –80°C for further testing. Phenotypic Detection of ESBL- and AmpC-Producing Isolates All samples were enriched in BPW at 37°C for 18–22 h before plating onto ESBL Chrom ID agar [17]. Based on colony morphology and appearance (presumptive ESBL/AmpC-producing E. coli colonies were colored purple/red) up to 3 colonies were subcultured by restreaking onto a fresh blood agar plate and incubated at 37°C for 18–22 h. The species of the isolates was confirmed by MALDI-TOF [18]. Presumptive ESBL/AmpC-producing E. coli isolates were tested for antimicrobial susceptibility using the microdilution method Sensititre™ [19]. All isolates were tested using the primary EUVSEC panel, containing essential first line antimicrobials. If the isolate was resistant to cefotaxime and/or ceftazidime, it was further tested using the second panel (EUVSEC2). This panel includes a cefoxitin, cefepime, and clavulanate synergy test in combination with cefotaxime and ceftazidime for detection of ESBL and AmpC production. The results were interpreted according to the protocol of the European Union Reference Laboratory for Antimicrobial Resistance based on EUCAST epidemiological cut-off values and clinical resistance breakpoints [20]. Molecular Confirmation of ESBL/AmpC-Producing Isolates and Identification of Resistance Gene Families All phenotypically confirmed ESBL/AmpC-producing isolates were further tested by PCR. DNA was released from bacterial cells by boiling [21]. All isolates were screened with multiplex PCR using primers specific for blaCTX-M groups as described by Woodford [22]. Briefly, each reaction mixture contained 2 μL of the bacterial lysate, 10 pg of each primer (blaCTX-M group 1F, blaCTX-M group 1R, blaCTX-M group 2F, blaCTX-M group 2R, blaCTX-M group 8F, blaCTX-M group 8R, blaCTX-M group 9F, blaCTX-M group 9R, blaCTX-M group 25F, blaCTX-M group 25R), 12,5 μL of DreamTaq PCR Master Mix [23], and 0.5 μL of sterile nuclease free water [23]. The cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, primer annealing at 52°C for 25 s, elongation at 72°C for 50 s, and the final elongation step for 6 min at 72°C. Genes blaTEM and blaSHV were first screened for with primer pairs blaTEM-F/blaTEM-R and blaSHV-F/blaSHV-R employing the protocol explained in detail by Dallenne et al. [24]. The blaTEM-R and blaSHV-F genes from positive isolates were subsequently multiplied for sequencing using primer pairs TEM seqF/TEM seqR and SHV seqF/SHV seqR [25] and the following cycling protocol: initial denaturation at 95°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, elongation at 72°C for 1.5 min, and the final elongation for 10 min at 72°C. For the detection of AmpC genes encoding beta-lactamases MOX, CMY, LAT, DHA, ACC, MIR, ACT, and FOX primers described by Perez-Perez and Hanson [26] were employed. The single PCR mixtures, with a total volume of 25 μL, which contained 10 pg of each primer, 12.5 μL of DreamTaq PCR Master Mix and sterile nuclease free water, were subjected to 30 cycles of denaturation at 94°C for 30 s, primer annealing at 64°C for 30 s and primer extension at 72°C for 1 min. The initial denaturation was performed at 95°C for 5 min and the final extension step for 10 min at 72°C. In positive isolates (only for CMY), the blaCMY gene was again amplified using primers CMY2seqF/CMY2seqR according Dierikx et al. [27]. Amplifications were done in a total volume of 50 μL, containing 3 μL of the bacterial lysate, 25 μL of DreamTaq PCR Master Mix, 20 pg of each primer and sterile nuclease free water. Initial denaturation was set at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, primer annealing at 58°C for 30 s, primer extension at 72°C for 1.5 min and completed with the final elongation step for 7 min at 72°C. All PCR sequencing reactions were performed at Macrogen [28] on our request. Primers, product sizes, and references are listed in Table 1. Table 1. Oligonucleotide Primers Used for PCR Amplification, PCR Product Size, and References. Primer name  Primer sequence  Size of PCR product (bp)  Reference  blaCTX-M group 1F  5΄-AAA AAT CAC TGC GCC AGT T C-3΄  415  [22]  blaCTX-M group 1R  5΄-AGC TTA TTC ATC GCC ACG TT-3΄      blaCTX-M group 2F  5΄-CGA CGC TAC CCC TGC TAT T-3΄  552  [22]  blaCTX-M group 2R  5΄-CCA GCG TCA GAT TTT TCA GG-3΄      blaCTX-M group 8F  5΄-TCG CGT TAA GCG GAT GAT GC-3΄  666  [22]  blaCTX-M group 8R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaCTX-M group 9F  5΄-CAA AGA GAG TGC AAC GGA TG-3΄  205  [22]  blaCTX-M group 9R  5΄-ATT GGA AAG CGT TCA TCA CC-3΄      blaCTX-M group 25F  5΄-GCA CGA TGA CAT TCG GG-3΄  327  [22]  blaCTX-M group 25R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaTEM-F  5΄-CAT TTC CGT GTC GCC CTT ATT C-3΄  800  [24]  blaTEM-R  5΄-CGT TCA TCC ATA GTT GCC TGA C-3΄      blaSHV-F  5΄-AGC CGC TTG AGC AAA TTA AA C-3΄  713  [24]  blaSHV-R  5΄-ATC CCG CAG ATA AAT CAC CAC-3΄      TEM seqF  5΄-ATT CTT GAA GAC GAA AGG GC-3΄  1150  [25]  TEM seqR  5΄-ACG CTC AGT GGA ACG AAA AC-3΄      SHV seqF  5΄-CAC TCA AGG ATG TAT TGT G-3΄  885  [25]  SHV seqR  5΄- TTA GCG TTG CCA GTG CTC G-3΄      MOXMF  5΄- GCT GCT CAA GGA GCA CAG GAT-3΄  520  [26]  MOXMR  5΄-CAC ATT GAC ATA GGT GTG GTG C-3΄      CITMF  5΄-TGG CCA GAA CTG ACA GGC AAA-3΄  462  [26]  CITMR  5΄-TTT CTC CTG AAC GTG GCT GGC-3΄      DHAMF  5΄-AAC TTT CAC AGG TGT GCT GGG T-3΄  405  [26]  DHAMR  5΄-CCG TAC GCA TAC TGG CTT TGC-3΄      ACCMF  5΄-AAC AGC CTC AGC AGC CGG TTA-3΄  346  [26]  ACCMR  5΄-TTC GCC GCA ATC ATC CCT AGC-3΄      EBCMF  5΄-TCG GTA AAG CCG ATG TTG CGG-3΄  302  [26]  EBCMR  5΄-CTT CCA CTG CGG CTG CCA GTT-3΄      FOXMF  5΄-AAC ATG GGG TAT CAG GGA GAT G-3΄  190  [26]  FOXMR  5΄-CAA AGC GCG TAA CCG GAT TGG-3΄      CMY2seqF  5΄-ATG ATG AAA AAA TCG TTA TGC TGC-3΄  1117  [27]  CMY2seqR  5΄-GCT TTT CAA GAA TGC GCC AGG-3΄      ChuA-F  5΄-GACGAACCAACGGTCAGGAT -3΄  279  [33]  ChuA-R  5΄-TGCCGCCAGTACCAAAGACA-3΄      YjA-F  5΄-TGAAGTGTCAGGAGACGCTG-3΄  211  [36]  YjA-R  5΄-ATGGAGAATGCGTTCCTCAAC-3΄      TspE4.C2-F  5΄-GAGTAATGTCGGGGCATTCA-3΄  152  [37]  TspE4.C2-R  5΄-CGCGCCAACAAAGTATTACG-3΄      Primer name  Primer sequence  Size of PCR product (bp)  Reference  blaCTX-M group 1F  5΄-AAA AAT CAC TGC GCC AGT T C-3΄  415  [22]  blaCTX-M group 1R  5΄-AGC TTA TTC ATC GCC ACG TT-3΄      blaCTX-M group 2F  5΄-CGA CGC TAC CCC TGC TAT T-3΄  552  [22]  blaCTX-M group 2R  5΄-CCA GCG TCA GAT TTT TCA GG-3΄      blaCTX-M group 8F  5΄-TCG CGT TAA GCG GAT GAT GC-3΄  666  [22]  blaCTX-M group 8R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaCTX-M group 9F  5΄-CAA AGA GAG TGC AAC GGA TG-3΄  205  [22]  blaCTX-M group 9R  5΄-ATT GGA AAG CGT TCA TCA CC-3΄      blaCTX-M group 25F  5΄-GCA CGA TGA CAT TCG GG-3΄  327  [22]  blaCTX-M group 25R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaTEM-F  5΄-CAT TTC CGT GTC GCC CTT ATT C-3΄  800  [24]  blaTEM-R  5΄-CGT TCA TCC ATA GTT GCC TGA C-3΄      blaSHV-F  5΄-AGC CGC TTG AGC AAA TTA AA C-3΄  713  [24]  blaSHV-R  5΄-ATC CCG CAG ATA AAT CAC CAC-3΄      TEM seqF  5΄-ATT CTT GAA GAC GAA AGG GC-3΄  1150  [25]  TEM seqR  5΄-ACG CTC AGT GGA ACG AAA AC-3΄      SHV seqF  5΄-CAC TCA AGG ATG TAT TGT G-3΄  885  [25]  SHV seqR  5΄- TTA GCG TTG CCA GTG CTC G-3΄      MOXMF  5΄- GCT GCT CAA GGA GCA CAG GAT-3΄  520  [26]  MOXMR  5΄-CAC ATT GAC ATA GGT GTG GTG C-3΄      CITMF  5΄-TGG CCA GAA CTG ACA GGC AAA-3΄  462  [26]  CITMR  5΄-TTT CTC CTG AAC GTG GCT GGC-3΄      DHAMF  5΄-AAC TTT CAC AGG TGT GCT GGG T-3΄  405  [26]  DHAMR  5΄-CCG TAC GCA TAC TGG CTT TGC-3΄      ACCMF  5΄-AAC AGC CTC AGC AGC CGG TTA-3΄  346  [26]  ACCMR  5΄-TTC GCC GCA ATC ATC CCT AGC-3΄      EBCMF  5΄-TCG GTA AAG CCG ATG TTG CGG-3΄  302  [26]  EBCMR  5΄-CTT CCA CTG CGG CTG CCA GTT-3΄      FOXMF  5΄-AAC ATG GGG TAT CAG GGA GAT G-3΄  190  [26]  FOXMR  5΄-CAA AGC GCG TAA CCG GAT TGG-3΄      CMY2seqF  5΄-ATG ATG AAA AAA TCG TTA TGC TGC-3΄  1117  [27]  CMY2seqR  5΄-GCT TTT CAA GAA TGC GCC AGG-3΄      ChuA-F  5΄-GACGAACCAACGGTCAGGAT -3΄  279  [33]  ChuA-R  5΄-TGCCGCCAGTACCAAAGACA-3΄      YjA-F  5΄-TGAAGTGTCAGGAGACGCTG-3΄  211  [36]  YjA-R  5΄-ATGGAGAATGCGTTCCTCAAC-3΄      TspE4.C2-F  5΄-GAGTAATGTCGGGGCATTCA-3΄  152  [37]  TspE4.C2-R  5΄-CGCGCCAACAAAGTATTACG-3΄      View Large Table 1. Oligonucleotide Primers Used for PCR Amplification, PCR Product Size, and References. Primer name  Primer sequence  Size of PCR product (bp)  Reference  blaCTX-M group 1F  5΄-AAA AAT CAC TGC GCC AGT T C-3΄  415  [22]  blaCTX-M group 1R  5΄-AGC TTA TTC ATC GCC ACG TT-3΄      blaCTX-M group 2F  5΄-CGA CGC TAC CCC TGC TAT T-3΄  552  [22]  blaCTX-M group 2R  5΄-CCA GCG TCA GAT TTT TCA GG-3΄      blaCTX-M group 8F  5΄-TCG CGT TAA GCG GAT GAT GC-3΄  666  [22]  blaCTX-M group 8R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaCTX-M group 9F  5΄-CAA AGA GAG TGC AAC GGA TG-3΄  205  [22]  blaCTX-M group 9R  5΄-ATT GGA AAG CGT TCA TCA CC-3΄      blaCTX-M group 25F  5΄-GCA CGA TGA CAT TCG GG-3΄  327  [22]  blaCTX-M group 25R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaTEM-F  5΄-CAT TTC CGT GTC GCC CTT ATT C-3΄  800  [24]  blaTEM-R  5΄-CGT TCA TCC ATA GTT GCC TGA C-3΄      blaSHV-F  5΄-AGC CGC TTG AGC AAA TTA AA C-3΄  713  [24]  blaSHV-R  5΄-ATC CCG CAG ATA AAT CAC CAC-3΄      TEM seqF  5΄-ATT CTT GAA GAC GAA AGG GC-3΄  1150  [25]  TEM seqR  5΄-ACG CTC AGT GGA ACG AAA AC-3΄      SHV seqF  5΄-CAC TCA AGG ATG TAT TGT G-3΄  885  [25]  SHV seqR  5΄- TTA GCG TTG CCA GTG CTC G-3΄      MOXMF  5΄- GCT GCT CAA GGA GCA CAG GAT-3΄  520  [26]  MOXMR  5΄-CAC ATT GAC ATA GGT GTG GTG C-3΄      CITMF  5΄-TGG CCA GAA CTG ACA GGC AAA-3΄  462  [26]  CITMR  5΄-TTT CTC CTG AAC GTG GCT GGC-3΄      DHAMF  5΄-AAC TTT CAC AGG TGT GCT GGG T-3΄  405  [26]  DHAMR  5΄-CCG TAC GCA TAC TGG CTT TGC-3΄      ACCMF  5΄-AAC AGC CTC AGC AGC CGG TTA-3΄  346  [26]  ACCMR  5΄-TTC GCC GCA ATC ATC CCT AGC-3΄      EBCMF  5΄-TCG GTA AAG CCG ATG TTG CGG-3΄  302  [26]  EBCMR  5΄-CTT CCA CTG CGG CTG CCA GTT-3΄      FOXMF  5΄-AAC ATG GGG TAT CAG GGA GAT G-3΄  190  [26]  FOXMR  5΄-CAA AGC GCG TAA CCG GAT TGG-3΄      CMY2seqF  5΄-ATG ATG AAA AAA TCG TTA TGC TGC-3΄  1117  [27]  CMY2seqR  5΄-GCT TTT CAA GAA TGC GCC AGG-3΄      ChuA-F  5΄-GACGAACCAACGGTCAGGAT -3΄  279  [33]  ChuA-R  5΄-TGCCGCCAGTACCAAAGACA-3΄      YjA-F  5΄-TGAAGTGTCAGGAGACGCTG-3΄  211  [36]  YjA-R  5΄-ATGGAGAATGCGTTCCTCAAC-3΄      TspE4.C2-F  5΄-GAGTAATGTCGGGGCATTCA-3΄  152  [37]  TspE4.C2-R  5΄-CGCGCCAACAAAGTATTACG-3΄      Primer name  Primer sequence  Size of PCR product (bp)  Reference  blaCTX-M group 1F  5΄-AAA AAT CAC TGC GCC AGT T C-3΄  415  [22]  blaCTX-M group 1R  5΄-AGC TTA TTC ATC GCC ACG TT-3΄      blaCTX-M group 2F  5΄-CGA CGC TAC CCC TGC TAT T-3΄  552  [22]  blaCTX-M group 2R  5΄-CCA GCG TCA GAT TTT TCA GG-3΄      blaCTX-M group 8F  5΄-TCG CGT TAA GCG GAT GAT GC-3΄  666  [22]  blaCTX-M group 8R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaCTX-M group 9F  5΄-CAA AGA GAG TGC AAC GGA TG-3΄  205  [22]  blaCTX-M group 9R  5΄-ATT GGA AAG CGT TCA TCA CC-3΄      blaCTX-M group 25F  5΄-GCA CGA TGA CAT TCG GG-3΄  327  [22]  blaCTX-M group 25R  5΄-AAC CCA CGA TGT GGG TAG C-3΄      blaTEM-F  5΄-CAT TTC CGT GTC GCC CTT ATT C-3΄  800  [24]  blaTEM-R  5΄-CGT TCA TCC ATA GTT GCC TGA C-3΄      blaSHV-F  5΄-AGC CGC TTG AGC AAA TTA AA C-3΄  713  [24]  blaSHV-R  5΄-ATC CCG CAG ATA AAT CAC CAC-3΄      TEM seqF  5΄-ATT CTT GAA GAC GAA AGG GC-3΄  1150  [25]  TEM seqR  5΄-ACG CTC AGT GGA ACG AAA AC-3΄      SHV seqF  5΄-CAC TCA AGG ATG TAT TGT G-3΄  885  [25]  SHV seqR  5΄- TTA GCG TTG CCA GTG CTC G-3΄      MOXMF  5΄- GCT GCT CAA GGA GCA CAG GAT-3΄  520  [26]  MOXMR  5΄-CAC ATT GAC ATA GGT GTG GTG C-3΄      CITMF  5΄-TGG CCA GAA CTG ACA GGC AAA-3΄  462  [26]  CITMR  5΄-TTT CTC CTG AAC GTG GCT GGC-3΄      DHAMF  5΄-AAC TTT CAC AGG TGT GCT GGG T-3΄  405  [26]  DHAMR  5΄-CCG TAC GCA TAC TGG CTT TGC-3΄      ACCMF  5΄-AAC AGC CTC AGC AGC CGG TTA-3΄  346  [26]  ACCMR  5΄-TTC GCC GCA ATC ATC CCT AGC-3΄      EBCMF  5΄-TCG GTA AAG CCG ATG TTG CGG-3΄  302  [26]  EBCMR  5΄-CTT CCA CTG CGG CTG CCA GTT-3΄      FOXMF  5΄-AAC ATG GGG TAT CAG GGA GAT G-3΄  190  [26]  FOXMR  5΄-CAA AGC GCG TAA CCG GAT TGG-3΄      CMY2seqF  5΄-ATG ATG AAA AAA TCG TTA TGC TGC-3΄  1117  [27]  CMY2seqR  5΄-GCT TTT CAA GAA TGC GCC AGG-3΄      ChuA-F  5΄-GACGAACCAACGGTCAGGAT -3΄  279  [33]  ChuA-R  5΄-TGCCGCCAGTACCAAAGACA-3΄      YjA-F  5΄-TGAAGTGTCAGGAGACGCTG-3΄  211  [36]  YjA-R  5΄-ATGGAGAATGCGTTCCTCAAC-3΄      TspE4.C2-F  5΄-GAGTAATGTCGGGGCATTCA-3΄  152  [37]  TspE4.C2-R  5΄-CGCGCCAACAAAGTATTACG-3΄      View Large Phylogenetic Group Assignment of ESBL/AmpC Positive E. coli Isolates The phylogenetic group of each isolate (A0, A1, B1, B22, B23, D1, and D2) was determined by triplex PCR [29, 30]. The reaction mixture containing 2 μL of the bacterial lysate, 20 pg of each primer (primer (ChuA-F, ChuA-R, YjA-F, YjA-R, TspE4.C2-F, and TspE4.C2-R), PCR DreamTaq Master mix, and sterile nuclease free water was first denatured at 94°C for 5 min and then subjected to 30 PCR cycles as follows: denaturation at 94°C for 30 s, primer annealing at 60°C for 30 s, and elongation at 72°C for 30 s. The final elongation step was set for 7 min at 72°C. Statistical Analysis To determine the influence of the antibacterial treatment on the presence of ESBL/AmpC-producing E. coli in broiler breeder flocks, a Chi-square test and a Z test with Bonferroni's correction were used. The similarity of proportion between the number of ESBL- and AmpC-producing E. coli isolates and different types of samples was tested using Fisher's exact test. For those analyses, the Statistical Package for the Social Sciences was utilized [31]. RESULTS AND DISCUSSION Eleven poultry flocks owned by different companies and from different locations were included in our investigation. ESBL/AmpC-producing E. coli strains were isolated from all breeder and broiler flocks, as well as from 2 turkey flocks (Tables 3–5). Contamination of barns, due to insufficient cleaning and disinfection, could be one of the causes for the high incidence of ESBL/AmpC-producing isolates [6, 32]. In our study, ESBL/AmpC-producing E. coli were not detected in samples taken from the farm environment before the start of production. In addition, all samples of feed, litter, and water also tested negative for ESBL/AmpC-producing E. coli (Table 2). The proportion of these samples containing E. coli was very low (9.2%), suggesting effective implementation of good hygiene management on the poultry farms. Table 2. The Prevalence of Escherichia coli in Environmental Samples Taken Before Placement of Chicks on Farms.   Number of positive samples/number of samples tested  Sample type  Broiler breeders  Broilers  Meat-type turkeys  Total (%1)  Swabs  3/50  6/40  3/35  12/125 (9.60)  Feed  2/5  0/4  0/4  2/13 (15.38)  Litter  1/5  0/4  0/4  1/13 (11.12)  Water  0/5  0/4  0/3  0/12 (0.0)  Total (%1)  6/65 (9.23)  6/52 (11.53)  3/46 (6.52)  15/163 (9.20)    Number of positive samples/number of samples tested  Sample type  Broiler breeders  Broilers  Meat-type turkeys  Total (%1)  Swabs  3/50  6/40  3/35  12/125 (9.60)  Feed  2/5  0/4  0/4  2/13 (15.38)  Litter  1/5  0/4  0/4  1/13 (11.12)  Water  0/5  0/4  0/3  0/12 (0.0)  Total (%1)  6/65 (9.23)  6/52 (11.53)  3/46 (6.52)  15/163 (9.20)  1Percentage of positive samples. View Large Table 2. The Prevalence of Escherichia coli in Environmental Samples Taken Before Placement of Chicks on Farms.   Number of positive samples/number of samples tested  Sample type  Broiler breeders  Broilers  Meat-type turkeys  Total (%1)  Swabs  3/50  6/40  3/35  12/125 (9.60)  Feed  2/5  0/4  0/4  2/13 (15.38)  Litter  1/5  0/4  0/4  1/13 (11.12)  Water  0/5  0/4  0/3  0/12 (0.0)  Total (%1)  6/65 (9.23)  6/52 (11.53)  3/46 (6.52)  15/163 (9.20)    Number of positive samples/number of samples tested  Sample type  Broiler breeders  Broilers  Meat-type turkeys  Total (%1)  Swabs  3/50  6/40  3/35  12/125 (9.60)  Feed  2/5  0/4  0/4  2/13 (15.38)  Litter  1/5  0/4  0/4  1/13 (11.12)  Water  0/5  0/4  0/3  0/12 (0.0)  Total (%1)  6/65 (9.23)  6/52 (11.53)  3/46 (6.52)  15/163 (9.20)  1Percentage of positive samples. View Large In Slovenia, all breeder flocks and meat-type turkeys are imported as day-old chicks. As some reports indicated that vertical or pseudo-vertical transmission may play an important role in the spread of ESBL/AmpC-producing E. coli [7, 33, 34], the second step was to investigate the presence of such strains in day-old chicks before placement on farms. Except 1 broiler breeder flock that was positive for AmpC-producing E. coli, ESBL/AmpC-producing E. coli were not detected in meconium or organ samples from other broiler breeders or turkey flocks. AmpC-producing E. coli were detected in meconium and 1 pooled liver sample from flock A (Tables 3 and 5). In broilers, ESBL/AmpC-producing E. coli was not detected in day-old chicks, although they were hatched from parent flocks from which ESBL/AmpC-producing E. coli had been isolated (Tables 3 and 4). Experience elsewhere suggests that the presence of ESBL-producing E. coli in day-old chicks will always result in subsequent isolation of these E. coli from the flock, while a flock can be negative when the chicks are placed and then become positive and subsequently negative again [7]. Table 3. Frequencies, phylogenetic group, and Extended-Spectrum Beta-Lactamases (ESBL)/AmpC resistance genes of Escherichia coli isolates obtained from different samples in broiler breeder flocks. Sampling    Flock A  Flock B  Flock C  Time  Sample type  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  Before housing  Meconium1  2/2  0/2  2/2a  0/2  0/2  0/2  1/2  0/2  0/2    Organs2  4/4  0/4  1/4a (Liver)  0/2  0/2  0/2  2/2  0/2  0/2  4 wk  Air3  3/3  3/3b  0/3  2/2  0/2  2/2a  2/2  0/2  0/2    Boot swabs1  2/2  0/2  0/2  2/2  1/2c  1/2d  2/2  0/2  0/2    Organs2  2/2  2/2b (Liver)  0/2  2/2  2/2c (Liver)  0/2  1/1  0/1  0/1  12 wk  Air3  2/2  1/2e  0/2  2/2  2/2f  2/2f  2/4  0/4  1/4g    Boot swabs1  2/2  0/2  0/2  2/2  2/2h  0/2  2/2  0/2  0/2    Organs2  2/2  2/2e,i (Liver)  0/2  2/2  1/2c (Liver)  0/2  2/2  0/2  1/2g  24 wk  Boot swabs1  2/2  2/2i,j  1/2j  2/2  2/2c  0/2  2/2  0/2  0/2    Organs2  2/2  1/2a (Liver)  0/2  4/4  1/4c (Liver)  0/4  3/4  0/4  0/4  36 wk  Boot swabs1  2/2  0/2  0/2  2/2  1/2k  0/2  3/3  0/3  1/3g    Organs2  2/2  0/2  0/2  3/4  0/4  2/4g (Liver, tarsometatarsal joints)  1/2  0/2  0/2  Total (%)    27/27 (100%)  11/27x (40.07%)  4/27z (14.81%)  23/28 (28.14%)  12/28x (42.86%)  7/28z (25.00%)  23/28 (82.14%)  0/28y (0.00%)  3/28z (10.71%)  Sampling    Flock A  Flock B  Flock C  Time  Sample type  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  Before housing  Meconium1  2/2  0/2  2/2a  0/2  0/2  0/2  1/2  0/2  0/2    Organs2  4/4  0/4  1/4a (Liver)  0/2  0/2  0/2  2/2  0/2  0/2  4 wk  Air3  3/3  3/3b  0/3  2/2  0/2  2/2a  2/2  0/2  0/2    Boot swabs1  2/2  0/2  0/2  2/2  1/2c  1/2d  2/2  0/2  0/2    Organs2  2/2  2/2b (Liver)  0/2  2/2  2/2c (Liver)  0/2  1/1  0/1  0/1  12 wk  Air3  2/2  1/2e  0/2  2/2  2/2f  2/2f  2/4  0/4  1/4g    Boot swabs1  2/2  0/2  0/2  2/2  2/2h  0/2  2/2  0/2  0/2    Organs2  2/2  2/2e,i (Liver)  0/2  2/2  1/2c (Liver)  0/2  2/2  0/2  1/2g  24 wk  Boot swabs1  2/2  2/2i,j  1/2j  2/2  2/2c  0/2  2/2  0/2  0/2    Organs2  2/2  1/2a (Liver)  0/2  4/4  1/4c (Liver)  0/4  3/4  0/4  0/4  36 wk  Boot swabs1  2/2  0/2  0/2  2/2  1/2k  0/2  3/3  0/3  1/3g    Organs2  2/2  0/2  0/2  3/4  0/4  2/4g (Liver, tarsometatarsal joints)  1/2  0/2  0/2  Total (%)    27/27 (100%)  11/27x (40.07%)  4/27z (14.81%)  23/28 (28.14%)  12/28x (42.86%)  7/28z (25.00%)  23/28 (82.14%)  0/28y (0.00%)  3/28z (10.71%)  1On arrival, 5 chicken box liners per pool. During the rearing and production period, 5 pairs of boot swabs per pool. 2Four liver samples per pool; where other organs (heart, ovaries, and tarsometatarsal joints) were affected, each type of tissue was examined separately. Type of organ sample positive to ESBL/AmpC is given in brackets. 3Two to 4 air samples were taken. 4Number of positive samples/number of samples. a–kDifferent letters indicated phylogenetic group and resistance gene/s of isolate/s. Where only 1 phylogenetic group/resistance gene is listed, all isolates were of the same group and contained the same resistance gene/s. aD1/blaCMY-2; bD1/blaSHV-12; cA1/blaCTX-M; dD2/blaCMY-2; eB1/blaSHV-2; f B1/blaCTX +blaCMY-2; gB1/blaCMY-2; hA1/blaCTX-M-1; iA1/blaSHV-12; jB1/blaSHV+blaCMY-2; kB23/blaCTX-M. x,yDifferent letters indicated statistical significance in ESBL detection between flocks at the 0.05 level using the Bonferroni's correction. zSame letter indicated that flocks did not differ significantly in AmpC detection from each other at the 0.05 level using the Bonferroni's correction. View Large Table 3. Frequencies, phylogenetic group, and Extended-Spectrum Beta-Lactamases (ESBL)/AmpC resistance genes of Escherichia coli isolates obtained from different samples in broiler breeder flocks. Sampling    Flock A  Flock B  Flock C  Time  Sample type  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  Before housing  Meconium1  2/2  0/2  2/2a  0/2  0/2  0/2  1/2  0/2  0/2    Organs2  4/4  0/4  1/4a (Liver)  0/2  0/2  0/2  2/2  0/2  0/2  4 wk  Air3  3/3  3/3b  0/3  2/2  0/2  2/2a  2/2  0/2  0/2    Boot swabs1  2/2  0/2  0/2  2/2  1/2c  1/2d  2/2  0/2  0/2    Organs2  2/2  2/2b (Liver)  0/2  2/2  2/2c (Liver)  0/2  1/1  0/1  0/1  12 wk  Air3  2/2  1/2e  0/2  2/2  2/2f  2/2f  2/4  0/4  1/4g    Boot swabs1  2/2  0/2  0/2  2/2  2/2h  0/2  2/2  0/2  0/2    Organs2  2/2  2/2e,i (Liver)  0/2  2/2  1/2c (Liver)  0/2  2/2  0/2  1/2g  24 wk  Boot swabs1  2/2  2/2i,j  1/2j  2/2  2/2c  0/2  2/2  0/2  0/2    Organs2  2/2  1/2a (Liver)  0/2  4/4  1/4c (Liver)  0/4  3/4  0/4  0/4  36 wk  Boot swabs1  2/2  0/2  0/2  2/2  1/2k  0/2  3/3  0/3  1/3g    Organs2  2/2  0/2  0/2  3/4  0/4  2/4g (Liver, tarsometatarsal joints)  1/2  0/2  0/2  Total (%)    27/27 (100%)  11/27x (40.07%)  4/27z (14.81%)  23/28 (28.14%)  12/28x (42.86%)  7/28z (25.00%)  23/28 (82.14%)  0/28y (0.00%)  3/28z (10.71%)  Sampling    Flock A  Flock B  Flock C  Time  Sample type  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  E. coli4  ESBL4  AmpC4  Before housing  Meconium1  2/2  0/2  2/2a  0/2  0/2  0/2  1/2  0/2  0/2    Organs2  4/4  0/4  1/4a (Liver)  0/2  0/2  0/2  2/2  0/2  0/2  4 wk  Air3  3/3  3/3b  0/3  2/2  0/2  2/2a  2/2  0/2  0/2    Boot swabs1  2/2  0/2  0/2  2/2  1/2c  1/2d  2/2  0/2  0/2    Organs2  2/2  2/2b (Liver)  0/2  2/2  2/2c (Liver)  0/2  1/1  0/1  0/1  12 wk  Air3  2/2  1/2e  0/2  2/2  2/2f  2/2f  2/4  0/4  1/4g    Boot swabs1  2/2  0/2  0/2  2/2  2/2h  0/2  2/2  0/2  0/2    Organs2  2/2  2/2e,i (Liver)  0/2  2/2  1/2c (Liver)  0/2  2/2  0/2  1/2g  24 wk  Boot swabs1  2/2  2/2i,j  1/2j  2/2  2/2c  0/2  2/2  0/2  0/2    Organs2  2/2  1/2a (Liver)  0/2  4/4  1/4c (Liver)  0/4  3/4  0/4  0/4  36 wk  Boot swabs1  2/2  0/2  0/2  2/2  1/2k  0/2  3/3  0/3  1/3g    Organs2  2/2  0/2  0/2  3/4  0/4  2/4g (Liver, tarsometatarsal joints)  1/2  0/2  0/2  Total (%)    27/27 (100%)  11/27x (40.07%)  4/27z (14.81%)  23/28 (28.14%)  12/28x (42.86%)  7/28z (25.00%)  23/28 (82.14%)  0/28y (0.00%)  3/28z (10.71%)  1On arrival, 5 chicken box liners per pool. During the rearing and production period, 5 pairs of boot swabs per pool. 2Four liver samples per pool; where other organs (heart, ovaries, and tarsometatarsal joints) were affected, each type of tissue was examined separately. Type of organ sample positive to ESBL/AmpC is given in brackets. 3Two to 4 air samples were taken. 4Number of positive samples/number of samples. a–kDifferent letters indicated phylogenetic group and resistance gene/s of isolate/s. Where only 1 phylogenetic group/resistance gene is listed, all isolates were of the same group and contained the same resistance gene/s. aD1/blaCMY-2; bD1/blaSHV-12; cA1/blaCTX-M; dD2/blaCMY-2; eB1/blaSHV-2; f B1/blaCTX +blaCMY-2; gB1/blaCMY-2; hA1/blaCTX-M-1; iA1/blaSHV-12; jB1/blaSHV+blaCMY-2; kB23/blaCTX-M. x,yDifferent letters indicated statistical significance in ESBL detection between flocks at the 0.05 level using the Bonferroni's correction. zSame letter indicated that flocks did not differ significantly in AmpC detection from each other at the 0.05 level using the Bonferroni's correction. View Large Table 4. Incidences, Phylogenetic Groups, and Extended-Spectrum Beta-Lactamases (ESBL)/AmpC Resistance Genes of Escherichia coli Isolates From Broiler Flocks.   All samples  Flock A/1  Flock A/2  Flock B/1  Flock B/2  Sampling time  ESBL1  AmpC2  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Before housing  0/8  0/8  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/13  3/13  +1a  +1a  +1b  +2c  +2d  +2d  –  –  –  –  –  –  Week 5  3/12  3/12  +1e  +1e  +1e  –  +2d  –  –  +2d  –  –  +2d  –    All samples  Flock A/1  Flock A/2  Flock B/1  Flock B/2  Sampling time  ESBL1  AmpC2  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Before housing  0/8  0/8  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/13  3/13  +1a  +1a  +1b  +2c  +2d  +2d  –  –  –  –  –  –  Week 5  3/12  3/12  +1e  +1e  +1e  –  +2d  –  –  +2d  –  –  +2d  –  1ESBL-producing E. coli positive samples/number of samples tested. 2AmpC-producing E. coli positive samples/number of samples tested. 3On arrival, meconium on chicken box liners was examined. At each visit during the fattening period, 2 pairs of boot swabs were collected per flock and pooled. 4Four liver samples per pool; in flock B/1, besides pooled liver sample 1 heart sample was tested at week 1. +Positive for ESBL- or AmpC-producing E. coli. –Negative for ESBL- or AmpC-producing E. coli. Samples were not taken. a–ePhylogenetic group and resistance gene of isolate; aD1/blaSHV-12; bA1/blaCTX-M; cD1/blaCMY-2; dD2/blaCMY-2; eB1/blaSHV-12. View Large Table 4. Incidences, Phylogenetic Groups, and Extended-Spectrum Beta-Lactamases (ESBL)/AmpC Resistance Genes of Escherichia coli Isolates From Broiler Flocks.   All samples  Flock A/1  Flock A/2  Flock B/1  Flock B/2  Sampling time  ESBL1  AmpC2  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Before housing  0/8  0/8  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/13  3/13  +1a  +1a  +1b  +2c  +2d  +2d  –  –  –  –  –  –  Week 5  3/12  3/12  +1e  +1e  +1e  –  +2d  –  –  +2d  –  –  +2d  –    All samples  Flock A/1  Flock A/2  Flock B/1  Flock B/2  Sampling time  ESBL1  AmpC2  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Air  Feces3  Organs4  Before housing  0/8  0/8  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/13  3/13  +1a  +1a  +1b  +2c  +2d  +2d  –  –  –  –  –  –  Week 5  3/12  3/12  +1e  +1e  +1e  –  +2d  –  –  +2d  –  –  +2d  –  1ESBL-producing E. coli positive samples/number of samples tested. 2AmpC-producing E. coli positive samples/number of samples tested. 3On arrival, meconium on chicken box liners was examined. At each visit during the fattening period, 2 pairs of boot swabs were collected per flock and pooled. 4Four liver samples per pool; in flock B/1, besides pooled liver sample 1 heart sample was tested at week 1. +Positive for ESBL- or AmpC-producing E. coli. –Negative for ESBL- or AmpC-producing E. coli. Samples were not taken. a–ePhylogenetic group and resistance gene of isolate; aD1/blaSHV-12; bA1/blaCTX-M; cD1/blaCMY-2; dD2/blaCMY-2; eB1/blaSHV-12. View Large The surveillance performed in our study showed an increase in ESBL/AmpC-producing E. coli in all 3 production types of poultry over the sampling period. In the breeder flocks, ESBL/AmpC-producing E. coli were detected in 2 flocks at week 4 and at 12 wk of age. AmpC-producing E. coli were also detected in the third flock. Two flocks were positive at the end of the observation period, at week 36 (Table 3). In broilers and turkeys, the first ESBL/AmpC-producing E. coli were detected in 3 of 8 flocks in the first week. At week 5, all 4 broiler flocks became positive (Table 4 and 5). Similar findings have been reported in studies on broiler and turkey farms in Germany, Great Britain, and the Netherlands [5, 33, 35], although those investigations were carried out on farms with a history of ESBL/AmpC-producing E. coli. Table 5. Number, Phylogenetic Group, and Extended-Spectrum Beta-Lactamases (ESBL) Resistance Genes of Escherichia coli Isolates Obtained From Different Samples in Meat-Type Turkey Flocks.   All samples  Flock D  Flock E  Flock F  Flock G  Sampling time  ESBL1  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Before housing  0/9  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/17  –  –  –  –  –  –  –  –  –  +a  +a  +b  Week 5  2/12  /  –  –  /  –  –  /  –  –  /  +a  +b  Week 9  3/17  –  –  –  +b  –  –  –  –  –  +b  –  +b  Week 16  4/15  /  –  –  /  –  –  /  –  –  /  +b  +b*    All samples  Flock D  Flock E  Flock F  Flock G  Sampling time  ESBL1  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Before housing  0/9  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/17  –  –  –  –  –  –  –  –  –  +a  +a  +b  Week 5  2/12  /  –  –  /  –  –  /  –  –  /  +a  +b  Week 9  3/17  –  –  –  +b  –  –  –  –  –  +b  –  +b  Week 16  4/15  /  –  –  /  –  –  /  –  –  /  +b  +b*  1ESBL-producing E. coli positive samples/number of samples tested. 2On arrival, meconium on chicken box liners was examined, in the fattening period 2 pairs of boot swabs per pool were tested. +Positive for ESBL-producing E. coli. –Negative for ESBL-producing E. coli. /Samples were not taken. a,bPhylogenetic group and resistance gene of isolate; aA0/blaCTX-M;bA1/blaSHV-12. *Three organ samples, e.g., pooled liver sample, tarsometatarsal joints and subcutis were positive for ESBL-producing E. coli. View Large Table 5. Number, Phylogenetic Group, and Extended-Spectrum Beta-Lactamases (ESBL) Resistance Genes of Escherichia coli Isolates Obtained From Different Samples in Meat-Type Turkey Flocks.   All samples  Flock D  Flock E  Flock F  Flock G  Sampling time  ESBL1  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Before housing  0/9  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/17  –  –  –  –  –  –  –  –  –  +a  +a  +b  Week 5  2/12  /  –  –  /  –  –  /  –  –  /  +a  +b  Week 9  3/17  –  –  –  +b  –  –  –  –  –  +b  –  +b  Week 16  4/15  /  –  –  /  –  –  /  –  –  /  +b  +b*    All samples  Flock D  Flock E  Flock F  Flock G  Sampling time  ESBL1  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Air  Feces2  Organs  Before housing  0/9  /  –  –  /  –  –  /  –  –  /  –  –  Week 1  3/17  –  –  –  –  –  –  –  –  –  +a  +a  +b  Week 5  2/12  /  –  –  /  –  –  /  –  –  /  +a  +b  Week 9  3/17  –  –  –  +b  –  –  –  –  –  +b  –  +b  Week 16  4/15  /  –  –  /  –  –  /  –  –  /  +b  +b*  1ESBL-producing E. coli positive samples/number of samples tested. 2On arrival, meconium on chicken box liners was examined, in the fattening period 2 pairs of boot swabs per pool were tested. +Positive for ESBL-producing E. coli. –Negative for ESBL-producing E. coli. /Samples were not taken. a,bPhylogenetic group and resistance gene of isolate; aA0/blaCTX-M;bA1/blaSHV-12. *Three organ samples, e.g., pooled liver sample, tarsometatarsal joints and subcutis were positive for ESBL-producing E. coli. View Large The proportion of ESBL-producing E. coli among all E. coli is needed to estimate the occurrence of ESBL populations in chicken flocks and other areas of production [7]. In our study, E. coli were found in 87.09% of samples taken during the production period. ESBL-producing E. coli were detected in 22.04% of samples, while AmpC-producing isolates were detected in 10.75% of samples, although initially the number of phenotypically confirmed isolates was higher due to the presence of inhibitor-resistant beta-lactamase TEM-32. Similar detection frequencies of ESBL- and AmpC-producing E. coli were found in organs of dead or culled birds, in feces and in air samples (Table 6). The fact that all 3 types of samples were positive at the same sampling time indicated a high rate of contamination and rapid spread of these strains among the birds within one poultry house. Table 6. Frequencies of Detection of E. coli, and Extended-Spectrum Beta-Lactamases (ESBL)- and AmpC-producing E. coli in Different Types of Samples. Type of sample  Number of E. coli-positive samples/number of samples tested (%)  Number of ESBL-positive samples/number of samples (%)  Number of AmpC-positive samples/number of samples (%)  Feces (boot swabs, meconium)  62/69 (89.86)  13/69 (18.84)  9/69 (13.04)  Organs of dead or culled animals  70/82 (85.36)  17/82 (20.73)  5/82 (6.09)  Air  30/35 (85.71)  11/35 (31.42)  6/35 (17.14)  Total (%1)  162/186 (87.09)  41/186 (22.04)  20/186 (10.75)  Type of sample  Number of E. coli-positive samples/number of samples tested (%)  Number of ESBL-positive samples/number of samples (%)  Number of AmpC-positive samples/number of samples (%)  Feces (boot swabs, meconium)  62/69 (89.86)  13/69 (18.84)  9/69 (13.04)  Organs of dead or culled animals  70/82 (85.36)  17/82 (20.73)  5/82 (6.09)  Air  30/35 (85.71)  11/35 (31.42)  6/35 (17.14)  Total (%1)  162/186 (87.09)  41/186 (22.04)  20/186 (10.75)  1Percentage of positive samples. View Large Table 6. Frequencies of Detection of E. coli, and Extended-Spectrum Beta-Lactamases (ESBL)- and AmpC-producing E. coli in Different Types of Samples. Type of sample  Number of E. coli-positive samples/number of samples tested (%)  Number of ESBL-positive samples/number of samples (%)  Number of AmpC-positive samples/number of samples (%)  Feces (boot swabs, meconium)  62/69 (89.86)  13/69 (18.84)  9/69 (13.04)  Organs of dead or culled animals  70/82 (85.36)  17/82 (20.73)  5/82 (6.09)  Air  30/35 (85.71)  11/35 (31.42)  6/35 (17.14)  Total (%1)  162/186 (87.09)  41/186 (22.04)  20/186 (10.75)  Type of sample  Number of E. coli-positive samples/number of samples tested (%)  Number of ESBL-positive samples/number of samples (%)  Number of AmpC-positive samples/number of samples (%)  Feces (boot swabs, meconium)  62/69 (89.86)  13/69 (18.84)  9/69 (13.04)  Organs of dead or culled animals  70/82 (85.36)  17/82 (20.73)  5/82 (6.09)  Air  30/35 (85.71)  11/35 (31.42)  6/35 (17.14)  Total (%1)  162/186 (87.09)  41/186 (22.04)  20/186 (10.75)  1Percentage of positive samples. View Large The only genes detected in the study were blaSHV-12, blaCMY-2, and blaCTX-M, which were detected in 15.4%, 12.3%, and 9.8% of E. coli strains isolated during the breeding period, respectively (Tables 3–5). These results are in line with reports from other countries [7, 33]. However, TEM-52, which, along with SHV-12, dominates in some European countries, was not detected in our study nor in previous studies that include screening of poultry meat for ESBL-producing E. coli (unpublished results). The relative proportion of blaSHV-12, blaCMY-2, and blaCTX-M genes differed between flocks. In some flocks, only 1 ESBL and/or AmpC gene was detected throughout the whole sampling period (e.g., blaSHV-12/blaCMY-2 in flock A or blaCTX-M/blaCMY-2 in flock B). Interestingly, these genes were harbored by E. coli assigned to different phylogenetic groups, thus anticipating the entry of different E. coli strains into the flocks from one or more unknown sources or horizontal gene transfer events between E. coli strains within most flocks. Vertical transmission was presumably involved in flock G, where blaSHV-12-encoding E. coli belonging to phylogenetic group A1 could be isolated from the first week onwards (Table 5). ESBL/AmpC-producing isolates belonged predominantly to phylogenetic group A1 (36.2%), followed by group D1 (24.1%), B1 (22.4%), and D2 (10.3%). Only 3 isolates from air and boot swabs (blaCTX-M) in flock G were from phylogenetic group A0 and 1 boot swab isolate from flock B was from phylogenetic group B23, which is rarely found among poultry, indicating a possible single externally sourced contamination event. An association between particular E. coli phylogroups and ESBL or AmpC genes could not be detected in our study [36]. The relative high proportion of ESBL/AmpC-producing E. coli from human pathogenic groups D1, D2, and B23 in air and fecal samples could pose a risk for people working on the farms. In the extensive study carried out in the Netherlands, a significantly greater prevalence of ESBL/AmpC-producing E. coli was seen among people working on broiler farms compared with the general population or patients. Moreover, an increased risk of transfer has been detected among individuals with a high level of contact with live poultry [4]. Antibiotic treatment can have a substantial influence on the prevalence of ESBL/AmpC-producing E. coli [33, 37–39]. The use of antibiotics in the flocks in our study was limited. Only 1 turkey flock was treated with enrofloxacin. The treatment was needed at the end of the observation period (at 112 d old) and, therefore, had no influence on our studies of the prevalence of ESBL-producing E. coli. Among breeder flocks, only flock B received sulfamonomethoxine, with trimethoprim in the first week and oxytetracycline after placement on the production farm. The frequency of detection of ESBL/AmpC seen in this flock was comparable to the untreated flock A. The lowest detection frequency of ESBL/AmpC was in untreated flock C, in which coccidiostats were included in the feed during the rearing period (Table 3). These results could suggest that the intake of antibiotics was less important for the occurrence/selection of ESBL/AmpC E. coli strains than horizontal or vertical transmission of resistance genes between E. coli in the flocks included in our study. In an investigation of broiler farms positive for ESBL/AmpC-producing E. coli, Laube et al. [6] detected ESBL-producing E. coli on boot swabs taken from the area around poultry houses as well as in ambient air samples at a distance of 50 m. The broiler breeder houses included in our study were located on larger farms, so it is very likely that airborne emission from the houses’ surroundings could take place. However, the fact that fattening flocks were placed in houses isolated from other poultry farms does not support such an explanation. Other sources such as rodents, wild birds, waste water, contaminated slurry, or flies could also have been responsible [40]. CONCLUSIONS AND APPLICATIONS Over the fattening period an increase of ESBL/AmpC-producing E. coli can be expected within the flock, although high hygienic measurements are implemented before the placement of the chicks. The entry of different E. coli carrying ESBL/AmpC determinants and rapid spread of these strains among the birds within 1 poultry house was confirmed. ESBL/AmpC-producing E. coli from human pathogenic groups D1, D2, and B23 in air and fecal samples could pose a risk for people working on the farms. 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PLoS One  10: e0135402. Google Scholar CrossRef Search ADS PubMed  Acknowledgements This work was financially supported by Slovene Ministry of Agriculture, Forestry and Food and Slovene Research Agency. All poultry companies and poultry veterinarians are gratefully acknowledged for their contributions to this study. © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Applied Poultry ResearchOxford University Press

Published: May 15, 2018

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