The emergence of extended-spectrum cephalosporin (ESC)-resistant Escherichia coli is a global concern. This study aimed to assess the prevalence and transmission of ESC-resistant E. coli in the Danish broiler production system. Samples from two vertically integrated Production Systems (1 and 2) and two slaughterhouses (A and B) were analyzed (n = 943) for the occurrence of ESC-resistant E. coli from 2015 to 2018. ESC-resistant E. coli isolates were whole-genome sequenced (WGS) for characterization of the multi-locus sequence type (MLST), antibiotic resistance genes, virulence genes, and plasmid replicon types. An ad hoc core genome (cg) MLST based on 2513 alleles was used to examine the genetic relatedness among isolates. The prevalence of ESC-resistant E. coli in the conventional Production System 1 was 2.7%, while in Production System 2 the prevalence was 26.7% and 56.5% for samples from the conventional and organic production, respectively. The overall prevalence of ESC-resistant E. coli in broiler thigh and fecal samples ranged from 19.3% in Slaughterhouse A to 22.4% in Slaughterhouse B. In total, 162 ESC-resistant E. coli were isolated and shown to belong to 16 different sequence types (STs). The most prevalent STs were ST2040 (n = 85) and ST429 (n = 22). Seven ESC resistance genes were detected: bla_CMY-2 (n = 119), bla_TEM-52B (n = 16), bla_CTX-M-1 (n = 5), bla_TEM-52C (n = 3), bla_CTX-M-14 (n = 1), bla_SHV-12 (n = 1), and up-regulation of ampC (n = 16), with an unknown resistance gene in one isolate (n = 1). The carriage of bla_CMY-2 in 119 isolates was primarily associated with IncI1 (n = 87), and IncK plasmids (n = 31). Highly similar bla_CMY-2 carrying E. coli isolates from ST429 were found in production systems as well as in slaughterhouses. In conclusion, findings from this study indicate that ESC-resistant E. coli are transferred vertically from farms in the production systems to slaughterhouses with the potential to enter the food supply.

Escherichia coli carrying IncK-bla_CMY-2 plasmids mediating resistance to extended-spectrum cephalosporins (ESC) has been frequently described in food-producing animals and in humans. This study aimed to characterize IncK-bla_CMY-2-positive ESC-resistant E. coli isolates from poultry production systems in Denmark, Finland, and Germany, as well as from Danish human blood infections, and further compare their plasmids. Whole-genome sequencing (Illumina) of all isolates (n = 46) confirmed the presence of the bla_CMY-2 gene. Minimum inhibitory concentration (MIC) testing revealed a resistant phenotype to cefotaxime as well as resistance to ≥3 antibiotic classes. Conjugative transfer of the bla_CMY-2 gene confirmed the resistance being on mobile plasmids. Pangenome analysis showed only one-third of the genes being in the core with the remainder being in the large accessory gene pool. Single nucleotide polymorphism (SNP) analysis on sequence type (ST) 429 and 1286 isolates showed between 0–60 and 13–90 SNP differences, respectively, indicating vertical transmission of closely related clones in the poultry production, including among Danish, Finnish, and German ST429 isolates. A comparison of 22 ST429 isolates from this study with 80 ST429 isolates in Enterobase revealed the widespread geographical occurrence of related isolates associated with poultry production. Long-read sequencing of a representative subset of isolates (n = 28) allowed further characterization and comparison of the IncK-bla_CMY-2 plasmids with publicly available plasmid sequences. This analysis revealed the presence of highly similar plasmids in ESC-resistant E. coli from Denmark, Finland, and Germany pointing to the existence of common sources. Moreover, the analysis presented evidence of global plasmid transmission and evolution. Lastly, our results indicate that IncK-bla_CMY-2 plasmids and their carriers had been circulating in the Danish production chain with an associated risk of spreading to humans, as exemplified by the similarity of the clinical ST429 isolate to poultry isolates. Its persistence may be driven by co-selection since most IncK-bla_CMY-2 plasmids harbor resistance factors to drugs used in veterinary medicine.