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.

Background/Objectives: Rapid surveillance tools are needed to monitor antimicrobial-resistant bacteria in the food chain. This study evaluated whether qPCR-based quantification of tetracycline resistance genes could replace culture-based quantification of tetracycline-resistant Escherichia coli in pig caecal content and carcass skin swabs. Methods: Caecal content (n = 89) and skin swab samples (n = 68) were collected across four Danish abattoirs and analysed by both qPCR and Petrifilm cultivation. Method comparisons were restricted to samples within the quantifiable range of Petrifilm. E. coli concentrations (uidA gene) and tetA/tetB gene concentrations were compared with Petrifilm counts of E. coli and culturable tetracycline-resistant E. coli. Method agreement was evaluated using paired t-tests, Bland–Altman analysis, and Spearman correlation. Results: In caecal samples (n = 84), no significant difference in mean log E. coli concentration was observed between uidA qPCR and Petrifilm counts (p = 0.33). However, Bland–Altman analysis showed poor agreement at the individual-sample level, with limits of agreement ranging from −1.09 to 1.21 log₁₀ CFU/g. In skin swab samples (n = 43), qPCR systematically overestimated E. coli counts compared with cultivation, with a significant bias of 0.75 log₁₀ CFU/1400 cm² (p < 0.001). No significant correlation was observed between tetA/tetB and culturable tetracycline-resistant E. coli in caecal (r_s = 0.091, p = 0.44) or skin swab samples (r_s = 0.13, p = 0.48). Conclusions: qPCR may be useful for population-level surveillance of tetracycline resistance genes in pig caecal samples but cannot replace culture-based methods for accurate quantification of tetracycline-resistant E. coli in individual samples.