Transmission of Extended-Spectrum Cephalosporin Resistant Escherichia coli from Poultry Production Systems and Slaughterhouses – a Food Safety Perspective

Extended-spectrum cephalosporins (ESCs) are classified as critically important antimicrobial agents by the World Health Organization (WHO). Resistance to these extended-spectrum cephalosporins (ESCs) is a global threat to both veterinary and human medicine. Resistance in Enterobacteriaceae, such as Escherichia coli, is caused by two main groups of inactivating enzymes: Extended Spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamase. In Europe, ESC-resistant E. coli has been reported in poultry products and poultry meat, even in the absence of the use of antimicrobials in poultry production environments. The work of this thesis aimed to fill in knowledge gaps about ESC-resistant E. coli isolates in the poultry production systems and slaughterhouses in Denmark, the transmission of ESC-resistant E. coli isolates and their antibiotic genes, and the risk of these isolates becoming potential pathogens in humans.

In Paper 1, the aim was to assess the prevalence and transmission of ESC-resistant E. coli in the Danish poultry production system and slaughterhouses during 2015-2018. A total of 162 ESC-resistant E. coli isolates were obtained and whole-genome sequenced (WGS) for characterization of their various multi-locus sequence types (MLST), resistance genes, virulence genes, and plasmid replicon types. We found that bla_CMY-2 carrying ESC-resistant E. coli isolates from ST429 existed both in production systems and slaughterhouses. Also, the findings indicate that ESC-resistant E. coli isolates are transferred vertically from farms in the production systems to slaughterhouses with the potential to enter the food supply.

In Paper 2, we aimed to continue to characterize IncK-bla_CMY‐2-positive ESC-resistant E. coli isolates from poultry production systems in Denmark (Paper 1) and other European countries (Finland and Germany), as well as from Danish human blood infections using long-reads and short-reads sequencing. We also further compared their IncK-bla_CMY‐2 plasmids with publicly available databases. This analysis indicated the existence of common sources of ST429 IncK-bla_CMY‐2 E. coli in poultry and poultry meat from Denmark, Finland, and Germany. Moreover, IncK-bla_CMY‐2 and their carriers appeared to have been circulating in the Danish production chain with an associated risk of spreading to humans.

In Paper 3, a selection of 30 ESC-resistant E. coli isolates, chosen to represent the ESC-resistant E. coli diversity observed in Paper 1, were assessed for their ability to invade human epithelial cells and environmental fitness in the context of poultry production, as well as analyzed for their virulence gene profiles to explore correlations between genotypes and phenotypes. The results show that the phenotypes and genotypes of these isolates are highly variable among individual isolates and cannot predicted based on virulence gene traits. Of interest, ST162 and ST429 were shown to have higher invasion rates and environment fitness (i.e., highest biofilm formation and growth rates at temperatures ≥10 °C), indicating a higher pathogenicity potential compared to other STs ESC-resistant E. coli poultry isolates.

In conclusion, this thesis has contributed to our understanding of the prevalence and transmission of ESC-resistant E. coli in the poultry production chain in Denmark. These findings suggest vertical transmission of clones and horizontal transmission are possible from the poultry production pyramid to humans from a food production chain perspective. While ESC-resistant E. coli isolates in general are unlikely to become potential pathogens, evidence presented in Paper 2 points to cases where poultry associated isolates may have caused human illness. Notably, this sequence type (ST429) was shown to be globally distributed (Paper 2) and also exhibited the highest invasiveness and environmental fitness (Paper 3). Hence, while current surveillance data is showing a decline in the occurrence of ESC-resistant E. coli in the Danish poultry industry, it is important to continue the strict industry biosecurity measures, and surveillance and monitoring to avoid the occurrence and spread of such resistance in the food-production chain to ensure food is safe for consumers.