Recombinant autobioluminescent Escherichia coli to monitor the progression of Escherichia coli infection in the embryonated chicken eggs

Avian pathogenic Escherichia coli (APEC) infections in poultry adversely affect health and production, with public health implications. This study assessed the potential of bioluminescence imaging for real-time, noninvasive tracking of microbial progression in 12-day-old chicken embryos inoculated with an APEC strain or its derivatives integrated either with luxABCDE or ilux2 operon. Eggs were imaged daily for bioluminescence detection, with dead embryos sampled immediately and survivors killed at 5 days post inoculation (dpi). The eggs were opened, and egg contents were imaged for bioluminescence. Yolks were sampled for E. coli isolation and quantification. Results showed lethality rates of 100%, 93.3% and 80% in embryos inoculated with native strain, luxABCDE or ilux2, respectively. Bioluminescence analysis showed increased bioluminescence signal strength over time preceding embryo death. Surviving embryos exhibited a sequential reduction in signal strength. A strong positive correlation was found between bioluminescence signal intensity in ovo and ex ovo, with ilux2-APEC infected eggs showing a higher luminoscore than luxABCDE-APEC. The E. coli load in yolks of APEC-inoculated eggs showed a positive trend over time. Overall, bioluminescence imaging of ilux2 operon labelled bacteria enabled more efficient real-time detection and monitoring of E. coli in ovo. Multiple imaging sessions on the same embryo throughout the experiment allow precise monitoring of infection progression without sequential culling. This offers a controlled platform for evaluating antimicrobial treatments’ efficacy in an in ovo model that closely resembles in vivo environment in chickens. It also bears the potential to study other pathogens’ infection patterns, especially those pose risks to public health. © 2025 The Author(s).