Recombinant auto-bioluminescent 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. The 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 allowed precise monitoring of infection progression without sequential culling. This offers a controlled platform for evaluating antimicrobial treatment efficacy in an in ovo model that closely resembles an in vivo chicken model. It can also be used to study infection patterns of other pathogens, especially those that pose risks to public health.RESEARCH HIGHLIGHTSBioluminescence imaging enabled real-time, noninvasive tracking of a bioluminescent APEC infection in embryonated chicken eggs over time.Bioluminescence signals showed contrasting patterns for dead and surviving embryos.The ilux2-APEC showed a higher luminoscore than luxABCDE-APEC in inoculated embryonated chicken eggs.The in ovo bioluminescent signal from intact eggs effectively reflects the ex ovo signal following the take out of yolk and embryo.