Recombinant auto-bioluminescent <i>Escherichia coli</i> to monitor the progression of <i>Escherichia coli</i> infection in the embryonated chicken eggs

Avian pathogenic <i>Escherichia coli</i> (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 <i>lux</i>ABCDE or <i>ilux2</i> 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 <i>E. coli</i> isolation and quantification. The results showed lethality rates of 100%, 93.3%, and 80% in embryos inoculated with native strain, <i>lux</i>ABCDE, or <i>ilux2,</i> 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 <i>in ovo</i> and <i>ex ovo</i>, with <i>ilux2-</i>APEC-infected eggs showing a higher luminoscore than <i>lux</i>ABCDE-APEC. The <i>E. coli</i> load in yolks of APEC-inoculated eggs showed a positive trend over time. Overall, bioluminescence imaging of <i>ilux2</i> operon-labelled bacteria enabled more efficient real-time detection and monitoring of <i>E. coli in ovo</i>. 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 <i>in ovo</i> model that closely resembles an <i>in vivo</i> chicken model. It can also be used to study infection patterns of other pathogens, especially those that pose risks to public health.<b>RESEARCH HIGHLIGHTS</b>Bioluminescence 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 <i>ilux2</i>-APEC showed a higher luminoscore than <i>lux</i>ABCDE-APEC in inoculated embryonated chicken eggs.The <i>in ovo</i> bioluminescent signal from intact eggs effectively reflects the <i>ex ovo</i> signal following the take out of yolk and embryo.