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. 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 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.禽致病性大肠杆菌（Avian pathogenic Escherichia coli, APEC）感染可对禽类健康与生产造成不利影响，同时兼具公共卫生意义。本研究评估了生物发光成像（bioluminescence imaging）用于实时、无创追踪12日龄鸡胚中微生物感染进程的潜力，实验所用鸡胚接种了APEC菌株，或其整合了luxABCDE操纵子、ilux2操纵子的衍生菌株。每日对鸡胚卵进行成像以检测生物发光信号，死亡胚胎即刻取样，存活胚胎则于接种后5天（dpi）处死后取样。剖解鸡胚卵后，对卵内容物进行生物发光成像。采集卵黄样本用于大肠杆菌分离与定量分析。结果显示，接种野生型菌株、luxABCDE-APEC、ilux2-APEC的胚胎致死率分别为100%、93.3%与80%。生物发光分析显示，在胚胎死亡前，生物发光信号强度随时间推移逐渐增强；存活胚胎的信号强度则呈逐步下降趋势。卵内（in ovo）与卵外（ex ovo）的生物发光信号强度呈显著正相关，且ilux2-APEC感染组卵的发光评分（luminoscore）高于luxABCDE-APEC感染组。接种APEC的鸡胚卵卵黄中的大肠杆菌载量随时间呈上升趋势。总体而言，经ilux2操纵子标记的细菌的生物发光成像可更高效地实现卵内大肠杆菌的实时检测与监测。本实验中对同一胚胎进行多次成像，可在无需反复剖杀的前提下精准追踪感染进程。该研究为在模拟体内鸡模型的卵内模型中评估抗菌治疗效果提供了可控平台。此外，该方法还可用于研究其他病原体的感染模式，尤其是那些具有公共卫生风险的病原体。 【研究亮点】 1. 生物发光成像可实现对受精鸡胚卵内生物发光标记APEC感染的长期实时无创追踪。 2. 死亡胚胎与存活胚胎的生物发光信号呈现截然不同的变化模式。 3. 接种ilux2-APEC的受精鸡胚卵的发光评分高于luxABCDE-APEC感染组。 4. 完整卵内的生物发光信号可有效反映取出卵黄与胚胎后的卵外信号。