Ultrasound Imaging of Engineered Bacteria Colonizing Tumors and the Gastrointestinal Tract

Genetically engineered bacteria have the potential to outperform traditional diagnostics and therapeutics. However, current tools to non-invasively monitor their activity and spatial distribution in vivo are limited. Optical imaging methods suffer from the poor penetration depth of light in tissue, while nuclear imaging methods rely on radioactive tracers. On the other hand, ultrasound is a widely available imaging modality that does not use ionizing radiation and has tissue penetration depth of several centimeters. Recently, the first genetically encoded ultrasound contrast agents based on gas vesicles were developed to link ultrasound to gene expression in bacteria. However, these first-generation acoustic reporter genes (ARGs) expressed too poorly under in vivo conditions to enable ultrasound imaging of bacteria in therapeutically relevant contexts.

Here we present a new and improved ARG construct that produces high levels of robust gas vesicle expression in the probiotic bacterium E. coli Nissle (EcN). This second-generation ARG construct, bARG_Ser, uses genes derived from Serratia sp. ATCC 39006 and was optimized for expression in EcN. We demonstrate that with bARG_Ser, we can use ultrasound to visualize the spatial distribution of engineered EcN after they home to and colonize tumors upon systemic administration. We also demonstrate that the engineered EcN can be imaged with ultrasound when colonizing the gastrointestinal tract and can be used to non-invasively report on biomarkers of gastrointestinal inflammation. By enabling monitoring of engineered probiotic bacteria inside the body, this technology could greatly improve the development and eventual clinical use of this emerging class of microbial cell-based theranostics.