Near-infrared
in vivo
imaging system for dynamic visualization of lung-colonizing bacteria in mouse pneumonia
Authors: Yamaguchi Daiki , Kamoshida Go , Kawakubo Syun , Azuma Saki , Tsuji Takamitsu , Kitada Nobuo , Saito-Moriya Ryohei , Yamada Noriteru , Tanaka Rentaro , Okuda Ayane , Ueyama Keisuke , Isaka Shingo , Tomita Manaha , Nakano Ryuichi , Morita Yuji , Yano Hisakazu , Maki Shojiro A. , Yahiro Kinnosuke , Kato Shinichi 
Published: September 2024

In vivo
imaging of bacterial infection models enables noninvasive and temporal analysis of individuals, enhancing our understanding of infectious disease pathogenesis. Conventional
in vivo
imaging methods for bacterial infection models involve the insertion of the bacterial luciferase LuxCDABE into the bacterial genome, followed by imaging using an expensive ultrasensitive charge-coupled device (CCD) camera. However, issues such as limited light penetration into the body and lack of versatility have been encountered. We focused on near-infrared (NIR) light, which penetrates the body effectively, and attempted to establish an
in vivo
imaging method to evaluate the number of lung-colonizing bacteria during the course of bacterial pneumonia. This was achieved by employing a novel versatile system that combines plasmid-expressing firefly luciferase bacteria, NIR substrate, and an inexpensive, scientific complementary metal-oxide semiconductor (sCMOS) camera. The D-luciferin derivative “TokeOni,” capable of emitting NIR bioluminescence, was utilized in a mouse lung infection model of
Acinetobacter baumannii
, an opportunistic pathogen that causes pneumonia and is a concern due to drug resistance. TokeOni exhibited the highest sensitivity in detecting bacteria colonizing the mouse lungs compared with other detection systems such as LuxCDABE, enabling the monitoring of changes in bacterial numbers over time and the assessment of antimicrobial agent efficacy. Additionally, it was effective in detecting
A. baumannii
clinical isolates and
Klebsiella pneumoniae
. The results of this study are expected to be used in the analysis of animal models of infectious diseases for assessing the efficacy of therapeutic agents and understanding disease pathogenesis.

IMPORTANCE

Conventional animal models of infectious diseases have traditionally relied upon average assessments involving numerous individuals, meaning they do not directly reflect changes in the pathology of an individual. Moreover, in recent years, ethical concerns have resulted in the demand to reduce the number of animals used in such models. Although
in vivo
imaging offers an effective approach for longitudinally evaluating the pathogenesis of infectious diseases in individual animals, a standardized method has not yet been established. To our knowledge, this study is the first to develop a highly versatile
in vivo
pulmonary bacterial quantification system utilizing near-infrared luminescence, plasmid-mediated expression of firefly luciferase in bacteria, and a scientific complementary metal-oxide semiconductor camera. Our research holds promise as a useful tool for assessing the efficacy of therapeutic drugs and pathogenesis of infectious diseases.