Near-infrared in vivo imaging system for dynamic visualization of lung-colonizing bacteria in mouse pneumonia

Abstract

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.

Keywords: Acinetobacter baumannii; In vivo imaging; TokeOni; antibacterial therapy; bacterial pneumonia; near-infrared bioluminescence; scientific complementary metal-oxide semiconductor (sCMOS) camera.

Fig 1

Optimization of a NIR emission imaging system for bacterial pneumonia. (A) A bacterial solution (ATCC 17978) with OD₆₀₀ = 0.5 was prepared and mixed with an equal volume of saline for the LuxCDABE-expressing strain or an equal volume of 10 µM substrate for the Luc2- and Akaluc-expressing strains, and the luminescence signal was measured. Experiments were performed in triplicate. Mean values are shown and error bars indicate standard deviations (SDs). Immunodeficient mice were intratracheally administered with 5 × 10⁷ CFU/mouse luminescent-enzyme-expressing ATCC 17978 strains. In vivo imaging was conducted 24 h post-infection. Experiments were performed with three mice per group, and representative images are shown (B). The mean value of the luminescence signal obtained from all three images is shown (C). Means are indicated and error bars indicate SDs. (D) The linearity between the number of bacteria and luminescence signal was evaluated by mixing a 4-fold dilution series (OD₆₀₀ = 0.5 to 4⁻³) of the bacterial solution (strain ATCC 17978-Luc) for measuring the luminescence signal. Experiments were independently performed in triplicate. Means are shown and error bars indicate SDs. (E) Immunodeficient mice were intratracheally administered with 1 × 10⁷ CFU/mouse of the ATCC 17978-Luc strain, and the bacterial load in the lungs was assessed. At 24 h post-infection, in vivo imaging quantification of lung-colonization bacteria was conducted using TokeOni. Experiments were performed on nine mice. Red dots represent plots of individuals for which luminescence was detected, and crosses indicate plots of individuals for which luminescence was not detected. Mean values are shown and error bars indicate SDs. ***P < 0.001, **P < 0.01, *P < 0.05 (One-way ANOVA and Dunnett’s test; A, C), n.s., not significant.

Fig 2

Temporal in vivo imaging of the number of lung-colonizing bacteria using TokeOni. (A) Immunodeficient mice were intratracheally administered ATCC 17978-Luc strain at a density of 5 × 10⁷ CFU/mouse, intraperitoneally administered TokeOni at the indicated time points post-infection, and in vivo imaging was performed. Experiments were performed on three mice per group. Representative images are shown in (B). Each signal extracted from all images was plotted for each individual imaging time point (C). The red symbols represent the results at the last time point that imaging was possible. (D) Immunodeficient mice were intratracheally administered 5 × 10⁷ CFU/mouse of the ATCC 17978-Luc strain, followed by intraperitoneal administration of TokeOni at 4, 24, and 48 h post-infection; in vivo imaging was then conducted. Six animals were used for each time point. At 48 h post-infection, two mice had died; hence, the experiment was performed with four surviving mice. The number of bacteria colonizing the lungs was measured after imaging at each time point, and the results were plotted alongside the corresponding image signals. The black, blue, and red symbols represent the results at 4, 24, and 48 h, respectively. The correlation between the luminescence signal in the lungs and the number of bacteria colonizing the lungs was evaluated. Correlation analysis was also performed using Pearson correlation coefficients, and the R² and P values are shown on the graph.

Fig 3

In vivo imaging for efficacy evaluation of antibacterial drugs using TokeOni. (A) Immunodeficient mice were intratracheally administered the ATCC 17978-Luc strain at a density of 5 × 10⁷ CFU/mouse. Subsequently, they were intraperitoneally administered TokeOni at the indicated time points, followed by in vivo imaging. Saline or imipenem/cilastatin (IPM/CS) was intraperitoneally administered at the indicated time points post-infection. Experiments were performed on five mice per group. (B) Kaplan–Meier plots. Representative images obtained via imaging are presented in (C). The signal obtained from all images was plotted for each imaging time point (D). The red symbols represent the results at the final imaging time point. ** P < 0.01 (Log-rank test; B).

Fig 4

Evaluation of the number of lung-colonizing bacteria of A. baumannii clinical isolates and its application to the assessment of the therapeutic efficacy of antimicrobial agents. Clinical isolates of A. baumannii (NR1127-Luc, a carbapenem-sensitive strain, and NR4001-Luc, a carbapenem-resistant strain) were intratracheally administered to immunodeficient mice at 5 × 10⁷ and 1 × 10⁸ CFU/mouse, respectively. TokeOni was then administered intraperitoneally for in vivo imaging, and saline (i.p.), imipenem/cilastatin (IPM/CS) (i.p.), or levofloxacin (LVFX) (s.c.) was administered for treatment at the indicated time points post-infection. Experiments were performed using five mice per group (A). Kaplan–Meier plot of the NR1127-Luc strain (B) and NR4001-Luc strain (E). Representative images of the NR1127-Luc strain (C) and NR4001-Luc strain (F) acquired using in vivo imaging. The signal obtained from all images of the NR1127-Luc strain (D) and NR4001-Luc strain (G) was plotted for each time point. Red symbols represent the results at the final imaging time points.

Fig 5

In vivo imaging of the clearance process of lung-colonizing bacteria in immunocompetent hosts. Immunocompetent mice were intratracheally administered ATCC 17978-Luc strain at a density of 5 × 10⁸ CFU/mouse. Subsequently, TokeOni was intraperitoneally administered at the indicated time points, followed by in vivo imaging. Experiments were performed on eight mice. Representative images of both surviving and deceased mice are shown in (A). The signal (P/sec/cm²/sr) obtained from all images was plotted for each time point (B). Black lines represent changes over time in surviving mice, whereas blue lines represent changes over time in deceased mice. Red dots indicate the results at the last imaging time point.

Fig 6

In vivo imaging of lung-colonizing bacteria in K. pneumoniae pneumonia. (A) Immunocompetent mice were intratracheally administered the Kp-GNLuc strain at a density of 5 × 10⁸ CFU/mouse. TokeOni was then administered intraperitoneally for in vivo imaging, and saline or imipenem/cilastatin (IPM/CS) was intraperitoneally administered for treatment at the indicated time points post-infection. Experiments were performed using five mice per group. (B) Kaplan–Meier plots. Representative images obtained via imaging are shown in (C). The signal obtained from all images was plotted for each time point (D). Red symbols represent the results at the final imaging time points. ** P < 0.01 (Log-rank test; B).