123I-BMIPP, a Radiopharmaceutical for Myocardial Fatty Acid Metabolism Scintigraphy, Could Be Utilized in Bacterial Infection Imaging

In this study, we evaluated the use of 15-(4-123I-iodophenyl)-3(R,S)-methylpentadecanoic acid (123I-BMIPP) to visualize fatty acid metabolism in bacteria for bacterial infection imaging. We found that 123I-BMIPP, which is used for fatty acid metabolism scintigraphy in Japan, accumulated markedly in Escherichia coli EC-14 similar to 18F-FDG, which has previously been studied for bacterial imaging. To elucidate the underlying mechanism, we evaluated changes in 123I-BMIPP accumulation under low-temperature conditions and in the presence of a CD36 inhibitor. The uptake of 123I-BMIPP by EC-14 was mediated via the CD36-like fatty-acid-transporting membrane protein and accumulated by fatty acid metabolism. In model mice infected with EC-14, the biological distribution and whole-body imaging were assessed using 123I-BMIPP and 18F-FDG. The 123I-BMIPP biodistribution study showed that, 8 h after infection, the ratio of 123I-BMIPP accumulated in infected muscle to that in control muscle was 1.31 at 60 min after 123I-BMIPP injection. In whole-body imaging 1.5 h after 123I-BMIPP administration and 9.5 h after infection, infected muscle exhibited a 1.33-times higher contrast than non-infected muscle. Thus, 123I-BMIPP shows potential for visualizing fatty acid metabolism of bacteria for imaging bacterial infections.


Introduction
Bacteria exhibiting antimicrobial resistance (AMR), such as fluoroquinolone-resistant Escherichia coli and penicillin-resistant Streptococcus pneumoniae, have emerged due to the Pharmaceutics 2022, 14 inappropriate use of antimicrobial agents [1][2][3]. It is predicted that, as the number of bacteria resistant to existing antimicrobial agents increases, conventional treatment methods will no longer be applicable, and treatment will thus become more difficult. The World Health Organization has advocated the development of international AMR countermeasure action plans detailing the investigation and monitoring of AMR trends, the appropriate use of antimicrobial agents, and research and development strategies for new antimicrobial agents [4,5].
With the increasing awareness of the seriousness of infectious diseases involving AMR, molecular imaging techniques have attracted attention for bacterial infection imaging. Nuclear medicine imaging is particularly useful as a new non-invasive diagnostic method that can be used to identify the site of an infection and follow up on the effects of treatment [6,7]. Various radiopharmaceuticals have been investigated for bacterial infection imaging. The development and application of radiopharmaceuticals, such as 99m Tc-based derivatives, 99m Tc-labeled antibiotics, 11 C-labeled D-amino acids, and 2-deoxy-2-[ 18 F]fluoro-D-glucose ( 18 F-FDG), which target components of the bacterial cell wall, are still in progress [6,8]. These compounds include not only carbon sources (D-glucose), nitrogen sources (amino acids), and minerals but also fatty acids, which are essential for bacterial growth [9,10]. However, to date, no radiopharmaceutical for bacterial infection imaging that targets fatty acid metabolism has been developed. We examined the use of 15-(4-123 I-iodophenyl)-3(R,S)-methylpentadecanoic acid ( 123 I-BMIPP), a radiopharmaceutical commonly used in single-photon emission computed tomography (SPECT) myocardial fatty acid metabolism scintigraphy in Japan, for bacterial imaging in comparison with 18 F-FDG, which is already widely used in imaging.

Materials and Methods
123 I-BMIPP was purchased from Nihon Medi-Physics Co., Ltd. (Tokyo, Japan). 18 F-FDG was synthesized at the PET facility of Kanazawa University.

Accumulation of Radiopharmaceuticals in E. coli EC-14
EC-14 (1.2 × 10 8 CFU/100 µL) was seeded in 5 mL of amino-acid-free DMEM and incubated for 1, 3 and 6 h. After incubation, 37 kBq/10 µL of 123 I-BMIPP and 18 F-FDG was added and incubated for 5 min at 37 • C with shaking. For in vitro experiments using 18 F-FDG, the concentration of glucose in the medium was adjusted to 0.1 mg/mL, since among the three glucose concentrations examined (4.5, 1.0 and 0.1 mg/mL), the highest accumulation of 18 F-FDG was observed at a concentration of 0.1 mg/mL. EC-14 was collected by centrifugation at 7000× g for 10 min at 4 • C and then washed three times with 5 mL of phosphate-buffered saline (PBS; Medical & Biological Laboratories Co., Ltd., Aichi, Japan). Samples were suspended in 1 mL of 0.1 M NaOH and measured using a gamma counter (AccuFLEX ARC-γ7010, Aloka Medical, Tokyo, Japan).

Accumulation of 123 I-BMIPP in E. coli EC-14 under Low-Temperature Conditions
EC-14 (1.2 × 10 8 CFU/100 µL) was seeded in 5 mL of amino-acid-free DMEM and incubated under ice-cold conditions at 4 • C for 3 h. After incubation, 37 kBq/10 µL of 123 I-BMIPP was added to the solution, and they were incubated for 5 min at 4 • C with shaking. The radioactivity accumulated in the EC-14 was then measured as described in the Section 2.2. Accumulation of Radiopharmaceuticals in E. coli EC-14.
2.4. Accumulation of 123 I-BMIPP in E. coli EC-14 in the Presence of a CD36 Inhibitor EC-14 was cultured under the same conditions as described in Section 2.2. Accumulation of Radiopharmaceuticals in E. coli EC-14. After incubation at 37 • C, 37 kBq/50 µL of 123 I-BMIPP and 50 µL of 1.0 mM sulfosuccinimidyl oleate (SSO, Cayman Chemical, Ann Arbor, MI, USA), an inhibitor of CD36, were mixed and added to the cell culture. CD36 binds long-chain fatty acids and promotes their transport into cells. The radioactivity accumulated in EC-14 was measured using the same method as described in the Section 2.2.

Mouse Model of E. coli EC-14 Infection
All experiments were conducted in accordance with the ethical standards of our university (Animal Care Committee of Kanazawa University, AP-183983) and with international standards for animal welfare and institutional guidelines. EC-14 was cultured in Luria-Bertani broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) for 12 h and then seeded into new broth and incubated at 37 • C for 12-14 h with shaking. Subsequently, EC-14 was collected by centrifugation at 8000× g for 5 min at 4 • C and suspended for inoculation into mice. Male Jcl:ICR mice (n = 4) (4 weeks old; CLEA Japan, Tokyo, Japan) were purchased 7 days prior to the experiments and subjected to immunosuppression treatment with 150 mg/kg and 100 mg/kg of endoxan (Shionogi) at 4 days and 1 day prior to infection, respectively.
EC-14 (approximately 5 × 10 6 CFU/100 µL) was injected into the muscle of the hind leg of mice under anesthesia. Mice were anesthetized using a mixture of butorphanol tartrate, midazolam, and medetomidine hydrochloride (Fujifilm Wako Pure Chemical Corp., Osaka, Japan). Mice were euthanized at 2, 6, 8 and 24 h after infection. Subsequently, the infected muscle was collected and homogenized in PBS. The number of CFU in the homogenized samples was determined by dilution in PBS and plating on agar medium.

Biological Distribution of 123 I-BMIPP and 18 F-FDG in E. coli EC-14 Infection Model Mice
EC-14 (approximately 5 × 10 6 CFU/100 µL) was injected into the muscle of the hind leg of mice (n = 4) as described in the Section 2.5. At 2 and 8 h after infection, mice were injected intravenously with 370 kBq/110 µL of 123 I-BMIPP and 10-17 MBq/90 µL of 18 F-FDG. The mice injected with 123 I-BMIPP and 18 F-FDG were different individuals. Before administration, the mice were fasted for 4 h. Mice were euthanized at 15 and 60 min after administration, and blood, heart, lung, liver, kidney, infected perineal muscle, and contralateral non-infected perineal muscle (control muscle) tissues were collected. The collected organs were dissolved by adding 1.0 mL of solubilizer, Solvable (PerkinElmer, Waltham, MA, USA), and crushed with a disposable homogenizer, BioMasher ® (Nippi, Tokyo, Japan, 49118-71). These samples and initial 123 I-BMIPP-and 18 F-FDG-associated radioactivity were measured using a gamma counter (AccuFLEX ARC-8001; Hitachi Aloka Medical, Tokyo, Japan). In heart, lung and kidney, the radioactivity and weight of whole organs were measured. In liver, a part of the liver was cut out, and the radioactivity and weight were measured. Weight of whole liver was also measured, and the radioactivity of whole liver was calculated using radioactivity of a part of the liver. Data are reported as percent injected dose per gram of tissue (%ID/g). Imaging conditions were set as follows: matrix, 512 × 512 pixels; magnification, 100%. The mice were anesthetized, and a gamma camera (MiniCam, Inter Medical, Lübbecke, Germany) was used to image the mice.
Planar image was acquired for 5 min (1 frame) at 1.5 h after 123 I-BMIPP administration and 9.5 h after EC-14 infection. Images were analyzed using AMIDE data analysis software (ver. 1.0.4).

Statistical Analysis
Data are presented as means and standard deviation and analyzed using the F-test and Student's t-test. All analyses were conducted using GraphPad Prism 8 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). A p value of less than 0.05 was considered indicative of a statistically significant difference. Table 1 shows the accumulation of 123 I-BMIPP and 18 F-FDG in EC-14 5 min after each addition. 123 I-BMIPP accumulated in EC-14 at 6.65%ID/ng protein at 1 h of incubation, 3.52%ID/ng protein at 3 h and 2.68% ID/ng protein at 6 h. 18 F-FDG accumulated in EC-14 at 6.32%ID/ng protein at 1 h of incubation, 15.9%ID/ng protein at 3 h and 11.8%ID/ng protein at 6 h. At a culture time of 1h, the accumulation of 123 I-BMIPP was higher than that of 18 F-FDG, whereas it was lower at culture times of 3 and 6 h. The accumulation of 123 I-BMIPP in EC-14 5 min after injection and the growth curve of EC-14 are shown in Figure 1. 123 I-BMIPP exhibited marked accumulation in E. coli EC-14 during the early growth phase.

Planar Imaging with 123 I-BMIPP in E. coli EC-14 Infection Model Mice
At 8 h post-infection, 123 I-BMIPP (18.5 MBq/300 µL/mouse) was injected into the tail vein of EC-14 infection model mice under lead shielding from head to bladder (n = 3). Imaging conditions were set as follows: matrix, 512 × 512 pixels; magnification, 100%. The mice were anesthetized, and a gamma camera (MiniCam, Inter Medical, Lübbecke, Germany) was used to image the mice.
Planar image was acquired for 5 min (1 frame) at 1.5 h after 123 I-BMIPP administration and 9.5 h after EC-14 infection. Images were analyzed using AMIDE data analysis software (ver. 1.0.4).

Statistical Analysis
Data are presented as means and standard deviation and analyzed using the F-test and Student's t-test. All analyses were conducted using GraphPad Prism 8 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). A p value of less than 0.05 was considered indicative of a statistically significant difference. Table 1 shows the accumulation of 123 I-BMIPP and 18 F-FDG in EC-14 5 min after each addition. 123 I-BMIPP accumulated in EC-14 at 6.65%ID/ng protein at 1 h of incubation, 3.52%ID/ng protein at 3 h and 2.68% ID/ng protein at 6 h. 18 F-FDG accumulated in EC-14 at 6.32%ID/ng protein at 1 h of incubation, 15.9%ID/ng protein at 3 h and 11.8%ID/ng protein at 6 h. At a culture time of 1h, the accumulation of 123 I-BMIPP was higher than that of 18 F-FDG, whereas it was lower at culture times of 3 and 6 h. The accumulation of 123 I-BMIPP in EC-14 5 min after injection and the growth curve of EC-14 are shown in Figure  1. 123 I-BMIPP exhibited marked accumulation in E. coli EC-14 during the early growth phase.

Accumulation of 123 I-BMIPP in E. coli EC-14 under Low-Temperature Conditions
The accumulation of 123 I-BMIPP in E. coli EC-14 under low-temperature conditions is summarized in Figure 2. The accumulation rate was 0.28-fold lower than that of the control incubated at 37 • C.

Accumulation of 123 I-BMIPP in E. coli EC-14 under Low-Temperature Conditions
The accumulation of 123 I-BMIPP in E. coli EC-14 under low-temperature conditions is summarized in Figure 2. The accumulation rate was 0.28-fold lower than that of the control incubated at 37 °C. Figure 2. Accumulation of 123 I-BMIPP in E. coli EC-14 under low-temperature conditions. EC-14 was incubated at 4 °C in amino-acid-free DMEM for 3 h. After incubation, 123 I-BMIPP was added and incubated for 5 min at 4 °C. The accumulation rate of EC-14 at low temperature is significantly lower than that of the control at 37 °C.     The accumulation of 123 I-BMIPP in E. coli EC-14 under low-temperature conditions is summarized in Figure 2. The accumulation rate was 0.28-fold lower than that of the control incubated at 37 °C.     Figure 4 shows a growth curve for EC-14 in the leg muscle of model mice. On average, approximately 1.9 × 10 6 CFU of EC-14 were present in the muscle tissue 2 h after infection, and the number of bacteria increased to 4.9 × 10 8 and 1.8 × 10 10 CFU at 8 and 24 h after infection, respectively.

Growth of E. coli EC-14 in Infection Model Mice
Pharmaceutics 2022, 14, x FOR PEER REVIEW Figure 4 shows a growth curve for EC-14 in the leg muscle of model mice. O age, approximately 1.9 × 10 6 CFU of EC-14 were present in the muscle tissue 2 infection, and the number of bacteria increased to 4.9 × 10 8 and 1.8 × 10 10 CFU at 8 h after infection, respectively.  Tables 2 and 3 summarize the biological distribution of 123 I-BMIPP and 18 F-F spectively, in EC-14 infection model mice. In comparison with the accumulatio FDG at 2 and 8 h after infection, the accumulation of 123 I-BMIPP was higher in th lung, liver and kidney.    Table 4 summarizes the accumulation of 123 I-BMIPP and 18 F-FDG in the EC-14-infected muscle and normal, uninfected muscle (control). The accumulation of both 123 I-BMIPP and 18 F-FDG was higher in the control mice than in the infected mice. 123 I-BMIPP accumulation tended to be higher than that of 18 F-FDG at both 2 and 8 h post-infection, and the rate of accumulation in the infected muscle was significantly higher 8 h post-infection. In this study, contrast means the ratio of the infected area to the contralateral normal area.   3.6. Planar Imaging with 123 I-BMIPP in E. coli EC-14 Infection Model Mice Figure 5 shows the planar images acquired 1.5 h after 123 I-BMIPP injection and 9.5 h after infection with E. coli EC-14. The infected area was visualized, and the contrast was approximately 1.33 times higher than that of the control area.  Table 4 summarizes the accumulation of 123 I-BMIPP and 18 F-FDG in the EC fected muscle and normal, uninfected muscle (control). The accumulation of bo BMIPP and 18 F-FDG was higher in the control mice than in the infected mice. 123 I-B accumulation tended to be higher than that of 18 F-FDG at both 2 and 8 h post-inf and the rate of accumulation in the infected muscle was significantly higher 8 h p fection. In this study, contrast means the ratio of the infected area to the contralater mal area.  Figure 5 shows the planar images acquired 1.5 h after 123 I-BMIPP injection an after infection with E. coli EC-14. The infected area was visualized, and the contra approximately 1.33 times higher than that of the control area.  from head to bladder, planar images were acquired for 5 min (1 frame) at 1.5 h after 123 I-BMIPP administration and 9.5 h after EC-14 infection. The infected muscle (arrow of right legs) is visualized, and the contrast is approximately 1.33 times higher than that of the control muscle (left legs).

Discussion
In this study, we investigated bacterial imaging using a radiopharmaceutical in common clinical use for fatty acid metabolism scintigraphy. Since fatty acids are components of the bacterial cell membrane [9,10], we explored the use of 123 I-BMIPP, a long-chain fatty acid analog used for myocardial fatty acid metabolism scintigraphy in Japan [11][12][13][14], for bacterial imaging. 123 I-BMIPP exhibited marked accumulation in E. coli EC-14 during the early growth phase (Figure 1), similar to the accumulation of 18 F-FDG (Table 1). This specific accumulation suggests that EC-14 actively metabolizes fatty acids. In addition, the accumulation of 123 I-BMIPP in EC-14 was higher than that of 18 F-FDG at the culture time of 1h, whereas it was lower at the culture times of 3 and 6 h because glucose concentration was regulated to 0.1 mg/mL in the culture medium for in vitro study with 18 F-FDG, and the accumulation of 18 F-FDG increased the most at the culture time of 3 h.
The mechanism of 123 I-BMIPP accumulation in E. coli EC-14 was elucidated under the conditions of low temperature ( Figure 2) and incubation in the presence of SSO, a CD36 inhibitor ( Figure 3). In our group's study, we have previously confirmed the inhibitory effect of SSO on the uptake of BMIPP in cancer cells (data not shown). Therefore, SSO was used in this study because it might also be relevant to bacterial accumulation. The rate of accumulation in EC-14 under low-temperature conditions was significantly lower than that of the control incubated at 37 • C, potentially due to a reduction in bacterial metabolic activity at the lower temperature, thereby impeding the uptake of 123 I-BMIPP. This result indicates that the accumulation of 123 I-BMIPP in EC-14 depends on both bacterial growth and metabolic activity. In the presence of SSO, an inhibitor of the fatty acid transport membrane protein CD36 [15,16], the accumulation of 123 I-BMIPP was significantly reduced at 3 and 6 h of incubation compared with the control. This suggests that the mechanism of 123 I-BMIPP uptake by EC-14 is sensitive to SSO and involves a fatty acid transport membrane protein such as CD36, which is present in human cells. CD36 binds longchain fatty acids and promotes their transport into cells [17]. The lack of significance of the difference between the control and SSO treatment in the present study at 1 h of incubation was possibly due to a large measurement error. In addition, during the early stage of bacterial growth, other uptake mechanisms and/or metabolic activity could play a significant role in uptake. As the incubation time increased, the effect of SSO became more notable, which suggests that the bacterial uptake of 123 I-BMIPP is sensitive to transporter selection. In addition, during the early stage of bacterial growth, other uptake mechanisms and/or metabolic activity could play a significant role in uptake. As the incubation time increased, the effect of SSO became more notable, which suggests that the bacterial uptake of 123 I-BMIPP is sensitive to transporter selection. In addition, SSO inhibits the mitochondrial respiratory chain, and reduced mitochondrial activity may reduce 123 I-BMIPP uptake. This is particularly important at later time points where significance is shown. The possibility that 123 I-BMIPP is also taken up by bacteria through other pathways such as endocytosis cannot be excluded. However, we assume that the CD36-like uptake mechanism is at least one of the mechanisms of 123 I-BMIPP uptake by bacteria.
Biological distribution was examined in the E. coli EC-14 infection model mice using 123 I-BMIPP (Table 2) and 18 F-FDG (Table 3). Compared with 18 F-FDG, the accumulation of 123 I-BMIPP in the blood, lung, liver, and kidney was higher at 2 and 8 h post-infection, indicating that 123 I-BMIPP has a longer retention time than 18 F-FDG in the primary organs. As for the blood clearance result, the lack of clearance from blood was likely affected by proteins in the mouse body. Studies have shown that serum albumin plays a role in binding and transporting fatty acids in the blood [18]. 123 I-BMIPP, a derivative of fatty acid, binds to albumin in the mouse body, causing its retention in the blood. This may have resulted in the delayed excretion of 123 I-BMIPP in this study. Moreover, in the in vivo experiment, the mice were fully anesthetized and not moving at about 2 h post-infection, but at 8 h post-infection, the mice were physically active, and the radioactivity in the blood was thought to have been transferred to the myocardium. An increased myocardial accumulation of 18 F-FDG after exercise has been reported [19]. One can assume that this may have led to the increased 18 F-FDG uptake in the heart. In addition, 123 I-BMIPP accumulation was higher than that of 18 F-FDG in non-infected control muscle (Table 4) because 123 I-BMIPP may have had a greater impact on walking and/or running than 18 F-FDG during the time between the infection and sacrifice of the mice. 123 I-BMIPP exhibited a tendency toward greater imaging contrast at both 2 and 8 h after infection compared with 18 F-FDG. A significant increase in accumulation in the infection area was observed at 8 h after infection (Table 4). Since 123 I-BMIPP accumulation reportedly decreases in myocarditis [20], it can be inferred that 123 I-BMIPP accumulated in the bacteria rather than in the inflamed tissues in this study.
123 I-BMIPP imaging in the E. coli EC-14 infection model mice was performed with lead shielding from head to bladder ( Figure 5). In this study, the right leg of the mouse was the E. coli-infected side, and the left leg was the non-infected side (control); the contrast indicates the ratio of the infected area to the contralateral normal area. Thus, the image of one mouse shows both infected and non-infected sides at the same time. The infected muscle imaged 1.5 h after 123 I-BMIPP injection and 9.5 h after infection exhibited higher contrast than the non-infected control muscle. The infected area, as determined by the 123 I-BMIPP signal, was approximately 1.33 times larger than the 123 I-BMIPP control area. Analysis of the biodistribution of 123 I-BMIPP 8 h after infection (Table 4) showed that the ratio of 123 I-BMIPP accumulation in the infected muscle to that in control muscle was 1.29 at 15 min and 1.31 at 60 min after 123 I-BMIPP injection. Thus, the lead shield had a negligible effect on planar imaging.
Clinical SPECT imaging in humans may show less 123 I-BMIPP accumulation in control muscle than imaging in mice, because humans can rest before SPECT imaging. This may result in a higher image contrast at the infection site, making infected areas more clearly visible in clinical SPECT imaging.
As a limitation of this study, there may be difficulty when using 123 I-BMIPP to detect bacterial infections in the trunk of the body, because of its accumulation and retention, but it could be very useful in infections involving the upper and lower limbs. 123 I-BMIPP accumulates in normal muscle, but it has better contrast ratios than 18 F-FDG at sites of bacterial infection (Table 4).

Conclusions
123 I-BMIPP can take up E. coli EC-14 via a fatty acid transport membrane protein such as CD36 and accumulate by fatty acid metabolism. 123 I-BMIPP has the potential to visualize fatty acid metabolism in bacteria for bacterial infection imaging, especially in upper and lower limbs.