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

Abstract

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.

1. Introduction

Bacteria exhibiting antimicrobial resistance (AMR), such as fluoroquinolone-resistant Escherichia coli and penicillin-resistant Streptococcus pneumoniae, have emerged due to the 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 ^99mTc-based derivatives, ^99mTc-labeled antibiotics, ¹¹C-labeled D-amino acids, and 2-deoxy-2-[¹⁸F]fluoro-d-glucose (¹⁸F-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-(4-¹²³I-iodophenyl)-3(R,S)-methylpentadecanoic acid (¹²³I-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 ¹⁸F-FDG, which is already widely used in imaging.

2. Materials and Methods

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

2.1. Bacterial Strain and Culture Conditions

Escherichia coli EC-14 was used as the clinical isolate strain. EC-14 was obtained from Shionogi & Co., Ltd. (Osaka, Japan). For pre-cultivation, stock EC-14 in 50% glycerol was mixed with THY medium consisting of Todd-Hewitt Broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and 0.2% yeast extract (Becton, Dickinson and Co.) at a ratio of 1:100. EC-14 was incubated at 37 °C for 12–14 h with shaking. Subsequently, EC-14 (1.2 × 10⁸ colony-forming units (CFU)/100 µL) was seeded in amino-acid-free Dulbecco’s Modified Eagle’s Medium (DMEM; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan; 048-33575) and incubated at 37 °C with shaking. Bacterial protein was measured using a Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA; 23227).

2.2. Accumulation of Radiopharmaceuticals in E. coli EC-14

EC-14 (1.2 × 10⁸ 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 ¹²³I-BMIPP and ¹⁸F-FDG was added and incubated for 5 min at 37 °C with shaking. For in vitro experiments using ¹⁸F-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 ¹⁸F-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).

2.3. Accumulation of ¹²³I-BMIPP in E. coli EC-14 under Low-Temperature Conditions

EC-14 (1.2 × 10⁸ 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 ¹²³I-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 ¹²³I-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 ¹²³I-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.

2.5. 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 × 10⁶ 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.

2.6. Biological Distribution of ¹²³I-BMIPP and ¹⁸F-FDG in E. coli EC-14 Infection Model Mice

EC-14 (approximately 5 × 10⁶ 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 ¹²³I-BMIPP and 10–17 MBq/90 µL of ¹⁸F-FDG. The mice injected with ¹²³I-BMIPP and ¹⁸F-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 ¹²³I-BMIPP- and ¹⁸F-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).

2.7. Planar Imaging with ¹²³I-BMIPP in E. coli EC-14 Infection Model Mice

At 8 h post-infection, ¹²³I-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 ¹²³I-BMIPP administration and 9.5 h after EC-14 infection. Images were analyzed using AMIDE data analysis software (ver. 1.0.4).

2.8. 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.

3. Results

3.1. Accumulation of Radiopharmaceuticals in E. coli EC-14

Table 1. Accumulation of radiopharmaceuticals in E. coli EC-14.

	Accumulation (%ID/ng Protein)
Culture Time (h)	123I-BMIPP	18F-FDG (0.1 mg/mL Glucose Concentration)
1	6.65 ± 0.49	6.32 ± 2.31
3	3.52 ± 0.20	15.9 ± 1.37
6	2.68 ± 0.23	11.8 ± 0.49

%ID/ng protein indicates percent injected dose per ng of protein.

shows the accumulation of ¹²³I-BMIPP and ¹⁸F-FDG in EC-14 5 min after each addition. ¹²³I-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. ¹⁸F-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 ¹²³I-BMIPP was higher than that of ¹⁸F-FDG, whereas it was lower at culture times of 3 and 6 h. The accumulation of ¹²³I-BMIPP in EC-14 5 min after injection and the growth curve of EC-14 are shown in Figure 1. ¹²³I-BMIPP exhibited marked accumulation in E. coli EC-14 during the early growth phase.

3.2. Accumulation of ¹²³I-BMIPP in E. coli EC-14 under Low-Temperature Conditions

The accumulation of ¹²³I-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.

3.3. Accumulation of ¹²³I-BMIPP in E. coli EC-14 in the Presence of a CD36 Inhibitor

Figure 3 summarizes the accumulation of ¹²³I-BMIPP in E. coli EC-14 incubated in the presence of a CD36 inhibitor. Under these conditions, ¹²³I-BMIPP accumulated in EC-14 at 10.8%ID/ng protein at 1 h of incubation, 2.41%ID/ng protein at 3 h and 1.07%ID/ng protein at 6 h. The difference between the control and SSO treatment at 1 h of incubation was not significant. At 3 and 6 h of incubation, however, the rate of ¹²³I-BMIPP accumulation was significantly lower than that of the control.

3.4. Growth of E. coli EC-14 in Infection Model Mice

Figure 4 shows a growth curve for EC-14 in the leg muscle of model mice. On average, approximately 1.9 × 10⁶ CFU of EC-14 were present in the muscle tissue 2 h after infection, and the number of bacteria increased to 4.9 × 10⁸ and 1.8 × 10¹⁰ CFU at 8 and 24 h after infection, respectively.

3.5. Biological Distribution of ¹²³I-BMIPP and ¹⁸F-FDG in EC-14 Infection Model Mice

Table 2. Biological distribution of ¹²³I-BMIPP in E. coli EC-14 infection model mice.

Accumulation of 123I-BMIPP (%ID/g)
Time after Infection (h)	2		8
Time after 123I-BMIPP Injection (min)	15	60	15	60
Blood	16.69 ± 0.93	16.10 ± 1.88	13.65 ± 3.57	19.43 ± 2.91
Heart	25.86 ± 6.46	29.11 ± 4.48	30.37 ± 3.90	30.66 ± 6.40
Lung	13.02 ± 2.00	11.84 ± 1.02	11.99 ± 1.10	12.01 ± 1.65
Liver	17.31 ± 3.86	11.04 ± 1.92	20.84 ± 3.32	12.02 ± 1.91
Kidney	14.01 ± 1.30	12.18 ± 1.27	12.00 ± 1.32	11.79 ± 1.88

%ID/g indicates percent injected dose per gram of tissue.

and

Table 3. Biological distribution of ¹⁸F-FDG in E. coli EC-14 infection model mice.

Accumulation of 18F-FDG (%ID/g)
Time after Infection (h)	2		8
Time after 18F-FDG Injection (min)	15	60	15	60
Blood	2.83 ± 0.90	0.69 ± 0.09	0.93 ± 0.11	0.18 ± 0.03
Heart	13.95 ± 5.47	15.05 ± 3.35	30.02 ± 7.33	28.57 ± 5.98
Lung	2.91 ± 0.76	2.96 ± 1.04	3.44 ± 0.39	4.04 ± 0.88
Liver	2.95 ± 1.07	1.10 ± 0.26	2.81 ± 0.49	2.10 ± 0.65
Kidney	5.43 ± 1.95	2.15 ± 0.52	5.77 ± 1.55	2.71 ± 0.67

%ID/g indicates percent injected dose per gram of tissue.

summarize the biological distribution of ¹²³I-BMIPP and ¹⁸F-FDG, respectively, in EC-14 infection model mice. In comparison with the accumulation of ¹⁸F-FDG at 2 and 8 h after infection, the accumulation of ¹²³I-BMIPP was higher in the blood, lung, liver and kidney.

Table 4. Accumulation of ¹²³I-BMIPP and ¹⁸F-FDG in E. coli EC-14 infection model mice.

After Infection (h)	After Injection (min)		123I-BMIPP		18F-FDG
			Accumulation (%ID/g)	Contrast	Accumulation (%ID/g)	Contrast
2	15	Infected	6.09 ± 3.72	1.15	1.69 ± 0.50	1.05
		Control	5.30 ± 0.72		1.61 ± 0.53
	60	Infected	6.13 ± 1.14	1.00	2.47 ± 0.69	0.97
		Control	6.10 ± 1.03		2.54 ± 0.81
8	15	Infected	6.82 ± 2.02 *	1.29	4.25 ± 0.92	0.93
		Control	5.29 ± 0.63		4.58 ± 1.53
	60	Infected	8.64 ± 1.80 *	1.31	3.14 ± 1.16	1.03
		Control	6.59 ± 0.80		3.05 ± 1.93

%ID/g indicates percent injected dose per gram of tissue. * p < 0.01 vs. control muscle at about 8 h after infection.

summarizes the accumulation of ¹²³I-BMIPP and ¹⁸F-FDG in the EC-14-infected muscle and normal, uninfected muscle (control). The accumulation of both ¹²³I-BMIPP and ¹⁸F-FDG was higher in the control mice than in the infected mice. ¹²³I-BMIPP accumulation tended to be higher than that of ¹⁸F-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 ¹²³I-BMIPP in E. coli EC-14 Infection Model Mice

Figure 5 shows the planar images acquired 1.5 h after ¹²³I-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.

4. 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 ¹²³I-BMIPP, a long-chain fatty acid analog used for myocardial fatty acid metabolism scintigraphy in Japan [11,12,13,14], for bacterial imaging. ¹²³I-BMIPP exhibited marked accumulation in E. coli EC-14 during the early growth phase (Figure 1), similar to the accumulation of ¹⁸F-FDG (Table 1). This specific accumulation suggests that EC-14 actively metabolizes fatty acids. In addition, the accumulation of ¹²³I-BMIPP in EC-14 was higher than that of ¹⁸F-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 ¹⁸F-FDG, and the accumulation of ¹⁸F-FDG increased the most at the culture time of 3 h.

The mechanism of ¹²³I-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 ¹²³I-BMIPP. This result indicates that the accumulation of ¹²³I-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 ¹²³I-BMIPP was significantly reduced at 3 and 6 h of incubation compared with the control. This suggests that the mechanism of ¹²³I-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 long-chain 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 ¹²³I-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 ¹²³I-BMIPP is sensitive to transporter selection. In addition, SSO inhibits the mitochondrial respiratory chain, and reduced mitochondrial activity may reduce ¹²³I-BMIPP uptake. This is particularly important at later time points where significance is shown. The possibility that ¹²³I-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 ¹²³I-BMIPP uptake by bacteria.

Biological distribution was examined in the E. coli EC-14 infection model mice using ¹²³I-BMIPP (Table 2) and ¹⁸F-FDG (Table 3). Compared with ¹⁸F-FDG, the accumulation of ¹²³I-BMIPP in the blood, lung, liver, and kidney was higher at 2 and 8 h post-infection, indicating that ¹²³I-BMIPP has a longer retention time than ¹⁸F-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]. ¹²³I-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 ¹²³I-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 ¹⁸F-FDG after exercise has been reported [19]. One can assume that this may have led to the increased ¹⁸F-FDG uptake in the heart. In addition, ¹²³I-BMIPP accumulation was higher than that of ¹⁸F-FDG in non-infected control muscle (Table 4) because ¹²³I-BMIPP may have had a greater impact on walking and/or running than ¹⁸F-FDG during the time between the infection and sacrifice of the mice. ¹²³I-BMIPP exhibited a tendency toward greater imaging contrast at both 2 and 8 h after infection compared with ¹⁸F-FDG. A significant increase in accumulation in the infection area was observed at 8 h after infection (Table 4). Since ¹²³I-BMIPP accumulation reportedly decreases in myocarditis [20], it can be inferred that ¹²³I-BMIPP accumulated in the bacteria rather than in the inflamed tissues in this study.

¹²³I-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 ¹²³I-BMIPP injection and 9.5 h after infection exhibited higher contrast than the non-infected control muscle. The infected area, as determined by the ¹²³I-BMIPP signal, was approximately 1.33 times larger than the ¹²³I-BMIPP control area. Analysis of the biodistribution of ¹²³I-BMIPP 8 h after infection (Table 4) showed that the ratio of ¹²³I-BMIPP accumulation in the infected muscle to that in control muscle was 1.29 at 15 min and 1.31 at 60 min after ¹²³I-BMIPP injection. Thus, the lead shield had a negligible effect on planar imaging.

Clinical SPECT imaging in humans may show less ¹²³I-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 ¹²³I-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. ¹²³I-BMIPP accumulates in normal muscle, but it has better contrast ratios than ¹⁸F-FDG at sites of bacterial infection (Table 4).

5. Conclusions

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