International Journal of

Molecular Sciences
ArticleComparison of L- and D-Amino Acids for Bacterial Imaging inLung Infection Mouse Model
Yuka Muranaka 1 , Asuka Mizutani 2 , Masato Kobayashi 2 , Koya Nakamoto 1 , Miki Matsue 3 , Kodai Nishi 4 ,Kana Yamazaki 5 , Ryuichi Nishii 5 , Naoto Shikano 6 , Shigefumi Okamoto 2,7 and Keiichi Kawai 2,8, *
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Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, 5-11-80 Kodatsuno,
Kanazawa 920-1192, Ishikawa, Japan; yukarisa93@stu.kanazawa-u.ac.jp (Y.M.);
kou.nakamoto@sage.ocn.ne.jp (K.N.)
Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University,
5-11-80 Kodatsuno, Kanazawa 920-1192, Ishikawa, Japan; mizutani.a@staff.kanazawa-u.ac.jp (A.M.);
kobayasi@mhs.mp.kanazawa-u.ac.jp (M.K.); sokamoto@mhs.mp.kanazawa-u.ac.jp (S.O.)
Ishikawa Prefectural Institute of Public Health and Environmental Science, 1-11, Taiyogaoka,
Kanazawa 920-1154, Ishikawa, Japan; mmiikkii_0804@pref.ishikawa.lg.jp
Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto,Nagasaki 852-8523, Nagasaki, Japan; koudai@nagasaki-u.ac.jp
Department of Molecular Imaging and Theranostics, Institute for Quantum Medical Science, Quantum Life
and Medical Science Directorate, National Institutes for Quantum Science and Technology, 4-9-1 Anagawa,Inage, Chiba 263-8555, Chiba, Japan; yamazaki.kana@qst.go.jp (K.Y.); nishii.ryuichi@qst.go.jp (R.N.)
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami,
Inashiki 300-0394, Ibaraki, Japan; sikano@ipu.ac.jp
Advanced Health Care Science Research Unit, Innovative Integrated Bio-Research Core Institute for Frontier
Science Initiative, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Ishikawa, Japan
Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji 910-1193, Fukui, Japan
Correspondence: kei@mhs.mp.kanazawa-u.ac.jp; Tel.: +81-76-265-2527; Fax: +81-76-234-4366

Citation: Muranaka, Y.; Mizutani, A.;
Kobayashi, M.; Nakamoto, K.;
Matsue, M.; Nishi, K.; Yamazaki, K.;
Nishii, R.; Shikano, N.; Okamoto, S.;et al. Comparison of L- and D-Amino
Acids for Bacterial Imaging in Lung
Infection Mouse Model. Int. J. Mol.
Sci. 2022, 23, 2467. https://doi.org/
10.3390/ijms23052467
Academic Editor: Nicola Scichilone
Received: 7 February 2022
Accepted: 21 February 2022
Published: 23 February 2022
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Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/

Abstract: The effectiveness of L- and D-amino acids for detecting the early stage of infection in bacterial imaging was compared. We evaluated the accumulation of 3 H-L-methionine (Met), 3 H-D-Met,
3 H-L-alanine (Ala), and 3 H-D-Ala in E. coli EC-14 and HaCaT cells. Biological distribution wasassessed in control and lung-infection-model mice with EC-14 using 3 H-L- and D-Met, and 18 F-FDG.
A maximum accumulation of 3 H-L- and D-Met, and 3 H-L- and D-Ala occurred in the growth phaseof EC-14 in vitro. The accumulation of 3 H-L-Met and L-Ala was greater than that of 3 H-D-Met and
D-Ala in both EC-14 and HaCaT cells. For all radiotracers, the accumulation was greater in EC-14
than in HaCaT cells at early time points. The accumulation was identified at 5 min after injectionin EC-14, whereas the accumulation gradually increased in HaCaT cells over time. There was little
difference in biodistribution between 3 H-L-and D-Met except in the brain. 3 H-L- and D-Met weresensitive for detecting areas of infection after the spread of bacteria throughout the body, whereas
18 F-FDG mainly detected primary infection areas. Therefore, 11 C-L- and D-Met, radioisotopes thatdiffer only in terms of 3 H labeling, could be superior to 18 F-FDG for detecting bacterial infection inlung-infection-model mice.
Keywords: bacterial imaging; nuclear medicine imaging; bacterial infection; amino acids; methionine

1. Introduction
Bacterial infections remain a threat to human life, despite the advancement of medical
technology. In recent years, the appearance of drug-resistant strains of various pathogens including methicillin-resistant Staphylococcus aureus and fluoroquinolone-resistant Escherichia coli [1,2]has heightened the sense of urgency regarding treatment of infectious diseases. Bacterial
infections are caused by infection of the host with pathogenic bacteria, and the pathogenesis varies depending on the type of causative bacteria [3]. If there is a dysregulated host4.0/).

Int. J. Mol. Sci. 2022, 23, 2467. https://doi.org/10.3390/ijms23052467

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response, the bacterial infection can lead to severe sepsis, organ dysfunction, and ultimately
death [4,5].
Reducing the risk associated with infection by these bacteria requires a diagnostic
method able to detect a rapid increase in the growth activity of pathogenic bacteria. Bacterial
culture is the main method currently used to identify the bacterial type and thus enableestablishment of an effective treatment strategy [6]. Polymerase chain reaction (PCR) andother methods are also clinically available [7]; however, these are invasive because theyrequire specimens to be collected, and there is a delay before the results are available.
Nuclear medicine imaging is a noninvasive diagnostic method for whole-body scanning in a short time. Pathogen-specific imaging agents target the metabolism of carbohydrates, bacterial folate biosynthesis, iron transport systems, components of and selective binders to the bacterial cell wall, substrates for intracellular bacterial proteins, andantimicrobial peptides as specific bacterial pathways [8,9]. In the metabolism of carbohydrates and components of the bacterial cell wall, 2-deoxy-2-[18 F]fluoro-D-glucose (18 F-FDG)
and [S-methyl-11 C]-L-methionine (11 C-L-Met), radiotracers in clinical use for tumor imaging [10], have already been applied for bacterial imaging [11,12]. These nutrients include
carbon sources (D-glucose), nitrogen sources (amino acids), and minerals that are essential
requirements for bacterial growth [13]. Amino acids of organic nitrogen sources in particular are a major source of nutrients for bacteria and components of cell walls. Therefore, we
consider that amino acid imaging would enable the detection of increased growth activityof pathogenic bacteria. Among amino acids, L-amino acids are incorporated as nutrients inhuman cells, whereas D-amino acids are rarely used in human cells but are used as cell wallcomponents in bacteria [14]. The aim of this study was to compare the suitability of L- andD-amino acids for bacterial nuclear medicine imaging to detect the early stage of infection.
As we focused on L-methionine (L-Met) and D-Met, an optical isomer of L-Met, and α-[Nmethyl-11 C]-methylaminoisobutyric acid ([11 C]MeAIB), a chemical analog of alanine (Ala)in human tumor cells [15–17], L-Met, D-Met, L-Ala, and D-Ala were evaluated.
2. Materials and Methods[S-methyl-3 H]-L-Met (3 H-L-Met), [S-methyl-3 H]-D-Met (3 H-D-Met), [2, 3-3 H]-L-Ala (3 HL-Ala), and [2, 3-3 H]-D-Ala (3 H-D-Ala) (American Radiolabeled Chemicals, St. Louis, MO,
USA) were used in the in vitro studies. 2-Deoxy-2-[18 F]fluoro-D-glucose (18 F-FDG), which
was synthesized at our PET facility, was used in the in vivo biological distribution study.
2.1. Bacterial Strain and Culture Conditions
Escherichia coli EC-14 (Shionogi, Osaka, Japan) was isolated as the clinical isolate strain.
In pre-cultivation, EC-14 stock solutions with 50% glycerol were mixed with THY medium
consisting of Todd–Hewitt Broth (Becton, Dickinson and Company, Franklin Lakes, NJ,
USA) and 0.2% yeast extract (Becton, Dickinson and Company) in the ratio of 1:100 and incubated at 37 ◦ C for 12–14 h with shaking. After incubation, EC-14 (1.2 × 108 CFU/100 µL)
was seeded in Dulbecco’s modified Eagle’s medium (DMEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), which does not contain amino acids (amino-acid-freeDMEM) and incubated at 37 ◦ C with shaking, for the experiments. Bacterial protein wasmeasured using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham,
MA, USA).
2.2. Human Cell Line and Culture Conditions
The human keratinocyte cell line (HaCaT cells; Boukamp et al., 1988) [18] was used inaccumulation experiments. As normal cells are difficult to culture, HaCaT cells establishedfrom adult male skin were used after considering technical issues in the research setup.
HaCaT cells was cultured in DMEM containing 8% fetal bovine serum (FBS; Biosera, Kansas,
MO, USA) and 100 U/mL penicillin/100 µg/mL streptomycin/250 ng/mL amphotericin B(FUJIFILM Wako Pure Chemical Corporation) at 37 ◦ C in an atmosphere of <5% CO2 .

Int. J. Mol. Sci. 2022, 23, 2467

3 of 112.3. Accumulation of 3 H-L- and D-Met and Ala in E. coli EC-14
E. coli EC-14 at 1.2 × 108 CFU/100 µL were seeded in 5 mL of amino-acid-free DMEM
and incubated for 1, 2, 4, 6, 8, 12, and 24 h. After incubation, 37 kBq/10 µL of 3 H-L-Met, 3 HD-Met, 3 H-L-Ala, and 3 H-D-Ala were added to the bacterial solution and incubated for 5, 30,
and 60 min at 37 ◦ C with gentle shaking. After conducting this time-course experiment inwhich the bacterial incubation time was varied, we investigated the accumulation of 3 H-LMet and 3 H-D-Met in each of the E. coli EC-14 and HaCaT cells, for various incubation times
after 3 H-L-Met and 3 H-D-Met were added. EC-14 was incubated in amino-acid-free DMEM
for 4 h. EC-14 was then collected by centrifugation at 7000× g for 10 min at 4 ◦ C, washedthree times with 5 mL of phosphate-buffered saline (PBS; Medical & Biological Laboratories
Co., Ltd., Aichi, Japan), and lysed by 1 mL of 0.1 M NaOH. Five hundred microliters ofbacterial lysate was mixed with 5 mL of ULTIMA GOLD (Perkin Elmer, Waltham, MA,
USA) and the radioactivity of the mixture was measured using a liquid scintillation counter(LSC-5100; Hitachi Aloka Medical, Tokyo, Japan). In measuring the incorporation rate of
3 H-L- and D-Met into the protein fraction, another 190 µL of the bacterial lysate consistingof 0.1 M NaOH was mixed with 10 µL of 100 w/v% trichloroacetic acid (Nacalai Tesque,
Kyoto, Japan). After mixing, the precipitated protein was collected on a glass fiber filter(GC-50; Advantec Toyo Kaisha, Tokyo, Japan), washed three times with 1 mL of ice-cold 5%
trichloroacetic acid solution, and fixed by heating at 100 ◦ C for 1 h. The fixed protein was
mixed with 5 mL of ULTIMA GOLD and the radioactivity was measured and evaluated as
the incorporation rate into the protein fraction.
2.4. Accumulation of 3 H-L- and D-Met and Ala in HaCaT Cells
HaCaT cells were seeded at 5 × 104 per cell in a 24-well plate in DMEM and cultured
at 37 ◦ C in an atmosphere of 5% CO2 . For 3 H-L-Met, 3 H-D-Met, 3 H-L-Ala, and 3 H-D-Ala
accumulation assay, DMEM was removed and 450 µL of amino-acid-free DMEM wasadded to the HaCaT cells followed by incubation at 37 ◦ C for 10 min. We then added
37 kBq/50 µL of 3 H-L-Met, 3 H-D-Met, 3 H-L-Ala, or 3 H-D-Ala to the HaCaT cells, whichwere incubated at 37 ◦ C for 5, 30, and 60 min with gentle shaking. After incubation, thecells were washed twice with 500 µL of ice-cold amino-acid-free DMEM and lysed by 1 mL
of 0.1 M NaOH. Five hundred microliters of cell lysate was mixed with 5 mL of ULTIMAGOLD and the radioactivity of the mixture was measured by LSC-5100. The incorporation
rates of each radiotracer into the protein fraction were measured the same as that describedin the section “Accumulation of 3 H-L- and D-Met in E. coli EC-14”.
2.5. E. coli EC-14 Lung-Infection-Model Mice
All applicable institutional guidelines of Kanazawa University for the care and useof animals were followed. All procedures were performed in accordance with the ethicalstandards of Kanazawa University (Animal Care Committee of Kanazawa University,
AP-173851) and with international standards for animal welfare and institutional guidelines.
EC-14 was pre-cultured in Luria–Bertani (LB) broth (Becton, Dickinson and Company) for
12 h followed by seeding into new LB broth in the ratio of EC-14 solution (LB broth = 1:100)
and incubation at 37 ◦ C for 12–14 h with shaking. After pre-incubation, EC-14 was collectedby centrifugation at 8000× g for 5 min at 4 ◦ C and suspended in amino-acid-free DMEM
for inoculation to mice. Jcl:ICR mice (n = 3; male; age, 4 weeks; CLEA Japan, Tokyo, Japan)
were purchased 7 days prior to infection and treated intraperitoneally with 150 mg/kg and
100 mg/kg of Endoxan (Shionogi) at 4 days and 1 day prior to infection, respectively. The
lungs of mice were infected with EC-14 at 3–7 × 107 colony forming units (CFU)/80 µL
under isoflurane (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) anesthesia.
At 1, 2, 4, 8, and 24 h after infection, the infected mice were euthanized with isoflurane andthe lung tissue was collected and homogenized with PBS. CFUs in homogenized solution
were measured by serial dilution in PBS and plating on LB agar plates.

Int. J. Mol. Sci. 2022, 23, 2467

4 of 112.6. Biological Distribution of E. coli EC-14 Lung-Infection-Model Mice Using 3 H-L-Met,
3 H-D-Met, and 18 F-FDG

EC-14 at 3–7 × 107 CFU/80 µL was infected to the lung of immunosuppressed mice,as described in the “E. coli EC-14 lung-infection-model mice” section. At 4 and 24 h after
infection, 50 kBq/50 µL of 3 H-L-Met and 3 H-D-Met, and 17 MBq/90 µL of 18 F-FDG wereadministered intravenously to the mice (n = 4), which were fasted for 4–5 h prior toadministration. Mice were euthanized at 15 and 60 min after administration and the blood,
heart, lung, liver, spleen, pancreas, kidney, brain, bladder, infected lung, and contralateralnoninfected lung were collected. Organ samples were adjusted to approximately 100 mg
and mixed with 1 mL of ULTIMA GOLD, followed by the initial measurement of 18 F-FDG
radioactivity using a γ-counter (AccuFLEX ARC-8001; Hitachi Aloka Medical). The samples
were incubated overnight at room temperature, mixed with 3 mL of ULTIMA GOLD and100 µL of hydrogen peroxide, and then incubated for 1–2 more days. After incubation, theradioactivity of 3 H-L-Met and 3 H-D-Met was measured by LSC-5100.
Data are presented as means and standard deviation. p values were calculated using
the two-tailed unpaired Mann–Whitney t-test for comparison between two groups using
GraphPad Prism 8 statistical software (GraphPad Software, La Jolla, CA, USA). A p valueless than 0.01 or 0.05 was considered to indicate statistical significance.
3. Results
3.1. Accumulation of 3 H-L- and D-Met and -Ala in E. coli EC-14
The accumulation of 3 H-L-Met, 3 H-D-Met, 3 H-L-Ala, and 3 H-D-Ala in E. coli EC-14 at
5 min after each injection are shown in Figs 1 and 2. 3 H-L-Met and 3 H-D-Met accumulation
was greatest at 4 h after incubation (Figure 1). The accumulation of 3 H-L-Met and 3 H-D-Met
in the protein fraction was 4.4 and 2.4 nmol/g protein, respectively. 3 H-L-Ala accumulation
was greatest at 6 h after incubation (Figure 2). 3 H-D-Ala accumulation was greatest after
12 h of incubation, in the stationary phase, but the amount of accumulation was littlechanged at 4 h or later after incubation. The accumulation of 3 H-L-Ala and 3 H-D-Ala in
the protein fraction was 3.0 and 1.1 nmol/g protein, respectively.

Figure 1. Accumulation of 3 H-L-Met and 3 H-D-Met in E. coli EC-14 at 5 min after each injection.
EC-14 was incubated in amino-acid-free DMEM for 1, 2, 4, 6, 8, and 12 h. The accumulation of
3 H-L-Met and 3 H-D-Met was greatest at 4 h after incubation. The incorporation rate of 3 H-L-Met
was higher than that of 3 H-D-Met at all EC-14 incubation times.

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Figure 2. Accumulation of 3 H-L-Ala and 3 H-D-Ala in E. coli EC-14 at 5 min after each injection.
EC-14 was incubated in amino-acid-free DMEM for 1, 2, 4, 6, 8, and 12 h. The accumulation of
3 H-L-Ala was greatest at 6 h after incubation, whereas the accumulation of 3 H-D-Ala was greatest
after an incubation time of 12 h; however, there was little change in accumulation at 4 h or later
after incubation. The incorporation rate of 3 H-L-Ala was higher than that of 3 H-D-Ala at all EC-14
incubation times.

3.2. Accumulation of 3 H-L- and D-Met and -Ala in E. coli EC-14 and HaCaT Cells
The accumulation of 3 H-L- and D-Met in E. coli EC-14 and HaCaT cells at 5 min, 30 min,and 60 min after injection is shown in Figure 3. The accumulation of 3 H-L-Met in EC-14 at
5 min after incubation was 13.8 nmol/g protein including a protein fraction of 6.0 nmol/g
protein (Figure 3a), whereas that in HaCaT cells was 1.71 nmol/g protein including aprotein fraction of 0.19 nmol/g protein (Figure 3b). The accumulation of 3 H-L-Met in EC-14
was stable over 60 min, whereas that in HaCaT cells increased to 15.9 nmol/g protein at
60 min after injection. In contrast, at 5 min after injection, 3 H-D-Met showed 4.3 nmol/g
protein including a protein fraction of 1.90 nmol/g protein, whereas that in HaCaT cells
was 0.21 nmol/g protein including a protein fraction of 0.01 nmol/g protein. At 60 min
after injection, the accumulation of 3 H-D-Met in EC-14 increased to 10.3 nmol/g protein
and the accumulation in HaCaT cells was 1.28 nmol/g protein.

Figure 3. Accumulation of 3 H-L-Met and 3 H-D-Met in (a) E. coli EC-14 at 4 h after incubation and in(b) HaCaT cells at 5, 30, and 60 min after 3 H-L-Met and 3 H-D-Met injection. The accumulation of
3 H-L-Met and 3 H-D-Met was greater in EC-14 at 5 min and later after incubation than in HaCaT cells.
Similar incorporation rates of 3 H-L-Met and 3 H-D-Met were identified in accumulation into EC-14.

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The accumulation of 3 H-L- and D-Ala in E. coli EC-14 and HaCaT cells at 5 min,30 min, and 60 min after injection is shown in Figure 4. The accumulation of 3 H-L-Alain EC-14 at 5 min after incubation was 17.1 nmol/g protein, which was maintained over
60 min (Figure 4a), whereas that in HaCaT cells was 3.57 nmol/g protein at 5 min after
injection, which increased to 13.0 nmol/g protein at 60 min (Figure 4b). In comparison, theaccumulation of 3 H-D-Ala in EC-14 was 9.87 nmol/g protein at 5 min after injection, which
was maintained until 60 min; and that in HaCaT cells was 0.24 nmol/g protein at 5 minafter injection and 1.70 nmol/g protein at 60 min after injection.

Figure 4. Accumulation of 3 H-L-Ala and 3 H-D-Ala in (a) E. coli EC-14 at 4 h after incubation and(b) accumulation of 3 H-L-Ala and 3 H-D-Ala in HaCaT cells at 5, 30, and 60 min after 3 H-L-Met
and 3 H-D-Met injection. The accumulation of 3 H-L-Ala and 3 H-D-Ala in EC-14 at 5 min or later
after incubation was higher than that in HaCaT cells. Similar incorporation rates of 3 H-L-Ala and3 H-D-Ala were identified in accumulation into EC-14.

3.3. Growth Curve of E. coli EC-14 in Lung of Lung-Infection-Model Mice
The growth curve for E. coli EC-14 in lung-infection-model mice with E. coli EC-14 isshown in Figure 5. The average CFU of EC-14 was approximately 4.3 × 106 at 1 h after
infection, which increased to 1.4 × 107 and 3.7 × 109 at 4 and 24 h after infection, respectively.

Figure 5. Growth curve of E. coli EC-14 in lung-infection-model mice. EC-14 at 3–7 × 107 CFU/80 µL
was infected to the lung of mice. At 1, 2, 4, 8, and 24 h after infection, the mean CFU of EC-14 wasapproximately 4.3 × 106 , 9.2 × 106 , 1.4 × 107 , 7.8 × 107 , and 3.7 × 109 , respectively.

3.4. Biological Distribution of 3H-L-Met, 3H-D-Met, and 18F-FDG in EC-14 Lung-Infection-Model Mice
Table 1 shows the biological distribution of 3 H-L-Met in EC-14 lung-infection-model
mice. Compared with the noninfected control mice, the radioactivity in all harvested tissues
except the heart, brain, and bladder was significantly greater at 4 h after infection, 15 min
and 60 min after 3 H-L-Met injection. The ratios of lung to heart were significantly higher inInt. J. Mol. Sci. 2022, 23, 2467

7 of 11comparison with those of the controls at 4 h after infection, 60 min after 3 H-L-Met injection;
and also significantly higher in comparison with the controls at 24 h after infection, 15 min
and 60 min after 3 H-L-Met injection. The ratios of lung to liver were significantly higher
in comparison with those of the controls at 4 h after infection, 15 min after 3 H-L-Met
injection; and were lower at 24 h after infection, 15 min and 60 min after 3 H-L-Met injection.
The ratios of infected lung to control lung were higher at 4 h after infection than at 24 hafter infection.
Table 1. Biological distribution of 3 H-L-Met in lung-infection-model mice.
Time after 3 H-L-Met
Injection

15 minOrgan (%ID/g)
Blood
Heart
Lung
Liver
SpleenPancreas
KidneyBrain
Bladder
Lung/heart
Lung/liver
Infected lung /control lung

60 min15 min0.50 ± 0.43
1.89 ± 0.31
1.57 ± 0.26
2.09 ± 0.84
2.47 ± 0.63
12.47 ± 4.68
3.17 ± 0.49
0.51 ± 0.10
0.35 ± 0.27
0.83 ± 0.03
0.85 ± 0.38–

1.61 ± 0.82 *2.96 ± 0.96
4.16 ± 0.60 †3.16 ± 0.20 †7.06 ± 1.81 †22.01 ± 6.38 †5.13 ± 2.83 *2.80 ± 3.39
0.55 ± 0.36
1.51 ± 0.51
1.32 ± 0.20 *2.80

Control
0.50 ± 0.18
1.65 ± 0.74
1.49 ± 0.19
1.66 ± 0.53
1.39 ± 0.57
4.29 ± 4.68
1.67 ± 0.62
4.10 ± 4.61
0.20 ± 0.17
1.03 ± 0.39
0.94 ± 0.20–

60 min4 h after Infection
1.51±0.07 †3.74± 0.10 †4.06 ± 0.12 †4.74 ± 0.14 †6.09 ± 2.79 *21.69 ± 3.40 *5.92± 0.73 †1.11 ± 0.48
1.15± 0.42 *1.09 ± 0.02 †0.86 ± 0.03
2.58

15 min60 min24 h after Infection
2.74 ± 0.86 †1.35 ± 0.19
3.13 ± 1.09 *6.19 ± 0.93 †3.01 ± 0.55 †14.39 ± 2.53 †5.15 ± 0.40 †1.41 ± 0.17
0.46 ± 0.41
2.27 ± 0.47 †0.50 ± 0.13 *2.11

1.24 ± 0.86 *1.40 ± 0.27
2.47 ± 0.10 †9.43 ± 0.82 †3.23 ± 1.81
11.94 ± 0.94
4.43 ± 0.72 *1.36 ± 0.18 †0.46 ± 0.41 *1.82 ± 0.41 †0.26 ± 0.04 *1.57%ID/g indicates percent injected dose per gram of tissue. † p < 0.01 and * p < 0.05 compared with control at 15 min
or 60 min after injection of 3 H-L-Met.

Table 2 shows the biological distribution of 3 H-D-Met in EC-14 lung-infection-model
mice. Compared with the noninfected control mice, the radioactivity in all harvested tissues
except the heart and bladder was significantly greater in all infected mice. The ratios oflung to heart were significantly higher in comparison with those of the controls at 24 h after
infection, 15 min after 3 H-D-Met injection. The ratios of lung to liver were significantly
lower in comparison with those of the controls at 24 h after infection, 15 min and 60 min
after 3 H-D-Met injection. The ratios of infected lung to control lung were higher at 4 h after
infection than at 24 h after infection.
Table 2. Biological distribution of 3 H-D-Met in lung-infection-model mice.
Time after 3 H-D-Met
Injection

15 minOrgan (%ID/g)
Blood
Heart
Lung
Liver
SpleenPancreas
KidneyBrain
Bladder
Lung/heart
Lung/liver
Infected lung /control lung

60 min15 min0.27 ± 0.07
0.92 ± 0.31
1.02 ± 0.33
1.64 ± 0.80
2.01 ± 0.30
8.77 ± 3.45
3.10 ± 0.42
0.59 ± 0.13
0.70 ± 0.49
1.20 ± 0.50
0.68 ± 0.24–

1.71 ± 0.17 †2.32 ± 0.07 †2.87 ± 0.28 †3.01 ± 0.26 †4.70 ± 0.37 †19.14 ± 2.18 †8.79 ± 0.67 †1.21 ± 0.17 †1.94 ± 0.41 †1.24 ± 0.14
0.96 ± 0.10
2.84

Control
0.34 ± 0.14
1.01 ± 0.25
1.01 ± 0.10
1.14 ± 0.13
1.29 ± 0.25
7.70 ± 1.25
3.36 ± 0.89
0.44 ± 0.06
0.62 ± 0.36
1.06 ± 0.33
0.91 ± 0.20–

60 min4 h after Infection
1.09 ± 0.08 †2.18 ± 0.38 †2.52 ± 0.55 †3.80 ± 0.26 †6.78 ± 2.16 †16.56 ± 4.03 *8.15 ± 1.26 †1.28 ± 0.36 *0.69 ± 0.13
1.17 ± 0.23
0.67 ± 0.18
2.48

15 min60 min24 h after Infection
2.54 ± 0.83 †1.25 ± 0.09
2.34 ± 0.09 †4.32 ± 0.24 †2.06 ± 0.42 *12.07 ± 1.57 †9.11 ± 1.55 †0.92 ± 0.15 †2.01 ± 0.85 *1.88 ± 0.18 †0.54 ± 0.03 *2.31

2.27 ± 0.66 †1.28 ± 0.30
2.34 ± 0.62 †7.12 ± 0.75 †4.05 ± 1.12 *15.68 ± 3.51 *10.25 ± 1.43 †1.60 ± 0.15 †–1.88 ± 0.52
0.33 ± 0.10 *2.30%ID/g indicates percent injected dose per gram of tissue. † p < 0.01 and * p < 0.05 compared with control at 15 min
or 60 min after injection of 3 H-D-Met/ The bladders of some mice could not be collected.

Table 3 shows the biological distribution of 18 F-FDG in EC-14 lung-infection-model
mice. Compared with the noninfected control mice, the radioactivity in the lung, liver,spleen, and brain was significantly greater at 24 h after infection, 15 min and 60 min after
18 F-FDG injection. The ratios of lung to heart were significantly higher in comparison with

Int. J. Mol. Sci. 2022, 23, 2467

8 of 11those of the controls at 24 h after infection, 15 min and 60 min after 18 F-FDG injection. The
ratios of lung to liver were significantly higher in comparison with those of the controls
at 4 h and 24 h after infection and at 15 min after 18 F-FDG injection. The ratios of infectedlung to control lung were higher at 24 h after infection than at 4 h after infection.
Table 3. Biological distribution of 18 F-FDG in lung-infection-model mice.
Time after 18 F-FDG Injection
Organ (%ID/g)
Blood
Heart
Lung
Liver
SpleenPancreas
KidneyBrain
Bladder
Lung/heart
Lung/liver
Infected lung /control lung

15 min60 min15 min0.45 ± 0.09
11.23 ± 6.51
3.57 ± 0.80
0.99 ± 0.21
3.42 ± 0.47
1.69 ± 0.31
1.90 ± 0.51
6.39 ± 1.03
2.04 ± 1.19
0.43 ± 0.33
3.86 ± 1.60-

1.84 ± 0.85
18.28 ± 8.61
4.58 ± 0.91 †2.47 ± 0.54
3.44 ± 0.63 *1.64 ± 0.30
5.40 ± 2.77
6.97 ± 1.46
1.86 ± 0.87
0.36 ± 0.34
1.88 ± 0.31 †1.34

Control
1.98 ± 0.58
13.14 ± 7.34
3.41 ± 0.34
2.85 ± 1.39
2.68 ± 0.68
2.42 ± 1.57
5.97 ± 3.73
5.12 ± 2.35
1.50 ± 0.84
0.36 ± 0.22
1.34 ± 0.37-

60 min4 h after Infection
0.41 ± 0.06
17.07 ± 7.99
3.99 ± 0.71
0.89 ± 0.13
3.53 ± 1.24
1.61 ± 0.11
3.30 ± 1.82
5.11 ± 1.49
1.76 ± 1.13
0.30 ± 0.18
4.52 ± 0.84
1.12

15 min60 min24 h after Infection
2.59 ± 0.72
5.72 ± 3.23 *7.82 ± 1.21 †4.65 ± 0.94 †5.92 ± 2.90 †2.66 ± 1.79
6.69 ± 0.74
12.46 ± 1.53 †1.67 ± 1.13
1.72 ± 0.76 †1.71 ± 0.23 *2.29

0.49 ± 0.15
9.85 ± 5.13
9.31 ± 1.79 †2.34 ± 1.04 †9.86 ± 6.01 †2.16 ± 0.35 *3.14 ± 0.54 †11.51 ± 2.78 †1.15 ± 0.52 †4.49 ± 1.49
2.61

%ID/g indicates percent injected dose per gram of tissue. † p < 0.01 and * p < 0.05 compared with control at 15 min
or 60 min after injection of 18 F-FDG. The bladders of some mice could not be collected.

4. Discussion
In this study, we conducted in vitro and in vivo studies to compare the L- and D-aminoacid accumulation of bacterial infection. In the in vitro study, 3 H-L-Met and 3 H-D-Met
showed the maximum accumulation in EC-14 at 4 h after incubation, in the early growthphase (Figure 1). 3 H-L-Ala accumulation was greatest at 6 h after incubation, whereas
3 H-D-Ala accumulation was greatest at 12 h after incubation, but there was little change inaccumulation at 4 h or later after incubation (Figure 2). These findings suggest that L- andD-Met and Ala accumulation can indicate the growth activity of bacteria.
We compared the accumulation of 3 H-L-Met, 3 H-D-Met, 3 H-L-Ala, and 3 H-D-Ala
in EC-14 at 4 h after incubation and in HaCaT cells with different incubation times after
3 H-L-Met, 3 H-D-Met, 3 H-L-Ala, and 3 H-D-Ala injection (Figs 3 and 4). The accumulationof 3 H-L-Met and 3 H-L-Ala was greater than that of 3 H-D-Met and 3 H-D-Ala in both EC-14
and HaCaT cells. In both D-amino acids, the accumulation was higher in EC-14 than in
HaCaT cells. Avid accumulation was identified at 5 min after 3 H-L- and D-Met and 3 H-Land D-Ala injection in EC-14, whereas the accumulation gradually increased in HaCaT cells
over time. In EC-14, the proportion of accumulation of 3 H-L-Met and 3 H-L-Ala into theprotein fraction was similar to that of 3 H-D-Met and 3 H-D-Ala. Human cells generally takein L-amino acids and then synthesize proteins to produce energy but take in few D-aminoacids [19]. In contrast, the present results indicate that bacteria use both L-amino andD-amino acids. As D-amino acids are known to regulate the composition and strength of
peptidoglycan layers and cell wall reinforcement [15,20,21], it may be possible that bacteria
utilize both L-amino acids and D-amino acids as constituents. In addition, bacteria convert
L-amino acids to D-amino acids and reverse in the bacterial body using the enzyme andamino acid racemase [22]. Therefore, D-amino acids may be converted to L-amino acids inbacteria, where they are used for protein synthesis. 11 C-MeAIB, which is an analog of Ala,
showed little accumulation in an in vitro study with EC-14 (no data), possibly because of
the slight difference in its chemical structure compared with Ala. For this reason, we did
not use 3 H-L- or D-Ala in our biological distribution study.
Muscle-infection-model mice have generally been used in bacterial infection imaging
studies. However, we used lung-infection-model mice (Figure 5) for the reasons that sepsisand organ-dysfunction-model mice are ideal for the bacterial infection imaging conductedin this study and because the lung infection model is highly applicable to clinical practice.

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The results of the study confirmed that the growth of EC-14 increased in the lungs of themice with continuing time after infection.
Based on the results of the in vitro experiments, we conducted in vivo distribution
experiments because we considered that the high accumulation contrast of labeled aminoacids between E. coli and HaCaT cells could be applied to bacterial imaging. Using lunginfection-model mice, we conducted a biological distribution study in EC-14-infected-model
mice at two injection time points, 4 h after infection as the early post-infection period and
24 h after infection as the late post-infection period (Tables 1–3). When we comparedradiotracer accumulation in control mice and lung-infection-model mice at 4 h and 24 hafter infection, at 15 min after 3 H-L-Met injection, the radioactivity in all organs except theheart, brain, and bladder was significantly higher at 15 min after 3 H-L-Met injection thanat 60 min (Table 1). In the biological distribution study of 3 H-D-Met, the radioactivity of
all organs except the heart at 24 h after infection was significantly higher at 15 min after
3 H-D-Met injection than at 60 min (Table 2). Although there is usually little accumulationof D-Met in the brain, accumulation in the brain was greater at 4 h and 24 h after infection
compared with control mice. This finding suggests the spread of bacteria throughout the
body. We confirmed bacteria of some colonies and approximately 1 × 105 CFU in the blood
at 4 h and 24 h, respectively, after infection of immunosuppressed mice. A previous study
that introduced E. coli HB-101 into lung-infection-model mice in an immunosuppressedstate following suppression of Lipocalin 2 (an effector molecule of the innate immune
system) reported infection from the lung to other organs at 48 h after infection [23]. It canbe assumed that bacteria in the lungs spread to the whole body via the bloodstream [24].
Overall, there was little difference in biological distribution between 3 H-L-Met and3 H-D-Met except in the brain. Neumann et al. performed bacterial imaging using 11 CL-Met and D-Met in a murine myositis model at 12 h after infection of E. coli strain
ATCC 25922 [12] and reported the superiority of 11 C-D-Met compared with 11 C-L-Met
in imaging at about 60 min after each radiotracer injection. However, their use of a different
strain of E. coli than that in the present study (EC-14) might have influenced their results,
and the better contrast ratios of 11 C-D-Met may have been due to the faster excretion of
3 H-D-Met than 3 H-L-Met in control mice (Tables 1 and 2).
In the biological distribution of 18 F-FDG, significant differences between the control
mice and the mice at 4 h after infection were found in the lung and spleen at 15 min after
18 F-FDG administration (Table 3). 18 F-FDG accumulation is reported to indicate areas ofinflammation after infection, as well as bacterial infection [25]. The 18 F-FDG results of thepresent study suggested minimal areas of inflammation in the whole body because theaccumulation of 18 F-FDG at 4 h after infection was not significantly different to that incontrols, except in the brain and spleen. Therefore, 3 H-L- and D-Met are sensitive for thedetection of areas of infection after the spread of bacteria throughout the body, whereas
18 F-FDG can detect areas of primary infection.
It is difficult to evaluate the ratios of accumulation between the infected lung andother organs (heart and liver), which are commonly calculated in tumor imaging, using
3 H-L- and D-Met because, in this mouse model, there was the whole-body spread ofbacteria (Tables 1 and 2); however, almost all of these ratios showed a significant increase
compared with controls at 24 h after infection. Although these ratios were also significantly
increased compared with controls at 24 h after infection when using 18 F-FDG (Table 3),
there was no significant difference in these ratios between the control and model mice at
4 h after infection, despite no significantly increased accumulation of 18 F-FDG in the organs
compared to the control. Therefore, 3 H-L- and D-Met are more sensitive than 18 F-FDG for
detection of bacterial infection.
At 4 h after infection (early post-infection period), the ratios of accumulation in theinfected lung/control lung were 2.80, 2.84, and 1.34 at 15 min after 3 H-L-Met, 3 H-D-Met,and 18 F-FDG injection, respectively. At 24 h after infection (late post-infection period),
these ratios were 2.11, 2.31, and 2.29; and for each radiotracer, the ratios at 60 min after
injection were similar to those at 15 min after injection. In a clinical oncology study, 18 F-

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FDG accumulation in non-small-cell lung cancer showed a more-than-two-fold increase
compared with the background [26]. Thus, we can confirm that a two- to three-foldaccumulation ratio in the infected lung/control lung in this study is sufficient for clinicalapplication. Therefore, 3 H-L- and D-Met showed comparable performance in in vivo
experiments in EC-14 lung-infection-model mice, and they could be superior to 18 F-FDG interms of detecting bacterial infection at the early stage of infection.
As a limitation of this study, we could not perform 11 C-Met imaging, for the reason that
our facility is not equipped with a cyclotron or an automatic synthesizer for 11 C labeling.
However, the results of the whole-body biodistribution are considered equivalent to thoseof whole-body imaging.
5. Conclusions
11 C-L-Met and D-Met, which differ from 3 H-L-Met and D-Met only in terms of theirradioisotopic labeling, showed comparable performance in in vivo experiments in EC-14
lung-infection-model mice, and are more sensitive than 18 F-FDG for detecting bacterial
infection at the early stage of infection.
Author Contributions: Conceptualization, Y.M., M.K. and K.K.; methodology, K.N. (Kodai Nishi)
N.S. and S.O.; investigation, Y.M., A.M., K.N. (Koya Nakamoto) and M.M.; writing—original draft
preparation, Y.M.; writing—review and editing, K.Y., R.N., M.K. and K.K. All authors have read andagreed to the published version of the manuscript.
Funding: This study was funded in part by Grants-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (No. 18K19508, 18K07747, 21H02865 and 21K15774) andSHIONOGI & Co., Ltd., Osaka, Japan.
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board of Kanazawa University (protocol code AP-173851 and date of approval 27 April 2021).
Informed Consent Statement: Not applicable.
Acknowledgments: The authors would like to thank Shusei Ohata, Takeaki Kiritani, Rika Terada,
Yoshie Yuasa, Akane Yonezawa, and staff of the Faculty of Health Sciences, Kanazawa University, fortheir valued contributions to the study.
Conflicts of Interest: The authors declare no conflict of interest.

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