In vivo monitoring of viable bacteria by SPECT using 99mTc-HYNIC(GH)2-UBI 29-41 and 99mTc-HYNIC(Tricine)2-UBI 29-41

In vivo monitoring of viable bacteria by SPECT using 99mTc-HYNIC(GH)2-UBI 29-41 and 99mTc-HYNIC(Tricine)2-UBI 29-41 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In vivo monitoring of viable bacteria by SPECT using 99m Tc-HYNIC(GH) 2 -UBI 29-41 and 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 Yuri Nishiyama, Tomoya Uehara, Kenichi Okazaki, Hideki Maki, Kohji Abe, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-439672/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The number of bacterial infections that—for various reasons—are challenging to cure continues to increase. One such reason is persister cell infection. To investigate persister formation and persister infections, viable bacteria must be evaluated in the same animal over time. In this study, the feasibility of monitoring viable bacteria by SPECT using two labeled peptides was evaluated. Results: Two types of ubiquicidin (UBI) 29-41 labeled with technetium-99m, 99m Tc-HYNIC(GH) 2 -UBI 29-41 and 99m Tc-HYNIC(Tricine) 2 -UBI 29-41, were synthesized. The in vitro binding of these labeled peptides to Staphylococcus aureu s was measured. For the in vivo study, each labeled peptide was injected into S. aureus infected mouse thigh after treatment with various doses of ciprofloxacin (CPFX). Two hours after injection, the accumulation of each labeled peptide at the infection site was assessed by SPECT, and then the number of viable bacteria was determined from the accumulation detected. The peptide labeling was successful, and the radiochemical purity was 91±9% (GH, n=8) and 100% (Tricine, n=8). The in vitro binding of the labeled peptides to S. aureus (5×10 8 cfu) without serum was 78.9% (GH) and 85.5% (Tricine) of the total 99m Tc activity. With serum, the binding rate was 67.5% (GH) and 13.3% (Tricine). The accumulation of labeled peptide was calculated from the SPECT images, and that in the bacterial infection site (left thigh) was higher than that in the non-infection site (right thigh) for both peptides. Good correlation was found between the target-to-non-target (T/NT) ratios of each labeled peptide and the viable bacterial count at the infection site, and 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 had a wider range than 99m Tc-HYNIC(GH) 2 -UBI 29-41. Conclusion: Using the SPECT/labeled peptide method, it was possible to monitor viable bacterial count in the range 10 3 –10 8 cfu, which is appropriate for tracking viable bacterial counts in the same animal over time. Bacteriology antimicrobial peptides Staphylococcus aureus monitoring viable bacteria SPECT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Bacterial infections continue to pose a serious global health threat. In addition to the development of bacterial resistance, there are many cases where infection-causing bacteria that are sensitive to antibiotics are not completely eradicated and therefore cause relapse. Some reports suggest that the bacteria in such cases include persisters. Maisonneuve et al. reported that there are multiple mechanisms that underlie the bacterial persister phenomenon, for example, environmental insults (i.e., starvation, oxidative and acid stress, and heat shock) provoke persister cell formation, biofilm microenvironments enhance persistence, and host-pathogen interactions, such as those of macrophages, induce the formation of persisters [1]. They also reported that when bacteria were treated with a bactericidal antibiotic, the bacterial count at persistent status was approximately 10 3 cfu [1]. The maximum growth of most bacteria in vivo is approximately 10 8 –10 9 cfu. Based on these values, we believe that the ability to quantify 10 3 –10 8 cfu is necessary for persister research. To investigate persister formation and infections, bacterial behavior in the same animal must be traced over time. With conventional acute infection models it is only possible to count viable bacteria by euthanizing animals at each time point, making it difficult to trace persister formation in the same animal. In contrast, imaging technology is useful for evaluating the same animal over time. There are two imaging methods that show potential for evaluating infections. One is optical imaging. van Oosten et al. reported using luciferase-engineered S. aureus and fluorescently labeled vancomycin (vanco-800CW) in a mouse myositis model [2]. The bioluminescence was used to indicate the localization of S. aureus , and allowed the overlap with vanco-800CW to be determined. The detected bacterial count in their report was 10 7 cfu/thigh. Ning et al. demonstrated in vivo detection of Escherichia coli , S. aureus , Pseudomonas aeruginosa , and Bacillus subtilis using maltodextrin-based imaging probes (MDPs) in a rat myositis model [3]. The MDPs enabled the imaging of as few as 10 5 cfu/thigh. Tang et al. reported infection imaging using Concanavalin A (Con A) as a bacteria-targeting ligand, a nanoparticle carrier, and a near infrared fluorescent dye [4]. The detected count was 10 6 –10 7 cfu/wound in a mouse wound model. Optical imaging is considered useful for detecting bacterial infection; however, it can only be used if the infection model is close to the surface, such as in subcutaneous and thigh infections. Furthermore, the optical imaging sensitivity of the bacterial count was reported to be approximately 10 5 –10 7 cfu at the infection site; therefore, the sensitivity was insufficient to detect persistent status. The other imaging technology is based on radioactivity. Radioactivity-based methods can be used to evaluate deep infections such as lung infections, and they have high sensitivity. We therefore selected a radioactivity-based method so that the 10 3 cfu expected lower limit of persisters could be detected. To monitor the same animal using radioactivity, we used single photon emission computed tomography (SPECT) and 99m Tc-labeled probes. Many radiolabeled agents, such as antibodies, antibiotics, and peptides, have been evaluated for imaging infections [5]. Antimicrobial peptides were designed to kill a broad spectrum of Gram-positive and Gram-negative bacteria, and fungi. The most widely investigated peptide is ubiquicidin (UBI) 29-41. UBI 29-41 is a cationic human antimicrobial peptide fragment with six positively charged residues (5 Arg + 1 Lys) that accumulates at the negatively charged surfaces of microorganisms. Nibbering et al. reported in vivo studies using UBI 29-41 in mice and rats infected with S. aureus [6]. They monitored the efficacy of antibiotics in mice and rats with S. aureus infections and reported good correlation between the accumulation of UBI 29-41 at the infection site and the dose of antibiotics administered. There are many reports describing the use of the bifunctional chelator hydrazine nicotinamide (HYNIC) as an intermediary with UBI [7, 8]. The HYNIC moiety allows the use of a variety of coligands. It has been reported that the nature of the coligand affects the biodistribution of 99m Tc-labeled HYNIC-chemotactic peptide [9]. We selected α-D-glucoheptonic acid (GH) and tricine as coligands. GH is more hydrophobic than tricine and shows high distribution in organs [10]. Therefore, high SPECT detection sensitivity was expected for GH-containing labels. Tricine has been reported to show low distribution in organs, but has been confirmed to show high accumulation at the infection site in animal models [10]. Using these two labeled peptides, we investigated the correlation between the accumulation of labeled peptide and the viable bacterial count at the infection site and determined which peptide is most suitable for detection by SPECT. Methods Antibiotics and Synthetic Antimicrobial Peptide Ciprofloxacin (CPFX; Hydrochloride, ≥98% activity) was purchased from LKT Laboratories, Inc. (MN, USA). UBI 29-41 denotes TGRAKRRMQYNRR (1693 Da). 6-Hydrazinonicotinic acid (HYNIC)-UBI 29-41 was synthesized at Toray Research Center (Tokyo, Japan, Fig. 1). Labeling Procedure and Quality Control 99m Tc-HYNIC(GH) 2 -UBI 29-41 was labeled as follows: 2 mg of GH kit (freeze-dried α-D-glucoheptonic acid [GH] 2.0 mg and SnCl 2 /2H 2 O 1.2 µg) was dissolved in 1 mL of 99m TcO 4 − (185 MBq/mL, Nihon Medi-Physics Co. Ltd.) and the sample was incubated for 10 min at room temperature to synthesize 99m Tc-GH. Then, 200 µL of the 99m Tc-GH solution and 200 µL of 400 µM HYNIC-UBI aqueous solution were mixed and incubated for 60 min at room temperature. 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 was labeled as follows: 125 µL of 99m TcO 4 − was mixed with 125 µL of 40 mg/mL tricine in 10 mM acetate buffer (pH 4). Then, 10 µL of 25 µM HYNIC-UBI aqueous solution and 6 µL of 1 mg/mL SnCl 2 /2H 2 O in 0.1 N HCl were added, and the sample was incubated for 10 min at 90°C [11]. Following labeling, each reaction mixture was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). The sample was applied to an XBridge® C18 5 µm column (4.6 × 150 mm, Waters) attached to a chromatograph equipped with an on-line UV set at 254 nm and a NaI (Tl) crystal gamma detection system (Gabi star, Raytest, Straubenhardt, Germany). 99m Tc-HYNIC(GH) 2 -UBI 29-41 was detected using a linear gradient of two eluents, 0.1% (v/v) trifluoroacetic acid (TFA)/water (solvent A) and 0.1% TFA/acetonitrile (solvent B), at a flow rate of 1.2 mL/min. The gradient was applied as follows: 95%–20% A in 15 min and 95% A for 5 min. 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 was detected using a linear gradient of two eluents, 0.01% (v/v) trifluoroacetic acid (TFA)/water (solvent A) and 0.01% TFA/acetonitrile (solvent B), at a flow rate of 1.0 mL/min. The gradient was applied as follows: 95%–70% A in 15 min and 95% A for 5 min. Microorganism Staphylococcus aureus 25923 (American Type Culture Collection) susceptible to CPFX (minimum inhibitory concentration [MIC] < 1 µg/mL) was used. S. aureus 25923 was cultured on Brain–Heart Infusion Agar (BHIA) for 24 h at 37°C. The suspension of colony was washed, counted by optical density, and used in the in vitro binding assay. For the in vivo assay, a stock solution of S. aureus 25923 stored at −80°C was used. In Vitro Binding to S. aureus Ten microliters of the preparation containing each labeled peptide and 10 µL of suspension containing 4 × 10 11 cfu/mL S. aureus were added to 80 µL of a binding buffer (20 mM phosphate buffered saline [PBS] containing 0.01% Tween80 and 5 mM acetic acid, pH=5). For the serum conditions, instead of 10 µL of the binding buffer, 10 µL of mouse serum were added. The suspensions were gently mixed using a vortex mixer and incubated at 37°C for 1 h. After incubation, the tubes were centrifuged at 5000 ×g for 10 min. The supernatant was removed, and the pellet was resuspended in 100 µL of binding buffer and centrifuged again using the conditions described above. The supernatant was again removed and the radioactivity of the pellet was counted using a γ-counter. The radioactivity associated with the bacteria pellet was expressed as percentage of the total 99m Tc activity added. Animals All of the procedures for the animal studies were approved by the Institutional Animal Care and Use Committee of Shionogi & Co., Ltd. (Osaka, Japan). Specific-pathogen-free (SPF) male ICR mice (CLEA Japan Inc., 5 weeks) were used in the infection and SPECT studies. Biodistribution A 0.2-mL solution containing 30 kBq of 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 or 10 kBq of 99m Tc-HYNIC(GH) 2 -UBI 29-41 was administered via the tail vein of mice. Animals were euthanized by exsanguination and opening of the thoracic cavity at 0.5, 2, and 3 h post injection. Organs were excised and weighed, and their activity was counted using a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/organ, Table 1). Treatment of Animal Infections with Antibacterial Agents Mice were anesthetized with isoflurane, and 6.0 × 10 6 –2.0 × 10 7 cfu of bacteria in 0.1 mL saline were aseptically injected into the left thigh muscle of each mouse. Mice were initially subcutaneously administered CPFX 100 mg/kg q.d. or t.i.d. for 1 or 2 days. To evaluate the correlation between the accumulation of each labeled peptide and the viable bacterial count, mice received 0–100 mg/kg of CPFX for 2 days after infection q.d. (2, 24 h) or t.i.d. (2, 4, 6, 24, 26, 28 h) (Fig. 3). SPECT imaging At 46 h after infection, 0.2 mL of solution containing 10–30 MBq of each labeled peptide was administered via the tail vein of mice. The accumulation of each labeled peptide in the bacteria-infected site in mice was assessed by SPECT/CT (Triumph II SPECT 2H/XO SRI CT, TriFoil Imaging). Two hours after the labeled peptide injection, mice were anesthetized with isoflurane. The mice were then arranged lying face down on a SPECT/CT bed with both hind legs spread out and fixed with surgical tape. Whole body images were acquired under the following conditions; energy window: 20% at 140 keV, projection limit: 20 s, projection count: 64, rotation angle: 360 degrees. After whole body imaging, the mice were euthanized and the infected (left) and normal (right) legs were extracted. Leg-only images were then acquired using the same conditions. For the image processing, adjusted regions of interest (ROI) were drawn over the entire infected muscle (target [T]) and contralateral muscle (nontarget [NT]). The accumulation of each labeled peptide at the infection site was expressed as the ratio of the counts in the target and the nontarget muscles (T/NT). Determining the Number of Viable Bacteria After SPECT imaging, the entire infected thigh muscles were removed and individually homogenized in Mueller–Hinton broth. Serial dilutions of the thigh homogenate were plated on Brain–Heart Infusion Agar. The plates were then incubated for 24 h at 37°C, and the number of cfu was counted. Statistical Analysis The differences between log cfu before and after treatment of mice with CPFX were evaluated using the Student t test. The P values were calculated, and statistical significance was accepted within 95% confidence limits. All results were reported as means and SD. The Pearson correlation coefficient (r) was used to assess the correlation between the accumulation of each labeled peptide and the viable bacterial count. Results Labeling and Quality Control RP-HPLC analysis of 99m Tc-HYNIC(GH) 2 -UBI 29-41 showed two major peaks (4.0 and 4.5 min) and that of 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 showed a single peak (4.2 min). Leading and trailing shoulders were detected for both peptides; however, a previous report indicated that derivatives with GH and tricine as coligands existed in many isomeric forms [12]. The radiochemical purity of 99m Tc-HYNIC(GH) 2 -UBI 29-41 was 91±9% (n=8), and that of 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 was 100% (n=8). In Vitro Binding to S. aureus The binding of 99m Tc-HYNIC(GH) 2 -UBI 29-41 to S. aureus (5×10 8 cfu) without serum was 78.9% of the total 99m Tc activity, and in the presence of serum the binding was 67.5%. The binding of 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 was 85.5% without serum, while binding was reduced to 13.3% in the presence of serum (Fig. 2). Biodistribution The biodistributions of 99m Tc-HYNIC(GH) 2 -UBI 29-41 and 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 in normal mice at 0.5, 2, and 3 h are summarized Table 1. The data show that the highest concentrations of radioactivity were measured in the kidney for both peptides. As was found for different peptides in a previous report [10], 99m Tc-HYNIC(GH) 2 -UBI 29-41 showed a high organ distribution and was 2–10 times more distributed than 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 in all organs at all time points. This difference in organ distribution is attributed to the difference in coligand. The activity of the thigh—which was the infection site—was found to show no further increase after 2 h; therefore, the SPECT images were acquired 2 h after peptide injection. Effect of Antibiotic Administration For 1-day administration the viable bacteria count reached a minimum of 10 4 cfu/thigh; however, for 2-day administration the number reached 10 3 cfu/thigh, which was the desired target (Fig. 4). Therefore, 2-day treatments were used to evaluate the correlation between the accumulation of the labeled peptide and the viable bacterial count. Effect of Antibiotics on Viable Bacterial Counts To establish a variety of bacterial counts, 10–100 mg/kg of CPFX was administrated to the mice. The decrease in viable bacteria count was found to correlate with the CPFX dose (Fig. 5). Detection of Accumulation at the Infection Site using SPECT The bacterial infection site could be imaged 2 h after the injection of each labeled peptide. Typical images for each labeled peptide in S. aureus infected mice are shown in Fig. 6. For 99m Tc-HYNIC(GH) 2 -UBI 29-41, when the viable bacterial count was 10 7 cfu/thigh, T/NT was 2.4, while when the viable bacterial count was 10 3 cfu/thigh, T/NT decreased to 1.2. For 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 T/NT was significantly higher when the viable bacterial count was 10 8 cfu/thigh, with a value of 10.0; however, T/NT was 1.3 at 10 3 cfu/thigh, which is similar to the value for 99m Tc-HYNIC(GH) 2 -UBI 29-41 with the same bacterial count. The correlations between the accumulation of each labeled peptide and the bacterial counts are shown in Fig. 7. The accumulation of each labeled peptide showed good correlation with the viable bacterial count; for 99m Tc-HYNIC(GH) 2 -UBI 29-41 r = 0.906, P = 0.002; for 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 r = 0.857, P = 0.001. Discussion The in vitro binding study indicated that both peptides showed high binding in the absence of serum. In the presence of serum, the binding of 99m Tc-HYNIC(Tricine) 2 -UBI 29–41 was significantly lower than that of 99m Tc-HYNIC(GH) 2 -UBI 29–41. Several research groups have reported that small biomolecules labeled with tricine as a coligand were unstable and existed as multiple species in solution; and that they may react with the imidazole group of the histidine residues of circulating blood proteins such as albumin [ 13 , 14 ]. The decrease in 99m Tc-HYNIC(Tricine) 2 -UBI 29–41 binding in the presence of serum is therefore attributed to similar interactions. Further investigation of the behavior and properties of the labeled peptide in the presence of serum will form part of future work. The biodistribution of 99m Tc-HYNIC(Tricine) 2 -UBI 29–41 showed fast elimination from the blood and poor distribution in all organs. The clearance of 99m Tc-HYNIC(GH) 2 -UBI 29–41 was slower than that of 99m Tc-HYNIC(Tricine) 2 -UBI 29–41, and the distribution of 99m Tc-HYNIC(GH) 2 -UBI 29–41 in organs was higher than that of the other peptide. The distributions of both peptides were in the following order: kidney > > liver > lung > left thigh. The distributions in muscle were the lowest of the organs measured in this study. One reason for the low distribution of 99m Tc-HYNIC(Tricine) 2 -UBI 29–41 in organs could be that it is trapped by blood proteins as described above. As detection by SPECT requires a certain amount of accumulation owing to the detection limit of the device, the higher organ distribution of 99m Tc-HYNIC(GH) 2 -UBI 29–41 is expected to make it easier to detect than 99m Tc-HYNIC(Tricine) 2 -UBI 29–41. Finally, we aimed to quantify the viable bacteria by measuring the amount of labeled peptide accumulated by the bacteria using SPECT. To do this we investigated the correlation between the accumulation of each labeled peptide and the viable bacterial count. To the best of our knowledge, this correlation has only been investigated by Lupetti et al. using 99m Tc labeled fluconazole with Candida albicans at 10 6 –10 8 cfu/g tissue [ 15 ], for which good correlation was observed. In this study, we set a dynamic range of bacterial count of 10 3 –10 8 cfu/thigh. In light of a previous report that quantified the bacterial count at persistent status [ 1 ], a lower limit of 10 3 cfu was our target for demonstrating the feasibility of the labeled peptide/SPECT system for future investigation of bacterial persisters. To achieve this bacterial count we used CPFX. Following 1-day administration the bacterial count reached a minimum of 10 4 cfu/thigh; however, after 2-day administration the count reached our target of 10 3 cfu/thigh (Fig. 4 , 5 ). The accumulation of each labeled peptide was first evaluated at high bacterial count (10 7 –10 8 cfu/thigh) using SPECT. The T/NT value of 99m Tc-HYNIC(GH) 2 -UBI 29–41 was 2.4 and that of 99m Tc-HYNIC(Tricine) 2 -UBI 29–41 was 10.0 as shown in Fig. 6 . We then investigated peptide accumulation at the detection target of 10 3 cfu/thigh using the same method and determined T/NT values of 1.2 and 1.3 for 99m Tc-HYNIC(GH) 2 -UBI 29–41 and 99m Tc-HYNIC(Tricine) 2 -UBI 29–41, respectively. The T/NT range of 99m Tc-HYNIC(GH) 2 -UBI 29–41 was 1.2–3.3 and that of 99m Tc-HYNIC(Tricine) 2 -UBI 29–41 was 1.2–10.0. This difference is thought to be due to the differences in the biodistributions of each peptide and their binding activity to the bacteria. Based on this thigh infection system, 99m Tc-HYNIC(Tricine) 2 -UBI 29–41 is more suitable for quantifying bacteria than 99m Tc-HYNIC(GH) 2 -UBI 29–41 owing to its wide operation range. Our results show that SPECT imaging is able to quantify viable bacteria in the range 10 3 –10 8 cfu by measuring the accumulation of labeled peptides. This is the most important finding of this study as it demonstrates the feasibility of monitoring viable bacterial counts in the same animal over time. Using this method, we will elucidate about persistent bacterial infections and persister cells, and search for ways to reduce such infections. Conclusions SPECT imaging can be used to quantify viable bacterial counts ranging from 10 3 to 10 8 cfu by measuring the accumulation of labeled antimicrobial peptides. This will enable the monitoring of viable bacterial count in the same animal over time, which required for the investigation of persistent infection. Declarations Ethics approval All of the procedures for the animal studies were approved by the Institutional Animal Care and Use Committee of Shionogi & Co., Ltd. (Osaka, Japan). Consent for publication Not applicable Availability of data and material All data generated or analyzed during this study are included in this published article. Competing interests The authors declare that they have no conflicts of interest. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Authors’ contributions TU participated in the design of the labeled peptide and drafted the manuscript. KO and HM participated in the design of the infection study and drafted the manuscript. KA and YA were the supervisors of the study. SM participated in the study coordination, the design of the study, and drafted the manuscript. All authors read and approved the final manuscript. Acknowledgments We thank Yumi Sato for helping with the 99m Tc labeling and Masaaki Izawa for establishing the mouse infection model. References Maisonneuve E, Gerdes K. Molecular mechanisms underlying bacterial persisters. Cell. 2014;157:539–48. van Oosten M, Schafer T, Gazendam JAC, et al. Real-time in vivo imaging of invasive- and biomaterial-associated bacterial infections using fluorescently labelled vancomycin. Nat Commun. 2013;4:2584. Ning X, Lee S, Wang Z, et al. Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity. Nat Mater. 2011;10:602–7. Tang E, Nair A, Baker DW, et al. In vivo imaging of infection using a bacteria-targeting optical nanoprobe. J Biomed Nanotechenol. 2014;10:856–63. van Oosten M, Hahn M, Crane LM, et al. Targeted imaging of bacterial infections: advances, hurdles and hopes. FEMS Microbiol Rev. 2015;39:892–916. Nibbering PH, Welling MM, Paulusma-Annema A, et al. 99m Tc-Labeled UBI 29–41 peptide for monitoring the efficacy of antibacterial agents in mice infected with Staphylococcus aureus. J Nucl Med. 2004;45:321–6. Gandomkar M, Najafi R, Mazidi M, et al. New peptide based freeze-dried kit [ 99m Tc-HYNIC]-UBI 29–41 as a human specific infection imaging agent. Iran J Nucl Med. 2008;16:25–30. Welling MM, Visentin R, Feitsma HIJ, et al. Infection detection in mice using 99m Tc-labeled HYNIC and N 2 S 2 chelate conjugated to the antimicrobial peptide UBI 29–41. Nucl Med Biol. 2004;31:503–9. Babich JW, Fischman AJ. Effect of “co-ligand” on the biodistribution of 99m Tc-labeled hydrazine nicotinic acid derivatized chemotactic peptides. Nucl Med Biol. 1995;22:25–30. Babich JW, Coco WG, Barrow S, et al. 99m Tc-labeled chemotactic peptides: influence of coligand on distribution of molecular species and infection imaging properties. Synthesis and structural characterization of model complexes with the {Re(η 2 -HNNC 5 H 4 N)(η 1 -HNNC 5 H 4 N)} core. Inorg Chim Acta. 2000;309:123–36. Welling MM, Korsak A, Gorska B, et al. Kit with technetium-99m labelled antimicrobial peptide UBI 29–41 for specific infection detection. J Label Compd Radiopharm. 2005;48:683–91. Edwards DS, Liu S, Ziegler MC, et al. RP463: A stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging. Bioconjugate Chem. 1999;10:884–91. Meszaros LK, Dose A, Biagini SCG, et al. Hydrazinonicotinic acid (HYNIC) – Coordination chemistry and applications in radiopharmaceutical chemistry. Inorg Chim Acta. 2010;363:1059–69. Purohit A, Liu S, Ellars CE, et al. Pyridine-containing 6-hydrazinonicotinamide derivatives as potential bifunctional chelators for 99m Tc-labeling of small biomolecules. Bioconjugate Chem. 2004;15:728–37. Lupetti A, Welling MM, Mazzi U, et al. Technetium-99m labelled fluconazole and antimicrobial peptides for imaging of Candida albicans and Aspergillus fumigatus infections. Eur J Nucl Med. 2002;29:674–9. Tables Table 1 Biodistribution of 99m Tc-HYNIC(Tricine) 2 -UBI 29-41 and 99m Tc-HYNIC(GH) 2 -UBI 29-41 in mice Tissue Time after injection 0.5 h (Mean ± SD) 2 h (Mean ± SD) 3 h (Mean ± SD) Tricine GH Tricine GH Tricine GH lung 1.77±0.16 4.07±0.50 0.55±0.04 3.55±0.43 0.36±0.03 3.75±0.24 liver 2.11±0.12 5.07±0.51 1.96±0.02 7.37±0.77 2.08±0.26 8.18±0.07 kidney 83.89±5.83 46.16±1.46 84.88±16.08 56.53±9.20 73.07±9.81 65.45±4.33 left thigh 0.84±0.06 1.41±0.18 0.22±0.01 1.17±0.25 0.18±0.03 1.11±0.24 blood 2.01±0.19 3.43±0.11 0.40±0.05 2.41±0.29 0.22±0.03 2.34±0.11 Expressed as % injected dose per gram. Each value represents mean ± SD for three animals at each interval. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-439672","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":22089576,"identity":"619bdfce-54d0-400f-b7d1-e43797714747","order_by":0,"name":"Yuri Nishiyama","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYJCCAwwMEgwGzGC2DYwhASYNiNCSRpwWZMnD+FWBVd7IfXiAcYeFvTk7j+GnGzXngQzmYw8+/LJg4G9vYCguwKYl3eAA4xmJxJ3NPMbSOcduAxls6YYz+yQYJM4cYDCegU1LGsMBxjaJBIPDPAbSOWy3QQwzad4eoO8kEhiMeXBrsQeqNP6d8++cPdFaGDeAVOa2HYAweH7g1iJ55hnDgcQ2icQNh9nKrHP7kkGMNMmZDRI8EmcONmDzC9/xNOYPH9vq7A3OH958O+ebHYhxTOLDnzo5/vbmY8ZYQkzhAJBIADM5kGKEsY2BB0QaY+pgkG+AM9kfIIn/AZPMj7FoGQWjYBSMghEHAI6CY0TYY5DNAAAAAElFTkSuQmCC","orcid":"","institution":"Shionogi Co Ltd Pharmaceutical Research Center: Shionogi Seiyaku Kabushiki Kaisha Iyaku Kenkyu Center","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yuri","middleName":"","lastName":"Nishiyama","suffix":""},{"id":22089577,"identity":"82c7572f-4020-4ae7-a471-2c2758a10188","order_by":1,"name":"Tomoya Uehara","email":"","orcid":"","institution":"Chiba Daigaku Daigakuin Yakugaku Kenkyuin Yakugakubu","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tomoya","middleName":"","lastName":"Uehara","suffix":""},{"id":22089578,"identity":"e48db32e-7788-4559-bc0a-89ebbd8b1849","order_by":2,"name":"Kenichi Okazaki","email":"","orcid":"","institution":"Shionogi and Co Ltd Pharmaceutical Research Center: Shionogi Seiyaku Kabushiki Kaisha Iyaku Kenkyu Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kenichi","middleName":"","lastName":"Okazaki","suffix":""},{"id":22089579,"identity":"e8477fa6-dab4-4a7e-bf2a-355d55a838b1","order_by":3,"name":"Hideki Maki","email":"","orcid":"","institution":"Shionogi and Co Ltd Pharmaceutical Research Center: Shionogi Seiyaku Kabushiki Kaisha Iyaku Kenkyu Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hideki","middleName":"","lastName":"Maki","suffix":""},{"id":22089580,"identity":"a2624327-db0b-4841-ad15-11144b888f52","order_by":4,"name":"Kohji Abe","email":"","orcid":"","institution":"Shionogi and Co Ltd Pharmaceutical Research Center: Shionogi Seiyaku Kabushiki Kaisha Iyaku Kenkyu Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kohji","middleName":"","lastName":"Abe","suffix":""},{"id":22089581,"identity":"ecbe2687-8cc5-424a-8c60-7073d95e79d0","order_by":5,"name":"Yasushi Arano","email":"","orcid":"","institution":"Chiba Daigaku Daigakuin Yakugaku Kenkyuin Yakugakubu","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasushi","middleName":"","lastName":"Arano","suffix":""},{"id":22089582,"identity":"b62510f6-71d6-4c43-af6d-5ecc99a97c90","order_by":6,"name":"Sotaro Momosaki","email":"","orcid":"","institution":"Shionogi and Co Ltd Pharmaceutical Research Center: Shionogi Seiyaku Kabushiki Kaisha Iyaku Kenkyu Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sotaro","middleName":"","lastName":"Momosaki","suffix":""}],"badges":[],"createdAt":"2021-04-19 12:05:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-439672/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-439672/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":8246892,"identity":"ac9c4fc3-2972-47ae-85e4-5d12f72c9e99","added_by":"auto","created_at":"2021-04-20 21:04:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7045,"visible":true,"origin":"","legend":"Structural formula of HYNIC-UBI 29-41.","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-439672/v1/2b2815d4c103a716187785b2.png"},{"id":8247209,"identity":"c6bf0d84-f0cd-4e51-bb21-35a8bf48d18f","added_by":"auto","created_at":"2021-04-20 21:07:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11821,"visible":true,"origin":"","legend":"In vitro bacterial binding of 99mTc-HYNIC(Tricine)2-UBI 29-41 and 99mTc-HYNIC(GH)2-UBI 29-41 to S. aureus. ","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-439672/v1/78f93a29f74c10ccb716e35b.png"},{"id":8247210,"identity":"942b49b3-99d6-4efa-b926-8baf8cfffa51","added_by":"auto","created_at":"2021-04-20 21:07:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5812,"visible":true,"origin":"","legend":"Timeline of SPECT imaging of t.i.d. administrations.","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-439672/v1/780f193fa34392a27a5902b7.png"},{"id":8247331,"identity":"ee81d891-1ee9-4149-a963-20b1dd4d44ec","added_by":"auto","created_at":"2021-04-20 21:10:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16470,"visible":true,"origin":"","legend":"Viable bacterial count in thigh infected with S. aureus for mice treated with CPFX for 1 or 2 days. Results are expressed as the mean ± SD for three animals. *Values that were significantly (P \u003c 0.05) different to the control.","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-439672/v1/074df8b67ea53dbc8c7fa103.png"},{"id":8247332,"identity":"8c944568-19cd-4d78-83fc-86f28ac77f45","added_by":"auto","created_at":"2021-04-20 21:10:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":15072,"visible":true,"origin":"","legend":"Viable bacterial count in thigh infected with S. aureus for mice treated with CPFX for 2 days. Results are expressed as the mean ± SD for three animals. *Values that were significantly (P \u003c 0.05) different to the control.","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-439672/v1/014c9329a7ec9954ab34834a.png"},{"id":8246898,"identity":"17b1062e-e4a7-4588-a5e5-a73827d6f724","added_by":"auto","created_at":"2021-04-20 21:04:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":267078,"visible":true,"origin":"","legend":"SPECT images of mouse legs infected in the left thigh (red circle) with S. aureus, acquired 2 h after the administration of 99mTc-HYNIC(Tricine)2-UBI 29-41 or 99mTc-HYNIC(GH)2-UBI 29-41.","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-439672/v1/a34374d302127792a55749ba.png"},{"id":8247212,"identity":"94482e89-db1c-4138-86d7-76a8647b925f","added_by":"auto","created_at":"2021-04-20 21:07:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":17766,"visible":true,"origin":"","legend":"Correlation between viable S. aureus counts and the accumulation of 99mTc-HYNIC(Tricine)2-UBI 29-41 (circle) and 99mTc-HYNIC(GH)2-UBI 29-41 (square), expressed as the target-to-non-target (T/NT) ratio.","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-439672/v1/2cdfee91ccf47d6f53a25156.png"},{"id":15672341,"identity":"d04e1ed8-b0cf-4016-9df6-dbaebaf3f93c","added_by":"auto","created_at":"2021-11-18 14:11:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":669800,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-439672/v1/6a532d42-ef06-4a5b-bfdb-b88c9d7ce7a7.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cem\u003eIn vivo \u003c/em\u003emonitoring of viable bacteria by SPECT using \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 and \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eBacterial infections continue to pose a serious global health threat. In addition to the development of bacterial resistance, there are many cases where infection-causing bacteria that are sensitive to antibiotics are not completely eradicated and therefore cause relapse. Some reports suggest that the bacteria in such cases include persisters. Maisonneuve \u003cem\u003eet al.\u003c/em\u003e reported that there are multiple mechanisms that underlie the bacterial persister phenomenon, for example, environmental insults (i.e., starvation, oxidative and acid stress, and heat shock) provoke persister cell formation, biofilm microenvironments enhance persistence, and host-pathogen interactions, such as those of macrophages, induce the formation of persisters [1]. They also reported that when bacteria were treated with a bactericidal antibiotic, the bacterial count at persistent status was approximately 10\u003csup\u003e3\u003c/sup\u003e cfu [1]. The maximum growth of most bacteria \u003cem\u003ein vivo\u003c/em\u003e is approximately 10\u003csup\u003e8\u003c/sup\u003e\u0026ndash;10\u003csup\u003e9\u003c/sup\u003e cfu. Based on these values, we believe that the ability to quantify 10\u003csup\u003e3\u003c/sup\u003e\u0026ndash;10\u003csup\u003e8\u003c/sup\u003e cfu is necessary for persister research.\u003c/p\u003e\n\u003cp\u003eTo investigate persister formation and infections, bacterial behavior in the same animal must be traced over time. With conventional acute infection models it is only possible to count viable bacteria by euthanizing animals at each time point, making it difficult to trace persister formation in the same animal. In contrast, imaging technology is useful for evaluating the same animal over time.\u003c/p\u003e\n\u003cp\u003eThere are two imaging methods that show potential for evaluating infections. One is optical imaging. van Oosten\u003cem\u003e et al.\u003c/em\u003e reported using luciferase-engineered \u003cem\u003eS. aureus\u003c/em\u003e and fluorescently labeled vancomycin (vanco-800CW) in a mouse myositis model [2]. The bioluminescence was used to indicate the localization of \u003cem\u003eS. aureus\u003c/em\u003e, and allowed the overlap with vanco-800CW to be determined. The detected bacterial count in their report was 10\u003csup\u003e7\u003c/sup\u003e cfu/thigh. Ning \u003cem\u003eet al.\u003c/em\u003e demonstrated \u003cem\u003ein vivo\u003c/em\u003e detection of \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eBacillus subtilis\u003c/em\u003e using maltodextrin-based imaging probes (MDPs) in a rat myositis model [3]. The MDPs enabled the imaging of as few as 10\u003csup\u003e5\u003c/sup\u003e cfu/thigh. Tang \u003cem\u003eet al.\u003c/em\u003e reported infection imaging using Concanavalin A (Con A) as a bacteria-targeting ligand, a nanoparticle carrier, and a near infrared fluorescent dye [4]. The detected count was 10\u003csup\u003e6\u003c/sup\u003e\u0026ndash;10\u003csup\u003e7\u003c/sup\u003e cfu/wound in a mouse wound model.\u003c/p\u003e\n\u003cp\u003eOptical imaging is considered useful for detecting bacterial infection; however, it can only be used if the infection model is close to the surface, such as in subcutaneous and thigh infections. Furthermore, the optical imaging sensitivity of the bacterial count was reported to be approximately 10\u003csup\u003e5\u003c/sup\u003e\u0026ndash;10\u003csup\u003e7\u003c/sup\u003e cfu at the infection site; therefore, the sensitivity was insufficient to detect persistent status.\u003c/p\u003e\n\u003cp\u003eThe other imaging technology is based on radioactivity. Radioactivity-based methods can be used to evaluate deep infections such as lung infections, and they have high sensitivity. We therefore selected a radioactivity-based method so that the 10\u003csup\u003e3\u003c/sup\u003e cfu expected lower limit of persisters could be detected. To monitor the same animal using radioactivity, we used single photon emission computed tomography (SPECT) and \u003csup\u003e99m\u003c/sup\u003eTc-labeled probes.\u003c/p\u003e\n\u003cp\u003eMany radiolabeled agents, such as antibodies, antibiotics, and peptides, have been evaluated for imaging infections [5]. Antimicrobial peptides were designed to kill a broad spectrum of Gram-positive and Gram-negative bacteria, and fungi. The most widely investigated peptide is ubiquicidin (UBI) 29-41. UBI 29-41 is a cationic human antimicrobial peptide fragment with six positively charged residues (5 Arg + 1 Lys) that accumulates at the negatively charged surfaces of microorganisms. Nibbering \u003cem\u003eet al.\u003c/em\u003e reported \u003cem\u003ein vivo\u003c/em\u003e studies using UBI 29-41 in mice and rats infected with \u003cem\u003eS. aureus\u003c/em\u003e [6]. They monitored the efficacy of antibiotics in mice and rats with \u003cem\u003eS. aureus\u003c/em\u003e infections and reported good correlation between the accumulation of UBI 29-41 at the infection site and the dose of antibiotics administered.\u003c/p\u003e\n\u003cp\u003eThere are many reports describing the use of the bifunctional chelator hydrazine nicotinamide (HYNIC) as an intermediary with UBI [7, 8]. The HYNIC moiety allows the use of a variety of coligands. It has been reported that the nature of the coligand affects the biodistribution of \u003csup\u003e99m\u003c/sup\u003eTc-labeled HYNIC-chemotactic peptide [9]. We selected \u0026alpha;-D-glucoheptonic acid (GH) and tricine as coligands. GH is more hydrophobic than tricine and shows high distribution in organs [10]. Therefore, high SPECT detection sensitivity was expected for GH-containing labels. Tricine has been reported to show low distribution in organs, but has been confirmed to show high accumulation at the infection site in animal models [10]. Using these two labeled peptides, we investigated the correlation between the accumulation of labeled peptide and the viable bacterial count at the infection site and determined which peptide is most suitable for detection by SPECT.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAntibiotics and Synthetic Antimicrobial Peptide\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCiprofloxacin (CPFX; Hydrochloride, \u0026ge;98% activity) was purchased from LKT Laboratories, Inc. (MN, USA). UBI 29-41 denotes TGRAKRRMQYNRR (1693 Da). 6-Hydrazinonicotinic acid (HYNIC)-UBI 29-41 was synthesized at Toray Research Center (Tokyo, Japan, Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLabeling Procedure and Quality Control\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 was labeled as follows: 2 mg of GH kit (freeze-dried \u0026alpha;-D-glucoheptonic acid [GH] 2.0 mg and SnCl\u003csub\u003e2\u003c/sub\u003e/2H\u003csub\u003e2\u003c/sub\u003eO 1.2 \u0026micro;g) was dissolved in 1 mL of \u003csup\u003e99m\u003c/sup\u003eTcO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (185 MBq/mL, Nihon Medi-Physics Co. Ltd.) and the sample was incubated for 10 min at room temperature to synthesize \u003csup\u003e99m\u003c/sup\u003eTc-GH. Then, 200 \u0026micro;L of the \u003csup\u003e99m\u003c/sup\u003eTc-GH solution and 200 \u0026micro;L of 400 \u0026micro;M HYNIC-UBI aqueous solution were mixed and incubated for 60 min at room temperature. \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 was labeled as follows: 125 \u0026micro;L of \u003csup\u003e99m\u003c/sup\u003eTcO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e was mixed with 125 \u0026micro;L of 40 mg/mL tricine in 10 mM acetate buffer (pH 4). Then, 10 \u0026micro;L of 25 \u0026micro;M HYNIC-UBI aqueous solution and 6 \u0026micro;L of 1 mg/mL SnCl\u003csub\u003e2\u003c/sub\u003e/2H\u003csub\u003e2\u003c/sub\u003eO in 0.1 N HCl were added, and the sample was incubated for 10 min at 90\u0026deg;C [11]. Following labeling, each reaction mixture was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). The sample was applied to an XBridge\u0026reg; C18 5 \u0026micro;m column (4.6 \u0026times; 150 mm, Waters) attached to a chromatograph equipped with an on-line UV set at 254 nm and a NaI (Tl) crystal gamma detection system (Gabi star, Raytest, Straubenhardt, Germany). \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 was detected using a linear gradient of two eluents, 0.1% (v/v) trifluoroacetic acid (TFA)/water (solvent A) and 0.1% TFA/acetonitrile (solvent B), at a flow rate of 1.2 mL/min. The gradient was applied as follows: 95%\u0026ndash;20% A in 15 min and 95% A for 5 min. \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 was detected using a linear gradient of two eluents, 0.01% (v/v) trifluoroacetic acid (TFA)/water (solvent A) and 0.01% TFA/acetonitrile (solvent B), at a flow rate of 1.0 mL/min. The gradient was applied as follows: 95%\u0026ndash;70% A in 15 min and 95% A for 5 min.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroorganism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e 25923 (American Type Culture Collection) susceptible to CPFX (minimum inhibitory concentration [MIC] \u0026lt; 1 \u0026micro;g/mL) was used. \u003cem\u003eS. aureus\u003c/em\u003e 25923 was cultured on Brain\u0026ndash;Heart Infusion Agar (BHIA) for 24 h at 37\u0026deg;C. The suspension of colony was washed, counted by optical density, and used in the \u003cem\u003ein vitro\u003c/em\u003e binding assay. For the \u003cem\u003ein vivo\u003c/em\u003e assay, a stock solution of \u003cem\u003eS. aureus\u003c/em\u003e 25923 stored at \u0026minus;80\u0026deg;C was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn Vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e Binding to \u003cem\u003eS. aureus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTen microliters of the preparation containing each labeled peptide and 10 \u0026micro;L of suspension containing 4 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e cfu/mL \u003cem\u003eS. aureus\u003c/em\u003e were added to 80 \u0026micro;L of a binding buffer (20 mM phosphate buffered saline [PBS] containing 0.01% Tween80 and 5 mM acetic acid, pH=5). For the serum conditions, instead of 10 \u0026micro;L of the binding buffer, 10 \u0026micro;L of mouse serum were added. The suspensions were gently mixed using a vortex mixer and incubated at 37\u0026deg;C for 1 h. After incubation, the tubes were centrifuged at 5000 \u0026times;g for 10 min. The supernatant was removed, and the pellet was resuspended in 100 \u0026micro;L of binding buffer and centrifuged again using the conditions described above. The supernatant was again removed and the radioactivity of the pellet was counted using a \u0026gamma;-counter. The radioactivity associated with the bacteria pellet was expressed as percentage of the total \u003csup\u003e99m\u003c/sup\u003eTc activity added.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the procedures for the animal studies were approved by the Institutional Animal Care and Use Committee of Shionogi \u0026amp; Co., Ltd. (Osaka, Japan). Specific-pathogen-free (SPF) male ICR mice (CLEA Japan Inc., 5 weeks) were used in the infection and SPECT studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiodistribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 0.2-mL solution containing 30 kBq of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 or 10 kBq of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 was administered via the tail vein of mice. Animals were euthanized by exsanguination and opening of the thoracic cavity at 0.5, 2, and 3 h post injection. Organs were excised and weighed, and their activity was counted using a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/organ, Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment of Animal Infections with Antibacterial Agents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were anesthetized with isoflurane, and 6.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e\u0026ndash;2.0 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cfu of bacteria in 0.1 mL saline were aseptically injected into the left thigh muscle of each mouse. Mice were initially subcutaneously administered CPFX 100 mg/kg q.d. or t.i.d. for 1 or 2 days. To evaluate the correlation between the accumulation of each labeled peptide and the viable bacterial count, mice received 0\u0026ndash;100 mg/kg of CPFX for 2 days after infection q.d. (2, 24 h) or t.i.d. (2, 4, 6, 24, 26, 28 h) (Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSPECT imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 46 h after infection, 0.2 mL of solution containing 10\u0026ndash;30 MBq of each labeled peptide was administered via the tail vein of mice. The accumulation of each labeled peptide in the bacteria-infected site in mice was assessed by SPECT/CT (Triumph II SPECT 2H/XO SRI CT, TriFoil Imaging). Two hours after the labeled peptide injection, mice were anesthetized with isoflurane. The mice were then arranged lying face down on a SPECT/CT bed with both hind legs spread out and fixed with surgical tape. Whole body images were acquired under the following conditions; energy window: 20% at 140 keV, projection limit: 20 s, projection count: 64, rotation angle: 360 degrees. After whole body imaging, the mice were euthanized and the infected (left) and normal (right) legs were extracted. Leg-only images were then acquired using the same conditions. For the image processing, adjusted regions of interest (ROI) were drawn over the entire infected muscle (target [T]) and contralateral muscle (nontarget [NT]). The accumulation of each labeled peptide at the infection site was expressed as the ratio of the counts in the target and the nontarget muscles (T/NT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermining the Number of Viable Bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter SPECT imaging, the entire infected thigh muscles were removed and individually homogenized in Mueller\u0026ndash;Hinton broth. Serial dilutions of the thigh homogenate were plated on Brain\u0026ndash;Heart Infusion Agar. The plates were then incubated for 24 h at 37\u0026deg;C, and the number of cfu was counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe differences between log cfu before and after treatment of mice with CPFX were evaluated using the Student t test. The P values were calculated, and statistical significance was accepted within 95% confidence limits. All results were reported as means and SD. The Pearson correlation coefficient (r) was used to assess the correlation between the accumulation of each labeled peptide and the viable bacterial count.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLabeling and Quality Control\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRP-HPLC analysis of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 showed two major peaks (4.0 and 4.5 min) and that of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 showed a single peak (4.2 min). Leading and trailing shoulders were detected for both peptides; however, a previous report indicated that derivatives with GH and tricine as coligands existed in many isomeric forms [12]. The radiochemical purity of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 was 91\u0026plusmn;9% (n=8), and that of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 was 100% (n=8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn Vitro \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eBinding to \u003cem\u003eS. aureus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe binding of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 to \u003cem\u003eS. aureus\u003c/em\u003e (5\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu) without serum was 78.9% of the total \u003csup\u003e99m\u003c/sup\u003eTc activity, and in the presence of serum the binding was 67.5%. The binding of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 was 85.5% without serum, while binding was reduced to 13.3% in the presence of serum (Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiodistribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biodistributions of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 and \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 in normal mice at 0.5, 2, and 3 h are summarized Table 1. The data show that the highest concentrations of radioactivity were measured in the kidney for both peptides. As was found for different peptides in a previous report [10], \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 showed a high organ distribution and was 2\u0026ndash;10 times more distributed than \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 in all organs at all time points. This difference in organ distribution is attributed to the difference in coligand. The activity of the thigh\u0026mdash;which was the infection site\u0026mdash;was found to show no further increase after 2 h; therefore, the SPECT images were acquired 2 h after peptide injection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of Antibiotic Administration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor 1-day administration the viable bacteria count reached a minimum of 10\u003csup\u003e4\u003c/sup\u003e cfu/thigh; however, for 2-day administration the number reached 10\u003csup\u003e3\u003c/sup\u003e cfu/thigh, which was the desired target (Fig. 4). Therefore, 2-day treatments were used to evaluate the correlation between the accumulation of the labeled peptide and the viable bacterial count.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of Antibiotics on Viable Bacterial Counts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo establish a variety of bacterial counts, 10\u0026ndash;100 mg/kg of CPFX was administrated to the mice. The decrease in viable bacteria count was found to correlate with the CPFX dose (Fig. 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Accumulation at the Infection Site using SPECT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bacterial infection site could be imaged 2 h after the injection of each labeled peptide. Typical images for each labeled peptide in \u003cem\u003eS. aureus\u003c/em\u003e infected mice are shown in Fig. 6. For \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41, when the viable bacterial count was 10\u003csup\u003e7\u003c/sup\u003e cfu/thigh, T/NT was 2.4, while when the viable bacterial count was 10\u003csup\u003e3\u003c/sup\u003e cfu/thigh, T/NT decreased to 1.2. For \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 T/NT was significantly higher when the viable bacterial count was 10\u003csup\u003e8\u003c/sup\u003e cfu/thigh, with a value of 10.0; however, T/NT was 1.3 at 10\u003csup\u003e3\u003c/sup\u003e cfu/thigh, which is similar to the value for \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 with the same bacterial count.\u003c/p\u003e\n\u003cp\u003eThe correlations between the accumulation of each labeled peptide and the bacterial counts are shown in Fig. 7. The accumulation of each labeled peptide showed good correlation with the viable bacterial count; for \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 r = 0.906, P = 0.002; for \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 r = 0.857, P = 0.001.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e binding study indicated that both peptides showed high binding in the absence of serum. In the presence of serum, the binding of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 was significantly lower than that of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41. Several research groups have reported that small biomolecules labeled with tricine as a coligand were unstable and existed as multiple species in solution; and that they may react with the imidazole group of the histidine residues of circulating blood proteins such as albumin [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. The decrease in \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 binding in the presence of serum is therefore attributed to similar interactions. Further investigation of the behavior and properties of the labeled peptide in the presence of serum will form part of future work.\u003c/p\u003e\n\u003cp\u003eThe biodistribution of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 showed fast elimination from the blood and poor distribution in all organs. The clearance of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 was slower than that of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41, and the distribution of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 in organs was higher than that of the other peptide. The distributions of both peptides were in the following order: kidney\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;liver\u0026thinsp;\u0026gt;\u0026thinsp;lung\u0026thinsp;\u0026gt;\u0026thinsp;left thigh. The distributions in muscle were the lowest of the organs measured in this study. One reason for the low distribution of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 in organs could be that it is trapped by blood proteins as described above. As detection by SPECT requires a certain amount of accumulation owing to the detection limit of the device, the higher organ distribution of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 is expected to make it easier to detect than \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41.\u003c/p\u003e\n\u003cp\u003eFinally, we aimed to quantify the viable bacteria by measuring the amount of labeled peptide accumulated by the bacteria using SPECT. To do this we investigated the correlation between the accumulation of each labeled peptide and the viable bacterial count. To the best of our knowledge, this correlation has only been investigated by Lupetti \u003cem\u003eet al.\u003c/em\u003e using \u003csup\u003e99m\u003c/sup\u003eTc labeled fluconazole with \u003cem\u003eCandida albicans\u003c/em\u003e at 10\u003csup\u003e6\u003c/sup\u003e\u0026ndash;10\u003csup\u003e8\u003c/sup\u003e cfu/g tissue [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e], for which good correlation was observed. In this study, we set a dynamic range of bacterial count of 10\u003csup\u003e3\u003c/sup\u003e\u0026ndash;10\u003csup\u003e8\u003c/sup\u003e cfu/thigh. In light of a previous report that quantified the bacterial count at persistent status [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e], a lower limit of 10\u003csup\u003e3\u003c/sup\u003e cfu was our target for demonstrating the feasibility of the labeled peptide/SPECT system for future investigation of bacterial persisters. To achieve this bacterial count we used CPFX. Following 1-day administration the bacterial count reached a minimum of 10\u003csup\u003e4\u003c/sup\u003e cfu/thigh; however, after 2-day administration the count reached our target of 10\u003csup\u003e3\u003c/sup\u003e cfu/thigh (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe accumulation of each labeled peptide was first evaluated at high bacterial count (10\u003csup\u003e7\u003c/sup\u003e\u0026ndash;10\u003csup\u003e8\u003c/sup\u003e cfu/thigh) using SPECT. The T/NT value of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 was 2.4 and that of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 was 10.0 as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. We then investigated peptide accumulation at the detection target of 10\u003csup\u003e3\u003c/sup\u003e cfu/thigh using the same method and determined T/NT values of 1.2 and 1.3 for \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 and \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41, respectively. The T/NT range of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 was 1.2\u0026ndash;3.3 and that of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 was 1.2\u0026ndash;10.0. This difference is thought to be due to the differences in the biodistributions of each peptide and their binding activity to the bacteria. Based on this thigh infection system, \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 is more suitable for quantifying bacteria than \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29\u0026ndash;41 owing to its wide operation range.\u003c/p\u003e\n\u003cp\u003eOur results show that SPECT imaging is able to quantify viable bacteria in the range 10\u003csup\u003e3\u003c/sup\u003e\u0026ndash;10\u003csup\u003e8\u003c/sup\u003e cfu by measuring the accumulation of labeled peptides. This is the most important finding of this study as it demonstrates the feasibility of monitoring viable bacterial counts in the same animal over time. Using this method, we will elucidate about persistent bacterial infections and persister cells, and search for ways to reduce such infections.\u003c/p\u003e"},{"header":"Conclusions","content":" \u003cp\u003eSPECT imaging can be used to quantify viable bacterial counts ranging from 10\u003csup\u003e3\u003c/sup\u003e to 10\u003csup\u003e8\u003c/sup\u003e cfu by measuring the accumulation of labeled antimicrobial peptides. This will enable the monitoring of viable bacterial count in the same animal over time, which required for the investigation of persistent infection.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the procedures for the animal studies were approved by the Institutional Animal Care and Use Committee of Shionogi \u0026amp; Co., Ltd. (Osaka, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTU participated in the design of the labeled peptide and drafted the manuscript. KO and HM participated in the design of the infection study and drafted the manuscript. KA and YA were the supervisors of the study. SM participated in the study coordination, the design of the study, and drafted the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Yumi Sato for helping with the \u003csup\u003e99m\u003c/sup\u003eTc labeling and Masaaki Izawa for establishing the mouse infection model.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMaisonneuve E, Gerdes K. Molecular mechanisms underlying bacterial persisters. Cell. 2014;157:539\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Oosten M, Schafer T, Gazendam JAC, et al. Real-time in vivo imaging of invasive- and biomaterial-associated bacterial infections using fluorescently labelled vancomycin. Nat Commun. 2013;4:2584.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNing X, Lee S, Wang Z, et al. Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity. Nat Mater. 2011;10:602\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang E, Nair A, Baker DW, et al. In vivo imaging of infection using a bacteria-targeting optical nanoprobe. J Biomed Nanotechenol. 2014;10:856\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Oosten M, Hahn M, Crane LM, et al. Targeted imaging of bacterial infections: advances, hurdles and hopes. FEMS Microbiol Rev. 2015;39:892\u0026ndash;916.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNibbering PH, Welling MM, Paulusma-Annema A, et al. \u003csup\u003e99m\u003c/sup\u003eTc-Labeled UBI 29\u0026ndash;41 peptide for monitoring the efficacy of antibacterial agents in mice infected with Staphylococcus aureus. J Nucl Med. 2004;45:321\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGandomkar M, Najafi R, Mazidi M, et al. New peptide based freeze-dried kit [\u003csup\u003e99m\u003c/sup\u003eTc-HYNIC]-UBI 29\u0026ndash;41 as a human specific infection imaging agent. Iran J Nucl Med. 2008;16:25\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWelling MM, Visentin R, Feitsma HIJ, et al. Infection detection in mice using \u003csup\u003e99m\u003c/sup\u003eTc-labeled HYNIC and N\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e chelate conjugated to the antimicrobial peptide UBI 29\u0026ndash;41. Nucl Med Biol. 2004;31:503\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBabich JW, Fischman AJ. Effect of \u0026ldquo;co-ligand\u0026rdquo; on the biodistribution of \u003csup\u003e99m\u003c/sup\u003eTc-labeled hydrazine nicotinic acid derivatized chemotactic peptides. Nucl Med Biol. 1995;22:25\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBabich JW, Coco WG, Barrow S, et al. \u003csup\u003e99m\u003c/sup\u003eTc-labeled chemotactic peptides: influence of coligand on distribution of molecular species and infection imaging properties. Synthesis and structural characterization of model complexes with the {Re(η\u003csup\u003e2\u003c/sup\u003e-HNNC\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eN)(η\u003csup\u003e1\u003c/sup\u003e-HNNC\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eN)} core. Inorg Chim Acta. 2000;309:123\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWelling MM, Korsak A, Gorska B, et al. Kit with technetium-99m labelled antimicrobial peptide UBI 29\u0026ndash;41 for specific infection detection. J Label Compd Radiopharm. 2005;48:683\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdwards DS, Liu S, Ziegler MC, et al. RP463: A stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging. Bioconjugate Chem. 1999;10:884\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeszaros LK, Dose A, Biagini SCG, et al. Hydrazinonicotinic acid (HYNIC) \u0026ndash; Coordination chemistry and applications in radiopharmaceutical chemistry. Inorg Chim Acta. 2010;363:1059\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePurohit A, Liu S, Ellars CE, et al. Pyridine-containing 6-hydrazinonicotinamide derivatives as potential bifunctional chelators for \u003csup\u003e99m\u003c/sup\u003eTc-labeling of small biomolecules. Bioconjugate Chem. 2004;15:728\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLupetti A, Welling MM, Mazzi U, et al. Technetium-99m labelled fluconazole and antimicrobial peptides for imaging of Candida albicans and Aspergillus fumigatus infections. Eur J Nucl Med. 2002;29:674\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:justify;line-height:150%;'\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 150%; font-family: Calibri, sans-serif; color: black; font-size: 15px;\"\u003eTable 1\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 150%; color: black;\"\u003eBiodistribution of \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 and \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 in mice\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left:-22.95pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 53.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:justify;vertical-align: middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eTissue\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" style=\"width: 405.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eTime after injection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width:133.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e0.5 h (Mean \u0026plusmn; SD)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width:139.3pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e2 h (Mean \u0026plusmn; SD)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width:133.1pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e3 h (Mean \u0026plusmn; SD)\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:66.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eTricine\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:72.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eTricine\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eTricine\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eGH\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:53.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:justify;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003elung\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e1.77\u0026plusmn;0.16\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e4.07\u0026plusmn;0.50\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:72.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e0.55\u0026plusmn;0.04\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e3.55\u0026plusmn;0.43\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e0.36\u0026plusmn;0.03\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e3.75\u0026plusmn;0.24\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:53.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:justify;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eliver\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e2.11\u0026plusmn;0.12\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e5.07\u0026plusmn;0.51\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:72.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e1.96\u0026plusmn;0.02\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e7.37\u0026plusmn;0.77\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e2.08\u0026plusmn;0.26\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e8.18\u0026plusmn;0.07\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:53.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:justify;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003ekidney\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: 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style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e2.01\u0026plusmn;0.19\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e3.43\u0026plusmn;0.11\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:72.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e0.40\u0026plusmn;0.05\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e2.41\u0026plusmn;0.29\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e0.22\u0026plusmn;0.03\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:center;vertical-align:middle;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003e2.34\u0026plusmn;0.11\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:justify;line-height:150%;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"line-height: 150%; color: black;\"\u003eExpressed as % injected dose per gram. Each value represents mean\u0026nbsp;\u0026plusmn;\u0026nbsp;SD for three animals at each interval.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-right:0in;margin-left:0in;font-size:14px;font-family:\"Century\",serif;margin:0in;text-align:justify;line-height:150%;'\u003e\u003cstrong\u003e\u003cspan style=\"line-height: 150%; font-family: Calibri, sans-serif; color: black; font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"antimicrobial peptides, Staphylococcus aureus, monitoring viable bacteria, SPECT","lastPublishedDoi":"10.21203/rs.3.rs-439672/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-439672/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The number of bacterial infections that—for various reasons—are challenging to cure continues to increase. One such reason is persister cell infection. To investigate persister formation and persister infections, viable bacteria must be evaluated in the same animal over time. In this study, the feasibility of monitoring viable bacteria by SPECT using two labeled peptides was evaluated. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eTwo types of ubiquicidin (UBI) 29-41 labeled with technetium-99m, \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 and \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41, were synthesized. The \u003cem\u003ein vitro\u003c/em\u003e binding of these labeled peptides to \u003cem\u003eStaphylococcus aureu\u003c/em\u003es was measured. For the \u003cem\u003ein vivo\u003c/em\u003e study, each labeled peptide was injected into \u003cem\u003eS. aureus\u003c/em\u003e infected mouse thigh after treatment with various doses of ciprofloxacin (CPFX). Two hours after injection, the accumulation of each labeled peptide at the infection site was assessed by SPECT, and then the number of viable bacteria was determined from the accumulation detected. The peptide labeling was successful, and the radiochemical purity was 91±9% (GH, n=8) and 100% (Tricine, n=8). The \u003cem\u003ein vitro\u003c/em\u003e binding of the labeled peptides to \u003cem\u003eS. aureus\u003c/em\u003e (5×10\u003csup\u003e8\u003c/sup\u003e cfu) without serum was 78.9% (GH) and 85.5% (Tricine) of the total \u003csup\u003e99m\u003c/sup\u003eTc activity. With serum, the binding rate was 67.5% (GH) and 13.3% (Tricine). The accumulation of labeled peptide was calculated from the SPECT images, and that in the bacterial infection site (left thigh) was higher than that in the non-infection site (right thigh) for both peptides. Good correlation was found between the target-to-non-target (T/NT) ratios of each labeled peptide and the viable bacterial count at the infection site, and \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(Tricine)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41 had a wider range than \u003csup\u003e99m\u003c/sup\u003eTc-HYNIC(GH)\u003csub\u003e2\u003c/sub\u003e-UBI 29-41.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eUsing the SPECT/labeled peptide method, it was possible to monitor viable bacterial count in the range 10\u003csup\u003e3\u003c/sup\u003e–10\u003csup\u003e8\u003c/sup\u003e cfu, which is appropriate for tracking viable bacterial counts in the same animal over time.\u003c/p\u003e","manuscriptTitle":"In vivo monitoring of viable bacteria by SPECT using 99mTc-HYNIC(GH)2-UBI 29-41 and 99mTc-HYNIC(Tricine)2-UBI 29-41","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-20 21:04:07","doi":"10.21203/rs.3.rs-439672/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"31deac09-be18-43e5-98ad-0eab48781616","owner":[],"postedDate":"April 20th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3784416,"name":"Bacteriology"}],"tags":[],"updatedAt":"2021-05-02T13:54:18+00:00","versionOfRecord":[],"versionCreatedAt":"2021-04-20 21:04:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-439672","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-439672","identity":"rs-439672","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}