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. 2026 Apr 1;4(9):2074–2083. doi: 10.1021/cbmi.5c00303

Pathogen-Specific PET Imaging of Pulmonary Infection Using [18F]Fluoromannitol

Spenser R Simpson †, Tyler Simmons ‡, Haley Echlin ‡, Shyra Wilde ‡, Shashika S Perera †, Paulina Villanueva †, Amy Iverson ‡, Amber Schuster †, Peter Vogel §, Amanda Green ∥,*, Jason Rosch ‡,*, Kiel D Neumann †,*
PMCID: PMC13625748  PMID: 42820011

Abstract

Pneumonia remains a leading cause of global morbidity and mortality, with diagnostic uncertainty frequently complicating clinical management, particularly in patients with chronic lung disease, immunocompromise, or healthcare-associated infections. Conventional imaging modalities and microbiologic testing lack specificity for distinguishing bacterial pneumonia from viral infection, sterile inflammation, or noninfectious lung disease, often resulting in empiric antibiotic overuse and delayed alternative therapies. In this study, we evaluated [18F]­fluoromannitol, a pathogen-specific Positron Emission Tomography (PET) tracer, in well-established murine models of pulmonary infection caused by Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa. Compared with controls, including heat-killed bacterial pneumonitis, influenza A virus–induced pneumonia, and noninfectious Poly­(I:C) induced pneumonia. [18F]­fluoromannitol PET enabled robust, rapid, and anatomically localized detection of active bacterial pneumonia, with tracer uptake correlating with bacterial burden and disease progression. These findings demonstrate that [18F]­fluoromannitol PET can noninvasively distinguish bacterial lung infection from viral and noninfectious etiologies. By directly targeting bacterial metabolism rather than host inflammation, this approach offers a promising strategy to improve etiologic classification of lower respiratory tract infections and support antimicrobial stewardship.

Keywords: PET, fluoromannitol, pneumonia, radiotracer, pulmonary, F18, infection

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Introduction

Pneumonia is a major cause of global morbidity and mortality, responsible for an estimated 2.5 million deaths annually and imposing substantial strain on healthcare resources. Disease burden is greatest among the very young, the elderly, and those with underlying chronic illness or immunocompromised. − In the United States, community-acquired pneumonia (CAP) is most commonly caused by viruses, followed by bacterial, bacterial-viral coinfection, and less frequently, fungal or mycobacterial pathogens, , with a causative pathogen identified in only 38% of adults requiring hospitalization. In contrast, healthcare-associated pneumonia (HAP) is more likely to be caused by bacterial and fungal pathogens, including multidrug-resistant organisms, and disproportionately affects critically ill patients. ,, S. pneumoniae, S. aureus, and P. aeruginosa account for a large share of complicated infections, such as necrotizing pneumonia, empyema, lung abscess, and recurring infections in cystic fibrosis (CF). ,, Patients with chronic lung disease, HAP, or immunocompromised face increased risk of severe disease, atypical pathogens or presentations, and noninfectious causes of lower respiratory tract illnesses (LRTIs), highlighting a critical need for specific, noninvasive, and rapid diagnostic tools capable of differentiating the causes of LRTI. −

Current diagnostic imaging modalities are limited in their ability to distinguish among LRTI etiologies. Chest radiography and computed tomography (CT) are highly sensitive for detecting pulmonary infiltrates but lack specificity, as patterns of consolidation, ground-glass opacities, and nodularity overlap substantially among bacterial, viral, fungal, inflammatory, and malignant processes. − As a result, imaging findings must be interpreted alongside clinical judgment and laboratory testing. Microbiological and molecular testing of noninvasive specimens such as blood, sputum, or nasopharyngeal swabs exhibit widely variable sensitivity and specificity depending on host factors and etiology, and may be difficult to interpret in the setting of bacterial colonization or asymptomatic viral shedding. ,,, This diagnostic uncertainty is particularly problematic in populations with a high pretest probability for multiple etiologies, such as those with underlying lung disease, cancer, sickle cell disease, or other immunodeficiency. In such settings, empiric broad-spectrum antibiotics are frequently initiated despite the absence of confirmed bacterial infection, contributing to antimicrobial overuse, selection for resistance, and antibiotic-associated toxicities. , Conversely, delays in identifying nonbacterial or alternative causes of pulmonary disease may postpone appropriate antiviral, antifungal, immunomodulatory, or oncologic therapies. Definitive diagnosis may require invasive procedures such as bronchoalveolar lavage (BAL) or lung biopsy, which carry procedural risks and may not be feasible in unstable patients. −

Molecular imaging approaches capable of distinguishing active bacterial infection from sterile inflammation or viral pneumonia could substantially improve diagnostic precision and antimicrobial stewardship. Positron Emission Tomography (PET) is a highly evolved molecular imaging modality with an expansive catalog of targeted imaging agents reported in the literature. Recent advances (PET-MR, Explorer (whole body, high sensitivity), and time-of-flight (TOF) technology) , that allow simultaneous acquisition of metabolic and anatomic data, with submm resolution and very low radioactive dose, are further increasing the utility of PET. Recently, we developed [18F]­fluoromannitol, a radiopharmaceutical analogue of mannitol that was demonstrated to possess high specificity for both Gram-positive and Gram-negative bacteria in vivo in several models of bacterial infection. Mannitol metabolism is minimal to absent in mammalian cells, providing an opportunity to exploit a pathogen-specific metabolic pathway to address this unmet clinical need.

In this study, we evaluated the ability of [18F]­fluoromannitol to detect and differentiate pulmonary bacterial infections in vivo using well-established murine models of bacterial pneumonia. To rigorously assess specificity, tracer uptake was compared with nonbacterial controls, including heat-killed bacterial pneumonitis, influenza A virus–induced pneumonia, and noninfectious Poly­(I:C) induced pneumonia. Our findings demonstrate that [18F]­fluoromannitol enables noninvasive visualization of active bacterial pneumonia with delineation from sterile inflammation and viral lung infection, supporting its potential utility as a pathogen-specific imaging biomarker in pulmonary infectious diseases.

Results

Imaging Streptococcus pneumoniae Lung Infection with [18F]­Fluoromannitol

We leveraged a well-characterized pulmonary infection model using S. pneumoniae strain BHN97 (serotype 19F) to assess whether [18F]­fluoromannitol can differentiate sterile inflammation from active infection in a clinically relevant model. The BHN97 strain is known to disseminate from the nasal passages into the lungs and middle ear over approximately 48 h. Pulmonary infection was established by inoculating one group of mice with 1 × 106 CFU of live S. pneumoniae BHN97 and a separate group with a 10-fold excess of heat-killed bacteria.

At 24 h postinoculation, [18F]­fluoromannitol demonstrated a significant increase in %ID/mL within the nasal passages of infected mice compared with heat-killed controls (Figure A, B; p < 0.0001). Interestingly, [18F]­fluoromannitol %ID/mLAVG at 24 h was not significantly different in the lungs of mice inoculated with live S. pneumoniae or heat-killed bacteria. (Figure B; p = 0.9517).

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[18F]­fluoromannitol detects S. pneumoniaein a murine lung infection model. A. [18F]­fluoromannitol PET signal is significantly elevated in the nasal passages of S. pneumoniae-infected mice compared to heat killed bacterial controls at 24 h post challenge (75–90 min PET/CT). B. Summation of [18F]­fluoromannitol PET signal in the lungs and nasal passages, expressed as %ID/mLAVG, measured 75–90 min post injection at each time point (n = 6 for 24- and 48-h infection, n = 4 for heat killed controls). Data presented as mean ± SEM; two way-ANOVA with Tukey post hoc test for significance. C. Pulmonary [18F]­fluoromannitol signal is significantly elevated in infected mice compared to heat killed controls at 48 h post challenge (75–90 min PET/CT). D. Two hour time activity curve of pulmonary [18F]­fluoromannitol signal in infected and heat killed control mice at 48 h post challenge. E. Ex vivo gamma counts demonstrate significant accumulation of [18F]­fluoromannitol exclusively in infected organs only (n = 4 for each group). Data presented as mean ± SEM; two way-ANOVA with Tukey post hoc test for significance. F. Bacterial burden, quantified as CFU, in selected organs from infected and heat killed control mice (n = 4 for all organs in both groups). Data presented as mean ± SEM; two way-ANOVA with Sidak post hoc test for significance. G. Representative confocal microscopy images of lung tissue from heat killed control (left) and S. pneumoniae (right) mice stained by IHC; bacteria are indicated by brown staining. **** = P < 0.0001.

At 48 h postinoculation, [18F]­fluoromannitol activity in the nasal passages had decreased to a 3-fold difference relative to heat-killed controls (Figure B; p < 0.001). In contrast, [18F]­fluoromannitol %ID/mLAVG was significantly increased in the lungs of infected mice compared with heat-killed controls (Figure B, C; p < 0.0001). Maximum Intensity Projection (MIP) shown in Figures S1A and S1B. The current clinical workhorse [18F]­FDG was predictably unable to distinguish between infection and inflammation (Figures S2A, S2B; p = 0.6656). MIP shown in Figures S1C and S1D. Time–activity analysis at 48 h postinoculation showed that differences in pulmonary tracer uptake between infected and heat-killed animals emerged within 20 min of injection (Figure D), while [18F]­FDG at no time point in the 2-h dynamic window showed a statistically significant difference between the infected and inflamed lungs (Figure S2C).

Ex vivo biodistribution of [18F]­fluoromannitol confirmed that radioactive signal originating from the lungs was significantly higher in the infected lungs than in the heart (Figure E; p < 0.0001). In contrast, radioactivity in the hearts of infected mice was not significantly different from radioactivity observed in the hearts or lungs of heat-killed controls (Figure E; p = 0.9998 (infected heart vs heat killed heart) and p = 0.9970 (infected heart vs heat killed lungs)). Ex vivo biodistribution of [18F]­FDG demonstrated no significant difference between the infection and sterile inflammation model in the lungs but did show a difference in hearts (Figure S2D; p = 0.895 lungs, p < 0.0001).

Bacterial burden was confirmed by homogenizing organs of interest and enumerating colony forming units (CFU) by serial dilution on agar. CFU analysis confirmed significant increases of log10 CFU in lungs and nasal cavities compared to heart and liver (Figure F; p < 0.0001). Histological analysis further confirmed the presence of bacteria within the lung tissue (Figure G). Together, these findings demonstrate that [18F]­fluoromannitol can noninvasively track the anatomical progression of S. pneumoniae infection over time in mice via PET.

Two other S. pneumoniae strains that are antibiotic resistant were tested for [18F]­fluoromannitol uptake in order to help determine if [18F]­fluoromannitol PET may be used track antibiotic efficacy. Uptake in these two strains demonstrated similar uptake to a variety of bacteria strains previously reported (Figure S3).

Imaging Staphylococcus aureus Lung Infection with [18F]­Fluoromannitol

We next evaluated [18F]­fluoromannitol in a model of S. aureus lung infection, a more treatment-resistant pathogen and a common culprit of CAP, HAP, and lung infection in CF. Infection was generated by inoculating one group of mice with 5 × 106 CFU of live S. aureus and a separate group with a 10-fold excess of heat-killed bacteria.

At 24 h post-inoculation, [18F]­fluoromannitol PET signal was elevated in the infected mouse compared to heat-killed controls (Figure A). Time–activity analysis demonstrated rapid pulmonary tracer accumulation, with significant differences between infected and control groups apparent within 10 min of injection (Figure B). Infected mice showed significant increases in %ID/mLAVG in the lungs of infected mice compared to the control lungs inoculated with heat-killed bacteria (Figure C; p < 0.001).

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[18F]­fluoromannitol detects S. aureusin a murine lung infection model. A. Pulmonary [18F]­fluoromannitol PET signal is significantly elevated in S. aureus infected mice compared to heat killed bacteria controls at 24 h post-challenge (75–90 min PET/CT). B. Two hour time activity curve of pulmonary [18F]­fluoromannitol signal in infected and heat killed control mice at 24 h post-challenge. C. Summed pulmonary [18F]­fluoromannitol PET signal, expressed as %ID/mLAVG, measured 75–90 min post injection (n = 6 for infection, n = 4 for heat killed). Data presented as mean ± SEM; two way-ANOVA with Tukey post hoc test for significance. D. Ex vivo gamma counts demonstrate significant accumulation of [18F]­fluoromannitol exclusively in infected organs (n = 4 for each group). Data presented as mean ± SEM; two way-ANOVA with Tukey post hoc test for significance. E. Bacterial burden, quantified as CFU, in selected organs from infected and heat killed control mice (n = 4 for all organs in both groups). Data presented as mean ± SEM; two way-ANOVA with Sidak post hoc test for significance. **** = P < 0.0001.

Ex vivo biodistribution confirmed that radioactivity was localized to the lungs, with infected lungs displaying significantly higher %ID/g than the hearts of infected animals (Figure D; p < 0.001). [18F]­fluoromannitol-associated radioactivity was not significantly different in the hearts of mice inoculated with live or heat-killed bacteria (Figure D; p = 0.9997). CFU plating of selected organs demonstrated significant increases of bacterial burden exclusively in the lungs (Figure E; p < 0.0001).

Two S. aureus (USA300 and USA400) strains that are antibiotic resistant were tested for [18F]­fluoromannitol uptake to help determine if [18F]­fluoromannitol PET may be used track antibiotic efficacy. Uptake in these two strains demonstrated similar uptake to a variety of bacteria strains previously reported (Figure S3).

Imaging Pseudomonas aeruginosa Lung Infection with [18F]­Fluoromannitol

We next focused on P. aeruginosa, a common cause of pneumonia in patients with CF. Mice were inoculated with 5 × 105 CFU of live P. aeruginosa or a 10-fold excess of heat-killed bacteria. At 24 h post-inoculation, [18F]­fluoromannitol PET signal was elevated in the lungs of infected mice compared to heat-killed controls (Figure A). MIP shown in Figure S1E. PET images summed from 45–60 min postinjection revealed significantly higher %ID/mLAVG in infected mouse lungs compared to heat-killed controls (Figure B; p < 0.001). Ex vivo biodistribution demonstrated significantly increased tracer accumulation in infected lungs and infected hearts compared to lungs and hearts inoculated with heat-killed bacteria (Figure C; p < 0.0001 for both comparisons). CFU plating revealed bacterial dissemination beyond the lungs, with significantly increased log10 CFU detected in the lungs, heart, and liver of infected animals compared to mice inoculated with heat-killed bacteria (Figure D).

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[18F]­fluoromannitol detects P. aeruginosain a murine lung infection model. A. Pulmonary [18F]­fluoromannitol PET signal is significantly elevated in P. aeruginosa infected mice compared to heat killed bacteria controls at 24 h post challenge (45–60 min PET/CT). B. Summed pulmonary [18F]­fluoromannitol PET signal, expressed as %ID/mLAVG, measured 45–60 min post injection (n = 4 per group). Data presented as mean ± SEM; Welch’s t test assuming Gaussian distribution for significance. C. Ex vivo gamma counts demonstrate significantly greater [18F]­fluoromannitol accumulation in organs from infected mice compared to heat killed controls (n = 4 per group). Data presented as mean ± SEM; two way-ANOVA with Tukey post hoc test for significance. D. Bacterial burden, quantified as CFU, in selected organs from infected and heat killed control mice (n = 4 per group). Data presented as mean ± SEM; two way-ANOVA with Sidak post hoc test for significance. *** = P < 0.001, **** = P < 0.0001.

Imaging Viral Lung Infection with [18F]­Fluoromannitol

While bacterial pneumonia represents a major clinical problem in the hospital setting, viral pneumonias account for a substantial proportion of pulmonary infections. At 72 h postviral inoculation, [18F]­fluoromannitol PET scans demonstrated equivocal PET signal in mice inoculated with H1N1 virus compared to heat killed bacteria controls (Figure A). MIP shown in Figure S1F. Dynamic time activity curves showed no significant differences in [18F]­fluoromannitol %ID/mLAVG compared to heat killed bacteria controls (Figure B). Dynamic scans summed from 75 to 90 min post-[18F]­fluoromannitol injection showed no significant differences in %ID/mLAVG in mice infected with H1N1 compared with mice exhibiting sterile inflammation (Figure A, C; p = 0.3884). Ex vivo biodistribution revealed no significant differences in %ID/g between H1N1-infected and sterile inflammation groups in either the lungs or heart (Figure D; lungs: p = 0.2040; heart: p = 0.2040). Following imaging, lung homogenates demonstrated an average viral burden of 1.35 × 108 TCID50. High-resolution CT imaging of bacterial lung infection, viral lung infection, and sterile inflammation revealed equivocal anatomic results between groups (Figure E).

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[18F]­fluoromannitol uptake in a viral murine lung infection model. A. Representative PET/CT image demonstrating minimal pulmonary [18F]­fluoromannitol signal in a mouse infected with H1N1 influenza virus at 72 h post-challenge (75–90 min PET/CT). B. Two hour time activity curve of pulmonary [18F]­fluoromannitol signal at 72 h post-challenge. C. Summed pulmonary [18F]­fluoromannitol signal, expressed as %ID/mLAVG, measured at 75–90 min post injection in H1N1 infected mice and heat killed controls (n = 6 for infection, n = 4 for heat killed). Data presented as mean ± SEM; two way-ANOVA with Tukey post hoc test for significance. D. Ex vivo gamma counts demonstrate no significant difference in [18F]­fluoromannitol accumulation between virally infected lungs to sterile inflammatory controls (n = 4 for each group). Data presented as mean ± SEM; two way-ANOVA with Tukey post hoc test for significance. E. Representative CT images from mice with lung inflammation induced by live S. pneumoniae, heat-killed bacteria, or H1N1 infection demonstrate minimal gross morphological differences despite distinct underlying etiologies.

Imaging Noninfectious Inflammation with [18F]­Fluoromannitol

Because the tracer is selectively taken up by live and not heat killed bacteria, a noninfectious Poly­(I:C) induced sterile inflammation model was tested to further exclude nonspecific uptake driven by immune activation. Poly­(I:C) is an immunostimulant known for simulating viral infections. Imaging of mice with [18F]­fluoromannitol and [18F]­FDG was done after 4 consecutive days of exposure to Poly­(I:C) as previously described to induce inflammation. PET scans demonstrated differential uptake of [18F]­fluoromannitol and [18F]­FDG. Notably, for each tracer, signal intensity in Poly­(I:C)-induced inflammation was equivalent to that observed in the heat-killed bacteria inflammation model (Figures S4A and S4B). MIPs shown in Figures S1G and S1H. Summed images from 75 to 90 min post-tracer injection showed no significant differences in %ID/mLAVG in the Poly­(I:C) inflammation model compared with mice exhibiting inflammation from heat killed bacteria (Figure S4B; p = 0.9589). Dynamic time activity curves showed no significant differences in either tracer %ID/mLAVG measurement compared to heat killed control (Figure S4D). Ex vivo biodistribution revealed no significant differences in %ID/g between either sterile inflammation groups for their respective tracer (Figure S4E; [18F]­fluoromannitol p = 0.9937, [18F]­FDG p = 0.349).

Biodistribution of [18F]­Fluoromannitol in C57Bl/6J Mice

Previous work using [18F]­fluoromannitol to delineate active infection from inflammation in myositis utilized CBA/J mice. It was found that the pulmonary infection models worked best in C57Bl/6J mice and due to this change, biodistribution was ran to confirm similar pharmacokinetics. As with the previously disclosed biodistribution in CBA/J mice, [18F]­fluoromannitol is excreted through the kidneys with the tracer reaching background levels between 2 and 3 h (Figure S5). Maximum Intensity Projection of a healthy mouse 90 min post-injection is included in Figure S1I for time comparison to the infectious and inflammation models presented.

Discussion

Pulmonary infections represent a major clinical burden across diverse patient populations. A fundamental challenge in the evaluation of LRTI is the marked symptom, imaging, and laboratory overlap among infectious (typical and atypical bacterial, fungal, and viral) and noninfectious etiologies (malignancy, autoimmune or other inflammatory disorders, hemorrhage, and toxic exposures such as vaping-associated lung injury). This overlap limits the ability of noninvasive testing to accurately identify the underlying etiology and frequently necessitates invasive diagnostic procedures to establish even broad disease categories. ,,,, Accurate identification of pathogen(s) causing LRTI can be elusive even when invasive specimens are obtained, and procedures such as BAL and lung biopsies are frequently undesirable or infeasible due to critically ill or unstable patients. − , Because most pneumonias are caused by a relatively narrow spectrum of pathogens, standardized empiric antibiotics are usually successful at treating LRTIs. However, in cases where patients do not respond to empiric therapy, are immunocompromised, or have chronic lung, autoimmune or other inflammatory disorders, or are found to have likely granulomatous or necrotic pulmonary lesions, the list of potential LRTI etiologies – and corresponding appropriate therapies – becomes quite broad. ,,, In these cases, determining even a causative category, such as bacterial, viral, malignancy or other noninfectious, may significantly improve their clinical outcome. Current laboratory and imaging approaches often struggle to distinguish bacterial pneumonia from viral infection or noninfectious LRTI, leading to diagnostic uncertainty, potentially delayed or inappropriate treatment, and antimicrobial overuse.

Conventional imaging modalities play an important role in evaluating LRTI but lack specificity. Chest radiography, CT, and magnetic resonance imaging (MRI) provide sensitive detection of LRTI and structural lung abnormalities yet cannot reliably differentiate bacterial from noninfectious etiologies. Functional imaging with the commonly used [18F]­FDG PET improves sensitivity to inflammation but remains nonspecific, as tracer uptake (glycolytic flux) may reflect host cell activation rather than infection. In LRTI cases marked by poor response to empiric antimicrobial therapy, atypical clinical features, or host chronic comorbidities, greater diagnostic specificity from imaging is needed to distinguish infectious from noninfectious disease and inform appropriate treatment decisions. By contrast, [18F]­fluoromannitol targets bacterial-specific mannitol metabolism, providing a fundamentally different imaging strategy that directly reports on the presence of metabolically active bacteria rather than host-driven inflammatory responses.

Three bacterial species, S. pneumoniae, S. aureus and P. aeruginosa, were chosen for this study to investigate clinically significant pneumonia pathogens with a high rate of complications, with a range of characteristics (Gram-positive nonencapsulated and encapsulated, and Gram-negative). The use of broad-spectrum antibiotics targeting these pathogens remains high in hospitalized patients and continues to promote the emergence of drug-resistant organisms. In the S. pneumoniae BHN97 model, [18F]­fluoromannitol PET captured the temporal progression of infection from the upper airway to the lungs over 48 h, consistent with the known dissemination kinetics of this clinically relevant CAP pathogen. Early tracer accumulation in the nasal passages followed by delayed pulmonary uptake highlights the ability of [18F]­fluoromannitol to localize bacterial burden with both spatial and temporal resolution. Notably, tracer uptake was minimal in the lungs at 24 h despite bacterial inoculation, suggesting that [18F]­fluoromannitol signal reflects established infection rather than transient exposure or inflammation alone. This capability to track infection progression noninvasively may be particularly valuable for monitoring disease evolution or treatment response.

We extended this approach to S. aureus, an important cause of both community- and hospital-acquired pneumonia, and a pathogen frequently targeted empirically due to concerns regarding antibiotic resistance. Although the magnitude of tracer uptake in this model was lower than that observed with S. pneumoniae, [18F]­fluoromannitol reliably distinguished infected lungs from inflamed lungs inoculated with heat killed bacteria and demonstrated rapid pulmonary accumulation detectable within minutes of tracer administration. These findings indicate that [18F]­fluoromannitol PET can detect active S. aureus infection despite interspecies differences in bacterial metabolism and burden, supporting its applicability across Gram-positive pathogens relevant to CAP and HAP.

Importantly, [18F]­fluoromannitol also detected P. aeruginosa infection, a Gram-negative pathogen that is a major contributor to pulmonary morbidity in CF and a frequent target of broad-spectrum antibiotics in hospitalized and neutropenic patients. In our mouse model of P. aeruginosa lung infection, tracer uptake was observed not only in the lungs but also in extrapulmonary tissues, including the heart and liver. CFU analysis confirmed systemic bacterial dissemination, indicating that elevated tracer signal reflected the presence of viable bacteria rather than nonspecific uptake. These findings highlight the sensitivity of [18F]­fluoromannitol PET for detecting Gram-negative bacteria, as well as both localized and disseminated bacterial infection, while emphasizing the importance of interpreting whole-body tracer distribution in the context of disease severity. Furthermore, as P. aeruginosa pathogenicity is well-associated with mucosal surface biofilm formation, these results suggest [18F]­fluoromannitol may be sensitive to this unique bacterial population and warrant further investigation.

A critical finding of this study is the absence of significant [18F]­fluoromannitol uptake in a viral lung infection model despite robust pulmonary inflammation. In mice infected with H1N1 influenza virus, tracer accumulation did not differ from sterile inflammation controls caused by either heat killed bacteria or Poly­(I:C) by PET imaging, time–activity analysis, or ex vivo biodistribution. High-resolution CT similarly failed to distinguish bacterial from viral lung disease, underscoring the limited specificity of anatomic imaging alone. Together, these results demonstrate that [18F]­fluoromannitol PET can differentiate bacterial pneumonia from viral-induced pulmonary inflammation, a distinction with important implications for antibiotic stewardship and clinical decision-making.

Several limitations should be acknowledged. Bacterial burden, metabolic activity, and dissemination varied across models and likely contributed to differences in the magnitude of tracer accumulation. As shown in Figure S6, lungs with no bacterial CFUs did show measurable background signal. Although a formal limit of detection was not established in this study, a bacterial burden of 104 CFU appears to approach the sensitivity threshold of the tracer. Any lower bacterial loads, [18F]­fluoromannitol uptake may be insufficient to reliably distinguish bacterial burden from inflammation. Systemic infection, as observed in the P. aeruginosa model, may potentially complicate interpretation of pulmonary signal in advanced disease. Murine viral infections were directly compared to sterile inflammation generated by heat-killed bacteria or Poly­(I:C) and not inflammation induced by heat-killed virus. Additionally, while murine models provide essential proof-of-concept data, further studies will be required to define sensitivity thresholds, optimize imaging windows, and evaluate performance in heterogeneous clinical populations.

Collectively, these findings address a common and clinically consequential diagnostic dilemma in the evaluation of lower respiratory tract illness, where overlapping clinical, laboratory, and imaging features often obscure the underlying etiology. While precise pathogen identification is not feasible with [18F]­fluoromannitol PET, the ability to distinguish infectious from noninfectious LRTI has substantial implications for management, particularly in patients who fail empiric therapy, present with granulomatous or mass-like pulmonary lesions, are chronically or severely ill, or are at heightened risk for noninfectious complications. In such settings, improved noninvasive etiologic classification may guide antimicrobial decision-making, reduce unnecessary treatment escalation, and, in some cases, obviate the need for invasive diagnostic procedures that may be unsafe, infeasible, or of limited yield.

Conclusion

This study demonstrates that [18F]­fluoromannitol is a pathogen-specific PET tracer capable of imaging pulmonary bacterial infections in vivo while discriminating bacterial infection from sterile inflammation and viral pneumonia. Across multiple murine models with high clinical relevance, [18F]­fluoromannitol uptake correlated with the presence, anatomical localization, and progression of live bacteria, including clinically relevant Gram-positive and Gram-negative pathogens commonly associated with community-acquired pneumonia and cystic fibrosis-related lung disease. The absence of tracer uptake in heat-killed bacterial controls and viral infection models further supports its specificity for metabolically active bacteria.

By directly targeting bacterial metabolism rather than host inflammatory responses, [18F]­fluoromannitol addresses key limitations of conventional anatomic imaging and inflammation-based PET approaches. These findings establish proof-of-concept for metabolically targeted PET imaging as a strategy to improve the noninvasive detection and characterization of bacterial lung infections. Together, this work supports continued development of [18F]­fluoromannitol and related pathogen-specific radiotracers for advancing the diagnosis and management of pulmonary infectious diseases.

Methods

Synthesis of [18F]­Fluoromannitol

[18F]­Fluoromannitol was synthesized as previously described using a commercially available precursor on either the Trasis All-In-One or ELIXYS Flex/Chem automated synthesis platforms. , The final product was formulated in sterile saline for injection.

Microbial Strains and Culture Conditions

S. pneumoniae strain BHN97 was cultured in complete medium supplemented with yeast (C+Y). , S. aureus (Strain XEN36; Revvity) and P. aeruginosa (Strain XEN41; Revvity) were cultured in Brain-Heart Infusion broth supplemented with yeast extract (BD). All bacterial strains were grown at 37 °C with 5% CO2 and aeration when appropriate.

To prepare bacteria for infection, S. pneumoniae was grown to mid logarithmic phase (OD600 nm = 0.4, ≈108 colony forming units/mL), while S. aureus and P. aeruginosa were grown overnight (OD600 nm = 1.0–1.3, ≈2 × 109 colony forming units/mL). Cultures were washed in PBS and diluted to 107 CFU/mL unless otherwise specified. Heat-killed bacteria were prepared by concentrating a subset of the inoculum 10× and heating at 95 °C for 30 min.

For viral pulmonary infection models, a mouse-adapted strain of A/California/04/2009 (H1N1; CA/09) was propagated in the allantoic cavity of 10-day-old specific-pathogen-free embryonated chicken eggs following animal passage. Viral titers were determined by 50% tissue culture infectious dose (TCID50). Virus was thawed and diluted in PBS to the target inoculum immediately prior to infection.

In Vitro Assays

S. aureus (USA300 and USA400) were obtained from American Type Culture Collection. S. pneumoniae (DAW30 and DAW57) bacterial strains were received internally from St. Jude collaborators and demonstrated resistance to clindamycin, erythromycin, and penicillin. Each strain was aerobically grown to absorbance at 600 nm 0.6 to 0.8 in 20 mL of Lysogeny Broth (LB). Bacteria were pelleted from 0.5 mL of the bacterial cultures. The LB was removed, and the pellet was resuspended in 1 mL of PBS media. Radiotracer uptake assays were performed by incubating the resuspended bacteria with [18F]­Fluoromannitol (37 kBq/mL) at 37 °C with rapid agitation. As a control, heat-killed (90 °C for 30 min) bacteria were similarly incubated with the probe. Bacteria were pelleted by centrifuge and washed with PBS. The activity for each pellet was measured using an automated γ counter (Hidex). Six replicates were used for each assay. Counts for each sample were corrected for background and normalized to CFU.

Murine Lung Infection

Female C57Bl/6J mice (8–12 weeks old; Jackson Laboratory, Strain #000664) were used in all studies. Experiments were performed in accordance with Protocol 3176 which underwent review and approved by the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee and the Institutional Biosafety Committee.

For bacterial infections, mice were lightly anesthetized (2%) with isoflurane, and 50 μL of bacterial suspension (∼106 CFU) was administered via intranasal inhalation unless otherwise noted. A separate group of mice received heat-killed bacteria as controls via intranasal inhalation. The true administered CFU was confirmed by plating serial dilutions on appropriate agar.

For viral infections, 102 TCID50 of CA/09 was administered in 50 μL. Lung infections were allowed to progress for 72 h prior to imaging. Following PET/CT scanning, mice were euthanized via carbon dioxide asphyxiation and cervical dislocation.

For biodistribution and CFU quantification, tissues were analyzed in an automated gamma counter (Hidex) and homogenized in 0.5 mL PBS using a hand-held homogenizer (Omni International).

Noninfectious Lung Inflammation Model

To stimulate production of protease in vivo, 7-week-old female Balb/cJ mice were treated with an inflammatory stimulant, Poly­(I:C). 50 μL of 1 mg/mL Poly­(I:C) HMW (Invivogen) intranasal daily for 4 days prior to imaging. Following PET/CT scanning, mice were euthanized via carbon dioxide asphyxiation and cervical dislocation. For biodistribution tissues were analyzed in an automated gamma counter (Hidex).

PET/CT Imaging

Animals were transferred to a Bruker Si78 PET/CT scanner and positioned in a three-mouse bed. 4.6 ± 0.9 MBq of [18F]­Fluoromannitol was administered intravenously via a lateral tail-vein catheter at the start of a 2-h dynamic PET acquisition. PET scans were preceded by a 5 min CT scan for attenuation correction and anatomical coregistration. Dynamic PET frames were acquired as follows: 10 × 15 s, 5 × 30 s, 3 × 60 s, 4 × 180 s, 8 × 300 s, 4 × 900 s.

Skyscan CT

Computed Tomography (CT) was performed on a Bruker Skyscan 1276 system (Bruker Biospin CT GmbH, Ettlingen, Germany). The mouse was placed in the scanner under 3% isoflurane, and a scout scan was taken to position the mouse. The CT scan was taken over 2 bed positions and had the following parameters: 70 kV voltage, 200 μA current, 0.5 mm Al filter, 0.2° rotation steps over 360°, and 2 frame averaging. The total acquisition time was about 5 min, and the reconstructed image had 18.6 μm isotropic resolution.

Histology

Following PET/CT imaging, select mice were euthanized and lungs excised for histological analysis. Tissues were fixed in 10% neutral-buffered formalin (ThermoFisher) for 2 weeks, embedded in paraffin, sectioned at 4 μm, and mounted on SuperFrost Plus slides (ThermoFisher). Sections were stained with hematoxylin and eosin (H&E) or subjected to immunohistochemical (IHC) staining to detect S. pneumoniae.

For IHC, antigen retrieval was performed in prediluted Cell Conditioning Solution (CC1; Ventana Medical Systems) for 32 min on a Discovery Ultra immunostainer. A rabbit polyclonal antibody against S. pneumoniae (NB100-64502; Novus Biologicals; 1:1000) was used as the primary probe. Binding was detected using OmniMap anti-Rabbit (760-4311) and visualized with DISCOVERY ChromoMap DAB (Ventana Medical Systems). Stained sections were evaluated by a pathologist blinded to experimental groups.

PET Quantification

Imaging data were analyzed using PMOD 4.4. Volumes of interest (VOIs) were manually defined on CT by outlining the pleural or nasal cavity. Time–activity curves were extracted as mean %ID/mL over each VOI.

Statistical Analysis

Quantitative data are presented as mean ± SEM unless otherwise stated. Group comparisons were performed using one-way or two-way analysis of variance (ANOVA) with appropriate post hoc multiple-comparison tests (e.g., Tukey, Sidak, Bonferroni), depending on the experimental design. A p-value <0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism version 10.6.1.

Supplementary Material

im5c00303_si_001.pdf (627.7KB, pdf)

Acknowledgments

We would like to thank the Molecular Imaging Core at St. Jude Children’s Research Hospital for the use of their facilities and the production of [18F]fluoromannitol. We would like to thank the Center for In Vivo Imaging and Therapeutics at St. Jude Children’s Research Hospital for the use of their preclinical imaging facilities.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.5c00303.

  • Maximum intensity projections of PET scans; [18F]­FDG PET imaging of S. pneumoniae lung infection; uptake of [18F]­Fluoromannitol in vitro in antibiotic-resistant organisms; PET imaging of noninfectious Poly­(I:C)-induced lung inflammation; [18F]­Fluoromannitol biodistribution in C57BL/6 mice; and CFU vs %ID/mL correlation plot (PDF)

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Hickman Hybrid Systems Lab, University of Central Florida, Orlando, FL 32826

K.D.N., J.R., and S.R.S. proposed and supervised the overall project. S.R.S. and S.S.P. conducted or supported radiochemistry. S.R.S., T.S., S.S.P., Pa.V., A.S., and S.W. conducted in vivo imaging and collected ex vivo data. A.S. conducted the in vitro experiments. T.S., H.E., S.W., and A.I. developed and provided the mouse models. Data analysis was performed by S.R.S., T.S., S.S.P., and Pa.V. Histology data was interpreted by Pe.V. The manuscript was written and edited by S.R.S., K.D.N., and A.G. Funding was provided by K.D.N. and J.R. Clinical connection and interpretation were provided by A.G.

This work is supported by NIH/NIAID R01AI192221 and the American Syrian Lebanese Associated Charities (ALSAC).

The authors declare the following competing financial interest(s): Spenser R. Simpson and Kiel D. Neumann hold a patent for the use of [18F]­fluoromannitol. Systems and Methods for Imaging Diverse Pathogenic Bacteria In Vivo with [18F]­fluoromannitol Positron Emission Tomography. PCT/US23/26841, WO/2024/006582 Published March 1st, 2024. PCT.

Published as part of Chemical & Biomedical Imaging special issue “New Chemistry and Emerging Technologies for Molecular Imaging”.

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Supplementary Materials

im5c00303_si_001.pdf (627.7KB, pdf)

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