Introduction

Infectious diseases are a major cause of morbidity and mortality worldwide. Better diagnosis and treatment of infections has motivated the recent development of pathogen-specific PET tracers such as [2-¹⁸F]maltose (1). One example of a successful non-invasive method already used clinically is breath testing, whereby a ¹³C-eniched substrate is administered orally to a patient followed by detection of exhaled [¹³C]CO₂ (2). Breath testing has traditionally been used to detect bacteria within the gastrointestinal tract especially H. pylori. The presence of the urease-producing H. pylori is suggested by the conversion of orally administered [¹³C]urea to [¹³C]CO₂ (2). In this study, we expand the scope of breath testing to include other ¹³C-enriched metabolites, that are administered intravenously rather than orally, with [¹³C]CO₂ production detected rapidly via a laser-based technology (3). We also show that [¹³C]CO₂ production by methicillin-resistant S. aureus MRSA clinical isolates is correlated with [2-¹⁸F]maltose accumulation in vivo.

Methods

Overnight cultures of bacterial strains were diluted to an optical density at 600 nm of 0.05 and grown to exponential phase. Bacterial cultures were then incubated with 1 mM of different ¹³C-enriched compounds at 37 °C for 120 minutes. After incubation, samples were sent for [¹³C]CO₂analysis using a Sable Systems Isotope Analyzer. 

In vivo murine myositis model

Healthy and infected CBA/J mice were used to study the metabolism of various universally ¹³C-enriched metabolites. After metaboilte administration, the mice were kept inside a metabolic chamber attached to a positive/negative pump for the duration of the experiment. Exhaled gas was collected using vacutainer tubes and sent for analysis.

Results/Discussion

S. aureus, E. coli, S. typhimurium, E. Faecalis and E. cloacae showed [¹³C]CO₂ production after incubation with ¹³C-glucose,¹³C-mannitol and ¹³C-maltose. In addition, E. coli showed [¹³C]CO₂production after incubation with ¹³C-sorbitol, ¹³C-xylose and ¹³C-arabinose. To evaluate background production of [¹³C]CO_2, healthy mice were injected with ¹³C-enriched glucose, sorbitol, arabinose, xylose, urea, maltotriose, mannitol or maltose. Glucose and sorbitol are used in the metabolic pathways of mammalian cells and therefore showed [¹³C]CO₂production. In contrast, ¹³C-arabinose, ¹³C-xylose, ¹³C-maltotriose, ¹³C-mannitol, and ¹³C-maltose showed no background metabolism. ¹³C-mannitol and ¹³C-maltose were further evaluated in a S. aureus and/or E. coli infection model. [¹³C]CO₂ production by MRSA after incubation with ¹³C-maltose showed similar tracer uptake profile when compared to [2-¹⁸F]maltose.

Conclusion

¹³C-enriched metabolites could be used for the noninvasive detection and diagnose of bacterial infections in vivo. [¹³C]CO₂ production in different MRSA clinical isolates using [U-¹³C] maltose were positively correlated with [2-¹⁸F]maltose accumulation in vivo.

Novelty

[2-¹⁸F]maltose is a pathogen-targeted PET tracer recently synthesized chemoenzymatically via reverse phosphorolysis from [¹⁸F]FDG, a method with broad implications for point-of-care diagnostics. This is also the first demonstration of bacteria-dependent ¹³C-enriched metabolite conversion to [¹³C]CO₂ in vivo for a variety of new substrates.

Impact

Pathogen-targeted PET and intravenous [¹³C]CO₂ breath testing complement each other and are straightforward to translate. These methods may be used to better diagnose and treat infected patients.

Author