Understanding of the gut microbiome's protective mechanism is limited. Fecal sampling and ex vivo strategies offer little real-time information and need many animals for longitudinal studies. Metabolic engineering permitted species-specific labeling and fluorescence imaging of Bacteroides fragilis (Bf) in vivo (1), albeit with shallow depth and low resolution. Further work using positron emission tomography (PET) showed promise but was limited by time constraints due to in vitro labeling with short-living radioisotopes (2). We aim to optimize the conditions of metabolic engineering-based protocols formerly established, "clicking" a radiotracer in vivo, to monitor modulation of Salmonella Typhimurium (STm) infection with Escherichia coli Nissle (EcN). Bacterial membranes were thus decorated with azide-modified sugars. Verification of labeling efficiency was performed by clicking alkyne conjugated fluorescent agents (AlexaFluor488) before fluorescence-activated cell sorting (FACS) analysis and in vitro fluorescent imaging. The bound fluorescent-agent was localized by fluorescent microscopy. Mean fluorescence intensity significantly differed from the negative control (fluorophore unconjugated cells) for STm and EcN. Yet, this was non-significant compared to control samples with fluorescent agent added without azidosugar pre-incubation. This indicates nonspecific intercalation of the fluorophore, with labeling not resulting from the desired click chemistry reaction. Compared to percent labeled cells for the published positive control (Bf) (1,2) neither EcN or STm were labeled significantly. Microscopy results supported this, where bound-fluorescent agent was localized to the membrane in Bf, but dispersed randomly for EcN and STm. Here, we have shown that metabolic engineering-click chemistry labeling is not presently translatable to all gram-negative species and success is dependent on fine-tuning conditions based on lipopolysaccharide structure and sugar metabolism of each. Further adaptation is vital before use in PET imaging of STm infection and modulation.

1. Geva-Zatorsky. Nat Med. 2015;21(9):1091-1100.

2.Wang. Eur J Nucl Med Mol Imaging. 2020;47(4):991-1002.