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Lactobacillus reuteri  is a symbiont that inhabits the gastrointestinal (GI) tract of mammals, and several strains are used as probiotics. After introduction of probiotic strains in a complex ecosystem like the GI tract, keeping track of them is a challenge. The main objectives of this study were to introduce reporter proteins that would enable  in vivo  and  in vitro  detection of  L .  reuteri  and increase knowledge about its interactions with the host. We describe for the first time cloning of codon-optimized reporter genes encoding click beetle red luciferase (CBRluc) and red fluorescent protein mCherry in  L .  reuteri  strains ATCC PTA 6475 and R2LC. The plasmid persistence of mCherry-expressing lactobacilli was evaluated by both flow cytometry (FCM) and conventional plate count (PC), and the plasmid loss rates measured by FCM were lower overall than those determined by PC. Neutralization of pH and longer induction duration significantly improved the mCherry signal. The persistency, dose-dependent signal intensity and localization of the recombinant bacteria in the GI tract of mice were studied with an  in vivo  imaging system (IVIS), which allowed us to detect fluorescence from 6475-CBRluc-mCherry given at a dose of 1×10 10  CFU and luminescence signals at doses ranging from 1×10 5  to 1×10 10  CFU. Both 6475-CBRluc-mCherry and R2LC-CBRluc were localized in the colon 1 and 2 h after ingestion, but the majority of the latter were still found in the stomach, possibly reflecting niche specificity for R2LC. Finally, an  in vitro  experiment showed that mCherry-producing R2LC adhered efficiently to the intra cellular junctions of cultured IPEC-J2 cells. In conclusion, the two reporter genes CBRluc and mCherry were shown to be suitable markers for biophotonic imaging (BPI) of  L .  reuteri  and may provide useful tools for future studies of  in vivo  and  in vitro  interactions between the bacteria and the host.

plos one

In Vivo and In Vitro Detection of Luminescent and Fluorescent Lactobacillus reuteri and Application of Red Fluorescent mCherry for Assessing Plasmid Persistence