Dual-Color Bioluminescence Imaging for Simultaneous Monitoring of the Intestinal Persistence of Lactobacillus plantarum and Lactococcus lactis in Living Mice.

Lactic acid bacteria are found in the gastrointestinal tract of mammals and have received tremendous attention due to their health-promoting properties. We report the development of two dual-color luciferase-producing Lactobacillus (Lb.) plantarum and Lactococcus (Lc.) lactis strains for noninvasive simultaneous tracking in the mouse gastrointestinal tract. We previously described the functional expression of the red luciferase mutant (CBRluc) from Pyrophorus plagiophthalamus in Lb. plantarum NCIMB8826 and Lc. lactis MG1363 (C. Daniel, S. Poiret, V. Dennin, D. Boutillier, and B. Pot, Appl Environ Microbiol 79:1086-1094, 2013, http://dx.doi.org/10.1128/AEM.03221-12). In this study, we determined that CBRluc is a better-performing luciferase for in vivo localization of both lactic acid bacteria after oral administration than the green click beetle luciferase mutant construct developed in this study. We further established the possibility to simultaneously detect red- and green-emitting lactic acid bacteria by dual-wavelength bioluminescence imaging in combination with spectral unmixing. The difference in spectra of light emission by the red and green click beetle luciferase mutants and dual bioluminescence detection allowed in vitro and in vivo quantification of the red and green emitted signals; thus, it allowed us to monitor the dynamics and fate of the two bacterial populations simultaneously. Persistence and viability of both strains simultaneously administered to mice in different ratios was studied in vivo in anesthetized mice and ex vivo in mouse feces. The application of dual-luciferase-labeled bacteria has considerable potential to simultaneously study the interactions and potential competitions of different targeted bacteria and their hosts.

Anti-inflammatory properties of Streptococcus salivarius, a commensal bacterium of the oral cavity and digestive tract.

Streptococcus salivarius is one of the first colonizers of the human oral cavity and gut after birth and therefore may contribute to the establishment of immune homeostasis and regulation of host inflammatory responses. The anti-inflammatory potential of S. salivarius was first evaluated in vitro on human intestinal epithelial cells and human peripheral blood mononuclear cells. We show that live S. salivarius strains inhibited in vitro the activation of the NF-κB pathway on intestinal epithelial cells. We also demonstrate that the live S. salivarius JIM8772 strain significantly inhibited inflammation in severe and moderate colitis mouse models. These in vitro and in vivo anti-inflammatory properties were not found with heat-killed S. salivarius, suggesting a protective response exclusively with metabolically active bacteria.

Bioluminescence imaging study of spatial and temporal persistence of Lactobacillus plantarum and Lactococcus lactis in living mice.

Lactic acid bacteria, especially lactobacilli, are common inhabitants of the gastrointestinal tract of mammals, for which they have received considerable attention due to their putative health-promoting properties. In this study, we describe the development and application of luciferase-expressing Lactobacillus plantarum and Lactococcus lactis strains for noninvasive in vivo monitoring in the digestive tract of mice. We report for the first time the functional in vitro expression in Lactobacillus plantarum NCIMB8826 and in Lactococcus lactis MG1363 of the click beetle luciferase (CBluc), as well as Gaussia and bacterial luciferases, using a combination of vectors, promoters, and codon-optimized genes. We demonstrate that a CBluc construction is the best-performing luciferase system for the noninvasive in vivo detection of lactic acid bacteria after oral administration. The persistence and viability of both strains was studied by bioluminescence imaging in anesthetized mice and in mouse feces. In vivo bioluminescence imaging confirmed that after a single or multiple oral administrations, L. lactis has shorter survival times in the mouse gastrointestinal tract than L. plantarum, and it also revealed the precise gut compartments where both strains persisted. The application of luciferase-labeled bacteria has significant potential to allow the in vivo and ex vivo study of the interactions of lactic acid bacteria with their mammalian host.

Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria.

AIM: To investigate the correlation between in vitro and in vivo immunomodulation potential of the probiotic strain and its ability to prevent experimental colitis in mice. METHODS: In vitro immunomodulation was assessed by measuring interleukin (IL)-12p70, IL-10, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) release by human peripheral blood mononuclear cells (PBMCs) after 24 h stimulation with 13 live bacterial strains. A murine model of acute TNBS-colitis was next used to evaluate the prophylactic protective capacity of the same set of strains. RESULTS: A strain-specific in vivo protection was observed. The strains displaying an in vitro potential to induce higher levels of the anti-inflammatory cytokine IL-10 and lower levels of the inflammatory cytokine IL-12, offered the best protection in the in vivo colitis model. In contrast, strains leading to a low IL-10/IL-12 cytokine ratio could not significantly attenuate colitis symptoms. CONCLUSION: These results show that we could predict the in vivo protective capacity of the studied lactic acid bacteria (LAB) based on the cytokine profile we established in vitro. The PBMC-based assay we used may thus serve as a useful primary indicator to narrow down the number of candidate strains to be tested in murine models for their anti-inflammatory potential.

Anti-inflammatory potential of the probiotic dietary supplement Lactibiane Tolérance: in vitro and in vivo considerations.

BACKGROUND & AIMS: Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. In inflammatory bowel disease (IBD), where major modifications of the intestinal microflora have been reported, there is an increasing interest in modulating the flora with probiotic products. This work addresses the anti-inflammatory potential of Lactibiane Tolérance, a probiotic dietary supplement (mixture of four strains) using in vitro and in vivo approaches. METHODS: Comparison of the four individual strains and the commercial product reconstituted from them was conducted by in vitro tests (cytokine release after 24h stimulation of human peripheral blood mononuclear cells (PBMC)). The potential immunomodulatory characteristics of Lactibiane Tolérance were determined in vivo in an acute mice model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Assessment of colitis included blinded histological and macroscopic scores. RESULTS: We showed that Lactibiane Tolérance has anti-inflammatory properties in vitro by stimulating IL-10 production and in vivo by conferring a significant protective effect in the TNBS-induced colitis model (more than 50% decrease of colitis symptoms, P<0.01). CONCLUSIONS: These results demonstrate that a probiotic dietary supplement, Lactibiane Tolérance, can significantly prevent the initial injury of TNBS and could stimulate the initiation of clinical trials in IBD.

Selecting lactic acid bacteria for their safety and functionality by use of a mouse colitis model.

Studies showed that specific probiotics might provide therapeutic benefits in inflammatory bowel disease. However, a rigorous screening of new probiotics is needed to study possible adverse interactions with the host, particularly when intended for administration to individuals with certain health risks. In this context, the objective of this study was to investigate the role of three lactobacilli (LAB) on intestinal inflammation and bacterial translocation using variations of the mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute colitis. We first compared the in vitro ability of LAB to survive gastrointestinal tract (GIT) conditions and their ability to persist in the GIT of mice following daily oral administration. As a control, we included a nonprobiotic Lactobacillus paracasei strain, previously isolated from an endocarditis patient. Feeding high doses of LAB strains to healthy and to TNBS-treated mice did not induce any detrimental effect or abnormal translocation of the bacteria. Oral administration of Lactobacillus salivarius Ls-33 had a significant preventive effect on colitis in mice, while Lactobacillus plantarum Lp-115 and Lactobacillus acidophilus NCFM did not. None of the three selected LAB strains translocated to extraintestinal organs of TNBS-treated mice. In contrast, L. paracasei exacerbated colitis under severe inflammatory conditions and translocated to extraintestinal organs. This study showed that evaluations of the safety and functionality of new probiotics are recommended. We conclude that not all lactobacilli have similar effects on intestinal inflammation and that selected probiotics such as L. salivarius Ls-33 may be considered in the prevention or treatment of intestinal inflammation.

Recommendations for improved use of the murine TNBS-induced colitis model in evaluating anti-inflammatory properties of lactic acid bacteria: technical and microbiological aspects.

Probiotic bacteria have been shown to exert promising beneficial effects in different types of intestinal disorders, including chronic inflammation. In this context, animal models of inflammatory bowel disease are useful in studying the possible prophylactic role of candidate probiotic strains. This study aimed at evaluating the critical technological and microbiological parameters as well as the robustness of the murine trinitrobenzene sulfonic acid (TNBS)-induced model of colitis, after intragastric administration of lactic acid bacteria (LAB) preparations. A standardized methodology was applied to assess the protective effect achieved by various bacterial concentrations and culture conditions of the reference strain Lactobacillus plantarum NCIMB 8826. Not only was protection found to vary in function in different levels of colitis, but also repeated experiments showed a clear bacterial dose-dependent attenuation of colitis. The physiological stage of bacteria was shown to impact as well, with substantial, mild, or reduced improvement of inflammatory scores for exponentially growing, stationary-phase, or killed bacteria, respectively. A recombinant strain, secreting murine interleukin-10 (IL-10) and previously reported to successfully treat colitis in two different models of murine colitis (dextran sulfate sodium [DSS] and IL-10-deficient mice), was used to validate the final experimental conditions. In conclusion, we identified and optimized some of the key parameters that need to be controlled in order to ensure reliable comparison of results generated over a long period of time or independent experiments. The recommendations for an improved model presented here will prove to be helpful for reproducible, independent comparison of the anti-inflammatory potential of wild-type or recombinant candidate probiotic strains, whether administered as pure cultures or as blends.