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Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5′-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1β and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-γ and TNF-α responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products.

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 1×1010 CFU and luminescence signals at doses ranging from 1×105 to 1×1010 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.

Dairy propionibacteria are widely used in starter cultures for Swiss type cheese. These bacteria can ferment glucose, lactic acid, and glycerol into propionic acid, acetic acid, and carbon dioxide. This research examined the antifungal effect of dairy propionibacteria when glycerol was used as carbon source for bacterial growth. Five type strains of propionibacteria were tested against the yeast Rhodotorula mucilaginosa and the molds Penicillium commune and Penicillium roqueforti. The conversion of 13C glycerol by Propionibacterium jensenii was followed with nuclear magnetic resonance. In a dual culture assay, the degree of inhibition of the molds was strongly enhanced by an increase in glycerol concentrations, while the yeast was less affected. In broth cultures, decreased pH in glycerol medium was probably responsible for the complete inhibition of the indicator fungi. NMR spectra of the glycerol conversion confirmed that propionic acid was the dominant metabolite. Based on the results obtained, the increased antifungal effect seen by glycerol addition to cultures of propionibacteria is due to the production of propionic acid and pH reduction of the medium.

Background: Daily routine ventilator-filter exchange interrupts the integrity of the ventilator circuit. We hypothesized that this might reduce positive airway pressure in mechanically ventilated ICU patients, inducing alveolar collapse and causing impaired oxygenation and compliance of the respiratory system., Methods: We studied 40 consecutive ICU subjects (P(aO2)/F(IO2) ratio ≤ 300 mm Hg), mechanically ventilated with pressure-regulated volume control or pressure support and PEEP ≥ 5 cm H2O. Before the filter exchange, (baseline) tidal volume, breathing frequency, end-inspiratory plateau pressure, and PEEP were recorded. Compliance of the respiratory system was calculated; F(IO2), blood pressure, and pulse rate were registered; and P(aO2), P(aCO2), pH, and base excess were measured. Measurements were repeated 15 and 60 min after the filter exchange. In addition, a bench test was performed with a precision test lung with similar compliance and resistance as in the clinical study., Results: The exchange of the filter took 3.5 ± 1.2 s (mean ± SD). There was no significant change in P(aO2) (89 ± 16 mm Hg at baseline vs 86 ± 16 mm Hg at 15 min and 88 ± 18 mm Hg at 60 min, P = .24) or in compliance of the respiratory system (41 ± 11 mL/cm H2O at baseline vs 40 ± 12 mL/cm H2O at 15 min and 40 ± 12 mL/cm H2O at 60 min, P = .32). The bench study showed that auto-triggering by the ventilator when disconnecting from the expiratory circuit kept the tracheal pressure above PEEP for at least 3 s with pressure controlled ventilation., Conclusions: This study showed that a short disconnection of the expiratory ventilator circuit from the ventilator during filter exchange was not associated with any significant deterioration in lung function 15 and 60 min later. This result may be explained by auto-triggering of the ventilator with high inspiratory flows during the filter exchange, maintaining airway pressure.