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New biological strategies for the treatment of Salmonella infection are needed in response to the increase in antibiotic-resistant strains. Escherichia coil L1000 and Bifidobacterium thermophilum RBL67 were previously shown to produce antimicrobial proteinaceous compounds (microcin B17 and thermophilicin B67, respectively) active in vitro against a panel of Salmonella strains recently isolated from clinical cases in Switzerland. In this study, two three-stage intestinal continuous fermentation models of Salmonella colonization inoculated with immobilized faeces of a two-year-old child were implemented to study the effects of the two bacteriocinogenic strains compared with a bacteriocin-negative mutant of strain L1000 on Salmonella growth, as well as gut microbiota composition and metabolic activity. Immobilized E. coli L1000 added to the proximal colon reactor showed a low colonization, and developed preferentially in the distal colon reactor independent of the presence of genetic determinants for microcin B17 production. Surprisingly, E. coli L1000 addition strongly stimulated Salmonella growth in all three reactors. In contrast, B. thermophilum RBL67 added in a second phase stabilized at high levels in all reactors, but could not inhibit Salmonella already present at a high level (>[10.sup.7] c.f.u, [ml.sup.-1]) when the probiotic was added. Inulin added at the end of fermentation induced a strong bifidogenic effect in all three colon reactors and a significant increase of Salmonella counts in the distal colon reactor. Our data show that under the simulated child colonic conditions, the microcin B17 production phenotype does not correlate with inhibition of Salmonella but leads to a better colonization of E. coli L1000 in the distal colon reactor. We conclude that in vitro models with complex and complete gut microbiota are required to accurately assess the potential and efficacy of probiotics with respect to Salmonella colonization in the gut. DOI 10.1099/mic.0.042036-0

Abstract Background Accurate assessment of probiotics with targeted anti-Salmonella activity requires suitable models accounting for both, microbe-microbe and host-microbe interactions in gut environments. Here we report the combination of two original in vitro intestinal models closely mimicking the complex in vivo conditions of the large intestine. Effluents from continuous in vitro three-stage fermentation colonic models of Salmonella Typhimurium infection inoculated with immobilized child microbiota and Salmonella were directly applied to confluent mucus-secreting HT29-MTX cell layers. The effects of Salmonella, addition of two bacteriocinogenic strains, Bifidobacterium thermophilum RBL67 (thermophilicin B67) and Escherichia coli L1000 (microcin B17), and inulin were tested on Salmonella growth and interactions with epithelial cell layers. Salmonella adhesion and invasion were investigated and epithelial integrity assessed by transepithelial electrical resistance (TER) measurements and confocal microscopy observation. Data from complex effluents were compared with pure Salmonella cultures. Results Salmonella in effluents of all reactors of the colonic fermentation model stabilized at mean values of 5.3 ± 0.8 log10 cfu/ml effluent. Invasion of cell-associated Salmonella was up to 50-fold lower in complex reactor samples compared to pure Salmonella cultures. It further depended on environmental factors, with 0.2 ± 0.1% being measured with proximal, 0.6 ± 0.2% with transverse and 1.3 ± 0.7% with distal reactor effluents, accompanied by a similar high decrease of TER across cell monolayers (minus 45%) and disruption of tight junctions. Subsequent addition of E. coli L1000 stimulated Salmonella growth (6.4 ± 0.6 log10 cfu/ml effluent of all 3 reactors) and further decreased TER, but led to 10-fold decreased invasion efficiency when tested with distal reactor samples. In contrast, presence of B. thermophilum RBL67 revealed a protective effect on epithelial integrity compared to previous E. coli L1000 periods, as reflected by a significant mean increase of TER by 58% in all reactors. Inulin addition enhanced Salmonella growth and invasion when tested with distal and proximal reactor samples, respectively, but induced a limited decrease of TER (minus 18%) in all reactors. Conclusions Our results highlight the benefits of combining suitable cellular and colonic fermentation models to assess strain-specific first-level host protection properties of probiotics during Salmonella infection, providing an efficient system biology tool for preclinical development of new antimicrobials.