Protective effect of probiotics on Salmonella infectivity assessed with combined in vitro gut fermentation-cellular models

Annina Zihler 0 Mlanie Gagnon 0 Christophe Chassard 0 Christophe Lacroix 0 0 Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich , Schmelzbergstrasse 7, 8092 Zurich , Switzerland 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 mucussecreting 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. - Background The human colon constitutes a protective and nutrientrich habitat to trillions of bacteria living in symbiosis with the host [1]. This complex consortium constantly competes with exogenous microbes for attachment sites in the brush border of intestinal epithelial cells, thus preventing pathogens from entering specific ecological niches and gut tissues [2]. Pathogens may however overcome this line of defense, leading to different manifestations of disease. Infectious gastroenteritis caused by non-typhoidal strains of Salmonella enterica spp. enterica is an important cause of morbidity and mortality worldwide [3]. Due to the increasing incidence of antibiotic resistant and more virulent serovars [4], the use of probiotics with specific anti-Salmonella activities is a prevailing interest. Mechanisms by which probiotics inhibit pathogens include competition for nutritional substrates and adhesion sites on intestinal epithelial cells, secretion of antimicrobial substances as well as toxin inactivation and host immunity stimulation [5]. However, in vivo mechanistic studies of probiotics and gut microbiota are hindered by ethical considerations, compliance issues and high costs. A variety of in vitro gut models have been applied to separately investigate microbe-microbe and simple microbe-host interactions [6-8]. Owing to the complexity of the intestinal environment, suitable models accounting for all intestinal parameters including both the gut microbiota and their substrates and metabolic products as well as the presence of epithelial intestinal cells, represent an indispensable platform for preclinical probiosis assessment. To investigate the complex gut microbiota in vitro, continuous intestinal fermentation models utilizing immobilized fecal microbiota have been developed for the controlled long-term cultivation of gut microbiota with conserved biodiversity [9-11]. Such models allow independent testing of different experimental treatments on both gut microbiota composition and metabolic activity within a single experimental period, using the same microbiota under controlled environmental conditions, which are designed to simulate the proximal, transverse and distal colon of healthy and infected subjects [9-14]. More recently, a three-stage in vitro colonic fermentation model of Salmonella infection in child colon was used to assess the effects of probiotic and prebiotic treatments on gut microbial behavior and on S. Typhimurium infection [15]. The activity of microcin B17-producing Escherichia coli L1000 wt [16] and bacteriocinogenic Bifidobacterium thermophilum RBL67, both exhibiting strong anti-Salmonella activity in simple in vitro tests [17,18], as well as the microcin B17-negative mutant strain MccB17-, were tested in two threestage models inoculated with the same fecal inoculum. When added to the colonic model, E. coli L1000 unexpectedly stimulated Salmonella growth in all reactors independently of the microcin B17-phenotype, partly due to a low colonization of the strain in the complex intestinal environment. In contrast, thermophilicin RBL67-producing Bifidobacterium thermophilum RBL67 revealed high competitiveness and colonized at high levels but did not reduce Salmonella counts, most likely a function of the presence of a very high Salmonella population in the in vitro model prior to probiotic addition. Most data available on the mechanistic effects of probiotics on the host are derived from in vitro studies with intestinal cells [19]. Such models have also been used to investigate bacterial interactions with the intestinal epithelium during enteric infection [20]. Salmonella pathogenesis, for example, has been studied in pure cultures using epithelial Caco-2 and HT-29 cell models [21,22], both of which lack the ability to produce mucus. The mucus-secreting HT29-MTX cell line however, represents more accurate physiological conditions of the gastrointestinal tract for investigating pathogenic behavior during infection, as the presence of mucus has been shown to enhance pathogenicity of pathogens such as Campylobacter jejuni [23]. All interaction studies of pathogens and prob (...truncated)