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Infection and Immunity, July 2007, p. 3490-3497, Vol. 75, No. 7
0019-9567/07/$08.00+0     doi:10.1128/IAI.00119-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Commensal Bacterial Strains That Modulate Yersinia enterocolitica and Dextran Sodium Sulfate-Induced Inflammatory Responses: Implications for the Development of Probiotics{triangledown}

Julia S. Frick,* Kerstin Fink, Frauke Kahl, Maria J. Niemiec, Matteo Quitadamo, Katrin Schenk, and Ingo B. Autenrieth

Institute of Medical Microbiology and Hygiene, University of Tübingen, Tübingen, Germany

Received 24 January 2007/ Returned for modification 13 March 2007/ Accepted 22 April 2007


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ABSTRACT
 
An increasing body of evidence suggests that probiotic bacteria are effective in the treatment of enteric infections, although the molecular basis of this activity remains elusive. To identify putative probiotics, we tested commensal bacteria in terms of their toxicity, invasiveness, inhibition of Yersinia-induced inflammation in vitro and in vivo, and modulation of dextran sodium sulfate (DSS)-induced colitis in mice. The commensal bacteria Escherichia coli, Bifidobacterium adolescentis, Bacteroides vulgatus, Bacteroides distasonis, and Streptococcus salivarius were screened for adhesion to, invasion of, and toxicity for host epithelial cells (EC), and the strains were tested for their ability to inhibit Y. enterocolitica-induced NF-{kappa}B activation. Additionally, B. adolescentis was administered to mice orally infected with Y. enterocolitica and to mice with mucosae impaired by DSS treatment. None of the commensal bacteria tested was toxic for or invaded the EC. B. adolescentis, B. distasonis, B. vulgatus, and S. salivarius inhibited the Y. enterocolitica-induced NF-{kappa}B activation and interleukin-8 production in EC. In line with these findings, B. adolescentis-fed mice had significantly lower results for mean pathogen burden in the visceral organs, intestinal tumor necrosis factor alpha mRNA expression, and loss of body weight upon oral infection with Y. enterocolitica. In addition, the administration of B. adolescentis decelerated inflammation upon DSS treatment in mice. We suggest that our approach might help to identify new probiotics to be used for the treatment of inflammatory and infectious gastrointestinal disorders.


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INTRODUCTION
 
Commensal bacteria play a crucial role in maintaining gastrointestinal homeostasis. For example, some commensal bacteria, such as lactobacilli (5, 10, 33) bifidobacteria (5, 31), or Escherichia coli Nissle 1917 (5, 31), are known to have beneficial effects on the host mucosal immune system. The available data suggest that probiotics possess the ability to modulate the immune system by promoting the endogenous host defense systems (40). Probiotics are thought to modify various immune parameters, including innate and adaptive immunity, and seem to enhance the activity of natural killer cells in the elderly (40). In contrast, commensal bacteria may trigger an inflammatory reaction in genetically predisposed individuals (39, 45).

Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a beneficial effect on the health of the host (40). Lactobacillus rhamnosus GG, Bifidobacterium bifidum, and Streptococcus thermophilus revealed a beneficial effect on children with rotavirus infection (12). A significant reduction in the duration of diarrhea and earlier hospital discharge were demonstrated using L. rhamnosus GG (30, 41). Furthermore, several studies, summarized by Nomoto et al., report a decrease in the incidence of antibiotic-induced diarrhea with the administration of Saccharomyces boulardii, L. rhamnosus, Bifidobacterium longum, and Enterococcus faecium (27).

In recent years, there have been reports on isolated cases of opportunistic infections caused by probiotics (40). The most commonly reported side effects of probiotics are endocarditis and mycosis associated with the application of Saccharomyces boulardii (27). Moreover, risks of nosocomial infection with vancomycin-resistant Enterococcus and dissemination of the drug resistance gene in the intestine have been reported, suggesting limitations in the application of enterococci as probiotics (27, 40).

To select probiotic strains out of a vast number of commensal bacteria, different criteria have been proposed, such as survival of gastric and jejunal pH conditions (8) and antimicrobial activity (8). In an attempt to select protective probiotic strains that might interfere with specific events in pathogen-triggered inflammation, we tested commensals for nontoxicity towards epithelial cells (EC) and inhibition of inflammatory EC responses induced by enteric pathogens. For this purpose, we selected Yersinia enterocolitica as a model pathogen. Upon the engagement of EC via its integrins, Y. enterocolitica triggers the activation of NF-{kappa}B and the production of proinflammatory cytokines, such as interleukin-8 (IL-8) (21, 37, 38). IL-8 is a cytokine belonging to the CXC chemokine family, with leukocyte chemotactic properties (3), and plays a crucial role in intestinal inflammatory responses (9). Hence, we investigated whether and how commensal bacteria may inhibit Yersinia-induced IL-8 production in EC. Furthermore, we determined in vivo whether strains modulating host responses upon Y. enterocolitica infection in vitro influence the course of Y. enterocolitica infection in the mouse model of oral yersiniosis. Additionally, we tested in vivo whether strains modulating inflammatory responses in vitro could prevent dextran sodium sulfate (DSS)-induced acute colitis in mice. The aim of the present study was to determine criteria for the identification of commensal bacteria with anti-inflammatory properties.


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MATERIALS AND METHODS
 
Cell culture and bacterial culture conditions. The following bacteria were used: Y. enterocolitica WA-314 serotype O8 (Y. enterocolitica pYV+) (15), Y. enterocolitica pYV– (14), E. coli K-12, Bacteroides vulgatus mpk (45), Bifidobacterium adolescentis (DSM 20086), Bacteroides distasonis (PZ 4013), and Streptococcus salivarius subsp. salivarius (PZ 86) (Ardeypharm GmbH). E. coli strain K-12 is commonly used in laboratories and considered a prototypic commensal strain; B. vulgatus mpk (45) is a commensal Bacteroides strain isolated out of the feces of healthy specific-pathogen-free mice; and B. distasonis and S. salivarius were isolated out of the feces of healthy adult human individuals. Plasmid-cured Y. enterocolitica pYV– (14) and Y. enterocolitica WA-314 serotype O8 (pYV+) were grown in Luria-Bertani medium under aerobic conditions at 27°C, and the E. coli strain was grown at 37°C. B. adolescentis, B. vulgatus mpk, B. distasonis, and S. salivarius were grown in brain heart infusion medium under anaerobic conditions at 37°C. For the infection experiments, bacteria in the exponential growth phase were used. HeLa 229 cells were cultured in very-low-endotoxin RPMI 1640 medium containing 10% fetal calf serum, 1% glutamine, 1% penicillin/streptomycin, and HT29 cells (DSMZ accession no. ACC 299) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 1% glutamine, and 1% penicillin/streptomycin. For the infection assays, antibiotic-free medium was used.

Infection assays. Totals of 1 x 105 HT29 cells were seeded 24 h prior to infection. The cells were infected with Y. enterocolitica pYV– (at a multiplicity of infection [MOI] of 20) or a different MOI (2 to 200) of the selected bacterial strains, or coinfected simultaneously with Y. enterocolitica and the selected bacterial strains. Tumor necrosis factor alpha (TNF-{alpha}) (100 ng/ml) was added to unstimulated (positive control) or coinfected (viability control) cells. One hour after infection, the cells were washed and cell culture medium containing 1% gentamicin was added. After another 3 h, the cell culture supernatants were harvested and the IL-8 levels were determined. For all assays using aerobic or anaerobic bacteria, the viability of the bacteria after incubation in the cell culture medium was confirmed by culture techniques.

Adhesion and invasion assays. Totals of 1 x 105 HT29 cells were infected with Y. enterocolitica pYV– or the selected bacterial strains or coinfected with Y. enterocolitica pYV– and the selected bacterial strains. After 30 min, the cells were washed and harvested for determination of the numbers of CFU of adherent bacteria. For determination of the numbers of CFU of anaerobic bacteria, the cells were grown on brain heart agar at 37°C under anaerobic conditions. Y. enterocolitica pYV– was grown at 27°C under aerobic conditions on cefsulodine-irgasan-novobiocine (CIN) agar. For the invasion assays, the cells were infected and then washed after 1 h and fresh medium containing 1% gentamicin was added. After another hour of incubation, the cells were harvested and lysed in ice-cold phosphate-buffered saline (PBS) for determination of the numbers of CFU of invasive bacteria. Coinfected cells were plated on CIN agar and incubated for 48 h at 27°C under aerobic conditions to determine the CFU of adherent or invasive Y. enterocolitica pYV– in coinfected cells.

Determination of levels of IL-8 production. The amount of IL-8 secreted into the supernatant was determined as previously described (36) by using an enzyme-linked immunosorbent assay with optimal concentrations of a mouse anti-human IL-8 monoclonal antibody (G265-5; PharMingen, San Diego, CA) and a biotinylated mouse anti-human IL-8 monoclonal antibody (G265-8; PharMingen) as detecting antibodies. The IL-8 concentrations were calculated from the straight-line portion of a standard curve by using recombinant human IL-8 (PharMingen).

Transient transfection. Totals of 5 x 104 HeLa cells seeded in 24-well plates for 24 h were cotransfected with 125 ng pNF-{kappa}B-(5x) luc (Stratagene, La Jolla, CA) and 125 ng pCMVß Gal (Clonetech, Mountain View, CA) (23), using ExGen 500 transfection reagent (Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions, and incubated for 20 h at 37°C. HeLa cells were infected with viable bacteria as described above; after 1 h, gentamicin was added and the culture was incubated for another 5 h. The HeLa cells were washed twice and lysed with luciferase lysis buffer according to the manufacturer's instructions (Roche, Mannheim, Germany). The lysates were centrifuged, and the supernatants were removed for determination of the protein levels and ß-galactosidase and luciferase activities. The luciferase activity was normalized to the ß-galactosidase activity and protein concentration (relative light units). The degree of induction was determined as the ratio of luciferase activity of Y. enterocolitica-infected cells to that of cells coinfected with Y. enterocolitica and the commensals.

Infection of mice. A plasmid harboring Y. enterocolitica WA-314 of serotype O8 (Y. enterocolitica pYV+) (15) was passaged in mice and grown as described previously (1). Female C57BL/6 mice, 6 to 8 weeks old with an initial weight of 18.3 g ± 1.7 g, were administered 1 x 1010/ml viable B. adolescentis in their drinking water and orogastrically infected by the injection of 200 µl of a suspension containing 1 x 108 to 5 x 108 yersiniae. Mice infected only with Yersinia were used as a control. The mice were starved for 3 h prior to and 3 h after infection. The actual number of bacteria was controlled for each experiment by plating 200 µl of serial dilutions of the suspensions on CIN agar and counting the CFU after an incubation at 27°C for 48 h. The mice were weighed prior to and every day after infection. At 5 days after infection, the mice were killed and the spleen of each mouse was aseptically removed. The entire small intestine was removed and extensively washed with cold PBS to remove bacteria associated with the mucosal surface of Peyer's patches. Then, the Peyer's patches of each mouse were carefully excised and pooled. The number of bacteria in the Peyer's patches and spleen was determined by homogenizing these organs in PBS and plating serial dilutions of the homogenates on CIN agar. The limit of detection was 50 CFU.

Determination of mRNA expression in intestinal tissue by quantitative real-time reverse transcription-PCR. Mucosal scrapings of the distal ileum were transferred into 1.2 ml RNeasy lysis buffer containing 0.1% ß-mercaptoethanol (QIAGEN, Hilden, Germany), homogenized, and finally snap-frozen in liquid nitrogen. RNA isolation was performed according to the manufacturer's instructions. The extracted RNA was dissolved in water containing 0.1% diethyl pyrocarbonate. For reverse transcription, 4 µg of RNA was mixed with 0.5 µg of oligo(dT)12-18 primers (Invitrogen Life Technologies), and diethyl pyrocarbonate-treated water was added to a final volume of 10 µl, followed by incubation at 65°C for 10 min. After the addition of 10 µl of a solution containing 5x first-strand buffer, 20 nmol/liter dithiothreitol, 200 U Superscript II (Invitrogen Life Technologies), 40 U RNase Out (Invitrogen Life Technologies), and 2 mmol/liter deoxynucleoside triphosphate (Roth), the mixture was incubated at 37°C for 60 min. Finally, the samples were heated at 90°C for 5 min, diluted with diethyl pyrocarbonate-treated water, and stored at –20°C until further use. Real-time reverse transcription-PCR was carried out in duplicate experiments in a 96-well format on a GeneAmp 5700 sequence detection system (Applied Biosystems/Applera, Darmstadt, Germany). Each 20-µl reaction mixture contained 10 µl TaqMan universal PCR master mix (No AmpErase UNG; Applied Biosystems), 1 µl target gene-specific assay-on-demand gene expression assay mix (Applied Biosystems), 4 µl PCR-grade water, and 5 µl cDNA. The thermal cycling conditions for all reactions were as follows: 2 min at 50°C, 10 min at 95°C, and then 40 cycles of 15 s at 95°C and 1 min at 60°C. The standard-curve method was used for semiquantitative data analysis, whereas 10-fold dilutions of pooled cDNA from all samples were used as standards. The data were normalized by dividing the values for the target gene by the values for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Determination of numbers of fecal and mucosa-associated CFU of Y. enterocolitica. During the infection period, feces was collected daily and homogenized and serial dilutions of the feces were plated on CIN agar. For the detection of mucosa-associated yersiniae, the feces were carefully removed and the intestine was flushed with 10 ml PBS. Serial dilutions of the fluid were plated on CIN agar. The CIN agar plates were incubated for 48 h at 27°C; afterwards, the number of CFU of Y. enterocolitica pYV+ was counted and normalized per mg feces or per ml fluid. The limit of detection was 50 CFU.

Induction of DSS colitis. Female C57BL/6 mice, 6 to 8 weeks old with an initial weight of 17.6 g ± 3.2 g, were housed in groups of five mice per cage. At day 2, colonic inflammation was induced in mice that received 4% DSS in their drinking water for 6 days. B. adolescentis (BIF) was administered to the DSS+/BIF+ group over the complete course of the experiment by adding 1 x 1010/ml viable B. adolescentis to the drinking water twice a day. The viability of B. adolescentis is not influenced by DSS, and the effectiveness of DSS in inducing inflammation is not decreased by mixing with B. adolescentis. Control groups received only drinking water or drinking water with B. adolescentis. The colonization of mice with B. adolescentis was controlled daily by the cultivation of fresh stool samples. During the experimental period, body weight was measured every day. Macroscopic symptoms of inflammation (stool consistency, the presence of blood in the stool, the presence of blood at the anus, the presence of relieving posture, and the appearance of the fur) were assessed daily during the course of the experiment. The following scores were given to stool consistency: 0, formed stool; 1, formed and soft stool; and 3, diarrhea. For blood in the stool, the scores were 0, no presence of fecal blood, and 3, presence of fecal blood. Anal blood scores were 0, no blood; 1, inflamed anus; 2, slight bleeding; and 3, bleeding and edema. Relieving posture was scored with 0 to 3 points, and unkempt fur was given 1 point. The scores were added, and the disease activity index was calculated.

Statistics. For the in vitro experiments, statistical analyses were performed using the paired Student t test. P values < 0.05 were considered significant. For the in vivo experiments, statistical analyses were performed using the analysis of variance one-way test and, as the posttest, the Bonferroni test. P values < 0.05 were considered significant.


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RESULTS
 
Bacterial adhesion to or invasion into EC. As a common virulence mechanism, pathogenic bacteria adhere to or invade EC (22, 26). To exclude the possibility that the tested commensal strains feature these virulence mechanisms, we determined whether E. coli K-12, Bifidobacterium adolescentis, Bacteroides vulgatus, Bacteroides distasonis, or Streptococcus salivarius adheres to or invades host EC. EC were infected with Y. enterocolitica (positive control) or commensal bacterial strains, and the number of CFU of adherent or invasive bacteria was determined. Neither B. adolescentis, B. vulgatus, B. distasonis, nor S. salivarius was adherent to HT29 cells; only E. coli K-12 showed slight adhesion. Accordingly, none of the commensals turned out to be invasive, whereas enteric Y. enterocolitica adhered to and invaded EC (Fig. 1).


Figure 1
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  		FIG. 1. Adhesion to or invasion of EC. Totals of 1 x 105 HT29 cells were infected with Y. enterocolitica pYV– (MOI, 20) or Bifidobacterium adolescentis, Streptococcus salivarius, Bacteroides vulgatus, Bacteroides distasonis, or E. coli K-12, and the number of adherent (A) or invasive (B) bacteria was determined. The number of adherent or invasive Yersinia was defined as 100% and the percentage of adherent or invasive commensals was calculated proportionately. Each experiment was performed in triplicate. The error bars represent the means ± standard deviations of the results. *, P < 0.05.

Induction of apoptosis and IL-8 secretion as markers of bacterial toxicity. The induction of apoptosis in host cells is a frequent characteristic of pathogenic bacteria (19, 25). To exclude bacteria with toxic effects, we tested whether the commensal strains induce apoptosis in EC. HeLa cells were stimulated with the selected commensal bacteria; staurosporine was used as a positive control. The number of apoptotic cells was determined by flow cytometry analysis and confocal microscopy. None of the selected commensal bacteria induced apoptosis in EC. These results were confirmed by confocal microscopy. Only cells stimulated with staurosporine were stained by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay and exhibited fragmentation of nuclei, as demonstrated by staining with 4', 6'-diamidino-2-phenylindole (data not shown).

Induction of the proinflammatory cytokine IL-8 was the second criterion for exclusion of commensal bacterial strains with toxic effects on EC. EC were infected with Y. enterocolitica or stimulated with the commensal bacteria. Y. enterocolitica induced high levels of IL-8, whereas none of the commensals induced IL-8 secretion by HT29 cells. As HT29 cells express Toll-like receptor 4 (10), E. coli K-12 induced a slight IL-8 secretion by HT29 cells (Fig. 2). In contrast, B. vulgatus and B. distasonis did not activate Toll-like receptor 4 signaling. This might be due to the fact that Bacteroides spp. are phylogenetically closely related to Porphyromonas gingivalis; P. gingivalis lipopolysaccharide (LPS) has an attenuated potential in the induction of proinflammatory responses compared to that of E. coli LPS (16). Moreover, the binding of P. gingivalis LPS to the LPS binding protein is 100-fold less than that observed for E. coli LPS (17).


Figure 2
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  		FIG. 2. Induction of IL-8 secretion by commensal bacteria. HT29 cells were infected with Y. enterocolitica pYV– or incubated with Bifidobacterium adolescentis, Streptococcus salivarius, Bacteroides vulgatus, Bacteroides distasonis, or E. coli K-12. The cell culture supernatants were harvested, and the levels of IL-8 secretion were determined by enzyme-linked immunosorbent assays. Each experiment was performed in triplicate. The error bars represent the means ± standard deviations of the results. *, P < 0.05.

In summary, E. coli K-12 turned out to be slightly adherent to EC and induced a minor proinflammatory response in HT29 cells. In contrast, none of the commensal, putatively anti-inflammatory strains (Bifidobacterium adolescentis, Bacteroides vulgatus, Bacteroides distasonis, or Streptococcus salivarius) was adherent or invasive or revealed toxic effects on human EC (Fig. 2).

Modulation of Y. enterocolitica-induced IL-8 secretion. To investigate whether the commensal strains may exert protective, anti-inflammatory effects on host EC, we performed coinfection experiments with enteropathogenic Y. enterocolitica and the selected commensal bacteria. The IL-8 concentrations in the supernatants of coinfected EC were analyzed. Two different kinds of effects were observable:

(i) E. coli K-12 had no effect on Y. enterocolitica-induced IL-8 secretion.

(ii) In contrast, B. adolescentis, S. salivarius, B. distasonis, and B. vulgatus inhibited Y. enterocolitica-induced IL-8 secretion by approximately 60%, suggesting that these strains may mediate anti-inflammatory effects (Fig. 3).


Figure 3
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  		FIG. 3. Modulation of Y. enterocolitica-induced IL-8 secretion by commensal bacteria. HT29 cells were coinfected with Y. enterocolitica pYV– and commensal bacteria as shown. The results show the reduction of IL-8 secretion in coinfected cells compared to that in Y. enterocolitica-monoinfected cells. Each experiment was performed in triplicate. The error bars represent the means ± standard deviations of the results. *, P < 0.05.

To exclude the possibility that the anti-inflammatory effect was due to inhibition of adhesion to or invasion of EC by Y. enterocolitica, we performed adhesion and invasion assays in cells coinfected with commensal bacteria and Y. enterocolitica. These experiments revealed that neither adhesion to nor invasion of the EC by Y. enterocolitica was significantly impaired by coincubation with B. adolescentis, S. salivarius, B. distasonis, or B. vulgatus (Fig. 4).


Figure 4
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  		FIG. 4. Adhesion and invasion of Y. enterocolitica in coinfection experiments with commensal bacteria. Totals of 1 x 105 HT29 cells were coinfected with Y. enterocolitica pYV– (MOI, 20) and Bifidobacterium adolescentis, Streptococcus salivarius, Bacteroides vulgatus, Bacteroides distasonis, or E. coli K-12, and the numbers of adherent (A) or invasive (B) CFU of Y. enterocolitica were determined. Each experiment was performed in triplicate. The error bars represent the means ± standard deviations of the results. *, P < 0.05.

B. adolescentis, B. distasonis, and S. salivarius decrease NF-{kappa}B activation by Y. enterocolitica. The Y. enterocolitica invasin protein triggers the activation of NF-{kappa}B and subsequent IL-8 secretion (13). To characterize the inhibitory effects of B. adolescentis, B. distasonis, and S. salivarius on Y. enterocolitica-induced NF-{kappa}B activation, we performed NF-{kappa}B reporter assays. HeLa cells were transiently transfected with pNF-{kappa}B-(5x) luc and pCMVß Gal and infected with Y. enterocolitica, B. adolescentis, B. distasonis, or S. salivarius or coinfected with both Y. enterocolitica and the commensal bacteria. The levels of luciferase activity were determined and normalized to the protein contents and levels of ß-galactosidase activity of the samples.

Y. enterocolitica induced NF-{kappa}B activation, whereas stimulation with B. adolescentis, B. distasonis, B. vulgatus, or S. salivarius did not. Furthermore, B. adolescentis, B. distasonis, and S. salivarius inhibited the Y. enterocolitica-induced NF-{kappa}B activation (Fig. 5).


Figure 5
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  		FIG. 5. Commensal bacteria inhibit Y. enterocolitica-induced NF-{kappa}B activation. HeLa cells were transfected transiently with pNF-{kappa}B-(5x) luc and pCMVß Gal. The transfected cells were infected with B. adolescentis, B. distasonis, B. vulgatus, S. salivarius, or Y. enterocolitica pYV– or coinfected with Y. enterocolitica pYV– and B. adolescentis, B. distasonis, B. vulgatus, or S. salivarius. Cell extracts were prepared for the determination of luciferase activities. The luciferase activities were normalized to the ß-galactosidase activities and protein concentrations. The data are expressed as relative light units. Each experiment was performed in triplicate. The error bars represent the means ± standard deviations of the results. *, P < 0.05.

B. adolescentis increases the resistance of mice to Y. enterocolitica infection. In orally infected mice, we investigated whether B. adolescentis increases the resistance of mice to Y. enterocolitica. The mice were fed with B. adolescentis 2 days before oral infection with Y. enterocolitica (YERS) and over the entire experimental period (BIF+/YERS+ mice). The control mice were only infected with Y. enterocolitica (BIF–/YERS+ mice). The animals were weighed daily, and on day 5 sacrificed for determination of the numbers of visceral CFU.

BIF+/YERS+ mice were significantly protected in terms of weight loss compared to BIF–/YERS+ mice (Fig. 6A). The determination of the numbers of CFU of Yersinia in the Peyer's patches revealed no differences between BIF+/YERS+ and BIF–/YERS+ mice (Fig. 6B). However, the BIF+/YERS+ mice were protected from the dissemination of Yersinia infection, as indicated by the significantly reduced numbers of CFU of Yersinia in the spleens of B. adolescentis-treated Y. enterocolitica-infected mice compared to the numbers in BIF–/YERS+ mice (Fig. 6C).


Figure 6
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  		FIG. 6. B. adolescentis inhibits generalization of Y. enterocolitica infection. The results show the effect of viable B. adolescentis on weight loss, dissemination of Y. enterocolitica pYV+ to Peyer's patches and the spleen, and the expression of TNF-{alpha} mRNA in mucosal tissue. C57BL/6 mice were administered viable B. adolescentis and infected intragastrally with 5 x 108 viable Yersinia enterocolitica (BIF+/YERS+) pYV+ or were only infected intragastrally with 5 x 108 viable Yersinia enterocolitica pYV+ (BIF–/YERS+). Body weight was measured daily (A), and the numbers of CFU of Y. entercolitica in Peyer's patches (B) and the spleen (C) were determined on day 5 of the infection. The data are the means ± standard deviations of the results. An asterisk indicates significant differences between the BIF+/YERS+ and the BIF–/YERS+ group (P < 0.05). n > 10 animals per group. TNF-{alpha} mRNA expression was measured in mucosal scrapings of BIF+/YERS+ and BIF–/YERS+ mice (D). Each symbol represents one animal (n ≥ 5 animals). An asterisk indicates significant differences between the BIF+/YERS+ and the BIF–/YERS+ group (P < 0.05).

Furthermore, the expression of TNF-{alpha} mRNA in the mucosal tissue of BIF+/YERS+ mice was significantly diminished compared to the expression in mice infected only with Yersinia, indicating that the treatment with B. adolescentis might also contribute to an attenuated inflammatory process in the mucosal tissue (Fig. 6D).

To exclude the possibility that B. adolescentis reduces the growth of Y. enterocolitica in the intestine, we determined the CFU of Y. enterocolitica in the feces of BIF+/YERS+ and BIF–/YERS+ mice on a daily basis. In both groups, we found an increase of Yersinia CFU from day 1 to day 2 of infection, but no differences in the Yersinia CFU in B. adolescentis-treated and untreated mice was observed (Fig. 7A). To determine the number of CFU of mucosa-adherent Yersinia, the feces were carefully removed and the small intestine was flushed with PBS. The number of CFU of Yersinia in the fluid was determined. In line with the bacterial loads in the feces, the same numbers of Yersinia were detected in the intestines of BIF+/YERS+ and BIF–/YERS+ mice (Fig. 7B).


Figure 7
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  		FIG. 7. Colonization with B. adolescentis does not reduce the intestinal growth of Y. enterocolitica. Mice were treated with B. adolescentis and infected with Y. enterocolitica pYV+ (BIF+/YERS+) or only infected with Y. enterocolitica pYV+ (BIF–/YERS+). The number of CFU of Yersinia in the feces (A) and the number of CFU of mucosa-associated Yersinia (B) were determined by cultural techniques. The data are the means ± standard deviations of the results. No significant differences between the BIF+/YERS+ and the BIF–/YERS+ group were observed. n > 10 animals per group.

B. adolescentis decelerates DSS-induced colitis development. We addressed the question of whether B. adolescentis, identified in vitro as an anti-inflammatory commensal bacterium, also inhibits inflammatory processes in vivo. C57BL/6 mice received 4% DSS in their drinking water (DSS+/BIF–) or were additionally administered viable B. adolescentis (DSS+/BIF+). The control groups received only drinking water (DSS–/BIF–) or drinking water charged with B. adolescentis (DSS–/BIF+). During the course of the experiment, body weight was measured (Fig. 8A) and clinical symptoms of inflammation were assessed daily (Fig. 8B).


Figure 8
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  		FIG. 8. B. adolescentis decelerates DSS colitis. The results show the effect of viable B. adolescentis on weight loss (A) and the disease activity index (B) in mice with acute colitis. C57BL/6 mice were fed with Bifidobacterium adolescentis, and acute colitis was induced with 4% DSS. DSS–/BIF– mice received drinking water only, DSS–/BIF+ mice received drinking water supplemented with B. adolescentis, DSS+/BIF– mice received 4% DSS, and DSS+/BIF+ mice received 4% DSS and B. adolescentis. The data are the means ± standard deviations of the results. An asterisk indicates significant differences between the DSS+/BIF+ and the DSS+/BIF– group (P < 0.05). n ≥ 8 animals per group.

Until day 4, DSS+/BIF– mice gained significantly less weight than DSS+/BIF+ and control mice. Additionally, DSS+/BIF– mice showed the first symptoms of colitis on day 4, whereas DSS+/BIF+ mice did not. On day 7, DSS+/BIF– mice presented significant weight loss and they presented significantly more clinical signs of inflammation than the DSS+/BIF+ group. However, B. adolescentis decelerated the induction of, but was not able to prevent, inflammation, as on day 8, the DSS+/BIF+ mice developed a weight loss comparable to that of the DSS+/BIF– group but still had significantly reduced symptoms of inflammation. These findings might lead to the notion that B. adolescentis attenuates inflammatory host responses, not only in vitro, but also in vivo.


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DISCUSSION
 
In this study, we evaluated commensal bacterial strains for putative probiotic effects. We tested commensal bacteria for adhesion, invasion, toxicity, and modulation of inflammatory host responses in vivo and in vitro. We chose different commensal strains representing bacteria of the mucosal microbiota. B. distasonis and B. vulgatus were chosen as representatives of gram-negative anaerobic bacteria; furthermore, the genus Bacteroides comprises 30% of bacteria in the feces and the mucus overlying the intestinal epithelium and represents one of the dominant bacterial divisions (2). E. coli K-12 was chosen as an aerobic, gram-negative rod, representing the commensal Enterobacteriaceae, and B. adolescentis represents gram-positive rods and lactic acid-producing bacteria, often considered to be properties of probiotics. S. salivarius is a member of the streptococcus family; another subspecies of S. salivarius is contained in the probiotic VSL#3 cocktail which combines eight strains of lactic acid-producing bacteria (44). None of the tested commensal strains was adherent to or invasive in EC. Likewise, none of the strains exerted toxic effects or induced apoptosis in EC (data not shown). Four commensal strains (B. adolescentis, S. salivarius, B. vulgatus, and B. distasonis) exerted an anti-inflammatory potential and led to a reduction in the Yersinia invasin-triggered IL-8 production by the EC. In the in vivo mouse model of oral Yersiniosis, the administration of viable B. adolescentis protected mice from the generalization of Y. enterocolitica infection. The treatment of mice with B. adolescentis resulted in an improved control of infection, as indicated by reduced weight loss and the absence of Yersinia in the spleens of B. adolescentis-treated, Y. enterocolitica-infected mice. In mice with DSS-induced colitis, B. adolescentis attenuated the development of colitis, indicating anti-inflammatory properties in vivo also.

Y. enterocolitica causes a broad range of gastrointestinal syndromes, ranging from acute enteritis and enterocolitis to lymphadenitis. The triggering of IL-8 production in host EC by Yersinia may be part of its pathogenic strategy (13, 37). Y. enterocolitica triggers IL-8 production in host cells by the engagement of ß1 integrins via its outer membrane protein invasin. The commensal bacteria B. distasonis, B. vulgatus, S. salivarius, and B. adolescentis significantly reduced the Y. enterocolitica-induced IL-8 secretion, whereas E. coli K-12 did not. We were able to exclude the possibility that the reduction of IL-8 secretion in the EC was due to reduced cell viability exerted by the commensal strains or Y. enterocolitica, and the commensal strains did not affect the growth of Y. enterocolitica. Neither adhesion to nor invasion of EC by Y. enterocolitica was impaired in coinfected cells. Therefore, we can exclude the possibility that the inhibitory effect of the commensal bacteria on Y. enterocolitica-induced IL-8 secretion is due to proteolytic cleavage of Yersinia invasin or inhibition of the Yersinia invasin-mediated host cell integrin activation.

We demonstrated that the reduction of Y. enterocolitica-induced IL-8 secretion was due to the inhibition of NF-{kappa}B activation by B. adolescentis, B. distasonis, B. vulgatus, or S. salivarius. To investigate the specificity of the commensal-mediated inhibition of Yersinia invasin-induced IL-8 production, we used TNF-{alpha} as an alternative stimulus. In fact, TNF-{alpha} is known to induce IL-8 production in EC upon engagement of the TNF receptor, which leads to the activation of NF-{kappa}B. Interestingly, TNF-{alpha}-induced IL-8 secretion was not affected by those commensals that inhibited Yersinia-induced IL-8 secretion (data not shown). These findings suggest that B. adolescentis, S. salivarius, B. vulgatus, and B. distasonis specifically interact with the Y. enterocolitica invasin-activated ß1 integrin signaling pathway.

We did not observe significant adhesion of the commensal strains to EC. Studies investigating the spatial distribution of the mucosal microbiota in humans revealed adherent commensals in patients with inflammatory bowel disease when compared to healthy controls (43). In healthy wild-type mice, the spatial organization of the mucosal microbiota differs within the ileum (no signs of adhesion), cecum (direct contact with the epithelium) and colon (42). However, phenomena such as shrinkage due to fixation and mechanical damage of the tissue have to be considered. Whether apathogenic commensals adhere to enterocytes in vivo or whether they are located luminally, separated by the intestinal mucus layer from the enterocytes, remains a topic of ongoing discussion.

Out of the commensals that presented anti-inflammatory mechanisms in vitro, B. adolescentis, was selected, and the ability of this strain to modulate inflammatory processes was evaluated in two different in vivo systems. Numerous probiotic supplementation trials have been carried out to assess the efficacies of probiotics against colonic inflammation. Among them, administration of the VSL#3 cocktail, containing various lactic acid-producing bacteria, decreased the intensity of colitis in IL-10–/–- and DSS-treated mice (24, 29). We tested the anti-inflammatory potential of B. adolescentis in DSS-treated mice and found delayed weight loss and delayed incidence of clinical colitis symptoms in mice treated with B. adolescentis. However, B. adolescentis was not able to inhibit DSS colitis, although it significantly decelerated colitis development. This supports the notion that B. adolescentis may affect intestinal homeostasis, probably by mediating anti-inflammatory effects on gut EC, and confirms our in vitro findings.

To investigate the impact of B. adolescentis on defense against Y. enterocolitica infection, mice were treated with viable B. adolescentis prior to orogastric infection with Y. enterocolitica. The data presented herein suggest that B. adolescentis attenuated the course of Y. enterocolitica infection by reducing clinical symptoms and the dissemination of Yersinia, as well as the Y. enterocolitica-induced mucosal inflammation. The administration of B. adolescentis led to equal numbers of Yersinia in the Peyer's patches but significantly decreased numbers of bacteria in the spleen compared to those in mice infected with Yersinia only. Furthermore, the treatment of mice with B. adolescentis significantly reduced the levels of Y. enterocolitica-induced TNF-{alpha} mRNA expression in mucosal tissue. At present, however, it is not clear which effect of B. adolescentis accounts for the reduced susceptibility of B. adolescentis-treated mice to Y. enterocolitica infection.

Several recent studies discuss mechanisms by which probiotic microbial agents and their components exert protective effects. Probiotic bacteria suppress the growth of conventional or potential pathogens, as well as their attachment and/or invasion, by secreting antimicrobial substances or by stimulating host expression of protective molecules. Furthermore, probiotics enhance the mucosal barrier function and can stimulate host production of immunosuppressive molecules that downregulate inflammatory responses or stimulate host-protective immunologic mechanisms that can prevent or accelerate the clearance of pathogenic infections (7, 11, 34, 35).

We can exclude the possibility that B. adolescentis suppresses the growth of Y. enterocolitica, as the numbers of fecal and mucosal CFU were equal in both groups, and the invasiveness of Y. enterocolitica in the Peyer's patches was not altered. Recently, it was demonstrated that Y. pseudotuberculosis, causing disease in the spleens and livers of mice upon oral infection, was derived from Yersinia populations located outside the intestinal lymph nodes and Peyer's patches, and replication of the bacteria in the intestine seemed to be critical for their dissemination (4). This study provided strong evidence that liver and spleen colonization with Y. pseudotuberculosis is independent of the preliminary replication of Yersinia in the Peyer's patches and mesenteric lymph nodes (4). Our results also suggest that routes other than the infection of the mucosa-associated lympoid tissue are modulated by the administration of B. adolescentis: there are at least three possible translocation mechanisms known that bypass the Peyer's patches. First, host or bacterial processes may cause local microdamage in the intestinal epithelium (6), permitting the translocation of bacteria. Second, villous-associated M cells (18) are hypothetical portals across the epithelium. Finally, phagocytic or dendritic cells have been demonstrated to sample luminal bacteria, and evidence exists for the phagocytic routing of Salmonella enterica serovar Typhimurium across the intestine (32). Which mechanisms account for the protective effect of B. adolescentis in inflammatory and infectious diseases is a topic of ongoing studies.

In summary, the system described herein provides an approach to screening commensal bacteria for their ability to inhibit the pathogenic functions of virulent enteric bacteria, such as invasion or the induction of inflammatory responses, and furthermore, offers information about possible molecular mechanisms affected by the commensals.


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ACKNOWLEDGMENTS
 
We thank Ulrich Sonnenborn for helpful discussion and critical reading of the manuscript.

This work was supported by the Landesstifung Baden-Württemberg GmbH.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Microbiology and Hygiene, University of Tübingen, Elfriede-Aulhorn-Str. 6, D-72076 Tübingen, Germany. Phone: 49 7071 29 81528. Fax: 49 7071 29 4972. E-mail: julia-stefanie.frick{at}med.uni-tuebingen.de Back

{triangledown} Published ahead of print on 7 May 2007. Back

Editor: J. B. Bliska


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Infection and Immunity, July 2007, p. 3490-3497, Vol. 75, No. 7
0019-9567/07/$08.00+0     doi:10.1128/IAI.00119-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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