Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus

Letters in Applied Microbiology ISSN 0266-8254 ORIGINAL ARTICLE Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus M.C. Collado 1, J. Meriluoto 2 and S. Salminen 1 1 Functional Foods Forum, University of Turku, Turku, Finland 2 Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland Keywords Mucus, adhesion, probiotics, pathogens. Correspondence M.C. Collado, Functional Foods Forum, University of Turku, Itäainen Pitkäkatu 4A 5th floor, FI-20520 Turku, Finland. E-mail: marcol@utu.fi 2007 0552: received 7 April 2007, revised and accepted 4 June 2007 doi:10.1111/j.1472-765x.2007.02212.x Abstract Aims: The aims of this study present were to assess and to evaluate in vitro the abilities of commercial probiotic strains derived from fermented milk products and related sources currently marketed in European countries, to inhibit, compete and displace the adhesion of selected potential pathogens to immobilized human mucus. Methods and Results: The adhesion was assessed by measuring the radioactivity of bacteria adhered to the human mucus. We tested 12 probiotic strains against eight selected pathogens. All strains tested were able to adhere to mucus. All probiotic strains tested were able to inhibit and displace (P < 0.05) the adhesion of Bacteroides,, Staphylococcus and Enterobacter. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting that several complementary mechanisms are implied in the processes. Conclusions: Our results indicate the need for a case-by-case assessment in order to select strains with the ability to inhibit or displace a specific pathogen. Probiotics could be useful to correct deviations observed in intestinal microbiota associated with specific diseases and also, to prevent pathogen infections. Significance and Impact of the Study: The competitive exclusion properties of probiotics as well as their ability to displace and inhibit pathogens are the most importance for therapeutic manipulation of the enteric microbiota. The application of such strategies could contribute to expand the beneficial properties on human health against pathogen infection. Introduction Specific strains of Lactobacillus, Bifidobacterium and also, some Propionibacterium strains have been introduced as probiotics in food products due to their observed healthpromoting effects (Lee and Salminen 1995; Huis in t veld et al. 1998; Salminen et al. 1999). The selection of probiotics is frequently based on the ability to adhere to the gastrointestinal mucosa and competitive exclusion of pathogens (Salminen et al. 1998, 1999; Ouwehand et al. 2002; Ouwehand and Salminen 2003). The protective role of probiotic bacteria against gastrointestinal pathogens and the underlying mechanisms have received special attention. Adhesion to and colonization of the mucosal surfaces are possible protective mechanisms against pathogens through competition for binding sites and nutrients (Ouwehand and Salminen 2003) or immune modulation (Schiffrin et al. 1997; Salminen et al. 1998). Pathogen inhibition by probiotic may provide significant protection against pathogen infection via a natural barrier against pathogen exposure in the gastrointestinal tract and this would enhance human health. The most extensive studies and clinical applications of probiotics have been related to the management of gastrointestinal infections caused by pathogenic micro-organisms. Due the importance of probiotics in the prevention of infections, the aim of this study was to assess the properties of commercial probiotic strains belonging to different genera and species derived from fermented milk 454 Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 45 (2007) 454 460

products and related sources currently marketed in European countries. The studied characteristics included the adhesion properties and the abilities to inhibit the adhesion, to displace and to compete with pathogens. We selected an immobilized human intestinal mucus model as an experimental model because good correlations have been previously reported between this model and the enterocyte-like caco-2 model (Aissi et al. 2001; Ouwehand and Salminen 2003; Vesterlund et al. 2005). Materials and methods Bacterial strains and culture conditions Commercial probiotic strains used in this study were: Lactobacillus rhamnosus GG (ATCC 53103, Valio Ltd., Helsinki, Finland), L. rhamnosus LC705 (Valio Ltd.), L. casei Shirota (Yakult Singapore Pty. Ltd., Singapore), L. fermentum ME3 (University of Tartu, Estonia), L. acidophilus NCFM (Danisco USA Inc., Madison, WI), L. plantarum Lp-115 (Danisco USA Inc), L. salivarius Ls-33 (Danisco USA Inc), Bifidobacterium longum 46 (Probiotical srl, Novara, Italy), B. lactis Bb12 (Chr. Hansen Ltd., Hørsholm, Denmark), B. lactis 420 (Danisco Deutschland GmbH, Niebüll, Germany), B. breve 99 E8 (Valio Ltd.), Propionibacterium freudenreichii spp. shermanii JS (Valio Ltd.). All these strains were obtained from Functional Foods Forum Culture Collection, University of Turku. The bacterial pathogens used in this study were Bacteroides vulgatus DSM 1447, histolyticum DSM 627, difficile DSM 1296, Escherichia coli K2, Enterobacter aerogenes DSM 30053, Listeria monocytogenes ATCC 15313, Salmonella enterica serovar Typhimurium ATCC 12028, Staphylococcus aureus DSM 20231. For assays, the lactobacilli were cultured in MRS broth and bifidobacteria in MRS (deman RogosaSharpe; MRS, Oxoid Ltd, Hampshire, UK) supplemented with 0.05% cysteine-hcl. Pathogens and propionibacteria were grown in Gifu anaerobic medium (GAM Nissui Pharmaceutical, Tokyo, Japan). All bacteria were incubated for 18 h at 37 C under anaerobic conditions (10% H 2, 10% CO 2 and 80% N 2 ; Concept 400 anaerobic chamber, Ruskinn Technology, Leeds, UK). All micro-organisms were metabolically labelled by addition to the media of 10 ll ml )1 tritiated thymidine (5-3 H-thymidine 1.0 mci ml )1 ; Amersham Biosciences, Little Chalfont, UK). In vitro adhesion assay to intestinal human mucus Human intestinal mucus was collected from the healthy part of resected colonic tissue (Ouwehand et al. 2002). Mucus was dissolved (0.5 mg protein ml )1 ) in HEPES (N-2-hydroxyethylpiperazine-N-2-ethanosulphonic acid)-hanks buffer (HH; 10 mmol l )1 HEPES, ph 7.4) and 100 ll of the mucus solution were immobilized on polystyrene microtitre plate wells (Maxisorp, Nunc, Denmark) during overnight incubation at 4 C. The adhesion assessment was carried out as previously described (Collado et al. 2005). Absorbance (A 600nm ) was adjusted to 0.25 ± 0.05 (10 8 cells ml) and 100 ll of suspension were added into the wells and incubated for 1 h at 37 C. The wells were washed to remove unattached bacteria. Adhered bacteria were recovered after lysis with 1% SDS in 0.1 mol l )1 NaOH (200 ll per well) after incubation at 65 C for 1 h. The radioactivity was measured by liquid scintillation (OptiPhase HiSafe 3 ; Turku, Finland) with a 1450 Microbeta Liquid Scintillation Counter (Wallac Oy., Turku, Finland). Adhesion was calculated as the percentage of radioactivity recovered after adhesion relative to the radioactivity of the bacterial suspension added to the mucus. Inhibition of pathogen adhesion to intestinal mucus To test the ability of the LAB (Lactic Acid Bacteria) strains to inhibit the adhesion of pathogens the procedure described by Collado et al. (2005) was used. The adhesion inhibition was calculated as the difference between the adhesion of the pathogen in the absence and presence of probiotic strains. Displacement of pathogens adhered to intestinal mucus The ability of the studied probiotic strains to displace already adhered pathogens was assessed according to Collado et al. (2005). Displacement of pathogens was calculated as the difference between the adhesion after the addition of the probiotic strains and the corresponding control buffer. Competence between pathogens and probiotic strains to adhere to intestinal mucus Competitive exclusion of the pathogens by tested probiotics was determined as described previously (Rinkinen et al. 2003). Competitive exclusion was calculated as the percentage of pathogens bound after the combination with probiotic strains relative to pathogens bound in the absence of probiotic (control). In addition, we analysed the possible coaggregation between pathogens and probiotic strains by means of previous incubation during 120 min at 37 C and after that, we calculated the % of competitive exclusion as described above. Statistical analysis Adhesion experiments were determined in three independent experiments, and each assay was performed in Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 45 (2007) 454 460 455

quadruplicate to calculate intra-assay variation. Statistical analysis was done using the SPSS 11.0 software (SPSS Inc, Chicago, IL, USA). Data were subjected to one-way anova. Results In vitro adhesion assay to intestinal human mucus The adhesion of commercial probiotic strains showed a great variability depending on the strain, specie and genera (Fig. 1) and varied from 0.6% to 19.7%. The most adhesive strains were L. rhamnosus GG (19.7%), L. acidophilus NCFM (16.5%) and B. lactis Bb12 (10.7%), while the least adhesive strains were L. casei Shirota (0.6%), L. salivarius LS-33 (1.0%) and P. freudenreichii ssp (a) 25 shermanii JS (1.0%). With regard to the pathogenic bacteria, E. coli showed the highest adhesion ability (13.8%), while the other pathogens tested showed adhesion values ranging from 4.6% to 12.6%. The least adhesive pathogens were L. monocytogenes and S. Typhimurium which showed a 0.5% and 0.6% of adhesion to human intestinal mucus, respectively. Inhibition of pathogen adhesion to intestinal mucus The inhibition of the adhesion of pathogens by commercial probiotic strains was dependent on the probiotic strain and the pathogen assayed (Table 1). All commercial probiotic strains were able to inhibit the adhesion (P < 0.05) of B. vulgatus (24.7 46.7%), C. histolyticum (12.0 43.1%), C. difficile (20.9 40.3%), St. aureus (27.8 48.0%) and Ent. aerogenes (28.5 44.1%). Most of the probiotic strains tested were not able to inhibit the adhesion of E. coli, L. monocytogenes and S. Typhimurium. (%) adhesion 20 15 10 5 0 L. rhamnosus GG B. lactis Bb12 (b) 25 (%) adhesion 20 15 10 5 0 L. rhamnosus LC-705 L. casei Shirota L. fermentum ME3 L. acidophilus NCFM L. plantarum Lp-115 L. salivarius LS-33 B. longum 46 B. lactis 420 B. breve 99 Propionibacterium PJS B. vulgatus C. histolyticum C. difficile E. coli Ent. aerogenes L. monocytogenes S. Typhimurium St. aureus Figure 1 Adherence of commercial probiotic strains (a) and pathogen strains (b) to human intestinal mucus. Results were expressed % adhesion average ± standard deviation. Displacement of pathogens adhered to intestinal mucus The results (Table 2) showed that at least six pathogens can be displaced by specific probiotic strains, but significant differences were found between strains. All commercial probiotic strains were able to displace (P < 0.05) B. vulgatus (54.8 70.4%), C. histolyticum (15.2 28.8%), C. difficile (51.0 64.5%), St. aureus (20.5 32.4%), Ent. aerogenes (48.9 60.0%) and L. monocytogenes (29.2 65.6%). None of the probiotic strains tested were able to displace E. coli already adhered to intestinal mucus and only six probiotic strains were able to inhibit S. Typhimurium. Competence between pathogens and probiotic strains to adhere to intestinal mucus The previous incubation step at 37 C for 2 h increased the percentage of reduction in the pathogen adhesion to intestinal mucus (Table 3), but the results demonstrate strain specificity to both probiotic strain and the pathogen assayed. Most of tested probiotic strains could compete and inhibit pathogen adhesion although differences were found between strains. Only three pathogens were inhibited by competition without pre-incubation by all probiotic strains tested, these were St. aureus (7.4 36.2%, P < 0.05), C. difficile (34.9 46.5%, P < 0.05) and Ent. aerogenes (28.3 52.5%, P < 0.05). Most of the probiotic strains tested increased the adhesion of C. histolyticum (7.1 21.9%, P < 0.05), E. coli (6.7 26.9%, P < 0.05), S. Typhimurium (7.5 22.7%, P < 0.05), L. monocytogenes (7.4 55.9%, P < 0.05). Previous incubation during 2 h at 37 C reduced these increments. These results could 456 Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 45 (2007) 454 460

Table 1 Inhibition of pathogen adhesion by commercial probiotic strains. Results are shown as median ± standard deviation and they are expressed as percentages compared to adhesion of pathogen strains without commercial probiotic strains (taken as 0%) % adhesion inhibition Strains Bacteroides vulgatus histolyticum Staphylococcus aureus difficile Salmonella Typhimurium Listeria monocytogenes Enterobacter aerogenes Escherichia coli L. rhamnosus GG 32.8 ± 9.9 43.1 ± 5.10 41.2 ± 7.0 40.2 ± 12.5 3.7 ± 1.9* )10.9 ± 7.3 42.8 ± 5.2 )20.6 ± 13.0 B. lactis Bb12 24.73 ± 4.6 12.0 ± 3.0 48.0 ± 3.6 27.8 ± 6.4 5.2 ± 3.8 25.7 ± 8.0 42.7 ± 9.0 )27.9 ± 4.5 L. rhamnosus LC705 37.9 ± 6.9 32.4 ± 5.7 32.3 ± 4.6 23.3 ± 9.3 25.6 ± 12.0 )10.8 ± 8.5* 32.1 ± 7.0 )35.4 ± 13.0 L. casei Shirota 44.33 ± 1.1 22.1 ± 5.6 28.8 ± 8.0 23.5 ± 1.8 )5.8 ± 4.0* )6.9 ± 3.0* 35.5 ± 2.8 )44.4 ± 12.0 L. fermentum ME3 35.7 ± 15.0 29.3 ± 3.7 28.6 ± 6.9 29.4 ± 8.1 12.3 ± 9.4 )19.1 ± 5.5 36.7 ± 7.6 )44.6 ± 14.1 L. acidophilus NCFM 46.7 ± 5.6 29.1 ± 8.3 45.7 ± 6.2 33.5 ± 10.0 )9.2 ± 6.5 15.4 ± 4.2 41.3 ± 6.6 )42.6 ± 15.2 L. plantarum Lp-115 30.8 ± 1.5 20.52 ± 4.5 33.4 ± 6.2 35.7 ± 13.6 )5.6 ± 3.5* 7.4 ± 2.2 30.0 ± 4.0 )46.2 ± 11.0 L. salivarius LS-33 35.9 ± 6.6 21.2 ± 9.0 37.0 ± 7.0 40.3 ± 16.0 )4.8 ± 4.0* 6.0 ± 1.8 36.1 ± 4.9 )35.0 ± 12.0 B. longum 46 28.5 ± 8.0 30.9 ± 9.8 41.4 ± 7.8 28.6 ± 8.0 )5.9 ± 3.0* )16.2 ± 9.0 44.1 ± 4.0 3.5 ± 1.5* B. lactis 420 32.8 ± 6.9 24.3 ± 4.5 39.1 ± 11.0 32.5 ± 13.0 )10.3 ± 7.5 10.5 ± 5.5 29.8 ± 5.5 )10.1 ± 7.2 B. breve 99 35.1 ± 3.5 23.3 ± 3.7 27.8 ± 2.2 26.6 ± 14.0 )33.0 ± 10.0 )12.7 ± 7.0 28.5 ± 6.3 )14.5 ± 8.8 P. freudenreichii JS 45.1 ± 7.5 30.1 ± 8.0 33.5 ± 5.4 20.9 ± 8.0 )9.5 ± 5.0 )5.6 ± 3.0* 30.5 ± 4.5 )13.2 ± 9.2 * Not significantly different from the corresponding control (P > 0.05) taken as 0% of pathogen adhesion inhibition. Table 2 Displacement of pathogens by commercial probiotic strains. Results are shown as median ± standard deviation and they are expressed as percentages compared to adhesion of pathogen strains without commercial probiotic strains (taken as 0%) % pathogen displaced Strains Bacteroides vulgatus histolyticum Staphylococcus aureus difficile Salmonella Typhimurium Listeria monocytogenes Enterobacter aerogenes Escherichia coli L. rhamnosus GG 65.2 ± 8.8 28.8 ± 8.0 26.5 ± 9.4 64.5 ± 11.0 )5.2 ± 3.5* 47.6 ± 10.6 59.6 ± 5.5 )12.3 ± 9.5* B. lactis Bb12 54.8 ± 7.9 22.2 ± 1.3 25.7 ± 7.5 56.7 ± 7.4 7.4 ± 2.8 43.8 ± 7.3 51.9 ± 2.5 )5.3 ± 4.0* L. rhamnosus LC705 62.3 ± 1.8 28.7 ± 7.8 29.0 ± 4.3 56.1 ± 8.6 11.9 ± 4.6 48.6 ± 7.5 50.6 ± 9.3 )9.4 ± 4.3 L. casei Shirota 70.4 ± 6.0 15.2 ± 4.0 26.7 ± 7.3 61.5 ± 12.3 )1.7 ± 1.3* 40.9 ± 11.0 53.2 ± 7.4 )6.8 ± 4.0* L. fermentum ME3 64.8 ± 5.1 23.8 ± 4.4 32.4 ± 6.9 56.7 ± 13.0 22.9 ± 4.8 65.6 ± 7.2 57.1 ± 6.3 )19.4 ± 12.1 L. acidophilus NCFM 60.6 ± 9.3 16.1 ± 3.5 20.5 ± 2.5 52.9 ± 12.0 )7.8 ± 5.0* 51.9 ± 6.0 55.4 ± 3.5 )27.7 ± 12.0 L. plantarum Lp-115 63.1 ± 12.0 24.0 ± 7.8 26.8 ± 8.1 54.2 ± 11.0 10.2 ± 7.4 36.8 ± 4.1 48.9 ± 6.3 )23.6 ± 6.3 L. salivarius LS-33 58.4 ± 12.0 22.5 ± 8.6 22.6 ± 7.5 51.9 ± 16.4 5.1 ± 3.1* 29.2 ± 8.4 52.8 ± 1.4 )17.1 ± 8.5 B. longum 46 69.0 ± 3.8 24.5 ± 6.4 27.4 ± 6.3 51.0 ± 11.0 )7.0 ± 2.0 47.1 ± 9.8 60.0 ± 9.6 )6.0 ± 2.3 B. lactis 420 61.3 ± 9.5 27.1 ± 10.0 27.5 ± 7.0 55.5 ± 9.6 )5.7 ± 1.6 48.7 ± 4.4 53.8 ± 8.2 )2.8 ± 1.8* B. breve 99 61.3 ± 9.0 27.0 ± 8.6 27.4 ± 6.2 59.0 ± 6.8 )3.3 ± 3.0* 53.4 ± 4.3 52.6 ± 5.4 )3.2 ± 1.5* P. freudenreichii JS 60.5 ± 9.2 22.4 ± 1.3 22.5 ± 1.5 58.7 ± 7.3 6.6 ± 2.3 50.7 ± 7.0 50.6 ± 2.2 )16.1 ± 1.1 *Not significantly different from the corresponding control (P > 0.05) taken as 0% pathogen displaced. suggest the possible coaggregation between pathogens and probiotic strains, and this coaggregation could avoid or reduce the pathogen adhesion. Discussion Probiotic bacteria selected for commercial use in foods and pharmaceutical preparations must retain the characteristics for which they were originally selected (Salminen et al. 1998; Tuomola et al. 2001). Adhesion to epithelial cells is an important step for both pathogenic bacteria and probiotics indicating a potential interaction between them. Adhesion of probiotics to the mucosa has been related to many of the health benefits attributed to probiotics such as antagonism against pathogens by interference mechanisms. In the current study the adhesion of commercial probiotic strains used in fermented milk products was assessed using the human intestinal mucus model. At the same time the abilities of the probiotics to inhibit the adhesion or to compete with pathogens and to displace previously adhered pathogens were characterized. This study demonstrates that the currently used probiotic microbes have different properties in adherence and competitive exclusion of model pathogens. The results indicate that specific probiotics or probiotic Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 45 (2007) 454 460 457

Table 3 Competence between pathogens and commercial probiotic strains to adhere to intestinal mucus with and without pre-incubation during 2 h at 37ºC. Results are shown as median ± standard deviation. Adhesion of pathogen strains (taken as 0%) Strains Bacteroides vulgatus histolyticum Staphylococcus aureus difficile Salmonella Typhimurium Listeria monocytogenes Enterobacter aerogenes Escherichia coli % reduction of adhesion pathogen competence without previous incubation L. rhamnosus GG 16.5 ± 5.6 15.3 ± 5.6 20.4 ± 14.2 36.2 ± 9.4 5.6 ± 3.7* )7.4 ± 4.5* 49.4 ± 5.5 )14.6 ± 10.0 B. lactis Bb12 28.7 ± 6.2 )20.6 ± 5.2 31.0 ± 6.2 42.4 ± 9.9 6.7 ± 3.9* )15.5 ± 6.5 48.7 ± 9.1 )16.5 ± 11.0 L. rhamnosus LC705 42.0 ± 15.0 )21.9 ± 13.1 33.9 ± 9.9 34.9 ± 9.5 6.9 ± 2.5 )55.9 ± 9.3 28.3 ± 10.3 )6.7 ± 0.6 L. casei Shirota )12.7 ± 4.0 )11.7 ± 7.1 9.0 ± 3.8 39.0 ± 8.0 )7.5 ± 3.7* )15.7 ± 9.7 38.6 ± 7.7 )18.5 ± 9.7 L. fermentum ME3 23.8 ± 4.0 )19.7 ± 6.3 7.4 ± 3.2 45.3 ± 3.3 )22.7 ± 6.6 )32.2 ± 11.0 44.2 ± 8.7 )18.1 ± 9.7 L. acidophilus NCFM 45.8 ± 9.7 )14.5 ± 3.2 30.4 ± 10.4 46.2 ± 3.4 5.2 ± 3.3* )18.4 ± 8.5 50.6 ± 1.8 )14.1 ± 9.9 L. plantarum Lp-115 42.0 ± 3.8 10.1 ± 6.4 12.7 ± 4.0 42.2 ± 8.8 )13.6 ± 2.5 39.5 ± 9.8 41.0 ± 3.3 )21.8 ± 5.2 L. salivarius LS-33 34.0 ± 4.0 )17.8 ± 9.8 36.2 ± 4.1 41.7 ± 7.4 7.8 ± 3.5 )37.3 ± 19.0 42.9 ± 9.5 )26.9 ± 8.6 B. longum 46 26.4 ± 5.4 33.0 ± 8.7 26.7 ± 2.0 46.5 ± 6.7 4.4 ± 2.5* )30.9 ± 6.1 52.5 ± 5.3 12.94 ± 8.1 B. lactis 420 20.2 ± 6.2 7.2 ± 1.0 20.1 ± 2.7 42.5 ± 3.2 12.7 ± 4.0 )35.6 ± 6.6 46.2 ± 1.6 9.8 ± 1.4 B. breve 99 35.8 ± 4.0 10.9 ± 1.3 10.3 ± 6.2 44.5 ± 5.6 3.9 ± 0.5 )13.9 ± 9.5 48.6 ± 5.8 )19.6 ± 8.1 P. freudenreichii JS 13.8 ± 8.3 )7.1 ± 0.2 31.9 ± 9.1 44.3 ± 5.0 7.5 ± 2.2 )25.6 ± 3.8 48.5 ± 2.1 )9.2 ± 8.8* % reduction of adhesion pathogen competence with previous incubation L. rhamnosus GG 17.9 ± 6.6 18.1 ± 7.3 50.2 ± 14.8 45.8 ± 8.6 7.2 ± 4.0 53.6 ± 3.6 52.9 ± 1.3 )5.5 ± 0.5 B. lactis Bb12 32.2 ± 8.3 24.4 ± 9.6 60.6 ± 12.0 49.2 ± 2.0 6.7 ± 4.6* 48.5 ± 9.7 54.8 ± 0.8 )2.4 ± 0.6 L. rhamnosus LC705 48.7 ± 7.8 24.5 ± 5.8 50.2 ± 10.0 42.0 ± 2.3 4.2 ± 3.0* 46.2 ± 8.7 40.8 ± 1.8 )4.4 ± 2.8* L. casei Shirota 35.7 ± 12.1 16.4 ± 9.0 64.1 ± 11.0 41.4 ± 7.0 12.4 ± 7.8 19.6 ± 9.6 47.7 ± 6.2 )10.8 ± 6.0 L. fermentum ME3 38.4 ± 8.7 28.6 ± 5.1 44.2 ± 8.8 45.7 ± 0.8 )17.7 ± 9.0 18.7 ± 11.0 46.0 ± 8.6 )10.2 ± 7.0 L. acidophilus NCFM 36.5 ± 7.1 29.5 ± 3.3 54.6 ± 6.4 44.5 ± 7.6 11.3 ± 3.8 38.1 ± 12.0 44.8 ± 9.2 5.9 ± 0.1 L. plantarum Lp-115 57.4 ± 6.9 24.0 ± 7.6 53.2 ± 12.0 42.9 ± 3.3 )6.1 ± 2.9* 37.9 ± 9.2 41.6 ± 5.6 )5.0 ± 3.2* L. salivarius LS-33 32.5 ± 9.5 22.0 ± 6.9 53.1 ± 9.9 42.7 ± 6.3 17.9 ± 1.9 47.5 ± 12.0 41.0 ± 0.5 10.4 ± 2.7 B. longum 46 48.0 ± 10.3 4.4 ± 2.0 59.4 ± 9.8 49.6 ± 7.4 5.5 ± 2.5* 45.2 ± 5.5 44.8 ± 6.8 )3.3 ± 3.5* B. lactis 420 51.6 ± 6.1 33.4 ± 4.7 60.1 ± 7.4 48.6 ± 2.2 8.5 ± 0.7 42.1 ± 2.4 41.3 ± 9.2 14.5 ± 3.2 B. breve 99 46.9 ± 7.0 22.0 ± 7.1 57.9 ± 6.9 47.6 ± 1.1 )16.7 ± 7.8 40.1 ± 8.1 39.8 ± 7.3 )5.22 ± 3.1* P. freudenreichii JS 44.9 ± 17.0 27.5 ± 4.5 59.7 ± 6.0 44.8 ± 1.8 9.1 ± 3.5 47.2 ± 11.2 48.8 ± 5.2 )2.5 ± 2.2* * Not significantly different from the corresponding control (P > 0.05) taken as 0% pathogen adhesion. combinations should be selected for combating some of the model pathogens. For this reason, the assessment of adhesion properties and characterization of competitive exclusion properties of specific probiotics remains an important task prior to planning and conducting clinical intervention studies in human volunteers. The adhesion levels obtained for some of commercial strains tested were comparable to the levels reported earlier (Ouwehand et al. 1999; He et al. 2001). The adhesion abilities of probiotic strains showed a great variability depending on the strain, species and genus. Interestingly, all the pathogens tested showed a high adherence to intestinal mucus, with the exception of L. monocytogenes and S. Typhimurium. These results suggest that they have the capacity to bind the intestinal mucus, which could at worst even assist the pathogens in the invasion into the human intestinal mucosa. In this context, to find appropriate probiotic strain with the ability to prevent the adhesion of these pathogenic bacteria is important. The pathogen adhesion inhibition by commercial probiotics strains showed a high variability and it was clearly a strain dependent property. Some of probiotic strains showed high inhibition ability with values of inhibition over 40% for some of the pathogens tested. In accordance with our results it has been previously reported that certain bifidobacteria and lactobacilli are able to inhibit mucosal adhesion of enteropathogens by competitive exclusion (Bernet et al. 1993; Collado et al. 2005). The ability to inhibit the adhesion of pathogens appears to depend on both the specific probiotic strains and the pathogen tested, indicating a very high specificity. Interestingly, some commercial probiotic strains increased the adhesion of E. coli, L. monocytogenes and S. Typhimurium. Increases in the adhesion of certain pathogens to mucus pre-treated with specific Lactobacillus (Tuomola et al. 1999; Gueimonde et al. 2006) or Bifidobacterium (He et al. 2001; Collado et al. 2005) strains have been previously reported. Although the biological importance of these increases in pathogen adhesion is unknown at this time it may be considered a factor worth further assessment with those strains. These results, in accordance with previous reports (Bibiloni et al. 1999; Chouraqui et al. 2004), indicate that the inhibition was not directly related to the adhesion ability of the strains. The inhibition could 458 Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 45 (2007) 454 460

thus be related with the specific adhesions and receptors that probiotic and pathogen are competing for (Lee and Puong 2002) or other factors such as coaggregation of both strains (Reid et al. 1988; Gueimonde et al. 2006). The displacement of pre-adhered pathogens was also found to be probiotic strain and pathogen dependent and no direct correlation was found between adhesion of probiotics and displacement of pathogen. Nevertheless, adhesion seems to be one of the factors involved. The displacement profiles were very different from those observed for the inhibition of pathogens. In general, the pre-incubation with probiotic strains reduced pathogen adhesion, is consistent with coaggregation between pathogen and LAB strains shown in previous studies (Tuomola et al. 1999). These results suggest that coaggregation mechanisms, between pathogen and probiotic strains, could be involved in the reduction of pathogen adhesion to mucus. Several reports attest that the coaggregation abilities of probiotic strains may enable the formation of a barrier to prevent colonization by pathogens (Reid et al. 1988). In addition, adherence of bacterial cells is usually related to cell surface characteristics (Bibiloni et al. 1999; Gueimonde et al. 2006). These results, together with previous observations (Lee et al. 2003; Collado et al. 2005), suggest that different mechanisms are likely to be involved in the two processes. No relation was found between the results obtained for the adhesion inhibition and displacement of pathogens, further indicating that different mechanisms prevail in both processes. No correlation was found between the adhesion and the pathogen inhibition ability of probiotic strains tested, indicating that, in addition to the adhesion to mucus, other factors such as coaggregation with the pathogen could be involved. Probiotic strains that inhibit and displace pathogens are excellent candidates for use in specific conditions involving the tested pathogens. Our results demonstrate that all commercial probiotic strains tested in this study and used in fermented milk products show probiotic characteristics and they can inhibit, displace and compete with several pathogens. However, it is important to take into account the high specificity of these processes. It is therefore important to characterize the properties of both the specific strains and the pathogens involved with the target groups in order to select the best strains or strain combinations. This would allow the development of probiotics for specific disease risk reduction or treatment. The results report a very high specificity in the inhibition of the adhesion and displacement of enteropathogens by different probiotic strains, belonging to different genera, species and strains. Thus, there is a need of a case-by-case characterization of the probiotic strains. This should allow the selection of strains with potential application in the prevention or treatment of specific gastrointestinal infections. Further testing using the specific pathogens identified in the target populations or target microbiota aberrancies is required to select the best probiotic or probiotic combination prior to intervention studies in human volunteers. Acknowledgements This work was supported by the Academy of Finland, Research Council for Biosciences and Environment (decision numbers 210309 to Åbo Akademi and 210310 to University of Turku). M.C. Collado is the recipient of Excellence Postdoctoral grant from Conselleria Empresa, Universidad y Ciencia de la Generalitat Valenciana, Spain (BPOSTDOC 06 016). References Aissi, E.A., Lecocq, M., Brassart, C. and Buoquelet, S. (2001) Adhesion of some Bifidobacteria strains to human enterocyte-like cells and binding to mucosal glycoproteins. Microb Ecol Health Dis 13, 32 39. Bernet, M.F., Brassart, D., Nesser, J.R. and Servin, A.L. (1993) Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enterophatogen-cell interactions. Appl Environ Microbiol 59, 4121 4128. Bibiloni, R., Perez, P.F. and de Antoni, G.L. (1999) Will a high adhering capacity in a probiotic strain guarantee exclusion of pathogens from intestinal epithelia? Anaerobe 5, 519 524. Chouraqui, J.P., Van Egroo, L.D. and Fichot, M.C. (2004) Acidified milk formula supplemented with Bifidobacterium lactis: impact on infant diarrhea in residential care settings. J Pediatr Gastroenterol Nutr 38, 288 292. Collado, M.C., Gueimonde, M., Hernandez, M., Sanz, Y. and Salminen, S. (2005) Adhesion of selected Bifidobacterium strains to human intestinal mucus and the role of adhesion in enteropathogen exclusion. J Food Prot 68, 2672 2678. Gueimonde, M., Jalonen, L., He, F., Hiramatsu, M. and Salminen, S. (2006) Adhesion and competitive inhibition and displacement of human enteropathogens by selected lactobacilli. Food Res Int 39, 467 471. He, F., Ouwehand, A.C., Hashimoto, H., Isolauri, E., Benno, Y. and Salminen, S. (2001) Adhesion of Bifidobacterium spp. to human intestinal mucus. Microbiol Immunol 45, 259 262. Huis in t veld, J.H., Bosschaert, J. and Shortt, C. (1998) Health aspects of probiotics. Food Sci Technol Today 12, 46 49. Lee, Y.-K. and Puong, K.-Y. (2002) Competition for adhesion between probiotics and human gastrointestinal pathogens in presence of carbohydrate. Br J Nutr 88, 101 108. Lee, Y.-K. and Salminen, S. (1995) The coming of age of probiotics. Trends Food Sci Technol 6, 241 246. Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 45 (2007) 454 460 459

Lee, Y.-K., Puong, K.Y., Ouwehand, A.C. and Salminen, S. (2003) Displacement of bacterial pathogens from mucus and Caco-2 cell surface by lactobacilli. J Med Microbiol 52, 925 930. Ouwehand, A.C. and Salminen, S. (2003) In vitro adhesion assays for probiotics and their in vivo relevance: a review. Microb Ecol Health Dis 15, 175 184. Ouwehand, A.C., Isolauri, E., Kirjavainen, P.V. and Salminen, S.J. (1999) Adhesion of four Bifidobacterium strains to human intestinal mucus from subjects in different age groups. FEMS Microbiol Lett 172, 61 64. Ouwehand, A.C., Salminen, S., Tölkkö, S., Roberts, P., Ovaska, J. and Salminen, E. (2002) Resected human colonic tissue: new model for characterizing adhesion of lactic acid bacteria. Clin Diag Lab Immuno 9, 184 186. Reid, G., McGroarty, J.A., Angotti, R. and Cook, R.L. (1988) Lactobacillus inhibitor production against Escherichia coli and coaggregation ability with uropathogens. Canadian J Microbiol 34, 344 351. Rinkinen, M., Westermarck, E., Salminen, S. and Ouwehand, A.C. (2003) Absence of host specificity for in vitro adhesion of probiotic lactic acid bacteria to intestinal mucus. Vet Microbiol 97, 55 61. Salminen, S., Bouley, C., Boutron-Ruault, M.-C., Cummings, J.H., Franck, A., Gibson, G.R., Isolauri, E., Moreau, M.-C. et al. (1998) Functional food science and gastrointestinal physiology and function. Br J Nutr 80, S147 S171. Salminen, S., Ouwehand, A.C., Benno, Y. and Lee, Y.K. (1999) Probiotics: how should they be defined? Trends Food Sci Technol 10, 107 110. Schiffrin, E.J., Brassard, D., Servin, A.L., Rochat, F. and Donnet-Hughes, A. (1997) Immune modulation of blood leukocytes in humans by lactic acid bacteria: criteria for strain selection. Am J Clin Nutr 66, 515 520. Tuomola, E.M., Ouwehand, A.C. and Salminen, S. (1999) The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus. FEMS Immunol Med Microbiol 26, 137 142. Tuomola, E., Crittenden, R., Playne, M., Isolauri, E. and Salminen, S. (2001) Quality assurance criteria for probiotic bacteria. Am J Clin Nutr 73, 393 398. Vesterlund, S., Paltta, J., Karp, M. and Ouwehand, A.C. (2005) Measurement of bacterial adhesion in vitro evaluation of different methods. J Microbiol Methods 60, 225 233. 460 Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 45 (2007) 454 460