Role of F1C Fimbriae, Flagella, and Secreted Bacterial Components inthe Inhibitory Effect of Probiotic Escherichia coli Nissle 1917 onAtypical Enteropathogenic E. coli Infection

Sylvia Kleta,a Marcel Nordhoff,a Karsten Tedin,a Lothar H. Wieler,a Rafal Kolenda,b,c Sibylle Oswald,d Tobias A. Oelschlaeger,d

Wilfried Bleiß,e Peter Schieracka,c

Zentrum für Infektionsmedizin, Institut für Mikrobiologie und Tierseuchen, Fachbereich Veterinärmedizin, Freie Universität Berlin, Berlin, Germanya; Department ofBiochemistry, Pharmacology and Toxicology, Wrocław University of Environmental and Life Sciences, Wrocław, Polandb; Fakultät für Naturwissenschaften,Brandenburgische Technische Universität Cottbus-Senftenberg, Senftenberg, Germanyc; Institut für Molekulare Infektionsbiologie, Universität Würzburg, Würzburg,Germanyd; Lehrstuhl für Molekulare Parasitologie, Humboldt-Universität zu Berlin, Berlin, Germanye

Enteropathogenic Escherichia coli (EPEC) is recognized as an important intestinal pathogen that frequently causes acute andpersistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. Theprobiotic E. coli strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. Whileboth in vitro and in vivo studies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including patho-genic E. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect ofEcN on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli (aEPEC) with respect to single infec-tion steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drasticallyreduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attachingand effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly medi-ated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interferewith the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to bespecific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of theinhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression.

Enteropathogenic Escherichia coli (EPEC) frequently causesacute and persistent diarrhea in animals and humans. EPEC

infection leads to serious acute diarrhea in weaned pigs (1, 2). Inhumans, EPEC infections are particularly serious for infants andtoddlers and are often accompanied by high lethality in develop-ing countries (3, 4). However, it is now recognized that atypicalEPEC (aEPEC) strains are more frequent in humans than typicalEPEC (tEPEC; expression of bundle-forming pili) strains in bothdeveloping and developed countries (5, 6). In animals, aEPECstrains are much more prevalent than EPEC strains, and aEPECstrains isolated from cattle, sheep, and pigs belong to the sameserotypes found in humans (7, 8). Furthermore, recent studiesshowed a close clonal relationship between human and animalaEPEC isolates, suggesting a possible zoonotic potential of animalaEPEC strains that could serve as a reservoir for human infections(9).

Infection of intestinal epithelial cells by EPEC is a complexmultistage process. Initially, EPEC loosely adheres to epithelialcells by diverse adhesins and subsequently translocates effectormolecules, including the translocated intimin receptor (Tir), intohost cells using a type 3 secretion system (T3SS). After integrationof Tir into the host cell membrane, EPEC binds tightly through theadhesin intimin with Tir. EPEC is able to intimately adhere toepithelial cells and to form microcolonies with resulting typicallyassociated histopathological alterations of the host cell surfaceknown as attaching and effacing (AE) lesions. Rearrangement andmassive accumulation of actin and other cytoskeletal proteins be-neath the site of bacterial attachment lead to the formation of

pedestal structures and destruction of microvilli (effacement).The pathogenesis is further characterized by loss of tight-junctionintegrity and barrier functions of the gut epithelium and destruc-tion of microvilli (effacement) and the brush border that leads todiarrhea (4, 10–13).

The nonpathogenic E. coli strain Nissle 1917 (EcN) is a widelyemployed probiotic strain, and several in vivo studies have dem-onstrated its promising probiotic activity in humans and animals,including the treatment of acute, chronic, or frequent recurringdiarrhea and inflammatory bowel disease (14–19). Proposed pro-biotic actions of EcN include effects on pathogens, host epithelialcells, host smooth muscle cell activity, and the host immune sys-tem (20–28). In vitro, EcN has been shown to inhibit invasion ofhost cells by several enteric pathogens, including Salmonella, Yer-sinia, Shigella, Legionella, Listeria, and adherent-invasive E. coli(29, 30). However, the underlying molecular mechanisms remainlargely unknown. Here, we characterize the effects of EcN onaEPEC infection of IPEC-J2 cells by means of confocal laser scan-

Received 13 November 2013 Returned for modification 20 December 2013Accepted 7 February 2014

Published ahead of print 18 February 2014

Editor: B. A. McCormick

Address correspondence to Peter Schierack, peter.schierack@hs-lausitz.de.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.01431-13

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ning microscopy and scanning electron microscopy, as well asmolecular and protein biochemical methods. Our data providenew insights into host-bacterium and interbacterial interactionsand show that EcN might be a promising tool in prophylacticdefense against EPEC infections.

MATERIALS AND METHODSCell line and bacterial strains. The porcine intestinal epithelial cell lineIPEC-J2 (31) was grown to confluence in Dulbecco’s modified Eagle me-dium (DMEM)–Ham’s F-12 (1:1) (Biochrom, Berlin, Germany) supple-mented with 5% fetal calf serum (FCS) and maintained in an atmosphereof 5% CO2 at 37°C. The bacterial strains used in this study are listed inTable 1. EcN was kindly provided by G. Breves (Hannover, Germany).The EcN mutant EcN �fliA (this study) was generated using the methodof Datsenko and Wanner (32). The fliA gene was replaced by a kanamycinresistance (kan) antibiotic cassette generated using the plasmid pKD4 asthe template and primer pair fliAH1P1 (5=-GTGAATTCACTCTATACCGCTGAAGGTGTAATGGATAAACAGTGTAGGCTGGAGCTGCTTC-3=) and fliAH2P2 (5=-ACTTACCCAGTTTAGTGCGTAACCGTTTAATGCCTGGCTGTGCATATGAATATCCTCCTTAG-3=). EcN �fliA wascomplemented using the plasmid pACYC177 harboring the sequence offliA (strain EcN �fliA � fliA). E. coli MG1655 was a laboratory strainoriginally obtained from C. A. Cross (San Francisco, CA, USA). E. coliIMT13962 was from the collection of the Institute of Microbiology andEpizootics (Berlin, Germany) and was originally isolated from the colonof a clinical healthy piglet and chosen for its strong adherence to IPEC-J2cells. Strain IMT13962(pCosF1C6) was generated from strain IMT13962by complementation with the foc operon cloned into the pSuperCos1vector (Stratagene, Heidelberg, Germany). Strain P2005/03 (kindly pro-vided by R. Bauerfeind, Gießen, Germany) was isolated from a piglet withdiarrhea and classified as aEPEC. Human EPEC E2348/69 was kindly pro-vided by J. B. Kaper (Baltimore, MD, USA). E. coli strain H5316 is amicrocin-sensitive indicator strain kindly provided by K. Hantke (Tübin-gen, Germany). Uropathogenic E. coli (UPEC) strain RZ525 was kindlyprovided by U. Dobrindt (Würzburg, Germany). Unless otherwise indi-cated, bacterial strains were grown in LB broth at 37°C with agitation at200 rpm. For cultivation of strains P2005/03 and E2348/69, LB broth wassupplemented with 20 �g/ml tetracycline and 30 �g/ml nalidixic acid,respectively.

Preparation of bacterial supernatants. Bacterial strains were grownin LB broth at 37°C with agitation at 200 rpm to an optical density at 600nm (OD600) of 1.0, diluted 1:100 in DMEM–Ham’s F-12 cell culture me-dium containing 5% fetal calf serum, and grown again to an OD600 of 1.0.The bacterial cultures were centrifuged at 8,000 � g at 4°C for 15 min. The

supernatants (SN) were sterile filtered with 0.22-�m filters and kept untiluse at �20°C.

Infection assay. aEPEC P2005/03 and EPEC E2348/69 were grown toan OD600 of 1.0, washed by centrifugation, resuspended in cell culturemedium, and adjusted by dilution to provide a multiplicity of infection(MOI) of 100:1 (bacteria to host cells) in wells of 12- or 24-well cell cultureplates. Confluent monolayers of IPEC-J2 cells were infected withP2005/03 or E2348/69 and incubated at 37°C. After 3 h, nonadherentbacteria were removed by three washes with phosphate-buffered saline(PBS). For P2005/03, incubation was continued for an additional 3 h.Efficiencies of infection of epithelial cells were determined by washing andlysing the cells with 0.1% Triton X-100 in double-distilled H2O (ddH2O)and plating serial dilutions on LB agar plates containing 5 �g/ml tetracy-cline or 30 �g/ml nalidixic acid, which allowed the selective growth ofP2005/03 and E2348/69, respectively. The plates were incubated over-night at 37°C, and the resulting numbers of CFU were determined. Forpreincubation experiments, epithelial cells were first incubated with thestrains indicated for 2 h and washed three times with PBS prior to EPECinfections. For co- and postincubation experiments, strains were addedsimultaneously or 1 h after aEPEC infection. For growth kinetics of ad-herent aEPEC on epithelial cells, the cell culture medium was changedevery 30 min 2 h after the beginning of aEPEC infection to remove non-adherent bacteria and to exchange exhausted cell culture medium.

Adhesion assay. Bacterial strains were prepared and added to IPEC-J2cell cultures as described for infection assays. At the indicated time points,nonadherent bacteria were removed by washing the cells three times withPBS. Numbers of adherent bacteria were determined by lysing the cellswith 0.1% Triton X-100 and plating serial dilutions on LB agar plates.

Fluorescence microscopy. For fluorescence microscopy, epithelialcells were grown to confluence on glass coverslips and fixed with acetonefor 2 min at �20°C (confocal laser scanning microscopy) or 4% parafor-maldehyde for 30 min at 4°C (epifluorescence microscopy) after perform-ing infection or adhesion experiments. All incubation steps during stain-ing with fluorescent dyes were performed in the dark. Strain P2005/03 wasdetected by immunohistochemical staining. Samples were incubated withpolyclonal antibodies against serotype O108 raised in rabbit (Federal In-stitute for Risk Assessment, Berlin, Germany; diluted 1:50 in PBS with0.5% bovine serum albumin [BSA]) for 30 min at room temperature,followed by incubation with goat anti-rabbit tetramethyl rhodamine iso-cyanate (TRITC)-labeled secondary monoclonal antibodies (Sigma-Al-drich, Munich, Germany; diluted 1:200 in PBS with 0.5% BSA) for 30 minat room temperature. Samples were washed three times with PBS aftereach antibody labeling.

The ability to produce AE lesions was indirectly examined by fluores-

TABLE 1 Bacterial strains used in this study

Bacterial strain Description Source Reference

EcN E. coli Nissle 1917 (DSM 6601); probiotic; O6:K5:H1; ST 73 Human 47EcN �fim EcN; deletion of fim operon Human 29EcN �fim foc EcN; deletion of fim and foc operons Human 29EcN �foc EcN; deletion of foc operon Human 29EcN �fliA EcN; deletion of fliA (bp 41–672); Kmr Human This studyEcN �fliA � fliA EcN; deletion of fliA (bp 41–672); pACYC177 fliA; Kmr Human This studyE2348/69 EPEC; O127:H6; ST 15; Nalr HumanH5316 E. coli K-12 MC4100; �lac entC::MudX; negative for the gene fur; 31zbd::Tn10 Human 37IMT13962 E. coli 140815; O15:NT; fim�; negative for the genes foc, eae, stx2e, faeG, fanA, fasA, fedA,

fimF41a, est-Ia, and eltB-IpPorcine 34

IMT13962 pCosF1C6 IMT13962; pCosF1C6; foc� Porcine This studyMG1655 E. coli K-12; OR:H48; ST 98;, F� ��; negative for the gene ilvG; rfb-50 rph-1 Human 64P2005/03 aEPEC; O108:H9; ST 302; intimin � eae� lerA�; negative for the genes perA, bfp, stx, est-II,

est-Ia, and eltB-Ip, astA; Tetr

Porcine 34

RZ525 UPEC; O6:K5:H1; foc�; negative for the gene hlyA Human 65

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cent actin staining (FAS) according to the method of Knutton et al. (33).F-actin was stained with fluorescein isothiocyanate (FITC)-phalloidin (5�g/ml; Invitrogen, Karlsruhe, Germany) for 30 min at room temperature.Samples were washed three times with PBS. Cell nuclei and bacteria ingeneral were visualized by staining DNA with 0.3 �g/ml propidium iodide(Invitrogen, Karlsruhe, Germany) for 3 min at room temperature. Beforestaining, paraformaldehyde (PFA)-fixed cells were first incubated with1 ml ice-cold 0.1% Triton X-100 in PBS for 4 min on ice and washed threetimes with PBS in order to permeabilize the cells. Images were acquiredwith the confocal laser scanning microscope DMIRE 2 TCS SP2 or theepifluorescence microscope DMBL (both from Leica, Wetzlar, Germany).

EcN flagella were stained with polyclonal antibodies against flagellumH1, kindly provided by L. Beutin (National Reference Laboratory for E.coli, Federal Institute for Risk Assessment, Berlin, Germany). Antibodieswere diluted 1:50 in PBS with 0.5% BSA and applied for 30 min at roomtemperature, followed by incubation with FITC-labeled secondary mono-clonal antibodies against rabbit (Sigma-Aldrich, Munich, Germany) for30 min at room temperature. Samples were washed three times with PBSafter each antibody labeling.

Scanning electron microscopy. Infected cells grown on glass coverslipswere fixed with 2% glutaraldehyde and 0.05% calcium chloride in 0.1 Msodium cacodylate buffer, pH 7.4, for 24 h at 4°C. Then, the samples wererinsed three times with ice-cold 0.1 M sodium cacodylate buffer, pH 7.4; fixeda second time with 1% osmium tetroxide in 0.1 M sodium cacodylate bufferfor 3 h at 4°C; and subsequently rinsed again. For raster preparation, sampleswere dehydrated in a graduated series of ethanol solutions and finally in 100%acetone, critical-point dried with liquid carbon dioxide using the point dryerCPD 030 (Bal-Tec, Witten, Germany), and sputtered with 20-nm gold parti-cles using the sputter coater SCD 005 (Bal-Tec, Witten, Germany). Sampleswere examined with a Leo 1430 scanning electron microscope (Leo Elek-tronenmikroskopie, Oberkochen, Germany).

Microcin test. The expression, as well as the sensitivity, of E. colistrains versus microcins was tested as described by Kleta et al. (34).

Isolation of secreted and intracellular EPEC proteins. The humanEPEC strain E2348/69 was grown in 6 ml DMEM–Ham’s F-12 cell culturemedium to an OD600 of approximately 1.0. The bacteria were centrifugedfor 5 min at 8,000 � g at room temperature, and the pellet was resus-pended in 6 ml DMEM–Ham’s F-12 cell culture medium. The bacterialsuspension was then diluted 1:100 in 50 ml DMEM–Ham’s F-12 in a250-ml Erlenmeyer flask and grown again to an OD600 of 1.0 with shakingat 200 rpm at 37°C. The bacterial culture (45 ml) was centrifuged (15 min;8,000 � g; 4°C), and the resulting supernatants were sterile filtered using0.22-�m filters. Secreted proteins in the supernatants were precipitatedwith trichloroacetic acid (TCA) (final concentration, 10%) overnight at4°C. The next day, the TCA precipitates were centrifuged for 30 min at10,000 � g at 4°C. The resulting protein pellet was dried for 5 min at roomtemperature; resuspended in 1 ml 0.2% SDS in 25 mM Tris-HCl, pH 8.0;mixed with ice-cold acetone (1:4); and incubated for 1 h at �20°C. Aftercentrifugation (30 min; 10,000 � g; 4°C), the protein pellet was dried (5min; room temperature) and diluted in 25 mM Tris-HCl (pH 8.0) and 4M urea. The secreted proteins (1,000-fold concentrated) were stored at�20°C.

In order to extract proteins from EPEC bacteria, 1 ml of the 50-mlbacterial suspension in the 250-ml Erlenmeyer flask was centrifuged for 5min at 8,000 � g at room temperature. The bacterial pellet was resus-pended in 4� sample buffer (1 M Tris-HCl, pH 6.8, 4% SDS, 8% �-mer-captoethanol, 20% glycerin, 0.025% bromophenol blue). The proteinswere denatured by heating at 99°C for 5 min. The tubes were incubated onice for 5 min, centrifuged for 5 s, and kept at �20°C until further use.

To determine the effects of EcN and MG1655 on protein secretion byEPEC, E2348/69 was grown in culture supernatants of EcN and MG1655,respectively. Bacterial supernatants were obtained from DMEM–Ham’sF-12 medium as described above but without added FCS. For dilution ofbacterial supernatants, DMEM–Ham’s F-12 cell culture medium wasused.

Western blot analysis. Proteins were separated by SDS-PAGE accord-ing to the method of Laemmli (35). For nonspecific protein detection,SDS-PAGE gels were stained with the Silver Stain Plus Kit (Bio-Rad, Mu-nich, Germany) according to the manufacturer’s instructions. The pro-teins EspA, EspB, and Tir were detected using Western blot analysis ac-cording to the method of Towbin et al. (36). Briefly, proteins weretransferred to nitrocellulose membranes (Sartorius, Göttingen, Germany)using a tank blot apparatus (Bio-Rad Laboratories, Munich, Germany).The membrane was blocked in 5% skim milk powder in Tris-bufferedsaline (TBS) buffer for 1 h. Primary antibodies against EspA, EspB, and Tirwere kindly provided by J. B. Kaper (University of Maryland, Baltimore,MD, USA). The membranes were incubated with primary antibodies di-luted 1:5,000 in blocking buffer overnight at 4°C with shaking, washedthree times with TBS for 10 min each time, incubated with 1:2,000-dilutedsecondary antibodies labeled with horseradish peroxidase (Sigma-Al-drich, Munich, Germany) in blocking buffer for 1 h, and washed as de-scribed above. The proteins were visualized by enhanced chemilumines-cence.

Statistical methods. Statistical calculations were performed usingSPSS 11.5 (SPSS, Chicago, IL, USA). Significances (P values) were calcu-lated using Student’s t test for normally distributed data or the Mann-Whitney U test for nonnormally distributed data. A P value of less than0.05 was considered to indicate a statistically significant difference.

RESULTSEffects of EcN on aEPEC adhesion, microcolony formation, andformation of attaching and effacing lesions. The probiotic strainEcN and two nonpathogenic E. coli control strains (IMT13962and MG1655) were tested for the ability to inhibit infection ofIPEC-J2 cells with the aEPEC strain P2005/03. In monoinfectionexperiments, the infection efficiencies of aEPEC strain P2005/03varied from approximately 2.2 � 104 to 1.7 � 105 bacteria per wellor 0.1 to 0.65 bacteria per epithelial cell, respectively. Preincuba-tion of EcN resulted in a reduction of aEPEC infection by 83%(P 0.001). Preincubation with the control strains IMT13962and MG1655 did not affect aEPEC infection (Fig. 1). In contrast to

FIG 1 aEPEC infection after preincubation with EcN. IPEC-J2 cells were pre-incubated with EcN, IMT13962, and MG1655 for 2 h, followed by aEPECinfection for 6 h. Numbers of adherent aEPEC bacteria were determined byserial dilutions of cell lysates. Shown are relative infection rates compared toaEPEC monoinfection (set as 100%) as mean values and standard deviations(SD) of at least 3 independent tests in duplicate wells. *, P 0.0001 comparedto monoinfection.

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preincubation, co- and postincubation experiments with EcN didnot decrease aEPEC infection efficiencies (data not shown).

Using confocal laser scanning microscopy, we evaluated in de-tail EcN effects on single aEPEC infection steps. aEPEC P2005/03was distinguishable from EcN by staining with specific antibodiesagainst serotype O108. In monoinfection, aEPEC P2005/03formed compact microcolonies, whereas preincubation with EcNreduced both the numbers and the size of aEPEC microcolonies,as indicated by single adherent aEPEC bacteria or by very smalland loose microcolonies. The formation of AE lesions was notinhibited. Bacteria that had attached as microcolonies alwaysformed AE lesions. Only very few single adherent bacteria did notform AE lesions (Fig. 2).

Growth of nonadherent aEPEC in the EcN–IPEC-J2 cell cul-ture supernatant. The reduction of aEPEC infection might havebeen due to a reduction of growth of nonadherent aEPEC in thesupernatant of EcN–IPEC-J2 cells. To test this, infection assayswith or without preincubation of EcN were performed, and thenumber of aEPEC bacteria in the cell culture supernatant wasdetermined every hour. In preincubation experiments with EcN,IMT13962, and MG1655, the growth of nonadherent aEPEC wasnot significantly affected in cell culture supernatants (data notshown).

Effects of EcN on growth of adherent aEPEC. Confocal laserscanning microscopy showed that both the numbers and the sizeof aEPEC microcolonies were reduced in preincubation experi-ments with EcN compared to monoinfection (Fig. 2). In order toestimate the growth of epithelial-cell-adherent aEPEC in micro-colonies, we performed growth kinetic experiments. In these ex-periments, we changed the cell culture medium every 30 min be-ginning 2 h after aEPEC infection to remove nonadherent bacteriaand to exchange exhausted cell culture medium. Thirty minutescorresponded to the doubling time of aEPEC strain P2005/03 incell culture medium, and thus, adhesion of new bacteria from thecell culture medium was minimized. We found in preincubationexperiments with EcN that the number of adherent aEPEC bacte-ria was reduced by 90% within 2 h after infection. Washing theepithelial cells 2 h after infection resulted in detachment of adher-

ent aEPEC bacteria in both monoinfection and preincubation ex-periments with EcN (Fig. 3, compare 2- and 3-h time points). Thisphenomenon was not observed in the subsequent washing steps.We therefore included only the times 3.0 to 4.5 h after aEPECinfection for determination of the effects of EcN on growth ofcell-adherent aEPEC. As shown in Fig. 3, preincubation with EcNresulted in reduced growth of adherent aEPEC on IPEC-J2 cells by31.5%, which was not statistically significant (P 0.056).

Effects of EcN microcins on aEPEC growth. EcN produces themicrocins H47 and M (37), low-molecular-weight peptides thathave antibacterial activity against other E. coli strains (38). Wetested for possible effects of EcN microcins on aEPEC growth inagar plate inhibition tests. The E. coli strain H5316 served as amicrocin-sensitive control strain. The production of microcins byEcN was indicated by a zone of inhibition around the filter paperdisc with EcN. In contrast to EcN, E. coli strains IMT13962 andMG1655 did not produce microcins. However, EcN microcins didnot affect aEPEC growth (data not shown).

Adhesion of EcN on IPEC-J2 cells and correlation with inhib-itory effects. An inhibitory effect on aEPEC infection was ob-served in preincubation experiments with EcN. In contrast, pre-incubation with the control strains IMT13962 and MG1655 didnot affect the infection levels of aEPEC. To determine whether theadhesion levels of these strains could affect aEPEC infection, as inthe preincubation experiments, epithelial cells were washed di-rectly prior to aEPEC infection to remove nonadherent bacteria,and the abilities of strains to adhere to epithelial cells were deter-mined by counting attached cells during aEPEC infection. Adhe-sion experiments with EcN, IMT13962, and MG1655 were per-formed for 2 h on epithelial cells. In contrast to control strains,EcN adhered well to epithelial cells. Using an MOI of 100, approx-imately 20 bacteria adhered to one epithelial cell (Fig. 4). EcNadhered 7-fold and 400-fold more strongly than IMT13962 andMG1655, respectively (P 0.05).

Role of F1C fimbriae in adhesion of EcN on IPEC-J2 cells andcorrelation with inhibitory effect. Adhesion of EcN to IPEC-J2cells was mediated by F1C fimbriae but not by type 1 fimbriae.Deletion of the foc operon (coding for F1C fimbriae) resulted in

FIG 2 Effect of EcN on aEPEC microcolony formation. IPEC-J2 cells were preincubated with or without EcN for 2 h, followed by aEPEC infection for 6 h.Nonadherent bacteria were removed by washing. The cells and adherent bacteria were fixed and stained, and images were taken by confocal laser scanningmicroscopy. (A) aEPEC monoinfection. (B) EcN preincubation. Scale bars 10 �m. aEPEC P2005/03 bacteria were stained using primary anti-O108 antiseraand secondary anti-rabbit-IgG–TRITC antibodies (red), actin was stained with FITC phalloidin (green), and cell nuclei and bacteria were stained with propidiumiodide (blue).

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a reduction of adhesion by 99.9% (P 0.001). Furthermore,the reduction of EcN adhesion correlated with a reduction ofthe inhibitory effect against aEPEC infection (Fig. 5). In con-trast, deletion of the fim operon (coding for type 1 fimbriae)did not affect adhesion to IPEC-J2 cells and did not inhibitaEPEC infection. We attempted to complement the foc mutantwith the cloned foc operon. However, several cloning strategiesusing three different cloning vectors failed. All the clones testedshowed abnormal cell lengths and growth defects and thus werenot further evaluated. The control strain IMT13962, whichdoes not harbor the gene for F1C fimbriae, adhered sparingly toIPEC-J2 cells and had no inhibitory effect on aEPEC infection.To determine the role of F1C fimbriae, we also complementedthe absence of F1C fimbriae in IMT13962 by introduction ofthe cosmid pCosF1C6 harboring the foc operon for productionof F1C fimbriae and tested its role in adhesion to IPEC-J2 cells.The complemented strain IMT13962(pCosF1C6) expressedfimbriae (Fig. 6A), showed higher levels of adherence toIPEC-J2 cells, and resulted in an inhibitory effect on aEPECinfection (Fig. 6B). In addition, we also tested another F1C

fimbria-positive E. coli strain, a nonhemolytic UPEC strain,RZ525, for adherence to IPEC-J2 cells and the subsequent inhib-itory effect. Strain RZ525 adhered well to IPEC-J2 cells and alsoreduced aEPEC infection efficiencies (Fig. 6B).

Role of flagella in adhesion of EcN to IPEC-J2 cells and cor-relation with inhibitory effect. Scanning electron and epifluores-cence microscopy showed that EcN formed a filamentous network

FIG 3 Growth kinetics of adherent aEPEC on epithelial cells. IPEC-J2 cellswere preincubated with EcN for 2 h, followed by aEPEC infection for 4.5 h.Numbers of adherent aEPEC bacteria were determined by plating serial dilu-tions of cell lysates on agar plates every 30 min beginning 2 h after aEPECinfection. aEPEC was selectable due to tetracycline resistance. (A) AdherentaEPEC bacteria as mean values � SD of 2 separate experiments in duplicatewells. (B) Regression and growth parameters. Calculations were as follows:td t/n, where t is the time of determination and n is the number of divisions;n (log N � log N0)/log2, where N is the number of bacteria after n divisionsand N0 is the number of bacteria at the beginning of the experiment; � n/t;� ln2/td.

FIG 4 Adhesion of EcN to IPEC-J2 cells. EcN, IMT13962, and MG1655 wereincubated with IPEC-J2 cells for 2 h in 12-well plates (2.6 � 105 epithelialcells/well). Nonadherent bacteria were removed by washing, and numbers ofadherent bacteria were determined by plating serial dilutions of cell lysates onagar plates. The data are given as mean values and SD of three separate exper-iments in duplicate wells. *, P 0.05 compared to EcN.

FIG 5 Roles of F1C and type 1 fimbriae in adhesion of EcN and in aEPECinfection. For adhesion, the EcN wild type and mutants were incubated onIPEC-J2 cells for 2 h. For aEPEC infection, IPEC-J2 cells were first incubatedwith the EcN wild type and mutants for 2 h, followed by aEPEC infection for 6h. IPEC-J2 cells were washed, and the numbers of adherent bacteria weredetermined by plating serial dilutions of cell lysates on agar plates. The data aregiven as relative adhesion rates compared to EcN or as infection rates in rela-tion to monoinfection, respectively, as mean values and SD of three separateexperiments in duplicate wells. **, P 0.001, and *, P 0.05 compared to EcNadhesion or aEPEC monoinfection, respectively.

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on IPEC-J2 cells (Fig. 7A and B). This network consisted of fla-gella, as shown by deletion of the fliA gene encoding the flagellarsigma factor 28, which resulted in loss of the filamentous network(Fig. 7C). Flagella were formed during the adhesion process andgrowth on IPEC-J2 cells, in which the length and the number offlagella increased with increasing incubation times (Fig. 7D).

Deletion of the fliA gene resulted in reduction of adhesion by50% (P 0.001). This resulted in a distinct but smaller (by 29%)inhibitory effect of EcN �fliA than of wild-type EcN (P 0.001).Complementation with the plasmid pACYC177 harboring se-quence including fliA resulted in restored adhesion and inhibitionof aEPEC infection to EcN wild-type levels (Fig. 8).

Role of EcN culture supernatants in aEPEC infection. Theinhibitory effect of EcN against aEPEC infection might have beendue to secreted EcN components. We therefore also tested theeffects of bacterium-free EcN SN compared to supernatants ofcontrol strains. For comparison with the initial experiments withbacteria (for which EcN was present in the pre- and coincubationperiods), we also performed pre- and coincubation experiments.Preincubation with bacterial supernatants had no effect on aEPECinfection, whereas coincubation led to a reduction of aEPEC in-fection by 75% (P 0.01), indicating the presence of an inhibitorycomponent in the supernatant and a direct effect on aEPEC bac-teria, but not on epithelial cells (Fig. 9). Interestingly, this compo-nent was not specific to EcN but was also found in strainsIMT13962 and MG1655. Supernatants of both strains inhibitedaEPEC infection to an extent similar to that with EcN (76% and85%; P 0.01). In contrast to the supernatants of MG1655 andfresh culture medium, the pH values of the supernatants of EcNand IMT13962 were slightly lower (pH 7.1 to 7.3 versus pH 7.7).Furthermore, the growth of aEPEC bacteria in cell culture me-dium was reduced by 30% to 35% in coincubation experiments

with the supernatants of EcN, IMT13962, and MG1655 3 h afterinfection (P 0.05) (data not shown). However, preincubation ofaEPEC for 2 h at 37°C in bacterial supernatant of EcN, IMT13962,or MG1655 or in cell culture medium alone (negative control),followed by addition to epithelial cells with fresh cell culture me-dium, showed that the aEPEC infection rates were not affectedcompared to the negative control (data not shown).

Effects of EcN on the secretion of virulence-associated pro-teins of EPEC by T3SS. To verify whether EcN might affect thesecretion of virulence-associated EPEC proteins by the T3SS, weexamined the influence of EcN SN on the secretion of EspA, EspB,and Tir. EspA could mediate the initial adherence to and the trans-location of other effector molecules into host cells (10, 12). EspB isinvolved in pore formation in host cell membranes, supports thetranslocation of other effector molecules into host cells, and sup-ports destruction of microvilli (39, 40). Tir is the translocatedreceptor for the aEPEC adhesin intimin (11).

In our experiments, aEPEC P2005/03 was found to secrete onlyvery small amounts of proteins, which were not sufficient for fur-ther analysis (data not shown). To overcome this, we chose toexamine the typical human EPEC strain E2348/69 (O127:H6).Strain E2348/69 had previously been shown to secrete largeamounts of proteins in DMEM–Ham’s F-12 cell culture medium(12, 41). First, we tested whether strain E2348/69 showed the samephenotypes as aEPEC P2005/03, i.e., infection of IPEC-J2 cells,microcolony formation on IPEC-J2 cells, and inhibition by EcN.Similar to aEPEC P2005/03, EPEC E2348/69 formed compact mi-crocolonies (localized adherence pattern) on IPEC-J2 cells, butepithelial cells were infected considerably faster by E2348/69 thanby P2005/03 and appeared more adherent. However, in preincu-bation experiments with EcN, EPEC E2348/69 infection was in-hibited by 77%, which was comparable to that seen in aEPEC

FIG 6 (A) Scanning electron micrograph of IMT13962(pCosF1C6) on IPEC-J2. The F1C fimbria-negative E. coli control strain IMT13962 was complementedwith the cosmid pCosF1C6 carrying the foc operon for production of F1C fimbriae. The strain was incubated with IPEC-J2 cells for 4 h, and images of bacteriaon cells were taken by scanning electron microscopy. IMT13962(pCosF1C6) is shown to express F1C fimbriae. Scale bar 1 �m. (B) Adhesion ofIMT13962(pCosF1C6) and UPEC RZ525 to IPEC-J2 cells and effects on aEPEC infection. For adhesion, strains were incubated on IPEC-J2 cells for 2 h. ForaEPEC infection, IPEC-J2 cells were first incubated with IMT13962 or RZ525 for 2 h, followed by aEPEC infection for 6 h. The IPEC-J2 cells were washed, andnumbers of adherent bacteria were determined by plating serial dilutions of cell lysates on agar plates. The data are given as relative adhesion rates compared toEcN or as rates in relation to monoinfection, respectively, as mean values and SD of three separate experiments in duplicate wells. *, P 0.05 compared to EcNadhesion or aEPEC monoinfection, respectively.

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P2005/03 infections. E. coli strains MG1655 and IMT13962 didnot inhibit EPEC E2348/69 infection, as was already shown foraEPEC P2005/03 infection (data not shown).

Having verified that EcN and control strains had similar effectson aEPEC P2005/03 and EPEC E2348/69 adhesion and micro-colony formation, we used EPEC E2348/69 to determine the ef-fects of EcN supernatants on the secretion of virulence-associatedEPEC proteins. EPEC E2348/69 bacteria were grown in EcN orMG1655 supernatants to an OD600 of 1. The bacteria were thenremoved by centrifugation, and the supernatant proteins wereprecipitated with TCA, followed by SDS-PAGE and detection oftotal proteins by silver staining. EcN and MG1655 did not producedetectable amounts of secreted proteins in cell culture medium(data not shown). As shown in Fig. 10A, EcN supernatants inhib-ited the secretion of almost all proteins by EPEC. Inhibition wasdependent on the EcN supernatant concentration but not on pH.MG1655 supernatants also inhibited EPEC protein secretion, butto a lesser extent.

Western blot analysis of protein gels showed that EcN super-natants inhibited secretion of EspA, EspB, and Tir. In contrast,MG1655 supernatants inhibited the secretion of Tir but showedlittle or no effect on the secretion of EspA and EspB (Fig. 10B).

In addition to secreted-protein levels, we also determined theexpression of EspA, EspB, and Tir in the EPEC bacterial-cell totalprotein preparations, including cytosolic proteins, which may nothave been secreted. EPEC E2348/69 bacteria were grown in EcNand MG1655 supernatants to an OD600 of 1. The bacteria werepelleted and lysed. After SDS-PAGE, EspA, EspB, and Tir weredetected by Western blot analysis. EcN and MG1655 supernatanttreatments showed only slight reductions in intracellular EspA,

FIG 7 Scanning electron micrographs and fluorescence microscopy of EcNand EcN �fliA on IPEC-J2 cells. (A and B) EcN homogeneously adhered toIPEC-J2 cells via F1C fimbriae (white arrows) and formed a filamentous net-work via flagella (black arrows). (C) EcN �fliA did not express flagella and didnot form a filamentous network but still adhered to IPEC-J2 cells via F1Cfimbriae. Adhesion assays were performed for 4 h. Scale bars 10 �m (A) and1 �m (B and C). (D) Expression of flagella by EcN on IPEC-J2 cells. IPEC-J2cells were incubated with EcN over a period of 4 h. At the indicated time points,cells and bacteria were fixed and flagella were stained by polyclonal anti-H1antibodies and by secondary anti-rabbit IgG FITC-labeled antibodies. Imageswere taken by epifluorescence microscopy. Scale bar 10 �m. Note that theEcN wild type expressed flagella depending on the incubation time on epithe-lial cells.

FIG 8 Role of flagella in adhesion of EcN to IPEC-J2 and in aEPEC infection.The EcN wild type, EcN �fliA, and EcN �fliA � fliA were incubated withIPEC-J2 cells for 2 h. For aEPEC infection, IPEC-J2 cells were first incubatedwith the EcN wild type, EcN �fliA, and EcN �fliA � fliA for 2 h, followed byaEPEC infection for 6 h. The IPEC-J2 cells were washed, and the numbers ofadherent bacteria were determined by plating serial dilutions of cell lysates onagar plates. The data are given as relative adhesion rates compared to EcN or asrates in relation to monoinfection, respectively, as mean values and SD of atleast three separate experiments in triplicate wells. *, P 0.001 compared toEcN adhesion or aEPEC monoinfection.

FIG 9 Infection efficiencies of aEPEC P2005/03 on IPEC-J2 in pre- and coin-cubation experiments with bacterial supernatants. Infection was performed atan MOI of 100 for 6 h. IPEC-J2 cells were preincubated for 2 h or coincubatedwith bacterial supernatants. The data are given in relation to monoinfection asmean values and SD of three separate experiments in duplicate wells. *, P 0.01 compared to monoinfection.

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EspB, and Tir concentrations, with MG1655 showing a smallereffect than EcN supernatants (Fig. 10C). These results suggestedthat the EcN supernatants affected the protein secretion of EPECE2348/69 rather than intracellular protein expression.

DISCUSSION

Probiotic bacteria might affect or interfere with aEPEC infectionsdue to direct effects against pathogens or due to stabilizing effectson the intestinal microbiota, mucosa, or mucosal immunity. EcNhas been successfully applied against different porcine and humanintestinal disorders, including infection by enterotoxigenic E. coli,acute nonspecific diarrhea, or chronic inflammatory bowel dis-eases (14–18). In vitro, EcN has been shown to inhibit the invasionof porcine and human intestinal epithelial cell lines by entero-pathogens (29, 30, 42), although the underlying mechanisms haverarely been defined. As there is currently no information aboutprobiotic effects of EcN and other probiotics against aEPEC infec-tion— either in animals or in humans—we determined the effectsof EcN on aEPEC infection using a porcine intestinal-epithelial invitro model.

In the present study, preincubation of IPEC-J2 cells with EcN

resulted in a drastic reduction of cell-adherent EPEC (aEPEC, aswell as tEPEC). EcN affected the attachment and growth of cell-adherent aEPEC and therefore the growth of microcolonies, butnot the formation of attaching and effacing lesions of adherentEPEC. We conclude that EcN interferes with the EPEC infectionprocess very early and that this interference is a complex multi-stage process. Furthermore, culture supernatants of EcN were alsofound to be very effective against aEPEC infection. This is consis-tent with the observations of Altenhoefer et al. (29) and Schieracket al. (42), which showed that supernatants are effective againstinvasion of enteropathogens. The bacterial supernatants appear toact directly on aEPEC bacteria and not on host epithelial cells, asaEPEC infection was not inhibited by preincubation of IPEC-J2cells with bacterial supernatants; only coincubation resulted inreduced infection efficiencies of aEPEC. However, we cannot ex-clude the possibility that other bacterial factors from the superna-tant may have effects on host cells. Furthermore, the effects ofsupernatants on aEPEC infection were not EcN specific, as thesupernatants of control E. coli strains also inhibited aEPEC infec-tion. This is in accordance with a recent study on the effects of EcNagainst Salmonella invasion (42) but in contrast to another studythat showed a specific effect of EcN supernatants against entero-invasive pathogens (29). The discrepancy might be based on theuse of E. coli strain DH5 in the latter study. The strain grows veryslowly in LB, as well as in cell culture medium (our unpublishedobservation), suggesting metabolic deficiencies, and might not se-crete sufficient amounts of inhibitory supernatant factor(s). Inaddition, these prior studies used different intestinal epithelial celllines and experimental conditions, which could also have beenresponsible for divergent results. Although there are several re-ports on the effects of bacterial supernatants on infection by en-teropathogens, to date, a clearly defined inhibitor has not beenidentified.

The probiotic effect of EcN is likely to depend in part on thevery high adhesion rate of EcN compared to the control strains.Only EcN was still present in high numbers after numerous wash-ing steps of IPEC-J2 cell monolayers. In contrast, poorly adherentcontrol bacteria were inevitably removed. As a result, only EcNwas still able to grow to sufficient densities at host cell surfaces andpossibly secrete sufficient amounts of putative inhibitors. Success-ful probiotic action of a bacterial strain is often associated with itscolonization of the intestine. The colonization of hosts by EcN canbe very successful but is likely to be specific for individual hosts(34, 43).

Our results suggest that the inhibitory factor secreted by EcN iseffective only when present at sufficient concentrations prior toaEPEC adhesion. Inhibitory effects were observed only in prein-cubation experiments where sufficient EcN bacteria (approxi-mately 20 adherent bacteria per epithelial cell) were present closeto the host cell surface, and thus the interaction site of aEPEC withepithelial cells. This suggestion is also supported by our observa-tions that dilution of EcN supernatants reduced the inhibitoryeffect on secretion of virulence-associated effector proteins byEPEC.

EcN expresses several fimbrial-gene clusters, including (i) type1 fimbriae (66), expressed by most E. coli strains and involved inthe infection processes of many pathogens, such as UPEC or ad-herent invasive E. coli (AIEC) (44, 45); (ii) F1C fimbriae, whichhave usually been associated with uropathogenic E. coli; and (iii)curli fimbriae (46, 47). The type 1 fimbriae do not appear to play a

FIG 10 Detection of proteins expressed in and secreted by EPEC E2348/69.EPEC E2348/69 bacteria were grown to an OD600 of 1.0 in pure cell culturemedium (lanes 1), in undiluted or 4:1- or 1:1-diluted supernatants of EcN(lanes 2, 3, and 4, respectively), or in undiluted supernatant of EcN with cor-rected pH (lanes 5), as well as in undiluted or 4:1- or 1:1-diluted supernatantsof MG1655 (lanes 6, 7, and 8, respectively). (A and B) E2348/69-secretedproteins were extracted by TCA precipitation, concentrated 1,000-fold, andanalyzed by SDS-PAGE, followed by silver staining (A) or Western blotting(B). (C) Intracellular proteins were detected by SDS-PAGE, followed by West-ern blotting. Western blotting included detection of EspA, EspB, or Tir. Theamounts of loaded proteins corresponded to the amounts of secreted proteinsin 10 ml bacterial culture at an OD600 of 1.0 for silver staining (A) and 3 ml forWestern blot analysis (B), and the amounts of intracellular proteins corre-sponded to approximately 2.5 � 107 E2348/69 bacteria each (C).

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predominant role in EcN adhesion or in mediating an inhibitoryeffect of EcN. An EcN �fim mutant adhered as well as the EcNwild-type strain to IPEC-J2 cells, and adhesion of EcN to IPEC-J2cells was not inhibited by -D-mannose (data not shown), indi-cating that type 1 fimbriae play only a subordinate role in EcNadhesion.

Recently, it was demonstrated that F1C fimbriae play an im-portant role in EcN biofilm formation, adherence to intestinalepithelial cells in vitro, and intestinal colonization of mice and aprobiotic effect against Salmonella invasion (42, 48, 49). In thepresent study, we showed that F1C fimbriae were expressed andappeared to play a prominent role in adhesion of EcN to IPEC-J2epithelial cells and contributed to the inhibitory effects on aEPECinfection. The notion that F1C fimbriae were indeed an importantadhesin involved in these effects was further strengthened by com-plementation studies of E. coli strain IMT13962 with the focoperon and adhesion assays with the UPEC strain RZ525 carryingthe F1C fimbriae. Both strains adhered at levels comparable tothose of the EcN �fliA mutant, and both inhibited aEPEC infec-tion.

A second important EcN adhesion determinant we identifiedwas the flagellum. We showed for the first time that EcN expressesH1 flagella during the adhesion process at epithelial cell surfaces.Time course experiments showed elongation of flagella after ad-hesion and the formation of a tight network on epithelial cellmonolayers. Flagella contributed to the strong adherence of EcNto IPEC-J2 cells through interactions between single bacteria, aswell as apparent anchorage to the host cell surface. We suggest thatflagella may also prevent detachment of EcN after bacterial celldivision, as well as facilitate adhesion of further EcN bacteria fromthe cell culture medium. Flagella, therefore, may contribute to theinhibitory effects on pathogen infection by enhancing EcN adhe-sion to host cell surfaces.

The role of flagella in adhesion might be of much greater im-portance in vivo, as flagella enable bacteria to direct their move-ment toward the host cell by chemotaxis and thus support subse-quent adhesion (50). Moreover, flagella can also serve as directadhesion determinants, as shown for enteropathogens, includingtypical EPEC strains (51). The cross-linking of EPEC bacteria onthe surfaces of human epithelial HeLa cells via flagella contributedto a three-dimensional structure but required a eukaryotic signal,similar to the expression of flagella by EcN in our present study. Asscanning electron microscopy did not show expression of flagellaby EcN on human intestinal Caco-2 cells in another study (52),expression of EcN flagella might depend on the epithelial cell line,suggesting host or tissue specificity.

A prerequisite for successful infection of epithelial cells byenteropathogens is their adhesion to receptors on host cell sur-faces. Receptors are predominantly carbohydrates of trans-membrane or transmembrane-associated glycolipids and gly-coproteins, as well as proteins or glycoproteins of extracellularmatrices. The specificity of binding is determined by the se-quence of carbohydrate chains (53). The blocking of host cellsurface receptors by probiotics (competitive exclusion), due todirect binding either to similar epitopes or to adjacent struc-tures, has been proposed to be a potential mechanism for in-hibiting pathogenic infections (54, 55).

In the present study, aEPEC infection was inhibited by EcNadhesion, as well as by EcN supernatants that were isolated fromshaking cultures and thus could also contain fimbrial components

serving as potential ligands for host cell receptors. It might bepossible that EcN and aEPEC use similar host cell receptors foradhesion, supporting competitive exclusion as one mechanismfor the inhibitory effects. F1C fimbriae mediating adhesion of EcNto IPEC-J2 cells exhibit a high binding affinity to glycosphingolip-ids (e.g., asialo-GM1 and asialo-GM2), with the disaccharide se-quence GalNac�1-4Gal� as a minimal binding epitope and loweraffinity for other ceramides (56, 57). In contrast, EPEC host cellreceptors have only rarely been defined, but in in vitro studies ofEPEC bacteria showing a localized adherent phenotype, they werefound to bind to glycosphingolipids, suggesting that both EcN andEPEC might use the same host cell receptor (58). Our experimentsin which IPEC-J2 receptors were blocked with antibodies directedagainst asialo-GM2 indicated the expression of asialo-GM2 byIPEC-J2 and the binding of aEPEC to this receptor. In contrast toaEPEC, EcN adhesion levels were not affected by blocking asialo-GM2 (data not shown). In order to verify whether both EcN andEPEC bind to the same host cell receptors, more detailed studiesidentifying receptors present on IPEC-J2 cell surfaces and thebinding of EcN and EPEC are necessary.

The results of the present study also show that the inhibitoryfactor might not affect aEPEC infection indirectly via epithelialcells, as preincubation of supernatants had no effect on aEPECinfection. The binding of ligands to receptors is in general verystable, and thus, the blocking of receptor structures used by patho-gens to bind to cell surfaces would also result in inhibition ofpathogen adhesion in preincubation experiments with superna-tants, as shown for probiotic bifidobacteria (59, 60). Our results,therefore, do not appear to support competitive exclusion as apredominant mechanism for the inhibitory effects of EcN.

EPEC infection of intestinal epithelial cells is mediated by anumber of virulence-associated proteins that are secreted ortranslocated by the T3SS. Inhibition of protein expression, secre-tion, or translocation results in decreased EPEC virulence (4, 61).In the present study, we tested the effects of EcN supernatants onprotein expression and secretion. As the porcine aEPEC strainsecreted protein concentrations insufficient for extensive SDS-PAGE and Western blot analysis, we examined the effects of EcNon secretion of virulence proteins in the human tEPEC strainE2348/69. The well-characterized tEPEC strain E2348/69 secreteddetectable amounts of proteins common for EPEC adherence fac-tor (EAF) plasmid-positive (tEPEC) compared to EAF plasmid-negative (aEPEC) EPEC strains (41, 62). Protein secretion ofstrain E2348/69 was almost completely inhibited by EcN superna-tants, including inhibition of secretion of EspA, EspB, and Tir.Supernatants of the control strain E. coli MG1655 inhibited pro-tein secretion to a much lesser extent. A reduction in secretion ofall three proteins indicated that the T3SS or expression of locus ofenterocyte effacement (LEE) genes encoding the secreted proteinswas affected. T3SSs are used by a number of pathogenic Gram-negative bacteria, including E. coli, Yersinia, Shigella, Salmonella,and Chlamydia. Inhibition of the T3SS would therefore be an ef-fective mechanism for prevention of infection. It has previouslybeen shown that a low-molecular-weight polyketide compoundisolated from cultures of Streptomyces sp. was able to inhibit theT3SS (63). However, there are no data available on whether andhow probiotics might block the T3SS. As intracellular cytosolicprotein concentrations of EPEC were only slightly reduced, weconclude that protein secretion via the T3SS was the major targetof inhibition, rather than bacterial protein expression. Further

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studies with respect to the expression of virulence genes areneeded to confirm this.

In conclusion, a number of previous studies have indicatedthat EcN is effective against a range of pathogenic bacteria thatpossess numerous different infection strategies with diversemechanisms to adhere to host cells. Our data give insights intopotential mechanisms by which EcN could affect infection due toone of these pathogens, aEPEC. However, it appears unlikely thatEcN will be found to inhibit infections by different specific mech-anisms. The probiotic mode of action might rather be based oninterfering with global mechanisms of bacterial pathogenesis, assuggested by this study. Although this mode of action might not bespecifically limited to EcN, we assume that the strong colonizationcapabilities of EcN, both in vitro and in vivo, together with itsfitness characteristics, support its probiotic effect.

ACKNOWLEDGMENTS

We thank R. Bauerfeind (Gießen, Germany), L. Beutin (Berlin, Ger-many), G. Breves (Hannover, Germany), U. Dobrindt (Würzburg, Ger-many), K. Hantke (Tübingen, Germany), J. B. Kaper (Baltimore, MD,USA), and H. Steinrück (Berlin, Germany) for providing strains and an-tibodies.

This work was supported by the German Research Foundation (DFG)through the Collaborative Research Center 852 (grant no. SFB852/1) andgrant FOR 438/1-1, by InnoProfile IP 03 IP 611 funded by the FederalMinistry of Education and Research (BMBF, Germany), by Berlin Fund-ing for Graduates (NaföG Grants), and by the FAZIT Foundation.

REFERENCES1. Bertschinger HU, Fairbrother JM. 1999. Escherichia coli infections, p

431– 468. In Straw BE, D’Allaire S, Mengeling WL, Taylor DJ (ed), Dis-eases of swine, 8th ed. Blackwell Science, Oxford, United Kingdom.

2. Zhu C, Harel J, Jacques M, Desautels C, Donnenberg MS, Beaudry M,Fairbrother JM. 1994. Virulence properties and attaching-effacing activ-ity of Escherichia coli O45 from swine postweaning diarrhea. Infect. Im-mun. 62:4153– 4159.

3. Clarke SC. 2001. Diarrhoeagenic Escherichia coli—an emerging problem?Diagn. Microbiol. Infect. Dis. 41:93–98. http://dx.doi.org/10.1016/S0732-8893(01)00303-0.

4. Nataro JP, Kaper JB. 1998. Diarrheagenic Escherichia coli. Clin. Micro-biol. Rev. 11:142–201.

5. Ochoa TJ, Barletta F, Contreras C, Mercado E. 2008. New insights intothe epidemiology of enteropathogenic Escherichia coli infection. Trans. R.Soc. Trop. Med. Hyg. 102:852– 856. http://dx.doi.org/10.1016/j.trstmh.2008.03.017.

6. Trabulsi LR, Keller R, Tardelli Gomes TA. 2002. Typical and atypicalenteropathogenic Escherichia coli. Emerg. Infect. Dis. 8:508 –513. http://dx.doi.org/10.3201/eid0805.010385.

7. Aktan I, Sprigings KA, La Ragione RM, Faulkner LM, Paiba GA,Woodward MJ. 2004. Characterisation of attaching-effacing Escherichiacoli isolated from animals at slaughter in England and Wales. Vet. Micro-biol. 102:43–53. http://dx.doi.org/10.1016/j.vetmic.2004.04.013.

8. Krause G, Zimmermann S, Beutin L. 2005. Investigation of domesticanimals and pets as a reservoir for intimin- (eae) gene positive Escherichiacoli types. Vet. Microbiol. 106:87–95. http://dx.doi.org/10.1016/j.vetmic.2004.11.012.

9. Moura RA, Sircili MP, Leomil L, Matte MH, Trabulsi LR, Elias WP,Irino K, Pestana de Castro AF. 2009. Clonal relationship among atypicalenteropathogenic Escherichia coli strains isolated from different animalspecies and humans. Appl. Environ. Microbiol. 75:7399 –7408. http://dx.doi.org/10.1128/AEM.00636-09.

10. Cleary J, Lai LC, Shaw RK, Straatman-Iwanowska A, Donnenberg MS,Frankel G, Knutton S. 2004. Enteropathogenic Escherichia coli (EPEC)adhesion to intestinal epithelial cells: role of bundle-forming pili (BFP),EspA filaments and intimin. Microbiology 150:527–538. http://dx.doi.org/10.1099/mic.0.26740-0.

11. Kenny B, DeVinney R, Stein M, Reinscheid DJ, Frey EA, Finlay BB.1997. Enteropathogenic E. coli (EPEC) transfers its receptor for intimate

adherence into mammalian cells. Cell 91:511–520. http://dx.doi.org/10.1016/S0092-8674(00)80437-7.

12. Knutton S, Rosenshine I, Pallen MJ, Nisan I, Neves BC, Bain C, WolffC, Dougan G, Frankel G. 1998. A novel EspA-associated surface organelleof enteropathogenic Escherichia coli involved in protein translocation intoepithelial cells. EMBO J. 17:2166 –2176. http://dx.doi.org/10.1093/emboj/17.8.2166.

13. Moon HW, Whipp SC, Argenzio RA, Levine MM, Giannella RA. 1983.Attaching and effacing activities of rabbit and human enteropathogenicEscherichia coli in pig and rabbit intestines. Infect. Immun. 41:1340 –1351.

14. Henker J, Laass MW, Blokhin BM, Maydannik VG, Bolbot YK, Elze M,Wolff C, Schreiner A, Schulze J. 2008. Probiotic Escherichia coli Nissle1917 versus placebo for treating diarrhea of greater than 4 days duration ininfants and toddlers. Pediatr. Infect. Dis. J. 27:494 – 499. http://dx.doi.org/10.1097/INF.0b013e318169034c.

15. Krammer HJ, Kamper H, von Bunau R, Zieseniss E, Stange C, SchliegerF, Clever I, Schulze J. 2006. Probiotic drug therapy with E. coli strainNissle 1917 (EcN): results of a prospective study of the records of 3,807patients. Z. Gastroenterol. 44:651– 656. (In German.). http://dx.doi.org/10.1055/s-2006-926909.

16. Kruis W, Fric P, Pokrotnieks J, Lukas M, Fixa B, Kascak M, Kamm MA,Weismueller J, Beglinger C, Stolte M, Wolff C, Schulze J. 2004. Main-taining remission of ulcerative colitis with the probiotic Escherichia coliNissle 1917 is as effective as with standard mesalazine. Gut 53:1617–1623.http://dx.doi.org/10.1136/gut.2003.037747.

17. Schroeder B, Duncker S, Barth S, Bauerfeind R, Gruber AD, Deppen-meier S, Breves G. 2006. Preventive effects of the probiotic Escherichia colistrain Nissle 1917 on acute secretory diarrhea in a pig model of intestinalinfection. Dig. Dis. Sci. 51:724 –731. http://dx.doi.org/10.1007/s10620-006-3198-8.

18. Schultz M, Strauch UG, Linde HJ, Watzl S, Obermeier F, Gottl C, Dunger N,Grunwald N, Scholmerich J, Rath HC. 2004. Preventive effects of Escherichiacoli strain Nissle 1917 on acute and chronic intestinal inflammation in twodifferent murine models of colitis. Clin. Diagn. Lab. Immunol. 11:372–378. http://dx.doi.org/10.1128/CDLI.11.2.372-378.2004.

19. von Buenau R, Jaekel L, Schubotz E, Schwarz S, Stroff T, Krueger M.2005. Escherichia coli strain Nissle 1917: significant reduction of neonatalcalf diarrhea. J. Dairy Sci. 88:317–323. http://dx.doi.org/10.3168/jds.S0022-0302(05)72690-4.

20. Arribas B, Rodriguez-Cabezas ME, Camuesco D, Comalada M, BailonE, Utrilla P, Nieto A, Concha A, Zarzuelo A, Galvez J. 2009. A probioticstrain of Escherichia coli, Nissle 1917, given orally exerts local and systemicanti-inflammatory effects in lipopolysaccharide-induced sepsis in mice.Br. J. Pharmacol. 157:1024 –1033. http://dx.doi.org/10.1111/j.1476-5381.2009.00270.x.

21. Bar F, Von Koschitzky H, Roblick U, Bruch HP, Schulze L, SonnenbornU, Bottner M, Wedel T. 2009. Cell-free supernatants of Escherichia coliNissle 1917 modulate human colonic motility: evidence from an in vitroorgan bath study. Neurogastroenterol. Motil. 21:559 –566. http://dx.doi.org/10.1111/j.1365-2982.2008.01258.x.

22. Bickert T, Trujillo-Vargas CM, Duechs M, Wohlleben G, Polte T, Hansen G,Oelschlaeger TA, Erb KJ. 2009. Probiotic Escherichia coli Nissle 1917 sup-presses allergen-induced Th2 responses in the airways. Int. Arch. AllergyImmunol. 149:219 –230. http://dx.doi.org/10.1159/000199717.

23. Helwig U, Lammers KM, Rizzello F, Brigidi P, Rohleder V, Cara-melli E, Gionchetti P, Schrezenmeir J, Foelsch UR, Schreiber S,Campieri M. 2006. Lactobacilli, bifidobacteria and E. coli Nissle inducepro- and anti-inflammatory cytokines in peripheral blood mononuclearcells. World J. Gastroenterol. 12:5978 –5986.

24. Kamada N, Maeda K, Inoue N, Hisamatsu T, Okamoto S, Hong KS,Yamada T, Watanabe N, Tsuchimoto K, Ogata H, Hibi T. 2008.Nonpathogenic Escherichia coli strain Nissle 1917 inhibits signal transduc-tion in intestinal epithelial cells. Infect. Immun. 76:214 –220. http://dx.doi.org/10.1128/IAI.01193-07.

25. Otte JM, Podolsky DK. 2004. Functional modulation of enterocytes by gram-positive and gram-negative microorganisms. Am. J. Physiol. Gastrointest. LiverPhysiol. 286:G613–G626. http://dx.doi.org/10.1152/ajpgi.00341.2003.

26. Reissbrodt R, Hammes WP, dal Bello F, Prager R, Fruth A, Hantke K,Rakin A, Starcic-Erjavec M, Williams PH. 2009. Inhibition of growth ofShiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli.FEMS Microbiol. Lett. 290:62– 69. http://dx.doi.org/10.1111/j.1574-6968.2008.01405.x.

Kleta et al.

1810 iai.asm.org Infection and Immunity

on October 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

27. Ukena SN, Singh A, Dringenberg U, Engelhardt R, Seidler U, HansenW, Bleich A, Bruder D, Franzke A, Rogler G, Suerbaum S, Buer J,Gunzer F, Westendorf AM. 2007. Probiotic Escherichia coli Nissle 1917inhibits leaky gut by enhancing mucosal integrity. PLoS One 2:e1308. http://dx.doi.org/10.1371/journal.pone.0001308.

28. Zyrek AA, Cichon C, Helms S, Enders C, Sonnenborn U, Schmidt MA.2007. Molecular mechanisms underlying the probiotic effects of Esche-richia coli Nissle 1917 involve ZO-2 and PKCzeta redistribution resultingin tight junction and epithelial barrier repair. Cell Microbiol. 9:804 – 816.http://dx.doi.org/10.1111/j.1462-5822.2006.00836.x.

29. Altenhoefer A, Oswald S, Sonnenborn U, Enders C, Schulze J, HackerJ, Oelschlaeger TA. 2004. The probiotic Escherichia coli strain Nissle 1917interferes with invasion of human intestinal epithelial cells by differententeroinvasive bacterial pathogens. FEMS Immunol. Med. Microbiol. 40:223–229. http://dx.doi.org/10.1016/S0928-8244(03)00368-7.

30. Boudeau J, Glasser AL, Julien S, Colombel JF, Darfeuille-Michaud A. 2003.InhibitoryeffectofprobioticEscherichia coli strain Nissle 1917 on adhesion toand invasion of intestinal epithelial cells by adherent-invasive E. colistrains isolated from patients with Crohn’s disease. Aliment. Pharmacol.Ther. 18:45–56. http://dx.doi.org/10.1046/j.1365-2036.2003.01638.x.

31. Schierack P, Nordhoff M, Pollmann M, Weyrauch KD, Amasheh S,Lodemann U, Jores J, Tachu B, Kleta S, Blikslager A, Tedin K, WielerLH. 2006. Characterization of a porcine intestinal epithelial cell line for invitro studies of microbial pathogenesis in swine. Histochem. Cell Biol.125:293–305. http://dx.doi.org/10.1007/s00418-005-0067-z.

32. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomalgenes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci.U. S. A. 97:6640 – 6645. http://dx.doi.org/10.1073/pnas.120163297.

33. Knutton S, Baldwin T, Williams PH, McNeish AS. 1989. Actin accumu-lation at sites of bacterial adhesion to tissue culture cells: basis of a newdiagnostic test for enteropathogenic and enterohemorrhagic Escherichiacoli. Infect. Immun. 57:1290 –1298.

34. Kleta S, Steinrück H, Breves G, Duncker S, Laturnus C, Wieler LH,Schierack P. 2006. Detection and distribution of probiotic Escherichia coliNissle 1917 clones in swine herds in Germany. J. Appl. Microbiol. 101:1357–1366. http://dx.doi.org/10.1111/j.1365-2672.2006.03019.x.

35. Laemmli UK. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680 – 685. http://dx.doi.org/10.1038/227680a0.

36. Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedure andsome applications. Proc. Natl. Acad. Sci. U. S. A. 76:4350 – 4354. http://dx.doi.org/10.1073/pnas.76.9.4350.

37. Patzer SI, Baquero MR, Bravo D, Moreno F, Hantke K. 2003. The colicin G, Hand X determinants encode microcins M and H47, which might utilize the cat-echolate siderophore receptors FepA, Cir, Fiu and IroN. Microbiology 149:2557–2570. http://dx.doi.org/10.1099/mic.0.26396-0.

38. Duquesne S, Destoumieux-Garzon D, Peduzzi J, Rebuffat S. 2007.Microcins, gene-encoded antibacterial peptides from enterobacteria. Nat.Prod. Rep. 24:708 –734. http://dx.doi.org/10.1039/b516237h.

39. Ide T, Laarmann S, Greune L, Schillers H, Oberleithner H, SchmidtMA. 2001. Characterization of translocation pores inserted into plasmamembranes by type III-secreted Esp proteins of enteropathogenic Esche-richia coli. Cell. Microbiol. 3:669 – 679. http://dx.doi.org/10.1046/j.1462-5822.2001.00146.x.

40. Iizumi Y, Sagara H, Kabe Y, Azuma M, Kume K, Ogawa M, Nagai T,Gillespie PG, Sasakawa C, Handa H. 2007. The enteropathogenic E. colieffector EspB facilitates microvillus effacing and antiphagocytosis by in-hibiting myosin function. Cell Host. Microbe 2:383–392. http://dx.doi.org/10.1016/j.chom.2007.09.012.

41. Kenny B, Finlay BB. 1995. Protein secretion by enteropathogenic Esche-richia coli is essential for transducing signals to epithelial cells. Proc. Natl.Acad. Sci. U. S. A. 92:7991–7995. http://dx.doi.org/10.1073/pnas.92.17.7991.

42. Schierack P, Kleta S, Tedin K, Babila JT, Oswald S, Oelschlaeger TA,Hiemann R, Paetzold S, Wieler LH. 2011. E. coli Nissle 1917 affectssalmonella adhesion to porcine intestinal epithelial cells. PLoS One6:e14712. http://dx.doi.org/10.1371/journal.pone.0014712.

43. Prilassnig M, Wenisch C, Daxboeck F, Feierl G. 2007. Are probioticsdetectable in human feces after oral uptake by healthy volunteers? WienKlin. Wochenschr. 119:456 – 462. http://dx.doi.org/10.1007/s00508-007-0808-1.

44. Boudeau J, Barnich N, Darfeuille-Michaud A. 2001. Type 1 pili-mediated ad-

herence of Escherichia coli strain LF82 isolated from Crohn’s disease is in-volved in bacterial invasion of intestinal epithelial cells. Mol. Microbiol.39:1272–1284. http://dx.doi.org/10.1111/j.1365-2958.2001.02315.x.

45. Connell I, Agace W, Klemm P, Schembri M, Marild S, Svanborg C.1996. Type 1 fimbrial expression enhances Escherichia coli virulence forthe urinary tract. Proc. Natl. Acad. Sci. U. S. A. 93:9827–9832. http://dx.doi.org/10.1073/pnas.93.18.9827.

46. Blum G, Marre R, Hacker J. 1995. Properties of Escherichia colistrains of serotype O6. Infection 23:234 –236. http://dx.doi.org/10.1007/BF01781204.

47. Grozdanov L, Raasch C, Schulze J, Sonnenborn U, Gottschalk G,Hacker J, Dobrindt U. 2004. Analysis of the genome structure of thenonpathogenic probiotic Escherichia coli strain Nissle 1917. J. Bacteriol.186:5432–5441. http://dx.doi.org/10.1128/JB.186.16.5432-5441.2004.

48. Monteiro C, Saxena I, Wang X, Kader A, Bokranz W, Simm R, NoblesD, Chromek M, Brauner A, Brown RM, Jr, Romling U. 2009. Charac-terization of cellulose production in Escherichia coli Nissle 1917 and itsbiological consequences. Environ. Microbiol. 11:1105–1116. http://dx.doi.org/10.1111/j.1462-2920.2008.01840.x.

49. Lasaro MA, Salinger N, Zhang J, Wang Y, Zhong Z, Goulian M, Zhu J.2009. F1C fimbriae play an important role in biofilm formation and in-testinal colonization by the Escherichia coli commensal strain Nissle 1917.Appl. Environ. Microbiol. 75:246 –251. http://dx.doi.org/10.1128/AEM.01144-08.

50. Ramos HC, Rumbo M, Sirard JC. 2004. Bacterial flagellins: mediators ofpathogenicity and host immune responses in mucosa. Trends Microbiol.12:509 –517. http://dx.doi.org/10.1016/j.tim.2004.09.002.

51. Giron JA, Torres AG, Freer E, Kaper JB. 2002. The flagella of enteropathogenicEscherichia coli mediate adherence to epithelial cells. Mol. Microbiol. 44:361–379. http://dx.doi.org/10.1046/j.1365-2958.2002.02899.x.

52. Ukena SN, Westendorf AM, Hansen W, Rohde M, Geffers R, ColdeweyS, Suerbaum S, Buer J, Gunzer F. 2005. The host response to the probi-otic Escherichia coli strain Nissle 1917: specific up-regulation of the pro-inflammatory chemokine MCP-1. BMC Med. Genet. 6:43. http://dx.doi.org/10.1186/1471-2350-6-43.

53. Ofek I, Doyle RJ. 1994. Bacterial adhesion to cells and tissues. Chapman& Hall, New York, NY.

54. Collado MC, Meriluoto J, Salminen S. 2007. Role of commercial probioticstrains against human pathogen adhesion to intestinal mucus. Lett. Appl. Micro-biol. 45:454–460. http://dx.doi.org/10.1111/j.1472-765X.2007.02212.x.

55. Mack DR, Lebel S. 2004. Role of probiotics in the modulation of intestinalinfections and inflammation. Curr. Opin. Gastroenterol. 20:22–26. http://dx.doi.org/10.1097/00001574-200401000-00006.

56. Backhed F, Alsen B, Roche N, Angstrom J, von Euler A, Breimer ME, West-erlund-Wikstrom B, Teneberg S, Richter-Dahlfors A. 2002. Identification oftarget tissue glycosphingolipid receptors for uropathogenic, F1C-fimbriatedEscherichia coli and its role in mucosal inflammation. J. Biol. Chem. 277:18198–18205. http://dx.doi.org/10.1074/jbc.M111640200.

57. Khan AS, Kniep B, Oelschlaeger TA, Van Die I, Korhonen T, Hacker J.2000. Receptor structure for F1C fimbriae of uropathogenic Escherichiacoli. Infect. Immun. 68:3541–3547. http://dx.doi.org/10.1128/IAI.68.6.3541-3547.2000.

58. Jagannatha HM, Sharma UK, Ramaseshan T, Surolia A, Balganesh TS.1991. Identification of carbohydrate structures as receptors for localisedadherent enteropathogenic Escherichia coli. Microb. Pathog. 11:259 –268.http://dx.doi.org/10.1016/0882-4010(91)90030-E.

59. Fujiwara S, Hashiba H, Hirota T, Forstner JF. 1999. Purification andcharacterization of a novel protein produced by Bifidobacterium longumSBT2928 that inhibits the binding of enterotoxigenic Escherichia coliPb176 (CFA/II) to gangliotetraosylceramide. J. Appl. Microbiol. 86:615–621. http://dx.doi.org/10.1046/j.1365-2672.1999.00705.x.

60. Zhong SS, Zhang ZS, Wang JD, Lai ZS, Wang QY, Pan LJ, Ren YX.2004. Competitive inhibition of adherence of enterotoxigenic Escherichiacoli, enteropathogenic Escherichia coli and Clostridium difficile to intestinalepithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis1027. World J. Gastroenterol. 10:1630 –1633.

61. Jarvis KG, Giron JA, Jerse AE, McDaniel TK, Donnenberg MS, KaperJB. 1995. Enteropathogenic Escherichia coli contains a putative type IIIsecretion system necessary for the export of proteins involved in attachingand effacing lesion formation. Proc. Natl. Acad. Sci. U. S. A. 92:7996 –8000. http://dx.doi.org/10.1073/pnas.92.17.7996.

62. Deng W, Li Y, Hardwidge PR, Frey EA, Pfuetzner RA, Lee S, GruenheidS, Strynakda NC, Puente JL, Finlay BB. 2005. Regulation of type III

Effects of E. coli Nissle 1917 on aEPEC Infection

May 2014 Volume 82 Number 5 iai.asm.org 1811

on October 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

secretion hierarchy of translocators and effectors in attaching and effacingbacterial pathogens. Infect. Immun. 73:2135–2146. http://dx.doi.org/10.1128/IAI.73.4.2135-2146.2005.

63. Kimura K, Iwatsuki M, Nagai T, Matsumoto A, Takahashi Y, ShiomiK, Omura S, Abe A. 2011. A small-molecule inhibitor of the bacterialtype III secretion system protects against in vivo infection with Citro-bacter rodentium. J. Antibiot. 64:197–203. http://dx.doi.org/10.1038/ja.2010.155.

64. Blattner FR, GPlunkett, III, Bloch CA, Perna NT, Burland V, Riley M,Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, DavisNW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y. 1997. The

complete genome sequence of Escherichia coli K-12. Science 277:1453–1462. http://dx.doi.org/10.1126/science.277.5331.1453.

65. Zingler G, Ott M, Blum G, Falkenhagen U, Naumann G, Sokolowska-Kohler W, Hacker J. 1992. Clonal analysis of Escherichia coli serotype O6strains from urinary tract infections. Microb. Pathog. 12:299 –310. http://dx.doi.org/10.1016/0882-4010(92)90048-S.

66. Troge A, Scheppach W, Schroeder BO, Rund SA, Heuner K, WehkampJ, Stange EF, Oelschlaeger TA. 2012. More than a marine propeller—theflagellum of the probiotic Escherichia coli strain Nissle 1917 is the majoradhesin mediating binding to human mucus. Int. J. Med. Microbiol. 302:304 –314. http://dx.doi.org/10.1016/j.ijmm.2012.09.004.

Kleta et al.

1812 iai.asm.org Infection and Immunity

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