Bacteroides vulgatus protects against escherichia coli-induced colitis in gnotobiotic interleukin-2-deficient mice

GASTROENTEROLOGY 2003;125:162–177

Bacteroides vulgatus Protects Against Escherichia coli–Induced Colitis in Gnotobiotic Interleukin-2–Deficient Mice MARC WAIDMANN,* OLIVER BECHTOLD,* JULIA–STEFANIE FRICK,* HANS–ANTON LEHR,‡ ¨ REN SCHUBERT,§ ULRICH DOBRINDT,㛳 JU ¨ RGEN LO ¨ FFLER,¶ ERWIN BOHN,* and SO INGO B. AUTENRIETH* *Institut fu¨r Medizinische Mikrobiologie und Krankenhaushygiene, Eberhard Karls-Universita¨t, Tu¨bingen, Germany; ‡Institut fu¨r Pathologie, Johannes Gutenberg-Universita¨t, Mainz, Germany; §Max von Pettenkofer-Institut, Ludwig Maximilians-Universita¨t, Mu¨nchen, Germany; 㛳Institut fu¨r Molekulare Infektionsbiologie, Bayerische Julius Maximilians-Universita¨t, Wu¨rzburg, Germany; ¶Medizinische Klinik, Abteilung II, Eberhard Karls-Universita¨t, Tu¨bingen, Germany

Background & Aims: The microflora plays a crucial role in inflammatory bowel diseases (IBDs). Specific pathogen-free (SPF), but not germ-free, interleukin (IL)-2– deficient (IL-2ⴚ/ⴚ) mice develop colitis. The colitogenicity of commensal bacteria was determined. Methods: Gnotobiotic IL-2ⴚ/ⴚ and IL-2ⴙ/ⴙ mice were colonized with Escherichia coli mpk, Bacteroides vulgatus mpk, or both bacterial strains, or with E. coli strain Nissle 1917. DNA arrays were used to characterize E. coli mpk. Colitis was analyzed by histology and real-time reverse-transcription polymerase chain reaction (RT-PCR) for interferon (IFN)-␥, tumor necrosis factor (TNF)-␣, IL-10, and CD14 messenger RNA (mRNA) expression. Bacterial numbers in feces and bacterial localization in the colon were determined by culture and fluorescence in situ hybridization (FISH). Results: IL-2ⴚ/ⴚ but not IL-2ⴙ/ⴙ mice monocolonized with E. coli mpk developed colitis, whereas mono-association with B. vulgatus mpk, or E. coli Nissle, or co-colonization with E. coli mpk and B. vulgatus mpk, did not induce colitis. DNA array experiments and cellular studies revealed that E. coli mpk is a nonpathogenic strain. FISH and culture methods revealed that the anticolitogenic effect of B. vulgatus mpk on E. coli mpk cannot be explained by a significant reduction in numbers of E. coli in the colon. E. coli mpk–induced colitis was associated with increased IFN-␥, TNF-␣, CD14, and IL-10 mRNA expression in the colon. Conclusions: In IL-2ⴚ/ⴚ mice, B. vulgatus mpk protects against E. coli mpk–triggered colitis by an unknown mechanism. E. coli Nissle does not induce colitis. Various bacterial species common to the microflora differ in their ability to trigger IBD.

nflammatory bowel diseases (IBDs) such as ulcerative colitis and Crohn’s disease are chronic immunologic disorders that afford striking disability to the affected individuals. Until recently, research on this group of diseases has been limited owing to the lack of suitable animal models. In the past decade, various models have

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been presented and have reinforced research activity in this field.1 The current pathophysiologic concept of IBD proposes a multifactorial event with environmental, immunologic, and genetic contributory factors.2,3 Among the environmental factors are the bacterial flora that clearly play a central role in the development of IBD. This is underlined by the therapeutic benefit of several antibiotics, especially in Crohn’s disease,4,5 and also by the finding that animals prone to develop IBD, such as interleukin-2– deficient (IL-2⫺/⫺),6,7 IL10⫺/⫺8 and ␣␤T-cell receptor⫺/⫺ mice,9 as well as HLA-B27/␤2m transgenic rats,10 remain healthy when maintained in a germ-free environment. An infection hypothesis has been proposed to explain IBD pathogenesis,11,12 however, despite great efforts, no convincing evidence has been presented to sustain the hypothesis. Mycobacterium avium subsp. paratuberculosis notably has been implicated in the disease development over the years,13–15 although other findings were controversial.16 –18 Therefore, it currently is assumed that the physiologic flora of the intestine, rather than nonindigenous pathogenic bacteria, are the main trigger of an abnormal immune response in the pathogenesis of IBD.10,19 –23 The normal intestinal microflora comprises more than 400 bacterial species of which only a few are well characterized.24 Several species are beneficial to the host (e.g., probiotic Lactobacillus spp. and Bifidobacterium spp.) Abbreviations used in this paper: CFU, colony-forming unit; FISH, fluorescence in situ hybridization; FP, forward primer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase (phosphorylating); IFN-␥, interferon ␥; IL, interleukin; KC, cytokine-induced neutrophil chemoattractant (CXCL1); ORF, open reading frame; PCR, polymerase chain reaction; RP, reverse primer; RT, reverse transcription; SPF, specific pathogen-free; TNF, tumor necrosis factor. © 2003 by the American Gastroenterological Association 0016-5085/03/$30.00 doi:10.1016/S0016-5085(03)00672-3

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whereas others, such as Clostridium difficile, are pathogenic in certain clinical settings.25,26 Beneficial functions of the indigenous microflora include the stimulation of secretory immunoglobulin A,27,28 competition for adhesion sites with enteropathogenic bacteria,29 and the secretion of antimicrobial compounds.30 Intestinal bacterial strains have been considered as treatment options for IBD. For example, Escherichia coli strain Nissle 1917 is effective in maintaining remission in ulcerative colitis.31,32 To date, little is known about the molecular basis of this therapeutic effect.33 Evidence from animal experiments is contradictory, with some reports revealing E. coli to be well tolerated in HLA-B27/␤2m transgenic rats and IL-10⫺/⫺ mice,34,35 whereas other studies performed in the transgenic rats showed a dramatic increase in cecal E. coli numbers in severely diseased animals.36 Adhesion to epithelial cells is considered to be a prerequisite for pathogenic enteric bacteria. In vitro data have suggested that E. coli strains from patients with IBD adhered more frequently when compared with E. coli strains isolated from healthy subjects,37–39 however, this finding was not confirmed by others.40 It also was shown that perinuclear antineutrophil cytoplasmic autoantibodies, a serologic marker of ulcerative colitis, are reactive with the E. coli outer membrane porin OmpC.41 The purpose of this study was to investigate the colitis-inducing potential of various bacterial strains indigenous to the intestinal flora of mice compared with the well-defined E. coli Nissle. Gnotobiotic IL-2⫺/⫺ mice were colonized exclusively with one strain of E. coli or B. vulgatus isolated from the fecal flora of specific pathogen-free (SPF) IL-2⫺/⫺ mice, or in combination with both strains. Alternatively, mice were monocolonized with E. coli Nissle.

Materials and Methods Animals Heterozygous mice from a mixed C57BL/6 ⫻ 129/Ola background were crossed to obtain IL-2⫺/⫺ and wild-type (IL-2⫹/⫹) mice. They were genotyped using the tip of their tails at 4 weeks of age as previously described.6 Mice were bred either under SPF conditions in a barrier sustained facility, or under gnotobiotic conditions in isolators at the University of Ulm, Germany. Gnotobiotic mice were maintained in a germfree environment or were colonized exclusively with 1 or 2 bacterial strains previously isolated from IL-2⫺/⫺ mice of the SPF colony with flourishing colitis, or monocolonized with the E. coli strain Nissle 1917 (kindly provided by Dr. Sonnenborn, Ardeypharm, Germany). Initial colonization of breeding couples was performed by feeding them a suspension of the respective bacterial strain(s). Successful colonization was controlled and monitored as detailed later, and was first confirmed

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several days after the introduction of the bacterial suspension into the isolators. The isolators with the mono- or co-colonized mice were kept for 2 years. The offspring were colonized after birth by contamination via the parents or the feces-contaminated environment. The gnotobiotic state was controlled weekly and at the time of necropsy, and this involved culturing for aerobic and anaerobic bacteria, gram stain examinations of feces and intestinal contents, as well as broad-range eubacterial 16S ribosomal DNA polymerase chain reaction (PCR) of stool samples from mice, as detailed later. The presence of Helicobacter spp. therefore was excluded.

Culture and Biochemical Typing of Intestinal Bacterial Species Bacteria from fecal and colon samples were grown on Columbia, MacConkey II, and Schaedler agar (Becton Dickinson, Heidelberg, Germany) at 37°C, in aerobic and anaerobic conditions, respectively, after which gram stains were performed. Biochemical typing was performed using the API-20E and API Rapid ID kits (BioMe´ rieux, Marcy l’E´ toile, France) according to the manufacturer’s instructions.

16S Ribosomal DNA Sequencing Bacterial DNA was extracted according to standard laboratory procedures. A hypervariable region of 16S ribosomal DNA was amplified by PCR using forward primers (FP) and reverse primers (RP) specific for eubacterial sequences (FP, 5⬘-GAG TAC CAG GGT ATC TAA TCC-3⬘; RP, 5⬘-AGA GTT TGA TCC TGG CTC AG-3⬘), and resulting PCR amplicons were sequenced with an ABIprism 377 DNA Sequencer (Applied Biosystems, Weiterstaadt, Germany). Homology searches were performed using Basic Local Alignment Search Tool, National Center for Biotechnology Information, National Institutes of Health (Bethesda, MD), and European Molecular Biology Laboratory (Heidelberg, Germany) databases, and sequence homologies greater than 95% were used to identify the bacterial species.

Characterization of E. coli Virulence Factors E. coli mpk (O not typable: H8) isolated from SPF IL-2⫺/⫺ mice was tested by PCR for the presence of different pathogenicity factors characteristic for enteropathogenic, enterohemorrhagic, enteroinvasive, enterotoxic, and enteroaggregative E. coli. Genomic DNA (50 pg) or 1 ␮L of cells (104 to 105 colony-forming units [CFUs]) from E. coli mpk was used as a template in PCR with oligonucleotides (Metabion, Munich, Germany; Roth, Karlsruhe, Germany).42– 46 The amplification mixtures consisted of either Taq DNA Polymerase (Applied Biosystems) or Pfu DNA Polymerase (Stratagene, Heidelberg, Germany), 200 ␮mol/L deoxynucleoside triphosphates, 1.5 ␮mol/L MgCl2 , and 0.2 ␮mol/L primers. PCR was performed in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) (Table 1). Products from these reactions were resolved by agarose gel electrophoresis and ethidium bromide staining. To group the E. coli strain phylogenetically,

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Table 1. PCR Primers and Conditions for Characterization of E. coli mpk PCR conditions Primer designation

Nucleotide sequence of primers (5⬘-3⬘)

EHEC-1 (FP) (RP) EHEC-2 (FP) (RP) EHEC-3 (FP) (RP) EIEC-1 (FP) (RP) EIEC-2 (FP) (RP) EPEC-1 (FP) (RP) EPEC-2 (FP) (RP) ETEC-1 (FP) (RP) ETEC-2 (FP) (RP) EaggEC (FP) (RP) ChuA.1 ChuA.2 YjaA.1 YjaA.2 TspE4C2.1 TspE4C2.2

CCC GGA TCC ATG AAA AAA ACA TTA TTA ATA GC CCC GAA TTC AGC TAT TCT GAG TCA ACG ATG AAG AAG ATG TTT ATG TCA GTC ATT ATT AAA CTG GGT GCA GCA GAA AAA GTT GTA G TCT CGC CTG ATA GTG TTT GGT A 5⬘GTT CCT TGA CCG CCT TTC CGA TAC CGT GCC GGT CAG CCA CCC TCT GAG AGT AC CTG GTA GGT ATG GTG AGG CCA GGC CAA CAA TTA TTT CAG GGT AAA AGA AAG ATG ATA A TAT GGG GAC CAT GTA TTA TCA CCC GAA TTC GGC ACA AGC ATA AGC CCC GGA TCC GTC TCG CCA GTA TTC G GCG TTA CTA TCC TCT CTA TG ATT GGG GGT TTT ATT ATT CC TCC CTC AGG ATG CTA AAC GCA ACA GGT ACA TAC GTT CTG GCG AAA GAC TGT ATC AT CAA TGT ATA GAA ATC CGC TGT T GAC GAA CCA ACG GTC AGG AT TGC CGC CAG TAC CAA AGA CA TGA AGT GTC AGG AGA CGC TG ATG GAG AAT GCG TTC CTC AAC GAG TAA TGT CGG GGC ATT CA CGC GCC AAC AAA GTA TTA CG

Positive controls

Annealing

Extension

Length of PCR product (bp)

stxI

A

52°C, 60 s

72°C, 40 s

285

Schmidt et al.45

stx-II

A

52°C, 60 s

72°C, 40 s

260

Gunzer et al.42

hlyAa

A

57°C, 60 s

72°C, 90 s

1600

ipaH

B

68°C, 60 s

72°C, 45 s

603

ial

B

50°C, 60 s

72°C, 45 s

320

Karch et al. (unpublished data) Karch et al. (unpublished data) Sethabutr et al.43

EAF-locus

C

60°C, 60 s

72°C, 45 s

397

Franke et al.44

eaeA

C

52°C, 60 s

72°C, 60 s

800

Schmidt et al.45

LT

D

49°C, 60 s

72°C, 40 s

313

ST1

D

48°C, 60 s

72°C, 40 s

244

pCVD432

E

53°C, 60 s

72°C, 60 s

630

Karch et al. (unpublished data) Karch et al. (unpublished data) Schmidt et al.46

chuA

ECOR 55

55°C, 30 s

72°C, 30 s

279

Clermont et al.47

yjaA

ECOR 55

55°C, 30 s

72°C, 30 s

211

Clermont et al.47

TspE4-C2

ECOR 58

55°C, 30 s

72°C, 30 s

152

Clermont et al.47

Target

Reference

NOTE. Denaturing conditions for PCR cycle were usually 94°C, 30 seconds with exception for EIEC-1 (60 s), and EPEC-1 and EaggEC (40 s). A, E. coli O157:H7 strain EDL933; B, E. coli E12860 clinical isolate; C, EPEC strain 12-1 and EPEC strain E2348/69 (Nataro); D, E. coli H 166/82 clinical isolate; E, plasmid pCVD432 and E. coli strain 17-2. ahlyA specfic for EHEC.

triplex PCR was performed targeting the genes chuA, yjaA, and TspE4-C2 (Table 1).47 In addition, total genomic DNA from E. coli mpk was used to probe E. coli DNA arrays.48 This array contains probes specific for the majority of open reading frames (ORFs) located on 5 pathogenicity islands of uropathogenic E. coli strain 536 as well as of all other extraintestinal pathogenic E. coli–specific virulence genes. Probes specific for several typical virulence-associated genes of intestinal pathogenic E. coli and Shigella have been included as well. Two ␮g of total genomic DNA was used as a template for direct incorporation of [33P]-dATP (Amersham Pharmacia, Freiburg, Germany) by a randomly primed polymerization reaction using 0.75 ␮g random hexamer primers (New England Biolabs, Frankfurt (Main), Germany) and 10 U Klenow fragment of the DNA polymerase I (New England Biolabs) according to the manufacturer’s recommendations. Unincorporated nucleotides were removed with Microspin GS 50 spin columns (Amersham Pharmacia). Before hybridization, the DNA arrays were rinsed in 2⫻ sodium chloride sodium phosphate EDTA (0.3 mol/L NaCl, 20 mmol/L NaH2PO4, 2.5 mmol/L EDTA, pH 7.4) solution and subsequently prehybridized for 3 hours at 65°C in 5 mL hybridization solution (5⫻ sodium chloride sodium phosphate EDTA, 2% sodium dodecyl sulfate, 1⫻ Denhardt’s, 100 ␮g/mL sheared, denatured herring sperm DNA). After the addition of the probe denatured in 3 mL of hybridization solution, the arrays were incubated for 12–18 hours at 65°C. After hybridization, the blots

were washed twice with wash solution (0.5⫻ SSPE, 0.2% sodium dodecyl sulfate) for 2–3 minutes at room temperature followed by 3 wash steps for 20 minutes at 65°C. Washed filters were air dried and exposed overnight to a phosphoimager screen (super resolution type) before scanning on a Typhoon 8600 Variable Mode Imager (Amersham Pharmacia). DNA arrays were hybridized in 4 different experiments using independently labeled DNA probes. The scanned arrays were analyzed with ArrayVision software (Imaging Research, St. Catharine’s, Canada) followed by visual inspection. Calculation of normalized intensity values of the individual spots was performed using the overall-spot-normalization function of ArrayVision. Background values were measured in the 4 corners of every spot. The mean of the normalized intensity values of the duplicate spots of each gene was used for further analysis. To avoid extreme intensity ratios for genes close to or below the detection limit, signal intensity values corresponding to a signal to noise ratio less than 1.0 were scaled up to a value corresponding to a signal to noise ratio equal to 1.0. ORFs were recorded as lacking/not detectable if the signal to noise ratio was below 1.0 in at least 3 of the 4 hybridization experiments.

Quantification of Fecal Bacteria The animals were killed by CO2 asphyxiation and their colon was removed aseptically. The most distal fecal pellet was transferred into 1 mL Luria-Bertani medium (LB; Oxoid Ltd.,

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Basingstoke, UK), vortexed for 15 minutes at room temperature, and allowed to settle. Ten-fold serial dilutions were performed in phosphate-buffered saline (PBS) and 100 ␮L of the appropriate dilutions were spread onto agar plates to give ⬃50 –100 colonies per plate, from which the CFU/g feces was calculated. Bacterial identity was confirmed as described earlier.

Histologic Staining and Evaluation of Sections Rings of transverse colon were fixed in 4% buffered formaldehyde and embedded in paraffin for histologic analysis. Sections (5 ␮m) were stained with H&E. Grading of colon inflammation was determined as described previously49 in a blinded fashion by one pathologist: rare inflammatory cells in the lamina propria (0); increased numbers of inflammatory cells (1); confluence of inflammatory cells extending into the submucosa (2); transmural extension of the inflammatory cell infiltrate (3); nil mucosal damage (0); discrete lymphoepithelial lesions (1); surface mucosal erosion (2); and widespread mucosal ulceration and extension through deeper bowel wall structures (3).

Fluorescence In Situ Hybridization At the time of necropsy, a portion of the distal part from the colon of each animal was fixed in 4% paraformaldehyde (4°C, overnight), washed with PBS, snap-frozen in Tissue-Tek (Sakura Finetek Europe BV, Zoeterwoude, The Netherlands), and stored at ⫺80°C. Sections (5 ␮m) were cut on a kryotom and air dried on glass slides at room temperature. Each section was stained by incubating the slides for 90 minutes at 46°C in a humidified chamber with 50 ␮L hybridization mix (0.9 mol/L NaCl, 0.02 mol/L TRIS-HCl, 20% formamide, 0.01% Na–sodium dodecyl sulfate, and 5 nmol/L specific oligonucleotide probe). The detection of Enterobacteriaceae used a labeled oligonucleotide (Cy3, Ent-Cy3; fluorescein isothiocyanate, Ent–fluorescein isothiocyanate) with the sequence 5⬘-CCC CCW CTT TGG TCT TGC-3⬘ as a 16S ribosomal RNA targeted probe,50 and B. vulgatus was detected specifically by hybridization with the labeled oligonucleotide 5⬘-TCC ATA CCC GAC TTT ATT CCT T-Cy3-3⬘ (B. vulgatus-Cy3) (accession number: AB050111). This latter probe was tested for specificity using different Bacteroides species such as B. vulgatus, B. urealyticus, B. distasonis, and B. fragilis, and also Enterobacteriaceae, Prevotella spp., and Porphyromonas spp. Sections stained without probe were used as negative controls, and suspensions of the respective bacteria were hybridized for use as positive controls. Thereafter, slides were washed with buffer (0.225 mol/L NaCl, 0.02 mol/L TRIS-HCl, 0.01% Na–sodium dodecyl sulfate) at 46°C for 15 minutes, rinsed with PBS, and counterstained with 1 ␮g/mL di-amino-phenylindol. Sections were examined under an epifluorescence microscope and images were captured using a CCD camera (Spot; Visitron Systems, Heidelberg, Germany).

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Determination of Messenger RNA Expression in Intestinal Tissue by Semiquantitative Real-Time ReverseTranscription PCR The colon including the cecum was freed grossly from the containing feces, cut transversally into pieces, transferred into 3 mL cold TRIZOL reagent (Invitrogen Life Technologies, Karlsruhe, Germany), and homogenized by repeated passage through a 15-gauge needle and finally snap-frozen in liquid nitrogen. RNA isolation was performed according to the manufacturer’s instructions. Extracted RNA was dissolved in water containing 0.1% diethyl-pyrocarbonate. For reverse transcription, 4 ␮g of RNA was mixed with 0.5 ␮g of oligo(dT)12–18 primers (Invitrogen Life Technologies), and diethyl-pyrocarbonate–treated water was added to a final volume of 10 ␮L, followed by incubation at 65°C for 10 minutes. After adding 10 ␮L of a solution containing 5⫻ first-strand buffer, 20 mmol/L dithiothreitol, 200 U SuperScript II (Invitrogen Life Technologies), 40 U RNaseOut (Invitrogen Life Technologies), and 2 mmol/L deoxynucleoside triphosphate (Roth), the mixture was incubated at 37°C for 60 minutes. Finally, the samples were heated at 90°C for 5 minutes, diluted 1:10 with diethyl-pyrocarbonate–treated water, and stored at ⫺20°C until further use. Quantification of glyceraldehydes-3-phosphate dehydrogenase (phosphorylating) (GAPDH), interferon (IFN)-␥, tumor necrosis factor (TNF)-␣, IL-10, and CD14 messenger RNA (mRNA) was performed by real-time reverse-transcription (RT)-PCR using a LightCycler System (Roche Diagnostics, Mannheim, Germany). CD14 and IL-10 mRNA was quantified by first generating standard plasmid complementary DNA (cDNA): 5 ␮L of murine splenocyte cDNA was added to 20 ␮L of a PCR reaction mixture containing 1 U of Taq DNA polymerase, 2.5 ␮L 10⫻ PCR buffer, 2.5 mmol/L MgCl2 (Roche Diagnostics), 0.2 mmol/L deoxynucleoside triphosphate (Roth), and 0.625 ␮mol/L of FPs and RPs (CD14 FP: 5⬘-TCTACCGACCATGGAGCGT-3⬘, RP: 5⬘-CCGCCGTACAATTCCACA-3⬘; IL-10 FP: 5⬘-AGCTGGACAACATACTGCTAAC-3⬘, RP: 5⬘-CTCCTTATTTTCACAGGGGAGAA3⬘; TIB-Molbiol, Berlin, Germany). Conventional PCR was performed with the following cycle conditions for CD14 and IL-10 (modifications in IL-10 PCR are indicated in brackets): 29 cycles of 1 minute (30 s) denaturation at 94°C, 45 seconds (30 s) annealing at 58°C (62°C), 1 minute (30 s) extension at 72°C, and a final extension step for 5 minutes at 72°C. The PCR products were separated on a 1.5% agarose gel and visualized using ethidium bromide staining, and the sizes were compared against a GeneRuler DNA Ladder Mix (MBI Fermentas, St. Leon-Rot, Germany). The correct PCR product was cloned using the TOPO TA Cloning Kit (Invitrogen Life Technologies) and plasmids were purified using the Qiagen Miniprep reagent set (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The concentration of purified plasmid DNA was measured in a spectrophotometer, copy numbers were calculated, and dilutions of the plasmid cDNA

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were used as standards in a LightCycler RT-PCR for detecting IL-10 and CD14 mRNA. Real-time RT-PCR was performed in glass capillaries in a volume of 20 ␮L with a LightCycler System. mRNA for GAPDH, IFN-␥, and TNF-␣ was quantified using LightCycler Primer Sets (including standards; Search-LC, Heidelberg, Germany) according to the manufacturer’s instructions. The reaction master mix for the quantification of CD14 mRNA contained 2 ␮L cDNA, 1 ␮L FastStart DNA Master SYBR Green I (Roche Diagnostics), 4 mmol/L MgCl2 , and 0.5 ␮mol/L of FPs and RPs (see earlier). The amplification conditions consisted of an initial 10-minute denaturation step at 95°C, followed by 45 cycles of denaturation at 95°C for 10 seconds, annealing at 68°C decreasing to 58°C for 10 seconds (step size, 0.5°C; step delay, 1 cycle) and extension at 72°C for 16 seconds. For IL-10 LightCycler RT-PCR the reaction master mix contained 10 ␮L cDNA, 1 ␮L FastStart DNA Master Hybridization Probes (Roche Diagnostics), 3.6 mmol/L MgCl2 , 2.5 pmol of FPs and RPs (see earlier), and 3 pmol FRET hybridization probes (fluorescein-labeled 5⬘-GGATCATTTTCCGATAAGGCTTGGCA X, LC-Red 640 labeled 5⬘-CCCAAGTAACCTTAAAGTCCCTGCATT p; TIB-Molbiol). Cycle conditions were 9 minutes at 95°C followed by 45 cycles of 3 seconds at 95°C, 15 seconds at 54°C, and 25 seconds at 72°C. Dilutions (100-fold) of plasmid-cloned PCR products were performed from 108 copies to 102 copies and were used as standards. The standards and the samples were amplified simultaneously using the same reaction master mix. Data were normalized by dividing the copy number of IFN-␥, TNF-␣, IL-10, or CD14, respectively, by the copy number of the housekeeping gene GAPDH. The specificity of the amplified PCR product was confirmed by performing a melting curve analysis.

Epithelial Cell Infection Protocols The E. coli strains were grown in LB medium under aerobic conditions, and B. vulgatus was cultured in brain heart infusion broth (Merck Eurolab, Bruchsal, Germany) under anaerobic conditions. For infection experiments, overnight cultures of E. coli strains were diluted 1:10 in LB and incubated for a further 3 hours. These cultures and overnight cultures of B. vulgatus were harvested by centrifugation and washed 3 times with PBS. After determination of the optical density at 600 nm, the concentrations of cells were calculated and appropriate dilutions of the bacteria in PBS were used for the in vitro assays. The murine intestinal epithelial cell line MODE-K51 was maintained as adherent monolayers in a humidified 5% CO2 atmosphere at 37°C in Dulbecco’s modified Eagle’s medium (Invitrogen Life Technologies) supplemented with 10% heatinactivated fetal bovine serum (Sigma-Aldrich, Taufkirchen, Germany), 4 mmol/L L-glutamine (Invitrogen Life Technologies), 100 U/mL penicillin, and 100 ␮g/mL streptomycin (Invitrogen Life Technologies). For in vitro assays approximately 105 MODE-K cells per well were seeded in 24-well tissue culture plates (Greiner Bio-One, Frickenhausen, Ger-

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many) and cultured overnight. Two hours before infection, the wells were washed 2 times with PBS and Dulbecco’s modified Eagle’s medium without antibiotics was added. Cells were infected at a bacterium-to-cell ratio of 100 (multiplicity of infection 100) or as indicated by centrifuging the bacteria to the MODE-K monolayers. For the adhesion assay, the medium was removed after 30 minutes and the cells were washed 3 times with PBS. Cells with adherent bacteria were lysed by adding 1 mL of ice-cold sterile distilled water and bacterial numbers were detected by plating serial dilutions on agar plates. Adhesion was expressed as the percentage of the initial inoculums recovered in lysates. To analyze the cytokine-induced neutrophil chemoattractant, CXCL1 (KC) secretion, MODE-K cells and bacteria were incubated for 1 hour. After removal of the medium, the cells were washed 3 times with PBS and incubated for a further 3 hours in Dulbecco’s modified Eagle’s medium with 100 ␮g/mL gentamicin (Sigma-Aldrich). Culture supernatants were harvested and stored at ⫺20°C. To determine the cytotoxic potential of the bacteria, MODE-K cells were grown on glass cover slips, and the infection protocol followed the procedure for induction of KC. Then, the cells on cover slips were washed, fixed with ice cold methanol, stained with Giemsa, and mounted on microscope slides.

Determination of KC Production by EnzymeLinked Immunosorbent Assay The amount of KC secreted by MODE-K cells into the supernatant was determined by enzyme-linked immunosorbent assay. Microtiter plates (Nunc, Wiesbaden, Germany) were coated overnight with a rabbit antimurine KC polyclonal antibody (PeproTech, Rocky Hill, NY). After nonspecific binding sites were blocked, the wells were incubated successively, separated by washing steps, with the supernatants, a biotinylated goat antimouse KC polyclonal antibody (R&D Systems, Wiesbaden-Nordenstadt, Germany), and a streptavidin-biotin-horseradish-peroxidase complex (StreptABComplex/HRP kit; DAKO Diagnostika, Hamburg, Germany). Then, a tetramethylbenzidine/hydrogen peroxide substrate solution (TMB Substrat Reagent Set; Becton Dickinson) was added to the wells. Within 30 minutes the reaction was terminated by adding 1 mol/L H2SO4 , and the optical density was determined at wavelengths of 450 and 570 nm. KC concentrations were calculated from the straight-line portion of standard curves with recombinant murine KC (PeproTech).

Statistics The unpaired, 2-tailed Student t test was used (JMP Version 4.0; SAS Institute Inc., Cary, NC) to compare differences between mean CFU and semiquantitative real-time RTPCR data. The nonparametric Wilcoxon test was used to determine differences in histologic scores. Survival rates were analyzed using Kaplan–Meier graphs and the log-rank test. The level of significance was set at less than 0.05.

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Results Monocolonization With E. coli mpk but not B. vulgatus mpk Results in Colitis in IL-2– Deficient Mice, Which Can Be Prevented by Co-colonization With B. vulgatus mpk IL-2⫺/⫺ mice kept under SPF conditions die from severe colitis between the age of 15 and 25 weeks,6 whereas IL-2⫺/⫺ mice kept under germ-free conditions develop only mild focal intestinal inflammation but no severe colitis.7 HLA-B27/␤2m transgenic rats kept in a germ-free environment do not develop colitis at all.34 In these rats, monocolonization with B. vulgatus, but not E. coli, induces colitis. Based on these observations we wanted to investigate whether colonization with E. coli and/or B. vulgatus might also have an effect on colitis in IL-2⫺/⫺ mice. For this purpose, E. coli and B. vulgatus were isolated from SPF IL-2⫺/⫺ mice, and designated E. coli mpk and B. vulgatus mpk, respectively. Germ-free mice were monocolonized with either E. coli mpk or B. vulgatus mpk, or they were co-colonized with both bacteria. Sadlack et al.6 reported that 50% of IL-2⫺/⫺ mice kept under SPF conditions died from severe anemia in the first 4 –9 weeks of life, and of the remaining animals, 100% developed colitis and died between 10 –25 weeks of age. To determine the influence of the intestinal microflora on the survival of germ-free and gnotobiotic IL-2⫺/⫺ mice, the life span of mice was monitored for a period of 33 weeks after birth. Within the first 20 weeks, 20% of IL-2⫺/⫺ mice kept under germ-free conditions died, and approximately 50% survived longer than 33 weeks (Figure 1). However, 80% (n ⫽ 8 of 10) of the B. vulgatus mpk mono-associated mice survived the entire observation period of 33 weeks, suggesting that B. vulgatus mpk monocolonization improved survival of IL-2⫺/⫺ compared with germ-free IL-2⫺/⫺ mice. In contrast, survival of E. coli mpk mono-associated IL-2⫺/⫺ mice was reduced significantly when compared with germ-free B. vulgatus mpk mono-associated and E. coli mpk/B. vulgatus mpk cocolonized IL-2⫺/⫺ mice (P ⬍ 0.01). Fifty percent of E. coli mpk mono-associated mice died within the first 22 weeks and only 18% (n ⫽ 6 of 34) reached the age of 33 weeks. The survival rate of IL-2⫺/⫺ mice co-colonized with B. vulgatus mpk and E. coli mpk was not significantly different when compared with B. vulgatus mpk monocolonized mice. In comparison with E. coli mpk monocolonized IL-2⫺/⫺ mice, the life span of co-colonized mice was extended by approximately 10 weeks. In addition, mice were monocolonized with E. coli Nissle. The results in Figure 1 showed that IL-2⫺/⫺ mice colonized with E. coli Nissle reached a comparable sur-

Figure 1. Influence of the intestinal microflora on the survival rate of IL-2⫺/⫺ mice. Mice were kept under germ-free conditions ({), or were colonized with E. coli mpk (■), B. vulgatus mpk (‚), both E. coli mpk and B. vulgatus mpk (䊐), or E. coli Nissle (E). Survival rate was monitored between 5 and 33 weeks of age (n ⫽ at least 11 for each group). #P ⬍ 0.01 compared with all other groups (log-rank test).

vival rate as germ-free or co-colonized mice. These data showed that the survival of IL-2⫺/⫺ mice was strongly influenced by the microflora. To study the development of colitis, histologic analyses were performed on colon specimens (Figures 2 and 3). Histologic examination of the colon from germ-free IL-2⫺/⫺ and IL-2⫹/⫹ mice, at the age of 20 and 33 weeks, respectively, showed no pathologic changes and no signs of colitis. As indicated in Figure 3, the histologic score of the colon of all germ-free IL-2⫺/⫺ mice was less than 0.5. In contrast, 75% (13 of 17 mice) of E. coli mpk mono-associated IL-2⫺/⫺, but not IL-2⫹/⫹ mice, showed significant signs of colitis that was most pronounced in the distal part of the colon. In greater than 50% of these animals the histologic score for tissue alterations associated with colitis ranged from 2 to 3 (Figure 3). As shown in Figure 2B, we observed pronounced inflammatory cell infiltrations in the lamina propria, focal neutrophil accumulation, and occasional formation of crypt abscesses in E. coli mono-associated IL-2⫺/⫺ mice. The maximum injury we observed was limited to lymphoepithelial lesions. However, despite the pronounced inflammatory cell infiltrate, the surface epithelium remained intact in all cases, with no formation of focal erosions or ulcerations. Only 1 of 9 IL-2⫺/⫺ mice mono-associated with B. vulgatus mpk and none of the E. coli Nissle mono-associated mice developed significant colitis. Similarly, we observed signs of colitis in less than 10% of IL-2⫺/⫺ mice that were colonized with both E. coli mpk and B. vulgatus mpk. E. coli mpk, B. vulgatus mpk, and E. coli mpk/B. vulgatus mpk colonized IL-2⫹/⫹ mice were examined for colitis and histologic scores were determined for control purposes. In 93% (14

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mice with B. vulgatus mpk did not lead to significant tissue changes. Therefore, E. coli mpk triggered the development of colitis whereas B. vulgatus mpk protected against E. coli mpk–triggered colitis. In contrast to E. coli mpk, monocolonization of IL-2⫺/⫺ mice with E. coli Nissle did not cause any signs of colitis. Molecular Characterization of E. coli mpk Strain To assign E. coli mpk to one of the established enteric pathotypes including enteroinvasive, enterohemorrhagic, enteropathogenic, enterotoxic, and enteroaggregative, a PCR survey was conducted using defined pathotype-specific primers for the detection of stxI, stxII, hlyA, ipaH, ial, EAF-locus, eaeA, LT, ST1, pCVD432, chuA, yjaA, and TspE4-C2 as previously described (Table 1).42– 46 Moreover, DNA array experiments using an E. coli DNA array were performed.48 The probes of the array enabled detection of genes encoding typical toxins, siderophores, fimbrial, and nonfimbrial adhesins, as well as other genes that have been described to be involved in virulence of extraintestinal pathogenic E. coli and/or intestinal pathogenic E. coli, or that are present on pathogenicity islands and other mobile genetic elements frequently present in pathogenic E. coli variants. The results revealed that only 8% of probes specific for virulenceassociated or pathogenicity island– encoded genes of extraintestinal pathogenic E. coli or homologues hybridized with genomic DNA of E. coli mpk (data not shown). The corresponding group of genes included the type 1 fimbriae-encoding operon, some ORFs of the E. coli capsule determinant, as well as several ORFs coding for trans-

Figure 2. Representative colonic sections of 33-week-old gnotobiotic IL-2⫹/⫹ and IL-2⫺/⫺ mice. All sections were stained with H&E. (A) E. coli mpk monocolonized IL-2⫹/⫹ and (B) IL-2⫺/⫺ mice; (C) B. vulgatus mpk monocolonized IL-2⫹/⫹ and (D) IL-2⫺/⫺ mice; (E) co-colonization of IL-2⫹/⫹ and (F ) IL-2⫺/⫺ mice with E. coli mpk; and (G) B. vulgatus mpk monocolonization of IL-2⫹/⫹ and (H ) IL2⫺/⫺ mice with E. coli Nissle. Sections in A, C–H show normal architecture of colonic mucosa with no significant inflammatory infiltrates and no mucosal injury. B exhibits pronounced inflammation of mucosa and submucosa with focal crypt abscesses and destruction of crypt epithelium in association with neutrophilic and mononuclear infiltrates. Original magnification, ⫻200.

of 15) of E. coli mpk mono-associated and 85% (17 of 20) of E. coli mpk/B. vulgatus mpk associated IL-2⫹/⫹ mice, no significant signs of colitis were observed (histologic score ⬍ 0.5). Likewise, monocolonization of IL-2⫹/⫹

Figure 3. Inflammation of the colon in IL-2⫹/⫹ and IL-2⫺/⫺ mice at 20 (Œ) and 33 (●) weeks of age. Histology slides were scored blinded by one pathologist as described in the Materials and Methods section. Each triangle or circle represents one animal. *P ⬍ 0.01 compared with mice of the same colonization status (Wilcoxon test). #P ⬍ 0.01 compared with all other IL-2⫺/⫺ groups.

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posases or bacteriophage integrases. Three percent of the probes specific for virulence-associated or pathogenicity island– encoded genes of intestinal pathogenic E. coli or homologues thereof gave significant hybridization signals. These probes were specific for several putative ORFs described in the O157:H7 strain EDL933, serine protease– encoding genes, and a gene of the diffuse adherence fibrilla determinant (dafa) of intestinal E. coli. Positive hybridization signals do not necessarily indicate the presence of complete and functional genes. Typical virulence-associated genes, for example, genes coding for adhesins (sfa, pap, afa, fae, fan), toxins (hly, cnf, cdt, lt, st, stx, set), or iron uptake systems (iuc, iro, fyuA) that frequently are present in pathogenic E. coli strains were not detectable in E. coli mpk. Both PCR and DNA array data were in accordance with the distribution of small amounts of virulence-associated genes among nonpathogenic, commensal E. coli isolates48 and strongly suggested that E. coli mpk is a nonpathogenic strain. Recently, a simple and rapid phylogenetic grouping technique based on a triplex PCR assay was described,47,52 and using this assay, the E. coli mpk strain was assigned to the phylogenic group B1 (data not shown). Members of this phylogenic group have been shown to represent commensals that rarely express virulence factors encoded by pathogenicity islands.53,54 Microbiologic Investigations in Colons of Specific Pathogen-Free and Gnotobiotic Mice To analyze whether the anticolitogenic effect of B. vulgatus mpk on E. coli mpk was owing to a reduction in the number of E. coli mpk in the colon by mechanisms such as colonization resistance, the number of bacteria in the colonic feces of SPF and gnotobiotic IL-2⫺/⫺ and IL-2⫹/⫹ mice was determined by culture methods and fluorescence in situ hybridization (FISH). As shown in Figure 4, the numbers of B. vulgatus mpk or E. coli mpk in colonic feces of monocolonized as well as co-colonized mice were comparable with and independent of the genetic haplotype of these mice. Data indicated that co-colonization with B. vulgatus mpk did not interfere with the ability of E. coli mpk to colonize gnotobiotic mice, and vice versa. Moreover, the presence of colitis was not associated with, and did not affect, bacterial numbers in the feces. This suggested that the protective effect of B. vulgatus mpk on E. coli mpk–induced colitis was not mediated by a reduction of E. coli mpk in the colon. Comparison of E. coli mpk and E. coli Nissle colonized mice revealed that both strains colonized the colon of mice at the same level.

Figure 4. Number of bacteria (A, B. vulgatus; B, E. coli) in colonic feces of IL-2⫹/⫹ (black bars) and IL-2⫺/⫺ (grey bars) SPF and gnotobiotic mice (A, B, n ⫽ 3–18 animals). Each value represents the mean log10 CFU ⫾ SD. *P ⬍ 0.05 compared with SPF mice of the same haplotype (Student t test). ND, not detected.

Comparison of gnotobiotic and SPF mice showed that the number of B. vulgatus mpk was slightly higher in gnotobiotic mice, however, this result was not significant (Figure 4). In contrast, the number of E. coli mpk in the

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Figure 5. Detection of E. coli mpk and B. vulgatus mpk in transverse colon sections of 20- or 33-week-old IL-2⫺/⫺ mice by FISH. All sections were counterstained with di-aminophenylindol. L, Lumen; V, villi; C, crypts. (A) Colon from B. vulgatus mpk monocolonized mouse hybridized with probe (B) B. vulgatus-Cy3, and (C) colons from E. coli mpk monocolonized mice hybridized with probe EntCy3, (D) colon from E. coli mpk/B. vulgatus mpk co-colonized mouse hybridized with probes Ent–fluorescein isothiocyanate and B. vulgatus-Cy3. Arrows indicate stained bacteria. Original magnification, 640⫻.

colonic feces of monocolonized and co-colonized IL2⫹/⫹ and IL-2⫺/⫺ mice was at least 100-fold higher than in SPF mice (P ⬍ 0.05). Therefore, under SPF conditions the ability of E. coli mpk to colonize is much lower than under gnotobiotic conditions. The total bacterial count in the feces was similar in gnotobiotic mice and in SPF mice (9.8 ⫾ 0.4 log10 CFU; data not shown). FISH was used to determine whether B. vulgatus mpk might have affected the spatial distribution of E. coli mpk at the colon epithelium, and to assess adherence of E. coli mpk and B. vulgatus mpk to the colon mucosa. Figure 5A and 5B show B. vulgatus mpk and E. coli mpk adhering predominantly in clusters to the luminal part of the mucosal surface of monocolonized mice. Occasionally, both bacteria also were observed to adhere to the crypt epithelium (Figure 5C). In co-colonized mice, mixed clusters consisting of both bacterial species adhered mainly to the luminal aspect of the mucosal surface (Figure 5D). Semiquantitative determination of bacterial adherence to the mucosa was performed by counting the number of bacteria on the epithelium, in crypts or in the lamina

propria, in 50 sections per colon of each mouse (Table 2). The number of adherent bacteria was highly variable in all groups of mice and usually ranged between 1 and 100 bacteria per section. However, there was no significant difference between colonization of IL-2⫹/⫹ and IL2⫺/⫺ mice. Moreover, in co-colonized mice the ratio of E. coli to B. vulgatus, which adhered to the mucosa, was about 1:1.5 and highly reproducible, and thus corresponded well with the CFU data determined from fecal samples by culture methods. Although the number of E. coli mpk adherent to the mucosa was higher in monocolonized mice than in co-colonized mice, this difference was not statistically significant. Furthermore, similar results were obtained for B. vulgatus mpk. Therefore, it appears that the reduced bacterial numbers at the colon epithelium may reflect the co-colonization status of mice. Taken together, the data showed that neither the total number nor the spatial distribution of E. coli mpk was influenced significantly by the simultaneous presence of B. vulgatus mpk and E. coli mpk. To address the interaction of E. coli mpk, E. coli Nissle, or B. vulgatus mpk with epithelial cells in more detail, we analyzed the ability of these strains to adhere to and to

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Table 2. Number of Bacteria Adherent to the Colon Mucosa as Determined by FISH Colonization status E. coli mpk B. vulgatus mpk E. coli mpk and B. vulgatus mpk

Specificity of FISH probe

na

Medianb

Rangeb

E. coli B. vulgatus E. coli B. vulgatus E. coli B. vulgatus

7 7 4 4 7 7

16.8 0 0 14.7 1.8 2.7

0.9–100 0 0 1.1–26.4 0.2–70.3 0.3–69.3

each animal about 150 sections from the colon (primarily distal part of the colon), 10-␮m each, were made. About every third section, at least 50 sections per animal, were used for FISH analysis. bNumber of bacteria adherent to the colon mucosa per section. The median and range were calculated from the values of 50 sections per mouse. aFrom

induce cytokine production in epithelial cell monolayers in vitro. For this purpose, MODE-K cells were cocultured with these bacterial strains and the percentage of adherent bacteria as well as the quantities of KC in the cell culture supernatants was assessed (Figure 6). The data indicate that E. coli Nissle was most adherent to the cells whereas E. coli mpk and B. vulgatus mpk were less

adherent. Thus, adhesion of these bacterial strains to epithelial cells in vitro appears not to be associated with the colitogenic or probiotic ability of these strains. KC levels determined in the culture supernatants revealed that B. vulgatus induced about 100- to 1000-fold less KC at a multiplicity of infection of 0.1 and 1, compared with both E. coli strains. Moreover, E. coli mpk and E. coli

Figure 6. (A) Adhesion of bacteria to epithelial cells. The indicated bacterial strains were centrifuged onto the cell monolayers, and the number of adherent bacteria was determined after 30 minutes of incubation. Adhesion is expressed as the percentage (means of triplicate wells ⫾ SD) of the initial inoculum recovered and representative for 3 independent experiments. (B) Production of KC by epithelial cell monolayers on 4 hours of coculture with the indicated bacterial strains at a multiplicity of infection of 0.1 (left hatched bars), 1 (black bars), 10 (right hatched bars), and 100 (open bars). The data are representative for 4 independent experiments. (C–F) Giemsa stain of epithelial cell monolayers cocultured with bacteria for 4 hours. (C) Not infected, (D) E. coli mpk, (E) E. coli Nissle, and (F ) B. vulgatus mpk. Original magnification, 20⫻.

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Table 3. Quantification of IFN-␥, TNF-␣, CD14, and IL-10 mRNA Expression in the Colon of IL-2⫺/⫺ and IL-2⫹/⫹ Mice by Semiquantitative Real-Time RT-PCR Ratio IFN-␥/GAPDH [⫻ 10⫺6] Colonization status

20 wk

33 wk

Ratio TNF-␣/GAPDH [⫻ 10⫺5] 20 wk

33 wk

Ratio CD14/GAPDH [⫻ 10⫺3] 20 wk

33 wk

Germ free ⫹/⫹ 1.7 ⫾ 2.4 1.0 ⫾ 0.3 7.3 ⫾ 3.5 5.8 ⫾ 0.9 5.0 ⫾ 1.8 10.7 ⫾ 2.7 Germ free ⫺/⫺ 3.8 ⫾ 3.9 8.3 ⫾ 1.9a 11.4 ⫾ 7.1 10.8 ⫾ 0.3a 5.9 ⫾ 2.7 14.9 ⫾ 2.1a SPF ⫹/⫹ 2.2 ⫾ 2.5 2.7 ⫾ 2.5 23.7 ⫾ 4.8 29.4 ⫾ 1.8 3.2 ⫾ 2.2 14.6 ⫾ 6.0 SPF ⫺/⫺ 148.2 ⫾ 73.6b ND 434.7 ⫾ 37.2c ND 111.5 ⫾ 69.2b ND E. coli mpk ⫹/⫹ 1.0 ⫾ 0.3 1.1 ⫾ 0.1 15.8 ⫾ 9.6 13.5 ⫾ 2.7 5.3 ⫾ 1.5 10.9 ⫾ 4.2 E. coli mpk ⫺/⫺ 20.5 ⫾ 30.5 85.8 ⫾ 18.8c,d 59.4 ⫾ 66.7 141.9 ⫾ 52.8c,d 15.9 ⫾ 12.2 56.7 ⫾ 28.1c,d B. vulgatus ⫹/⫹ 1.9 ⫾ 0.8 1.5 ⫾ 1.1 23.9 ⫾ 7.2 10.7 ⫾ 3.6 5.9 ⫾ 4.0 5.2 ⫾ 4.3 B. vulgatus ⫺/⫺ 1.1 ⫾ 0.5 5.0 ⫾ 5.6 18.4 ⫾ 4.2 16.4 ⫾ 7.6 6.6 ⫾ 4.0 12.0 ⫾ 9.5 E. coli and B. vulgatus ⫹/⫹ 1.8 ⫾ 0.7 0.9 ⫾ 0.2 20.1 ⫾ 8.9 12.2 ⫾ 2.8 3.7 ⫾ 3.8 14.9 ⫾ 3.3 E. coli and B. vulgatus ⫺/⫺ 13.8 ⫾ 19.9 8.9 ⫾ 11.0 37.0 ⫾ 38.4 32.2 ⫾ 20.5 7.7 ⫾ 7.3 17.9 ⫾ 9.9 E. coli Nissle ⫹/⫹ ND 5.9 ⫾ 4.1 ND 17.4 ⫾ 10.4 ND 25.7 ⫾ 6.5 E. coli Nissle ⫺/⫺ ND 19.2 ⫾ 6.2b ND 38.6 ⫾ 32.6 ND 32.1 ⫾ 5.0e

Ratio IL-10/GAPDH [⫻ 10⫺6] 20 wk

33 wk

4.0 ⫾ 2.3 3.9 ⫾ 1.7 4.2 ⫾ 3.7 9 ⫾ 4.1 9.9 ⫾ 5.3 23.3 ⫾ 4.9 312.0 ⫾ 203.9b ND 243.3 ⫾ 368.5 14.8 ⫾ 5.3 484.4 ⫾ 500.6 677.9 ⫾ 860.0b 9.6 ⫾ 6.9 9.9 ⫾ 7.8 246.5 ⫾ 383.5 21.3 ⫾ 25.2 53.9 ⫾ 105.0 6.3 ⫾ 2.1 17.6 ⫾ 6.6 28.5 ⫾ 36.4 ND 57.0 ⫾ 32.6 ND 122.1 ⫾ 79.7

NOTE. Data were normalized by dividing the copy number of IFN-␥, TNF-␣, CD14, or IL-10 mRNA by the copy number of the housekeeping gene GAPDH. Each value represents the mean ratio ⫾ SD of tested mRNA and GAPDH. Each group consisted of 3– 8 mice. ND, not determined. an ⫽ 2 mice per group, not included in statistical analyses. bP ⬍ 0.05 and cP ⬍ 0.01 compared with mice of same colonization status and age (Student t test). dP ⬍ 0.05 compared with all other IL-2⫺/⫺ mice groups of same age with e1 exception as indicated.

Nissle induced comparable quantities of KC production in MODE-K cells. Together, this data suggest that both adhesion to and cytokine induction of bacterial strains in epithelial cells in vitro may not necessarily be associated with their differential colitogenic or probiotic potential in IL-2⫺/⫺ mice. In addition, the microscopic analysis of epithelial cell monolayers after 4 hours co-incubation with the various bacterial strains indicated that none of the strains exerted a toxic effect to the epithelial cell monolayers (Figure 6). Altered Gene Expression in the Colon Is Associated With Colitis in Gnotobiotic IL-2–Deficient Mice The development of colitis in SPF IL-2⫺/⫺ mice is associated with a Th1-type inflammatory response.1 For this reason, we investigated whether colitis in gnotobiotic mice is associated with gene expression patterns similar to that of diseased SPF mice. IFN-␥, TNF-␣, IL-10, and CD14 mRNA expression were measured by real-time RT-PCR in 20- and 33-week-old mice. Twenty-week-old IL-2⫹/⫹ mice expressed comparable levels of IFN-␥, TNF-␣, and CD14 mRNA in the colon, independent of the associated microflora (Table 3). A significant increase in the expression of IFN-␥, TNF-␣, CD14, and IL-10 mRNA was observed in 20week-old IL-2⫺/⫺ mice compared with wild-type mice (IFN-␥: 74-fold, P ⬍ 0.05; TNF-␣: 18-fold, P ⬍ 0.01; CD14: 35-fold, P ⬍ 0.05; IL-10: 30-fold, P ⬍ 0.05), only under SPF conditions. Additionally, the mRNA

expression of IFN-␥, TNF-␣, and CD14 was slightly higher in E. coli mpk monocolonized, and in B. vulgatus mpk/E. coli mpk co-colonized IL-2⫺/⫺ mice, compared with germ-free or B. vulgatus mpk monocolonized IL-2⫺/⫺ mice. In 33-week-old IL-2⫺/⫺ compared with wild-type mice, IFN-␥, TNF-␣, CD14, and IL-10 mRNA expression was increased significantly in E. coli mpk monocolonized mice (P ⬍ 0.01; IL-10 P ⬍ 0.05), but not in B. vulgatus mpk or E. coli Nissle (except IFN-␥) monocolonized or in co-colonized mice. In E. coli mpk monoassociated IL-2⫺/⫺ mice, IFN-␥, TNF-␣, as well as CD14 mRNA expression also was increased significantly compared with B. vulgatus mpk colonized and co-colonized IL-2⫺/⫺ mice (P ⬍ 0.05), and IFN-␥ and TNF-␣ mRNA levels were increased compared with E. coli Nissle colonized IL-2⫺/⫺ mice (P ⬍ 0.05). Given that SPF IL-2⫺/⫺ mice died from severe colitis before they reached the age of 33 weeks, gene expression in these mice could not be analyzed. The data indicates that IFN-␥, TNF-␣, and CD14 mRNA expression levels are not only associated with colitis development in SPF IL-2⫺/⫺ mice, but also in E. coli mpk mono-associated IL-2⫺/⫺ mice. The late increase of IFN-␥, TNF-␣, and CD14 mRNA expression in the colon of E. coli mpk mono-associated mice reflects the delayed development of colitis in these mice compared with SPF IL-2⫺/⫺ mice. IL-2⫺/⫺ mice monocolonized by E. coli mpk expressed the highest IL-10

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mRNA levels. However, there was a high variation of IL-10 mRNA expression in the single mice. IL-2⫺/⫺ mice colonized with E. coli Nissle showed a significant increase in IFN-␥, but not TNF-␣, IL-10, and CD14 mRNA expression compared with IL-2⫹/⫹ mice (P ⬍ 0.05). Nevertheless, the relative mRNA expression levels of all genes tested were much lower in E. coli Nissle monocolonized IL-2⫺/⫺ mice than those in E. coli mpk monocolonized IL-2⫺/⫺ mice.

Discussion Commensal bacteria in the gut modulate important intestinal functions such as nutrient absorption, mucosal barrier function, angiogenesis, and intestinal maturation.55 The intestinal flora also plays a crucial role in the development of colitis in animal models,6,8 –10 as well as in human IBD.4,5,56 However, little is known about the nature of the interaction of intestinal microbes with each other and with their host, or of their role in the pathogenesis of IBD. Therefore, we investigated the colitogenic potential of single bacterial species indigenous to the colonic flora. The exclusive presence of B. vulgatus mpk in the intestine of IL-2⫺/⫺ mice is generally well tolerated without signs of inflammation, whereas E. coli mpk elicits a pronounced inflammatory response. Furthermore, co-colonization with both bacterial species does not induce bowel inflammation, and the data also argue against competition for adhesion sites and nutritional factors as possible probiotic mechanisms of B. vulgatus mpk. In contrast to E. coli mpk, E. coli Nissle does not induce colitis. B. vulgatus has been implicated in the pathogenesis of bowel inflammation in several animal models of IBD21,57 as well as in humans.58,59 IL-2⫺/⫺ mice monocolonized with B. vulgatus mpk were free of colitis at 20 weeks of age, and even 33-week-old mice very rarely developed colitis. Therefore, in monocolonized mice the bowel inflammation is not simply delayed. Taken together, our results did not confirm the colitogenic properties of B. vulgatus postulated by others. It appears to be unlikely that nonphysiologic bacterial counts in gnotobiotic mice may account for this discrepancy because the numbers of Bacteroides spp. in colonic feces were comparable in SPF and in gnotobiotic mice. Rather, specific differences between the B. vulgatus strains applied by others and by us may explain the lack of colitis in B. vulgatus mpk monocolonized mice. In contrast to other pathogens such as E. coli, the genome and pathogenicity factors of B. vulgatus are characterized only partially, and considerable work must be performed before this question can be addressed.

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Although we cannot exclude that the bacterial strains may lose their colitogenic determinants during the isolation procedure, the strains used in this study were isolated initially from our SPF colony of IL-2⫺/⫺ mice, which develop flourishing colitis. It is also interesting to note that one strain of B. vulgatus was observed to be an inducer of severe colitis in HLA-B27/␤2m transgenic rats,21,34 but not in IL-10⫺/⫺ mice.8 This points toward a critical role of the host genetic background for the induction and development of colitis in response to a particular bacterial stimulus. In general, IL-2⫺/⫺ mice monocolonized with E. coli mpk developed pronounced colitis, and compared with SPF IL-2⫺/⫺ mice, the disease developed more slowly and was accompanied by a delayed increase in mRNA expression levels of colitis markers such as IFN-␥, TNF-␣, and CD14. It is unclear if the delayed onset of disease in E. coli mpk mono-associated mice is caused by the lack of a synergistic effect of various components of the normal microflora. The role of E. coli in IBD is discussed controversially in the literature.60 On one hand there are reports of increased numbers of E. coli in patients with relapses,17 and on the other hand there are reports outlining the therapeutic benefit of E. coli Nissle.32 Monocolonization with E. coli Nissle did not cause colitis in IL-2⫺/⫺ mice. The data support recent studies in IBD patients that argue for a probiotic effect of E. coli Nissle. Whether the different observations for various E. coli strains reflect specific strain characteristics or different host conditions is unclear. As discussed for the differential ability of B. vulgatus strains in colitis induction earlier, it is not yet possible to draw conclusions about the general probiotic or pathogenic properties of a given bacterial species. To address this important issue in more detail, genetic studies including investigations of defined bacterial mutant strains in a mouse model of colitis are required. Such studies currently are performed in our laboratory and might reveal detailed information about single bacterial genes that are associated with probiotic or colitogenic properties. In a primary attempt to elucidate the mechanism of E. coli mpk–induced colitis, we analyzed our strain for the presence of known virulence factors typically expressed by enterohemorrhagic, enteropathogenic, enteroinvasive, enterotoxic, and enteroaggregative by PCR and DNA array analysis. We found that E. coli mpk does not belong to any of these pathotypes, and after further analysis it was assigned to the phylogenetic B1 group, which largely represents nonpathogenic commensals. The genetic markers of human pathogenic E. coli may not be

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relevant for mice, and in fact most E. coli pathotypes in humans are not pathogenic in mice. Therefore, it might be that different pathotypes expressing other pathogenicity factors are relevant for disease in mice. Nevertheless, DNA array analysis confirmed that only a small number of genes encoding putative virulence factors are present in E. coli mpk, and recent work showed that several putative virulence-associated genes usually are present in commensal, nonpathogenic E. coli strains such as E. coli K1248 or E. coli Nissle (unpublished data). There is no evidence that E. coli mpk exhibits the adherent and invasive characteristics reported for E. coli strains isolated from IBD patients.8,39,61 The data obtained from FISH suggest that epithelial cell adhesion of microorganisms are rather rare events, possibly attributing an important role to soluble factors in the pathogenesis of microflora-triggered IBD. This speculation is supported by the observation that the noncolitogenic E. coli Nissle exhibited the highest adhesion rate to epithelial cell cultures in vitro compared with E. coli mpk and B. vulgatus mpk. It was surprising to find that B. vulgatus mpk exerted probiotic properties by abolishing the colitogenic properties of E. coli mpk in co-colonized mice because both bacterial strains were isolated from SPF IL-2⫺/⫺ mice with colitis. This indicated that in the presence of complete SPF flora, B. vulgatus cannot prevent the development of colitis. The nature of the probiotic effect of B. vulgatus mpk in co-colonized animals is obscure although several possible explanations could be ruled out: a significant reduction in the number of E. coli in the gut by B. vulgatus did not occur, therefore mechanisms proposed for probiotics such as direct inhibition of growth by antibiotic factors30 or competition for nutritional factors, do not appear to be involved in our system. Competition for colonization sites on the epithelial surface appears to play a minor or no role because bacterial adhesion to the epithelium only rarely was observed by FISH and large areas of the epithelium remained free of attached bacteria. Moreover, the reduction of mucosa-associated bacteria in co-colonized mice was not statistically significant and was found for both E. coli mpk and B. vulgatus mpk. Whether the reduced bacterial adherence accounts for the protective effect of B. vulgatus mpk in co-colonized mice is not yet clear. However, although others found that the adherent mucosal flora is not different in the proximal and distal colon,62 we cannot exclude that at different intestinal sites the distribution of E. coli mpk was affected by B. vulgatus mpk.

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The onset or development of colitis in SPF IL-2⫺/⫺ mice is associated with a CD4⫹ T-cell driven Th1-type inflammatory response, including an increased expression of IFN-␥ as well as proinflammatory cytokines (IL-1␣, IL-1␤, IL-6, TNF-␣), chemokines (KC, JE) and other factors associated with inflammation such as CD14.63– 66 E. coli mpk–induced colitis coincided with an increased expression of IFN-␥, TNF-␣, and CD14 mRNA, suggesting that monocolonization with E. coli mpk also triggered a Th1 inflammatory response. This is in line with reports showing that colitis in IL-2⫺/⫺ mice is dependent on CD4 Th1 cells.1,63 The expression of CD14 mRNA in colonic tissue of IL-2⫺/⫺ mice might reflect a hypersensitivity against lipopolysaccharide and thus a loss of tolerance to parts of the normal flora. The cell types actually expressing CD14 were not determined, but macrophages and the colonic epithelial cells66 are likely main sources. Determination of IL-10 mRNA levels confirmed previous reports, indicating an increased IL-10 mRNA expression in SPF IL-2⫺/⫺ mice with colitis compared with IL-2⫹/⫹ mice.64 In gnotobiotic mice, only monocolonization with E. coli mpk resulted in a significant difference in IL-10 expression between IL-2⫺/⫺ and IL-2⫹/⫹ mice. Owing to high variations within some groups, significant differences of E. coli mpk colonized IL-2⫺/⫺ mice compared with the other groups of IL-2⫺/⫺ mice without colitis were not detected. Considering the fact that IL-10 is regarded to have antiinflammatory properties, the data point to a complex yet unclear role of IL-10 in inflammatory processes.1,8 Although recent reports point toward a role of bacterial superantigens in IBD,67,68 based on our data we favor the hypothesis that microorganisms initially trigger proinflammatory cell responses (e.g., in epithelial cells or dendritic cells). Although it is highly speculative, we hypothesize that E. coli mpk might elicit a signaling cascade in certain host cells leading to a proinflammatory reaction that, on the genetic basis and immunologic defect of IL-2⫺/⫺ mice, leads to a Th1-cell–mediated colitis. B. vulgatus mpk might elicit a signaling in host cells that inhibits initial proinflammatory signals or that might inhibit a Th1 response. In the presence of both bacteria B. vulgatus could be able to antagonize the Th1 response and thus the colitis-inducing potential of E. coli mpk. Whether E. coli mpk and B. vulgatus mpk may regulate I␬B/nuclear factor ␬ B complexes, as has been shown for nonvirulent Salmonella strains,69 remains to be investigated. Both E. coli mpk and E. coli Nissle as well as B. vulgatus mpk induced production of KC in epithelial cell cultures. Because KC is regulated by nuclear

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factor ␬ B we assume that these bacteria may in principle activate nuclear factor ␬ B.70 Furthermore, because both E. coli strains induced high KC levels at a 100- to 1000-fold lower multiplicity of infection than B. vulgatus, we can speculate that KC induction by these bacterial strains is associated with their differential LPS activity.71 The presence of both E. coli mpk and B. vulgatus mpk might alter the gene expression of the bacteria. There are reports that showed that microbial pathogenicity factors may not only act on the host but may also have an ecologic role with effects on other members of a microbial community.72 We cannot rule out that the presence of B. vulgatus mpk regulates gene expression in E. coli mpk, which may result in repression of colitogenic factors in E. coli. In conclusion, our results together with those from other studies, strongly suggest that different bacterial species of the indigenous microflora might dramatically differ in their colitogenic or probiotic potential. Furthermore, we have described a newly isolated B. vulgatus strain with possible probiotic properties that is able to ameliorate E. coli mpk–induced colitis development, by an as yet unknown mechanism. Future experiments will address whether particular interbacterial interactions play a role in the prevention of E. coli mpk–triggered colitis by B. vulgatus mpk. We will focus on the interaction of these newly described bacterial strains with host cells to define early molecular mechanisms involved in colitis induction. We hope that the reduced complexity of the flora in gnotobiotic mice developing colitis will help to define the roles of cells such as epithelial and T cells in a scenario in which only a limited set of bacterial antigens operate in the intestinal flora.

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Received September 17, 2002. Accepted April 10, 2003. Address requests for reprints to: Professor Ingo B. Autenrieth, M.D., Institut fu ¨r Medizinische Mikrobiologie und Krankenhaushygiene, Universita ¨tsklinikum Tu ¨bingen, Elfriede-Aulhorn-Strasse 6, D-72076 Tu ¨bingen, Germany. email: [email protected]; fax: (49) 7071-295440. Supported by a grant from the Deutsche Forschungsgemeinschaft and by the Bundesministerium fu ¨r Bildung und Forschung. O.B. was the recipient of a scholarship from the Graduiertenkolleg Infektionsbiologie, Tu ¨bingen. The authors thank Burkhart Jilge and Sabine Schmidt, University of Ulm, for expert gnotobiotic animal breeding facilities; Antonietta Rastiello and Claudia Braun (Mainz) for their technical support with the histologic studies; Marija Markulin (Tu ¨ bingen) for technical support with the IL-10 RT-PCR; Helge Karch (Mu ¨ nster) for providing unpublished PCR primer sequences for detection of E. coli virulence factors; Jo ¨ rg Hacker (Wu ¨ rzburg) for stimulating discussions; and Pierre Kyme (Tu ¨ bingen) for critical reading of the manuscript. M.W., O.B., and J.-S.F. contributed equally to this work.