The role of type 1 and type 3 pili in mediating Klebsiella pneumoniae colonization of inert surfaces was assessed by analysis of 26 clinical, 7 water distribution system and four reference strains. Amongst the 33 wild type strains tested, 57.6% expressed type 1 pili, 54.5% expressed type 3 pili, 42.4% were highly hydrophobic, 69.7% were highly adherent to both glass and polypropylene surfaces, and 48.5% were efficient biofilm-forming strains on polystyrene dishes. High adherence and biofilm formation were positively correlated with bacterial surface hydrophobicity, type 3 fimbriae expression but not with type 1 pili expression and were not dependent upon the strain's origin. The K. pneumoniae CF3051 reference strain expressing only type 1 fimbriae adhered slightly to glass and polypropylene and did not form biofilm on polystyrene. K. pneumoniae IA565 and CF3097 reference strains producing type 1 and type 3 fimbriae showed efficient adherence to both glass and polypropylene and biofilm formation on polystyrene. Moreover, transformation of the afimbriated, weakly-adherent CF3172 strain with the recombinant pFK10 plasmid carrying the mrk gene cluster resulted in type 3 fimbriae expression, increased surface hydrophobicity, increased adherence to abiotic surfaces and biofilm formation. Thus, type 3 pili constitute the main K. pneumoniae adhesive factor, facilitating adherence and biofilm formation on abiotic surfaces of strains of different origins.

ABSTRACT To identify Shiga toxin-producing Escherichia coli genes associated with severe human disease, a genomic subtraction technique was used with hemolytic-uremic syndrome-associated O91:H21 strain CH014 and O6:H10 bovine strains. The method was adapted to the Shiga toxin-producing E. coli genome: three rounds of subtraction were used to isolate DNA fragments specific to strain CH014. The fragments were characterized by genetic support analysis, sequencing, and hybridization to the genome of a collection of Shiga toxin-producing E. coli strains. A total of 42 fragments were found, 19 of which correspond to previously identified unique DNA sequences in the enterohemorrhagic E. coli EDL933 reference strain, including 7 fragments corresponding to prophage sequences and others encoding candidate virulence factors, such a SepA homolog protein and a fimbrial usher protein. In addition, the subtraction procedure yielded plasmid-related sequences from Shigella flexneri and enteropathogenic and Shiga toxin-producing E. coli virulence plasmids. We found that lateral gene transfer is extensive in strain CH014, and we discuss the role of genomic mobile elements, especially bacteriophages, in the evolution and possible transfer of virulence determinants.

Twenty-nine Enterococcus faecalis isolates from patients with endocarditis or bacteremia or from stools of healthy volunteers were investigated for their ability to adhere to Int-407 and Girardi heart cell lines and for the presence of known enterococcal virulence factors. Eight strains (27.6%) adhered predominantly to Int-407 cells. The adherence of enterococci was enhanced by proteolytic digestion, suggesting that some cell binding components become surface-exposed after treatment with trypsin. The occurrence of known potential virulence factors of enterococci among these strains was determined and was as follows: enterococcal surface protein (72.4%), gelatinase (58.6%), aggregation substance (48.3%) and cytolysin (17.2%). Bacterial adherence was not significantly associated with any of these virulence factors.

The interest of probiotics as remedies for a broad number of gastrointestinal and other infectious diseases has gained wide interest over the last few years, but little is known about their underlying mechanism of action. In this study, the probiotic activities of a human isolate of Lactobacillus casei subsp. rhamnosus strain (Lcr35) were investigated. Using intestinal Caco-2 cell line in an in vitro model, we demonstrated that this strain exhibited adhesive properties. The inhibitory effects of Lcr35 organisms on the adherence of three pathogens, enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli (ETEC) and Klebsiella pneumoniae, were determined. A decrease in the number of adhering pathogens was observed, using either preincubation, postincubation or coincubation of the pathogens with Lcr35. Moreover, the antibacterial activities of cell-free Lcr35 supernatant was examined against nine human pathogenic bacteria, ETEC, EPEC, K. pneumoniae, Shigella flexneri, Salmonella typhimurium, Enterobacter cloacae, Pseudomonas aeruginosa, Enterococcus faecalis and Clostridium difficile. The growth of all strains was inhibited, as measured by determining the number of viable bacteria over time, but no bactericidal activity was detected in this in vitro assay. Together, these findings suggest that this probiotic strain could be used to prevent colonization of the gastrointestinal tract by a large variety of pathogens.

Some strains of Escherichia coli are involved in enteric infections in both adults and children. However the classical diagnostic methods can not differentiate pathogenic from nonpathogenic E. coli, because of the lack of phenotypic differences. In this study, we developed multiplex PCR in order to amplify fragments of specific virulence genes of the five main E. coli pathotypes. Fragments of the expected size were obtained using previously or newly designed primers and allowed identification of 10 virulence genes in only 5 reactions. This method was applied to the detection of pathogenic E. coli isolated from 90 patients' stools specimens during an 18-month survey. Patients were suffering from diarrhea or hemolytic uremic syndrome and in 13 cases (14.4%), an enterovirulent E. coli strain was detected. This diagnostic method could therefore represent an important technique in clinical laboratories which lack standard tests for these pathogens.

Escherichia coli strains isolated from patients with Crohn's disease (CD) with chronic ileal lesions (n=14), early endoscopic recurrent lesions (n=20), without endoscopic recurrence (n=7), and controls (n=21) were compared by ribotyping. The dendrogram generated by 50 ribotype profile analysis revealed a large cluster of genetically linked E coli strains isolated significantly more frequently from patients with chronic and recurrent CD (24/33 patients) than from controls (9/21) (p<0.05). Most patients operated on for chronic ileal lesions (78.5%) harboured E coli strains belonging to cluster A (p<0.002 v controls). The prevalence of patients with early recurrent lesions harbouring E coli strains belonging to this cluster was high but not significant, although 16 strains isolated from eight patients presented the same ribotype profile. In this cluster, 21 of 26 strains isolated from patients with active CD demonstrated adherent ability to differentiated Caco-2 cells, indicating that most of the genetically related strains share a common virulence trait. Comparison of E coli strains recovered from ulcerated and healthy mucosa of patients operated on for CD demonstrated in each patient that a single strain colonised the intestinal mucosa. Our results suggest that although a single E coli isolate was not found in Crohn's ileal mucosa, some genotypes were more likely than others to be associated with chronic or early recurrent ileal lesions.

Verotoxin producing Escherichia coli (VTEC) have been associated with disease outbreaks of diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome in humans. Contamination occurs mainly by ingestion of beef and dairy products, but water and person to person transmission have also been described. Most of the clinical signs are due to the production of Stx1 and/or Stx2 Shiga toxins, also called verotoxins. Other virulence factors include enterohemolysin, and the product of the eae gene, intimin, involved in the attaching and effacing adherence phenotype. The predominant serotype is O157:H7, but VTEC strains of more than one hundred serotypes can cause human disease. in order to determine the prevalence of VTEC infections among children in the central part of France, stool samples from hospitalized children were examined for stx1 and stx2 genes by using a polymerase chain reaction (PCR) technique. From October 1997 to September 1998, 658 stool samples were analysed: among them 19 (3 %) were stx-PCR positive. Only 8 children out of 19 had diarrhea, and for 5 of them, an enteric pathogen other than VTEC was isolated. VTEC strains were isolated from 10 samples: most of the isolates did not produce verotoxins at a high level, and they did not belong to serotypes associated with pathogenicity, which might explain the absence of relationship between VTEC isolation and pathogenicity in our study (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Verotoxin producing Escherichia coli (VTEC) have been associated with disease outbreaks of diarrhea hemorrhagic colitis and hemolytic-uremic syndrome in humans. Contamination occurs mainly by ingestion of beef and dairy products, but water and person to person transmission have also been described. Most of the clinical signs are due to the production of Stx1 and/or Stx2 Shiga toxins, also called verotoxins. Other virulence factors include enterohemolysin, and the product of the eae gene, intimin, involved in the attaching and effacing adherence phenotype. The predominant serotype is O157:H7, but VTEC strains of more than one hundred serotypes can cause human disease. In order to determine the prevalence of VTEC infections among children in the central part of France, stool samples from hospitalized children were examined for stx1 and stx2 genes by using a polymerase chain reaction (PCR) technique. From October 1997 to September 1998, 658 stool samples were analysed: among them 19 (3%) were stx-PCR positive. Only 8 children out of 19 had diarrhea, and for 5 of them, an enteric pathogen other than VTEC was isolated. VTEC strains were isolated from 10 samples: most of the isolates did not produce verotoxins at a high level, and they did not belong to serotypes associated with pathogenicity, which might explain the absence of relationship between VTEC isolation and pathogenicity in our study.

Les Escherichia coli (E. coli) producteurs de vérotoxines (VTEC) sont responsables de diarrhées aqueuses, de colites hémorragiques, qui peuvent se compliquer d'un syndrome hémolytique et urémique. Le réservoir de ces bactéries est le tube digestif des bovins; la contamination humaine se fait par l'intermédiaire d'aliments, principalement viande de bœuf et lait cru. Les symptômes cliniques sont dus à la production de vérotoxines également appelées toxines Shiga-like (Stx). Les E. coli entérohémorragiques de sérotype O157:H7 sont impliqués dans la plupart des grandes épidémies, mais un nombre croissant d'infections est attribué aux souches appartenant à d'autres sérogroupes. Afin d'obtenir des données sur la prévalence des infections à VTEC chez l'enfant dans la région Auvergne, nous avons entrepris une étude prospective et choisi d'utiliser une technique d'amplification multiple des gènes codant les toxines Shiga-like. Sur une période d'un an (octobre 1997 à septembre 1998), nous avons analysé 658 prélèvements de selles d'enfants hospitalisés, parmi lesquels 255 présentaient une diarrhée. Un total de 19 prélèvements (3%) a donné un résultat positif pour la recherche des gènes stx, et dans 10 cas (1,5 %) une souche VTEC a été isolée. Dans 8 cas sur 19, les enfants avaient des symptômes diarrhéiques, avec une association à un autre pathogène entérique dans 5 cas. La plupart des souches isolées ne produisent pas ou peu de toxines, et n'appartiennent pas à des sérotypes associés à la pathogénicité, ce qui pourrait expliquer l'absence de relation entre présence de ces souches et pathogénicité chez ces enfants.

Myeloperoxidase (MPO) is a heme enz~me abund~t in monocytes .andmacrophages which catalyses the generation of reactive oxygen and mtrogen species.It is present at many sites of inflammation and is postulated to play a key role in free radical mediated tissue damage in human Inflammatory Bowel Disease (IBD).To asses its importance in gut inflammation and tissue damage, we induced acute colitis in wild-type and MPO deficient C57IBL6 mice by continuous feeding of a 5% aqueous solution of Dextran Sodium Sulfate (DSS).Although both sets of animals lost weight in a similar manner over the course of the experiment there was substantially improved survival in the MPO K.O.animals 92% at day 6 compared to 60% for the wild type.They also had substantially higher numbers of circulating lymphocytes and neutrophils compared to the wild-types.The MPO K.O.mice had less peri-anal blood staining and post-mortem examination revealed substantially less hemorrhage in the colon.However, the length of the large intestine was generally shorter in these animals, indicating more severe disease.Interestingly, histologic examination of the LI tissue from both sets of animals revealed similar degrees of inflammation and tissue destruction.These results demonstrate that tissue damage and inflammation can still occur in the absence of myeloperoxidase in this acute model of colitis.However, its role in these processes is likely complex and the mechanisms leading to the improved clinical outcomes will require further study.

ABSTRACT During a 1-year survey of Shiga toxin-producing Escherichia coli (STEC) prevalence in central France, 2,143 samples were investigated by PCR for Shiga toxin-encoding genes. A total of 330 (70%) of 471 fecal samples collected from healthy cattle at the Clermont-Ferrand slaughterhouse, 47 (11%) of 411 beef samples, 60 (10%) of 603 cheese samples, and 19 (3%) of 658 stool specimens from hospitalized children with and without diarrhea were positive for the stx gene(s). A STEC strain was isolated from 34% (162 of 471) of bovine feces, 4% (16 of 411) of beef samples, 1% (5 of 603) of cheese samples, and 1.5% (10 of 658) of stool specimens. Of the 220 STEC strains isolated, 34 (15%) harbored the stx 1 gene, 116 (53%) harbored the stx 2 gene, and 70 (32%) carried both the stx 1 and stx 2 genes. However, 32 (14.5%) were not cytotoxic for Vero cells. The eae gene, found in 12 (5%) of the 220 strains, was significantly associated with the stx 1 gene and with isolates from children. Sequences homologous to ehxA were found in 102 (46%) of the 220 strains. Thirteen serotypes, OX3:H2, O113:H21, O113:H4, OX3:H21, O6:H10, OX178:H19, O171:H2, O46:H38, O172:H21, O22:H16, O91:H10, O91:H21, and O22:H8, accounted for 102 (55%) of 186 typeable isolates, and only one strain (0.5% of the 186 STEC isolates from cattle), belonged to the O157:H7 serotype. We showed that the majority of the STEC isolates from cattle, beef, and cheese were not likely to be pathogenic for humans and that the STEC strains isolated from children in this study were probably not responsible for diarrheal disease. Finally, the strains associated with hemolytic-uremic syndrome in the same geographical area were shown to belong to particular subsets of the STEC population found in the bovine reservoir.

Crohn's disease (CD) is an inflammatory bowel disease in which Escherichia coli strains have been suspected of being involved. We demonstrated previously that ileal lesions of CD are colonized by E. coli strains able to adhere to intestinal Caco-2 cells but devoid of the virulence genes so far described in the pathogenic E. coli strains involved in gastrointestinal infections. In the present study we compared the invasive ability of one of these strains isolated from an ileal biopsy of a patient with CD, strain LF82, with that of reference enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteraggregative (EAggEC), enterohemorrhagic (EHEC), and diffusely adhering (DAEC) E. coli strains. Gentamicin protection assays showed that E. coli LF82 was able to efficiently invade HEp-2 cells. Its invasive level was not significantly different from that of EIEC and EPEC strains (P > 0.5) but significantly higher than that of ETEC (P < 0.03), EHEC (P < 0. 005), EAggEC (P < 0.004) and DAEC (P < 0.02) strains. Strain LF82 also demonstrated efficient ability to invade intestinal epithelial cultured Caco-2, Intestine-407, and HCT-8 cells. Electron microscopy examination of infected HEp-2 cells revealed the presence of numerous intracellular bacteria located in vacuoles or free in the host cell cytoplasm. In addition, the interaction of strain LF82 with epithelial cells was associated with the elongation of microvillar extensions that extruded from the host cell membranes and engulfed the bacteria. This internalization mechanism strongly resembles Salmonella- or Shigella-induced macropinocytosis. The use of cytochalasin D and colchicine showed that the uptake of strain LF82 by HEp-2 cells was mediated by both an actin microfilament-dependent mechanism and microtubule involvement. In addition, strain LF82 survived for at least 24 h in HEp-2 and Intestine-407 cells and efficiently replicated intracellularly in HEp-2 cells. PCR and hybridization experiments did not reveal the presence of any of the genetic determinants encoding EIEC, EPEC, or ETEC proteins involved in bacterial invasion. Thus, these findings show that LF82, which colonized the ileal mucosa of a patient with CD, is a true invasive E. coli strain and suggest the existence of a new potentially pathogenic group of E. coli, which we propose be designated adherent-invasive E. coli.

Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes. Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction. None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon. Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E. coli 17.2. No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E. coli 17.2.

Most Klebsiella pneumoniae clinical isolates are fully encapsulated and adhere in vitro to intestinal cell lines with an aggregative pattern. In this study, the influence of the capsule on interactions with epithelial cells was investigated by creating an isogenic mutant defective in the synthesis of the capsule. Determination of the uronic acid content of bacterial extracts confirmed that the mutant did not produce capsular polysaccharides whereas, with the wild-type strain, the level of encapsulation was growth phase dependent and reached a maximum during the lag and early log phases. Assays performed with different epithelial cell lines, Int-407, A-549, and HEp-2, showed that the capsule-defective mutant demonstrated greater adhesion than did the wild-type strain and that the aggregative pattern was maintained, indicating that the capsule was not related to the adhesion phenotype. In contrast, when the mucus-producing HT-29-MTX cells were used, the encapsulated wild-type strain adhered more strongly than did the capsule-defective mutant. No invasion properties were observed with any of the capsular phenotypes or cell lines used. The K. pneumoniae adhesin CF29K was detected by Western blot analysis and enzyme-linked immunosorbent assay on the surface of transconjugants obtained after transfer of a conjugative plasmid harboring the CF29K-encoding genes into both the wild-type and the capsule-defective strains. The amounts of adhesin detected were greater in the capsule-defective background strain than in the wild-type encapsulated strain and were associated with an increase in the level of adhesion to Caco-2 cells. Moreover, RNA slot blot experiments showed that transcription of the adhesin-encoding gene was markedly increased in the capsule-defective mutant compared to the wild-type encapsulated background. These results suggest (i) that the capsule plays an active role during the initial steps of the pathogenesis by interacting with mucus-producing cells but is subsequently not required for the adhesin-related interaction with the epithelial cell surface and (ii) that the expression of the adhesin is modulated by the presence of a capsule at a transcriptional level.

From August 1996 to May 1997, six verotoxin-producing Escherichia coli (VTEC) strains were isolated from stool specimens of adults suffering from hemolytic-uremic syndrome (HUS). All the isolates were stx(2) positive and belonged to different serotypes: O6:H4, O91:H10, O91:H21, O rough:H16, OX3:H-, and O nontypeable: H-. The enterohemolysin (Ehly)-encoding genes were detected in two isolates, and none of the isolates harbors the intimin (Eae) encoding gene. These findings suggest that stx(2)-positive non O157:H7 VTEC is a major cause of HUS in adults and that several sources of pathogens are responsible for local endemic infections.

The effect of subinhibitory concentrations of roxithromycin on the adhesion of three strains of Klebsiella pneumoniae to Int-407 cells was studied. Adherence was markedly inhibited and the effect was increased when roxithromycin was added to the cell culture medium rather than to the bacterial growth medium. Several assays were performed in order to understand the mechanism by which roxithromycin exerted this inhibitory effect. The greatest effect was obtained when roxithromycin was concentrated in the extracellular compartment; when roxithromycin was concentrated in the intracellular compartment, the inhibitory effect was reduced. The analysis of adhesion factors of bacteria showed that exposure to roxithromycin did not alter their apparent structure or quantity. Roxithromycin appears to interfere in the interaction between bacteria and eukaryotic receptors.

BACKGROUND & AIMS:Infectious agents are suspected of being involved in the pathogenesis of Crohn's disease. This study was designed to look for the presence of virulent Escherichia coli strains associated with the ileal mucosa of patients with Crohn's disease.METHODS:E. coli strains were recovered from resected chronic ileal lesions (n = 20), neoterminal ileum after surgery from patients with (n = 19) and without (n = 11) endoscopic recurrence, and controls (n = 13). Bacterial adhesion was determined in vitro using intestinal cell lines; other associated virulence factors were assessed by DNA hybridization and polymerase chain reaction experiments.RESULTS:None of the strains harbored any of the virulence factor-encoding genes of E. coli involved in acute enteric diseases. However, mannose-resistant adhesion to differentiated Caco-2 cells was found for 84.6% and 78.9% of the E. coli strains isolated from chronic and early recurrent lesions, respectively, compared with 33% of controls (P < 0.02). In addition, 21.8% of the strains induced a cytolytic effect by synthesis of an alpha-hemolysin.CONCLUSIONS:E. coli strains isolated from the ileal mucosa of patients with Crohn's disease adhere to differentiated intestinal cells and may disrupt the intestinal barrier by synthesizing an alpha-hemolysin.

CS31A is a K88-related capsule-like surface protein that mediates Escherichia coli and Klebsiella pneumoniae adhesion to the human Caco-2 and Intestine-407 cell lines. In this study, we demonstrate that ClpG, the major subunit of CS31A, contains the adhesive domain of the polymerized structure. We mapped this domain within the ClpG protein by performing adhesion inhibition experiments with Intestine-407 cells with nine synthetic peptides (CLP1 to CLP9) covering the dominant antigenic regions of ClpG and with the corresponding rabbit antipeptide antibodies. The peptides CLP1 (amino acid positions in parentheses) (5-18), CLP2 (44-56), CLP3 (82-96), CLP7 (174-190), CLP8 (185-199), and CLP9 (235-249) and corresponding antipeptide antibodies targeting parts of the N- and C-terminal regions of ClpG had no effect on the adhesion of the TCFF15 recombinant strain expressing CS31A. Only the CLP5 (132-146) peptide, corresponding to the central part of the protein, and relevant antibodies inhibited bacterial adhesion to intestinal epithelial cells. Anti-CLP4 (97-109) and anti-CLP6 (148-162) antibodies targeting regions surrounding the CLP5 sequence also inhibited bacterial adhesion. Site-directed mutagenesis experiments inducing changes in the amino acid sequence of the ClpG protein corresponding to the CLP5 peptide resulted in the expression of nonadhesive CS31A antigen. These findings indicate that the ClpG receptor-binding domain is located in the central variable V2 region.