Fimbriation ofEscherichia coli in urinary tract infections. Comparisons between bacteria in the urine and subcultured bacterial isolates

Midstream urine samples from 37 patients with urinary tract infections were studied by electron microscopy, hemagglutination, and the salt aggregation test (SAT) to measure the hydrophobicity of the bacterial surface.Escherichia coli subcultured from these urine samples were tested in the same way. Fimbriae were visualized onE. coli in the urine of 31 specimens, and all these urines containedE. coli that expressed pronounced surface hydrophobicity and aggregated in ammonium sulfate of 0.1–1.6 M final concentration. Hemagglutination of human and/or guinea pig erythrocytes was expressed by 21E. coli in the urine. TheE. coli strains subcultured from these 31 urine samples were also fimbriated, but the number of fimbriae per bacterium as well as the percentage of fimbriated bacteria varied compared with the directly collected strains. The surface hydrophobicity and hemagglutination were similar to the results with the directly collected bacteria. However, after serial transfer in CFA-broth under static conditions, all non-hemagglutinating strains expressed mannose-sensitive hemagglutination of guinea pig erythrocytes, and three strains also expressed weak mannose-resistant hemagglutination of human erythrocytes. Following serial transfer, fimbriae were also visualized on the sixE. coli strains that appeared non-fimbriate in the urine. It is thus concluded thatE. coli causing urinary tract infection are often fimbriated and express surface hydrophobicity in the urine. Based on these findings, a rapid method to isolate hydrophobic, possibly fimbriated bacteria was tried in which the urine was mixed with a hydrophobic gel. Hydrophobic bacteria bound to the gel and could be eluted from the sedimented gel.     

Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation

The influence of type 1 fimbriae, mannose-sensitive structures, on biofilm development and maturation has been examined by the use of three isogenic Escherichia coli K12 strains: wild type, fimbriated, and non-fimbriated. Experiments with the three strains were done in minimal medium or Luria–Bertani broth supplemented with different concentrations of d-mannose. The investigation consisted of: (1) characterizing the bacterial surface of the three strains with respect to hydrophilicity and surface charge, (2) investigating the effect of type 1 fimbriae on bacterial adhesion rate and reversibility of initial adhesion on glass surfaces, and (3) verifying the role of type 1 fimbriae and exopolysaccharides (EPS) in biofilm maturation. The results suggest that type 1 fimbriae are not required for the initial bacterial adhesion on glass surfaces as the non-fimbriated cells had higher adhesion rates and irreversible deposition. Type 1 fimbriae, however, are critical for subsequent biofilm development. It was hypothesized that in the biofilm maturation step, the cells synthesize mannose-rich EPS, which functions as a ‘conditioning film’ that can be recognized by the type 1 fimbriae.     

Genomic heterogeneity in Chlorobium limicola: chromosomic and plasmidic differences among strains

Abstract The purpose of this study is to certify the importance of the fimbriae as an attachment factor of Actinobacillus actinomycetemcomitans , a human periodontopathic bacterium, and the significance of anti-fimbrial antibody function as an attachment inhibitor. Fimbrial antigen was prepared from the A. actinomycetemcomitans 310-a strain. Oligopeptides were synthesized according to the amino acid sequence of the fimbrial protein. The peptide antigen was conjugated with branched lysine polymer resin beads. The peptide antigen was suspended in PBS emulsified with incomplete Freund's adjuvant and used to immunize rabbits. A rabbit antiserum reacted with an approximately 54 kDa protein of the fimbriae protein from A. actinomycetemcomitans 310-a and with those of other fimbriated strains. This antiserum strongly inhibited the attachment of fimbriated A. actinomycetemcomitans strains to saliva-coated hydroxyapatite beads, buccal epithelial cells, and a fibroblast cell line, Gin-1. Such a synthetic fimbrial peptide antigen may be effective in inducing antibodies which inhibit adhesion and subsequent colonization by A. actinomycetemcomitans .     

Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin

Adherence of type-1-fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type-1-fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non-fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside-inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(plSF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae-coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type-1 -fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type-1-fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type-1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(plSF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type-1 fimbriae of S. enterica and E. coli bind to oMgomannoside chains of the lamjnjn network in basement membranes.     

The DraC usher in Dr fimbriae biogenesis of uropathogenic E. coli Dr+ strains

Biogenesis of Dr fimbriae encoded by the dra gene cluster of uropathogenic Escherichia coli strains requires the chaperone-usher pathway. This secretion system is based on two non-structural assembly components, the DraB periplasmic chaperone and DraC outer-membrane usher. The DraB controls the folding of DraE subunits, and DraC forms the assembly and secretion platform for polymerization of subunits in linear fibers. In this study, mutagenesis of the DraC N-terminus was undertaken to select residues critical for Dr fimbriae bioassembly. The DraC-F4A, DraC-C64, DraC-C100A and DraC-W142A significantly reduced the adhesive ability of E. coli strains. The biological activity of the DraC mutants as a assembly platform for Dr fimbriae polymerization was verified by agglutination of human erythrocytes and adhesion to DAF localized at the surface of CHO-DAF⁺ and HeLa cells. The residue F4 of the DraC usher conserved among FGL and FGS chaperone-assembled adhesive organelles can be used to design pillicides blocking the biogenesis of Dr fimbriae. Because the draC and afaC-III genes share 100% identity the range of the virulence determinant inhibitors could also be extended to E. coli strains encoding afa-3 gene cluster. The investigations performed showed that the usher N-terminus plays an important role in biogenesis of complete fiber.     

Isolation and characterisation of dog uropathogenic Escherichia coli strains and their fimbriae

A number of Escherichia coli strains have been isolated from dogs with urinary tract infections. These strains have been characterised with respect to their O, K, H, and fimbrial antigens, colicin production, antibiotic resistance, plasmid content and their ability to haemagglutinate erythrocytes from various species. Crossed immunoelectrophoresis of fimbrial extracts, as well as the reaction of partly purified fimbriae of a number of these strains with monoclonal antibodies revealed homology or a strong crossereaction with an F12 fimbrial subunit protein of human uropathogenic E. coli strains. Unlike human F12 fimbriae producing strains, the dog isolates did agglutinate dog erythrocytes in the presence of D-mannose but not human erythrocytes, indicating that the adhesin carried by these strains is different from the adhesin on fimbriae of human uropathogenic E. coli. Similar indications were obtained from experiments with latex beads coated with the receptor for P-fimbriae. These beads were agglutinated by Escherichia coli strains from human urinary tract infections, but not by the dog isolates described here. Preliminary adhesion experiments of human and dog Escherichia coli to human bladder epithelial and canine kidney epithelial cells also showed differences in adhesion depending on the origin of the strain tested.     

Salivary film expresses a complex, macromolecular binding site for Streptococcus sanguis

Teeth in the oral cavity are coated with a salivary film or pellicle, which lacks apparent intermolecular organization. This heterogeneous film facilitates binding of early commensal colonizing bacteria, including Streptococcus sanguis. To test the hypothesis that sufficient intermolecular organization exists in salivary films to form binding sites for S. sanguis, an in vitro model of saliva-coated teeth was probed with murine anti-idiotypical monoclonal antibodies (mAb2, anti-ids). The anti-ids were harvested from hybridomas that were developed in response to first generation murine hybridomas that produced anti-S. sanguis adhesin monoclonal antibodies (mAb1). The anti-ids (i) reacted with experimental salivary films and inhibited S. sanguis adhesion in a dose-dependent fashion. In Western blots, the anti-ids (ii) recognized a high molecular weight salivary antigen and (iii) secretory IgA (sIgA) light chain and alpha-amylase. After isolation by gel filtration from whole saliva or mixed secretory IgA and alpha-amylase, the high molecular weight component, containing amylase activity and sIgA, bound to hydroxyapatite to promote adhesion of S. sanguis. Therefore, a complex enriched in secretory immunoglobulin A and alpha-amylase forms a S. sanguis-binding site.     

Establishment of a mouse model of cystitis and roles of type 1 fimbriated Escherichia coli in its pathogenesis.

The role of type 1 fimbriae in promoting bladder colonization and the course of Escherichia coli cystitis were examined with type 1 fimbriated strains of clinically isolated E. coli. In the experiments of mice in vivo, intact bladder epithelium showed natural resistance to the adherence of type 1 fimbriated and non-fimbriated E. coli. However, the exfoliation of bladder superficial cells by trypsinization before the bacterial inoculation promoted the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium. After colonization of E. coli, maximum numbers of E. coli and leukocytes were observed 3 days after inoculation. Nine days after inoculation, both of E. coli and leukocytes disappeared and the regeneration of superficial cells was observed. On the other hand, superficial cells in mice injected with phosphate-buffered saline or non-fimbriated E. coli regenerated 5 days after trypsinization. The present study demonstrated that the removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium in vivo and a new model of E. coli cystitis in mice was established. The model which we established is valuable for histopathological, immunological, and therapeutic studies.     

The influence of artificial colonization withE. coli strain O83 on the intestinal flora in infants

Dominant bacterial strains present in stool (with particular emphasis onE. coli strains) were examined in 4 groups of healthy infants: breast-fed and bottle-fed, colonized withE. coli O83, and control (non-colonized) breast-fed and bottle-fed newborns. The presence of fimbriae was examined by hemagglutination, the P-fimbriae-bearing strains were tested by the PPA latex test. In addition, adherence to cell line HT-29 and serotyping was performed in selected strains. TheE. coli strain O83 was found to possess type 1 fimbriae. Fewer bacterial strains possessing type 1 fimbriae were found inE. coli O83-colonized infants (except the O83 serotype) than in control infants. TheE. coli O83 strain colonized significantly better the breast-fed than the bottle-fed infants; its higher adherence activity was demonstrated even in cell line HT-29. Finally, colonization withE. coli O83 influenced the character of microbial intestinal flora: the frequency of positiveE. coli isolates was significantly higher in colonized (both breast- and bottle-fed) than noncolonized infants.     

Aggregation of Streptococcus sanguis biotypes I and II by parotid saliva: a comparison between peritrichously fibrillar and tufted strains

Twelve strains of Streptococcus sanguis biotype I and seven strains of Streptococcus sanguis biotype II carrying either peritrichous fibrils or tufts of fibrils, were examined for their susceptibility to aggregation by parotid saliva. Salivary aggregation was evaluated using a spectrophotometric measurement of sedimentation to assess clump size. A clear distinction emerged between structural sub-groups. Irrespective of biotype, strains carrying peritrichous fibrils aggregated strongly whilst tufted strains were little affected. The one strain with peritrichous fimbriae as well as fibrils, was not aggregated by saliva. Pre-treatment of two peritrichously fibrillar strains with parotid saliva reduced their ability to adhere to parotid saliva-coated hydroxyapatite, whereas adhesion of two tufted strains was not inhibited. Inhibition of adhesion may have been due to steric hindrance, but blocking of bacterial adhesins by saliva components could not be discounted.     

Adhesion of canine and human uropathogenicEscherichia coli andProteus mirabilis strains to canine and human epithelial cells

Escherichia coli strains causing urinary tract infections in dogs produce fimbriae composed of fimbrial subunits closely related to the F12 and F13 fimbriae of human uropathogenic strains [4]. The adhesins carried by the fimbriae of human and canine isolates differ, however, as concluded from a different hemagglutination pattern and from the fact that the dog strains do not agglutinate latex beads coated with P-fimbriae receptor. This possible difference in adhesive specificity was confirmed by experiments in which the adhesion of human and dog isolates to dog kidney epithelial cells (MDCK cells) and human bladder epithelial cells (T24 cells) was compared. Dog uropathogenic strains, in contrast to human uropathogenicE. coli strains, adhere to MDCK cells but hardly to T24 cells. Adhesion to MDCK cells correlates with the presence of F12 or F13 fimbriae on the dog strains. These results suggest that homologous fimbrial subunits can carry different adhesin molecules and that these adhesin molecules can be responsible for species-specific adherence. On the contrary, adhesion of a number of dog uropathogenicProteus mirabilis strains to MDCK and T24 cells was not species specific; it depended on the mere presence of fimbriae.     

Virulence factors of Escherichia coli isolated from urine of diabetic women with asymptomatic bacteriuria: correlation with clinical characteristics

Since Escherichia coli isolated from compromised patients with symptomatic urinary tract infections (UTIs) express fewer virulence factors than those isolated from healthy controls, the question arises whether this is also the case for diabetic patients with asymptomatic bacteriuria (ASB). Polymerase chain reaction (PCR) assays were conducted on 111E. coli strains, isolated from the urine of diabetic women with ASB, using primers for the major subunit A and the G-adhesin (I, II, and III) of P fimbriae, type 1 fimbriae, S fimbriae, afimbrial adhesin, cytotoxic necrotizing factor (CNF), and aerobactin. Phenotypically, hemolysis, mannose-sensitive hemagglutination, mannose-resistant hemagglutination and O:K:H-serotypes were determined. Furthermore, we investigated the associations between virulence factors and patient characteristics (including deterioration of renal function). Type 1 fimbriae were the most prevalent virulence factor (86% by genotyping and 59% phenotypically). Except for a lower prevalence of known uropathogenic O-serotypes, we found the same number of virulence factors in our compromised patient group as listed in the literature in noncompromised patients with ASB. Certain virulence factors (type 1 and S fimbriae and CNF) of the causative E. colicorrelated with the risk of a decline in renal function. In conclusion, the number of virulence factors in E. coli isolated from the urine of diabetic women with ASB are comparable with the results found in other (noncompromised) patients with ASB. Furthermore, certain virulence factors of E. colimight contribute to a decline in renal function.     

Epitope Specificity of a Monoclonal Antibody Generated against the Dissociated CFA/I Fimbriae of Enterotoxigenic Escherichia coli

A monoclonal antibody (MAb 84) raised against the dissociated CFA/I fimbriae of enterotoxigenic Escherichia coli was characterized with regard to antigen binding and epitope specificity. Enzyme-linked immunosorbent assay (ELISA) showed that MAb 84 had higher affinity to CFA/I subunits than to intact CFA/I fimbriae and recognized a Salmonella flagellin carrying an insert corresponding to amino acids 32 to 45 of the CFA/I subunit. Fine epitope mapping based on the Pepscan technique showed that the peptide ³⁹TFESY⁴³, derived from the sequence of the mature CFA/I subunit, was specifically recognized by MAb 84. The ³⁹TFESY⁴³ sequence is probably not accessible on the surface of the native CFA/I fimbriae since MAb 84 did not bind to intact fimbriae as evaluated in inhibition ELISA tests. Moreover, MAb 84 did not agglutinate fimbriated ETEC cells nor inhibit CFA/I-mediated hemagglutination or the adhesion to Caco-2 cells.     

Fimbriae in Escherichia Coli Isolated from the Small Intestine of Piglets

Ninety E. coli strains, isolated from piglets which had died from neonatal diarrhea, were tested for the presence of K88, K99, 987P and type 1 fimbriae. Two or more types of fimbriae were demonstrated in 14 of the strains, a single fimbria! type in 44 strains while in 32 strains no fimbriae were detected. Of the 14 E. coli strains with more than 1 type of fimbriae, 12;, 10, 8 and 4 strains showed K88, K99, 987P and type 1, respectively. Twelve E. coli strains were isolated from piglets which had died in the neonatal period without showing signs of neonatal diarrhea at necropsy. One strain showed 987P and 3 strains showed type 1 fimbriae, while the remaining 8 strains were unfimbriated. Sixteen fimbriated E. coli strains were subcultured in order to examine the stability of fimbrial expression in the strains. The K88 and the type 1 fimbriae were regularly expressed, while the K99 and 987P were inconsistently demonstrated.     

Effects of Lactobacillus reuteri PTA 5289 and L. paracasei DSMZ16671 on the Adhesion and Biofilm Formation of Streptococcus mutans

Probiotics have decreased the counts of salivary mutans streptococci (MS) in clinical studies. The aim of this study was to compare the effects of Lactobacillus reuteri PTA 5289 and L. paracasei DSMZ16671 on the adhesion of a reference strain and a clinical isolate of Streptococcus mutans and on the counts of MS in a biofilm. The adhesion of S. mutans Ingbritt and the clinical isolate S. mutans 2366 to a smooth glass surface and saliva-coated hydroxyapatite (SHA) were studied in the presence of and without the lactobacilli. A three-species biofilm formed on saliva-coated hydroxyapatite discs was used in the biofilm experiments. The lactobacilli did not affect adhesion to the glass surface but interfered with binding to SHA. No effects of the lactobacilli were detected on the MS levels in the three-species biofilms. The results of the SHA binding experiments best reflected the results of the existing clinical studies.     

Expression of functional Porphyromonas gingivalis fimbrillin polypeptide domains on the surface of Streptococcus gordonii.

Genetically engineering bacteria to express surface proteins which can antagonize the colonization of other microorganisms is a promising strategy for altering bacterial environments. The fimbriae of Porphyromonas gingivalis play an important role in the pathogenesis of periodontal diseases. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to be an important virulence factor, which likely promotes adherence of the bacterium to saliva-coated oral surfaces and induces host responses. Immunization of gnotobiotic rats with synthetic peptides based on the predicted amino acid sequence of fimbrillin has also been shown to elicit a specific immune response and protection against P. gingivalis-associated periodontal destruction. In this study we engineered the human oral commensal organism Streptococcus gordonii to surface express subdomains of the fimbrillin polypeptide fused to the anchor region of streptococcal M6 protein. The resulting recombinant S. gordonii strains expressing P. gingivalis fimbrillin bound saliva-coated hydroxyapatite in a concentration-dependent manner and inhibited binding of P. gingivalis to saliva-coated hydroxyapatite. Moreover, the recombinant S. gordonii strains were capable of eliciting a P. gingivalis fimbrillin-specific immune response in rabbits. These results show that functional and immunologically reactive P. gingivalis fimbrillin polypeptides can be expressed on the surface of S. gordonii. The recombinant fimbrillin-expressing S. gordonii strains may provide an effective vaccine or a vehicle for replacement therapy against P. gingivalis. These experiments demonstrated the feasibility of expressing biologically active agents (antigens or adhesin molecules) by genetically engineered streptococci. Such genetically engineered organisms can be utilized to modulate the microenvironment of the oral cavity.     

Escherichia coli K‐12 possesses multiple cryptic but functional chaperone–usher fimbriae with distinct surface specificities

Commensal and pathogenic Escherichia coli adherence to host and environmental surfaces is mediated by a variety of adhesins. Although extensively studied as a model bacterium, 34% of the genes in the E. coli K‐12 genome have no known function. We hypothesized that some of them may correspond to functional adhesins. We characterized E. coli K‐12 ycb, ybg, yfc, yad, yra, sfm and yeh operons, which display sequence and organizational homologies to type 1 fimbriae exported by the chaperone/usher pathway. We showed that, although these operons are poorly expressed under laboratory conditions, six of them are nevertheless functional when expressed, and promote adhesion to abiotic and/or epithelial cell surfaces. While the studied fimbriae display different binding specificities, we obtained evidence of synergy/interference with other adhesins such as Ag43 or type 1 fimbriae. We showed that their expression is under the negative control of H‐NS and, except for yad, subjected to cAMP receptor protein‐mediated activation and carbon catabolite repression. These results therefore demonstrate that ycb, yfc, yad, yra, sfm and yeh operons encode cryptic but functional fimbriae adhesins whose expression following environmental modifications could contribute to E. coli's ability to adhere to and colonize a wide diversity of surfaces in its various ecological niches.     

Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion

Escherichia coli was used as a model to study initial adhesion and early biofilm development to abiotic surface. Tn10 insertion mutants of Escherichia coli K-12 W3110 were selected for altered abilities to adhere to a polystyrene surface. Seven insertion mutants that showed a decrease in adhesion harbored insertions in genes involved in lipopolysaccharide (LPS) core biosynthesis. Two insertions were located in the rfaG gene, two in the rfaP gene, and three in the galU gene. These adhesion mutants were found to exhibit a deep-rough phenotype and to be reduced, at different levels, in type 1 fimbriae production and motility. The loss of adhesion exhibited by these mutants was associated with either the affected type 1 fimbriae production and/or the dysfunctional motility. Apart from the pleiotropic effect of the mutations affecting LPS on type 1 fimbriae and flagella biosynthesis, no evidence for an involvement of the LPS itself in adhesion to polystyrene surface could be observed. Received: 1 December 1998 / Accepted: 3 April 1999