Mutations in cloned Escherichia coli P fimbriae genes that makes fimbriae-production resistant to suppression by trimethoprim.

The effect of sublethal concentrations of trimethoprim on the expression of P fimbriae was tested in Escherichia coli HB101 recombinant strains. Fimbriation was inhibited at trimethoprim concentrations down to at least 1/64 of the minimal inhibitory concentration. However, the expression of the P fimbrillin by recombinant plasmids containing deletions in front of the fimbrillin gene did not respond to the inhibitory effect of trimethoprim indicating that trimethoprim may act at the level of gene regulation.

Nucleotide sequence analysis of a P fimbrial regulatory element of the uropathogenic Escherichia coli strain KS71 (04:K12).

The nucleotide sequence of a trans-acting P-fimbrial regulatory element obtained from the uropathogenic Escherichia coli strain KS71 (04:K12) was determined. The regulatory element was found to contain an open reading frame of 231 nucleotide residues that showed 95.2% homology with papl, a functionally analogous regulatory gene of E. coli strain J 96.

Location of adhesion sites for P-fimbriated and for 075X-positive Escherichia coli in the human kidney.

High-affinity binding sites for P-fimbriated and for 075X-positive Escherichia coli were located in the human kidney. Frozen sections of normal human kidney were double-stained first with fluorochrome-labelled bacteria and then with fluorochrome-labelled nephron site-specific lectins or antibodies. The P-fimbriate recombinant E. coli strain used showed specific adherence to glomerular structures, to the lumen of proximal and sital tubules and to vascular endothelium but did not adhere to collecting ducts or to peritubular sites. Two E. coli strains having the 075X adhesin showed specific adherence to renal interstitium, to glomerular elements and to Bowman's capsule. The method described allows the detailed determination of tissue-substructure specificity of bacterial adhesion. Our results demonstrate tissue tropism in the adhesion of E. coli to human kidneys and suggest a pathogenetic role for X adhesins.

Genetic relationships and clonal structure of strains of Escherichia coli causing neonatal septicemia and meningitis.

Genetic diversity and relationships among 63 isolates of Escherichia coli from infants in Finland with septicemia or meningitis were assessed by analyzing electrophoretic variation in 21 enzymes encoded by chromosomal genes. Thirty-nine multilocus genotypes (electrophoretic types) were distinguished, 23 of which formed a closely related, distinctive subset (group 1) of five or six clones represented by 40 (63%) of the isolates. The remaining isolates represented a second subset of 16 electrophoretic types (group 2) that were, on the average, rather more distantly related to one another. Although the number of electrophoretic types causing neonatal systemic disease is smaller than that occurring in healthy intestinal floras, the pathogenic electrophoretic types are only slightly less diverse genetically. Isolates of group 1 were characterized by relatively high incidences of hemolysin production and S, type 1, type 1C, and P fimbriae. However, because phenotypic characters, considered individually or in combination, did not adequately reflect the overall genetic relationships of isolates, it is recommended that the genetic structure of populations be defined on the basis of multilocus chromosomal genotypes.

Aerobactin-mediated iron uptake by Escherichia coli isolates from human extraintestinal infections.

A total of 516 strains of Escherichia coli were screened for the presence and expression of the aerobactin iron uptake system. The incidence was markedly higher among clinical isolates from patients with septicemia (68.8%), pyelonephritis (74.6%), and symptomatic (59.8%) and asymptomatic (63.2%) lower urinary tract infections than among normal human fecal isolates (34.3%).

Analysis of P fimbriae on Escherichia coli O2, O4, and O6 strains by immunoprecipitation.

P fimbriae on Escherichia coli O2, O4, and O6 strains were analyzed by immunoprecipitation. Fimbrial extracts were prepared from a total of 35 strains and tested for precipitation with four anti-P-fimbria sera. The overall fimbrial composition of the strains was related to the O:K:H serotype, and two to three P fimbrial variants per strain were found on most of the O4 and some of the O6 strains. The O2 strains, in contrast, showed only one antigenic variant of P fimbriae per strain, which was serologically unrelated to those of the O4 and O6 strains. The results stress the multiplicity and serological complexity of E. coli P fimbriae.

Fimbriation and P-antigen recognition of Escherichia coli strains harbouring mutated recombinant plasmids encoding fimbrial adhesins of the uropathogenic E. coli strain KS71.

Deletion mutants of recombinant plasmids encoding the KS71B fimbrial antigens of the uropathogenic Escherichia coli strain KS71 (O4:K12) were constructed. The effects of these mutations were tested by transforming the mutated plasmids into non-fimbriated E. coli HB101 cells and testing the transformants for fimbriation and haemagglutination. A deletion transcriptionally upstream from the fimbrial subunit gene increased the expression of KS71B fimbriae. Deletion of the fimbrial subunit gene resulted in non-fimbriated but haemagglutinating transformants, whereas a deletion 6 kb transcriptionally downstream from the subunit gene resulted in non-haemagglutinating but fimbriate transformants, indicating that fimbriation and haemagglutination were genetically separable. We also present evidence suggesting that the fimbrillin and haemagglutinin are physically associated in the wild-type KS71 strain.

Clonal analysis of Escherichia coli O2:K1 isolated from diseased humans and animals.

Forty-six Escherichia coli isolates of serotype O2:K1 from human urinary tract infections, chicken sepsis, and bovine mastitis were obtained from laboratories in England, Denmark, Sweden, and Finland. The bacteria were compared for outer membrane protein (OMP) pattern, lipopolysaccharide pattern, electrophoretic mobilities of enzymes, and flagellar serotype and were tested for fimbriation, biotype, hydroxamate production, hemolysin production, antibiotic resistance, plasmid content, colicin production, and virulence in neonatal rats. Isolates from humans were assigned to two clonal groups; poultry isolates belonged to one of these clonal groups, whereas bovine isolates belonged to the other. Poultry and human isolates of the same clonal group could be distinguished only by their plasmid content. Strains within this group were heterogeneous with respect to biotype, fimbriation, virulence, and flagellar serotype. Human and bovine isolates of the second clonal group were distinguished by a minor change in OMP pattern and by their plasmid content. It is concluded that meaningful clonal groupings are best recognized by the combination of OMP and electrophoretic enzyme patterns. The O:K serotype can aid in the recognition of important subclones, whereas the other microbiological properties tested can vary widely within clonal groupings. Furthermore, we conclude that certain O:K serotypes can contain very different clonal groupings having little genetic relatedness.

Organization of genes expressing the blood-group-M-specific hemagglutinin of Escherichia coli: identification and nucleotide sequence of the M-agglutinin subunit gene.

The organization of genes encoding the blood group M-specific hemagglutinin (M-agglutinin) of Escherichia coli strain IH11165 was studied with a cloned 6.5-kb DNA segment. This DNA segment contains at least five genes which code for the polypeptides of 12.5, 30, 80, 18.5 and 21 kDa. The 30-, 80- and 21-kDa polypeptides are synthesized as precursors that are approximately 2 kDa larger. The 21-kDa polypeptide was identified as the M-agglutinin subunit by its reactivity with anti-M-agglutinin serum. Nucleotide sequence analysis of the corresponding gene showed that the M-agglutinin precursor had a 24-amino acid (aa) signal sequence, while the mature protein is 146 aa residues long. Although the organization of the M-agglutinin gene cluster resembles those of other E. coli adhesins, there is no significant sequence homology between the M-agglutinin subunit and the subunits of the other potentially related proteins in E. coli.

Occurrence of type-1C fimbriae on Escherichia coli strains isolated from human extraintestinal infections.

Two monoclonal antibodies specific for type-1C fimbriae of Escherichia coli were produced. In enzyme-linked immunosorbent assay and immunoblotting the antibodies, which were of the IgG1 isotype, reacted with type-1C, but not with P or type-1 fimbriae of E. coli strain KS71. Immunoblotting and immunoprecipitation of crude fimbrial extracts from 25 strains invariably gave an apparent molecular weight of 17 000 for the type-1C fimbrillin. A total of 313 E. coli strains, isolated from patients with extraintestinal infection or from faeces of healthy children, were screened for the presence of type-1C fimbriae using both the monoclonal and polyclonal antibodies. Of these, 45 (14%) strains had type-1C fimbriae, with the highest frequency (27%) on strains isolated from patients with pyelonephritis. No faecal strain had type-1C fimbriae, and the frequency on the other diagnostic groups ranged from 11 to 15%. Thus, no direct correlation between type-1C fimbriae and bacterial virulence in human extraintestinal infections was found. Type-1C fimbriae were detected on only a few E. coli serotypes, notably on all O6:K2:H1 and O22:K13:H1 strains tested.

Serotypes, hemolysin production, and receptor recognition of Escherichia coli strains associated with neonatal sepsis and meningitis.

Sixty-three Escherichia coli strains isolated from neonatal sepsis or meningitis were studied and compared with previous data on fecal or urinary pyelonephritis-associated isolates from children. Characteristics significantly associated with neonatal infection were capsular type K1 (54%), O group 18 (27%), rough-type lipopolysaccharide together with K1 capsule (19%), and S fimbriae (29%). Within the neonatal infection group, the K1 capsule and rough lipopolysaccharide were most common among the youngest infants (0 to 21 days old) and in meningitis. Hemolysin production, P fimbriae, and X adhesions (adhesions not identifiable as type 1, P, or S) were significantly more common in the two materials from infections as compared with the fecal isolates. One large clone of 11 strains (O18:K1:H7, with both type 1 and S fimbriae) and three smaller ones (O7:K1:H1 and O6:K2:H1, both with type 1 and P fimbriae and X adhesions; and R:K1:H33 with no adhesions) were identified among the strains from neonatal infections. Only O6:K2:H1 strains were also common among the strains from pyelonephritis.

Organization of fimbriate cells in colonies of Escherichia coli strain 3040.

Immunofluorescence staining with fimbria-specific antibodies was used to study the organization of fimbriate cells in colonies of Escherichia coli strain 3040. The strain has both type-1 and S fimbriae and shows fast phase variation between the fimbrial types. Colonies stained in sectors whose length and number per colony were dependent on the fimbrial phase of progeny cells. It is proposed that such sectors result from fimbrial phase variation.

Identification of the major human sialoglycoprotein from red cells, glycophorin AM, as the receptor for Escherichia coli IH 11165 and characterization of the receptor site.

The pyelonephritogenic Escherichia coli strain 1 H 11165 specifically agglutinates human erythrocytes carrying the M blood group antigen. The polymorphic forms of this antigen, M and N, are located in the NH2-terminal region of the major human red-cell sialoglycoprotein, glycophorin A. Radioactively labeled glycophorin A from M cells specifically bound to the bacteria. Purified glycophorin AM, but not glycophorin AN, efficiently inhibited for binding. Mild periodate treatment oxidized the NH2-terminal serine in glycophorin AM and this resulted in loss of binding to the bacteria. High concentrations of serine and alkali-labile oligosaccharides derived from glycophorin AM inhibited the binding, whereas the synthetic M-specific NH2-terminal pentapeptide Ser-Ser-Thr-Thr-Gly did not. Neuraminidase treatment of glycophorin AM did not destroy the binding. The most efficient inhibition of binding was observed with the N-terminal glyco-octapeptide obtained from glycophorin AM by CNBr cleavage. This peptide contains both the essential serine residue and the alkali-labile oligosaccharides, which both are recognized by the bacterium.

Complementation and regulatory interaction between two cloned fimbrial gene clusters of Escherichia coli strain KS71.

Complementation experiments with cloned DNA fragments encoding either the KS71A, the KS71B or the KS71C fimbriae of the pyelonephritogenic Escherichia coli strain KS71 were used to localise the P-fimbrillin genes and to demonstrate regulatory interactions between the cloned genes. The structural genes of the KS71A and KS71B fimbriae were located within a common 1.1 kilobase pair ClaI-SmaI fragment, and it was shown that the gene clusters for these fimbriae could complement each other in trans. The gene cluster encoding the KS71C fimbriae did not complement for the other KS71 fimbriae. A DNA fragment, located near the KS71A fimbrillin gene, was found to enhance the production of the KS71B fimbriae in trans.

Fimbria-like hemagglutinin of Escherichia coli O75 strains.

Fifteen strains of Escherichia coli O75 from human feces and patients with urinary tract infections were analyzed for their hemagglutinative properties, production of hemolysin and colicin, and plasmid contents. Fourteen strains produced type-1 fimbriae in broth culture. Nine of the strains agglutinated human P1 and p erythrocytes, i.e., possessed an X adhesin (X hemagglutinin). All but one of the X+ strains agglutinated human but not sheep or rabbit erythrocytes. Of the 15 strains, 4 had P fimbriae; one strain had both X hemagglutinin and P fimbriae. A coli-like structure was found by electron microscopy on the surface of the X+ strains. This structure was purified from two strains and characterized chemically and serologically. It was a protein consisting of subunits with an apparent molecular weight of 16,000. The amounts of hydrophobic amino acids in proteins purified from strains IH11033 and IH11128 were 35 and 39%, respectively. The amino acid content of the proteins was quite similar; only glutamine-glutamate and tyrosine contents were different. The radiolabeled protein bound to human erythrocytes, but it differed morphologically from typical E. coli fimbriae. Several methods showed the hemagglutinin, termed O75-X, to be serologically related in all the O75 X+ strains. The eight X+ O75:K5:H- strains had type-1 fimbriae and an identical outer membrane protein pattern, lacked P fimbriae and hemolytic activity, and are proposed to represent a clonal group.

Immunofluorescence study of fimbrial phase variation in Escherichia coli KS71.

An immunofluorescence assay was developed to study fimbrial phase variation in a pyelonephritogenic Escherichia coli strain, KS71. By using fluorochrome-labeled antibodies specific for either P, type-1C, or type-1 fimbriae of strain KS71, it was shown that in a broth culture of strain KS71 the fimbrial types mostly occurred on different cells. Only 9% of the cells carried more than one fimbrial type. The KS71 cell population was fractionated into subpopulations expressing only one of the fimbrial types or lacking fimbriae. Immunofluorescence assay of the subpopulations revealed a rapid phase variation in fimbrial synthesis. Kinetic analyses of a nonfimbriated cell population suggested that a change from one fimbrial phase to another was not totally random.

Escherichia coli fimbriae recognizing sialyl galactosides.

Fimbriae recognizing sialyl galactosides (S fimbriae) were purified from an Escherichia coli strain. The S fimbriae were morphologically identical to type 1 and P fimbriae of E. coli and showed a hemagglutination that was abolished when erythrocytes were treated with neuraminidase. Hemagglutination by the purified fimbriae was inhibited by orosomucoid but not by its desialylated derivative. Of the oligosaccharides tested, sialyl-(alpha 2-3)-lactose and sialyl-(alpha 2-3)-N-acetyllactosamine had the strongest inhibitory activities. It was concluded that S fimbriae have the strongest affinity for (alpha 2-3)-linked sialyl galactosides. In the enzyme-linked immunosorbent assay, the hyperimmune serum to the S fimbriae reacted strongly with the homologous antigen but not with type 1, P, or nonhemagglutinating KS71C fimbriae of E. coli. Analogously, the hyperimmune sera to the other E. coli fimbriae did not react with the purified S fimbriae. The immunoprecipitation assay showed that S fimbriae on different E. coli serotypes shared immunological cross-reactivity.

P-fimbriated clones among uropathogenic Escherichia coli strains.

A total of 237 Escherichia coli strains isolated from the urine of patients with various forms of urinary tract infection or from feces of healthy children were analyzed for O group, possession of K1 capsule, type 1 fimbriae, P fimbriae, X adhesin, and production of hemolysin. Some of the strains were also analyzed for K and H antigens, outer membrane protein pattern, and plasmid content. P fimbriation, hemolysin production, and certain O groups were found to be significantly correlated with pyelonephritogenicity. Possession of type 1 fimbriae or of K1 capsule or plasmid content did not significantly correlate with virulence. Outer membrane protein patterns in 139 strains of the more common O groups were analyzed. Only one to three patterns, which varied between serotypes, were usually found within any one O group. Distinctive groups (clones) were found when the strains were grouped according to complete serotype, fimbriation, hemolysin production, and outer membrane protein pattern; also, the mean number of plasmids was typical of the strains in a given clone. Seven clones associated with pyelonephritis were found; together they accounted for 57% of the O serotypable strains from the pyelonephritis patients. The seven clones were P fimbriated but differed in their serotypes as follows: O1:K1:H7, O4:K12:H1, O4:K12:H5, O6:K2:H1, O16:K1:H6, or O18ac:K5:H7. All O1:K1:H7 strains observed fell into two clones according to the presence or absence of type 1 fimbriae and hemolysin production. One clone associated with cystitis was also found; this consisted of O6:K13:H1 strains lacking P fimbriae. Not a single representative of these eight clones was found among the fecal strains from the healthy children. They are proposed to represent virulent clones with special ability to cause human urinary tract infection.

Novel cell-binding activity specific for N-acetyl-D-glucosamine in an Escherichia coli strain.

Escherichia coli strains isolated from patients with different levels of urinary tract infection and from healthy persons were tested for their ability to haemagglutinate endo-beta-galactosidase-treated human erythrocytes. Among the 104 strains studied one revealed a strong agglutination reaction with the enzyme-treated erythrocytes. From the monosaccharides tested N-acetyl-D-glucosamine inhibited agglutination most effectively. Orosomucoid and asialo-orosomucoid had no effect on the haemagglutination whereas beta-galactosidase treated asialo-orosomucoid was inhibitory. These findings indicate that the E. coli strain studied contains a novel cell-binding activity with specificity for terminal N-acetyl-D-glucosamine residues.