Clonal differentiation of uropathogenic Escherichia coli isolates of serotype O6:K5 by fimbrial antigen typing and DNA long-range mapping techniques.

Escherichia coli isolates of serotype O6:K5 are the most common causative agents of cystitis and pyelonephritis in adults. To answer the question, as to whether strains of this particular serotype represent one special clonal group, out of a collection of 34 serotype O6:K5 isolates [Zingler et al. (1990) Zentralbl. Bakteriol Mikrobiol Hyg [A] 274:372-381] 15 strains were selected and analyzed in detail. The flagellar (H) antigen and the outer membrane protein (OMP) pattern were determined. Further serum resistance properties and the genetic presence and expression of other virulence factors, including hemolysin, aerobactin, P fimbriae, S/F1C fimbriae and type 1 fimbriae was evaluated. In addition the XbaI-macrorestriction pattern of ten representative isolates was elaborated and the fimbrial (F) antigen type of the P fimbriae was determined, to obtain the complete O:K:H:F pattern. These analyses could clearly show that the O6:K5 isolates do not represent one clonal group. The XbaI-macrorestriction profiles were heterogeneous and marked differences in the hybridization patterns, using virulence-associated gene probes in Southern hybridization of long-range-separated genomic DNA, were observed among the strains. However, some of strains showed similarities in the genomic profiles, arguing for clonal groupings among the O6:K5 isolates. Interestingly the strains grouped together exhibited the same fimbrial F type that many indicate a coincidence of this phenotypic trait with clonality.

Is Escherichia coli invading tubuloepithelial cells?

The adhesion of Escherichia coli to host epithelium cells is the very first step of urinary tract infections followed by the internalization of the bacteria into these cells. These steps are influenced by several surface antigens or products of the pathogen, e.g. fimbriae or adhesins, K antigen, and hemolysin. The bacterial adherence and the internalization of several isogenic E. coli O18 strains differing in the expression of K5 antigen, hemolysin, and type of fimbriae were measured by using a permanent line of porcine tubuloepithelial cells (LLC-PK1). Strains with K5 antigen were reduced in their adherence and internalizability as compared to the K-negative strains. The expression of hemolysin by these strains lead to an increase of adherence and internalization. The internalization of bacteria is influenced mainly by their adherence to the epithelial cells. Thus, the engorgement of attached bacteria is rather a kind of endocytosis than an invasion of bacteria.

Clonal analysis of Escherichia coli serotype O6 strains from urinary tract infections.

A total of 36 Escherichia coli urinary tract isolates (UTI) of serotype O6, with different combinations of capsule (K) and flagellin (H) antigens, were analysed according to the outer membrane pattern (OMP), serum resistance properties, mannose-resistant hemagglutination using various types of erythrocytes, and also for the genetic presence and the expression of P-fimbriae, S fimbriae/F1C fimbriae, Type 1 fimbriae, aerobactin and hemolysin. Twenty selected strains were further analysed by pulsed field gel electrophoresis (PFGE), elaborating genomic profiles by XbaI cleavage and subsequent Southern hybridization to virulence-associated DNA probes. It could be shown that O6 UTI isolates represent a highly heterogeneous group of strains according to the occurrence and combination of these traits. Relatedness on the genetic and the phenotypic level was found for some of the strains exhibiting the same O:K:H:F serotype. DNA long-range mapping further indicated some interesting features, according to the copy number and the genomic linkage of virulence genes.

Biochemical and serological patterns of Escherichia coli O2 strains isolated from patients with urinary tract infections.

We examined the results of two biochemical test systems for their ability to discriminate a series of 58 Escherichia coli O2 strains collected from patients with urinary tract infections. The O:K:H serotypes and O antigen factors of the strains were also determined. The strains could be assigned to 13 distinct serological patterns by means of O2 antigen factors as well as K and H antigens. The combination of O:K:H-serotyping with biotyping enabled very fine strain discrimination. These data indicate that the biotyping system of Achtman (BTA) is more precise than the Enterobacteriaceae-code-system described by Reiske (ECR) and that the determination of BTA types supports the identification of bacterial clones.

Serum resistance in different serotypes of Escherichia coli.

Resistance to complement-mediated serum activity is an important virulence factor in E. coli isolated from extraintestinal infections. Because there are no reports about the percentage of serum-resistant E. coli strains in common O serogroups, the study was carried out using Taylor's method (75% serum) for the determination of serum resistance of 253 E. coli strains, which had been isolated from urinary tract infections. The strains belonged to 8 common serogroups (O1, O2, O4, O6, O9, O16, O18, and O75) with a frequency of 6 to 24%, 218 (86%) were encapsulated. Among 26 different K antigens, K1 and K5 could be found in 32 and 33%. 25% of all strains investigated were found to be serum-resistant. The percentage of serum-resistant strains was between 11% and 63% in the different O serotypes, the highest frequency was found in O6 (63%) and O2 strains (43%). Among all serum-resistant strains carrying 13 different K antigens, K1 and K5 were the most common ones, with a percentage of 62% altogether. Serum resistance can be expected in strains from urinary tract infections, with a quite varying frequency depending on the O serotype, certain K antigens and other factors.

K-antigen identification, hemolysin production, and hemagglutination types of Escherichia coli O6 strains isolated from patients with urinary tract infections.

Serotyping of 1918 Escherichia coli strains isolated in significant cell numbers from the urine of patients with urinary tract infections (UTI) revealed the presence of 117 O6 strains. The K antigens were identified by means of K-specific phages and serological methods. The phages used included a K1 phage pool (phi 1, A-E) and the separate phages phi 2, phi 5, phi 7, phi 12 and phi 13. The presence of H antigens, type 1 fimbriae formation, hemolysin production and mannose-resistant hemagglutination (MRHA) ability with human A, sheep, calf and pig erythrocytes were also analyzed. Six different MRHA types were defined and discussed in relation to the O6:K:H serotype. Remarkably, E. coli O6 strains were found to possess a whole arsenal of virulence factors (K antigens, MRHA, hemolysin). The most common serotypes - O6:K2:H1/H- (26), O6:K5:H1/H- (35) and O6:K13:H1/H- (20) - differed from each other in some cases in both MRHA type and hemolysin production.

[Hemagglutinating ability and fimbrial antigens of Escherichia coli strains isolated from urine]. / Hämagglutinationsvermögen und Fimbrien-Antigene von aus Urin isolierten Escherichia coli-Stämmen.

The close connection between mannose-resistant hemagglutination (MRHA) and adhesion to uroepithelial cells of urinary E. coli with regard to the pathogenesis of urinary tract infection (UTI) prompted us to examine the hemagglutinating ability of 1499 E. coli strains from urine using human blood group OP1 erythrocytes. In 317 strains (21.2%), an MRHA was found. There were no significant differences in the prevalence of MRHA related to the isolation time and admitting hospital. A correlation was found between MRHA and the presence of P fimbriae in the strains investigated. Another association appears to exist between certain O:K:H serovars and distinct fimbrial antigens which had been serologically identified. The F11 antigen was detected most frequently and proved to be present in strains of serovars O1:K1:H-, O1::K1:H7, O2:K1:H-, O2:K1:H4, O2:K1:H7, and O15:K1:H7. The F8 antigen was strongly associated with serovar O18:K5:H-. O18:K5:H1 and O6:K5:H1 were apparently related to cross-reacting F14 antigens.

[Rapid typing of Escherichia coli K antigens using bacteriophages]. / Schnelltypisierung von Escherichia coli-K-Antigenen mit Hilfe von Bakteriophagen.

E. coli capsular (K) antigens are important virulence factors contributing to the development of urinary tract infections (UTI). Serotyping of these antigens is laborious and depends on the availability of respective antisera which are difficult to prepare because of the low immunogenicity of these polysaccharide antigens. The application of specific K phages are a big advantage. Two collections of E. coli strains (500 from Rostock, 214 from Erfurt) isolated from patients with UTI have been investigated with a set of ten K specific bacteriophages including phi 1, phi 2, phi 5, phi 7, phi 9, phi 12, phi 13, phi 20, phi 30 and phi 36. The K1 antigen has been found to be the most frequent capsular antigen (16.4% in Rostock; 12.2% in Erfurt) followed by K13/K20 in Erfurt (8.9%) and K5 in Rostock (8%) and Erfurt (7.5%). The collection of phages proved to be suitable for the detection of UTI associated K antigens covering the most common O serogroups found in UTI. The method appears to be simple, non-laborious and applicable for routine use.

[Excretion of antibody-coated bacteria in chronic pyelonephritis]. / Zur Ausscheidung antikörperbeladener Bakterien bei chronischer Pyelonephritis.

Of 168 urine sediments, which were obtained from 55 patients with chronic pyelonephritis in the course of 3 years when a significant bacteriuria with E. coli was present, we demonstrated antibody-coated bacteria in 81 cases (48.21%). In the active stage of the disease with 54.10% were found significantly more than in the inactive with 32.61%. In obstructive chronic pyelonephritis the positive rate was 54.79% in contrast to 43.16% in non-obstructive chronic pyelonephritis. While in the active stage of the obstructive chronic pyelonephritis with 57.41% more antibody-coated bacteria were excreted only ACB+-, 34.29% only ACB-- and 40% ACB+- and ACB--germs in the course of the disease. The ACB-positive quote was in rough forms with 62.50% statistically significantly increased in contrast to 45.54% in O-typable and 42.86% in non-O-typable strains. In the ACB+-group the immunofluorescence titres to the homologous strain and the C3-activator in the serum as well as the urine lysozyme were significantly higher than in the ACB--group.

[O- and H-antigens of Escherichia coli K5 isolates in the urine]. / O- und H-Antigene von Escherichia coli-K5-Isolaten aus Urin.

88 E. coli-K5-strains identified by a K5-phage were serotyped with regard to O- and H-antigens and 19 different O:K5:H-serotypes were registered. Further 11 isolates were rough strains and 2 other strains could not be O-typed. The following frequency of O:K:H-serotypes (number) was determined: O75:K5:H- (21), O18:K5:H- (11), O6:K5:H1 (9), and O18:K5:H1 (6). Seven other O:K:H-serotypes were detected once only. The results of typing are different in dependence of the geographic area and the time of isolation, respectively.

Studies on clonal assignment of urinary Escherichia coli O1:K1 strains.

Fifty-nine Escherichia coli strains belonging to two clonal groupings were investigated for major outer membrane proteins, colicin production, and partly for plasmid DNA content. The membrane protein patterns of the 01:K1:H7(H-):F11 and O1:K1:H-:F9 strains obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were distinctly different from each other and, therefore, are useful for clonal assignment. All of the F11 isolates had one plasmid of about 85 Md in common which is suggested to be characteristic for the clone. Their content of smaller plasmid DNA was heterogeneous and showed geographical differences. The F9 strains showed a plasmid pattern different from the F11 strains. A plasmid of 2.6 Md was found in all of these isolates. Colicin production was found to be useful for clonal assignment only in two thirds of the F9 and not in the F11 strains. Some strains with identical properties seem to be of epidemic importance.

[Defense mechanisms of the macro-organism in bacteriuria in chronic pyelonephritis]. / Abwehrmechanismen des Makroorganismus zum Zeitpunkt der Bakteriurie bei chronischer Pyelonephritis.

In the course of 4 years we isolated 193 E. coli strains of 55 patients with chronic pyelonephritis. In patients with obstructive chronic pyelonephritis the mean value of the immunofluorescence titre (in the serum) to the E. coli strain excreted in the urine as well as the total complement were significantly increased, the serum lysozyme was significantly lower than in patients with non-obstructive chronic pyelonephritis. A relation to the activity of the disease was existing only in the non-obstructive chronic pyelonephritis, where in the active stage the total complement was significantly decreased, the complement factors C3 and C4 as well as the urine lysozyme were significantly increased in comparison to the inactive stage. 94.64% of all immunofluorescence titres obtained to the homologous strain in the patients' serum were above the border of the normal area of 1:40. A relation between level of the titre and activity of the disease could not be established. No significant differences could be proved between the titres taken to serum-sensitive and serum-resistant strains. In 32.73% of the patients we observed disturbances of the serum bactericidia against the homologous serum-sensitive E. coli urine strain at one or several points. They fall to equal shares to patients with obstructive and non-obstructive chronic pyelonephritis and were found at 66.67% in the active stage of the two forms of the disease. In patients with and without disturbances of bactericidia no significant differences in the total complement, in the complement factors C3 and C4, the C3-activator, the serum lysozyme and the immunofluorescence titres could be proved.

Stimulation of particle-induced chemiluminescence in human granulocytes by various Escherichia coli strains.

The patterns of neutrophil chemiluminescence stimulated by groups of E. coli strains opsonized with pooled normal human serum were compared. All strains of E. coli were obtained from patients with chronic pyelonephritis. 'O' serovars which lacked capsular (K) antigen produced significantly higher chemiluminescence than strains possessing a variety of K antigens. Chemiluminescence produced by a range 'O' serovars bearing the K1 antigen did not differ significantly from those containing other K antigens. The lowest chemiluminescence values were obtained with a group of O2:K1 strains. Rough strains containing K1 antigen were also poor stimulators of chemiluminescence.

[Occurrence of Escherichia coli K1 and K5 strains in patients with urinary tract infections]. / Vorkommen von Escherichia-coli-K1- und -K5-Stämmen bei Patienten mit Harnwegsinfektionen.

1499 E. coli isolates from patients with urinary tract infections were investigated for the presence of K1 and K5 strains using specific phages. K1 antigen was detected in 10,5% and K5 in 6,1% of the strains examined. The strains were isolated in two laboratories with different hospitals and outpatient departments. With respect to the isolation area and isolation time the frequency of K1 and K5 strains was found to be different.

[Serum resistance of Escherichia coli and serum bactericidal activity in chronic pyelonephritis]. / Serumresistenz von Escherichia coli und Serumbakterizidie bei chronischer Pyelonephritis.

123 patients with chronic pyelonephritis of the renal dispensary of the clinic of internal medicine of the Department of Medicine Wilhelm Pieck University Rostock were controlled microbiologically for one to four years. Of 59 patients of this group with significant bacteriuria we cultivated 170 strains of E. coli, which were standardised O, K, and H. Of all causative organisms the serum resistance was determined in a human serum pool. All sera of patients were examined for their bactericidal activity to the homologous E. coli urine strain as well as to a serum-sensitive E. coli reference strain (E. coli O 22:K-:H+). In 11.8% we found in the sera investigated at the time of the excretion of causative organisms disturbances of the serum bactericidal action, which rendered possible the survival of the sensitive in the human serum pool causative organism. In 8.82% of the sera of patients at the time of bacteriuria there was a decreased bactericidal action against the E. coli reference strain, only two of these sera did also not kill the urine strain of the patients. In individual cases sera of patients were examined for bactericidal activity with the homologous germ after absorption. By this absorption the antibactericidal activity of the sera could be removed. The proof of a disturbance of a bactericidal action may be an important reference for the course of the chronic pyelonephritis.

Fimbrial antigens of Escherichia coli 01:K1:H7 and 01:K1:H- strains isolated from patients with urinary tract infections.

Thirty E. coli strains belonging to serotypes 01:K1:H7 and 01:K1:H- were examined for the presence of fimbrial (F) antigens by crossed immunoelectrophoresis. All strains exhibited a mannose-resistant haemagglutination of human and monkey erythrocytes. This ability was found to be highly correlated with the existence of F antigens. Another correlation was found between O:K:H serotype and F antigen. Eleven 01:K1:H7 strains were associated with fimbrial antigen F11 which was also demonstrated in five immotile variants of this serotype. In other fourteen strains of serotype 01:K1:H-, however, the F9 antigen could be detected. The F9 and F11 antigens belong to the group of P fimbriae. The 01:K1:H7:F11 and 01:K1:H-:F9 isolates were supposed to be of different clonal origin.