Degradation studies on Escherichia coli capsular polysaccharides by bacteriophages.

The serologically and structurally related Eschrichia coli capsular polysaccharides (K antigens) K13, K20, and K23 were found to be depolymerized by the bacteriophages phi K13 and phi K20 to almost similar oligomer profiles as shown by polyacrylamide gel electrophoresis. The phage-polysaccharide interactions were followed by an increase of reducing 2-keto-3-deoxyoctulosonic acid due to a phage-associated glycanase that catalyzed the hydrolytic cleavage of common beta-ketopyranosidic 2-keto-3-deoxyoctulosonic acid linkages. The related E. coli K antigens K18, K22, and K100 as well as the Haemophilus influenzae type b capsular polysaccharide were degraded by bacteriophage phi K100 with different efficacy. It is suggested that phi K100 enzymatically cleaves ribitol-5-phosphate bonds as the only structural feature present in all the polysaccharides investigated.

The structure of the capsular polysaccharide from Klebsiella type 52, using the computerised approach CASPER and NMR spectroscopy.

The structure of the capsular polysaccharide from Klebsiella type 52 has been elucidated using an improved and extended version of the computerised approach CASPER and NMR spectroscopy as principal methods. A previous suggestion to the structure but without the anomeric prefixes, could be shown correct [H. Björndal et al., Carbohydr. Res., 31 (1973) 93-100]. The polysaccharide has a hexasaccharide repeat with the following structure: [formula: see text]

Characterization of urinary Escherichia coli O75 strains.

Forty-four Escherichia coli O75 strains from patients with urinary tract infections were characterized by a variety of methods to obtain evidence of their clonal distribution and uropathogenic properties. By K and H antigen typing, the strains were divided into the following serotypes: O75:K5:H- (18 strains), O75:K95:H- (10 strains), O75:K95:H5 (7 strains), O75:K100:H5 (4 strains), and O75:K-:H55 (5 strains). Generally, biotyping proved to be of no discriminative value. With two exceptions the strains were found to be sensitive to the bactericidal effect of normal human serum. As shown by multilocus enzyme electrophoresis, the whole-cell protein profile (WCPP), and the patterns of the outer membrane proteins and lipopolysaccharides, all but the five O75:H55 strains were genetically closely related to each other and could be classified into one clonal group. The O75:K-:H55 strains proved to be quite different and lacked type 1 fimbriae. All 17 K95 (H-, H5) strains produced hemolysin and P fimbriae. Five of the O75:K5:H- strains were different from the other K5 strains by showing hemagglutinating properties, on the basis of the presence of the OX adhesin. The last two groups are suggested to be uropathogenic and are proposed to represent separate clonal groups or subgroups.

Structural studies of the capsular polysaccharide from Klebsiella type 7.

The structure of the capsular polysaccharide elaborated by Klebsiella type 7 has been investigated. NMR spectroscopy together with sugar and methylation analysis have been the main methods used. A uronic acid degradation was also employed. The polysaccharide consists of hexasaccharide repeating units having the following structure. [formula: see text]

Structure of the capsular polysaccharide from the Klebsiella K8 reference strain 1015.

The structure of the capsular polysaccharide from the Klebsiella K8 reference strain 1015 has been elucidated. The structure was deduced from sugar analysis, different methylation analyses, a uronic acid degradation, and NMR spectroscopy. It is concluded that the polysaccharide is composed of pentasaccharide repeating units with the structure: [formula: see text] The structure differs from that of the previously published structure of the capsular polysaccharide from Klebsiella K8, which originates from another strain and has the following structure: [formula: see text] The serological similarity between the two strains is most likely derived from a common tetrasaccharide which is substituted in different ways in the two strains. Since the strain in the present investigation originates from the Klebsiella K reference strain collection of the International Escherichia and Klebsiella Centre, Copenhagen, Denmark, it is suggested that it should keep the designation K8. The other polysaccharide with Klebsiella K8 specificity should be renamed as K8,52,59 based on the cross-reactivity of the strain (I. Orskov, unpublished).

Detection of Escherichia coli K95 strains by bacteriophages.

A set of three bacteriophages (phi K5, phi K20, and phi K95) was used to discriminate between Escherichia coli K5 and K95 strains isolated from patients with urinary tract infections. All the strains tested were detected primarily by phi K5 and thought to carry the capsular antigen K5. Ten O2, 33 O6, and 32 O18 strains proved to be sensitive to the three phages. Eleven of 44 O75:H- strains, all 8 O75:H5 strains, 1 O17 strain, and 1 OR strain were not lysed by phage phi K20, the only phage without activity toward the K95 test strain. It has been suggested that the phi K20-resistant strains carry K95. These findings were confirmed by countercurrent immunoelectrophoresis with K-antigen extracts and anti-K95 serum. These preliminary data indicate the usefulness of these phages to detect K95 strains among K5 isolates.

Bacteriophages specifically recognizing the lipopolysaccharide antigens O4, O5, O6, and O7 of Escherichia coli.

Four bacteriophages recognizing the Escherichia coli lipopolysaccharide (LPS) antigens O4, O5, O6, and O7, respectively, were isolated from pooled sewage samples. Electron microscopic investigations revealed icosahedral phage structures. Phages phi O4, phi O5, and phi O7 belonged to Bradley's morphology group C, while phi O6 had a tail and resembles phages of group A of Bradley. The nucleic acid of the phages was identified as double-stranded DNA of different genomic sizes. Host range studies showed that only E. coli strains with homologous O antigens were attacked. No lysis of encapsulated and rough E. coli strains was observed. The phages specifically depolymerized the homologous LPS of their host strains; they may be useful for detecting respective non-capsulated E. coli strains in epidemiological studies as simple alternative to the laborious serological typing. Diagnostic application is restricted, however, as strains carrying K antigens have not been detected. The high specificity of the phage-associated enzymes provides a mild method for the preparation of oligosaccharides from the LPS for structural studies.

Structural studies of the capsular polysaccharide from Klebsiella type 38: a reinvestigation.

The structure of the capsular polysaccharide from Klebsiella type K38 has been reinvestigated. It is composed of pentasaccharide repeating units of the structure given below. In this structure, Sug stands for a 4-deoxy-threo-hex-4-enopyranosyluronic acid group, most probably having the beta-L configuration. 1H NMR studies further indicate that this group assumes the 1H2 conformation. [formula: see text]

The structure of the capsular polysaccharide from Klebsiella K43.

The structure of the capsular polysaccharide from Klebsiella type K43 has been investigated using sugar and methylation analysis, uronic acid degradation, and NMR spectroscopy on the native and the O-deacetylated polysaccharide. It is concluded that the polysaccharide is composed of pentasaccharide repeating units with the structure [formula: see text] The polysaccharide contains approximately 0.4 equiv of O-acetyl group per repeating unit, located at a primary position.

Binding studies of a monoclonal antibody specific for 3-deoxy-D-manno-octulosonic acid with a panel of Klebsiella pneumoniae lipopolysaccharides representing all of the O serotypes.

A monoclonal antibody (MAb) raised against Salmonella minnesota R595 and specific for alpha-3-deoxy-D-manno-octulosonic acid (alpha-Kdo) of the inner core was tested for binding to lipopolysaccharides (LPS) of Klebsiella pneumoniae. The MAb was tested in several assay systems (enzyme-linked immunosorbent assay, passive hemolysis, and inhibition of passive hemolysis) with a large panel (n = 23) of K. pneumoniae LPS representing all nine currently known O serotypes. MAb 20 showed reactivity with almost all O serotypes of K. pneumoniae LPS, and this reactivity could be inhibited by synthetic Kdo. This suggests an epitope in the cores of these Klebsiella LPS much like that in the inner core of LPS of S. minnesota. Large differences in reactivity between LPS of different strains belonging to the same O serotype were observed. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPS followed by immunoblotting, reactivity of MAb 20 was observed only with the fast-moving fraction possibly representing the nonsubstituted core. No binding was seen with the high-molecular-weight fraction that contained core material substituted with several units of O-antigen building blocks. The chemical basis for these differences in reactivity remains to be established. As far as we know, this is the first report containing comprehensive immunochemical data on the LPS core of K. pneumoniae.

Isolation and characterization of bacteriophages specific for capsular antigens K3, K7, K12, and K13 of Escherichia coli.

Four bacteriophages recognizing the Escherichia coli capsular antigens K3, K7, K12, and K13, respectively, were isolated from pooled sewage samples. The nucleic acid of these phages was identified as double-stranded DNA of different size (phi K3, 71.3; phi K7, 32.8; phi K12, 42.9; phi K13, 43.4 kbp). Three of these phages belonged to Bradley's morphology group C and were specific for K3, K7, and K12 antigens, respectively. The phage phi K13 (Bradley's group A) attacked not only E. coli K13 strains but also E. coli producing the closely related K20 and K23 antigens. It is suggested that the common basic repeating units occurring in these capsular polysaccharides are the primary receptor of phi K13. It could be demonstrated that the four phages were able to depolymerize enzymatically the capsular polysaccharides isolated from the respective host strains.

Two different Escherichia coli capsular polysaccharide depolymerases each associated with one of the coliphage phi K5 and phi K20.

The Escherichia coli capsular polysaccharides (K antigens) K5 and K20 are known as primary receptors for the coliphage phi K5 and phi K20, respectively. A host range study of the phage revealed that E. coli K5 strains were not only lysed by phi K5 but also by phi K20, and furthermore that the E. coli K95 test strain was attacked by phi K5 in addition to K5 strains. In order to find out whether the phage can degrade the K antigens, the interaction of the phage with isolated polysaccharides was studied. It could be demonstrated that phi K5 was able to depolymerize the K5 and K95 polysaccharides and that phi K20 showed degrading activity towards the antigens K20 and K5. Obviously, each of the phages was associated with two different enzyme systems which enabled them to recognize and depolymerize chemically unrelated polysaccharides.

Structural studies of the extracellular polysaccharide elaborated by Azotobacter vinelandii strain 1484.

The structure of the extracellular polysaccharide from Azotobacter vinelandii strain 1484 has been investigated, specific degradations and n.m.r. spectroscopy being the main methods used. It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the following structure, [sequence: see text] in which Sug is 3-deoxy-D-threo-hexulosonic acid. The polysaccharide also contains a non-stoichiometric amount of O-acetyl groups, distributed over at least two positions.

[Determining the pattern of outer membrane proteins of gram-negative bacteria as a contribution to complex typing]. / Die Bestimmung des Musters der Proteine der äusseren Membran gramnegativer Bakterien als Beitrag zur komplexen Typisierung.

The aim of these investigations was to study relations between the serotype of E. coli strains and the pattern of their outer membrane proteins ("OMP") in sodium dodecylsulfate polyacrylamide gel electrophoresis. Three groups of strains being well characterized at least serologically (01, 02, 018ac containing different K, H, and in part F antigens) were submitted to this analysis. In all cases a nearly complete paralellity between OMP pattern and O:K:H(F:) type was observed, provided that the strains were epidemiologically related. The possibility is discussed that the OMP type could be used as a guide marker for the complex typing of E. coli strains.

Bacteriophage-associated glycan hydrolases specific for Escherichia coli capsular serotype K12.

Four bacteriophages were identified, which carry glycan hydrolases specific for the Escherichia coli K12 capsular polysaccharide. All these glycanases catalyze the hydrolysis of the alpha-L-rhamnosyl-1,5-beta-3-deoxy-D-manno-2-octulosonic acid linkage as demonstrated with a special thiobarbituric acid assay procedure, which discriminates between the C5 substituted and unsubstituted 3-deoxy-D-manno-2-octulosonic acid (dOclA). This assay, together with gel filtration, 1H-NMR and 13C-NMR spectroscopy showed that depolymerization led to the dimer of the K12 repeating unit, (,5-beta-dOcl1Ap-2,3-alpha-LRhap-1,2-alpha LRhap-1,)2, as the primary degradation product. The phages (phi 12-W, phi 12-S, phi 82-W1, phi 82-W2) were tested for their ability to infect Escherichia coli strains Su65-42 (O4:K12:H-) and CDC63-57 [O139:K82(12):H1]. phi 12-W and phi 12-S, respectively, infected strain Su65-42 only, phi 82-W2 CDC63-57 only, and phi 82-W1 both bacterial strains. These distinct host specificities cannot be explained by differences in the action of the glycanases, which depolymerize the capsules of both strains.

[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.

Isolation and characterization of the non-fimbrial adhesin NFA-4 from uropathogenic Escherichia coli O7:K98:H6.

The non-fimbrial adhesin NFA-4 from uropathogenic Escherichia coli O7:K98:H6 mediates the agglutination of human red cells (RBC), notably of blood group MM. The adhesin can be separated from the bacteria by heat extraction and was purified to homogeneity by ammonium sulphate precipitation and anion exchange chromatography in the presence of 8 M urea. NFA-4 consists of non-covalently linked 28 kDa subunits which tend to form aggregates of an apparent molecular weight in excess of 10(6) Da. The first 23 amino-terminal amino acids were sequenced, and no homology of this region was found with that of the blood group M specific non-fimbrial adhesin of an unrelated uropathogenic E. coli. It has, however, an about 70% homology to the corresponding region of the K88 antigen from animal-pathogenic enterotoxic E. coli. Both polyclonal and monoclonal antibodies against NFA-4 were prepared. One of the monoclonal antibodies strongly inhibits the hemagglutinating activity of both whole bacteria and purified NFA-4.