Purification and immunochemical properties of Escherichia coli B polysaccharide cross-reacting with Salmonella typhi Vi antigen: preliminary evidence for cross-reaction of the polysaccharide with Escherichia coli K1 antigen.

An acidic polysaccharide of Escherichia coli B was isolated by a mild procedure and purified to homogeneity. The polysaccharide was found to react in Salmonella typhi Vi antisera and E. coli K1 antisera. Serological analysis and preliminary chemical characterization of the polysaccharide indicated that it is an aminouronic acid polymer which, although not structurally identical to either Vi or K1, appears more like the Vi antigen, both immunochemically and chemically.     

Simple technique for detecting K1 antigen of Escherichia coli.

The K1 antigen is a poor immunogen, its detection by serological means is difficult, and previously described methods using K1 specific bacteriophages require standardised suspensions of five different bacteriophages. A simple technique was developed which uses an unstandardised suspension of a single K1 specific bacteriophage applied with a 1 mm wire loop to bacteria streaked on to cystine lactose electrolyte deficient (CLED) medium. The technique correctly identified all 99 known K1 strains tested, including 14 strains negative with the serological method. Among 71 clinical isolates from urinary tract infections, the single bacteriophage method distinguished 30 K1 strains from 41 strains without this antigen. Suspensions of the bacteriophage were shown to remain fully active for at least two years when stored at 4 degrees C. It is concluded that this technique required so little in materials, time, or equipment that it could be routinely used in most laboratories.     

Identification of Escherichia coli K1 antigen

We compared the use of bacteriophage sensitivity, seroagglutination with polyclonal antisera raised in rabbits or horses, seroagglutination with murine monoclonal antibody, and the serum agar precipitin technique for the detection of K1 capsular polysaccharide among clinical isolates of Escherichia coli obtained from blood stream infections. Some E. coli isolates failed to yield agreement among these tests, indicating that reliable detection of K1 antigen may require the use of multiple tests.     

Protection against Escherichia coli K1 infection in newborn rats by antibody to K1 capsular polysaccharide antigen

The protective value of antibody to the K1 capsular polysaccharide antigen of Escherichia coli was investigated in a newborn rat model of E. coli K1 infection. Pregnant rats were immunized intravenously with E. coli, and the agglutinating titer to meningococcal group B polysaccharide, which is identical to K1 polysaccharide, was measured in the serum of rats and their offspring. Convalescent serum from rat mothers showed an increased antibody titer in animals injected twice but not once with E. coli K1. Although no agglutinating antibody was detected in the serum of rat pups, animals suckled by mothers having a meningococcal group B agglutinating titer of 1:8 or greater had reduced infection and mortality rates after intraperitoneal injection with E. coli K1 compared with animals suckled by mothers having a low titer of agglutinating antibody (P less than 0.05). In addition, greater protection could be conferred on rat sucklings by oral supplementation with a horse serum rich in antibody to meningococcal group B polysaccharide, suggesting that antibody was abosorbed from the gastrointestinal tract and by itself could be protective. These studies demonstrated that antibody to the capsular polysaccharide of E. coli K1 altered the severity of E. coli K1 infection. Final clearance of bacteria from the blood appeared to await the maturation of other host defense systems in the newborn rat.     

Chemical characterization of Escherichia coli K 1 capsular polysaccharide

The exact chemical characterization of the prepared Escherichia coli K 1 capsule polysaccharide is necessary and a prerequisite for using this antigen as a screening antigen in the production of monoclonal anti-K 1-antibodies. The K 1-antigen, prepared by phenol-water-extraction, was analyzed by protein, RNA, and neuraminic acid determination. An addition, the antigen was subjected to elementary analysis, infrared- and 13C-NMR-spectroscopy, and gelchromatography. After testing the serological specificity, the K 1-antigen was identified as a high molecular polyneuraminic acid.     

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.     

Monoclonal antibodies specific for the phase-variant O-acetylated K1 capsule of Escherichia coli.

Two monoclonal antibodies, one each of the immunoglobulin M and G2b types, were produced from mouse spleen cells. These monoclonal antibodies only reacted with approximately 50% of the Escherichia coli K1 strains and not against group B meningococci. No reaction was observed after the strains were boiled. E. coli K1 strains that reacted with the monoclonal antibodies could become nonreactive after subculture. Based on these findings, we conclude that the monoclonal antibodies react with the O-acetylated K1 capsule.     

Isolation and characterization of specialized transducing bacteriophages for the recA gene of Escherichia coli.

Mg²⁺ stabilized potato virus Y helper component during partial purification by precipitation using polyethyleneglycol. In solutions containing 0.02 M Mg²⁺ the helper component retained most of its activity for 2 days at 4° and for at least 8 months at ?15°. Activity was destroyed on incubation with pronase or trypsin, or by heating for 5 min at 55°, but not by incubation with ribonuclease. Incubation with its own antiserum strongly inhibited helper component activity, but antisera to potato virus Y coat protein or inclusion protein had no more effect than a control serum showing that the component was unrelated to these two proteins. Filtration through a Sephadex G-200 column resulted in a broad peak of activity which produced many protein-staining bands when electrophoresed on polyacrylamide gel. Gel filtration and ultrafiltration experiments both indicated a molecular weight of 100,000–200,000. Some helper component activity was retained by aphids allowed to probe into a sucrose solution for 20 min showing that the helper component is more firmly bound to the aphid than is the tobacco mosaic viruspoly-l-ornithine complex.     

Breast milk lymphocyte response to K1 antigen of Escherichia coli.

Comparison milk and blood lymphocyte blastogenic responses to the K1 antigen of Escherichia coli and lipopolysaccharide (LPS) from E. coli O127,B8 were examined in 16 postpartum women by [3H]thymidine uptake. Rabbit hemolysincoated sheep erythrocyte monolayers were used to deplete macrophages from milk lymphocyte preparations and to enrich for T lymphocytes in order to make milk preparations more comparable to blood preparations. Response was defined as a stimulation index of greater than or equal to 2.0. There was no evidence of selective response to K1 antigen by milk lymphocytes, since both blood and milk lymphocytes responded in four women and neither blood nor milk lymphocytes responded in nine. Milk lymphocytes alone responded to K1 in one woman, whereas blood lymphocytes alone responded in two women. Additional nonpaired milk or blood cultures were available from three women. None of these responded to K1 antigen. Corresponding lymphocyte cultures were stimulated with LPS. A positive K1 response was always accompanied by an LPS response, and the LPS response correlated with the K1 response in 17 of 19 women. Stool cultures examined with an antiserum agar showed no correlation between the presence of K1 E. coli in the stool and milk or blood lymphocyte response to K1 antigen. In the system used here, no selectivity of response of breast milk lymphocytes to K1 antigen was noted.     

Structure and serological specificity of the K13-antigenic polysaccharide (K13 antigen) of urinary tract-infective Escherichia coli.

The primary structure of the K13-antigenic polysaccharide (K13 antigen) of Escherichia coli O6:K13:H1 was elucidated by composition, periodate oxidation, Smith degradation, and methylation analysis. The polysaccharide consists of a repeating sequence of 3-linked ribofuranose and 7-linked 3-deoxymannooctulosonic acid (KDO). About 50% of the KDO residues are O-acetylated at position 4 or 5. Measurement of the optical rotary dispersion indicated that in aqueous solution the K13 polysaccharide assumes a secondary structure in which the carboxyl groups of KDO are engaged. The serological specificity of the K13 polysaccharide is expressed through KDO and its O-acetyl substituent, the ribose unit being antigenically silent. There are two populations of anti-K13 antibodies one directed against the charged region of the KDO and the other against the O-acetyl groups.     

Contribution of capsular polysaccharide and surface properties to virulence of Escherichia coli K1.

We examined the surface properties, susceptibility to the bactericidal activity of serum, and susceptibility to phagocytosis of Escherichia coli K1, a laboratory strain of E. coli (LE392), and strain LE392 carrying a plasmid (pKT274) incorporating a 17-kilobase insert of DNA that encodes the ability to produce surface K1 antigen. As determined by electron microscopy, LE392 was nonencapsulated but both E. coli K1 and LE392(pKT274) possessed a thin capsule. By using charged aqueous two-phase polymer systems, both E. coli K1 and LE392(pKT274) were shown to be significantly more negatively charged than LE392. E. coli K1 was resistant to the bactericidal activity of serum, but both LE392 and LE392(pKT274) were completely inhibited by neonatal serum at a concentration of 20% (vol/vol). As measured by counting endocytosed and nonendocytosed bacteria and by chemiluminescent response, E. coli K1 was highly resistant to phagocytic uptake by polymorphonuclear leukocytes, whereas LE392 was rapidly taken up by such cells; LE392(pKT274) was resistant to endocytosis, although less so than E. coli K1. Most intraphagocytic E. coli LK1 remained structurally intact for up to 60 min, whereas both LE392 and LE392(pKT274) were rapidly degraded.     

Isolation and partial characterization of temperature bacteriophages from enteropathogenic strains of Escherichia coli 0111:K58

We report studies on ten strains of Escherichia coli 0111:K58 isolated from children with acute diarrhea. Our results show that these E. coli strains do not produce the pathogenic factors of enterotoxic E. coli (ETEC) and are lysogenic for phages belonging to two groups that differ for host range, kinetics of thermal inactivation, antigenicity and morphology. These data support the hypothesis that these phages may in vivo contribute to reduction of the number of common E. coli strains by lytic infection favouring the development of the enteropathogenic strain of E. coli.     

K antigen and serum sensitivity of rough Escherichia coli.

We prepared bacterial hybrids which express both K1 and K27 antigens and examined the relative contributions of these capsules to serum resistance. Escherichia coli Hfr strain F639 (rough, K27+, serum sensitive) was conjugated with E. coli recipient strain E412 (rough, K1+, His- Trp- Strr, serum resistant). Transconjugants which inherited both the his and trp linked genes for K27 antigen synthesis were analyzed. These hybrids retained and expressed the K1 antigen since the K1 locus is nonallelic with K27 gene loci. Hybrid strains which express both K1 and K27 antigens exhibit serum resistance, but not at the level of the K1+ parental strain. An isogenic K1 derivative of a hybrid which expressed only K27 antigen was serum sensitive (greater than 99% kill, 60 min). These findings indicate that the presence of the K1 capsular antigen can protect some rough strains of E. coli from serum bactericidal activity, whereas K27 and perhaps other K antigens fail to provide such a protective effect.     

Relationship of K1 antigen to biotype in clinical isolates of Escherichia coli.

Two hundred and ninety-four isolates of Escherichia coli, including 105 from blood cultures, 94 from stools of hospital inpatients, and 96 from rectal cultures of healthy young adults, were biotyped by using the API-20E system and tested for the presence of K1 antigen. The overall frequency of K1 strains was 14.2% and was similar among the three sources. Forty-eight biotypes were observed, but two-thirds of all isolates, including two-thirds of the K1 strains, belonged to only five biotypes. Among the five commonest biotypes, the distribution of K1 strains was nonrandom, since 23 of the 27 K1 strains belonged to only two biotypes. Analysis of the O and H antigens of K1 strains indicated that this correlation of biotype with K1 antigen was due to a restricted number of serovars ("clones") that were repeatedly isolated from the population studied. These serovas included O18:K1:H7, O1:121:H6 and O16:K1:H6. Although a statistically significant correlation between biotype and K1 antigen was observed, the correlation was not sufficiently great to alow biotyping to be of significant predictive value as a marker for the K1 antigen.     

Screening for cross-reacting capsular polysaccharide K antigens of Escherichia coli using antiserum agar.

Agar plates containing antiserum against group B meningococcus or Haemophilus influenzae type b were used to determine the prevalence of cross-reacting K1 and K100 capsular polysaccharide antigens in 265 isolates of disease-causing Escherichia coli. K1 antigen was found in 22% of isolates from various sites. K100 antigen was found in only three isolates. This technique is a convenient method to detect specific E. coli K antigens for evaluation as possible factors important in the virulence of the organism.     

Escherichia coli O18ac antigen: structure of the O-specific polysaccharide moiety.

The O-specific polysaccharide moiety (O18ac polysaccharide) of the O18ac antigen (lipopolysaccharide) from Escherichia coli 2980 (O18ac:K5:Fim+:H-) was isolated in pure form by degradation of the lipopolysaccharide and chromatography on Sephadex G-50. The primary structure of the O18ac polysaccharide was elucidated by composition, fragmentation procedures, methylation analysis, and nuclear magnetic resonance spectroscopy. The polysaccharide consists of repeating units of the pentasaccharide: (formula; see text) which are joined in the polymer by alpha-1,2 linkages.     

K99 surface antigen of Escherichia coli: antigenic characterization.

K99 prepared by acid precipitation hemagglutinated guinea pig erythrocytes, whereas K99 prepared by chromatography on diethylaminoethyl-Sephadex did not. K99 purified by either procedure hemagglutinated horse erythrocytes. K99 prepared by acid precipitation contained a second antigen not presnet in the K99 prepared by chromatography on diethylaminoethyl-Sephadex. This antigen could be detected by immunoprecipitation with some, but not all, sera prepared against K99-positive Escherichia coli strains. It was assumed that this second antigen is not K99 and is responsible for the guinea pig erythrocyte hemagglutination reaction. Furthermore, the second antigen has an isoelectric point of 4.2, which has been reported by Morris and co-workers to be the isoelectric point of K99.     

Escherichia coli K antigen in relation to serum-induced lysis and phagocytosis.

The presence of capsular polysaccharides (K antigens) and their relation to phagocytosis and sensitivity to the lytic action of serum of 26 strains of E. coli isolated from stools of healthy volunteers and from blood cultures were studied. Four of 12 strains isolated from stool cultures and 12 (86%) of the 14 strains isolated from blood cultures possessed K antigen. Three of the 12 strains isolated from stool cultures and seven of the 14 isolated from blood cultures were resistant to uptake by polymorphonuclear leucocytes; these resistant strains contained large amounts of K antigen. By contrast 10 strains, three with low amounts of K antigen and seven without detectable amounts of K antigen, were readily phagocytosed. Thus it appears that K antigen renders E. coli resistant to phagocytosis. Only four (15%) of the 26 strains were sensitive to serum lysis and there was no correlation between the presence of K antigen and the resistance to serum lysis.