Properties and participation of "free" endotoxin in the toxic activity of Morganella morganii.

"Free" and "bound" Morganella morganii endotoxin was characterized by chemical (determination of proteins, saccharides and 3-deoxy-2-octulosonic acid) and immunochemical (double-diffusion test, immunoelectrophoresis, tandem crossed immunoelectrophoresis) methods. Chemical analysis showed that "free" endotoxin contains more protein and phosphorus and less saccharides than bound endotoxin. Immunochemical tests revealed differences in the structure of polysaccharide portions of both endotoxins, and, on the other hand, identity of certain antigenic determinants. Free endotoxin possessed a higher biological activity.

Adherence of intestinal and extraintestinal Pseudomonas aeruginosa to tissue culture cells.

The adherence pattern of Pseudomonas aeruginosa strains to HeLa, Vero and CHO cells was studied. The diffuse type of adherence was found to prevail on HeLa cells. It was characteristic for intestinal and environmental strains. Urinary strains revealed more often a localized adherence. A similar pattern was obtained with CHO cells. Experiments with Vero cells showed an equal distribution of intestinal strains regarding the diffuse, localized and mixed adherence. Urinary strains revealed mostly a localized adherence of a similar pattern as was observed on HeLa and CHO cells.

Characterization of adhesion associated surface properties of uropathogenic Escherichia coli.

Escherichia coli was isolated from the urine of patients with pyelonephritis, with urinary tract infections other than pyelonephritis and with asymptomatic bacteriuria. Surface properties of the strains were analyzed by the salting-out aggregation test (SAT), hydrophobic interaction chromatography (HIC), Congo red binding (Crb), agglutination of erythrocytes (MRHA) and latex particles covered by digalactoside (PF) and by adherence to tissue culture cells. In addition, a DNA probe for the pap gene was used. The DNA probe detected the highest proportion of strains with pap gene in the group of patients with pyelonephritis, lower in the urinary tract infections other than pyelonephritis and the lowest in the group with asymptomatic bacteriuria. Tests for P-fimbriae (PF, MRHA) showed a similar distribution. Hydrophobicity measured by SAT and by HIC did not show differences among the tested groups of strains. The results suggest that factors other than the P-fimbriae and hydrophobicity may contribute to the persistence of E. coli in the urinary tract.

[Methods of detection of P-fimbria in uropathogenic E. coli]. / Metodické aspekty dôkazu P-fimbrií uropatogénnych E. coli.

We characterized the surface properties of E. coli strains isolated from urine of children with urinary tract infections. The PF test and haemagglutination reaction were used for detection of P-fimbriae. P-fimbriae were detected in 30-54% of the pyelonephritic strains. All these strains expressed P-fimbriae in vitro. Pap genes were determined by a DNA probe in 85% of the strains isolated from pyelonephritis. These strains showed diffuse and localized adherence to tissue culture cells without any association to the expression of P-fimbriae on agar medium.

Adherence pattern of non-pilated Aeromonas hydrophila strains to tissue cultures.

Adherence of non-pilated Aeromonas hydrophila strains to HEp-2, HeLa, CHO and Vero tissue culture cells was studied. The strains were isolated from intestinal and extra-intestinal human infections and from the environment. The environmental isolates revealed a diffuse type of adherence to all types of cell lines while clinical isolates showed localized adherence (50%). Strains revealing the diffuse type of adherence showed high levels of adhesion i.e. > 20 bacteria per cell. This activity correlated with hydrophobicity of the strains tested. Haemagglutination assay with human erythrocytes was negative, showing no association with plasmid profile, Congo red binding and adherence to tissue culture cells. Thus the experiments showed that in non-pilated strains, hydrophobicity may be the major factor responsible for adherence to epithelial cells.

Properties of free and bound Citrobacter freundii lipopolysaccharides.

Culture medium content of free lipopolysaccharide (LPS) components spontaneously released from a Citrobacter freundii culture grown in minimum synthetic medium was determined during early (8-hr culture) and late (24-hr culture) phases of growth. As judged by Limulus-lysate test, free LPS occurred in the medium as early as after 8 hrs of incubation, i.e. at the beginning of log growth phase. As the culture continued to grow the LPS amount released into culture medium kept rising, reaching 30% of endotoxin present in 24-hr Citrobacter culture. The released LPS complex was isolated by separation and its physicochemical, immunochemical and biological properties were determined and compared with those of cell-bound endotoxin recovered from cells by phenol extraction. Comparisons revealed distinct differences in the chemical composition and the degree of heterogeneity; free LPS was less heterogeneous. Immunologically, free LPS differed from bound LPS in the structure of macromolecules, but was identical with it in some antigenic determinants. The biological activity of free LPS preparation was greater than that of cell-bound LPS.

Toxic exoproducts of citrobacter freundii.

A strain of Citrobacter freundii isolated from the feces of a patient with diarrhoea was examined for growth kinetics and toxic exoproduct formation using the complete (BHI) and synthetic culture media. It was found that the test organism in synthetic medium grew distinctly slower than in BHI. Fractionations on Sephadex G-100 column yielded 3 fractions from the complete medium culture filtrate and 2 fractions from the culture filtrate obtained from synthetic medium. The first culture filtrate fractions (F1) were represented by components of the molecular weight over 100,000, the respective second fractions (F2) from complete and synthetic medium were of the molecular weights of about 40,000 and 10,000. In the early skin test on rabbits the toxicity of culture filtrates and their fractions manifested itself by an increased permeability of blood vessels, in the late skin test by a hemorrhagic reaction associated with dilatation of blood vessels and induration of the skin tissue. In a test on mouse foot pad all separated filtrate fractions gave a positive edematous reaction. In cultured Vero cells samples of synthetic medium fractions gave a distinct cytotoxic reaction. Immunochemically, the presence of LPS in culture filtrates as well as some variations in the antigenicity of components from the complete and synthetic medium fractions were found. Apart from LPS some additional high-molecular-weight components were also present in the toxic complex of both first filtrate fractions (F1). Much more attention should be given to analysis of these first fraction complexes as well as to toxinogenicity of second fractions (F2) using some additional tests.

Crossed immunoelectrophoretic analysis of antigenic composition of B-subunit/whole-cell and whole-cell only killed oral cholera vaccines.

Crossed immunoelectrophoresis was used to identify antigens preserved in the whole-cell component of oral cholera vaccines tested in the field trial in Bangladesh. The composition and immunogenicity of the vaccine antigens were compared with those of antigens obtained from live cells of Vibrio cholerae 01 of both biovars and serovars. The whole-cell component of the vaccine contained ten antigens in comparison with the live Vibrio cells which revealed the presence of 30 antigens. The whole-cell component contained lipopolysaccharide, flagellar antigen, one cell-bound haemagglutinin and at least six outer membrane protein antigens.