Bactericidal Action Of Human Serum Research Articles

Sialic Acid-Containing Lipopolysaccharides of Salmonella O48 Strains—Potential Role in Camouflage and Susceptibility to the Bactericidal Effect of Normal Human Serum

Sialic acid (N-acetylneuraminic acid, NeuAc) plays an essential role in protecting gram-negative bacteria against the bactericidal activity of serum and may contribute to the pathogenicity of bacteria by mimicking epitopes that resemble host tissue components (molecular mimicry). The role of sialic acid (NeuAc)-containing lipopolysaccharides (LPS) of Salmonella O48 strains in the complement activation of normal human serum (NHS) was investigated. NeuAc-containing lipooligosaccharides cause a downregulation of complement activation and may serve to camouflage the bacterial surface from the immunological response of the host. Serotype O48 Salmonella strains have the O-antigen structure containing NeuAc while its serovars differ in outer membrane protein composition. In this study, the mechanisms of complement activation responsible for killing Salmonella O48 serum-sensitive rods by NHS were established. Four of such mechanisms involving pathways, which are important in the bactericidal mechanism of complement activation, were distinguished: only the classical/lectin pathways, independent activation of the classical/lectin or alternative pathway, parallel activation of the classical/lectin and alternative pathways, and only the alternative pathway important in the bactericidal action of human serum. To further study the role of NeuAc, its content in bacterial cells was determined by gas-liquid chromatography-mass spectrometry in relation to 3-deoxy-D-manno-2-octulosonic acid (Kdo), an inherent constituent of LPS. The results indicate that neither the presence of sialic acid in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of complement and that the presence of sialic acid in the structure of LPS is not sufficient to block the activation of the alternative pathway of complement. We observed that for three strains with a very high NeuAc/Kdo ratio the alternative pathways were decisive in the bactericidal action of human serum. The results indicated that those strains are not capable of inhibiting the alternative pathway very effectively. As the pathogenicity of most Salmonella serotypes remains undefined, research into the interactions between these bacterial cells and host organisms is indispensable.

Composition of the outer membrane of Proteus mirabilis in relation to serum sensitivity in progressive stages of cell form defectiveness

A serum-resistant strain of Proteus mirabilis was used to determine whether changes in the composition of surface components could be detected following induction of progressive stages of cell form defectiveness by beta-lactam antibiotics. The critical stage was the conversion from filaments to the spheroplast form, which was accompanied by increased susceptibility to the bactericidal action of human serum. Inner and outer membranes of the bacterium, its filament form and its spheroplast form were separated by sucrose density-gradient centrifugation after digestion of peptidoglycan, followed by osmotic lysis of the cells. Outer membranes of the bacterial and the filament forms sedimented at the same density, whilst the outer membrane fraction of the spheroplast form sedimented in a region of lesser density. In addition, the amounts of two major outer-membrane proteins as well as the O-polysaccharide content of the lipopolysaccharide were reduced in the spheroplast form. These results indicate a general disorganization in structure and assembly of components in regard to their interactions with one another in the outer membrane of the spheroplast form.

Phenotypic modification of the reactivity in human serum of escherichia coli K1 solates

Three Escherichia coli strains producing the K1 antigen and shown to be resistant to the complement-mediated bactericidal action of human serum when grown in batch culture, were cultivated in the chemostat under conditions of carbon-, nitrogen-, magnesium- and phosphate-limitation. All strains were fully serum resistant when grown under carbon-limiting conditions but became phenotypically serum sensitive when limited by magnesium. One strain, belonging to serogroup O7:K1, also displayed serum sensitivity when nitrogen limited and showed an intermediate serum response when phosphate was used as the limiting nutrient.

Release of endotoxic lipopolysaccharide by sensitive strains of Escherichia coli submitted to the bactericidal action of human serum.

Free endotoxin was assayed in filtered samples of E. coli suspensions submitted to the bactericidal and bacteriolytic action of 10% human serum. The Limulus amoebocyte lysate test, a passive hemolysis inhibition assay based on O antigenic specificity and the determination of 3-OH-myristic acid by mass spectrometry were used as assay methods differing from one another with regard to the part of the endotoxin macromolecule involved in the reaction. The biological activity of endotoxin was assessed in a mouse lethality test. The bactericidal and bacteriolytic action of human serum on sensitive strains of E. coli released quantities of endotoxic lipopolysaccharide (LPS) amounting to 3,000-12,000 ng/ml, for an inoculum of 1--3 x 10(8) colony-forming units. The material thus appearing in the medium was shown to react with the Limulus amoebocyte lysate, to be lethal for actinomycin D-sensitized mice and to bear O antigen, as well as 3-OH-myristic acid, a marker of lipid A. Samples of serum depleted of lysozyme by adsorption onto bentonite, and displaying a strictly bactericidal effect, released approximately 80% of the quantity of LPS appearing in the medium in a control experiment performed with untreated serum. The LPS release is therefore mainly linked to the bactericidal effect of antibody and complement. The amount of LPS released depended on the concentration of divalent cations in the medium, being reduced by an increase in the concentration of calcium and magnesium beyond the values optimal for complement activity. This effect was already observed for an increase in the concentration of divalent cations too low to alter the bactericidal or bacteriolytic effects. The significance of the release of endotoxin by complement dependent bactericidal reactions occurring in vivo is discussed.

Effect of subinhibitory concentrations of mecillinam on the serum susceptibility of Escherichia coli strains.

For serum-resistant clinical isolates of Escherichia coli were grown in the presence of various subinhibitory concentrations of mecillinam or pivmecillinam and then exposed to the bactericidal action of human serum. All strains became more serum susceptible as a result of pregrowth in medium containing mecillinam, but the concentration of antibiotic needed to produce the effect varied according to the strain being used. Production of ovoid or round cells was a prerequisite for sensitization to serum. Growth in the presence of mecillinam did not alter the response to serum of a serum-susceptible E. coli strain.

Determinants that increase the serum resistance of Escherichia coli.

The rfb locus, determining biosynthesis of O8-specific lipopolysaccharide side chains, was transferred to a rough mutant of Escherichia coli; recombinants producing a complete lipopolysaccharide were more resistant to the complement-mediated bactericidal action of human serum than the rough recipient. Inheritance of the his-linked genes for K27 antigen production did not alter the response to serum. The serum resistance of strains carrying O8 side chains, but not of strains with incomplete lipopolysaccharides, was further increased by inheritance of plasmids R1 and NR1.20

Morphological, biological and antigenic properties of Neisseria gonorrhoeae adapted to growth in guinea-pig subcutaneous chambers.

Gonococci (strain BS3) passaged three times and harvested directly from plastic chambers implanted subcutaneously in guinea pigs were compared with the parent strain (BS) grown in vitro. The strain grown in vivo produced smaller colonies than that grown in vitro and when examined directly in chamber fluid was sometimes not pilated. It was more resistant to the bactericidal action of human serum and more infective for guinea-pig chambers. In gel diffusion, extracts of the organisms adapted in vivo and cultured once on agar appeared to contain one or two antigens that were different from those in extracts of the in vitro grown organisms; and on polyacrylamide gels, electrophoresis of similar extracts showed one or more protein components for strain BS3 which were not seen for strain BS. Gonococci grown in guinea-pig subcutaneous chambers appear to be suitable for studies on the determinants of gonoccal pathogenicity.

Immunochemical Studies on Human Serum

Summary The bactericidal action of human serum against V. comma, strain 82, is a result of the combined action of antibody and complement. The bactericidal action of human complement against this vibrio is destroyed by the inactivation of any one of the four complement components. Such a complement is said to be specifically inactivated. Bactericidal action is restored to a specifically inactivated complement (siC′) by the addition to it of the particular complement component which it lacks. Bactericidal action of human complement against this vibrio is fortified in particular by the addition of the “pH=5.4—μ 0.02 supernate.” All evidence in this paper points to the identity of bactericidal and hemolytic complements in human serum.

Bactericidal Action of Human Serum on Hemolytic Streptococci. Active Principle Obtained by Fractionation of Sera

The present report is part of an investigation concerning the capacity of serum from patients who are acutely ill to destroy hemolytic streptococci of the beta type. Previous reports have demonstrated that the bactericidal property under consideration was demonstrable in the serum derived from patients during the period of active disease due to a variety of infections, but that following recovery from illness the streptococcidal activity was greatly diminished, as measured by the methods which were employed. Furthermore, normal sera have been found to be essentially devoid of streptococcidal activity and have served as controls throughout the observations. The previous articles have described the technical procedures. In the present study a strain of Streptococcus hemolyticus, which has been found to be uniformly highly sensitive to the killing effect of patients sera, has been used in all of the experiments. The samples of sera were derived from patients who were acutely, and usually severely ill from diseases such as pneumonia, or pyogenic infections due to different kinds of organisms. Studies have been carried out in an attempt to isolate the active principle in serum which is responsible for the streptococcidal activity. Observations have been made with a protein-fraction and a non-protein constituent. The materials have been used separately and in combination. The protein-fraction most regularly employed in the tests has been obtained by precipitation of serum at low temperatures with alcohol according to the method described by Felton. Sufficient 95 % alcohol was added to serum to make a final concentration of 20%. The material was kept in the icebox overnight in order to obtain the maximal yield and was then spun down in a cold centrifuge.