The mechanisms of activation of normal human serum complement by<Emphasis Type="Italic">Escherichia coli</Emphasis> strains with K1 surface antigen

Ten E. coli K1 strains isolated from the urine of children with urinary tract infections were sensitive to the bactericidal action of normal human serum (NHS). The role of the particular mechanisms of complement activation was determined in the process of killing these strains, showing variable sensitivity to the bactericidal action of NHS; three mechanisms of activation of human complement were observed. Important role of alternative pathway activation in the bactericidal action of NHS against E. coli K1 strains independent of the classical and lectin pathways was not established.     

Bactericidal activity of C9-deficient human serum.

Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes.     

Lysis of Neisseria gonorrhoeae initiated by binding of normal human IgM to a hexosamine-containing lipooligosaccharide epitope(s) is augmented by strain-specific, properdin-binding-dependent alternative complement pathway activation.

We studied the specificity of naturally acquired IgM bactericidal for strains of Neisseria gonorrhoeae that varied in sensitivity to the lytic action of normal human serum (NHS) and the relative ability of these strains to deplete the classical (CP) and alternative (ACP) C pathways. Lysis of both highly sensitive and relatively insensitive strains was inhibited by the same gonococcal lipooligosaccharides (LOS), as well as by Salmonella minnesota Re LOS and three hexosamine-containing glycose polymers. A polymer of N-acetylgalactosamine phosphate was the most inhibitory; a polymer of N-acetylglucosamine phosphate only partially inhibited. Neither 3-deoxy-D-manno-octulosonic acid (dOc1A) nor a polymer that contained dOc1A but not hexosamine inhibited NHS lysis. A co-polymer of N-acetylgalactosamine-dOc1A inhibited both bactericidal activity and the binding of IgM to the LOS of a highly serum-sensitive (sers) gonococcal strain. Carboxyl reduction of the dOc1A in this polymer did not affect its inhibitory capacity for gonococcal antibody, but abolished its binding to homologous antibody induced by vaccination. CP activity was not affected by vaccination. CP activity was not affected by absorption of NHS with gonococcal strains, whereas ablation of CP activity markedly but variously diminished lytic activity for highly sers strains. ACP activity was variously depleted by gonococcal strains, and the proportion of bacteria that could be lysed through the ACP varied among strains and among different populations of a given strain. The titer at which a strain was sensitive to NHS lysis was a function of its ACP consumption (p = 0.006), which accounted for 70% of the differences in titer among strains. Analyses of the absorbed sera revealed that the gonococci had variously depleted properdin from NHS as assessed by using an Ag-capture solid-phase RIA. Addition of purified properdin to absorbed sera restored ACP activity to normal levels. Western immunoblots of gonococcal lysates showed that purified properdin bound directly to a 39-kDa outer membrane protein. We conclude that both CP activation by IgM binding to LOS epitopes, one of which contains hexosamine, and ACP activation, which is a function of strain-specific direct binding of properdin, can initiate lysis of sers strains and that ACP activation, also enhances lysis and accounts for variations in sensitivity of sers strains.     

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.     

Reptiles as a Source of Salmonella O48��Clinically Important Bacteria for Children: The Relationship Between Resistance to Normal Cord Serum and Outer Membrane Protein Patterns

Bacteria of the Salmonella O48 somatic antigen group are clinically important strains causing intestinal dysfunction and diarrhoea, especially in children. The susceptibility of Salmonella O48 strains containing sialic acid (N-acetylneuraminic acid (NeuAc)) in lipopolysaccharide (LPS) to the bactericidal action of normal cord serum (NCS) was determined. The authors' previous results published in Microbial Ecology in 2010 indicated that neither the presence of NeuAc 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 normal human serum (NHS), and that the presence of NeuAc in the LPS structure is not sufficient to block the activation of the alternative pathway of complement in NHS. The current results showed that the tested strains showed various sensitivities also to the bactericidal action of NCS. The authors postulate that the presence of certain outer membrane proteins (OMPs) are characteristic of the resistant and sensitive phenotypes of Salmonella O48 strains. To establish a possible relationship between resistance to NCS and OMPs band patterns, ten Salmonella O48 strains were studied as follows: susceptibility to the bactericidal effect of NCS, the mechanisms of NCS activation and OMP band patterns obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.     

Anti-MRSA activity of alkyl gallates.

A series of alkyl gallates (3,4,5-trihydroxybenzoates) was found to show antibacterial activity against Gram-positive bacteria including methicillin resistant Staphylococcus aureus (MRSA) strains. For example, dodecyl (C(12)) gallate (1) exhibited bactericidal activity against MRSA ATCC 33591 strain with the minimum bactericidal concentration (MBC) of 25 microg/mL (74 microM). The time-kill curve study showed that dodecyl gallate is bactericidal against this MRSA strain. This bactericidal activity comes in part from its ability to inhibit respiratory electron transport systems. The length of the alkyl chain is not a major contributor but plays an important role in eliciting the activity.     

Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28,400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000-2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine.     

Bactericidal activity of human sera with a physiologic or genetic defect of the complement system

The serum of patient suffering genetically conditioned C2 defect showed a weak bactericidal activity against Pseudomonas aeruginosa strains. Out of 23 tested strains one was susceptible comparing with 16 ones sensitive to normal human serum. The normal cord serum exerted a bactericidal effect on 8 strains. All sera were found to be active against Salmonella strains tested.     

Antigenicity of bdellovibrios.

Antigenic relationships between 12 locally isolated bdellovibrios and 3 established reference strains (109D, 6-5-S, and UKi2) were investigated. Antigenicity of the strains was examined by use of the micro-complement fixation test, the serum and complement bactericidal test, and the immunodiffusion test. Antisera were prepared against one of the local strains (MS7) and against one of the established reference strains (UKi2). The complement fixation titers suggest a close relationship among all strains. Immunodiffusion tests produced lines of identity between the homologous strain MS7 and all other strains. It is suggested on the basis of these results that bdellovibrio may possess a common antigen.     

Antigenicity of bdellovibrios.

Antigenic relationships between 12 locally isolated bdellovibrios and 3 established reference strains (109D, 6-5-S, and UKi2) were investigated. Antigenicity of the strains was examined by use of the micro-complement fixation test, the serum and complement bactericidal test, and the immunodiffusion test. Antisera were prepared against one of the local strains (MS7) and against one of the established reference strains (UKi2). The complement fixation titers suggest a close relationship among all strains. Immunodiffusion tests produced lines of identity between the homologous strain MS7 and all other strains. It is suggested on the basis of these results that bdellovibrio may possess a common antigen.     

Human serum bactericidal activity against Haemophilus influenzae type b

We examined bactericidal and opsonizing activity of pooled adult 'immune' serum against Haemophilus influenzae type b with and without the addition of phagocytes. Four type b strains from cerebrospinal fluid (CSF) and three such strains from the nasopharynx (NP) of healthy children were examined. Duplicate reaction mixtures contained organisms in exponential (E) or stationary phase (S) of growth, serum, a complement source (human agammaglobulinaemic serum), and culture medium (bactericidal assay); separate assays contained the above components and polymorphonuclear leucocytes (opsonization system). A decrease in bacterial density of greater than or equal to 1 log10 unit was considered significant. All four S-CSF strains, three of four E-CSF strains and one of three S-NP strains were sensitive to the bactericidal activity of pooled serum. The other E-CSF strain, two S-NP strains and all three E-NP strains were resistant to the bactericidal activity of pooled serum. Two of three E-NP strains were opsonized by pooled serum; the other strains resistant to the bactericidal activity of pooled serum were also resistant to opsonization. Bactericidal and opsonizing activity of serum from an immunized adult was greater than or equal to that of pooled serum against each strain. Assuming normal adults are immune to invasive H. influenzae type b infection, an experimental test reflecting this immunity is the bactericidal activity against CSF isolates tested in stationary phase. We conclude that protection against invasive disease due to H. influenzae type b appears more complex than the presence of bactericidal and opsonizing activity in serum.     

Characterization of fourteen strains of Neisseria gonorrhoeae: structural analyses and serum reactivities

Resistance to normal human serum (NHS) killing in Neisseria gonorrhoeae has been associated with particular types of Protein I (PI) and lipopolysaccharide (LPS), but many exceptions exist, and the role of these structures in determining serum reactivities remains controversial. In reality, the response of the gonococcus to NHS is probably governed by several parameters involving a number of outer-membrane (OM) components. We surveyed the serum reactivities of 14 strains of N. gonorrhoeae and characterized each of their major OM components. The strains presented a spectrum of sensitivity to pooled NHS. As assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping, the strains were also quite heterogeneous in terms of PI, H.8 antigen, and LPS type, and the presence of the 2-1-L8 epitope. Five of the strains had identical PIAs in varying LPS and H.8 backgrounds, and four had identical PIBs in varying LPS and H.8 backgrounds. As assessed by electrophoretic migration and monoclonal antibody binding, Protein III and the 44,000 Dalton protein were identical in these strains. We found no association between PI subclass and serum sensitivity, while H.8 and LPS variation appeared to be related to bactericidal responses. The diversity and close interaction of gonococcal components in the OM are undoubtedly involved in differential abilities to survive NHS killing.     

Bactericidal action of oleuropein extracted from green olives against Lactobacillus plantarum

The phenolic compound oleuropein extracted from green olives was shown to be bactericidal against nine strains of Lactobacillus plantarum isolated from green olive fermentation brines. Heat-treated oleuropein also demonstrated a strong bactericidal effect but not alkali-treated oleuropein, which allowed survival of most of the strains tested. The bactericidal effect was accompanied by changes in the typical bacillary structure and Gram-positive stain of L. plantarum.     

Inhibition of adhesion of food-borne pathogens to Caco-2 cells by Lactobacillus strains

AIMS: To select adhesive strains among strains of Lactobacillus and to apply them to inhibit adhesion of food-borne pathogens. METHODS AND RESULTS: Twelve Lactobacillus strains (10 from intestine) were examined for adhesion using Caco-2 cell cultures. The two most adhesive strains, Lactobacillus crispatus JCM 8779 and Lact. reuteri JCM 1081, were used to test antiadhesion activity against enterotoxigenic Escherichia coli, Salmonella typhimurium and Enterococcus faecalis strains. Adhesion of the pathogens was inhibited by both Lactobacillus strains. Adhesion of Ent. faecalis was especially strongly inhibited by JCM 8779. Although antimicrobial activity was not detected in the culture supernatant fluid by agar well diffusion assay, the supernatant fluid obtained from the harvested JCM 8779 cell suspension showed bactericidal activity against Ent. faecalis. CONCLUSION: The strong antiadhesion activity of JCM 8779 against Ent. faecalis appears to be due to the combined effect of both bactericidal activity and competition for attachment site. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that Lact. crispatus produces a bactericidal substance.     

Combined Action of Carbenicillin and Gentamicin on Pseudomonas aeruginosa In Vitro

The minimal inhibitory and minimal bactericidal concentrations of carbenicillin and gentamicin were determined for 10 strains of Pseudomonas aeruginosa isolated from the urinary tract. Combinations of the two drugs were tested for possible enhanced activity. Such enhancement was demonstrated in the inhibitory activity of combinations for eight strains. Striking bactericidal activity against five strains was achieved by the combination, whereas neither drug alone in low dosage was capable of bactericidal action. The possible mode of action and the possible merit of pursuing these preliminary findings are discussed.     

Impaired bactericidal activity of serum of a child suffering from focal proliferative glomerulonephritis.

The serum of a child with focal proliferative glomerulonephritis was found to exhibit a weaker bactericidal activity against Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enteritidis and Escherichia coli strains as compared with sera of the child's parents. The child's serum showed a low haemolytical activity of complement as well as a low C3 concentration. The authors believe that the abnormal complement concentration could cause the impaired bactericidal activity of the patient serum.     

Variability of the lytic susceptibility of Neisseria gonorrhoeae to human sera.

The bactericidal activity of human sera for Neisseria gonorhoeae was studied. Sera were obtained from a group of patients with gonococcal infections who had acute urethritis, acute pelvic inflammatory disease, disseminated gonococcal infection, or who were asymptomatic carriers. The homologous and heterologous strains were tested with these sera. The development of serum bactericidal antibodies was not a universal event. With few exceptions, the susceptibility of a particular strain to human antibody and complement appeared to be largely independent of the particular person from whom the serum was obtained and was due instead to antigenic properties intrinsic to each individual strain. Lipopolysaccharide appeared to be the predominant antigen against which bactericidal antibodies were directed. The principal bactericidal antibody class was IgM. Blocking antibodies were not found to account for the lack of lytic activity. A correlation of bactericidal antibodies with protection from developing gonococcal infection could not be demonstrated in three pateints.     

Bactericidal activity of human, swine and cattle serum against pseudomonas aeruginosa strains

The aim of this study was to assess of bactericidal activity of human, swine and cattle serum against 136 of Pseudomonas aeruginosa strains isolated from people, fishes, domestic and fur animals. The mechanism of the bactericidal activity of serum against gram-negative bacteria is complex and involves the participation of complement, antibodies and lysozyme (1, 5, 7, 8, 10, 11, 13, 14, 24, 25, 27, 30). The susceptibility of gram-negative rods to serum is differentiated. Pseudomonas aeruginosa strains are the most resistant (17, 25, 30). This opportunistic pathogen produce proteases that destroy complement components and immunoglobulins (3, 18, 19). The bactericidal activity of serum was determined after 3 hours incubation of bacteria in 50% serum by the method of Jankowski (1981) (5). The results of this study indicate that 71% of this strains were resistant to swine serum action, 68% of this strains were resistant to bovine serum and 57% of the Pseudomonas aeruginosa strains were sensitive to human serum. The P. aeruginosa strains isolated from fishes were the most sensitive to serum action and the strains isolated from people and cattle were most resistant to the bactericidal activity of serum.     

Design of synthetic bactericidal peptides derived from the bactericidal domain P(18-39) of aprotinin.

A bactericidal domain, P(18-39), of the proteinase inhibitor aprotinin, possesses the structural feature of two antiparallel beta-sheets connected by a short turn. In order to understand the structural requirements for antibacterial activity, two peptides, each having the sequence corresponding to a single beta-sheet structure of P(18-39), were synthesized and their antibacterial properties investigated. One peptide, P(18-28), with the sequence IIRYFYNAKAG, was active against almost all the bacterial strains investigated. However, the bactericidal activity of P(18-28) was reduced compared to the parent molecule, P(18-39). The other peptide, P(29-39), with the sequence LCQTFVYGGCR, was only weakly bactericidal against Pseudomonas aeruginosa. A peptide, P(18-26), devoid of the C-terminus dipeptide Ala-Gly of P(18-28), retained the bactericidal activity of P(18-28) against most of the bacterial strains investigated. Only Klebsiella pneumoniae, P. aeruginosa and Staphylococcus aureus were resistant to P(18-26). Replacement of lysine 26 by arginine in P(18-26) (IIRYFYNAR) improved the bactericidal activity. The retropeptide, RANYFYRII, retained the antibacterial activity of IIRYFYNAR toward Gram-negative bacteria, but it was less active against Gram-positive bacteria. The random peptide, IANRIYRYF, was as bactericidal as IIRYFYNAR. Moreover, the random peptide possessed, in contrast to IIRYFYNAR, a strong antifungal activity against Candida albicans. Elimination of the N-hydrophobic terminal Ile-Ile from P(18-26) (RYFYNAK) strongly reduced the bactericidal potency of the peptide. Attaching the hydrophobic peptide, FFVAP, to the C-terminal of P(18-26) (IIRYFYNAKFFVAP) increased the bactericidal potency of the peptides considerably. We concluded that the order of the amino acids in the sequence of the peptides is not, per se, a critical feature for bactericidal activity. Hydrophobic interaction between peptide and bacterial membrane is probably the most important feature involved in the bactericidal mechanism of the antibiotic peptides.     

Bacteriostatic and bactericidal activity of antituberculosis drugs against Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium intracellulare complex and Mycobacterium kansasii in different growth phases.

Bacteriostatic and bactericidal activities of rifampicin, isoniazid, streptomycin, enviomycin and ethambutol against Mycobacterium tuberculosis, Mycobacterium avium--M. intracellulare complex and Mycobacterium kansasii were studied in different growth phases. Bacteriostatic activities of the drugs were similar in different growth phases, except isoniazid. M. tuberculosis was much less susceptible to isoniazid in the lag phase than in the log and the stationary phases. In contrast, bactericidal activity was influenced by the growth phase. M. tuberculosis was killed by isoniazid, streptomycin and rifampicin. The bactericidal activity of isoniazid was strongest. The bactericidal activity of isoniazid and streptomycin was most marked in the log phase. M. avium complex and M. kansasii resisted the bactericidal activity, but some strains of M. avium complex were killed by streptomycin and enviomycin, and the activities of these two drugs were most marked in the lag phase.