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Electron microscopic and microcalorimetric investigations of the possible mechanism of the antibacterial action of a defined propolis provenance.

Microcalorimetric and electron microscopic studies on the mode of the antibacterial action of propolis were performed on Streptococcus agalactiae. It was shown that propolis inhibits bacterial growth by preventing cell division, thus resulting in the formation of pseudo-multicellular streptococci. In addition, propolis disorganized the cytoplasm, the cytoplasmic membrane, and the cell wall, caused a partial bacteriolysis, and inhibited protein synthesis. It was evident that the mechanism of action of propolis on bacterial cells is complex and a simple analogy cannot be made to the mode of action of any classic antibiotics.

Anti-Bacterial Agents↗

[Recurring meningococcal meningitis in hereditary C 5 deficiency].

Inherited deficiency of the 5th complement component (C5) was the cause of recurrent meningococcal meningitis in a woman aged 20 years. Familial investigations confirmed an autosomal co-dominant type of inheritance for the defect. Whereas additional loss of chemotaxis only exists with a genetic defect in C5 complement, bacteriolytic activity of serum is lacking in all genetic defects of the terminal complement components C5 to C8. An intact bacteriolysis, however, is necessary for resistance against Neisseria meningitidis and Neisseria gonorrhoeae. Neisseria infections, particularly recurrent meningococcal meningitis or disseminated gonococcal infections, suggest the presence of a congenital or acquired deficiency in one of the complement components C5 to C8.

Adult↗

Bactericidal activity of human milk: stability during storage.

Human milk provides infants with defensive factors against many illnesses. This study aims to analyse global bactericidal activity in fresh human milk and evaluate its stability in relation to milk manipulation and its possible alteration following refrigeration. Nineteen milk samples (mature milk) from 19 healthy women are analysed. Viability testing involving a strain of Escherichia coli NCTC 9111, serovar O111:K58(B4):H- was used to determine the bactericidal effect of human milk. Degree of bacteriolysis is calculated as the difference between E. coli counts in controls and in milk samples, expressed as a percentage of the control sample counts. An evaluation of the effect of refrigeration at 4-6 degrees C after 24, 48 and 72 hours, and at -20 degrees C for seven days on bactericidal capacity is made. Bactericidal activity was detected in all milk samples analysed (77.33 +/- 15.14%). This activity persisted after refrigeration for 48 hours and after freezing for 10 days, but showed a significant decrease after refrigeration for 72 hours. In conclusion, maternal milk has bactericidal capacity, providing defence and protection against infection for newborn infants. This property can be altered during the storage of milk. Consequently, if storage in excess of 48 hours is required, freezing is preferable to refrigeration.

Anti-Bacterial Agents↗

Bactericidal activity of the alternative complement pathway generated from 11 isolated plasma proteins.

Exposure of E. coli K12 W1485 to the cytolytic alternative pathway assembled from the 11 isolated pathway proteins resulted in killing of the bacteria, as evidenced by loss of viability. Lysis of the bacteria required introduction of lysozyme into the reaction mixture. The time-course and dose dependency of bacteriolysis in the isolated system were identical to those in C4-depleted serum. The bacteriolytic activity of the pathway was highly dependent on the concentration of the pathway proteins and became insignificant at 1:16 physiological concentration. Electron microscopic visualization of killed and of lysed bacteria revealed numerous complement membrane lesions and partial disintegration of the outer phospholipid membrane. Scanning electron microscopy showed that killed bacteria were enlarged, partially collapsed and exhibited irregular surface protrusions. Lysed bacteria were fragmented and appeared polymorphic. This study demonstrates that the alternative pathway, in absence of immunoglobulins, has the potential or eradicating gram-negative bacteria.

Blood Bactericidal Activity↗

Molecular interplay of murein synthases and murein hydrolases in Escherichia coli.

Affinity chromatography using different lytic transglycosylases as a specific ligand revealed an interaction of both murein hydrolases and murein synthases. This interaction is taken as evidence for the assemblage into a multienzyme complex that could function as a murein replicase precisely copying the given three-dimensional structure of the murein sacculus. The sacculus of the mother cell would function as a template, which is identically replicated by copying the lengths of the existing glycan strands and the pattern of crosslinkages. A hypothetical enzyme complex specifically involved in cell division and a complex specifically involved in cell elongation are presented. It is postulated that PBPs 1a and/or 1b are present in both complexes, whereas the presence of PBP2 or PBP3 defines the specificity of the murein-synthesizing machinery as being involved in either cell elongation or septation. Moreover, the proposed "holoenzyme" suprastructure could explain why the specific inhibition of PBPs 1a/1b results in bacteriolysis and why inhibition of PBP2 and PBP3 causes the well-known morphological alterations, spherical growth, and filamentation, respectively.

Escherichia coli↗

A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli.

The first gene of a family of prokaryotic proteases with a specificity for L,D-configured peptide bonds has been identified in Escherichia coli. The gene named ldcA encodes a cytoplasmic L, D-carboxypeptidase, which releases the terminal D-alanine from L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine containing turnover products of the cell wall polymer murein. This reaction turned out to be essential for survival, since disruption of the gene results in bacteriolysis during the stationary growth phase. Owing to a defect in muropeptide recycling the unusual murein precursor uridine 5'-pyrophosphoryl N-acetylmuramyl-tetrapeptide accumulates in the mutant. The dramatic decrease observed in overall cross-linkage of the murein is explained by the increased incorporation of tetrapeptide precursors. They can only function as acceptors and not as donors in the crucial cross-linking reaction. It is concluded that murein recycling is a promising target for novel antibacterial agents.

Amino Acid Sequence↗

Immunoepidemiology of meningococcal disease in military recruits. II. Blocking of serum bactericidal activity by circulating IgA early in the course of invasive disease.

The potential role of IgA antibody to Neisseria meningitidis, which blocks bacteriolysis by IgG and IgM, in producing the susceptible state in military recruits, a population at greater than expected risk, was investigated in 28 patients with meningococcal disease. IgA was removed from acute-phase sera by immunoadsorption; IgG was separated by ion-exchange, and IgM by gel-filtration chromatography. The bactericidal activity of acute-phase sera, before and after removal of IgA, and of IgG and IgM fractions from the sera, was tested against each of the homologous infecting strains. Bactericidal activity, the correlate of protective immunity, was deficient in 24 of 28 unabsorbed sera, but uniformly present after the removal of IgA in a median titer of 1:16. IgM accounted for all or nearly all of the bactericidal activity. IgG was largely inactive. Susceptibility to meningococcal disease may be affected by the blocking of bactericidal IgM by circulating IgA.

Blood Bactericidal Activity↗

Role of immunity in the clearance of bacteremia due to Haemophilus influenzae.

The role of antibodies to capsular and somatic antigens in the clearance of Haemophilus influenzae was investigated by active and passive immunization. The clearance index (k) and the proportion of strain b organisms cleared 30 min after intravenous administration (deltaY30) were greater in eight-week-old actively immunized rats (k = 0.693, deltaY30 = 4.07) than in nonimmune animals (k = 0.075, deltaY30 = 0.95)(P less than 0.025 for all); however, clearance correlated imprecisely with titers of bactericidal or anticapsular antibody. In three-week-old rats, intranasal immunization with strain b or U significantly increased (P less than 0.005) the rate of clearance of strains b and U. Passive immunization with antibodies to somatic or capsular antigens significantly increased the rate of clearance (P less than 0.001) and the proportion of bacteria cleared (P less than 0.05) with all test strains. The increased clearance associated with passive immunization correlated with increased splenic uptake of 32P-labeled H. influenzae (r = 0.83, P less than 0.025). Analysis of the disappearance of viable organisms and bacterial 32P suggested that bacteriolysis of H. influenzae did not occur during clearance of the bacteremia. Either antibody to capsular antigen or antibody to somatic antigen, administered or evoked in rats, accelerates intravenous clearance of H. influenzae by promotion of reticuloendothelial phagocytosis.

Animals↗

Antibacterial mechanisms of antibody to mannose-sensitive pili of Escherichia coli.

To identify mechanisms whereby antibody to mannose-sensitive pili of Escherichia coli might enhance host defenses, we evaluated the activity of antibody to pili in four antibacterial immune processes. Antiserum to pili and Fab' fragments of IgG antibody to pili inhibited the ability of homologous piliated organisms to adhere to buccal epithelial cells. However, this antiserum did not enhance intravascular clearance, complement-dependent bacteriolysis, or opsonophagocytosis. The addition of antiserum to pili to polymorphonuclear leukocytes and piliated E. coli reduced the rate of killing from 52% to 5%. The addition of complement restored the rate to 52%, but this was much less than the 99% rate achieved with polymorphonuclear leukocytes and either fresh serum or antibody to the whole bacteria. These observations suggest that the principal anti-bacterial property of antibody to mannose-sensitive pili of E. coli is inhibition of bacterial attachment. Whether the anti-opsonic effect of antibody to pili is detrimental to host defense remains to be determined.

Adhesiveness↗

Complement-mediated early clearance of Haemophilus influenzae type b from blood is independent of serum lytic activity.

The role of complement-mediated serum lytic activity in the clearance of encapsulated Haemophilus influenzae type b was studied by comparing clearance of bacteria from blood in normal mice with that in congeneic C5-deficient animals and mice depleted of complement by using cobra-venom factor. The clearance of organisms from blood by C5-deficient and C5-sufficient normal animals was similar. C3 depletion and the inability to fix C3 to the surface of bacteria were, however, associated with impaired clearance of organisms during the first 24 h after intravenous bacterial challenge. These studies suggest that complement-mediated opsonization rather than bacteriolysis is important in determining early clearance of H. influenzae type b from the bloodstream.

Animals↗

In-vitro activity of ofloxacin against Mycobacterium tuberculosis and its clinical efficacy in multiply resistant pulmonary tuberculosis.

The in-vitro susceptibilities to ofloxacin of 159 clinical sputum isolates of Mycobacterium tuberculosis, comprising 95 isolates sensitive to all drugs, 31 isolates resistant to streptomycin or isoniazid or both, 27 isolates resistant to streptomycin, isoniazid and rifampicin and six isolates resistant to rifampicin (and in three cases to other drugs) were determined. Favourable MICs of ofloxacin (0.63-1.25 mg/l) were demonstrated for 147 isolates (92%). Twenty-two patients with resistant strains (including one patient with rifampicin intolerance) were studied: ten were given 300 mg ofloxacin and ten were given 800 mg ofloxacin, once daily in both cases, together with second-line accompanying drugs, for nine months to one year. Two received 800 mg of ofloxacin once daily alone for similar periods. In the 300 mg-ofloxacin group and the 800 mg-ofloxacin group, five and eight patients, respectively, achieved culture conversion; the rest failed. In the former group, the peak serum ofloxacin concentrations were 3.71-8.08 mg/l and the mean sputum/serum ratio was 0.85. In the latter group, the corresponding values were 10-18.7 mg/l, and 0.76, respectively. All patients tolerated the drugs well. Analysing only patients with accompanying drugs, those on ofloxacin 800 mg once daily had more rapid sputum culture conversion than those on ofloxacin 300 mg once daily (Mann-Whitney Wilcoxon Rank Sum Test: P less than 0.05), indicating more rapid bacteriolysis and implying the definite efficacy of ofloxacin when used together with second-line accompanying drugs in the management of resistant tuberculosis.

Adult↗

The effect of three broad-spectrum antimicrobials on mononuclear cell responses to encapsulated bacteria: evidence for down-regulation of cytokine mRNA transcription by trovafloxacin.

The effect of trovafloxacin, ciprofloxacin and ceftriaxone on cytokine production of human peripheral blood mononuclear cells (PBMCs) was examined. PBMC responses were measured after stimulation with lipopolysaccharide (LPS), lipoteichoic acid (LTA) or killed or viable Streptococcus pneumoniae and Haemophilus influenzae. Trovafloxacin inhibited the production of tumour necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6 and IL-8 by PBMCs after stimulation with either LPS or LTA by 83%. Similar inhibition occurred in PBMCs incubated with killed or live bacteria and trovafloxacin, but not with ciprofloxacin or ceftriaxone. The relevance of this in vitro observation was explored by examining TNF-alpha and IL-6 responses in trovafloxacin-treated mice. Serum concentrations of both cytokines 1 h after LPS challenge were 95% less than serum concentrations in mice that were not given trovafloxacin. Reverse transcription- polymerase chain reaction studies of the mechanisms determining cytokine down-regulation demonstrated that trovafloxacin reduced TNF-alpha, IL-1beta and IL-6 mRNA to levels similar to those of unstimulated cells. These observations indicate that trovafloxacin can consistently and significantly reduce production of cytokines that play an important role in sepsis. In vitro, this effect can occur in the presence of bacteriolysis and is associated with inhibition of transcription of cytokine genes.

Animals↗

Gram-negative shock in rats depends on the presence of capsulated bacteria and is modified by laparotomy.

To develop a hyperdynamic sepsis model in rats, four Escherichia coli strains were used, which differed in the presence or absence of a capsule or K antigen (K1 and K-, respectively) and/or in O serogroup (O9 and O18). Of the two clinical isolates, O9K- did not survive in rat serum, whereas O18K1 and two isogenic laboratory strains (O18K1 and O18K-) were able to resist serum bacteriolysis. Pentobarbital-anesthetized rats (n = 21) received an intravenous bolus of 10(9) bacteria. In contrast to the two noncapsulated strains, both capsulated strains induced hyperdynamic shock; arterial lactate rose from a mean value of .91 to 3.09 mmol.L-1, systemic vascular resistance dropped from 1.15 to .78 mmHg.min.mL-1, and cardiac output transiently increased from 98 to 115 mL.min-1; renal plasma flow remained at 3-4 mL.min-1, whereas glomerular filtration rate decreased from 1.3 to .7 mL.min-1. Laparotomy, which is often performed to study kidney function, completely abolished the hyperdynamic condition, while glomerular filtration rate was still decreased. We conclude that in rats, in contrast to humans, capsulated bacteria are required to induce a hyperdynamic septic shock; the hyperdynamic characteristics of the shock do not occur in animals subjected to a laparotomy.

Animals↗

Persistence of group C anticapsular antibodies two to three years after immunization with an investigational quadrivalent Neisseria meningitidis-diphtheria toxoid conjugate vaccine.

BACKGROUND: An investigational quadrivalent (A, C, Y and W-135) meningococcal conjugate (MC-4) vaccine was reported to be more immunogenic in 2-year-olds than the currently licensed meningococcal polysaccharide vaccine, but persistence of serum antibody beyond 6 months after conjugate vaccination is unknown. OBJECTIVE: Determine persistence and the immunologic basis of protective activity of group C anticapsular antibodies in sera obtained 2-3 years after MC-4 vaccination. DESIGN: Group C antibody concentrations, bactericidal activity and passive protective activity were measured in sera from 48 children, ages 4-5 years, who had been immunized 2-3 years earlier with an MC-4 vaccine and from 47 children who had not been previously vaccinated. RESULTS: Serum antibody concentrations were higher in the vaccinated than the unvaccinated children (geometric means, 0.30 and 0.09 mug/mL, respectively, P < 0.0001). Bactericidal titers > or =1/4 (considered protective) were infrequent in both vaccinated and unvaccinated children (14.6 and 6.4%, respectively, P = 0.3). Passive protective activity against bacteremia in the infant rat model was more frequent in sera from vaccinated (37.5%) than sera from unvaccinated children (12.5%, P < 0.02). The proportion of sera with passive protective activity increased with increasing anticapsular antibody concentrations (P < 0.0001). INTERPRETATION: Serum group C antibody concentrations remained elevated for 2-3 years after MC-4 vaccination, and passive protective activity was more frequent in vaccinated than unvaccinated children. However, serum antibody concentrations in many vaccinated children were no longer sufficient to activate complement-mediated bacteriolysis in vitro or to confer passive protection against experimental group C disease.

Animals↗

The effect of purified lipopolysaccharide on the bactericidal reaction of human serum complement.

Escherichia coli ML308 225 was killed and lysed by human serum. Bacterial sensitivity to serum was maximal for organisms in the early-exponential phase of batch culture and declined as growth proceeded. Bacterial killing was dependent upon the complete complement sequence and the subsequent action of serum lysozyme caused bacteriolysis. Purified lipopolysaccharide from this organism bound to serum-sensitive bacteria and inhibited the bactericidal reaction.

Blood Bactericidal Activity↗

D-alanyl-lipoteichoic acid in Lactobacillus casei: secretion of vesicles in response to benzylpenicillin.

Vesicles containing lipoteichoic acid (LTA) have been isolated from Lactobacillus casei ATCC 7469 grown in the presence of either benzylpenicillin or D-cycloserine. These cell wall antibiotics enhanced the rate of LTA and lipid secretion 6.7 times, whereas chloramphenicol inhibited their release. The formation of these vesicles from peripheral and septal wall regions did not appear to be the result of bacteriolysis. The vesicle composition of LTA and lipid was similar to that of the cytoplasmic membrane whereas the protein composition was dissimilar. The size of these vesicles ranged from 20 to 40 nm and the length of LTA ranged from 5 to 50 glycerol phosphate residues. The isolation of these vesicles provides a potential in vitro acceptor system for studying the D-alanylation of lipoteichoic acid.

Chloramphenicol↗

Peptidoglycan biosynthesis in Escherichia coli: variations in the metabolism of alanine and D-alanyl-D-alanine.

The in vivo functioning of the alanine/D-alanyl-D-alanine pathway of Escherichia coli was investigated by determining precursor pool levels and specific enzyme activities under various growth conditions. Cells grown on D- or L-alanine showed several remarkable features compared with cells grown on other carbon sources: 10-fold higher values of the D-alanyl-D-alanine and the UDP-MurNAc-pentapeptide pools, a 240-fold increase of the alanine racemase activity, and the absence of bacteriolysis after treatment with D-cycloserine at high concentrations (50 micrograms ml-1). In cells grown on glucose, D-cycloserine (1 micrograms ml-1) led to depletion of the D-alanyl-D-alanine pool and to lysis, which was efficiently antagonized by chloramphenicol. A threefold increase of the dipeptide pool was observed when cells were treated with chloramphenicol alone. The alanine racemase activity was lowest in glucose-grown cells and the D-alanine:D-alanine ligase and D-alanyl-D-alanine-adding activities were the same whatever the carbon source. Molecular masses of 53-56 kDa and 56-60 kDa were estimated for the partially purified inducible alanine racemase and D-alanine:D-alanine ligase respectively.

Alanine↗

The pathogenic role of fimbriae of Haemophilus influenzae type b in murine bacteraemia and meningitis.

Complement activation and development of murine bacteraemia and meningitis following intranasal instillation of cell-bound fimbriated or non-fimbriated organisms were compared to clarify the role of fimbriae in the pathogenesis of illnesses caused by Haemophilus influenza type b (Hib). In-vitro resistance of non-fimbriate bacteria to the bactericidal effects of normal human serum was at least 400 times greater than that of fimbriate bacteria. The amount of C3 bound to fimbriate Hib was more than that to non-fimbriate Hib. When mice were infected with fimbriate bacteria, 11.5% died. When mice were infected with non-fimbriate bacteria, the mean number of viable organisms gradually increased or was constant up to day 7; 38.5% of these mice died. These in-vivo results were coincident with the in-vitro data. However, the content of polyribosyl ribitol phosphate (PRP) in fimbriate organisms was the same as in non-fimbriate organisms. These results indicate that fimbriate Hib may be less likely to produce bacteraemia and meningitis, correlating with the greater susceptibility to complement-mediated bacteriolysis and the lower mortality seen with this type of organism, although fimbriae increase adherence to epithelial cells (mucosal surface).

Animals↗