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Scanning electron microscopic study of Streptococcus mutans BHT lysed by lysozyme.

The effects of purified salivary lysozyme (HSL) and hen egg white lysozyme (HEWL) on the surface structure of Streptococcus mutans BHT were studied with the aid of scanning electron microscopy. In parallel experiments bacteriolysis was monitored by liberation of 3H-thymidine incorporated into the bacteria. Control cells maintained their shape and had intact cell walls during the experimental period. Exposure of the cells to HSL (5.0 U/ml) or HEWL (5.0 micrograms/ml) for 3 and 18 h resulted in progressive destruction of cell structure. Some cells exhibited ruptures of the cell walls on top of spherical swellings, predominantly located at the ends of the bacteria. After 18 h the majority were disrupted in the septal area leaving numerous empty cup shaped cell walls in the preparations. The findings of the electron microscopic examination were confirmed in the biochemical assay.

Animals↗

Target for bacteriostatic and bactericidal activities of beta-lactam antibiotics against Escherichia coli resides in different penicillin-binding proteins.

The relationship between cell-killing kinetics and penicillin-binding protein (PBP) saturation has been evaluated in the permeability mutant Escherichia coli DC2 in which the antimicrobial activity of beta-lactams has been described as being directly related to the extent of saturation of the PBP target(s). Saturation of a single PBP by cefsulodin (PBP 1s), mecillinam (PBP 2), and aztreonam (PBP 3) resulted in a slow rate of killing (2.5-, 1.5-, and 0.8-log-unit decreases in the number of CFU per milliliter, respectively, in 6 h). Saturation of two of the three essential PBPs resulted in a marked increase in the rate of killing, which reached the maximum value when PBPs 1s and 2 were simultaneously saturated by a combination of cefsulodin and mecillinam (4.7-log-unit decrease in the number of CFU per milliliter in 6 h). Inactivation of all three essential PBPs by the combination of cefsulodin, mecillinam, and aztreonam further increased the killing kinetics (5.5-log-unit decrease in the number of CFU per milliliter), and this was not significantly changed upon additional saturation of the nonessential PBPs 5 and 6 by cefoxitin. Similar relationships between PBP saturation and killing kinetics were obtained with imipenem and meropenem at concentrations which inhibited only one PBP (PBP 2), only two PBPs (PBP 1s and 2), or all three essential PBPs. Saturation of one or more PBPs also resulted in a different rate of bacteriolysis, the highest rate being obtained by the cefsulodin-mecillinam combination and by 5 micrograms of either imipenem or meropenem per ml. All of these conditions caused saturation of PBP 2 and saturation or extensive binding of PBP 1s. However, none of these conditions caused determined the fastest possible rate of killing, which occurred only when all three essential PBPs were saturated. It was concluded that the actual killing effect of beta-lactams is reflected by killing rates that approach the fastest possible rate for the given microorganism and that the targets for the bactericidal activity are precisely those PBPs whose saturation or binding occurs under conditions.

Anti-Bacterial Agents↗

Requirement of autolytic activity for bacteriocin-induced lysis.

The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co(2+) and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmADelta1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmADelta1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.

Bacteriocins↗

Sequential metabolic expressions of the lethal process in human serum-treated Escherichia coli: role of lysozyme.

Several metabolic parameters indicative of Escherichia coli function and integrity were kinetically examined in response to treatment with normal human serum in the presence and absence of functional human lysozyme. Specific inhibition of this enzyme in bacteriolytic and bactericidal reactions was accomplished by using purified rabbit anti-human lysozyme immunoglobulin G. Initiation of the complement-mediated alterations of cytoplasmic membrane integrity, as judged by the leakage of 86Rb from prelabeled cells or the hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by a cryptic strain, was found to be independent of lysozyme action. Furthermore, inhibition of macromolecular synthesis by E. coli in response to serum treatment occurred at the same time regardless of the functional state of lysozyme. Although the rate and extent of bacteriolysis were reduced in the absence of lysozyme, the bactericidal kinetics was unaffected. These results demonstrate that the lethal events associated with the action of antibody and complement on gram-negative bacteria are independent of lysozyme, suggesting an accessory role for this enzyme in immune reactions. A possible temporal sequence of complement-induced effects occurring at the cell surface is presented.

Bacterial Proteins↗

Immunity in experimental salmonellosis. II. Basis for the avirulence and protective capacity of gal E mutants of Salmonella typhimurium.

Salmonella typhimurium strains which are deficient in uridine diphosphate (UDP)-galactose-4-epimerase (gal E mutants) owe their outstanding protective capacity when used as live vaccine to the fact that when galactose is supplied exogenously, such as occurs in vivo, smooth cell wall lipopolysaccharides are synthesized. The mutants lose most of their protective capacity when this phenotypic curing is prevented by a second mutation of the kind found in strains LT(2)M(1)A (deficient in galactokinase) or E(32) (deficient in UDP-galactose-lipopolysaccharide transferase). Despite such phenotypic reversion, the gal E mutants are rendered avirulent as a result of galactose-induced bacteriolysis. Secondary mutants have been isolated which differ from each other with respect to the extent of galactose-induced lysis. The differences in galactose sensitivity are attributable to different activities of the other Leoloir pathway enzymes, namely, galactokinase and galactose-1-phosphate-uridyl transferase. The influence of these enzymes on lipopolysaccharide composition and galactose sensitivity and thus on virulence and immunogenicity of gal E mutants has been studied.

Adsorption↗

Analysis of murein and murein precursors during antibiotic-induced lysis of Escherichia coli.

Lysis of Escherichia coli induced by either D-cycloserine, moenomycin, or penicillin G was monitored by studying murein metabolism. The levels of the soluble murein precursor UDP-N-acetylmuramyl-L-alanyl-D-glutamyl-m-diaminopimelyl-D-alanyl- D-alanine (UDP-MurNAc-pentapeptide) and the carrier-linked MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol as well as N-acetylglucosamine-beta-1,4-MurNAc-(pentapeptide)-pyrophosphoryl- undecaprenol varied in a specific way. In the presence of penicillin, which is known to interfere with the cross-linking of murein, the concentration of the lipid-linked precursors unexpectedly decreased before the onset of lysis, although the level of UDP-MurNAc-pentapeptide remained normal. In the case of moenomycin, which specifically blocks the formation of the murein polysaccharide strands, the lipid-linked precursors as well as UDP-MurNAc-pentapeptide accumulated as was expected. D-Cycloserine, which inhibits the biosynthesis of UDP-MurNAc-pentapeptide, consequently caused a decrease in all three precursors. The muropeptide composition of the murein showed general changes such as an increase in the unusual DL-cross bridge between two neighboring meso-diaminopimelic acid residues and, as a result of uncontrolled DL- and DD-carboxypeptidase activity, an increase in tripeptidyl and a decrease in tetrapeptidyl and pentapeptidyl moieties. The average length of the glycan strands decreased. When the glycan strands were fractionated according to length, a dramatic increase in the amount of single disaccharide units was observed not only in the presence of penicillin but also in the presence of moenomycin. This result is explained by the action of an exo-muramidase, such as the lytic transglycosylases present in E. coli. It is proposed that antibiotic-induced bacteriolysis is the result of a zipperlike splitting of the murein net by exo-muramidases locally restricted to the equatorial zone of the cell.

Bacteriolysis↗

The importance of a lipopolysaccharide-initiated, cytokine-mediated host defense mechanism in mice against extraintestinally invasive Escherichia coli.

Extraintestinally invasive Escherichia coli (EC) that possess both a complete LPS and K1 capsule evade both complement-mediated bacteriolysis and neutrophil-mediated killing. Since C3H/HeJ mice that are hyporesponsive to LPS were uniquely susceptible to lethal infection with EC of this phenotype, we speculated there was an LPS-initiated host defense mechanism against this pathogenic phenotype. The LPS-normoresponsive C3H/HeN as well as the C3H/HeJ mice cleared these EC from the circulation within 4 h of intravenous administration. Whereas electron micrographs of the liver demonstrated these EC undergoing degeneration within the phagolysosomes of of both macrophages and Kupffer cells of C3H/HeN mice, these EC replicated within these cells of the C3H/HeJ mice. Restoration of anti-EC activity of C3H/HeJ mice occurred with activation of Kupffer cells and peritoneal macrophages in vivo with BCG and in vitro with IFN-gamma, but not with LPS. Pretreatment of C3H/HeJ mice with a combination of recombinant murine IL-1 and TNF-alpha also restored the killing of K1(+)-EC but did not enhance the killing of a K1(-)-EC mutant. These data are consistent with the hypothesis that (a) there is no intrinsic inability of C3H/HeJ phagocytes to kill EC, but (b) an LPS-initiated, cytokine-mediated host defense mechanism is required for such killing. These studies emphasize the importance of bacterial surface characteristics in the interaction with specific host defenses.

Animals↗

A bactericidal monoclonal antibody elicits a change in its antigen, OspB of Borrelia burgdorferi, that can be detected by limited proteolysis.

mAb CB2, directed against outer surface protein B (OspB), causes bacteriolysis of Borrelia burgdorferi in the absence of complement. How this happens is unknown. We examined the effect of mAb binding on OspB tertiary structure by using limited proteolysis to probe changes in protein conformation. Truncated OspB (tOspB) that lacked N-terminal lipid was cleaved by four enzymes: trypsin, endoproteinase Arg-C, endoproteinase Asp-N, and endoproteinase Glu-C. CB2 affected the cleavage by trypsin and Arg-C, but not by AspN or Glu-C. None of the enzymes cleaved CB2 under these conditions. Both trypsin and Arg-C cleaved tOspB near the N-terminus; CB2 slowed the rate of cleavage, but did not affect the identity of the sites cleaved. Irrelevant mAb had no effect, indicating that the effect was specific. CB2 was active against tOspB of strain B31, but not against tOspB of strain BEP4, to which it does not bind, suggesting that binding was required to elicit the effect on cleavage. With trypsin, CB2 showed a maximal effect at 8 mol of tOspB to 1 mol of mAb. At this ratio, not enough CB2 was present to bind all the tOspB; therefore, either CB2 shows turnover or CB2 acts by binding tOspB and effecting a change in this tOspB such that it, in turn, propagates the effect in other molecules of tOspB. Regardless of the mechanism, these data show that CB2 elicits a change in tOspB that can be measured by its reduced susceptibility to protease cleavage.

Amino Acid Sequence↗

Synergism of cephalosporins at subinhibitory concentrations and polymorphonuclear leukocytes on phagocytic killing of Escherichia coli and its mode of action.

The interactions between subinhibitory concentrations of cephalosporins and polymorphonuclear leukocytes in the killing of a strain of Escherichia coli are described and an attempt is made to define the responsible mechanism. Ceftizoxime was the most potent agent tested. Pretreatment of the E. coli strain with subinhibitory concentrations of ceftizoxime increased the susceptibility to both; phagocytic killing activity of the polymorphonuclear leukocytes and bactericidal activities of the oxygen metabolites and the granule extracts. A most interesting result was the increased susceptibility of the ceftizoxime-treated E. coli to killing by beta-glucuronidase which normally is not bactericidal. It is suggested that the augmented killing could be due to bacteriolysis by beta-glucuronidase.

Animals↗

[Role of lipopolysaccharide in the action of complement on gram negative bacteria].

In this review a short description of the methods for the activation of the complement system and data on the role of different structures of lipopolysaccharide of gram negative bacteria in this process are presented. Variants of complement-induced bacteriolysis are considered. Special attention is given to cholera infection and the role of Vibrio cholerae O139 polysaccharide in interaction with the complement.

Animals↗

Comparison of four immune variables and pulmonary lesions of goats with intrapulmonary exposure and subsequent intrathoracic challenge exposure with Pasteurella haemolytica.

A comparison of immune variables following lung sensitization with live Pasteurella haemolytica serotype 1 (Ph1)-impregnated agar beads was done in 2 separate trials. The Ph1 immune variables studied were blood bactericidal activity, serum bacteriolysis, total classical complement, and indirect hemagglutination antibody. Each trial had 16 male weanling goats: 6 controls and 10 principals. In trial 1, each goat was surgically catheterized through the trachea, then the material was deposited in a bronchus. The controls received only agar beads and the principals received agar beads impregnated with live Ph1. These goats were studied for 32 days, euthanatized, and necropsied. In trial 2, the controls were each transthoracically injected with agar beads into the left lung and the principals were similarly injected with agar beads impregnated with live Ph1. These goats were studied for 35 days, then challenge exposed transthoracically by injection of Ph1 in saline solution (1.2 x 10(7) CFU/ml) into the right lung. Four days later, they were euthanatized and necropsied. The volume of lung consolidated tissue was an excellent measure of Ph1 immunity. Principal goats generated solid protective immunity to subsequent challenge exposure because minimal or no lung consolidation was observed, whereas large volumes of lung consolidation were seen in the controls. The principal goats in trial 1 gave a weak serum indirect hemagglutination Ph1 antibody response, which was attributed to the bronchial method of depositing the Ph1. The corresponding response of the control group remained negative. The Ph1 agar beads (1 x 10(6) CFU in 0.5 ml) protected the bacteria from immediate phagocytosis and lysis as indicated by the induced pneumonic deaths of 2 principals 5 days later.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Anti-bacterial activity of peritoneal cells from transgenic mice producing high levels of GM-CSF.

Two lines of transgenic mice carrying the gene for granulocyte-macrophage colony-stimulating factor (GM-CSF) produce vastly increased numbers of macrophages with abundant foamy cytoplasm resembling classical activated macrophages. Cells from both lines were negative for myeloperoxidase, a bactericidal enzyme found in monocytes as well as neutrophils, but not mature macrophages. Cells from the so called 'male line' produced greatly increased levels of oxygen degradation products in response to phagocytosis, compared with cells from the 'female line' or from normal littermates. The ability of the cells to phagocytose and lyse the intracellular bacterium Listeria monocytogenes was tested in vitro using radiolabelled organisms. Although the cells from transgenic mice were more highly phagocytic than cells from normal littermates, cells from either line were no more efficient than normal at lysing the bacteria they had phagocytosed. Nevertheless, because of the high phagocytic rate, more bacteria were exposed to lysis in the cells of transgenic mice, and the final outcome was a higher rate of bacteriolysis.

Animals↗

[Epidemiological study of a group of staphylococci isolated in a hospital environment].

A survey has been carried out on 336 strains of staphylococci isolated in a hospital environment in order to assess the validity of the lyogroup classifying method, to know their epidemiological behavior in our territory and to test their sensitivity to various antibiotics. The findings of the survey can be summed up as follows: the practical test for bacteriolysis activity appears to be a valid method of identifying staphylococci in so far as it allows to classify them, but also to differentiate them from micrococci; lyogroup VI appears to be prevalent both as a whole and in nearly all materials of origin; the incidence of resistance is highest for piperacillin (47%), gentamicin (20%), tobramycin (19%); it is lowest for netilmicin (1.2%) and amikacin (2.6%); among meticilline resistant strains (18%) there is a marked resistance towards nearly all antibiotics tested.

Anti-Bacterial Agents↗

[Bactericidal activity of aspoxicillin in an in vitro model simulating human serum levels].

Bactericidal activities of aspoxicillin (ASPC) against E. coli (2 strains) and K. pneumoniae (1 strain) were compared with those of piperacillin (PIPC) using in vitro kinetic models simulating human serum levels. In the model of intravenous injection, both drugs exhibited bactericidal action against the strains of E. coli. The activity of ASPC was found to superior to PIPC and ASPC could decrease viable cell counts from 10(7) cells/ml to 10(8) cells/ml. On the other hand, the bactericidal activity of ASPC against K. pneumoniae was weaker than that of PIPC. In the model of intravenous drip infusion, ASPC showed a high level of bactericidal activity comparable to that observed in the intravenous model against E. coli. Interestingly, the bactericidal activity of ASPC against K. pneumoniae in this model was similar to that of PIPC, though the MIC value of ASPC was higher than that of PIPC. In the intravenous model, the effects of ASPC and PIPC on the morphology of E. coli KC-14 were examined with a phase contrast microscope. Exposure to PIPC caused only an elongation of the cells, but the treatment with ASPC resulted in the formation of spheroplast-like structure of the cells, which were finally subjected to bacteriolysis.

Amoxicillin↗

Cytotoxic antibody dependent cells in mice experimentally infected with Brucella abortus.

In this study we investigated the cytotoxic activity mediated by antibody dependent cells (K cell) in C57BL/6 mice experimentally infected with Brucella abortus. The involvement of the K cells in experimental infection from Brucella was studied by evaluating the bacteriolysis in the presence of specific antibodies. After 3 h of incubation, approximately 30 +/- 3% of the labeled Brucellae undergo lysis, whereas the presence of lymphocytic populations alone, or of a specific decomplemented serum, determines a lysis of 5 +/- 3%. Cytotoxic antibody dependent activity of purified lymphomonocytic populations (ADCC) evaluated against cells labeled with 51Cr sensitized with specific antibodies obtained from mice is considerably increased in mice experimentally infected with Brucellae with respect to that present in non-infected mice. The increased activity begins to manifest itself 5 to 7 days after inoculation of the Brucellae reaching a maximum on the 10th day. The same results were obtained in the lymphocytic populations prepared from normal animals and those infected by Brucellae searching for K cells with the ability to form rosettes (EA-RFC).

Animals↗

[Attempt to express the spontaneous global bacteriolytic power of water].

The utilization of a technique for the determination of spontaneous bacteriolytic power in surface waters has led us to propose a new way of expressing the spontaneous bacteriolytic power of water: PBSG. PBSG allows us to evaluate the intensity of global spontaneous bacteriolysis and to compare the global spontaneous bacteriolytic powers of different water samples.

Bacteriolysis↗

Lysozyme, an abomasal enzyme in the ruminants.

A strong lytic activity against Micrococcus luteus was demonstrated in abomasal secretions from calf, adult cattle, goat and sheep. This bacteriolytic activity was undetectable in other secretions. Bacteriolysis was caused by a glycosidase displaying endo-N-acetylmuramoylhydrolase specificity (EC 3.2.1.17) and was further characterized in the calf. This lysozyme also displayed significant chitinase activity. Immunofluorescence microscopy confirmed the secretion of lysozyme by abomasal gastric glands exclusively. Electrofocusing revealed multiple molecular forms, the predominant one (more than 80%) being characterized by Mr approx. 15,000, pH optimum 5.0, pl 7.5 and remarkable conformational stability. The lytic activity of lysozyme was ionic strength dependent and competitive inhibition was observed with both N-acetyl glucosamine and N-acetyl-muramic acid. Amino-acid analysis demonstrated common characteristics with known lysozymes, i.e. four disulphide bridges, two proline and N-terminal lysine. Structural homology between the three ruminant lysozymes was established by immunological cross-reactivity.

Abomasum↗

[Various aspects of humoral immunity in meningococcal infections. II. Formation of antibodies to group A meningococci in adults with generalized meningococcal infections].

Altogether 258 serum samples obtained from 102 patients with generalized meningococcal infection were studied by means of the immune bacteriolysis test and the indirect hemagglutination test. This study revealed a pronounced increase in the titer of bactericidal and hemagglutinating antibodies to group A meningococcus in the process of the disease. The dynamics and intensity of antibody formation, revealed by means of the two tests, were found to be in complete correspondence. Antibody formation was most intensive in meningitis accompanied by meningococcemia. In most cases these antibodies were found to belong to the class of IgM on account of their physico-chemical nature. The highest bactericidal activity was found in the sera with high hemagglutinin titers; the summary titers of these sera were 4-64 times higher than the titers of cystein-resistant antibodies.

Antibodies, Bacterial↗