Quantitative characteristics of human antibodies to the peptidoglycans of different Staphylococcus species

The content of antibodies to peptidoglycans of 13 staphylococcal species was studied in the ELISA with the IgG fraction isolated from commercial human immunoglobulin. The content of antibodies to S. aureus peptidoglycan (calculated per mg of protein) was 3.0 micrograms and that of antibodies to peptidoglycans of other species amounted to 1.8-4.1 micrograms. The maximal value was obtained for S. xylosus and the minimal value, for S. lentus. No relationship between the content of antibodies to peptidoglycans and the degree of their antigenic affinity to S. aureus peptidoglycan was noted: the scatter of the values in species with relatively low and even minimal affinity did not exceed the range of variance in the group with a high degree of homology (2.2-2.4 micrograms). The production of antibodies to staphylococcal peptidoglycans was supposed to result from the action of a complex of antigenic determinants, both common and specific for each staphylococcal species.     

Reactivity to bacterial peptidoglycans in a phagocytosis system. 1. The range of immunological specificity of Staphylococcus aureus peptidoglycan

The antigenic features of S. aureus peptidoglycan (PG) were studied in the reaction of stimulation of oxygen-dependent neutrophil metabolism, mediated by the IgG opsonins of normal human serum. The study was carried out at different taxonomic levels: the species (S. aureus), the genus (Staphylococcus), the family (Micrococcaceae), as well as in relation to remote taxons (organisms belonging to the families Streptococcaceae, Enterobacteriaceae, Neisseriaceae, to the genus Corynebacterium). All S. aureus strains were identical with respect to the specificity of their PG, essentially differing from other bacteria in this regard. After the removal of antibodies to different PG the effectiveness of PG opsonization decreased by 10.4-44.7%. Such decrease was most pronounced in experiments with the PG of streptococci (S. pyogenes, S. faecalis, S. salivarius) and Micrococcus luteus.     

Structural studies of peptidoglycans in Campylobacter species.

Peptidoglycans (PG) from Campylobacter coli, Campylobacter jejuni, and Campylobacter fetus were composed of muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid in a molar ratio of 1.1:1:1.7:1.1:09. Thirty percent of the amino groups of diaminopimelic acid were involved in cross-linkages between peptides. During cultivation, C. coli and C. jejuni changed from a spiral to a coccoid form. In C. coli, we could isolate PG only from the spiral forms in yields of 0.8-1.2% by dry weight. C. fetus did not change to a coccoid form, and always contained PG. Thus, it is possible that the morphological transformation from the spirals to the coccoid forms of C. coli and C. jejuni is accompanied by, and probably due to, the degradation of PG.     

Effects of gentamycin on interactions of neutrophils with Staphylococcus

The authors studied the effect of gentamicin on abilities of neutrophils to uptake and killing of extracellular Staphylococcus. Gentamicin had not changed naturally the ability of healthy men and burn patients neutrophils to uptake Staphylococcus, but intensified the ability of neutrophils to killing of these bacteria.     

Heterogeneity in chemotaxis of neutrophils with different densities

We investigated N-formyl-Methionyl-Leucyl-Phenylalanine (fMLP)-induced chemotaxis of neutrophils with different densities. FMLP-induced (10(-8)M) chemotaxis of neutrophils with lower density were significantly reduced when compared to neutrophils with higher density (p less than 0.05). These findings imply a relationship between the neutrophil density and chemotaxis.     

Genetic analysis of morphological variability in three Plantago species with different mating systems

Summary Morphological variability in three Plantago species possessing different mating systems — P. Major, P. coronopus, and P. lanceolata — was analyzed. From each species several populations were analyzed. In comparing the species, it appeared that both the inbreeding P. major and the outbreeding P. lanceolata showed a high degree of morphological differentiation between populations. It was hypothesized that the absence of population differentiation for the species with a mixed mating system, P. coronopus, was caused by high adaptive plasticity. Plasticity is probably the predominant mechanism by which this species copes with different environments. There were considerable differences between populations within each species for their levels of variability and their heritabilities. It appeared that besides the influence of the mating system, selection may diminish morphological variability in the case of strong directional selection, especially in P. major and P. lanceolata.Grassland Species Research Group Publication No. 135     

Synergy of human neutrophils with fluconazole in killing Candida species

The killing of Candida species by human neutrophils in a long-term 24-h assay and possible synergy with fluconazole (FCZ) for killing was investigated. The test medium (TM) consisted of RPMI-1640, penicillin and streptomycin (P/S), and 10% fresh autologous serum. TM alone was highly fungistatic for Candida species compared to TM without serum. When neutrophils were cocultured in TM with Candida species for 24 h the inoculum colony-forming units (CFU) were always significantly reduced (killing) by 58 to 99%. FCZ was tested over a range of 1–500 g/ml, and though almost always fungistatic itself, it synergized with neutrophils for significantly increased killing of C. albicans (isolate Sh27) (P<0.01) and C. albicans (isolate 94-20) (P<0.05). Killing of non-albicans Candida species was so efficient in the absence of FCZ that demonstration of synergy with FCZ was difficult.     

Stimulation of the respiratory burst of the neutrophils from healthy subjects by different Staphylococcus aureus concentrations

Preopsonized live and heat-killed S. aureus stimulated, without the washing of serum, the luminol-dependent chemiluminescence of human neutrophils obtained from healthy donors. The intensity of chemiluminescence was evaluated by the index of stimulation with staphylococci, with due consideration for their concentration. With the microbe/phagocyte ratio equal to 10:1, these indices had the maximum values when both live and killed staphylococci were used. At high concentrations of staphylococci, especially live ones, all indices were low (those for live staphylococci had negative values) and uniform. As the concentration of the antigen decreased, individual features in the reaction of each donor became apparent. With the microbe/phagocyte ratio equal to 100:1, stimulation with live and killed staphylococci induced the identical fluorescence of neutrophils. The capacity of nonopsonized staphylococci for inducing chemiluminescence was poorly pronounced. For this reason, the test system using S. aureus at low concentrations was proposed for the prognostication of this infection, while the ratio 100:1 can be used for the evaluation of the opsonin-phagocytic system in case of a developed purulent process.     

A comparative study of the immunological properties of peptidoglycans of different origins

The action of peptidoglycans (PG) of different origin has been experimentally studied in vivo. In these experiments PG of bacterial origin, such as blastolysin (BL), and synthetic PG, viz. muramyldipeptide (MDP) and its analog glucosaminylmuramyldipeptide (GMDP) have been used. Their toxicity, allergenic action, their effect on the phagocytic activity of peritoneal exudate macrophages (PEM), the accumulation of antibody-producing cells in the spleen, antibody titer in the blood serum and delayed hypersensitivity to nonbacterial antigens have been determined. As revealed in this study, BL does not differ from MDP in its toxicity and allergenic action. The phagocytic activity of PEM under the influence of BL only insignificantly differs from their activity under the influence of MDP, but is lower than under the influence of GMDP. The adjuvant action of BL is somewhat higher than that of synthetic PG.     

Oxygen-dependent killing of Staphylococcus aureus by human neutrophils

Luminol-dependent chemiluminescence was used as a monitor of reactive oxidant generation during phagocytosis of Staphylococcus aureus by human neutrophils. Reactive oxidants play a crucial role in the killing of this organism because: (a) S. aureus was killed most rapidly when the rate of increase of chemiluminescence was greatest; (b) neutrophils which had been activated to generate reactive oxidants by re-aeration of anaerobic suspensions killed this bacterium more efficiently than control suspensions; and (c) neutrophils from a patient with chronic granulomatous disease could neither generate reactive oxidants nor kill S. aureus.     

Evaluation of the usefulness of selected commercial systems for identification of Staphylococcus species

The aim of the study was the assessment of the usefulness of commercially available systems for rapid identification of staphylococci. API STAPH (bioMerieux, France), ID 32 STAPH (bioMerieux, France), GPL (HTL, Poland) and Staph-Zym (Rosco, Denmark). The identifications were carried out according to producer's instruction. The material for the study comprised 76 staphylococcal strains, coagulase-positive and coagulase-negative. The strains were isolated from throat, nasal, wound, bone slivers, pus and blood of inpatients and from throat and nasal swabs of outpatients. Besides that, for the study staphylococcal strains were obtained from the American Collection of Typical Cultures (ATCC) and from the Polish Collection of Microorganisms (PCM). All tested strains were identified on the basis of classic biochemical tests. In the light of obtained results it is concluded that the commercial system most suitable for identification of staphylococci was ID 32 STAPH (bioMerieux), which has a wide spectrum of species identifiable with it and the highest percent (95%) of correctly identified species. The least suitable system was the GPL 15 (HTL, Poland).     

The characteristics of Staphylococcus aureus peptidoglycan in the spectrum of the reactivity of human lymphocytes to bacterial peptidoglycans]

The sensitivity of lymphocytes of healthy persons to S. aureus peptidoglycan as compared with that to the polyclonal stimulator zymosan C3b and peptidoglycans of other bacteria (Streptococcus faecalis, Escherichia coli, Bacterium bifidum) was analyzed with a test system permitting the determination of specific reactivity to peptidoglycans. The analysis showed that at the peak of luminol-dependent chemiluminescence (25-30 minutes) individual reactivity to S. aureus peptidoglycan varied within wide limits (the coefficient of lymphocyte stimulation was 1.4-9.6, 3.5 +/- 0.6), exceeding sensitivity to other bacteria, as well as the values obtained in the negative control. The conclusion of the wide spread of sensitization to S. aureus peptidoglycan and the possibility of using this preparation for the study of cell-mediated immunity reactions was made.     

Survival of Staphylococcus aureus inside neutrophils contributes to infection

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar-) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.     

Reactivity in human cerebral artery: species variation

It is becoming obvious that the reactivity of vascular smooth muscle to vasoactive agents is not homogeneous in different arteries (cerebral vs. other arteries) from the same animal species or in cerebral arteries from different species. In this communication, the reactivity of human cerebral arteries to norepinephrine (NE), dopamine (DA), small amounts of K+ and ouabain and their mechanisms of action are compared with those in monkey and dog cerebral arteries. NE produces moderate contractions in the primate arteries, mediated by alpha 1 adrenoceptors, and slight contractions in the dog arteries, possibly mediated by alpha 2 receptors. DA relaxes human and monkey cerebral arteries but contracts the dog arteries. The primate artery relaxation mediated by dopaminergic receptors is large enough to predominate over the alpha 1 receptor-mediated contraction. Minute amounts of K+ preferentially relax cerebral arteries from humans, monkeys, and dogs, possibly activating the electrogenic Na+ pump. Ouabain, a Na+ pump inhibitor, contracts these cerebral arteries with low concentrations. Human and monkey cerebral arteries respond similarly to vasoactive agents presented so far, and appear to share the same mechanisms of action; however, the responsiveness of dog cerebral arteries differs.     

Reactivity of different oxidases with lignins and lignin model compounds

Oxidase activities toward lignins and lignin model compounds failed to produce low molecular weight products. Peroxidase and laccase react similarly, and can be distinguished when both enzymes are present on the basis of pH activity differences with syringaldazine as substrate.     

A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species

D evriese , L.A. 1984. A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species. Journal of Applied Bacteriology 56 , 215–220.
A biotyping system for Staphylococcus aureus strains is proposed which is a simplified version of biotyping procedures described in the literature. It differentiates Staph. aureus strains from man and animals into host-specific ecovars and biotypes which are not host-specific. With the help of tests for βhaemolysin, staphylokinase, coagulation of bovine plasma and the crystal-violet reaction, the origin of many but not all Staph. aureus strains can be determined: 604 of 809 strains from man. poultry, cattle, pigs, goats, rabbits and foods could be alloted to four ecovars which are typically associated with man, poultry, sheep and goats and cattle. The other strains belonged to five non-host specific biotypes.     

Controlled pseudopod extension of human neutrophils stimulated with different chemoattractants

The formation of pseudopods and lamellae after ligation of chemoattractant sensitive G-protein coupled receptors (GPCRs) is essential for chemotaxis. Here, pseudopod extension was stimulated with chemoattractant delivered from a micropipet. The chemoattractant diffusion and convection mass transport were considered, and it is shown that when the delivery of chemoattractant was limited by diffusion there was a strong chemoattractant gradient along the cell surface. The diffusion-limited delivery of chemoattractant from a micropipet allowed for maintaining an almost constant chemoattractant concentration at the leading edge of single pseudopods during their growth. In these conditions, the rate of pseudopod extension was dependent on the concentration of chemoattractant in the pipet delivering chemoattractant. The pseudopod extension induced using micropipets was oscillatory even in the presence of a constant delivery of chemoattractant. This oscillatory pseudopod extension was controlled by activated RhoA and its downstream effector kinase ROCK and was abolished after the inhibition of RhoA activation with Clostridium botulinium C3 exoenzyme (C3) or the blocking of ROCK activation with Y-27632. The ability of the micropipet assay to establish a well-defined chemoattractant distribution around the activated cell over a wide range of molecular weights of the used chemoattractants allowed for comparison of the effect of chemoattractant stimulation on the dynamics of pseudopod growth. Pseudopod growth was stimulated using N-formylated peptide (N-formyl-methionyl-leucyl-phenylalanine (fMLP)), platelet activating factor (PAF), leukotriene B4 (LTB(4)), C5a anaphylotoxin (C5a), and interleukin-8 (IL-8), which represent the typical ligands for G-protein coupled chemotactic receptors. The dependence of the rate of pseudopod extension on the concentration of these chemoattractants and their equimolar mixture was measured and shown to be similar for all chemoattractants. The inhibition of the activity of phosphoinositide-3 kinase (PI3K) with wortmannin showed that 72%-80% of the rate of pseudopod extension induced with N-formyl-methionyl-leucyl-phenylalanine, platelet activating factor, and leukotriene B4 was phosphoinositide-3 kinase-dependent, in contrast to 55% of the rate of pseudopod extension induced with interleukin-8. The dependence of the rate of pseudopod extension on the concentration of individual chemoattractants and their equimolar mixture suggests that there is a common rate-limiting mechanism for the polymerization of cytoskeletal F-actin in the pseudopod region induced by G-protein coupled chemoattractant receptors.