Cell wall degradation ofStaphylococcus aureus by lysozyme

In contrast to former findings lysozyme was able to attack the cell walls ofStaphylococcus aureus under acid conditions. However, experiments with¹⁴C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. attack from the inside). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but stabilized protoplasts were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.     

Effect of physiological conditions on the autolysis ofStaphylococcus aureus strains

The effect of physiological conditions on autolysis and autolytic activity in various strains ofStaphylococcus aureus was determined. The rate of whole cell autolysis ofS. aureus was growth phase dependent and a maximum rate was observed in early stationary phase cultures. However, the autolysins extracted by the freeze-thaw method (cell-wall bound autolytic activity) did not show any significant increase in activity. The addition of NaCl to the growth medium enhanced the rate of autolysis with the highest rate being displayed by cultures grown in 1.5 M NaCl. However, lower autolytic activity was found in the freeze-thaw extracts of cultures grown at higher concentrations of NaCl. The rate of autolysis of cultures grown at 30°C was higher than cultures grown at 37 or 43°C. Thus, the rate of autolysis seems to be independent of the bacterial growth rate. Cultures grown in slightly acidic conditions showed a faster rate of autolysis compared to cultures grown under alkaline conditions. SDS-polyacrylamide gel containing 0.2% crude cell-wall ofS. aureus did not show any obvious correlation with the appearance of any particular lytic band in the zymogram to autolytic activity or rate of autolysis of cultures grown under various environmental conditions. A nonhemolytic phenotype, mutations in the accessory gene regulator, and lysogeny (phages ø11, ø12, ø13) had no obvious effect either on the rate of autolysis or on the pattern of lytic bands in the zymograms.     

Effect of photoactivated hematoporphyrin derivative on the viability ofStaphylococcus aureus

The photodynamic effect of hematoporphyrin derivative (HPD) on the viability of penicillin-resistantStaphylococcus aureus is described. Growth rate of the bacteria was markedly reduced by exposure to light and HPD.Staphylococcus viability was decreased by 80% in 3 h of growth even at low HPD concentration (12 g/ml). A synergistic killing effect of HPD, light, and penicillin (10 g/ml) onStaphylococcus aureus was demonstrated, although the bacteria were originally resistant to 100 g/ml penicillin. A residual viability of only 3% was found by growth in medium containing this drug combination. The surviving bacteria after 3 h of treatment were sensitive even to 1 g penicillin/ml. The mechanism of HPD action onStaphylococcus is composed of two steps: i) penetration of HPD into the bacterial cell, which may be accomplished in the dark with no harm to the cells; ii) damaging of the bacterial cell upon photoactivation. The photoactivated HPD completely inhibited thymidine incorporation into the bacterial DNA. This effect was accompanied by inhibition of RNA and protein synthesis, which was parallel in extent to the reduction in growth rate. Electron microscopic examination ofS. aureus exposed for 3 h to HPD and light showed the appearance of well-developed mesosomes in the bacterial cells. None of these effects was observed on Gram-negative bacteria.     

Chemical and ultrastructural studies on the cell wall ofStaphylococcus simulans biovarstaphylolyticus

Cell walls of strains ofStaphylococcus simulans biovarstaphylolyticus andS. aureus FDA 209P were compared ultrastructurally and chemically to investigate the mechanism of resistance of this strain ofS. simulans to its own staphylolytic endopeptidase. Chemical analysis of the peptidoglycans of the various strains examined showed that cells that were more resistant to lysis by the endopeptidase had lower glycine/serine ratios in their cross bridges and that, within a species, the more resistant cells had either fewer residues in these cross bridges or fewer cross bridges. Ultrastructural studies showed that cell wall thickness was not involved in resistance to the enzyme. Comparisons of the endopeptidase susceptibility of intact cells and isolated peptidoglycans from these cells suggested that the three-dimensional structure of the cell wall may play a role in resistance to lysis by the endopeptidase.     

Effect of specific growth limitations on cell wall composition of Staphylococcus aureus H

Conditions are described for the continuous culture of a derivative of Staphylococcus aureus H in a fully defined minimal medium in which cysteine is the sole amino acid. The effects of growth under various nutrient limitations on the composition and properties of the cell wall have been studied. The proportion of ribitol teichoic acid present in the wall, and the extent to which it is substituted with N-acetylglucosamine, varies in bacteria grown under different conditions as does the composition and extent of cross-linking of the peptidoglycan. Neither the derivative nor the original strain H produced teichuronic acid when grown under phosphate limitation.Non-Standard Abbreviation SDS Sodium dodecyl sulphate     

Contribution on the diagnosis ofStaphylococcus aureus rosenbach

Возможность трансформации прогестерона, стероида ?S? (11-дезокси-17-гидроксикортикостерон), дегидроэпиандростерона и

     

Inhibition of coagulase activity and growth ofStaphylococcus aureus by garlic extracts

Garlic extract (1 mg dry weight/ml) produced an inhibition of the coagulase reaction and increased the time of coagulation by a factor of 1.5, whereas 4 mg dry weight/ml increased the coagulation time factor by 2.75. At this latter concentration of garlic, coagulation was only partially complete at 4 h. Garlic extract (1.4 mg dry weight/ml) reduced growth in nutrient broth whereas 5.6 mg dry weight/ml was completely inhibitory. These effects were not observed until 8 h after exposure of the organisms to the garlic extracts. There were 5.9% more survivors among garlic treated mice compared to non-treated animals, but this difference is not significant.     

Reciprocal regulation of anaerobic and aerobic cell wall mannoprotein gene expression in Saccharomyces cerevisiae

The DAN/TIR genes encode nine cell wall mannoproteins in Saccharomyces cerevisiae which are expressed during anaerobiosis (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3, TIR4, and TIP1). Most are expressed within an hour of an anaerobic shift, but DAN2 and DAN3 are expressed after about 3 h. At the same time, CWP1 and CWP2, the genes encoding the major mannoproteins, are down-regulated, suggesting that there is a programmed remodeling of the cell wall in which Cwp1 and Cwp2 are replaced by nine anaerobic counterparts. TIP1, TIR1, TIR2, and TIR4 are also induced during cold shock. Correspondingly, CWP1 is down-regulated during cold shock. As reported elsewhere, Mox4 is a heme-inhibited activator, and Mot3 is a heme-induced repressor of the DAN/TIR genes (but not of TIP1). We show that CWP2 (but not CWP1) is controlled by the same factors, but in reverse fashion-primarily by Mot3 (which can function as either an activator or repressor) but also by Mox4, accounting for the reciprocal regulation of the two groups of genes. Disruptions of TIR1, TIR3, or TIR4 prevent anaerobic growth, indicating that each protein is essential for anaerobic adaptation. The Dan/Tir and Cwp proteins are homologous, with the greatest similarities shown within three subgroups: the Dan proteins, the Tip and Tir proteins, and, more distantly, the Cwp proteins. The clustering of homology corresponds to differences in expression: the Tip and Tir proteins are expressed during hypoxia and cold shock, the Dan proteins are more stringently repressed by oxygen and insensitive to cold shock, and the Cwp proteins are oppositely regulated by oxygen and temperature.     

Production and purification ofStaphylococcus aureus toxic-shock toxin

Staphylococcus aureus strain 5761, isolated from a patient with toxic-shock syndrome, was used for the production of toxic-shock toxin. The medium used contained 4% bio-Trypcase and 1% yeast extract adjusted to pH 7. Production of 50 μg of toxic-shock toxin/ml of culture supernatant was obtained. The purification method involves removal of the toxin from the culture supernatant with Biorex 70 resin and purification by isoelectric focusing, on 2% (pH 3–10) ampholine-sucrose gradients, and gel filtration on Sephadex G-100. Three antigenically similar entities were isolated after electrofocusing, with a major component at isoionic point pH 7.4. The purified toxin migrated as a homogeneous protein with a molecular weight of 23,700 when tested by gel electrophoresis. Specific antibodies to toxic-shock toxin in rabbits were obtained after one subcutaneous injection of 5 μg enterotoxin.     

Extraction and characterization ofStaphylococcus aureus h2 antigen

Staphylococcus aureus antigen h₂, one of the few heat-stable type antigens of this bacteria, has been extracted from bacteria by autolysis and purified by alcohol precipitation, ion exchange chromatography, and gel filtration. Purified h₂ is a precipitating antigen, reacting neither with antisera against α and β ribitol teichoic acids, nor with antisera against Staphylococcal lipoteichoic acid. Infrared spectrometry and chemical analysis reveal a teichoic acidlike composition with glycerol, phosphorus, N-acetylated glucosamine, and alanine as major constituents. These findings confirm the validity of the Oeding-Haukenes scheme of serotyping.     

Effect of anaerobic and aerobic exercise of equal duration and work expenditure on plasma growth hormone levels

Growth hormone (GH) and lactic acid levels were measured in five normal males before, during and after two different types of exercise of nearly equal total duration and work expenditure. Exercise I (aerobic) consisted of continuous cycling at 100 W for 20 min. Exercise II (anaerobic) was intermittent cycling for one minute at 285 W followed by two minutes of rest, this cycle being repeated seven times. Significant differences (P less than 0.01) were observed in lactic acid levels at the end of exercise protocols (20 min) between the aerobic (I) and anaerobic (II) exercises (1.96 +/- 0.33 mM X 1(-1) vs 9.22 +/- 0.41 mM X 1(-1), respectively). GH levels were higher in anaerobic exercise (II) than in aerobic (I) at the end of the exercise (20 min) (2.65 +/- 0.95 micrograms X 1(-1) vs 0.8 +/- 0.4 micrograms X 1(-1); P less than 0.10) and into the recovery period (30 min) (7.25 +/- 6.20 micrograms X 1(-1) vs 2.5 +/- 2.9 micrograms X 1(-1); P less than 0.05, respectively).     

The antibacterial activity of vancomycin towards Staphylococcus aureus under aerobic and anaerobic conditions

AIMS: To investigate the antibacterial efficacy of vancomycin towards Staphylococcus aureus under aerobic and anaerobic conditions, and to assess the influence of oxygen on the duration of the post-antibiotic effect (PAE) after exposure to vancomycin. METHODS AND RESULTS: Culture-based techniques and flow cytometric measurements of 5-cyano-2,3-ditolyl tetrazolium chloride (an indicator of redox activity) and the membrane potential-sensitive fluorophore Sytox Green, were used to test four staphylococcal strains. The MICs for all strains, and the duration of PAE, were similar whether tested with or without oxygen. However, a fivefold logarithmic reduction in cell counts was observed in 10-15 h aerobically, depending on strain, compared with longer than 60 h in an anaerobic environment. Flow cytometric data correlated well with counts of colony-forming units under both aerobic and anaerobic conditions. CONCLUSIONS: The death rate of Staph. aureus exposed to vancomycin was greater in the presence of oxygen, although MIC values and PAE durations were similar whether tested aerobically or anaerobically. Also, flow cytometry provided a rapid and sensitive alternative to plate counts for the assessment of antibiotics in oxygen-free conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study underlines the need for further anaerobic testing using different strain/antibiotic combinations, the results of which will have clinical significance due to the anaerobic nature of some sites of infection.     

A rapid method for the differentiation ofStaphylococcus aureus hemolysins

A rapid method for the differentiation of hemolytic staphylococci is described. Instead of a β-hemolysin monoproducingStaphylococcus culture, a test strip, soaked in a stabilized product of theS. aureus strain CCM 6188 with defined cytolytic activity, is used. The results of the rapid method can be read one day earlier than those of the conventional method. A set of 137 strains ofS. aureus from various sources, including 46 enterotoxin-producing (SE-positive) ones, were examined by both methods. A higher proportion of α- and σ-hemolysin-producing strains was found among the SE-positive strains, while (α+β)-hemolysin production prevailed among the SE-negative ones.     

Effect of papulacandin B on the cell wall and growth of Geotrichum lactis

Addition of the antifungal antibiotic papulacandin B to an exponential culture of Geotrichum lactis inhibited incorporation of glucose into the alkali-insoluble and alkali-soluble glucan fractions of the hyphal wall, although the rate of growth was practically unaltered. Synthesis of other cell wall components (i.e. galactomannan and chitin) was not affected. Papulacandin B also induced the proliferation of branches along the hyphae which continued to branch dichotomously resulting in a 'colonial' pattern of growth. Aculeacin A, another antifungal antibiotic that inhibited beta-glucan synthesis also caused morphological alterations similar to those described for papulacandin B. Inhibition of beta-glucan synthesis and the altered growth pattern persisted for several hours after removal of the antibiotic. Recovery of beta-glucan synthesis and restoration of the normal pattern of growth occurred simultaneously. Growth of G. lactis in L-sorbose medium also led to inhibition of beta-glucan synthesis and dichotomous branching.     

Immunological examination of the glycocalyces ofStaphylococcus aureus strains Wiley and Smith

The immunological examination of the glycocalyces ofStaphylococcus aureus has been concerned with capsular elements while essentially neglecting slime layers. We have found bacterial slime layers to be prevalent in many natural bacterial environments and, in particular, in recently isolatedS. aureus strains Wiley and Smith [1]. Growth in modified staphylococcus 110 medium induces slime layer production in these strains, and investigation of this material has revealed the two slime layers to be immunogenically and antigenically identical. The slime layer of the Smith strain is immunologically distinct from the tight, integral capsule that also comprises the glycocalyx of this strain. The Wiley strain glycocalyx is composed of only a slime layer.     

Temperature-sensitive mutants ofStaphylococcus aureus: Isolation and preliminary characterization

Temperature-sensitive (ts) mutants ofStaphylococcus aureus were isolated after mutagenesis with nitrosoguanidine and two cycles of enrichment with Penicillin G and D-Cycloserine. The mutants expressed tight, coasting, and leaky phenotypes on solid media. In broth, however, most exhibited coasting for a limited number of generations. The reversion frequency of selected ts mutants was less than 10^–6. Intraperitoneal (i.p.) immunization with ts mutant G/1/2 conferred significant protection (0 dead/6 total vs. 7/7, immunized vs. control; p=0.0006) from lethal i.p. challenge with the parental wild-type (wt)S. aureus suspended in 5% porcine mucin, performed 28 days after i.p. administration of 10⁸ colony-forming units. Protection induced by mutants of coasting phenotype was higher and lasted longer than that induced by mutants of the tight phenotype. The results of this study demonstrate that ts mutants ofS. aureus can be obtained and that ts mutants are able to induce protective immunity from subsequent challenge with the parental wt strain.     

Distribution of amino acids in the cellular fractions ofStaphylococcus aureus

WhenStaphylococcus aureus cells were labeled with a single radioactive amino acid for 20 minutes, the highest activity, except for alanine, leucine, and glycine, was found in the free pool. Significant amounts of the above amino acids and also valine and methionine were incorporated into the protein — cell wall fraction.Cells previously labeled with a single amino acid underwent a net loss of radioactivity when transferred to buffer, glucose, or complete medium. An exception was glycine. The greatest loss in activity occurred in the free pool.While some amino acids (alanine, cystine) were transferred from the free pool to the protein — cell wall fraction under all conditions tested, others (glutamic acid, proline) were transferred only under conditions of growth.Cells labeled with certain single amino acids and then transferred to a complete medium lost a significant portion of the label. The most extreme case noted was proline, but other amino acids also effluxed from the cell under these conditions.