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.     

The use of bacteriophages in eliminating polyresistant strains ofStaphylococcus aureus andStreptococcus agalactiae

Temperate bacteriophages were induced in and released from isolates of Staphylococcus aureus and Streptococcus agalactiae using mitomycin C. Various specific indicator cultures were tested for providing clear plaques after phage infection. Specific lytic mixture of bacteriophages was prepared using the induced, modified and laboratory variants of phages. Under laboratory conditions, the mixture eliminated all isolates from the tested collection of microorganisms. The restriction barrier of some bacterial isolates to bacteriophage infection was overcome either by UV irradiation or in vitro modification of bacteriophage DNA with specific methyltransferases. Conjugative R plasmids, capable of replication in G+ and G- bacteria, were detected and isolated from S. aureus and S. agalactiae antibiotic-resistant strains.     

Immunological characterization of an exopolysaccharide from the Staphylococcus aureus strain Smith diffuse

Exopolysaccharides (EXPs) of Staphylococcus aureus are associated with virulence in animal models. An EXP from the S. aureus strain Smith diffuse was previously detected in 64.3% of S. aureus clinical isolates. EXP was isolated from culture supernatants of this strain after DNAase, RNAase, phosphodiesterase I and lysostaphin treatment, and was further purified by cation-exchange and molecular-sieve chromatography. Isoelectric focusing revealed a pI of 3.6 for the EXP while the pI of teichoic acid was less than 2.7. Crossed immunoelectrophoresis with homologous Smith diffuse antisera indicated that the EXP contained two immunological components. A major precipitin line persisted after the antisera had been absorbed with the non-EXP-producing variant strain, Smith compact, while the second component was removed. Tandem immunoelectrophoresis also demonstrated that the EXP was distinct from teichoic acid. The EXP contained 2-amino-2-deoxyglucuronic acid, glucose, mannose and galactose. No fatty acids or nucleic acids were present and total protein content was less than 2%. Teichoic acid could not be demonstrated in the EXP, thus further substantiating the immunological studies. S. aureus EXP isolated by the present method can be used for further serological and virulence studies.     

Contribution on the diagnosis ofStaphylococcus aureus rosenbach

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

     

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.     

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.     

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.     

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.     

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.     

Glycine betaine and proline are the principal compatible solutes ofStaphylococcus aureus

The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.     

A new method of the reporting of phage patterns ofStaphylococcus aureus

Summary A new method of recording the phage pattern ofStaphylococcus aureus is suggested. The phage numbers are given in order of strength of the various reactions. The stability of minor reactions gives valuable information for epidemiological purposes. Working with a grant from the Gezondheidsorganisatie T.N.O.     

Effect of aerobic and anaerobic growth on the cell wall ofStaphylococcus aureus

Electron micrographs ofStaphylococcus aureus 7167 which had been grown anaerobically showed that the cell wall was approximately 5 times thicker than the wall of bacteria after aerobic growth. Cell walls prepared from anaerobically grownS. aureus were more sensitive to the bacteriolytic enzymes: lysostaphin, lysozyme, and the wall-associated autolytic enzyme ofB. subtilis 168 I^?. Our findings are interpreted as evidence that the cell wall or surface of anaerobically grownS. aureus 7167 is different from that of aerobically grownS. aureus 7167. The findings suggest that the cell wall peptidoglycan of the anaerobe is a more loosely formed network, resulting in a more rapid solubilization by the bacteriolytic enzymes.     

The mechanism of photodynamic inactivation ofStaphylococcus aureus by deuteroporphyrin

The mechanism ofStaphylococcus aureus inactivation by deuteroporphyrin (DT) and light was studied with singlet oxygen quenchers or hydroxyl radical scavengers. The light-activated DT (10 /ml) reduced the viability of the culture to less than 1%, whereas methionine, tryptophan, and 1,4-diazabicyclo-2,2,2-octane (DBCO) used as singlet oxygen quenchers provided almost 60% protection. Propylgallate, which is a hydroxyl free radical scavenger, also provided 60% protection. The presence of a singlet oxygen quencher and propylgallate provided almost complete protection from inactivation (96%). Photoinactivation in the absence of culture media (in saline) increased the killing rate and decreased the ability of the singlet oxygen quenchers to protect. In the same conditions damage from hydroxl free radicals was well protected by propyl gallate. The present results indicate thatS. aureus photoinactivation by DT and light is mediated by both singlet oxygen and hydroxyl free radicals.     

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.     

Morphological stabilization of the glycocalyces of 23 strains of five Bacteroides species using specific antisera

Cells of five Bacteroides species were examined following treatment with homologous antisera and staining with ruthenium red. They were enveloped by glycocalyces and these extensive fibrous exopolysaccharide matrices were fully retained as an integral "capsule" by some cells, while other cells showed "capsule" as well as detached glycocalyx components forming an intercellular "slime.". These extensive glycocalyces collapsed during dehydration for electron microscopy and formed electron-dense accretions on cell surfaces and electron-dense reticula in intercellular spaces when the cells were treated with heterologous antiserum or when antibody stabilization was omitted. The glycocalyces of all strains, both stabilized and unstabilized, were observed outside the outer membranes of cell walls that showed the "classic" gram-negative structural organization. Appropriate modifications of the indirect fluorescent antibody test demonstrated an integral "capsule" on all strains examined; detached glycocalyx and varying amounts of slime were demonstrated after stabilization with homologous, but not heterologous, antiserum.     

Identification and functional reconstitution of phosphate: Sugar phosphate antiport ofStaphylococcus aureus

Summary Resting cells ofStaphylococcus aureus displayed a phosphate (P_i) exchange that was induced by growth with glucose 6-phosphate (G6P) orsn-glycerol 3-phosphate (G3P). P_i-loaded membrane vesicles from these cells accumulated³²P_i, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of³²P_i (andl-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (dl-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both P_i anddl-lactate to establish an internal pool of P_i. Thistrans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal P_i.Reconstitution of membrane protein allowed a quantitative analysis of P_i-linked exchange. P_i-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous³²P_iP_i exchange (K _i's of 2.2 and 1.4mm;V _max's of 180 and 83 nmol P_i/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (K _i=27 m) over G3P (K _i=1.3mm) and P_i (K _i=2.2mm), suggesting that the same antiporter was induced in both cases. We conclude that³²P_iP_i exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate. In this respect, P_i-linked antiport inS. aureus resembles other examples in a newly described family of bacterial transporters that use anion exchange as the molecular basis of solute transport.