Growth of Staphylococcus aureus with nafcillin in vitro induces alpha-toxin production and increases the lethal activity of sterile broth filtrates in a murine model.

The morbidity and mortality of Staphylococcus aureus infections remain high despite antibiotic therapy. To investigate further the observation that penicillins increase the hemolytic activity of staphylococcal cultures, 37 strains were grown in broth with and without subinhibitory nafcillin. Nafcillin stimulated hemolytic activity in nafcillin-susceptible and -resistant isolates. Sterile broth filtrates of nafcillin-associated cultures injected intraperitoneally in mice were more rapidly lethal than filtrates of the same strain grown without nafcillin. Lethality was neutralized by anti-alpha-toxin antisera. DNA-RNA hybridization revealed a nafcillin-associated increase in alpha-toxin mRNA during the postexponential growth phase after the activation of agr. Isolates grown in slightly inhibitory nafcillin concentrations had more alpha-toxin mRNA than did nafcillin-free cultures, whereas agr RNAIII levels were comparable. This suggests that nafcillin-induced alpha-toxin production is not entirely attributable to agr. A supplemental regulatory mechanism may be involved.

Diaper type and fecal contamination in child day care.

In this study, modern all-in-one, front closure, reusable cloth diapers were compared with single-use, disposable paper diapers for their effect on fecal contamination in the child day care environment. Four licensed child day care centers were surveyed from which 1722 bacterial samples were cultured. The frequency of isolation of fecal organisms ranged from a low of 12% of the total bacterial isolates at a center using cloth diapers to a high of 46% and 45%, respectively, obtained at a center using first paper and then cloth diapers. Diaper type, cloth versus paper, when the method of application and the handling are made comparable, showed no significant difference in the frequency or the intensity of fecal contamination in child day care centers, as measured in the play/sleep area, the diaper change area, or on the hands of the care givers and children. Future studies to control microbial contamination in child day care centers should focus on effective ways of reducing contamination of sink faucets, hands of the caregivers, and hands of the children.

Fecal contamination in child day care centers: cloth vs paper diapers.

OBJECTIVES: Cloth diapers with front closure and all-in-one design were compared with paper diapers containing absorbent gel material for their influence on fecal contamination of the environment in licensed child day care centers. METHODS: One infant room and two toddler rooms in each of four day care centers were monitored for the presence of fecal bacteria. Microbial samples were taken from the play/sleep area, the diaper-changing area, and the hands of the caregivers and the children. Sampling was done twice weekly for two 4-week periods. Each center used either cloth or paper diapers during the first period, changing to the other diaper type during the second period. RESULTS: A total of 1722 samples were cultured, 881 during the first 4 weeks and 841 during the second 4 weeks. The frequency of isolation of fecal organisms ranged from a low of 12% of the total bacteria isolates at a center using cloth diapers, to highs of 46% and 45%, respectively, at a center using first paper and then cloth diapers. Sink faucets and the hands of the caregivers and the children were often contaminated. CONCLUSIONS: Analysis of the results of comparisons between cloth and paper diapers showed no significant difference in the frequency (F = .380, P < .535) or the intensity of fecal contamination in child day care centers.

Induction of muscle-relaxing factor by staphylococcal alpha-toxin.

Brain tissue and serum from mice intracerebrally injected with 1 microgram of staphylococcal alpha-toxin contained elevated amounts of a naturally occurring brain tissue component(s) called muscle-relaxing factor (MRF). MRF induced reversible, generalized, flaccid paralysis of mice after intracerebral but not intraperitoneal or intravenous administration. MRF (i) was soluble in Hanks balanced salt solution and in acidified (pH 2) Hanks balanced salt solution, in which it partitions into ethyl acetate, acetone, and methanol; (ii) was separated from some pigments by thin-layer chromatography on silica gel plates; (iii) did not comigrate with prostaglandin and leukotriene standards during high-pressure liquid chromatography with a mu Bondapak fatty acid column; and (iv) did not contain amino acids, exhibit absorption maxima at a wavelength range of 210 to 600 nm, or fluoresce when exposed to UV light. MRF has been detected in rabbit brain that has been stored frozen at -70 degrees C and has been enhanced in vitro in slices of both mouse and rabbit brain following incubation of the brain slices with staphylococcal alpha-toxin. Studies to identify the chemical nature of MRF and the mechanism by which, in mice, it induces reversible, flaccid paralysis of voluntary muscle are continuing.

Staphylococcal alpha toxin: a study with chronically instrumented awake sheep.

The in vivo responses to staphylococcal alpha toxin are reported for 15 chronically instrumented awake yearling sheep. The data obtained from a total of 30 experiments are grouped into four categories of response: no response, noted in seven experiments done on 5 sheep; pressor response, obtained seven times in 4 sheep; fluid and solute exchange, noted on six occasions in 3 sheep; and acute heart failure and death, which occurred in 10 of the 15 sheep. "No response" denoted no change in any of the measured outcome variables. The group of sheep labeled as showing "pressor response" responded to alpha toxin infusion with an increase in pulmonary artery pressure, unaccompanied by changes either in lung lymph flow or in lung mechanics. "Changes in lung fluid and solute exchange" involve increases in lung lymph flow. The harbinger of the last category, acute left heart failure leading to death, was a marked elevation in left atrial pressure. The threshold response dose in sheep is approximately 21 micrograms/kg. A very steep dose-response curve is observed, with only a narrow window of doses, 15 to 25 micrograms/kg, between the group showing no response and the group showing death from acute heart failure. The data obtained in these studies indicate that the lethal effects of alpha toxin in sheep include acute heart failure, which may be due to direct toxicity to heart muscle and/or the coronary vasculature endothelium.

Reaction of staphylococcal alpha-toxin with peptide-induced antibodies.

Two peptides representing separate 13-amino-acid sequences of staphylococcal alpha-toxin have been synthesized and acrylamide gel-purified alpha-toxin monomer and hexamer forms have been prepared and used to produce antisera in rabbits. We report here that each synthetic peptide, P-I and P-II, induces the formation of a specific precipitating antiserum. Moreover, these sera also react with the toxin monomer and sometimes with the hexamer, indicating that each peptide has more than one epitope. The purified toxin monomer can induce antibodies to fragments of toxin but is significantly less potent than the hexamer in inducing antibodies to the toxin monomer and almost not effective in inducing a response to the toxin hexamer. The purified toxin hexamer induces responses that are almost the reciprocals of the monomers, with the antihexamer and -monomer responses dominating and almost no responses to fragments of toxin being induced. These responses are interpreted in terms of the stability of the toxin hexamer to proteolytic degradation, compared with the relative sensitivity of the monomer to proteases. In assays of toxin-neutralization activity, only those sera containing antihexamer antibodies can block toxin hemolytic activity. This is true for both peptide- and toxin-induced antisera. The basis for this apparent association between toxin-neutralizing potency and antihexamer reactivity is being studied. Peptide P-I contains the uniquely reactive tyrosine residue and may be involved in monomer-to-monomer associations required to form hexamers. Peptide P-II is near the carboxyl terminus of alpha-toxin and may be involved in the binding of toxin to membranes. In a study of the ability of each peptide to inhibit the rate of hexamer formation induced by membrane lipoprotein, peptide P-I (as expected) proves to be more efficient than peptide P-II. Finally, one rabbit immunized with the toxin hexamer produces antibodies to peptides P-I and P-II. This finding suggests that the two synthetic peptides selected for study are relevant to the in vivo immunoprocessing of staphylococcal alpha-toxin.

Staphylococcal alpha-toxin: a study of membrane penetration and pore formation.

Cell lysis by staphylococcal alpha-toxin, a potent virulence factor of most pathogenic strains of Staphylococcus aureus, follows a three-step sequence: binding of toxin to the membrane, leaking of ions caused by membrane injury, and rupturing of the membrane caused by osmotic swelling. The membrane injury step is composed of two separate events, membrane penetration and membrane perturbation. The membrane penetration event involves conversion of the soluble toxin monomer into an amphipathic molecule, which inserts into the lipid bilayer of the membrane. The membrane perturbation event involves association of the toxin monomers, in the plane of the membrane, to form hexameric transmembrane pores. In this study, we demonstrate that, in an asolectin liposome system, controlling the pH of the external buffer permits these two events to be temporally resolved. Using Controlled-Pore Glass bead-purified alpha-toxin, four events are measured as a function of pH: (a) release of potassium from prelabeled asolectin vesicles, (b) conversion of the toxin to a globally hydrophobic molecule, (c) binding of detergent by the toxin, and (d) labeling of the toxin with photoactivable, radiolabeled, hydrophobic probes. Two of these events, potassium release and conversion to a net hydrophobic state, are paired in that, for the event to occur, each requires a pH of 4.6 or less. In contrast, photolabeling with the membrane probes PC I and PC II (where PC represents phosphatidylcholine) is easily detectable at pH values as high as 5.0 and 6.0. These results demonstrate that, as the pH is lowered, two distinct changes in the physical properties of alpha-toxin occur. The first, which occurs under mild acidic conditions, converts the toxin from a water-soluble molecule into an amphipathic molecule. The second, requiring relatively more acidic conditions, converts the amphipathic toxin molecule into a globally hydrophobic molecule. Correlated with these physical changes in the alpha-toxin molecule is the acquisition of two new biological properties. The conversion of alpha-toxin into an amphipathic conformation correlates with the acquisition of the biological property of the reversible penetration into the bilayer of the asolectin liposome membrane, as evidenced by labeling with the photoactivable probes. At lower pH, the conversion of the toxin into a globally hydrophobic molecule correlates with the biological property of causing damage to the cell membrane, as measured by the release of internal potassium ions, presumably by the formation of transmembrane hexamer pores.(ABSTRACT TRUNCATED AT 400 WORDS)

Staphylococcal alpha-toxin: a structure-function study using a monoclonal antibody.

A monoclonal antibody (A-Tox-653.1) selected for its reactivity in a dot immunoblot assay with denatured staphylococcal alpha-toxin has been isolated and its capacity to block the hemolytic and lethal activities of alpha-toxin measured. In addition, 'reactivity with monomer, hexamer, 125I-monoiodinated and CNBr peptides of alpha-toxin was studied. In all cases the reactions of the monoclonal antibody were compared to those obtained with anti-alpha-toxin rabbit hyperimmune serum. We find that while both the monoclonal antibody and the rabbit antiserum react with all forms of alpha-toxin, only the rabbit antiserum blocks hemolytic or lethal activity. Further, the rabbit antiserum reacts with CNBr fragments IV, V ad VII, whereas the monoclonal antibody reacts only with the carboxy terminal CNBr peptide VII. We conclude that, in solution, the carboxy terminal segment of alpha-toxin is relatively free and reaction with the monoclonal antibody neither impedes its binding to the specific receptor on the membrane nor interferes with formation of the hexamer complex.

Susceptibility to staphylococcal alpha-toxin of Friend virus-infected murine erythroblasts during differentiation.

Splenic erythroblasts obtained from BALB/c mice infected with the anemia strain of Friend virus were compared with "matured" cells and adult erythrocytes for their sensitivity to staphylococcal alpha-toxin. Matured cells were obtained by treating erythroblasts in culture with erythropoietin for 48 h. Sensitivity to staphylococcal alpha-toxin, measured both by release of 86Rb and by cell lysis, failed to demonstrate significant differences among the cell types. Since maturation of erythroblasts to matured cells or erythrocytes is associated with synthesis of band 3, hemoglobin, and spectrin and the loss of transferrin receptors, we conclude that none of these compounds serves as the specific receptor for staphylococcal alpha-toxin in BALB/c mice.

Effect of calcium ions on staphylococcal alpha-toxin-induced hemolysis of rabbit erythrocytes.

Calcium in millimolar concentrations protected rabbit erythrocytes from hemolysis caused by staphylococcal alpha-toxin. This effect was maximal at 30 mM CaCl2 and required the continued presence of calcium. The protection was not absolute and could be overcome by increased concentrations of alpha-toxin. Calcium did not block the binding of alpha-toxin to erythrocytes but inhibited the alpha-toxin-induced release of small ions from the cell as measured by 86Rb release. The transient removal of calcium was sufficient to abrogate its protective effect, suggesting that its action involves a reversible alteration in the state of the membrane. The three steps of the alpha-toxin-induced hemolytic sequence are: (i) binding to specific receptors, (ii) formation of transmembrane pores, and (iii) cell lysis. We concluded that calcium acted at step ii by impeding the lateral movement of alpha-toxin necessary to form the transmembrane hexamer pores.

Staphylococcal alpha toxin induced changes in the electroencephalogram of the rat.

Staphylococcal alpha-toxin at 1 microgram and 10 micrograms was injected into the right lateral ventricle of the brain of conscious, unrestrained rats. Clinical behavior and changes in EEG patterns were monitored. Clinical behavior attributed to alpha-toxin intoxication consisted of intermittent periods of stretching, tremors, convulsions and 'barrel rolling'. The EEG patterns, selected from recordings obtained during quiescent periods of behavior, demonstrate focal spiking, with and without recruitment, slow waves, spindling and complex spikes. We conclude that the central nervous system is a critical target for the lethal action of alpha-toxin.

Disruption of myelin sheaths in mouse brain in vitro and in vivo by staphylococcal alpha-toxin.

In recent studies we have demonstrated that staphylococcal alpha-toxin can specifically bind to rabbit vagus nerves and cause disruption of myelin sheaths in this peripheral nerve in vitro. We report here that staphylococcal alpha-toxin, incubated in vitro with brain slices or injected intracerebrally into mice, can induce disruption of myelin sheaths in central nervous tissue. Intracerebral injection of alpha-toxin is followed by a characteristic and reproducible syndrome involving ataxia followed by a severe contraction of the limbs on the side contralateral to the injection and a maximal extension of the opposing limbs. At 1.1 micrograms of toxin injected, death occurs within 20 min. Histopathologic examination reveals extensive demyelination with minimal involvement of the axons. It is possible that staphylococcal alpha-toxin may play a role in the etiology of multiple sclerosis.

Toxicity of staphylococcal alpha toxin for rabbit alveolar macrophages.

Highly purified staphylococcal alpha toxin was toxic in vitro for rabbit alveolar macrophages. Cytotoxicity, manifested by loss of the ability to exclude trypan blue dye and by morphological evidence of cell necrosis and lysis, was observed after exposure for 4 h to 1 microgram of toxin preparation per ml and after exposure for 8 h to 0.1 microgram of toxin per ml. In addition, exposure to toxin under conditions which did not kill more than 10% of the cells (1 microgram/ml for 1.5 to 2 h) significantly reduced the phagocytic activity of the cells and their ability to respond to an activator of hexose monophosphate shunt activity.

Increase in plasma membrane phosphodiesterase activity in contact-inhibited 3T3 cells and in phenotypically reverted SV-3T3 cells.

Contact-inhibited 3T3 mouse fibroblast cells, in contrast to logarithmically growing 3T3 cells and SV-3T3 transformed cells, have increased levels of plasma membrane-bound phosphodiesterase (oligonucleotidase, E.C.3.1.4.19; nucleotide pyrophosphatase, E.C. 3.6.1.9) activity. The increase in enzyme, recorded as increased specific activity, is reversible, as evidenced by the return to normal values following dilution of confluent 3T2 cells and re-initiation of growth. Increased enzyme activity is induced again when the cells regain the confluent state. Transformed SV-3T3 cells can be induced to mimic the contact inhibited state, including increased plasma membrane phosphodiesterase activity, by exposure to a combination of: (i) agents that are known to induce increased intracellular cAMP levels and (ii) additions of purified 3T3 or SV-3T3 plasma membranes. Additions of either alone fails to induce the increase in membrane phosphodiesterase activity, although each alone can significantly suppress cell growth, as measured by incorporation of 3H amino acids. We suggest that the elevation of plasma membrane phosphodiesterase activity may serve as a measure of conversion to the contact-inhibited state in both normal cells and phenotypically reverted transformed cells.

Uncapping of viral messenger RNA by phosphodiesterase of fibroblast plasma membranes.

Isolated plasma membranes from mouse fibroblast lines 3T3 and its tranformant SV-3T3 contain a phosphodiesterase (oligonucleotidase, E.C. 3.1.4.19; nucleotide pyrophosphatase, E.C. 3.6.1.9) that splits capped and methylated messenger RNA obtained from both reovirus and vesicular stomatitis virus. The isolated membranes are free of demonstrable ribonuclease activity and split the mRNA to produce 7-methyl guanosine diphosphate as a product. With ATP as substrate for the phosphodiesterase enzyme, the product is AMP. Synthetic caps, AMP, ADP and ATP, but not cyclic AMP, can compete with the substrate p-nitrophenyl thymidilic acid. A possible regulatory role on messenger translation is proposed.

Action of staphylococcal alpha-toxin on membranes: some recent advances.

Recent developments in the area of Staphylococcal alpha-toxin studies are presented which modify the concepts previously held with respect to both biological and physical properties of alpha-toxin. New data concerning the nature of the binding site for alpha-toxin on rabbit erythrocyte membranes and a model to explain the various observed complexes of alpha-toxin and membrane receptor are discussed. Finally, evidence suggesting that Staphylococcal alpha-toxin is a potent demyelinating agent is presented.