delta-hemolysin-protein--staphylococcus-aureus and Hemolysis

We assessed a new screening method, based on δ-hemolysin production in the presence of 6 mg/liter vancomycin, to distinguish heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) and vancomycin-intermediate S. aureus (VISA) from vancomycin-susceptible S. aureus (VSSA). On 37 clinical methicillin-resistant S. aureus (MRSA) isolates, hVISA and VISA displayed no δ-hemolysis whereas VSSA displayed strong δ-hemolysis, showing 91.6% sensitivity. These data, supported by real-time reverse transcription PCR (real-time RT-PCR) highlighting an hld downregulation, i.e., VSSA>hVISA>VISA, define this new assay as a valid screening method.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Bacteriological Techniques; Culture Media; Hemolysin Proteins; Hemolysis; Humans; Mass Screening; Methicillin-Resistant Staphylococcus aureus; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Staphylococcal Infections; Vancomycin; Vancomycin Resistance

Twenty coagulase-negative Staphylococcus strains displaying alpha-haemolysis (delta-haemolysin) on sheep-blood agar were isolated from the noses of different pigs in Switzerland. The strains were Gram-stain-positive, non-motile cocci, catalase-positive and coagulase-negative. Sequence analysis of the 16S rRNA gene, sodA, rpoB, dnaJ and hsp60 and phylogenetic characteristics revealed that the strains showed the closest relatedness to Staphylococcus microti CCM 4903(T) and Staphylococcus muscae DSM 7068(T). The strains can be differentiated from S. microti by the absence of mannose fermentation and arginine arylamidase and from S. muscae by the absence of beta-glucuronidase activity and production of alkaline phosphatase. The chosen type strain ARI 262(T) shared 20.1 and 31.9 % DNA relatedness with S. microti DSM 22147(T) and S. muscae CCM 4903(T), respectively, by DNA-DNA hybridization. iso-C(15 : 0), anteiso-C(15 : 0) and iso-C(17 : 0) were the most common fatty acids. Cell-wall structure analysis revealed the peptidoglycan type A3alpha l-Lys-Gly(2)-l-Ser-Gly (type A11.3). The presence of teichoic acid was determined by sequencing the N-acetyl-beta-d-mannosaminyltransferase gene tarA, which is involved in biosynthesis of ribitol teichoic acid. Menaquinone 7 (MK-7) was the predominant respiratory quinone. The G+C content of ARI 262(T) was 38.8 mol%. The isolated strains represent a novel species of the genus Staphylococcus, for which we propose the name Staphylococcus rostri sp. nov. The type strain is ARI 262(T) (=DSM 21968(T) =CCUG 57266(T)) and strain ARI 602 (=DSM 21969 =CCUG 57267) is a reference strain.

Topics: Animals; Bacterial Proteins; DNA, Bacterial; DNA, Ribosomal; Fatty Acids; Hemolysin Proteins; Hemolysis; Molecular Sequence Data; Nose; Phylogeny; RNA, Ribosomal, 16S; Staphylococcus; Sus scrofa; Switzerland

Legionella pneumophila, the causative agent of Legionnaires' disease, is a waterborne bacteria. It can multiply in man-made water systems and infect people who inhale contaminated droplets. We have previously reported a Staphylococcus warneri strain that display an anti-Legionella activity. In this work, we characterized three anti-Legionella peptides that are produced by S. warneri. One peptide, warnericin RK, is original, while the two others are delta-lysin I and delta-lysin II, whose genes were previously described. Due to high sequence similarity of the two delta-lysins, further characterization was performed only on delta-lysin I. Warnericin RK and delta-lysin I displayed the same antibacterial spectrum, which is almost restricted to the Legionella genus. Also, both peptides have a hemolytic activity. These results led to the hypothesis that warnericin RK and delta-lysin I share a similar mode of action, and that Legionella should have a specific feature that may explain the high specificity of these antibacterial peptides.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Dose-Response Relationship, Drug; Erythrocytes; Hemolysin Proteins; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Legionella pneumophila; Microbial Sensitivity Tests; Molecular Sequence Data; Peptides; Protein Structure, Secondary; Sequence Homology, Amino Acid; Staphylococcus

The majority of infections with glycopeptide intermediate-level resistant Staphylococcus aureus (GISA) originate in biomedical devices, suggesting a possible increased ability of these strains to produce biofilm. Loss of function of the accessory gene regulator (agr) of S. aureus has been suggested to confer an enhanced ability to bind to polystyrene. We studied agr in GISA, hetero-GISA, and related glycopeptide-susceptible S. aureus isolates. All GISA strains from diverse geographic origins belong to agr group II. All GISA strains were defective in agr function, as demonstrated by their inability to produce delta-hemolysin. Hetero-GISA isolate A5940 demonstrated a nonsense mutation in agrA that was not present in a pulsed-field gel electrophoresis-indistinguishable vancomycin-susceptible isolate from the same patient. Various other agr point mutations were noted in several clinical GISA and hetero-GISA isolates. A laboratory-generated agr-null strain demonstrated a small but reproducible increase in vancomycin heteroresistance after growth in vitro in subinhibitory concentrations of vancomycin. This was not seen in the isogenic agr group II parent strain in which agr was intact. The in vitro bactericidal activity of vancomycin was attenuated in the agr-null strain compared to the parent strain. These findings imply that compromised agr function is advantageous to clinical isolates of S. aureus toward the development of vancomycin heteroresistance, perhaps through the development of vancomycin tolerance.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Hemolysin Proteins; Hemolysis; Humans; Microbial Sensitivity Tests; Molecular Sequence Data; Polymorphism, Restriction Fragment Length; Sequence Analysis, DNA; Staphylococcal Infections; Staphylococcus aureus; Trans-Activators; Vancomycin; Vancomycin Resistance

Beta (beta) and delta (delta)-hemolysin of Staphylococcus aureus strains were cultured in vitro in milk lactoserum (whey) prepared from both healthy and mastitis bovine milk. Production of beta- and delta-hemolysins were detected in 12 out of 50 strains studied. The association between N-acetyl-beta-D-glucosaminidase (NAGase) activity, plasmin activity (PL) and trypsin inhibitory capacity (TIC), known as inflammatory indicators for mastitis, and hemolytic activity were also studied. Mastitic milk decreased directly the lytic effect of both beta-and delta-hemolysins of S. aureus on hemolytical blood agar plates. S. aureus in healthy milk samples produced more beta-hemolysin (3 times) and delta-hemolysin (2 times) when compared to S. aureus supernatants in milk from infected quarters. Furthermore, beta- and delta-hemolysis correlated negatively with TIC and NAGase and PL activities. Addition of reduced glutathione (GSH) or beta-mercaptoethanol into the artificial medium enhanced hemolysins activity.

Topics: Acetylglucosaminidase; Adenosine Triphosphatases; Animals; ATP-Binding Cassette Transporters; Bacterial Proteins; Cattle; Culture Media; Fibrinolysin; Glutathione; Hemolysin Proteins; Hemolysis; In Vitro Techniques; Mastitis; Mercaptoethanol; Milk; Staphylococcus aureus; Trypsin Inhibitors

Fibronectin-binding proteins (FnBPs) are thought to be important for the attachment of Staphylococcus aureus during infection. The regulation of the genes fnbA and fnbB by the global regulatory loci sar and agr was examined using site-specific regulatory mutants of S. aureus strain Newman. The results from binding assays using both aqueous and solid-phase fibronectin as well as ligand blotting with biotinylated fibronectin showed that the expression of FnBPA is enhanced in the agr mutant but inhibited in the sar mutant and the sar-agr double mutant. The same regulatory pattern was observed in Northern blot analysis using fnbA-specific probes. The introduction of sar on a multicopy plasmid increased the already enhanced fnbA transcription of the agr mutant. FnBPB was not detectable by ligand blotting and the fnbB promoter activity in promoter fusion assays was not affected by either sar or agr. The sequence encompassing ORF3 located upstream of sarA was found to be essential for the activation of fnbA transcription. We hypothesize that this sequence may modulate SarA expression and/or activity on the post-transcriptional level. Gel shift assays demonstrated that SarA binds to the fnbA promoter fragments, probably as a dimer. DNase I footprinting assays with SarA revealed a protected area of 102 bp upstream of fnbA.

Topics: Adhesins, Bacterial; Bacterial Adhesion; Bacterial Proteins; Base Sequence; Binding Sites; Carrier Proteins; Dimerization; Fibronectins; Gene Expression Regulation, Bacterial; Genes, Reporter; Green Fluorescent Proteins; Hemolysin Proteins; Hemolysis; Ligands; Luminescent Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; Staphylococcal Protein A; Staphylococcus aureus; Trans-Activators; Transcription Factors; Transcriptional Activation

The antimicrobial activity of a synthetic peptide corresponding to delta-hemolysin had been examined. The peptide did not exhibit antimicrobial activity against gram negative and gram positive micro-organisms unlike other hemolytic peptides like melittin. This lack of antibacterial activity arises due to the inability of delta-hemolysin to perturb the negatively charged bacterial cell surface and permeabilize the bacterial plasma membrane. However, the red blood cell surface has a structure considerably different from bacteria and does not act as a barrier to molecules reaching the lipid membrane. Hence delta-toxin can lyse erythrocytes. Thus, the specificity in biological activity has been rationalized in terms of differences in the interaction of the toxin with the bacterial and red blood cell surfaces.

Topics: Amino Acid Sequence; Bacillus subtilis; Bacteria; Bacterial Proteins; Cell Membrane; Cell Membrane Permeability; Escherichia coli; Hemolysin Proteins; Hemolysis; Microbial Sensitivity Tests; Molecular Sequence Data; Staphylococcus aureus

The effects of the lytic peptides, melittin and delta-haemolysin, are compared in vesicles of gel-phase dipalmitoylphosphatidylcholine (DPPC), using calcein as trapped marker. At low concentration, both toxins cause vesicles to lose contents in 5 mM phosphate buffer near neutral pH, with melittin being the more active. As phosphate concentration is increased, the kinetics of melittin-induced leakage change from a slow, sustained loss to a rapid 'burst' of leakage when melittin is present mainly as tetramer in solution, under conditions where it is reported to lose haemolytic activity towards erythrocytes. At low phosphate concentration, the leakage induced by delta-haemolysin is preceded by a lag phase, though fluorescence measurements show that binding of toxin is rapid. At higher phosphate concentration, the toxin binds rapidly to vesicles, but causes no leakage of entrapped calcein. Steady-state fluorescence spectra show no obvious differences in tryptophan emission for delta-haemolysin bound to lipid in high- or low-phosphate buffer. Spin-label fluorescence-quenching studies show that the single tryptophan residue of delta-haemolysin is buried within the lipid bilayer at all phosphate concentrations used. In gel-phase DPPC, delta-haemolysin shows no tendency to cause vesicle aggregation over several hours, as judged by light scattering, though a slow non-linear effect is seen above the lipid phase transition temperature. These effects are contrasted with those of melittin under similar conditions.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Bacterial Proteins; Bee Venoms; Cell Membrane; Fluoresceins; Hemolysin Proteins; Hemolysis; Hydrogen-Ion Concentration; Kinetics; Lipid Bilayers; Melitten; Phosphatidylcholines; Scattering, Radiation; Spectrometry, Fluorescence

The synergistic hemolysis reactions of 61 reference strains and 189 clinical isolates representing 17 species of staphylococci were examined on plates of Trypticase soy blood agar (BBL Microbiology Systems, Cockeysville, Md.). Some or all of the strains of Staphylococcus aureus, S. epidermidis, S. capitis, S. cohnii, S. haemolyticus, S. hyicus, S. simulans, S. warneri, and S. xylosus produced a delta-hemolysin that gave synergistic, complete hemolysis of washed human, sheep, and ox blood cells in an area of beta-lysin activity from strains of S. aureus and S. intermedius. Strains of the same nine species were positive with a commercial beta-lysin paper disk designed for presumptive identification of group B streptococci; most of these strains also gave synergistic, complete hemolysis with exotoxin from a strain of Corynebacterium pseudotuberculosis. None of the strains of S. auricularis, S. carnosus, S. caseolyticus, S. hominis, S. intermedius, S. saprophyticus, S. sciuri, or S. lentus were positive by any of these tests for synergistic hemolysis. These results indicate that a synergistic hemolysis test could prove very useful for differentiating these species; they also suggest that one role of some of these organisms in human infections could be that of a synergist. Further studies of synergism may clarify the clinical significance of these results.

Topics: Antimicrobial Cationic Peptides; Bacterial Proteins; Bacterial Toxins; Blood Proteins; Corynebacterium; Hemolysin Proteins; Hemolysis; Humans; Protein Biosynthesis; Proteins; Sphingomyelin Phosphodiesterase; Staphylococcal Infections; Staphylococcus; Staphylococcus aureus; Staphylococcus epidermidis

Topics: Bacterial Proteins; Chemical Phenomena; Chemistry, Physical; Chromatography, Gel; Hemolysin Proteins; Hemolysis; Melitten; Molecular Weight; Protein Conformation; Spectrometry, Fluorescence

Haque, Riaz-Ul (The Ohio State University, Columbus), and Jack N. Baldwin. Types of hemolysins produced by Staphylococcus aureus as determined by the replica plating technique. J. Bacteriol. 88:1442-1447. 1964.-The type of hemolysins produced by a representative number of cells of a culture of Staphylococcus aureus was determined by the replica plating technique, with the use of sheep, rabbit, human, and horse blood agar plates. Unless horse and human red blood cells were used, a distinction between alpha-and delta-hemolysins was difficult to make. Strains producing alpha-beta-, beta-delta-, and alpha-delta-hemolysins were recognized by the replica plating technique. A few colonies produced lysis of rabbit, horse, and human cells without lysis of sheep red blood cells. This pattern of lysis was not detected by the radial streak or the tube titration procedures when mixed populations were examined, and may be due to a lytic factor not hitherto described.

Topics: Animals; Bacterial Proteins; Bacteriological Techniques; Cell Death; Culture Media; Erythrocytes; Hemolysin Proteins; Hemolysis; Horses; Humans; Metabolism; Rabbits; Research; Sheep; Sheep, Domestic; Staphylococcal Infections; Staphylococcus; Staphylococcus aureus

Topics: Bacterial Proteins; Exotoxins; Hemolysin Proteins; Hemolysis; Micrococcus

Topics: Bacterial Proteins; Exotoxins; Hemolysin Proteins; Hemolysis; Micrococcus; Staphylococcus aureus

Topics: Bacterial Proteins; Exotoxins; Hemolysin Proteins; Hemolysis; Micrococcus

Topics: Bacterial Proteins; Hemolysin Proteins; Hemolysis; Micrococcus; Staphylococcus; Staphylococcus aureus

Topics: Bacterial Proteins; Cell Death; Hemolysin Proteins; Hemolysis; Humans; Staphylococcus