Whole genome sequencing of meticillin-resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS: Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS: The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION: The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

A novel action of the proton pump inhibitor rabeprazole and its thioether derivative against the motility of Helicobacter pylori.

The motility of Helicobacter pylori was maximum at 37 degrees C and at pH 6. A newly developed proton pump inhibitor, rabeprazole (RPZ), and its thioether derivative (RPZ-TH) markedly inhibited the motility of H. pylori. The concentrations of the drug necessary to inhibit 50% of the motility were 0.25, 16, 16, and >64 microgram/ml for RPZ-TH, RPZ, lansoprazole, and omeprazole, respectively. No such inhibitory effects were observed with H(2) blockers or anti-H. pylori agents. The motilities of Campylobacter jejuni and C. coli-but not those of Vibrio cholerae O1 and O139, Vibrio parahaemolyticus, Salmonella enterica serovar Typhimurium, and Proteus mirabilis-were also inhibited. Prolonged incubation with RPZ or RPZ-TH inhibited bacterial growth of only H. pylori, except for a turbid colony mutant. The results indicate that RPZ and RPZ-TH have a characteristic inhibitory effect against the motility of H. pylori (spiral-shaped bacteria), which is distinguished from that against bacterial growth.

Cell wall-affecting antibiotics induce expression of a novel gene, drp35, in Staphylococcus aureus.

A novel gene, drp35, of Staphylococcus aureus, which was inducible especially with cell wall-affecting antibiotics, has been cloned. Analysis of differential hybridization with mRNAs enhanced in the presence of beta-lactams resulted in two positive clones that harbored a new gene encoding a 35,845-Da protein (Drp35) and the penicillin-binding protein 2 (PBP2). Immunoblot analysis revealed that the Drp35 protein band was evidently enhanced after 30 min in the presence of beta-lactams. The Drp35 expression was also enhanced with not only beta-lactams, but also vancomycin, bacitracin, and fosfomycin. Homology search revealed that Drp35 was a new protein. Our results revealed that it was specific in S. aureus and respondent to these agents in both methicillin-resistant and -sensitive strains of S. aureus.

Assignment of circular dichroism spectra of 2-(4'-hydroxyphenylazo)benzoic acid induced by bovine serum albumin to the azo form.

In the displacement study, the effects of tolbutamide on the absorption spectra and induced CD spectra of 2-(4'-hydroxyphenylazo)benzoic acid(HABA)-bovine serum albumin (BSA) complex were investigated using the samples obtained from equilibrium dialysis at pH 7.40. The binding of tolbutamide to BSA enhanced the binding of the azo form in spite of the displacement of the hydrazone form of bound HABA at the molar ratio of HABA to BSA of 0.65, causing the increase in the negative ellipticity at 445 nm, which was termed the C band. The C band could be assigned to the azo form of bound HABA.

Effects of phenylbutazone and mefenamic acid on two classes of binding sites of 2-(4'-hydroxyphenylazo)benzoic acid on bovine serum albumin characterized by absorption spectra and circular dichroism spectra.

The competitive binding of phenylbutazone (PB) and mefenamic acid (MF) with 2-(4'-hydroxyphenylazo)benzoic acid (HABA) on bovine serum albumin (BSA) was studied by the investigation of the effects of these drugs on two bound forms of HABA, the azo and hydrazone forms. PB displaced preferentially the hydrazone form. MF displaced both the azo and hydrazone forms, though the azo form was preferentially displaced at the small molar ratio of MF to BSA. The binding constants of PB and MF obtained from the convenient spectrophotometry were dependent on the concentration of added drugs. These facts reflect the selective displacement of the azo and hydrazone forms. In connection with the absorption spectra of bound HABA, induced circular dichroism (CD) spectra of HABA-BSA complex were investigated. The induced CD spectra varied with the change of the molar ratio of HABA to BSA, indicating the presence of at least three different perturbed HABA molecules by BSA. The addition of PB and MF caused dramatic changes of the induced CD spectra of HABA-BSA complex. These spectral changes were discussed with the displacement of the azo and hydrazone forms.

Binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin characterized by optical spectroscopy and equilibrium dialysis.

Binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin (BSA) was studied by equilibrium dialysis, by the spectrophotometric method and by the absorption spectra of bound HABA. Comparison of equilibrium dialysis with the spectrophotometric method clarified the presence of a undisclosed class of binding sites, which do not cause the spectral change at about 480 nm to HABA, on BSA. Two bands observed on the absorption spectra of bound HABA were attributed to differently perturbed two HABA molecules by BSA, the azo and hydrazone forms, and these two forms correspond to HABA molecules bound to non-metachromasy sites and metachromasy sites, respectively. The concentration of each from was calculated by using estimated molar extinction coefficient.

Model of interaction of ajmaline with polyvinylpyrrolidone.

The chemical shifts in the Fourier transform 13C-NMR spectrum of ajmaline were assigned from consideration of the acetylation shifts and multiplet splitting in the off-resonance decoupled spectrum. On the basis of the assigned signals, the changes of chemical shifts in ajmaline induced by polyvinylpyrrolidone were investigated, and an interaction model of ajmaline with polyvinylpyrrolidone in chloroform was proposed.