Virulence factors produced by strains of Staphylococcus aureus isolated from urinary tract infections.

Staphylococcus aureus infections are widely prevalent in West Africa and are often associated with urinary tract infections (UTIs). Virulence factors from S. aureus have rarely been described for such infections. The purpose of the current study was to determine the prevalence of toxins and adhesion factors obtained from S. aureus isolated from presumed primary UTIs at the Cotonou University Hospital (CUH) in Benin as compared with the Strasbourg University Hospital (SUH) in France. Both ambulatory and hospitalised patients were included in the study. Sixty-five independent strains of S. aureus from CUH and 35 strains from SUH were obtained over a four-month period. Virulence factors were characterised by immunodetection or multiplex polymerase chain reaction, and meticillin susceptibility was recorded. Approximately 50% of all isolates produced at least one enterotoxin. No isolate from SUH produced Panton-Valentine leucocidin (PVL), whereas 21.5% of the S. aureus isolates from CUH produced PVL (P<0.01). Six of 14 (43%) PVL-positive isolates were meticillin-resistant. At SUH, the incidence of MRSA (57%) was significantly higher (P<0.01) than at CUH (14%). Genes encoding clumping factor B, and elastin and laminin binding proteins were detected in almost all isolates (80%), irrespective of the geographical origin. The results for elastin binding protein differed significantly from published data regarding isolates from other clinical origins. Staphylococcal toxins and adhesion factors may be important in the physiopathology of UTI.

[Monitoring of antibiotic resistance of gram negative anaerobes]. / Surveillance de la résistance aux antibiotiques des anaérobies stricts à Gram négatif.

MATERIAL AND METHOD: Using an agar reference method (Norma M11-A5, National Committee for Clinical and Laboratory Standards) the minimal inhibitory concentrations of nine antibiotics were determined for 376 anaerobic strains. The following strains were investigated: 254 Bacteroides fragilis group (including 143 B. fragilis), 122 other gram-negative anaerobes (Bacteroides spp., Prevotella, Fusobacterium, Porphyromonas, Suterella, Desulfomonas, Veillonella). RESULTS: In the B. fragilis group resistance rates were: coamoxyclav 2.8%, ticarcillin 27.5%, ticarcillin-clavulanic acid 1.9%, piperacillin-tazobactam 1.9%, cefoxitin 6.2%, imipenem 0.8%, clindamycin 28.3%, respectively. Based on previous studies, resistance to imipenem remained low in 2003 and was only observed for B. fragilis. Resistance to clindamycin was maintained around 25%. No metronidazole resistance was observed, but decreased susceptibility was found for B. fragilis, B. merdae and Prevotella, as in 4.3% of gram-negative anaerobes. DISCUSSION: This study confirms the high resistance rate of gram-negative anaerobes to clindamycin, the efficient activity of imipenem, beta-lactam/beta-lactamase inhibitor combinations and metronidazole. However, reduced metronidazole susceptibility seems to be increasing.

Detection of Chlamydia trachomatis DNA using MagNA Pure DNA extraction and Cobas Amplicor CT/NG amplification.

The automated MagNA Pure DNA extraction method for Chlamydia trachomatis was compared with the manual Cobas Amplicor protocol using 100 microL of input sample volume from 964 specimens. Agreement between protocols was 96.1%. The automated extraction method had a sensitivity of 99% and a specificity of 100%. Amplification inhibition observed after manual preparation of samples (3.8%) was not apparent following automated extraction. Using 200 microL of sample in the automated extraction process lowered the detection limit without raising the inhibition rate. Furthermore, the automated extraction method halved the hands-on time required for the procedure.

The structure of a Staphylococcus aureus leucocidin component (LukF-PV) reveals the fold of the water-soluble species of a family of transmembrane pore-forming toxins.

BACKGROUND: Leucocidins and gamma-hemolysins are bi-component toxins secreted by Staphylococcus aureus. These toxins activate responses of specific cells and form lethal transmembrane pores. Their leucotoxic and hemolytic activities involve the sequential binding and the synergistic association of a class S and a class F component, which form hetero-oligomeric complexes. The components of each protein class are produced as non-associated, water-soluble proteins that undergo conformational changes and oligomerization after recognition of their cell targets. RESULTS: The crystal structure of the monomeric water-soluble form of the F component of Panton-Valentine leucocidin (LukF-PV) has been solved by the multiwavelength anomalous dispersion (MAD) method and refined at 2.0 A resolution. The core of this three-domain protein is similar to that of alpha-hemolysin, but significant differences occur in regions that may be involved in the mechanism of pore formation. The glycine-rich stem, which undergoes a major rearrangement in this process, forms an additional domain in LukF-PV. The fold of this domain is similar to that of the neurotoxins and cardiotoxins from snake venom. CONCLUSIONS: The structure analysis and a multiple sequence alignment of all toxic components, suggest that LukF-PV represents the fold of any water-soluble secreted protein in this family of transmembrane pore-forming toxins. The comparison of the structures of LukF-PV and alpha-hemolysin provides some insights into the mechanism of transmembrane pore formation for the bi-component toxins, which may diverge from that of the alpha-hemolysin heptamer.

Pharmacokinetic and preliminary metabolic fate of navelbine in humans as determined by high performance liquid chromatography.

The pharmacokinetics and metabolism of Navelbine (NVB) were investigated in 20 patients by a specific high performance liquid chromatographic methodology allowing the monitoring of NVB, deacetyl-NVB, and N-oxide NVB. After the i.v. (15 min) administration of 30 mg/m2 of drug, blood and urine samples were collected for, respectively, 144 and 48 h. NVB is characterized by a three compartmental kinetics, with a Cmax of 1130 +/- 139 (SEM) ng/ml. The total body clearance and apparent volume of distribution, as defined by high performance liquid chromatography, are 1.26 +/- 0.09 liter/h/kg (48.6 +/- 4.1 liters/h/m2) and 75.6 +/- 9.2 liters/kg (2918.4 +/- 307.2 liters/m2). No metabolite could be detected in serum; the urinary excretion of NVB represented 11% of the administered dose. Deacetyl-NVB could be identified as a minor urinary metabolite when no N-oxide NVB appeared in the urine samples. Two additional peaks appeared in most of urinary chromatograms as trace amounts. Thus, the major pathway of NVB, as for other Vinca alkaloids, should be hepatic clearance, as biliary elimination and/or hepatic biotransformation.

Membrane ATPase of Bacillus subtilis. I. Purification and properties.

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step. The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s20,w of 13,4 and an amino acid composition very similar to bacterial ATPases already studied. After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (alpha) and 57 000 (beta) with different charges. Kinetic studies showed that Ca2+ or Zn2+ are required for ATPase activity, although Mg2+ was uneffective. At optimal Ca2+ concentration, the Mg2+ has an inhibitory effect. The Km for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na+ + K+)-ATPase are studied.

Membrane ATPase of Proteus L-forms. Solubilization and molecular properties.

The Mg2+ -dependent ATPase (EC 3.6.I.3) of Proteus L-form membrane has been solubilized according to various procedures (Tris - HCL shock-wash with or without MG2+, EDTA, Triton X-100). The best results were obtained by the same 33mM Tris-HCL (pH 7.5) shock-wash without MG2+ used for ATPase of protoplasts from Streptococcus faecalis. The solubilized enzyme after 105 000 times g centrifugation was purified on acrylamide/agarose. The molecular weight was established to be 360 000 by gel filtration and by sedimentation coefficient (12.5 S). Polyacrylamide disc-gel electrophoresis in sodium dodecylsulphate revealed two classes or subunit of mol. wt. 64 000 (alpha) and 58 000 (beta), associated in ratio 1:1. We propose a formula alpha-3beta-3 for the native ATPase of Proteus L-forms. Structural similarities to ATPase of various origins are discussed.

Isolation and purification of dicyclohexylcarbodiimide-reactive proteolipid from Bacillus subtilis membrane.

The membrane-bound ATPase activity of Bacillus subtilis was inhibited by dicyclohexylcarbodiimide (DCCD). The DCCD-reactive proteolipid of B. subtilis was extracted, from labelled or untreated membranes containing F1 or depleted of F1, with neutral or acidic chloroform/methanol. Purification of the [14C]DCCD-binding proteolipid was attempted by column chromatography on methylated Sephadex G-50 and on DEAE-cellulose. The maximal amount of DCCD which could be bound to the purified proteolipid was found to exceed the amount bound by the purified proteolipid extracted from membranes labelled with the lowest [14C]DCCD concentration required for maximal inhibition of the membrane-bound ATPase activity. The radioactive protein peaks eluted by gel filtration and ion-exchange chromatography were analysed by urea-SDS polyacrylamide slab gel electrophoresis and autoradiography. Radioactivity was incorporated into two components of Mr 18 000 and 6000 when proteolipid was purified by methylated Sephadex. The 6000 polypeptide was always present, whatever the extraction and purification procedures. However, the 18 000 polypeptide was present in largest quantity only when proteolipid was extracted from membranes containing F1 and purified by methylated Sephadex. When proteolipid was purified on DEAE-cellulose this [14C]DCCD binding component of Mr 18 000 was absent.

A predicted beta-sheet from class S components of staphylococcal gamma-hemolysin is essential for the secondary interaction of the class F component.

Site-directed mutagenesis was performed on genes encoding HlgA and HlgC, two of the three proteins expressed from the staphylococcal y-hemolysin locus, which originate two pore-forming toxins (HlgA + HlgB, HlgC + HlgB). As related proteins, HlgA and HlgC were found to bind first to cell membranes. Amino acid substitutions concerned residues that would predictably disrupt a 13 amino acid conserved beta-sheet of the Chou and Fasman secondary structure prediction. The mutation of a threonin into an aspartic acid residue from HlgA (T28D) and from HlgC (T30D) that would break this predicted N-terminal structure lowered dramatically the biological activities on purely lipidic vesicles, erythrocytes and polymorphonuclear cells. The change in secondary structure was confirmed by Fourier Transformed Infrared spectroscopy. The binding of mutated and native proteins at the same kind of sites onto polymorphonuclear cells was evidenced with flow cytometry and fluorescein-labelled anti-class S antibodies or wild type HlgA or HlgC. However, the subsequent binding of fluorescein-labelled HlgB to membrane-bound mutated HlgA or HlgC complexes was inhibited. In conclusion, the first binding of class S components is essential for the subsequent binding of class F components, and a predicted beta-sheet seems to be at least one of the functional domains involved.

Prevalence and pathogenicity of Clostridium difficile in hospitalized patients. A French multicenter study.

BACKGROUND: Although Clostridium difficile is the main agent responsible for nosocomial diarrhea in adults, its prevalence in stool cultures sent to hospital microbiology laboratories is not clearly established. OBJECTIVES: To determine the prevalence of C difficile in inpatient stools sent to hospital microbiology laboratories and to assess the relationship between serotypes and toxigenicity of the strains isolated and the clinical data. METHODS: From January 18, 1993, to July 31, 1993, the presence of C difficile was systematically investigated in a case-control study on 3921 stool samples sent for stool culture to 11 French hospital microbiology laboratories. The prevalence of C difficile in this population (cases) was compared with that of a group of 229 random hospital controls matched for age, department, and length of stay (controls). Stool culture from controls was requested by the laboratory although not prescribed by the clinical staff. Serotype and toxigenesis of the strains isolated were compared. RESULTS: The overall prevalence of C difficile in the cases was twice the prevalence in the controls (9.7% vs 4.8%; P < .001) and was approximately 4 times as high in diarrheal stools (ie, soft or liquid) as in normally formed stools from controls (11.5% vs 3.3%; P < .001). The strains isolated from diarrheal stools were more frequently toxigenic than those isolated from normally formed stools. Serogroup D was never toxigenic, and its proportion was statistically greater in the controls than in the cases (45% vs 18%; chi 2 = 5.2; P < .05). Conversely, serogroup C was isolated only from the cases. Clostridium difficile was mainly found in older patients ( > 65 years), suffering from a severe disabling disease, who had been treated with antibiotics and hospitalized for more than 1 week in long-stay wards or in intensive care. CONCLUSIONS: This multicenter period prevalence study clearly supports the hypothesis of a common role of C difficile in infectious diarrhea in hospitalized patients. Disease associated with C difficile should therefore be systematically evaluated in diarrheal stools from inpatients.

Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L.

Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.

Cloning and characterization of the cytotoxin L-encoding gene of Clostridium sordellii: homology with Clostridium difficile cytotoxin B.

Hybridization of an oligodeoxyribonucleotide (oligo) probe, designed from a repeated sequence ('oligo rep') at the C terminus of the Clostridium difficile (Cd) cytotoxin (Cyt), revealed that homologies exist between the Cd cyt gene and the genomes of several other clostridia, including Clostridium sordellii (Cs), suggesting a common ancestral cyt amongst the Clostridium genus. This Cd 'oligo rep' probe was used to clone the Cs (strain 6018) cyt. The sequenced (7095 bp) region encodes 2364 amino acids (aa) and corresponds to a protein of 270,614 Da. Cs Cyt has 76% identity with the Cd Cyt and 47% identity with the Cd enterotoxin (Ent). The latter third of the protein consists of repeated units, similar to those found for Cd Cyt. A highly conserved hydrophobic domain can be delineated. Few structural differences are evident between Cd and Cs Cyt to explain their different cellular and sub-cellular effects. A small open reading frame (ORF) encoding a protein of 16,484 Da is located 210 bp downstream from cyt. No homology was evident with any known sequence. The first 30 aa of this ORF may correspond to a signal peptide.

Characterisation of a synergohymenotropic toxin produced by Staphylococcus intermedius.

Staphylococcal synergohymenotropic (SHT) toxins damage membranes of host defence cells and erythrocytes by the synergy of two secreted and non-associated proteins: class S and class F components. Whereas Panton-Valentine leucocidin (PVL), gamma-hemolysin and Luk-M from Staphylococcus aureus are members of this toxin family, a new bi-component toxin (LukS-I + LukF-I) from Staphylococcus intermedius, a pathogen for small animals, was characterised and sequenced. It is encoded as a luk-I operon by two cotranscribed genes, like PVL, LukS-I + LukF-I shares a strong leukotoxicity of various PMNs, but only slight haemolytic properties on rabbit erythrocytes. When intradermally injected into rabbit skin, a 100 ng dose caused acute inflammatory reaction leading to tissue necrosis. The new SHT seemed to be largely distributed among various Staphylococcus intermedius strains.

Characterization of a novel structural member, LukE-LukD, of the bi-component staphylococcal leucotoxins family.

A new member of the staphylococcal bi-component leucotoxins family, LukE (32 kDa) and LukD (34.3 kDa) has been characterized from Staphylococcus aureus strain Newman. LukE was 58-68% identical with the class S proteins, whereas LukD was 71-77% identical with the class F proteins of the family. A partial immunoreactivity with the various affinity-purified antibodies specific for the other proteins was observed. Immunoprecipitation assay and gene probing confirmed a 30% frequency among human clinical isolates, differing from the distribution of the other known leucotoxins (P<0.005). LukE+LukD was as effective as the Panton-Valentine leucocidin for inducing dermonecrosis when injected in the rabbit skin, but not hemolytic and poorly leucotoxic compared to other leucotoxins expressed by Staphylococcus aureus.

Ion channels and bacterial infection: the case of beta-barrel pore-forming protein toxins of Staphylococcus aureus.

Staphylococcus aureus strains causing human pathologies produce several toxins, including a pore-forming protein family formed by the single-component alpha-hemolysin and the bicomponent leukocidins and gamma-hemolysins. The last comprise two protein elements, S and F, that co-operatively form the active toxin. alpha-Hemolysin is always expressed by S. aureus strains, whereas bicomponent leukotoxins are more specifically involved in a few diseases. X-ray crystallography of the alpha-hemolysin pore has shown it is a mushroom-shaped, hollow heptamer, almost entirely consisting of beta-structure. Monomeric F subunits have a very similar core structure, except for the transmembrane stem domain which has to refold during pore formation. Large deletions in this domain abolished activity, whereas shorter deletions sometimes improved it, possibly by removing some of the interactions stabilizing the folded structure. Even before stem extension is completed, the formation of an oligomeric pre-pore can trigger Ca(2+)-mediated activation of some white cells, initiating an inflammatory response. Within the bicomponent toxins, gamma-hemolysins define three proteins (HlgA, HlgB, HlgC) that can generate two toxins: HlgA+HlgB and HlgC+HlgB. Like alpha-hemolysin they form pores in planar bilayers with similar conductance, but opposite selectivity (cation instead of anion) for the presence of negative charges in the ion pathway. gamma-Hemolysin pores seem to be organized as alpha-hemolysin, but should contain an even number of each component, alternating in a 1:1 stoichiometry.

Discoupling the Ca(2+)-activation from the pore-forming function of the bi-component Panton-Valentine leucocidin in human PMNs.

The consecutive cell activation, including Ca(2+)-channel opening, and pore formation leading to human neutrophil lysis were the two functions of the staphylococcal Panton-Valentine leucocidin attempted to be discoupled by site-directed mutagenesis. In a first approach consisting in deletions of the cytoplasmic extremity of the transmembranous domain, we produced a LukF-PV DeltaSer125-Leu128 with a slightly reduced Ca(2+) induction but with a significantly lowered lytic activity when combined with its synergistic protein LukS-PV. The second approach consisted in the modification of charges and/or introduction of a steric hindrance inside the pore, which also led to interesting mutated proteins: LukF-PV G131D, G131W and G130D. The latter had an intact Ca(2+) induction ability while the lytic one was 20-fold diminished. Binding properties and intrinsic pore diameters of these discoupled toxins remained comparable to the wild-type protein. The mutated proteins promoted interleukin-8 secretion, but they were rather inactive in an experimental model. New insights are brought concerning the role of the two functions in the virulence of this bi-component leucotoxin.

CD14 expression by human mononuclear phagocytes is modulated by Clostridium difficile toxin B.

Toxin B, an exotoxin produced by the anaerobic Gram-positive bacteria Clostridium difficile, is responsible for pseudomembranous colitis in humans. It deeply modifies morphology of cultured cells and enhances their membrane surface area, which suggests a possible alteration of membrane receptor distribution. Since toxin B and bacterial lipopolysaccharide can act synergistically on TNF-alpha production by mononuclear phagocytes, the effect of toxin B on CD14 expression was investigated using flow cytometric analysis. It was shown that monocytes overexpressed CD14 after 5 h of treatment with toxin B. In contrast, after 24 h of treatment, the percentage of CD14 monocytes decreased, although, most frequently, the remaining positive cells expressed high levels of CD14 compared with untreated cells. Macrophages treated for 5 h with toxin B overexpressed CD14, but this effect persisted for at least 24 h. Both the percentage of positive macrophages and the mean level of CD14 per cell were increased. Thus toxin B can modulate expression of CD14 and its modulation depends on the differentiation status and maybe on the activation state, since some individual variations were observed in monocyte response to toxin.

Clinical pharmacokinetics of cefotiam.

Cefotiam, a semisynthetic parenteral cephalosporin of the aminothiazole group, exhibits interesting properties: stability against hepatic metabolism and excellent solubility, accounting for an apparent volume of distribution 2 to 3 times higher than that of most other cephalosporins. Its degree of protein binding is about 40%. High concentrations of cefotiam are observed in several tissues (kidney, heart, ear, prostate and genital tract) as well as in fluids and secretions (bile, ascitic fluid). In healthy subjects, the serum elimination half-life is about 1 hour. The pharmacokinetics are linear only for doses lower than 1g. Cefotiam is mostly (and rapidly) eliminated in unchanged form in urine; 50 to 70% of the dose is recovered during the 12 hours following administration, and only severe renal failure, with creatinine clearance less than 5 ml/min, significantly alters the elimination half-life. Although the drug has no proven nephrotoxicity in man, a reduction of the dose is recommended when creatinine clearance is less than 30 ml/min.

New serotypes of Pseudomonas cepacia.

O and H serotyping of Pseudomonas cepacia has provided a suitable procedure for epidemiological studies. Our previous reports have described 7 O and 5 H antigens. The study of strains from another geographical origin led us to prepare antisera against those which could not be serotyped and thus to determine 2 new O and 2 new H specificities (O:8 and O:9, H:4 and H:8).

Phenylmercuric acetate biodegradation by environmental strains of Pseudomonas species.

Organomercurial pollution occurring in the Rhine river in 1986 led us to study the possibility of depollution by mercury-resistant environmental aquatic strains. Four species of Pseudomonas were investigated for their ability to biotransform phenylmercuric acetate (PMA). Such biological depollution was demonstrated to be due to an enzymatic activity in whole cells and in cell-free extracts from Pseudomonas fluorescens and other Pseudomonas species. PMA biotransformation was followed by high-performance liquid chromatography. Some of those bacteria growing between 4 and 41 degrees C probably represent a natural means of organomercurial depollution, which acts slowly in interaction with other organisms and non-organic porous surfaces.