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Presence of fibrinogen-binding adhesin gene in Staphylococcus epidermidis isolates from central venous catheters-associated and orthopaedic implant-associated infections.

Attention has recently been paid to identify and elucidate those pathogenetic mechanisms, which play a significant role in sustaining the early phases of Staphylococcus epidermidis colonisation and infection development. Several analogies with the physiology of Staphylococcus aureus, a more thoroughly investigated pathogen, have lead to carefully consider all bacterial surface components that mediate cell adhesion. This study aimed at investigating the presence of the fbe gene encoding for a fibrinogen-binding protein in a collection of 107 S. epidermidis strains isolated from orthopaedic infections and 67 from central venous catheter-associated infections. The strains isolated from orthopaedic infections were in large part associated to four different classes of orthopaedic devices, respectively: internal fixation devices, external fixation devices, knee arthroprostheses and hip arthroprostheses. The molecular epidemiology analysis performed by PCR enlightened a statistically significant difference in the prevalence of this adhesion mechanism between orthopaedic infections and catheter-related infections, respectively, of 78% and 91%. The prevalence of fbe ranged from 67% to 91%, suggesting that, even though this adhesin is not strictly necessary for the development of infection, nevertheless it represents a rather common characteristic of strains causing clinical infections, this independently on the presence or the absence of implant materials.

Bacterial Proteins↗

Immunomagnetic separation and MS/SPR end-detection combined procedure for rapid detection of Staphylococcus aureus and protein A.

The aim of this study was to establish an IMS-MS/SPR technique for the detection of Staphylococcus aureus (S. aureus) and Staphylococcus protein A (SPA) at the same time, which consists of isolating S. aureus and trapping-enrichmenting its SPA by IMS, and the end point is determined by using either MS or SPR measurements. Magnetic bead (MB) containing aldehyde group was synthesized with latex-polymerization and immunomagnetic bead (IMB) was fabricated by modifying its surface with an oriented layer of human IgG in covalent linkage. As soon as sample of pulverator-treated bacterial cell lysate (10(8) cfu/mL) was incubated with IMB at 4 degrees C for 30 min, SPA was captured and separated from the mixed solution in a few minutes by the IMB and then detected with mass spectrometry after washing. SPR was used to detect S. aureus quantitatively in situ at the end-detection procedure. All in all, this technique can be employed to detect rapidly SPA and S. aureus within 2h and also be applied to detect other cells or their membrane proteins with changed modified antibodies.

Aldehydes↗

The effect of essential oils on methicillin-resistant Staphylococcus aureus using a dressing model.

Patchouli, tea tree, geranium, lavender essential oils and Citricidal (grapefruit seed extract) were used singly and in combination to assess their anti-bacterial activity against three strains of Staphylococcus aureus: Oxford S. aureus NCTC 6571 (Oxford strain), Epidemic methicillin-resistant S. aureus (EMRSA 15) and MRSA (untypable). The individual essential oils, extracts and combinations were impregnated into filter paper discs and placed on the surface of agar plates, pre-seeded with the appropriate strain of Staphylococcus. The effects of the vapours of the oils and oil combinations were also assessed using impregnated filter paper discs that were placed on the underside of the Petri dish lid at a distance of 8mm from the bacteria. The most inhibitory combinations of oils for each strain were used in a dressing model constructed using a four layers of dressings: the primary layer consisted of either Jelonet or TelfaClear with or without Flamazine; the second was a layer of gauze, the third a layer of Gamgee and the final layer was Crepe bandage. The oil combinations were placed in either the gauze or the Gamgee layer. This four-layered dressing was placed over the seeded agar plate, incubated for 24h at 37 degrees C and the zones of inhibition measured. All experiments were repeated on three separate occasions. No anti-bacterial effects were observed when Flamazine was smeared on the gauze in the dressing model. When Telfaclear was used as the primary layer in the dressing model compared to Jelonet, greater zones of inhibition were observed. A combination of Citricidal and geranium oil showed the greatest-anti-bacterial effects against MRSA, whilst a combination of geranium and tea tree oil was most active against the methicillin-sensitive S. aureus (Oxford strain). This study demonstrates the potential of essential oils and essential oil vapours as antibacterial agents and for use in the treatment of MRSA infection.

Anti-Bacterial Agents↗

Revised structures for the capsular polysaccharides from Staphylococcus aureus Types 5 and 8, components of novel glycoconjugate vaccines.

Glycoconjugate vaccines based on the capsular polysaccharides (CPSs) from Staphylococcus aureus serotypes 5 and 8 conjugated to genetically detoxified recombinant exoprotein A (rEPA) from Pseudomonas aeruginosa have been shown, in Phase 3 clinical trials, to elicit a strong bactericidal immune response in end-stage renal disease patients. Such vaccines have the potential to reduce morbidity and mortality due to methicillin-resistant Staphylococcus aureus (MRSA), a major cause of hospital-acquired infection. The serotype 5 and 8 polysaccharides have been fully characterized by NMR spectroscopy and full structural analyses carried out. Published structures were found incorrect and the revised structures of the repeat units of the two polysaccharides are: [carbohydrate structure: see text]. Resonances indicative of the presence of peptidoglycan were observed in the spectra of both CPSs, consistent with reports that the CPS is covalently linked to peptidoglycan.

Acetylation↗

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain.

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.

Biofilms↗

Clinical isolates of Staphylococcus aureus from 1987 and 1989 demonstrating heterogeneous resistance to vancomycin and teicoplanin.

Fifty isolates of Staphylococcus aureus, obtained during a multicenter clinical trial evaluating the efficacy of teicoplanin that was performed between 1987 and 1992, underwent glycopeptide susceptibility testing, and 2 isolates were found to be capable of growth on agar containing 4 or 8 mg/L of vancomycin. Both of these isolates were from patients that had received prolonged teicoplanin therapy and were deemed clinical failures. Extended susceptibility testing combined with mecA gene probing revealed that one isolate was susceptible to oxacillin, the other was resistant, and both were susceptible to a variety of nonglycopeptide agents. Population analyses revealed heterogeneous vancomycin- and teicoplanin-susceptibility profiles. Both strains were differentiated from recent glycopeptide intermediately resistant Staphylococcus aureus isolates by pulse-field analysis and by the fact that the resistance phenotype was stable to multiple serial passages. To our knowledge, this is the earliest report of S. aureus clinical isolates having reduced vancomycin and teicoplanin susceptibility.

Analysis of Variance↗

Current antimicrobial resistance profiles among methicillin-resistant Staphylococcus aureus encountered in the outpatient setting.

The acquisition of the mec gene complex by methicillin-susceptible Staphylococcus aureus in the community and the increased spread of methicillin-resistant Staphylococcus aureus (MRSA) from the health care setting to the community underscore a need to monitor the resistance phenotypes likely to be encountered among outpatient MRSA. Data from the LEADER 2004 surveillance program were analyzed to evaluate current resistance profiles among outpatient MRSA. Outpatient MRSA exhibited 26 different resistance phenotypes; the 4 most common were resistance to erythromycin only (40.8%), multidrug resistance to erythromycin, clindamycin, and levofloxacin (21.5%), double drug resistance to erythromycin and levofloxacin (11.3%), and double drug resistance to clindamycin and erythromycin (5.1%). These phenotypes were also the most common among inpatient MRSA (n = 946), but multidrug resistance to erythromycin, clindamycin, and levofloxacin (43.7%) was most common. Fifty percent (256) of the outpatient MRSA were resistant to 2 or more agents, whereas resistance to either vancomycin or linezolid was not encountered. The extensive similarities in resistance profiles between inpatient and outpatient MRSA have important implications for establishing outpatient management and treatment guidelines for staphylococcal infections.

Ambulatory Care↗

Molecular identification and characterization of mannitol-negative methicillin-resistant Staphylococcus aureus.

We report on the isolation, molecular identification, and characterization of 5 mannitol-negative methicillin-resistant Staphylococcus aureus (MRSA) from clinical samples in KwaZulu-Natal (KZN) province, South Africa. Identification based on phenotypic testing and polymerase chain reaction detection of the S. aureus species-specific nuc gene and the coagulase gene indicated that the mannitol-negative isolates were S. aureus. Furthermore, they were mecA positive, and SCCmec typing demonstrated that all the isolates harbored type IV SCCmec. API STAPH (Biomerieux, Marcy-l'Etoile, France) misidentified 2 mannitol-negative MRSA that belonged to the major clone in KZN province, as Staphylococcus lugdunensis. Although the prevalence and mechanism of mannitol-negative MRSA is unknown, laboratories are encouraged to investigate S. aureus with atypical characteristics.

Bacterial Proteins↗

Molecular epidemiology and household transmission of community-associated methicillin-resistant Staphylococcus aureus in Hong Kong.

This study evaluated the clinical and epidemiologic features of individuals with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Hong Kong from January 2004 through December 2005. Twenty-four episodes of skin and soft tissue infections and 1 episode of meningitis due to CA-MRSA were identified. CA-MRSA infections or carriage was found in 6 (13%) of 46 household contacts. A total of 29 isolates were analyzed by the Staphylococcus cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. In addition, polymerase chain reaction detection of the genes encoding Panton-Valentine leukocidin was also carried out. It was observed that 24 had SCCmec IV/IVA and 5 had SCCmec V, and 23 were pvl positive. PFGE analysis clustered all except 1 isolate into 3 pulsed-field types (PFTs), HKU100 through HKU300. The HKU100 isolates had genotype ST30-IV identical to the Southwest Pacific clone. The HKU200 isolates belonged to ST59-V and were multiresistant, including an ermB-mediated macrolide resistance trait, which is characteristic of the predominant CA-MRSA clone in Taiwan. The HKU300 isolates had unique features (ST8, Panton-Valentine leukocidin negative, and SCCmec IVA) typical of CA-MRSA in Japan. In conclusion, CA-MRSA has a propensity to spread within families. Our findings showed that CA-MRSA strains in Hong Kong have diverse genetic backgrounds.

Adolescent↗

5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate.

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway, and is the target of the broad-spectrum herbicide glyphosate. Kinetic analysis of the cloned EPSPS from Staphylococcus aureus revealed that this enzyme exerts a high tolerance to glyphosate, while maintaining a high affinity for its substrate phosphoenolpyruvate. Enzymatic activity is markedly influenced by monovalent cations such as potassium or ammonium, which is due to an increase in catalytic turnover. However, insensitivity to glyphosate appears to be independent from the presence of cations. Therefore, we propose that the Staphylococcus aureus EPSPS should be classified as a class II EPSPS. This research illustrates a critical mechanism of glyphosate resistance naturally occurring in certain pathogenic bacteria.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

A fibrinogen-binding protein of Staphylococcus lugdunensis.

A gene called fbl, encoding a Staphylococcus lugdunensis fibrinogen-binding protein, was identified by phage display. The encoded protein, Fbl, is a member of the Sdr-family, a group of staphylococcal cell surface proteins containing a characteristic serine-aspartate repeat region. The fibrinogen-binding domain was mapped to 313 amino acids, and shows 62% identity to the corresponding region in clumping factor (ClfA) from Staphylococcus aureus. Anti-serum against ClfA cross-reacted with Fbl, and blocked S. lugdunensis adherence to fibrinogen. Twelve clinical isolates of S. lugdunensis analysed by Southern blot all had an fbl-like gene.

Amino Acid Sequence↗

Methicillin-resistant Staphylococcus aureus prosthetic aortic valve endocarditis with paravalvular abscess treated with daptomycin.

Prosthetic valves have been used extensively for severe cardiac valvular dysfunction for the past 3 decades. Prosthetic cardiac valves may be infected with organisms causing bacteremia, particularly gram-positive cocci. Staphylococcus epidermidis (coagulase negative staphylococci) and Staphylococcus aureus , both methicillin-susceptible S. aureus and methicillin-resistant S. aureus (MRSA) strains, are the most frequent pathogens causing prosthetic valve endocarditis (PVE). Vancomycin has been the cornerstone of therapy for serious MRSA infections including bacteremia and endocarditis. Clinicians have noted that MRSA bacteremias treated with vancomycin often fail to clear even with prolonged therapy. Persistent or prolonged MRSA bacteremia unresponsive to vancomycin therapy has led to the treatment of these infections by other agents, that is, quinupristin, dalfopristin, linezolid, or daptomycin. These antibiotics have been found particularly useful in treating MRSA bacteremias unresponsive to vancomycin therapy. We report a case of a patient who presented with MRSA PVE complicated by perivalvular aortic abscess with persistent MRSA bacteremia unresponsive to vancomycin therapy. The patient's MRSA bacteremia was cleared with daptomycin therapy (6 mg/kg/d). Because the patient refused surgery, daptomycin therapy was continued in hopes of curing the endocarditis and sterilizing the perivalvular aortic abscess. Transesophageal echocardiogram revealed a decrease in abscess in the aortic perivalvular abscess after 1 week of daptomycin therapy. The patient made an uneventful recovery. The cure of PVE and perivalvular abscesses usually requires removal of the prosthetic device and abscess drainage. In this case, in which surgery was not an option, medical therapy of PVE and a decrease in size of the aortic perivalvular abscess were accomplished with daptomycin therapy. Daptomycin is an alternative to vancomycin therapy in patients with prolonged or persistent MRSA bacteremia secondary to endocarditis or abscess.

Abscess↗

Daptomycin cure after cefazolin treatment failure of Methicillin-sensitive Staphylococcus aureus (MSSA) tricuspid valve acute bacterial endocarditis from a peripherally inserted central catheter (PICC) line.

Right-sided acute bacterial endocarditis (ABE) is an infrequent complication of central intravenous (IV) lines. We report a case of methicillin-sensitive Staphylococcus aureus tricuspid valve (TV) ABE related to a peripherally inserted central catheter line (PICC). Patients with right-sided ABE present with symptoms of fever and chills, and symptoms and signs of pulmonary emboli. In the patient presented, the PICC line was removed and high-dose cefazolin therapy, 2 g (IV) every 8 hours, was initiated. Although the patient's blood cultures became negative during the third week of cefazolin therapy, her erythrocyte sedimentation rate and teichoic acid antibody titers remained high. Pulmonary emboli developed. A large TV vegetation (1 x 2 cm) remained unchanged after 4 weeks of cefazolin therapy. For these reasons, cefazolin treatment was considered a treatment failure. Therapy with daptomycin was initiated at a dose of 6 mg/kg (IV) every 24 hours. During daptomycin therapy, the patient's erythrocyte sedimentation rate and teichoic acid antibody titers gradually returned to normal. Repeat transthoracic echocardiograph revealed the TV vegetation was gone and the methicillin-sensitive Staphylococcus aureus ABE was cured with daptomycin. We conclude daptomycin is a rapidly bactericidal antistaphylococcal antibiotic reliably effective even when other usually effective antistaphylococcal antibiotics have failed.

Acute Disease↗

Immunohistochemical evidence of Clostridium sp, Staphylococcus aureus, and group A Streptococcus in severe soft tissue infections related to injection drug use.

Severe soft tissue infections are caused by either single or multiple microorganisms. We performed a retrospective immunohistochemical (IHC) study on formalin-fixed, paraffin-embedded soft tissue samples from 20 injection drug users who were part of a cluster of severe illness and death after skin and soft tissue infections in Scotland and Ireland in 2000. The IHC assays used antibodies against Clostridium sp, Staphylococcus aureus, group A streptococci, and Bacillus anthracis. Intact bacilli and granular Clostridium antigen staining in areas with necrosis, edema, and inflammation were observed in skin, fascia, or muscle samples of 12 (60%) patients. A variety of clostridia were isolated from affected soft tissues in 10 IHC-positive cases. Staphylococcus aureus antigens were observed in 3 cases including 1 where S aureus was isolated, 1 with negative cultures, and 1 where mixed cultures were obtained. Group A streptococcal antigens were observed in 1 case in which Streptococcus pyogenes and S aureus were isolated. By using IHC, we detected different bacteria in archival soft tissue samples from patients with severe skin and soft tissue infections. Immunohistochemical assays can be of great diagnostic value, particularly for bacteria such as Clostridium sp, which are difficult to isolate because of their anaerobic fastidious growth requirements.

Clostridium Infections↗

Ecology and characterization by molecular methods of Staphylococcus species isolated from fresh sausages.

Staphylococcus spp. ecology in fresh sausages stored at 4 degrees C for a period of 10 days was investigated. Microbiological analyses to assess the quality and safety of the products studied were carried out, and strains on MSA agar were isolated. A total of 85 strains were identified, by traditional methods, as Staphylococcus spp. and a PCR-DGGE method, together with a S. xylosus-specific PCR, were used to determine the species of the strains isolated. Almost 50% of them were recognized as S. xylosus, but strains of S. pasteuri, S. warneri, S. equorum and S. succinus, were also found. Interesting population dynamics were observed, characterized by a succession of S. pasteuri and S. warneri at 0 day, with S. xylosus at 3 days and S. equorum at 6 and 10 days. Molecular characterization of S. xylosus strains and cluster analysis highlighted the presence of six main populations in the fresh sausages studied. However, the clusters were formed by strains isolated at different days of storage, implying a homogeneous distribution of the six subpopulations throughout the period in the products followed.

Animals↗

Identification of Bacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. with 16S ribosomal DNA-based oligonucleotide array hybridization.

Rapid identification of the genus and species of bacteria in foods and clinical specimens is important. In this report, DNA sequences of bacterial 16S rDNA were used to develop the oligonucleotide array for the identification of bacterial strains of Bacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. Most of these bacterial strains may cause food-borne outbreaks or sporadic cases. A rapid (<4 h) detection method that used universal PCR primers to amplify the variable regions of bacterial 16S rDNA, followed by reverse hybridization of the PCR products, which were biotin labeled, to the oligonucleotides arrayed on the chip was developed. Fifteen oligonucleotide probes were selected and spotted on the nylon strip to determine the array hybridization patterns. It was successful in discriminating Bacillus spp., E. coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. with identification, in general, to the genus level, not species level. As 182 randomly selected strains were assayed, the detection rate was found higher than 98%. Except for 3 strains, the remaining 179 strains were correctly identified and no cross reactions were observed. These 179 strains generated five hybridization patterns. Adding more oligonucleotide probes to the array may allow the detection of more bacterial genera and species without significantly increasing the complexity or cost.

Bacillus↗

Staphylococcus quorum sensing in biofilm formation and infection.

Cell population density-dependent regulation of gene expression is an important determinant of bacterial pathogenesis. Staphylococci have two quorum-sensing (QS) systems. The accessory gene regulator (agr) is genus specific and uses a post-translationally modified peptide as an autoinducing signal. In the pathogens Staphylococcus aureus and Staphylococcus epidermidis, agr controls the expression of a series of toxins and virulence factors and the interaction with the innate immune system. However, the role of agr during infection is controversial. A possible second QS system of staphylococci, luxS, is found in a variety of Gram-positive and Gram-negative bacteria. Importantly, unlike many QS systems described in Gram-negative bacteria, agr and luxS of staphylococci reduce rather than induce biofilm formation and virulence during biofilm-associated infection. agr enhances biofilm detachment by up-regulation of the expression of detergent-like peptides, whereas luxS reduces cell-to-cell adhesion by down-regulating expression of biofilm exopolysaccharide. Significant QS activity in staphylococci is observed for actively growing cells at a high cell density, such as during the initial stages of an infection and under optimal environmental conditions. In contrast, the metabolically quiescent biofilm mode of growth appears to be characterized by an overall low activity of the staphylococcal QS systems. It remains to be shown whether QS control in staphylococci represents a promising target for the development of novel antibacterial agents.

Animals↗