Epidemiological characterization of methicillin-resistant Staphylococcus aureus isolated in the North West of England by protein A (spa) and coagulase (coa) gene polymorphisms.
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In a comparative study, isolates of methicillin-resistant Staphylococcus aureus (MRSA) with known pulsed-field gel electrophoresis (PFGE) and bacteriophage type were analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) for additional discriminatory subtyping information. PFGE was previously performed using standardized, commercially available kits and pre-programmed software. Isolates were examined for coagulase (coa) and protein A (spa) gene polymorphisms following PCR amplification of the coa hypervariable and spa repeat regions. Coa gene RFLPs produced a total of 38 distinct combined patterns after digestion with HaeIII and AluI and identified the predominant epidemic (EMRSA) types 15 and 16. A unique HaeIII restriction site was identified by RFLP and sequence analysis in the coa gene for EMRSA 15 but not EMRSA 16. The spa gene PCR yielded a total of 14 different profiles ranging from 3-18 repeats with the 2 predominant EMRSA types falling into 2 distinct groups. PCR detection of coa and spa polymorphisms offer a rapid preliminary strain identification and discriminatory subtyping information for surveillance of MRSA. (+info)
Changing susceptibilities of coagulase-negative staphylococci to teicoplanin in a teaching hospital.
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The susceptibility of two collections of coagulase-negative staphylococci (CNS) isolated from clinical specimens for teicoplanin and vancomycin were compared. They comprised 91 and 101 isolates, collected in 1985 and 1994 respectively, from different departments of a teaching hospital. MICs of vancomycin and teicoplanin were determined by a modified Etest method. Additionally, a disc diffusion test was performed for teicoplanin. All isolates were susceptible to vancomycin (MIC < or = 4 mg/L). Two of the 91 isolates collected in 1985 were intermediate to teicoplanin (MIC between 8 and 32 mg/L), whereas in 1994 the number of intermediate isolates was 20 out of 101 (P < 0.01). The correlation between MICs, as determined by the modified Etest assay, and disc diffusion zones was poor (r = -0.35). Results show that resistance to teicoplanin in CNS has increased in the study hospital over a period of 9 years. This increase is likely to be correlated with the introduction of teicoplanin. Furthermore, a disc diffusion method does not appear to be the first method of choice for detection of strains of CNS with diminished susceptibility to teicoplanin. (+info)
Rapid identification of Staphylococcus aureus by using fluorescent staphylocoagulase assays.
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Two rapid (1-h) assays for the detection of Staphylococcus aureus staphylocoagulase were developed by using the fluorogenic thrombin substrates N-t-boc-Val-Pro-Arg-7-amido-4-methylcoumarin (VPA) and N-t-boc-beta-benzyl-Asp-Pro-Arg-7-amido-4-methylocoumarin (BB). The assays were compared to the tube coagulase test and latex agglutination (LA) (Sanofi Diagnostics Pasteur, Guildford, Surrey, United Kingdom) by using 406 clinical isolates of staphylococci, and they produced positive and negative predictive values of 99.2 and 99. 1% for LA, 98.9 and 92.7% for VPA, and 98.9 and 99.1% for BB. Fluorescent assays used colonies from solid media, thereby eliminating the need for broth cultures, and were performed in microtiter trays, thus making them suitable for large-scale screening. (+info)
The Staphylococcus aureus rsbW (orf159) gene encodes an anti-sigma factor of SigB.
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SigB, a newly discovered alternative sigma factor of Staphylococcus aureus, has been shown to play an important role in stress responses and the regulation of virulence factors. The rsbW (orf159) gene is immediately upstream of sigB. Its gene product is homologous to Bacillus subtilis RsbW which under appropriate conditions binds to B. subtilis SigB and functions as an anti-sigma factor or negative posttranslational regulator. To define the function of S. aureus RsbW, both the S. aureus SigB and RsbW proteins were expressed in Escherichia coli and purified. Cross-linking experiments with these purified proteins revealed that RsbW was capable of specific binding to SigB. In an in vitro transcription runoff assay, RsbW prevented SigB-directed transcription from the sar P3 promoter, a known SigB-dependent promoter, and the inhibitory activity of RsbW was found to be concentration dependent. We also identified SigB promoter consensus sequences upstream of the genes encoding alkaline shock protein 23 and coagulase and have demonstrated SigB and RsbW dependence for the promoters in vitro. These results show that RsbW is a protein sequestering anti-sigma factor of S. aureus SigB and suggest that SigB activity in S. aureus is regulated posttranslationally. (+info)
Use of semi-quantitative and quantitative culture methods and typing for studying the epidemiology of central venous catheter-related infections in neonates on parenteral nutrition.
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To study the epidemiology - especially the impact of contaminated stopcocks - on central venous catheter (CVC) infection and catheter-related sepsis (CRS), semi-quantitative (SQ) and quantitative (Q) culture methods and typing of coagulase-negative staphylococci (CNS) were employed in 49 neonates with clinical signs of sepsis while receiving parenteral nutrition in the paediatric intensive care unit. The patients were divided into two groups according to stopcock contamination: group A consisted of 18 patients (36%) with contaminated stopcocks and group B consisted of 31 patients (64%) with sterile stopcocks. Five specimens were obtained from each patient, in addition to that from the stopcock: a swab taken from the skin surrounding the catheter puncture site; the CVC tip; the intradermal segment (IDC); and samples of parenteral fluid and blood. A total of 294 specimens (392 sites) was cultured and micro-organisms were identified. All CNS isolated were typed by biotyping, antibiogram, plasmid analysis and pulsed-field gel electrophoresis (PFGE), and the discriminatory power of the typing methods was compared. The CVC tips were infected in 25 patients (51%); 15 (83%) in group A and 10 (32%) in group B. Sepsis was detected in 24 neonates (49%), 13 in group A and 11 in group B. This was catheter-related in 15 patients (63%), 12 in group A and 3 in group B. CNS were recovered from 13 (52%) of 25 infected CVCs, nine in group A and four in group B. Sixty-five CNS isolates were recovered from these patients and belonged to 14 biotypes, 22 antibiograms, 22 plasmid profiles and 26 PFGE types. Typing showed that in six of nine patients in group A, CNS of the same type were recovered from the catheter tip and the stopcock, in one patient the catheter tip and skin isolates were the same and in two others the catheter tip isolates were different from stopcock and skin isolates. In all four patients in group B, different CNS types were recovered from CVC tips and skin. Bacteraemia was caused by CNS in 14 patients (58%), six in group A and eight in group B. Typing confirmed that nine cases (six in group A and three in group B) were catheter-related but five were not. SQ and Q culture methods and typing, especially by PFGE, allowed the study to determine that bacteria from contaminated stopcocks were frequently the source of CVC infection and CRS. (+info)
Oxacillin susceptibility testing of staphylococci directly from Bactec Plus blood cultures by the BBL Crystal MRSA ID system.
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The BBL Crystal MRSA ID test (Becton Dickinson) was applied directly to blood culture vials containing clusters of gram-positive cocci. The sensitivity and specificity of the test were 84 and 100% and 54 and 100% for vials containing Staphylococcus aureus and coagulase-negative staphylococci, respectively. This test is a reliable method for direct detection of methicillin resistance in positive blood culture vials when S. aureus is identified in parallel by rapid identification procedures. (+info)
Analysis of genomic diversity within the Xr-region of the protein A gene in clinical isolates of Staphylococcus aureus.
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Protein A of Staphylococcus aureus contains a polymorphic Xr-region characterized by a tandem repeat of eight amino acid units. In this study, the diversity of genes encoding the repeat regions and their relatedness among S. aureus strains was analyzed. Ten different protein-A types characterized by repeat numbers 4-13 were identified in a total of 293 clinical isolates. The protein-A type with 10 repeat units (10 repeats) in the Xr-region was most frequently detected in methicillin-resistant S. aureus, whereas the majority of methicillin-susceptible strains were distributed almost evenly into protein-A types with 7-11 repeats. Strains that belonged to a single coagulase type were classified into multiple protein-A types, e.g. strains with the common coagulase types II and VII were differentiated into 7 and 8 protein-A types, respectively. Nucleotide sequence analysis of the Xr-region of 42 representative strains revealed the presence of 37 different genotypes (spa types), which were constituted by a combination of several of 24 different repeat unit genotypes. Based on the similarity in arrangement of repeat unit genotypes, 34 strains with different repeat numbers were classified into 5 genetic clusters (C1-C5). The clusters C1, C2 and C3 consisted exclusively of strains with identical coagulase types II, III, and IV, respectively. These findings suggested that the protein-A gene of S. aureus has evolved from a common ancestral clone in individual clusters independently. (+info)
Coagulase gene polymorphism of Staphylococcus aureus isolates from dairy cattle in different geographical areas.
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The objectives of this study were to investigate the coagulase gene polymorphism of Staphylococcus aureus isolates obtained from bovine mastitic milk and to determine the resistance of predominant and rare coagulase genotypes to bovine blood neutrophil bactericidal activities. A total of 453 isolates were collected from four countries: the Czech Republic, France, Korea and the United States. The isolates were subtyped into 40 types by restriction fragment length polymorphism (RFLP) of the coagulase gene. Twenty-three strains from predominant and rare genotypes were evaluated for their ability to resist neutrophil bactericidal activities. There were significant (P < 0.01) differences in the average percent neutrophil killing of the predominant (16.7%) and rare (39.7%) genotypes when bacteria were opsonized with antiserum. The results indicate that the profiles of coagulase genotype differ among geographic locations, and only a few genotypes prevail in each location. In addition, the predominant genotypes were more resistant to neutrophil bactericidal activities than rare genotypes. (+info)