Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.     

The investigation of Staphylococcus aureus and coagulase-negative staphylococci nasal carriage among patients undergoing haemodialysis

The frequency of nasal staphylococcal colonization among haemodialysed patients was investigated. The swabs were collected in 1998 and 2004 from 28 and 43 patients, respectively.

Staphylococcus aureus colonization rates were 57.1% and 27.9% in 1998 and 2004, respectively. Twenty-six coagulase-negative staphylococci (CNS) isolates were cultured: S. epidermidis (21), S. lugdunensis (2), single S. haemolyticus, S. warneri, and S. capitits isolates. One S. aureus and 10 CNS isolates were methicillin resistant. The methicillin-resistant S. aureus (MRSA) was resistant to β-lactams, tetracycline, and harbored the pvl gene encoding the Panton-Valentine leukocidin.

The decrease in S. aureus colonization at 6-year interval was observed. The presence of the pvl gene and a favorable antibiotic susceptibility pattern of the MRSA suggest that the isolate was a member of community-acquired MRSA (CA-MRSA). Concluding, screening of haemodialysed patients for staphylococcal colonization accompanied by characterization of cultured isolates is important to understand its epidemiology and to develop infection prevention measures and treatment strategies.     

Macrolide resistance genotypes and phenotypes among erythromycin-resistant clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, Italy

One hundred macrolide-resistant staphylococcal isolates from clinically relevant infections in Italy during a 19-month period were studied. Four distinct resistance phenotypes were observed using the triple-disk induction test (erythromycin, clindamycin, telithromycin): the cMLS_B phenotype (24 isolates); the iMLS_B phenotype (41 isolates); the MS phenotype (three isolates); and the iMTS phenotype (erythromycin-induced telithromycin resistance) (32 isolates). ermC and ermA genes predominated within erythromycin-resistant Staphylococcus aureus isolates with iMLS_B phenotype and cMLS_B phenotype, respectively. Among erythromycin-resistant CoNS isolates, half of the strains showed the iMTS or MS/ msrA association, and ermC gene predominated among isolates with MLS_B phenotype. By pulsed-field gel electrophoresis, high genetic heterogeneity was observed among the isolates studied. Both independent acquisition of macrolide resistance genes and spread of specific resistant clones were observed. Association between certain clonal types and specific types of infection could be detected. To our knowledge, this is the first report on characterization of erythromycin-resistant staphylococci in Italy.     

Pulsed-field gel electrophoresis of genomic restriction fragments as a tool for the epidemiological analysis of Staphylococcus aureus and coagulase-negative staphylococci

Thirteen Staphylococcus aureus and S. epidermidis strains obtained from nose and hand of two employees and one patient of a medical ward as well as two S. hemolyticus strains were analysed according to their restriction fragment length patterns (RFLP) by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI and SstII. Species identification of the isolates was performed by a system which includes 20 biochemical reactions. Furthermore, the antibiotic resistance patterns of the strains were determined. While several isolates exhibited identical antibiotic susceptibilities and biochemical profiles, differences in the RFLP were obtained. In three cases, S. epidermidis strains colonizing the skin showed an identical restriction profile as isolates from the mucous membranes of the same person. We concluded that the analysis of staphylococcal strains by PFGE is an important epidemiological tool with high discrimination power.     

Binding of type-I and type-II collagens to Staphylococcus aureus strains isolated from patients with toxic shock syndrome compared to other staphylococcal infections

Abstract Toxic shock syndrome toxin-1 (TSST-1) producing strains of Staphylococcus aureus isolated from 18 patients with toxic shock syndrome (TSS) and from 56 patients with other diagnoses were compared for capacity to interact with various serum and connective tissue proteins. TSS associated isolates showed significantly stronger binding of Type-I collagen (Cn-I) and Cn-II than non-TSS strains, in a particle agglutination assay (PAA) as well as in ¹²⁵I labelled Cn uptake experiments. ¹²⁵I Cn-IV binding, was similar between the two groups, whereas in PAA, a stronger interaction was observed for non-TSS than TSS associated strains. The median binding of ¹²⁵I Cn to TSS-associated strains were 52.2 (Cn-I), 30.6 (Cn-II) and 20.0 (Cn-IV) compared to 20.0 (Cn-I), 14.4 (Cn-II) and 24.4 (Cn-IV) values of non-TSS strains. A saturation with ¹²⁵I Cn-I and Cn-II binding was established for TSS (30 min) and non-TSS (15 min) strains. ¹²⁵I Cn-IV binding reached a saturation in 10 min and 90 min with TSS and non-TSS strains respectively. Finally, the binding profiles of TSS associated and non-TSS strains to fibronectin, fibrinogen, laminin and IgG did not differ in both PAA and radioisotope assays. In scanning electron microscopy, cells of TSS associated strains bound to the reprecipitated native Cn-I fibrils. In contrast, most cells of non-TSS strains were localized to the distal end or were trapped between the Cn fibrils. The stronger interaction with Cn-I and II in particular, shown by TSS associated strains, might enhance submucosal localization, thereby facilitating entry of toxins into the blood and establishment of TSS.     

Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: epidemiological and molecular aspects

Infections caused by the genus Staphylococcus are of great importance for human health. Staphylococcus species are divided into coagulase-positive staphylococci, represented by S. aureus, a pathogen that can cause infections of the skin and other organs in immunocompetent patients, and coagulase-negative staphylococci (CNS) which comprise different species normally involved in infectious processes in immunocompromised patients or patients using catheters. Oxacillin has been one of the main drugs used for the treatment of staphylococcal infections; however, a large number of S. aureus and CNS isolates of nosocomial origin are resistant to this drug. Methicillin resistance is encoded by the mecA gene which is inserted in the SCC mec cassette. This cassette is a mobile genetic element consisting of five different types and several subtypes. Oxacillin-resistant strains are detected by phenotypic and genotypic methods. Epidemiologically, methicillin-resistant S. aureus strains can be divided into five large pandemic clones, called Brazilian, Hungarian, Iberian, New York/Japan and Pediatric. The objective of the present review was to discuss aspects of resistance, epidemiology, genetics and detection of oxacillin resistance in Staphylococcus spp., since these microorganisms are increasingly more frequent in Brazil.     

Catecholamine inotropes as growth factors for Staphylococcus epidermidis and other coagulase-negative staphylococci

Drugs commonly used in intensive care settings were assayed for their ability to affect the growth of Staphylococcus epidermidis in a minimal salts medium containing 30% serum. Of 28 compounds tested, the inotropic catecholamines adrenaline, dobutamine, dopamine, isoprenaline and noradrenaline significantly stimulated bacterial growth. These drugs, but not structurally similar compounds lacking a dihydroxybenzoyl moiety (such as tyramine, phenylephrine and salbutamol), were able to remove iron from iron-saturated transferrin and to supply transferrin-bound 55Fe to S. epidermidis cells. Similar results were observed with a range of coagulase-negative staphylococci associated with line infections, but not with Staphylococcus aureus (including MRSA).     

Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci

Two shuttle vectors containing the gene for protein A (spa) from Staphylococcus aureus have been constructed to study expression of the gene in various strains of S. aureus and in the coagulase-negative species Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus xylosus. One plasmid, pSPA15, contains the complete structural gene for protein A, which binds to the cell wall in various Staphylococcus species. The other plasmid, pSPA16, codes for a truncated protein A lacking the C-terminal part called region X. The latter is exclusively extracellular in all Staphylococcus species tested, which confirms the importance of region X for cell wall binding. The expression of the plasmid-coded protein A in various strains of S. aureus is strongly correlated to the expression of the chromosomal spa gene. The coagulase-negative species expressing plasmid-encoded protein A produce 12 to 30% of the amount coded by the chromosomal spa gene in S. aureus strains Cowan I and A676.     

Cytidine deamination assay to differentiate Staphylococcus aureus from other staphylococci

AIMS: Adoption of the property of cytidine (cytosine-beta-d-riboside) deamination in staphylococci to distinguish Staphylococcus aureus from other staphylococci. METHODS AND RESULTS: A total of 560 staphylococcal strains were examined. The test demonstrated a sensitivity of 97.1% and a specificity of 98.8%. Of the 249 S. aureus strains (115 oxacillin-resistant) 58 strains were coagulase-negative S. aureus and another 16 strains were clumping factor-negative S. aureus. The 74 deficient S. aureus strains were identified by 16S rRNA gene sequencing and further investigated by spa typing and 13 spa types were found. CONCLUSIONS: The cytidine deaminase test (CDT) is useful especially for distinguishing coagulase- and clumping factor-negative S. aureus from other staphylococci and the results correlated well with 16S rRNA sequencing and the polymerase chain reaction (PCR) amplification of the nuc gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Cytidine deamination assay differentiates S. aureus from other staphylococci. This method is fast (6 h) and reliable in distinguishing between non-S. aureus and the defective (coagulase-negative, clumping factor-negative) S. aureus isolates which could have major consequences for therapy.     

Pathogenic conversion of coagulase-negative staphylococci

Humans and animals are colonized by members of the genus Staphylococcus, however only some of these species evolved to cause invasive disease. The genetic basis for conversion of commensal staphylococci into pathogens is not known. We hypothesized that Staphylococcus aureus genes for coagulation and agglutination in vertebrate blood (coa, vwb and clfA) may support pathogenic conversion. Expression of coa and vwb in Staphylococcus epidermidis or Staphylococcus simulans supported a coagulase-positive phenotype but not the ability to cause disease in a mouse model of bloodstream infection. However, the simultaneous expression of coa, vwb and clfA in coagulase-negative staphylococci enabled bacterial agglutination in plasma and enhanced survival of S. simulans in human whole blood. Agglutination of S. simulans in the bloodstream of infected mice upon expression of coa, vwb and clfA provided also a mean for dissemination and replication in distal organs. Thus, the acquisition of genes for bacterial agglutination with fibrin appear sufficient for the conversion of commensal staphylococci into invasive pathogens.     

Isolation and characterization of a DNA probe for Staphylococcus aureus subspecies aureus biovar

The gene encoding for polynucleotide phosphorylase (pnp) of a new biovar of Staphylococcus aureus subsp. aureus (NBSA) has been isolated from a genomic library of strain M280(0). The coding region consisted of a 1094-bp HindIII-HindIII DNA fragment encoding for a protein of 277 amino acids with a calculated molecular mass of 29.5 kDa. The nucleotide sequence of the structural gene, contained a continuous open reading frame of 836 bp, showed significant homology with the genes of bacterial polynucleotide phosphorylase from Bacillus subtilis (67.7% identity), from Haemophilus influenzae (62.4% identity), from Pseudomonas luminescens (61.6% identity), and from Escherichia coli (59.7% identity). DNA-DNA and DNA-colony slot-blot hybridizations demonstrated that the pnp gene, employed as a molecular probe, is specific for the identification of NBSA strains.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37 degrees C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Vaccination with Staphylococcus aureus fibrinogen binding proteins (FgBPs) reduces colonisation of S. aureus in a mouse mastitis model

Abstract A mouse mastitis model was used to study the effect of vaccination with fibrinogen binding proteins and collagen binding protein from Staphylococcus aureus against challenge infection with S. aureus . The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibronogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. Histopathological examination of tissue sections from challenged glands showed that most glands from mice vaccinated with fibrinogen binding protein developed disseminated necrosis or had no pathological changes. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. In a similar study, immunization of mice with collagen binding protein did not induce protection against challenge infection with S. aureus .     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37°C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Different effects of fibronectin on the phagocytosis of Staphylococcus aureus and coagulase-negative staphylococci by murine peritoneal macrophages

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase-negative staphylococci (CNS) strains for comparison. Fibronectin-reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose-dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin-binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.     

Cloning and restriction analysis of DNA conferring new quinolone antimicrobial agent resistance from Staphylococcus aureus and other coagulase-negative Staphylococcus species

DNA conferring resistance to new quinolone antimicrobial agents (NQR) in clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CNS), S. epidermidis and S. haemolyticus, was analyzed after cloning in Escherichia coli. The NQR phenotypes were expressed in E. coli at lower levels. NQR gene(s)-carring HindIII fragments were very similar to each other among S. aureus or CNS, although the S. aureus fragments differed from the CNS fragments in terms of the NQR phenotypes and restriction endonuclease maps. The data suggest the possibility that the NQR genes have disseminated in evolutionarily distinct routes among S. aureus and CNS.     

The slime of coagulase-negative staphylococci: Biochemistry and relation to adherence

Abstract In recent years, infections of implanted plastic devices by coagulase-negative staphylococci have become a major cause of septicaemia in human patients. The causal bacterial species is usually Staphylococcus epidermidis and these organisms grow as a biofilm adherent to a solid surface. Several methods have been introduced to assess the mass of adherent bacteria and the slimy matrix in which they are embedded. Some methods measure total biofilm, others measure the organisms or the slime alone. In vitro , the type of medium, the atmosphere during incubation, and the nature of the solid surface, affect the quantity of biofilm that is formed. In most studies on the chemistry of the slime, the material used was formed on complex media solidified with agar. Contamination by ingredients of the media or by agar may not always have been recognised. Recent work with chemically defined medium (liquid or solidified with silica gel) shows that the slime is a mixture of about 80% (w/w) teichoic acid and 20% protein. Growth as a biofilm may protect the staphylococci from antibiotics. At present, the greatest success in preventing infection has come from improved surgical techniques during the insertion of implants.