Detection of fibronectin-binding protein genes in staphylococcal strains from peri-prosthesis infections.

Several studies have been devoted to identify the adhesion mechanisms of Staphylococcus aureus and Staphylococcus epidermidis, which are the most frequent causes of prosthesis-associated infections. Recently, in particular for Staphylococcus aureus, considerable attention has been given to the host protein receptors as mediators for bacterial adherence. Fibronectin, in important matrix protein, seems to be a major ligand for bacterial adherence in the early stages of infection. To determine the importance of the fibronectin adhesin as virulence factor in Staphylococcus-induced prosthesis infection, a simple and reliable method using a polymerase chain reaction (PCR) was devised to identify fibronectin adhesin genes (fnbA and fnbB). Results obtained by this method were in accordance with those obtained by the in vitro phenotypic characterization of binding ability to fibronectin of Staphylococcus strains.

Slime production by Staphylococci isolated from prosthesis-associated infections.

Clinical isolates of Staphylococcus epidermidis are frequently referred to produce a biofilm, known as slime, involved in adherence to medical devices and in resistance to host defences. A high frequency of slime producing Staphylococcus aureus strains was never reported, at least in the case of human isolates. In the present study the production of slime by clinical isolates of S. aureus and S. epidermidis from catheter associated infections and from post-surgical infections was studied by a sensitive method based on culturing the isolates on Congo red agar. The study demonstrates that in nosocomial surgical infections, considered separately from catheter-associated infections, S. aureus emerges as a more prevalent etiologic agent than S. epidermidis, with a proportion of slime producing strains markedly high.

Presence and expression of collagen adhesin gene (cna) and slime production in Staphylococcus aureus strains from orthopaedic prosthesis infections.

Prosthesis-associated infections still represent one of the most serious complications in the clinical use of biomaterials. The most frequent causes are Staphylococcus aureus and Staphylococcus epidermidis. Several studies have been devoted to identify adhesion mechanisms for these bacteria. Slime in particular has been extensively investigated. Recently, in Staphylococcus aureus species, considerable attention has been given to the host protein receptors that have been shown in in vitro assays to serve as substrates for bacterial adhesion. Collagen-rich tissues, as bone and cartilage, that are the preferential sites of staphylococcal infections, are also the tissues that harbour orthopaedic implants. These can be easily coated in vivo by collagen and thus become prone to adhesion of Staphylococci strains which carry the collagen adhesin gene (cna). In this study the frequency of cna was determined within a collection of 35 Staphylococcus aureus strains from orthopaedic prosthesis infections by a PCR method. Also the collagen-binding ability and slime forming capacity was evaluated. 29% of the strains were cna-positive and also able to bind collagen in vitro. 83% of the strains were slime forming. The results indicate that in the examined bacterial population slime-positive strains predominate over the cna-positive strains, with a striking association of the two adhesion mechanisms in cna-positive strains.

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells.

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.

A polymerase chain reaction (PCR) method for the identification of collagen adhesin gene (CNA) in Staphylococcus-induced prosthesis infections.

Staphylococci are well-recognized pathogens of foreign body-associated infections. The pathogenesis of such infections involves an initial step of contact between the colonizing bacterium and the biomaterial, with subsequent colony formation. Several studies have been devoted to identify adhesion mechanisms for these bacteria. Slime in particular has been extensively investigated. Recently, considerable attention has been given to the host protein receptors that have been shown in in vitro assays to serve as substrates for bacterial adherence. To determine the importance of the collagen adhesin as virulence factor in Staphylococcus-induced prosthesis infection, a simple and reliable method using a polymerase chain reaction (PCR) was devised to identify collagen adhesin gene (cna). By using lysates of ten strains from orthopedic prostheses (5 Staphylococcus aureus and 5 Staphylococcus epidermidis) and two 20-oligonucleotides as primers, a 192-bp region of the cna gene was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were in accordance with those obtained by the in vitro phenotypic characterization of binding ability to collagen of Staphylococcus strains.

Surface plasmon resonance for real-time detection of molecular interactions between chromomycin and target DNA sequences.

DNA-binding drugs interfere with the activity of a large variety of transcription factors, leading to an alteration of transcription. This and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases. The analysis of sequence selectivity of DNA-binding drugs by footprinting, gel retardation studies, polymerase chain reaction and in vitro transcription does not allow an easy study of kinetics of binding and dissociation. The recent development of biosensor technologies for biospecific interaction analysis (BIA) enables the monitoring of a variety of molecular reactions in real-time by surface plasmon resonance (SPR). In this report we demonstrate that molecular interactions between the DNA-binding drug chromomycin and a biotinylated GC-rich Ha-ras oligonucleotide probe immobilized on a sensor chip is detectable by SPR technology using the BIAcore(TM) biosensor. This approach appears of interest in the development of drugs exhibiting differential affinity for target DNA sequences for the following reasons: a) results are obtained within one hour; b) unlike footprinting and gel retardation studies, this technology does not require P-32-labelled probes; c) BIA allows kinetic studies of both association and dissociation.