Many identification and relatedness studies methods had been commonly used for epidemiological studies in microbiological laboratories. Apart from phenotypic methods, genotypic are also often used. The aim of this study was to compare, obtained by PFGE chromosomal DNA patterns of methicillin-resistant S. epidermidis strains isolated from clinical material. 46 methicillin-resistant S. epidermidis strains were included in this study. Most of them were isolated from wound swabs (65.2%) and catheters (19.6%) from different surgical clinics (76.1%). To identify strains and receive biochemical profiles, ID 32 Staph tests and GPI cards of Vitek 1 system were used. Pulsed-field gel electrophoresis and Tenover et al. interpretation were used to compare chromosomal DNA patterns of examined strains. 44 and 42 PFGE patterns of chromosomal DNA were received, using visual interpretation classifying two pairs of strains as the same, two pairs as closely related and three pairs as probably related. Strains classified as identical and similar in visual evaluation were indistinguishable in Molecular Analyst DST interpretation, probably due to tolerance in bands location pattern. Strains probably related in visual interpretation represent at least 96% similarity in Molecular Analyst DST but different susceptibility and biochemical profiles obtained by ID 32 Staph and Vitek 1. PFGE analysis had foremost capacity to distinguish methicillin-resistant S. epidermidis strains using visual interpretation and Molecular Analyst DST (Bio-Rad) program and seems to be useful method in epidemiological studies. Strains with the same PFGE pattern, had different susceptibility and biochemical profiles.

The calcium binding L1 protein was found to inhibit growth of blood culture isolates of Candida spp and cerebrospinal fluid isolates of Cryptococcus neoformans. Minimum inhibitory concentrations (MIC) were 4-128 mg/l, and concentrations 2-4 times the MIC were fungicidal. Blood culture isolates of Escherichia coli, Klebsiella spp, Staphylococcus aureus, and Staphylococcus epidermidis had MIC values of 64-256 mg/l. Antibacterial activity was strongly influenced by the nature of the culture medium. In view of the biological activity of L1, the name calprotectin is proposed to describe this antimicrobial protein with calcium binding properties.

Previously, Staphylococcus epidermidis and other coagulase-negative staphylococci isolated from the blood of hospitalized patients were often considered contaminants. Now, coagulase-negative staphylococci are among the leading causes of nosocomial blood infections. Multidrug resistance could predict a true nosocomial infection rather than a blood culture contaminant. Recent studies indicated the emergence of resistance to the quinolones, particularly to ciprofloxacin. Tolerance and occasional resistance to vancomycin have been reported recently. In addition, several reports indicated that vancomycin and other glycopeptide antibiotics lose their effectiveness against S. epidermidis organisms embedded in the biofilm environment on the surface of medical devices. Alternative agents have been proposed in the prevention and treatment of device-related and glycopeptide-tolerant S. epidermidis infections. These agents include minocycline, rifampin, and, more recently, quinupristin/dalfopristin and the oxazolidinones.

Peritonitis is a major complication of continuous ambulatory peritoneal dialysis. Relapsing peritonitis after the cessation of antimicrobial therapy is frequently reported and often involves Staphylococcus epidermidis. To investigate the potential role of catheter-associated biofilm in the pathogenesis of relapsing peritonitis, we describe an in vitro model permitting the development of an S. epidermidis biofilm on silicone elastomer biomaterial. This model has been used to investigate the ability of vancomycin hydrochloride to kill biofilm-encased organisms by using an antibiotic regimen typical of peritonitis therapy. No significant differences were seen between vancomycin-exposed and control groups in biofilm viable and total cell counts after 10 days. Vancomycin-exposed silicone-associated biofilm populations decreased by only 0.5 log10 CFU/cm2 over the study period. MICs and MBCs for the original S. epidermidis suspension were 3.125 and 6.25 micrograms/ml, respectively. For biofilm homogenate suspensions, MICs were 3.125 micrograms/ml, but MBCs were greater than 400 micrograms/ml. These data indicate that the biofilm organisms associated with an indwelling peritoneal catheter may display a form of tolerance, thereby suggesting one possible mechanism behind relapsing peritonitis.

Clinical isolates of Staphylococcus aureus (a total of 206) and S. epidermidis (a total of 188) from various countries were tested with multiplex PCR assays to detect clinically relevant antibiotic resistance genes associated with staphylococci. The targeted genes are implicated in resistance to oxacillin (mecA), gentamicin ¿aac(6')-aph(2"), and erythromycin (ermA, ermB, ermC, and msrA). We found a nearly perfect correlation between genotypic and phenotypic analysis for most of these 394 strains, showing the following correlations: 98% for oxacillin resistance, 100% for gentamicin resistance, and 98.5% for erythromycin resistance. The discrepant results were (i) eight strains found to be positive by PCR for mecA or ermC but susceptible to the corresponding antibiotic based on disk diffusion and (ii) six strains of S. aureus found to be negative by PCR for mecA or for the four erythromycin resistance genes targeted but resistant to the corresponding antibiotic. In order to demonstrate in vitro that the eight susceptible strains harboring the resistance gene may become resistant, we subcultured the susceptible strains on media with increasing gradients of the antibiotic. We were able to select cells demonstrating a resistant phenotype for all of these eight strains carrying the resistance gene based on disk diffusion and MIC determinations. The four oxacillin-resistant strains negative for mecA were PCR positive for blaZ and had the phenotype of beta-lactamase hyperproducers, which could explain their borderline oxacillin resistance phenotype. The erythromycin resistance for the two strains found to be negative by PCR is probably associated with a novel mechanism. This study reiterates the usefulness of DNA-based assays for the detection of antibiotic resistance genes associated with staphylococcal infections.

Seven septicemias in neutropenic leukemia patients (two with fatal outcome) caused by ciprofloxacin-resistant coagulase-negative staphylococci were diagnosed in the hematologic unit of Turku University Central Hospital in 1988 soon after the introduction of the drug. Coagulase-negative staphylococcal skin flora of 28 neutropenic patients receiving ciprofloxacin prophylaxis and therapy for gram-negative bacterial infections were compared with those of 31 untreated patients and 33 hospital personnel working in the same unit. In ciprofloxacin-treated patients the flora were almost completely ciprofloxacin-resistant, whereas in the control groups resistant flora were detected only occasionally. Similarities in the plasmid profile patterns were found in 91% of the ciprofloxacin-resistant coagulase-negative staphylococci, suggesting an epidemiologic relation between these strains. It seems evident that cross-infection is responsible for the spread of ciprofloxacin-resistant coagulase-negative staphylococci in these patients.

Of 41 methicillin-resistant coagulase-negative staphylococcal clinical isolates collected during a 5-month period between late 1995 and early 1996, 28 showed tube dilution teicoplanin MICs of 4 to 8 microg/ml which increased to 16 to 32 microg/ml upon prolonged incubation. Cultures of such bacteria were heterogeneous; they contained subpopulations with frequencies of 10(-5) to 10(-4) that could grow on up to 50 microg of teicoplanin per ml. The same cultures were also heterogeneous with respect to susceptibility to vancomycin; while the MICs for the majority of cells were 2 to 4 microg/ml, subpopulations that could grow on 6 to 12 microg of vancomycin per ml were also present at frequencies of 10(-5) to 10(-7). Selective enrichment of such cultures for the resistant subpopulation occurred with relative ease under laboratory conditions. Heterogeneous phenotypes for teicoplanin (but not for vancomycin) susceptibility were also identified in several Staphylococcus epidermidis isolates collected during the preantibiotic era. The addition of half the MIC of teicoplanin inhibited autolysis and caused formation of cellular aggregates which disintegrated to individual bacteria in the stationary phase when the titer of teicoplanin in the medium fell to undetectable levels, indicating removal of the antibiotic from the culture medium by the bacteria.

Discitis after discography is due to bacterial penetration into the intervertebral disc by a contaminated needle and has an incidence of 1% to 4%. We have examined the prophylactic role of cephazolin administered at the time of discography. An experimental study in sheep using radiographic contrast containing Staphylococcus epidermidis showed that either adding the antibiotic to the intradiscal suspension or giving it intravenously 30 minutes before intradiscal inoculation of bacteria prevented any radiographic, macroscopic or histological signs of discitis; all the intervertebral disc cultures were negative. In a prospective clinical study of 127 consecutive patients having lumbar discography, the injected contrast contained cephazolin 1 mg per ml. None of the patients developed clinical or radiographic signs of discitis. We recommend the use of a suitable broad spectrum antibiotic in a single prophylactic dose whenever the intervertebral disc is entered.

Indolicidin, an antimicrobial peptide with a unique amino acid sequence (ILPWKWPWWPWRR-NH(2)) is found in bovine neutrophils. A derivative of indolicidin, CP10A, has alanine residues substituted for proline residues and has improved activity against Gram-positive organisms. Transmission electron microscopy of Staphylococcus aureus and Staphylococcus epidermidis treated with CP10A showed mesosome-like structures in the cytoplasm. The peptide at 2-fold the minimal inhibitory concentration did not show significant killing of S. aureus ISP67 (a histidine, uridine, and thymidine auxotroph) but did show an early effect on histidine and uridine incorporation and, later, an effect on thymidine incorporation. Upon interaction with liposomes, detergents, and lipoteichoic acid, CP10A was shown by circular dichroism spectroscopy to undergo a change in secondary structure. Fluorescence spectroscopy indicated that the tryptophan residues were located at the hydrophobic/hydrophilic interface of liposomes and detergent micelles and were inaccessible to the aqueous quencher KI. The three-dimensional structure of CP10A in the lipid mimetic dodecylphosphocholine was determined using two-dimensional NMR methods and was characterized as a short, amphipathic helical structure, whereas indolicidin was previously shown to have an extended structure. These studies have introduced a cationic peptide with a unique structure and an ability to interact with membranes and to affect intracellular synthesis of proteins, RNA, and DNA.

A Staphylococcus epidermidis plasmid conferring inducible resistance to 14-membered ring macrolides and type B streptogramins has been analysed and the DNA sequence of the gene responsible for resistance determined. A single open reading frame of 1.464 kbp, preceded by a complex control region containing a promoter and two ribosomal binding sites, was identified. The deduced sequence of the 488-amino-acid protein (MsrA) revealed the presence of two ATP-binding motifs homologous to those of a family of transport-related proteins from Gram-negative bacteria and eukaryotic cells, including the P-glycoprotein responsible for multidrug resistance. In MsrA, but not these other proteins, the two potential ATP-binding domains are separated by a Q-linker of exceptional length. Q-linkers comprise a class of flexible interdomain fusion junctions that are typically rich in glutamine and other hydrophilic amino acids and have a characteristic spacing of hydrophobic amino acids, as found in the MsrA sequence. Unlike the other transport-related proteins, which act in concert with one or more hydrophobic membrane proteins, MsrA appears to function independently when cloned in a heterologous host (Staphylococcus aureus RN4220). MsrA might, therefore, interact with and confer antibiotic specificity upon other transmembrane efflux complexes of staphylococcal cells. The active efflux of [14C]-erythromycin from cells of S. aureus RN4220 containing msrA has been demonstrated.

The bactericidal activities of vancomycin against two reference strains and two clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis were studied with five different concentrations ranging from 2x to 64x the MIC. The decrease in the numbers of CFU at 24 h was at least 3 log10 CFU/ml for all strains. No concentration-dependent killing was observed. The postantibiotic effect (PAE) was determined by obtaining viable counts for two of the reference strains, and the viable counts varied markedly: 1.2 h for S. aureus and 6.0 h for S. epidermidis. The determinations of the PAE, the postantibiotic sub-MIC effect (PA SME), and the sub-MIC effect (SME) for all strains were done with BioScreen C, a computerized incubator for bacteria. The PA SMEs were longer than the SMEs for all strains tested. A newly developed in vitro kinetic model was used to expose the bacteria to continuously decreasing concentrations of vancomycin. A filter prevented the loss of bacteria during the experiments. One reference strain each of S. aureus and S. epidermidis and two clinical isolates of S. aureus were exposed to an initial concentration of 10x the MIC of vancomycin with two different half-lives (t1/2s): 1 or 5 h. The post-MIC effect (PME) was calculated as the difference in time for the bacteria to grow 1 log10 CFU/ml from the numbers of CFU obtained at the time when the MIC was reached and the corresponding time for an unexposed control culture. The difference in PME between the strains was not as pronounced as that for the PAE. Furthermore, the PME was shorter when a t1/2 of 5 h (approximate terminal t1/2 in humans) was used. The PMEs at t1/2s of 1 and 5 h were 6.5 and 3.6 h, respectively, for S. aureus. The corresponding figures for S. epidermidis were 10.3 and less than 6 h. The shorter PMEs achieved with a t1/2 of 5 h and the lack of concentration-dependent killing indicate that the time above the MIC is the parameter most important for the efficacy of vancomycin.