SummaryBiofilm formation of Staphylococcus epidermidis on smooth polymer surfaces has been shown to be mediated by the ica operon. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized that supports bacterial cell-to-cell contacts and triggers the production of thick, multilayered biofilms. Thus, the ica gene cluster represents a genetic determinant that significantly contributes to the virulence of specific Staphylococcus epidermidis strains. PIA synthesis has been reported recently to undergo a phase variation process. In this study, biofilm-forming Staphylococcus epidermidis strains and their PIA-negative phase variants were analysed genetically to investigate the molecular mechanisms of phase variation. We have characterized biofilm-negative variants by Southern hybridization with ica-specific probes, polymerase chain reaction and nucleotide sequencing. The data obtained in these analyses suggested that in Ϸ30% of the variants the missing biofilm formation was due to the inactivation of either the icaA or the icaC gene by the insertion of the insertion sequence element IS256. Furthermore, it was shown that the transposition of IS256 into the ica operon is a reversible process. After repeated passages of the PIA-negative insertional mutants, the biofilm-forming phenotype could be restored. Nucleotide sequence analyses of the revertants confirmed the complete excision of IS256, including the initially duplicated 8 bp target sites. These results elucidate, for the first time, a molecular mechanism mediating phase variation in staphylcocci, and they demonstrate that a naturally occurring insertion sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor.

Biofilm production is an important step in the pathogenesis of Staphylococcus epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin. A chromosomally encoded reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from Escherichia coli was constructed and used to investigate the influence of both environmental factors and subinhibitory concentrations of different antibiotics on ica expression in S. epidermidis. It was shown that S. epidermidis biofilm formation is induced by external stress (i.e., high temperature and osmolarity). Subinhibitory concentrations of tetracycline and the semisynthetic streptogramin antibiotic quinupristin-dalfopristin were found to enhance ica expression 9-to 11-fold, whereas penicillin, oxacillin, chloramphenicol, clindamycin, gentamicin, ofloxacin, vancomycin, and teicoplanin had no effect on ica expression. A weak (i.e., 2.5-fold) induction of ica expression was observed for subinhibitory concentrations of erythromycin. The results were confirmed by Northern blot analyses of ica transcription and quantitative analyses of biofilm formation in a colorimetric assay.Staphylococcus epidermidis is a major cause of medical device-associated infections, especially in immunocompromised patients, and the treatment of these infections is complicated by the emergence of multiresistant strains (10,37,41,50). The ability of S. epidermidis to generate biofilms on smooth surfaces is believed to contribute significantly to the pathogenesis of polymer-associated infections. S. epidermidis biofilm formation depends on the production of a polysaccharide intercellular adhesin (PIA). It mediates the contact of the bacterial cells with each other, resulting in the accumulation of a multilayered biofilm (24,28). PIA is a sugar polymer consisting of a beta-1,6-linked glucosaminoglycan backbone substituted with different side groups (28, 30). The enzymes involved in PIA synthesis were found to be encoded by the ica operon comprising the icaA, icaD, icaB, and icaC genes (20,24). PIA is suggested to be an important virulence factor of S. epidermidis, and the ica operon is known to be widespread in S. epidermidis isolates causing polymer-associated infections (18, 55). Recently, it has also been detected in Staphylococcus aureus and a range of other staphylococcus species (16, 31). The expression of the ica operon and, as a result, the formation of biofilms seems to be highly variable among staphylococci (32, 55). In S. epidermidis, ica expression undergoes a phase variation process which, at least in a significant part of the variants, is caused by the alternating insertion and precise excision of an IS element (56). Apart from this phase variation mechanism that mediates the complete on or off switch of gene expression, our knowledge of factors involved in the modulation of ica expression is very limited. Since biofilm formation represents a useful target for the prevention of...

Background: Progress in contemporary medicine is associated with an increasing number of immunocompromised individuals. In this vulnerable group, the underlying disease together with long-term hospitalization and the use of medical devices facilitate infections by opportunistic pathogens, of which coagulase-negative staphylococci (CoNS) represent a prime example. Objectives: The diversity of CoNS with species-and strain-specific differences concerning virulence and clinical impact is highlighted. A focus is on the ability of CoNS to generate biofilms on biotic and abiotic surfaces, which enables skin and mucosa colonization as well as establishment of CoNS on indwelling foreign bodies. Sources: Literature about the virulence of CoNS listed in PubMed was reviewed. Content: Most catheter-related and prosthetic joint infections as well as most other device-related infections are caused by CoNS, specifically by Staphylococcus epidermidis and Staphylococcus haemolyticus. A common theme of CoNS infections is a high antibiotic resistance rate, which often limits treatment options and contributes to the significant health and economic burden imposed by CoNS. Implications: Breaching the skin barrier along with the insertion of medical devices offers CoNS opportunities to gain access to host tissues and to sustain there by forming biofilms on foreign body surfaces. Biofilms represent the perfect niche to protect CoNS from both the host immune response and the action of antibiotics. Their particular lifestyle, combined with conditions that facilitate host colonization and infection, has led to the growing impact of CoNS as pathogens. Moreover, CoNS may serve as hidden reservoirs for antibiotic resistance and virulence traits.

Osmotic stress was found to induce biofilm formation in a Staphylococcus aureus mucosal isolate. Inactivation of a global regulator of the bacterial stress response, the alternative transcription factor B , resulted in a biofilm-negative phenotype and loss of salt-induced biofilm production. Complementation of the mutant strain with an expression plasmid encoding B completely restored the wild-type phenotype. The combined data suggest a critical role of B in S. aureus biofilm regulation under environmental stress conditions.For numerous pathogenic bacteria, biofilms represent a source of persisting and relapsing infections and thus contribute significantly to pathogenesis (4). Biofilms seem to protect bacteria from unfavorable external conditions, and, in some bacterial ecosystems, the conversion of planktonic cells into a biofilm-producing community is triggered by environmental stress factors (6,12,13). In the human pathogen Staphylococcus aureus, biofilm formation is mediated by the production of the extracellular polysaccharide adhesin PIA, whose synthesis depends on the expression of the icaADBC-encoded enzymes (5, 15). The regulation of biofilm expression in this organism is poorly understood. All S. aureus strains analyzed so far contain the entire ica gene cluster, but only a few express the operon and produce biofilms in vitro (5). In this study, we investigated whether the alternative transcription factor B is involved in the regulation of ica expression.B is known to be a global regulator of the stress response in S. aureus and also influences various virulence-associated genes (7,11,16,20). To elucidate the possible role of B in biofilm formation, we used a genetic approach and constructed an S. aureus sigB::ermB insertion mutant of the biofilm-forming, methicillin-sensitive mucosal isolate S. aureus MA12 (18). Biofilm formation and ica expression of the mutant were compared with the phenotypes of the corresponding wild-type strain and a complemented strain that carried a sigB copy on an expression vector.Construction of a sigB insertion mutant and complementation of the mutation. The inactivation of sigB was done by insertion of an erythromycin resistance cassette into the sigB gene of S. aureus MA12 by double-crossover integration. For this purpose, the temperature-sensitive shuttle vector pSK8, which carries a sigB::ermB mutation, was constructed. A 937-bp fragment containing the entire sigB gene was amplified by PCR from S. aureus MA12 by using the primers 5Ј CGG GAT CCG GTG TGA CAA TCA GTA TGA C 3Ј and 5Ј CGG AAT TCG CGA CAT TTA TGT GGA TAC AC 3Ј. The DNA fragment was inserted into the shuttle vector pBT1 (17), resulting in pSK7. Then the ermB cassette of pEC1 (1) was excised by XbaI-HindIII digestion, treated with the Klenow fragment of Escherichia coli DNA polymerase, and ligated with EcoRVdigested pSK7, resulting in plasmid pSK8. Following passage through the restriction-negative strain S. aureus RN4220, pSK8 was reisolated and transformed into S. aureus MA12 by electroporation (19). Replacem...

Bacteria are a major cause of infection. To fight disease and growing resistance, research interest is focused on understanding bacterial metabolism. For a detailed evaluation of the involved mechanisms, a precise knowledge of the molecular composition of the bacteria is required. In this article, various vibrational spectroscopic techniques are applied to comprehensively characterize, on a molecular level, bacteria of the strain Staphylococcus epidermidis, an opportunistic pathogen which has evolved to become a major cause of nosocomial infections. IR absorption spectroscopy reflects the overall chemical composition of the cells, with major focus on the protein vibrations. Smaller sample volumes-down to a single cell-are sufficient to probe the overall chemical composition by means of micro-Raman spectroscopy. The nucleic-acid and aromatic amino-acid moieties are almost exclusively explored by UV resonance Raman spectroscopy. In combination with statistical evaluation methods [hierarchical cluster analysis (HCA), principal component analysis (PCA), linear discriminant analysis (LDA)], the protein and nucleic-acid components that change during the different bacterial growth phases can be identified from the in vivo vibrational spectra. Furthermore, tip-enhanced Raman spectroscopy (TERS) provides insight into the surface structures and follows the dynamics of the polysaccharide and peptide components on the bacterial cells with a spatial resolution below the diffraction limit. This might open new ways for the elucidation of host-bacteria and drug-bacteria interactions.