Search

Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.

EDIN-B (epidermal cell differentiation inhibitor-B; also termed C3Stau) is an exotoxin of Staphylococcus aureus which ADP-ribosylates and inactivates Rho GTP binding proteins. The EDIN-B gene (edin-B) and the gene for exfoliative toxin D (etd) make up the central part of a recently described pathogenicity island. Here we evaluated the prevalence and genetic organization of the edin-B/etd pathogenicity island in invasive S. aureus isolates, and characterized edin-B transcription and EDIN-B production using artificial constructs transduced in S. aureus strains RN6390 and Newman. We found that eight out of 121 (7 %) S. aureus blood culture isolates harbour edin-B, which is organized in three novel variants of the original edin-B/etd pathogenicity island. In the serum of patients infected with edin-B-positive S. aureus, significant titres of anti-EDIN-B antibodies could be detected. Regulation of edin-B transcription depended on the sarA but not on the agr regulatory system. Furthermore, retrieval of EDIN-B protein secreted by S. aureus RN6390 required the presence of alpha2-macroglobulin to inhibit the activity of extracellular proteases. These data suggest that the EDIN-B toxin is produced during human infection, is part of a highly variable pathogenicity island and can be controlled by the sarA gene regulon and secreted bacterial proteases.

Virulence of nosocomial pathogen Staphylococcus epidermidis is essentially related to formation of adherent biofilms, assembled by bacterial attachment to an artificial surface and subsequent production of a matrix that mediates interbacterial adhesion. Growing evidence supports the idea that proteins are functionally involved in S. epidermidis biofilm accumulation. We found that in S. epidermidis 1585v overexpression of a 460 kDa truncated isoform of the extracellular matrix-binding protein (Embp) is necessary for biofilm formation. Embp is a giant fibronectin-binding protein harbouring 59 Found In Various Architectures (FIVAR) and 38 protein G-related albumin-binding (GA) domains. Studies using defined Embp-positive and -negative S. epidermidis strains proved that Embp is sufficient and necessary for biofilm formation. Further data showed that the FIVAR domains of Embp mediate binding of S. epidermidis to solid-phase attached fibronectin, constituting the first step of biofilm formation on conditioned surfaces. The binding site in fibronectin was assigned to the fibronectin domain type III12. Embp-mediated biofilm formation also protected S. epidermidis from phagocytosis by macrophages. Thus, Embp is a multifunctional cell surface protein that mediates attachment to host extracellular matrix, biofilm accumulation and escape from phagocytosis, and therefore is well suited for promoting implant-associated infections.

Staphylococcus epidermidis is of major importance in infections associated with indwelling medical devices. The tight pathogenic association is essentially linked to the species ability to form adherent biofilms on artificial surfaces. Aiming at identifying novel targets for vaccination or therapy much effort has been made to unravel the molecular mechanisms leading to S. epidermidis biofilm formation. At present, polysaccharide intercellular adhesin (PIA) is the best studied factor involved in S. epidermidis biofilm accumulation. PIA is a glycan of beta-1,6-linked 2-acetamido-2-deoxy-D-glucopyranosyl residues of which 15 % are non-N-acetylated. PIA-producing S. epidermidis are widespread in clinical strain collections and PIA synthesis has been shown to be essential for S. epidermidis virulence. Moreover, PIA homologues have been identified in many other staphylococcal species, including the major human pathogen Staphylococcus aureus, and also Gram-negative human pathogens, suggesting that it might represent a more general pathogenicity principle in biofilm-related infections. In this review the current knowledge about the structure and biosynthesis of PIA is summarized. Additionally, information on its role in pathogenesis of biomaterial-related and other type of infections and the potential use of PIA and related compounds for prevention of infection is discussed., (Copyright (c) 2009 Elsevier GmbH. All rights reserved.)

Biofilm formation is a major pathogenetic factor of Staphylococcus epidermidis. In S. epidermidis the alternative sigma factor sigma B was identified to regulate biofilm formation in S. epidermidis 1457. In S. aureus sigma B dependent regulation plays a minor role, whereas sarA (Staphylococcus accessory regulator) is an essential regulator. Therefore, we investigated the impact of sigma B on sarA transcription and biofilm formation in three independent S. epidermidis isolates. Mutants with dysfunctional sigma B displayed a strongly reduced biofilm formation, whereas in mutants with constitutive sigma B activity biofilm formation was increased. Transcriptional analysis revealed that icaA transcription was down-regulated in all sigma B negative mutants while icaR transcription was up-regulated. However, transcriptional differences varied between individual strains, indicating that additional sigma B-dependent regulators are involved in biofilm expression. Interestingly, despite the presence of a sigma B promoter beside two sigma A promoters no differences, or only minor ones, were observed in sarA transcription, indicating that sigma B-dependent sarA transcript has no influence on the phenotypic changes. The data observed in independent clinical S. epidermidis isolates suggests that, in contrast to S. aureus, regulation of biofilm formation by sigma B is a general feature in S. epidermidis. Additionally, we were able to demonstrate that the sarA- dependent regulation is not involved in this regulatory pathway.

The skin commensal and opportunistic pathogen Staphylococcus epidermidis is an important cause of nosocomial infections. Virulence is attributable to formation of biofilm, which provides a microenvironment that protects the bacterium from attack by the host immune system and by chemotherapy. In this study we extended to S. epidermidis strategies previously aimed at treatment of S. aureus biofilms using photodynamic treatment (PDT) combined with chemotherapy or phagocytosis. A significant reduction in bacterial survival was observed when structurally distinct biofilms were exposed to the cationic porphyrin, tetra-substituted N-methyl-pyridyl-porphine (TMP), and simultaneously to visible light. Of note, the extent of biofilm clearance depended on its maturation stage: developing, young biofilms, were more sensitive towards PDT than mature biofilms. Furthermore, PDT-treated biofilms exposed to vancomycin or subjected to phagocytic action of whole blood were almost completely eradicated. The data we obtained establish that PDT combined with antibiotics or host defenses may also be a useful approach for the inactivation of S. epidermidis biofilms.

Chronic infections are commonly associated with biofilms formed by bacteria such as Staphylococcus epidermidis. With the increase in antibiotic resistant bacteria, maggot debridement therapy has been reintroduced for the treatment of chronic wounds. Studies have shown that the excretion/ secretions (ES) of Lucilia sericata larvae (maggots) contain many bioactive compounds which may contribute to the efficacy of maggot therapy. The present study evaluates the effect of L. sericata ES on the formation and disruption of S. epidermidis 1457 and 5179-R1 biofilms. These strains employ either polysaccharide intercellular adhesin (PIA) or accumulation associated protein (Aap) for intercellular adhesion. A semiquantitative biofilm assay was used to measure the formation/disruption of S. epidermidis 1457 and 5179-R1 biofilms by ES. ES activity was characterized according to concentration, incubation time and temperature, thermal stability, and size. Immunofluorescence microscopy was used to ascertain the effect of ES on PIA and Aap. In the presence of ES, S. epidermidis 1457 and 5179-R1 nascent biofilm formation was inhibited, and pre-formed biofilms disrupted. ES activity was temperature and time dependent, inactivated by heat treatment, and disruption depended on the mechanism of intercellular adhesion. The molecule(s) responsible was >10 kDa in size and appeared to have protease or glucosaminidase activity. ES interferes with S. epidermidis biofilm formation, specifically degrading factors employed in biofilm accumulation, which would increase bacterial susceptibility to antibiotics and the host's immune system. In purified form, ES-factors may have general applicability for the treatment or prevention of chronic biofilm infections caused by staphylococci.

Infections due to coagulase-negative staphylococci (CoNS) most frequently occur after the implantation of medical devices and are attributed to the biofilm-forming potential of CoNS. Staphylococcus haemolyticus is the second most frequently isolated CoNS from patients with hospital-acquired infections. There is only limited knowledge of the nature of S. haemolyticus biofilms. The aim of this study was to characterize S. haemolyticus biofilm formation. We analyzed the biofilm-forming capacities of 72 clinical S. haemolyticus isolates. A detachment assay with NaIO(4), proteinase K, or DNase was used to determine the main biofilm components. Biofilm-associated genes, including the ica operon, were analyzed by PCR, and the gene products were sequenced. Confocal laser scanning microscopy (CLSM) was used to elucidate the biofilm structure. Fifty-three isolates (74%) produced biofilms after growth in Trypticase soy broth (TSB) with glucose, but only 22 (31%) produced biofilms after growth in TSB with NaCl. It was necessary to dissolve the biofilm in ethanol-acetone to measure the optical density of the full biofilm mass. DNase, proteinase K, and NaIO(4) caused biofilm detachment for 100%, 98%, and 38% of the isolates, respectively. icaRADBC and polysaccharide intercellular adhesin (PIA) production were found in only two isolates. CLSM indicated that the biofilm structure of S. haemolyticus clearly differs from that of S. epidermidis. We conclude that biofilm formation is a common phenotype in clinical S. haemolyticus isolates. In contrast to S. epidermidis, proteins and extracellular DNA are of functional relevance for biofilm accumulation, whereas PIA plays only a minor role. The induction of biofilm formation and determination of the biofilm mass also needed to be optimized for S. haemolyticus.

Phosphoinositides (PPIn) are important regulators of cellular processes like intracellular protein transport, cellular proliferation, apoptosis, and cytoskeletal organization. The amount and localization of these membrane-bound second messengers are regulated through a set of specific phospholipases, lipid kinases, and phosphatases. The elucidation of PPIn-phosphatases and their cellular function has gained much attention because phosphatase dysregulation is often associated with human genetic diseases. Our laboratory has identified the 3'-PPIn-phosphatase myotubularin 1 (MTM1) mutated in X-linked myotubular myopathy (XLMTM). In addition, a whole family of myotubularin-related proteins (MTMR1-MTMR13) has been discovered. Some of them display phosphatase activity, whereas for other family members no enzymatic activity could be detected. Nevertheless, these "dead phosphatases" myotubularins are conserved throughout evolution and probably exert regulatory function by heteromeric interaction with active phosphatase members. It was shown that MTM1 and related phosphatases act on PtdIns3P and PtdIns(3,5)P2; both PPIn species are important regulators of endocytic pathways. We describe two methods to determine phosphatase activity and substrate specificity of myotubularins. One is an immunoprecipitation-phosphatase assay, testing the activity of myotubularin immunoprecipitated from overexpressing cells on artificial PPIn. The other method analyzes phosphatase activity indirectly ex vivo in transiently transfected mammalian cells. The presence and subcellular localization of the myotubularin substrate PtdIns3P were determined using a specific binding domain (2xFYVE) produced recombinantly as a biosensor.

Medical device-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. The formation of adherent, multilayered bacterial biofilms is crucial in the pathogenesis of these infections. Polysaccharide intercellular adhesin (PIA), a homoglycan of β-1,6-linked 2-acetamido-2-deoxy-D: -glucopyranosyl residues, of which about 15% are non-N-acetylated, is central to biofilm accumulation in staphylococci. It transpires that polysaccharides - structurally very similar to PIA - are also key to biofilm formation in a number of other organisms including the important human pathogens Escherichia coli, Aggregatibacter (Actinobacillus) actinomycetemcomitans, Yersinia pestis, and Bordetella spp. Apparently, synthesis of PIA and related polysaccharides is a general feature important for biofilm formation in diverse bacterial genera. Current knowledge about the structure and biosynthesis of PIA and related polysaccharides is reviewed. Additionally, information on their role in pathogenesis of biomaterial-related and other type of infections and the potential use of PIA and related compounds for prevention of infection is evaluated.

Tight Junction (TJ) proteins have been shown to exert a barrier function within the skin. Here, we study the fate of TJ proteins during the challenge of the skin by bacterial colonization and infection. We investigated the influence of various exfoliative toxin-negative Staphylococcus strains on TJ, adherens junction (AJ), desmosomal proteins, and actin in a human keratinocyte infection culture and in a porcine skin infection model. We found that the pathogen Staphylococcus aureus downregulates TJ and subsequently AJ and desmosomal proteins, including atypical protein kinase C, an essential player in TJ formation, at the cell-cell borders of keratinocytes in a time and concentration dependent manner. Little changes in protein and RNA levels were seen, indicating redistribution of proteins. In cultured keratinocytes, a reduction of transepithelial resistance was observed. Staphylococcus epidermidis shows only minor effects. All strains induced enhanced expression of occludin and ZO-1 at the beginning of colonization/infection. Thus, we demonstrate that TJ are likely to be involved in skin infection of exfoliative toxin-negative S. aureus. As we did not find a change in actin, and as changes of TJ preceded alterations of AJs and desmosomes, we suggest that S. aureus targets TJ.

When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I-mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor-induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery.

While coagulase-negative staphylococci (CoNS), with their ability to form a thick, multilayered biofilm on foreign bodies, have been identified as the major cause of implant-associated infections, no data are available about biofilm formation by staphylococcal small-colony variants (SCVs). In the past years, a number of device-associated infections due to staphylococcal SCVs were described, among them, several pacemaker infections due to SCVs of CoNS auxotrophic to hemin. To test the characteristics of SCVs of CoNS, in particular, to study the ability of SCVs to form a biofilm on foreign bodies, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in Staphylococcus epidermidis. In fact, this mutant displayed a stable SCV phenotype with tiny colonies showing strong adhesion to the agar surface. When the incubation time was extended to 48 h or a higher inoculum concentration was used, the mutant produced biofilm amounts on polystyrene similar to those produced by the parent strain. When grown under planktonic conditions, the mutant formed markedly larger cell clusters than the parental strain which were completely disintegrated by the specific beta-1,6-hexosaminidase dispersin B but were resistant to trypsin treatment. In a dot blot assay, the mutant expressed larger amounts of polysaccharide intercellular adhesin (PIA) than the parent strain. In conclusion, interrupting a hemin biosynthetic gene in S. epidermidis resulted in an SCV phenotype. Markedly larger cell clusters and the ability of the hemB mutant to form a biofilm are related to the augmented expression of PIA.

Staphylococcus epidermidis and Staphylococcus aureus are leading causes of hospital-acquired infections that have become increasingly difficult to treat due to the prevalence of antibiotic resistance in these organisms. The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere to living and artificial surfaces and to resist host immune factors and antibiotics. Here, we show that the icaADBC locus, which synthesizes the biofilm-associated polysaccharide intercellular adhesin (PIA) in staphylococci, is required for the formation of a lethal S. epidermidis infection in the intestine of the model nematode Caenorhabditis elegans. Susceptibility to S. epidermidis infection is influenced by mutation of the C. elegans PMK-1 p38 mitogen-activated protein (MAP) kinase or DAF-2 insulin-signaling pathways. Loss of PIA production abrogates nematocidal activity and leads to reduced bacterial accumulation in the C. elegans intestine, while overexpression of the icaADBC locus in S. aureus augments virulence towards nematodes. PIA-producing S. epidermidis has a significant survival advantage over ica-deficient S. epidermidis within the intestinal tract of wild-type C. elegans, but not in immunocompromised nematodes harboring a loss-of-function mutation in the p38 MAP kinase pathway gene sek-1. Moreover, sek-1 and pmk-1 mutants are equally sensitive to wild-type and icaADBC-deficient S. epidermidis. These results suggest that biofilm exopolysaccharide enhances virulence by playing an immunoprotective role during colonization of the C. elegans intestine. These studies demonstrate that C. elegans can serve as a simple animal model for studying host-pathogen interactions involving staphylococcal biofilm exopolysaccharide and suggest that the protective activity of biofilm matrix represents an ancient conserved function for resisting predation.

Staphylococcus epidermidis is both a human skin commensal and an opportunistic pathogen, causing infections linked to implanted medical devices. This paper describes localized tufts of fibrillar appendages on a subpopulation (25%) of wild-type (WT) S. epidermidis NCTC 11047 cells. The fibrils (122.2 +/- 10.8 nm long) are usually in a lateral position on the cells. Fibrillar (Fib(+)) and nonfibrillar (Fib(-)) subpopulations were separated (enriched) by 34 sequential partitions of WT cells between a buffer phase and a hexadecane phase. Following enrichment, hydrophobic cells from the hexadecane phase comprised 70% Fib(+) cells and the less hydrophobic cells from the buffer phase entirely comprised Fib(-) cells. The Fib(+) and Fib(-) subpopulations did not revert on subculture (34 times) on solid medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell surface proteins from WT, Fib(+), and Fib(-) cells revealed two high-molecular-mass proteins (280 kDa and 230 kDa) on the WT and Fib(+) cells that were absent from the Fib(-) cells. Amino acid sequencing revealed that fragments of both the 280- and 230-kDa proteins had 100% identity to the accumulation-associated protein (Aap). Aap is known to cause biofilm formation if it is truncated by loss of the terminal A domain. Immunogold staining with anti-Aap antibodies labeled tuft fibrils of the WT and Fib(+) cells but not the cell surface of Fib(-) cells. The tufts were labeled with N-terminally directed antibodies (anti-A domain), showing that the fibrillar Aap was not truncated on the cell surface. Thus, the presence of full-length Aap correlated with the low biofilm-forming abilities of both WT and Fib(+) S. epidermidis NCTC 11047 populations. Reverse transcription-PCR showed that aap was transcribed in both Fib(+) and Fib(-) cells. We therefore propose that full-length Aap is expressed on cells of S. epidermidis NCTC 11047 as tufts of short fibrils and that fibril expression is regulated at a posttranscriptional level.

Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. As the use of biofilm-associated factors as vaccines is critically restricted by their prevalence in natural staphylococcal populations we studied the distribution of genes involved in biofilm formation, the biofilm phenotype and production of polysaccharide intercellular adhesin (PIA) in clonally independent Staphylococcus aureus and Staphylococcus epidermidis strains isolated from prosthetic joint infections after total hip or total knee arthroplasty. Biofilm formation was detected in all S. aureus and 69.2% of S. epidermidis strains. Importantly, 27% of biofilm-positive S. epidermidis produced PIA-independent biofilms, in part mediated by the accumulation associated protein (Aap). Protein-dependent biofilms were exclusively found in S. epidermidis strains from total hip arthroplasty (THA). In S. aureus PIA and proteins act cooperatively in biofilm formation regardless of the infection site. PIA and protein factors like Aap are of differential importance for the pathogenesis of S. epidermidis in prosthetic joint infections (PJI) after THA and total knee arthroplasty (TKA), implicating that icaADBC cannot serve as a general virulence marker in this species. In S. aureus biofilm formation proteins are of overall importance and future work should focus on the identification of functionally active molecules.

Medical device-associated infections, most frequently caused by coagulase-negative staphylococci, especially Staphylococcus epidermidis, are of increasing importance in modern medicine. The formation of adherent, multilayered bacterial biofilms is the most important factor in the pathogenesis of these infections, which regularly fail to respond to appropriate antimicrobial therapy. Progress in elucidating the factors functional in elaboration of S. epidermidis biofilms and the regulation of their expression with a special emphasis on the role of quorum sensing are reviewed. Significant progress has been made in recent years, which provides the rationale for developing better preventive, therapeutic and diagnostic measures.

In eukaryotic cells, phosphoinositides are lipid second messengers important for many cellular processes and have been found dysregulated in several human diseases. X-linked myotubular (centronuclear) myopathy is a severe congenital myopathy caused by mutations in a phosphatidylinositol 3-phosphate (PtdIns3P) phosphatase called myotubularin, and mutations in dominant centronuclear myopathy (CNM) cases were identified in the dynamin 2 gene. The genes mutated in autosomal recessive cases of CNMs have not been found. We have identified a novel phosphoinositide phosphatase (hJUMPY) conserved through evolution, which dephosphorylates the same substrates as myotubularin, PtdIns3P and PtdIns(3,5)P(2), in vitro and ex vivo. We found, in sporadic cases of CNMs, two missense variants that affect the enzymatic function. One of these appeared de novo in a patient also carrying a de novo mutation in the dynamin 2 gene. The other missense (R336Q) found in another patient changes the catalytic arginine residue of the core phosphatase signature present in protein tyrosine/dual-specificity phosphatases and in phosphoinositide phosphatases and drastically reduces the enzymatic activity both in vitro and in transfected cells. The inheritance of the phenotype with regard to this variant is still unclear and could be either recessive with an undetected second allele or digenic. We propose that impairment of hJUMPY function is implicated in some cases of autosomal CNM and that hJUMPY cooperates with myotubularin to regulate the level of phosphoinositides in skeletal muscle.

To evaluate the role of the polysaccharide intercellular adhesin as an energy-storage molecule, we investigated the effect of nutrient limitation on S. epidermidis biofilms. The stability of established biofilms depends on sigma(B) activity; however, the slow decay of biofilms under conditions of nutrient limitation reveal its use as an energy-storage molecule to be unlikely.

Because of its biofilm forming potential Staphylococcus epidermidis has evolved as a leading cause of device-related infections. The polysaccharide intercellular adhesin (PIA) is significantly involved in biofilm accumulation. However, infections because of PIA-negative strains are not uncommon, suggesting the existence of PIA-independent biofilm accumulation mechanisms. Here we found that biofilm formation in the clinically significant S. epidermidis 5179 depended on the expression of a truncated 140 kDa isoform of the 220 kDa accumulation-associated protein Aap. As expression of the truncated Aap isoform leads to biofilm formation in aap-negative S. epidermidis 1585, this domain mediates intercellular adhesion in a polysaccharide-independent manner. In contrast, expression of full-length Aap did not lead to a biofilm-positive phenotype. Obviously, to gain adhesive function, full-length Aap has to be proteolytically processed through staphylococcal proteases as demonstrated by inhibition of biofilm formation by alpha(2)-macroglobulin. Importantly, also exogenously added granulocyte proteases activated Aap, thereby inducing biofilm formation in S. epidermidis 5179 and four additional, independent clinical S. epidermidis strains. It is therefore reasonable to assume that in vivo effector mechanisms of the innate immunity can directly induce protein-dependent S. epidermidis cell aggregation and biofilm formation, thereby enabling the pathogen to evade clearance by phagocytes.

Biofilms are composed of bacterial cells embedded in an extracellular polysaccharide matrix. A major component of the Escherichia coli biofilm matrix is PGA, a linear polymer of N-acetyl-D-glucosamine residues in beta(1,6) linkage. PGA mediates intercellular adhesion and attachment of cells to abiotic surfaces. In this report, we present genetic and biochemical evidence that PGA is also a major matrix component of biofilms produced by the human periodontopathogen Actinobacillus actinomycetemcomitans and the porcine respiratory pathogen Actinobacillus pleuropneumoniae. We also show that PGA is a substrate for dispersin B, a biofilm-releasing glycosyl hydrolase produced by A. actinomycetemcomitans, and that an orthologous dispersin B enzyme is produced by A. pleuropneumoniae. We further show that A. actinomycetemcomitans PGA cross-reacts with antiserum raised against polysaccharide intercellular adhesin, a staphylococcal biofilm matrix polysaccharide that is genetically and structurally related to PGA. Our findings confirm that PGA functions as a biofilm matrix polysaccharide in phylogenetically diverse bacterial species and suggest that PGA may play a role in intercellular adhesion and cellular detachment and dispersal in A. actinomycetemcomitans and A. pleuropneumoniae biofilms.

Because of their biofilm-forming capacity, invasive Staphylococcus epidermidis isolates, which cause the majority of nosocomial catheter-related bloodstream infections (BSIs), are thought to be selected at the time of catheter insertion from a population of less virulent commensal strains. This fact allows the prediction that invasive and contaminating strains can be differentiated via detection of virulence-associated genes. However, the hospital environment may pave the way for catheter-related infections by promoting a shift in the commensal bacterial population toward strains with enhanced virulence. The distribution of virulence-associated genes (icaADBC, aap, atlE, bhp, fbe, embp, mecA, IS256, and IS257), polysaccharide intercellular adhesin synthesis, and biofilm formation were investigated in S. epidermidis strains from independent episodes of catheter-related BSIs in individuals who have received bone marrow transplantation (BMT). The results were compared with those obtained for commensal S. epidermidis isolates from hospitalized patients after BMT and from healthy individuals, respectively. The clonal relationships of the strains were investigated by pulsed-field gel electrophoresis. icaADBC, mecA, and IS256 were significantly more prevalent in BSI isolates than in commensal isolates from healthy individuals. However, the prevalence of any of the genes in clonally independent, endogenous commensal strains from BMT patients did not differ from that in invasive BSI strains. icaADBC and methicillin resistance, factors important for the establishment of catheter-related infections, already ensure survival of the organisms in their physiological habitat in the hospital environment, resulting in a higher probability of contamination of indwelling medical devices with virulent S. epidermidis strains. The dynamics of S. epidermidis populations reveal that detection of icaADBC and mecA is not suitable for discriminating invasive from contaminating S. epidermidis strains.

The new BD PHOENIX automated microbiology system (Becton Dickinson Diagnostic Systems, Sparks, Md.) is designed for automated rapid antimicrobial susceptibility testing and identification of clinically relevant bacteria. In our study, the accuracy and speed of the BD PHOENIX oxacillin MIC determination for detecting methicillin resistance was evaluated for 200 clinical isolates of coagulase-negative staphylococci (CoNS). Compared to mecA PCR, the BD PHOENIX system detected methicillin resistance with a sensitivity of 99.2%. According to the actual NCCLS oxacillin MIC breakpoint of > or =0.5 microg/ml, the specificity was only 64.9%, attributable to false-positive results in 26 mecA-negative strains, including 16 non-Staphylococcus epidermidis strains. Alternative oxacillin breakpoints of > or =1, > or =2, and > or =4 microg/ml resulted in increased specificities of 83.8, 94.6, and 100% and high sensitivities of 99.2, 99.2, and 96.7%, respectively. Similarly, NCCLS broth microdilution oxacillin MICs exhibited a sensitivity of 100% but a low degree of specificity. However, the previous oxacillin MIC breakpoint of > or =4 microg/ml performed with a sensitivity of 98.4% and a specificity of 98.7%. BD PHOENIX oxacillin MIC results were available after 9 h for 40.5% of the examined CoNS strains and were completed after 17 h. Our results revealed the high reliability of the BD PHOENIX system as a phenotypic method for detection of resistance to oxacillin in mecA-positive CoNS. However, for the improvement of specificity, reevaluation of the optimal oxacillin MIC breakpoint for CoNS appears to be necessary.

Biomaterial-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. Regularly, antimicrobial therapy fails without removal of the implanted device. The most important factor in the pathogenesis of biomaterial-associated staphylococcal infections is the formation of adherent, multilayered bacterial biofilms. In this review, recent insights regarding factors functional in biofilm formation of S. epidermidis, their role in pathogenesis, and regulation of their expression are presented. Similarly, in S. aureus the biofilm mode of growth affects gene expression and the overall metabolic status. Experimental approaches for analysis of differential expression of genes involved in these adaptive responses and evolving patterns of gene expression are discussed.

Transposon mutagenesis of rsbU leads to a biofilm-negative phenotype in Staphylococcus epidermidis. However, the pathway of this regulatory mechanism was unknown. To investigate the role of RsbU in the regulation of the alternative sigma factor sigma(B) and biofilm formation, we generated different mutants of the sigma(B) operon in S. epidermidis strains 1457 and 8400. The genes rsbU, rsbV, rsbW, and sigB, as well as the regulatory cascade rsbUVW and the entire sigma(B) operon, were deleted. Transcriptional analysis of sarA and the sigma(B)-dependent gene asp23 revealed the functions of RsbU and RsbV as positive regulators and of RsbW as a negative regulator of sigma(B) activity, indicating regulation of sigma(B) activity similar to that characterized for Bacillus subtilis and Staphylococcus aureus. Phenotypic characterization of the mutants revealed that the dramatic decrease of biofilm formation in rsbU mutants is mediated via sigma(B), indicating a crucial role for sigma(B) in S. epidermidis pathogenesis. However, biofilm formation in mutants defective in sigma(B) or its function could be restored in the presence of subinhibitory ethanol concentrations. Transcriptional analysis revealed that icaR is up-regulated in mutants lacking sigma(B) function but that icaA transcription is down-regulated in these mutants, indicating a sigma(B)-dependent regulatory intermediate negatively regulating IcaR. Supplementation of growth media with ethanol decreased icaR transcription, leading to increased icaA transcription and a biofilm-positive phenotype, indicating that the ethanol-dependent induction of biofilm formation is mediated by IcaR. This icaR-dependent regulation under ethanol induction is mediated in a sigma(B)-independent manner, suggesting at least one additional regulatory intermediate in the biofilm formation of S. epidermidis.

Based on molecular typing methods, we identified a rare case of a recurrent L. monocytogenes infection resulting from an infected aortic prosthesis as detected by 18-F-Fluoro-d-deoxyglucose positron emission tomography (FDG PET). Our case highlights the usefulness of molecular typing and nuclear imaging methods for understanding L. monocytogenes pathogenesis and epidemiology.

The Saccharomyces cerevisiae SAC1 gene encodes an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus. Yeast SAC1 mutants display a wide array of phenotypes including inositol auxotrophy, cold sensitivity, secretory defects, disturbed ATP transport into the ER, or suppression of actin gene mutations. At present, it is not clear how these phenotypes relate to the finding that SAC1 displays polyphosphoinositide phosphatase activity. Moreover, it is still an open question whether SAC1 functions similarly in mammalian cells, since some phenotypes are yeast-specific. Potential protein interaction partners and, connected to that, possible regulatory circuits have not been described. Therefore, we have cloned human SAC1 (hSAC1), show that it behaves similar to ySac1p in terms of substrate specificity, demonstrate that the endogenous protein localizes to the ER and Golgi, and identify for the first time members of the coatomer I (COPI) complex as interaction partners of hSAC1. Mutation of a putative COPI interaction motif (KXKXX) at its C terminus abolishes interaction with COPI and causes accumulation of hSAC1 in the Golgi. In addition, we generated a catalytically inactive mutant, demonstrate that its lipid binding capacity is unaltered, and show that it accumulates in the Golgi, incapable of interacting with the COPI complex despite the presence of the KXKXX motif. These results open the possibility that the enzymatic function of hSAC1 provides a switch for accessibility of the COPI interaction motif.

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.

Biofilm formation in Staphylococcus epidermidis depends, in the majority of the strains, on the activity of the icaADBC locus. The expression of the operon that encodes the synthetic enzymes of the intercellular polysaccharide adhesin (PIA) depends on a variety of exogenic environmental conditions and is, at least in part, regulated by the alternative sigma factor sigma(B). We investigated the transcriptional regulation of the ica operon and the respective phenotypes expressed under growth conditions differing in the content of glucose in the growth medium. In the presence of glucose, S. epidermidis exhibited a PIA- and biofilm-positive phenotype whereas ica transcription was down-regulated in the postexponential and stationary phases of growth. Surprisingly, maximum transcription of ica was detectable in the stationary phase of growth in the absence of glucose despite the expression of a PIA- and biofilm-negative phenotype. In vitro enzymatic assays and phenotypic characterization showed that the abundant amount of ica mRNA was functionally active because induction of stationary-phase cells with glucose led to immediate PIA synthesis. Induction of biofilm formation could be completely inhibited by chloramphenicol, which, given at a later stage of biofilm accumulation, also inhibited further development of preformed biofilm, indicating that continuous translation of an additional, icaADBC-independent factor is required for the expression of a biofilm-positive phenotype.

Due to its ability to attach to polymeric surfaces Staphylococcus epidermidis is a common pathogen in chronic, medical device-associated infections. Attached S. epidermidis displays reduced susceptibility against a variety of antimicrobial substances, and little correlation between standard susceptibility test results and clinical outcome of antibiotic treatment is observed. In this study we established a new, versatile, and easy method of antimicrobial susceptibility testing for attached Staphylococcus epidermidis, suitable for both biofilm-negative and biofilm-positive attached bacteria using readily available equipment. For three biofilm-positive wild-type strains and their biofilm-negative mutants minimal attachment killing concentrations (MAK) of penicillin, oxacillin, vancomycin, and gentamicin were determined. Depending on strain and investigated antibiotics, a heterogeneous MAK (MAK(hetero)) could be differentiated from a homogeneous resistance (MAK(homo)), favoring a model of few persisters within attached cells under antibiotic treatment. For the biofilm-negative mutants, a lower MAK(homo) was detected than for the corresponding wild types for some of the tested antibiotics, which probably resulted from higher bacterial inocula of wild-type strains, whereas the MAK(hetero) were comparable for mutants and wild types for most of the tested antibiotics and strains. These data indicate that biofilm formation is not a necessary prerequisite for persistence of attached S. epidermidis cells under antibiotic treatment, which could explain therapeutic failure in foreign body-associated infections due to biofilm-negative S. epidermidis isolates. The highly individual resistance phenotypes of the investigated strains with different antibiotics suggests that MAK determination could help to predict the therapeutic outcome of foreign body-associated infections with both biofilm-positive and biofilm-negative S. epidermidis.

The icaADBC gene locus of Staphylococcus aureus and its polysaccharide intercellular adhesin (PIA/PNSG) were recently identified, but biofilm formation has rarely been detected in vitro. In this study we evaluated a tissue culture plate (TCP) assay and a tube test, as well as Congo red agar, using the two basic media trypticase soy broth (TSB) and brain heart infusion (BHI) broth with different sugar supplements for detection of biofilm formation in 128 ica-positive S. aureus isolates. Of the S. aureus strains, 57.1% displayed a biofilm-positive phenotype under optimized conditions in the TCP test. The tube test correlated well with the TCP test for strongly biofilm-producing strains, whereas weak producers were not safely discriminated from biofilm-negative strains. Screening on Congo red agar displayed a strong correlation with the TCP and the tube test for only 3.8%, and is therefore not recommended for investigation of biofilm formation in S. aureus.

The aim of the present study was to evaluate the accuracy of the new VITEK 2 system (bioMérieux, Marcy l' Etoile, France) for the detection of methicillin resistance in coagulase-negative staphylococci (CoNS) by using AST-P515 and AST-P523 test cards. Analyses of the VITEK 2 oxacillin MIC determination evaluated according to the actual breakpoint (>/=0.5 micro g/ml) of the National Committee for Clinical Laboratory Standards resulted in a high sensitivity of 99.2% but a moderate specificity of 80%. The newly included oxacillin resistance (OR) test of the VITEK 2 system displayed a high sensitivity and a high specificity of 97.5 and 98.7%, respectively. Concordance between the results of the mecA PCR and the VITEK 2 oxacillin MIC was observed for almost all Staphylococcus epidermidis strains, but the reduced specificity was attributable to higher oxacillin MICs for mecA-negative non-S. epidermidis strains, especially S. saprophyticus, S. lugdunensis, and S. cohnii. Evaluation of alternative oxacillin MIC breakpoints of 1, 2, or 4 micro g/ml resulted in improved degrees of specificity of 84, 90.7, and 97.3%, respectively. Only minor changes occurred in the corresponding sensitivity values, which were 98.4, 97.5, and 97.5%, respectively. Methicillin resistance in CoNS was detected after 7 and 8 h in 91.1 and 93.5% of the mecA-positive strains, respectively, by the VITEK 2 OR test and in 86.3 and 89.5% of the mecA-positive strains, respectively, by VITEK 2 oxacillin MIC determination. After 7 and 8 h the VITEK 2 OR test classified 59.2 and 78.9% of the mecA-negative strains, respectively, as susceptible to oxacillin, whereas comparable values were obtained 2 h later by VITEK 2 oxacillin MIC determination. The results of our study encourage the use of the VITEK 2 system, which proved to be a highly reliable and rapid phenotypic method for the detection of methicillin resistance in CoNS.

The pathogenesis of Staphylococcus epidermidis is correlated with biofilm formation. We investigated the effect of three common alcoholic skin disinfectants, ethanol, n-propanol and isopropanol, on the biofilm formation of 37 clinical, icaADBC-positive S. epidermidis isolates. In alcohol-supplemented media 18 strains displayed increased biofilm expression. Sixteen of 19 strains were generally incapable of biofilm formation. In three representative isolates, the increase in biofilm formation was paralleled by increased polysaccharide intercellular adhesin synthesis. Regarding the widespread use of alcoholic skin disinfectants, it is possible that the alcohol-inducible biofilm phenotype of S. epidermidis could add to the development of foreign body-related infections.

Biofilm formation mediated by polysaccharide intercellular adhesin (PIA) is the major virulence factor of Staphylococcus epidermidis and is often associated with methicillin resistance. Transposon Tn917 insertions leading to a biofilm-negative phenotype in the biofilm-producing S. epidermidis strain 1457 (mecA-negative) were transferred into the methicillin-resistant, biofilm-producing S. epidermidis 1057 (mecA-positive) by transduction. According to their phenotypes and genotypes, the mutants could be separated into genetic classes I to IV (D. Mack, H. Rohde, S. Dobinsky, J. Riedewald, M. Nedelmann, J. K. M. Knobloch, H.-A. Elsner, and H. H. Feucht, Infect. Immun. 68:3799-3807, 2000). All transductants of S. epidermidis 1057 had phenotypes for biofilm formation similar to those of the corresponding mutants of S. epidermidis 1457. With a mecA-specific probe, identical hybridization patterns were observed for wild-type S. epidermidis 1057 and all the transductants. There were minor changes in oxacillin MICs for Class II and III transductants compared to those for wild-type S. epidermidis 1057. On population analysis, S. epidermidis 1057 displayed a heterogeneous expression type of resistance with an oxacillin MIC of > or =6 microg/ml for more than 90% of the cells. An almost identical profile was observed with biofilm-negative class I mutants, where the transposon insertions inactivate the icaADBC gene locus essential for PIA synthesis. In contrast, class III mutants were more sensitive to oxacillin with a MIC of < or =1 microg/ml for more than 90% of the cells. The class IV mutant displayed homogeneous resistance with a MIC of > or =50 microg/ml for more than 90% of the cells. On oxacillin gradient plates, the class II mutant displayed decreased resistance. Apparently, different independent mutations leading to a biofilm-negative phenotype of S. epidermidis by influencing expression of icaADBC on the level of transcription significantly influence the expression of methicillin resistance. However, transcription of mecA was not significantly altered in the different transductants compared to the wild type, independent of mecA induction with oxacillin, indicating that other mechanisms influencing phenotypic expression of methicillin resistance are involved.