Characterization of the Atl-mediated staphylococcal internalization mechanism.

Staphylococcus aureus internalization by non-professional phagocytes is considered a main pathogenicity mechanism leading to chronic infections. The well-established mechanism of Staphylococcus aureus internalization is mediated by fibronectin (Fn)-binding proteins (FnBPs), Fn as a bridging molecule and the host cell α5ß1 integrin. We previously identified a novel alternative internalization mechanism in Staphylococcus aureus, which involves the major autolysin Atl and the host cell heat shock cognate protein 70 (Hsc70). Atl-dependent internalization is also employed by the coagulase-negative Staphylococcus epidermidis, where it might represent the major or even sole internalization mechanism, because of the lack of FnBP-homologous proteins. In this study, we aimed to further characterize the Atl-dependent staphylococcal internalization mechanism. We performed biomolecular interaction analysis (BIA) to quantify the adhesive properties of Atl and found multivalent and high affinity interactions of Atl with Fn and Hsc70. Confocal laser scanning microscopy (CLSM) and a flow-cytometric internalization assay in combination with different pharmacological inhibitors suggested an involvement of the α5ß1 integrin, Fn and Hsc70 and subsequent signaling events mediated by Src and phosphoinositide 3 (PI3) kinases in the Atl-dependent staphylococcal uptake by EA.hy 926 cells. Further characterization of the endocytic machinery implicated a role for clathrin-dependent receptor-mediated endocytosis involving actin cytoskeletal rearrangements and microtubules. In conclusion, Atl ubiquitous among staphylococcal species may substitute for the FnBPs ensuring low-level internalization via a mechanism that seems to share important features with the FnBP-mediated staphylococcal uptake potentially being the prerequisite for the development of therapy-resistant chronic infections by staphylococcal strains that lack FnBPs.

Emergence of coagulase-negative staphylococci.

Introduction: Compared to Staphylococcus aureus, coagulase-negative staphylococci (CoNS) are characterized by a lower capacity to cause acute, live-threatened infections. CoNS are, however, of ever increasing importance as pathogens causing infections in immunocompromised patients and after foreign-material implantation. Typically, antibiotics fail to cure foreign body-related infections and removal of the implanted device is inevitable.Areas covered: This review focuses on the emergence of CoNS species, their pathogenic potential in particular due to their ability to form therapy-refractory biofilms on biotic and abiotic surfaces and evasion strategies to resist host response and antibiotic treatment. Their medical significance and proven and novel therapy strategies are discussed.Expert opinion: CoNS contribute significantly to morbidity and socio-economic costs. The anticipated developments in modern medicine, in particular the increasing use of foreign materials and the rising numbers of immunocompromised patients, as well as the changing demographic and hospital-related factors will inevitably contribute to further emergence of CoNS infections. Increasing rates of (multi-)resistant CoNS strains will limit the therapeutic armamentarium and aggravate treatment strategies. Increased research is necessary to understand their role as resistance and virulence gene reservoir and to reduce CoNS infections by the development of innovative colonization-preventing materials and other CoNS-tailored treatment strategies.

A new host cell internalisation pathway for SadA-expressing staphylococci triggered by excreted neurochemicals.

Staphylococcus aureus is a facultative intracellular pathogen that invades a wide range of professional and nonprofessional phagocytes by triggering internalisation by interaction of surface-bound adhesins with corresponding host cell receptors. Here, we identified a new concept of host cell internalisation in animal-pathogenic staphylococcal species. This new mechanism exemplified by Staphylococcus pseudintermedius ED99 is not based on surface-bound adhesins but is due to excreted small neurochemical compounds, such as trace amines (TAs), dopamine (DOP), and serotonin (SER), that render host cells competent for bacterial internalisation. The neurochemicals are produced by only one enzyme, the staphylococcal aromatic amino acid decarboxylase (SadA). Here, we unravelled the mechanism of how neurochemicals trigger internalisation into the human colon cell line HT-29. We found that TAs and DOP are agonists of the α2-adrenergic receptor, which, when activated, induces a cascade of reactions involving a decrease in the cytoplasmic cAMP level and an increase in F-actin formation. The signalling cascade of SER follows a different pathway. SER interacts with 5HT receptors that trigger F-actin formation without decreasing the cytoplasmic cAMP level. The neurochemical-induced internalisation in host cells is independent of the fibronectin-binding protein pathway and has an additive effect. In a sadA deletion mutant, ED99ΔsadA, internalisation was decreased approximately threefold compared with that of the parent strain, and treating S. aureus USA300 with TAs increased internalisation by approximately threefold.

Panton-Valentine Leukocidin associated with S. aureus osteomyelitis activates platelets via neutrophil secretion products.

Globalization and migration promote the spread of Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus strains. The toxin PVL is linked to the development of thrombosis in association with osteomyelitis. The mechanisms by which PVL drives thrombosis development are however still unknown. We demonstrate that PVL-damaged neutrophils activate platelets via neutrophil secretion products, such as α-defensins and the myeloperoxidase product HOCl, as well as the formation of HOCl-modified proteins. Neutrophil damage by PVL is blocked by anti-PVL-antibodies, explaining why especially young osteomyelitis patients with a low antibody titre against PVL suffer from thrombotic complications. Platelet activation in the presence of PVL-damaged neutrophils is prevented by α-defensin inhibitors and by glutathione and resveratrol, which are both inhibitors of HOCl-modified protein-induced platelet activation. Remarkably, intravenously infused glutathione also prevents activation of human platelets in an ex vivo assay. We here describe a new mechanism of PVL-neutrophil-platelet interactions, which might be extrapolated to other toxins that act on neutrophils. Our observations may make us think about new approaches to treat and/or prevent thrombotic complications in the course of infections with PVL-producing S. aureus strains.

The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA) Is a Glycoprotein.

Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff's staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa) and two (>300 and ∼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl residues can stimulate biofilm formation. Thus, sugar modifications may represent promising new targets for novel therapeutic or prophylactic measures against life-threatening S. aureus infections.

Important contribution of the novel locus comEB to extracellular DNA-dependent Staphylococcus lugdunensis biofilm formation.

The coagulase-negative species Staphylococcus lugdunensis is an emerging cause of serious and potentially life-threatening infections, such as infective endocarditis. The pathogenesis of these infections is characterized by the ability of S. lugdunensis to form biofilms on either biotic or abiotic surfaces. To elucidate the genetic basis of biofilm formation in S. lugdunensis, we performed transposon (Tn917) mutagenesis. One mutant had a significantly reduced biofilm-forming capacity and carried a Tn917 insertion within the competence gene comEB. Site-directed mutagenesis and subsequent complementation with a functional copy of comEB verified the importance of comEB in biofilm formation. In several bacterial species, natural competence stimulates DNA release via lysis-dependent or -independent mechanisms. Extracellular DNA (eDNA) has been demonstrated to be an important structural component of many bacterial biofilms. Therefore, we quantified the eDNA in the biofilms and found diminished eDNA amounts in the comEB mutant biofilm. High-resolution images and three-dimensional data obtained via confocal laser scanning microscopy (CSLM) visualized the impact of the comEB mutation on biofilm integrity. The comEB mutant did not show reduced expression of autolysin genes, decreased autolytic activities, or increased cell viability, suggesting a cell lysis-independent mechanism of DNA release. Furthermore, reduced amounts of eDNA in the comEB mutant biofilms did not result from elevated levels or activity of the S. lugdunensis thermonuclease NucI. In conclusion, we defined here, for the first time, a role for the competence gene comEB in staphylococcal biofilm formation. Our findings indicate that comEB stimulates biofilm formation via a lysis-independent mechanism of DNA release.

The adhesive properties of the Staphylococcus lugdunensis multifunctional autolysin AtlL and its role in biofilm formation and internalization.

Although it belongs to the group of coagulase-negative staphylococci, Staphylococcus lugdunensis has been known to cause aggressive courses of native and prosthetic valve infective endocarditis with high mortality similar to Staphylococcus aureus. In contrast to S. aureus, only little is known about the equipment of S. lugdunensis with virulence factors including adhesins and their role in mediating attachment to extracellular matrix and plasma proteins and host cells. In this study, we show that the multifunctional autolysin/adhesin AtlL of S. lugdunensis binds to the extracellular matrix and plasma proteins fibronectin, fibrinogen, and vitronectin as well as to human EA.hy926 endothelial cells. Furthermore, we demonstrate that AtlL also plays an important role in the internalization of S. lugdunensis by eukaryotic cells: The atlL-deficient mutant Mut17 adheres to and becomes internalized by eukaryotic cells to a lesser extent than the isogenic wild-type strain Sl253 and the complemented mutant Mut17 (pCUatlL) shows an increased internalization level in comparison to Mut17. Thus, surface localized AtlL that exhibits a broad binding spectrum also mediates the internalization of S. lugdunensis by eukaryotic cells. We therefore propose an internalization pathway for S. lugdunensis, in which AtlL plays a major role. Investigating the role of AtlL in biofilm formation of S. lugdunensis, Mut17 shows a significantly reduced ability for biofilm formation, which is restored in the complemented mutant. Thus, our data provide evidence for a significant role for AtlL in adherence and internalization processes as well as in biofilm formation of S. lugdunensis.

Coagulase-negative staphylococci.

The definition of the heterogeneous group of coagulase-negative staphylococci (CoNS) is still based on diagnostic procedures that fulfill the clinical need to differentiate between Staphylococcus aureus and those staphylococci classified historically as being less or nonpathogenic. Due to patient- and procedure-related changes, CoNS now represent one of the major nosocomial pathogens, with S. epidermidis and S. haemolyticus being the most significant species. They account substantially for foreign body-related infections and infections in preterm newborns. While S. saprophyticus has been associated with acute urethritis, S. lugdunensis has a unique status, in some aspects resembling S. aureus in causing infectious endocarditis. In addition to CoNS found as food-associated saprophytes, many other CoNS species colonize the skin and mucous membranes of humans and animals and are less frequently involved in clinically manifested infections. This blurred gradation in terms of pathogenicity is reflected by species- and strain-specific virulence factors and the development of different host-defending strategies. Clearly, CoNS possess fewer virulence properties than S. aureus, with a respectively different disease spectrum. In this regard, host susceptibility is much more important. Therapeutically, CoNS are challenging due to the large proportion of methicillin-resistant strains and increasing numbers of isolates with less susceptibility to glycopeptides.

Functional and structural analysis of the major amidase (Atl) in Staphylococcus.

The cytoplasmic membrane of most bacteria is surrounded by a more or less thick murein layer (peptidoglycan) that protects the protoplast from mechanical damage, osmotic rupture and lysis. When bacteria are dividing processes are initiated stepwise that involve DNA replication, constriction of the membranes, cell growth, biosynthesis of new murein, and finally the generation of two daughter cells. As the daughter cells are still covalently interlinked by the murein network they must be separated by specific peptidoglycan hydrolases, also referred to as autolysins. In staphylococci, the major autolysin (Atl) and its processed products N-acetylmuramoyl-l-alanine amidase (AM) and endo-ß-N-acetylglucosaminidase (GL) have been in the research focus for long time. This review addresses phenotypic consequences of atl mutants, impact of Atl in virulence, the mechanism of targeting to the septum region, regulation of atl, the structure of the amidase and the repeat regions, as well as the phylogeny of Atl and its use in Staphylococcus genus and species typing.

Early Staphylococcus aureus-induced changes in endothelial barrier function are strain-specific and unrelated to bacterial translocation.

The vascular endothelium provides the critical barrier during hematogenous spreading of bacteria, a phenomenon that might contribute to severe diseases in humans including endocarditis and sepsis as known from infections by Staphylococcus aureus. Here we aimed to uncover early responses of the endothelium to S. aureus infection with respect to (a) inflammatory reactions such as paracellular endothelial barrier function and expression of cell adhesion molecule-1 (ICAM-1) and (b) translocation through the endothelium. After infection of the cultured endothelium with 22 different clinical isolates of S. aureus and two well-characterized lab strains a diverse and strain-specific change in para- and transcellular endothelial barrier function was observed. Bayesian data analysis revealed positive correlation of paracellular barrier function decrease followed by expression of ICAM-1 while these parameters negatively correlated with transcellular bacterial translocation. Translocating bacteria largely blocked TNFα-induced ICAM-1 expression indicating an active anti-inflammatory effect mediated by those strains probably due to intracellularly released virulence factors. Furthermore, the underlying background of barrier function decrease was investigated in more detail using two well-characterized lab strains, ls 8325-4 and ls 6850 and respective mutants. Barrier function decrease was found to be independent of early cell death and early release of virulence factors into the medium, but require internalization of live bacteria. The data show for the first time that endothelial cells respond diversely to infection with different strains of S. aureus and that translocating strains downregulate inflammatory response of the endothelium. Furthermore, data indicate that S. aureus-mediated activation of the endothelium reduces bacterial translocation.

Role for the fibrinogen-binding proteins coagulase and Efb in the Staphylococcus aureus-Candida interaction.

Staphylococcus aureus and Candida species are increasingly coisolated from implant-associated polymicrobial infections creating an incremental health care problem. Synergistic effects between both genera seem to facilitate the formation of mixed S. aureus-Candida biofilms, which is thought to play a critical role in coinfections with these microorganisms. To identify and characterize S. aureus factors involved in the interaction with Candida species, we affinity-panned an S. aureus phage display library against Candida biofilms in the presence or absence of fibrinogen. Repeatedly isolated clones contained DNA fragments encoding portions of the S. aureus fibrinogen-binding proteins coagulase or Efb. The coagulase binds to prothrombin in a 1:1 ratio thereby inducing a conformational change and non-proteolytic activation of prothrombin, which in turn cleaves fibrinogen to fibrin. Efb has been known to inhibit opsonization. To study the role of coagulase and Efb in the S. aureus-Candida cross-kingdom interaction, we performed flow-cytometric phagocytosis assays. Preincubation with coagulase reduced the phagocytosis of Candida yeasts by granulocytes significantly and dose-dependently. By using confocal laser scanning microscopy, we demonstrated that the coagulase mediated the formation of fibrin surrounding the candidal cells. Furthermore, the addition of Efb significantly protected the yeasts against phagocytosis by granulocytes in a dose-dependent and saturable fashion. In conclusion, the inhibition of phagocytosis of Candida cells by coagulase and Efb via two distinct mechanisms suggests that S. aureus might be beneficial for Candida to persist as it helps Candida to circumvent the host immune system.

Characterization of the modular design of the autolysin/adhesin Aaa from Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. CONCLUSIONS/SIGNIFICANCE: We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections.

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases.

OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase and endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Adhesion mechanisms of staphylococci.

Staphylococcal adherence to an either biotic or abiotic surface is the critical first event in the establishment of an infection with these serious pathogens. Especially Staphylococcus aureus harbours a variety of proteinaceous and non-proteinaceous adhesins that mediate attachment to a multitude of host factors, such as extracellular matrix and plasma proteins and human host cells, or intercellular adhesion, which is essential for biofilm accumulation. Proteinaceous adhesins may be classified in covalently surface-anchored proteins of the MSCRAMM (microbial surface components recognizing adhesive matrix molecules) family or in proteins that are surface-associated by different means, such as ionic or hydrophobic interactions. Non-covalently surface-associated proteins include the autolysin/adhesins, proteins of the SERAM (secretable expanded repertoire adhesive molecules) family, or membrane-spanning proteins. Non-proteinaceous adhesins comprise the polysaccharide PIA (polysaccharide intercellular adhesin) and wall teichoic and lipoteichoic acids. The features and functions of surface and surface-associated protein adhesins as well as of non-proteinaceous adhesins are discussed.

A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor.

Staphylococcus aureus and Staphylococcus epidermidis can cause serious chronic and recurrent infections that are difficult to eradicate. An important pathogenicity factor in these infections caused by S. aureus is its ability to be internalized by non-professional phagocytes thereby evading the host immune system and antibiotic treatment. Here, we report a novel mechanism involved in staphylococcal internalization by host cells, which is mediated by the major autolysin/adhesins Atl and AtlE from S. aureus and S. epidermidis respectively. In a flow cytometric internalization assay, atl and atlE mutants are significantly reduced in their capacities to be internalized by endothelial cells. Moreover, pre-incubation of endothelial cells with recombinant Atl dose-dependently inhibited internalization. As putative Atl-host cell receptor, the heat shock cognate protein Hsc70 was identified by mass spectrometry. The importance of Hsc70 in internalization was demonstrated by the inhibition of S. aureus internalization with anti-Hsc70 antibodies. In conclusion, this novel Atl- or AtlE-mediated internalization mechanism may represent a 'back-up' mechanism in S. aureus internalization, while it may represent the major or even sole mechanism involved in the internalization of coagulase-negative staphylococci and thus may play an important role in the pathogenesis of chronic and relapsing infections with these serious pathogens.

Molecular characterization of a novel Staphylococcus aureus surface protein (SasC) involved in cell aggregation and biofilm accumulation.

BACKGROUND: Staphylococci belong to the most important pathogens causing implant-associated infections. Colonization of the implanted medical devices by the formation of a three-dimensional structure made of bacteria and host material called biofilm is considered the most critical factor in these infections. To form a biofilm, bacteria first attach to the surface of the medical device, and then proliferate and accumulate into multilayered cell clusters. Biofilm accumulation may be mediated by polysaccharide and protein factors. METHODOLOGY/PRINCIPAL FINDINGS: The information on Staphylococcus aureus protein factors involved in biofilm accumulation is limited, therefore, we searched the S. aureus Col genome for LPXTG-motif containing potential surface proteins and chose the so far uncharacterized S. aureus surface protein C (SasC) for further investigation. The deduced SasC sequence consists of 2186 amino acids with a molecular mass of 238 kDa and has features typical of gram-positive surface proteins, such as an N-terminal signal peptide, a C-terminal LPXTG cell wall anchorage motif, and a repeat region consisting of 17 repeats similar to the domain of unknown function 1542 (DUF1542). We heterologously expressed sasC in Staphylococcus carnosus, which led to the formation of huge cell aggregates indicative of intercellular adhesion and biofilm accumulation. To localize the domain conferring cell aggregation, we expressed two subclones of sasC encoding either the N-terminal domain including a motif that is found in various architectures (FIVAR) or 8 of the DUF1542 repeats. SasC or its N-terminal domain, but not the DUF1542 repeat region conferred production of huge cell aggregates, higher attachment to polystyrene, and enhanced biofilm formation to S. carnosus and S. aureus. SasC does not mediate binding to fibrinogen, thrombospondin-1, von Willebrand factor, or platelets as determined by flow cytometry. CONCLUSIONS/SIGNIFICANCE: Thus, SasC represents a novel S. aureus protein factor involved in cell aggregation and biofilm formation, which may play an important role in colonization during infection with this important pathogen.

Pneumococcal association to platelets is mediated by soluble fibrin and supported by thrombospondin-1.

Platelets and coagulation are involved in bacterial colonisation of the host. Streptocococcus pneumoniae (pneumococcus) are important etiologic agents of respiratory tract infections in humans. The formation of pneumococci-platelet associations may facilitate haematogenous dissemination of pneumococci by providing an adhesive surface on damaged endothelium. However, the formation of platelet-pneumococci associations and the factors involved in this process have not been described so far. The formation of platelet-pneumococci associates was analysed and quantified using flow cytometry. Binding of pneumococci to platelets was significantly increased after activation of platelets with thrombin, while platelet activation by ADP or collagen did not promote formation of platelet-pneumococci associates. In addition to be a platelet agonist, thrombin cleaves fibrinogen, which results in the generation of fibrin. The simultaneous formation of fibrin and activation of platelets was shown to be a prerequisite for a high number of platelet-pneumococci associates. Moreover, exogenously added human thrombospondin-1 (TSP-1) significantly enhanced the association of pneumococci with activated platelets. Soluble fibrin and TSP-1 are key co-factors of platelet-pneumococci-association. Similar results were recently demonstrated for S. aureus-platelet adhesion. Consequently, we hypothesise that the described mechanism of platelet-bacteria-association might represent a general and important strategy of Gram-positive bacteria during development of invasive diseases.

Augmented expression of polysaccharide intercellular adhesin in a defined Staphylococcus epidermidis mutant with the small-colony-variant phenotype.

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.

Systematic survey of nonspecific agglutination by Candida spp. in latex assays.

In order to demonstrate that cells of Candida spp. may show considerable nonspecific agglutination in latex agglutination tests, 150 clinical and reference isolates of 12 yeast species were systematically studied by applying various test parameters. In fact, 40 (26.7%) of these isolates revealed nonspecific results, significantly associated with the time allowed for agglutination.

Very low ethanol concentrations affect the viability and growth recovery in post-stationary-phase Staphylococcus aureus populations.

Pharmaceuticals, culture media used for in vitro diagnostics and research, human body fluids, and environments can retain very low ethanol concentrations (VLEC) (< or =0.1%, vol/vol). In contrast to the well-established effects of elevated ethanol concentrations on bacteria, little is known about the consequences of exposure to VLEC. We supplemented growth media for Staphylococcus aureus strain DSM20231 with VLEC (VLEC(+) conditions) and determined ultramorphology, growth, and viability compared to those with unsupplemented media (VLEC(-) conditions) for prolonged culture times (up to 8 days). VLEC(+)-grown late-stationary-phase S. aureus displayed extensive alterations of cell integrity as shown by scanning electron microscopy. Surprisingly, while ethanol in the medium was completely metabolized during exponential phase, a profound delay of S. aureus post-stationary-phase recovery (>48 h) was observed. Concomitantly, under VLEC(+) conditions, the concentration of acetate in the culture medium remained elevated while that of ammonia was reduced, contributing to an acidic culture medium and suggesting decreased amino acid catabolism. Interestingly, amino acid depletion was not uniformly affected: under VLEC(+) conditions, glutamic acid, ornithine, and proline remained in the culture medium while the uptake of other amino acids was not affected. Supplementation with arginine, but not with other amino acids, was able to restore post-stationary-phase growth and viability. Taken together, these data demonstrate that VLEC have profound effects on the recovery of S. aureus even after ethanol depletion and delay the transition from primary to secondary metabolite catabolism. These data also suggest that the concentration of ethanol needed for bacteriostatic control of S. aureus is lower than that previously reported.