连翘苷和黄芩苷对表皮葡萄球菌生物膜抑制作用的研究

目的通过中药有效成分连翘苷和黄芩苷分别对表皮葡萄球菌生物膜抑制作用的研究,为表皮葡萄球菌生物膜引起的相关感染提供新的治疗途径。方法体外构建表皮葡萄球菌生物膜,XTT减低法评价连翘苷、黄芩苷对表皮葡萄球菌初始黏附及生物膜内细菌代谢的影响,显微镜下观察用药后表皮葡萄球菌生物膜形态和结构改变。结果连翘苷和黄芩苷对表皮葡萄球菌生物膜的早期黏附均无抑制作用;连翘苷对表皮葡萄球菌生物膜菌的SMIC50为31.25μg/ml,而黄芩苷对表皮葡萄球菌生物膜菌的代谢无影响;在显微镜下观察,连翘苷使部分表皮葡萄球菌被膜的形态发生改变,而黄芩苷对其形态影响不显著。结论连翘苷对表皮葡萄球菌生物膜的初始黏附阶段无抑制作用,对生物膜菌的代谢和生物膜形态均有显著影响;黄芩苷对表皮葡萄球菌生物膜无显著作用。     

Monoclonal antibodies against accumulation-associated protein affect EPS biosynthesis and enhance bacterial accumulation of Staphylococcus epidermidis

Because there is no effective antibiotic to eradicate Staphylococcus epidermidis biofilm infections that lead to the failure of medical device implantations, the development of anti-biofilm vaccines is necessary. Biofilm formation by S. epidermidis requires accumulation-associated protein (Aap) that contains sequence repeats known as G5 domains, which are responsible for the Zn(2+)-dependent dimerization of Aap to mediate intercellular adhesion. Antibodies against Aap have been reported to inhibit biofilm accumulation. In the present study, three monoclonal antibodies (MAbs) against the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5) were generated. MAb(18B6) inhibited biofilm formation by S. epidermidis RP62A to 60% of the maximum, while MAb(25C11) and MAb(20B9) enhanced biofilm accumulation. All three MAbs aggregated the planktonic bacteria to form visible cell clusters. Epitope mapping revealed that the epitope of MAb(18B6), which recognizes an identical area within AapBrpt constructs from S. epidermidis RP62A, was not shared by MAb(25C11) and MAb(20B9). Furthermore, all three MAbs were found to affect both Aap expression and extracellular polymeric substance (EPS, including extracellular DNA and PIA) biosynthesis in S. epidermidis and enhance the cell accumulation. These findings contribute to a better understanding of staphylococcal biofilm formation and will help to develop epitope-peptide vaccines against staphylococcal infections.     

SdrF, a Staphylococcus epidermidis surface protein, binds type I collagen

Staphylococcus epidermidis is the leading cause of device-related infections. These infections require an initial colonization step in which S. epidermidis adheres to the implanted material. This process is usually mediated by specific bacterial surface proteins and host factors coating the foreign device. Some of these surface proteins belong to the serine-aspartate repeat (Sdr) family, which includes adhesins from Staphyloccus aureus and S. epidermidis. Using a heterologous expression system in Lactococcus lactis to overcome possible staphylococcal adherence redundancy we observed that one of these Sdr proteins, SdrF, mediates binding to type I collagen when present on the lactococcal cell surface. We used lactococcal recombinant strains, a protein-protein interaction assay and Western ligand blot analysis to demonstrate that this process occurs via the B domain of SdrF and both the alpha1 and alpha2 chains of type I collagen. It was also found that a single B domain repeat of S. epidermidis 9491 retains the capacity to bind to type I collagen. We demonstrated that the putative ligand binding N-terminal A domain does not bind to collagen which suggests that SdrF might be a multiligand adhesin. Antibodies directed against the B domain significantly reduce in vitro adherence of S. epidermidis to immobilized collagen.     

The capacity of bovine endothelial cells to eliminate intracellular Staphylococcus aureus and Staphylococcus epidermidis is increased by the proinflammatory cytokines TNF-alpha and IL-1beta

Staphylococcus aureus is a pathogenic bacterium causing clinical and subclinical bovine mastitis. Infections of the udder by S. aureus are frequently associated with the presence of Staphylococcus epidermidis, an opportunistic pathogen. We reported previously that the capacity of bovine endothelial cells (BEC) to endocytize S. aureus is associated with the activation of NF-kappaB and modulated by the proinflammatory cytokines TNF-alpha and IL-1beta. In this work, we explore the ability of BEC to eliminate intracellular S. aureus and S. epidermidis and their response to these cytokines. Time-kinetics survival experiments indicated that BEC eliminate intracellular S. epidermidis more efficiently. Replication of S. aureus, but not S. epidermidis, inside BEC was evident by an increase in intracellular bacteria recovered at 2 h postinfection. Afterwards, the intracellular number of staphylococci decreased gradually, reaching the lowest value at 24 h. Treatment of BEC with TNF-alpha or IL-1beta potentiated the capacity of BEC to eliminate both Staphylococcus species at the times tested. These results indicate that activation of the intrinsic antistaphylococcal response in BEC, enhanced by TNF-alpha and IL-1beta, is effective to eliminate S. aureus and S. epidermidis and suggest that endothelial cells may play a prominent role in the defense against infections caused by these bacteria.     

Slime production by Staphylococci isolated from prosthesis-associated infections.

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

Is the GehD lipase from Staphylococcus epidermidis a collagen binding adhesin?

The opportunistic human pathogen Staphylococcus epidermidis is the major cause of nosocomial biomaterial infections. S. epidermidis has the ability to attach to indwelling materials coated with extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and collagen. To identify the proteins necessary for S. epidermidis attachment to collagen, we screened an expression library using digoxigenin-labeled collagen as well as two monoclonal antibodies generated against the Staphylococcus aureus collagen-adhesin, Cna, as probes. These monoclonal antibodies recognize collagen binding epitopes on the surface of S. aureus and S. epidermidis cells. Using this approach, we identified GehD, the extracellular lipase originally found in S. epidermidis 9, as a collagen-binding protein. Despite the monoclonal antibody cross-reactivity, the GehD amino acid sequence and predicted structure are radically different from those of Cna. The mature GehD circular dichroism spectra differs from that of Cna but strongly resembles that of a mammalian cell-surface collagen binding receptor, known as the alpha(1) integrin I domain, suggesting that they have similar secondary structures. The GehD protein is translated as a preproenzyme, secreted, and post-translationally processed into mature lipase. GehD does not have the conserved LPXTG C-terminal motif present in cell wall-anchored proteins, but it can be detected in lysostaphin cell wall extracts. A recombinant version of mature GehD binds to collagens type I, II, and IV adsorbed onto microtiter plates in a dose-dependent saturable manner. Recombinant, mature GehD protein and anti-GehD antibodies can inhibit the attachment of S. epidermidis to immobilized collagen. These results provide evidence that GehD may be a bi-functional molecule, acting not only as a lipase but also as a cell surface-associated collagen adhesin.     

Erratum to "Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron". [FEMS Immunol. Med Microbiol. 37 (2003) 45-51

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.     

Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.     

Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases

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.     

Adhesion dependence of coagulase-negative staphylococci to biomaterials and PIA polysaccharide synthesis on icaADBC operon presence

Both Staphylococcus epidermidis and Staphylococcus haemolyticus are important causes of infections associated with catheters and other medical devices. This infections result in significant morbidity, mortality and economic cost. It has recently been shown that not only S. epidermidis but also S. haemolyticus can produce slime and carries the ica operon responsible for and slime production. In the operon, coexpression of icaA and icaD is required for full slime synthesis. This study is focused on detecting icaA and icaD genes in S. haemolyticus and comparison of these two species. It turned out that strain representatives within the same species behave very differently and a single tested strain from each species is unlikely to be representative of the species as a whole. Contrary to S. epidermidis, S. haemolyticus strain appeared to carry no icaA-like and icaD-like genes, but was able to form biofilm in vitro.     

Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain

Staphylococcus aureus is an opportunistic pathogen and the major causative agent of numerous hospital- and community-acquired infections. Staphylococcus epidermidis has emerged as a causative agent of infections often associated with implanted medical devices. We have sequenced the approximately 2.8-Mb genome of S. aureus COL, an early methicillin-resistant isolate, and the approximately 2.6-Mb genome of S. epidermidis RP62a, a methicillin-resistant biofilm isolate. Comparative analysis of these and other staphylococcal genomes was used to explore the evolution of virulence and resistance between these two species. The S. aureus and S. epidermidis genomes are syntenic throughout their lengths and share a core set of 1,681 open reading frames. Genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria appears to have shaped their virulence and resistance profiles. Integrated plasmids in S. epidermidis carry genes encoding resistance to cadmium and species-specific LPXTG surface proteins. A novel genome island encodes multiple phenol-soluble modulins, a potential S. epidermidis virulence factor. S. epidermidis contains the cap operon, encoding the polyglutamate capsule, a major virulence factor in Bacillus anthracis. Additional phenotypic differences are likely the result of single nucleotide polymorphisms, which are most numerous in cell envelope proteins. Overall differences in pathogenicity can be attributed to genome islands in S. aureus which encode enterotoxins, exotoxins, leukocidins, and leukotoxins not found in S. epidermidis.     

GST-Fbe can recognize beta-chains of fibrin(ogen) on explanted materials

Staphylococcus epidermidis, a coagulase-negative staphylococcus (CoNS), is one of the leading pathogens of nosocomial infections, particularly associated with foreign body infections. Adherence of S. epidermidis to fibrinogen deposited on the surfaces of implants is important for the development of foreign body infections. A gene (fbe) encoding a fibrinogen-binding protein from S. epidermidis (Fbe) was identified by shotgun phage display. A portion of fbe was cloned into a GST-fusion vector. Affinity to glutathione-Sepharose by the GST-tag and affinity to fibrinogen-Sepharose by the Fbe part were applied to purify the recombinant Fbe. The purity and efficacy of the methods used in protein purification was compared. Furthermore, the potential physiological role of Fbe was studied by the interaction between GST-Fbe and components extracted from explanted materials in vitro.     

In vitro activity of allicin against Staphylococcus epidermidis and influence of subinhibitory concentrations on biofilm formation

AIMS: The aim of this study is to determine the in vitro activity of allicin against Staphylococcus epidermidis and to evaluate the influence of allicin on biofilm formation. METHODS AND RESULTS: In vitro activity of allicin (diallyl thiosulphinate) against 38 strains of S. epidermidis was investigated. The activity of allicin was similar against S. epidermidis methicillin susceptible and methicillin resistant strains [minimum inhibitory concentration (MIC)90=8 mg l(-1)]. In general, subinhibitory concentrations (sub-MIC) of allicin diminished biofilm formation in the five strains analysed. CONCLUSION: The results confirm the antibacterial effect of allicin. Sub-MICs of allicin also diminished the biofilm formations by S. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that allicin is active in vitro against S. epidermidis and that sub-MICs of allicin may play a role in the prevention of adherence of this bacteria to medical devices.     

Confocal laser scanning microscopy analysis of S. epidermidis biofilms exposed to farnesol, vancomycin and rifampicin

ABSTRACT: BACKGROUND: Staphylococcus epidermidis is the major bacterial species found in biofilm-related infections on indwelling medical devices. Microbial biofilms are communities of bacteria adhered to a surface and surrounded by an extracellular polymeric matrix. Biofilms have been associated with increased antibiotic tolerance to the immune system. This increased resistance to conventional antibiotic therapy has lead to the search for new antimicrobial therapeutical agents. Farnesol, a quorum-sensing molecule in Candida albicans, has been described as impairing growth of several different microorganisms and we have previously shown its potential as an adjuvant in antimicrobial therapy against S. epidermidis. However, its mechanism of action in S. epidermidis is not fully known. In this work we better elucidate the role of farnesol against S: epidermidis biofilms using confocal laser scanning microscopy (CLSM). FINDINGS: 24 h biofilms were exposed to farnesol, vancomycin or rifampicin and were analysed by CLSM, after stained with a Live/Dead stain, a known indicator of cell viability, related with cell membrane integrity. Biofilms were also disrupted by sonication and viable and cultivable cells were quantified by colony forming units (CFU) plating. Farnesol showed a similar effect as vancomycin, both causing little reduction of cell viability but at the same time inducing significant changes in the biofilm structure. On the other hand, rifampicin showed a distinct action in S. epidermidis biofilms, by killing a significant proportion of biofilm bacteria. CONCLUSIONS: While farnesol is not very efficient at killing biofilm bacteria, it damages cell membrane, as determined by the live/dead staining, in a similar way as vancomycin.. Furthermore, farnesol might induce biofilm detachment, as determined by the reduced biofilm biomass, which can partially explain the previous findings regarding its role as a possible chemotherapy adjuvant.     

Discovery of quinoline small molecules with potent dispersal activity against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis biofilms using a scaffold hopping strategy

Staphylococcus aureus and Staphylococcus epidermidis are recognized as the most frequent cause of biofilm-associated nosocomial and indwelling medical device infections. Biofilm-associated infections are known to be highly resistant to our current arsenal of clinically used antibiotics and antibacterial agents. To exacerbate this problem, no therapeutic option exists that targets biofilm-dependent machinery critical to Staphylococcal biofilm formation and maintenance. Here, we describe the discovery of a series of quinoline small molecules that demonstrate potent biofilm dispersal activity against methicillin-resistant S. aureus and S. epidermidis using a scaffold hopping strategy. This interesting class of quinolines also has select synthetic analogues that demonstrate potent antibacterial activity and biofilm inhibition against S. aureus and S. epidermidis.     

TNF-α reduces the level of Staphylococcus epidermidis internalization by bovine endothelial cells

Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor α (TNF-α). Therefore, this work was designed to explore the effect of TNF-α on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-α caused a marked reduction in the number of intracellular S. epidermidis , suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-κB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-κB inhibitors, SN50 or BAY 11–7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis . These results suggest that activation of the signaling pathway initiated by TNF-α could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-κB was shown not to be important for the internalization process of S. epidermidis .     

Staphylococcus epidermidis strategies to avoid killing by human neutrophils

Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs) contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs), including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS). These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus.     

Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system

The skin commensal and opportunistic pathogen Staphylococcus epidermidis is the leading cause of nosocomial and biofilm-associated infections. Little is known about the mechanisms by which S. epidermidis protects itself against the innate human immune system during colonization and infection. We used scanning electron microscopy to demonstrate that the exopolysaccharide intercellular adhesin (PIA) resides in fibrous strands on the bacterial cell surface, and that lack of PIA production results in complete loss of the extracellular matrix material that has been suggested to mediate immune evasion. Phagocytosis and killing by human polymorphonuclear leucocytes was significantly increased in a mutant strain lacking PIA production compared with the wild-type strain. The mutant strain was also significantly more susceptible to killing by major antibacterial peptides of human skin, cationic human beta-defensin 3 and LL-37, and anionic dermcidin. PIA represents the first defined factor of the staphylococcal biofilm matrix that protects against major components of human innate host defence.     

Antimicrobial activities of YycG histidine kinase inhibitors against Staphylococcus epidermidis biofilms

Staphylococcus epidermidis has become a significant pathogen causing infections due to biofilm formation on surfaces of indwelling medical devices. Biofilm-associated bacteria exhibit enhanced resistance to many conventional antibiotics. It is therefore, important to design novel antimicrobial reagents targeting S. epidermidis biofilms. In a static chamber system, the bactericidal effect of two leading compounds active as YycG inhibitors was assessed on biofilm cells by confocal laser scanning microscopy combined with viability staining. In young biofilms (6-h-old), the two compounds killed the majority of the embedded cells at concentrations of 100 microM and 25 microM, respectively. In mature biofilms (24-h-old), one compound was still effectively killing biofilm cells, whereas the other compound mainly killed cells located at the bottom of the biofilm. In contrast, vancomycin was found to stimulate biofilm development at the MBC (8 microg mL(-1)). Even at a high concentration (128 microg mL(-1)), vancomycin exhibited poor killing on cells embedded in biofilms. The two compounds exhibited faster and more effective killing of S. epidermidis planktonic cells than vancomycin at the early stage of exposure (6 h). The data suggest that the new inhibitors can serve as potential agents against S. epidermidis biofilms when added alone or in concert with other antimicrobial agents.     

Biomaterial-associated staphylococcal peritoneal infections in a neutropaenic mouse model

Abstract Adhesion of staphylococcal cells to polyethylene with end point-attached heparin was quantified by bioluminescence. Staphylococcus epidermidis 3380 and the slime-producing S. epidermidis RP12 adhered to the highest extent, and S. lugdunensis 2342 to the least extent. Preincubation of the polymer with dialysis fluid reduced adhesion of S. epidermidis 3380 and RP12 but enhanced that of S. aureus , and preadsorption of the surface with fibronectin decreased subsequent adhesion of S. epidermidis and S. haemolyticus strains. When staphylococci were grown in the presence of a biomaterial their ability to activate peritoneal cells was decreased. The bactericidal activity was impaired, whereas ingestion of opsonized coagulase-negative staphylococci (CNS) strains was unaffected. With S. epidermidis RP12 the presence of biomaterial did not influence either phagocytosis or bactericidal effect of peritoneal cells. After intra-peritoneal challenge with staphylococcal strains, the organ uptake of S. aureus Cowan 1 was increased in normal mice whereas immunosuppressed mice died. CNS strains increased mainly in the peritoneal cavity of immunosuppressed mice. The uptake of bacteria in liver and kidneys was increased with S. epidermidis 3380, S. lugdunensis 2343 and S. schleiferi 667-88. Generally, CNS strains persisted in the peritoneal cavity of both normal and immunosuppressed mice. These data indicate that host defense mechanisms, mainly polymorphonuclear neutrophils, fail to eliminate CNS infections in the peritoneum, and that initial adhesion to an implanted biomaterial may be of lesser importance in the peritoneal cavity than in e.g. catheter-associated infections. There are strain-specific virulence factors of bacteria, and slime producing strains evade the host defense mechanisms more efficiently than non-slime producing strains.