Effect of sub-inhibitory concentrations of antibiotics on the virulence of Staphylococcus aureus

The production of virulence factors by various bacteria can be influenced by sub-inhibitory concentrations of antibiotics. The effect of six antibiotics on the production of representative extracellular enzymes and toxins produced by Staphylococcus aureus was investigated. The production of the virulence determinants coagulase, protein A, alpha and delta haemolysin was monitored in the presence of ciprofloxacin, enoxacin, chloramphenicol, gentamicin, tetracycline and methicillin. The protein synthesis inhibitors reduced the production of coagulase and protein A, and almost completely inhibited the production of the haemolysins. Haemolysin production was also reduced by ciprofloxacin and enoxacin, but these antibiotics had little effect on the production of coagulase and protein A. Methicillin stimulated the production of alpha and delta haemolysins but had no effect on the production of coagulase and protein A.     

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein‐like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat‐inducible and appears to play a major role in Lpl1 interaction. Pre‐incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1‐Hsp90 interaction induces F‐actin formation, thus, triggering an endocytosis‐like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl‐Hsp90 interaction.     

Phorbol ester stimulation of fibronectin-mediated cell adhesion

Stimulation of Chinese hamster ovary cells with 12-O-tetradecanoyl 13-acetate increases the rate of adhesion to fibronectin-coated substratum. The EC50 of the phorbol ester that initiates the change in kinetics of adhesion is approximately 8 nM and is specific to those phorbol esters which activate protein kinase C. When compared to control cells, cells stimulated with active phorbol esters require a significantly lower amount of fibronectin to support their adhesion, and exhibit 50% adhesion inhibition by a log-fold higher concentration of PB1, a monoclonal antibody which specifically blocks fibronectin-mediated adhesion. These results indicate that stimulation of cells with phorbol esters results in a modification of the fibronectin receptor leading to an apparent increase in the interaction of the receptor with fibronectin.     

Therapeutic effect of some antibiotics on experimental staphylococcal infection and its correlation with in vitro activity of antibiotics in sub-inhibitory concentration against Staphylococcus aureus strains

The aim of the study was to demonstrate of whether the therapeutic effects of antibiotics depend on their in vitro activity in sub-inhibitory concentrations against staphylococci. Cloxacillin, gentamicin and lincomycin were used in the study. Groups of S. aureus strains, containing 6 strains with similar MIC values each but different sensitivity to sub-inhibitory antibiotic concentrations (sub-MIC) were selected (a total of 36 trains): i. strains increasing their sensitivity to phagocytosis and bactericidal activity of rabbit leukocytes after incubation with an antibiotic in 0.1 MIC concentration, ii. strains with sensitivity to the above factors unaffected by incubation with an antibiotic in 0.5 MIC concentration. The doses of staphylococci causing death of 90-100% of Swiss albino mice 10 days after i.p. infection were determined. The injected doses (LD 90-100) and various doses of antibiotics were used to determine ED50 values as well as the survival rate of the mice with experimental staphylococcal infections after treatment with these antibiotics. It was demonstrated that effective doses (ED 50) of the antiboitics were significantly lower when the antibiotics were administered once to mice infected with strains S. aureus sensitive to sub-MIC concentrations of the investigated antibiotics than for mice infected with strains resistant to their sub-MIC concentrations. Similar correlations were observed in mice which were given the antibiotics several times (for 7 days): the percentage of the surviving mice was higher in the group infected with sub-MIC sensitive strains. The therapeutic effect of cloxacillin, gentamicin and lincomycin demonstrated a significant correlation with the S. aureus strains used to induce the infections and their sensitivity, or lack of sensitivity in vitro, to phagocytosis and bactericdal activity of leukocytes in the presence of antibiotics in sub-MIC concentrations.     

Development and validation of a high-throughput whole cell assay to investigate Staphylococcus aureus adhesion to host ligands

Staphylococcus aureus adhesion to the host''s skin and mucosae enables asymptomatic colonization and the establishment of infection. This process is facilitated by cell wall-anchored adhesins that bind to host ligands. Therapeutics targeting this process could provide significant clinical benefits; however, the development of anti-adhesives requires an in-depth knowledge of adhesion-associated factors and an assay amenable to high-throughput applications. Here, we describe the development of a sensitive and robust whole cell assay to enable the large-scale profiling of S. aureus adhesion to host ligands. To validate the assay, and to gain insight into cellular factors contributing to adhesion, we profiled a sequence-defined S. aureus transposon mutant library, identifying mutants with attenuated adhesion to human-derived fibronectin, keratin, and fibrinogen. Our screening approach was validated by the identification of known adhesion-related proteins, such as the housekeeping sortase responsible for covalently linking adhesins to the cell wall. In addition, we also identified genetic loci that could represent undescribed anti-adhesive targets. To compare and contrast the genetic requirements of adhesion to each host ligand, we generated a S. aureus Genetic Adhesion Network, which identified a core gene set involved in adhesion to all three host ligands, and unique genetic signatures. In summary, this assay will enable high-throughput chemical screens to identify anti-adhesives and our findings provide insight into the target space of such an approach.     

Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell beta1 integrins

Although Staphylococcus aureus is primarily considered an extracellular pathogen, recent evidence suggests that this bacterium can invade a variety of nonprofessional phagocytic cells. Here we investigate the early stages of cellular invasion by S. aureus and determine the bacterial and host components that are required for this process. S. aureus expresses two cell surface-associated fibronectin (FN)-binding proteins (FnbpA and FnbpB) that mediate the interaction of the bacteria with both soluble and solid-phase FN in vitro. Using a mutant of S. aureus that lacks the expression of both Fnbps, we show that the expression of either protein is necessary for efficient uptake by the mouse fibroblast line GD25beta1A. Invasion could be inhibited by soluble recombinant proteins encompassing either the FN-binding D repeat region or the A region (and B repeats) of FnbpA, suggesting that the activities of both regions are important in this process. We demonstrate that FN is also required for invasion of this cell line. In the presence of FN-depleted fetal bovine serum, the invasion level was reduced by approximately 40% compared to in the presence of whole fetal bovine serum. Invasion could be further reduced by the addition of anti-mouse FN antibodies to the assay. Finally, we utilize a mutant mouse fibroblast line, which lacks beta1 integrin expression, to demonstrate that host cell beta1 integrins are necessary for efficient cellular invasion. The level of invasion of the mutant cell line GD25 was reduced by approximately 97% compared to the beta1-expressing complemented cell line GD25beta1A. In addition, invasion of the GD25beta1A cell line could be inhibited by an RGD-containing peptide, further implicating a role for integrins in this process. Based on these observations, we put forward a model of S. aureus invasion in which host FN forms a bridge between the bacterial Fnbps and host cell beta1 integrins, leading to bacterial uptake.     

Plasmid curing in Staphylococcus aureus by antibiotics affecting the bacterial cell wall

Abstract Strains of Staphylococcus aureus were converted into L-forms with β-lactam antibiotics, vancomycin and lysostaphin. Reverted bacteria obtained after several transfers in protoplast forms (unstable L-forms) as well as stable L-forms lost their plasmids. Curing was obtained whatever the plasmid size (from 3.4 to 28.2 kb) but complete curing required cell division. Elimination of plasmids in wall-less organisms could be the result of an inhibition of new rounds of plasmid replication following the loss of DNA-envelope interactions.     

Impact of antibiotics on conjugational resistance gene transfer in Staphylococcus aureus in sewage

The growing rate of microbial pathogens becoming resistant to standard antibiotics is an important threat to public health. In order to assess the role of antibiotics in the environment on the spread of resistance factors, the impact of subinhibitory concentrations of antibiotics in sewage on gene transfer was investigated using conjugative gentamicin resistance (aacA-aphD) plasmids of Staphylococcus aureus. Furthermore, the concentration of antibiotics in hospital sewage was measured by high-performance liquid chromatography (HPLC)-electrospray tandem mass spectrometry. Several antibiotics were found to be present in sewage, e.g. ciprofloxacin up to 0.051 mgl(-1) and erythromycin up to 0.027 mgl(-1). Resistance plasmid transfer occurred both on solidified (dewatered) sewage and in liquid sewage in a bioreactor with a frequency of 1.1x10(-5)-5.0x10(-8). However, low-level concentrations of antibiotics measured in sewage are below concentrations that can increase plasmid transfer frequencies of gentamicin resistance plasmids of staphylococci.     

Intracellular Staphylococcus aureus eludes selective autophagy by activating a host cell kinase

Autophagy, a catabolic pathway of lysosomal degradation, acts not only as an efficient recycle and survival mechanism during cellular stress, but also as an anti-infective machinery. The human pathogen Staphylococcus aureus (S. aureus) was originally considered solely as an extracellular bacterium, but is now recognized additionally to invade host cells, which might be crucial for persistence. However, the intracellular fate of S. aureus is incompletely understood. Here, we show for the first time induction of selective autophagy by S. aureus infection, its escape from autophagosomes and proliferation in the cytoplasm using live cell imaging. After invasion, S. aureus becomes ubiquitinated and recognized by receptor proteins such as SQSTM1/p62 leading to phagophore recruitment. Yet, S. aureus evades phagophores and prevents further degradation by a MAPK14/p38α MAP kinase-mediated blockade of autophagy. Our study demonstrates a novel bacterial strategy to block autophagy and secure survival inside the host cell.     

Diverse modulation of spa transcription by cell wall active antibiotics in Staphylococcus aureus

ABSTRACT: BACKGROUND: The aim of this study was to investigate the effect of various classes of clinically relevant antibiotics at sub-lethal concentrations on virulence gene expression and biofilm formation in Staphylococcus aureus. FINDINGS: LacZ promoter fusions of genes related to staphylococcal virulence were used to monitor the effects of antibiotics on gene expression in a disc diffusion assay. The selected genes were hla and spa encoding alpha-hemolysin and Protein A, respectively and RNAIII, the effector molecule of the agr quorum sensing system. The results were confirmed by quantitative real-time PCR. Additionally, we monitored the effect of subinhibitory concentrations of antibiotics on the ability of S. aureus to form biofilm in a microtiter plate assay. The results show that sub-lethal antibiotic concentrations diversely modulate expression of RNAIII, hla and spa. Consistently, expression of all three genes were repressed by aminoglycosides and induced by fluoroquinolones and penicillins. In contrast, the beta-lactam sub-group cephalosporins enhanced expression of RNAIII and hla but diversely affected expression of spa. The compounds cefalotin, cefamandole, cefoxitin, ceftazidime and cefixine were found to up-regulate spa, while down-regulation was observed for cefuroxime, cefotaxime and cefepime. Interestingly, biofilm assays demonstrated that the spa-inducing cefalotin resulted in less biofilm formation compared to the spa-repressing cefotaxime. CONCLUSIONS: We find that independently of the cephalosporin generation, cephalosporins oppositely regulate spa expression and biofilm formation. Repression of spa expression correlates with the presence of a distinct methyloxime group while induction correlates with an acidic substituted oxime group. As cephalosporines target the cell wall penicillin binding proteins we speculate that subtle differences in this interaction fine-tunes spa expression independently of agr.     

Staphylococcus aureus host range and human-bovine host shift

Staphylococcus aureus is a major agent of bovine mastitis. The concomitant emergence of pig-associated methicillin-resistant S. aureus (MRSA) in human carriage and infection requires a reexamination of the host range and specificity of human- and cow-associated S. aureus strains, something which has not been systematically studied previously. The genetic relatedness of 500 S. aureus isolates from bovine mastitis cases, 57 isolates from nasal carriage of farmers, and 133 isolates from nonfarmers was determined by amplified fragment length polymorphism (AFLP) analysis and spa typing. Multilocus sequence typing (MLST) was conducted on a subset of isolates to match AFLP clusters with MLST clonal complexes (CCs). This data set allowed us to study host range and host specificity and to estimate the extent of bovine-to-human transmission. The genotype compositions of S. aureus isolates from farmers and nonfarmers were very similar, while the mastitis isolates were quite distinct. Overall, transmission was low, but specific genotypes did show increased cow-to-human transmission. Unexpectedly, more than one-third of mastitis isolates belonged to CC8, a lineage which has not been considered to be bovine mastitis associated, but it is well known from human carriage and infection (i.e., USA300). Despite the fact that we did detect some transmission of other genotypes from cows to farmers, no transmission of CC8 isolates to farmers was detected, except for one tentative case. This was despite the close genetic relatedness of mastitis CC8 strains to nonfarmer carriage strains. These results suggest that the emergence of the new bovine-adapted genotype was due to a recent host shift from humans to cows concurrent with a loss of the ability to colonize humans. More broadly, our results indicate that host specificity is a lineage-specific trait that can rapidly evolve.     

Staphylococcus aureus produces membrane-derived vesicles that induce host cell death

Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs) produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.     

Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death

Staphylococcus aureus is a common bacterial etiology of serious infectious diseases. S. aureus can invade various types of non-professional phagocytes to produce host cell death. We show here that shortly after invasion of HeLa cells S. aureus transit to autophagosomes was characterized by double membranes and co-localization with LC3. S. aureus were not able to replicate and produce cell death in autophagy-deficient atg5-/- mouse embryonic fibroblasts. S. aureus-containing autophagosomes do not acidify nor do they acquire lysosome-associated membrane protein-2, indicating that S. aureus inhibits autophagosome maturation and fusion with lysosomes. Eventually, S. aureus escape from autophagosomes into the cytoplasm, which results in caspase-independent host cell death. S. aureus strains deficient for agr, a global regulator of S. aureus virulence, were not targeted by autophagy and did not produce host-cell death. Autophagy induction by rapamycin restored both replication and cytotoxicity of agr-deficient S. aureus strains, indicating that an agr-regulated factor(s) is required for autophagy-mediated cytotoxicity. The results of this study suggest that rapid induction of autophagy is essential for S. aureus replication, escape into the cytoplasm, and host cell killing.     

Enhanced fibronectin-mediated cell adhesion of human osteoblast by fibroblast growth factor, FGF-2

Using specific recombinant human fibronectin peptide (hFNIII9-10) that contains the binding site for integrin, we found that the fibroblast growth factor, FGF-2, enhances fibronectin-mediated adhesion in human osteoblast-like MG63 cells. The mechanism of the synergistic adhesion was due to the activation of extracellular-regulated kinase (ERK)-type MAPK upon interaction of integrin to hFNIII9-10 and its downstream activation of signaling pathways.     

金黄色葡萄球菌逃逸宿主免疫的研究进展

金黄色葡萄球菌(Staphylococcus aureus)是一种能引起人和动物发生多种疾病的革兰阳性菌(Gram positive bacterium),并且金黄色葡萄球菌免疫逃逸(immune escap)是导致宿主持续性感染以及诱导细胞死亡的主要原因。现就金黄色葡萄球菌阻止自噬溶酶体(lysosome)的形成、阻碍中性粒细胞(neutrophils)的募集、吞噬作用及抑制巨噬细胞(macrophage)的免疫功能等作一概述。     

Hepatocyte adhesion on plastic : Different mechanisms for serum- and fibronectin-mediated adhesion

Hepatocytes adhere well on plastic in the presence of serum or fibronectin and subsequent spreading is not prevented when protein synthesis was blocked by cycloheximide. Protein synthesis-independent spreading was also observed in cultures containing serum depleted of fibronectin by affinity chromatography. This indicates that serum-mediated adhesion is independent of fibronectin and suggests the existence of an adhesion factor other than fibronectin in serum. The involvement of different membrane components for fibronectin- and serum-mediated adhesion was demonstrated by experiments where the different adhesion-inhibiting activities of antisera raised against plasma membranes of rat liver and Morris hepatoma 7777 (Neumeier et al., FEBS lett 168 (1984) 241-244) were used. Whereas anti-liver antibodies inhibited both types of adhesion, anti-hepatoma antibodies were only able to prevent fibronectin-mediated adhesion. This indicates again that two different mechanisms are responsible for fibronectin- and serum-mediated adhesion. Fractionation of fetal calf serum (FCS) by size exclusion HPLC revealed that proteins of molecular weights of 60-80 kD promoted attachment and spreading of hepatocytes. Spreading was not perturbated by anti-hepatoma antibodies, indicating that an adhesion factor of 60-80 kD is responsible for serum-mediated adhesion. 'Serum-spreading factor', also called vitronectin, from human plasma has been described as having a similar molecular weight. The purified factor was found to mediate hepatocyte adhesion which was not inhibited by anti-hepatoma antibodies. This suggests that serum-mediated adhesion depends on an adhesion factor present in FCS, which is similar to or identical with vitronectin.