Thickening of the cell wall in macrolide-resistant Staphylococcus aureus

Macrolides are widely used at low dosage for long-term therapy of chronic sinusitis. Twenty clinical macrolide-resistant Staphylococcus aureus strains were morphologically compared with 10 clinical macrolide-sensitive strains. PCR amplification was performed to determine the presence of four known macrolide resistance genes. Transmission electron microscopy revealed significantly thicker cell walls in clinical macrolide-resistant strains. Even though the ultrastructural characteristics were shared by all macrolide-resistant strains, they were not associated with the presence or absence of the known macrolide-resistance genes. We also demonstrated that macrolide-resistant mutant strains derived in vitro from a macrolide-sensitive parent strain had thickened cell walls and did not harbor the known macrolide-resistance genes. These results, therefore, revealed that macrolide-resistant S. aureus strains have thickened cell walls as a common ultrastructural characteristic and that cell wall thickening is likely mediated by an unknown gene which is unrelated to any known macrolide resistance gene.     

金黄色葡萄球菌胞壁成分在细菌性眼内炎中的致病作用

目的:观察并比较金黄色葡萄球菌胞壁及其成分在大鼠眼内的致炎特性。方法:将大鼠随机分为热灭活细菌组(96只,注射热灭活细菌10μg)、热灭活细菌碎片组(96只,注射热灭活细菌碎片10μg)、肽聚糖组(96只,注射肽聚糖10μg)和对照组(96只,注入等量无菌生理盐水)。在玻璃体腔注入药物后6、12、24、48、72h和5d,裂隙灯显微镜下进行眼部炎症评分,组织病理学切片进行眼内浸润白细胞计数,并测定玻璃体内肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β及中性粒细胞趋化因子(CINC)-1的表达水平和前房水蛋白质浓度。结果:在注射后6-72h,热灭活细菌组、热灭活细菌碎片组及肽聚糖组眼内均可见严重的炎症反应,至术后5d炎症基本消退。各组眼内白细胞的浸润数量在术后24h达到高峰[热灭活细菌组(207.1±33.7)细胞/眼;热灭活细菌碎片组(514.2±116)细胞/眼,与热灭活细菌组比较,P0.05;肽聚糖组(412.6±191)细胞/眼,与热灭活细菌组比较,P0.05],至术后3d浸润细胞数量迅速下降。各组TNF-α的浓度在术后24h均达到高峰[热灭活细菌组(178.3±76.5)ng/L;热灭活细菌碎片组(353.2±141.3)ng/L,与热灭活细菌组比较,P0.05;肽聚糖组(311.2±99.8)ng/L,与热灭活细菌组比较,P0.05],并持续至术后48h,随后迅速下降,并在术后5d回复至基线水平。各组IL-1β浓度在术后24h均达到高峰[热灭活细菌组(637.8±277.7)ng/L;热灭活细菌碎片组(1922.0±670.3)ng/L,与热灭活细菌组比较,P0.05;肽聚糖组(1517.0±420.2)ng/L,与热灭活细菌组比较,P0.05],并持续至术后48h,随后迅速下降,并在术后5d回复至基线水平。各组CINC-1的浓度在术后12h均达到高峰[热灭活细菌组(1173.3±221.5)ng/L;热灭活细菌碎片组(1864.0±403.4)ng/L,与热灭活细菌组比较,P0.05;肽聚糖组(2536.0±386.3)ng/L,与热灭活细菌组比较,P0.05],并持续至术后24h,随后迅速下降,并在术后3d回复至正常水平。在各观察时点,均可观察到眼内炎组房水蛋白质浓度较对照组显著增高(P0.01)。结论:金黄色葡萄球菌胞壁及其成分可在SD大鼠诱导出典型的实验性眼内炎。大量白细胞眼内浸润、血眼屏障的破坏、TNF-α和IL-1β的高水平表达是实验性眼内炎模型的主要病理特点。与完整的热灭活金黄色葡萄球菌比较,细菌碎片及其成分肽聚糖具有更强烈的眼内促炎能力。     

Role of Staphylococcus aureus cell wall antigens in the stimulation of delayed hypersensitivity after staphylococcal infection.

In Staphylococcus aureus-infected mice, prior treatment with cyclophosphamide enhanced delayed hypersensitivity to cell walls and peptidoglycan. S. aureus did not cross react with S. epidermidis or S. saprophyticus.     

Ultrastructural cell wall characteristics of clinical gentamycin-resistant Staphylococcus aureus isolates

The frequent use of gentamycin (GM) ointment for the treatment of skin infections has led to an increase in the number of GM-resistant clinical isolates of Staphylococcus aureus. We examined the ultrastructural characteristics of 14 clinical strains of S. aureus by transmission electron microscopy. Seven of these isolates were GM-resistant, and seven isolates were GM-sensitive. We found that the cell wall of GM-resistant strains (32.24 ± 5.99 nm) was significantly thicker than that of GM-sensitive strains (19.02 ± 2.72 nm). We genetically characterized these isolates by polymerase chain reaction, targeting the genes for three aminoglycoside-modifying enzymes, aac(6′)-aph(2′′), aph(3′)-III, and ant(4′)-I. All GM-resistant strains tested carried the gene encoding aac(6′)-aph(2′′). However, we were unable to establish a link between a specific gene and cell wall thickening, because one GM-resistant strain was also positive for aph(3′)-III. We also demonstrated that a GM-resistant mutant strain, derived in vitro from a GM-sensitive S. aureus parent strain (209P), also exhibited a thickened cell wall. These results strongly suggest that a thickened cell wall is a common ultrastructural characteristic of GM-resistant S. aureus clinical strains.     

Interactions between human plasma proteins and cell wall components of Staphylococcus aureus

Staphylococcus aureus has surface structures with affinity to human IgG, fibrinogen, and fibronectin. Besides the binding of the Fc-terminal part of IgG from a range of mammalian species, S. aureus protein A binds some IgM, IgA, and IgE molecules. Furthermore, it seems also able to bind immunoglobulins via their Fab-terminal parts. Protein A (Mr 42,000) is the only well-characterized S. aureus cell wall protein, and its structure is known in detail. A considerable number of biological properties of protein A has been demonstrated. Most of these properties seem to be a consequence of the complement activation induced by protein A-IgG complexes. The role of protein A in the phagocytosis of S. aureus is complex. By complement consumption protein A has been found to inhibit the phagocytosis of staphylococci by polymorphonuclear leucocytes. However, it has been demonstrated that protein A-containing staphylococci bind to surface IgG on human alveolar and peritoneal macrophages and thereby promote phagocytosis by these cells. This phenomenon might explain the increased virulence of S. aureus in the presence of human IgG in experimental peritonitis in mice. Fibrinogen binds to a surface structure on S. aureus, designated clumping factor as the binding results in clumping of whole bacteria. Recently, a glycoprotein (Mr of about 400,000) has been isolated from S. aureus. This glycoprotein seems to be the clumping factor. It binds to fibrinogen, inhibits the fibrinogen induced clumping, and seems to be a S. aureus specific, surface component. The isolated component activates human complement in vitro. Also, it induces protection against S. aureus peritonitis in immunized mice. The presence of fibrinogen and an unknown human plasma component increases the virulence of S. aureus in experimental peritonitis in mice, but the role of fibrinogen in human S. aureus infection is unknown. Fibronectin binds to a surface protein on S. aureus, and this binding also results in the clumping of the bacteria. The binding site(s) for fibronectin is different from the binding sites for fibrinogen and IgG. A fibronectin-binding protein (Mr 197,000) has been isolated from S. aureus by affinity chromatography. This protein binds fibronectin and inhibits the fibronectin induced S. aureus clumping. No other biological properties of this protein have yet been demonstrated. The binding of fibronectin to S. aureus opsonize the bacteria for polymorphonuclear leucocytes. The opsonic capacity is, however, low compared to other serum opsonins. It has been suggested that fibronectin plays a role in the attachment of S. aureus, but further studies are needed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Staphylococcus aureus antigens and challenges in vaccine development

Staphylococcus aureus is an important cause of nosocomial and community-acquired infections in humans and animals, as well as mastitis in dairy cattle. Methicillin-resistant S. aureus is increasingly recognized as a cause of staphylococcal infection and, therefore, immunotherapeutics have received new interest in both human and veterinary medicine. Vaccines aimed at preventing S. aureus infection in humans and mastitis in dairy cattle have been studied for many years. While some formulations have shown promise in ameliorating clinical disease, few, if any, of the S. aureus vaccines developed have adequately prevented new infection. The antigens targeted by S. aureus vaccines and potential reasons for the lack of success of vaccination against S. aureus are reviewed in this article.     

Arylamine N-acetyltransferase activity in Staphylococcus aureus

N-Acetyltransferase (NAT) activities were determined by incubation of Staphylococcus aureus cytosols with p-aminobenzoic acid (PABA) or 2-aminofluorene (2-AF) followed by high pressure liquid chromatography assays. The NAT activities from S. aureus were found to be 0.67 +/- 0.04 nmol/min/mg protein for the acetylation of 2-AF and 0.46 +/- 0.02 nmol/min/mg protein for the acetylation of PABA. The apparent K(m) and Vmax values obtained were 2.85 +/- 0.65 mM and 7.51 +/- 0.86 nmol/min/mg protein for 2-AF, and 2.35 +/- 0.39 mM and 9.43 +/- 0.78 nmol/min/mg protein for PABA, respectively. The optimal pH value for the enzyme activity was 7.0 for both substrates tested. The optimal temperature for enzyme activity was 37 degrees C for both substrates. The NAT activity was inhibited by iodoacetamide at 0.25 mM, and activity was reduced 50%. At 1.0 mM iodoacetamide activity was inhibited more than 90%. Among a series of divalent cations and salts, Zn2+, Ca2+, and Fe2+ were demonstrated to be the most potent inhibitors. The molecular weight of NAT from S. aureus was found to be 44.9 kDa. This report is the first demonstration of acetyl CoA: arylamine NAT activity in S. aureus.     

Lipoprotein is a predominant Toll-like receptor 2 ligand in Staphylococcus aureus cell wall components

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of Toll-like receptor 2 (TLR2). In this study, we demonstrated that lipoproteins obtained from S. aureus are potent activators of TLR2. A fraction obtained by Triton X-114 phase partitioning activated cells through TLR2. The fraction contained proteins and LTA. The activity was detected in compounds in a mass range of 12-40 kDa. Proteinase K digested the active compounds into lower molecular weight active materials <10 kDa. In contrast, hydrofluoric acid treatment, which decomposes LTA, did not alter the molecular mass of the active compounds. Further, most of the activity was abrogated by lipoprotein lipase digestion. These results suggested that lipoproteins are predominant TLR2 ligands in S. aureus cell wall components.     

Defining Staphylococcus epidermidis cell wall proteins

Three Staphylococcus epidermidis isolates of differing bacteriophage types were studied to define proteins confined to the cell wall, which were surface exposed and thus available to interact with the host. Three major proteins of 37, 41, and 51 kDa were identified in all whole-cell lysates and cell wall extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Two additional proteins of 18 and 25 kDa became evident by using 125I labeling to delineate surface-exposed proteins. A classification scheme using P1 to P5 to delineate the 51-, 41-, 37-, 25- and 18-kDa proteins is proposed. Additionally, murine immune sera were used to identify two immunodominant proteins of 51 and 25 kDa (P1 and P4, respectively).     

Detection of cell surface rubella virus antigens in Vero cells with Staphylococcus aureus rich in protein A

The presence of cell surface rubella antigen was used to verify and monitor viral replication in Vero cell monolayers. Viral antigen was observed in infected cells by the adherence of Staphylococcus aureus sensitized with immune anti-rubella sera. The staphylococci specifically bound to infected cells were Gram-stained and observed using light microscopy. The minimum titer of IgG antiviral hemagglutinin required for sensitizing the bacteria was 3.9 IU/ml. The specificity of the assay was demonstrated by treating the infected cells with bacteria sensitized with normal sera, by treating the mock-infected cells with staphylococci sensitized with either immune or normal sera, and by sensitizing the bacteria with immune sera from which anti-rubella antibodies had been removed. Viral antigens were detected from day 2-9 post-infection. The sensitivity of the assay in verifying and monitoring viral propagation was comparable to the titration of viral particles of hemagglutination. The assay is specific, rapid, simple and can be performed in laboratories with minimal equipment.     

The wall teichoic acid and lipoteichoic acid polymers of Staphylococcus aureus

Staphylococci and most other Gram-positive bacteria incorporate complex teichoic acid (TA) polymers into their cell envelopes. Several crucial roles in Staphylococcus aureus fitness and cell wall maintenance have been assigned to these polymers, which are either covalently linked to peptidoglycan (wall teichoic acid, WTA) or to the cytoplasmic membrane (lipoteichoic acid, LTA). However, the exact TA structures, functions, and biosynthetic pathways are only superficially understood. Recently, most of the enzymes mediating TA biosynthesis have been identified and mutants lacking or with defined changes in WTA or LTA have become available. Their characterization has revealed crucial roles of TAs in protection against harmful molecules and environmental stresses; in control of enzymes directing cell division or morphogenesis and of cation homeostasis; and in interaction with host or bacteriophage receptors and biomaterials. Accordingly, several in vivo studies have demonstrated the importance of WTA and LTA in S. aureus colonization, infection, and immune evasion. TAs and enzymes required for TA biosynthesis represent attractive candidates for novel vaccines and antibiotics and are targeted by recently developed antibacterial therapeutics.     

Bactericidal activity of antiseptics against methicillin-resistant Staphylococcus aureus.

Various commonly used antiseptics were tested against three strains of methicillin-resistant Staphylococcus aureus (MRSA) at stock strength and in serial 10-fold dilutions. The stock solutions of 4% chlorhexidine gluconate-alcohol (Hibiclens), 1% p-chloro-m-xylenol (Acute-Kare), and 3% hexachlorophene (Phisohex) produced 2-log reductions of MRSA after a 15-s exposure, but even after 240 s, these solutions failed to kill all the MRSA. Povidone-iodine (Betadine) solution was maximally effective at the 1:100 dilution, killing all the MRSA within 15 s; other dilutions were less effective, though each killed the MRSA within 120 s. Similar results were obtained with three different strains of methicillin-susceptible S. aureus. Thus, of the four most commonly used antiseptics, povidone-iodine, when diluted 1:100, was the most rapidly bactericidal against both MRSA and methicillin-susceptible S. aureus.     

Staphylococcus aureus host cell invasion and post-invasion events

Staphylococcus aureus is now recognized as a facultative intracellular pathogen. The aim of this review is to discuss novel data regarding the invasion mechanism and post-invasion events with a focus on the fate of the infected phagosome in non-professional phagocytes and the role of S. aureus α-toxin.     

Comparative bactericidal activity of fourteen antibiotics against Staphylococcus aureus

The bactericidal activity of LY 146032 (LY), Oxacillin (OXA), Cefamandole (CEF), Rifampin (RIF), Gentamicin (GEN) or Tobramycin (TOB), Pefloxacin (PEF), Vancomycin (VAN), Teicoplanin (TEI), Pristinamycin (PRI) was compared against 8 strains of S. aureus (4 Meth. sensitive, 4 Meth. resistant). Kill Kinetics studies were done: bacteria were incubated with antibiotics at their MICs, 2 X MIC, 4 X MIC and at concentrations obtained in vivo with usual therapeutic doses. With 4 X MIC, a 3 Log reduction of the initial inoculum was observed only at 24 h with OXA, CEF (MSSA), RIF, PEF, VAN, at 30 h with TEI and PRI. With LY, GEN or TOB at 2 X MIC the 3 Log reduction was observed at 3 h or 4 h, a greater than or equal to 5 Log reduction at 24 h: LY and Aminoglycosides are the most bactericidal antibiotics against S. aureus.     

Variation in the expression of cell wall proteins of Staphylococcus aureus grown on solid and liquid media.

To evaluate the variation in the expression of cell wall antigens between Staphylococcus aureus grown in liquid medium and solid support, bacteria were harvested from liquid chemically defined medium and chemically defined medium in a 1% agar base. Cell wall proteins were then extracted by lysostaphin in a protoplast-stabilizing medium (30% raffinose). After separation of the cell wall antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots, they were probed with chicken antiserum to an S. aureus strain grown on a solid support. For each of the 15 clinical strains analyzed, high-molecular-size bands (molecular size range, 120 to 220 kilodaltons) were either enhanced or distinctly present when compared with those from the cell wall extract of the same strain grown in liquid medium. Results of enzymatic treatment of whole staphylococci grown on solid medium suggested the proteinaceous nature and the surface location of these antigens. Limited passage studies demonstrated the ability of the staphylococci to alter these surface proteins when passaged alternately on liquid and solid media. These observations suggested the importance of the microenvironment to the expression of cell wall proteins in S. aureus. Correlations with observations in vivo may help identify the determinants of microbial pathogenicity in S. aureus.     

Specific and cross-reacting antigens of Staphylococcus aureus of human and canine origins.

Biotype -specificity of Staphylococcus aureus of human and canine origins has been found to be associated with thermolabile agglutinogens represented in S. aureus strains 17 and 61218, respectively. Both strains also have exhibited a common thermostable antigen. On that basis, absorbed antisera have been developed for the differentiation of S. aureus of the two biotypes. In the present study, still another thermostable agglutinogen was established, shared by strain 17 and some S. aureus strains of canine origin, as represented by S. aureus strain 887. These findings led to modification and enhanced specificity of the serological method of distinguishing S. aureus of the human biotype from S. aureus of the canine biotype.     

A method of solid-phase analysis of antigens using Staphylococcus aureus as the label

A new method of solid-phase analysis of viral antigens in which St. aureus, strain Cowan 1, was used as a label is described. The result of analysis is read visually by bacterial agglutination. The degree of agglutination of the staphylococcal diagnosticum corresponds to the amount of the antigen bound with antibodies immobilized on the solid phase. Staining of Staphylococcus with methylene blue simplifies reading of the results. The method has been shown to be sufficiently simple, specific, sensitive, and may be used for diagnosis of different infections diseases.