金黄色葡萄球菌凝集因子A的免疫原性

为研究金黄色葡萄球菌(Staphylococcus aureus)凝集因子A(ClfA)免疫原性及免疫保护作用,应用PCR方法扩增出金黄色葡萄球菌Newman、Wood46和HLJ23-1株的clfa基因并进行序列分析,再将Newman株的clfa基因插入到pQE-30载体上,导入宿主菌Escherichia coli M15(pREP4)并诱导表达和纯化ClfA重组蛋白。用纯化的ClfA免疫小鼠,检测血清中抗体和细胞因子水平,首次免疫后35 d时用金黄色葡萄球菌Wood46、HLJ23-1、Newman株对小鼠攻毒。结果发现:clfa基因序列高度保守;ClfA重组蛋白在E.coli M15中获得表达;在首次免疫后35 d时血清抗体效价和细胞因子浓度与对照组相比,均显著升高(P<0.05);攻毒结果为蛋白免疫组小鼠获得一定的免疫保护。由此表明,ClfA重组蛋白有较好的免疫原性和免疫保护力。     

New method for typing Staphylococcus aureus resistant to methicillin based on sulphur-35 methionine labelled proteins: its application in an outbreak.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis of sulphur-35 methionine labelled cellular proteins of Staphylococcus aureus resistant to methicillin was used as a typing method during an outbreak on a cardiothoracic ward. This showed that the outbreak strain was indistinguishable from the epidemic strain of methicillin resistant S aureus prevalent in London. In contrast, 44 epidemiologically separate strains of S aureus gave individually distinct radiolabelled protein profiles. This method permitted rapid confirmation that an epidemic strain was responsible and indicated the need for urgent control measures.     

Structural evidence of α-aminoacylated lipoproteins of Staphylococcus aureus

Bacterial lipoproteins are known to be diacylated or triacylated and activate mammalian immune cells via Toll-like receptor 2/6 or 2/1 heterodimer. Because the genomes of low G+C content gram-positive bacteria, such as Staphylococcus aureus, do not contain Escherichia coli-type apolipoprotein N-acyltransferase, an enzyme converting diacylated lipoproteins into triacylated forms, it has been widely believed that native lipoproteins of S. aureus are diacylated. However, we recently demonstrated that one lipoprotein SitC purified from S. aureus RN4220 strain was triacylated. Almost simultaneously, another group reported that another lipoprotein SA2202 purified from S. aureus SA113 strain was diacylated. The determination of exact lipidated structures of S. aureus lipoproteins is thus crucial for elucidating the molecular basis of host-microorganism interactions. Toward this purpose, we intensively used MS-based analyses. Here, we demonstrate that SitC lipoprotein of S. aureus RN4220 strain has two lipoprotein lipase-labile O-esterified fatty acids and one lipoprotein lipase-resistant fatty acid. Further MS/MS analysis of the lipoprotein lipase digest revealed that the lipoprotein lipase-resistant fatty acid was acylated to α-amino group of the N-terminal cysteine residue of SitC. Triacylated forms of SitC with various length fatty acids were also confirmed in cell lysate of the RN4220 and Triton X-114 phase in three other S. aureus strains, including SA113 strain and one Staphylococcus epidermidis strain. Moreover, four other major lipoproteins including SA2202 in S. aureus strains were identified as N-acylated. These results strongly suggest that lipoproteins of S. aureus are mainly in the N-acylated triacyl form.     

Cloning of lysozyme and lysostaphin genes of Staphylococcus aureus and their expression in Bacillus subtilis cells

The gene of microbial lysozyme (lyz) of S. aureus 118 and the gene of lysostaphin (lzf) of S. aureus RN 3239 were cloned and their expression in B. subtilis cells was shown. Lysozyme production in B. subtilis recombinant clone pLF14-Lyz, obtained as the result of cloning, was 2.5-fold greater than lysozyme production in S. aureus wild strain 118. Lysostaphin production in B. subtilis recombinant strain pLF14-Lzf which had inherited the cloned genes was approximately equal to lysostaphin production observed in S. aureus initial strain RN 3239. The production of lysozyme and lysostaphin in the cells of B. subtilis recombinant strains was observed at 30 degrees C and pH 5.5, while in S. aureus initial strains 118 and RN 3239 bacteria produced lysozyme and lysostaphin at 37 degrees C and pH 7.5 respectively.     

产SEB金黄色葡萄球菌α-溶血毒素基因缺失菌株的构建

构建产肠毒素B(Staphylococcal enterotoxin B ,SEB)的金黄色葡萄球菌α-溶血毒素（α-hemolysin, α-HL)缺失菌株。首先构建用于α-HL基因敲除的同源重组质粒pMHL-α,经金黄色葡萄球菌RN4220修饰后再通过原生质体转入金黄色葡萄球菌SM-01。含重组质粒pMHL-α的金黄色葡萄球菌SM-01在42℃诱导条件下培养多代,最终筛选出α-溶血毒素基因缺失菌株。经序列分析和血平板溶血实验结果证明最终获得产SEB金黄色葡萄球菌α-HL缺失菌株。为野生型金黄色葡萄球菌的体内遗传操作及构建产超抗原药物金黄色葡萄球菌基因工程菌株提供了一定的理论基础和方法。     

A comparative study of the IgG-binding components of the membrane in different strains of Staphylococcus aureus using the indirect hemagglutination reaction and an immunofluorescence method

A comparative estimation of IgG-binding activity of 85 S. aureus clinical strains was carried out by the method of indirect hemagglutination reaction. The S. aureus strain selected as a result of screening was found to exceed by more than an order the Cowan I strain obtained from the L. A. Tarasevich State Institute of Standards and Control of the Medical Biological Drugs in the IgG-binding activity. It was established that the ratio of two types of IgG-binding sites located on the S. aureus surface, varied depending on the strain, composition and quality (liquid or solid) of the culture medium.     

Whole-genome sequence of Staphylococcus aureus strain LCT-SA112

Staphylococcus aureus is a facultative anaerobic Gram-positive coccal bacterium. S. aureus is the most common species of Staphylococcus to cause staphylococcal infections, which are very common in clinical medicine. Here we report the genome sequence of S. aureus strain LCT-SA112, which was isolated from S. aureus subsp. aureus CGMCC 1.230.     

金黄色葡萄球菌hmp基因缺失突变株的构建及抗一氧化氮能力分析

摘要：【目的】:构建金黄色葡萄球菌RN6390黄素血红蛋白(flavohaemoglobin, HMP)基因缺失突变株,研究其抗一氧化氮(Nitric Oxide, NO) 能力及其在细菌生物被膜形成中的作用。【方法】：根据同源重组技术的原理,利用PCR扩增RN6390的hmp基因上下游同源臂,经过抗生素和温度交替培养筛选hmp基因缺失突变株,利用基因组PCR、定量PCR对突变菌株进行鉴定。以硝普钠（SNP）为NO供体,检测了hmp基因缺失菌株的抗NO能力,并初步研究了hmp基因在生物被膜形成中的作用。【结果】：成功构建了RN6390的hmp基因缺失突变株,外源NO能够诱导菌株hmp基因的表达,hmp基因缺失菌株抗NO能力明显下降,但其生物被膜形成能力有明显提高。【结论】：获得了RN6390的hmp基因缺失突变株,该突变株的获得为进一步研究hmp基因的生物功能,以及细菌内源性NO的作用奠定了良好的技术平台。     

Mercury and tetracycline resistance genes and flanking repeats associated with methicillin resistance on the chromosome of Staphylococcus aureus

Sections of a cloned 27 kb segment of chromosomal DNA, associated with resistance to four antimicrobial agents in a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), were tested for their ability to determine resistance when transformed into a sensitive laboratory strain of S. aureus. This was achieved by inserting the sections into a newly constructed shuttle vector, amplifying the recombinant DNA in E. coli, and transforming protoplasts of the sensitive S. aureus strain. Two sections of the cloned DNA were found to determine resistance separately to mercuric ion and to tetracycline, in both S. aureus and Escherichia coli.     

Characterization of Staphylococcus aureus in a Pediatric Burn Unit

A one-year study on an endemic strain of Staphylococcus aureus phage type 84/85 in a children's burn unit is described. The endemic strain rapidly colonized the burns and nares of acute patients after admission but was not isolated from a patient on admission. Nonendemic strains of S. aureus found on some new patients were mostly non-phage typable and did not prevail in burns. The endemic strain was rarely isolated from the nares and skin of reconstructive patients or from the nares of hospital personnel. The endemic strain did colonize the oral cavity, normal skin, and intestinal tract of some acute patients. Endemic and nonendemic strains of S. aureus from the burned children were compared in their biochemical activities and antibiotic sensitivities to two groups of S. aureus from one other local and one Danish burns unit. The latter groups of strains represented different combinations of staphylococcal phage group III strains. Each of the four groups of strains differed in production of hemolysins, Tween 80 hydrolysis, egg yolk reaction, and proteolysis of casein and gelatin. All of the strains were uniformly sensitive to gentamicin, oxacillin, and cephalothin. Only 4 of 162 strains tested were methicillin resistant. The endemic S. aureus strains of phage type 84/85 were uniformly resistant to eight other antibiotics including lincomycin and clindamycin. The endemic strain was not the known cause of a clinically documented infection in a group of 82 acute patients studied. The possible role of S. aureus strains of phage group III in burn grafting problems is discussed.     

Two thermostable nucleases coexisted in Staphylococcus aureus: evidence from mutagenesis and in vitro expression

Thermostable nuclease is known to be an important pathogenic factor unique to Staphylococcus aureus and it is commonly presumed to have had the same genetic origin. However, two ORFs in S. aureus genomes were predicted to encode nucleases. One encoded an unnamed nuclease A (SNase) (termed nuc1 ), and the other encoded a thermonuclease (TNase) named nuc (termed nuc2 ). In order to verify whether the two thermostable nuclease proteins are coexpressed in S. aureus , the nuc1 and nuc2 genes were cloned and expressed in Escherichia coli , and both of the recombinant proteins showed thermostable nuclease activity in a toluidine blue-DNA assay. Furthermore, a nuc1 -deleted mutant of S. aureus strain RN4220 (termed RNΔ nuc1 ) was successfully constructed by homologous recombination. Selection and characterization of this mutant strain revealed that it still exhibited thermostable nuclease activity, but at a relative lower level than that of the parent strain. The nucleases secreted by the parent strain and nuc1 -deleted strain still showed functional activity after 30 min at 121 °C. The findings indicated that two types of thermostable nucleases, encoded by two different genes, coexisted in S. aureus .     

Different effects of fibronectin on the phagocytosis of Staphylococcus aureus and coagulase-negative staphylococci by murine peritoneal macrophages

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase-negative staphylococci (CNS) strains for comparison. Fibronectin-reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose-dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin-binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.     

Identification of Staphylococcal Hemolysins by an Electrophoretic Localization Technique

A technique for identifying and characterizing staphylococcal hemolysins by first separating them electrophoretically in barbital-buffered agar gel (pH 8.4) at 5 ma/cm for 2 hr and then determining their hemolytic activities by exposing them to human, horse, rabbit, and sheep erythrocytes is described. The alpha-hemolysin produced by a White variant of the Wood 46 strain of Staphylococcus aureus migrated 18 mm towards the cathode, and it lysed horse, rabbit, and sheep erythrocytes, whereas a Clear variant of the Wood 46 strain of S. aureus produced a lysin which migrated similarly to the alpha-hemolysin but lysed only rabbit cells. This latter lysin was tentatively named alpha(1)-lysin. This strain of S. aureus also produced beta-hemolysin which migrated 36 mm towards the cathode and lysed sheep cells. beta-Hemolysin produced by some strains of S. aureus showed considerable tailing during electrophoresis, whereas beta-hemolysin produced by other strains of S. aureus migrated as a well-defined peak. A lysin migrating 11 mm towards the anode was probably delta-lysin. It was, however, not produced in sufficient concentration when the cultures were grown in semisolid medium.     

The combined effects of plasma and hydrogel coating on adhesion of Staphylococcus epidermidis and Staphylococcus aureus to polyurethane catheters

Abstract The adhesion of three Staphylococcus epidermidis and three S. aureus clinical isolates, to uncoated and hydrogel-coated polyurethane catheters was tested, following pretreatment of catheters with human plasma. Plasma significantly decreased the adhesion of S. epidermidis strains to uncoated polyurethane catheters, but had no significant effect on the adhesion to hydrogel-coated catheters. The influence of plasma on adhesion of S. aureus strains to catheters was strain dependent. Plasma significantly increased the adhesion of one strain (SA6) to uncoated catheters. For two other strains (SA3 and SA14) plasma produced no clear effect on their adhesion to uncoated catheters; adhesion values for each strain showed either a small but significant increase or a replicate-dependent increase or decrease. However, plasma significantly increased the adhesion of all S. aureus strains to hydrogel-coated polyurethane catheters. Overall, with the exception of one batch culture of S. epidermidis strain SE3 tested, attachment to plasma-treated hydrogel-coated catheters was statistically significantly lower, by up to 85%, than attachment to plasma-treated uncoated catheters for both S. epidermidis and S. aureus .     

安徽不同地区奶站牛奶中金黄色葡萄球菌及其肠毒素污染状况评估

目的评定安徽地区各奶站牛奶中金黄色葡萄球菌以及肠毒素的污染情况。方法通过从安徽省不同地区30所奶站采集乳样,进行乳源性金黄色葡萄球菌的分离与生化鉴定,并采用PCR技术对分离出的菌株进行金黄色葡萄球菌肠毒素血清型鉴定。结果安徽省30个奶站中有4个地区奶站的牛奶中污染金黄色葡萄球菌;从污染牛奶中共分离出5株金黄色葡萄球菌,检出率为16.7%。经鉴定,所分离出的金黄色葡萄球菌中2株为肠毒素A型,1株为肠毒素C型,2株为同时产肠毒素A和肠毒素C。结论安徽省不同地区奶站中的牛奶污染的金黄色葡萄球菌产肠毒素类型以肠毒素A为主。     

Characterisation of Staphylococcus aureus nasal and skin carriage among patients undergoing haemodialysis treatment

The aim of the study was to investigate the rate of Staphylococcus aureus nasal and skin carriage in patients undergoing haemodialysis. The cultured staphylococcal isolates were subsequently characterized by molecular methods. The study group comprised 43 haemodialysed patients from whom nasal and skin swabs from the vascular access sites were collected. The identification of staphylococcal isolates and antibiotic susceptibility testing were performed on the basis of conventional diagnostic procedures. The staphylococci were further characterized using Pulsed-Field Gel Electrophoresis (PFGE). S. aureus was cultured from 12 (27.9%) patients. Only one (8.3%) patient was colonized with the microorganism both in the anterior nares and the vascular access site representing a single strain, as evidenced by PFGE analysis. Antibiotic susceptibility testing identified one (7.6%) methicillin-resistant S. aureus (MRSA) strain. PFGE typing identified several S. aureus genotypes with the lack of one specific strain responsible for colonization. However, it should be noted that among two (A and D) PFGE patterns genetically indistinguishable and closely related isolates (two isolates for each pattern) were identified. The obtained results revealed a relatively low rate of S. aureus carriage accompanied by low methicillin resistance rate and a significant genetic diversity of cultured isolates with the lack of one predominant strain responsible for colonization.     

Prevention of diseases caused by Staphylococcus aureus using the peptide RIP

Staphylococcus aureus causes many diseases including cellulitis, keratitis, osteomyelitis, septic arthritis and mastitis. The heptapeptide RIP has been shown to prevent cellulitis in mice, which was induced by S. aureus strain Smith diffuse. Here we show that RIP can also significantly reduce the overall pathology and delay the onset of disease symptoms in several other models of S. aureus infections, including: keratitis (tested in rabbits against S. aureus 8325-4), osteomyelitis (tested in rabbits against S. aureus MS), mastitis (tested in cows against S. aureus Newbould 305, AE-1, and environmental infections) and septic arthritis (tested in mice against S. aureus LS-1). These findings substantiate that RIP is not strain specific in its inhibitory activity and that RIP is an effective inhibitor of bacterial pathology at multiple body sites following diverse routes and doses of administration. These findings strongly evidence the potential value of RIP as a chemotherapeutic agent.     

Chemical characterization of a new surface antigenic polysaccharide from a mutant of Staphylococcus aureus

A mutant of Staphylococcus aureus H was isolated by virtue of its inability to agglutinate with antibodies against teichoic acid of S. aureus. Immunological studies revealed that the mutant, S. aureus T, possessed a new surface antigen in addition to having the antigenic determinant of the wild-type strain, the ribitol teichoic acid. The presence of this additional surface component rendered strain T resistant to staphylococcal typing phages, presumably by masking the phage-receptor sites. The polymer was separated from teichoic acid by chromatography on diethylaminoethyl cellulose and was shown to be composed of two amino sugars, N-acetyl-d-fucosamine and N-acetyl-d-mannosamin uronic acid.     

Functional characterization of lipase in the pathogenesis of Staphylococcus aureus

During infection, Staphylococcus aureus produces multiple enzymes that enable it to invade and destroy host tissues and metastasize to other sites. One such enzyme, lipase, has been recognized for its relationship in the virulence of S. aureus. However, a direct involvement of lipase in the pathogenesis of S. aureus remains to be demonstrated. Our prior study indicated that anti-lipase serum inhibits biofilm formation in S. aureus clinical strains. The aim of this study was to further characterize the roles of lipase in the pathogenesis in S. aureus. We found that deletion of the lipase-coding gene reduced biofilm formation relative to the wild-type strain. This was shown by culture in 96-well plates coated with collagen to resemble the in vivo infection process. Intraperitoneal inoculation of mice with a lipase mutant strain showed defective formation of peritoneal abscesses, and bacterial loads in different organs were much lower compared with the wild-type. Importantly, active immunization with recombinant lipase protected mice against a lethal challenge with S. aureus. Altogether, our data provide evidence that S. aureus lipase plays important roles in the pathogenesis of S. aureus.