用序列比对设计的茎环结构探针检测金黄色葡萄球菌

基于金黄色葡萄球菌16S rRNA基因序列,采用序列比对设计了一种茎环结构的寡聚核苷酸探针。探针的环序列即为金黄色葡萄球菌16S rRNA基因序列的其中一个片段,同其他菌种的16S rRNA基因序列误配2个以上的核苷酸,因此能高度专一、灵敏的检测金黄色葡萄球菌16S rRNA。根据分子信标技术和酶联免疫分析的原理,评估一个实验方法,即利用能构象转换的、固定化的茎环结构探针酶联检测靶核酸。由于探针的特异性加强,这个检测系统能有效的排除假阳性即不会出现误配一个核苷酸的情况。采用微量浓度测定分析,最低下限可检测出大约4ng的金葡球菌16SrRNA。这种方法的灵敏度比其他常规检测方法高出了至少一个数量级。     

Sensitive enzyme-amplified electrical immunoassay for protein A-bearing Staphylococcus aureus in foods

An amperometric electrochemical immunoassay specific for protein A-bearing Staphylococcus aureus was developed. The method was based on a sandwich immunosorbent assay and incorporated an enzyme amplification step, using a NAD-specific redox cycle generating NADH (C. H. Stanley, A. Johannsson, and C. H. Self, J. Immunol. Methods 83:89-95, 1985). Reduction of the mediator, ferricyanide, was dependent on the initial concentration of antigen. The final potential was measured by using a Pt disk electrode polarized at +0.8 V to the Ag/AgCl reference electrode. The assay was rapid (4 h) and generated protein A- and cell (S. aureus)-dependent signals. The system was highly sensitive and could detect 10 pg of protein A ml-1 and less than 100 CFU of S. aureus ml-1. Similar sensitivities were observed with S. aureus cultures inoculated into beef and milk, but the sensitivity was reduced slightly (ca. 10(3) g-1) with samples of Cheddar cheese.     

Sensitive enzyme-amplified electrical immunoassay for protein A-bearing Staphylococcus aureus in foods.

An amperometric electrochemical immunoassay specific for protein A-bearing Staphylococcus aureus was developed. The method was based on a sandwich immunosorbent assay and incorporated an enzyme amplification step, using a NAD-specific redox cycle generating NADH (C. H. Stanley, A. Johannsson, and C. H. Self, J. Immunol. Methods 83:89-95, 1985). Reduction of the mediator, ferricyanide, was dependent on the initial concentration of antigen. The final potential was measured by using a Pt disk electrode polarized at +0.8 V to the Ag/AgCl reference electrode. The assay was rapid (4 h) and generated protein A- and cell (S. aureus)-dependent signals. The system was highly sensitive and could detect 10 pg of protein A ml-1 and less than 100 CFU of S. aureus ml-1. Similar sensitivities were observed with S. aureus cultures inoculated into beef and milk, but the sensitivity was reduced slightly (ca. 10(3) g-1) with samples of Cheddar cheese.     

Identification of Staphylococcus aureus exotoxins by combined sodium dodecyl sulfate gel electrophoresis and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry

Staphylococcus aureus is an important human pathogen whose pathogenesis involves the synthesis of cell wall associated virulence factors and secreted toxins with damaging effects on the host cells. Most of these pathogenic factors are synthesized in a growth-phase dependent manner as a response to environmental stress like heat, lack of nutrients or other deleterious conditions. Conventional identification of these pathogenic factors is based on Western blot analysis or enzyme-linked immunosorbent assay (ELISA) and is limited by the commercial availability of antibodies against these toxins. We report here the use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry for monitoring the pathogenic factors of S. aureus. For the identification of pathogenic factors, a methicillin sensitive strain of S. aureus, ATCC-29213, was grown at 37 degrees C or 42 degrees C in brain-heart infusion broth and harvested during the early stationary phase of growth. Secreted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzymatically digested with trypsin and analyzed by MALDI-TOF mass spectrometry. When grown at 42 degrees C, alpha- and beta-hemolysins were found to accumulate in S. aureus supernatants while the concentration of protein A was slightly decreased. The identity of some of these toxins was confirmed by Western-blot analysis. MALDI-TOF mass spectrometry combined with sodium dodecyl sulfate gel electrophoresis represents a rapid and simple approach to characterize the virulence of S. aureus strains which seems to be particularly valuable for the identification of S. aureus exotoxins for which ELISA is not established.     

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。     

Antibody response to Staphylococcus aureus whole cell, lipase and staphylolysin in patients with S. aureus infections

Abstract Three assays to measure antibodies against Staphylococcus aureus whole cells, lipase and staphylolysin were used to try to discriminate between complicated and uncomplicated S. aureus septicaemia. Sera were examined from 8 patients with S. aureus endocarditis, 23 patients with complicated S. aureus septicaemia, 12 patients with uncomplicated S. aureus septicaemia and 93 febrile non-septicaemic controls. No single assay could distinguish between complicated and uncomplicated S. aureus septicaemia. If the criterion for a positive result is defined as positive antibody level in the anti-lipase ELISA as well as in at least 1 of the other 2 assays, 10/31 patients with S. aureus endocarditis or complicated septicaemia were positive compared to 0/93 non-septicaemic patients and 0/12 patients with uncomplicated S. aureus septicaemia. Therefore, the combined use of serological assays in the diagnosis of complicated S. aureus septicaemia, one of which is the anti-lipase ELISA, is recommended.     

Direct detection of nasal Staphylococcus aureus carriage via helicase-dependent isothermal amplification and chip hybridization

ABSTRACT: BACKGROUND: The bacterium Staphylococcus aureus constitutes one of the most important causes of nosocomial infections. One out of every three individuals naturally carries S. aureus in their anterior nares, and nasal carriage is associated with a significantly higher infection rate in hospital settings. Nasal carriage can be either persistent or intermittent, and it is the persistent carriers who, as a group, are at the highest risk of infection and who have the highest nasal S. aureus cell counts. Prophylactic decolonization of S. aureus from patients' noses is known to reduce the incidence of postsurgical infections, and there is a clear rationale for rapid identification of nasal S. aureus carriers among hospital patients. FINDINGS: A molecular diagnostic assay was developed which is based on helicase-dependent target amplification and amplicon detection by chip hybridization to a chip surface, producing a visible readout. Nasal swabs from 70 subjects were used to compare the molecular assay against culturing on "CHROMagar Staph aureus" agar plates. The overall relative sensitivity was 89%, and the relative specificity was 94%. The sensitivity rose to 100% when excluding low-count subjects (<100 S. aureus colony-forming units per swab). CONCLUSIONS: This molecular assay is much faster than direct culture and has sensitivity that is appropriate for identification of high-count (>100 S. aureus colony-forming units per swab) nasal S. aureus carriers who are at greatest risk for nosocomial infections.     

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods

A multiplex PCR assay was devised and compared with standard conventional methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples which were artificially contaminated with <10 colony forming units of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species and possibly contaminated samples were incubated for 16 h with different enrichment media. Primers that deduce 559 bp fragment of the 16S rRNA gene was employed in amplifying E. coli species, similarly invasion protein gene with 275 bp fragment size was used as target for detecting Salmonella spp., in case of S. aureus a 461 bp amplicon from m-RNA nuclease gene, and an 709 bp fragment from oprL gene was used for amplifying P. aeruginosa. The detection limits for artificially contaminants by multiplex PCR was 1 CFU/g, where as in case of conventional method the detection limit was >2 CFU/g. Similarly, when tested with possibly contaminated samples, 35% were detected for E. coli, Salmonella spp., S. aureus and P. aeruginosa species with multiplex PCR, while only 21% were detected with standard conventional microbial methods. Multiplex PCR assay provides sensitive and reliable results and allows for the cost-effective detection of all four bacterial pathogens in single reaction tube.     

Immunoquantitative real-time PCR for detection and quantification of Staphylococcus aureus enterotoxin B in foods

A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. iqPCR was compared to an in-house enzyme-linked immunosorbent assay (ELISA) using the same couple of capture-detection antibodies and to commercial kits for detection of S. aureus enterotoxins (SE). The iqPCR was approximately 1,000 times more sensitive (<10 pg ml(-1)) than the in-house ELISA and had a dynamic range of approximately 10 pg ml(-1) to approximately 30,000 pg ml(-1). iqPCR was not inhibited by any of the foods tested and was able to detect SEB present in these foods. No cross-reactivity with SE other than SEB was observed. Application of iqPCR for detection of SEB in cultures of S. aureus revealed the onset of SEB production after 4 h of incubation at 22, 37, and 42 degrees C, which was in the first half of the exponential growth phase. The total amounts of SEB produced by the two strains tested were larger at 42 degrees C than at 37 degrees C and were strain dependent.     

Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks

Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5' nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay.     

Quantitative spectrophotometric assay for staphylococcal lipase.

We report the development of a specific spectrophotometric assay for the quantitative determination of lipase activity in Staphylococcus aureus. The assay is based on the rate of clearance of a tributyrin emulsion, and it can detect as little as 1.0 micrograms of purified Pseudomonas lipase per ml. By comparison with the reaction rates obtained with Pseudomonas lipase, we calculated that S. aureus PS54C and S6C produce approximately 15 and 60 micrograms of extracellular lipase per ml, respectively. Neither PS54, which is lysogenized with the converting bacteriophage L54a and is consequently lipase negative (Lip-), nor KS1905, a Lip- transpositional mutant of strain S6C, was positive in our spectrophotometric assay. The specificity of the spectrophotometric tributyrin assay was confirmed with a triolein plate assay; supernatants from S6C and PS54C hydrolyzed triolein, while supernatants from PS54 and KSI905 did not. In contrast to the results of the spectrophotometric tributyrin assay, all enzyme preparations tested (including commercially purified esterase) were positive when examined by a tributyrin plate assay. The lack of specificity in the tributyrin plate assay emphasizes the need to interpret the results of tributyrin lipolysis kinetically for assessing lipase activity in S. aureus.     

Quantitative spectrophotometric assay for staphylococcal lipase.

We report the development of a specific spectrophotometric assay for the quantitative determination of lipase activity in Staphylococcus aureus. The assay is based on the rate of clearance of a tributyrin emulsion, and it can detect as little as 1.0 micrograms of purified Pseudomonas lipase per ml. By comparison with the reaction rates obtained with Pseudomonas lipase, we calculated that S. aureus PS54C and S6C produce approximately 15 and 60 micrograms of extracellular lipase per ml, respectively. Neither PS54, which is lysogenized with the converting bacteriophage L54a and is consequently lipase negative (Lip-), nor KS1905, a Lip- transpositional mutant of strain S6C, was positive in our spectrophotometric assay. The specificity of the spectrophotometric tributyrin assay was confirmed with a triolein plate assay; supernatants from S6C and PS54C hydrolyzed triolein, while supernatants from PS54 and KSI905 did not. In contrast to the results of the spectrophotometric tributyrin assay, all enzyme preparations tested (including commercially purified esterase) were positive when examined by a tributyrin plate assay. The lack of specificity in the tributyrin plate assay emphasizes the need to interpret the results of tributyrin lipolysis kinetically for assessing lipase activity in S. aureus.     

Detection of methicillin- and aminoglycoside-resistant genes and simultaneous identification of S. aureus using triplex real-time PCR Taqman assay

In this study we describe a triplex real-time PCR assay that enables the identification of S. aureus and detection of two important antibiotic resistant genes simultaneously using real-time PCR technology in a single assay. In this triplex real-time PCR assay, the mecA (methicillin resistant), femA (species specific S. aureus) and aacA-aphD (aminoglycoside resistant) genes were detected in a single test using dual-labeled Taqman probes. The assay gives simultaneous information for the identification of S. aureus and detection of methicillin and aminoglycoside resistance in staphylococcal isolates. 152 clinical isolates were subjected to this triplex real-time PCR assay. The results of the triplex real-time PCR assay correlated with the results of the phenotypic antibiotic susceptibility testing. The results obtained from triplex real-time PCR assay shows that the primer and probe sets were specific for the identification of S. aureus and were able to detect methicillin- and aminoglycoside-resistant genes. The entire assay can be performed within 3 h which is a very rapid method that can give simultaneous information for the identification of S. aureus and antibiotic resistance pattern of a staphylococcal isolate. The application of this rapid method in microbiology laboratories would be a valuable tool for the rapid identification of the S. aureus isolates and determination of their antibiotic resistance pattern with regards to methicillin and aminoglycosides.     

一株金黄色葡萄球菌小菌落突变株的分离鉴定

摘要：【目的】由于金黄色葡萄球菌（金葡菌）小菌落突变株（small colony variants,简称SCVs ）可引起持续复发性感染,且对氨基糖苷类有抗药性,在临床诊断和治疗上造成很大的困扰。我国国内尚无金葡菌SCVs的报道,本研究旨在分离鉴定出金葡菌SCVs菌株,为国内进行SCVs的相关研究提供生物学材料。【方法】通过细菌的形态鉴定、种特异性基因（nuc）的PCR扩增鉴定以及系列生化实验,从人源、动物源及环境源共104株金葡菌分离株中筛选得到金葡菌SCVs,并通过甲萘醌、硫胺素、胸腺嘧啶和血红素等补     

Staphylococcus aureus pneumonia in hamsters with elastase-induced emphysema--the virulence enhancing activity of mucin

Staphylococcus aureus pneumonia was studied in hamsters with elastase-induced emphysema and in saline-treated controls. Emphysematous animals cleared endotracheally administered inocula of S. aureus in saline as rapidly as controls. After infection with S. aureus in 1% mucin, emphysematous animals had impaired clearance compared with controls; after infection with S. aureus in 5% mucin, emphysematous animals had decreased survival at 96 hr compared to controls (6/24 vs 15/24, P less than 0.01 Fisher's exact test). Bronchoalveolar lavage of uninfected elastase-treated hamsters yielded twice as many cells per animal as uninfected controls (P less than 0.0001, paired t test), and the cells contained a higher percentage of polymorphonuclear leukocytes (37.8% vs 3.8%, P less than 0.0001). Lavage cells from both groups of animals were equally efficient per cell at killing opsonized S. aureus in an in vitro bactericidal assay. Hamsters with elastase-induced emphysema were resistant to infection with S. aureus alone despite marked structural abnormalities in the lung, possibly due in part to increased numbers of resident phagocytic cells. After infection with S. aureus in mucin as a virulence enhancing factor emphysematous animals had impaired clearance and decreased survival compared to controls.     

Efficacy of acidic and basic electrolyzed water in eradicating Staphylococcus aureus biofilm

Staphylococcus aureus is a major pathogen. It can form biofilm on the surfaces of medical devices and food equipment, which makes it more difficult to eradicate. To develop a novel method to eradicate S. aureus biofilm, the effects of electrolyzed water on removing and killing S. aureus biofilm were investigated in this study. By using a biofilm biomass assay with safranin staining and visualization of biofilm architecture with scanning electron microscopy, it was shown that basic electrolyzed water (BEW) could effectively remove established biofilm. The pH of electrolyzed water affected removal efficacy. Using a biofilm viability assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining, acidic electrolyzed water (AEW) efficiently killed biofilm-imbedded S. aureus. The available chlorine in AEW may be a main contributing factor for bactericidal activity. Additionally, BEW had a removal efficacy for S. aureus biofilm equivalent to 2% NaOH, and AEW had a bactericidal capability for S. aureus in biofilm equivalent to 2% HCl. These data suggested that AEW and BEW could be applied as a bactericide and removing agent for S. aureus in biofilm, respectively.     

Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese.

Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/microl) than using a fluorigenic TaqMan probe (6 nuc gene copies/microl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 x 10(2) to 6.4 x 10(2) copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.     

Immunological detection of penicillin-binding protein 2'' in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.     

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.     

Bacteriocin of Enterococcus from lactoserum able to cause oxidative stress in Staphylococcus aureus

The effect of a bacteriocin of Enterococcus on the oxidative metabolism of sensitive bacteria was investigated through the detection of oxidative stress by chemiluminescence (CL). The bacteriocin named EntB was purified to study the action on Staphylococcus aureus isolated from cosmetic. Chromatographic separation of EntB indicated different states of oligomerization with molecular weights multiple of 12,000Da monomeric form. The monomer purified by ion exchange was studied in its capacity to affect the oxidative metabolism of S. aureus, which showed increase of anion superoxide (O(2)(-)) when incubated with EntB. This effect was compared to the action of EntB on leukocytes as an assay of toxicity. EntB did not generate significant oxidative stress in leukocytes. Pyoverdin, a leukotoxic pigment of Pseudomonas fluorescens, was taken as reference, and it was found that this pigment caused similar oxidative stress to EntB in S. aureus; however, pyoverdin generated high production of anion superoxide (O(2)(-)) in leukocytes, while EntB did not increase the level of O(2)(-).