A dual-affinity gene fusion system to express small recombinant proteins in a soluble form: expression and characterization of protein A deletion mutants

A novel gene fusion system to express and purify small recombinantproteins in Escherichia coli has been constructed. The conceptallows for affinity purification of soluble gene products bysequential albumin- and Zn²+-affinity chromatography. The dual-affinitysystem is well suited for expression of unstable proteins asonly full-length protein is obtained after purification andproteins gain proteolytic stability in the fusion protein. Herewe show that the dual-affinity approach can be used for theexpression of various unstable derivatives of a single IgG-bindingdomain based on staphylococcal protein A. Analysis of the proteolyticstabilities and the IgG-binding properties of the differentmutant proteins suggest that the model for the structure ofan IgG-binding domain must be re-evaluated.     

Staphylococcus aureus—A Known Opponent against Host Defense Mechanisms and Vaccine Development—Do We Still Have a Chance to Win?

The main purpose of this review is to present justification for the urgent need to implement specific prophylaxis of invasive Staphylococcus aureus infections. We emphasize the difficulties in achieving this goal due to numerous S. aureus virulence factors important for the process of infection and the remarkable ability of these bacteria to avoid host defense mechanisms. We precede these considerations with a brief overview of the global necessitiy to intensify the use of vaccines against other pathogens as well, particularly in light of an impasse in antibiotic therapy. Finally, we point out global trends in research into modern technologies used in the field of molecular microbiology to develop new vaccines. We focus on the vaccines designed to fight the infections caused by S. aureus, which are often resistant to the majority of available therapeutic options.     

金黄色葡萄球菌肠毒素在食源性微生物中的研究进展

由细菌污染引起的食源性疾病依然是影响人类公共健康和食品安全的最大问题之一。其中,金黄色葡萄球菌是人类的一种重要病原菌,引起许多严重感染。金黄色葡萄球菌属于革兰氏阳性球菌,广泛分布于自然界,是引起化脓性疾病的重要病原菌,也是引起食品污染和细菌性食物中毒的一种重要细菌。食品受金黄色葡萄球菌污染后,不仅会腐败变质,而且部分菌株产生金黄色葡萄球菌肠毒素（Staphylococcal enterotoxins,SEs）而引起食物中毒,由金黄色葡萄球菌肠毒素引起的食物中毒占整个细菌性食物中毒的首位。所以,对SEs的研究以及快速、精确的检测和筛查,成为关键环节。本文拟对金黄色葡萄球菌肠毒素生物学性状、致病．洼、检验方法等方面的研究进展进行综述。当然,对金黄色葡萄球菌及其主要致病因子肠毒素的研究有待进一步深入与发展。     

硅烷法固定ssDNA制作的压电式DNA传感器

利用硅烷法将ssDNA固化T方向切割、3.58MHz和10MHz石英晶体金电极上,构建了压电DNA传感器,建立了快速检测葡萄球菌肠毒素B(SEB)基因的方法。优化了固定SEB基因的条件,并用动态法、静态法和斑点杂交法进行了固化效果观察。结果表明:该传感器2h后,有较好响应;对不同长度DNA探针固化效果进行比较,169bp(即169个碱基)SEB探针固定和杂交效果较好;利用0.5mol/LNaOH进行电极洗脱,时间为45min,电极可以再生,且传感器可以重复使用3次;利用1.2mol/LNaOH和1.2mol/LHCl洗涤电极,该电极可多次再利用;固化好的电极于4℃条件下可以保存三周之久。     

Graduate Student Literature Review: Enterotoxigenic potential and antimicrobial resistance of staphylococci from Brazilian artisanal raw milk cheeses

More than 30 types of artisanal cheeses are known in Brazil; however, microorganisms, such as Staphylococcus spp., can contaminate raw milk cheeses through different sources, from milking to processing. Staphylococcal food poisoning results from the consumption of food in which coagulase-positive staphylococci, mostly Staphylococcus aureus, have developed and produced enterotoxins. In addition, an emerging public health concern is the increasing antimicrobial resistance of some Staphylococcus strains. Furthermore, the ability of Staphylococcus spp. in sharing antibiotic resistance-related genes with other bacteria increases this problem. In light of these observations, this review aims to discuss the presence of, enterotoxins of, and antibiotic-resistant of Staphylococcus spp. in Brazilian artisanal cheese produced with raw milk.     

The olive compound 4-hydroxytyrosol inactivates Staphylococcus aureus bacteria and Staphylococcal Enterotoxin A (SEA)

The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single chain protein which consists of 233 amino acid residues with a molecular weight of 27078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, diarrhea, atopic dermatitis, arthritis, and toxic shock) syndromes. Changes in the native structural integrity may inactivate the toxin by preventing molecular interaction with cell membrane receptor sites of their host cells. In the present study, we evaluated the ability of the pure olive compound 4-hydroxytyrosol and a commercial olive powder called Hidrox-12, prepared by freeze-drying olive juice, to inhibit S. aureus bacteria and SEA's biological activity. Dilutions of both test substances inactivated the pathogens. Two independent cell assays (BrdU incorporation into newly synthesized DNA and glycyl-phenylalanyl-aminofluorocoumarin proteolysis) demonstrated that the olive compound 4-hydroxytyrosol also inactivated the biological activity of SEA at concentrations that were not toxic to the spleen cells. However, efforts to determine inhibition of the toxin by Hidrox-12 were not successful because the olive powder was cytotoxic to the spleen cells at concentrations found to be effective against the bacteria. The results suggest that food-compatible and safe antitoxin olive compounds can be used to inactivate both pathogens and toxins produced by the pathogens. Practical Application: The results of this study suggest that food-compatible and safe antitoxin olive compounds can be used to reduce both pathogens and toxins produced by the pathogens in foods.     

一起金黄色葡萄球菌食物中毒事件的病原检测和溯源分析

目的 对上海市浦东新区一起食物中毒事件进行病原检测和溯源分析,为此类事件的处置和判定提供依据。方法 对病例和从业人员肛拭子以及留样食品样品进行病原分离和鉴定;对分离株进行肠毒素检测、药物敏感性试验、脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)分子分型和溯源。结果 从9份病例肛拭子、1份从业人员肛拭子及3份留样食品样品中分离到13株金黄色葡萄球菌。10株人源分离株产A型和E型肠毒素,3株食源分离株产A型、D型和E型肠毒素。13株分离株均对青霉素耐药,PFGE条带相似度为100%,MLST分型均为ST6型。结论 本起事件是由金黄色葡萄球菌污染引起的食物中毒事件,需要进一步加强对食品加工企业、食堂等的宣传教育和管理,防止类似事件的发生。     

Carbon nanotubes based optical immunodetection of Staphylococcal Enterotoxin B (SEB) in food

Staphylococcal enterotoxins (SEs) are a major cause of food-borne diseases, traditionally SEs assayed immunologically with ELISA. Carbon nanotubes' (CNT) unique mechanical and electronic properties combined with a large specific surface area make them attractive for biosensing. To investigate whether CNT could improve the sensitivity of ELISA assays, we developed an optical CNT immunosensor for the detection of Staphylococcal Enterotoxin B (SEB) in food. Anti-SEB antibodies were immobilized onto a CNT surface through electrostatic adsorption and then the antibody-nanotube mixture was bound onto a polycarbonate film. SEB was then detected by a "sandwich-type" ELISA assay on the polycarbonate film. The use of CNT increased the sensitivity of the immunosensor by at least 6-fold, lowering the detection limit of SEB. The CNT immunosensor was also able to detect SEB various foods, suggesting the utility of CNT for this and other optical-based immunological detection methods.