致病性大肠埃希菌血清型分布及对抗生素的敏感性分析

目的了解临床病例中致病性大肠埃希菌的主要血清型和对抗生素的敏感性。方法致病性大肠埃希菌的鉴定使用血清学的方法,药敏试验采用纸片扩散法,WHONET 5.0软件分析药敏结果。结果致病性大肠埃希菌的检出率为5.93%,共分离到7种血清型。在分离到的菌株中,ESBLs的检出率达45%。结论致病性大肠埃希菌是引起小儿腹泻的一种重要致病菌,应开展对致病性大肠埃希菌的检测,根据药敏结果选用合适药物。     

合肥地区动物源性大肠埃希菌的血清型分布与耐药性分析

目的了解安徽省合肥地区动物源性大肠埃希菌的血清型分布和耐药状况,以期筛选出菌苗株和指导临床合理用药。方法对46份疑似大肠埃希菌病病料进行细菌分离培养、生化编码鉴定和致病性测定。采用玻片凝集试验对分离到的46株致病性大肠埃希菌进行血清型鉴定。同时分别采用K-B纸片琼脂扩散法和双纸片增效法检测致病性大肠埃希菌的耐药性和ESBLs阳性菌株。结果46株致病性大肠埃希菌中,除7株细菌未能定型外,其余39株细菌分布于10个血清型,O127：K63血清型为优势血清型,占定型菌株的33．33％。46株致病性大肠埃希菌对21种抗菌药物均呈现不同程度的耐药性,15个ESBLs阳性菌株表现为多重耐药,对各种抗菌药物的耐药率均高于ESBLs阴性菌株。结论O127：K63血清型为优势血清型,可作为菌苗株。合肥地区动物源性大肠埃希菌耐药性较为严重,尤其是产ESBLs大肠埃希菌多重耐药更为突出。     

噬菌体phiYe-F10的裂解谱的测定以及裂解能力与宿主毒力基因间关系分析

目的为测定小肠结肠炎耶尔森菌噬菌体phiYe-F10的裂解谱,分析噬菌体phiYe-F10裂解能力与宿主毒力基因间的关系。方法采用双层平板法观察噬菌体phiYe-F10对213株小肠结肠炎耶尔森菌,36株假结核耶尔森菌和1株鼠疫耶尔森菌的裂解能力;同时对不同来源不同地区的小肠耶尔森菌进行黏附侵袭位点基因(ail)、肠毒素基因(ystA、ystB)、黏附素基因A(yadA)、毒力因子基因F(virF)、O∶3血清型特异性基因(rfbc)检测和血清型别鉴定。结果表明213株小肠结肠炎耶尔森菌包括O∶3血清型84株(其中rfbc+78株),O∶5血清型10株,O∶8血清型13株,O∶9血清型34株,其它及未分型的共72株。携带毒力质粒的典型致病性小肠耶尔森菌(ail+,ystA+,ystB-,yadA+,virF+)77株,毒力质粒丢失的致病性小肠结肠炎耶尔森菌(ail+、ystA+、ystB-、yadA-、virF-)15株,其它非致病基因型121株。噬菌体phiYe-F10裂解71株O∶3小肠结肠炎耶尔森氏菌,其中致病性小肠结肠炎耶尔森菌52株,非致病性小肠结肠炎耶尔森菌19株。对O∶5,O∶9血清型和其它菌株均不裂解,对O∶3的裂解率高达84.5%(71/84)。噬菌体phiYe-F10对假结核耶尔森菌和鼠疫耶尔森氏菌均不裂解。结论:phiYe-F10噬菌体是一株小肠结肠炎耶尔森菌的裂性噬菌体,在25℃时表现出一个典型的窄裂解谱,高度专一性裂解O∶3血清型小肠结肠炎耶尔森菌。phiYe-F10对典型致病性小肠结肠炎耶尔森菌的裂解能力明显高于非致病性的小肠结肠炎耶尔森菌     

金黄色葡萄球菌荚膜多糖研究进展

金黄色葡萄球菌作为引起人和动物发病的一种主要的致病菌,已严重影响到人们的身体健康和畜牧业的发展。致病性金黄色葡萄球菌多数含有荚膜成分,并且这种荚膜成分与金黄色葡萄球菌的毒力和抗噬菌作用有关。主要从金黄色葡萄球菌荚膜多糖的5型和8型血清学分类、分子结构、影响因素、表达调控等方面简要介绍了目前国内外关于金黄色葡萄球菌荚膜多糖的研究进展。     

两种方法纯化临床分离阿萨希毛孢子菌荚膜多糖GXMCSCD

目的使用两种方法分离和纯化临床分离阿萨希毛孢子菌的荚膜多糖葡萄糖醛酸木糖甘露聚糖(GXM)。方法对临床分离的阿萨希毛孢子菌进行增菌,采用无水乙醇沉淀荚膜多糖,通过十六烷基三甲基溴化铵(CTAB)对GXM特异性沉淀,分别采用透析法和萃取法分离GXM-CTAB复合物从而纯化GXM。使用苯酚硫酸法测定GXM的浓度。结果通过透析法从200 mL培养上清中获得了约6.6 mg的GXM,阿萨希毛孢子菌GXM的获得率(GXM/GXM和GalXM混合多糖)为13.9%(6.6/47.4)。通过萃取法从200 mL培养上清中获得了约8.4 mg的GXM,阿萨希毛孢子菌GXM的获得率为14.2%(8.4/59)。结论两种方法都成功地提取和纯化了阿萨希毛孢子菌荚膜多糖GXM,萃取法较透析法更为高效。     

肺炎克雷伯氏菌荚膜多糖表达量评价指标的建立及发酵条件优化

建立了特异性强的肺炎克雷伯氏菌荚膜多糖全菌ELISA检测方法,检测结果与多糖表达量相关性好;以全菌ELISA值结合菌数为评价指标,对影响荚膜多糖表达的培养基组成及发酵条件进行了优化,优化后的摇瓶培养条件下发酵液活性和生物量分别比优化前提高72.7和33倍,并经7L罐放大实验,绘制发酵动力学曲线,为肺炎克雷伯氏菌荚膜多糖进一步开发打下基础。     

细菌荚膜多糖的化学结构

荚膜是一些细菌所具有的表层结构,与多种疾病有着密切联系。细菌荚膜多糖不仅结构复杂,而且在免疫活性方面发挥着重要的作用。同一种细菌根据其荚膜多糖的抗原性不同可分为不同的血清型,不同血清型细菌荚膜多糖的化学结构也存在差异。以荚膜多糖为基础的疫苗正在积极研究开发当中,对不同致病细菌荚膜多糖具体化学结构的掌握是疫苗得到许可的必备条件之一。本文对致病细菌荚膜多糖的化学结构进行了归纳和总结,以期为荚膜多糖的化学结构研究和疫苗开发提供参考。     

金黄色葡萄球菌引起食物中毒的作用机制与其耐药性的研究进展

葡萄球菌广泛分布于自然界中,如空气、土壤、水以及物体的表面,在人和动物的皮肤表面部、鼻咽、肠道也常可发现葡萄球菌。大部分葡萄球菌是非致病菌,少数可引起人或动物致病,金黄色葡萄球菌（Staphylococcus aureus,金葡菌）即为最主要的致病性葡萄球菌。金葡菌是一种革兰氏阳性球菌,是医院感染常见的病原体之一,同时也是引起食品污染和细菌性食物中毒的一种重要细菌,其产生的毒素可使人中毒,带来非常严重的公共卫生负担。本文拟对金葡菌的病原与病理学特性,金葡菌与食物中毒,抗生素滥用与金葡菌耐药性等方面做简要综述。     

2011至2012年石家庄地区沙门菌食物中毒分离株分子流行病学特征

研究沙门菌食物中毒分离株invA基因、血清型和基因型的分子流行病学特征。收集2011至2012年发生于石家庄地区6起食物中毒分离出的16株沙门菌,参照GB 4789.4-2010方法确定其血清型;用实时荧光PCR法检测分离株侵袭蛋白A(invA)基因;应用中国细菌性传染病分子分型实验室监测网络(PulseNet China)推荐的脉冲场凝胶电泳技术对分离株进行基因型研究。16株沙门菌食物中毒分离株共分为5种血清型,分别为都柏林沙门菌2株、鼠伤寒沙门菌2株、肠炎沙门菌8株、圣保罗沙门菌3株、依麦克沙门菌1株;所有分离株invA基因均为阳性;血清型不同的分离株基因型也不相同,其中2起食物中毒分离株的基因型相同,相似性系数为100%,符合同一起暴发的一般规律。石家庄地区沙门菌食物中毒分离株致病性较强,某些血清型存在暴发的风险,应加强措施进行防范。     

金黄色葡萄球菌引起食物中毒的作用机制与其耐药性的研究进展

葡萄球菌广泛分布于自然界中,如空气、土壤、水以及物体的表面,在人和动物的皮肤表面部、鼻咽、肠道也常可发现葡萄球菌.大部分葡萄球菌是非致病菌,少数可引起人或动物致病,金黄色葡萄球菌(Staphylococcus aureus,金葡菌)即为最主要的致病性葡萄球菌.金葡菌是一种革兰氏阳性球菌,是医院感染常见的病原体之一,同时也是引起食品污染和细菌性食物中毒的一种重要细菌,其产生的毒素可使人中毒,带来非常严重的公共卫生负担.本文拟对金葡菌的病原与病理学特性,金葡菌与食物中毒,抗生素滥用与金葡菌耐药性等方面做简要综述.     

Turnover of cell surface-bound capsular polysaccharide in Staphylococcus aureus

The capsular polysaccharide (CPS) of Staphylococcus aureus strain Smith was labelled by growth of bacteria in the presence of radioactive N-acetylglucosamine and was separated from labelled cell wall components by affinity chromatography on wheat germ agglutinin following dissolution of the cells by lysostaphin. The products were partially characterised chemically and immunochemically. Similar labelled components were found in the culture fluid during growth. In a pulse-chase experiment, cell-bound CPS was released continuously into the culture fluid at the same rate as cell wall turnover and there was no evidence of direct excretion of CPS.     

Revised structures for the capsular polysaccharides from Staphylococcus aureus Types 5 and 8, components of novel glycoconjugate vaccines

Glycoconjugate vaccines based on the capsular polysaccharides (CPSs) from Staphylococcus aureus serotypes 5 and 8 conjugated to genetically detoxified recombinant exoprotein A (rEPA) from Pseudomonas aeruginosa have been shown, in Phase 3 clinical trials, to elicit a strong bactericidal immune response in end-stage renal disease patients. Such vaccines have the potential to reduce morbidity and mortality due to methicillin-resistant Staphylococcus aureus (MRSA), a major cause of hospital-acquired infection. The serotype 5 and 8 polysaccharides have been fully characterized by NMR spectroscopy and full structural analyses carried out. Published structures were found incorrect and the revised structures of the repeat units of the two polysaccharides are: [carbohydrate structure: see text]. Resonances indicative of the presence of peptidoglycan were observed in the spectra of both CPSs, consistent with reports that the CPS is covalently linked to peptidoglycan.     

PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802

AIMS: Evaluation of the occurrence of most known staphylococcal enterotoxin (SE) genes, egc (enterotoxin gene cluster) and TSST1 (toxic shock syndrome toxin 1) gene in both coagulase-positive (CPS) and coagulase-negative (CNS) staphylococcal strains isolated from meat and dairy products. METHODS AND RESULTS: Specificity and reliability of the PCR detection methods used were ascertained by using nine reference strains of Staphylococcus (S. aureus) harbouring SE genes (seA to seE; seG, seH, seI, seM, seJ, seN and seO) and egc (containing the following sequence of genes: seO, seM, seI, phient1, phient2, seN and seG). Of 109 wild Staphylococcus spp. strains analysed, only 11 S. aureus strains were SE and/or TSST1 PCR-positive. The last 11 strains also appeared to harbour the egc. Restriction endonuclease analysis of part of the egc of both reference and wild strains showed that different variants of the egc exist. Moreover, nucleotide sequences of seG and seI indicate that the egc of the strain AB-8802 is characterized by the presence of variants of these enterotoxins (seGv and seIv). CONCLUSIONS: The occurrence of SE genes in CNS and other non-S. aureus species isolated from Napoli-type salami, raw water buffalo milk and natural whey cultures used for mozzarella cheese manufacturing is very rare. SIGNIFICANCE AND IMPACT OF THE STUDY: During this study it was shown that at least five different egc may exist in S. aureus. A thorough study of egc polymorphism should provide further insight into the phylogenetics of the egc.     

Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives.

The agr quorum-sensing system in Staphylococci controls the production of surface proteins and exoproteins. In the pathogenic species Staphylococcus aureus, these proteins include many virulence factors. The extracellular signal of the quorum-sensing system is a thiolactone-containing peptide pheromone, whose sequence varies among the different staphylococcal strains. We demonstrate that a synthetic Staphylococcus epidermidis pheromone is a competent inhibitor of the Staphylococcus aureus agr system. Derivatives of the pheromone, in which the N-terminus or the cyclic bond structure was changed, were synthesized and their biological activity was determined. The presence of a correct N-terminus and a thiolactone were absolute prerequisites for an agr-activating effect in S. epidermidis, whereas inhibition of the S. aureus agr system was less dependent on the original structure. Our results show that effective quorum-sensing blockers that suppress the expression of virulence factors in S. aureus can be designed based on the S. epidermidis pheromone.     

Crystal structure and substrate specificity of the beta-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

beta-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 A resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- > acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.     

DGGE法分析慢性化脓性中耳炎耳道菌群变化及药敏试验研究

目的解析正常成人及慢性化脓性中耳炎（CSOM）患者耳道内菌群结构的差别,寻找有效抑制致病菌同时对皮肤主导菌群影响不大的抗生素,指导临床合理用药。方法取20例正常成人及20例CSOM患者耳道分泌物行血琼脂平板划线法分离鉴定细菌,选择典型病例进行DGGE法分析耳道菌群多样性,对培养的常见细菌进行药物敏感试验,分析药敏结果。结果（1）20例正常成人耳道分泌物中18例培养分离出表皮葡萄球菌（90％）,2例培养阴性（10％）;20例CSOM患者耳道分泌物中离出金黄色葡萄球菌8例（40％）,铜绿假单胞菌5例（25％）,溶血性葡萄球菌、表皮葡萄球菌各2例（各10％）,阴沟肠杆菌、洋葱伯克霍尔德菌各1例（各5％）,无菌生长1例（5％）。（2）DGGE分析显示,正常成人耳道菌群种类比CSOM明显增多。（3）金黄色葡萄球菌、表皮葡萄球菌、铜绿假单胞菌对环丙沙星均敏感。表皮葡萄球菌对阿莫西林、头孢唑林、左旋氧氟沙星耐药而金黄色葡萄球菌对其敏感。结论（1）正常人耳道正常菌群多样性高于CSOM。（2）目前培养出的正常成人主要耳道菌群为表皮葡萄球菌,CSOM主要菌群为金黄色葡萄球菌、铜绿假单胞菌。（3）环丙沙星同时抑制正常菌群和致病菌的生长。阿莫西林、头孢唑林、左旋氧氟沙星在抑制金黄色葡萄球菌等致病菌的同时,可以在一定程度上保护正常菌群成员表皮葡萄球菌。     

金黄色葡萄球菌所致肺部感染的耐药特点及其Panton—Valentine杀白细胞素基因的携带状况分析

目的分析金黄色葡萄球菌所致肺部感染的耐药性特点及其Panton—Valentine杀白细胞素基因的携带状况。方法回顾性调查了温州医学院第一附属医院2005年1月至2006年1月医院感染的金黄色葡萄球菌所致肺部感染患者132例,对其体外药敏试验进行分析;并利用多重PCR检测其PVL基因,应用多位点基因序列分型（multilocus sequence typing,MLST）技术对PVL基因阳性的菌株进行序列分型。耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus,MRSA）的SCCmec基因分型采用多重聚合酶链反应。结果致肺部感染的132株金黄色葡萄球菌的耐药现象较为严重,仅对万古霉素、呋喃妥因及复方新诺明等药物的敏感率较高;其中经多重PCR筛选出10株携带PVL基因的金葡菌,全部为MRSA菌株,3株为ST239-SCCⅢ,2株为ST398-SCCmecⅢ,2株为ST398-SCCmecⅣ,ST25-SCCmecⅢ、ST59-SCCmecⅠ和ST88-SCCmecⅢ各1株。结论肺部感染的金黄色葡萄球菌对多种抗生素耐药,呈多重耐药性;其携带PVL基因占一定比例。     

IsdG and IsdI, heme-degrading enzymes in the cytoplasm of Staphylococcus aureus

Staphylococcus aureus requires iron for growth and utilizes heme as a source of iron during infection. Staphylococcal surface proteins capture hemoglobin, release heme from hemoglobin and transport this compound across the cell wall envelope and plasma membrane into the bacterial cytoplasm. Here we show that Staphylococcus aureus isdG and isdI encode cytoplasmic proteins with heme binding properties. IsdG and IsdI cleave the tetrapyrrol ring structure of heme in the presence of NADPH cytochrome P450 reductase, thereby releasing iron. Further, IsdI complements the heme utilization deficiency of a Corynebacterium ulcerans heme oxygenase mutant, demonstrating in vivo activity of this enzyme. Although Staphylococcus epidermidis, Listeria monocytogenes, and Bacillus anthracis encode homologues of IsdG and IsdI, these proteins are not found in other bacteria or mammals. Thus, it appears that bacterial pathogens evolved different strategies to retrieve iron from scavenged heme molecules and that staphylococcal IsdG and IsdI represent examples of bacterial heme-oxygenases.     

乳杆菌抑制金黄色葡萄球菌对HeLa细胞的粘附

对弯曲乳杆菌Lactobacillus crispatus T79-3和T90-1、詹氏乳杆菌Lactobacillus jensenii T118-3和T231-1四株乳杆菌对金黄色葡萄球菌生长的抑制效果以及抑菌成分进行了分析,比较乳杆菌排除、竞争、置换3种不同作用方式对金黄色葡萄球菌粘附HeLa细胞的抑制作用。结果表明4株乳杆菌皆能抑制金黄色葡萄球菌的生长及其粘附HeLa细胞的能力,分析发现4株乳杆菌发挥抑制作用的主要成分是有机酸,同时比较分析乳杆菌3种不同作用方式发现它们对金黄色葡萄球菌粘附HeLa细胞的抑制效果不同,其中,排除作用方式效果最好。另外,乳杆菌对金黄色葡萄球菌粘附HeLa细胞的抑制作用具有浓度依赖性,随着乳杆菌浓度增大,抑制作用增强并逐渐达到饱和。4株乳杆菌中,T79-3粘附能力最强,对金黄色葡萄球菌的抑制作用最强,排除作用方式抑制金黄色葡萄球菌粘附HeLa细胞作用效果较好,提示乳杆菌T79-3有可能作为益生菌防治妇女泌尿生殖道感染。     

金黄色葡萄球菌蛋白质相互作用网络及功能

【目的】金黄色葡萄球菌是一种革兰氏阳性菌,是目前最难以对付的病菌之一。它能引起多种感染,特别是在医院环境中。近年来,抗药性金黄色葡萄球菌传染更加严重,已成为公共卫生威胁。由于以前对于金黄色葡萄球菌的实验性研究大都是基于单个基因或者蛋白进行的,为了更好的研究这个物种,有必要从整体上把握金黄色葡萄球菌的蛋白作用机理。【方法】采用系统发生谱、操纵子法、基因融合法、基因邻近法、同源映射法等五种计算方法预测金黄色葡萄球菌蛋白质相互作用网络。【结果】从蛋白组的角度构建了金黄色葡萄球菌蛋白相互作用网络,并对网络进行功能分析。【结论】网络的分析表明金黄色葡萄球菌的蛋白质相互作用网络也服从scale-free属性,发现了SA0939、SA0868、rplD等重要的蛋白。通过对金黄色葡萄球菌的重要的细胞壁合成和信号转导调控蛋白局部网络分析,发现了一些对这两个系统十分重要的蛋白分子,这些信息将为更好的了解金黄色葡萄球菌的致病机理和开发新的药物靶点提供指导。