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1.
目的 使用重叠PCR方法构建构建△A146Ply突变体,原核可溶性表达△A146Ply蛋白,并明确其毒力变化情况;分析肺炎链球菌溶血素(pneumolysin,Ply)在不同血清型肺炎链球菌(streptococcus pneumoniae,SPN)中的表达情况.方法 以SPN D39型基因组DNA为模板设计合成构建突变体ply基因所需引物;利用重叠PCR方法扩增合成△A146ply突变体.通过溶血实验分析其溶血活性,利用中和试验验证△A146Ply诱导产生的特异性抗体中和野生Ply毒素溶血能力,并利用Western印迹检测5株不同血清型肺炎链球菌流行菌株中Ply蛋白表达情况.结果 突变体基因测序结果显示,Ply146位密码子GCT 3个碱基被缺失,△A146ply突变体构建成功,并实现了△A146Ply的可溶性表达,得到纯度>90 %的重组蛋白.△A146Ply蛋白浓度为100 000 ng/ml亦未表现出溶血活性.△A146Ply蛋白诱导产生的特异性抗体能够中和野生Ply毒素的溶血活性.Western印迹结果显示,△A146Ply诱导产生的多克隆抗体可与国内临床常见4株肺炎链球菌有交叉反应.结论 △A146Ply蛋白是一种安全的肺炎链球菌疫苗候选分子,可刺激机体产生具有中和作用的特异性抗体.  相似文献   

2.
目的了解目前从中国住院治疗肺炎患儿分离到的肺炎链球菌的血清型分布,及几种蛋白多糖结合疫苗的覆盖率,评估应用蛋白多糖结合疫苗预防肺炎链球菌感染的价值。方法选择2006年2月16日至2007年2月16日在首都医科大学附属北京儿童医院、复旦大学附属儿科医院、广州市儿童医院和深圳市儿童医院呼吸科住院治疗的肺炎患儿为研究对象,采用一次性吸痰管收集全部病例的呼吸道分泌物标本分离肺炎链球菌,部分患儿进行脑脊液、血液和胸腔积液中肺炎链球菌的分离。采用荚膜肿胀实验进行血清型分析。对4家儿童医院肺炎链球菌分离率和血清型进行分析,率的比较采用χ2检验或Fisher精确概率法。结果研究期间共纳入2865例肺炎患儿,2865例呼吸道吸取物标本中分离到肺炎链球菌279株,其中有2株不同血清型菌株分离自同一病例,分离阳性率为9.7%(278/2865)。3/8例胸腔积液中分离到肺炎链球菌,其中2例同时从呼吸道分泌物分离到肺炎链球菌,取其一进行血清分型,另1株从胸腔积液中分离的肺炎链球菌复苏失败,未进行血清分型。脑脊液和血液标本中未分离到肺炎链球菌。共有279株肺炎链球菌进行了血清型分析,以19F型最常见(60.6%,169/279),其次为19A(9.7%,27/279)、23F(9.3%,26/279)和6B(5.4%,15/279),上述4种血清型占全部菌株的84.9%(237/279)。肺炎链球菌7价结合疫苗(PCV7)覆盖率为81.0%,但在北京仅为46.0%,明显低于上海(80.0%)、广州(98.4%)和深圳(94.4%)。9价、10价和11价疫苗的覆盖率与PCV7相比并没有明显增加。13价疫苗的覆盖率(92.8%)较PCV7明显升高。结论4家儿童医院肺炎住院患儿分离的肺炎链球菌以19F、19A、23F和6B型常见。PCV7覆盖率为87%。  相似文献   

3.
目的研究临床分离肺炎克雷伯菌的耐药机制及基因多态性。方法用WHONET5.4分析菌株药敏情况。PCR检测菌株I类整合酶、ISCR和ESBLs基因;ERIC—PCR检测基因型,SPSS分析其多态性。结果除亚胺培南和美洛培南,产ESBLs菌的耐药率明显高于不产ESBLs菌(P〈0.005)。对I类整合酶、ISCR、blaTEM、blaSHV、blaCTX-M,88株产ESBLs株的阳性率分别为59.1%、43.2%、69.3%、4.5%、45.5%;45株不产ESBLs株的阳性率分别为40.0%、15.6%、22.2%、4.4%、6.7%。根据指纹图谱把133株肺炎克雷伯菌分为126种,经SPSS系统聚类分析,归为20个大类。结论南方医院产ESBLs肺炎克雷伯菌主要是CTX-M基因型。整合子和ISCR可以共存;ERIC—PCR是一种效果较好的基因分型方法,该院肺炎克雷伯菌呈散发存在。  相似文献   

4.
目的 构建肺炎链球菌(Streptococcus pneumoniae,Sp)溶血素基因(pneumolysin,ply)缺陷菌株,并对其毒力作初步研究,为进一步探索宿主对溶血素的防御应答奠定基础.方法 采用长臂同源多聚酶链反应(LFH-PCR)技术将ply基因替换为红霉素耐药基因(erm)后同源重组于肺炎链球菌,在含红霉素的血平板上筛选出ply缺陷菌株.用PCR鉴定缺陷菌株,观察体外缺陷菌株生长情况,并在小鼠体内感染模型研究其毒力侵袭变化.结果 PCR结果显示ply基因完全被erm基因所替代,构建ply缺陷菌成功;单个菌落培养基生长情况表明ply基因缺陷并未对细菌的体外生长造成影响;但在小鼠鼻腔感染模型中,缺陷菌株入血时间(6 h)明显晚于野生菌株(2 h),且各时间点的菌量均显著低于野生菌株,两者比较差异有统计学意义(P<0.01);小鼠腹膜感染模型显示野生菌株半数致死时间为3 d,而缺陷菌株半数致死时间为18d,两者比较差异有统计学意义(P<0.01).结论 采用LFH-PCR技术作基因突变完全替代ply基因,方法简便快捷;ply的缺陷不影响细菌在体外的生长,但可显著降低细菌在宿主体内的毒力和侵袭.  相似文献   

5.
目的 研究耐药基因ermB、mefA、tetM与转座子整合酶基因intTn在携带肺炎链球菌的北京儿童中分布特点。方法对185株呼吸道感染患儿鼻咽部分离的肺炎链球菌进行以下检测:E-test、琼脂稀释或纸片扩散法测定对大环内酯类、四环素、β-内酰胺类及头孢类等15种抗生素的药物敏感性。PCR检测大环内酯类耐药基因ermB和mef/A,四环素耐药基因tetM以及转座子Tn1545的整合酶基因intTn。结果185株肺炎链球菌的药敏结果显示,对红霉素、克林霉素、四环素和复方磺胺甲基异嗯唑的耐药率较高,分别为78.9%、76.2%、86.0%和78.7%,对阿莫西林,克拉维酸、头孢克洛、头孢曲松和头孢呋辛耐药率较低,分别为2.2%、15.5%、2.8%和14.1%。所有红霉素耐药株均检出ermB和/或mefA,其中79.5%为ermB阳性,17.8%为ermB和mefA同时阳性,2.7%为mefA阳性。tetM基因在分离株中的阳性率是87%,四环素耐药组的tetM基因携带率是96.9%,高于敏感组(26.9%)。四环素耐药株的红霉素耐药率(90.0%)亦高于敏感株组(11.5%)。87.6%的肺炎链球菌存在intTn基因,intTn基因阳性组的红霉素、四环素、氯霉素、复方磺胺甲基异嗯唑和环丙沙星的耐药率较intTn基因阴性组高。分离株最常见的基因组合是intTn+terM+ermB,占58.4%。结论北京地区呼吸道感染儿童鼻咽部肺炎链球菌耐大环内酯类抗生素的主要原因是ermB编码的23S rRNA甲基化酶致靶位改变。tetM基因编码蛋白质的核糖体保护作用,是肺炎链球菌四环素耐药的重要机制。接合性转座子Tn1545的存在与菌株的红霉素和四环素耐药关系密切,可能是肺炎链球菌多重耐药的重要机制之一。  相似文献   

6.
目的 研究氨基糖苷乙酰胺转移酶基因的突变形式aac(6’) -Ib -cr在肺炎克雷伯菌和大肠埃希菌临床株中的分布状况;分析携带该基因菌株对环丙沙星、氨基糖苷类和头孢菌素类的耐药特征.方法 利用多聚酶链式反应检测肺炎克雷伯菌和大肠埃希菌临床分离株中aac(6’)-Ib基因携带情况,采用BtsC I酶切消化,经DNA测序确认aac(6 ’)-Ib-cr基因;统计分析aac(6’) -Ib-cr基因与环丙沙星、头孢菌素类和氨基糖苷类耐药性关系.结果 aac(6’)-Ib-cr基因在肺炎克雷伯菌和大肠埃希菌的检出率分别为12.6% (30/238)和18.8% (13/69);aac(6 ’)-Ib-cr阳性株与阴性株对环丙沙星的耐药率分别为72.1% (31/43)和48.1%(127/264),差异有统计学意义(X2=8.52,P<0.05).肺炎克雷伯菌aac(6’)-Ib-cr基因阳性株对阿米卡星的耐药率高于aac(6’) -Ib-cr基因阴性株(X2=4.25,P<0.05).aac(6’)- Ib-cr基因阳性株和阴性株对庆大霉素的耐药率、常用的头孢菌素类的耐药率和多重耐药率均较高,无统计学差异.结论 本地区肺炎克雷伯菌和大肠埃希菌临床分离株中,aac(6’) -Ib-cr基因均有检出;aac(6’)-Ib-cr基因阳性株对环丙沙星耐药率明显高于阴性株,肺炎克雷伯菌中aac(6’)-Ib-cr基因阳性株对阿米卡星的耐药率明显高于阴性株,具有统计学意义.aac(6’) - Ib-cr基因阳性株和阴性株对常用头孢菌素类的耐药率和多重耐药率均较高,无统计学意义.  相似文献   

7.
目的 调查我院产ESBL肺炎克雷伯菌临床分离株16S rRNA甲基化酶基因的分布以及与耐药谱的关系,并初步探讨其在分子流行病学分析中的作用.方法 收集我院临床2010年3月至9月分离出69株非重复产ESBL肺炎克雷伯菌,采用PCR法检测16S rRNA 甲基化酶基因,并对阳性菌株进行ESBL基因及整合子基因分析,通过DNA直接测序确定.质粒接合试验和质粒消除试验确定16S rRNA甲基化酶基因的传播途径,利用ERIC-PCR技术进行基因分型.结果 69株产ESBL肺炎克雷伯菌中有rmtB阳性菌株20株(28.9%),其中2株同时携带有rmtB和armA.在20株产16SrRNA甲基化酶菌株中,均携带有CTX-M基因,测序显示14株CTX-M-14基因,6株CTX-M-15基因;14株携带有TEM-1基因;8株携带有SHV基因,测序显示5株SHV-12基因,3株SHV-11基因;3株携带有OXA-10基因;3株携带有VBE-1基因.另有12株携带有int1阳性,含有5种不同的耐药基因盒,分别携带drfA25、drfA1、drfA12、aadA1、aadA2、sat和blaVEB-1基因.ERIC-PCR法显示20株16SrRNA甲基化酶基因阳性的肺炎克雷伯菌主要分为5型,A型为优势流行克隆株.质粒接合和消除试验发现A型克隆株KP5和KP16 rmtB均位于一质粒上并通过接合传播.结论 本院产ESBL肺炎克雷伯菌临床分离株中存在16S rRNA甲基化酶基因rmtB的普遍流行,导致对多种氨基糖苷类抗生素高水平耐药.rmtB可通过水平基因传播和克隆传播的两种方式进行播散,并且存在同时产ESBLs、16S rRNA甲基化酶和Ⅰ类整合子的肺炎克雷伯菌的传播.  相似文献   

8.
目的了解广州地区肺炎住院的患儿感染肺炎链球菌的耐药性及血清型分布情况。方法用吸痰法采集患儿痰标本进行涂片,革兰氏染色镜检,合格痰标本划线接种血平板,用E-test法检测分离到的肺炎链球菌对青霉素、阿莫西林、头孢曲松、头孢呋辛、亚胺培南、氧氟沙星、万古霉素、红霉素和克林霉素9种药物的耐药性,采用K-B法检测四环素、复方新诺明的耐药性,并采用荚膜肿胀技术对分离到的79株肺炎链球菌进行血清分型。结果79株肺炎链球菌中青霉素耐药肺炎链球菌(PRSP)11.4%,青霉素中介肺炎链球菌(PISP)77.2%,青霉素敏感肺炎链球菌(PSSP)11.4%,对红霉素、克林霉素的耐药率分别为100%和93.7%,对阿莫西林、氧氟沙星、万古霉素的耐药率均为0,对头孢曲松、头孢呋辛、亚胺培南的耐药率分别为3.8%、72.2%、2.5%。79株肺炎链球菌中只有1株PSSP仅对红霉素耐药.78株肺炎链球菌对两种以上药物耐药.多重耐药率为98.7%(78/79),同时对克林霉素、红霉素、四环素、复方新诺明耐药的菌株65株,占82.3%(65/79)。79株肺炎链球菌血清型分别为19F(70.9%),23F(16.5%),6B(5.1%),4(2.5%),15B(2.5%),不能分型(2.5%),7价疫苗涵盖率为94.9%(75/79)。结论广州地区肺炎住院患儿肺炎链球菌对大环内脂类抗生素红霉素和林可酰胺类抗生素克林霉素耐药情况严重,对二代头孢菌素头孢呋辛耐药率居高,临床治疗儿童肺炎链球菌感染的肺炎应首选阿莫西林和三代头孢菌素。7价疫苗覆盖率高。预防儿童肺炎链球菌感染所致的肺炎,采用7价疫苗可以达到很好效果。  相似文献   

9.
目的了解儿童急性呼吸道感染(ARTIS)鼻咽部常见的病原菌及其耐药性.方法对285例取鼻咽拭子进行培养,分离鉴定病原菌,同时对肺炎链球菌和流感嗜血杆菌(Hi)进行耐药性监测.结果 285例中共检出病原菌161株,肺炎链球菌、流感嗜血杆菌、卡他莫拉氏菌、金黄色葡萄球菌的分离率分别为20.4%、16.5%、13.0%、6.7%.肺炎链球菌对青霉素耐药率高达57.5%,同时对非β-内酰胺类抗生素复方新诺明、四环素、红霉素的耐药率已高达79.2%~89.5%,对头孢曲松、阿莫西林/棒酸尚具有较好的敏感性(85.1%~93.3%).Hi对氨苄西林的耐药率为13.3%,对头孢克洛、头孢曲松、头孢呋新、阿莫西林/棒酸均有很好的敏感性(96.4%~100%),对四环素、复方新诺明的耐药范围分别为25.3%~56.6%.结论肺炎链球菌、流感嗜血杆菌、卡他莫拉氏菌和金黄色葡萄球菌是广州地区儿童急性呼吸道感染的常见病原菌.肺炎链球菌和流感嗜血杆菌耐药形势严峻,抗生素合理应用尤为重要.  相似文献   

10.
致肾盂肾炎大肠杆菌特异性片段R049聚集性分布的研究   总被引:2,自引:2,他引:0  
目的 研究致肾盂肾炎大肠杆菌(UPEC)132特异性片段R049在UPEC菌株和正常人粪便分离的大肠杆菌菌株中的分布情况.方法 对20株UPEC和加株粪便分离大肠杆菌采用PCR方法扩增R049片段,对R049阳性菌株检测编码5种毒力因子的基因(papC、fimH、hly、aer、cnf1),并对其基因组DNA用Xba Ⅰ酶切,进行脉冲场凝胶电泳分析,继而用毛细管法将DNA片段转移至尼龙膜,利用地高辛标记的R049 ORF探针片段进行Southem杂交,分析杂交条带的分布特征.结果 20株UPEC中有8株(40%)、40株粪便分离的大肠杆菌中有3株(7.5%)R049片段扩增阳性,两组差异有统计学意义(P<0.01).R049阳性菌株中,R049片段与5种UPEC毒力因子密切相关.Southem杂交结果表明3株粪便分离大肠杆菌R049阳性杂交带分别为150 kb、15 kb与240 kb;8株UPEC中有6株阳性杂交带均为350 kb,其余2株分别为280 kb和25 kb.结论 UPEC132特异性片段R049在国内分离的UPEC菌株中具有共有性和聚集性分布的特征.  相似文献   

11.
The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.  相似文献   

12.
Although the polysaccharide capsule of Streptococcus pneumoniae has been recognized as a sine qua non of virulence, much recent attention has focused on the role of pneumococcal proteins in pathogenesis, particularly in view of their potential as vaccine antigens. The individual contributions of pneumolysin (Ply), the major neuraminidase (NanA), autolysin (LytA), hyaluronidase (Hyl), pneumococcal surface protein A (PspA), and choline-binding protein A (CbpA) have been examined by specifically mutagenizing the respective genes in the pneumococcal chromosome and comparing the impact on virulence in a mouse intraperitoneal challenge model. Mutagenesis of either the ply, lytA, or pspA gene in S. pneumoniae D39 significantly reduced virulence, relative to that of the wild-type strain, indicating that the respective gene products contribute to pathogenesis. On the other hand, mutations in nanA, hyl, or cbpA had no significant impact. The virulence of D39 derivatives carrying a ply deletion mutation as well as an insertion-duplication mutation in one of the other genes was also examined. Mutagenesis of either nanA or lytA did not result in an additional attenuation of virulence in the ply deletion background. However, significant additive attenuation in virulence was observed for the strains with ply-hyl, ply-pspA, and ply-cbpA double mutations.  相似文献   

13.
The capsular serotype has long been associated with the virulence of Streptococcus pneumoniae. Here we present an in-depth study of phenotypic and genetic differences between serotype 3 and serogroup 11 S. pneumoniae clinical isolates from both the general and indigenous populations of Australia. Both serotypes/groups included clonally unrelated strains with differences in well-known polymorphic virulence genes, such as nanA and pspA, as demonstrated by multilocus sequence typing and Western blot analysis. Nonetheless, the serotype 3 strains were consistently and significantly more virulent in mice than the serogroup 11 strains. Despite extensive genomic analysis, noncapsular genes common to one serotype/group but not the other were not identified. Nevertheless, following the conversion of a serotype 11A isolate to serotype 3 and subsequent analysis in an intranasal infection model, it was evident that both capsular and noncapsular factors determine the virulence phenotype in mice. However, it appears that these noncapsular factors vary from strain to strain.  相似文献   

14.
The lytA gene encoding the autolysin of Streptococcus pneumoniae may be a virulence determinant. Single-strand conformational polymorphism analysis demonstrated heterogenicity throughout the gene in clinical isolates and strains from the clonal serotypes 7 and 14. Sequence analysis of part of the choline-binding domain showed that in two isolates four amino acid substitutions occurred.  相似文献   

15.
Streptococcus pneumoniae is a major cause of bacteremia, meningitis, pneumonia, sinusitis, and acute otitis media in children. Although optochin susceptibility, bile solubility, and Quellung testing are the standards for identifying and differentiating pneumococci, there are several reports of nontypeable pneumococci that give inconsistent results with one or more of these tests. We characterized 52 isolates previously labeled as nontypeable pneumococci. Microbiological methods included repeating the Quellung reaction using a new and expanded group of antisera, optochin susceptibility and bile solubility tests, and automated Vitek 2 identification. Molecular methods included PCR detection of ply and psaA genes, multilocus sequence typing (MLST), 16S rRNA gene sequencing, and pyrosequencing. Of the 52 isolates, 38 (73%) were optochin susceptible, were psaA and ply positive, and could be serotyped by the Quellung reaction. The remaining 14 isolates, isolated from patients with otitis media (n = 6), bacteremia (n = 6), meningitis (n = 1), and pneumonia (n = 1), underwent further analysis. Three of these 14 isolates were nontypeable due to autoagglutination but were pneumococci by all tests and represented pneumococcal sequence types previously recognized by MLST. The 11 remaining isolates were optochin resistant, and 6 of these were bile soluble. Three of 11 were both psaA and ply positive and clustered with pneumococci by MLST (2 were bile soluble); 8 lacked psaA (5 ply positive, 4 bile soluble) and likely belonged to other Streptococcus species. In conclusion, few isolates were truly nontypeable by Quellung reaction, and MLST and the presence of psaA proved useful in distinguishing between atypical pneumococci and other streptococcal species.  相似文献   

16.
Eight optochin-susceptible (Opt(s)) alpha-hemolytic (viridans) streptococcus isolates were characterized at the molecular level. These isolates showed phenotypic characteristics typical of both viridans streptococci and Streptococcus pneumoniae. Comparison of the sequence of housekeeping genes from these isolates with those of S. pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae suggested that the Opt(s) isolates corresponded to streptococci of the mitis group. Besides, the Opt(s) streptococci were negative by a Gen-Probe AccuProbe pneumococcus test and hybridized with specific pneumococcal probes (lytA and ply) but also with ant, a gene not present in most S. pneumoniae strains. Moreover, the isolates were insoluble in 1% sodium deoxycholate but completely dissolved in 0.1% deoxycholate. Sequence analysis of the lytA gene revealed that the Opt(s) streptococci carried lytA alleles characteristic of those present in nonpneumococcal streptococci of the mitis group. The determination of the partial nucleotide sequence embracing the atp operon encoding the F(o)F(1) H(+)-ATPase indicated that the optochin susceptibility of the isolates was due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae by horizontal gene transfer.  相似文献   

17.
It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulence-factor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.  相似文献   

18.
Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from "pneumococcus-like" oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search for differences between Streptococcus pneumoniae strain WU2 and the most closely related oral streptococcus, Streptococcus mitis strain 903. We identified 19 clones that contained S. pneumoniae-specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of S. mitis and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of S. pneumoniae were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four S. pneumoniae clinical isolates and from 20 atypical organisms classified as S. mitis or S. oralis, which harbored genes encoding the pneumococcal virulence factors autolysin (lytA) or pneumolysin (ply), as templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of S. pneumoniae infections.  相似文献   

19.
Although often considered a strict human pathogen, Streptococcus pneumoniae has been reported to infect and cause pneumonia in horses, although the pathology appears restricted compared to that of human infections. Here we report on the molecular characterization of a group of S. pneumoniae isolates obtained from horses in England and Ireland. Despite being obtained from geographically distinct locations, the isolates were found to represent a tight clonal group, virtually identical to each other but genetically distinguishable from more than 120 divergent isolates of human S. pneumoniae. A comprehensive analysis of known pneumococcal virulence determinants was undertaken in an attempt to understand the pathogenicity of equine pneumococci. Surprisingly, equine isolates appear to lack activities associated with both the hemolytic cytotoxin pneumolysin, often considered a major virulence factor of pneumococci, and the major autolysin gene lytA, also considered an important virulence factor. In support of phenotypic data, molecular studies demonstrated a deletion of parts of the coding sequences of both lytA and ply genes in equine pneumococci. The implications of these findings for the evolution and pathogenicity of equine S. pneumoniae are discussed.  相似文献   

20.
PspA is anchored to the surface of all pneumococci by the C-terminal end of the molecule. The N-terminal half of PspA is known to be serologically variable and to be able to elicit protective immune responses. Molecular analysis with DNA probes spanning different regions of pspA was carried out to identify homologous sequences among pneumococcal isolates. At high stringency, DNA probes derived from the 3'-half of pspA (encoding the C-terminal half of PspA) hybridized to all of 37 pneumococcal isolates tested, representing 20 capsular serotypes and 12 PspA serotypes. Most strains had two sequences highly homologous to this region of pspA. Using derivatives of strain Rx1, with insertion mutations in pspA, it was possible to identify the functional pspA sequence. At 50% stringency, the 3' pspA probes also detected lytA and additional sequences. lytA encodes autolysin and shares homology with the 3' portion of pspA. A probe derived from the 5'-half of pspA (encoding the N-terminal half of PspA) hybridized with only 75% of strains and generally detected only one of the two sequences recognized by the 3' probes. Thus, the 3'-half of pspA appears to contain more highly conserved sequences than the 5'-half of pspA and shares homology with several additional sequences, suggesting that the pneumococcus might make several proteins that interact with the surface by the same mechanism as PspA.  相似文献   

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