非O157产志贺毒素大肠杆菌分离株的志贺毒素基因变种及黏附相关基因分析

目的 了解我国部分地区非O157产志贺毒素大肠杆菌分离株的志贺毒素基因变种及其黏附相关基因,为进一步研究致病机制提供依据。 方法 采用聚合酶链反应(polymerase chain reaction,PCR)方法对29株分离菌株的stx1、stx2基因全长扩增并测序,通过与GenBank中已公布的变种序列比对确定菌株stx1、stx2基因的变种类型。对位于LEE毒力岛上的eaeA、escF、escC、tir、espA、espB、espD基因及LEE以外其他黏附相关基因iha、toxB、efa1、sfpA、lpfAO157/OI-141、 lpfAO157/OI-154、saa、lpfAO113、eibG进行PCR检测。 结果 25株stx1阳性菌株中有13株携带stx1a原毒素,12株携带stx1c变种;10株stx2阳性菌株中,7株携带stx2d变种,1株为stx2a原毒素,1株携带stx2g变种,1株携带与stx2e A亚单位、stx2d B亚单位最接近的stx2变种。LEE岛上的7个基因检测结果均为阴性,黏附相关基因iha阳性率为89.7%(26/29),saa阳性菌株3株、eibG阳性菌株1株,其余6个黏附相关基因均为阴性。 结论 我国部分非O157 STEC菌株的志贺毒素基因以stx1c、stx2d变种为主,LEE毒力岛不存在,而黏附相关基因iha广泛存在于不同血清型的产志贺毒素大肠杆菌菌株中。     

非O157产志贺毒素大肠埃希菌分离株血清型鉴定及主要毒力基因分析

目的 了解中国不同来源非O157产志贺毒素大肠埃希菌分离株血清型及主要毒力基因的流行情况。方法 采用O抗原基因簇特异性聚合酶链反应（PCR）结合全套O抗血清凝聚法确定O血清群,以PCR扩增和测序fliC基因确定H型;采用PCR方法对所有菌株进行stx1、stx2、eae及ehxA基因检测。结果 434株非O157 STEC分离株中,除不可分型菌株外,共检测出82种O血清群和28种H型,其中O20:H30、O2:H32和O2:H45为优势血清型。stx1、stx2和stx1+stx2 3种志贺毒素基因型的检出率分别为25.35%、64.98%和9.68%,而eae和ehxA基因的阳性率分别为3.92%和32.95%。仅15株菌同时携带3种毒力基因,且主要为血清型O26:H11的腹泻患者分离株。结论 我国非O157 STEC分离株血清型复杂多样,同时检测eae和ehxA基因对于发现高致病潜力的菌株具有重要参考价值。     

产志贺毒素大肠埃希菌的鉴定及分布特征

目的 探讨吉林地区产志贺毒素大肠埃希菌（STEC）分离株的分布和特征。方法 采集牛羊粪便及其肉类食品、蔬菜，分离大肠埃希菌，PCR鉴定毒力因子及血清学分型。结果 380份标本中鉴定出12株STEC,3株为产志贺毒素O157：H7型，9株为产志贺毒素非O157：H17型。结论 STEC存在于不同来源的标本中，菌株血清型与毒力因子存在一定差异。     

网状分枝扩增技术检测产志贺样毒素大肠埃希菌

目的 通过检测产志贺毒素大肠埃希菌中的志贺样毒素,确定网状分枝扩增技术的灵敏度和特异性,从而进一步探讨该方法用于检测食品和临床标本中0157:H7和STEC的可行性。方法 用网状分枝扩增技术检测合成的志贺样毒素2的基因和临床分离的菌株,确定该方法的灵敏度和特异性,与聚合酶链反应进行比较。结果 网状分枝扩增技术最少能检测10个志贺样毒素DNA分子,与PCR灵敏度一样。E.coli0157:H7和志贺痢疾杆菌志贺样毒素阳性,而非致病性大肠埃希菌为阴性。用网状分枝扩增技术与PCR对分离的菌株进行检测,结果也一致。结论 网状分枝扩增技术具有高度的灵敏度和特异性,操作简便,而且在等温条件下扩增核酸,因此可替代PCR检测食品和临床标本中的产志贺样毒素大肠埃希菌。     

产志贺毒素大肠埃希菌的特殊类型

产志贺毒素大肠埃希菌（STEC）的菌种以O157：H7为代表，但现在已扩展至近70个血清型。STEC的重要生化反应特征是不发酵山梨醇，现已在德国，捷克发现能在24h内发酵山梨醇的菌株，且其H抗原缺失。少数不产生志贺毒素，stx基因为阴性的发酵山梨醇的O157：H菌株也有欧洲发现。以上新情况及STEC的检出更具挑战性。     

多重实时PCR检测致泻性大肠埃希菌志贺毒素和紧密素基因

目的 建立多重实时PCR检测志贺毒素（stx1、stx2）基因和紧密素基因（eae）的方法。方法 优化多重实时PCR反应条件，检测系列稀释的阳性菌DNA提取物及纯化阳性质粒。检测42株携带已知毒力基因的大肠埃希菌株，并比较其阳性和阴性符合率。对比3种粪便样品处理和DNA提取方法，选出最适方法。同时用该方法对36份腹泻患者粪便标本进行直接检测且对阳性标本进行菌株分离和鉴定。结果 多重实时PCR方法最低可以检测到10^1拷贝／pJ的毒力基因和10。CFU／p．1的DEL933大肠埃希菌。检测42株携带已知毒力基因的大肠埃希菌的阳性和阴性符合率均为100％。粪便样品的DNA提取以BP肉汤增菌6h后煮沸提取效果最好。36份腹泻患者粪便中2份eae阳性，均鉴定为大肠埃希菌。结论 建立的同时检测stx1、stx2、eae基因的多重实时PCR方法，具有较高的敏感性，可用于产志贺毒素大肠埃希菌（STEC）和肠致病性大肠埃希菌（EPEC）毒力基因鉴定及临床腹泻粪便标本的快速筛检。     

产志贺毒素大肠埃希菌的特殊类型

产志贺毒素大肠埃希菌 (STEC)的菌种以O15 7∶H7为代表 ,但现在已扩展至近 70个血清型。STEC的重要生化反应特征是不发酵山梨醇 ,现已在德国、捷克发现能在 2 4h内发酵山梨醇的菌株 ,且其H抗原缺失。少数不产生志贺毒素、stx基因为阴性的发酵山梨醇的O15 7∶H菌株也在欧洲发现。以上新情况使STEC的检出更具挑战性。     

产志贺毒素大肠埃希菌生物被膜形成能力与毒力基因表达的关系

目的探讨临床腹泻患者产志贺毒素大肠埃希菌(STEC)生物被膜形成能力与致病毒力基因表达的关系。方法收集从520例临床腹泻患者标本中检测分离到的STEC,进行体外药敏试验和生物被膜形成能力定量分析;应用多重聚合酶链反应(PCR)检测产志贺毒素相关基因stx1、stx2,实时定量逆转录PCR(RT-PCR)检测eae、ehx A基因表达。以健康体检人群粪便标本中分离的98株大肠埃希菌作为对照菌株。结果从520例腹泻患者标本中分离出328株大肠埃希菌,其中stx1、stx2基因阳性菌(即STEC)78株(23.8%)。78株STEC中产生物被膜菌57株(73.1%),eae和ehx A基因的相对表达量分别为6.57(2.82~27.8)、1.81(1.07~5.28);不产生物被膜菌21株(26.9%),eae和ehx A基因的相对表达量分别为3.41(0.96~12.1)、1.76(0.89~6.06);另分离出副溶血弧菌18株[其中产生物被膜菌10株(55.6%)]、奇异变形杆菌3株。98株对照菌中产生物被膜菌39株(39.7%),eae和ehx A基因的相对表达量分别为1.02(0.86~3.47)、1.03(0.88~3.03)。产生物被膜STEC对氨苄西林/舒巴坦、复方磺胺甲噁唑、环丙沙星的耐药率明显高于不产生物被膜STEC和对照菌株。结论STEC生物被膜形成率明显高于健康人群定植大肠埃希菌,产生物被膜STEC的eae基因表达明显提高;ehx A基因表达与不产生物被膜的STEC比较无明显变化。     

一株不产志贺毒素的肠出血性大肠杆菌O157:H7菌株的鉴定

目的 对一株不产志贺毒素的肠出血性大肠杆菌O157:H7菌株进行血清生化特征及毒力基因鉴定.方法 对154菌株进行常规血清生化鉴定,使用聚合酶链反应(PCR)方法检测该菌的志贺毒素基因及其他毒力因子,同时以southern杂交、Hela细胞毒实验进行证实.结果 菌株154的志贺毒素PCR检测、shouthern杂交为阴性,Hela细胞毒定量结果显示该菌株不产生志贺毒素,毒力因子eae,hly检测为阳性.结论 菌株154是不产志贺毒素的肠出血性大肠杆菌O157:H7菌株,且具有O157:H7菌株特异性的毒力因子.     

实时定量PCR检测胆囊结石患者胆汁中华支睾吸虫DNA

摘要：目的：探讨用实时定量PCR技术检测胆囊结石患者胆汁标本中的华支睾吸虫DNA。 方法：随机选取40例实施内镜取石保胆手术胆囊结石患者的胆汁标本,对胆汁沉渣分别进行显微镜检及实时定量PCR检测。同时收集患者的血清、粪便标本,分别进行免疫学检测及粪便镜检。 结果：建立的实时定量PCR法特异性良好,对华支睾吸虫DNA的最低检测限为0.1 pg/μL;40例胆囊胆汁标本中有21例镜检发现虫卵（阳性率为52.5%）,实时定量PCR检测结果与镜检结果一致;免疫学检测抗体阳性20例（阳性率为50%）,与前二者的符合率为82.5%(33/40);粪便镜检虫卵阳性9例（阳性率为22.5%）,与其他3种方法比较,差异有统计学意义（χ²=9.12,P＜0.01）。 结论: 建立的实时定量PCR技术可用于胆囊结石患者华支睾吸虫感染的检测。     

Evaluation of the Meridian Premier EHEC assay as an indicator of Shiga toxin presence in direct faecal specimens

Molecular testing for stx1 and/or stx2 is a reliable way of detecting Shiga toxigenic Escherichia coli (STEC) when faecal specimens can be cultured; however, detection of Shiga toxin in unculturable specimens is also of public health importance. The Meridian Premier EHEC assay was evaluated against the gold standard Vero cell cytotoxic assay for Shiga toxin detection in direct faecal specimens. An initial study of 817 patient specimens submitted for routine STEC detection was conducted, evaluating positive faeces detected by the Meridian assay with the Vero cell assay. Twenty-nine percent of 136 Meridian-positive faeces were confirmed as containing Shiga toxin. A further 62 faecal specimens were evaluated for statistical purposes, with all specimens tested by both Meridian and Vero cell assays. On direct faeces, the Meridian assay gave high specificity (76.95%) but low sensitivity (40%). This study confirmed that testing by Meridian assay on cultures is preferential to testing direct faeces for Shiga toxin.     

A multiple protocol to improve diagnosis and isolation of Shiga toxin-producing Escherichia coli from human stool specimens

Many infections caused by Shiga toxin-producing Escherichia coli (STEC) are undiagnosed, particularly non-O157 STEC. We evaluated the use of a multiple protocol approach to improve diagnosis, isolation, and characterization of STEC strains. Among 18 presumptive STEC-positive stool samples received by the INOVA Fairfax Hospital, Falls Church, VA, in 2006, 16 were Shiga toxin positive. From these 16 stool samples, 8 O157:H7 and 5 non-O157 STEC were isolated by plating onto sorbitol MacConkey (SMAC) agar. The remaining 5 stool samples that did not yield colonies on SMAC agar plates were enriched. All enriched samples were Shiga toxin positive, and 2 O157:H7 and 1 non-O157 STEC were subsequently isolated. The 2 remaining enriched samples did not yield isolates; however, based on polymerase chain reaction (PCR) analysis, both samples contained STEC genes. Based on PCR analysis of non-O157 strains, 3 strain types were identified. Samples from 3 patients, received within 2 days of one another, had a similar gene profile-eae and stx(1) negative and stx(2) positive-suggesting that these patients were likely infected with the same strain. Our results indicate that a multiple protocol approach is necessary to reliably diagnose and isolate STEC strains, and that PCR profiling of strains could allow for more rapid identification of outbreaks.     

Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR Green I in a real-time multiplex PCR

Enterohemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen capable of causing diarrhea and vomiting, but more serious complications such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) can result. A real-time PCR method to detect the presence of Shiga toxin producing E. coli (STEC) and E. coli O157:H7 was investigated using SYBR Green I (SG). Primers were designed to target the Shiga toxin genes (stx1 and stx2) and a highly conserved base substitution at +93 of the beta-glucuronidase gene (uidA) unique to E. coli O157:H7. An initial test panel of five E. coli and non-E. coli isolates was tested with individual primer sets (simplex assay) and all primer sets including stx1, stx2, and uidA (multiplex assay). All strains were correctly identified in both assays. Average melt temperatures (Tm's, degrees C) for PCR products were 85.42--stx1, 81.93--stx2, and 88.25--uidA in simplex assays and 85.20--stx1, 81.20--stx2, and 88.16--uidA when multiplexed. Each of the three gene targets in one multiplex reaction could be distinguished by melt curve data with significantly different Tm's. The assay was expanded to a panel of 138 isolates consisting of STEC, E. coli O157:H7, non-toxigenic E. coli, and non-E. coli isolates with melt peaks consistent with those stated above.     

Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome.

BACKGROUND: The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. MATERIALS AND METHODS: We measured cell surface TF activity in response to tumor necrosis factor-alpha (TNF-alpha) (20 ng mL(-1), 2-144 h), Stx-1 (10(-11) mol L(-1), 4-144 h), or their combination (TNF-alpha 22 h and Stx-1 for the last 0.5-4 h of TNF-alpha incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). RESULTS AND CONCLUSIONS: We observed that while TNF-alpha caused an increase in cell surface TF activity on both cell types, the combination of TNF-alpha and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 +/- 0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx-1 binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-alpha markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-alpha and Stx-1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine-activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS.     

Shiga toxin binds to activated platelets

Summary. Hemolytic uremic syndrome (HUS) is associated with acute renal failure in children and can be caused by Shiga toxin (Stx)‐producing Escherichia coli. Thrombocytopenia and formation of renal thrombi are characteristic of HUS, suggesting that platelet activation is involved in its pathogenesis. However, whether Shiga toxin directly activates platelets is controversial. The present study evaluates if potential platelet sensitization during isolation by different procedures influences platelet interaction with Shiga toxin. Platelets isolated from sodium citrate anticoagulated blood were exposed during washing to EDTA and higher g forces than platelets prepared from acid‐citrate‐dextrose (ACD) plasma. Platelet binding of Stx was significantly higher in EDTA‐washed preparations relative to ACD‐derived platelets. Binding of Stx was also increased with ACD‐derived platelets when activated with thrombin (1 U mL^?1) and exposure of the Gb3 Stx receptor was detected only on platelets subjected to EDTA, higher g forces or thrombin. EDTA‐exposed platelets lost their normal discoid shape and were larger. P‐selectin (CD62P) exposure was significantly increased in EDTA‐washed preparations relative to ACD‐derived platelets, suggesting platelet activation. Taken together, these results suggest that direct binding of Stx occurs only on ‘activated’ platelets rather than on resting platelets. The ability of Stx to interact with previously activated platelets may be an important element in understanding the pathogenesis of HUS.     

基于夹心法的DNA生物传感器用于检测大肠埃希菌O157：H7 stx2基因

目的构建DNA生物传感器用于大肠埃希菌O157:H7的快速检测。方法基于双探针夹心法,结合酶标生物电催化反应技术和计时电流的电化学法检测大肠埃希菌O157:H7的stx2基因。结果在最佳反应条件下,DNA探针能较好地固定于金电极表面;探针标记链酶亲合素后,检测信号明显增强。DNA生物传感器具有良好的特异性,其值可达6.25×10-8mol/L。结论构建的DNA生物传感器特异性和灵敏度高,可为大肠埃希菌O157:H7的快速检测提供新的方法。     

产志贺毒素大肠埃希菌分离株多位点可变数目串联重复序列分析

目的 探讨多位点可变数目串联重复序列分析[multiple-locus VNTR (variable-number tandem repeats) analysis,MLVA]分型方法对产志贺毒素大肠埃希菌分离株的分型效果,初步了解菌株分子流行病学特征。 方法 采用7个VNTR位点对70株产志贺毒素大肠埃希菌分离株进行分析,并根据多态性位点的重复数目利用BioNumerics软件进行聚类分析。 结果 70株菌株被分为46种MLVA型别,O157与非O157菌株可分为A、B两个明显不同的群。除少部分菌株外,不同宿主来源、志贺毒素型别及血清型的非O157菌株与MLVA型别存在较好的相关性。 结论 7个VNTR位点的MLVA分型方案对于产志贺毒素大肠埃希菌的菌株分型、暴发溯源等具有一定的参考价值,但仍需进一步完善。