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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Detection of Shiga Toxin-Producing Escherichia coli Serotypes O26:H11 O103:H2 O111:H8 O145:H28 and O157:H7 in Raw-Milk Cheeses by Using Multiplex Real-Time PCR
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Detection of Shiga Toxin-Producing Escherichia coli Serotypes O26:H11 O103:H2 O111:H8 O145:H28 and O157:H7 in Raw-Milk Cheeses by Using Multiplex Real-Time PCR

机译：使用多重实时PCR检测生乳奶酪中产志贺毒素的大肠杆菌血清型O26：H11O103：H2O111：H8O145：H28和O157：H7

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Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzxO26-eae-β1 (4.8%; 19 samples), stx-wzxO103-eae-ɛ (1.3%; five samples), stx-ihp1O145-eae-γ1 (0.8%; three samples), and stx-rfbEO157-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeβ1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.
机译：产生志贺毒素（Stx）的大肠杆菌（STEC）菌株是多种食源性病原体，对人类的毒力各不相同。在这项研究中，我们描述了结合使用多种实时PCR分析方法筛选400种生乳奶酪中的5种主要致病性STEC血清型（O26：H11，O103：H2，O111：H8，O145：H28 ，以及O157：H7）。对stx，内膜蛋白编码基因（eae）和五个O组遗传标记中至少一个阳性的样本的患病率分别为29.8％，37.3％和55.3％。 H2，H7，H8，H11和H28 fliC等位基因非常普遍，不能用作可靠的筛选靶标。通过实时PCR在6.5％的奶酪（26个样品）中检测到stx，eae变体和O基因标记的组合，这是五个目标STEC血清型的典型特征，其中包括stx-wzxO26-eae-β1（4.8） ％； 19个样本），stx-wzxO103-eae-ɛ（1.3％；五个样本），stx-ihp1O145-eae-γ1（0.8％；三个样本）和 stx - rfbE O157- eae -γ 1 （0.3％；一个样品）。对这些组合呈阳性的样品进行了28次免疫磁分离（IMS）分析，可以回收到7个 eae β 1 阳性STEC O26：H11分离株，而没有STEC O103 ：H2，O145：H28或O157：H7菌株可以分离出来。三个 stx 阴性和 eae β 1 阳性 E。还通过IMS从干酪中分离出了大肠杆菌O26：[H11]菌株。菌落杂交使我们可以从 stx 阳性样品中回收15种STEC阳性样品中的15种，这突出了从乳制品中分离STEC所遇到的困难。 STEC O26：H11分离株与肠出血性 E具有相同的毒力遗传特征。大肠杆菌（EHEC）O26：H11，即它们携带了与毒力相关的基因EHEC- hlyA ， katP 和 espP ，以及基因组O岛71和122。除了一个菌株外，它们都只包含 stx1 变体，据报道与人类病例相关的频率比 stx2 。脉冲场凝胶电泳（PFGE）分析显示它们具有很高的遗传多样性。它们均没有与此处研究的人O26：H11菌株相同的模式。

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