【作者】 申进玲；

【导师】 孟江洪；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2013， 博士

【摘要】 非O157产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli, STEC）和单增李斯特菌是近年来导致人类疾病的两大主要病原菌。本文研究了359株食品（n=99）、人（n=105）和动物源（n=155）非O157产志贺毒素大肠杆菌的主要毒力基因（志贺毒素基因stx1和stx2，凝集素基因eae和溶血素基因hlyA）、血清型和猴肾细胞毒性（所选菌株）。然后从此359株菌株中选取272株进行PCR电喷雾电离质谱（PCR Coupled to Electrospray Ionization-Mass Spectrometry, PCR-ESI-MS）检测，测定看家基因（mdh和mutS）的五个片段和毒力基因（stx1、stx2、eae、hlyA和aggA）的六个基因片段的碱基组成，从而对它们进行检测鉴定和分型。另外，对来源于2008年零售肉中的单增李斯特菌进行了耐药性和分子血清组测定；通过对单增李斯特菌中的主要毒力基因包括inlC、inlJ、llsX和inlA以及流行克隆（EC）的研究，并结合脉冲场凝胶电泳（Pulsed-Field Gel Electrophoresis, PFGE）探讨了它们以及另外来源于2003年马里兰地区鸡肉和蔬菜中的30株单增李斯特菌的致病潜力。得出以下主要结果：（1）在359株非O157STEC中共检出18种stx基因型，前六种分别为stx_1a（61%）、stx_2a（9%）、stx_2dact（8%）、stx_1a+stx_2a（7%）、stx2c（2%）和复合变体stx_2a+stx_2dact（2%）。与O157STEC相比，stx2c和复合变体的比例较低。主要有四种eae变体eaeβ、eaeε1、eaeγ1和eaeγ2/θ，并且eae变体和血清型尤其是鞭毛抗原类型有很大的关系。stx₂变体在eae阳性和阴性菌株中分布有很大差别：eae阳性菌株中主要含stx_2a，而stx₂变体在eae阴性菌株中较多样化，且stx_2dact主要存在于eae阴性菌株中。猴肾细胞毒性测试结果表明含stx_2b、stx_2e或stx_2g的菌株的毒性较低，而含stx_2a、stx₂c或stx_2dact的菌株的毒性普遍较高。（2）不同来源的菌株所含的血清型和毒力基因有很大差异。人源菌株中“六大O抗原”O26、O111、O103、 O145、 O121和O45占84%。动物源（主要为牛）菌株中O111、O103和O26占91%。而对于食源性菌株，不属于“六大O抗原”的菌株占98%。关于毒力基因，在99株食品源菌株中，大多数只含Stx2（67%），21%只含Stx1，11%同时含有Stx1和Stx2。只有一株含有eae,将近一半（45%）的菌株含有hlyA。相反，105株人源菌株中， Stx1占67%，而Stx2只占21%， Stx1+Stx2占12%，但大多数含有eae （85%）和hlyA （82%）。关于毒力基因型， stx_1a+eae+hlyA在人源菌株中最流行占57%；而食源菌株中，毒力基因型呈现多样化，最常见的是stx_2dact，只占不到18%。（3）食源性菌株中发现曾引起人类疾病的血清型： O113:H21、O91:H21、O104:H21、O22:H8、O45:H2、O73:H18、O113:H21、O116:H21、O121:H19、ONT:H2、ONT: H10和O91:H14，且这些菌株大多含有stx_2a和（或）stx_2dact+hlyA，有些菌株与临床菌株表现出一样高的猴肾细胞毒性。这些菌株具有致病潜力，需要引起重视。（4）关于PCR-ESI-MS，首先选取164株典型菌株在96孔板（Foodborne Bacteriav2）上测试从而建立毒力基因（变体）和血清型的数据库，然后对剩余的108株典型菌株进行测试，从而评估板子的检测和分型能力。对108株非O157STEC毒力基因的检测特异性如下：100%（stx1、eae和aggA），对于stx2和hlyA来说分别是99%和96%。另外，此方法还能够分辨出stx1的主要变体(stx_1a和stx1c)，对于stx2基因来说，它能够把与严重疾病高度相关的stx2变体(stx_2a/stx2c/stx_2dact)和其他变体(stx_2b、stx_2e和stx_2g)区分开。对于存在于同一株菌中的多个不同stx1或stx2基因变体也能够检测出来。此外，由于以下几种重要血清型含有独特的看家基因碱基组成，因此此方法还能将它们分别辨别出来： O91:H14、O103:H25、O145:H28/NM、O113:H21和O104:H4。另外，由于菌株所含有的看家基因和毒力基因碱基组成差异，此方法能够将菌株分为具有不同致病潜力的组。这种高通量的方法在STEC的检测、鉴定和分型中非常有前景，尤其是对具有高致病潜力的STEC的筛选中更加有优势。（5）来源于2008年近700份生肉中，李斯特菌占20%，共检出三个种：单增李斯特菌（10%）、威尔斯李斯特氏菌（9%）和英诺克李斯特氏菌（2%）。关于单增李斯特菌，最常见的血清型为1/_2a,3a （33%），以下依此为1/2c,3c （27%）、1/_2b,3b,7（22%）、4b,4d,4e （16%）和4a,4c （2%）。世系II菌株（60%）比世系I菌株（39%）常见，只发现一株（1%）属于世系III。大多数单增李斯特菌株对所测的抗生素敏感，但表面出对四环素（4.5%）、环丙沙星（4.5%）和呋喃咀啶（10.5%）较低的耐药率。另外，61%和85%的菌株对环丙沙星和呋喃咀啶中介耐药。未发现多重耐药（耐两种及以上抗生素）菌株。世系I（3/26）中比世系II （9/40）中含有较少的耐药菌株。（6）对来源于2008年马里兰零售肉中的67株单增李斯特菌和来源于2003年马里兰州的30株单增李斯特菌（24株来源于生鸡肉，6株来源于蔬菜）的毒力基因分析结果表明，除了一株世系III菌株，所有单增李斯特菌都含有inlC和inlJ；李斯特菌溶血素S （LLS）阳性菌株中大多数（11/12）属于世系I，而另外的一株菌株属于世系III。五个属于分子血清组4b,4d,4e的菌株（两个来源于火鸡肉、三个来源于蔬菜）属于流行I （ECI）。共发现有四种与分子血清组相关的突变类型，可导致inlA终止密码子提前。PFGE和inlA序列分析结果高度相似，且分别在分子血清组1/_2a,3a和4b,4d,4e中发现有不同致病潜力的菌株组。来源于零售肉和蔬菜中的一些菌株属于ECI，含有inlC、 inlJ和LLS，且具有功能性的InlA，这表明他们可能会引起人类疾病。更多还原

【Abstract】 Non-O157Shiga toxin-producing Escherichia coli （STEC） and Listeria monocytogenesare two important emerging foodborne pathogens that cause human illnesses. In this study,the main virulence factos （stx1, stx2, eae and hlyA and their subtypes）, the serotypes and verocell cytotoxicities （for selected isolates） of359non-O157STEC isolates from food, humanand animals were studied. In the mean time,272isolates were selected from the359STECisolates for PCR Coupled to Electrospray Ionization-Mass Spectrometry （PCR-ESI-MS）analysis; a new plate was developed to identify and subtype them by testing the basecompositions of fragments from two housekeeping genes （mdh and mutS） and five virulencegenes （stx1, stx2, eae, hlyA and aggA）. In addition, the antimicrobial susceptibility andmolecular serogroups of Listeria monocytogenes from retail meats in2008were studied;furthermore, combined with pulsed-field gel electrophoresis （PFGE）, the virulence potentialof them together with extra30isolates from chicken and produce in Maryland from2003were explored by testing the presence of inlC, inlJ and llsX, whether belonging to epidemicclones （EC）, or having full length InlA. Major findings and conclusions were summarized asfollowing:（1） Among the359non-O157STEC isolates,18stx genotypes were observed, and thetop six were stx_1a（61%）, stx_2a（9%）, stx_2dact（8%）, stx_1a+stx_2a（7%）, stx2c（2%） and stx_2a+stx_2dact（2%）. Compared with O157STECs, the percentages of stx2cand multiple alleles were lower.Four eae subtypes eaeβ,eaeε1,eaeγ_1and eaeγ2/θwere observed. Association between eaesubtype and serotype was found and some eae subtypes are flagella associated. Eae positiveisolates and negative isolates differed a lot in their virulence factors in that Shiga toxin type2subtypes in the latter were more diverse than in the former, and stx_2awas the main subtype ineae positive isolates, whereas stx_2dactwas mainly found in eae negative isolates. Isolates withstx_2b, stx_2eand stx_2gshowed lower cell cytotoxicity, whereas isolates with stx_2a, stx2cor stx_2dactshowed higher cell cytotoxicity.（2） The serogroups and virulence genes in isolates from different sources differed a lot.The “top six” non-O157O serogroups O26, O111, O103, O145, O121and O45took up to84% of the STEC serogroups from human. O111, O103and O26encompassed91%of isolatesfrom animals. O serogroups which were other than the top six non-O157accounted for98%of all isolates from food. As for virulence genes, among the99food isolates, the majoritywere Stx2positive （67%）, and21%carried only Stx1, or Stx1and Stx2in combination （11%）.Of those only one isolate carried eae, however almost half （45%） were hlyA positive.Contrary, the majority of human isolates （67%） carried Stx1, and less frequently Stx2（21%）or Stx1+Stx2（12%）, but most were eae （85%）, and hlyA （82%） positive. As for virulencegenotypes, stx_1a+eae+hlyA was prevalent in human （57%）; in food isolates, the genotypeswere more diverse and the most frequently found one was stx_2dact, which was less than18%.（3） The following important serotypes found in food isolates have been reported inhuman illnesses: O113:H21, O91:H21, O104:H21, O22:H8, O45:H2, O73:H18, O113:H21,O116:H21, O121:H19, ONT: H2, ONT: H10and O91:H14. Most of the isolates had stx_2aand/or stx_2dact+hlyA, and some even had vero cell cytotoxicities as high as human isolates.These isolates may have the potential to cause future human diseases and should attach greatimportance.（4） As for PCR-ESI-MS, a total of164well characterized STEC isolates were firstexamined with the assay to build a DNA base composition database. Another panel of108diverse STEC isolates was tested with the established database to evaluate the assay’sidentification capability. Among the108isolates, the assay specificity was100%for three（stx1, eae, and aggA） out of five tested virulence genes, but99%for stx_2and96%for hlyA,respectively. Main stx1/stx2subtypes and multiple alleles of stx1/stx2could be differentiated.The assay successfully identified several clinically significant serotypes, including O91:H14,O103:H25, O145:H28/NM, O113:H21and O104:H4, due to their unique base compositionpatterns of housekeeping genes. Meanwhile, it was able to group isolates with different levelsof pathogenic potential due to the difference in the base compositons of housekeeping genesand virulence genes. The results suggest that this high-throughput method may be useful inclinical and regulatory laboratories for STEC identification, particularly strains with increasedpathogenic potential.（5） Recovery of Listeria spp from about the700raw meat samples was20%, with threedifferent species L. monocytogenes （10%）, L. welshimeri （9%） and L. innocua （2%）. As for L.monocytogenes, the most common molecular serogroup found in the meat samples from2008was1/_2a,3a （33%）, followed by1/_2b,3b,7（22%）,4b,4d,4e （16%）, and4a,4c （2%）. Ingeneral, lineage II isolates （60%） were more commonly found than lineage I isolates （39%）,and only one isolate belonged to lineage III （1%）. Most L.monocytogenes isolates weresusceptible to the antimicrobials tested and low resistance rates to tetracycline （4.5%）, ciprofloxacin（4.5%） and nitrofurantoin （10.5%） were found.61%and85%of isolates wereintermediate resistant to ciprofloxacin and nitrofurantoin respectively. No multiple resistantisolates （resistant to two or more antimicrobials） were found. More resistant isolates werefound in lineage II （9/40） isolates than in lineage I isolates （3/26）.（6） The virulence gene analysis of all97L. monocytogenes isolates including67isolatesfrom retail meats in2008and30isolates from chicken meat and produce in2003showed thatall carried inlC and inlJ except for a lineage III isolate110-1. Most Listeriolysin S （LLS）-carrying isolates （11/12） belonged to lineage I, whereas the remaining one isolate belonged tolineage III. Five4b,4d,4e isolates including two from turkey and three from producebelonged to Epidemic Clone I （ECI）. Four molecular serogroup associated mutation types thatlead to premature stop codons （PMSCs） in inlA were identified. PFGE and inlA sequenceanalysis results were concordant, and different virulence potential within1/_2a,3a and4b,4d,4e isolates were observed. The study revealed that a subset of isolates from meat and producebelonged to ECI, harbored inlC, inlJ and LLS, and produced full length InlA, suggesting thatthey be capable of causing human illness.更多还原