鉴定单增李斯特菌四个进化家系及两个亚系的多PCR方法的建立

目的利用多重PCR（multiplexPCR）技术,建立针对单增李斯特菌4个进化家系（LineageⅠ,LineageⅡ、LineageⅢ、LineageⅣ）及2个亚系（LineageⅢA、LineageⅢC）的鉴定方法。方法以InlA、Lmo0733、Lmo0038、Lmo0735和LmaA作为靶基因设计引物,建立同时鉴别LineageⅠ、LineageⅡ、LineageⅢA、LineageⅢC和LineageⅣ菌株的多重PCR反应体系,并对387株单增李斯特菌进行了所属进化家系及亚系的鉴定。结果387株单增李斯特菌可用本方法进行所属家系和亚系的分型,包括LineageⅠ246株、LineageⅡ123株、LineageⅢA4株、LineageⅢC13株和LineageⅣ1株。结论本方法能准确鉴定单增李斯特菌的4个进化家系及2个亚系的菌株,可用于食品、临床标本中单增李斯特菌的楠原学诊断和疫情暴发时的溯源调查。     

单增李斯特菌1/2a血清型菌株生物膜形成能力及相关基因差异研究

摘要： 目的 检测5株1/2a血清型单增李斯特菌生物膜形成能力并分析其单增李斯特菌生物膜形成相关基因的携带情况,为进一步探究单增李斯特菌生物膜形成影响因素及致病机制提供线索。方法 依据96孔微孔板结晶紫染色方法对5株单增李斯特菌进行了生物膜形成能力检测,并用扫描电镜观察生物膜形成情况。使用聚合酶链反应（PCR）技术对5株菌进行了生物膜形成相关的12对基因（sigB,prfA,yneA,hpt,relA,flaA,recA,agrA,luxS,degU,motB,bapL）全长的扩增及测序分析,通过与GeneBank中已公布的单增标准菌株EGD-e序列比对确定基因变异情况。 结果 5株1/2a血清型单增李斯特菌形成生物膜的能力有所不同,结晶紫染色观察结果与扫描电镜观察结果较为一致。11种生物膜相关基因在5株单增李斯特菌中的检测结果全阳性,测序结果发现部分基因出现突变位点（多为同义突变,部分为非同义突变）。bapL基因在生物膜形成能力较低两株菌中为阳性,而在生物膜形成能力较高的两株菌中为阴性。结论 不同的1/2a血清型单增李斯特菌间生物膜形成能力有所差异,5株1/2a血清型单增李斯特菌bapL基因携带与其生物膜形成能力并不一致,11株生物膜相关基因中出现多处碱基位点及氨基酸变化等同义突变与非同义突变。     

3例单增李斯特菌感染的病原学、临床及流行病学特征分析

目的 分析四川省自贡市2014年3例单增李斯特菌感染的临床特征及其病原学特征。方法 搜集临床病例信息;采用血清分型,脉冲场凝胶电泳技术和多位点序列分型方法对单增李斯特菌进行分子分型。结果 3例患者均为免疫力低下人群,有发热和败血症症状;3株单增李斯特菌中2株为1/2a血清型、ST8型,1株为1/2b血清型、ST778型,3株PFGE带型均不同。结论 免疫力低下人群易受单增李斯特菌侵袭性感染。通过与国内外食品及病人单增李斯特菌相关流行学资料比较,提示国外流行克隆群以及国内某些流行型菌株可引起国内李斯特菌病散发,甚至成为将来暴发的隐患。     

3例单增李斯特菌感染的病原学、临床及流行病学特征分析北大核心CSCD

目的分析四川省自贡市2014年3例单增李斯特菌感染的临床特征及其病原学特征。方法搜集临床病例信息;采用血清分型,脉冲场凝胶电泳技术和多位点序列分型方法对单增李斯特菌进行分子分型。结果 3例患者均为免疫力低下人群,有发热和败血症症状;3株单增李斯特菌中2株为1/2a血清型、ST8型,1株为1/2b血清型、ST778型,3株PFGE带型均不同。结论免疫力低下人群易受单增李斯特菌侵袭性感染。通过与国内外食品及病人单增李斯特菌相关流行学资料比较,提示国外流行克隆群以及国内某些流行型菌株可引起国内李斯特菌病散发,甚至成为将来暴发的隐患。     

1例产后哺乳期腹泻患者分离单增李斯特菌的生物学特征分析

目的 分析1例单增李斯特菌所致产后哺乳期腹泻患者的临床特征、病原学、菌株的毒力基因携带及分子特征。方法 搜集病例的临床相关信息;采用选择性增菌和显色培养基分离单增李斯特菌,用VITEK- 2 COMPACT30 全自动细菌分析仪鉴定分离菌株,采用单增李斯特菌诊断血清对分离株进行血清分型,使用脉冲场凝胶电泳技术及多位点序列分型技术进行分子分型,应用PCR检测毒力基因,并用E-test方法检测分离菌株对5种抗生素的药物敏感性。结果 产后哺乳期患者食用甜瓜和冰箱冷藏的酸奶后引起不能自限的腹泻,检测结果证实病原为4b血清型、ST145序列型单增李斯特菌。分离菌株携带毒力基因plcB、act、hly、iap、prfA、inlA,对青霉素、氨苄西林、美罗培南、红霉素和复方磺胺均敏感。结论 单增李斯特菌引起的腹泻患者容易漏诊,加强孕妇等高危人群腹泻患者粪便标本中单增李斯特菌的检测,对于单增李斯特菌病病人的及时治疗,预防不良预后具有重要的公共卫生意义,尤其对这一特殊型别单增李斯特菌在我国引起的病例感染应引起重视,其可能的感染来源和致病风险值得进一步的研究。     

EMA实时荧光PCR技术检测食品中单核增生李斯特活菌方法研究

目的利用叠氮溴化乙錠(EMA)实时荧光PCR技术,建立直接检测食品中单增李斯特活菌的方法。方法根据单核李斯特菌inlA基因设计特异性引物和探针。用不同EMA浓度、不同光照次数优化样品前处理条件。用平板计数法验证该方法的检出限及对死菌的抑制率。用35株单增李斯特菌、25株非单核李斯特菌、92株非李斯特菌验证该方法特异性。用添加了不同剂量的单增李斯特死菌、活菌及金黄色葡萄球菌的15件饮料,15件熟肉制品进行模拟实样检测。结果单增李斯特活菌EMA实时荧光PCR方法的Ct=(38.46-3.30)×log(菌量)(R2=0.999)。最低检测的活菌浓度为55cfu/反应。对死菌DNA抑制效率≥99.98%。35株单增李斯特菌Ct值最低16.21,最高29.38,而25株非单单增李斯特菌、92株非单增李斯特菌的Ct值均大于35或无Ct值。重复试验Ct值变异系数小于5%。30件模拟实样单增李斯特菌的检测结果与常规方法完全一致。且EMA实时荧光PCR方法检出时间仅需10h左右,而常规分离培养方法需要5~7d。结论 EMA实时荧光PCR检测技术是一种快速、简便、特异性强、灵敏度高、仅检测单增李斯特菌活菌的有效方法,可作为食品中检测单增李斯特活菌的方法推广使用。     

北京市一些地区生肉标本中单增李斯特菌的分离及其分子流行病学特征分析

目的 了解北京市一些地区生肉中单增李斯特菌的污染情况及其分子流行病学特征。方法 采用北欧食品分析标准(NMKL)对北京市一些地区采集的340份牛、羊、猪、鸡和鸭生肉标本进行单增李斯特菌的分离鉴定,并对分离菌株进行血清学分型(Serotyping)、脉冲场凝胶电泳(Pulsed Field Gel Electrophoresis, PFGE)分型及多位点序列分型(Multilocus Sequence Typing, MLST)分析。结果 生肉标本中单增李斯特菌污染率为6.2%(21/340)。分离菌株含3种血清型(1/2a,1/2b和1/2c),以1/2c为优势血清型(占47.6%)。PFGE分析中,使用内切酶AscI酶切电泳将21株单增李斯特菌分为12个型别,可聚类为3个群。MLST分析将21株单增李斯特菌分为7个ST型,其中 ST9型最多(10株)。结论 北京市一些地区的生肉类食品中存在6.2%的单增李斯特菌污染率,这些单增李斯特菌以家系II菌株占据绝对优势(80.1%),其中1/2c型,GX6A16.CN0004和ST9型分别为血清型、PFGE带型以及MLST序列型的优势型别,与我国食源性单增李斯特菌的优势菌群特征一致。     

单增李斯特菌P60单克隆抗体的制备及初步应用

目的 制备并筛选单增李斯特菌P60特异性单克隆抗体,初步建立其快速检测方法。方法 以体外表达并纯化的李斯特菌P60蛋白为抗原免疫Balb/c 小鼠, 采用杂交瘤技术制备抗李斯特菌P60的McAb,鉴定其抗体亚型,并对单抗的腹水效价、稳定性及亲和力进行分析。用纯化的单克隆抗体包被酶标板,结合生物素化处理的P60多克隆抗体,夹心ELISA筛选单增李斯特菌特异性单抗,并以重组P60为标准品,建立标准曲线。结果 获得了4株可稳定分泌P60单克隆抗体的杂交瘤细胞株。ELISA检测表明,筛选的6C2单克隆抗体株可特异性与单增李斯特菌(4b)P60反应。结论 建立的夹心ELISA方法可对单增李斯特菌进行特异性鉴别和定量检测。     

单增李斯特菌三重实时荧光PCR检测的建立及其毒力基因在分离菌株中分布

目的建立同时检测单增李斯特菌(Listeria monocytogenes,Lm)溶血素基因hlyA、内化素基因inlA和磷脂酶C基因plcB的TaqMan-MGB三重实时荧光PCR检测方法,并对101株单增李斯特菌的hlyA、inlA和plcB基因进行了检测,分析3个毒力基因在多位点序列分型(Multilocus sequence typing,MLST)聚类群组中的分布情况。方法根据单增李斯特菌毒力基因hlyA、inlA和plcB的保守序列设计引物和小沟结合物(minor groove binder,MGB)探针建立三重荧光PCR方法,对其特异性、灵敏度和可重复性进行了测试,并对分离的101株单增李斯特菌进行三重PCR检测。结果建立的三重实时荧光PCR方法特异于单增李斯特菌的检测,重复性良好,组内Ct值变异系数均小于1.5%,灵敏度可达100CFU/mL。对101株单增李斯特菌的hlyA、inlA和plcB的检出率分别为98%、75%和98%。在可被MLST分型的89株菌株中,groupI的30株菌株有20株均缺失inlA基因;groupII的57株菌株中有56株三个基因均检出;groupIII的菌株hlyA、inlA和plcB三个基因均未检出。结论本文建立的三重实时荧光PCR方法能准确、快速、稳定的检测单增李斯特菌,101株单增李斯特菌的hlyA、inlA、plcB三个毒力基因的检出情况与单增李斯特菌的MLST分型聚类有较一致的关系,对分析单增李斯特菌毒力的强弱有重要参考意义。     

2011—2021年福建省即食食品中单增李斯特菌的监测及菌株特征分析

目的 了解2011—2021年福建省即食食品中单增李斯特菌(Listeria monocytogenes,Lm)污染状况、毒力基因、耐药及分子分型特征。方法 参照GB 4789.30检测Lm;采用PCR方法对Lm的13种毒力基因进行检测及开展血清学、MLST分型；应用微量肉汤稀释法，选择15种抗生素进行药敏试验。结果 2011—2021年共采集8 166份即食食品，Lm总检出率为1.25%,以寿司、中式凉拌菜及熟肉制品检出率较高，分别为6.26%、5.60%、1.87%;毒力基因plcB、llsX、ptsA的检出率分别为55.42%、16.87%、22.89%,prfA等其余10个毒力基因检出率均为100%;83株Lm对头孢西丁的耐药率为100%,苯唑西林为15.66%、四环素为13.25%;寿司、中式凉拌菜、熟肉制品中检出多重耐药株，多重耐药率达13.25%;83株Lm分4种血清型，1/2a、1/2b为优势血清型；83株Lm MLST分型得到16种序列型(ST),ST87为优势型，其次是ST8,ST101。结论 2011—2021年福建省即食食品存在不同程度的单增李斯特菌污染，寿司、...     

Genotypic characterization of Listeria monocytogenes isolated from humans in India

Listeria monocytogenes is a foodborne pathogen associated with severe diseases in humans and animals. The genotypic analysis of 17 L. monocytogenes isolates recovered from humans in India during 2006-2009 using multiplex serotyping PCR allowing serovar predictions, conventional serology and by pulsed field gel electrophoresis (PFGE) is presented. The isolates were recovered from patients exhibiting various clinical conditions. A multiplex-PCR based serotyping assay revealed 88·24% (15/17) of the strains belonging to the serovar group 4b, 4d, 4e and 11·76% (2/17) to the serovar group 1/2b, 3b. Conventional serology indicated that 13 (76·47%) L. monocytogenes isolates to be of serotype 4b, 2 (11·76%) serotype 4d, and 2 (11·76%) serotype 1/2b. Ten ApaI and nine AscI pulsotypes were recognized among the 17 human isolates. PFGE analysis allowed discrimination among isolates of the same serotype and among isolates from the same sampling areas or those isolated from different areas. Thus, PFGE together with multiplex-PCR serotyping allows rapid discrimination of L. monocytogenes strains. In addition, the predominance of L. monocytogenes serotype 4b is of concern, as this serotype has been most frequently associated with human listeriosis outbreaks.     

Characterization of a Novel Group of Listeria Phages That Target Serotype 4b Listeria monocytogenes

Listeria monocytogenes serotype 4b strains are the most prevalent clinical isolates and are widely found in food processing environments. Bacteriophages are natural viral predators of bacteria and are a promising biocontrol agent for L. monocytogenes. The aims of this study were to characterize phages that specifically infect serotype 4b strains and to assess their ability to inhibit the growth of serotype 4b strains. Out of 120 wild Listeria phages, nine phages were selected based on their strong lytic activity against the model serotype 4b strain F2365. These nine phages can be divided into two groups based on their morphological characteristics and host range. Comparison to previously characterized phage genomes revealed one of these groups qualifies to be defined as a novel species. Phages LP-020, LP-027, and LP-094 were selected as representatives of these two groups of phages for further characterization through one-step growth curve and inhibition of serotype 4b L. monocytogenes experiments. Listeria phages that target serotype 4b showed an inhibitory effect on the growth of F2365 and other serotype 4 strains and may be useful for biocontrol of L.monocytogenes in food processing environments.     

Human listeriosis in Peru.

In a survey on listeriosis in Peru a study was made of 653 stool samples; material from 8 cases of perinatal death, in which a macroscopic diagnosis of listeriosis had been made at autopsy; 1 sample of vaginal secretion from a woman with abortion due to listeriosis; and 1 case of abscess which turned out to be an hematometra. The following results have been obtained: 1. From the faeces 7 isolates of Listeria (1.07%) were obtained: 3 belonged to L. monocytogenes serovar 4ab, 3 to L. innocua (the former "4g"--nownow serovar 6b) and 1 to L. monocytogenes serovar 4a. 2. The 8 strains of autopsy materials belonged to L. monocytogenes: 6 serovar 4b and 2 serovar 4d. 3. From the vaginal secretion L. monocytogenes serovar 4b was cultured. 4. The strain from the hematometra was identified as L. monocytogenes serovar 4b. The serovars 4a, 4ab and 4d are at this time the only ones identified in Peru as cause of human listeriosis. The serovars of serogroup 1/2 frequent in Western and primarily in Central Europe, were so far never cultivated.     

Listeria monocytogenes cross-contamination in a nursery [corrected

Molecular evidence of Listeria monocytogenes cross-contamination in a nursery is presented. Listeria monocytogenes serotype 4b was isolated from the blood and the conjunctiva of a baby with neonatal sepsis who was born after septic amnionitis and premature rupture of membrane. Nine days later, the same bacterium was isolated from the cerebrospinal fluid of a second baby presenting with meningitis. Cervical cultures from the second baby's healthy mother were negative for Listeria sp. An in-depth epidemiologic investigation revealed that the same nurse administered routine treatments to both babies in the nursery during a 1-hour interval of time [corrected]. Pulse-field gel electrophoresis analysis of both strains with 2 different restriction enzymes demonstrated that they were identical and differ from other wild strains of L monocytogenes serotype 4b isolated in Israel. This fact strongly suggests that the second baby was infected during admittance to the nursery as a result of a hospital cross-contamination.     

A molecular marker for evaluating the pathogenic potential of foodborne Listeria monocytogenes

BACKGROUND: Internalin mediates entry of Listeria monocytogenes into some human cultured cell lines and crossing of the intestinal barrier in transgenic mice expressing its receptor, human E-cadherin, in enterocytes. The relevance of these findings for humans is challenged by the observation that some L. monocytogenes isolates express a truncated nonfunctional form of internalin. METHODS: We investigated expression of internalin by use of immunoblot assay in 300 clinical strains obtained in France in a single year and a representative set of 150 strains obtained from food products during the same period. RESULTS: Clinical strains expressed full-length internalin far more frequently (288/300 strains [96%]) than did strains recovered from food products (98/150 strains [65%]; odds ratio, 12.73; 95% confidence interval, 6.27-26.34; P<1 x 10(-7)). All 61 strains (100%) from pregnancy-related cases, 55 (98%) of 56 strains from patients with central nervous system infections, and 151 (93%) of 162 strains from patients with bacteremia expressed full-length internalin. All 110 strains belonging to serovar 4b, the most frequently implicated serovar in human listeriosis, expressed full-length internalin. CONCLUSIONS: This study demonstrates the critical role of internalin in the pathogenesis of human listeriosis. It provides a molecular explanation for the predominance of serovar 4b among clinical strains and supports the usefulness of studying the expression of internalin as a marker of virulence in humans.     

Listeria monocytogenes septicemia associated with consumption of salted mushrooms

Listeria monocytogenes septicemia in an 80-year-old man is described. On the day before clinical symptoms appeared the patient had eaten homemade salted mushrooms, rufous milkcap (Lactarius rufus Fr.). L. monocytogenes serotype 4b was isolated in blood cultures. The mushrooms which had been stored in cold for 5 months before consumption contained the same listeria serotype at a level of 10(6) CFU/g. Salt content (NaCl) of the mushrooms was 7.5%. Fever and diarrhea disappeared with penicillin therapy and the patient was discharged after 4 weeks in the hospital.     

4b型单核细胞增生李斯特菌内化素基因NTSN_0462的功能初探

目的单核细胞增生性李斯特菌(Listeria monocytogenes,Lm)对宿主的粘附、侵袭作用与其多种内化素蛋白密切相关。本研究以4b型菌株LmNTSN的內化素基因NTSN_0462为研究对象,初步探究其致病作用。方法利用同源重组技术构建该基因缺失的突变株,研究0462基因对NTSN在生长、细胞侵袭和体内定植中的作用。人结肠腺癌细胞Caco-2、人肝癌细胞HepG2的侵袭率,以及BALB/c小鼠体内肝、脾脏中定植能力的差异。结果缺失株的生长曲线结果显示,NTSN_0462基因对Lm在BHI培养基中的生长及代谢未造成明显影响。以人结肠腺癌细胞Caco-2、人肝癌细胞HepG2进行的体外试验表明,缺失株的侵袭能力低于野生株(P0.001)。以BALB/c小鼠进行的体内试验显示,缺失株在肝脏中定殖的能力显著低于野生株(P0.001),在脾脏中无统计学差异。结论 NTSN_0462基因在LmNTSN入侵肝脏和定植中发挥重要作用,是侵袭相关的重要毒力因子。     

2016年福建省单核细胞增生李斯特菌临床病例及食品来源菌株的分子特征分析

目的 研究福建省单核细胞增生李斯特菌感染病例的临床特征及分离菌株的分子型别,为食源性疾病溯源和防控提供参考依据。方法 通过监测网报告的感染病例进行个案访谈调查;对2016年临床病例和即食食品来源的单核细胞增生李斯特菌分离株进行血清分型和PFGE分子分型。结果 感染病例均为免疫力低下的孕妇和新生儿,3株病例分离株血清型为2株1/2a和1株4b。8株食品分离株血清型为5株1/2a、2株4b和1株1/2b。11株PFGE分为10个型别。结论 2016年福建存在散发的单核细胞增生李斯特菌病例,PT型各异,无同源性。应加强监测、流行病学调查和饮食卫生宣教,保护孕妇和新生儿等高危人群。