采用高级微生物基因分型系统对食品中单核细胞增生李斯特菌进行同源性分析

采用高级微生物基因分型系统(DiversiLab系统)对食品中单核细胞增生李斯特菌进行基因型分析,将分型结果与血清型和菌株的耐药性进行比对研究。方法 DiversiLab系统是基于rep-PCR原理对微生物进行分型,将分型结果与菌株的血清型进行比较研究,同时对菌株的耐药性与rep-PCR型别进行研究。结果 采用DiversiLab系统将46株单核细胞增生李斯特菌分为9个型别A～I。血清型为1/2a的分离株以A型为主,所测试的6株血清型为4b的分离株均为H型;14株呋喃妥因耐受菌株9株为A型、1株为B型、3株为C型、1株为F型,除2株血清型为3a外,其余均为1/2a。结论 DiversiLab系统分析结果,揭示了46株从食品中分离的单增细胞增生李斯特菌间的亲缘关系,rep-PCR分型结果与H抗原结合位点存在一定联系,呋喃妥因耐受菌株的DNA指纹图谱相似度高,与耐药基因有关。     

应用DiversiLab分型系统对沙门氏菌进行同源性分析

目的:为研究食品污染中沙门氏菌间的同源性关系,建立起沙门氏菌基于重复序列的分型技术。通过建立DiversiLab基因图谱数据库,以便对将来不同来源的沙门氏菌的基因图谱进行即时比较,确定其同源性,进而追溯污染来源,切断传播途径,为预测预警保障食品安全提供科学依据,为防止食源性疾病的发生提供技术支持。同时进行血清分型,比较分析2种方法的优劣和之间的关系。方法:对2002～2010年福建省出入境检验检疫局从各类食品及饲料中分离出的沙门氏菌先进行血清分型,然后进行DiversiLab分型,其中包括:DNA提取,rep-PCR和微电泳芯片分离检测。rep-PCR条件:94℃预变性2min;94℃变性30s,50℃退火30s,70℃延伸90s,35个循环,最后70℃延伸3min。结果:23株沙门氏菌被分成6个血清型,DiversiLab分型能将相同血清型的菌株分为不同的亚型。同一企业来源的菌株具有同源性。结论:DiversiLab分型系统是一种快速、易于操作、高重复性、高分辨率的基因分型方法,可作为食源性疾病同源性分型工具。结论:DiversiLab分型的分辨率高于血清型,2种分型方法密切相关。     

分型方法在单核细胞增多性李斯特菌监测溯源中的应用研究进展

单增李斯特菌(LM)是引起人和动物李斯特菌病的重要食源性病原菌,具有较高的病死率,严重危害人类公共卫生安全和畜牧业发展。快速准确的鉴定LM污染来源,对有效控制李斯特菌病的暴发和蔓延发挥重要作用。建立高效快捷的分型方法是LM溯源的关键。目前LM分型方法主要分为表型分型和分子分型方法,每种分型方法各有优劣,具有适用不同的流行病学调查范围。本文就LM分型方法的研究进展进行综述,以期为开展LM的暴发确认和溯源检测方法的选择提供参考依据,对防御并控制LM引起的食源性疾病的暴发和传播具有重要意义。     

食源性单增李斯特菌的ERIC-PCR和Sau-PCR分型研究

对从广州市天河区超市和菜市场及厦门某食品加工厂分离得到的单增李斯特菌进行ERIC-PCR和Sau-PCR检测,进行溯源研究及遗传多样性分析。ERIC-PCR将22株单增李斯特菌共分为8个基因型,其中以h型为主,共有8株菌。Sau-PCR方法将这批菌分为7个基因型,主要型别为E型和G型,二者共占50%。两种分型方法的结果呈现出一定的差异与关联,使用两种不同分型方法进行综合分析,有助于更好地认识单核细胞增生李斯特菌(LM)菌株间的遗传关系和流行病学特点。     

应用DiversiLab分型系统对奶制品中的阪崎肠杆菌进行同源性分析

为研究奶制品污染中阪崎肠杆菌的同源性关系,并建立起阪崎肠杆菌基于重复序列的分型技术。对2005-2006年福建省出入境检验检疫局从各类奶制品中分离出的阪崎肠杆菌先进行API鉴定,然后进行DiversiLab分型。其中DiversiLab分型包括:DNA提取,rep-PCR和微电泳芯片分离检测。rep-PCR条件为:94℃预变性2 min;94℃变性30 s,55℃退火30 s,70℃延伸90 s,35个循环;最后70℃延伸3 min。结果表明:31株阪崎肠杆菌被API分成了5种不同的生物型,DiversiLab分型能将阪崎肠杆菌分为不同的亚型,但它们之间没有必然的联系。部分同一企业来源的菌株具有同源性,样品的同源性与采样地无关。DiversiLab分型系统是一种快速,高效,易于操作,高重复性,高分辨率的基因分型方法,可作为食源性污染的同源性分型工具。     

出入境动物源性食品中单增李斯特菌的多位点序列分型分析

对出入境动物源性食品中分离的单增李斯特菌进行多位点序列分型（mutilocus sequence typing,MLST）分析,了解其序列型分布特点及不同菌株之间的亲缘关系。提取单增李斯特菌基因组DNA,选择其7 个管家基因进行聚合酶链式反应扩增并测序。将测序结果截成标准序列的长度后上传到MLST数据库进行比对分析,获得7 个管家基因的等位基因谱和序列分型编码,并将结果采用不加权算术平均组对（unweighted pair group method usingarithmetic averages,UPGMA）法进行聚类分析。89 株单增李斯特菌共获得51 个STs,其中26 个为新获得的STs（STnew1～STnew26）;数量最多的5 个STs为ST8（9.0%）,ST121（9.0%）、ST7（5.6%）、ST87（5.6%）及新发现的STnew3（7.8%）;其中ST456、ST34、ST343、ST19、ST517、ST201、ST98、ST330和ST73为在国内首次获得。采用UPGMA算法得到的进化树可将89 株菌株分为3 大类群,分类的结果与单增李斯特菌血清学家系分类结果一致。MLST结果对了解出入境动物源性食品中分离的单增李斯特菌的亲缘关系及流行病学溯源有重要意义。     

临沂市食源性单增李斯特菌分子特征分析

目的：基于全基因组测序分析临沂市食源性单增李斯特菌的毒力基因、耐药基因携带情况及分子分型特征。方法：利用测序数据对27株食源性单增李斯特菌株进行多位点序列分型、毒力基因和耐药基因分析,并构建基于cg-SNP的系统发育树。结果：27株分离菌株共分为10个ST型,其中ST121为优势型别。27株单增李斯特菌有26株菌携带磷霉素（FosX）耐药基因,占96.3%,2株菌携带四环素类（tetM）和甲氧苄氨嘧啶类（dfrG）2种耐药基因,仅有1株菌不耐药。27株菌出现两种毒力缺失,第一种是毒力岛LIPI-1缺失,缺失率为7.4%。第二种是毒力岛LIPI-2中inlF单一缺失,缺失率为40.7%。系统发育树分析显示,27株菌株距离较近,聚集于3个分支中。绝大部分菌株分离自肉制品,占74.1%(20/27)。结论：食源性单增李斯特菌存在多种耐药基因,毒力基因分布以毒力岛（LIPI-1、LIPI-2）为主,具有潜在的致病性。建议临沂市单增李斯特菌专项监测工作在牲畜肉制品的基础上,应加大对冷冻饮品及其乳制品原料、生禽肉类、水果蔬菜类样本的监管,进一步降低单增李斯特菌所致食源性疾病流行风险。     

食源性单增李斯特菌检测技术研究进展

由单核细胞增生李斯特氏菌(简称单增李斯特菌)(Listeria monocytogenes,L. monocytogenes)引起的李斯特菌病,被认为是世界范围内主要的食源性疾病之一,致死率可高达20%～30%。单增李斯特菌普遍存在于各类食品中,其在低温、有氧或无氧条件下、以及较宽的p H和渗透压范围内均可生长繁殖。因此,迫切需要在整个食物链条中对其进行有效监测和控制,以更好地预防食品污染和食源性疾病的爆发。食品中的单增李斯特菌是低细胞数量致病菌,且与其他非致病性李斯特菌在菌落形态、生化特性等方面具有诸多相似之处,这增加了对单增李斯特菌检测的难度。本文对传统培养方法及免疫分析法、分子生物测定法、生物传感器、噬菌体等新兴替代方法进行了系统全面综述,并比较其各自优缺点,以期为食品中单增李斯特菌的快速检测方法研究提供参考。     

单核细胞增生李斯特菌生物被膜交叉污染评估进展

单核细胞增生李斯特菌（Listeria monocytogenes）（以下简称单增李斯特菌）是一种引发李斯特菌病罹患者高住院率和高死亡率的食源性致病菌,其可在冷、热、干燥和消毒剂处理等不利条件下黏附于食品接触表面并进一步形成难以清除的生物被膜。交叉污染是单增李斯特菌传播的主要途径,生物被膜的形成提高了单增李斯特菌在工厂和厨房环境持续传播和污染的可能性,可引发相关食源性疾病暴发和食品召回等,从而造成健康和经济损失。本文首先介绍了单增李斯特菌生物被膜的胞外聚合物组分,并从外部生存环境和内部微生物自身因素两方面总结了影响单增李斯特菌生物被膜交叉污染转移的因素;进一步重点从研究类型和细菌收集两方面阐述了生物被膜交叉污染的相关研究进展;最后,归纳总结了针对单增李斯特菌生物被膜形成早期的防控策略,展望该领域的研究前景,以期为科学评估和早期精准防控单增李斯特菌生物被膜交叉污染的潜在风险提供理论依据。     

肉及肉制品中单核细胞增生李斯特菌交叉污染的研究进展

单核细胞增生李斯特菌（以下简称单增李斯特菌）是一种引起细菌性食物中毒的重要食源性病原菌,常因污染肉品而引发食物中毒事件,其中交叉污染是引起单增李斯特菌污染并导致食源性疾病的主要途径。本文综述了国内外肉及肉制品污染单增李斯特菌引发的流行病学情况、家庭厨房和肉品加工厂的交叉污染情况、交叉污染模型构建、交叉污染的预防与控制等,为维护家庭食品安全以及保障工厂食品加工卫生安全提供理论依据。     

速冻水饺中金黄色葡萄球菌的DiversiLab分型研究

目的:对速冻水饺中分离的20株金黄色葡萄球菌进行DiversiLab分型,研究其基因特征和聚类分布。方法:采用DiversiLab自动分型系统扩增金黄色葡萄球菌基因组中广泛分布的重复序列,然后在Agilent2100自动生物分析仪上对扩增片段进行微流体电泳分离,并使用DiversiLab Software软件实时分析和处理数据,对20株金黄色葡萄球菌进行分子分型。结果:DiversiLab分型系统将20株金黄色葡萄球菌分为13个型别,其中P1型包含菌株L17、L14、L11,P2型包含L16、L12、L19、L4菌株,P3型包含菌株L3、L18、L13,其余10株金黄色葡萄球菌的每一个菌株被分为一个单独的型别。结论:20株金黄色葡萄球菌从基因型来看有一定的聚集性,可能来自相近的污染源。同时也有一些金黄色葡萄球菌菌株之间的基因型差异较大,遗传学上关系较远,可能来自不同的污染途经。Diversilab自动化分型技术方便快捷,是监控食品及其生产企业细菌性污染的有力工具。     

Characterization of Listeria monocytogenes isolates in import food products of China from 8 provinces between 2005 and 2007

A total of 48 Listeria monocytogenes isolates of different import food products from 8 provinces between 2005 and 2008 were characterized. The serotype and virulence were confirmed for each strain and molecular subtyping were analyzed by multilocus sequence typing (MLST). Twenty five strains were assigned to serotype 1/2a, and 11 isolates to serotype 1/2b, serotype 4b were found in 7 isolate, and the remaining 5 strains were grouped into serotypes 1/2c, 4a, and 4e. Molecular subtyping schemes found thirty two sequence types (STs) among these isolates and the majority of L. monocytogenes strains belonged to lineage II (56%), followed by lineage I (38%), lineage III (6%). Two molecular subtype clusters, cluster A included all isolates of lineage II, while cluster B contained the isolates of lineages I and lineages III. Two L. monocytogenes strains were not grouped in either of the two clusters. Fifty three isolates were as virulent as L. monocytogenes reference strain EGD in mouse virulence assay, while the isolates 22213 and 22265 had low pathogenicity. These results provide the first molecular insight into the L. monocytogenes strains isolated from import food products of 8 provinces in China and indicate the potential risk to cause human disease if intake by contaminated foods. MLST could be used as a routine subtyping method of L. monocytogenes isolates. In China, inspection and quarantine strategies of imported foods should be strengthened. PRACTICAL APPLICATION: There is a potential risk of listeriosis in China and routine subtyping of L. monocytogenes isolates is important. It is necessary for food hygiene management to strengthen the supervision of imported foods.     

荧光假单胞菌、沙门氏菌和单增李斯特菌多重PCR检测方法的建立

针对肉制品中易污染的荧光假单胞菌、沙门氏菌和单增李斯特菌等有害微生物,通过3 种标准菌株及肉制品建立多重聚合酶链式反应方法,实现肉制品中这3 种菌的同时、快速检测。利用荧光假单胞菌的gyrB基因、沙门氏菌的invA基因和单增李斯特菌的hlyA基因设计3 对特异性引物,在确定引物特异性的基础上,对3 种标准菌株及在冷却肉上过夜富集后进行灵敏度检测。结果表明,该多重聚合酶链式反应方法对于同时检测这3 种有害微生物具有高度的特异性;同时检测这3 种菌时,纯菌DNA检测限可达1 pg/μL;将3 种菌一起接种到冷却肉中35 ℃过夜培养后,荧光假单胞菌、沙门氏菌和单增李斯特菌的检测限分别可达到9、5、70 CFU/mL。     

Incidence and susceptibility to antimicrobial agents of Listeria spp. and Listeria monocytogenes isolated from poultry carcasses in Porto,Portugal

The occurrence of Listeria spp. and Listeria monocytogenes in 63 samples of Portuguese poultry carcasses obtained from two local butcher shops and one canteen in the city of Porto, Portugal, and the susceptibility of these bacteria to antimicrobial agents allowed for use in human or animal therapeutics were evaluated. All poultry samples were contaminated with Listeria spp., and L. monocytogenes was isolated from 41% (26 of 63) of the samples. Other Listeria species, including L. innocua, L. welshimeri, and L. seeligeri, were also isolated from poultry samples. A multiplex polymerase chain reaction method was used for the identification of all of the Listeria isolates; this method showed total conformity with the conventional method of biochemical identification and proved to be more reliable, faster, and less arduous. In addition, high percentages of Listeria spp. (84%) and L. monocytogenes (73%) isolates were found to be resistant to one or more antimicrobial agents of different groups, and 12 different resistance profiles were recorded. The frequency of the resistance of L. monocytogenes isolates to enrofloxacin and clindamycin is notable. The results of this study suggest a high incidence of L. monocytogenes on Portuguese poultry products available for consumers and indicate that poultry could be a potential vehicle of foodborne infections due to strains of L. monocytogenes that are resistant to antimicrobial agents.     

实时荧光HDA法快速检测单核细胞增生李斯特菌

目的：建立一种用于单核细胞增生李斯特菌的实时荧光赖解旋酶恒温核酸扩增（helicase-dependentisothermal DNA amplification,HDA）快速检测方法。方法：针对单核细胞增生李斯特菌hly基因序列设计引物对,基于荧光定量聚合酶链式反应（polymerase chain reaction,PCR）仪的平台,提取单核细胞增生李斯特菌基因组DNA,以此作为模板,优化反应温度、反应时间及引物浓度。利用单核细胞增生李斯特菌及10 株对照菌株,并与实时荧光PCR对比,来验证实时荧光HDA方法的特异性和灵敏度,并初步用于样品检测。结果：实时荧光HDA体系的最适引物浓度为0.075 μmol/L,反应温度及反应时间为65 ℃、80 min（40 个循环）,具有良好的特异性和灵敏度。结论：建立了一种特异性强、灵敏度高的实时荧光HDA检测单核细胞增生李斯特菌的方法。     

Incidence of Salmonella, Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes in poultry carcasses and different types of poultry products for sale on the Belgian retail market.

From January 1997 to May 1998, 772 samples of poultry carcasses and poultry products for sale on the retail market in Belgium were analyzed for the presence of Salmonella spp., Salmonella Enteritidis, Campylobacter jejuni, C. coli, and Listeria monocytogenes per 100 cm2 or 25 g. Poultry samples were contaminated with Salmonella (36.5%), C. jejuni and C. coli (28.5%), and L. monocytogenes (38.2%). In about 12.3% of the poultry samples, the L. monocytogenes contamination level exceeded 1 CFU per g or cm2. Significant differences in pathogen contamination rates of poultry products were noticed between the poultry products originating from Belgian, French, and U.K. abattoirs. Poultry products derived from broiler chickens running free in pine woods until slaughtering age (12 to 13 weeks) had a significantly (P < 0.05) lower contamination rate of Salmonella than poultry products from enclosed broilers slaughtered at the age of 6 to 8 weeks. A significantly (P < 0.05) lower pathogen contamination rate was noted for Salmonella, C. jejuni, and C. coli for poultry cuts without skin compared to poultry cuts with skin on. An increase in pathogen contamination rate was noticed during cutting and further processing. To diminish C. jejuni, C. coli, Salmonella, and L. monocytogenes contamination rates, hygienic rules of slaughter and meat processing must be rigorously observed. At the moment, zero tolerance for these pathogens is not feasible, and there is a need to establish criteria allowing these pathogens to be present at reasonable levels in the examined poultry samples.     

IMMUNOLOGICAL AND CYTOPATHOGENIC PROPERTIES OF LISTERIA MONOCYTOGENES ISOLATED FROM NATURALLY CONTAMINATED MEATS

Meat products have been implicated as the potential source of Listeria monocytogenes infection in humans. Here, we investigated the incidence of this organism in raw beef and poultry meat products and assessed their biochemical, immunological and cytopathogenic properties. Forty meat samples (20 beef and 20 poultry) were analyzed and the isolates were tested for sugar fermentation, hemolysin production, phospholipase activity, serotype profile, abilities to react with Listeria- specific monoclonal antibodies (MAbs) EM-7G1 and C11E9, and cytotoxic effects on hybridoma Ped-2E9 cells. Thirteen (6 beef and 7 poultry) meat samples (32.5%) were positive for L. monocytogenes. A total of 276 Listeria isolates were obtained, of which 182 (66%) were confirmed to be L. monocytogenes, 80 (29%) were L. innocua, 12 (4.3%) were L. welshimeri and 2 (0.7%) were identified as L. grayi. Fifty six percent of the L. monocytogenes isolates were serotype 4, while 42% were serotype 1, and 2% were untypeable. All but two L. monocytogenes isolates were hemolytic and phospholipase positive (99%). In the ELISA assay, MAb C11E9 showed reaction with L. monocytogenes isolates from all 13 positive meat samples (100%), while MAb EM-7G1 reacted positively with 12 of 13 positive meat samples (92.3%). Hemolysin-positive L. monocytogenes isolates were cytopathogenic to Ped-2E9 cells, while hemolysin-negative strains showed no effect. This study demonstrated that 32.5% of commercially purchased raw meat products were contaminated with cytopathogenic L. monocytogenes strains, and could be a potential source for infection in susceptible populations if these meats were not processed or cooked properly.