鲜活贝类中单核细胞增生李斯特菌的分离鉴定及毒力基因与耐药性分析

目的研究采自产地的鲜活贝类中单核细胞增生李斯特菌(单增李斯特菌)的污染状况、毒力基因分布与耐药性。方法对采自产地的200份鲜活贝类,按照GB/T 4789.30—2010《食品安全国家标准食品卫生微生物学检验单核细胞增生李斯特氏菌检验》进行单增李斯特菌的分离与鉴定;采用Kirby-Bauer纸片扩散法测定分离株的耐药性;通过PCR法分析分离株9个毒力基因(包括prfA、plcB、hly、actA、iap、inlA、plcA、mpl、inlB)。结果在200份鲜活贝类中,共检出阳性样品4份(2.0%);有1株缺失inlB基因,1株缺失mpl基因;分离株对氨苄青霉素、庆大霉素等一线临床治疗药物敏感,但有2株对四环素和复方新诺明同时耐药,1株对复方新诺明和氧氟沙星耐药。结论采自产地的鲜活贝类存在单增李斯特菌的污染,分离株毒力基因有一定程度缺失,提示初级水产品中的单增李斯特菌污染需引起关注,并且需要继续加强食品中该菌的耐药性监测。     

部分市售农副产品李斯特菌污染情况调查及毒力基因检测

目的了解上海市闵行区农贸市场食品中李斯特菌的污染情况及其毒力基因的携带情况。方法按国标方法GB/T4789.30—2008《食品卫生微生物学检验单核细胞增生李斯特氏菌检验》,采用科玛嘉显色培养基,对食品进行李斯特菌的分离、API试剂条进行生化鉴定,PCR扩增进行hly、prfA、plcB、actA、inlA和iap6种毒力基因的检测。结果320份样品中共检出李斯特菌36株,其中单核细胞增生李斯特菌10株;英诺克李斯特菌23株,其余3株。10株单核细胞增生李斯特菌hly、prfA、plcB、iap、inlA、actA6种毒力基因携带率分别为100%、100%、100%、100%、100%和0,即10株菌株actA基因全部缺失。结论本地区农贸市场食品存在李斯特菌的污染,生肉禽类食品中李斯特菌污染比较严重,应加强监督管理;单核细胞增生李斯特菌菌株actA毒力基因均表现为缺失,是否为本地区特性,还有待研究。     

食品中单核细胞增生性李斯特氏菌PCR快速检测方法

为建立食品中单核细胞增生性李斯特氏菌 (单增李斯特氏菌 )的快速、敏感、特异的PCR检测方法 ,选取hlyA基因作为靶序列设计一对引物 ,用该引物对 6 3株从国内食品中分离的李斯特氏菌 (进行传统方法验证 )和 2 0株非李斯特氏菌进行PCR扩增 ,并用此方法对人工污染食品进行检测 ,扩增片段表现出极好的单增李斯特氏菌种特异性 ,人工污染的生肉、冷冻虾仁、卷心菜的检出限为 39cfu g ,为食品中单增李斯特氏菌的快速、敏感并且特异的检测方法。     

环介导等温扩增法检测动植物源性单核细胞增生李斯特氏菌

目的采用环介导等温扩增技术检测动植物源性单核细胞增生李斯特氏菌。方法在北京市范围内各业态餐厅购买600份凉菜样品为研究对象,以单核细胞增生李斯特氏菌的hlyA基因为目标基因,设计4条特异性引物,使用环比等温扩增法(loop-to-isothermal amplification, LAMP)创建凉菜中单核细胞增生李斯特氏菌快速检测方法并将其应用于鉴定凉菜中分离的单核细胞增生李斯特氏菌。结果通过细菌分离培养法对600份凉菜进行检测,一共检测分离出37株致病菌菌株,其中包括7株单核细胞增生李斯特氏菌、10株沙门氏菌和20株大肠埃希氏菌,单核细胞增生李斯特氏菌的检出率为1.2%。用LAMP对样品中的菌株进行测定,7株凉菜中分离的单核细胞增生李斯特氏菌为阳性,其他菌株为阴性。通过LAMP方法检测动植物源性凉菜中分离的单核细胞增生李斯特氏菌平均检出限为2.2*10 CFU/μL、特异性为100%。动物源性凉菜中的致病菌含量较高。结论环介导等温扩增法具备灵敏、快速、操作简便的特点,适合用于餐饮行业的现场快速检测领域。     

徐州市市售生肉中单核细胞增生李斯特菌污染及其分子特征分析

目的 分析2018年和2020年徐州市市售生肉中单核细胞增生李斯特菌检出率及其分子生物学特征。方法 随机采集市区零售鲜或冷冻生禽肉及调理肉，按照GB 4789.30—2016《食品安全国家标准 食品微生物学检验 单核细胞增生李斯特氏菌检验》对单核细胞增生李斯特菌进行分离、鉴定，并分析其血清型、耐药性、毒力基因型、脉冲场凝胶电泳（PFGE）分子分型。结果 142份市售生肉中有40份检出单核细胞增生李斯特菌，检出率为28.2%（40/142）；血清型主要为1/2a型，占50.0%（20/40）；40株单核细胞增生李斯特菌共分为15种PT型；对氨苄西林及青霉素耐药菌株分别有4株和2株；所有菌株均携带多种毒力基因。结论 徐州市市售生肉中单核细胞增生李斯特菌污染较严重，存在小规模同源菌株，需进一步加强监测管理，以防食源性疾病的发生。     

江干区部分食品中单增李斯特菌污染状况分析

目的了解江干区食品中单核细胞增生性李斯特菌污染状况。方法参考国标方法,采用进口显色培养基,对4类食品进行单核细胞增生性李斯特菌分离,生化及血清型鉴定。结果109份样品共检出单核细胞增生性李斯特菌15株,总检出率为13.8%;生肉类、生食果蔬类检出率分别为36.8%、5.9%,熟肉制品和水产品中未检出。15株单核细胞增生性李斯特菌分属2个血清型,1/2b血清型占73.3%,1/2a血清型占26.7%。结论江干区食品中存在单核细胞增生性李斯特菌的污染,特别是生肉。     

添加扩增内标的单增李斯特菌PCR检测方法的建立与应用

以单增李斯特菌inl A基因为靶基因,设计一对特异性引物,以16S rRNA为扩增内标对照,建立了一种含有扩增内标的单增李斯特菌PCR检测方法。优化了PCR反应体系,并对PCR检测方法的特异性、灵敏度、人工污染样品及食品样品检测效果进行了测试。对2株单增李斯特菌、1株英诺克李斯特菌以及19株非李斯特菌菌株进行PCR检测,结果显示,只有2株单增李斯特菌能被检出大小为826 bp的特异性片段,其余20株细菌只能检出1 500 bp的扩增内标片段。灵敏度实验结果显示,基因组DNA和纯培养物的最低检出限分别为1.80×10~2fg/μL和1.21×10~2CFU/m L。当人工污染牛乳样品中单增李斯特菌在最低接种量为0.48 CFU/m L时,经8 h增菌培养后可被该方法检出。采用该研究建立的检测方法对36种食品样品进行检测,证实了该检测方法可以指示检测过程中出现的假阴性现象。综上所述,该研究建立的PCR检测方法能特异性的检测单增李斯特菌,并可有效排除检测过程中出现的假阴性现象,提高检测的准确性。     

食品中单增李斯特氏菌检测PCR-免疫胶体金 试纸条方法的建立

目的 建立食品中单核增生李斯特氏菌快速检测PCR-免疫胶体金试纸条法。方法 通过设计特异性引物建立单增李斯特氏菌检测PCR方法并使用免疫胶体金技术建立PCR产物快速检测试纸条; 用试验菌株检测PCR-免疫胶体金试纸条方法的检测特异度与敏感度。使用新建方法对市售肉制品和乳制品中单核增生李斯特氏菌进行检测, 验证该方法在食品检测中的可行性。结果 PCR-免疫胶体金法具有良好的特异度, 敏感度比标准琼脂糖凝胶电泳法高100倍。采集乳品样品131份, 阳性样品1份, 检出率1.53%; 肉制品224份, 阳性样品4份, 检测率1.79%。结论 建立的单增李斯特氏菌检测PCR-免疫胶体金试纸条法特异度好, 敏感度高, 适用于食品中单增李斯特氏菌的检测。     

食品中单增李斯特氏菌检测PCR-免疫胶体金试纸条方法的建立

目的建立食品中单核增生李斯特氏菌快速检测PCR-免疫胶体金试纸条法。方法通过设计特异性引物建立单增李斯特氏菌检测PCR方法并使用免疫胶体金技术建立PCR产物快速检测试纸条;用试验菌株检测PCR-免疫胶体金试纸条方法的检测特异度与敏感度。使用新建方法对市售肉制品和乳制品中单核增生李斯特氏菌进行检测,验证该方法在食品检测中的可行性。结果 PCR-免疫胶体金法具有良好的特异度,敏感度比标准琼脂糖凝胶电泳法高100倍。采集乳品样品131份,阳性样品1份,检出率1.53%;肉制品224份,阳性样品4份,检测率1.79%。结论建立的单增李斯特氏菌检测PCR-免疫胶体金试纸条法特异度好,敏感度高,适用于食品中单增李斯特氏菌的检测。     

昆明市西山区食品中单核细胞增生李斯特菌的污染状况调查

了解昆明市西山区食品中单增李斯特菌的污染状况,预防其食物中毒的暴发和流行。方法 采用GB 4789.30—2010《食品卫生微生物检验 单核细胞增生李斯特菌检验》,并结合梅里埃仪器鉴定系统,对样品进行单增李斯特菌检验。结果 2012年5～9月间,分组随机抽取食品及食品加工工具样品406份进行单增李斯特菌的检验,从中检出10株单增李斯特菌,总检出率为2.46%。其中,食品加工工具检出率为2.50%;食品总检出率为2.46%,蔬菜类和生肉类检出率均为5.00%。结论 西山区食品及食品加工工具单增李斯特菌的污染较普遍,存在单增李斯特菌引起公共卫生问题的风险,应加强食品安全管理。     

Mini-VIDAS法和国标法检测单增李斯特菌的效果比较

目的比较两类方法检测单增李斯特菌的特异性、灵敏度和抗干扰性,并研究添加成分和样品基质对检测效果的影响。方法选取单增李斯特菌(CICC 21633/ATCC 19111)、斯氏李斯特菌(CICC 21671)、伊氏李斯特菌(CICC 21663/ATCC19119)和英诺克李斯特菌(CICC 10417/ATCC 33090)为试验对象,采用国标法和mini-VIDAS法为试验方法。结果两种检测方法的特异性较好,均能区分李斯特菌属和非李斯特菌属。国标法和mini-VIDAS法的灵敏度分别为104~105 cfu/m L和105 cfu/m L;同时,国标法的抗干扰性稍强,1/104混合干扰菌液可检出目标菌。添加次氯酸钠后两种方法对目标菌的检出都受到影响。添加成分对单增李斯特菌生长的抑制作用符合乙醇苯甲酸钠十三香料凉拌菜料次氯酸钠。当基质中单增李斯特菌污染水平较低并且背景微生物数量高时,目标菌的检测结果受基质严重干扰。不同增菌液对单增李斯特菌的增菌效果为Fraser肉汤LB2FB2。结论 mini-VIDAS法和国标法对单增李斯特菌的检测性能基本相当。采用国标法时需考虑微生物污染背景、添加成分等样品基质特性,以提高目标菌的检出率。mini-VIDAS法可作为初筛的良好工具。     

Listeria monocytogenes contamination pattern in pig slaughterhouses

Ten low-capacity slaughterhouses were examined for Listeria by collecting a total of 373 samples, of which 50, 250, and 73 were taken from carcasses, pluck sets, and the slaughterhouse environment, respectively. Six slaughterhouses and 9% of all samples were positive for Listeria monocytogenes. Of the samples taken from pluck sets, 9% were positive for L. monocytogenes, the highest prevalence occurring in tongue and tonsil samples, at 14% and 12%, respectively. Six of 50 (12%) carcasses were contaminated with L. monocytogenes. In the slaughterhouse environment, L. monocytogenes was detected in two, one, one, and one sample originating from the saws, drain, door, and table, respectively. Carcasses were contaminated with L. monocytogenes in those two slaughterhouses, where the mechanical saws, used for both brisket and back splitting, were also positive for L. monocytogenes. A total of 58 L. monocytogenes isolates were characterized by pulsed-field gel electrophoresis typing. The isolates were divided into 18 pulsotypes, 15 of which were detected in pluck sets. In two slaughterhouses, where the carcasses were contaminated with L. monocytogenes, the same pulsotypes were also recovered from splitting saws. In addition, identical pulsotypes were recovered from pluck sets. Our findings indicate that L. monocytogenes of tongue and tonsil origin may contaminate the slaughtering equipment that may in turn spread the pathogen to carcasses. Thus, it is of the utmost importance to follow good manufacturing practices and to have efficient cleaning and disinfection procedures to prevent equipment being contaminated with L. monocytogenes.     

Tracking of Listeria monocytogenes in smoked fish processing plants

Four smoked fish processing plants were used as a model system to characterize Listeria monocytogenes contamination patterns in ready-to-eat food production environments. Each of the four plants was sampled monthly for approximately 1 year. At each sampling, four to six raw fish and four to six finished product samples were collected from corresponding lots. In addition, 12 to 14 environmental sponge samples were collected several hours after the start of production at sites selected as being likely contamination sources. A total of 234 raw fish, 233 finished products, and 553 environmental samples were tested. Presumptive Listeria spp. were isolated from 16.7% of the raw fish samples, 9.0% of the finished product samples, and 27.3% of the environmental samples. L. monocytogenes was isolated from 3.8% of the raw fish samples (0 to 10%, depending on the plant), 1.3% of the finished product samples (0 to 3.3%), and 12.8% of the environmental samples (0 to 29.8%). Among the environmental samples, L. monocytogenes was found in 23.7% of the samples taken from drains, 4.8% of the samples taken from food contact surfaces, 10.4% of the samples taken from employee contact surfaces (aprons, hands, and door handles), and 12.3% of the samples taken from other nonfood contact surfaces. Listeria spp. were isolated from environmental samples in each of the four plants, whereas L. monocytogenes was not found in any of the environmental samples from one plant. Overall, the L. monocytogenes prevalence in the plant environment showed a statistically significant (P < 0.0001) positive relationship with the prevalence of this organism in finished product samples. Automated EcoRI ribotyping differentiated 15 ribotypes among the 83 L. monocytogenes isolates. For each of the three plants with L. monocytogenes-positive environmental samples, one or two ribotypes seemed to persist in the plant environment during the study period. In one plant, a specific L. monocytogenes ribotype represented 44% of the L. monocytogenes-positive environmental samples and was also responsible for one of the two finished product positives found in this plant. In another plant, a specific L. monocytogenes ribotype persisted in the raw fish handling area. However, this ribotype was never isolated from the finished product area in this plant, indicating that this operation has achieved effective separation of raw and finished product areas. Molecular subtyping methods can help identify plant-specific L. monocytogenes contamination routes and thus provide the knowledge needed to implement improved L. monocytogenes control strategies.     

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 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.     

Contamination patterns of Listeria monocytogenes in cold-smoked pork processing

Contamination patterns of Listeria monocytogenes were studied in a cold-smoked pork processing plant to identify the sources and possible reasons for the contamination. Environmental sampling combined with pulsed-field gel electrophoresis (PFGE) subtyping and serotyping were applied to investigate the genetic diversity of L. monocytogenes in the plant environment and ready-to-eat (RTE) cold-smoked pork products. A total of 183 samples were collected for contamination analyses, including samples of the product at different stages during manufacture (n = 136) and environmental samples (n = 47) in 2009. L. monocytogenes isolates, previously recovered from 73 RTE cold-smoked pork samples and collected from the same meat processing plant in 2004, were included in this study. The brining machine and personnel working with brining procedures were the most contaminated places with L. monocytogenes. The overall prevalence of L. monocytogenes in raw pork (18%) increased to 60% after the brining injections. The brining machine harbored six different PFGE types belonging to serotypes 1/2a, 1/2c, 4b, and 4d, which were found on the feeding teeth, smooth surfaces, and spaces of the machine, thus potentially facilitating dissemination of L. monocytogenes contamination. Two PFGE types (2 and 8) belonging to serotypes 1/2a and 1/2c were recovered from RTE cold-smoked pork collected in 2004, and from surfaces of the brining machine sampled in 2009, and may indicate the presence of persistent L. monocytogenes strains in the plant. Due to poor hygiene design, removal of the brining machine from the production of cold-smoked meat products should be considered to reduce L. monocytogenes contamination in the finished products.     

Comparative evaluation of culture- and BAX polymerase chain reaction-based detection methods for Listeria spp. and Listeria monocytogenes in environmental and raw fish samples

Two commercial polymerase chain reaction (PCR)-based Listeria detection systems, the BAX for Screening/Listeria monocytogenes and the BAX for Screening/Genus Listeria, and a culture-based detection system, the Biosynth L. monocytogenes Detection System (LMDS), were evaluated for their ability to detect L. monocytogenes and Listeria spp. in raw ingredients and the processing environment. For detection of L. monocytogenes from raw fish, enrichment was performed in Listeria enrichment broth (LEB), followed by plating on both Oxford agar and LMDS L. monocytogenes plating medium (LMPM). Detection of Listeria and L. monocytogenes from environmental samples was performed using LMDS enrichment medium, followed by plating on both Oxford agar and LMPM. A total of 512 environmental samples and 315 raw fish were taken from two smoked fish processing facilities and screened using these molecular and cultural Listeria detection methods. The BAX for Screening/L monocytogenes was used to screen raw fish and was 84.8% sensitive and 100% specific. The BAX for Screening/Genus Listeria was evaluated on environmental samples and had 94.7% sensitivity and 97.4% specificity. In conjunction with enrichment in LEB, LMPM had a sensitivity and specificity for detection of L. monocytogenes from raw fish of 97.8 and 100%, respectively. Use of LMDS enrichment medium followed by plating on LMPM allowed for sensitivity and specificity rates of 94.8 and 100%, respectively, for detection of L. monocytogenes from environmental samples. We conclude that both the BAX systems and the use of LMPM allow for reliable and rapid detection of Listeria spp. and L. monocytogenes. While the BAX systems provide screening results in about 3 days, the use of LMPM allows for L. monocytogenes isolation in 4 to 5 days.     

High incidence of Listeria monocytogenes in European red smear cheese

The incidence of Listeria and Listeria monocytogenes in European red smear cheese was determined in order to assess whether the lack of recent outbreaks of listeriosis associated with cheese is due to improved hygenic conditions in the dairies. Out of European red-smear cheese samples of various types, 15.8% contained organisms of the genus Listeria, 6.4% of the samples were contaminated with L. monocytogenes, 10.6% with L. innocua, and 1.2% with L. seeligeri. Six cheese samples contained two or more Listeria species, including at least one L. monocytogenes isolate. The incidences of L. monocytogenes in cheeses from various countries were: Italy 17.4%, Germany 9.2%, Austria 10%, and France 3.3%. Listeria were found most frequently in soft and semi-soft cheese. Eight samples contained more than 100 L. monocytogenes cfu/cm2 cheese surface, 2 samples had counts above 10(4) cfu/cm2 cheese surface. Surprisingly, a higher incidence of L. monocytogenes was observed in cheeses made from pasteurized milk (8.0%) than in cheeses manufactured from raw milk (4.8%). Phage-typing of isolated Listeria strains clearly confirmed that (i) contaminations within dairy plants were persistent over a period of several weeks to months and (ii) that cross-contamination within the dairy plant is and important factor. Comparison of our data with past surveys seems to indicate that contamination of red smear soft cheese with L. monocytogenes has not decreased sufficiently over the past 15 years. It is therefore strongly recommended that these products are monitored carefully by cheese-making companies.     

Listeria monocytogenes in foods in Norway

Three-hundred-and-eighty-two samples of different retail food items in Norway (imported soft cheese, raw chicken, minced meat, fermented sausages, vacuum-packed processed meat products, smoked salmon, peeled shrimps, raw minced fish) and 78 carcass samples (sheep, pig, cattle), were screened for Listeria monocytogenes. Of the 460 samples investigated, 78 were found to contain L. monocytogenes. Five of these contained greater than 10(3) cfu/g, four greater than 10(2) cfu/g, while the remainder were shown to contain L. monocytogenes only after enrichment. L. monocytogenes was isolated most frequently from raw chicken, sporadically from soft cheese, shrimps, processed meat products and smoked salmon, and not at all from carcasses and fermented sausages.     

Ecology of Listeria spp. in a fish farm and molecular typing of Listeria monocytogenes from fish farming and processing companies

This study focused on the ecology of Listeria monocytogenes in a fish farm by following the changes in its occurrence in different types of samples for a three year period. In addition, L. monocytogenes isolates from different seafood industry areas were compared with pulsed field gel electrophoresis (PFGE) typing to discover possible associations between primary production, further processing and final products. Weather conditions were found to have a strong influence on the probability of finding Listeria spp. in a fish farm environment. The number of samples contaminated with Listeria spp. was typically bigger after rainy periods. Brook and river waters as well as other runoff waters seemed to be the main contamination source at the farm studied. The farmed fish originally found to carry L. monocytogenes become gradually Listeria free. The time needed for the purification of the fish was several months. The sea bottom soil samples were the ones that preserved the L. monocytogenes contamination the longest time. It can be stated that the fish and fish farm equipment studied did not spread listeria contamination. On the contrary, they were found to suffer from listeria contamination coming from outside sources like the brook water. There was a wide range of different L. monocytogenes PFGE-pulsotypes (30) found at 15 Finnish fish farms and fish processing factories. L. monocytogenes isolates from the final products often belonged to the same pulsotypes as did the isolates from the processing environment as well as from the raw fish. This suggests that, in addition to the fish processing factory environment, the fish raw materials are important sources of L. monocytogenes contamination in final products.