单增李斯特氏菌MALDI-TOF-MS鉴定与分型研究

为建立单增李斯特氏菌的基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)快速鉴定与分型方法,实验收集37株单增李斯特氏菌分离株,应用MALDI-TOF-MS采集图谱,获取独特的蛋白质指纹图谱,汇总成标准图谱,建立单增李斯特氏菌鉴定数据库。采用单增李斯特氏菌标准菌株进行验证,表明鉴定结果的可信度很高。在数据库信息的基础上,对37株单增李斯特氏菌分离株进行聚类分型。分型结果表明,在蛋白质水平上,MALDI-TOF-MS可把37株单增李斯特氏菌分成9个型别。     

FAPAS乳粉中单核细胞增生李斯特菌能力验证结果分析与讨论

为了提升食品中单核细胞增生李斯特氏菌检测能力,本实验室参加了FAPAS分析实验室能力验证机构组织的实验室能力验证,分别运用国标方法、分子生物学方法和基质辅助激光解吸电离飞行时间质谱法对样品进行检测鉴定。结果表明,M224d02-A样品检出单核细胞增生李斯特氏菌,M224d02-B样品检出英诺克李斯特菌,属于李斯特氏菌属。2个样品检验结果均取得满意结果。     

食源性单增李斯特菌与英诺克李斯特菌基因组特征比较

目的 通过全基因组测序对甘肃省市售食品中分离的单增李斯特菌和英诺克李斯特菌基因组特征进行比较分析。方法 收集2021—2022年甘肃省市售食品中分离的25株单增李斯特菌和7株英诺克李斯特菌作为研究对象,对菌株进行全基因组测序,分析其系统发育谱系、克隆复合群（CC）、序列型（ST）、毒力基因、抗性基因及泛基因组。结果 32株李斯特菌分属单增李斯特菌谱系Ⅰ和Ⅱ及英诺克李斯特菌3个群,单增李斯特菌分为10个亚群,英诺克李斯特菌分为5个亚群,与CC型保持一致,核心基因组多位点序列分型能将各谱系中不同CC型的菌株明显分开,谱系Ⅰ与英诺克李斯特菌的进化关系更近。25株单增李斯特菌均携带李斯特菌毒力岛LIPI-1和内化素基因,不携带LIPI-3,有2株ST87型菌株携带LIPI-4;7株英诺克李斯特菌均不携带LIPI-1和内化素基因,均携带LIPI-4,有5株菌携带LIPI-3。单增李斯特菌有16株携带SSI-1、3株携带SSI-2,7株英诺克李斯特菌均不携带SSI-1,有6株携带SSI-2。李斯特菌的泛基因组大小随着测序基因组数目的增加呈现线性增多,25株单增李斯特菌当菌株数量达到15后核心基因数目稳定在2 272个,占泛基因组基因数目的46.2%,25株单增李斯特菌和7株英诺克李斯特菌共同的核心基因1 487个,当菌株数量达到10后数目趋于稳定。结论 核心基因组多位点序列分型可将不同谱系不同克隆复合群的李斯特菌进行区分,英诺克李斯特菌与单增李斯特菌生化特性相似与其亲缘关系相近有关,致病性差异与英诺克李斯特菌缺失单增李斯特菌特有的毒力基因相关。     

食品中单核增生性李斯特菌(Listeria monocytogenes) 污染状况研究

目的:了解食品中单核增生性李斯特菌(Listeria monocytogenes)的污染状况,为单增李斯特茵病的监控、检测以及追踪污染源提供依据.方法:对采自保定市各大超市及农贸市场的生内、熟肉制品、海产品、速冻食品、水果、蔬菜、奶及奶制品、豆制品、腌制品等9大类食品426份样品进行单增李斯特茵的分离与鏊定.结果:检出了单增李斯特茵79株,英诺克李斯特茵7株,格氏李斯特茵32株,西尔李斯特茵2株,威尔斯李斯特茵1株,绵羊李斯特茼1株.默氏李斯特茵2株.李斯特茵的总检出率为29.1%,其中单增李斯特茵的检出率为18.5%.结论:保定市9大类食品中存在不同程度的单增李斯特茵污染,尤其在生内中该茵的污染率高达32.4%.海产品中的污染率31.0%.应引起高度重视.     

基于高分辨率溶解曲线分析鉴别食品中的3种李斯特氏菌

本研究利用高分辨率熔解曲线的方法,以iap基因为靶标新设计一对引物同时鉴别单增李斯特氏菌、伊氏李斯特氏菌和英诺克李斯特氏菌,其余14种常见食源性病原微生物扩增为阴性结果,单增李斯特氏菌和伊氏李斯特氏菌的检出限为10个拷贝,英诺克李斯特氏菌的检测限为50个拷贝。本研究对78份样品进行HRM-realtimePCR法、GB4789.30-2016和SN/T1870-2016(荧光PCR法)的检测,并对3种方法的检测结果进行统计学分析。结果显示:HRM法和国标方法、HRM法和荧光PCR法的检测结果之间存在统计学差异,国标方法和荧光PCR法检测结果之间无统计学差异。本研究建立的基于HRM-real time PCR法检测3种李斯特氏菌的方法快速高效、特异性好、成本低,适用于食品中李斯特氏菌的日常检测和监管。     

单增李斯特氏菌快速检测方法的建立

建立单增李斯特氏菌的快速检测方法.针对单增李斯特氏菌virR基因序列设计特异性引物及探针,建立等温扩增法,并利用免疫金标试纸条对结果进行检测.用10株单增李斯特氏菌、6株李斯特菌属菌株及其他食源性致病菌24株进行特异性试验;通过定量DNA、纯菌液计数进行灵敏度验证.结果表明建立方法具有较好的特异性;增菌液检测灵敏度为102 cfu/test,DNA检测灵敏度为10°pg/test.建立的单增李斯特氏菌的快速检测方法特异性较好、灵敏度高,适合于单增李斯特氏菌快速筛选检测.     

食品中3种致病李氏菌MPCR-DHPLC检测方法的建立

应用复合PCR(multiplex PCR,MPCR)结合变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)技术建立食品中单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌的快速高通量检测方法。根据单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌的特异基因序列分别设计引物,MPCR扩增的产物经DHPLC技术进行快速检测。以94株参考菌株做特异性实验,并开展了重现性检测实验。MPCR-DHPLC方法同步检测到单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌的特异性阳性吸收峰,未检测到李斯特菌属其他近源种和非近源种参考菌株的阳性吸收峰,且重现性良好。该方法具有很好的特异性,可以快速、准确、高通量地检测食品中单核细胞增生李斯特菌、绵羊李斯特菌和英诺克李斯特菌,是食品微生物快速检测的新技术。     

食品中单核细胞增生李斯特氏菌能力验证结果分析与讨论

目的:通过检验食品中单增李斯特氏菌了解实验室关于食品中微生物的检验和检测水平,通过检验技术的更新和检测方法的不断优化,增强实验室整体竞争能力,提升食品检验检测机构检验能力,使微生物实验室外部质控水平得以维护和巩固。方法:利用BAX Q7对能力验证中的3个样品进行快速筛查,严格依据《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》(GB 4789.30—2016),对疑似菌进行鉴定。结果:编号为270的样品检出为单增李斯特氏菌,编号为318和159的这两个样品中均未检出该致病菌,经过进一步实验,发现这两个样品中的致病菌为英诺克李斯特氏菌。结论:本实验室顺利完成本次能力验证,结果较满意。综上表明本实验室具备检验检测单增李斯特氏菌的能力,且检测方法和结果具有一定的准确性。     

食品中李斯特氏菌的分离鉴定

英诺克李斯特氏菌与有毒性单核细胞增生李斯特氏菌的菌落特征和生化特征相似,仅溶血试验结果不同。本文通过对河南省濮阳市市售凉皮进行检验,分离纯化出一株李斯特氏菌,利用单增李斯特氏菌生化鉴定条进行检验,结果显示木糖和甘露醇两项生化试验结果为阴性,且溶血试验结果为阴性,符合英诺克李斯特氏菌的判定标准。同时,VITEK 2 Compact全自动微生物鉴定系统也准确鉴定出该菌为英诺克李斯特氏菌(概率为99%)。     

云南淡水鱼中单增李斯特氏菌污染状况及耐药性分析

目的了解云南淡水鱼中单增李斯特氏菌污染状况及耐药情况。方法从云南省昆明市、曲靖市、大理州、红河州4个淡水鱼主产区采集淡水鱼样品110件,参照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》分离鉴定单增李斯特氏菌,并进一步开展16S rRNA序列鉴定和同源性分析,对鉴定菌株采用微量肉汤稀释法分析抗生素敏感性。结果110件样品检出2株单增李斯特氏菌(DZ-054、DZ-101),检出率为1.82%。2株单增李斯特氏菌之间相似度达到99.64%。DZ-101菌株对氨苄西林、青霉素敏感(S),DZ-054菌株对抗生素不敏感。结论云南省淡水鱼中单增李斯特菌的污染率较低,但仍有一定的安全隐患,相关部门应加强监管。     

Identification of Listeria innocua surrogates for Listeria monocytogenes in hamburger patties

ABSTRACT:  Listeria innocua M1 has been used by many researchers as a nonpathogenic thermal processing surrogate for Listeria monocytogenes . However, L. innocua M1 has been criticized because its thermal survivability characteristics are not as closely parallel to L. monocytogenes as some would like in a variety of foods and processing conditions. The present study was conducted to compare multiple L. innocua and L. monocytogenes strains to validate L. innocua M1 as the ideal surrogate under high-temperature thermal processing conditions for L. monocytogenes . The D - and z -values of L. innocua M1, L. innocua strain SLCC 5639 serotype (6a), SLCC 5640 (6b), SLCC 2745 (4ab), and L. monocytogenes F4243 (4b) were calculated for raw hamburger patties. Hamburger patties were inoculated with 10^7–8 CFU/g of L. monocytogenes or L. innocua . Samples were heat treated at 4 temperatures (62.5 to 70 °C). At each temperature, the decimal reduction time ( D -value) was obtained by linear regression of survival curves. The D - and z -values were determined for each bacterium. The D -values of L. innocua and L. monocytogenes serotypes ranged from 3.17 to 0.13 min at 62.5 to 70 °C, and the z -values of L. innocua and L. monocytogenes were 7.44 to 7.73 °C. Two of the 4 L. innocua serotypes used in this experiment have the potential for use as surrogates in hamburger meat with varying margins of safety. L. innocua M1 should serve as the primary nonpathogenic surrogate with the greatest margin of safety in verifying a new thermal process to destroy L. monocytogenes .     

Survival of Listeria monocytogenes, Listeria innocua, and Lactic Acid Bacteria in Chill Brines

ABSTRACT:  Listeria monocytogenes is the pathogen of concern in ready-to-eat (RTE) meat products. Salt brines are used to chill processed meats. L. monocytogenes and lactic acid bacteria (LAB) can grow under saline conditions, and may compete with each other for nutrients. The objective of this study was to determine the effect of lactic acid bacteria ( Enterococcus faecalis , Carnobacterium gallinarum , and Lactobacillus plantarum ) on the survival of L. monocytogenes and Listeria innocua in brines stored under low temperatures for 10 d. Sterile tap water (STW) and 2 brine solutions (7.9% and 13.2% NaCl) were inoculated with 1 of 5 cocktails ( L. monocytogenes , L. innocua , LAB, L. monocytogenes + LAB, or L. innocua + LAB) at initial concentrations of 7 log CFU/mL. Brines were stored for 10 d at 4 or 12 °C. Three replications of each brine concentration/cocktail/temperature combination were completed. No significant reductions of L. monocytogenes occurred in 7.9%[w/v] or 13.2%[w/v] brines when LAB were present; however, there were significant reductions after 10 d of L. monocytogenes in the STW solution when LAB were present (1.43 log CFU/mL at 4 °C and 3.02 log CFU/mL at 12 °C). L. innocua was significantly less resilient to environmental stresses of the brines than L. monocytogenes , both with and without LAB present ( P ≤ 0.05). These strains of lactic acid bacteria are not effective at reducing L. monocytogenes in brines at low temperatures. Furthermore, use of L. innocua as a model for L. monocytogenes is not appropriate under these environmental conditions.     

Occurrence and characterization of Listeria spp. in ready-to-eat retail foods from Vancouver, British Columbia

The occurrence of Listeria spp. and Listeria monocytogenes in retail RTE meat and fish products in Vancouver, British Columbia (B.C.) was investigated. To assess potential consumer health risk, recovered L. monocytogenes isolates were subjected to genotypic and phenotypic characterization. Conventional methods were used to recover Listeria spp. from deli meat (n = 40) and fish (n = 40) samples collected from 17 stores. Listeria spp. were recovered only from fish samples (20%); 5% harboured Listeria innocua, 5% had L. monocytogenes and 10% contained Listeria welshimeri. L. monocytogenes isolates serotyped as 1/2a and 1/2b, possessed dissimilar PFGE patterns, and had full-length InlA. Three 1/2a clonal isolates encoded the 50 kb genomic island, LGI1. Antimicrobial resistance (AMR) profiling showed all Listeria spp. possessed resistance to cefoxitin and nalidixic acid. L. monocytogenes were resistant to clindamycin, two were resistant to streptomycin, and one to amikacin. Reduced susceptibility to ciprofloxacin was seen in all L. monocytogenes, L. innocua and three L. welshimeri isolates. Reduced susceptibility to amikacin and chloramphenicol was also observed in one L. monocytogenes and three L. welshimeri isolates, respectively. Recovery of L. monocytogenes in fish samples possessing AMR, full-length InlA, LGI1, and serotypes frequently associated with listeriosis suggest B.C. consumers are exposed to high-risk strains.     

基质辅助激光解吸电离飞行时间质谱在 李斯特菌检测和鉴定中的应用

为建立食品中各种李斯特菌的快速检测和鉴定方法,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)对单增李斯特菌、绵羊李斯特菌、英诺克李斯特菌、威尔斯李斯特菌和格氏李斯特菌5种李斯特菌进行检测,并与传统的生化鉴定方法(VITEK 2)和聚合酶链式反应(PCR)方法检测结果进行比较;用不同培养基和不同培养时间培养的单增李斯特菌对该方法进行检测,还对60份人工感染5种李斯特菌的食品样品分别进行MALDI-TOF-MS、PCR以及传统生化方法的检测和鉴定。结果表明：MALDI-TOF-MS能够快速、可靠地区分上述5种李斯特菌,而且具有很好的稳定性和重复性,在检测时间、重复性和准确性方面的总体表现优于传统生化鉴定方法。MALDI-TOF-MS技术适用于对李斯特菌等食源性病原菌进行高通量、低成本的快速鉴定。     

Evaluation of Listeria innocua as a suitable indicator for replacing Listeria monocytogenes during ripening of Camembert cheese

The suitability of Listeria innocua for use as an indicator for replacing Listeria monocytogenes during the cheese-making and ripening of Camembert cheese was evaluated. Pasteurized whole milk inoculated with either L. innocua or L. monocytogenes was used to make Camembert cheese, which were ripened in three stages. All cheese was ripened in three stages: room temperature (∼20 °C) and relative humidity of 60% for 36 h; 12 °C and relative humidity of 93% for 2 weeks; and 7 °C and relative humidity of 85% for 3 weeks. Results showed that population values of L. innocua and L. monocytogenes on day 1 were 7.16 and 6.11 log₁₀ CFU/g, respectively, which declined to 6.54 and 5.45 log₁₀ CFU/g, respectively, during subsequent 20 days. Thereafter, L. innocua and L. monocytogenes populations increased to 7.38 and 6.06 log₁₀ CFU/g on day 35 of ripening, respectively. During ripening, surface and interior of cheeses were analysed for populations of L. innocua and L. monocytogenes , respectively. The data were collected on day 1, 5, 10, 15, 20, 25, 30, and 35 of ripening. Generally, the growth of L. innocua and L. monocytogenes is faster in surface than in centre. Top centre, bottom centre and bottom surface locations had similar population values during ripening. There were no significant differences ( P  > 0.05) between batch and section of cheese. The ripening time and locations had significant effect ( P  < 0.05) on the survival and growth of L. innocua and L. monocytogenes . The trends of survival and growth of L. innocua and L. monocytogenes were similar. These results indicated that L. innocua can be considered as an indicator for L. monocytogenes during ripening of Camembert cheese.     

单核细胞增生李斯特菌的毒力基因检测及PFGE分型的研究

目的了解福建省食品中单核细胞增生李斯特菌携带hly、plcA、plcB和prfA毒力基因的情况及脉冲场凝胶电泳(PFGE)的分型情况。方法将hly、plcA、plcB和prfA基因作为靶序列选取4对引物,通过聚合酶链反应(PCR)检测61株单核细胞增生李斯特菌和2株可疑单核细胞增生李斯特菌的毒力基因,用PulseNet单核细胞增生李斯特菌标准方法进行7株单核细胞增生李斯特菌的PFGE分子分型。结果61株单核细胞增生李斯特菌毒力基因为hly 、plcA 、plcB 和prfA ,2株可疑单核细胞增生李斯特菌的毒力基因分别为hly-、plcA 、plcB-、prfA-和hly-、plcA-、plcB-、prfA-。7株单核细胞增生李斯特菌的PFGE分为5个型。结论实验结果表明福建省食品中分离到的单核细胞增生李斯特菌均含有hly、plcA、plcB和prfA基因,属于致病株。对2株可疑单核细胞增生李斯特菌进行了进一步的鉴定,排除了单核细胞增生李斯特菌,该毒力基因检测方法可用于可疑单核细胞增生李斯特菌的进一步鉴别。7株菌中有两对2株PFGE型别一致,一致的菌株来自不同年份不同销售地点的同一品牌,应用PFGE方法可以进一步调查该厂冻鸡肉中单核细胞增生李斯特菌的传播途径和污染源。     

傅立叶变换红外光谱技术对3种李斯特氏菌的快速分类鉴定

为建立李斯特氏菌属内单核细胞增生李斯特氏菌、绵羊李斯特氏菌、英诺克李斯特氏菌3种菌的傅立叶变换红外(Fourier transform infrared,FT-IR)光谱数据库及FT-IT分类鉴定方法,作者应用FT-IR技术对3种李斯特氏菌进行指纹图谱数据采集,建立了3种李斯特氏菌的标准FT-IR导数谱数据库,同时结合化学计量学分析方法,建立了基于主成分分析(principalcomponent analysis,PCA)和分级聚类分析(hierarchical cluster analysis,HCA)两种聚类分析模型。结果表明,两种聚类模型均可成功将3种李斯特氏菌进行区分,FT-IR光谱数据库可用于FT-IR技术对3种可疑李斯特氏菌进行鉴定。FT-IR分析方法简便、快速、易操作,结果重现性好,是一种区分3种李斯特氏菌的有效方法。     

Incidence and characterization of Listeria spp. from foods available in Korea

A total of 410 domestic Korean food samples were analyzed for the presence of Listeria spp. by the conventional U.S. Department of Agriculture protocol, and presumptive strains were identified by morphological, cultural and biochemical tests according to Bergey's manual and confirmed by API-Listeria kit. Among the total 410 food samples, 46 samples (11.2%) were found to be contaminated with Listeria species. Among the 46 strains of Listeria spp. isolates, 8 strains (17.42%) for Listeria monocytogenes, 3 strains (6.5%) for Listeria seeligeri, 33 strains (71.7%) for Listeria innocua, and 2 strains (4.4%) for Listeria welshimeri were identified, respectively. Also, only beef, chicken, pork, frozen foods, and sausage were contaminated with L. monocytogenes, and the other products were free of L. monocytogenes. Of 46 Listeria spp. isolates, L. innocua (71.7%) was the most predominantly isolated in a variety of foods compared to other Listeria spp. An in vitro virulence assay for Listeria spp. using myeloma and hybridoma cells from murine and human sources was performed. The result showed that only L. monocytogenes killed approximately 95 to 100% hybridoma cells after 6 h and the other Listeria species, such as L. innocua, L. seeligeri, and L. welshimeri strains had about 0 to 10% lethal effect on hybridoma cells. Also, an antibiotic susceptibility test showed that Listeria spp. isolates were very susceptible to the antibiotics tested, except for nalidixic acid. Also, serotyping results showed 75% of L. monocytogenes isolates from beef, chicken, and frozen pizza belonged to serotype 1 and 25% from sausage were type 4.