1例急性淋巴细胞白血病患者感染单核细胞增生李斯特菌病原学及分子分型特征分析

目的对北京市1名急性淋巴细胞白血病患者感染单核细胞增生李斯特菌病例进行病因溯源,对分离到的单核细胞增生李斯特菌进行血清学分型、耐药及分子分型研究。方法对患者不同时期外周血分离的2株单核细胞增生李斯特菌和1株环境涂抹样品单核细胞增生李斯特菌分离株进行血清学分型、耐药性分析、脉冲场凝胶电泳(PFGE)和多位点序列分析(MLST)。结果 3株单核细胞增生李斯特菌均为1/2a-3a血清型,耐药结果一致,均对青霉素、氨苄西林、复方新诺明、美罗培南及红霉素敏感,3株菌的PFGE带型一致,MLST型别均为ST155。结论本研究中患者生活环境中存在单核细胞增生李斯特菌的污染情况,高度怀疑患者感染单核细胞增生李斯特菌与其生活环境中分离到的菌株为同一来源。     

温州市单核细胞增生李斯特菌的毒力基因及血清学分型和分子分型研究

目的了解温州市近十年单核细胞增生李斯特菌分离株的血清型、毒力基因及分子分型特征。方法用聚合酶链式反应(PCR)方法对单核细胞增生李斯特菌进行血清型及毒力基因检测;用多位点序列分型(MLST)方法对单核细胞增生李斯特菌进行分子分型,并绘制MLST数据的最小生成树。结果 97株单核细胞增生李斯特菌分离株分为4种血清型,以血清型1/2b、1/2a为优势血清型,占比分别为48.45%(47/97)、35.05%(34/97);而毒力基因iap、prfA基因阳性率均为100.00%(97/97),hlyA、inlA基因阳性率均为97.94%(95/97),plcB基因阳性率为96.91%(94/97)。其中患者分离株5种毒力基因阳性率均为100.00%(6/6)。97株单核细胞增生李斯特菌分离株得到20个MLST型别,其中ST87型是优势型别,其次为ST121和ST9,ST1和ST779型是患者特有的,ST2、ST3、ST5型分布于食品和患者分离株。结论温州市不同来源的单核细胞增生李斯特菌分离株分子型别呈多态性,食品和患者分离株存在相同的ST型,且这些菌株大部分携带毒力基因,具有潜在的致病性,因此食品中单核细胞增生李斯特菌污染的潜在风险不容忽视。     

台州市食源性单核细胞增生李斯特菌分子特征研究

目的了解台州市食品中分离的单核细胞增生李斯特菌的血清型、毒力基因以及基因分型情况,建立食源性单核细胞增生李斯特菌的分子特征本底信息,为食源性疾病的防治提供技术支持。方法对近几年从食品中分离的37株单核细胞增生李斯特菌进行多重PCR血清分型、9种毒力基因(prf A、inl A、inl B、iap、fla A、hly A、plc B、mpl和act A)PCR检测、PFGE基因分型,用Bio Numerics 6.6软件对分型数据进行聚类分析。结果 37株食源性单核细胞增生李斯特菌的血清型以1/2a或3a型别为主;所有菌株均检出4种以上毒力基因,有15株菌携带所有9种毒力基因;37株菌经Apa I酶切PFGE分型后,共得到22种带型,每种带型包含1~5株不等,相似度区间为67%~100%。结论台州市食源性单核细胞增生李斯特菌存在致病流行风险,建立的指纹图谱数据库可为食源性疾病的防治提供技术支持。     

食品中单增李斯特菌的血清分型及脉冲场凝胶电泳分型

对进口食品和国内食品中的单增李斯特菌进行血清学分型和分子分型研究,探讨不同来源菌株之间的遗传相关性及不同分型方法之间的联系。对分离的单增李斯特菌进行血清学分型和脉冲场凝胶电泳分型。69株单增李斯特菌分为4种血清型,主要血清型为1/2a(3a)型,脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)分型分为55种带型,主要型别为GX6A12.CN0018。食品中的单增李斯特菌分子型别呈现多态性,进口食品分离株和国内食品分离株之间无相关性。     

2000—2018年福建省单核细胞增生李斯特菌分离株血清型及脉冲场凝胶电泳分型

目的 分析福建省食品、临床病例和环境中单核细胞增生李斯特菌分离株的血清型和脉冲场凝胶电泳(PFGE)分型,为食源性疾病的暴发识别和溯源提供参考依据。方法 采用多重聚合酶链式反应(PCR)血清学分型、免疫血清凝集和PFGE方法对2000—2018年分离的单核细胞增生李斯特菌进行分型。结果 117株单核细胞增生李斯特菌分为4组血清型,包括1/2a(3a)、1/2b(3b)、1/2c(3c)和4b(4d,4e),占比分别为67.5%(79/117)、23.1%(27/117)、5.1%(6/117)和4.3%(5/117)。9株病例分离株血清型为1/2a(6株)、4b(2株)和1/2b(1株)。采用Asc I限制性内切酶将117株单核细胞增生李斯特菌分为83种不同PFGE型别,其中10株具有独特单一的型别。9株病例分离株分为8种不同PFGE型别。结论 福建省食品和病例中分离的单核细胞增生李斯特菌,1/2a血清型占主导地位,4b血清型应引起关注。     

2012—2015年北京市西城区单核细胞增生李斯特菌多位点序列分型及耐药研究

探讨单核细胞增生李斯特菌多位点序列分型(MLST)与耐药性的关联性,确定某些具有高致病性潜能的流行克隆株的存在。方法 采用Kirby-Bauer(K-B)纸片扩散法和E-test药敏试条法对14种抗生素进行药物敏感性试验,以MLST技术对50株菌株进行基因分型。结果 单核细胞增生李斯特菌耐药率为22.00%(11/50),并出现多重耐药株。50株单核细胞增生李斯特菌MLST分析共获得12个型别,以ST9和ST121为优势型别。结论 特定ST型别在食品生产过程中存在特定菌株之间的传递,人源性和食源性单核细胞增生李斯特菌中均发现耐药株,可能存在耐药基因的传递,应加强对具有潜在致病性的ST型别的监测力度。     

北京市人源性单核细胞增生李斯特菌耐药特征及分子分型研究

目的研究北京市人源性单核细胞增生李斯特菌的血清学分型、耐药及分子特征。方法对2013—2015年北京市李斯特菌病专项监测中分离的32株单核细胞增生李斯特菌进行PCR法血清学分型和PFGE分型,并采用CLSI和EUCAST推荐的微量肉汤稀释法检测菌株对12种抗生素的敏感性。结果 32株单核细胞增生李斯特菌共分为4种血清型,其中位居前2位的血清型是1/2b和1/2a,分别为17株和12株,4b血清型2株,1/2c血清型1株。32株菌经Asc I酶切共分为23种PFGE带型,分为4个簇,相同血清型李斯特菌的PFGE带型聚集成簇,两种分型方法一致性为93.75%(30/32)。32株单核细胞增生李斯特菌均对青霉素、氨苄西林、复方新诺明及红霉素敏感,大部分菌株对其他8种抗生素的MIC值较小,但有少数菌株对苯唑西林和四环素的MIC值较大,高达32、64μg/ml。结论北京市人源性单核细胞增生李斯特菌血清型以1/2b和1/2a为主,PFGE图谱与血清学分型存在一定相关性,耐药率普遍较低,但需加强耐药趋势监测。     

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。     

食源性单核细胞增生李斯特菌的重金属镉抗性及其与抗生素耐药性的关系研究

研究了不同血清型食源性单核细胞增生李斯特菌对重金属镉的抗性及其与抗生素耐药性之间的关系。采用琼脂稀释法检测单核细胞增生李斯特菌对重金属镉的抗性,与菌株的抗生素耐药性进行比较分析。结果表明,73株实验菌株中,36株具有镉抗性,总体镉抗性率达49.3%(36/73)。分离到的5株多重耐药单核细胞增生李斯特菌均为镉抗性菌株,表明食品生产、加工及其储存环境中这种同时携带多重耐药性与重金属抗性的微生物从食品链及有关环境进入到人类,就会对人类健康构成构成潜在威胁。     

禽肉制品中单增李斯特菌的DiversiLab分型

目的：研究从同一类食品中分离出的单增李斯特菌间的同源性关系,为预测预警保障食品安全和食源性疾病溯源提供科学依据和技术支持。方法：应用DiversiLab分型系统对2001-2010年福建出入境检验检疫局从各类禽肉制品中分离出的25 株单增李斯特菌进行分型,其中包括：DNA提取、重复序列-聚合酶链式反应（repetitivesequence-based polymerase chain reaction,rep-PCR）、微电泳芯片分离检测及数据分析。结果：DiversiLab分型中,相似系数为95%时,25 株菌被分为了6 组（G）;相似系数为97%时,25 株菌被分为11 组（P）。结论：DiversiLab分型结果较准确地揭示了25 株菌株间的亲缘性关系。基于rep-PCR的DiversiLab分型系统,是一种操作简单、分辨率高、重复性好且高效快速的基因分型方法,可作为食源性病原体检测及食源性疾病溯源的工具。     

Characterization of Listeria monocytogenes isolated from retail foods

Listeria monocytogenes isolates recovered from retail ready-to-eat (RTE) meats, raw chickens and fresh produce were characterized by serogroup identification using PCR, genotyping using pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Five L. monocytogenes serogroups were identified. Of the 167 isolates 68 (41%) belonged to serogroup 1/2b, 3b; 53 (32%) belonged to serogroup 4b, 4d, 4e; 43 (26%) belonged to serogroup 1/2a, 3a; 2 (1.2%) belonged to serogroup 1/2c, 3c; and 1 (0.6%) belonged to serogroup 4a, 4c. PFGE generated 120 patterns which correlated well with PCR serogrouping. Most L. monocytogenes isolates were resistant to sulfonamide (73%) and some were resistant to tetracycline (8.4%) and ciprofloxacin (1.8%). Tetracycline resistance was conjugatively transferable and the tet(M) gene was identified in 14 tetracycline-resistant isolates as well as their transconjugants. These findings indicate that L. monocytogenes present in food were diverse, and that resistance to one or more antibiotics among these isolates was common. In addition, the presence of potential serotype 4b in all food categories is of public health concern, as serotype 4b has been the serotype most frequently associated with human listeriosis.     

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.     

Traceback identification of an ingredient (pork dewlap) as the possible source of Listeria monocytogenes serotype 4b contamination in raw chicken products

In surveys conducted on finished product samples from a single poultry processing plant in Spain, Listeria monocytogenes was found in 14 different uncooked products. To track contamination patterns, 77 L. monocytogenes isolates were characterized by PCR-based serotyping, pulsed-field gel electrophoresis (PFGE) restriction analysis, and PCR-based allelic analysis of the virulence gene actA. Serotyping revealed that 12 isolates (15.6%) were of the L. monocytogenes serotype 4b complex (serotype 4b or the closely related serotypes 4d and 4e). A combination of endonucleases AscI and ApaI PFGE patterns yielded 15 different pulsotypes among all 77 tested isolates. All the serotype 4b isolates belonged to one pulsotype. Sequencing of the actA gene confirmed that all serotype 4b isolates corresponded to the same allelic subtype. The subtype was recovered from five product types, but its presence was not correlated with the production line or the date of isolation, suggesting a possible association of this strain with a common ingredient. This traceback investigation established that pork dewlap, an ingredient common to all the products contaminated with this strain, was the most probable source of L. monocytogenes 4b. The same 4b strain was isolated from four samples of pork dewlap from one specific supplier. After replacement of this contaminated ingredient in the fresh products, this strain of L. monocytogenes serotype 4b was not detected. This study confirms the effectiveness of molecular subtyping to control contamination by specific strains of L. monocytogenes and the importance of testing the different ingredients added to the food products.     

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.     

Random amplification of polymorphic DNA typing of Listeria monocytogenes isolated from meat

To investigate the epidemiological characteristics of Listeria monocytogenes isolated from imported or domestic meats, L. monocytogenes was isolated and identified through biochemical and serological tests, and epidemiological analysis of the isolates was carried out through the random amplification of polymorphic DNA (RAPD) method. Fifty-four isolates were identified as L. monocytogenes through biochemical tests, of which 36 (67%) were confirmed as serotype 1, and 18 (33%) were serotype 4, through the microagglutination test. In the molecular epidemiological analysis using RAPD method, the isolates could be classified into 10, 6 and 6 types using three random primers, PB1, PB4, and HLWL74, respectively. Forty composite profiles were identified by a combination of the three primers. RAPD analysis demonstrated the relationships between the isolates from beef from Korea and the USA, pork from Korea and Denmark. These results suggested that RAPD could be a useful typing tool for the epidemiological study of L. monocytogenes and other bacteria.     

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。     

Listeria monocytogenes isolated from cold-smoked fish products in Osaka City, Japan

Listeria monocytogenes contamination of ready-to-eat seafood products commercially available in Osaka was examined between 1999 and 2000. L. monocytogenes was isolated from 12 (13%) of the 95 products tested. All positive samples were from cold-smoked fish with 9 being obtained during the summer. Thirteen isolates of L. monocytogenes were typed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR)-based typing methods. Isolates of the same serotype originating from the same manufacturer gave similar DNA profiles, irrespective of the type of sample or date of isolation. The finding suggest that persistent strains in each manufacturing facility proliferate during the summer and contaminate products during manufacturing processes.     

DiversiLab系统沙门氏菌分子分型评价

目的：评价DiversiLab(DVL)系统对沙门氏菌(SA)的分型能力。方法：用rep-PCR技术为原理的DVL系统对进出口食品中分离的44株SA进行分子分型,并与脉冲场凝胶电泳(PFGE)结果比较,探讨DVL对SA的种群分类能力和分辨率。结果：44株SA经rep-PCR分型分成22个群,分离自不同国家、不同食品中的相同血清型SA分布在同一个群,而且菌株之间相似性非常高。在rep-PCR结果中分在同一个群中的SA,在PFGE结果中除SA2和SA15外,其他菌株之间相关性较低。结论：DVL在SA的种群的分类能力上优于PFGE,但分辨率低于PFGE。     

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。