云南淡水鱼中副溶血性弧菌的污染情况及耐药性分析

目的了解云南省淡水鱼中副溶血性弧菌的污染和耐药情况。方法采集昆明、曲靖、大理、红河的淡水鱼110件,参照GB 4789.7-2013《食品安全国家标准食品微生物学检验副溶血性弧菌检验》,分离副溶血性弧菌的疑似菌株,通过生化试验和飞行时间质谱仪进行鉴定;采用革兰阴性需氧菌药敏检测板对分离鉴定到的副溶血性弧菌进行药物敏感试验。结果 110件淡水鱼中有15件样品检出副溶血性弧菌,总检出率为13.6%,说明云南淡水鱼中存在一定程度的副溶血性弧菌污染。所有的副溶血性弧菌分离株具有耐药性,对头孢唑林(cefazolin,CFZ)的耐药率最高为100.0%,存在多重耐药菌株。结论本研究可为淡水鱼类养殖和消费提供科学依据,建议相关部门加强对水产养殖业抗生素使用及流通过程的监督管理,适度增加淡水鱼的安全性检测。     

2005—2013年河北省即食食品中单增李斯特菌污染及耐药特征研究

研究2005—2013年河北省即食食品中单增李斯特菌污染状况及耐药特征。方法 单增李斯特菌检验参照(GB/T 4789.30),药物敏感性试验应用微量肉汤稀释法,对15种抗生素进行耐药检测。结果 单增李斯特菌总污染率为1.34%,凉拌菜及沙拉(2.62%)、生食水产品(2.51%)的检出率均高于所测其他食品。136株单增李斯特菌对15种抗生素耐药率为13.97%,以氯霉素耐药率最高(7.35%),其次是四环素和复方新诺明(均为4.41%),强力霉素和环丙沙星的耐药率均为2.94%,所有菌株对青霉素、亚胺培南、头孢噻吩、利福平、氨苄西林-舒巴坦酸敏感。结论 河北省即食食品中存在单增李斯特菌污染,分离菌株存在较严重的耐药情况,且耐药性有增长趋势,应加强即食食品的监管以保证食品安全及公众健康。     

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。     

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。     

云南乳饼中金黄色葡萄球菌污染状况及其耐药性分析

目的了解云南省乳饼中主要致病菌的污染状况及耐药情况。方法采集云南省乳饼主产地区样品62件,用传统方法结合基质辅助激光解吸电离飞行时间质谱进行分离鉴定,采用微量肉汤稀释法对金黄色葡萄球菌进行12种抗生素敏感性分析。结果共检出18株金黄色葡萄球菌,检出率为29.03%,沙门氏菌和单增李斯特氏菌均未检出。农贸市场、加工店和奶牛养殖户检出率分别为44.44%、26.67%和16.67%(P=0.321),超市和网店的样品均未检出;散装和包装样品检出率分别为34.38%、23.33%(P=0.433)。所有菌株的耐药性如下:青霉100%,红霉素44.44%,复方新诺明33.33%,苯唑西林、克林霉素和头孢西丁的耐药率均为27.78%,四环素22.22%,多重耐药率为72.22%。18株菌对环丙沙星、达托霉素、万古霉素、氯霉素和庆大霉素均为敏感。结论金黄色葡萄球菌为乳饼中的主要食源性致病菌,其对抗生素耐药性较普遍,需加强畜禽养殖及乳饼加工卫生条件监管。     

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。     

云南省牛乳中单核细胞增生李斯特氏菌的分布特征、耐药性及毒力研究

目的分析云南省牛乳中单核细胞增生李斯特氏菌(Listeria monocytogenes)的分布特征、耐药性及毒力现状。方法采集云南省主要奶产区牛乳样本,按照GB 4789.30—2016方法分离单核细胞增生李斯特氏菌疑似菌株,采用飞行时间质谱技术结合16SrDNA测序对疑似菌株进行鉴定;采用微量肉汤稀释法对分离菌株进行抗生素药敏实验;通过聚合酶链式反应(polymerasechainreaction,PCR)技术检测单核细胞增生李斯特氏菌中7种毒力基因的存在状况。结果158份样品共有4份检出单核细胞增生李斯特氏菌,检出率为2.53%;共分离获得8株菌。其中,生鲜乳中检出率为3.36%,巴氏杀菌乳中未检出。在生鲜乳中,荷斯坦牛乳、水牛乳、牦牛乳检出率分别为2.13%、0.00%和20.00%;标准化牛场、奶牛合作社和个体户的检出率分别为2.50%、1.51%和15.38%;手工挤奶和机械挤奶检出率为11.76%和1.96%。药敏实验结果显示8株菌均对青霉素耐药,对四环素、复方新诺明、红霉素耐药率分别为87.50%、75.00%和50.00%;多重耐药率为75.00%;但所有菌株对氨苄西林均敏感。8株菌对7种毒力基因的携带程度从12.50%~75.00%不等。结论云南省牛乳尤其生鲜乳中存在一定程度的单核细胞增生李斯特氏菌污染,且该菌具有普遍耐药性并携带一定程度的毒力基因,对消费者的安全具有潜在风险,应加强卫生监督管理。     

基于MALDI-TOF MS技术对李斯特氏菌属两种细菌的快速鉴定

为提高基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF MS)在李斯特氏菌属鉴定中的分辨能力,建立快速准确鉴定单增和英诺克李斯特氏菌的质谱学方法。通过采集79株单增和57株英诺克李斯特氏菌的指纹图谱,利用Clin Pro tools软件对数据进行统计学分析,建立数学判别模型并验证其准确性。峰统计结果显示,两组数据峰强度差异显著的特征峰有16个,推测出单增李斯特氏菌生物标志物6个,英诺克李斯特氏菌10个,发现在单增李斯特氏菌中质量峰3985/7970 u和3972/7942 u是独立且连锁存在。基于遗传算法的判别模型交叉验证率和检测识别能力最强,分别为99.44%和100.00%,经验证准确率达到96%以上,可实现对单增和英诺克李斯特氏菌的快速准确鉴定。同时,利用Bruker Biotyper软件将以上菌株建库形成了实验室内部李斯特氏菌谱库,对8株未测李斯特氏菌进行搜库鉴定,匹配分数均高于商品化数据库,提升了MALDI-TOF MS对李斯特菌属的自动鉴定能力。     

单核细胞增生李斯特氏菌国家标准检验方法的验证与探讨

本文旨在验证、分析和探索食品中单核细胞增生李斯特氏菌的检验方法。方法:依据GB4789.30-2016,首先将实验样品预增菌24 h后,之后,用李斯特氏菌显色培养基和PALCAM琼脂分离培养,将菌株进行染色显微镜观察、动力试验、生化鉴定、溶血试验、协同溶血试验、李斯特氏菌属鉴定试剂盒、全自动微生物生化鉴定仪等鉴别菌种的方法。结果:实验样品和单核细胞增生李斯特氏菌以及伊氏李斯特氏菌菌落均在李斯特氏菌显色培养基上呈蓝绿色,周围环绕着白色晕圈,实验样本及4种李斯特氏菌标准菌株均是革兰氏阳性,短杆状,不产芽孢和荚膜;生化反应中与葡萄糖、MRVP、麦芽糖、七叶苷、甘露醇等发酵反应结果一致。除了伊氏李斯特氏菌在木糖发酵管中反应呈黄色,为阳性外,其他李斯特氏菌菌株都呈绿色,为阴性。在溶血反应中,英诺克李斯特氏菌在血平板上没有溶血,为阴性,而其他菌株产生透明溶血圈,为阳性;由全自动微生物生化鉴定仪鉴定菌株均为李斯特氏菌属。结论:通过对国家标准测试方法—单增李斯特氏菌的验证,建议通过选择性培养基进行分离后,用木糖、鼠李糖生化反应区分单增李斯特氏菌与其他李斯特氏菌,最后用单增李斯特氏菌生化鉴定试剂盒验证,使检测效率提高同时也提升了检测的准确度。     

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。     

2017年楚雄地区餐饮业过桥米线微生物污染状况调查分析

目的 了解楚雄地区过桥米线微生物污染状况, 获得地域代表性数据。方法 按照《2017年云南省食品安全风险监测方案》, 采用国家标准规定的检测方法, 对60份过桥米线样品进行菌落总数、大肠菌群、霉菌计数、沙门氏菌、蜡样芽胞杆菌、变形杆菌、金黄色葡萄球菌、单核细胞增生李斯特杆菌8个微生物指标检测。结果 60份过桥米线样品中,菌落总数检出率83.33%(50/60), 大肠埃希氏菌计数检出率40.00%(24/60), 霉菌计数检出率65.00%(39/60); 致病菌总检出率33.33%(20/60), 共检出食源性致病菌23株, 检出菌株数(检出率)分别是: 蜡样芽胞杆菌9株(15.00%)、金黄色葡萄球菌6株(10.00%)、变形杆菌5株(8.33%)、沙门氏菌3株(5.00%), 单核细胞增生李斯特杆菌未检出。结论 楚雄地区过桥米线微生物污染严重, 应加强监管, 控制食用安全风险; 同时应尽快制定云南省地方特色食品过桥米线的食品质量或安全标准。     

Pulsed-field gel electrophoresis typing of Listeria monocytogenes isolated in two Finnish fish farms

The aim of this study was to find sources of Listeria monocytogenes contamination in fish products from a fish farm. The occurrence of L. monocytogenes also was compared in two freshwater fish farms with different types of fishponds. Samples collected from chilled rainbow trout (Oncorhynchus mykiss) and the slaughterhouse environment did not contain L. monocytogenes, but Listeria innocua was found in two samples from the slaughterhouses. Ten isolates of L. monocytogenes were discovered in sediment and water samples from farming tanks and earth ponds. Further characterization by serovar revealed the same serovar (1/2a) for all the isolates. Pulsed-field gel electrophoresis was used to divide the isolates into five different pulsotypes, three of which have been identified previously in fish products on the retail market. This finding supports the assumption that the primary production, and probably the raw fish, is a source of Listeria contamination in fish products. Some of the isolates were associated with a certain type of fishpond, indicating the need for hygienic analysis of the suitability of different types of farming ponds.     

Raw and processed fish show identical Listeria monocytogenes genotypes with pulsed-field gel electrophoresis

A total of 257 raw fish samples at two different sites were examined for the presence of Listeria monocytogenes. The prevalence of L. monocytogenes was 4%. From 11 positive samples, nine different L. monocytogenes pulsed-field gel electrophoresis genotypes were recovered. From nine pulsotypes recovered from raw fish and 32 pulsotypes shown by 101 fish product isolates, two raw fish and fish product pulsotypes were indistinguishable from each other. Although the prevalence of L. monocytogenes in raw fish is low, the range of L. monocytogenes strains entering the processing plant in large amounts of raw material is wide. This indicates that the raw material is an important initial contamination source of L. monocytogenes in fish processing plants. This postulation is supported by the identical pulsotypes recovered from both raw and processed fish. Some L. monocytogenes strains entering a plant may thus contaminate and persist in the processing environment, causing recurrent contamination of the final products via processing machines.     

Prevalence and survival of Listeria monocytogenes in Danish aquatic and fish-processing environments

Listeria monocytogenes contamination of ready-to-eat food products such as cold-smoked fish is often caused by pathogen subtypes persisting in food-processing environments. The purpose of the present study was to determine whether these L. monocytogenes subtypes can be found in the outside environment, i.e., outside food processing plants, and whether they survive better in the aquatic environment than do other strains. A total of 400 samples were collected from the outside environment, fish slaughterhouses, fish farms, and a smokehouse. L. monocytogenes was not detected in a freshwater stream, but prevalence increased with the degree of human activity: 2% in seawater fish farms, 10% in freshwater fish farms, 16% in fish slaughterhouses, and 68% in a fish smokehouse. The fish farms and slaughterhouses processed Danish rainbow trout, whereas the smokehouse was used for farm-raised Norwegian salmon. No variation with season was observed. Inside the processing plants, the pattern of randomly amplified polymorphic DNA (RAPD) types was homogeneous, but greater diversity existed among isolates from the outside environments. The RAPD type dominating the inside of the fish smokehouse was found only sporadically in outside environments. To examine survival in different environments, L. monocytogenes or Listeria innocua strains were inoculated into freshwater and saltwater microcosms. Pathogen counts decreased over time in Instant Ocean and remained constant in phosphate-buffered saline. In contrast, counts decreased rapidly in natural seawater and fresh water. The count reduction was much slower when the natural waters were autoclaved or filtered (0.2-microm pore size), indicating that the pathogen reduction in natural waters was attributable to a biological mechanism, e.g., protozoan grazing. A low prevalence of L. monocytogenes was found in the outside environment, and the bacteria did not survive well in natural environments. Therefore, L. monocytogenes in the outer environment associated with Danish fish processing is probably of minor importance to the environment inside a fish production plant.     

Efficiency of four secondary enrichment protocols in differentiation and isolation of Listeria spp. and Listeria monocytogenes from smoked fish processing chains

Four secondary enrichment protocols (conventional methods: UVM II, Fraser 24 h and Fraser 48 h: Impedimetric method: Listeria electrical detection medium) were studied for their ability to isolate Listeria spp. and Listeria monocytogenes from fish and environmental samples collected along the processing chain of cold-smoked fish. From all methods, Listeria spp. and L. monocytogenes were respectively present in 56 and 34 of 315 samples analysed. Fraser broth incubated for 48 h gave the fewest false negative Listeria spp. results [4/56; (7.1%)], but concurrently only 15/34 (44.1%) samples were correctly identified as containing L. monocytogenes, Listeria electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp. positive samples. Despite this lower isolation rate, LED identified 20/34 (58.8%) L. monocytogenes positive samples correctly and gave fewer false positive results. The overall conclusion was that more than one isolation method is needed to accurately estimate L. monocytogenes contamination rates.     

2015年吉林市食品安全风险监测结果分析

摘 要：目的 分析吉林市食源性致病菌污染情况,为预防和控制食源性疾病提供依据。方法 按照《2014年国家食品污染和有害因素风险监测工作手册方法》对食源性致病菌进行监测。结果 2015年吉林市共监测10类240份食品样本,检出19株致病菌,总检出率7.92%,其中检出7株蜡样芽孢杆菌,8株单核细胞增生李斯特氏菌,3株金黄色葡萄球菌,1株沙门氏菌;3类食品样本监测到食源性致病菌：流动早餐检出率57.14%,肉与肉制品检出率50%,调味品检出率2%;依据散装和预包装不同包装类型的食品样本中食源性致病菌的检出率差异有统计学意义。结论 吉林市流动早餐和肉与肉制品两类食品样本中检出食源性致病菌为金黄色葡萄球菌、蜡样芽胞杆菌、单核细胞增生李斯特菌,检出率均较高,相关部门需要加强对此类食品的监管。     

Molecular typing to trace Listeria monocytogenes isolated from cold-smoked fish to a contamination source in a processing plant

In this study, Listeria monocytogenes contamination in a cold-smoked fish processing plant in Osaka, Japan, was examined from 2002 to 2004. A total of 430 samples were collected and divided into five categories: raw fish, materials during processing, processing equipment, environment, and finished products. A total of 59 finished products were examined throughout this study. L. monocytogenes was isolated from four of these samples during summer and autumn but was not found during winter or spring. During the warmer seasons, L. monocytogenes was more prevalent on processing equipment, especially slicing machines (8 of 54 samples in summer and autumn versus 1 of 50 samples in winter and spring). L. monocytogenes was not detected on whole skins removed from 23 frozen raw fish. L. monocytogenes strains isolated from 56 samples were characterized by serotyping, pulsed-field gel electrophoresis, and three PCR-based methods. Seventy-seven L. monocytogenes strains were recognized as contaminants of the samples: 2 distinguishable strains were identified in each of 13 samples, 3 strains were identified in 2 samples, 5 strains were identified in 1 sample, and the other 40 strains were identified in 40 samples. Combining the results from these techniques, 77 strains were classified into 13 different types. Three of these types prevailed throughout the plant, and two of the three were also isolated from final products. The DNA subtype found in the product was also found on the slicing machines. Our findings suggest that the slicing machines at this plant were the source of the product contamination. Implementing an appropriate cleaning regime for the slicing machines was effective in preventing contamination.     

实时荧光PCR法和国标法检测乳粉中单增李斯特菌的比较研究

目的 提升实验室检测食品中单核细胞增生李斯特氏菌的速度和准确性。方法 依据《GBS4789.30-2016S食品安全国家标准S食品微生物学检验S单核细胞增生李斯特氏菌检验》和能力验证作业指导书对食品中单核细胞增生李斯特氏菌进行检测, 同时利用实时荧光PCR法对能力验证中的2个样品进行快速筛查,采用VITEK2 Compact30全自动微生物鉴定系统鉴定可疑菌落。结果 GB 4789.30国标法和实时荧光PCR快速筛查法检测,结果均为样品CODE:FC02220028单核细胞增生李斯特氏菌阳性, 样品CODE:FC02220098阴性。结论 本实验室参加了中国食品药品质量检定研究院组织的食品中单核细胞增生李斯特氏菌检测能力验证,取得了满意的结果。     

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.