牛乳中单核细胞增生李斯特菌的分离与鉴定

牛乳中单核细胞增生李斯特菌的分离与鉴定唐敏莉，刘敏娟，孙钊，黄金文（齐齐哈尔市卫生防疫站）前言李斯特菌是引起人、畜单核细胞增生的一种致病菌，已逐渐被人们注意，世界卫生组织１９８８年报告，在乳及乳制品、肉类、禽类等食品中均发现李斯特菌的存在［１］．近年...     

水产品中单核细胞增生李斯特氏菌检验研究

本文是对水产品中单核细胞增生李斯特氏菌常规检验的研究。结合国家标准、行业标准和FDA《细菌学检验手册》第八版中所述方法进行检验。自朝鲜进境的水产品23类、255批中检出李斯特氏菌7株。结果表明,由于食品加工过程对微生物损害极大,使大多数微生物处于濒死状态,因而加速受损细胞恢复是单核细胞增生李斯特氏菌检验前增菌的关键。在检验过程中最初分离时发现了可疑菌落并就此进行确认和得出结论,而不是延长增菌时间继续分离和鉴定,容易产生假阴性结果。     

盐水鸭中单核细胞增生李斯特菌生长预测模型的建立

为研究盐水鸭中单核细胞增生李斯特菌(Listeriamonocytogenes,Lm)的生长规律,通过测定4、10、16、25℃条件下的生长数据,选用4种常用的一级模型(Gompertz、Logistic、Richards及MMF模型)对数据进行拟合,比较各模型决定系数R^2和均方误差(MSE),确定最适一级模型,根据一级模型得到的最大比生长速率(μmax)和迟滞期(λ)建立与温度相关的二级模型。结果表明:Gompertz模型拟合的生长曲线R^2均达到0.99以上,为最适一级模型,在25℃条件下,Lm经0.78 h后即进入对数期,从4℃提高到10℃时,生长速率从0.02 1g(cfu/g)·h^-1增至0.05 1g(cfu/g)·h^-1,说明温度对盐水鸭中Lm的生长影响较大。选用Ratkowsky平方根模型建立的温度与μmax关系的二级模型R^2为0.98,偏差因子(Bf)、准确因子(Af)分别为0.99、1.01,二次多项式模型建立的温度与λ关系的R^2为0.99,Bf、Af分别为1.01、1.08,表明所建两种模型均能较好地描述盐水鸭中Lm的生长情况。本研究建立的生长模型可为监控盐水鸭的食品安全和风险评估提供参考。     

食品中单核细胞增生李斯特氏菌检测分析

目的:分析食品中单核细胞增生李斯特氏菌的检测结果,以及时发现食品安全隐患,为食品安全监管提供参考依据.方法:严格按照《全国食源性致病菌监测工作手册》中的单核细胞增生李斯特氏菌检验标准,对某市2021年1—12月定期采样的10类常用食品进行单核细胞增生李斯特氏菌检测,分析其阳性检出率.结果:897份10类常用食品中,检出...     

单核细胞增生李斯特菌菌膜形成突变株的筛选

通过对功能和调控基因的研究,探讨单核细胞增生李斯特菌菌膜的形成机制,建立防治该细菌污染食品的方法.使用电转化的方法,将携带转座子Tn917的质粒pTV1-OK转化到单核细胞增生李斯特菌中,诱导转座,获得1 880个突变株,加上前期构建的突变株,使突变库增加到2 200株.使用96孔细胞培养板对随机挑选的1 000余株突变株进行菌膜培养,结晶紫染色后测OD595值.以此值作为菌膜形成量的筛选依据,最终挑选出菌膜形成能力变小的突变株8株.目标突变获得率约8‰.对筛选的菌膜形成量变小的突变株进行荧光显微现察,证实该8株突变株菌膜形成能力弱于野生菌株.通过PCR验证,这8株突变株的基因组中插入了Tn917,这可能是它们的表型发生变化的原因.     

即食食品中单核细胞增生李斯特菌风险管理措施的研究

单核细胞增生李斯特菌是一种重要的食源性致病菌,由于该菌引发的疾病致死率较高,且其暴发常出现于工业加工食品中,从而引起世界范围的广泛关注.FAO/WHO微生物风险评估联合专家委员会(JEMRA)及美国、欧盟、澳大利亚和新西兰等国家分别对即食食品中单核细胞增生李斯特菌进行了风险评估,并根据风险评估采取适当的风险管理措施.本文对国际食品法典委员会及部分国家的风险管理措施进行了横向比较分析,提出进行完整的微生物风险评估是选择适当的微生物风险管理的必要前提,风险管理措施的有效实施需要国家主管机构、企业和消费者三方的共同参与.上述结论可为我国政府部门在现有法规框架内如何控制即食食品中单核细胞增生李斯特菌的风险提供参考.     

SPA-ELISA用于单核细胞增生性李斯特菌检验的应用研究

创造性地将SPA-ELISA应用于食品中单核细胞增生性李斯特菌(简称LM)的检验中,建立了快速检测该菌的ELISA方法。试验表明:该方法可特异性检测肉品中的LM,样品中最低检出量为5cfu/10g,经增菌后的培养液最低检测限可达到105cfu/mL,并可在48h内报告结果,比常规分离培养鉴定方法快5￣6d,适于LM的快速筛选。     

SPA-ELISA用于单核细胞增生性李斯特菌检验的应用研究

本研究创造性地将SPA-ELISA应用于食品中单核细胞增生性李斯特菌(简称LM)的检验中,建立了快速检测该菌的ELISA方法.试验表明:该方法可特异性检测肉品中的LM,样品中最低检出量为5个/10克,经增菌后的培养液最低检测限可达到105CFU/ml,并可在48小时内报告结果,比常规分离培养鉴定方法快5～6天,适于LM的快速筛选.     

食品中单核细胞增生李斯特菌的危害及其检测

单核细胞增生李斯特菌是一种能引起人畜共患的食源性致病菌之一。近年来 ,在许多国家 ,它被列为卫生部门重点检测的几种食源性致病菌之一。本文综述了单核细胞增生李斯特菌的生理生化特征、分布、污染途径、危害与流行病学概况和检测方法。     

多方法分离鉴定食品中单核细胞增生李斯特菌

参加中国检验检疫科学研究院编号为ACAS-PT1343 (2022)的能力验证,该能力验证考核能否从人工污染的样品中检出单核细胞增生李斯特菌检验。对随机人工污染样品22-D378、22-G483进行单核细胞增生李斯特菌检验,采用国标法、酶联免疫法和荧光定量PCR技术3种检测方法,探讨食品中单核增生李斯特菌的快速检测及鉴定。试验结果表明,编号22-D378样品检出单增李斯特菌,22-G483未检出。在国标方法的基础上,辅助酶联免疫和荧光定量PCR技术进行检测,缩短了检测时间,特异性和敏感性均较好,3种方法互相补充可确保试验结果的准确性。     

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。     

单核细胞增生李斯特菌毒力基因及其致病机制的研究进展

单核细胞增生李斯特菌是常见的食源性致病菌,广泛存在于环境中。单核细胞增生李斯特菌感染后主要表现为败血症、脑膜炎和单核细胞增多,也可导致孕妇流产、胎死宫内、新生儿死亡等。单核细胞增生李斯特菌致病性与其毒力基因及毒力岛密切相关,其机制是众多毒力因子在各调控因子复杂的网络调控下的结果。本综述旨在了解单核细胞增生李斯特菌毒力基因及其致病机制。     

Modelling the growth of Listeria monocytogenes in fresh green coconut (Cocos nucifera L.) water

The behaviour of Listeria monocytogenes in the fresh coconut water stored at 4 °C, 10 °C and 35 °C was studied. The coconut water was aseptically extracted from green coconuts (Cocos nucifera L.) and samples were inoculated in triplicate with a mixture of 5 strains of L. monocytogenes with a mean population of approximately 3 log₁₀ CFU/mL. The kinetic parameters of the bacteria were estimated from the Baranyi model, and compared with predictions of the Pathogen Modelling Program so as to predict its behaviour in the beverage. The results demonstrated that fresh green coconut water was a beverage propitious for the survival and growth of L. monocytogenes and that refrigeration at 10 °C or 4 °C retarded, but did not inhibit, growth of this bacterium. Temperature abuse at 35 °C considerably reduced the lagtimes. The study shows that L. monocytogenes growth in fresh green coconut water is controlled for several days by storage at low temperature, mainly at 4 °C. Thus, for risk population this product should only be drunk directly from the coconut or despite the sensorial alterations should be consumed pasteurized.     

一例孕产妇单核细胞增生李斯特菌感染的溯源调查及发病机制探讨

目的 针对一例孕产妇李斯特菌病开展病例调查和溯源分析,探讨感染来源和单增李斯特菌病的发病机制,为防控李斯特菌病提供依据。方法 开展现场流行病学调查,收集病例信息,采集病例血液标本、家庭冰箱内食品及厨房环境样本、家庭附近农贸市场的食品样本,针对不同来源样本中的单增李斯特菌进行检测。结果 病例经常食用从农贸市场购买的中式凉拌菜（5～7次/周）,在家自制或二次加工中式凉拌菜。其家庭冰箱冷藏室储存食物生熟不分,且生食水果在冰箱中出现腐烂现象。厨房的两块菜板生熟不分、清洗消毒不及时,厨房操作面存在交叉污染。检测结果显示,11份样本中共分离出3株单增李斯特菌,1株来自该病例的血液标本,2株来自厨房冰箱内食品涂抹和菜板涂抹。提示该病例由于食用污染食品导致感染并通过胎盘屏障感染胎儿。结论 本起事件是丰台区首次在食品和环境中尝试溯源单增李斯特菌病的感染来源。病例家庭冰箱内生肉和胡萝卜、厨房环境涂抹样本中均检出单增李斯特菌,明确了食品与环境交叉污染导致病例单增李斯特菌感染发病;医院的早期识别及处置是避免新生儿不良结局出现的重要保障。     

Characterization of Listeria monocytogenes strains isolated from Gorgonzola cheese rinds

Listeria monocytogenes is a foodborne pathogen frequently present in ripened soft cheeses. Forty-three strains of L. monocytogenes isolated from the rind of ripened Gorgonzola cheeses produced in 24 different dairy plants were characterized by biotyping, serotyping, and molecular typing. Biotyping was performed by studying two phenotypes closely associated with virulence, such as hemolytic and phospholipase C activities. Traditional typing techniques did not allow a discrimination among the 43 strains studied. All strains showed a good hemolytic activity on blood agar, and only slight differences were observed when titration of hemolytic activity of culture supernatants was performed. Also phospholipase activities were quite similar for all the strains. Concerning serotyping, all strains belonged to serotype 1/2a. The molecular characterization was performed by RAPD-PCR. Combined cluster analysis following PCR amplification experiments allowed to group L. monocytogenes strains into few distinguishable profiles. At a level of similarity of 80%, the 43 strains were grouped into only 5 composite profile groups. Although isolated in 24 different plants, the presence of a few closely related strains demonstrated a possible relationship between these cheese isolates; a special ability of these strains to adapt to Gorgonzola cheese processing environment could be suggested.     

Comparing predicting models for heat inactivation of Listeria monocytogenes and Pseudomonas aeruginosa at different pH

Under the same experimental conditions it has been demonstrated that whereas survival curves of Listeria monocytogenes in the range of temperatures from 54 to 62 °C followed a first-order kinetic, those of Pseudomonas aeruginosa in the range of temperatures from 50 to 56 °C were not linear showing a shoulder followed by a linear region. The first order kinetic model did not describe survival curves of P. aeruginosa. A model based on the Weibull distribution (Log₁₀(N_t/N₀)=(1/−2.303)*(t/b)ⁿ)) accurately described the inactivation kinetics of both microorganisms at the three pHs of 4, 5.5, 7.4 investigated. For both microorganisms, the b value depended on the treatment temperature and the pH of the treatment medium. Whereas for L. monocytogenes the n value was independent of the treatment conditions, for P. aeruginosa the n value depended on the pH of the treatment medium.The model based on the Weibull distribution was capable of accurately predicting the treatment time to inactivate five Log₁₀ cycles of both microorganisms at the three pHs investigated.     

The occurrence of Listeria monocytogenes in cheese from a manufacturer associated with a case of listeriosis

A case of listeriosis was associated with the consumption of a soft cheese produced in England. Goats cheese and other products from the same food manufacturer were examined for the presence of Listeria over the following 11 months. Listeria monocytogenes was isolated from 16 of 25 cheese samples on retail sale, 12 of 24 cheese samples obtained directly from the factory, and from shelving within the plant. Phage-typing of 68 isolates of L. monocytogenes from cheese samples and the factory showed that 66 (97%) were indistinguishable from the strain isolated from the patient's cerebrospinal fluid and stool. L. monocytogenes was not isolated from seven goats milk or two yoghurt samples. Listeria innocua was isolated from 10 cheese samples, two of which contained no other species of Listeria. Levels of L. monocytogenes shortly after production were low (<10/g), but were higher (10⁵–10⁷ cfu/g) in six of the 16 cheese samples obtained from retail outlets. Multiplication of L. monocytogenes was demonstrated in cheeses contaminated at the factory and held at 4°C in the laboratory.     

Evaluation of the effect of defrosting practices of ground beef on the heat tolerance of Listeria monocytogenes and Salmonella Enteritidis

The effect of common defrosting practices of ground beef, including (i) defrosting in the refrigerator (5 °C for 15 h), (ii) defrosting at room temperature (25 °C for 12 h) and (iii) defrosting in the microwave, on the heat tolerance of artificially inoculated Listeria monocytogenes and Salmonella Enteritidis, was studied. The thermal inactivation of S. Enteritidis was not, overall, affected by defrosting practices. In contrast, defrosting at room temperature resulted, overall, in an increased heat tolerance of L. monocytogenes compared to the rest tested defrosting practices. Inactivation kinetics of the two pathogens for the different defrosting practices were determined by fitting the data to the Weibull model. The δ parameter of the Weibull model (heat challenge time (min) required for the first 1-log reduction) for S. Enteritidis and for defrosting at 25 °C, microwave defrosting, defrosting at 5 °C and for the control (fresh ground beef inoculated with the pathogens just before the heat challenge trials) was 1.13, 1.62, 1.60 and 0.96, respectively, while the corresponding values for L. monocytogenes were 20.13, 10.82, 9.95 and 9.47, respectively. The findings of this study should be useful in risk assessments and in developing food handling guidelines for the consumers.     

Modelling the effect of a temperature shift on the lag phase duration of Listeria monocytogenes

The aim of this work is to study and model the effect of a temperature shift on h₀, the product of the growth rate by the lag phase duration (μλ). Our work is based on the data of Whiting and Bagi [Int. J. Food Microbiol. 73 (2002) 291], who studied the influence of both the pre-incubation temperature (T_prior) and the growth temperature (T_growth) on λ values of Listeria monocytogenes. We introduce a new model to describe the evolution of the parameter h₀ as a function of T_prior and T_growth, and compare it to Whiting and Bagi's published polynomial model that describes the influence of T_prior and T_growth on λ independently of μ. For exponential as well as stationary phase cells, h₀ increases almost linearly with the magnitude of the temperature shift. A simple linear model of h₀ turns out to be more suitable to predict λ values than a polynomial model of λ.     

The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: A comparison between raw milk and milk treated with high hydrostatic pressure

Camembert-type cheese was produced from: raw bovine milk; raw milk inoculated with 2 or 4 log CFU/ml Listeria monocytogenes; raw milk inoculated with L. monocytogenes and subsequently pressure-treated at 500 MPa for 10 min at 20 °C; or uninoculated raw milk pressure-treated under these conditions. Cheeses produced from both pressure-treated milk and untreated milk had the typical composition, appearance and aroma of Camembert. Curd and cheese made from inoculated, untreated milk contained large numbers of L. monocytogenes throughout production. An initial inoculum of 1.95 log CFU/ml in milk increased to 4.52 log CFU/g in the curd and remained at a high level during ripening, with 3.85 log CFU/g in the final cheese. Pressure treatment inactivated L. monocytogenes in the raw milk at both inoculum levels and the pathogen was not detected in any of the final cheeses produced from pressure-treated milk. Therefore high pressure may be useful to inactivate L. monocytogenes in raw milk that is to be used for the production of soft, mould-ripened cheese.

Industrial relevance

This paper demonstrates the potential of high pressure (HP) for treatment of raw milk to be used in the manufacture of soft cheeses. HP treatment significantly reduced the level of Listeria monocytogenes in the raw milk and so allowed the production of safer non-thermally processed camembert-like soft cheese.