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.     

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。     

Development of a membrane filtration method for enumeration of Listeria monocytogenes from soft cheese

A new and simple analytical method has been developed to quantify low levels (≤50 cfu g⁻¹) of Listeria monocytogenes in soft cheese. The technique allows the analysis of 1 g of cheese instead of 0·1 g or 0·01 g using the ISO 11290-2 standard method. The analysis protocol combines filtration of the decimally diluted cheese suspension through a 0·45-μm pore size cellulose ester membrane, and a culture of the filter on a Palcam agar. A tween 80–trypsin treatment used to increase filterability of cheese did not reduce L. monocytogenes counts. The tested method provided more precise results (nearer to the true value) compared to the ISO 11290-2 standard method in the enumeration of L. monocytogenes from artificially contaminated cheese. However, it improves neither repeatability nor reproducibility since the selected medium, Palcam, does not allow distinction between the different Listeria species.     

Schnellnachweis von Listeria monocytogenes mittels real-time PCR

Molecular biological methods like the real-time PCR are well suited for a safe and rapid detection of Listeria and especially of Listeria monocytogenes. The use of commercially available kits in combination with adapted sample preparation is advantageous for the routine testing in the food industry. In addition to its high specificity and sensitivity, its superior reproducibility and short time to result, the described method is easy to use. Depending on the food matrix, the result is available after 24 h to 48 h. The procedure consists of a short enrichment, a sample preparation to extract the DNA from the cells and the PCR. The data interpretation is carried out automatically and can be used directly for documentation purposes.     

成都市冷藏即食预包装熟肉制品中单核细胞增生李斯特菌定量检测及污染风险研究

目的 为探明了解冷藏即食预包装熟肉制品中单核细胞增生李斯特菌（单增李斯特菌）污染情况及潜在风险,本研究针对短保质期的冷藏即食预包装熟肉制品开展单增李斯特菌定量污染风险调查。方法 2022年5~8月,从成都市采集样品64份,依据采用传统培养分离鉴定和RT-PCR分子检测方法进行单增李斯特菌的定性和定量检测,分离菌株提取DNA后进行二代全基因组测序并开展相关流行病学特征分析。结果 检测结果发现64份即食熟肉样品中,6份样品检出单增李斯特菌,检出份数为6/64。6份阳性样品均由某同一工厂生产,其中5份为卤肉三拼样品。定量检测结果表明,卤肉三拼的单增李斯特菌污染水平为30~200 CFU/g;单增李斯特菌阳性的猪肉样品污染水平为<10 CFU/g。阳性样品中的6株分离菌株经全基因组测序分析鉴定为单增李斯特菌,均含有LIPI-1毒力岛相关基因,多序列位点分型发现主要的序列型（ST）为ST3（n=2）和ST121（n=2）,其次是ST8和ST9。单核苷酸多态性（SNP）结果分析发现同一工厂分离到的单增李斯特菌菌株LM22071911和LM22080802之间检测到23个SNP差异,单增李斯特菌株LM22062706和LM22080806之间检测到13个SNP差异,说明在该工厂加工环节存在两种克隆群的单增李斯特菌直接传播的风险。结论 成都市部分市售冷藏即食预包装熟肉制品污染多种李斯特菌,需加强源头控制。     

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.     

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。     

Efficacy of pulsed light for shelf-life extension and inactivation of Listeria monocytogenes on ready-to-eat cooked meat products

Pulsed light (PL) was tested for its utility to improve the microbial quality and safety of ready-to-eat cooked meat products. Vacuum-packaged ham and bologna slices were superficially inoculated with Listeria monocytogenes and treated with 0.7, 2.1, 4.2 and 8.4 J/cm². PL treatment at 8.4 J/cm² reduced L. monocytogenes by 1.78 cfu/cm² in cooked ham and by 1.11 cfu/cm² in bologna. The effect of PL on lipid oxidation and sensory properties was also investigated. The 2-thiobarbituric acid values were very low and chromaticity parameters were within the normal values reported for cooked meat products. PL at 8.4 J/cm² did not affect the sensory quality of cooked ham, while treatments above 2.1 J/cm² negatively influenced the sensory properties of bologna. The combination of PL and vacuum packaging provided ham with an additional shelf-life extension of 30 days compared with only vacuum packaging. The shelf-life of bologna was not extended by PL.

Industrial relevance

The efficacy of pulsed light for the decontamination of surfaces offers excellent possibilities to ensure food safety and to extend shelf-life of ready-to-eat (RTE) products. The results of this study indicate that Listeria monocytogenes can be reduced by approximately 2 log cfu/cm² in RTE cooked ham and 1 log cfu/cm² in bologna using a fluence of 8.4 J/cm². This dose does not affect the sensory properties of ham and triples its shelf-life when compared with conventional RTE products. On the contrary, fluences above 2.1 J/cm² are not suitable for the treatment of bologna since sensory quality is modified.     

The effect of nisin and garlic (Allium sativum L.) essential oil separately and in combination on the growth of Listeria monocytogenes

In the present study, the anti-listerial activity of the nisin and garlic (Allium sativum L.) essential oils (GO) alone and in combination was investigated at different temperatures (20 and 30 °C), pH (6.8, 5.6 and 4.8) and NaCl concentrations (0, 0.5, 2.5 and 4.5 g/100 mL). Minimum inhibitory concentrations (MICs) against Listeria monocytogenes were assessed for the nisin and essential oil. Furthermore, for combinations of the antimicrobials, the Differences in Population (DP) method were used to determine their effect. Both essential oil and nisin possessed considerable antimicrobial effects on the microorganism. The MICs for nisin and GO were 12.5 IU/mL and 100 μg/mL, respectively. Regardless of NaCl concentration and temperature, the anti-listerial activity of both GO and nisin was strongly influenced by pH. Moreover, at the same temperature, and regardless of pH value, the growth of the organism was also affected by increasing NaCl concentration. In combination, the DP was related strongly to the agent’s concentration and media pH. Meanwhile, among all combined systems, the effect of the combination (EC) of nisin with GO at 30 °C, pH 5.6 and 0 g/100 mL NaCl, showed significant anti-listerial activity (P ≤ 0.05).     

可生食蔬菜中单核细胞增生李斯特菌生存特征研究

目的 探究可生食蔬菜品种、温度、接种部位对单核细胞增生李斯特菌（以下简称单增李斯特菌）存活的影响,为可生食蔬菜中单增李斯特菌的风险评估和关键控制措施提供理论依据。方法 以冻干定量单增李斯特菌为菌株来源,以彩椒、洋葱、黄瓜、圣女果和生菜5种可生食蔬菜的表面和切面为单增李斯特菌的接种点,在4 ℃、25 °C条件下培养7 d,定期监测每份样本中的单增李斯特菌的菌量,对其生长情况进行分析。结果 单增李斯特菌冻干菌种不同瓶间菌量均匀（F=1.923,P<0.05）,-20 ℃储存28 d后的复苏率为93.3%±4.2%。在4 ℃条件下,除了彩椒表面、黄瓜切面、生菜表面和生菜切面外,单增李斯特菌在其他蔬菜上放置7 d后均未见显著生长（δ<0.5 log₁₀ CFU/mL）。在25 ℃条件下,单增李斯特菌在彩椒、洋葱、圣女果、生菜以及黄瓜切面上均呈现为支持生长［δ为（1.16±0.35）~（2.68±0.18）log₁₀ CFU/mL］。单增李斯特菌在黄瓜切面、生菜表面和切面放置7 d后,菌量仍持续增长,在生菜的表面和切面生长趋势和浓度基本一致。结论 单增李斯特菌在可生食蔬菜上的存活能力与蔬菜种类、表面与切面、储存温度等条件密切相关,温度的控制对降低其在可生食蔬菜中的风险至关重要。生菜和切后的黄瓜作为单增李斯特菌高风险食品,应引起风险评估的重视。     

Functional properties of novel protective lactic acid bacteria and application in raw chicken meat against Listeria monocytogenes and Salmonella enteritidis

In this study 635 lactic acid bacteria of food origin were evaluated for their potential application as protective cultures in foods. A stepwise selection method was used to obtain the most appropriate strains for application as protective cultures in chicken meat. Specifically, all strains were examined for antimicrobial activity against various Gram positive and Gram negative pathogenic and spoilage bacteria. Strains exhibiting anti-bacterial activity were subsequently examined for survival in simulated food processing and gastrointestinal tract conditions, such as high temperatures, low pH, starvation and the presence of NaCl and bile salts. Selected strains where then examined for basic safety properties such as antibiotic resistance and haemolytic potential, while their antimicrobial activity was further investigated by PCR screening for possession of known bacteriocin genes. Two chosen strains were then applied on raw chicken meat to evaluate their protective ability against two common food pathogens, Listeria monocytogenes and Salmonella enteritidis, but also to identify potential spoilage effects by the application of the protective cultures on the food matrix. Antimicrobial activity in vitro was evident against Gram positive indicators, mainly Listeria and Brochothrix spp., while no antibacterial activity was obtained against any of the Gram negative bacteria tested. The antimicrobial activity was of a proteinaceous nature while strains with anti-listerial activity were found to possess one or more bacteriocin genes, mainly enterocins. Strains generally exhibited sensitivity to pH 2.0, but good survival at 45 °C, in the presence of bile salts and NaCl as well as during starvation, while variable survival rates were obtained at 55 °C. None of the strains was found to be haemolytic while variable antibiotic resistance profiles were obtained. Finally, when the selected strains Enterococcus faecium PCD71 and Lactobacillus fermentum ACA-DC179 were applied as protective cultures in chicken meat against L. monocytogenes and S. enteritidis respectively, a significantly reduced growth of these pathogenic bacteria was observed. In addition, these two strains did not appear to have any detrimental effect on biochemical parameters related to spoilage of the chicken meat.     

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.     

胡椒单萜类化合物对单增李斯特菌抑菌机理的研究

本文研究了胡椒单萜类化合物对单增李斯特菌(L.monocytogenes)的抑菌机制,通过分析单增李斯特菌差异蛋白、呼吸链复合体以及三磷酸腺苷酶(ATP酶)等指标,根据同源建模与分子对接技术探索其作用靶点。添加胡椒单萜类化合物可显著抑制单增李斯特菌的生长(p<0.05),同时Na⁺-K⁺-ATPase、Ca²⁺-ATPase、呼吸链复合体I~V活力均显著低于对照组(p<0.05),其中复合体V在蛋白水平显著下调。胡椒单萜类化合物可使单增李斯特菌细胞膜通透性发生变化,抑制ATPase和呼吸链复合体活性,使得ATP合成减少,从而导致菌体衰亡。这为胡椒单萜类化合物应用于食品保鲜提供理论基础。     

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。     

乳酸钠对单增李斯特菌生物被膜形成的抑制作用

为初步研究乳酸钠对单增李斯特菌生物被膜形成的抑制作用,作者首先采用结晶紫染色法检测不同质量浓度乳酸钠(0、2.5、5、10、20 g/dL)对单增李斯特菌生物被膜形成的抑制效果,其次探究乳酸钠对其生物被膜结构、胞外多糖和胞外蛋白、膜内细菌细胞活性及细胞膜完整性的影响,并采用荧光实时定量PCR(quantitative real-time PCR,qRT-PCR)检测其生物被膜相关基因motB、mogR、degU、flgE、dnaK、prfA及sigB的表达水平。结果表明,随着乳酸钠浓度的增高(2.5%～20%),其对单增李斯特菌生物被膜形成的抑制效果显著增强(p0.05),抑制率分别为8.34%、32.2%、46.6%、55.2%。显微镜观察结果显示,经5 g/dL乳酸钠处理后,单增李斯特菌生物被膜结构明显变稀疏,厚度减少;同时胞外多糖和胞外蛋白质的形成量显著降低(p0.05)。此外,该质量浓度下的乳酸钠可抑制膜内细菌细胞活性及破坏细胞膜的完整性,从而抑制新生物被膜的形成;实时定量PCR结果进一步显示,与单增李斯特菌生物被膜形成相关基因的表达显著下调(p0.05)。本研究为乳酸钠可有效抑制单增李斯特菌生物被膜的形成提供科学依据。     

食品中单核细胞增生李斯特氏菌两种定量检测方法的比较

研究比较不同杂菌污染条件下平板计数(Plate counting)法和稀释培养计数(Most probable number counting)法检测食品中单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)含量的准确性,并探讨冷藏和冷冻食品,MPN保存过程中LM的数量增长情况。采用国标法中的平板计数法和MPN计数法对不同程度人工污染杂菌的牛奶、凉拌菜和盐水鸭分别进行LM含量的检测,并用MPN计数法对冷藏(2~8℃)和冷冻(-20℃)条件下保存的牛奶、凉拌菜和盐水鸭进行LM含量的检测。结果表明,杂菌染菌浓度较低时(LM含量与杂菌含量比为10:1), LM检出限较高(≥ 100 CFU/g (mL)),平板计数法检测LM含量的准确性较高,而杂菌染菌的初始浓度较高时(LM含量与杂菌含量比为1:10),LM检出限较低(< 100 CFU/g (mL)),MPN计数法检测LM含量的准确性较高;牛奶、凉拌菜和盐水鸭在经过不同冷藏和冷冻保存时间后LM数量对比差异有统计学意义(p<0.05),且食品中LM数量随着冷藏和冷冻保存时间的增加而增多。     

Effect of carbon dioxide pressure on Listeria monocytogenes in physiological saline and foods

The effect of 15,30 and 60 atm CO₂pressure at 25,35 and 45°C on Listeria monocytogenes was studied. A biphasic curve was observed in the CO₂treatments. A primary curve was characterized with very little killing and a secondary curve was showed straight-line inactivation kinetics. An increase of pressure and/or temperature enhanced the antimicrobial effects of CO₂. Listeria monocytogenes suspended in physiological saline containing 1% brain–heart infusion broth was completely inactivated under 60 atm CO₂treatment in 115,75 and 60 min at 25,35 and 45°C, respectively. A minimum D -value was obtained under 60 atm CO₂pressure at 45°C. High pressure CO₂treatment technique can possibly be applied to reduce microbiol load in whole and skim milk; and carrot, orange, peach, and apple juices.     

Effect of lactic acid on Listeria monocytogenes and Edwardsiella tarda attached to catfish skin

This study evaluated the use of lactic acid to decontaminate Listeria monocytogenes andEdwardsiella tarda attached to catfish skin with or without mucus. At the highest inoculum levels (10⁴–10⁵cfu skin⁻¹), lactic acid (0·5–2·0%) exposure for 10 min reduced counts of L. monocytogenes firmly attached to catfish skin by 0·9–>1·9 log₁₀cfu skin⁻¹and cells loosely attached by 2·7–>3·7 logs. Counts of E. tarda firmly attached to catfish skin were reduced by 0·9–>3·0 logs and cells loosely attached by 1·5–>3·5 logs. Overall bacterial numbers of lactic acid-treated cells that were firmly attached to skin with mucus were higher than on skin without mucus. Firmly attached L. monocytogenes was more resistant to lactic acid than was firmly attached E. tarda. Catfish skin mucus decreased the antimicrobial effect of lactic acid against attached L. monocytogenes and E. tarda.     

可视化跨越式滚环扩增技术检测食品中单核细胞增生李斯特氏菌

本研究以现代加工工艺条件下生产制作的金华火腿为研究对象,评估了因单增李斯特菌而引起食物中毒的风险。通过调查生猪肉中单增李斯特菌的初始污染率及污染水平,金华火腿生产及销售过程中影响单增李斯特菌生长的参数,如pH、水分活度、温度以及乳酸菌含量等,再结合单增李斯特菌生长模型,模拟其暴露水平,并评估了不同人群因食用即食金华火腿切片而患李斯特菌病的风险。结果显示：金华火腿零售时污染水平为−9.47~7.05 lg CFU/g（90%的置信区间）;健康成年人食用即食金华火腿切片的平均患病概率小于10⁻¹⁵,易感人群的平均患病概率小于10⁻¹³。食用即食金华火腿切片而患李斯特菌病的风险较低,金华火腿的现代加工工艺对于单增李斯特菌的控制水平可以与国际接轨。本研究首次定量模拟了金华火腿从生猪肉到腌制发酵至最终产品的全过程中单增李斯特菌的暴露情况,为发酵火腿中食源性致病菌的风险评估提供了参考模型。