Evidence of non-coccoid viable but non-culturableCampylobacter jejunicells in microcosm water by direct viable count,CTC-DAPI double staining,and scanning electron microscopy

Production of viable but non-culturable (VNC)Campylobacter jejunicells was studied using 36 strains resuspended in microcosm water. Cells in stationary phase were resuspended at 4°C, with gentle stirring at 100 r min⁻¹, in filtered surface water adjusted to pH 6.050. The culturability of starved cell suspensions was determined by spread plate counts, and the activity of cells by the direct viable count technique and CTC–DAPI double staining. Although mostC. jejunistrains died within a few days of suspension in microcosm water, three strains (Bf, 79 and 85) demonstrated a complete decrease of culturability within 15 days of suspension. A constant activity level was found in these three strains, even after 30 days of starvation in microcosm water; and an almost constant concomitant level of adenosine triphosphate was measured in VNC cell suspensions. Scanning electron microscopy ofC. jejuniVNC cells showed that the spiral-shaped cells of strain 85 cultures became coccoid in ageing microcosm-water cell suspensions, whereas most VNC cells of strains Bf and 79 remained spiral-shaped after 30 days of starvation.     

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。     

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。     

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。     

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。     

Differential fluorescent staining of Listeria monocytogenes and a whey food soil for quantitative analysis of surface hygiene

The accurate monitoring of surface cleanliness in terms of bacterial contamination is usually carried out using methods such as plate counts or replica plating. However these methods take at least eighteen hours to obtain results and do not determine the presence or amount of residual organic material on a surface, which may interfere with cleaning and disinfection. This work describes the application of fluorescent stains to cells (Listeria monocytogenes) and food soil (solubilized whey) to optimize a dual staining method that can be used in the quantitative analysis of surface cleanability. Seven different stains were tested at a range of concentrations (0.3%–0.001 mg/ml) and application methods. The best stain combination for differential staining of L. monocytogenes and whey food soil was 0.1 mg/ml rhodamine B with 0.1 g/ml DAPI. Differential staining of the cells and soil occurred regardless of the application method. This method has been successfully used to demonstrate the hygienic status of surfaces in an industrial situation. This novel work enables quantitative assessment of soils and cells on surfaces.     

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。     

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。     

Inhibitory effect of clove oil on Listeria monocytogenes in meat and cheese

The antilisteric activity of clove oil was examined in meat and cheese at both 30°C and 7°C. At concentrations of 0·5% and 1% clove oil restricted the growth of Listeria monocytogenes in the food items at both temperatures. The inhibitory activity of clove oil was more pronounced at a concentration of 1%.Listeria counts in treated samples were 1–3 log₁₀cfu g⁻¹less compared to controls at different intervals during storage. The results revealed the potential of clove oil as a natural preservative in meat and cheese.     

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。     

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.     

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。     

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.     

食品中单核细胞增生李斯特菌微滴数字PCR定量检测方法建立

目的 建立食品中单核细胞增生李斯特氏菌的微滴式数字聚合酶链式反应（ddPCR）快速定量检测方法。方法 筛选单核细胞增生李斯特氏菌的特异性引物和探针,通过对目标菌纯菌液及人工污染样品的检测,比较ddPCR方法和平板计数法的定值效果,对ddPCR结果进行特异性、灵敏性和重复性分析。结果 本研究建立的单核细胞增生李斯特氏菌ddPCR检测方法具有良好的特异性、灵敏性和重复性。单核细胞增生李斯特氏菌纯菌液中定量限（LOQ）和检出限（LOD）均为136 CFU/mL,在鱿鱼圈和香肠样品中定量限分别为240 CFU/g和155 CFU/g。ddPCR在各梯度水平上变异系数均小于25%,ddPCR和平板计数定值对数值相对偏差均小于30%。结论 本研究建立的ddPCR方法能够快速、准确、灵敏、特异地定量检测食品中单核细胞增生李斯特氏菌。     

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.     

广西单核细胞增生李斯特菌的流行率、毒力特征和分子分型研究

目的 比较广西不同种类食品中单核细胞增生李斯特菌（以下简称单增李斯特菌）的流行情况、毒力特征和分子分型特征,更好地了解单增李斯特菌的遗传特点及其传播的潜力。方法 对来自2002—2020年的210株单增李斯特菌进行全基因组测序（WGS）,分析其序列分型（ST）、克隆群（CC）型及毒力基因。结果 2002—2020年广西53.8%单增李斯特菌分离株来源于肉与肉制品,59.0%分离株来自于谱系Ⅱ,优势ST型为ST8和ST9。79.4%菌株携带LIPI-1基因（hly、prfA、plcA、plcB、mpl、actA）,83.7%菌株携带LIPI-2基因（inlC、inlF、inlJ、inlK）。17.6%菌株携带LIPI-3基因（llsA、llsB、llsD、llsG、llsH、llsP、llsX、llsY）。结论 肉与肉制品是广西单增李斯特菌检出率最高的食品类型,分子分型结果证实了单增李斯特菌种群具有高度的遗传多样性,WGS的高分辨力可用于监测食源性单增李斯特菌病的暴发。毒力基因的分布表明广西单增李斯特菌存在不同程度毒力基因的缺失,应警惕高致病性的菌株引起的食源性暴发事件。