基于MALDI-TOF MS技术快速检测产呕吐毒素蜡样芽胞杆菌

该研究探讨了基质辅助激光解吸电离飞行时间质谱仪快速鉴别产呕吐毒素蜡样芽胞杆菌的方法。通过对标准品进行分析,重新对特征峰进行定位并测定其灵敏度,并用正交试验分析不同培养条件下检验结果的差异,用优化后的培养条件对49株野生蜡样芽胞杆菌及3株标准菌株进行特异性检验。研究表明,MALDI-TOF MS可检测到产呕吐毒素蜡样芽胞杆菌中呕吐毒素相应m/z值为1 175的[M+Na]+和m/z值为1 191的[M+K]+加合物特征峰,具有较高的灵敏度（0.01 μg/mL）,经极差分析显示,选用MYP培养基30 ℃培养12 h后的菌落能获得最稳定、响应值高（>104）的检验结果;方法应用验证表明,49株野生菌株中2株含ces基因的蜡样芽胞杆菌均检出,其余未含有ces基因的菌株均未检出。该研究建立的MALDI-TOF MS检测方法能直接快速准确检出产呕吐毒素蜡样芽胞杆菌,该方法检测特异性强（100%）,灵敏度高（0.01 μg/mL）,对于食品安全事故快速精准分析研判有重要的意义。     

云南边贸进口即食食品中蜡样芽胞杆菌污染状况及其呕吐毒素基因型检测

为探究云南省边贸进口即食食品中蜡样芽胞杆菌的污染状况及其呕吐毒素基因型携带情况,从云南边境各口岸进口食品贸易集散点(边民互市点、农贸市场以及进出口产品店等地)共采集市售包装样品224份。通过VITEK 2 compact全自动微生物鉴定系统和双重实时荧光聚合酶链式反应(Double real-time fluorescent polymerase chain reaction,Dual real-time PCR)技术对分离得到的蜡样芽胞杆菌进行鉴定,并对阳性菌株中的呕吐毒素基因进行检测。结果表明:蜡样芽胞杆菌在所采集样品中的检出率为20.09%(45/224),天保、金水河、瑞丽以及畹町各口岸检出率均高于10%。检出率较高的食品类别为:酱腌菜60%(3/5)、调味品50%(15/30)、水产及其制品46.67%(7/15);从45份阳性样品中共分离出113株蜡样芽胞杆菌,其中呕吐型毒素基因cesB的携带率为0.88%(1/113),呕吐型毒素基因携带率较低,但是蜡样芽胞杆菌的污染风险仍不可忽视。该研究结果对边贸进口食品安全、食源性疾病的监测及预防提供了理论依据。     

基于环介导等温扩增技术的产呕吐毒素 蜡样芽胞杆菌特异性检测

本研究旨在建立食品中产呕吐毒素蜡样芽胞杆菌的快速检测方法.基于产呕吐毒素蜡样芽胞杆菌合成酶基因cesB靶基因,设计4条特异性引物(2条内引物、2条外引物),建立环介导等温扩增技术(LAMP).然后采用2株产呕吐毒素蜡样芽胞杆菌、19株蜡样芽胞杆菌和41株非蜡样芽胞杆菌验证了该LAMP具有很好的特异性.LAMP检测的灵敏...     

2012—2014年江西省市售婴幼儿配方食品中蜡样芽胞杆菌调查及呕吐毒素基因分析

目的了解2012—2014年江西省市售婴幼儿配方食品中蜡样芽胞杆菌的污染情况并对呕吐毒素基因进行分析。方法在全省市县中选取13个采样点,包括超市、百货商场、便利店、农贸市场、网店、批发市场,随机抽取婴幼儿配方食品397份,对蜡样芽胞杆菌进行检测和鉴定,同时应用叠氮溴化丙锭内参多重PCR方法检测分离株的呕吐毒素基因。结果 397份食品中蜡样芽胞杆菌阳性率为13.10%(52/397),其中2013年阳性率最高。不同采样点的阳性率差异有统计学意义(P0.05),不同产地、不同流通环节和不同年龄段的阳性率差异无统计学意义(P0.05),52份阳性食品中检出呕吐型蜡样芽胞杆菌2份,呕吐毒素阳性率为3.85%。结论江西省市售婴幼儿配方食品中存在蜡样芽胞杆菌及呕吐型蜡样芽胞杆菌的污染,存在一定的安全隐患,相关监管部门应继续加强监管,预防食源性疾病的发生。     

北京市某区2021年5—9月生鲜蔬菜中蜡样芽胞杆菌检出情况和耐药特征分析

目的 对生鲜蔬菜中蜡样芽胞杆菌的分布和耐药特征进行分析。方法 采集北京市顺义区2021年5—9月生鲜蔬菜样本120份,对样本进行蜡样芽胞杆菌平板计数检测、增菌培养及增菌液呕吐毒素基因(ces)、蜡样芽胞杆菌16S r DNA基因实时荧光PCR检测,对分离株进行14种抗生素敏感性检测。结果 生鲜蔬菜中蜡样芽胞杆菌检出率为84.17%(101/120)。根茎类和叶菜类生鲜蔬菜蜡样芽胞杆菌检出率差异有统计学意义(χ²=14.181,P_校正=0.000);超市、农贸市场和农户菜地3类采集地点蜡样芽胞杆菌检出率差异有统计学意义(χ²=11.050,P=0.004),基于样本增菌液的ces检出率差异有统计学意义(P_Fisher=0.001)。12件ces⁺的生鲜蔬菜增菌液检测蜡样芽胞杆菌16S rDNA基因和ces基因Ct值均值分别为19.96和31.80,但均未能分离到ces⁺蜡样芽胞杆菌菌落。蜡样芽胞杆菌分离株对氨苄西林、青霉素、复方磺胺耐药率分别为100%、99...     

米饭中蜡样芽孢杆菌剂量效应模型的构建

目的建立有效的米饭蜡样芽孢杆菌剂量效应模型。方法调研并选用2001-2011年我国9起米饭中蜡样芽孢杆菌导致食物中毒的数据,基于指数模型、泊松模型、对数模型、威布模型等及其修正形式分别构建了米饭中蜡样芽孢杆菌剂量效应模型。结果修正的指数模型、对数概率模型和Gamma威布模型三者在拟合效果上显著优于其他模型(R20.70),拟合曲线外延至低剂量预测中毒率时,Gamma威布模型低于前两者,进一步的数学检验表明,Gamma威布模型准确因子(Af=1.40)优于前两者,偏差因子(Bf)和预测标准误差(SEP)相近或略高于前两者。结论建议选用Gamma威布模型作为米饭中蜡样芽孢杆菌最优的剂量效应模型用于蜡样芽孢杆菌风险评估体系中的危害特征分析。     

婴幼儿奶粉和米粉中蜡样芽胞杆菌及其毒素、毒力基因的调查研究

了解婴幼儿奶粉及米粉中蜡样芽胞杆菌污染状况及其毒素、毒力基因的携带特点。方法 采用稀释培养计数(MPN计数)法分离蜡样芽胞杆菌,采用PCR技术检测10种蜡样芽胞杆菌的腹泻毒素及呕吐毒素基因,在流动相A为0.1%甲酸-乙腈溶液,流动相 B为0.1%甲酸-0.2 mmol/L乙酸铵溶液条件下,用Acquity BEH300 C₁₈色谱柱(100 mm×2.1 mm,1.7 μm)对样品进行分离,采用超高效液相色谱-串联质谱法检测样品中的呕吐毒素(cereulide)。结果 本研究共监测39份样品,28份检出蜡样芽胞杆菌,检出率为71.79%(28/39);2份检出呕吐毒素,检出率为5.13%(2/39)。检出的蜡样芽胞杆菌菌株大多属于携带复合型毒素的菌株,均携带3种以上的腹泻毒素基因,非溶血性的肠毒素 nhe基因(nheA、nheB 和nheC)和肠毒素FM基因(entFM)为主要的毒力基因,其中nheABC 基因携带率为100%,entFM基因携带率为35.71%(10/28),cytK基因是检测到的最少的一种毒力基因。结论 应加强婴幼儿奶粉及米粉中的蜡样芽胞杆菌污染监测及其毒力基因致病性研究,以科学评估蜡样芽胞杆菌对婴幼儿食品可能构成的食品安全风险。     

2011—2019年吉林省食品中蜡样芽胞杆菌污染状况分析

目的 了解2011—2019年吉林省食品中蜡样芽胞杆菌污染情况,为食品安全监管及食源性疾病防治提供理论依据。方法 采集2011—2019年吉林省九个地(市)级的餐饮服务环节和流通环节中的样品3 173份,参照GB 4789.14—2014《食品安全国家标准 食品微生物学检验 蜡样芽胞杆菌检验》中的方法,对食品中的蜡样芽胞杆菌进行检测并分析。结果 2011—2019年吉林省3 173份食品样品中,蜡样芽胞杆菌总检出率为23.6%(750/3 173),2015年检出率最高(38.5%, 62/161),2017年检出率最低(11.8%, 20/170);白山市检出率最高(35.8%, 139/388),其次为延边州(31.4%, 97/309),四平市检出率最低(15.3%, 76/496);蛋与蛋制品检出率最高(60.0%, 3/5),其次为乳与乳制品(39.3%, 114/290)及婴幼儿食品(31.1%, 185/595);百货商场中蜡样芽胞杆菌检出率最高(32.4%, 22/68),其次为小吃店及饮品店(30.9%, 43/139)、快餐店(29.1%, 25/86)。平板计数(CFU)法测得蜡样芽胞杆菌检出结果的中位数(四分位数间距)为5.8(2.9,8.7)CFU/g(mL),稀释培养测数(MPN)法测得结果的中位数(四分位数间距)为6.4(3.2,9.6)MPN/g(mL)。结论 吉林省各地市食品中存在不同程度的蜡样芽胞杆菌污染,以白山市最为严重,蛋与蛋制品、乳与乳制品是主要受污染食品,食品监管部门应加强百货商场、小吃店及饮品店等地点的安全监测与管理。     

石家庄市131株食源性蜡样芽胞杆菌毒力基因的分布

目的研究石家庄市食源性蜡样芽胞杆菌毒力基因的分布及毒力活性,了解蜡样芽胞杆菌的潜在威胁。方法采用PCR方法,对食品风险监测中分离到的131株蜡样芽胞杆菌进行肠毒素、呕吐毒素9种毒力基因扩增检测,用血平板检测的方法分析蜡样芽胞杆菌的毒力。结果毒力基因携带率较高,至少携带一个毒力基因的菌株达到检出菌总数的99.2%(130/131),溶血素BL基因(hbl ACD)和肠毒素FM基因(ent FM)是石家庄市食源性蜡样芽胞杆菌的主要毒力基因;检出的蜡样芽胞杆菌均产生溶血素BL,检出率为100%。结论腹泻型肠毒素在食品中的分布比较广泛,检出的蜡样芽胞杆菌均具有溶血素,对进食者存在潜在的危险性,今后应加强监控蜡样芽胞杆菌的污染,预防和控制蜡样芽胞杆菌食源性疾病的发生。     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

Properties of Bacillus cereus and other bacilli contaminating biomaterial-based industrial processes

This paper is an overview on bacilli in industrial processes, with focus on food grade paper and paperboard production. Paperboards mainly contain sporeforming bacteria belonging to the genera Bacillus, Paenibacillus and Brevibacillus, usually found in quantities from <50 to 250 cfu g⁻¹ homogenized paperboard. Of those frequently found, Bacillus cereus group, B. licheniformis, B. subtilis and Brevibacillus brevis are important for food hygiene because of their hydrolytic activities on food components and the ability of some strains to produce food poisoning toxins or to grow at refrigerated temperatures. We found that the phenotypic properties (lecithinase activity, nitrate reduction) used in standard methods (e.g., ISO, FDA, IDF) to recognize B. cereus, were unreliable for industrial isolates. Whole cell fatty acid composition of a group of the industrial isolates deviated so much from those in a widely used commercial database that the strains were not or only poorly recognized as B. cereus. Industrial isolates, including toxigenic ones, often missed one or more of these characters, even in cases where 100% 16S rDNA identity was found with B. cereus or with B. thuringiensis. 11-Methyldodecanoic acid and trans-9-hexadecenoic acid were found without exception in over 200 industrial B. cereus group isolates and in over 30 culture collection strains. The detection of these fatty acids is a secure method for the identification of B. cereus. Negative reaction for starch hydrolysis and for BCET-RPLA test and a specific ribotype were found in all B. cereus strains producing the emetic toxin.     

一起疑似由蜡样芽胞杆菌引起的食源性疾病事件的分子溯源研究

目的 对一起食源性疾病事件中食品分离蜡样芽胞杆菌（B. cereus）进行溯源分析,为确定污染源、切断传播途径提供技术支撑。方法 本研究建立脉冲场凝胶电泳（PFGE）方法,对12株B. cereus进行分子分型,同时对10株B. cereus进行全基因组测序（WGS）,利用BioNumerics软件对测序数据进行拼接组装、多位点序列分型（MLST）、毒力基因、核苷酸多态性（SNP）分析。结果 本起食源性疾病事件中分离自不同食品的12株B. cereus的PFGE分型显示为8种带型,其中3株ST1435型 B. cereus带型相同,且SNP分析显示这3株B. cereus只有3个碱基差异;2株ST24型B. cereus 的PFGE带型完全相同,且SNP分析显示只有1个碱基差异,提示3株ST1435和2株ST24菌株分别为克隆株。2株ST24型B. cereus携带溶血性肠毒素hlbA、hlbC和hlbD,另有4株B. cereus携带腹泻毒力基因（hlbA、hlbC和hlbD）。结论 B. cereus引起的食源性疾病事件比较复杂,污染源也比较复杂,因此加强原辅料监测、从业人员的卫生监测、环境和设备及环节的清洗消毒,对预防控制由其引起的食源性疾病事件非常重要。     

1株食源性蜡样芽胞杆菌分离株的分子特征毒性及耐药性研究

目的 研究株蜡样芽胞杆菌SCY分离株的毒力和耐药性。方法 使用选择性培养基从银川市某餐厅鱼肉菜肴中培养分离获得蜡样芽胞杆菌SCY分离株,经23S rRNA基因PCR测序鉴定后,通过二代全基因组测序技术分析SCY分离株的基因组特征,进而分别采用免疫组化技术和药敏纸片实验研究SCY分离株的毒性和耐药性。结果 SCY分离株的基因组大小为5.82 Mb,编码基因个数为5 767,编码区总长度占全基因组的85.50%。SCY分离株基因组中共含有11个基因岛、16个CRISPR和5个前噬菌体;其中,SCY编码基因分别在PHI和VFDB毒力因子数据库中注释到了14个和62个毒力基因（Identiy>80,Evalue<0.05）,在CARD耐药数据库中注释到了215个耐药基因（Best Hit evalue<0.05）。小鼠肠道组织切片免疫组化结果表明,炎性因子IL-1β、CASP1和Nlrp3的表达水平发生差异显著性上调。22种细菌抗生素药敏纸片试验结果显示,SCY分离株对氯霉素、克林霉素等高度敏感,对四环素、头孢唑啉、头孢哌酮、氨苄青霉素表现为中介,对青霉素、苯唑西林、哌拉西林、杆菌肽、头孢他啶等10种抗生素表现为耐药,SCY分离株多重耐药情况较为严重。结论 本研究发现的SCY分离株属于肠毒株,携带多种肠毒素毒力基因,且具有典型的多重耐药特征。研究结果为揭示蜡样芽胞杆菌分离株的致病机制提供参考依据。     

Changes in membrane fatty acids and murein composition of Bacillus cereus and Salmonella Typhi induced by gamma irradiation treatment

This present study was carried out to evaluate the effects of gamma irradiation on the fatty acids (FA) and mureins composition of two different radiotolerant bacteria. Bacillus cereus LSPQ 2872 and Salmonella Typhi ATCC 19430 were used for this study. The bacterial strains were treated with a sublethal radiation dose of 1 kGy to cause a cellular damage. Gas chromatography (GC) and high performance liquid chromatography (HPLC) analysis were performed to demonstrate respectively the modification of the FA composition and the changes in muropeptide profile. Results obtained show, for both bacteria, that this treatment had a significant effect (P ≤ 0.05) on the FA content with an increase of unsaturated FA percentage. Substantial changes were also noticed for the relative percentage and the number of the muropeptides. This study represents one of the few to demonstrate the modifications on bacterial membrane as a cellular response to survive the ionising radiation stress.     

Production of high degree polymerized chitooligosaccharides in a membrane reactor using purified chitosanase from Bacillus cereus

Crude chitosanase from Bacillus cereus NTU-FC-4 was separated by a cation exchanger to three fractions named CBCI, CBCII, and CBCIII. The CBCI hydrolyzed chitosan to yield dimers. The primary hydrolytic products of CBCII were low degree polymerized (DP) chitooligosaccharides. The CBCIII had the fastest reaction rate and yielded high DP chitooligosaccharides (heptamer and higher DP oligomers). When CBCIII was used in the ultrafiltration membrane reactor with enzyme/substrate ratio 0.06 unit/mg and 100 min of residence time (RT), the concentration of high DP oligomers was 9.78 mg/mL which occupied ca. 48% of total oligomers in the final product as compared to ca. 29% resulted from the crude enzyme. Decrease of RT to 50 min and 33 min, the high DP oligomers in the products were ca. 61% and 69%, respectively. This system could be operated for at least 24 h and kept a constant permeate flux and product output rate.     

2014—2019年岳阳市食源性蜡样芽胞杆菌溶血素hblA与磷脂酶C plc基因特征分析

目的 了解湖南省岳阳市2014－2019年食源性蜡样芽胞杆菌菌株病原学特征,并为蜡样芽胞杆菌引起的食物中毒事件的科学防控提供依据。方法 对2014—2019年分离自湖南省岳阳市城区餐饮门店的26株蜡样芽胞杆菌菌株的致病毒力因子溶血素BL的hblA基因和磷脂酶C的plc基因进行序列扩增和测序,并使用Seqman和MEGA X软件对蜡样芽胞杆菌的hblA和plc基因进行遗传进化分析。结果 从岳阳市分离到的蜡样芽胞杆菌hblA、plc毒力基因的同源性与GenBank中的蜡样芽胞杆菌群相比均大于93.0%。结论 岳阳市分离的蜡样芽胞杆菌的hblA和plc基因与GenBank中的蜡样芽胞杆菌群的同源性高,具有一定的亲缘关系。本研究为进一步了解和科学防控蜡样芽胞杆菌引起的食物中毒事件奠定了研究基础。     

蜡样芽孢杆菌高密度发酵条件与过程的优化

蜡样芽孢杆菌的生物量对其生物功能具有重要影响。为了提高蜡样芽孢杆菌的产量与芽孢率,通过单因素实验筛选出最适宜其生长的碳源与氮源,分别为质量比1∶1复配的葡萄糖与水溶性淀粉和质量比1∶1复配的大豆蛋白胨与酵母提取物。在此基础上,于7 L发酵罐中探究流加方式、p H、搅拌转速和通气量等因素对其生物量及芽孢率的影响。最优发酵条件为：在0～9 h之间,转速250 r/min,通气量3 L/min,pH恒定6.5;9～18 h时,转速升高至350 r/min,通气量升高至4.5 L/min,并以0.45 mL/min流量补加培养基浓缩液;18 h时,转速降低至150 r/min,通气量降低至2.25 L/min,pH调高至7.5,停止补料至发酵结束。基于以上发酵策略,在18 h时,蜡样芽孢杆菌活菌数达2.2×10¹⁰CFU/mL,是未优化前的4.88倍;发酵结束时,芽孢率超过90%。本研究结果为蜡样芽孢杆菌的工业化应用提供了一定基础。     

Production of diarrheal toxin by selected strains of Bacillus cereus

The production of diarrheal toxin by six selected strains of Bacillus cereus was monitored during growth at 32 °C, a temperature described as near-optimal for growth and toxin production. Toxic activity was measured in culture filtrates and cellular extracts sampled at three different times during growth. Two alternative methods, a cytotoxicity test on Chinese hamster ovary (CHO) cells and a commercial immunological test (BCET-RPLA, Oxoid) were used. Toxin titres were in agreement with epidemiological characteristics and toxicity demonstrated by using other systems in other examinations. A comparison of intra- and extracellular toxicities measured at the exponential and stationary growth phases showed that the toxin was essentially secreted during the exponential phase. For several strains, secretion peaked during the period from the middle exponential phase to the beginning of the stationary phase. There was no important overall increase of the toxicity during full and late stationary phase. The level was stable or even lower, thus indicating that diarrheal toxin production during stationary phase was small, if any, and that the toxin was unstable under these conditions. Statistical analysis of toxicities showed that the cytotoxicity test was correlated with the immunological test (significant at a 1% level). For routine determinations, a toxicologic laboratory may use any of the two methods, depending oft its facilities, the immunological test being relatively expensive.