26例蜡样芽胞杆菌败血症临床分析

本文报道２６例经血培养证实的蜡样芽胞杆菌败血症。婴儿占５０％,院内感染发生率８８．５％,全部病例均继发于基础病上,８８．５％病例发病前均使用过１种或２种以上抗生素,菌株对多种常用抗生素耐药。提示该菌为临床感染不可忽视的菌。     

血液脑脊液中检出蜡样芽胞杆菌

蜡样芽胞杆菌又称为坚实芽胞杆菌(Bacillus cereus),在实际工作中每年都能在血液、脑脊液、胸腹水、痰液等标本中检测到多例蜡样芽胞杆菌,而且纯度很高。但由于受教科书的影响,只有在食物中毒患者中检测到蜡样芽胞杆菌有临床意义,因而在临床标本中检测到都把它作为污染菌而漏检。从今年4月份以来,在血液和脑脊液中共检出4例蜡样芽胞杆菌,现将结果报道如下。病例一,男,80岁,职业,干部,2005年4月22日因脑梗死、右下肢淋巴管炎入住心血管内科,白细胞5.78×109/L,中性粒细胞0.74、生化全套、尿常规正常,血培养检出蜡样芽胞杆菌,用左旋氧氟沙星…     

致病性大肠杆菌和蜡样芽胞杆菌的分离鉴定

【背景】猪只消化道疾病是养猪业上一大重要疾病,给养猪业带来一定的经济损失。大肠杆菌是引起猪腹泻的一种常见病原菌,可以引起不同日龄的猪腹泻,但主要以幼龄猪为主。【目的】旨在分离鉴定引起四川省眉山市一规模化养猪场病猪大规模腹泻的病原菌。【方法】采用常规细菌分离方法结合16S rRNA基因序列的分析方法从发病猪肝脏、胃以及污染的饲料分离鉴定细菌,并对分离株进行小鼠致病性试验、16S rRNA基因遗传进化树分析、毒力基因的检测、药物敏感试验。【结果】从腹泻猪肝脏中分离到一株致病性大肠杆菌,胃中分离到一株蜡样芽胞杆菌,并且追溯到传染源是该猪场饲料。通过检测这两株菌相应的毒力基因发现大肠杆菌不属于肠外致病性型,蜡样芽胞杆菌检测到了nheA、nheB、nheC、bceT、entFM 5种毒力基因;药敏试验表明常规的氨基糖苷类和头孢类抗生素对大肠杆菌抑菌效果较好,红霉素、氟苯尼考、头孢氨苄、头孢哌酮对蜡样芽胞杆菌抑菌效果较好,而蜡样芽胞杆菌对青霉素、阿莫西林等常规药物不敏感。【结论】饲料存在大肠杆菌和蜡样芽胞杆菌的混合污染。     

蜡样芽胞杆菌制剂临床用药调查

为临床合理使用蜡样芽胞杆菌制剂提供参考。方法：收集我院2002年1～12月门诊西药房蜡样芽胞杆菌制剂的处方,对其临床合并用药、与抗菌药的合并使用、与其他胃肠道药物合并使用等三方面情况进行统计,并参考有关文献对其临床用药的合理性进行判断分析。结果：我院临床蜡样芽胞杆菌制剂的使用存在不少不合理的情况。结论：蜡样芽胞杆菌制剂的不合理使用应该引起临床医生的注意。     

蜡样芽胞杆菌B905 hblA基因的初步分析

采用血平板培养的方法对蜡样芽胞杆菌905菌株溶血素BL检测,并通过PCR方法克隆其基因,结果表明该菌株产生溶血环且含有hblA、hblC、hblD溶血素BL全部基因;采用同源重组法构建了该菌株hblA基因缺失突变体,结果该菌株的溶血活性并未发生改变,可能是由于该菌株溶血素基因的结构与Handelsman构建所用的菌株Bacillus cereus UW85有一定的差异,或者是由于突变位点在阅读框内后端,未能真正破坏其表达。还需要进一步对其进行研究。     

2016年辽宁省婴幼儿配方食品及谷类辅助食品中蜡样芽胞杆菌毒力基因的鉴定

目的 了解婴幼儿配方食品和谷类辅助食品中蜡样芽胞杆菌的毒力基因携带特点,对辽宁省婴幼儿配方食品和谷类辅助食品中蜡样芽胞杆菌的污染状况进行调查。方法 依据GB4789.14‒2014《食品安全国家标准 食品微生物学检验 蜡样芽胞杆菌检验》及采用PCR扩增技术和血平板检测的方法对2016年采自辽宁省15个监测点,收集的176份乳源性食品中检出的22株蜡样芽胞杆菌进行10种毒力基因检测。结果 婴幼儿配方食品和谷类辅助食品蜡样芽胞杆菌检出率为12.5%（22/176）,非溶血性的肠毒素Nhe基因、溶血素BL基因、肠毒素T基因和细胞毒素K基因是辽宁省乳源性蜡样芽胞杆菌的主要毒力基因,至少携带2种毒力基因的菌株达到检出菌总数的100.0%。结论 研究结果证实辽宁省婴幼儿配方食品及谷类辅助食品存在蜡样芽胞杆菌污染情况,严格监控婴幼儿配方食品及谷类辅助食品的蜡样芽胞杆菌污染,对于生产出优质婴幼儿配方食品及谷类辅助食品具有重要意义,以期提高婴幼儿食品的质量安全。     

苏云金芽胞杆菌肠毒素基因的PCR检测

采用多重引物PCR进行了 45株苏云金芽胞杆菌、2株蜡状芽胞杆菌和 2株球形芽胞杆菌溶血素BL ,肠毒素T和entS基因的检测 ,结果表明 95 6%苏云金芽胞杆菌含溶血素hblA基因 ,91 1 %含bceT基因 ,93 3%含entS基因。用两种商业化肠毒素检测试剂盒TECRA和RPLA进行所有菌株肠毒素的体外免疫测定 ,大部分苏云金芽胞杆菌和阳性蜡状芽胞杆菌都能产生不同水平的肠毒素活性 ,同hblA基因PCR检测结果基本相符。尽管DBT0 0 7和T2 4 0 0 1含有hblA基因 ,但用TECRA却检测不到肠毒素 ;Dmu39菌株不含肠毒素基因 ,但用TECRA却检测出高的肠毒素活性。苏云金芽胞杆菌BDT2 4 8和球性芽孢杆菌不含肠毒素基因和肠毒素。结果表明昆虫病原菌苏云金芽胞杆菌的安全性有待进一步研究     

蜡样芽胞杆菌对羔羊,仔猪和鸡腹泻控制的流行病学分析

本文报道了自中国土壤中分离出的一株蜡样芽胞杆菌(DM423)活菌制剂对羔羊、仔猪和鸡腹泻控制效果的流行病学分析,结果表明,蜡样芽胞杆菌的实验组的腹泻控制效果明显好于抗生素治疗组及未处置组,统计学处理具有显著意义。因此我们相信,利用蜡样芽胞杆菌制剂是一条控制人和动物腹泻的新途径。     

辽宁省2012‒2015年食源性蜡样芽胞杆菌污染检测结果分析

目的 调查辽宁省食品中蜡样芽胞杆菌污染状况,为国家标准的制订与修订提供数据。方法 在辽宁省各市建立监测点,定期对6类食品进行采样,参照历年国家食品安全风险监测工作手册蜡样芽胞杆菌检测标准操作程序对样品进行检测。结果 检测6类食品共2021份,检出蜡样芽胞杆菌183株,检出率达到9.02%。结论 市售婴幼儿食品与熟制米面制品中有一定程度的蜡样芽胞杆菌污染,希望引起政府相关部门的重视,亟待制定相关限量标准,改进检测方法,加大监管力度。     

中国食品中蜡样芽胞杆菌污染状况荟萃分析

蜡样芽胞杆菌（Bacillus cereus）是一种兼性厌氧的杆状革兰氏阳性菌,广泛分布于自然界中,对环境具有很强的适应性。文章通过对Web of Science数据库及中国知网中国期刊全文数据库的检索,根据文献纳入和排除标准,系统筛选出2014—2023年间发表的有关我国食品蜡样芽胞杆菌污染情况的文献,并展开荟萃分析。经筛查,文章共纳入26篇研究文献,涵盖2014—2022年间全国13个省级地区（省、自治区和直辖市）共18 499份样品。部分菌株测序发现,腹泻型毒素基因的整体检出率高于呕吐型毒素基因。蜡样芽胞杆菌检出率荟萃分析结果显示,在7种明确的食品类别中,乳及乳制品最高（25.50%,95%CI:18.61%～33.89%）;其次分别为生鲜产品（17.64%,95%CI:2.46%～64.48%）、豆制品（16.43%,95%CI:12.56%～21.21%）、餐饮及混合食品（14.67%,95%CI:9.05%～22.90%）、粮谷制品（14.26%,95%CI:9.29%～21.28%）、婴幼儿及儿童食品（12.10%,95%CI:8.72%～16.54%）;肉及肉制品（7.48%,95%CI:1.92%～24.99%）的检出率较低;食品总体合并检出率为14.92%(95%CI:12.24%～18.08%)。可见,利用荟萃分析方法能明确不同食品类别、食品采样环节、食品地点和蜡样芽胞杆菌污染情况的关联特征,为我国食品中蜡样芽胞杆菌污染的风险研判和管理提供参考。     

A new rapid and sensitive detection method for cereulide-producing Bacillus cereus using a cycleave real-time PCR

Aims:  To develop a rapid and sensitive detection method for cereulide-producing Bacillus cereus using a real-time PCR based on the sequence of the cereulide synthesis gene.
Methods and Results:  A total of 56 cereulide-producing B. cereus and 15 cereulide-negative strains were tested. We designed specific primers and probes for the detection of cereulide-producing B. cereus . The new cycleave real-time PCR assay gave positive detections for all of 56 cereulide-producing B. cereus strains, whereas all other strains including 10 systemic infectious disease strains were negative. No cross-reaction was observed and the internal control showed positive for all samples.
Conclusions:  The performance of the assay was highly reproducible and specific for cereulide-producing B. cereus . The positive detection was obtained within only 2 h for cereulide-producing strains. The detection limit of this assay was evaluated as 10⁴ CFU g⁻¹ food sample. The assay also confirmed that strains from systemic infectious cases were cereulide-negative.
Significance and Impact of the Study:  This assay is applicable for contaminated foods as well as specimens from infectious disease cases. We recommend this assay for routine examination of suspected B. cereus food poisonings.     

Development of a PCR assay for identification of the Bacillus cereus group species

Aims:  A PCR technique was developed as a reliable and rapid identification method for the Bacillus cereus group species, based on a unique conserved sequence of the motB gene (encoding flagellar motor protein) from B. cereus , Bacillus thuringiensis and Bacillus anthracis .
Methods and Results:  Primer locations were identified against eight strains of the B. cereus group spp. from nucleotide sequences available in the National Centre for Biotechnology Information database. The PCR assay was applied for the identification of 117 strains of the B. cereus group spp. and 19 strains from other microbial species, with special emphasis on foodborne pathogens.
Conclusion:  The designed cross-species primers are group specific and did not react with DNA from other Bacillus and non- Bacillus species either motile or not. The primers system enabled us to detect 10³ CFU of B. cereus cells per millilitre of sample.
Significance and Impact of the Study:  Bacillus cereus group spp. belongs to one of the most prevalent foodborne pathogens. Bacterial growth results in production of different toxins; therefore, consumption of food containing >10⁶ bacteria per gram may result in emetic and diarrhoeal syndromes. A rapid and sensitive bacterial detection method is significant for food safety.     

利用PCR技术检测致病性腊样芽孢杆菌的研究

目的：利用PCR技术对致病性蜡样芽孢杆菌（Bacillus cereus）进行检测。方法：对致病性蜡样芽孢杆菌溶血素HBLa基因序列进行分析设计一对特异引物,通过优化PCR反应条件,来实现对致病蜡样芽孢杆菌的快速检测,结果：该方法具有较强的灵敏性及特异性,能够对肠毒素型腊样芽孢杆菌进行有效的检测,其最低检出限可达9CFU／ml,用PCR技术对食物样品中致病性蜡样芽孢杆菌的检测取得与普通生化检测方法一致的结果。结论：利用PCR技术对食品中蜡样芽孢杆菌的检测较常规的生化检测方法具有省时省力的特点且灵敏性较高,具有较强的实际应用价值。     

Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.     

Rapid discrimination of cytK-1 and cytK-2 genes in Bacillus cereus strains by a novel duplex PCR system

Bacillus cereus is the causative agent of gastrointestinal diarrhoea. At least three known enterotoxins may be involved in this syndrome: nonhaemolytic (Nhe) enterotoxin, Hbl enterotoxin and cytotoxin K. Two different forms were recently described for cytotoxin K, encoded by cytK-1 and cytK-2 genes. The CytK-1 toxin appeared to carry a high toxicity, but there is currently no method available to rapidly detect and discriminate the B. cereus strains able to produce this CytK-1 form. In this study, a duplex PCR assay was developed and validated on 162 known cytotoxin-containing strains. This PCR method is the first molecular tool to provide rapid detection and discrimination of cytK-1- and cytK-2-carrying B. cereus strains.     

Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.     

Glycosylation of flavonoids with a glycosyltransferase from Bacillus cereus

Microbial glycosyltransferases can convert many small lipophilic compounds such as phenolics, terpenoids, cyanohydrins and alkaloids into glycons using uridine-diphosphate-activated sugars. The main chemical functions of glycosylation processes are stabilization, detoxification and solubilization of the substrates. The gene encoding the UDP-glycosyltransferase from Bacillus cereus, BcGT-1, was cloned by PCR and sequenced. BcGT-1 was expressed in Escherichia coli BL21 (DE3) with a his-tag and purified using a His-tag affinity column. BcGT-1 could use apigenin, genistein, kaempferol, luteolin, naringenin and quercetin as substrates and gave two reaction products. The enzyme preferentially glycosylated at the 3-hydroxyl group, but it could transfer a glucose group onto the 7-hydroxyl group when the 3-hydroxyl group was not available. The reaction products made by biotransformation of flavonoids with E. coli expressing BcGT-1 are similar to those produced with the purified recombinant enzyme. Thus, this work provides a method that might be useful for the biosynthesis of flavonoid glucosides and for the glycosylation of related compounds.     

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group

Aims:  To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores.
Methods and Results:  Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis , e.g. lack of β -haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis . PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis , but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an Alu I restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different.
Conclusions:  Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples.
Significance and Impact of the Study:  This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.     

Studies on the ATPase of Bacillus cereus.

The membrane ATPase (EC 3.6.1.3) of Bacillus cereus was solubilized by a 'shock-wash' process and purified. The non-specific phosphatase contaminant was separated by glycerol density gradient centrifugation. The optimum temperature was 39.5 degrees C and the pH optimum at 7.5. On SDS-polyacrylamide gel electrophoresis two classes of subunits were observed in equal proportions with molecular weights of 70 K and 83 K. The effect of various compounds on the enzymatic activity was studied. The enzyme was insensitive to NaN3, oligomycin and to divalent cations, but was inhibited by citrate and oxalate.