普洱茶中微生物的筛选及其安全性初步评价

目的在对普洱茶中微生物筛选和鉴定基础上对蜡样芽胞杆菌毒素基因的分布、普洱茶下调毒素基因的表达和改善肠上皮细胞的损伤进行研究。方法分别采用无菌水和沸水泡制普洱茶,获得分离株,通过16SrDNA测序以及生理生化试验确定其归属;对所筛选的菌株进行耐模拟胃肠液能力评价和毒力基因的检测;采用细胞实验和荧光定量PCR技术,研究普洱茶对蜡样芽胞杆菌毒素的抑制作用。结果无菌水浸泡普洱茶获得的45株菌中44株为芽胞杆菌属,沸水浸泡获得的7株菌均为蜡样芽胞杆菌(FBCE01、FBCE06、FBCE10、FBCE14、FBCE20、FBCE26和FBCE29),多重PCR技术结果表明其分别含有毒力基因cytK、nheA和hblD的2种或3种。耐模拟胃肠液实验表明,7株菌均具有很强的耐模拟胃肠液消化能力;细胞实验结果发现,普洱茶汤能显著降低蜡样芽胞杆菌对Caco-2细胞的粘附(P0.05);MTT实验结果显示,普洱茶能有效降低蜡样芽胞杆菌对细胞的损伤;荧光定量PCR技术结果进一步说明,普洱茶使蜡样芽胞杆菌肠毒素的mRNA表达水平下调。结论普洱茶具有抑制蜡样芽胞杆菌毒素的作用。     

PlcR在炭疽芽胞杆菌A16R中的功能研究

炭疽芽胞杆菌(Bacillus anthracis)、蜡样芽胞杆菌(B. cereus)和苏云金芽胞杆菌(B. thuringiensis)均属于蜡样芽胞杆菌群,在遗传学上有很高的相似性。PlcR (Phospholipase C regulator)在蜡样芽胞杆菌中是十分重要的调控因子,但plcR基因在炭疽芽胞杆菌中发生一个无义突变导致在炭疽芽胞杆菌中产生一个截短PlcR蛋白。为了研究plcR基因对炭疽芽胞杆菌功能的影响,文章以蜡样芽胞杆菌CMCC6330基因组为模板,构建重组表达质粒pBE2A-plcR后导入炭疽芽胞杆菌疫苗株A16R中获得重组菌株,对其进行表型分析。结果显示,炭疽芽胞杆菌重组菌株的溶血活性基本没有恢复,但恢复了部分神经鞘磷脂酶活性,表明将蜡样芽胞杆菌的plcR基因导入炭疽芽胞杆菌后,可以直接激活神经鞘磷脂酶活性。     

低温环境中T-2毒素降解菌的分离鉴定及特性研究

离筛选低温环境中T-2毒素的降解菌并探明其生化特性,探究T-2毒素降解微生物生化特性异同点,为T-2毒素降解微生物的检验提供生化判断参考。针对暴露于－20 ℃低温环境中的低浓度T-2毒素标准品,采用LC-MS/MS定量分析T-2毒素残留量,利用营养琼脂培养基（NA）和马铃薯葡萄糖琼脂培养基（PDA）共分离出5株降解菌,16S rDNA结合生化鉴定结果为死谷芽胞杆菌（Bacillus vallismortis）、蜡状芽胞杆菌（Bacillus cereus）、阴沟肠杆菌（Enterobacter cloacae）、弯曲假单胞菌（Pseudomonas geniculata）和尼泊尔葡萄球菌（Staphylococcus nepalensis）。这些分离株在－20 ℃条件下对低浓度T-2毒素均有不同程度的降解能力,其中蜡状芽胞杆菌（Bacillus cereus）降解能力最强,降解率为91%。它们的理化特性具有很多相似性,主要表现在5株菌均不能利用D 塔格糖(dTAG）,对ELLMAN都表现出阴性;Bacillus vallismortis Bp1234-7、Pseudomonas geniculata Bp24-4、〖WTBX〗Staphylococcus nepalensis Bp124-5和Enterobacter cloacae Bp123-7都能分解D-葡萄糖,而Bacillus cereus Bp1234-8不能。     

钙粘蛋白与苏云金芽胞杆菌晶体毒素 互作机制研究进展

昆虫钙粘蛋白是苏云金芽胞杆菌晶体毒素的重要受体之一,本文简述了昆虫钙粘蛋白种类、结构特征和在昆虫体内的分布,对昆虫钙粘蛋白与苏云金芽胞杆菌毒素相互结合位点进行了较深入讨论;详细论述了苏云金芽胞杆菌晶体毒素作用孔洞模式和信号转导模式,并简要介绍了钙粘蛋白与昆虫抗性的关系.     

不同品系小鼠对炭疽芽胞杆菌弱毒株芽胞的敏感性研究

通过比较四种品系小鼠对炭疽芽胞杆菌（Bacillus anthracis）（简称炭疽杆菌）弱毒株芽胞的敏感性，确定炭疽杆菌弱毒株芽胞攻毒合适的动物模型。采用炭疽杆菌弱毒株A16Q1（pXO1-、pXO2+）和A16PI2（pXO1+、pXO2-）的芽胞对四种品系小鼠（DBA/2、KM、ICR和BALB/c）进行腹腔攻毒，记录小鼠死亡时间，计算LD₅₀、绘制存活曲线并统计分析。运用较敏感的KM小鼠研究不同canSNP基因型毒素缺陷株（含pXO2拷贝数不同）芽胞的毒力差异。利用更为敏感的DBA/2小鼠评价S-层蛋白BA3338对荚膜缺陷株芽胞毒力的影响。结果表明，在四种品系小鼠中，毒素缺陷株芽胞的毒力均高于荚膜缺陷株芽胞的毒力。DBA/2小鼠对炭疽杆菌弱毒株芽胞的剂量依赖关系最好，最为敏感，其次是KM小鼠，而ICR小鼠和BALB/c小鼠对炭疽杆菌弱毒株芽胞不敏感。确定了DBA/2小鼠和KM小鼠在炭疽杆菌弱毒株芽胞研究中的适用性。使用KM小鼠评价了不同canSNP基因型炭疽杆菌芽胞的毒力差异，结果表明，不同canSNP基因型炭疽杆菌由于所含pXO2质粒拷贝数的差异导致芽胞的毒力不同。使用DBA/2小鼠评价了S-层蛋白BA3338缺失对炭疽杆菌芽胞毒力的影响，表明BA3338基因的缺失导致炭疽杆菌芽胞毒力降低。     

PCR法快速鉴定眼源性蜡样芽胞杆菌

目的建立一种快速、灵敏、特异的眼源性蜡样芽胞杆菌PCR检测方法,为蜡样芽胞杆菌性眼内炎患者的快速诊断提供依据。方法选择编码蜡样芽胞杆菌细胞毒素的cytK为靶基因设计引物,建立检测眼源性蜡样芽胞杆菌PCR;PCR产物用琼脂糖凝胶电泳鉴定,基因序列与GenBank比对验证扩增产物;将计数过的5株蜡样芽胞杆菌菌悬液,梯度稀释后分别提取DNA进行PCR扩增,确定检测方法的灵敏度;分别用眼部常见感染菌金黄色葡萄球菌、表皮葡萄球菌、甲型溶血性链球菌、化脓性链球菌、藤黄微球菌、铜绿假单胞菌、大肠埃希菌、普通变形杆菌和白假丝酵母菌以及枯草芽胞杆菌DNA进行特异性试验;进一步将该方法应用到人工污染致病蜡样芽胞杆菌的房水标本中,并分析其灵敏度。结果5株分离自眼内炎患者标本中的蜡样芽胞杆菌均扩增出360bp左右的DNA片段,测序结果与GenBank比对一致;该法检测在5h内完成,方法灵敏度达7．5～15．0CFU／mL;其他菌株检测未出现非特异性扩增;对模拟感染房水标本的PCR鉴定结果与分离培养对比,二者符合率为100％,模拟标本的检测灵敏度与纯菌结果一致。结论cytK基因为靶基因的PCR用于眼源性蜡样芽胞杆菌的快速检测,具有简便、快速、敏感、特异等特点,为眼内炎患者的快速诊断提供依据,在实际检验工作中有良好的应用前景。     

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

摘要：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。     

常见4种微生态制剂菌种产淀粉酶的比较

目的 探索常见4种微生态制剂菌种地衣芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌和纳豆芽胞杆菌产生淀粉酶的能力,以筛选和研制动物微生态制剂和饲料添加剂的使用菌种。方法 将各菌种接于淀粉酶试验培养基,培养后滴加稀碘溶液,观察透明圈,判定产酶能力。结果 地衣芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌和纳豆芽胞杆菌都能产生淀粉酶,以蜡样芽胞杆菌产生的淀粉酶较多。结论 4种常见芽胞杆菌产淀粉酶能力依次为：蜡样芽胞杆菌＞纳豆芽胞杆菌＞枯草芽胞杆菌＞地衣芽胞杆菌。     

几种芽胞杆菌溶血素BL基因及其溶血素的检测

用PCR方法对几种芽胞杆菌溶血素BL基因进行了检测,结果表明7株蜡样芽胞杆菌含有溶血素BL基因hblA、hblC、hblD,其他枯草芽胞杆菌、多粘类芽胞杆菌、地衣芽胞杆菌检测到部分溶血素基因;通过血平板培养的方法检测结果表明只有含有溶血素全部基因的菌株才会产生溶血环,从而为筛选不产生溶血素的有益芽胞杆菌奠定一定基础。     

1株来自海洋的芽胞杆菌dhs-330转座突变库的构建及表面活性相关基因的克隆

为研究产生物表面活性剂的海洋芽胞杆菌dhs-330合成活性产物的分子机制,应用质粒pIC333介导的mini-Tn10转座子随机突变技术,构建了芽胞杆菌dhs-330的突变体库。通过表面活性测定和反向PCR克隆,从300个突变株中筛选出产表面活性剂水平提高的突变株2株,分别在ycsG和yvkC基因发生插入突变;表面活性降低的突变株4株,分别在fenC、yrkF、kinE和sigD基因发生插入突变。这些基因可能与芽胞杆菌dhs-330中表面活性剂的合成代谢和调控有关。     

艰难拟梭菌毒素致病机制及毒素基因调控的研究进展

艰难拟梭菌(Clostridioides difficile)是一种产芽孢的革兰氏阳性专性厌氧杆菌,是引起抗生素相关性腹泻的主要致病菌。艰难拟梭菌产生的毒素A和毒素B在其致病过程中发挥关键作用。毒素发挥毒性作用依赖其4个功能结构域：葡萄糖基转移酶结合域、半胱氨酸蛋白酶结合域、易位域和受体结合域。毒素的受体结合域识别并结合细胞表面受体,介导毒素内吞并形成内体。经过自体催化切割,毒素将真正的毒性片段——葡萄糖基转移酶结合域释放到胞浆中,葡萄糖基转移酶能够失活宿主肠上皮细胞内的GTP酶导致细胞骨架解聚和坏死,进而引起腹泻和伪膜性结肠炎等临床症状。艰难拟梭菌毒素产生受致病基因座内及基因座外许多调控因子的调节。tcdR和tcdC基因位于致病基因座内,对毒素基因的表达分别起促进和抑制作用,而基因座外如spo0A、codY等基因则通过抑制tcdR的表达从而间接影响毒素蛋白产生。本文将重点介绍艰难拟梭菌毒素的致病过程和影响毒素基因表达的分子调控机制,以期为开发针对毒素的治疗手段提供新思路。     

Horizontal transfer of a mitochondrial plasmid

Direct evidence for horizontal transfer of a mitochodnrial plasmid from the discomyceteAscobolus immersus to the pyrenomycetePodospora anserina is presented. Southern blot hybridisation analysis, polymerase chain reaction (PCR) amplification, and DNA sequencing demonstrate transmission of a linear plasmid upon hyphal contact. DNA extraction from isolated organelles indicates a mitochondrial localisation for the plasmid inP. anserina. This is the first report of horizontal gene transfer among unrelated fungi. These results have important evolutionary implications for plasmid propagation in fungi.     

Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genusLoxosceles (Araneae)

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 ofLoxosceles gaucho, L. laeta, orL. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components, B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa inL. gaucho andL. intermedia, but 32 kDa inL. laeta venom. These toxins were isolated from venoms ofL. gaucho, L. laeta, andL. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of theL. reclusa (North American spider) toxin. A multiple sequence alignment of theLoxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho andL. reclusa) to 61.1% (L. intermedia andL. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping afterin situ partial hydrolysis of the blotted samples. The results obtained suggest thatL. intermedia protein is more similar toL. laeta toxin thanL. gaucho toxin and revealed a smaller homology betweenL. intermedia andL gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms ofLoxosceles species have a molecular mass of 32–35 kDa and are probably homologous proteins.     

Lineage‐specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co‐evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large‐scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co‐evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.     

Incidence of <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> endosymbionts in the <Emphasis Type="Italic">Osmia</Emphasis> community in Korea

Sex ratio distorting endosymbionts induce reproductive anomalies in their arthropod hosts. They have recently been paid much attention as firstly texts of evolution of host-symbiont relationships and secondly potential biological control agents to control arthropod pests. Among such organisms, Wolbachia and Cardinium bacteria are well characterized. This study aims at probing such bacteria in the Osmia community to evaluate their potential utilization to control arthropod pests. Among 17 PCR tested species, Osmia cornifrons and a parasitic fly are infected with Wolbachia and a mite species is infected with Cardinium. Phylogenetic tree analyses suggest that horizontal transfer of the bacteria occurred between phylogenetically distant hosts.     

Bacillus sphaericus Mtx1 and Mtx2 toxins co-expressed in Escherichia coli are synergistic against Aedes aegypti larvae

Mtx1 and Mtx2 are mosquitocidal toxins produced by some strains of Bacillus sphaericus during vegetative phase of growth. Mtx1 from B. sphaericus 2297 shows higher toxicity against Culex quinquefasciatus larvae than to Aedes aegypti larvae whereas Mtx2 from B. sphaericus 2297 shows lower toxicity against C. quinquefasciatus than to A. aegypti larvae. To test synergism of these toxins against A. aegypti larvae, mtx1 and mtx2 genes were cloned into a single plasmid and expressed in Escherichia coli. Cells producing both Mtx1 and Mtx2 toxins exhibited high synergistic activity against A. aegypti larvae approximately 10 times compared to cells expressing only a single toxin. Co-expression of both toxins offers an alternative to improve efficacy of recombinant bacterial insecticides. There is a high possibility to develop these toxins to be used as an environmentally friendly mosquito control agent.     

Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution

Virulence genes of pathogenic bacteria, which code for toxins, adhesins, invasins or other virulence factors, may be located on transmissible genetic elements such as transposons, plasmids or bacteriophages. In addition, such genes may be part of particular regions on the bacterial chromosome, termed‘pathogenicity islands’(Pais). Pathogenicity islands are found in Gram-negative as well as in Gram-positive bacteria. They are present in the genome of pathogenic strains of a given species but absent or only rarely present in those of non-pathogenic variants of the same or related species. They comprise large DNA regions (up to 200 kb of DNA) and often carry more than one virulence gene, the G+C contents of which often differ from those of the remaining bacterial genome. In most cases, Pais are flanked by specific DNA sequences, such as direct repeats or insertion sequence (IS) elements. In addition, Pais of certain bacteria (e.g. uropathogenic Escherichia coli, Yersinia spp., Helicobacter pylori) have the tendency to delete with high frequencies or may undergo duplications and amplifications. Pais are often associated with tRNA loci, which may represent target sites for the chromosomal integration of these elements. Bacteriophage attachment sites and cryptic genes on Pais, which are homologous to phage integrase genes, plasmid origins of replication or IS elements, indicate that these particular genetic elements were previously able to spread among bacterial populations by horizontal gene transfer, a process known to contribute to microbial evolution.     

Integration ofhup cosmid pHU52 into the chromosomal DNA ofCicer-Rhizobium using Tn5 as an homologous sequence

Cosmid pHU52, which carrieshup genes ofBradyrhizobium japonicum, has been integrated into theCicer-Rhizobium G36-84 genome via Tn5-mediated homologous recombination. Tn5 was inserted into both the cosmid pHU52 and the chromosome ofCicer-Rhizobium to provide a region of DNA homology, without affecting the expression of necessary genes. An incompatible plasmid, pPH1JI, was used to select those few cells that had undergone recombination. The integration of the cosmid was demonstrated by Southern blot analysis. Chromosomal integration of thehup genes maximized stability and minimized the potential for their horizontal transfer to other bacterial species. The integratedhup genes were found to expressex planta as well in nodules. The method described illustrates how a given gene can be stably integrated into the chromosome.     

Heptapeptide toxin production during the batch culture of two Microcystis species (Cyanobacteria)

Changes in the content of cyclic heptapeptide hepatotoxins called microcystins were investigated during batch culture of two Microcystis species using high performance liquid chromatography. After adsorption to ODS-silica gel cartridges and elution with methanol, the toxins were analyzed and quantified by HPLC. 35 μg per 100 mg dry cells of microcystin-RR, 34 μg of -YR and 43 μg of -LR were present at the beginning of the exponential growth phase of M. viridis. Microcystin-RR increased markedly towards the end of the exponential phase with the maximum content of 112 μg per 100 mg cells was measured at the late stage of the exponential phase. A remarkable increase of microcystin-YR from 130 μg per 100 mg cells to 1020 μg was observed during the exponential phase of a highly toxic strain of M. aeruginosa. However no clear differences were found in the pattern of change among the three toxins during the growth course.     

Evidence for phylogenetic inheritance in pathogenicity of Mycobacterium

In this study, we attempt to highlight part of the adaptive and phylogenetic constraints in mycobacterial pathogenicity. For this purpose, we first provide a phylogeny of Mycobacteria based on cladistic analyses of 64 different taxa. We then performed a comparative analysis, taking into account both ecological factors and phylogenetic relationships. The GLIM modelling analysis showed that different ecological and phylogenetic factors might be invoked to explain the variation in pathogenicity levels. Interestingly, the most harmful species were shown to be connected with the most diversified habitats. However, the independent contrast analysis revealed that once phylogeny was taken into account, none of the relationships between ecological factors and pathogenicity remained significant, and the pathogenicity appeared to be phylogenetically inherited among mycobacteria. The most pathogen were found in the slow-growing/long helix 18 group, and within this group in the most derived taxa. This revised version was published online in June 2006 with corrections to the Cover Date.