挥发性物质诱吸线虫的土壤细菌系统多样性特征及其活性挥发物鉴定

摘要:【目的】明确有机挥发物诱吸线虫的土壤细菌种群多样性及其活性挥发物,有助于了解线虫与土壤微生物间的互作关系,为利用诱吸性物质与杀线虫剂配合使用,提高防治寄生线虫效果奠定基础。【方法】通过双培养皿倒扣法筛选我国26 个省市的187 份农业土壤中挥发性诱吸线虫的细菌资源、测定其诱吸活性,并结合RFLP-16S rRNA 方法分析活性细菌的系统亲缘关系;采用SPME-GC /MS 方法鉴定其挥发物的类型,并通过纯品进行诱吸功能验证。【结果】从分离自187 份土样的3800 株细菌中,筛选出诱吸活性(AN)≥     

Resistance mechanisms of bacteria to antimicrobial compounds

A range of antimicrobial compounds (bactericides) commonly termed biocides, microbicides, sanitizers, antiseptics and disinfectants are available, all of which are claimed by their producers to kill bacteria. Resistance has been defined as the temporary or permanent ability of an organism and its progeny to remain viable and/or multiply under conditions that would destroy or inhibit other members of the strain. Bacteria may be defined as resistant when they are not susceptible to a concentration of antibacterial agent used in practice. Traditionally, resistance refers to instances where the basis of increased tolerance is a genetic change, and where the biochemical basis is known. Antimicrobial substances target a range of cellular loci, from the cytoplasmic membrane to respiratory functions, enzymes and the genetic material. However, different bacteria react differently to bactericides, either due to inherent differences such as unique cell envelope composition and non-susceptible proteins, or to the development of resistance, either by adaptation or by genetic exchange. At low concentrations bactericides often act bacteriostatically, and are only bacteriocidal at higher concentrations. For bactericides to be effective, they must attain a sufficiently high concentration at the target site in order to exert their antibacterial action. In order to reach their target site(s), they must traverse the outer membrane of the gram negative bacteria. Bacteria with effective penetration barriers to biocides generally display a higher inherent resistance than those bacteria which are readily penetrated. The rate of penetration is linked to concentration, so that a sufficiently high bactericide concentration will kill bacteria with enhanced penetration barriers. It has been indicated that susceptible bacterial isolates acquire increased tolerance to bactericides following serial transfer in sub-inhibitory concentrations. Whereas the basis of bacterial resistance to antibiotics is well know, that of resistance to antiseptics, disinfectants and food preservatives is less well understood.Three mechanisms of resistance that have been reported include:

• limited diffusion of antimicrobial agents through the biofilm matrix,

• interaction of the antimicrobial agents with the biofilm matrix (cells and polymer),

• enzyme mediated resistance,

• level of metabolic activity within the biofilm

• genetic adaptation

• efflux pumps and

• outer membrane structure.

     

Oligotrophic bacteria from rendzina forest soil

Oligotrophic bacteria were shown to exist abundantly in all layers of a rendzina forest soil throughout the year. Two-hundred-three oligotrophic bacteria were isolated from forest soil (Aoba, Sendai) at different layers (L, F, H and A layers) throughout the year, and their morphological and physiological characteristics were examined. A high proportion (95%) of the isolated oligotrophs were Gram-negative, non-spore forming bacteria. Based on the cell shape, the isolates were divided into four groups: regular rods, curved/spiral bacteria, irregular rods, and buddin and/or prosthecate bacteria. Each group of bacteria is discussed in relation to the physiological characteristics. Notably oligotrophic bacteria of different cell types showed a marked zonal distribution in respect to profile depth.     

星海链霉菌抑菌活性物质的分离纯化

星海链霉菌(Streptomyces xinghaiensis)是从大连星海湾海泥样品中分离的海洋链霉菌新种,其发酵液具有广谱抗菌活性。研究该菌株的发酵液对临床分离的耐药菌的抑制活性,发现星海链霉菌发酵液对鲍曼不动杆菌和耐甲氧西林金黄色葡萄球菌菌株具有抑制活性。采用SPE、大孔吸附树脂HP-20、葡聚糖凝胶Sephadex LH-20、HPLC、TLC等对抗耐甲氧西林金黄色葡萄球菌的物质进行了活性追踪,发现活性物质为弱碱性水溶性成分,无特征紫外吸收,可被茚三酮显色,推断活性物质可能为氨基糖苷类化合物。对星海链霉菌基因组序列进行初步分析,发现一个与核糖霉素生物合成基因簇相似性较高的基因簇,但TLC分析结果表明,活性化合物与核糖霉素不同。     

土壤产铁载体细菌的筛选及其对铁氧化物的活化与利用

【背景】土壤中铁主要以难溶态铁氧化物的形式存在,有效性较低,产铁载体细菌对铁氧化物的活化是提高铁利用效率的有效途径。【目的】从林木土壤筛选产铁载体细菌,并观察菌株对难溶性铁氧化物的利用效应,可为土壤微生物资源开发及其在养分调控中的作用提供理论依据。【方法】通过CAS检测法从林木根系附近表层土壤中分离产铁载体细菌,借助生物培养实验分析温度和pH对微生物生长和铁载体产生的影响,通过振荡平衡实验,探究细菌产铁载体对铁氧化物的活化效应。【结果】通过CAS检测法从林木根系附近表层土壤中分离得到12株产铁载体细菌,16S rRNA基因扩增子测序初步鉴定结果显示筛选细菌均为假单胞菌属。选取铁载体产生能力和生长活性较高的两株细菌ARSB02和CNRSB01作为重点研究对象,结果显示,不同条件下CNRSB01的生物量和铁载体产量均高于菌株ARSB02,22 h时菌株ARSB02和CNRSB01的铁载体活性单位分别达到67.07%和84.60%。pH 5.0-8.0的范围内两株细菌可以保持较好的铁载体产生能力,菌株ARSB02和CNRSB01在pH7.0时铁载体产生能力最强,铁载体活性单位分别达到38.9...     

Susceptibility of various purple and green sulfur bacteria to different antimicrobial agents

Abstract Several purple and green sulfur bacteria (genera Chromatium, Thiocapsa and Chlorobium ) were tested for their sensitivity to different antimicrobial agents by a disc diffusion assay, using thioacetamide as a source of hydrogen sulfide for plate growth. Chlorobium limicola strains were more sensitive to amoxicillin, erythromycin and nalidixic acid, whereas gentamicin and netilmicin were more active against the purple bacteria tested. None of the organisms were sensitive to oxacillin and trimethoprim + sulfamethoxazole. The critical concentrations at the edge of the inhibition zone were also calculated for three organisms and the antimicrobials colistin, mitomycin C, penicillin G, rifampicin, and streptomycin. The results obtained suggest that colistin, mitomycin C, penicillin G would provide selective conditions against the growth of Chlorobium limicola strains, while streptomycin and other aminoglycoside antibiotics would select against purple bacteria.     

蜘蛛抱蛋属植物内生细菌的多样性及其抗菌活性筛选

【背景】药用植物中蕴含多样性丰富的内生菌资源,这些微生物产生的多种新型物质在制药领域表现出较好的应用前景。【目的】研究蜘蛛抱蛋属(Aspidistra Ker-Gawl.)植物内生细菌的多样性,探索药用植物内生细菌在药用活性产物方面的开发潜力,以期发现具有抗菌活性的次级代谢产物。【方法】对9种13株新鲜的蜘蛛抱蛋植物进行表面消毒,采用5种分离培养基分离内生细菌;根据菌落形态特征排除重复菌株,并测定其16S rRNA基因序列,构建系统进化树分析内生细菌多样性;将菌株分别用2种培养基发酵,使用耻垢分枝杆菌(Mycobacterium smegmatis ATCC 700044)、水稻白叶枯菌(Xanthomonas oryzae PXO99A)、白色念珠菌(Candidaalbicans ATCC 10231)、肺炎雷伯菌(Klebsiella pneumoniae ATCC 700603)和耐药粪肠球菌HH22(Enterococcus faecalis HH22)5种检定菌对分离菌株的发酵液进行抑菌活性筛选。【结果】从植物组织中分离得到了234株内生细菌,根据形态初步排重得到156株植物内生细菌;基于16S rRNA基因序列构建的系统进化树显示它们分属于3门10目22科29属,其中链霉菌属(Streptomyces)、芽孢杆菌属(Bacillus)、微杆菌属(Microbacterium)、类芽孢杆菌属(Paenibacillus)和根瘤菌属(Rhizobium)的菌株广泛地分布在不同种的蜘蛛抱蛋植株中,且占据一定优势;发现可能的潜在新分类单元6个;156株内生细菌中38株菌的发酵液具有抑菌活性,初筛阳性率为23.7%。【结论】蜘蛛抱蛋植物组织中含有种类多样的内生细菌,它们可能是抗菌生物活性次级代谢产物的有效来源。     

辽北平原苏打盐渍土增施泥炭对土壤盐分的影响

对辽河北部平原苏打盐渍化土壤改良试验表明,在增施泥炭条件下,苏打草甸盐土当年玉米产量可达２５００～４４００ｋｇ·ｈｍ－２,苏打碱化草甸土为５５００～６８００ｋｇ·ｈｍ－２,增产率分别为４０～１４４％和１２７～１８１％;第２年后者的持续产量为６２００～８０００ｋｇ·ｈｍ－２,增产率仍高达６９～１１８％．相应地,土壤剖面特别是耕作层呈现为明显脱盐,ｐＨ及碱化度下降,土壤有机质、全氮及全磷含量增加．     

Phylogenetic identification of epibiotic bacteria possessing antimicrobial activities isolated from red algal species of Japan

We investigated the diversity of epibiotic bacteria possessing antimicrobial activity isolated from nine species of red algae, and identified their phylogenetic position. For the isolation of epibiotic bacteria, nine species of red algae, Pachymeniopsis lauceolata, Plocamium telfairiae, Gelidium amansii, Chondrus oncellatus, Grateloupia filicina, Ceramium kondoi, Lomentaria catenata, Schizymenia dubyi and Porphyra yezoensis, were collected from the intertidal zone of Awaji Island, Japan. In total 92 bacteria were collected from the above red algal species. Primary screening results using disc diffusion assay revealed that 33% of bacteria possess antibacterial activity. Ten bacteria that showed high antibacterial activity were further studied for their ability to inhibit a set of fouling bacteria, some luminescent Vibrio and Photobacterium species and a panel of pathogenic bacteria. In general, the inhibitory activities were high against fouling and luminescent bacteria, while low against various pathogenic bacteria tested. These results suggest that some epibiotic bacteria have adapted to defend their position in their surface environment through the production of antibacterial metabolites giving defense against a broad spectrum of bacterial competitors. The phylogenetic analysis using 16 S rRNA sequences identified 7 of the 10 strains as belonging to the genus Bacillus, and other strains each 1 belonging to genus Microbacterium, Psychrobacter, and Vibrio species.     

Phylogenetic analysis on the bacteria producing non-volatile fungistatic substances

This study characterized the soil bacteria producing non-volatile fungistatic substances. Among the 2,100 colonies of soil bacteria randomly isolated from seven agricultural soil samples, 518 isolates (24.67% of total) showed fungistatic activity toward nematophagous fungi Paecilomyces lilacinus and Trichoderma viride by producing non-volatile substances. A phylogenetic analysis based on amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequence placed the 518 bacteria in three groups of the domain Bacteria: Actinomycetales, Bacillales, and Gammaproteobacteria. Three genera, Arthrobacter, Bacillus, and Pseudomonas, were the most frequently encountered groups.     

高产铁载体棉田土壤细菌SS05的筛选与鉴定

【目的】研究从棉田土壤中筛选得到的高产铁载体细菌产铁载体能力、分类地位和抑菌活性。【方法】通过改良蔗糖-天冬氨酸培养基选择性筛选产铁载体细菌,通过分光光度计法测定铁载体活性,通过混菌法测定产铁载体细菌上清液对棉花枯萎病致病菌尖孢镰刀菌(Fusarium oxysporum)的抑菌效果,采用形态学、生理生化鉴定及16S rDNA序列系统发育分析对高产铁载体菌株进行鉴定。【结果】从棉田土壤中筛选到162株产铁载体细菌,30株产铁载体能力较强的细菌中21株具有较高产铁载体能力,菌株SS05的铁载体活性单位达到98.3%;在低铁条件下,SS05上清液对F.oxysporum具有显著的抑制作用;SS05与莫哈韦芽孢杆菌(Bacillus mojavensis)最为接近。【结论】SS05是高产铁载体菌株,与莫哈韦芽孢杆菌(Bacillus mojavensis)最为接近,在低铁培养条件下其上清液对F.oxysporum具有显著的抑制作用。     

青海湖裸鲤肠道乳酸菌多样性与抑菌活性

【目的】通过生理生化特性,结合16S r RNA基因序列分析研究青海湖裸鲤肠道乳酸菌分离株的多样性,并对这些代表株的抑菌活性进行初步探讨,以期筛选具有高效抑菌活性的鱼源益生菌。【方法】对分离的47株乳酸菌代表株进行p H、温度生长范围、耐盐性等生理生化特征检测,结合16S r RNA基因序列对已分离到的乳酸菌进行基因分型和菌种鉴定,采用牛津杯双层平板法检测乳酸菌代表株的抑菌活性。【结果】鉴定结果显示:23株为Lactobacillus fuchuensis(48.94%),12株为Lactobacillus curvatus(25.53%),3株为Leuconostoc fallax(6.38%),2株为Lactobacillus sakei(4.26%),2株为Weissella ceti(4.26%);2株为Lactococcus cremoris(4.26%),1株为Leuconostoc lactis(2.13%),1株为Weissella minor(2.13%),1株为Enterococcus devriesei(2.13%)。qz1217、qz1196、qz1220所在的A、B、C三组乳酸菌在5-50°C的温度范围内生长良好,qz1196、qz1220所在的B、C组在pH 3.0-10.0的范围内生长良好,几乎所有乳酸菌都具有耐6.5%盐浓度特性。13株乳酸菌菌株对6种病原菌都具有抑制作用。通过排除酸、过氧化氢实验,发现上清液仍然具有抑菌活性。对qz1251发酵液进行蛋白酶处理,抑菌活性消失,确定其抑菌物质属于蛋白类物质,是一种细菌素。【结论】青海湖裸鲤肠道附着乳酸菌的多样性为益生性乳酸菌的筛选提供优质资源及数据参考。     

Reuse of peat bags for tomatoes and cucumbers

Summary Growing tomatoes in low humified Sphagnum peat gave satisfactory results when using the peat bags in three successive seasons without any sterilization. Use of a complete nutrient solution showed an increase of phosphorus and boron from one season to the next. The pH varied with the electrical conductivity in the peat, dependent on the nutrient concentration used. The solution used for reused peat should have a lower content of phosphorus and boron compared to a solution used for one season peat.The air capacity is high and have to be considered as optimal also in reused peat. The effect of reusing the peat on physical properties as porosity and pore size distribution is therefore of no practical significance.     

Morphometric analysis of bacteria associated with soil millipedes

Scanning electron microscopy revealed that the average cell size of bacteria associated with the digestive tract of soil millipedes was 0.65 μm in diameter, 1.36 μm in length, and 0.60 μm³ in volume. An example of millipedes illustrated that the intestinal tract bacteria of soil invertebrates share the following features: (1) a high density level in this habitat; (2) existence mostly in the form of vegetative cells; (3) a cell size significantly smaller than that of bacteria functioning in soil; (4) a cell size closer to the lower limits of the size range characteristic for bacterial cultures grown in laboratory media. All this suggests that the bacterial community of the digestive tract differs from the typical soil community not only in composition but also in a higher level of physiological activity.     

基于K-B法筛选异质性耐药菌株及其对抗菌药物敏感试验结果的影响

目的 探讨K-B法抑菌圈内出现细菌单菌落的可能原因及其对药敏结果的影响。方法 采用头孢他啶和美罗培南药敏纸片对革兰阴性菌株进行筛选,对抑菌圈内出现的细菌单菌落进行鉴定及药敏试验,并与原鉴定结果和药敏结果进行比较。结果 从痰液,尿液,血液等临床标本分离的2 500株革兰阴性杆菌。55株在头孢他啶或美罗培南纸片抑菌圈内会出现肉眼可见的细菌单菌落,其中26株经过了菌落纯培养,出现异质性耐药23株,污染3株,29株未经过菌落纯培养,出现异质性耐药14株,污染15株。头孢他啶纸片筛选试验中,共有28株抑菌圈中菌株与原鉴定结果一致,但耐药表型及具体药敏结果均发生改变,其中7株阴沟肠杆菌氨曲南和头孢曲松药敏结果均从敏感变成耐药。美罗培南纸片筛选试验中,共有9株抑菌圈中菌株与与原鉴定结果一致,其中7株铜绿假单胞菌氨曲南从敏感变为中介,亚胺培南从中介变为耐药。结论 本研究中,1.48%革兰阴性杆菌分离菌株会出现异质性耐药,菌落传代纯培养可以有效降低药敏操作过程中的污染。异质性耐药菌株的存在以及实验操作过程中的可能污染对病原菌体外药敏结果的影响,需要引起临床,特别是临床微生物工作者的关注和重视。     

海洋抑菌放线菌Y15的筛选、鉴定及其代谢产物理化性质

【目的】筛选产广谱、高效抑菌活性物质的海洋放线菌,为新型抗生素的开发奠定基础。【方法】采用琼脂块法初筛,打孔扩散法复筛,以金黄色葡萄球菌、大肠埃希氏菌、枯草芽孢杆菌和单核细胞增生李斯特菌为指示菌从海泥样品中筛选目标菌株;利用20种指示菌株考查Y10、Y11、Y15、Y16和Y21的抑菌谱;通过形态观察和16S r RNA基因序列分析确定菌株分类地位;以单核细胞增生李斯特菌的OD600值为指标探讨Y15抑菌活性物质对该菌的作用方式;以抑菌活性为指标研究Y15抑菌活性物质的理化性质。【结果】共分离12株具抑菌活性的海洋放线菌,其中Y15抑菌谱最广,对20种指示菌株中的18种具有抑菌活性,并且在4种培养基中均能产生抑菌活性物质。16S r RNA基因序列分析表明Y15属于小单孢菌属,并与Micromonospora endolithica亲缘关系最近。Y15抑菌活性物质在144 h达到最高值为480 AU/m L,在168-216 h保持平衡为320 AU/m L。Y15抑菌活性物质对单核细胞李斯特菌作用方式为杀菌。Y15抑菌活性物质在-20-60°C抑菌活性保持稳定,在80-120°C活性逐渐下降,但在120°C处理30 min仍保留37.5%的活性;在p H 7.0-10.0抑菌活性稳定,在p H 2.0-6.0和p H 11.0-12.0抑菌活性均有所损失;Y15抑菌活性物质对紫外和4种酶(蛋白酶K、胰蛋白酶、木瓜蛋白酶和α-淀粉酶)处理均保持稳定,表明活性物质为非蛋白和非多肽类的抑菌物质。【结论】Y15产生的活性物质具有良好的抑菌谱和抑菌活性,且较为稳定,具有较高的应用价值。     

红树林土壤中产聚羟基脂肪酸酯细菌的分离及其评估

[背景] 细菌能通过合成聚羟基脂肪酸酯（Polyhydroxyalknoates,PHA）在细胞内储存物质和能量,提高对环境的适应能力。在红树林中,由于土壤周期性受海水浸没,形成营养物质种类丰富和含量波动大的特殊生境,为细菌进化出特殊的PHA合成途径提供了条件。[目标] 为了增加对红树林产PHA细菌资源的了解,获得产PHA细菌,使用纯培养方法分离和鉴定细菌,并评估菌株的产PHA能力。[方法] 采集红树植物海桑根系和红树滩涂土壤样品,连续5周培养、分离纯化获得细菌菌株;通过16S rRNA基因相似性及系统进化分析鉴定细菌分类地位,利用PHA合成酶基因（phaC）鉴定细菌合成PHA的能力;通过基因组草图测序,分析细菌的phaC基因种类、代谢通路及系统进化关系;通过气相色谱分析细菌产PHA的累积量及组成。[结果] 从红树林土壤样品中分离得到97株细菌,其中13株带有phaC基因,包括坚强芽孢杆菌（Cytobacillus firmus）、弯曲芽孢杆菌（Bacillus flexus）、除烃海杆菌（Marinobacter hydrocarbonoclasticus）和酯香微杆菌（Microbacterium esteraromaticum）。B. flexus MN15-19以丙酮酸盐为碳源,可累积细胞干重11%的PHA,同时具有固碳功能的还原性三羧酸循环通路,有开发成为固碳产PHA工程菌株的潜力。酯香微杆菌可产PHA,但是其phaC基因结构特殊,基因组注释未能识别出任何已知phaC基因。[结论] 研究发现红树林土壤可培养细菌中存在未知的PHA合成途径,说明红树林生态系统中的细菌具有资源挖掘的重要价值。     

宁夏枸杞内生细菌的多样性及其抑菌活性研究

【目的】对宁夏枸杞各药用部位内生细菌的分布特征、遗传多样性和抑菌活性进行分析。【方法】采用菌落计数和16S rRNA基因序列分析法研究枸杞内生细菌的分布特征、遗传多样性,采用琼脂扩散法测定其抑菌活性。【结果】从各药用组织器官中分离出内生细菌34株,隶属于7科11属,内生细菌的数量和群落组成存在明显的组织特异性,其数量表现为根皮>叶>花>果实,而多样性则表现为花>根皮>叶>果实。芽孢杆菌属为枸杞优势内生菌群,分布于所有组织中;抑菌实验结果表明有76.5%的内生菌对一种或多种病原菌的生长有抑制作用,芽孢杆菌属菌株R2、R7、L3和短波单胞菌属的R3拮抗番茄炭疽杆菌和玉米大斑病菌的能力较强,而多数菌株对大肠杆菌和金黄色葡萄球菌的抑制能力较弱。【结论】枸杞可培养内生细菌遗传多样性丰富,对植物病原菌有较强的抑制活性。     

Determination of antimicrobial susceptibilities of clinically isolated anaerobic bacteria by E-test, ATB-ANA and agar dilution

A total of 60 anaerobic strains were isolated from 322 clinical specimens. These isolates were tested for susceptibility to seven antibiotics (penicillin G, amoxicillin/clavulanic acid, cefoxitin, imipenem, chloramphenicol, metronidazole, clindamycin) by using ATB-ANA and Epsilometer test (E-test) strips and the results were compared with the gold standard agar dilution method. Imipenem was found as the most effective agent in vitro among the agents tested (100%). Susceptibility to penicillin G, amoxicillin/clavulanic acid, cefoxitin, chloramphenicol, metronidazole and clindamycin are 36.7%, 83.3%, 88.3%, 96.6%, 85% and 90%, respectively. E-test has showed a good correlation (r=0.62, p=0.001) statistically with the results of agar dilution (total agreement for all antibiotics changing between 90.01% and 98.45%) and a moderate correlation (r=0.45, p=0.048) with the results of ATB-ANA method (total agreement for all antibiotics changing between 75.46% and 98.76%). However, the routine use of agar dilution procedure is concluded to be cumbersome, whereas E-test method offers a reliable alternative.     

艾丁湖可培养嗜盐菌多样性及功能酶、抗菌活性筛选

【目的】探究艾丁湖可培养嗜盐菌的多样性、功能酶活性以及抗菌活性,进一步了解其次级代谢产物情况,为新型生物活性物质的发掘提供依据。【方法】选用以20种糖及糖的衍生物作为唯一碳源的寡营养培养基从艾丁湖5个样点中分离得到298株嗜盐菌,根据形态特征去重复后,选取62株菌运用16S r RNA基因系统发育分析的方法研究样品中嗜盐菌的多样性;从不同类群选取22株代表菌株,采用点接法进行3种功能酶的筛选,运用平板对峙法检测代表菌株对12种病原菌的抗菌活性。【结果】从5%、10%和15%3个盐浓度中分别分得221、54和23株嗜盐菌,获得的嗜盐菌分布在9个科18个属;其中放线菌分布于4个属,细菌分布于14个属;Nocardiopsis和Pontibacillus属为艾丁湖可培养嗜盐菌的优势类群,分别占17.7%和16.1%;有15株嗜盐菌相似性低于98.5%,可能为潜在新种。所选取的22株代表菌株中,分别有68.2%、22.7%和72.7%的实验菌株具有蛋白酶、淀粉酶和酯酶活性;45.5%的代表菌株对12种病原菌表现出了抗至少1种病原菌的活性,其中一株Nocardiopsis属放线菌能抗9种病原菌,表现出了广谱的抗菌活性。【结论】新疆艾丁湖土样中嗜盐菌的多样性较丰富,而且具有较好的生物活性,为后续进一步研究其次级代谢产物提供了依据。