枯草芽孢杆菌HS-A38高产抗菌肽突变株的筛选及其抑菌机理的研究

本研究通过采用多种诱变方法以提高枯草芽孢杆菌HS-A38产抗菌肽能力,并研究诱变株产抗菌肽活性物质对副溶血弧菌的抑制作用机理。原始菌株经过多种复合诱变后,最终获得一株抗菌活性明显提高的诱变菌株(mut HS-301)。进一步研究采用双倍稀释法确定最低抑菌浓度,并通过扫描电镜观察、胞膜通透性和胞膜完整性的测定,初步阐述该抗菌肽的抑菌机理。结果表明:诱变菌株mut HS-301对副溶血弧菌的抑菌活性较原始菌株提高了20%;抗菌肽成品对副溶血弧菌的最低抑菌浓度为0.625 mg/m L;抗菌肽破坏了菌体细胞的形态,出现形变;随着抗菌肽作用时间的增加,胞外紫外吸收物质和胞外蛋白含量明显增加,说明抗菌肽影响了副溶血弧菌细胞膜的通透性,其胞膜的完整性遭到破坏,影响其代谢活性,从而抑制副溶血弧菌的生长。该研究表明,微生物产抗菌肽主要作用于细菌细胞膜,该产品适宜添加到水产动物养殖饲料中,对提高水产动物免疫力和预防疾病起重要作用。     

副溶血弧菌EMA-PCR检测技术的建立

PCR技术被广泛应用于副溶血弧菌的检测中, 但传统的PCR技术无法区分样品中的死细菌与活细菌, 往往使检测结果出现较高的假阳性。因此, 将叠氮溴乙锭(Ethidium monoazide bromide, EMA)与PCR技术结合, 建立一种快速、准确的副溶血弧菌检测方法。以dnaJ基因为检测副溶血弧菌的靶基因, 分别用副溶血弧菌的纯培养细胞及其基因组DNA作模板进行PCR检测, 灵敏度分别为2.5×104 CFU/mL和6×102 fg/μL。在检测样品前处理过程中加入EMA, 当EMA的浓度小于5 mg/L时, EMA对活菌靶基因的扩增没有明显的抑制; 而终浓度为2 mg/L的EMA, 能有效抑制1×108 CFU/mL副溶血弧菌死菌的扩增。活菌和死菌混合体系的PCR结果表明, EMA-PCR能有效降低副溶血弧菌检测过程中的假阳性。     

副溶血弧菌高效消毒剂的筛选及PHMG防治对虾AHPNS的应用研究

为寻找凡纳滨对虾急性肝胰腺坏死综合症(AHPNS)重要致病菌——副溶血弧菌的高效低毒消毒剂, 选取了包括苯扎溴铵、聚六亚甲基胍盐酸盐(PHMG)等22种水产或医学上常用的消毒剂, 比较分析其对副溶血弧菌的杀灭效果。悬液定量杀菌试验发现, 22种消毒剂中PHMG对副溶血弧菌的杀灭效果最好。接着, 进一步分析PHMG的有效杀菌浓度, 结果显示, PHMG浓度达到0.2 mg/L时作用48h即能100%地杀死副溶血弧菌。对7种常见病原菌和凝结芽孢杆菌的杀灭试验显示, PHMG对弧菌的杀灭效果最好, 0.5 mg/L PHMG能100%地杀灭四种弧菌, 对迟钝爱德华菌、嗜水气单胞菌和海豚链球菌的杀灭效果较差, 对凝结芽孢杆菌的杀灭效果最差。急性毒性试验得出PHMG 24h的LC₅₀=80.28 mg/L; 48h的LC₅₀=23.32 mg/L, 安全浓度SC=0.59 mg/L。对攻毒副溶血弧菌凡纳滨对虾的保护试验显示, PHMG浓度达到0.5 mg/L时, 24h对对虾的相对保护率达(77.78±5.01)%左右, 且与三个高浓度组相对保护率差异不显著。随着PHMG浓度的升高, 凡纳滨对虾肝胰腺所含副溶血弧菌数量明显减少, 48h后1.1及1.4 mg/L组副溶血弧菌数量降为0。降解试验结果显示: 0.5 mg/L的PHMG在第5、第6天浓度有所下降, 1 mg/L的PHMG在试验7d中基本保持稳定。研究证明了PHMG具有高效、低毒、低降解的特性, 是防治凡纳滨对虾AHPNS的理想消毒剂。     

恩诺沙星对4种水产致病弧菌的抑杀菌效应

采用二倍稀释法测定恩诺沙星对溶藻弧菌、最小弧菌、哈维氏弧菌、创伤弧菌4种12株菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),结果表明:恩诺沙星对12株弧菌的MIC为0.1?0.8 mg/L,MBC为0.4?3.2 mg/L。在此基础上,从每种菌中选取一株对恩诺沙星较为敏感的菌株,研究恩诺沙星不同药物浓度(1 MIC、2 MIC、4 MIC)对4种弧菌的杀菌动力学和抗菌后效应(PAE),结果显示:各浓度恩诺沙星对溶藻弧菌X040625-R菌株均有较强的杀菌作用,而对哈维氏弧菌M071202-H、创伤弧菌Q050723-C、最小弧菌H010911-Z等3菌株却表现出了低浓度抑菌、高浓度缓慢杀菌的作用。恩诺沙星的抗菌后效应PAE与药物浓度及细菌与药物的接触时间成正相关,恩诺沙星对溶藻弧菌X040625-R的PAE最长,对哈维氏弧菌M071202-H的PAE最短。     

蒙脱石对细菌黏附Caco-2细胞的影响

采用Caco-2细胞培养模型,观察两歧双歧杆菌、嗜酸乳杆菌、嗜水气单胞菌、副溶血弧菌、大肠杆菌、鼠伤寒沙门菌的黏附率,并在培养液中加入蒙脱石,计算蒙脱石对细菌黏附的阻断率,探讨蒙脱石对上述细菌黏附作用的影响。结果表明:所试菌与Caco-2细胞均有不同程度的黏附作用;蒙脱石对细菌黏附Caco-2细胞均有不同程度的阻断作用,对病原菌黏附Caco-2细胞的阻断作用要明显大于其对益生菌的阻断效果,其中对大肠杆菌、鼠伤寒沙门菌、嗜水气单胞菌、副溶血弧菌黏附的阻断率分别为54.22%、48.41%、60.53%、50.64%,而对两歧双歧杆菌、嗜酸乳杆菌黏附的阻断率分别为25.64%和21.49%。结果提示蒙脱石可有效阻断病原菌黏附,从而防治肠道细菌感染和细菌移位。     

低温寡营养条件下副溶血弧菌形成活的非可培养状态及其复苏研究

为研究在低温寡营养条件下副溶血弧菌(Vibrio parahaemolyticus)能否进入活的非可培养状态(VBNC),将浓度为1×1010CFU/mL的副溶血弧菌HW799在陈海水中4℃保存,每隔5天取样分别用吖啶橙染色荧光显微镜直接计数法(AODC)、活菌直接计数法(DVC)和涂布平板法(PC)测定细菌总数、活细菌数和可培养细菌数.在第30天时总细菌数基本不变,仍保持在109CFU/mL,活菌数为106CFU/mL,比总菌数低了约三个数量级,可培养细菌数为零,表明绝大部分副溶血弧菌HW799进入了VBNC状态;用扫描电镜、流式细胞仪对副溶血弧菌HW799活的非可培养状态、正常状态以及复苏后的细胞形态的研究表明进入VBNC状态后副溶血弧菌HW799形状变为球状,体积比正常状态明显变小,活细胞数也略有减少;采用在培养液中添加营养物质升温培养的方法,使VBNC状态的副溶血弧菌细胞在48h内复苏为可培养状态,复苏后的副溶血弧菌HW799与正常状态的细菌形态相似.     

基于单克隆抗体的多元弧菌流式细胞术检测技术的研究

【目的】建立一种基于单克隆抗体的多元弧菌流式细胞仪检测技术。【方法】以副溶血弧菌表面蛋白r-OmpW的单克隆抗体(mAb)为基础,以细胞染色率为指标优化流式细胞仪检测副溶血弧菌时所需mAb的反应浓度和反应时间。通过菌落数对比评价在优化条件下流式细胞术方法的准确度、检出限和精密度。根据所建立的流式细胞术平台分析鉴定单抗对其它5种多元弧菌的特异性。【结果】流式细胞仪检测副溶血弧菌时所需r-OmpW单克隆抗体的优化反应浓度为20 mg/L,反应时间为60 min。当菌浓在104 107cells/mL范围时,检测值可信度较高,可特异性识别5种病原弧菌。对含不同菌体浓度的样品进行重复检测,变异系数均在7%以内。【结论】所建立的这种基于单克隆抗体的多元弧菌流式细胞仪检测技术可快速准确地检测多种病原弧菌。     

黄芩乙醇提取物通过下调NAD特异的谷氨酸脱氢酶抑制哈维氏弧菌生长北大核心CSCD

哈维氏弧菌(Vibrio harveyi)是水产动物的常见致病菌,对人类健康和水产经济带来巨大威胁。抗生素的滥用使得药物残留和耐药性问题变得日益严重。因此,迫切需要寻找新型、不易产生耐药性和低毒的抗菌物质。本文研究黄芩醇提物对哈维氏弧菌的抑制作用及抑菌机制。实验结果表明,黄芩醇提物对哈维氏弧菌的抑菌圈为18.33±0.58 mm,最低抑菌浓度(MIC)和最小杀菌浓度(MBC)分别为7.92 mg/m L和15.84 mg/m L。经分析型扫描电镜(SEM)观察和细菌胞内外蛋白质浓度测定,发现实验组菌体表面虽有细小破裂,但形态依然完整,表面光滑,菌体细胞膜仍保持相对完整性;通过SDS-PAGE、蛋白质质谱(MALDI-TOF-TOF-MS)和实时荧光定量PCR分析,显示黄芩醇提物抑制哈维氏弧菌体内NAD特异性的谷氨酸脱氢酶(NAD-specific glutamate dehydrogenase,NAD-GDH)及其mRNA的表达。本研究表明,黄芩醇提物通过下调NAD特异性的谷氨酸脱氢酶表达而抑制哈维氏弧菌的生长,为中药应用于水产养殖提供了新的证据。     

植物精油对大菱鲆弧菌的体外和体内抗菌活性

【目的】研究天然植物精油对大菱鲆弧菌的体外和体内抗菌活性。【方法】采用纸片扩散法和微量肉汤稀释法对14种植物精油或其组分的体外抑菌活性进行检测;通过细菌形态透射电镜观察、胞内乳酸脱氢酶及核酸释放研究山苍子精油对大菱鲆弧菌的膜损伤作用;采用大菱鲆人工攻毒感染实验研究山苍子精油的体内抗菌作用。【结果】14种植物精油或其组分对大菱鲆弧菌具有不同程度的抑制效果,其中肉桂醛的抗菌活性最强,最低抑菌浓度为0.25μL/m L;百里香酚、丁香酚、柠檬醛和山苍子的抗菌活性次之,最低抑菌浓度为0.5μL/m L;山苍子精油可破坏大菱鲆弧菌的细胞膜,并导致胞内蛋白酶和核酸外泄;经200μL/L山苍子精油浸浴后,大菱鲆攻毒后死亡率由对照组50%降至0。【结论】富含芳香醛、芳香酚和柠檬醛的植物精油对大菱鲆弧菌具有良好抗菌活性,有望替代抗生素用于大菱鲆弧菌病的防治。     

用基于TaqMan探针的Real-timePCR技术定量检测副溶血弧菌

副溶血弧菌是一种引起食源性疾病的重要病原菌,传统的鉴定方法费时费力且容易出现假阴性,建立一种定量检测副溶血弧菌基因的方法尤为重要。根据GenBank公布的副溶血弧菌的gyrB基因序列设计一对引物和TaqMan探针,建立了基于TaqMan探针的Realtime PCR方法。通过对9种细菌（12株菌株）的DNA进行扩增,结果所有4株副溶血弧菌均可产生扩增曲线,其他8株非副溶血弧菌均不产生扩增曲线,证明了引物和探针具有很高的特异性。细菌纯培养物品和人工布菌的检测敏感度分别为1 CFU/ PCR反应体系和10 CFU/PCR反应体系,相关系数均为0.99（r2＝0.99）,整个试验可在1h内完成。建立的方法可用于海产品中副溶血弧菌的快速定量检测。     

三种消毒剂对大黄鱼致病性弧菌的体外杀灭作用研究

为了解不同消毒剂对大黄鱼中致病性弧菌的抑菌效果。采用纸片扩散法比较了聚维酮碘(PVP-I)、聚六亚甲基双胍(PHMB)和过氧化氢(H2O2)对溶藻弧菌和副溶血性弧菌的抑菌效果,再以肉汤稀释法测定了三种消毒剂的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)及MBC/MIC比值,在此基础上,获得了抑菌效果相对较优的H2O2、PHMB对溶藻弧菌、副溶血性弧菌及其混菌的杀菌动力学曲线。结果表明,H2O2(3%)对两种弧菌具有最大的抑菌圈直径(19~21mm),PHMB(30mg/mL)次之(约为15mm),PVP-I(10%)的抑菌效果最弱(小于10mm)。3种消毒剂的MIC值由小到大依次为PHMB相似文献   

Impact of exposure to bacteria on metabolism in the penaeid shrimp Litopenaeus vannamei

We hypothesized that aggregation of bacteria and hemocytes at the gill, which occurs as part of the shrimp's antibacterial immune defenses, would impair normal respiratory function and thereby disrupt aerobic metabolism. Changes in oxygen uptake and lactate accumulation were determined in Litopenaeus vannamei, the Pacific white shrimp, following injection with either saline (control) or a strain of the gram-negative bacterium Vibrio campbellii that is pathogenic in crustaceans. The rate of oxygen uptake was determined during the first 4 h after injection and after 24 h. Injection of bacteria decreased oxygen uptake by 27% (from 11.0 to 8.2 micromol g-1 h-1) after 4 h, while saline-injected shrimp showed no change. Decreased oxygen uptake persisted 24 h after Vibrio injection. In well-aerated water, resting whole-animal lactic acid levels increased in shrimp injected with bacteria (mean=2.59 micromol lactate g-1+/-0.39 SEM, n=8) compared to saline-injected control shrimp, but this difference did not persist at 24 h. Exposure to hypercapnic hypoxia (PCO2=1.8 kPa, PO2=6.7 kPa) also resulted in significant whole-body lactic acid differences (mean=3.99 and 1.8 micromol g-1 tissue in Vibrio and saline-injected shrimp, respectively). Our results support the hypothesis that the crustacean immune response against invading bacteria impairs normal metabolic function, resulting in depression of oxygen uptake and slightly increased anaerobic metabolism.     

An analysis of the concentration change of intermediate metabolites by gene manipulation in fatty acid biosynthesis

In this report, concentration of malonic acid and acetic acid produced in Escherichia coli were investigated by the expression of acetyl-CoA carboxylase genes (accs) and a malonyl-CoA:ACP transacylase gene (fabD). Both malonyl-CoA and acetyl-CoA are essential intermediate metabolites in the fatty acid biosynthetic pathway, and are reversibly transformed to malonic acid and acetic acid, respectively in the cell. Acetyl-CoA is converted to malonic-CoA by acetyl-CoA carboxylases (Accs), which are composed of 3 different subunits (AccA, AccB, and AccC), and the resulting malonyl-CoA is then converted to malonyl-[acp] by malonyl-CoA:ACP transacylase (FabD). In this study, these genes were separately cloned, and the influences of overexpression of 4 different genes on the concentration of malonic acid and acetic acid were analyzed. Compared with the wild type E. coli, a recombinant strain containing 3 acc genes together showed a 41.03% enhanced malonic acid production, and a 4.29-fold increased ratio of malonic acid to acetic acid.     

牛樟芝发酵液提取物抗菌活性研究

牛樟芝作为一种珍稀食用和药用菌,具有极大的开发潜力。该研究以麦芽浸粉肉汤液体培养基(BD,美国BD公司)对牛樟芝菌丝体进行摇床培养60 d后,收获发酵液并用乙酸乙酯对其进行萃取,浓缩至干获得提取物;同时,采用抑菌圈法评价培养物对13种致病细菌抗菌活性(蜡样芽孢杆菌、缓慢芽孢杆菌、无乳链球菌、短小芽孢杆菌、福氏志贺氏菌、枯草芽孢杆菌、金黄色葡萄球菌、藤黄微球菌、副溶血性弧菌、溶血性葡萄球菌、铜尿假单胞菌、乙型副伤寒沙门氏菌、大肠埃希菌),并检测相应致病细菌的最低抑制浓度(MIC)。结果表明:牛樟芝麦芽浸粉肉汤发酵液提取物对供试的13种致病菌均有抑菌活性;在供试的13种致病菌中,提取物对缓慢芽孢杆菌、短小芽孢杆菌、枯草芽孢杆菌、副溶血性弧菌、藤黄微球菌5种致病菌的最低抑制浓度值均小于80μg·m L~(-1),其中对藤黄微球菌的最低抑制浓度最低为66.5μg·m~(-1);随着培养时间的增加,提取物的抗菌活性也增加。这说明牛樟芝菌丝体在液体培养条件下,能够产生广谱高效抑菌活性的次生代谢产物。该研究结果为牛樟芝进一步的有效利用开发奠定了理论基础。     

Assessment of Cu (II)-Bearing Montmorillonite on Cd Adsorption

The abilities of montmorillonite (MMT) and Cu (II)-bearing montmorillonite (Cu-MMT) for adsorption of Cd from aqueous solutions have been studied at different optimized conditions of shaking time, pH value, and initial concentration, respectively. The results showed that the adsorbability of Cu-MMT onto Cd is stronger than the MMT. The amount of Cu-MMT necessary to remove Cd from solution was about 0.5 g for 100–200 mg/L Cd solution at a pH of 4.0. The maximum adsorption (94%) was 20 min of shaking time using Cu-MMT as adsorbent. The maximum adsorption of Cd = 95% at a pH of 4.0. By increasing the initial concentration, the adsorption amount of Cd onto Cu-MMT increased. Animal experiment of this study indicated that the addition of Cu-MMT to the Cd-contaminated diet of pigs reduced significantly the Cd levels in tissues (p < 0.05) and increased contents in feces (p < 0.01), improving the safety of animal products. Thus, the overall results established the use of Cu-MMT as a heavy metal adsorbent in animal’s diets, implying a potential protective role against heavy metal toxicities.     

Antibacterial action of L-amino acid oxidase from the skin mucus of rockfish Sebastes schlegelii

L-amino acid oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H(2)O(2) generated in the oxidative process of L-amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H(2)O(2). SSAP inhibited potently the growth of Aeromonas salmonicida, Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 microg/mL, respectively. H(2)O(2) inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P. damselae subsp. piscicida and Escherichia coli, SSAP was demonstrated to bind to P. damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H(2)O(2) induced cell surface damage to A. salmonicida, remarkable elongation of P. damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.     

Distribution and significance of heterotrophic marine bacteria with antibacterial activity

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.     

Distribution and significance of heterotrophic marine bacteria with antibacterial activity.

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.     

Inhibition of C4 photosynthesis by (benzamidooxy)acetic acid

(Benzamidooxy)acetic acid (common name benzadox) which has herbicidal properties was evaluated as a potential inhibitor of photosynthesis in C₄ plants. Among enzymes of the C₄ pathway, it was a relatively strong inhibitor of alanine aminotransferase in in vitro experiments at concentrations of 5mM. In benzadox treated leaves of Panicum miliaceum, a NAD-malic enzyme type C₄ species, there was strong inhibition of both alanine and aspartate aminotransferase and of photosynthetic O₂ evolution within one hour. Consistent with the inhibition of these enzymes of the C₄ cycle, the pool sizes of metabolites of the cycle was altered: the aspartate level was increased two fold, while the levels of other metabolites such as pyruvate, alanine, oxalacetate and malate were decreased. Kinetic studies with partially purified alanine aminotransferase showed that benzadox is a competitive inhibitor with respect to alanine and a noncompetitive inhibitor with respect to 2-oxoglutarate. Comparisons between the structures and inhibitory actions of benzadox and (aminooxy)acetic acid, the latter a potent inhibitor of alanine and aspartate aminotransferases, suggest that in vivo, benzadox may exert its effect through metabolism to (aminooxy)acetic acid.Abbreviations benzadox (benzamidooxy)acetic acid - DTE dithioerythritol This research was supported in part by gift funds from Monsanto Agricultural Products Company. St. Louis, Missouri, and by NSF Grant PCM-8107953.