不同酚类成分及其组合对P.aeruginosa PAO1的抑制效果研究

为研究茶叶酚类成分及其复方对P.aeruginosa PAO1的体外抑制作用,筛选出用药量最低的最佳抑菌组方,采用倍比稀释法结合刃天青显色测定5种酚类成分对P.aeruginosa PAO1的最小抑菌浓度(MIC),并采用响应面法研究对P.aeruginosa PAO1具有较强抑制作用的最佳配伍。结果表明5种酚类成分对P.aeruginosa PAO1均有不同程度的抑制作用,5种茶叶活性成分对P.aeruginosa PAO1的抑菌性大小为:EGCG>TF60=ECG>EGC>EC。响应面分析结果表明,EGCG、EGC、ECG、TF60这4种酚类成分以1.0∶4.0∶4.81∶4.0的比例配伍时对P.aeruginosa PAO1具有最佳抑菌效果。

Detoxification strategy of Microcystis aeruginosa to the toxicity of Cd(Ⅱ):role of EPS in alleviating toxicity

Although many studies have found that cadmium(Cd)can be toxic to microalgae,only a few reports focused on the role of extracellular polymeric substances(EPS)in Cd(Ⅱ)detoxification.The biochemical and physiological endpoints of Microcystis aeruginosa,including the composition and functional groups of soluble EPS(SL-EPS),loosely bound EPS(LB-EPS),and tightly bound EPS(TB-EPS),were detected to elucidate the toxicity and detoxification mechanisms of Cd(Ⅱ)for cyanobacteria.Toxicological and physiological assays on M.aeruginosa showed that the 0.25-mg/L Cd(Ⅱ)resulted in a larger inhibition on growth and F_(v)/F_(m).Nevertheless,Cd(Ⅱ)significantly induced much higher contents of superoxide dismutase(SOD),intracellular microcystin LR(MC-LR),extracellular MC-LR,and EPS.Scanning electron microscopy with energy dispersive X-ray spectroscopy confirmed that Cd(Ⅱ)was absorbed into the EPS layer.Fourier transform infrared spectrum analysis revealed that the functional groups bound with Cd(Ⅱ)of algae biomass,SL-EPS,LB-EPS,and TB-EPS were somewhat different.The C=O/C=N groups ofδ-lactam or protein were their prominent functional groups,suggesting that amide or proteins in the EPS played a key role in the adsorption in Cd(Ⅱ).The concentration of 0.25 mg/L of Cd(Ⅱ)may change the chemical structure of EPS by altering the production of protein-like substances containing tryptophan.This study indicated that M.aeruginosa could detoxify Cd(Ⅱ)stress via induction of antioxidant capacity(higher SOD activity and MC synthesis),EPS production,and modification in chemical structure of EPS.

Microbiologically influenced corrosion resistance enhancement of coppercontaining high entropy alloy Fe_(x)Cu_(1−x)CoNiCrMn against Pseudomonas aeruginosa

To enhance the microbiologically influenced corrosion(MIC)resistance of FeCoNiCrMn high entropy alloy(HEAs),a series of Fe_(x)Cu_((1−x))CoNiCrMn(x=1,0.75,0.5,and 0.25)HEAs were prepared.Microstructural characteristics,corrosion behavior(morphology observation and electrochemical properties),and antimicrobial performance of Fe_(x)Cu_((1−x))CoNiCrMn HEAs were evaluated in a medium inoculated with typical corrosive microorganism Pseudomonas aeruginosa.The aim was to identify copper-containing FeCoNiCrMn HEAs that balance corrosion resistance and antimicrobial properties.Results revealed that all Fe_(x)Cu_((1−x))CoNiCrMn(x=1,0.75,0.5,and 0.25)HEAs exhibited an FCC(face centered cubic)phase,with significant grain refinement observed in Fe_(0.75)Cu_(0.25)CoNiCrMn HEA.Electrochemical tests indicated that Fe_(0.75)Cu_(0.25)CoNiCrMn HEA demonstrated lower corrosion current density(i_(corr))and pitting potential(E_(pit))compared to other Fe_(x)Cu_((1−x))CoNiCrMn HEAs in P.aeruginosa-inoculated medium,exhibiting superior resistance to MIC.Anti-microbial tests showed that after 14 d of immersion,Fe_(0.75)Cu_(0.25)CoNiCrMn achieved an antibacterial rate of 89.5%,effectively inhibiting the adhesion and biofilm formation of P.aeruginosa,thereby achieving resistance to MIC.

Molecular investigation of exoU and exoY virulence genes in Pseudomonas aeruginosa collected from hospitalized patients in North of Iran:A descriptive-analytical study

Objective:To investigate the frequency of exoU and exoY genes in patients with Pseudomonas aeruginosa infection.Methods:In this study,100 clinical isolates of Pseudomonas aeruginosa were collected from patients hospitalized in educational-therapeutic hospitals and were identified using standard microbiological tests.Then,the antibiotic resistance pattern of the isolates was determined by the disk agar diffusion method.The bacterial DNAs were extracted by the alkaline lysis method.Finally,the presence of exoU and exoY genes was evaluated by the PCR test.Results:In this study,47%,72%,29%,39%,40%,and 44%of the isolates were non-susceptible to piperacillin,aztreonam,ceftazidime,imipenem,tobramycin,and ciprofloxacin,respectively.In addition,95%and 93%of the clinical isolates carried the exoU and exoY genes.Blood and fecal isolates had both virulence genes,while only one wound isolate had neither genes.Meanwhile,all urinary isolates contained the exoY gene and only one isolate lacked the exoU gene.Also,88 isolates simultaneously had both exoU and exoY genes.Conclusions:High prevalence of exoU and exoY genes in this region indicates a significant role of typeⅢsecretion system in pathogenesis of Pseudomonas aeruginosa.The typeⅢsecretion system may be a suitable target to reduce the pathogenicity of this bacterium.

Efficacy of Pudilan Xiaoyan Oral Liquid in the treatment of pneumonia induced by drug-resistant Pseudomonas aeruginosa

Background:Pudilan Xiaoyan Oral Liquid(PDL)is a Chinese patent medicine with notable pharmacological properties,including anti-inflammatory and antibacterial effects.Drug-resistant Pseudomonas aeruginosa infection is a common and refractory bacterial infection in clinical practice.Due to its high drug resistance,it brings great challenges to treatment.This study aimed to assess the therapeutic efficacy of PDL in a murine model of pneumonia induced by drug-resistant Pseudomonas aeruginosa.Methods:Three different doses of PDL(11 mL/kg/d,5.5 mL/kg/d,2.75 mL/kg/d)were used to observe lung tissue pathology and inflammatory cytokine levels in pneumonia mouse models induced by multidrug-resistant Pseudomonas aeruginosa(MDR-PA).Additionally,the protective efficacy of PDL against mortality in infected mice was evaluated using a death model caused by MDR-PA.Finally sub-MIC concentration of levofloxacin was used to induce drug-resistant mice pneumonia model to evaluate the role of PDL in reversing drug resistance.Experimental data are expressed as mean±standard deviation.Statistical significance was determined by one-way analysis of variance followed by Tukey’s multiple-comparisons test.Results:Treatment effect of PDL on MDR-PA pneumonia:the medium and small doses of PDL can significantly reduce the lung index of multi-drug resistant bacteria infected pneumonia model mice(P<0.05),the lung index inhibition rates for these groups were 55.09%and 58.43%,and improve the degree of lung tissue lesions of mice;The expression of serum cytokines keratinocyte chemoattractant,tumor necrosis factor-αand monocyte chemoattractant protein-1 could be decreased in the three dosage groups of PDL(P<0.01).PDL treatment not only lowered the mortality but also extended the survival duration in mice infected with MDR-PA.It was found after sub-MIC concentration of levofloxacin induced resistance of Pseudomonas aeruginosa to pneumonia in mice.Compared with the model group,the lung index of mice in high and medium PDL doses was significantly reduced(P<0.05),with inhibition rates of 32.16%and 37.73%,respectively.Conclusion:PDL demonstrates protective effects against MDR-PA infection pneumonia,notably decreasing serum inflammatory factor levels.It shows promise in mitigating antibiotic resistance and offers potential for treating pneumonia resulting from Pseudomonas aeruginosa resistance.

Evaluation of Azadirachta indica cultivated in Egypt against biofilm formation by Pseudomonas aeruginosa: Insights from molecular docking studies targeting LasR

Background:Azadirachta indica(A.indica),commonly known as neem,is a widely distributed medicinal plant in Asia and Africa and is well known to have a wide spectrum of biological activity.A.indica is considered a skin food that was traditionally used in different cultures to treat a wide range of skin disorders.A.indica was reported to possess antibacterial activity against Pseudomonas aeruginosa(P.aeruginosa)which is considered the most common biofilm model organism.This study aims to investigate the ability of A.indica cultivated in Egypt to inhibit/reduce the biofilm formation by P.aeruginosa.Methods:The microtiter plate assay was used to evaluate the anti-biofilm activity of neem,cultivated in Egypt,leaves against P.aeruginosa as well as the ability to reduce the activity of P.aeruginosa.To investigate the phytocompounds responsible for their bioactivity and to explore potential interactions between their bioactive components and one of the quorum-sensing regulatory proteins of P.aeruginosa involved in biofilm formation,liquid chromatography-mass spectrometric and molecular docking studies were done.Results:Results showed that methanol extract of leaves can reduce the formation of P.aeruginosa biofilm at lower concentrations than those reported in other regions with 1.25 mg/mL as the optimum concentration.The two-way analysis of variance revealed the significance of the extract effect and its concentration on the reduction of biofilm formation(P<0.05).Liquid chromatography-mass spectrometric study revealed the presence of fourteen compounds that belong to limonoids and flavonoids.Molecular docking analysis against LasR,the quorum-sensing regulatory protein,of P.aeruginosa supported these findings.Nimbolinin,a limonoid,has achieved the highest Libdock score of 138.769.Conclusion:It was concluded that A.indica,cultivated in Egypt,leaves can target LasR as a new mechanism of action for biofilm control by A.indica and therefore could be a good source of leads for anti-biofilm medicine.

Prokaryotic Expression of Pseudomonas Aeruginosa Lipase Gene

[Objective] The aim of this study was to investigate the prokaryotic expression of pseudomonas aeruginosa Lipase gene.[Method]Lipase gene was amplified by PCR from the genome DNA of pseudomonas aeruginosa,and its nucleotide sequence was determined.The prokaryotic expression vector of Lipase gene was constructed by the gene recombination technique.The protein expression was induced for 4 hours by IPTG with the final concentration of 1.0 mmol/L,and then SDS-PAGE electrophoresis was analyzed.[Result]The sequence of mature peptides in Lipase gene cloned from pseudomonas aeruginosa had a 99.36% homology with that of pseudomonas aeruginosa lipase submitted in NCBI,so the prokaryotic expression vector of Lipase gene pET32a-Lip was successfully constructed.Furthermore,the results of SDS-PAGE electrophoresis showed that the target gene was expressed highly and effectively.[Conclusion]The cloned pseudomonas aeruginosa lipase with its signal peptide could be normally expressed in E.coli and also used for further study.

黑暗条件下不同氮源对铜绿微囊藻(Microcystis aeruginosa)生长和pH的影响

在黑暗条件下,利用不同形态的氮源(硝酸盐氮,氨氮,有机氮和硝酸盐氮,有机氮)培养蓝藻水华优势种铜绿微囊藻(Microcystis aeruginosa),分析其氮代谢和对水体pH的影响。研究结果表明,在不同氮源的培养液中铜绿微囊藻密度在最初的24h内出现波动,之后下降。培养液中pH值在试验最初的24h显著下降,之后趋于稳定,在硝态氮培养液中pH值下降最大,从8.18下降到7.19,其反硝化作用产生的NO2-浓度也最大。不同氮源培养液中总氮含量都有所下降,以混合氮源培养液中总氮减少量最大,说明化合态氮经过反硝化作用生成了氮气并溢出培养液,因此,在黑夜条件下藻华水体中存在反硝化作用。

好氧反硝化菌Pseudomonas aeruginosa NO62筛选分离与性质鉴定

从生活污水处理场活性污泥中筛选分离到1株具有好氧反硝化特性的菌株NO62,经过常规生理生化鉴定、Vitek-32细菌鉴定系统鉴定结合16S rDNA鉴定,确定其为铜绿假单胞菌(Pseudomonas aeruginosa)。菌株P.aeruginosa NO62在好氧培养(150 r/min)时在37℃生长最好,但其反硝化活性则在30℃培养时最好。反硝化活性与菌体生长成正相关,在菌体的对数生长期硝态氮迅速被还原成亚硝态氮,继续培养则亚硝态氮浓度也逐渐降低,表明亚硝态氮被进一步还原。通过PCR的方法在NO62的总DNA中也检测到硝酸盐还原酶的编码基因narG。对NO62菌株反硝化活性的研究,可为该菌应用于污水脱氮处理奠定良好的理论基础。

Study on the Phosphorus Metabolism of Microcystis aeruginosa and Adnascent Pseudomonas under Different pH Conditions

[Objective] The study aimed to discuss the effects of pH value on the growth metabolism of Microcystis aeruginosa and the phosphorus metabolism relationship with adnascent Pseudomonas.[Method] By the phosphorus uptake experiment of M.aeruginosa under different pH conditions(8.0-10.0) and the effect experiment on the phosphorus metabolism of M.aeruginosa and adnascent Pseudomonas under different pH conditions(7.0-9.0),the phosphorus uptake of M.aeruginosa in the short time and the growth curve of M.aeruginosa,the change of phosphorus concentration in the water,the change of total phosphorus content in M.aeruginosa in the longer time were measured.[Results] In the short time,pH value had the effects on the absorption phosphorus ability of M.aeruginosa.As pH value rose,the absorption phosphorus ability enhanced.During the longer time,the higher pH value was,the quicker the growth speed of M.aeruginosa was,and the better the growth situation was.M.aeruginosa had the ability of self regulation pH value and could use the phosphorus well in the water which was released from Pseudomonas.In the system of the algae,bacteria and water,the phosphorus in the bacteria played the role of phosphorus source which was released slowly.Though the phosphorus concentration was lower,it was favorable to the growth of algae.[Conclusions] pH value was the factor that affected the circle of the phosphorus element in the system of algae-bacteria-water.

两种外源性磷及振荡对铜绿微囊藻(Microcystis aeruginosa)生长的影响

为了探索富营养化水体“水华”形成机理 ,研究了甘油磷酸钠和磷酸二氢钾这两种外源性磷和振荡与否对铜绿微囊藻生长的影响 .试验结果表明 :在静止培养时 ,外源性磷能提高铜绿微囊藻的生长速率 ,提高的程度随着外源性磷浓度的上升而下降 .该有机磷比无机磷更有利于对数期铜绿微囊藻的生长 .同静止培养相比 ,振荡能大大降低铜绿微囊藻的生长速率 .在振荡时 ,只有添加高浓度的外源性磷才能促进铜绿微囊藻的生长 .因此 ,在“水华”暴发过程中 ,虽然水体中外源性磷数量是影响铜绿微囊藻生长的重要因素之一 ,但是其它的环境因子可能起到更重要的作用 ,模拟风搅动的振荡就是重要的环境因子之一 ,振荡对铜绿微囊藻生长产生的不利影响要远远大于外源性磷的种类和数量所产生的影响 .图 6表 2参

铜绿假单胞菌(Pseudomonas aeruginosa)BS-03的诱变育种及产鼠李糖脂类生物表面活性剂的摇瓶工艺初探

采用UV、UV+LiCl对铜绿假单胞菌(Pseudomonasaeruginosa)BS-03的野生菌株进行诱变,筛选出一株糖脂产量较高的菌株LY4,可将产量由41g/L提高至68g/L。并对其摇瓶发酵工艺进行了初步研究,较高产量(893g/L)的适宜发酵条件为植物油6%,尿素02%,一定量的无机盐,34℃,初始pH值为8,搅拌转速200～240r/min,发酵周期2d。

温度、pH和氮、磷含量对铜绿微囊藻(Microcystis aeruginosa)生长的影响

以南太湖水华蓝藻铜绿微囊藻为材料,研究了温度、pH和氮、磷浓度对铜绿微囊藻生长的影响,为阐述微囊藻水华形成的机制提供科学依据。研究结果表明,铜绿微囊藻对温度的适应范围较广,15—40℃温度范围内铜绿微囊藻均可生长,25—30℃为铜绿微囊藻的最适生长温度,当温度达到45℃时,铜绿微囊藻停止生长并逐渐死亡。在pH6.5—9.5范围内,铜绿微囊藻均有较高的生长量,最适pH为8.0—9.0,pH值高于9.0时,铜绿微囊藻的最终生长量略有下降。BG-11培养基中添加高浓度的氮(1g/LNH4Cl)后,铜绿微囊藻的生长被显著抑制(P<0.01),但同时添加不同浓度的磷(0.36—10mg/LKH2PO4)后,显著地提高了铜绿微囊藻的生长(P<0.01),不同磷浓度处理之间无极显著差异,氮磷平衡是影响铜绿微囊藻生长的重要因素。

Study on the Inhibitory Mechanism of Sophora japonica N-hexane Extract on Microcystis aeruginosa

[Objective] The research aimed to analyze the inhibitory mechanism of Sophora japonica n-hexane extract which significantly inhibited Microcystis aeruginosa in the prior research.[Method] S.japonica n-hexane extract was used to treat M.aeruginosa.By inspecting chlorophyll a content,protein content,cell membrane permeability and superoxide dismutase（SOD） activity,the inhibitory mechanism of S.japonica n-hexane extract on M.aeruginosa was analyzed initially.[Result] S.japonica n-hexane extract destroyed the cell membrane system of M.aeruginosa,and increased the cell membrane permeability.The contents of chlorophyll a and protein respectively declined to 10% and 50% of that in the control group after cultivated for 7 d,which indicated the photosynthetic reaction system of M.aeruginosa was destroyed.In addition,under the effect of S.japonica n-hexane extract,SOD activity of M.aeruginosa increased in the early period and decreased in the latter period.[Conclusion] The possible inhibitory mechanism of S.japonica n-hexane extract on M.aeruginosa was destroying the cell membrane to increase the membrane permeability;destroying the photosynthetic reaction system to decrease the contents of photosynthetic pigment and protein;making SOD activity showing the phased variation.

盐京九号水稻种植水对铜绿微囊藻(Microcystis aeruginosa)抑制作用的研究

以培养盐京九号水稻幼苗20 d的种植水配置培养液,培养铜绿微囊藻(Microcystis aeruginqsa),追踪测定微囊藻生长量、藻细胞水溶性蛋白含量、超氧化物岐化酶(SOD)活性、叶绿素含量等相关生理生化指标的变化,研究水稻种植水对铜绿微囊藻生长的抑制作用及其机制.实验结果显示,藻细胞生长量和叶绿素含量显著降低;可溶性蛋白含量和SOD酶活性呈先升高后急剧下降的趋势.对水稻种植水抑藻物质作初步分离和鉴定,表明乙醚萃取液中有机酸和酚类组分抑藻效果明显.对有机酸组分进一步做GC/MS分析鉴定,可确定其中含有1,2-二甲基十三酸、棕榈酸和十八烷酸等组分.

不同氮磷营养条件下铜绿微囊藻(Microcystis aeruginosa)对正磷酸盐的蓄积效果

铜绿微囊藻是中国湖泊、水库及其它水域生态系统发生、形成富营养化危害的主要藻类。采用了直接显色法对单细胞铜绿微囊藻(Microcystis aeruginosa)蓄积正磷酸盐的浓度进行测定,并与传统测定方法进行比较。直接显色法测定的磷浓度值减去溶液中可溶性磷浓度值即得到铜绿微囊藻蓄积的磷浓度值。对用不同处理方式处理过的铜绿微囊藻藻液进行过滤显色和直接显色测定,结果表明聚磷酸盐(正磷酸盐)在铜绿微囊藻体内不是游离存在,而很可能是与细胞内某一活性部分结合。在确立测定方法的基础上,研究了以修改了的MⅢ培养基为基本培养条件,改变氮磷浓度对产毒铜绿微囊藻(FACHB942)蓄积正磷酸盐效果的影响。结果显示,①氮磷比不变时,随着起始磷浓度的增加,铜绿微囊藻对正磷酸盐蓄积量也逐渐增加,成明显正相关;②氮浓度不变时,铜绿微囊藻对正磷酸盐的蓄积效果与磷浓度正相关;③而同一磷浓度下,铜绿微囊藻对正磷酸盐的蓄积效果与氮浓度负相关,但氮浓度超过21 mg/L时,氮浓度对于铜绿微囊藻蓄积磷的效果影响变得不规则。

Study on Induction of Microcystis aeruginosa Cell Apoptosis by Signal Molecule N-acetyl-homoserine Lactones(AHLs)

[Objective]The relationship between signal molecule N-acety-homoserine lactones（AHLs） and Microcystis aeruginosa cell apoptosis was studied.[Method]With M.aeruginosa as the test materials treated by 5 μmol/L N-acety-homoserine lactones（AHLs）,the morphology of cell apoptosis was observed through staining with DAPI.[Result]Microcystis aeruginosa cell apoptosis was induced by signal molecule N-acetyhomoserine lactones（AHLs） with the concentration of 1 μmol/L to inhibit the growth and proliferation of Microcystis aeruginosa.[Conclusion] The results provided the important scientific basis and new management ideas for the treatment of water bloom of Microcystis aeruginosa.

磷化氢及其氧化产物动态释放对铜绿微囊藻(Microcystis aeruginosa)生长的影响

就磷化氢释放对铜绿微囊藻的生长进行了研究.采用藻类生长潜力试验(AGP)的方法,研究了磷化氢及其氧化产物亚磷酸盐、次亚磷酸盐对水华暴发优势藻类(铜绿微囊藻)的生长的影响,结果表明:磷化氢及其氧化产物对铜绿微囊藻的生长有重要的影响.富营养水体磷化氢的释放可能在引起湖泊水华暴发中起到了重要的作用.

滇池铜绿微囊藻(Microcystis aeruginosa Küitz)毒素及其在水体中的变化

本文研究了昆明市滇池富营养化所形成水华的主要藻类———铜绿微囊藻 (MicrocystisaeruginosaK櫣ｉｔｚ)的毒素及其在水体的变化。经研究 ,滇池铜绿微囊藻的提取液有一个毒峰出现 ,用双波长紫外分光光度计测定 ,发现于 2 4 0nm出现一个毒峰。用色质谱联用扫描得出 ,其分子离子峰为1 0 4 0 M/Z。用小白鼠注入藻类提取液进行试验 ,发现其肝脏肿大 ,并呈紫褐色。再将肝脏匀浆提取液用高压液相色谱测定 ,发现有毒峰出现。在不同季节、不同地点 ,采取湖水 ,经过滤后 (去除藻类和碎屑 )进行测定 ,结果得出 :在藻类生长旺季及生长繁茂的地点 ,水体中藻毒素的含量较高。而在藻类生长淡季及分布较少的地点 ,水中藻毒素含量则较少。但总的看来 ,经过过滤后的湖水 。

苦草(Vallisneria spiralis)释放的酚酸类物质对铜绿微囊藻(Microcystis aeruginosa)的化感作用

采用广谱性固相萃取小柱富集以10 g(FW)/L的密度培养三天后的苦草(Vallisneria spiralis)种植水,不同溶剂洗脱得到的各组分对铜绿微囊藻(Microcystis aeruginosa)的生长表现出不同程度的抑制,其中甲醇洗脱组分抑藻活性最强.去除该组分中的酚酸后,其抑藻活性下降了22.8%,表明酚酸参与到苦草的化感抑藻作用中.甲醇组分通过液液萃取进一步分离,得到的乙酸乙酯组分进行GC-MS分析,从中检测到苯甲酸、对羟基苯甲酸、对羟基苯乙酸、邻苯二甲酸、对羟基苯丙酸、香草酸、原儿茶酸、阿魏酸和咖啡酸等九种酚酸.酚酸抑藻测试的结果显示其抑藻活性与本身的结构有关,不同酚酸以毒性效应比例多维混合表现出加和抑藻效应,且随着混合种数的增多,酚酸的加和效应增强.以上结果显示苦草可以释放酚酸,抑制铜绿微囊藻的生长,多种化感物质的联合作用可能是水生态系统中沉水植物抑制蓝藻生长的一个重要机制.