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.

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。

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.

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.

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。

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.

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 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.

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.

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.

耐有机溶剂蛋白酶高产菌Pseudomonas aeruginosa PT121的发酵条件

优化了耐有机溶剂蛋白酶产生菌株Pseudomonas aeruginosa PT121的产酶发酵条件。通过单因子试验探索了碳源、有机氮源对菌株发酵产酶的影响;通过正交试验对甘油、果糖、胰蛋白胨进行了水平组合优化。正交试验的方差分析表明,培养基中各成分显著性排列顺序为:胰蛋白胨>甘油>果糖。采用优化后培养基发酵72h,发酵液蛋白酶酶活达到12758U/mL,比优化前提高了6.3倍,为现有国内外报道中耐有机溶剂蛋白酶最高发酵产量。

Study on the Inhibitory Effect of Sophora japonica Extracts against the Growth of Microcystis aeruginosa

[Objective] The research aimed to discuss the inhibitory effect of Sophora japonica extracts against the growth of Microcystis aeruginosa.[Method] The inhibitory effect of extracted liquid of Sophora japonica leaf against the growth of M.aeruginosa was measured.Moreover,the active component was studied and analyzed initially.[Result] The absolute alcohol extract of Sophora japonica leaf was separated by n-hexane,ethyl acetate,n-butanol and water phases in turn.The polar fractions were found being the majority （〉60%）.The non-polar fraction in n-nexane （about 25%） was found significantly inhibiting the growth of M.aeruginosa.The inhibition rates of fraction in n-hexane at the concentrations of 25 and 50 mg/L against M.aeruginosa in 7 d were higher than 75% and 90% respectively.In addition,chlorophyll a of M.aeruginosa was also destroyed in the presence of the hexane fraction.[Conclusion] The research provided the theoretical basis for preventing and controlling the water bloom of M.aeruginosa.

Anti-bacterial mechanism of baicalin-tobramycin combination on carbapenem-resistant Pseudomonas aeruginosa

BACKGROUND Pseudomonas aeruginosa(P.aeruginosa)is an important cause of nosocomial infections,and contributes to high morbidity and mortality,especially in intensive care units.P.aeruginosa is considered a'critical'category bacterial pathogen by the World Health Organization to encourage an urgent need for research and development of new antibiotics against its infections.AIM To investigate the effectiveness of baicalin combined with tobramycin therapy as a potential treatment method for carbapenem-resistant P.aeruginosa(CRPA)infections.METHODS Polymerase chain reaction(PCR)and RT-PCR were used to detect the expression levels of drug-resistant genes(including VIM,IMP and OprD2)and biofilmrelated genes(including algD,pslA and lasR)in CRPA that confer resistance to tobramycin,baicalin and tobramycin combined with baicalin(0,1/8,1/4,1/2 and 1MIC).RESULTS There was a correlation between biofilm formation and the expression of biofilmrelated genes.In addition,VIM,IMP,OprD2,algD,pslA and lasR that confer biofilm production under different concentrations in CRPA were significantly correlated.The synergistic effect of baicalin combined with tobramycin was a significant down-regulation of VIM,IMP,algD,pslA and lasR.CONCLUSION Baicalin combined with tobramycin therapy can be an effective treatment method for patients with CRPA infection.

梳状聚苯乙烯环氧载体的制备及其对Pseudomonas aeruginosa LX1脂肪酶的柔性固定化

以氯磺化交联聚苯乙烯-二乙烯基苯(PS-SO_2Cl)树脂为基质,依次与壳聚糖(Chi)和环氧氯丙烷(ECH)反应,制得了具有柔性链的梳状聚苯乙烯环氧载体PS-SO_2-Chi-ECH,研究了缚酸剂用量和种类、反应时间、反应温度及ECH用量对载体制备的影响。结果表明,较优的制备条件为:用Na_2CO_3作缚酸剂,壳聚糖树脂、Na_2CO_3和ECH的物质的量比1∶5∶5.0,反应时间2 h,反应温度50℃。将该聚苯乙烯环氧载体用于Pseudomonas aeruginosa LX1脂肪酶的共价柔性固定化,固定化酶的热稳定性和贮藏稳定性均较游离酶明显提高。

NMR-based metabolomic responses of freshwater gastropod Bellamya aeruginosa to MC-producing and non MC-producing Microcystis aeruginosa

Molluscan metabolomic analysis is essential for the understanding of the regulatory mechanism of aquatic invertebrate in response to hepatotoxic microcystins(MCs)stress.To understand the system responses of the gastropod to MC exposure,metabolomic alterations caused by two strains(MC-producing and non MC-producing)of Microcystis aeruginosa were characterized indiff erent biological matrices(hepatopancreas and muscle)of Bellamya aeruginosa(Gastropoda)using 1 H nuclear magnetic resonance(NMR)spectroscopy combined with MCs detections after exposure for 1,7,and 14 d.Although ELISA analysis showed that no MCs was detected in both tissues after non MC-producing M.aeruginosa exposure,MCs concentrations were increasing in the hepatopancreas(from 1.29±0.48μg/g to 3.17±0.11μg/g)and foot muscle(from 0.07±0.02μg/g to 0.21±0.08μg/g)after 14-d exposure of MC-producing M.aeruginosa.Meanwhile,we observed that MC induced signifi cant increase in creatine,a variety of amino acids(leucine,isoleucine,valine,threonine,alanine,methionine,glutamate,aspartate,and lysine),carboxylic acids(lactate,acetate,and D-3-hydroxybutyrate),and choline and its derivatives(phosphocholine and glycerophosphocholine)but decreased the energy substance(lipids,glucose,and glycogen)in the hepatopancreas.However,no signifi cant metabolite diff erences were observed in the muscle between MC-producing and non MC-producing cyanobacteria treated groups.These results suggest that MC exposure may cause hepatic energy expenditure accompanied with various metabolic disorders that involve lipid metabolism,protein catabolism,osmoregulation,glycolysis,glycogenolysis,and tricarboxylic acid(TCA)cycle.Moreover,metabolic perturbation was aggravated as the level of accumulated MCs raised over time in the MC-producing cyanobacteria treatment.These fi ndings indicated that MCs accumulation might lead to oxidative-stress-mediated damage of mitochondria functions.

菌株Pseudomonas aeruginosa BC1发酵-渗透汽化耦合制备鼠李糖脂

采用发酵-渗透汽化耦合(fermentation coupling with pervaporation,FCP)系统和分批补料式发酵系统培养Pseudomonas aeruginosa BC1制备生物表面活性剂鼠李糖脂,两轮发酵过程分别持续124 h和107 h。在FCP系统中,最大细胞吸光度OD600为1.06;生物表面活性剂OD600为0.61;鼠李糖脂最终产量为1.3 g/L,相比于分批补料式发酵系统提高38%;对比纯水的表面张力67.52 m N/m,发酵第29 h的发酵上清液表面张力为22.56 m N/m。同时,该菌的发酵上清液对液体石蜡、机油、柴油、正己烷、十六烷均有较好的乳化能力。经GC-MS结合SPME分析发现,FCP系统分离出的渗透蒸汽冷凝液中含有乙醇、戊醇等有机物。实验结果表明,相比分批补料式发酵系统,FCP系统能够分离发酵过程中产生的一部分挥发性代谢产物,减轻这些物质对细胞生长的抑制,使细胞浓度和鼠李糖脂产量都有明显提高。

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。