Study on Distribution of Four Pseudomonas Species in Living Environment Using Multiplex PCR

Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.

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.

Thoracic spine infection caused by Pseudomonas fluorescens:A case report and review of literature

BACKGROUND The clinical incidence of spinal infection is gradually increasing,and its onset is insidious,easily leading to missed diagnosis and misdiagnosis,which may lead to serious complications such as nervous system dysfunction,spinal instability and/or deformity,and cause a huge burden on society and families.Early identification of the causative agent and precision medicine will greatly reduce the suffering of patients.At present,the main pathogenic bacteria that cause spinal infection are Staphylococcus aureus,Streptococcus,Pneumococcus,Escherichia coli,and Klebsiella.There are no reports of spinal infection caused by Pseudomonas fluorescens.CASE SUMMARY We report a 32-year-old female patient with spinal infection.She presented with flank pain,initially thought to be bone metastases or bone tuberculosis,and had a family background of tumors.Her clinical features and changes in imaging and laboratory tests led to the suspicion of thoracic spine infection.Histopathology of the lesion showed inflammation,tissue culture of the lesion was negative several times,and the possible pathogen-Pseudomonas fluorescens was found after gene sequencing of the lesion.The patient recovered completely after a full course of antibiotic treatment.CONCLUSION This report increases the range of pathogens involved in spinal infections,highlights the unique advantages of gene sequencing technology in difficult-todiagnose diseases,and validates conservative treatment with a full course of antibiotics for spinal infections without complications.

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.

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.

荧光假单胞菌Pseudomonas fluorescens RN-88抑菌特性研究

本研究旨在利用拮抗菌防治植物病害,为桃的病害生物防治提供更多可行性的方案。以前期筛选获得的一株桃褐腐病拮抗细菌荧光假单胞菌(Pseudomonas fluorescens)RN-88为试验材料,采用杯碟法测定发酵液对桃褐腐菌(Monilinia fructicol)的抑菌活性,并结合抑菌圈法,研究了该菌在pH、温度、紫外线(UV)等不同环境中的稳定性。实验结果表明,细菌RN-88能显著降低桃褐腐病的发病率,其发酵液对褐腐病菌(M.fructicol)的抑制率可达44.69%。在过滤除菌后,相对于高温灭菌,发酵液中抑菌物质的损失较小。在pH 7.5~9.0的碱性溶液中,发酵液的抑菌活性有较好的稳定性,但在酸性溶液(3.0≤pH≤6.5)中处理后,其抑菌能力减弱。发酵液的抑菌率几乎不受温度和紫外线(UV)的影响。Pseudomonas fluorescens RN-88细菌具有较好的环境稳定性,具有开发成桃褐腐病生物拮抗剂的潜在价值,本研究为荧光假单胞菌作为新型生防菌的应用提供了理论依据,同时为实现桃病害的生物防治提供了更多可行性的方案。

南海海洋细菌Pseudomonas sp.产生的一种抗肿瘤蓝色素

Pseudomonassp.是从大亚湾表面海水分离到的一株新的海洋细菌,在改变培养条件之后,能产生不同的次级代谢产物。从该菌培养液的脂溶性部分分离得到一种蓝色素Blue-1,它的结构通过NMR,2D-NMR,FABMS,元素分析得到确定,与从陆生菌Chromobacteriumviolacewn(Bacillusvidaceus)中分离得到紫色杆菌素(violacein)的结构一致。抗肿瘤活性试验结果表明,Blue-1对肿瘤细胞生长有很强的抑制作用,对MCG803的IG50为4.6μg/mL,对BEL-7402的IC50为6.8μg/mL。

大亚湾海洋细菌Pseudomonas sp.中的红色素

海洋细菌Pseudomonassp .,采集于大亚湾 ,从该菌的菌体提取物分离得到 2个红色素 :灵菌红素A和新的衍生物二甲基灵菌红素B。它们的结构通过NMR ,LC_MS等确定 ,并得出灵菌红素是具有强烈抗微生物和细胞毒性的化合物 ,同时研究了它的培养条件。

植物病原细菌Pseudomonas syringae pv.tomato基因组中的信号肽分析

应用SignaIP 3.0对植物病原细菌Pseudomonas syringae pv.tomato DC3000菌株基因组中的细菌染色体全部5 615个ORFs进行了分析,确定其中679个ORFs所编码蛋白质的N-端有信号肽序列,其中已经命名并有注释的有107个ORFs.信号肽的长度以19～31个氨基酸居多,其中最多的是23个氨基酸的信号肽.具有信号肽的ORFs编码蛋白的长度大多为101～400个氨基酸之间.同时,对组成信号肽的氨基酸种类作了系统的分析,发现组成信号肽的氨基酸中非极性氨基酸占48.54%,极性氨基酸占18.67%,带负电荷氨基酸占24.54%,带正电荷氨基酸仅占8.00%,出现最多的3种氨基酸依次为亮氨酸、丙氨酸和丝氨酸,最少的氨基酸是异亮氨酸,在切割位点-1端的氨基酸中83.21%均为丙氨酸,在切割位点后3位的氨基酸中最多的氨基酸也是丙氨酸.通过分析确定628个分泌类信号肽,36个信号肽具有RR-motif的保守区段,15个脂蛋白类信号肽,未发现Prepiln-like信号肽和Bacteriocin and Pheromone信号肽.

适冷菌Pseudomonas extremaustralis PF中海藻糖的合成途径及TreS基因的克隆

通过对一株高寒冰缘植物内生适冷假单胞菌Pseudomonas extremaustralis PF的研究,发现该菌中同时存在TPS/TPP,TreY/TreZ和TreS三种海藻糖合成途径,其中TreS途径的合成酶活性最高.对该菌的海藻糖合成酶TreS的基因进行克隆,得到一个新的TreS基因PFTreS,该基因与已报道的细菌TreS基因在核酸序列上表现出较高的同源性(最高达80.2%).根据基因序列预测的PFTreS氨基酸序列具有TreS酶的催化功能保守区,与假单胞菌P.Fluorescens Pf-5的TreS有很高的同源性.这些结果表明了Pseudomonas extremaustralis中海藻糖的合成特性,为进一步揭示海藻糖的合成与Pseudomonas extremaustralis PF的低温响应机制的关系奠定了基础.

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.

引起梨花枯和芽枯的Pseudomonas syringae pv.syringae病原细菌鉴定

【目的】明确在中国发生的梨花枯和芽枯的确切病原菌。【方法】用普通细菌学方法、电镜观察、Koch氏病原假说测定、Biolog、脂肪酸分析、PCR及与标准对照菌株的比较。【结果】从16个病样中分离获得12菌株,6株代表菌株显示出与Pseudomonas syringaepv.syringae3株标准对照菌株相似的致病反应,它们的Biolog和脂肪酸分析的相似度分别为0.57～0.86和0.58～0.81,PCR和序列测定结合上述结果证实了P.syringaepv.syringae为该病的病原菌。【结论】首次证实了中国梨树上的花枯和芽枯可由P.syringaepv.syringae引起。

梨灰霉病菌拮抗细菌Pseudomonas extremorientalis的鉴定、发酵条件优化及防效评价

灰霉病是梨贮藏期的主要病害,发病率高,为害严重。为了有效控制该病害发生,利用平皿稀释法从梨根际土壤中分离内生细菌,并采用琼脂平板扩散法进行抑菌活性初筛,筛选出1株拮抗细菌L21,基于形态特征、生理生化特性及16S rRNA和gyrB序列分析,鉴定为Pseudomonas extremorientalis。采用生长速率法测定菌株L21的无菌发酵液对12种病原真菌均有抑制作用,采用Salkowski比色法测定菌株L21能够分泌吲哚-3-乙酸(IAA),采用酪蛋白培养基平板检测菌株L21可以产生胞外蛋白酶。在单因素试验的基础上,通过Plackett-Burman试验设计筛选出pH、硫酸锰和转速3个影响发酵的重要因素,利用最陡爬坡试验和Box-Behnken试验设计,获得菌株L21的最佳发酵条件为蔗糖20 g/L、牛肉膏20 g/L、硫酸锰3.00 g/L、甘油1 g/L、K2HPO4 1.50 g/L、接种量8%、温度34℃、pH 7.31、转速200.66 r/min和培养时间15 h。处理96 h后,菌株L21对梨灰霉病的防效为74.23%,与多菌灵的防效差异不显著。本研究表明菌株L21具有广谱抑菌活性,为植物真菌病害的生物防治提供新的生防资源。

一株异养硝化-好氧反硝化菌Pseudomonas sp.GK-01的筛选及脱氮能力研究

从禽畜粪便发酵沼液中分离筛选出1株异养硝化-好氧反硝化菌株假单胞菌属(Pseudomonas sp.) GK-01,采用经16S rDNA同源性比对及系统发育分析方法鉴定该菌,通过单因素变量控制实验对该菌株生长和脱氮作用的影响因素进行优化,并在最优条件下考察其在单一和混合氮源中的脱氮效果。结果表明,该菌株为1株Pseudomonas sp.,最佳碳源为柠檬酸钠,最佳C/N为10,最佳初始pH为8~9,最佳培养温度为30~35℃。此外,当NH4+-N的初始浓度为400 mg·L-1时,该菌株在混合氮源体系中24 h对NH4+-N和NO3--N的去除率分别为99.08%和96.12%,表明其对高氨氮废水具有高效的异养硝化-好氧反硝化能力,在高氨氮废水生物脱氮等领域具有广泛的应用前景。

外源甜菜碱提高恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的研究

研究了外源甜菜碱对恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的影响并对其渗透保护机制进行了初步的探讨;结果表明培养基中添加甜菜碱可以改善DLL-1细胞在高盐培养基中的生长情况,添加150mg/L的甜菜碱可以使DLL-1在1.2mol/L NaCl的基础盐培养基中生长,添加10mg/L的甜菜碱就足以显著缩短渗透胁迫条件下DLL-1细胞的延滞期和代时,增加生长量;和不添加对照相比,延滞期由24h缩短到6h,代时由60min缩短到35.7min,最大生长量OD610由1.29增长到1.57。在渗透胁迫条件下,细胞从外界快速吸收外源甜菜碱来代替自身相容性溶质的合成。

对硝基苯酚降解菌Pseudomonas sp．PDS-7的降解特性及其降解相关基因的克隆

【目的】本研究的目的是分离对硝基苯酚(PNP)降解菌,研究其对PNP的降解特性;克隆其降解相关基因,并进行表达。【方法】本研究通过富集培养法和系列稀释平板涂布法分离PNP降解菌株;采用形态观察、生理生化特征测定和16S rDNA分析对菌株进行初步鉴定;通过摇瓶试验研究菌株降解特性;利用SEFA-PCR技术克隆降解相关基因,并亚克隆到表达载体pET29a中,构建重组表达质粒pETpnpC,再转入受体菌E.coli BL21(DE3)中进行诱导表达;通过分光光度法测定表达产物的酶活力。【结果】分离到一株PNP降解菌PDS-7,将该菌株鉴定为假单胞菌属(Pseudomonas sp.);该菌株能够以PNP作为唯一碳源、氮源和能源生长,菌株对PNP的最高耐受浓度为80mg/L,最适降解温度为30°C,偏碱性条件有利于菌株对PNP的降解;克隆了PNP降解过程中的偏苯三酚1,2-双加氧酶基因pnpC及马来酰醋酸还原酶基因pnpD(GenBank登陆号EU233791);将pnpC在E.coli BL21(DE3)菌株进行了诱导表达,表达产物对偏苯三酚和邻苯二酚均有邻位开环活性,比活力分别为0.45U/mg protein和0.37U/mg protein,表明偏苯三酚1,2-双加氧酶基因pnpC得到了活性表达。【结论】分离鉴定了一株PNP降解菌Pseudomonas sp.PDS-7,研究了该菌株的降解特性,克隆和表达了降解相关基因。

Pseudomonas mosselii E1铁载体合成相关基因cysI的克隆与功能初步分析

从棉花根际分离的铁载体产生菌E1,其16SrDNA与Pseudomonas mosselii ATCCBAA-99的同源性为100%。采用三亲本杂交方法将携带转座子Tn5-1063的质粒pRL1063a导入E1中进行转座子插入诱变。利用CAS法,从1000个突变株中,筛选到一株铁载体合成缺失突变株E1-185。利用TAIL-PCR方法,扩增位于Tn5-1063两端的侧翼序列。测序结果表明,转座子插入到E1的cysI基因内。该基因与Pseudomonas entomophila L48的cysI同源性为96%,其CysI氨基酸序列相似性为97%。该基因与半胱氨酸的合成密切相关,而在加有半胱氨酸的CAS平板上,突变株恢复了铁载体产生能力,证明cysI在E1铁载体合成过程中具有重要作用。据推测,cysI可能与铁载体合成途径中关键蛋白acyl-S-PCPs的形成有关。

1株Pseudomonas frederiksbergensis JW-SD2的解磷特性及解磷条件研究

为探讨解磷细菌Pseudomonas frederiksbergensis JW-SD2的解磷特性及条件,检测了该菌株对不同矿质磷酸盐的溶解能力,并研究了不同碳源、氮源、温度、p H、装液量及盐离子等营养、环境因素对其解磷能力的影响。结果表明,该菌株对Ca3（PO4）2的溶解能力强于Fe PO4和Al PO4,解磷量达7.43 mmol/L。以葡萄糖作为碳源,硫酸铵作为氮源该菌株具有最大的解磷能力。同时还发现,该菌株能够在温度20~35℃,p H 4~9,装液量1/5~4/5,Na Cl浓度0~3.0%保持3.0 mmol/L以上的解磷量,具有较强的环境适应能力。

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷（MP）高效降解菌株,同时对MP具有趋化性.cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异.通过土壤盆钵试验（MP浓度为50mg/kg）,发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%～30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用.