Identification and Distribution of the Clinical Isolates of Imipenem-resistant Pseudomonas aeruginosa Carrying Metallo-β-lactamase and/or Class 1 Integron Genes

To investigate the distribution of the genes of two major metallo-β-1actamases （MBL;i.e.,IMP and VIM） and class 1 integrons （intI） in the clinical imipenem-resistant Pseudomonas aeruginosa, a total of 65 isolates, from a university hospital in Sichuan between December 2004 and April 2005 were screened for MBL genes by PCR using primers specific for blaIMP-1, blaVIM and blaVIM-2 genes. The MBL-positive isolates were further assessed for class 1 integrons by PCRusing specific primers. The nucleotide sequences of several PCR products were also determined. The results revealed that the blaVIM gene was found in 81.5% （53/65） of all isolates, blaVIM-2 gene was found in only 1 isolate and the intl gene was observed in 45.3% （24/53） of blaVIM-positive isolates. One isolate carried simultaneously both blaIMP-1 and intl genes, and to the best of our knowledge this is the first report of such isolate in southwest China. These observations highlight that the genes for VIM β-1actamase and class 1 integrons were predominantly present among the imipenem-resistant P. aeruginosa tested, confirming the current widespread threat of imipenem-resistant, integron-borne P.aeruginosa.

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.

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.

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.

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。

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.

1株铜绿假单胞菌(Pseudomonas aeruginosa)1217石油降解特性及相关基因分析（英文）

从石油污染土壤中分离得到1株石油降解菌1217,经细菌形态学、生理生化及16S rD NA序列分析初步鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。在温度5~65℃,pH 2~10,盐度0~9%的条件下菌株能很好生长,在以正十二烷、正十八烷、苯、甲苯、二甲苯和萘为唯一碳源的培养基中生长良好。在10℃和30℃条件下培养7 d,对原油的降解率分别为21.57%和15.15%。菌株产生的生物表面活性剂可以将表面张力从72.20 mN/m降至35.14 mN/m。利用特异性PCR扩增,在菌株中检测到烷烃单加氧酶、甲苯双加氧酶、联苯双加氧酶、芳香烃双加氧酶和氧化还原酶基因,并成功克隆出烷烃单加氧酶和芳烃双加氧酶基因,同相关基因比对分析,与铜绿假单胞菌PAO1的相应基因相似度分别为99.91%和99.22%。研究表明,菌株在生物修复和石油烃污染环境中具有潜在的应用价值。

Pseudomonas aeruginosa菌的原油除蜡降黏性能

采用单细胞Raman-D2O同位素标记技术确定了5种原油嗜蜡菌中的优势菌种;考察了其最佳生长条件,并对其代谢产物及其对原油的除蜡降黏性能进行测定。结果表明:优势原油嗜蜡菌种为SS1,属于铜绿假单胞菌属(Pseudomonas aeruginosa);其最佳生长条件为温度36℃、pH值7、NaCl质量浓度5 g/L;该菌种在生长代谢过程中产生糖脂类生物表面活性剂,乳化系数高达63.75%;用该菌种对原油处理120 h后,原油蜡降解率为53.16%,析蜡点及析蜡高峰点均有所降低;在36℃下,其对原油降黏率达到72.84%。

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。

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。

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。

海洋Pseudomonas aeruginosa sp. DES-3的鉴定以及基因ap02对其产蛋白酶的影响研究

海洋是地球上最大的生态系统,其多样独特的生态环境造就了其有别于其他环境的微生物资源。本研究利用蛋白酶的活性筛选策略,从辽宁省营口市某海洋淤泥中分离得到一株产蛋白酶的优势菌株DES-3。通过微生物形态学特征、生理生化性质和16SrDNA基因鉴定以及系统发育进化关系的分析,将该菌株鉴定为铜绿假单胞菌属,命名为Pseudomonas aeruginosa sp.DES-3。其胞外蛋白酶的最适反应温度和最适pH分别为55℃和9.0。将来自于碱性污染土壤宏基因组文库的编码碱性蛋白酶基因ap02与广宿主表达载体pBBR1MCS-5连接,转化至野生型菌株P.aeruginosasp.DES-3细胞中,成功构建了基因工程菌株P.aeruginosasp.DES-3/pBBR1MCS-5-ap02。基因工程菌株的最适反应温度为60℃,较野生型菌株提高了5℃。本研究构建基因工程菌株的策略可为菌株的改造和相应工业应用提供一定的技术参考。

Reuse of waste frying oil for production of rhamnolipids using Pseudomonas aeruginosa zju.u1M

In this work,rhamnolipid production was investigated using waste frying oil as the sole carbon source. By culture in shaking flasks,a naturally isolated strain synthesized rhamnolipid at concentration of 12.47 g/L and its mutant after treatment by UV light increased this productivity to 24.61 g/L. Fermentation was also conducted in a 50 L bioreactor and the productivity reached over 20 g/L. Hence,with a stable and high productive mutant strain,it could be feasible to reuse waste frying oil for rhamnolipid production on industrial scale.

Isolation, Identification, and Herbicidal Activity of Metabolites Produced by Pseudomonas aeruginosa CB-4

CB-4, a bacterial strain with highly effective herbicidal activity, was isolated from infected corn leaves. Through morphology, physiological and biochemical tests, and 16 S ribosomal DNA gene sequencing methods, CB-4 was identified as Pseudomonas aeruginosa. We conducted activity-evaluation experiments in the laboratory to assess the herbicidal potential of metabolites produced by strain CB-4. Crude extracts of strain CB-4 have high inhibition activity on Digitaria sanguinalis. In general, the root and shoot growth parameters of D. sanguinalis were significantly reduced by metabolites of strain CB-4. The IC50 of the culture filtrate extracts for the radicula and coleoptile of D. sanguinalis were 0.299 and 0.210 mg mL-1, respectively. Component 2 of the herbicidal activity of the crude toxin from strain CB-4 was successfully purified for the first time by using high-speed counter current chromatography with a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water（4：5：4：5, v/v） and high-performance liquid chromatography. We concluded that the metabolites of strain CB-4 have the potential to be developed as a microbe-based herbicide.

Biodegradation of crude oil by Pseudomonas aeruginosa in the presence of rhamnolipids

The potential biodegradation of crude oil was assessed based on the development of a fermentative process with a strain of Pseudomonas aeruginosa which produced 15.4 g/L rhamnolipids when cultured in a basal mineral medium using glycerol as a sole carbon source. However, neither cell growth nor rhamnolipid production was observed in the comparative culture system using crude oil as the sole carbon source instead. As rhamnolipid, an effective biosurfactant, has been reported to stimulate the biodegradation of hydrocarbons, 1 g/L glycerol or 0.22 g/L rhamnolipid was initially added into the medium to facilitate the biodegradation of crude oil. In both situations, more than 58% of crude oil was degraded and further converted into accumulated cell biomass and rhamnolipids. These results suggest that Pseudomonas aeruginosa could degrade most of crude oil with direct or indirect addition of rhamnolipid. And this conclusion was further supported by another adsorption experiment, where the ad-sorption capacity of crude oil by killed cell biomass was negligible in comparison with the biologic activities of live cell biomass.

Kinetics and mechanisms of p-nitrophenol biodegradation by Pseudomonas aeruginosa HS-D38

The kinetics and mechanisms of p-nitrophenol （PNP） biodegradation by Pseudomonas aeruginosa HS-D38 were investigated. PNP could be used by HS-D38 strain as the sole carbon, nitrogen and energy sources, and PNP was mineralized at the maximum concentration of 500 mg/L within 24 h in an mineral salt medium （MSM）. The analytical results indicated that the biodegradation of PNP fit the first order kinetics model. The rate constant kpNp is 2.039 ×10^-2/h in MSM medium, KeNp＋N is 3.603 × 10^-2/h with the addition of ammonium chloride and KPNP＋c is 9.74 ×10^-3/h with additional glucose. The addition of ammonium chloride increased the degradation of PNP. On the contrary, the addition of glucose inhibited and delayed the biodegradation of PNP. Chemical analysis results by thin-layer chromatography （TLC）, UV-Vis spectroscopy and gas chromatography （GC） techniques suggested that PNP was converted to hydroquinone （HQ） and further degraded via 1,2,4-benzenetriol （1 ,2,4-BT） pathway.

Antibacterial activities of some plant extracts alone and in combination with different antimicrobials against multidrug-resistant Pseudomonas aeruginosa strains

Objective:To evaluate the possible in vitro interaction between ethanolic extracts of Rhus coriaria[R.coriaria)(seed),Sacropoterium spinosum(S.spinosum)(seed),Rosa damascena(R. damascene)(flower) and certain known antimicrobial drugs including oxytetracycline HCl, penicillin C,cephalexin,sulfadimethoxine as sodium,and enr of loxacin.This synergy study was carried out against 3 clinical strains of multidrug-resistant Pseudomonas aeruginosa (P.aeruginosa).Methods:Evaluation of synergy interaction between plant extracts and antimicrobial agents was carried out using microdilution method.Results:The results of this study showed that there is a decrease in the MIC in case of combination of ethanolic plant extracts and test antimicrobial agents.The most interesting result was that the combination between R. coriaria and these antibiotics,showed a high decrease in minimum inhibitory concentration(MIC), and a strong bactericidal activity against these strains.Conclusions:These results may indicate that combinations between R.coriaria extract and these antibiotics could be useful in fighting emerging drug-resistance P.aeruginosa,which may due to that R.coriaria extract contain natural inhibitors working by different mechanisms or inhibiting efflux pumps.Now we have experiments underway leading to the identification of the active molecules present in R.coriaria.Further,in vivo experiments are needed to confirm pseudomonal protection.