SYT09/PI荧光探针标记lasR rhlR基因缺陷对铜绿假单胞菌生物膜形成影响的体外实验研究

目的研究群体感应（QS）系统在铜绿假单胞菌生物膜（BF）形成中的作用。方法体外建立3 d QS系统完整的铜绿假单胞菌野生型PA01菌株与QS系统缺陷（lasRrhlR基因缺陷△lasR△rhlR）型菌株生物模型,通过SYT09/PI荧光探针标记,结合激光共聚焦显微镜摄取BF不同层面的图片,经图像结构分析（ISA）软件分析获得QS系统lasRrhlR缺陷株,PA01菌株BF相关空间结构参数。结果培养第3天PA01菌株可形成较厚、有孔状通道的成熟BF结构,而△lasR△rhlR菌株仅形成明显稀薄的早期BF结构,△lasR△rhlR菌株的3 d BF厚度为（7.36±0.2）μm,PA01菌株为（21.64±0.57）μm（P〈0.05）,区域孔率（areal porosity,AP）分别为：（0.902±0.006）、（0.928±0.002）;平均扩散距离（average diffusion distance,ADD）和结构熵（textural entropy,TE）在△lasR△rhlR菌株分别为：（1.503±0.029）和（5.706±0.190）;在PA01菌株分别为：（1.467±0.015）和（5.213±0.111）,△lasR△rhlR菌株AP较PA01菌株低（P〈0.05）;而ADD、TE较PA01菌株高（P〈0.05）。结论△lasR△rhlR基因缺陷明显影响铜绿假单胞菌的BF形成能力,QS系统lasRrhlR基因在铜绿假单胞菌BF形成中发挥重要作用。     

铜绿假单胞菌生物膜和浮游菌的毒力表达差异

目的探究铜绿假单胞菌生物膜和浮游菌状态下毒力因子的表达差异。方法使用铜绿假单胞菌标准菌株PAO1,分别在生物膜(静置)和浮游菌(摇床)状态下培养,收集上清液,检测总蛋白酶、LasA和LasB弹性蛋白酶、鼠李糖脂、绿脓素、溶血活性;通过荧光定量PCR检测群体感应(quorum sensing, QS)系统相关基因的表达;同时,通过活菌计数检测PAO1在生物膜和浮游菌状态下的生长曲线。结果生物膜状态下,铜绿假单胞菌PAO1的总蛋白酶、LasA、LasB弹性蛋白酶、鼠李糖脂、绿脓素表达均增高(均P0.05),溶血活性增高(P0.05),生物膜和浮游菌状态下细菌生长曲线差异无统计学意义,QS相关基因rhlI、rhlR、rhlA、lasI、lasR、pqsA、pqsR表达增高(均P0.05)。结论铜绿假单胞菌PAO1在生物膜状态下毒力因子表达较浮游菌状态下增高。     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

铜绿假单胞菌lasI,rhlI基因功能缺陷株的构建和生物学功能验证

构建铜绿假单胞菌lasI,rhlI基因功能缺陷株,为进一步阐明氦氧饱和高气压暴露条件诱导lasI,rhlI基因介导铜绿假单胞菌毒力调节的分子机制研究奠定基础。用双亲株接合转移法删除lasI,rhlI基因ORF编码区,通过RT-PCR方法验证目标基因编码序列mRNA的缺失;通过对细菌生长增殖能力、弹性蛋白酶代谢活性和细菌绿脓菌素分泌能力等表型的测定,验证目标基因编码序列缺失后的基因调节功能的缺陷。结果表明成功构建铜绿假单胞菌lasI,rhlI基因功能缺陷株,可作为进一步研究的基因工程菌。     

5-甲基间苯二酚对铜绿假单胞菌及其生物膜形成的影响

探讨5-甲基间苯二酚对铜绿假单胞菌(Pseudomonas aureginosa)及其生物膜形成的影响。通过微量肉汤稀释法检测铜绿假单胞菌对5-甲基间苯二酚的敏感性并绘制时间-杀菌曲线;通过微孔板培养生物膜结合结晶紫染色法检测5-甲基间苯二酚对铜绿假单胞菌生物膜形成和分散的影响。当5-甲基间苯二酚的浓度为512μg/mL时,可显著抑制铜绿假单胞菌PAO1生物膜的形成,而5-甲基间苯二酚对铜绿假单胞菌PA47生物膜的形成无影响。32μg/mL的5-甲基间苯二酚还能显著分散铜绿假单胞菌PAO1成熟生物膜,但无明显的剂量依赖性。不同临床菌株生物膜对5-甲基间苯二酚的敏感性各异。结果表明,5-甲基间苯二酚能抑制铜绿假单胞菌生物膜的形成并能分散已形成的生物膜。     

纳米银离子对铜绿假单胞菌生物被膜结构的影响的分析

目的探讨纳米银离子对细菌生物被膜（biofilm,BF）的空间结构的影响。方法采用摇床法,以纳米银离子含量不同的乙烯-醋酸乙烯酯（Ethylene-Vinyl acetate,EVA）塑料为细菌粘附载体,模拟体内铜绿假单胞菌（P.aeruginosa,PA）BF形成的微环境,建立体外BF模型;将培养3 d的空白标本分别在扫描电子显微镜（scanning electron microscopy,SEM）下及用FITC-ConA染色后荧光显微镜下观察不含纳米银EVA中BF的形成情况;将生长0.5、1、2、3、5 d的BF模型行SYTO9/PI染色,激光共聚焦扫描电镜（confocal laser scanning microscopy,CLSM）下摄取不同层面的图像,然后应用激光共聚焦显微镜TCS SP2自身具有的分析软件及ISA分析软件获得PAO1菌株BF的相关空间结构参数定量化数据。结果 （1）运用SEM及荧光显微镜的方法,在以不含纳米银EVA塑料为细菌粘附载体上培养3 d的标本中均观察到流线状的BF形成。（2）激光共聚焦显微镜TCS SP2自身具有的分析软件定量化分析显示,随着时间的延长,各含纳米银离子材料组PAO1菌株BF的平均厚度都呈先升高后降低的趋势,3天组都达最高值;纳米银离子的含量对BF厚度的影响差异无统计学意义（F=2.11,P＆gt;0.1）,作用时间对BF厚度的影响差异有统计学意义（F=985.81,P0.05）。随着培养时间的延长,各含纳米银离子材料组PAO1菌株BF的AP值、ADD值无明显的变化趋势,同一时间组的含有纳米银离子材料组PAO1菌株BF的AP值都高于空白对照组的AP值;同一时间组的含有纳米银离子材料组PAO1菌株BF的ADD值都低于空白对照组的ADD值。各含纳米银离子材料组PAO1菌株BF的TE值随着时间的延长,都呈先升高后降低的趋势,2天组都为最高值;同一时间组的TE值随着含纳米银离子的增加都呈降低趋势。结论运用摇床法成功建立了体外PAO1菌株BF模型;纳米银离子对PAO1菌株BF空间结构有显著的影响。     

生物膜定量分析仪对不同细菌早期生物膜形成能力的比较研究

目的通过生物膜定量分析仪来观察铜绿假单胞菌(Pseudomonas aeruginosa PAO1),变形链球菌(Streptococcus mutans UA159)以及大肠埃希菌(Escherichia coli MG1655)生物膜形成能力的不同,并以各菌株的吸光度值A600为参考,对3种菌株早期生物膜形成能力进行比较。方法通过向生物膜培养悬液中加入与细菌直径相近的磁性小珠,利用这些小珠在磁场中受到生物膜的位移约束力的原理,采用生物膜定量分析仪,定量比较3种菌株在生物膜形成上的差别。结果实验发现铜绿假单胞菌PAO1和大肠埃希菌MG1655的细菌增长速度基本相同,但铜绿假单胞菌PAO1的生物膜形成明显快于大肠埃希菌MG1655。大肠埃希菌MG1655和变形链球菌UA159的生物膜形成速度基本相同,但大肠埃希菌MG1655的细菌增长速度明显高于变形链球菌UA159。结论不同细菌有各自的生物膜形成模式。生物膜定量分析仪作为一种高效简便的检测手段,可用于生物膜早期形成的动态分析。     

大蒜素对铜绿假单胞菌生物膜形成的影响及结构定量化分析

目的利用激光共聚焦显微镜（Confocal Laser Scanning Microscopy, CLSM）观察大蒜素对铜绿假单胞菌PA01菌株生物膜（biofilms,BF）形成过程的影响,并用ISA（Image Structure Analyzer）软件对BF结构进行定量化分析。方法体外建立1d．3d和7d组PA01菌株BF模型,每组分3小组,分别用生理盐水、大蒜素128μg／mL、大蒜素10μg／mL作用6h。通过荧光探针SYT09／PI标记,利用CLSM进行动态观察,并用ISA软件对其BF结构进行定量分析。结果（1）生理盐水对照组铜绿假单胞菌生物膜厚度随时间的增加而增加,但在后3d增加的速度减慢;同时区域孔率（Areal Porosity,AP）呈下降趋势,平均扩散距离（Average Diffusion Distance,ADD）有逐渐增加趋势,结构熵（Textural Entropy,TE）有增加的趋势。（2）7d组模型中,大蒜素128μg／mL作用后,BF厚度由（28．83±0．67）μm减低到（16．50±0．69）μm;AP由0．76±0．10增加到0．92±0．02;TE由6．78±0．93减低到5．61±0．55。10μg／mL大蒜素干预后,有同样趋势,但效应不如高浓度明显。（3）1d、3d模型组,大蒜素作用后同对照组相比,厚度、AP、TE的差异均有统计学意义,趋势同7d组。结论通过ISA软件对干预前后的BF进行结构定量分析,大蒜素对各个时间段BF的形成均有影响,高浓度效果更显著。     

多重耐药铜绿假单胞菌群体感应系统与主动外排基因表达的研究

目的研究临床多重耐药铜绿假单胞菌群体感应(QS)系统与主动外排泵MexAB-OprM系统基因表达水平与抗生素耐药关系。方法收集苏州市立医院和上海市江湾医院2011年2月至6月间临床标本中分离的铜绿假单胞菌,定量分析细菌生物被膜形成能力;MIC法检测细菌抗生素耐药性,用多重聚合酶链反应(PCR)扩增群体感应系统lasI、lasR及主动外排泵系统mexA基因,实时定量逆转录RT-PCR检测lasI、lasR和mexA基因的相对表达量。结果临床样本分离出84株铜绿假单胞菌,其中产生物被膜菌58株,占比69%;多重耐药菌共24株,占比28.6%;多重耐药菌株中产生物被膜有11株,占45.8%;多重耐药菌中mexA基因表达上调有18株,占75%;lasI基因表达上调有8株,占33.3%。结论多重耐药菌株的生物被膜形成率显著低于非多重耐药组,多重耐药铜绿假单胞菌的主动外排泵MexAB-OprM系统基因表达出现显著上调,生物被膜菌的lasI基因表达显著上调而lasR基因的表达无明显变化。     

野生型铜绿假单胞菌和新型mucA突变铜绿假单胞菌生物膜形成的动态观察

目的观察新型mucA突变的粘液型菌株PA17和野生型菌株PAO1生物膜(biofilm,BF)形成的动态过程,并比较2株菌生物膜形成过程的差异。方法 SYTO9/PI荧光探针标记PAO1和PA17,体外建立1d,3d,5d,9d时间点PAO1及PA17的BF模型,激光共聚焦显微镜(CLSM)观察两株菌BF动态形成的过程。结果通过SYTO9/PI双染可以动态观察PAO1菌株和PA17菌株的BF形成过程;PAO1菌株和PA17菌株的BF形成过程有差异:PAO1菌株1d时已形成微菌落,3d时形成覆盖整个玻片的生物膜结构,而PA17菌株1d时仅有散在的不可逆粘附细菌,3d时才形成微菌落,5d时形成生物膜结构;随着时间的推移,2株菌生物膜形成的厚度均逐渐增加,且死菌的比例也逐渐增加。结论 PAO1和PA17BF的动态形成过程存在差异,而PA17与PAO1生物膜形成存在的差异可能与其mucA基因突变造成大量藻酸盐的产生有关。     

惰性材料表面细菌生物膜构建的研究

目的构建惰性材料塑料输液管内壁细菌生物膜体外模型,观察细菌生物膜的结构,探讨输液管内壁细菌生物膜形成影响因素。方法建立铜绿假单胞菌生物膜和铜绿假单胞菌、肺炎克雷伯菌混合生物膜,分别于培养1、3和7d用扫描电镜动态观察生物膜形成过程。结果混合菌生物膜的生长速度高于铜绿假单胞菌单独成膜。结论输液管是形成细菌生物膜的良好支持材料,混合细菌培养可以加速细菌形成生物膜。     

Pseudomonas aeruginosa-plant root interactions. Pathogenicity, biofilm formation, and root exudation

Pseudomonas aeruginosa is an opportunistic human pathogen capable of forming a biofilm under physiological conditions that contributes to its persistence despite long-term treatment with antibiotics. Here, we report that pathogenic P. aeruginosa strains PAO1 and PA14 are capable of infecting the roots of Arabidopsis and sweet basil (Ocimum basilicum), in vitro and in the soil, and are capable of causing plant mortality 7 d postinoculation. Before plant mortality, PAO1 and PA14 colonize the roots of Arabidopsis and sweet basil and form a biofilm as observed by scanning electron microscopy, phase contrast microscopy, and confocal scanning laser microscopy. Upon P. aeruginosa infection, sweet basil roots secrete rosmarinic acid (RA), a multifunctional caffeic acid ester that exhibits in vitro antibacterial activity against planktonic cells of both P. aeruginosa strains with a minimum inhibitory concentration of 3 microg mL(-1). However, in our studies RA did not attain minimum inhibitory concentration levels in sweet basil's root exudates before P. aeruginosa formed a biofilm that resisted the microbicidal effects of RA and ultimately caused plant mortality. We further demonstrated that P. aeruginosa biofilms were resistant to RA treatment under in vivo and in vitro conditions. In contrast, induction of RA secretion by sweet basil roots and exogenous supplementation of Arabidopsis root exudates with RA before infection conferred resistance to P. aeruginosa. Under the latter conditions, confocal scanning laser microscopy revealed large clusters of dead P. aeruginosa on the root surface of Arabidopsis and sweet basil, and biofilm formation was not observed. Studies with quorum-sensing mutants PAO210 (DeltarhlI), PAO214 (DeltalasI), and PAO216 (DeltalasI DeltarhlI) demonstrated that all of the strains were pathogenic to Arabidopsis, which does not naturally secrete RA as a root exudate. However, PAO214 was the only pathogenic strain toward sweet basil, and PAO214 biofilm appeared comparable with biofilms formed by wild-type strains of P. aeruginosa. Our results collectively suggest that upon root colonization, P. aeruginosa forms a biofilm that confers resistance against root-secreted antibiotics.     

大蒜素对铜绿假单胞菌生物膜群体密度感应系统调控毒力因子表达的影响

目的通过体外测定铜绿假单胞菌（Pseudomonas aeruginosa）群体密度感应（Quorum Sensing,QS）系统调控的毒力因子表达,比较大蒜素干预前后毒性因子表达的差异以及对铜绿假单胞菌PAO1成熟生物膜（biofilm,BF）的影响。方法应用ELISA法比较处理前后外毒素A的含量差异;利用弹性蛋白一刚果红染色的方法,测定处理前后弹性蛋白酶活性;用蒽酮一硫酸法测定鼠李糖脂;利用荧光酶标仪检测青脓素含量变化;利用激光共聚焦显微镜观察干预前后大蒜素对铜绿假单胞菌成熟BF的影响。结果大蒜素干预后与生理盐水对照组比较,外毒素A、弹性蛋白酶、鼠李糖脂和青脓素表达分别由（19．630±0．573）pg/μl、（0．467±0．003）、（2．009±0．063）g／L、（9325．833±367．675）下降到（6．529±0．289）pg／μl、（0．032±0．001）、（0．269±0．009）g／L、（7819．167±111．800）。激光共聚焦显微镜观察可见生理盐水对照组细菌菌落云集呈蘑菇状分布,干预组黏附的细菌稀疏散在平坦分布。结论大蒜素可抑制铜绿假单胞菌群体密度感应系统调控的毒力因子表达,减弱其毒力,干扰BF分化成熟。     

Human and murine paraoxonase 1 are host modulators of Pseudomonas aeruginosa quorum-sensing

The pathogenic bacterium Pseudomonas aeruginosa uses acyl-HSL quorum-sensing signals to regulate genes controlling virulence and biofilm formation. We found that paraoxonase 1 (PON1), a mammalian lactonase with an unknown natural substrate, hydrolyzed the P. aeruginosa acyl-HSL 3OC12-HSL. In in vitro assays, mouse serum-PON1 was required and sufficient to degrade 3OC12-HSL. Furthermore, PON2 and PON3 also degraded 3OC12-HSL effectively. Serum-PON1 prevented P. aeruginosa quorum-sensing and biofilm formation in vitro by inactivating the quorum-sensing signal. Although 3OC12-HSL production by P. aeruginosa was important for virulence in a mouse sepsis model, Pon1-knock-out mice were paradoxically protected. These mice showed increased levels of PON2 and PON3 mRNA in epithelial tissues suggesting a possible compensatory mechanism. Thus, paraoxonase interruption of bacterial communication represents a novel mechanism to modulate quorum-sensing by bacteria. The consequences for host immunity are yet to be determined.     

Effect of ovotransferrin, protamine sulfate and EDTA combination on biofilm formation by catheter-associated bacteria

AIMS: To determine the effect of a composition comprising ovotransferrin (OT), protamine sulfate (PS) and ethylenediaminetetraacetic acid (EDTA) on biofilm formation by catheter-associated bacteria. METHODS AND RESULTS: The in vitro activity of OT, PS and EDTA alone and in combinations against biofilm formation by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis and Staphylococcus epidermidis was investigated. All the three compounds either alone or in combinations failed to inhibit the growth completely at the concentrations tested. However, the subinhibitory concentrations of three compounds in a composition showed synergistic inhibitory effect on biofilm formation by K. pneumoniae, Ps. aeruginosa and S. epidermidis. Furthermore, 79-95% reduction in Ps. aeruginosa and S. epidermidis biofilm formation was observed in a clear vinyl urinary catheter treated with the composition. CONCLUSION: The subinhibitory concentrations of OT, PS and EDTA in a composition were effective in reducing biofilm formation by catheter-associated bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that a synergistic composition-comprising non-antibiotic generally regarded as safe (GRAS) compounds such as OT, PS and EDTA may be used in the prevention of catheter-related infections.     

Detection of Pseudomonas aeruginosa biofilm on medical biomaterials

The adhesion and biofilm formation of Pseudomonas aeruginosa strains on the surface of catheters made of various polymers (PU, SL, PCW) were determined in vitro. It was used the method by Richards et al. with modification of Rózalska et al. (1998), in which soluble colourless TTC is reduced to insoluble red formazan. The results of this study indicate that 80.3% of this strains adhered and 94.6% formed biofilm on the Nelaton catheter, 86% strains adhered and 76.1% formed biofilm on the polyurethane catheter, and 73.2% strains adhered, and 78.9% formed biofilm on the Foley catheter.     

Efficacy of silver-releasing rubber for the prevention of Pseudomonas aeruginosa biofilm formation in water

The aim of the present study was to evaluate the efficacy of Elastoguard silver-releasing rubber in preventing Pseudomonas aeruginosa biofilm formation in water. Biofilm formation by P. aeruginosa under various conditions in an in vitro model system was compared for silver-releasing and conventional rubber. Under most conditions tested, the numbers of sessile cells attached to silver-releasing rubber were considerably lower with reference to conventional rubber, although the effect diminished with increasing volumes. The release of silver also resulted in a decrease in planktonic cells. By exposing both materials simultaneously to conditions for biofilm growth, it became obvious that the antibiofilm effect was due to a reduction in the number of planktonic cells, rather than to contact-dependent killing of sessile cells. The data demonstrate that the use of silver-releasing rubber reduces P. aeruginosa biofilm in water and reduces the number of planktonic cells present in the surrounding solution.     

Contribution of cell elongation to the biofilm formation of Pseudomonas aeruginosa during anaerobic respiration

Pseudomonas aeruginosa, a gram-negative bacterium of clinical importance, forms more robust biofilm during anaerobic respiration, a mode of growth presumed to occur in abnormally thickened mucus layer lining the cystic fibrosis (CF) patient airway. However, molecular basis behind this anaerobiosis-triggered robust biofilm formation is not clearly defined yet. Here, we identified a morphological change naturally accompanied by anaerobic respiration in P. aeruginosa and investigated its effect on the biofilm formation in vitro. A standard laboratory strain, PAO1 was highly elongated during anaerobic respiration compared with bacteria grown aerobically. Microscopic analysis demonstrated that cell elongation likely occurred as a consequence of defective cell division. Cell elongation was dependent on the presence of nitrite reductase (NIR) that reduces nitrite (NO(2) (-)) to nitric oxide (NO) and was repressed in PAO1 in the presence of carboxy-PTIO, a NO antagonist, demonstrating that cell elongation involves a process to respond to NO, a spontaneous byproduct of the anaerobic respiration. Importantly, the non-elongated NIR-deficient mutant failed to form biofilm, while a mutant of nitrate reductase (NAR) and wild type PAO1, both of which were highly elongated, formed robust biofilm. Taken together, our data reveal a role of previously undescribed cell biological event in P. aeruginosa biofilm formation and suggest NIR as a key player involved in such process.