铜绿假单胞菌群体感应系统的研究进展

细菌的群体感应也称自身诱导,是指细菌通过产生和感应信号分子浓度的变化来监测其群体密度,协调群体行为的过程.自身诱导物随着细菌密度增高而增高,当自身诱导物达到某一阈值后,会与一些转录调节子结合,从而诱导或抑制多种基因的表达.群体感应系统内由多种信号分子和效应蛋白组成复杂的调节网络,调控包括细菌毒力因子产生与释放、生物膜形成、接合反应等,从而影响细菌的致病过程.本文主要对铜绿假单胞菌的群体感应系统及其与宿主关系、群体感应抑制剂等方面的研究进展进行综述.     

铜绿假单胞菌群体感应抑制剂研究进展

铜绿假单胞菌是一种常见的机会致病菌,可在人群中引起严重的急性和慢性感染,是病人在医院期间发生感染的第三大致病菌。铜绿假单胞菌多种毒力因子的分泌以及生物被膜的形成都是受一种被称为群体感应(Quorum Sensing,QS)的胞间信号传导系统调控的。QS使细菌能够根据细胞密度变化进行基因表达的调控。通过抑制QS来治疗铜绿假单胞菌感染是一个很有前景的发展方向。本文将就近年来铜绿假单胞菌群体感应抑制剂的研究进展进行综述。     

铜绿假单胞菌生物膜研究进展

生物膜（biofilm,BF）是细菌为了适应生存环境的需要而形成的与浮游细胞相对应的生存形式,是细菌生来具有的本领。不同的细菌形成生物膜的能力是不同的,铜绿假单胞菌极易形成生物膜,临床许多生物医学材料相关感染和某些慢性顽固性感染性疾病都与之密切相关,在生物膜中的细菌不仅耐抗生素还可耐抗体的杀菌作用,危害性严重。     

谷胱甘肽对铜绿假单胞菌exoS 和exoY基因的影响

摘要: 【目的】研究谷胱甘肽对铜绿假单胞菌exoS和exoY基因表达的影响。【方法】利用丁硫氨酸亚砜胺和马来酸二乙酯同时耗竭细胞内的谷胱甘肽,并构建包含被lacZGm破坏的谷胱甘肽合成酶基因的突变体。通过分别连有exoS 和exoY基因启动子的pMS402质粒上Lux报道子发光值大小检测exoS 和exoY基因表达变化情况。【结果】exoS和exoY基因的表达在用化学药品耗竭的细胞中或是在谷胱甘肽合成酶突变体中都降低。【结论】铜绿假单胞菌细胞内的谷胱甘肽可以促进exoS和exoY的表达。这将为进一步研究铜绿假单胞菌的感染以及致病性机理提供一定的理论基础。     

铜绿假单胞菌蹭行运动相关基因的研究

应用Mu转座重组技术研究铜绿假单胞菌 (Pseudomonasaeruginosa)蹭行运动 (Twitchingmotility)的相关基因。通过转座突变、表型筛选 ,得到 8个Twitchingmotility缺陷或减弱的突变子。经过基因克隆、核苷酸测序研究 ,鉴定转座子插入到基因组中的位置。结果表明 ,在其中 4个突变子中 ,转座子分别插入到与IV型菌毛生物合成和功能相关的 3个已知基因中 (其中有两个突变子转座子插入到同一基因的不同位置 ) ,它们是pilV ,pilQ ,algR。另外 4个突变子中 ,有 3个是转座子分别插入到基因pilL基因的前端 ,中部和后端 ,均引起Twitchingmotility功能缺失。另一个突变子中 ,转座子插入到基因PA1 82 1中 ,引起Twitchingmotility功能减弱。PilL和PA1 82 1的编码产物均属于 3 类蛋白质 ,它们的功能是根据其保守的氨基酸基序或基因序列与已知功能基因的相似性推测得出的。但缺乏详细的试验证据。研究结果为pilL控制Twichingmotility提供了有力的证据。并证实基因PA1 82 1与Twitchingmotility有关。将Mu转座重组技术应用到假单胞菌的研究中 ,国内外均未见报道。由于该技术具有随机单点插入的优点 ,克服了传统转座子能在染色体上迁移的缺点。保证了表型的改变与转座子插入位点的基因突变的一一对应关系。为进一步研究铜绿假     

铜绿假单胞菌体外对肠上皮细胞骨架的影响

为探索铜绿假单胞菌粘附肠上皮细胞后,细胞骨架特性的变化规律及可能的机制。采用微管吸吮实验技术并结合细胞ELISA、图像分析等方法研究体外绿脓杆菌粘附肠上皮细胞后细胞骨架的变化。结果显示细菌粘附后1h肠上皮细胞活力即开始下降,3h后IEC面积明显减小,而细胞周长无明显变化;胞内骨架蛋白减少,且随孵育时间的延长愈趋明显;细胞弹性系数K1、K2在粘附后3h明显降低,同时伴有粘性系数μ也明显下降。以上结果表明绿脓杆菌粘附肠上皮细胞后,细胞骨架成分改变,细胞骨架功能受损害,最终导致细胞损伤。     

氨溴索对铜绿假单胞菌生物膜早期黏附及胞外多糖的影响

目的研究氨溴索对铜绿假单胞菌PA01菌株BF早期黏附及胞外多糖复合物（Extracellular Pdymeric Substances,EPS）的影响。方法利用荧光多功能酶标仪检测各组不同时间点96孔板中pGFPuv转化PA01菌株的荧光强度,计算黏附率以表示干预对不同时间点细菌黏附的影响;利用罗丹明标记的麦胚凝集素（WGA）特异性结合细菌EPS,荧光显微镜下定性观察各组EPS的变化;利用硫酸-苯酚法定量各组细菌EPS的产量。结果8h组,氨溴索高浓度干预后细菌的黏附率由0．72±0．17下降至0．49±0．08,t=4．03,P〈0．05,与克拉霉素阳性对照组黏附率（0．50±0．06）相比,t=-1．19,P〉0．05;氨溴索低浓度干预后黏附率也有下降,但不及高浓度组明显;其余时间组趋势与8h组大致相似。罗丹明标记WGA可使胞外多糖显色,在荧光显微镜下观察可见氨溴索干预后EPS减少,稀薄;EPS定量实验,EPS总量（μg）／细菌干重（g）氨溴索干预组（477．82±7．90）较生理盐水对照组（523．76±10．12）有明显降低,t=8．76,P〈0．05。结论氨溴索可显著减少PA01菌株黏附及产EPS的能力。     

铜绿假单胞菌的双组分系统

双组分系统是存在于原核和少部分真核生物细胞中的信号转导系统,主要由组氨酸蛋白激酶和反应调节蛋白组成,通过感应外界环境信号、信号输入、磷酸基团传递、信号输出等环节调节基因表达,使细胞能更加适应环境变化。铜绿假单胞菌为条件致病菌,其双组分系统构成多样、功能复杂且参与介导耐药性产生,因此铜绿假单胞菌的双组分系统日益引起人们关注。本文对铜绿假单胞菌双组分系统的组成、信号转导机制、种类、研究方法及其临床意义进行了综述。     

铜绿假单胞菌外毒素A基因表达的研究进展

外毒素A(PEA)是铜绿假单胞菌最主要的一种毒力因子,它具有ADP-核糖基化转移酶活性,通过对延伸因子-2(eEF-2)的ADP-核糖基化抑制细胞的的蛋白质合成而导致细胞死亡。PEA由613个氨基酸组成,分子量66kD;其编码结构基因toxA位于细菌染色体上,为单一顺反子。本文着重综述近年来对PEA的基因表达及表达调控的研究进展,为利用基因工程手段获得高纯度PEA的工作理清一条思路。     

铜绿假单胞菌对11种抗生素的耐药性差异

目的探讨不同标本分离铜绿假单胞菌对11种抗生素耐药性的差异,指导临床合理使用抗生素。方法按常规方法培养分离后,经VITEK-AMS60或VITEK-II自动微生物鉴定分析仪的鉴定,用Kirty-Bauer法做药物敏感试验。结果尿液与血液之间,以及尿液与伤口分泌物之间,分离株均对所检测的11种抗生素的耐药性差异有非常显著性（P〈0.01）,尿液与穿刺液分离菌对除亚胺培南和美洛培南外的9种抗生素的耐药性差异有非常显著性（P〈0.01）,血液与穿刺液、血液与伤口分泌物和穿刺液与伤口分泌物间,除穿刺液与伤口分泌物分离菌对美洛培南的耐药性差异有显著性（P〈0.05）外,其他差异均无显著性（P〉0.05）。结论不同标本来源铜绿假单胞菌对哌拉西林等11种抗生素存在耐药性差异。     

Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa

Pseudomonas aeruginosa is capable of twitching, swimming, and swarming motility. The latter form of translocation occurs on semisolid surfaces, requires functional flagella and biosurfactant production, and results in complex motility patterns. From the point of inoculation, bacteria migrate as defined groups, referred to as tendrils, moving in a coordinated manner capable of sensing and responding to other groups of cells. We were able to show that P. aeruginosa produces extracellular factors capable of modulating tendril movement, and genetic analysis revealed that modulation of these movements was dependent on rhamnolipid biosynthesis. An rhlB mutant (deficient in mono- and dirhamnolipid production) and an rhlC mutant (deficient in dirhamnolipid production) exhibited altered swarming patterns characterized by irregularly shaped tendrils. In addition, agar supplemented with rhamnolipid-containing spent supernatant inhibited wild-type (WT) swarming, whereas agar supplemented with spent supernatant from mutants that do not make rhamnolipids had no effect on WT P. aeruginosa swarming. Addition of purified rhamnolipids to swarming medium also inhibited swarming motility of the WT strain. We also show that a sadB mutant does not sense and/or respond to other groups of swarming cells and this mutant was capable of swarming on media supplemented with rhamnolipid-containing spent supernatant or purified rhamnolipids. The abilities to produce and respond to rhamnolipids in the context of group behavior are discussed.     

Imaging and analysis of Pseudomonas aeruginosa swarming and rhamnolipid production

Many bacteria spread over surfaces by "swarming" in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacterium Pseudomonas aeruginosa. First, we quantify the temporal distribution of P. aeruginosa cells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming of P. aeruginosa and Salmonella enterica serovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of several P. aeruginosa strains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.     

Self-produced extracellular stimuli modulate the Pseudomonas aeruginosa swarming motility behaviour

Pseudomonas aeruginosa presents three types of motilities: swimming, twitching and swarming. The latter is characterized by rapid and coordinated group movement over a semisolid surface resulting from morphological differentiation and intercellular interactions. A striking feature of P. aeruginosa swarming motility is the formation of migrating tendrils producing colonies with complex fractal-like patterns. Previous studies have shown that normal swarming motility is intimately related to the production of extracellular surface-active molecules: rhamnolipids (RLs), composed of monorhamnolipids (mono-RLs) and dirhamnolipids (di-RLs), and 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). Here, we report that (i) di-RLs attract active swarming cells while HAAs behave as strong repellents, (ii) di-RLs promote and HAAs inhibit tendril formation and migration, (iii) di-RLs and HAAs display different diffusion kinetics on a surface as di-RLs spread faster than HAAs in agar, (iv) di-RLs and HAAs have no effect on swimming cells, suggesting that swarming cells are different from swimming cells not only in morphology but also at the regulatory level and (v) mono-RLs act as wetting agents. We propose a model explaining how HAAs and di-RLs together modulate the behaviour of swarming migrating cells by acting as self-produced negative and positive chemotactic-like stimuli.     

Surface hardness impairment of quorum sensing and swarming for Pseudomonas aeruginosa

The importance of rhamnolipid to swarming of the bacterium Pseudomonas aeruginosa is well established. It is frequently, but not exclusively, observed that P. aeruginosa swarms in tendril patterns--formation of these tendrils requires rhamnolipid. We were interested to explain the impact of surface changes on P. aeruginosa swarm tendril development. Here we report that P. aeruginosa quorum sensing and rhamnolipid production is impaired when growing on harder semi-solid surfaces. P. aeruginosa wild-type swarms showed huge variation in tendril formation with small deviations to the "standard" swarm agar concentration of 0.5%. These macroscopic differences correlated with microscopic investigation of cells close to the advancing swarm edge using fluorescent gene reporters. Tendril swarms showed significant rhlA-gfp reporter expression right up to the advancing edge of swarming cells while swarms without tendrils (grown on harder agar) showed no rhlA-gfp reporter expression near the advancing edge. This difference in rhamnolipid gene expression can be explained by the necessity of quorum sensing for rhamnolipid production. We provide evidence that harder surfaces seem to limit induction of quorum sensing genes near the advancing swarm edge and these localized effects were sufficient to explain the lack of tendril formation on hard agar. We were unable to artificially stimulate rhamnolipid tendril formation with added acyl-homoserine lactone signals or increasing the carbon nutrients. This suggests that quorum sensing on surfaces is controlled in a manner that is not solely population dependent.     

Inhibition of swarming motility of Pseudomonas aeruginosa by branched-chain fatty acids.

Pseudomonas aeruginosa is capable of moving by swimming, swarming, and twitching motilities. In this study, we investigated the effects of fatty acids on Pseudomonas aeruginosa PAO1 motilities. A branched-chain fatty acid (BCFA)--12-methyltetradecanoic acid (anteiso-C15:0)--has slightly repressed flagella-driven swimming motility and completely inhibited a more complex type of surface motility, i.e. swarming, at a concentration of 10 microg mL(-1). In contrast, anteiso-C15:0 exhibited no effect on pili-mediated twitching motility. Other BCFAs and unsaturated fatty acids tested in this study showed similar inhibitory effects on swarming motility, although the level of inhibition differed between these fatty acids. These fatty acids caused no significant growth inhibition in liquid cultures. Straight-chain saturated fatty acids such as palmitic acid were less effective in swarming inhibition. The wetness of the PAO1 colony was significantly reduced by the addition of anteiso-C15:0; however, the production of rhamnolipids as a surface-active agent was not affected by the fatty acid. In addition to motility repression, anteiso-C15:0 caused 31% repression of biofilm formation by PAO1, suggesting that BCFA could affect the multiple cellular activities of Pseudomonas aeruginosa.     

Long-range alteration of the physical environment mediates cooperation between Pseudomonas aeruginosa swarming colonies

Pseudomonas aeruginosa makes and secretes massive amounts of rhamnolipid surfactants that enable swarming motility over biogel surfaces. But how these rhamnolipids interact with biogels to assist swarming remains unclear. Here, I use a combination of optical techniques across scales and genetically engineered strains to demonstrate that rhamnolipids can induce agar gel swelling over distances >10,000× the body size of an individual cell. The swelling front is on the micrometric scale and is easily visible using shadowgraphy. Rhamnolipid transport is not restricted to the surface of the gel but occurs through the whole thickness of the plate and, consequently, the spreading dynamics depend on the local thickness. Surprisingly, rhamnolipids can cross the whole gel and induce swelling on the opposite side of a two-face Petri dish. The swelling front delimits an area where the mechanical properties of the surface properties are modified: water wets the surface more easily, which increases the motility of individual bacteria and enables collective motility. A genetically engineered mutant unable to secrete rhamnolipids (ΔrhlA), and therefore unable to swarm, is rescued from afar with rhamnolipids produced by a remote colony. These results exemplify the remarkable capacity of bacteria to change the physical environment around them and its ecological consequences.     

Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methyl-accepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.     

Effect of Carbenicillin on Pseudomonas aeruginosa

The activity of carbenicillin against 200 strains of Pseudomonas aeruginosa was measured by a quantitative agar dilution method. Minimal inhibitory concentrations (M.I.C.''s) for five graded inocula were measured in terms of complete inhibition (CI) and reduced growth (RG). The M.I.C. decreased progressively as inocula were reduced, median values for the 200 strains ranging from 100 to 37.5 μg. per ml. by the CI criterion, and from 75 to 25 μg. per ml. by the RG definition. Ratios of M.I.C. obtained for large and small inocula were usually small. Identical M.I.C.''s by both CI and RG criteria were most often obtained when the inoculum for the RG criterion was 1 or 2 logs higher than that for complete inhibition.Population analysis of 15 strains of Ps. aeruginosa showed that one specific drug concentration usually caused a sharp drop in proportion of viable cells, ranging from 3 to 5 logs. None of the populations were completely non-viable even at 150 μg. per ml. There was evidence that the viability of different-sized populations was reduced disproportionately by carbenicillin.Carbenicillin 300 μg. per ml. exerted appreciable bactericidal effect against nine of 15 strains of Ps. aeruginosa after a 24-hour contact period; after only six hours the bactericidal effect was very small.Quantitative sensitivity measurements for carbenicillin should include M.I.C. values for both CI and RG criteria, using a range of inocula for testing. Such M.I.C. values may well be useful in monitoring carbenicillin therapy of tissue infections.