铜绿假单胞菌生物膜分散的研究近况

细菌分泌胞外多糖附着在物体表面组成一个结构性群体即生物膜,导致对抗生素的强抵抗性和感染的迁延不愈。反过来,已形成的生物膜也可以分散为游离菌,许多环境物质能够促进该分散过程,并且这些物质与抗生素合用对生物膜有强大的对抗作用。从生物膜到浮游菌是个复杂的过程,目前关于铜绿假单胞菌生物膜分散的特征、机制、诱导分子等已经引起了学者的强烈兴趣,随着问题的深入研究必然会给人类治疗生物膜所致的难治性感染带来更大的意义。     

Involvement of the Pseudomonas aeruginosa MexAB–OprM efflux pump in the secretion of the metallophore pseudopaline

To overcome the metal restriction imposed by the host’s nutritional immunity, pathogenic bacteria use high metal affinity molecules called metallophores. Metallophore‐mediated metal uptake pathways necessitate complex cycles of synthesis, secretion, and recovery of the metallophore across the bacterial envelope. We recently discovered staphylopine and pseudopaline, two members of a new family of broad‐spectrum metallophores important for bacterial survival during infections. Here, we are expending the molecular understanding of the pseudopaline transport cycle across the diderm envelope of the Gram‐negative bacterium Pseudomonas aeruginosa. We first explored pseudopaline secretion by performing in vivo quantifications in various genetic backgrounds and revealed the specific involvement of the MexAB–OprM efflux pump in pseudopaline transport across the outer membrane. We then addressed the recovery part of the cycle by investigating the fate of the recaptured metal‐loaded pseudopaline. To do so, we combined in vitro reconstitution experiments and in vivo phenotyping in absence of pseudopaline transporters to reveal the existence of a pseudopaline modification mechanism, possibly involved in the metal release following pseudopaline recovery. Overall, our data allowed us to provide an improved molecular model of secretion, recovery, and fate of this important metallophore by P. aeruginosa.     

mvaT mutation modifies the expression of the Pseudomonas aeruginosa multidrug efflux operon mexEF-oprN

Pseudomonas aeruginosa carries several multidrug efflux operons, including mexEF-oprN, that contribute to its resistance to multiple antibiotics. mvaT affects the expression of several P. aeruginosa genes. In this study, we show that the mvaT mutant PAODeltamvaT is more resistant than its parent PAO1 strain to chloramphenicol and norfloxacin but more sensitive to imipenem; yet both were less resistant to chloramphenicol, norfloxacin, and imipenem than 'typical'nfxC-type mutants. Neither strain carries the deletion described for nfxC-type mutants in mexT, the mexEF-oprN regulatory gene. Expression of mexEF-oprN is increased by five- to sixfold in PAODeltamvaT, while the expression of oprD is reduced by approximately twofold. mvaT mutation had no effect on the expression of other multidrug resistance operons, although it increased the expression of several ATP-binding cassette transporter genes. We show that mvaT mutation does not affect mexEF-oprN expression through mexT or mexS. We also explored several other potential mechanisms.     

Effects of phenol and natural phenolic compounds on biofilm formation by Pseudomonas aeruginosa

A biofilm is formed as a result of adhesion of microorganisms to various surfaces with the production of extracellular polymers (polysaccharides and proteins). Biofilms cause serious problems in the chemical, medical and pharmaceutical industries. Recent findings indicate that some natural phenolic compounds found in plants have an anti-biofouling effect on biofilm formation by Gram-negative bacteria. The anti-biofouling activities of 14 selected phenol and natural phenolic compounds were tested against Pseudomonas aeruginosa, using a microtiter-plate. A modified microtiter-plate assay was used because it enabled indirect measurement of bacterial cells attached to the surface of the wells. This assay involved fixing the bacterial film with methanol, staining with crystal violet dye and then releasing the bound dye with 33% glacial acetic acid. The optical density (OD) of the solution was measured at 570 nm by using an automated ICN Flow Titertek Multiscan Plus reader. Phenol and natural phenolic compounds except ethyl linoleate and tocopherol showed a significant reduction in biofilm formation by P. aeruginosa.     

A bestrophin‐like protein modulates the proton motive force across the thylakoid membrane in Arabidopsis

During photosynthesis, photosynthetic electron transport generates a proton motive force(pmf) across the thylakoid membrane, which is used for ATP biosynthesis via ATP synthase in the chloroplast. The pmf is composed of an electric potential(△Ψ) and an osmotic component(△pH).Partitioning between these components in chloroplasts is strictly regulated in response to fluctuating environments.However, our knowledge of the molecular mechanisms that regulate pmf partitioning is limited. Here, we report a bestrophin-like protein(At Best), which is critical for pmf partitioning. While the Dp H component was slightly reduced in atbest, the △Ψ component was much greater in this mutant than in the wild type, resulting in less efficient activation of nonphotochemical quenching(NPQ) upon both illumination and a shift from low light to high light. Although no visible phenotype was observed in the atbest mutant in the greenhouse, this mutant exhibited stronger photoinhibition than the wild type when grown in the field. At Best belongs to the bestrophin family proteins, which are believed to function as chloride(Cl~-) channels. Thus, our findings reveal an Researimportant Cl~- channel required for ion transport and homeostasis across the thylakoid membrane in higher plants. These processes are essential for fine-tuning photosynthesis under fluctuating environmental conditions.     

Lactoferrin‐derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa

Aims: To investigate the bactericidal activity of lactoferrin‐derived peptides and a new LF‐derived peptides chimera (LFchimera) against P. aeruginosa and the influence on virulence factors of P. aeruginosa. Methods and Results: Lactoferricin (LFcin) and lactoferrampin (LFampin) are highly bioactive peptides isolated from the N‐terminal region of lactoferrin (LF) by pepsin digestion. In this study, we designed LFchimera containing LFcin amino acids 17‐30 and LFampin amino acids 268‐284. Pseudomonas aeruginosa cells were incubated in medium with peptides at different concentrations, and then the assays of viability, pyocyanin, elastase activity and biofilm formation of P. aeruginosa were performed. We found that the concentration‐dependent antibactericidal activity and down‐regulating pyocyanin, elastase and biofilm formation of LFchimera were significantly stronger than those of LF, LFcin, LFampin or LFcin plus LFampin. Conclusions: Our results indicated that LF, LFcin, LFampin and LFchimera were potential candidates to combat P. aeruginosa, and LFchimera was the most effective in them. Significance and Impact of the Study: The new LFchimera has better activity against P. aeruginosa than LF, LFcin and LFampin and may be a promising new compound for treatment of P. aeruginosa infection.     

Linoleic acid inhibits Pseudomonas aeruginosa biofilm formation by activating diffusible signal factor‐mediated quorum sensing

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor‐mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis‐2‐decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.     

镁离子对黏液型铜绿假单胞菌生物膜形成过程的影响

目的探讨镁离子对黏液型铜绿假单胞菌早期黏附和生物膜形成过程的影响。方法荧光多功能酶标仪检测各组不同时间点96孔板底部黏附细菌的荧光强度,荧光探针FTTC-ConA染细菌胞外多糖（Extracellular Polymeric Substances,EPS）、荧光显微镜下观察各组多糖差别;SYTO9／PI染生物膜内细菌、激光共聚焦显微镜观察结合BF图像结构分析软件（Image Structor Analyzer,ISA）对各组生物膜结构参数进行定量分析。结果2d时,空白组和1mmol／L镁组的黏附细菌的荧光强度分别为1845．67±45．3和2254．78±42．45,t=-9．96,P〈0．05;0．1mmol／L的镁浓度下荧光强度也有增加,其余各时间组趋势与2d组相似;在荧光显微镜下观察可见随着镁浓度增加,EPS增多;激光共聚焦显微镜下可见随着镁浓度增加,生物膜活菌增加、菌落变密集;ISA软件分析结果示：空白组和1mmol／L镁组的6d生物膜厚度分别为（25．80±1．16）μm和（34．87±1．59）μm,t=-13．85,P〈0．05;区域孔率分别为0．96±0．05和0．90±0．04,t=2．48,P〈0．05;平均扩散距离分别为1．54±0．15和1．92±0．16,t=5．23,P〈0．05;结构熵分别为3．64±0．57和4．70±1．09,t=-2．6,P〈0．05,3d组生物膜也有相同的趋势。结论镁离子可以增强黏液型铜绿假单胞菌的早期黏附,影响随后生物膜的形成及结构。     

纳米银水凝胶涂膜干预气管导管表面铜绿假单胞菌黏附及生物膜形成的实验研究

目的研究纳米银水凝胶涂膜对气管导管（endotracheal tube,ETT）表面铜绿假单胞菌粘附及细菌生物膜（biofilm,BF）形成的干预作用。方法实验共设6组,分别为空白对照组,涂纳米银3．5、7．0、10．5、14．0和17．5μg／cm^2组。参考Brown平板法,制备ETT、表面铜绿假单胞菌BF模型。通过超声振荡-平板菌落计数法检测体外培养6、12、18h时各组ETT表面BF中的活细菌粘附数量。借助激光共聚焦显微镜观察BF中的活死菌分布情况,并测量BF厚度。结果（1）与空白对照组相比,最小量涂膜组（3．5μg／cm^2）体外培养6h时,导管表面活细菌的粘附量显著减少（P〈0．05）,12h时差异无显著性（P〉0．05）;最大量涂膜组（17．5μg／cm^2）体外培养6h时,ETT表面几乎未见细菌粘附（P〈0．05）,18h时BF中的活菌数量及BF厚度均显著减少（P〈0．05）。（2）激光共聚焦显微镜观察可见,培养6h时,空白对照组ETT表面粘附的死活菌呈不规则散点样分布,未见明显的细菌菌落形成,而各实验组ETT表面仅有细菌零星分布,其数量少于空白组。18h时空白对照组表面可见大量活死菌堆积粘连,有小菌落形成并相互交通成地图状,可见典型BF结构,而此时最大量涂膜组（17．5μg／cm^2）表面仅见数量不等的菌落形成,菌落周围可见数量不等的细菌分布。结论纳米银水凝胶涂膜可有效减少ETT表面铜绿假单胞菌的粘附数量,延缓导管表面细菌BF形成,其作用强弱随培养时间及单位面积中的纳米银剂量的变化而变化。     

Influence of State-2 transition on the proton motive force across the thylakoid membrane in spinach chloroplasts

The proton motive force (pmf) across the thylakoid membrane is composed of the proton gradient and the membrane potential, which promotes millisecond-delayed light emission (ms-DLE). In this study, the time courses of LHC II phosphorylation and ms-DLE were investigated in spinach chloroplast during State-2 transition. Red light illumination resulted in an exponential rise in LHC II phosphorylation and a biphasic time course of ms-DLE. The phospho-LHC II appeared upon ∼ 1 min illumination. The phosphorylation level increased exponentially when illumination was elongated to 20 min. The t_&frac; of saturated LHC II phosphorylation was estimated 4–5 min under present illumination. During this process, the amplitudes of ms-DLE increased transiently to a maximal amplitude within 0.5 min illumination, and the reached maximum of the fast phase of ms-DLE was ∼ 140% of the dark control. Then, ms-DLE decreased from the maximum. After ≥3 min illumination, ms-DLE decreased to a lower level than the dark control. In the presence of uncouplers and inhibitors, the transient increase in the biphasic time course of ms-DLE was removed by nigericin and DCMU, and the sequential decrease was delayed by DCCD. The time course was not affected significantly by valinomycin and DBMIB. Moreover, the level of LHC II phosphorylation was enhanced by nigericin, valinomycin and DCCD, and was inhibited completely by DCMU and partially by DBMIB. Taken together, we proposed that the PS II photochemical activity remained unaffected even with a higher level of LHC II phosphorylation, which was reflected by the effect of DCCD on the time course of ms-DLE. Probably, the evidence of LHC II phosphorylation is the rearrangement of LHC II–PS II complex and the thylakoid, a feedback to light-exposure, rather than the redistribution of excitation energy from PS II to PS I.     

Effect of antimicrobial residues on early adhesion and biofilm formation by wild-type and benzalkonium chloride-adapted Pseudomonas aeruginosa

Antimicrobial residue deposition can change the physico-chemical properties of bacteria and surfaces and thus promote or impair bacterial adhesion. This study focuses on benzalkonium chloride (BC) deposition on polystyrene (PS) surfaces and the influence of this conditioning film on the physico-chemical properties of PS and on early adhesion and biofilm formation by Pseudomonas aeruginosa wild-type and its laboratory BC-adapted strain. The latter readily acquired the ability to grow in BC, and also exhibited physico-chemical surface changes. The existence of residues on PS surfaces altered their hydrophobicity and favoured adhesion as determined by the free energy and early adhesion characterization. Adapted bacteria revealed a higher ability to adhere to surfaces and to develop biofilms, especially on BC-conditioned surfaces, which thereby could enhance resistance to sanitation attempts. These findings highlight the importance of investigations concerning the antimicrobial deposition effect after cleaning procedures, which may encourage bacterial adhesion, especially of bacteria that have been previously exposed to chemical stresses.     

Effect of a low concentration of a cationic steroid antibiotic (CSA-13) on the formation of a biofilm by Pseudomonas aeruginosa

Aims: Cationic steroids like CSA‐13 have been designed by analogy with antimicrobial cationic peptides and have bactericidal properties. The purpose of this work was to evaluate the effect of a low concentration (1 mg l^?1) of CSA‐13 on the formation of a biofilm by eight strains of Pseudomonas aeruginosa (four mucoid and four nonmucoid strains) on an inert surface. Method and Results: The biofilm formation was measured with the Crystal Violet method. CSA‐13 inhibited the formation of a biofilm by three strains. The zeta potential varied among the strains. The inhibition by the cationic steroid analogue affected the populations of bacteria with the lowest zeta potential. P. aeruginosa bound a fluorescent, more hydrophobic analogue of CSA‐13 but there was no correlation between this binding and the inhibition by CSA‐13 of biofilm formation. The interaction of CSA‐13 with bacteria did not modify their ability to produce rhamnolipids. Conclusions: A low concentration of CSA‐13 inhibits the formation of a biofilm by P. aeruginosa through electrostatic interactions and without affecting the production of rhamnolipids. Significance and Impact of the Study: A low, nontoxic concentration of CSA‐13 might be beneficial for the prevention of biofilm formation.     

Molecular investigation of carbapenem resistance among multidrug‐resistant Pseudomonas aeruginosa isolated clinically in Thailand

Carbapenem resistant Pseudomonas aeruginosa were isolated among multidrug‐resistant (CR‐MDR) organisms from tertiary hospitals in Thailand. Decreased expression of oprD mRNA (93.65%) was predominant followed by increased expression of mexAB‐oprM mRNA (92.06%) and mexXY mRNA (63.49%). Interestingly, 23 of 126 (18.25%) isolates were susceptible to imipenem with down‐regulated oprD expression and non‐up‐regulated mexCD‐oprJ mRNA expression. Metallo‐β‐lactamases production was clearly positive in 24 isolates (18.46%) and weakly positive in 12 isolates (9.23%). Among both of these sets of isolates, imp‐1, imp‐14 and vim‐2 were identified. Hyperproduction of AmpC β‐lactamase had the lowest prevalence rate (3.97%). It was concluded that CR‐MDR P. aeruginosa clinical isolates in Thailand possess multifactorial resistance mechanisms.     

Inhibitors of the Pseudomonas aeruginosa quorum‐sensing regulator,QscR

QscR is a quorum‐sensing (QS) signal receptor that controls expression of virulence genes in the prevalent opportunistic pathogen, Pseudomonas aeruginosa. Unlike the previously reported LuxR‐type QS receptor proteins, that is, LasR and TraR, QscR can be obtained as an apo‐protein that can reversibly form an active complex in vitro with its cognate signal molecule, 3‐oxododecanoyl‐homoserine lactone (3OC12‐HSL), and subsequently bind to target promoter DNA sequences. To search for potential QS inhibitors, an in vitro gel retardation assay was developed using the purified QscR. Both the in vitro assay and the in vivo cell‐based assay using QscR‐overproducing recombinant strains were applied in the screening process. Furanones were chosen for testing the activity as QS inhibitors because they have been reported to strongly inhibit expression of QS‐related genes in Agrobacterium tumefaciens. Among more than a hundred furanones tested, three compounds showed strong and dose‐dependent inhibitory effects on QscR in both assays. One compound in particular, designated as F2, could completely inhibit the 3OC12‐HSL‐dependent QscR activity in vitro at a concentration of 50‐fold molar excess over 3OC12‐HSL. However, with the furanones F3 and F4, which are structurally similar to F2 but with a nitro group instead of the amine moiety, significantly decreased activities were observed. These results suggest that (i) the in vitro assay is a sensitive and reliable tool for screening QS inhibitors, and (ii) furanones are potentially important QS inhibitors for many LuxR‐type receptor proteins. Biotechnol. Bioeng. 2010; 106: 119–126. © 2010 Wiley Periodicals, Inc.     

Effects of localised,low-voltage pulsed electric fields on the development and inhibition of Pseudomonas aeruginosa biofilms

Abstract

This work describes the use of low-voltage (0.5 – 5 V) pulsed electric fields to prevent Pseudomonas aeruginosa biofilm development. Interdigitated electrodes (IDEs) with 29-μm spacing between 22-μm-wide electrodes, were used as a platform where the effect of localised, high-strength electric fields could be tested. Alternating current, square-wave pulses were applied to the IDEs in 1 sec intervals. A two-level, three-variable factorial design experiment was used to detect the effects of applied voltage, frequency, and pulse duty ratio (i.e. percentage of pulsing time over one cycle) on the inhibition of biofilm formation. The observations indicated that a pulse configuration of 1% duty ratio, 5 V, and 200 Hz frequency reduced the area of the electrodes covered by biofilm by 50%. In general, the application of low-duty ratio pulses had a positive effect on preventing biofouling. Comparatively, frequency and applied voltage were observed to have less influence on biofouling.