Role of polysaccharides in Pseudomonas aeruginosa biofilm development

During the past decade, there has been a renewed interest in using Pseudomonas aeruginosa as a model system for biofilm development and pathogenesis. Since the biofilm matrix represents a crucial interface between the bacterium and the host or its environment, considerable effort has been expended to acquire a more complete understanding of the matrix composition. Here, we focus on recent developments regarding the roles of alginate, Psl, and Pel polysaccharides in the biofilm matrix.     

Bacteriophage and phenotypic variation in Pseudomonas aeruginosa biofilm development

A current question in biofilm research is whether biofilm-specific genetic processes can lead to differentiation in physiology and function among biofilm cells. In Pseudomonas aeruginosa, phenotypic variants which exhibit a small-colony phenotype on agar media and a markedly accelerated pattern of biofilm development compared to that of the parental strain are often isolated from biofilms. We grew P. aeruginosa biofilms in glass flow cell reactors and observed that the emergence of small-colony variants (SCVs) in the effluent runoff from the biofilms correlated with the emergence of plaque-forming Pf1-like filamentous phage (designated Pf4) from the biofilm. Because several recent studies have shown that bacteriophage genes are among the most highly upregulated groups of genes during biofilm development, we investigated whether Pf4 plays a role in SCV formation during P. aeruginosa biofilm development. We carried out immunoelectron microscopy using anti-Pf4 antibodies and observed that SCV cells, but not parental-type cells, exhibited high densities of Pf4 filaments on the cell surface and that these filaments were often tightly interwoven into complex latticeworks surrounding the cells. Moreover, infection of P. aeruginosa planktonic cultures with Pf4 caused the emergence of SCVs within the culture. These SCVs exhibited enhanced attachment, accelerated biofilm development, and large regions of dead and lysed cells inside microcolonies in a manner identical to that of SCVs obtained from biofilms. We concluded that Pf4 can mediate phenotypic variation in P. aeruginosa biofilms. We also performed partial sequencing and analysis of the Pf4 replicative form and identified a number of open reading frames not previously recognized in the genome of P. aeruginosa, including a putative postsegregational killing operon.     

左氧氟沙星浸涂导管对铜绿假单胞菌生物膜的影响

目的评估左氧氟沙星（levofloxacin,LFX）浸涂导管抑制铜绿假单胞菌粘附、定植,防止生物膜形成的能力。方法体外部分：制备LFX浸涂导管。LFX浸涂导管、PVC导管分别浸没在5 mL 50%LB培养液中（含PAO1 108CFU/mL）,37℃孵育6、12、24和48 h,在各时间点,予导管表面和导管培养液进行细菌计数。体内部分：小鼠皮下植入LFX浸涂导管或PVC导管,沿着导管注射PAO1菌液50μL（107CFU）。第1、5天,对植入导管及导管周围组织进行细菌计数及扫描电镜（SEM）观察。结果 （1）LFX浸涂导管显示药物的快速释放。（2）在各孵育时间点,LFX浸涂导管及导管培养液的细菌数较PVC导管均明显减少（P〈0.05）。（3）小鼠感染第1、5天,LFX浸涂植入导管表面没有或很少细菌;LFX浸涂导管较PVC导管能明显减少植入导管周围组织的细菌量（P〈0.05）。（4）SEM观察：感染第1、5天,LFX浸涂导管表面散在单个细菌或者没有细菌;而第1天,PVC导管表面大量细菌分散存在。第5天,导管表面＂珊瑚状＂生物膜形成。结论 LFX浸涂导管能抑制铜绿假单胞菌粘附、定植,防止生物膜形成,从而有效降低导管生物膜相关感染的发生。     

Pseudomonas aeruginosa attachment and biofilm development in dynamic environments

Biofilm formation by Pseudomonas aeruginosa is hypothesized to follow a developmental pattern initiated by attachment to a surface followed by microcolony formation and mature biofilm development. Swimming and twitching motility are important for attachment and biofilm development in P. aeruginosa. However, it is clear that many P. aeruginosa strains lacking swimming motility exist as biofilms in the lungs of cystic fibrosis patients. Consequently, we have developed a dynamic attachment assay to identify motility-independent attachment-defective mutants. Using transposon mutagenesis, we identified 14 novel dynamic attachment-deficient (dad) mutants including four mutants specific to dynamic assay conditions (dad specific). Two of the dad-specific mutants contain insertions in genes involved in sensing and responding to external stimuli, implying a significant impact of external factors on the biofilm developmental pathway. Observations of initial attachment and long-term biofilm formation characterized our dad mutants into two distinct classes: biofilm delayed and biofilm impaired. Biofilm-delayed mutants form wild-type biofilms but are delayed at least 24 h compared with the wild type, whereas biofilm-impaired mutants never form wild-type biofilms in our assays. We propose a dynamic model for attachment and biofilm formation in P. aeruginosa including these two classes.     

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。     

Putative exopolysaccharide synthesis genes influence Pseudomonas aeruginosa biofilm development

An analysis of the Pseudomonas aeruginosa genomic sequence revealed three gene clusters, PA1381-1393, PA2231-2240, and PA3552-3558, in addition to the alginate biosynthesis gene cluster, which appeared to encode functions for exopolysaccharide (EPS) biosynthesis. Recent evidence indicates that alginate is not a significant component of the extracellular matrix in biofilms of the sequenced P. aeruginosa strain PAO1. We hypothesized that at least one of the three potential EPS gene clusters revealed by genomic sequencing is an important component of P. aeruginosa PAO1 biofilms. Thus, we constructed mutants with chromosomal insertions in PA1383, PA2231, and PA3552. The mutant with a PA2231 defect formed thin unstructured abnormal biofilms. The PA3552 mutant formed structured biofilms that appeared different from those formed by the parent, and the PA1383 mutant formed structured biofilms that were indistinguishable from those formed by the parent. Consistent with a previous report, we found that polysaccharides were one component of the extracellular matrix, which also contained DNA. We suggest that the genes that were inactivated in our PA2231 mutant are required for the production of an EPS, which, although it may be a minor constituent of the matrix, is critical for the formation of P. aeruginosa PAO1 biofilms.     

Multiple roles of biosurfactants in structural biofilm development by Pseudomonas aeruginosa

Recent studies have indicated that biosurfactants produced by Pseudomonas aeruginosa play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. Through the use of flow cell technology and enhanced confocal laser scanning microscopy, we have obtained results which suggest that the biosurfactants produced by P. aeruginosa play additional roles in structural biofilm development. We present genetic evidence that during biofilm development by P. aeruginosa, biosurfactants promote microcolony formation in the initial phase and facilitate migration-dependent structural development in the later phase. P. aeruginosa rhlA mutants, deficient in synthesis of biosurfactants, were not capable of forming microcolonies in the initial phase of biofilm formation. Experiments involving two-color-coded mixed-strain biofilms showed that P. aeruginosa rhlA mutants were defective in migration-dependent development of mushroom-shaped multicellular structures in the later phase of biofilm formation. Experiments involving three-color-coded mixed-strain P. aeruginosa biofilms demonstrated that the wild-type and rhlA and pilA mutant strains formed distinct subpopulations on top of each other dependent on their ability to migrate and produce biosurfactants.     

Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development

Bacteria form surface attached biofilm communities as one of the most important survival strategies in nature. Biofilms consist of water, bacterial cells and a wide range of self-generated extracellular polymeric substances (EPS). Biofilm formation is a dynamic self-assembly process and several distinguishable stages are observed during bacterial biofilm development. Biofilm formation is shown to be coordinated by EPS production, cell migration, subpopulation differentiation and interactions. However, the ways these different factors affect each other and contribute to community structural differentiation remain largely unknown. The distinct roles of different EPS have been addressed in the present report. Both Pel and Psl polysaccharides are required for type IV pilus-independent microcolony formation in the initial stages of biofilm formation by Pseudomonas aeruginosa PAO1. Both Pel and Psl polysaccharides are also essential for subpopulation interactions and macrocolony formation in the later stages of P. aeruginosa PAO1 biofilm formation. Pel and Psl polysaccharides have different impacts on Pseudomonas quinolone signal-mediated extracellular DNA release in P. aeruginosa PAO1 biofilms. Psl polysaccharide is more important than Pel polysaccharide in P. aeruginosa PAO1 biofilm formation and antibiotic resistance. Our study thus suggests that different EPS materials play distinct roles during bacterial biofilm formation.     

Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. We report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective ( sad ). Two classes of sad mutants were analysed: (i) mutants defective in flagellar-mediated motility and (ii) mutants defective in biogenesis of the polar-localized type IV pili. We followed the development of the biofilm formed by the wild type over 8 h using phase-contrast microscopy. The wild-type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time-lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As observed with the wild type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild-type strain, the type IV pili mutants did not develop microcolonies over the course of the experiments, suggesting that these structures play an important role in microcolony formation. Very few cells of a non-motile strain (carrying a mutation in flgK ) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell-to-surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.     

The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa

LecA (PA-IL) is a cytotoxic lectin and adhesin produced by Pseudomonas aeruginosa which binds hydrophobic galactosides with high specificity and affinity. By using a lecA-egfp translation fusion and immunoblot analysis of the biofilm extracellular matrix, we show that lecA is expressed in biofilm-grown cells. In static biofilm assays on both polystyrene and stainless steel, biofilm depth and surface coverage was reduced by mutation of lecA and enhanced in the LecA-overproducing strain PAO-P47. Biofilm surface coverage by the parent strain, PAO-P47 but not the lecA mutant on steel coupons was also inhibited by growth in the presence of either isopropyl-beta-D-thiogalactoside (IPTG) or p-nitrophenyl-alpha-D-galactoside (NPG). Furthermore, mature wild-type biofilms formed in the absence of these hydrophobic galactosides could be dispersed by the addition of IPTG. In contrast, addition of p-nitrophenyl-alpha-L-fucose (NPF) which has a high affinity for the P. aeruginosa LecB (PA-IIL) lectin had no effect on biofilm formation or dispersal. Planktonic growth of P. aeruginosa PAO1 was unaffected by the presence of IPTG, NPG or NPF, nor was the strain able to utilize these sugars as carbon sources, suggesting that the observed effects on biofilm formation were due to the competitive inhibition of LecA-ligand binding. Similar results were also obtained for biofilms grown under dynamic flow conditions on steel coupons, suggesting that LecA contributes to P. aeruginosa biofilm architecture under different environmental conditions.     

铜绿假单胞菌铁摄取与生物被膜形成研究进展

生物被膜是单细胞微生物通过其分泌的胞外多聚基质粘附于介质表面并将其自身包绕其中而成的膜样微生物细胞聚集物。生物被膜的形成使细菌具有更强的适应外界环境的能力,也是导致微生物产生耐药性及慢性感染性疾病难以治疗的重要原因之一。铜绿假单胞菌在肺部的定殖是肺囊性纤维化病患者发病和死亡主要原因,其造成的感染通常与形成抗生素抗性极强的生物被膜有关。铜绿假单胞菌生物被膜的形成受控于多种复杂的细菌调控体系之下,包括群体感应系统及参与调节胞外多聚基质合成的双组分调控系统等。此外,为了利用低浓度的环境铁来维持生存并完成各种生理功能,铜绿假单胞菌进化出了一系列铁摄取系统,这些系统对其毒力因子的释放和生物被膜的形成又起着重要的调控作用。本文主要对铜绿假单胞菌生物被膜的形成与调控机制及其铁摄取系统进行了综述,为进一步了解及清除铜绿假单胞菌引发的问题提供途径与思路。     

Pseudomonas aeruginosa biofilm: Potential therapeutic targets

Pseudomonas aeruginosa is a gram-negative pathogen that has become an important cause of infection, especially in patients with compromised host defense mechanisms. It is frequently related to nosocomial infections such as pneumonia, urinary tract infections (UTIs) and bacteremia. The biofilm formed by the bacteria allows it to adhere to any surface, living or non-living and thus Pseudomonal infections can involve any part of the body. Further, the adaptive and genetic changes of the micro-organisms within the biofilm make them resistant to all known antimicrobial agents making the Pseudomonal infections complicated and life threatening. Pel, Psl and Alg operons present in P. aeruginosa are responsible for the biosynthesis of extracellular polysaccharide which plays an important role in cell–cell and cell–surface interactions during biofilm formation. Understanding the bacterial virulence which depends on a large number of cell-associated and extracellular factors is essential to know the potential drug targets for future studies. Current novel methods like small molecule based inhibitors, phytochemicals, bacteriophage therapy, photodynamic therapy, antimicrobial peptides, monoclonal antibodies and nanoparticles to curtail the biofilm formed by P. aeruginosa are being discussed in this review.     

Pseudomonas aeruginosa: characteristics of biofilm process

Definition of the biofilm process as one of the types of intercellular bacterial communications is presented. The modern data concerning the structure of the Pseudomonas aeruginosa biofilm matrix and genetic mechanisms necessary for its production are described. Active and passive rejections of biofilm bacteria, which are the basis of bacterial spreading to new surfaces, are discussed. The complexity and chain type of the reactions associated with biofilm formation are emphasized.     

Use of in-biofilm expression technology to identify genes involved in Pseudomonas aeruginosa biofilm development

Mature Pseudomonas aeruginosa biofilms form complex three-dimensional architecture and are tolerant of antibiotics and other antimicrobial compounds. In this work, an in vivo expression technology system, originally designed to study virulence-associated genes in complex mammalian environments, was used to identify genes up-regulated in P. aeruginosa grown to a mature (5-day) biofilm. Five unique cloned promoters unable to promote in vitro growth in the absence of purines after recovery from the biofilm environment were identified. The open reading frames downstream of the cloned promoter regions were identified, and knockout mutants were generated. Insertional mutation of PA5065, a homologue of Escherichia coli ubiB, was lethal, while inactivation of PA0240 (a porin homologue), PA3710 (a putative alcohol dehydrogenase), and PA3782 (a homologue of the Streptomyces griseus developmental regulator adpA) had no effect on planktonic growth but caused defects in biofilm formation in static and flowing systems. In competition experiments, mutants demonstrated reduced fitness compared with the parent strain, comprising less than 0.0001% of total biofilm cells after 5 days. Therefore, using in-biofilm expression technology, we have identified novel genes that do not affect planktonic growth but are important for biofilm formation, development, and fitness.     

Alterations in extracellular substances during the biofilm development of Pseudomonas aeruginosa on aluminum plates

The chemical moieties during biofilm formation of Pseudomonas aeruginosa on aluminium plates were examined for a period of 17 days. The effect of fluid shearing upon biofilm formation has also been investigated. The Fourier transform infrared (FTIR) spectrum of the biofilm taken on the fifth day showed significant differences compared with the spectrum of the unattached bacterial cells, indicating that structural changes or modifications of the cell envelope had taken place during the development of the biofilm. Major changes were also observed in the spectrum during the subsequent development of the biofilm from day 5 to day 17. The increasing intensity of a band corresponding to the symmetric stretching mode of the carboxyl group indicated interactions between the carboxyl group and the aluminium surface. Increased bacterial colonization was also observed at the air-water interface of the aluminium plates when compared with the middle and the bottom parts. Changes in FTIR spectra of the biofilm at the bottom, at the middle, and at the air-water interface suggest that the mechanisms of bacterial attachment differed by a -COO(-) interaction at the air-water interface, and by both -COO(-) and NH3(+) groups beneath the water surface.     

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.     

In situ growth rates and biofilm development of Pseudomonas aeruginosa populations in chronic lung infections

The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.     

Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa

Bacterial biofilms at times undergo regulated and coordinated dispersal events where sessile biofilm cells convert to free-swimming, planktonic bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, we previously observed that dispersal occurs concurrently with three interrelated processes within mature biofilms: (i) production of oxidative or nitrosative stress-inducing molecules inside biofilm structures, (ii) bacteriophage induction, and (iii) cell lysis. Here we examine whether specific reactive oxygen or nitrogen intermediates play a role in cell dispersal from P. aeruginosa biofilms. We demonstrate the involvement of anaerobic respiration processes in P. aeruginosa biofilm dispersal and show that nitric oxide (NO), used widely as a signaling molecule in biological systems, causes dispersal of P. aeruginosa biofilm bacteria. Dispersal was induced with low, sublethal concentrations (25 to 500 nM) of the NO donor sodium nitroprusside (SNP). Moreover, a P. aeruginosa mutant lacking the only enzyme capable of generating metabolic NO through anaerobic respiration (nitrite reductase, DeltanirS) did not disperse, whereas a NO reductase mutant (DeltanorCB) exhibited greatly enhanced dispersal. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. We observed that exposure to SNP (500 nM) greatly enhanced the efficacy of antimicrobial compounds (tobramycin, hydrogen peroxide, and sodium dodecyl sulfate) in the removal of established P. aeruginosa biofilms from a glass surface. Combined exposure to both NO and antimicrobial agents may therefore offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.