Role of Mutation in Pseudomonas aeruginosa Biofilm Development

The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.     

Biofilm formation by hyperpiliated mutants of Pseudomonas aeruginosa

Under static growth conditions, hyperpiliated, nontwitching pilT and pilU mutants of Pseudomonas aeruginosa formed dense biofilms, showing that adhesion, not twitching motility, is necessary for biofilm initiation. Under flow conditions, the pilT mutant formed mushroom-like structures larger than those of the wild type but the pilU mutant was defective in biofilm formation. Therefore, twitching motility affects the development of biofilm structure, possibly through modulation of detachment.     

Chelator-Induced Dispersal and Killing of Pseudomonas aeruginosa Cells in a Biofilm

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.     

Biofilm culture of Pseudomonas aeruginosa expressing lux genes as a model to study susceptibility to antimicrobials

A simple in vitro model for culture of biofilm populations of self-bioluminescent Pseudomonas aeruginosa was used for real-time monitoring of the effects of ciprofloxacin. Biofilms of these organisms were established within Sorbarod filters, perfused with a chemically defined simple salts medium. The biofilm population was shown to achieve a pseudo-steady state which was reproducible and stable over several days. The viability of membrane-associated and eluted cells was assessed by spread plate viable counts and by monitoring bioluminescence as a measure of metabolic activity. Pseudo-steady state biofilms were exposed to 5x MIC ciprofloxacin (0.3 mg x l(-1)) in the perfusing medium for 1 h. Whilst both methods for viability assessment indicated an immediate reduction in viable cell numbers, the decline recorded with bioluminescence was greater. The use of bioluminescent bacteria proved to be a rapid and sensitive method for the measurement of real-time antibacterial effects on a bacterial biofilm.     

A Systems-Level Approach for Investigating Pseudomonas aeruginosa Biofilm Formation

Prevention of the initiation of biofilm formation is the most important step for combating biofilm-associated pathogens, as the ability of pathogens to resist antibiotics is enhanced 10 to 1000 times once biofilms are formed. Genes essential to bacterial growth in the planktonic state are potential targets to treat biofilm-associated pathogens. However, the biofilm formation capability of strains with mutations in these essential genes must be evaluated, since the pathogen might form a biofilm before it is eliminated. In order to address this issue, this work proposes a systems-level approach to quantifying the biofilm formation capability of mutants to determine target genes that are essential for bacterial metabolism in the planktonic state but do not induce biofilm formation in their mutants. The changes of fluxes through the reactions associated with the genes positively related to biofilm formation are used as soft sensors in the flux balance analysis to quantify the trend of biofilm formation upon the mutation of an essential gene. The essential genes whose mutants are predicted not to induce biofilm formation are regarded as gene targets. The proposed approach was applied to identify target genes to treat Pseudomonas aeruginosa infections. It is interesting to find that most essential gene mutants exhibit high potential to induce the biofilm formation while most non-essential gene mutants do not. Critically, we identified four essential genes, lysC, cysH, adk, and galU, that constitute gene targets to treat P. aeruginosa. They have been suggested by existing experimental data as potential drug targets for their crucial role in the survival or virulence of P. aeruginosa. It is also interesting to find that P. aeruginosa tends to survive the essential-gene mutation treatment by mainly enhancing fluxes through 8 metabolic reactions that regulate acetate metabolism, arginine metabolism, and glutamate metabolism.     

Heavy Metal Resistance of Biofilm and Planktonic Pseudomonas aeruginosa

A study was undertaken to examine the effects of the heavy metals copper, lead, and zinc on biofilm and planktonic Pseudomonas aeruginosa. A rotating-disk biofilm reactor was used to generate biofilm and free-swimming cultures to test their relative levels of resistance to heavy metals. It was determined that biofilms were anywhere from 2 to 600 times more resistant to heavy metal stress than free-swimming cells. When planktonic cells at different stages of growth were examined, it was found that logarithmically growing cells were more resistant to copper and lead stress than stationary-phase cells. However, biofilms were observed to be more resistant to heavy metals than either stationary-phase or logarithmically growing planktonic cells. Microscopy was used to evaluate the effect of copper stress on a mature P. aeruginosa biofilm. The exterior of the biofilm was preferentially killed after exposure to elevated concentrations of copper, and the majority of living cells were near the substratum. A potential explanation for this is that the extracellular polymeric substances that encase a biofilm may be responsible for protecting cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm.     

Biofilm and Quorum sensing mediated pathogenicity in Pseudomonas aeruginosa

The emergence of multidrug resistance has become an alarming and lifethreatening phenomenon for humans. Various mechanisms are involved in the development of resistance in bacteria towards antimicrobial compounds and immune system. Bacterial biofilm is a complicated, selfdefensive, rigid structure of bacteria crowded together to develop a selfrecessive nature, which enhances the ability to cause infections much easier in the living host. P. aeruginosa biofilm formation is supported by extracellular polymeric substances (EPS) such as exopolysaccharides, extracellular DNA (eDNA), proteins and biomolecules. Published evidences suggest that biofilm formation can also be the result of several other mechanisms such as cell signaling or communication. Bacterial biofilm is also regulated by strong intercellular communication known as Quorum Sensing (QS). It is a cellular communication mechanism involving autoinducers and regulators. In P. aeruginosa, Acyl Homoserine Lactone, the prime signaling molecule, controls approximately 300 genes responsible for various cellular functions, including its pathogenesis. The surrounding environment and metabolism have a specific effect on the biofilm and QS, thus, understanding the involvement of QS in the biofilm developing mechanism is still complicated and complex to understand. Therefore, this review will include basic knowledge of the biofilmforming mechanism and other regulatory factors involved in causing infections and diseases in the host organisms.     

Characterization of Membrane Lipidome Changes in Pseudomonas aeruginosa during Biofilm Growth on Glass Wool

Bacteria cells within biofilms are physiologically distinct from their planktonic counterparts. In particular they are more resistant to detrimental environmental conditions. In this study, we monitored the evolution of the phospholipid composition of the inner and outer membranes of P. aeruginosa during the biofilm formation (i.e., from 1-, 2-, to 6-day-old biofilm). Lipidome analyses were performed by electrospray ionization mass spectrometry. In addition to the lipidomic analysis, the fatty acid composition was analysed by gas chromatography/mass spectrometry. We found that the lipidome alterations of the inner and the outer membranes varied with the biofilm age. These alterations in phospholipid compositions reflect a higher diversity in sessile organisms than in planktonic counterparts. The diversity is characterized by the presence of PE 30∶1, PE 31∶0 and PG 31∶0 for the lower masses as well as PE 38∶1, 38∶2, 39∶1, 39∶2 and PG 38∶0, 38∶1, 38∶2, 39∶1, 39∶2 for the higher masses. However, this lipidomic feature tends to disappear with the biofilm age, in particular the high mass phospholipids tend to disappear. The amount of branched chains phospholipids mainly located in the outer membrane decreased with the biofilm age, whereas the proportion of cyclopropylated phospholipids increased in both membranes. In bacteria present in oldest biofilms, i.e., 6-day-old, the phospholipid distribution moved closer to that of planktonic bacteria.     

Phenotypes of non-attached Pseudomonas aeruginosa aggregates resemble surface attached biofilm

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations.     

PslD Is a Secreted Protein Required for Biofilm Formation by Pseudomonas aeruginosa

The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.     

Novel Multiscale Modeling Tool Applied to Pseudomonas aeruginosa Biofilm Formation

Multiscale modeling is used to represent biological systems with increasing frequency and success. Multiscale models are often hybrids of different modeling frameworks and programming languages. We present the MATLAB-NetLogo extension (MatNet) as a novel tool for multiscale modeling. We demonstrate the utility of the tool with a multiscale model of Pseudomonas aeruginosa biofilm formation that incorporates both an agent-based model (ABM) and constraint-based metabolic modeling. The hybrid model correctly recapitulates oxygen-limited biofilm metabolic activity and predicts increased growth rate via anaerobic respiration with the addition of nitrate to the growth media. In addition, a genome-wide survey of metabolic mutants and biofilm formation exemplifies the powerful analyses that are enabled by this computational modeling tool.     

Phomopsis tersa as Inhibitor of Quorum Sensing System and Biofilm Forming Ability of Pseudomonas aeruginosa

Indian Journal of Microbiology - Endophytic fungi provide rich reservoir for novel antimicrobial compounds. An endophytic fungus, from Carica papaya plant identified as Phomopsis tersa, was...     

Tannins Possessing Bacteriostatic Effect Impair Pseudomonas aeruginosa Adhesion and Biofilm Formation

Plants produce many compounds that are biologically active, either as part of their normal program of growth and development or in response to pathogen attack or stress. Traditionally, Anadenanthera colubrina, Commiphora leptophloeos and Myracrodruon urundeuva have been used by communities in the Brazilian Caatinga to treat several infectious diseases. The ability to impair bacterial adhesion represents an ideal strategy to combat bacterial pathogenesis, because of its importance in the early stages of the infectious process; thus, the search for anti-adherent compounds in plants is a very promising alternative. This study investigated the ability of stem-bark extracts from these three species to control the growth and prevent biofilm formation of Pseudomonas aeruginosa, an important opportunistic pathogen that adheres to surfaces and forms protective biofilms. A kinetic study (0–72 h) demonstrated that the growth of extract-treated bacteria was inhibited up to 9 h after incubation, suggesting a bacteriostatic activity. Transmission electron microscopy and fluorescence microscopy showed both viable and nonviable cells, indicating bacterial membrane damage; crystal violet assay and scanning electron microscopy demonstrated that treatment strongly inhibited biofilm formation during 6 and 24 h and that matrix production remained impaired even after growth was restored, at 24 and 48 h of incubation. Herein, we propose that the identified (condensed and hydrolyzable) tannins are able to inhibit biofilm formation via bacteriostatic properties, damaging the bacterial membrane and hindering matrix production. Our findings demonstrate the importance of this abundant class of Natural Products in higher plants against one of the most challenging issues in the hospital setting: biofilm resilience.     

The Use of New Methylene Blue in Pseudomonas aeruginosa Biofilm Destruction

A Pseudomonas aeruginosa biofilm was produced in a model system using the bacterial strain NCIMB 8295, grown on silicone tubing (bore size 0.75 cm). Destruction of the biofilm was attempted using either ampicillin or a combination of white light (light dose=7.2 J cm^{m 2}) and the phenothiazinium photosensitiser new methylene blue, and damage, both to extra-cellular polymeric substance (EPS) and to the organism, was monitored. It was found that although little damage to the EPS occurred with ampicillin, NMB caused both cell death and breakdown of the EPS, suggesting the use of photodynamic antimicrobial chemotherapy (PACT) in the disinfection of pathogenic biofilms, e.g. at external catheter surfaces.     

Biofilm formation by the small colony variant phenotype of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an ubiquitous environmental bacterium and an opportunistic human pathogen. Not only in most natural habitats but also within the human host, e.g. within the chronically infected cystic fibrosis lung, P. aeruginosa is associated with surfaces in structures known as biofilms. These functional communities represent a unique mode of bacterial growth where bacteria display particular phenotypes that are fundamentally different from planktonic cells. In this review the issue of the molecular mechanisms underlying the emergence of small colony variant (SCV) P. aeruginosa morphotypes that are especially capable of forming biofilms is addressed. It is assumed that the expression of the chaperone usher pathway (cup) genes encoding putative fimbrial adhesins is responsible for the phenotypic switch to an autoaggregative SCV phenotype. The elucidation of phenotypic switching in response to environmental stimuli will significantly increase our understanding of regulatory processes during bacterial adaptation and might be the basis for the initiation of the development of new antimicrobial treatment strategies.     

QS系统中LasR和RhlR蛋白对铜绿假单胞菌生物被膜形成的影响以及免疫原性研究

为探讨铜绿假单胞菌 PAO1 中 lasR 和 rhlR 基因表达产物的分子生物学特性,研究它们对铜绿假单胞菌生物被膜形成的影响以及对小鼠的免疫保护效果,采用聚合酶链式反应 (PCR) 方法扩增铜绿假单胞菌标准株 PAO1 中的 lasR 和 rhlR 基因,全自动荧光测序仪测序,并用 Blast 方法检测克隆片段. 利用 pGEX4T-1 载体分别构建 lasR/rhlR-pGEX4T-1 重组质粒,在大肠杆菌 BL21(DE3)中诱导表达,并经过免疫印迹实验验证其生物学活性. 用硅胶膜培养法建立生物被膜模型,诱导转入了pGFPuv 质粒的铜绿假单胞菌 PAG0305 形成生物被膜,并测定 LasR 蛋白和 RhlR 蛋白对生物被膜形成的影响. 同时用纯化的重组蛋白免疫小鼠,菌落计数法检测免疫组和对照组鼠肺对铜绿假单胞菌的清除率. 以 PAO1 染色体 DNA 为模板的 PCR 结果显示,lasR 的全基因序列为 720 bp,rhlR 基因序列为 726 bp,经序列分析和同源性比较分别与 GenBank 中 lasR/rhlR 基因(登录号:M59425; AE004768) 的同源性为 100%. 大肠杆菌 BL21 (DE3) 分别转化重组质粒 lasR/rhlR-pGEX4T-1 后,经 IPTG诱导和 SDS- 聚丙烯酰胺凝胶电泳分析,表达的融合蛋白分子质量均为 54 ku 左右,与预期蛋白质分子质量相同. 荧光显微镜观察和测定结果表明,在硅胶膜上 PAG0305 能够形成典型的发荧光的生物被膜,LasR 或 RhlR 蛋白 (10 mg/L) 存在的情况下,PAG0305 生物被膜的形成速度在前三天比对照组平均提高 40.77%,而且两蛋白单独存在与同时存在时的作用相同. 体内实验中,免疫小鼠肺部对铜绿假单胞菌的清除率显著高于未经免疫的正常组 (P < 0.05). 上述结果表明:构建的lasR/rhlR-pGEX4T-1 重组质粒能够在大肠杆菌 BL21(DE3)中成功地表达并具有生物学活性. LasR/RhlR 蛋白在体外模型中能够加快铜绿假单胞菌生物被膜的形成速度,是调节铜绿假单胞菌生物被膜形成的重要因素之一. 免疫结果表明,重组蛋白对小鼠表现出一定的保护作用,这为进一步开展疫苗研究奠定了基础.     

Biofilm formation by Pseudomonas aeruginosa in solid murine tumors - a novel model system

The ability of opportunistic bacterial pathogens to grow in biofilms is decisive in the pathogenesis of chronic infectious diseases. Growth within biofilms does not only protect the bacteria against the host immune system but also from the killing by antimicrobial agents. Here, we introduce a mouse model in which intravenously administered planktonic Pseudomonas aeruginosa bacteria are enriched in transplantable subcutaneous mouse tumors. Electron microscopy images provide evidence that such bacteria reside in the tumor tissue within biofilm structures. Immunohistology furthermore demonstrated that infection of the tumor tissue elicits a host response characterized by strong neutrophilic influx. Interestingly, the biofilm defective PA14 pqsA transposon mutant formed less biofilm in vivo and was more susceptible to clearance by intravenous ciprofloxacin treatment as compared to the wild-type control. In conclusion, we have established an experimentally tractable model that may serve to identify novel bacterial and host factors important for in vivo biofilm formation and to re-evaluate bactericidal and anti-biofilm effects of currently used and novel antibacterial compounds.     

The Mechanism of Formation of Pseudomonas aeruginosa Biofilm,a Type of Structured Population

The paper is an attempt to analyze and generalize molecular and cell biology data on the formation of polysaccharide matrix–based biofilms. The conception of biofilms as structured populations sharing the characteristics of uni- and multicellular organisms and population is proposed.