Pyocyanin Promotes Extracellular DNA Release in Pseudomonas aeruginosa

Bacterial adhesion and biofilm formation are both dependent on the production of extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA). eDNA promotes biofilm establishment in a wide range of bacterial species. In Pseudomonas aeruginosa eDNA is major component of biofilms and is essential for biofilm formation and stability. In this study we report that production of pyocyanin in P. aeruginosa PAO1 and PA14 batch cultures is responsible for promotion of eDNA release. A phzSH mutant of P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H₂O₂) generation, cell lysis, and eDNA release in comparison to its wildtype strain. A ΔphzA-G mutant of P. aeruginosa PA14 deficient in pyocyanin production generated negligible amounts of H₂O₂ and released less eDNA in comparison to its wildtype counterpart. Exogenous addition of pyocyanin or incubation with H₂O₂ was also shown to promote eDNA release in low pyocyanin producing (PAO1) and pyocynain deficient (PA14) strains. Based on these data and recent findings in the biofilm literature, we propose that the impact of pyocyanin on biofilm formation in P. aeruginosa occurs via eDNA release through H₂O₂ mediated cell lysis.     

Viscoelastic properties of Staphylococcus aureus and Staphylococcus epidermidis mono‐microbial biofilms

The viscoelastic properties of mono‐microbial biofilms produced by ocular and reference staphylococcal strains were investigated. The microorganisms were characterized for their haemolytic activity and agr typing and the biofilms, grown on stainless steel surface under static conditions, were analysed by Confocal Laser Scanning Microscopy. Static and dynamic rheometric tests were carried out to determine the steady‐flow viscosity and the elastic and viscous moduli. The analysed biofilms showed the typical time‐dependent behaviour of viscoelastic materials with considerable elasticity and mechanical stability except for Staphylococcus aureus ATCC 29213 biofilm which showed a very fragile structure. In particular, S. aureus 6ME biofilm was more compact than other staphylococcal biofilms studied with a yield stress ranging between 2 and 3 Pa. The data obtained in this work could represent a starting point for developing new therapeutic strategies against biofilm‐associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier.     

Influence of membrane surface properties on the behavior of initial bacterial adhesion and biofilm development onto nanofiltration membranes

In order to investigate biofouling problems, the fundamental behaviors of initial bacterial adhesion and biofilm development on four different nanofiltration (NF) membranes were evaluated using Pseudomonas aeruginosa PAO1 as a model bacterial strain. Initial cell adhesion was considerably higher on an aromatic polyamide-based NF membrane with a hydrophobic and rough surface, whereas cell aggregation on a polypiperazine-based NF membrane with a relatively hydrophilic and smooth surface was lower. Moreover, significant differences in the structural heterogeneity of the biofilms were observed among the four NF membranes. This study shows that the surface roughness and hydrophobicity of a membrane play an important role in determining initial cell adhesion, aggregation and favorable localization sites for colony formation. In addition, it was found that biofilm development was strongly influenced by the surface morphology of a membrane.     

Origin and Impact of Nitric Oxide in Pseudomonas aeruginosa Biofilms

The formation of the organized bacterial community called biofilm is a crucial event in bacterial physiology. Given that biofilms are often refractory to antibiotics and disinfectants to which planktonic bacteria are susceptible, their formation is also an industrially and medically relevant issue. Pseudomonas aeruginosa, a well-known human pathogen causing acute and chronic infections, is considered a model organism to study biofilms. A large number of environmental cues control biofilm dynamics in bacterial cells. In particular, the dispersal of individual cells from the biofilm requires metabolic and morphological reprogramming in which the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) plays a central role. The diatomic gas nitric oxide (NO), a well-known signaling molecule in both prokaryotes and eukaryotes, is able to induce the dispersal of P. aeruginosa and other bacterial biofilms by lowering c-di-GMP levels. In this review, we summarize the current knowledge on the molecular mechanisms connecting NO sensing to the activation of c-di-GMP-specific phosphodiesterases in P. aeruginosa, ultimately leading to c-di-GMP decrease and biofilm dispersal.     

The Effect of a Cationic Porphyrin on Pseudomonas aeruginosa Biofilms

Current studies have indicated the utility of photodynamic therapy using porphyrins in the treatment of bacterial infections. Photoactivation of porphyrins results in the production of singlet oxygen (¹O₂) that damages biomolecules associated with cells and biofilms, e.g., proteins, polysaccharides, and DNA. The effect of a cationic porphryin on P. aeruginosa PAO1 biofilms was assessed by exposing static biofilms to 5,10,15,20-tetrakis(1-methyl-pyridino)-21H,23H-porphine, tetra-p-tosylate salt (TMP) followed by irradiation. Biofilms were visualized using confocal laser scanning microscopy (CLSM) and cell viability determined using the LIVE/DEAD BacLight viability assay and standard plate counts. At a concentration of 100 μM TMP, there was substantial killing of P. aeruginosa PAO1 wild-type and pqsA mutant biofilms with little disruption of the biofilm matrix or structure. Exposure to 225 μM TMP resulted in almost complete killing as well as the detachment of wild-type PAO1 biofilms. In contrast, pqsA mutant biofilms that contain less extracellular DNA remained intact. Standard plate counts of cells recovered from attached biofilms revealed a 4.1-log₁₀ and a 3.9-log₁₀ reduction in viable cells of wild-type PAO1 and pqsA mutant strains, respectively. Our results suggest that the action of photoactivated TMP on P. aeruginosa biofilms is two-fold: direct killing of individual cells within biofilms and detachment of the biofilm from the substratum. There was no evidence of porphyrin toxicity in the absence of light; however, biofilms pretreated with TMP without photoactivation were substantially more sensitive to tobramycin than untreated biofilms.     

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.     

Homogenization of Pseudomonas aeruginosa PAO1 biofilms visualized by freeze‐substitution electron microscopy

A knowledge of the mechanical properties of bacterial biofilms is required to more fully understand the processes of biofilm formation such as initial adhesion or detachment. The main contribution of this article is to demonstrate the use of homogenization techniques to compute mechanical parameters of Pseudomonas aeruginosa PAO1 biofilms. For this purpose, homogenization techniques are used to analyze freeze substitution electron micrographs of the biofilm cross‐sections. The concept of a representative volume element and the study about his representativeness allows us to determine the optimal size in order to analyze these biofilm images. Results demonstrate significant heterogeneities with respect to stiffness and these can be explained by varying cell density distribution throughout the bacterial biofilms. These stiffness variations lead to different mechanical properties along the height of the biofilm. Moreover, a numerical shear stress test shows the impact of these heterogeneities on the detachment process. Several modes of detachment are highlighted according to the local strain energy in the different parts of the biofilm. Knowing where, and how, a biofilm may detach will allow better prediction of accumulation and biomass detachment. Biotechnol. Bioeng. 2013; 110: 1405–1418. © 2012 Wiley Periodicals, Inc.     

Biofilm formation and persistence on abiotic surfaces in the context of food and medical environments

The biofilm formation on abiotic surfaces in food and medical sectors constitutes a great public health concerns. In fact, biofilms present a persistent source for pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, which lead to severe infections such as foodborne and nosocomial infections. Such biofilms are also a source of material deterioration and failure. The environmental conditions, commonly met in food and medical area, seem also to enhance the biofilm formation and their resistance to disinfectant agents. In this regard, this review highlights the effect of environmental conditions on bacterial adhesion and biofilm formation on abiotic surfaces in the context of food and medical environment. It also describes the current and emergent strategies used to study the biofilm formation and its eradication. The mechanisms of biofilm resistance to commercialized disinfectants are also discussed, since this phenomenon remains unclear to date.     

Influence of O Polysaccharides on Biofilm Development and Outer Membrane Vesicle Biogenesis in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to contribute to the development and fitness of these bacterial communities. The goal of this study was to examine the relationships between changes in the cell surface expression of LPS O polysaccharides, biofilm development, and OMV biogenesis in P. aeruginosa. We compared wild-type P. aeruginosa PAO1 with three chromosomal knockouts. These knockouts have deletions in the rmd, wbpM, and wbpL genes that produce changes in the expression of common polysaccharide antigen (CPA), O-specific antigen (OSA), or both. Our results demonstrate that changes in O polysaccharide expression do not significantly influence OMV production but do affect the size and protein content of OMVs derived from both CPA⁻ and OSA⁻ cells; these mutant cells also exhibited different physical properties from wild-type cells. We further examined biofilm growth of the mutants and determined that CPA⁻ cells could not develop into robust biofilms and exhibit changes in cell morphology and biofilm matrix production. Together these results demonstrate the importance of O polysaccharide expression on P. aeruginosa OMV composition and highlight the significance of CPA expression in biofilm development.     

Influence of Hydrodynamics and Cell Signaling on the Structure and Behavior of Pseudomonas aeruginosa Biofilms

Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.     

Mechanisms of Cation Exchange by Pseudomonas aeruginosa PAO1 and PAO1 wbpL, a Strain with a Truncated Lipopolysaccharide

The ability of bacterial cells to sequester cations is well recognized, despite the fact that the specific binding sites and mechanistic details of the process are not well understood. To address these questions, the cation-exchange behavior of Pseudomonas aeruginosa PAO1 cells with a truncated lipopolysaccharide (LPS) (PAO1 wbpL) and cells further modified by growth in a magnesium-deficient medium (PAO1 wbpL − Mg²⁺) were compared with that of wild-type P. aeruginosa PAO1 cells. P. aeruginosa PAO1 cells had a negative surface charge (zeta potential) between pH 11 and 2.2, due to carboxylate groups present in the B-band LPS. The net charge on PAO1 wbpL cells was increasingly positive below pH 3.5, due to the influence of NH₃⁺ groups in the core LPS. The zeta potentials of these cells were also measured in Na⁺, Ca²⁺, and La³⁺ electrolytes. Cells in the La³⁺ electrolyte had a positive zeta potential at all pH values tested. Growing P. aeruginosa PAO1 wbpL in magnesium-deficient medium (PAO1 wbpL − Mg²⁺) resulted in an increase in its zeta potential in the pH range from 3.0 to 6.5. In cation-exchange experiments carried out at neutral pH with either P. aeruginosa PAO1 or PAO1 wbpL, the concentration of bound Ca²⁺ was found to decrease as the pH was reduced from 7.0 to 3.5. At pH 3.5, the bound Mg²⁺ concentration decreased sharply, revealing the activity of surface sites for cation exchange and their pH dependence. Infrared spectroscopy of attached biofilms suggested that carboxylate and phosphomonoester functional groups within the core LPS are involved in cation exchange.     

Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa

Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation.     

Does Streptococcus mitis,a neonatal oropharyngeal bacterium,influence the pathogenicity of Pseudomonas aeruginosa?

Microorganisms in a biofilm might promote or suppress each other. We previously found that Pseudomonas aeruginosa (P. aeruginosa) and the normal colonized bacteria in the oropharynx, Streptococcus mitis (S. mitis), were the most common bacteria in the biofilm found on newborns' endotracheal tubes. Here, we found that S. mitis enhanced the adhesion and biofilm formation of P. aeruginosa. Furthermore, it alleviated the immune response induced by P. aeruginosa. These findings remind us that we should not ignore the role of traditionally viewed non-pathogenic bacteria in biofilms and provide new insights into exploring bacterial interaction mechanisms in biofilm related infections.     

An in vitro urinary tract catheter system to investigate biofilm development in catheter-associated urinary tract infections

Biofilm development in urinary tract catheters is an often underestimated problem. However, this form of infection leads to high mortality rates and causes significant costs in health care. Therefore, it is important to analyze these biofilms and establish avoiding strategies. In this study a continuous flow-through system for the cultivation of biofilms under catheter-associated urinary tract infection conditions was established and validated. The in vitro urinary tract catheter system implies the composition of urine (artificial urine medium), the mean volume of urine of adults (1 mL min^-1), the frequently used silicone catheter (foley silicon catheter) as well as the infection with uropathogenic microorganisms like Pseudomonas aeruginosa. Three clinical isolates from urine of catheterized patients were chosen due to their ability to form biofilms, their mobility and their cell surface hydrophobicity. As reference strain P. aeruginosa PA14 has been used. Characteristic parameters as biofilm thickness, specific biofilm growth rate and substrate consumption were observed. Biofilm thicknesses varied from 105 ± 16 μm up to 246 ± 67 μm for the different isolates. The specific biofilm growth rate could be determined with a non invasive optical biomass sensor. This sensor allows online monitoring of the biofilm growth in the progress of the cultivation.     

Predicting biofilm deformation with a viscoelastic phase-field model: Modeling and experimental studies

Biofilms commonly develop in flowing aqueous environments, where the flow causes the biofilm to deform. Because biofilm deformation affects the flow regime, and because biofilms behave as complex heterogeneous viscoelastic materials, few models are able to predict biofilm deformation. In this study, a phase-field (PF) continuum model coupled with the Oldroyd-B constitutive equation was developed and used to simulate biofilm deformation. The accuracy of the model was evaluated using two types of biofilms: a synthetic biofilm, made from alginate mixed with bacterial cells, and a Pseudomonas aeruginosa biofilm. Shear rheometry was used to experimentally determine the mechanical parameters for each biofilm, used as inputs for the model. Biofilm deformation under fluid flow was monitored experimentally using optical coherence tomography. The comparison between the experimental and modeling geometries, for selected horizontal cross sections, after fluid-driven deformation was good. The relative errors ranged from 3.2 to 21.1% for the synthetic biofilm and from 9.1 to 11.1% for the P. aeruginosa biofilm. This is the first demonstration of the effectiveness of a viscoelastic PF biofilm model. This model provides an important tool for predicting biofilm viscoelastic deformation. It also can benefit the design and control of biofilms in engineering systems.     

Monitoring of microbial adhesion and biofilm growth using electrochemical impedancemetry

Electrochemical impedance spectroscopy was tested to monitor the cell attachment and the biofilm proliferation in order to identify characteristic events induced on the metal surface by Gram-negative (Pseudomonas aeruginosa PAO1) and Gram-positive (Bacillus subtilis) bacteria strains. Electrochemical impedance spectra of AISI 304 electrodes during cell attachment and initial biofilm growth for both strains were obtained. It can be observed that the resistance increases gradually with the culture time and decreases with the biofilm detachment. So, the applicability of electric cell-substrate impedance sensing (ECIS) for studying the attachment and spreading of cells on a metal surface has been demonstrated. The biofilm formation was also characterized by the use of scanning electron microscopy and confocal laser scanning microscopy and COMSTAT image analysis. The electrochemical results roughly agree with the microscope image observations. The ECIS technique used in this study was used for continuous real-time monitoring of the initial bacterial adhesion and the biofilm growth. It provides a simple and non-expensive electrochemical method for in vitro assessment of the presence of biofilms on metal surfaces.     

A simple method for quantifying biomass cell and polymer distribution in biofilms

Biofilms are ubiquitous and play an essential role in both environmental processes and hospital infections. Standard methods are not capable of quantifying biomass concentration in dilute suspensions. Furthermore, standard techniques cannot differentiate biomass composition. In this study, a user-friendly technique was developed for measuring biomass cell and polymer content in detached biofilms using a standard coulter counter. The method was demonstrated for an environmentally relevant strain of Pseudomonas aeruginosa (Schroeter) Migula grown in a bioreactor and also for a medically relevant strain of P. aeruginosa (PAO1) grown on standard growth pegs. Results were compared and validated by standard assays, including EPA method 1684 for measuring biomass, microscopic direct counts, and a crystal violet staining assay. The minimum detection limit for the coulter counter method (0.07 mg-biomass L^− 1) was significantly lower than the EPA method 1684 (1.9 ± 0.4 mg/L) and the crystal violet assay (1.1 ± 0.2 mg L^− 1). However, the coulter counter method is limited to dilute biomass samples (below 204 ± 16 mg L^− 1) due to clogging of the aperture tube. While biomass measurements are useful, the major advantage of the coulter counter method is the ability to directly determine EPS, cell, and aggregate fractions after mild chemical treatment. The rapid technique (4–5 min per sample) was used to measure biomass fractions in dispersed P. aeruginosa (Schroeter) and PAO1 biofilms. This technique will be critical for understanding biofilm formation/dispersal.     

Bacillus subtilis Bacteria Generate an Internal Mechanical Force within a Biofilm

A key issue in understanding why biofilms are the most prevalent mode of bacterial life is the origin of the degree of resistance and protection that bacteria gain from self-organizing into biofilm communities. Our experiments suggest that their mechanical properties are a key factor. Experiments on pellicles, or floating biofilms, of Bacillus subtilis showed that while they are multiplying and secreting extracellular substances, bacteria create an internal force (associated with a −80 ± 25 Pa stress) within the biofilms, similar to the forces that self-equilibrate and strengthen plants, organs, and some engineered buildings. Here, we found that this force, or stress, is associated with growth-induced pressure. Our observations indicate that due to such forces, biofilms spread after any cut or ablation by up to 15–20% of their initial size. The force relaxes over very short timescales (tens of milliseconds). We conclude that this force helps bacteria to shape the biofilm, improve its mechanical resistance, and facilitate its invasion and self-repair.     

The influence of flow cell geometry related shear stresses on the distribution,structure and susceptibility of Pseudomonas aeruginosa 01 biofilms

The effects of non-uniform hydrodynamic conditions resulting from flow cell geometry (square and rectangular cross-section) on Pseudomonas aeruginosa 01 (PAO1) biofilm formation, location, and structure were investigated for nominally similar flow conditions using a combination of confocal scanning laser microscope (CSLM) and computational fluid dynamics (CFD). The thickness and surface coverage of PAO1 biofilms were observed to vary depending on the location in the flow cell and thus also the local wall shear stress. The biofilm structure in a 5:1 (width to height) aspect ratio rectangular flow cell was observed to consist mainly of a layer of bacterial cells with thicker biofilm formation observed in the flow cell corners. For square cross-section (1:1 aspect ratio) flow cells, generally thicker and more uniform surface coverage biofilms were observed. Mushroom shaped structures with hollow centers and wall breaks, indicative of ‘seeding’ dispersal structures, were found exclusively in the square cross-section tubes. Exposure of PAO1 biofilms grown in the flow cells to gentamicin revealed a difference in susceptibility. Biofilms grown in the rectangular flow cell overall exhibited a greater susceptibility to gentamicin compared to those grown in square flow cells. However, even within a given flow cell, differences in susceptibility were observed depending on location. This study demonstrates that the spanwise shear stress distribution within the flow cells has an important impact on the location of colonization and structure of the resultant biofilm. These differences in biofilm structure have a significant impact on the susceptibility of the biofilms grown within flow channels. The impact of flow modification due to flow cell geometry should be considered when designing flow cells for laboratory investigation of bacterial biofilms.