Sessile State Research Articles

Characterization of Biofilm Formation by the Bioleaching Acidophilic Bacterium <i>Acidithiobacillus Ferrooxidans</i> by a Microarray Transcriptome Analysis

Bioleaching is the extraction of metals, such as copper or gold, from ore by microorganisms. Bacterial attachment increases leaching activities due to the formation of a "reaction space" between the metal sulfide surface and the cell. This process depends on abiotic characteristics such as purity and degree of crystallization of the metal sulfide, as well as biotic ones such as the capacity of the bacteria for detecting favourable attachment sites and synthesizing a suitable cell envelope (EPS), for adhesion. Planktonic and sessile cells should differ significantly in their metabolic activities and therefore in their gene expression patterns. To help to understand At. ferrooxidans biofilm formation, microarray transcript profiling was carried out to compare planktonic and sessile cells. The high contents of EPS and ferric iron of the biofilms are interfering with RNA extraction, causing inhibition of DNAse, reverse transcriptase and/or polymerase activities required to get labelled target cDNA. In order to have sufficient high quality RNA suitable for transcriptomic analysis, we have optimized the biofilm formation of At. ferrooxidans on pyrite (FeS2) and the RNA extraction from the sessile cell population. DNA microarrays have been hybridized with labelled cDNAs from sessile and planktonic cells and preliminary data suggest that some genes are differently expressed between these two subpopulations. The understanding of these differences may help us to shift populations of leaching bacteria from the planktonic state towards the sessile state in order to influence bioleaching.

Biofilm-detached cells, a transition from a sessile to a planktonic phenotype: a comparative study of adhesion and physiological characteristics in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a pathogenic bacterium widely investigated for its high incidence in clinical environments and its ability to form strong biofilms. During biofilm development, sessile cells acquire physiological characteristics differentiating them from planktonic cells. But after treatment with disinfectants, or to ensure survival of the species in hostile environments, biofilm cells can detach. This complicates disinfection procedures. This study aimed to physiologically characterize cells detached from a P. aeruginosa biofilm and to compare them with their sessile and planktonic counterparts. We first tested planktonic growth kinetics and capacities to form new biofilms. Then we investigated cell-surface properties. And finally, we tested in vitro susceptibility to antibiotics. The results first indicated that sessile and detached cells have similar planktonic growth kinetics and cell-surface properties, distinguishable from those of planktonic cells. Interestingly, the three populations exhibited different biofilm-forming capacities, suggesting that there is a transitional phenotype between sessile and planktonic states, at least during the first hours following cell detachment. It is important to consider this observation when developing treatments to optimize disinfection processes. Surprisingly, the three populations showed the same antibiotic susceptibility profile.

Persistence of Helicobacter pylori in heterotrophic drinking-water biofilms.

Although the route of transmission of Helicobacter pylori remains unknown, drinking water has been considered a possible transmission vector. It has been shown previously that, in water, biofilms are a protective niche for several pathogens, protecting them from stressful conditions, such as low carbon concentration, shear stress, and less-than-optimal temperatures. In this work, the influence of these three parameters on the persistence and cultivability of H. pylori in drinking-water biofilms was studied. Autochthonous biofilm consortia were formed in a two-stage chemostat system and then inoculated with the pathogen. Total numbers of H. pylori cells were determined by microscopy using a specific H. pylori 16S rRNA peptide nucleic acid probe, whereas cultivable cells were assessed by standard plating onto selective H. pylori medium. Cultivable H. pylori could not be detected at any time point, but the ability of H. pylori cells to incorporate, undergo morphological transformations, persist, and even agglomerate in biofilms for at least 31 days without a noticeable decrease in the total cell number (on average, the concentration was between 1.54 x 10(6) and 2.25 x 10(6) cells cm(-2)) or in the intracellular rRNA content may indicate that the loss of cultivability was due to entry into a viable but noncultivable state. Unlike previous results obtained for pure-culture H. pylori biofilms, shear stress did not negatively influence the numbers of H. pylori cells attached, suggesting that the autochthonous aquatic bacteria have an important role in retaining this pathogen in the sessile state, possibly by providing suitable microaerophilic environments or linking biomolecules to which the pathogen adheres. Therefore, biofilms appear to provide not only a safe haven for H. pylori but also a concentration mechanism so that subsequent sloughing releases a concentrated bolus of cells that might be infectious and that could escape routine grab sample microbiological analyses and be a cause of concern for public health.

Antagonism between Bacillus cereus and Pseudomonas fluorescens in planktonic systems and in biofilms

In the environment, many microorganisms coexist in communities competing for resources, and they are often associated as biofilms. The investigation of bacterial ecology and interactions may help to improve understanding of the ability of biofilms to persist. In this study, the behaviour of Bacillus cereus and Pseudomonas fluorescens in the planktonic and sessile states was compared. Planktonic tests were performed with single and dual species cultures in growth medium with and without supplemental FeCl3. B. cereus and P. fluorescens single cultures had equivalent growth behaviours. Also, when in co-culture under Fe-supplemented conditions, the bacteria coexisted and showed similar growth profiles. Under Fe limitation, 8 h after co-culture and over time, the number of viable B. cereus cells decreased compared with P. fluorescens. Spores were detected during the course of the experiment, but were not correlated with the decrease in the number of viable cells. This growth inhibitory effect was correlated with the release of metabolite molecules by P. fluorescens through Fe-dependent mechanisms. Biofilm studies were carried out with single and dual species using a continuous flow bioreactor rotating system with stainless steel (SS) substrata. Steady-state biofilms were exposed to a series of increasing shear stress forces. Analysis of the removal of dual species biofilms revealed that the outer layer was colonised mainly by B. cereus. This bacterium was able to grow in the outermost layers of the biofilm due to the inhibitory effect of P. fluorescens being decreased by the exposure of the cells to fresh culture medium. B. cereus also constituted the surface primary coloniser due to its favourable adhesion to SS. P. fluorescens was the main coloniser of the middle layers of the biofilm. Single and dual species biofilm removal data also revealed that B. cereus biofilms had the highest physical stability, followed by P. fluorescens biofilms. This study highlights the inadequacy of planktonic systems to mimic the behaviour of bacteria in biofilms and shows how the culturing system affects the action of antagonist metabolite molecules because dilution and consequent loss of activity occurred in continuously operating systems. Furthermore, the data demonstrate the biocontrol potential of P. fluorescens on the planktonic growth of B. cereus and the ability of the two species to coexist in a stratified biofilm structure.

Coccoid Form of Helicobacter pylori as a Morphological Manifestation of Cell Adaptation to the Environment

After characterization of preferred conditions for Helicobacter pylori survival in the sessile state, it was observed that the bacterium transforms from spiral to coccoid under mild circumstances, whereas under extreme ones it is unable to undergo shape modification. This strongly supports the view that transformation into the coccoid form is an active, biologically led process, switched on by the bacterium as a protection mechanism.

Assessing the Contamination Potential of Freshly Extracted Escherichia coli Biofilm Cells by Impedancemetry

Planktonic bacteria passing to a sessile state during the formation of a biofilm undergo many gene expression and phenotypic changes. These transformations require a significant time to establish. Inversely, cells extracted from a biofilm should also require a significant time before acquiring the same physiological characteristics as planktonic cells. Relatively few studies have addressed the kinetics of this inverse transformation process. We tested one aspect, namely, the contamination potential of freshly extracted Escherichia coli biofilm cells, precultured in a synthetic medium, in a rich liquid growth medium. We compared the time between inoculation and the beginning of the growth phase of freshly extracted biofilm cells, and suspended exponential and suspended stationary phase cells precultured in the same synthetic medium. Unexpectedly, the lag time for the extracted biofilm cells was the same as the lag time of the suspended exponential phase cells and significantly less than the lag time of the suspended stationary phase cells. The lag times were determined by an impedance technique. Cells extracted from biofilms, i.e., biofilms formed in canalizations and broken up by hydrodynamic forces, are an important source of contamination. Our work shows, in the case of E. coli, the high potential of freshly extracted biofilm cells to reinfect a new medium.

Characterization of starvation‐induced dispersion in Pseudomonas putida biofilms

The biofilm lifestyle, where microbial cells are aggregated because of expression of cell-to-cell interconnecting compounds, is believed to be of paramount importance to microbes in the environment. Because microbes must be able to alternate between sessile and planktonic states, it is anticipated that they must be able to regulate their ability to form biofilm and to dissolve biofilm. We present an investigation of a biofilm dissolution process occurring in flow-chamber-grown Pseudomonas putida biofilms. Local starvation-induced biofilm dissolution appears to be an integrated part of P. putida biofilm development that causes characteristic structural rearrangements. Rapid global dissolution of entire P. putida biofilms was shown to occur in response to carbon starvation. Genetic analysis suggested that the adjacent P. putida genes PP0164 and PP0165 play a role in P. putida biofilm formation and dissolution. PP0164 encodes a putative periplasmic protein of previously unknown function, and PP0164 mutant bacteria are sticky, and unable to reduce their adhesiveness and dissolve their biofilm in response to carbon starvation. PP0165 encodes a putative transmembrane protein containing GGDEF and EAL domains, and PP0165 mutant bacteria are unable to increase their adhesiveness and form biofilm. We suggest that the PP0164 and PP0165 proteins are involved in the regulation of the adhesiveness of the bacteria; the PP0165 protein through c-di-GMP signalling, and the PP0164 protein as a transducer of the signal.

A master regulator for biofilm formation by Bacillus subtilis

Wild strains of Bacillus subtilis are capable of forming architecturally complex communities of cells known as biofilms. Critical to biofilm formation is the eps operon, which is believed to be responsible for the biosynthesis of an exopolysaccharide that binds chains of cells together in bundles. We report that transcription of eps is under the negative regulation of SinR, a repressor that was found to bind to multiple sites in the regulatory region of the operon. Mutations in sinR bypassed the requirement in biofilm formation of two genes of unknown function, ylbF and ymcA, and sinI, which is known to encode an antagonist of SinR. We propose that these genes are members of a pathway that is responsible for counteracting SinR-mediated repression. We further propose that SinR is a master regulator that governs the transition between a planktonic state in which the bacteria swim as single cells in liquid or swarm in small groups over surfaces, and a sessile state in which the bacteria adhere to each other to form bundled chains and assemble into multicellular communities.

Dynamics and Control of Biofilms of the Oligotrophic Bacterium Caulobacter crescentus

Caulobacter crescentus is an oligotrophic alpha-proteobacterium with a complex cell cycle involving sessile-stalked and piliated, flagellated swarmer cells. Because the natural lifestyle of C. crescentus intrinsically involves a surface-associated, sessile state, we investigated the dynamics and control of C. crescentus biofilms developing on glass surfaces in a hydrodynamic system. In contrast to biofilms of the well-studied Pseudomonas aeruginosa, Escherichia coli, and Vibrio cholerae, C. crescentus CB15 cells form biphasic biofilms, consisting predominantly of a cell monolayer biofilm and a biofilm containing densely packed, mushroom-shaped structures. Based on comparisons between the C. crescentus strain CB15 wild type and its holdfast (hfsA; DeltaCC0095), pili (DeltapilA-cpaF::Omegaaac3), motility (motA), flagellum (flgH) mutants, and a double mutant lacking holdfast and flagellum (hfsA; flgH), a model for biofilm formation in C. crescentus is proposed. For both biofilm forms, the holdfast structure at the tip of a stalked cell is crucial for mediating the initial attachment. Swimming motility by means of the single polar flagellum enhances initial attachment and enables progeny swarmer cells to escape from the monolayer biofilm. The flagellum structure also contributes to maintaining the mushroom structure. Type IV pili enhance but are not absolutely required for the initial adhesion phase. However, pili are essential for forming and maintaining the well-defined three-dimensional mushroom-shaped biofilm. The involvement of pili in mushroom architecture is a novel function for type IV pili in C. crescentus. These unique biofilm features demonstrate a spatial diversification of the C. crescentus population into a sessile, "stem cell"-like subpopulation (monolayer biofilm), which generates progeny cells capable of exploring the aqueous, oligotrophic environment by swimming motility and a subpopulation accumulating in large mushroom structures.

Dynamics of the stochastic jerky motion of dislocations with application to a description of the yield strength anomaly of materials

A theory is presented describing dislocation motion in the presence of spontaneous transitions between glissile and sessile states. Different regimes of the dislocation motion are predicted and used to interpret the most prominent features of anomalous plastic deformation of materials. A procedure of thermal-activation analysis of experimental data of mechanical testing within the anomalous temperature range is reconsidered.

Dynamics of the stochastic jerky motion of dislocations with application to a description of the yield strength anomaly of materials

A theory is presented describing dislocation motion in the presence of spontaneous transitions between glissile and sessile states. Different regimes of the dislocation motion are predicted and used to interpret the most prominent features of anomalous plastic deformation of materials. A procedure of thermal-activation analysis of experimental data of mechanical testing within the anomalous temperature range is reconsidered.

Penetrating the plaque biofilm: impact of essential oil mouthwash.

The interaction between saliva-coated tooth surfaces and pathogenic bacteria is partly governed by electrostatic and hydrophobic interactions, providing a solid rationale for using chemical agents as part of a plaque-control routine. Chlorhexidine works in several ways. For example, it binds to salivary mucins on the bacterial cell membrane, and penetrates the plaque biofilm. Essential oil (EO) mouthwashes kill micro-organisms by disrupting their cell walls and inhibiting their enzymic activity. They prevent bacterial aggregation, slow multiplication and extract endotoxins. Recent studies have shown that bacterial phenotypes are altered when organisms change from a planktonic to a sessile state. This suggests that an effective mouthwash must also penetrate the plaque biofilm. Two studies have demonstrated the ability of an EO mouthwash to penetrate the plaque biofilm.

Thigmo responses in plants and fungi

Thigmo mechanisms are adaptations that permit a plant to alter growth rates, change morphology, produce tropisms, avoid barriers, control germination, cling to supporting structures, infect a host plant, facilitate pollination, expedite the movement of pollen, spores, or seeds, and capture prey. Through these varied functions, plant thigmo systems have evolved impressive controls of cell differentiation, localized growth rates, regulated synthesis of novel products, and some elegant traps and projectile systems. For most thigmo events, there will be a dependence upon transmission of a signal from the cell wall through the plasmalemma and into the cytoplasm. We propose the possible involvement of integrin-like proteins, Hechtian strands, and cytoskeletal structures as possible transduction components. Many thigmo mechanisms may use some modification of the calcium/calmodulin signal transduction system, though the details of transduction systems are still poorly understood. While transmission of thigmo signals to remote parts of a plant is associated with the development of action potentials, hormones may also play a role. Thigmo mechanisms have facilitated an enormous array of plant and fungal adaptations that make major contributions to their success despite their relatively sessile or immobile states.

The dependence of anomalous temperature peak of a deforming stress on microstructural parameters

The temperature dependence of a deforming stress (including both low-temperature decreasing branch and an anomalous high-temperature peak) has been calculated on the basis of one mechanism. The theory developed takes into account spontaneous thermally activated processes of dislocation locking and unlocking and describes the transition from conventional dislocation gliding to the jumpwise motion of dislocations with the change of the temperature (in full accordance with the in situelectron microscopy observations). The dependences of the plasticity characteristics (flow stress, strain-rate sensitivity, etc.) on the microstructural parameters such as free dislocation path and the energy barriers that should be overcome by dislocations for transformation of their cores from the “gliding” to the “sessile states” and back have also been determined.

Apparent surface associated lag time in growth of primary biofilm cells.

The ability of microorganisms to form biofilms has been well documented. Bacterial cells make a transition from a planktonic state to a sessile state, replicate, and subsequently populate a surface. In this study, organisms that initially colonize a ``clean'' surface are referred to as ``primary'' biofilm cells. The progeny of the first generation of sessile cells are known as ``secondary'' biofilm cells. This study examined the growth of planktonic, primary, and secondary biofilm cells of a green fluorescent protein producing (GFP+) Pseudomonas aeruginosa PA01. Biofilm experiments were performed in a parallel plate flow cell reactor with a glass substratum. Individual cells were tracked over time using a confocal scanning laser microscope (CSLM). Primary cells experience a lag in their growth that may be attributed to adapting to a sessile environment or undergoing a phenotypic change. This is referred to as a surface associated lag time. Planktonic and secondary biofilm cells both grew at a faster rate than the primary biofilm cells under the same nutrient conditions.

Planktonic/sessile dimorphism of polysaccharide-encapsulated sphingomonads.

Sphingomonads have acquired diverse metabolic activities to inhabit a wide range of environments. Several strains of Sphingomonas display phenotypic dimorphism and can adopt either a planktonic or sessile behavior in liquid media. The sessile state is marked by the presence of a viscous exopolysaccharide capsule. Specific types of these capsular polysaccharides are harvested from large-scale fermentations for use as rheology modifiers in many industrial and food applications. Sensing of environmental stimuli and genetic control over synthesis of the capsule are key events in alternating between these two phenotypes.

The effect of strain rate on the anomalous peak of yield stress in β-CuZn alloy

A theory is presented describing dislocation motion in the presence of spontaneous transitions between glissile and sessile states. Different regimes of the dislocation motion are predicted and used to interpret the most prominent features of anomalous plastic deformation of materials. A procedure of thermal-activation analysis of experimental data of mechanical testing within the anomalous temperature range is reconsidered.

Direct and rapid induction of migration in human CD4+ T lymphocytes within three-dimensional collagen matrices mediated by signalling via CD3 and/or CD2.

Specific activation of T cells requires stable cell-cell interaction; however, little is known how the transition from a previously motile state into a sessile state following activation is achieved. We investigated the direct effect of T-cell receptor (TCR)/CD3 complex engagement and/or stimulation of the accessory molecule CD2 on the locomotion of peripheral human T cells within three-dimensional (3-D) collagen lattices. Simultaneous engagement of CD3 and CD2 very potently stimulated T-cell migration, resulting in the recruitment of previously sessile cells (about 24% of the total population was additionally recruited) as well as an increase in the mean duration of active locomotion. This induction of migration was accompanied by an increased tyrosine phosphorylation of a 125 000 MW substrate corresponding to the focal adhesion kinase. Using confocal laser scanning microscopy we detected antibody-induced receptor capping into the uropod of migrating T cells whereas untreated control cells displayed an even distribution of CD3 and CD2 on the cell surface. Less pronounced induction of locomotion was achieved following triggering of CD3 or CD2 alone. Thus, in 3-D collagen lattices specific T-cell activation did not lead to cessation of cellular migration but rather induced cytoskeletal activity that ultimately resulted in vigorous locomotory activity.

The antimicrobial activity of an aqueous extract of Brassica negra

An aqueous extract from the seeds of Brassica negra was shown to be effective in reducing the numbers of viable planktonic and sessile cells of a Pseudomonas sp., the fungus Aspergillus fumigatus and a mixed sulphate-reducing bacteria (SRB) culture, all isolated from contaminated diesel oil. The most resistant organism in the planktonic state was A. fumigatus, which required 24 h treatment with 0.04% mustard seed to reduce the cell numbers to below the detection level of 10 cells/ml. In the sessile state SRB were the most resistant, their numbers being reduced significantly only after 1 h treatment with 0.25% mustard seed. Planktonic Pseudomonas sp. and SRB were undetectable after 24 h contact time with 0.01%. Sessile Pseudomonas and A. fumigatus cell numbers were substantially reduced after 1 hours' contact with 0.05%. These results show that the natural biocide, like other antibiotics and synthetic antimicrobial agents, is less effective against sessile than planktonic cells; nevertheless this aqueous extract of Brassica negra seeds has potential for use against microorganisms in industrial biofilms.