First Step In Biofilm Formation Research Articles

Assessment of Polydopamine to Reduce Streptococcus mutans Adhesion to a Dental Polymer.

Bacterial adhesion to the surface of materials is the first step in biofilm formation, which will lead to conditions that may compromise the health status of patients. Recently, polydopamine (PDA) has been proposed as an antibacterial material. Therefore, the objective of the current work was to assess and compare the adhesion of Streptococcus mutans to the surface of poly(methyl methacrylate) (PMMA) discs that were modified using PDA following a biomimetic approach versus smooth PDA-coated PMMA surfaces. In addition, an assessment of the growth inhibition by PDA was performed. PMMA discs were manufactured and polished; soft lithography, using the topography from the Crocosmia aurea leaf, was used to modify their surface. PDA was used to smooth-coat PMMA discs by dip-coating. The growth inhibition was measured using an inhibition halo. The surfaces were characterized by means of atomic force microscopy (AFM), the contact angle (CA), and Fourier-transform infrared spectroscopy (FTIR). Polydopamine exhibited a significant antibacterial effect when used directly on the S. mutans planktonic cells, but such an effect was not as strong when modifying the PMMA surfaces. These results open the possibility of using polydopamine to reduce the adhesion and growth of S. mutans, which might have important consequences in the dental field.

Bacteria-on-a-bead: probing the hydrodynamic interplay of dynamic cell appendages during cell separation

Surface attachment of bacteria is the first step of biofilm formation and is often mediated and coordinated by the extracellular appendages, flagellum and pili. The model organism Caulobacter crescentus undergoes an asymmetric division cycle, giving rise to a motile “swarmer cell” and a sessile “stalked cell”, which is attached to the surface. In the highly polarized predivisional cell, pili and flagellum, which are assembled at the pole opposite the stalk, are both activated before and during the process of cell separation. We explored the interplay of flagellum and active pili by growing predivisional cells on colloidal beads, creating a bacteria-on-a-bead system. Using this set-up, we were able to simultaneously visualize the bacterial motility and analyze the dynamics of the flagellum and pili during cell separation. The observed activities of flagellum and pili at the new cell pole of the predivisional cell result in a cooperating interplay of the appendages during approaching and attaching to a surface. Even in presence of a functioning flagellum, pili are capable of surface attachment and keeping the cell in position. Moreover, while flagellar rotation decreases the average attachment time of a single pilus, it increases the overall attachment rate of pili in a synergetic manner.

Bacterial Competition for the Anode Colonization under Different External Resistances in Microbial Fuel Cells

This study investigated the effect of external resistance (Rext) on the dynamic evolution of microbial communities in anodic biofilms of single-chamber microbial fuel cells fueled with acetate and inoculated with municipal wastewater. Anodic biofilms developed under different Rext (0, 330 and 1000 ohms, and open circuit condition) were characterized as a function of time during two weeks of growth using 16S rRNA gene sequencing, cyclic voltammetry (CV) and fluorescence microscopy. The results showed a drastic difference in power output of MFCs operated with an open circuit and those operated with Rext from 0 to 1000 ohms. Two steps during the bacterial community development of the anodic biofilms were identified. During the first four days, nonspecific electroactive bacteria (non-specific EAB), dominated by Pseudomonas, Acinetobacter, and Comamonas, grew fast whatever the value of Rext. During the second step, specific EAB, dominated by Geobacter and Desulfuromonas, took over and increased over time, except in open circuit MFCs. The relative abundance of specific EAB decreased with increasing Rext. In addition, the richness and diversity of the microbial community in the anodic biofilms decreased with decreasing Rext. These results help one to understand the bacterial competition during biofilm formation and suggest that an inhibition of the attachment of non-specific electroactive bacteria to the anode surface during the first step of biofilm formation should improve electricity production.

Cold plasma surface treatments to prevent biofilm formation in food industries and medical sectors.

Environmental conditions in food and medical fields enable the bacteria to attach and grow on surfaces leading to resistant bacterial biofilm formation. Indeed, the first step in biofilm formation is the bacterial irreversible adhesion. Controlling and inhibiting this adhesion is a passive approach to fight against biofilm development. This strategy is an interesting path in the inhibition of biofilm formation since it targets the first step of biofilm development. Those pathogenic structures are responsible for several foodborne diseases and nosocomial infections. Therefore, to face this public health threat, researchers employed cold plasma technologies in coating development. In this review, the different factors influencing the bacterial adhesion to a substrate are outlined. The goal is to present the passive coating strategies aiming to prevent biofilm formation via cold plasma treatments, highlighting antiadhesive elaborated surfaces. General aspects of surface treatment, including physico-chemical modification and application of cold plasma technologies, were also presented.Key points • Factors surrounding pathogenic bacteria influence biofilm development. • Controlling bacterial adhesion prevents biofilm formation. • Materials can be coated via cold plasma to inhibit bacterial adhesion.

Evaluation of Streptococcus mutans Adhesion to Stainless Steel Surfaces Modified Using Different Topographies Following a Biomimetic Approach

Bacterial adhesion to surfaces is the first step in biofilm formation, which leads to the development of conditions that may compromise the health status of patients. Surface modification has been proposed to reduce bacterial adhesion to biomaterials. The objective of this work was to assess and compare Streptococcus mutans adhesion to the surface of biomimetically-modified stainless steel using different topographies. Stainless steel plates were modified using a soft lithography technique following a biomimetic approach. The leaves from Colocasia esculenta, Crocosmia aurea and Salvinia molesta were used as surface models. Silica sol was synthesized using the sol-gel method. Following a soft lithography technique, the surface of the leaves were transferred to the surface of the SS plates. Natural and modified surfaces were characterized by means of atomic force microscopy and contact angle. Streptococcus mutans was used to assess bacterial adhesion. Contact angle measurements showed that natural leaves are highly hydrophobic, but such hydrophobicity could not be transferred to the metallic plates. Roughness varied among the leaves and increased after transference for C. esculenta and decreased for C. aurea. In general, two of the surface models used in this investigation showed positive results for reduction of bacterial adhesion (C. aurea and C. esculenta), while the other showed an increase in bacterial adhesion (S. molesta). Therefore, since a biomimetic approach using natural surfaces showed opposite results, careful selection of the surface model needs to be taken into consideration.

Analysis of the Phospholipid Profile of the Collection Strain PAO1 and Clinical Isolates of Pseudomonas aeruginosa in Relation to Their Attachment Capacity.

Bacteria form multicellular and resistant structures named biofilms. Biofilm formation starts with the attachment phase, and the molecular actors involved in this phase, except adhesins, are poorly characterized. There is growing evidence that phospholipids are more than simple structural bricks. They are involved in bacterial adaptive physiology, but little is known about their role in biofilm formation. Here, we report a mass spectrometry analysis of the phospholipid (PL) profile of several strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients. The aim of our study was to evaluate a possible link between the PL profile of a strain and its attachment phenotype. Our results showed that PL profile is strongly strain-dependent. The PL profile of P. aeruginosa PAO1, a collection strain, was different from those of 10 clinical isolates characterized either by a very low or a very high attachment capacity. We observed also that the clinical strain’s PL profiles varied even more importantly between isolates. By comparing groups of strains having similar attachment capacities, we identified one PL, PE 18:1-18:1, as a potential molecular actor involved in attachment, the first step in biofilm formation. This PL represents a possible target in the fight against biofilms.

Salivary Pellicle Modification with Grape-seed Extract: In Vitro Study on the Effect on Bacterial Adhesion and Biofilm Formation.

Grape-seed extract (GSE) contains polyphenols that readily adhere to proteins and modify the acquired enamel pellicle (AEP). The first step in biofilm formation is bacterial adhesion to the AEP-covered enamel. The aim of this in vitro study was to test whether AEP modification with GSE, fluoride (F-), or their combination (GSE+F-) modulates bacterial adhesion, biofilm metabolism and composition, or cariogenic demineralisation of the enamel. The study comprised 3 parts: 1) single-strain Streptococcus gordonii species, 2) a five-species biofilm model, or 3) biofilm (re-)formation using the five-species biofilm model after removal of initial biofilm with toothbrushing. Human whole-mouth stimulated saliva was used to form an AEP on human enamel specimens. The AEP was incubated in water (control), or modified with GSE, F-, or GSE+F-. Bacterial adhesion, biofilm diversity, metabolic activity, biofilm mass, and cariogenic demineralisation (surface hardness) of enamel were assessed after incubation in bacterial broths after 4 h or 22 h. Differences between groups were analysed with one-way ANOVA and post-hoc Bonferroni tests. GSE and GSE+F- statistically significantly decreased single-strain S. gordonii adhesion, but had no relevant influence when the five-species biofilm model was used. In the biofilm (re-)formation model, GSE reduced bacterial adhesion compared to GSE+F-, while F- caused less cariogenic demineralisation than was found in the control group. AEP modified with GSE retards S. gordonii adhesion, but it does not influence the formation, metabolism and composition of a cariogenic multi-species biofilm.

Advances in Biomaterials for the Prevention and Disruption of Candida Biofilms.

Candida species can readily colonize a multitude of indwelling devices, leading to biofilm formation. These three-dimensional, surface-associated Candida communities employ a multitude of sophisticated mechanisms to evade treatment, leading to persistent and recurrent infections with high mortality rates. Further complicating matters, the current arsenal of antifungal therapeutics that are effective against biofilms is extremely limited. Antifungal biomaterials are gaining interest as an effective strategy for combating Candida biofilm infections. In this review, we explore biomaterials developed to prevent Candida biofilm formation and those that treat existing biofilms. Surface functionalization of devices employing clinically utilized antifungals, other antifungal molecules, and antifungal polymers has been extremely effective at preventing fungi attachment, which is the first step of biofilm formation. Several mechanisms can lead to this attachment inhibition, including contact killing and release-based killing of surrounding planktonic cells. Eliminating mature biofilms is arguably much more difficult than prevention. Nanoparticles have shown the most promise in disrupting existing biofilms, with the potential to penetrate the dense fungal biofilm matrix and locally target fungal cells. We will describe recent advances in both surface functionalization and nanoparticle therapeutics for the treatment of Candida biofilms.

Initial Deposition and Pioneering Colonization on Polymeric Membranes of Anaerobes Isolated from an Anaerobic Membrane Bioreactor (AnMBR).

Membrane biofouling constitutes a great challenge in anaerobic membrane bioreactor (AnMBR). Here, we studied the initial deposition of anaerobes, the first step in biofilm formation, with a consortium isolated from an AnMBR on membranes with different surface properties and under two shear rate conditions without filtration. We found that the cell transfer coefficient, calculated from the initial deposition experiments, was similar under the two shear rates for the hydrophobic membranes, but much higher under low shear rate and much lower under high shear rate, for the hydrophilic membrane. The cell transfer coefficient measured under filtration mode and at a higher shear rate showed a similar trend. The pioneer bacteria and archaea (without filtration) were identified by next-generation sequencing. The results showed that the selective force for the dissimilarity of the pioneer bacterial and archaeal diversity was the shear rate and the membrane surface properties, respectively. However, statistical analyses revealed minor changes in the pioneer bacteria (class level) and archaea (order level) populations under the various conditions. These results shed light on the first step of biofilm formation on the membranes in AnMBRs and emphasize the importance of hydrodynamic shear and membrane surface properties on the initially deposited anaerobes.

Spatio-temporal formation of biofilms and extracellular matrix analysis in Azospirillum brasilense.

Elucidation of biofilm structure formation in the plant growth-promoting rhizobacterium Azospirillum brasilense is necessary to gain a better understanding of the growth of cells within the extracellular matrix and its role in the colonization of plants of agronomic importance. We used immunofluorescence microscopy and confocal laser scanning microscopy to study spatio-temporal biofilm formation on an abiotic surface. Observations facilitated by fluorescence microscopy revealed the presence of polar flagellin, exopolysaccharides, outer major membrane protein (OmaA) and extracellular DNA in the Azospirillum biofilm matrix. In static culture conditions, the polar flagellum disaggregated after 3 days of biofilm growth, but exopolysaccharides were increasing. These findings suggest that the first step in biofilm formation may be attachment, in which the bacterium first makes contact with a surface through its polar flagellum. After attaching to the surface, the long flagella and OmaA intertwine the cells to form a network. These bacterial aggregates initiate biofilm development. The underlying mechanisms dictating how the biofilm matrix components of A. brasilense direct the overall morphology of the biofilm are not well known. The methods developed here might be useful in further studies that analyze the differential spatial regulation of genes encoding matrix components that drive biofilm construction.

How Functionalized Surfaces Can Inhibit Bacterial Adhesion and Viability.

Device-associated infections (DAI) remain a serious concern in modern healthcare. Bacterial attachment to a surface is the first step in biofilm formation, which is one of the main causes of DAIs. The development of materials capable of preventing or inhibiting bacterial attachment constitutes a promising approach to deal with this problem. The multifactorial nature of biofilm maturation and antibiotic resistance directs the research for multitargeted or combinatorial therapeutic approaches. One attractive strategy is the modification or the engineering of surfaces in order to provide antiadhesive and/or antimicrobial properties. Currently, several different approaches that involve physical and chemical surface modification deliver some possible alternatives to achieve this goal. The engineered surfaces can be coated with molecules capable of inhibiting the bacterial adhesion or with active agents that kill microorganisms. In addition, surfaces can also be modified in order to be stimuli-responsive, responding to a particular trigger and then delivering the consequent antimicrobial outcome. Here, we review the prevailing strategies to modify surfaces in order to create an antimicrobial surface and discuss how different surface functionalization can affect bacterial adhesion and/or viability.

Visualizing Biomaterial Degradation by Candida albicans Using Embedded Luminescent Molecules To Report on Substrate Digestion and Cellular Uptake of Hydrolysate.

Microbial pathogens use hydrolases as a virulence strategy to spread disease through tissues and colonize medical device surfaces; however, visualizing this process is a technically challenging problem. To better understand the role of secreted fungal hydrolases and their role in Candida albicans virulence, we developed an in situ model system using luminescent Re(I) and Ir(III) containing probe molecules embedded in a biodegradable (poly(lactic-co-glycolic acid), PLGA) polymer and tracked their uptake using epifluorescent imaging. We found that secretion of esterases can explain how physically embedded probes are acquired by fungal cells through the degradation of PLGA since embedded probes could not be liberated from nonbiodegradable polystyrene (PS). It was important to verify that epifluorescent imaging captured the fate of probe molecules rather than naturally occurring fungal autofluorescence. For this, we exploited the intense luminescent signals and long spectral relaxation times of the Re and Ir containing probe molecules, resolved in time using a gated imaging system. Results provide a visual demonstration of a key virulence trait of C. albicans: the use of hydrolases as a means to degrade materials and acquire hydrolysis products during fungal growth and hyphal development. These results help to explain the role of nonspecific hydrolases using a degradable material that is relevant to the study of fungal pathogenesis on biotic (tissues) surfaces. Additionally, understanding how fungal pathogens condition surfaces by using nonspecific hydrolases is important to the study of fungal attachment on abiotic surfaces, the first step in biofilm formation on medical devices.

The trimeric autotransporter adhesin BadA is required for in vitro biofilm formation by Bartonella henselae

Bartonellahenselae (Bh) is a Gram-negative rod transmitted to humans by a scratch from the common house cat. Infection of humans with Bh can result in a range of clinical diseases including lymphadenopathy observed in cat-scratch disease and more serious disease from persistent bacteremia. It is a common cause of blood-culture negative endocarditis as the bacterium is capable of growing as aggregates, and forming biofilms on infected native and prosthetic heart valves. The aggregative growth requires a trimeric autotransporter adhesin (TAA) called Bartonella adhesin A (BadA). TAAs are found in all Bartonella species and many other Gram-negative bacteria. Using Bh Houston-1, Bh Houston-1 ∆badA and Bh Houston-1 ∆badA/pNS2PTrcbadA (a partial complement of badA coding for a truncated protein of 741 amino acid residues), we analyze the role of BadA in adhesion and biofilm formation. We also investigate the role of environmental factors such as temperature on badA expression and biofilm formation. Real-time cell adhesion monitoring and electron microscopy show that Bh Houston-1 adheres and forms biofilm more efficiently than the Bh Houston-1 ∆badA. Deletion of the badA gene significantly decreases adhesion, the first step in biofilm formation in vitro, which is partially restored in Bh Houston-1 ∆badA/pNS2PTrcbadA. The biofilm formed by Bh Houston-1 includes polysaccharides, proteins, and DNA components and is susceptible to enzymatic degradation of these components. Furthermore, both pH and temperature influence both badA expression and biofilm formation. We conclude that BadA is required for optimal adhesion, agglutination and biofilm formation.

Comparative study of virulence factors among methicillin resistant Staphylococcus aureus clinical isolates

BackgroundMethicillin resistant Staphylococcus aureus (MRSA) is recognized worldwide as a leading cause of hospital and community infections. Biofilm formation by MRSA is an extremely important virulence factor to be understood. Our aim was to establish phenotypic and genotypic characterization of virulence factors among 43 MRSA clinical isolates in a Tunisian hospital.MethodsWe investigated enzymatic profiles, biofilm production and prevalences of genes encoding intracellular adhesion molecules (icaA and icaD), Microbial Surface Components Recognizing Adhesive Matrix Molecules genes (fnbA, fnbB and cna) and exoenzymes genes (geh, sspA and sspB).ResultsOur findings revealed that caseinase, gelatinase, lipase and lecithinase activities were detected in 100%, 100%, 76.6% and 93.3% of cases respectively. This study showed that 23 strains (76.7%) were slime producers on Congo red medium. Furthermore, 46.5% and 53.5% of isolates were respectively highly and moderately biofilm-forming on polystyrene. Significant association was found between both biofilm tests. PCR detection showed that 74.4%, 18.6%, 69.8%, 65.1% and 74.4% of isolates harbored fnbA, fnbB, icaA, icaD and cna genes respectively. In addition, 34.9%, 18.6% and 30.2% of MRSA strains were found positive for sspA, sspB and geh genes respectively. Further, statistical data showed that the presence of the fnbA and fnbB genes was significantly associated with a high biofilm production on polystyrene. However, no statistical association was observed for the icaA, icaD and cna genes.ConclusionsThis study indicates that the detection of fnbA and fnbB contributing to the first step of biofilm formation has been predictable of high biofilm production. As studied factors contribute to MRSA virulence, this research could be of value in orienting towards the development of new preventive and therapeutic measures.

Zirconium Nitride Coating Reduced Staphylococcus epidermidis Biofilm Formation on Orthopaedic Implant Surfaces: An In Vitro Study.

One of the most commonly identified pathogens responsible for orthopaedic implant infection is Staphylococcus epidermidis, which can form biofilms on surfaces. Currently, orthopaedic implants made of various surface materials are available, each with features influencing osseointegration, biocompatibility, and adherence of bacteria to the surface, which is the first step in biofilm formation. The aim of this experimental study was to investigate the effect of a high tribologic-resistant 2.5-µm zirconium nitride top coat on an antiallergic multilayer ceramic-covered cobalt-chromium-molybdenum surface on the formation of S. epidermidis biofilm compared with other commonly used smooth and rough orthopaedic implant surface materials. (1) When evaluating the surfaces of a cobalt-chromium-molybdenum (CoCrMo) alloy with a zirconium (Zr) nitride coating, a CoCrMo alloy without a coating, titanium alloy, a titanium alloy with a corundum-blasted rough surface, and stainless steel with a corundum-blasted rough surface, does a Zr coating reduce the number of colony-forming units of S. epidermidis in an in vitro setting? (2) Is there quantitatively less biofilm surface area on Zr-coated surfaces than on the other surfaces tested in this in vitro model? To determine bacterial adhesion, five different experimental implant surface discs were incubated separately with one of 31 different S. epidermidis strains each and subsequently sonicated. Twenty test strains were obtained from orthopaedic patients undergoing emergency hip prosthesis surgeries or revision of implant infection and 10 further strains were obtained from the skin of healthy individuals. Additionally, one reference strain, S. epidermidis DSM 3269, was tested. After serial dilutions, the number of bacteria was counted and expressed as colony-forming units (CFUs)/mL. For biofilm detection, discs were stained with 0.1% Safranin-O for 15 minutes, photographed, and analyzed with computer imaging software. The lowest bacterial count was found in the CoCrMo + Zr surface disc (6.6 x 10 CFU/mL ± 4.6 x 10 SD) followed by the CoCrMo surface (1.1 x 10 CFU/mL ± 1.9 x 10 SD), the titanium surface (1.36 x 10 CFU/mL ± 1.8 x 10 SD), the rough stainless steel surface (2.65 x 10 CFU/mL ± 3.8 x 10 SD), and the rough titanium surface (2.1 x 10 CFU/mL ± 3.0 x 10 SD). The mean CFU count was lower for CoCrMo + Zr discs compared with the rough stainless steel surface (mean difference: 2.0 x 10, p = 0.021), the rough titanium alloy surface (mean difference: 1.4 x 10, p = 0.002), and the smooth titanium surface (mean difference: 7.0 x 10, p = 0.016). The results of biofilm formation quantification show that the mean covered area of the surface of the CoCrMo + Zr discs was 19% (± 16 SD), which was lower than CoCrMo surfaces (35% ± 23 SD), titanium alloy surface (46% ± 20 SD), rough titanium alloy surface (66% ± 23 SD), and rough stainless steel surface (58% ± 18 SD). These results demonstrate that a multilayer, ceramic-covered, CoCrMo surface with a 2.5-µm zirconium nitride top coat showed less S. epidermidis biofilm formation compared with other surface materials used for orthopaedic implants. CoCrMo with a 2.5-µm zirconium nitride top coat seems to be a promising surface modification technology able to reduce bacterial attachment on the surface of an implant and, hence, may further prevent implant infection with S. epidermidis biofilm formation.

New Thiazole Nortopsentin Analogues Inhibit Bacterial Biofilm Formation.

New thiazole nortopsentin analogues were conveniently synthesized and evaluated for their activity as inhibitors of biofilm formation of relevant Gram-positive and Gram-negative pathogens. All compounds were able to interfere with the first step of biofilm formation in a dose-dependent manner, showing a selectivity against the staphylococcal strains. The most active derivatives elicited IC50 values against Staphylococcus aureus ATCC 25923, ranging from 0.40–2.03 µM. The new compounds showed a typical anti-virulence profile, being able to inhibit the biofilm formation without affecting the microbial growth in the planktonic form.

Antiadhesion activity of juniper (Juniperus communis L.) preparations against Campylobacter jejuni evaluated with PCR-based methods.

The food-borne pathogen Campylobacter jejuni can cause bacterial gastrointestinal infections. Biofilm formation amplifies the risk of human infection by improving survival and persistence of C.jejuni in food processing environments and its transmission through the food chain. We aimed to control C.jejuni using an alternative strategy of low doses of Juniperus communis fruit preparations to target bacterial adhesion properties in the first step of biofilm formation. First, we defined the anti-Campylobacter activity of a juniper fruit crude extract and its fractionated biflavonoids, flavone glycosides, and purified amentoflavone, of juniper fruit essential oil and of juniper fruit postdistillation waste material extract. For accurate quantification of adherent C.jejuni, we optimised digital Polymerase Chain Reaction (PCR) and quantitative real-time PCR for construction of standard curves and quantification. We show for the first time that juniper fruit formulations can effectively inhibit adhesion of C.jejuni to polystyrene. Furthermore, ≥94% of the antiadhesion activity of juniper fruit crude extract and juniper fruit essential oil remained under food-related conditions: modified culture medium with glucose, or a stainless steel surface, or mixed co-cultures of C.jejuni and Listeria monocytogenes. This study indicates that addition of juniper fruit formulations can control growth and adhesion of C.jejuni and thus limit food chain transmission of campylobacters.

Adhesion forces of the sea-water bacterium Paracoccus seriniphilus on titanium: Influence of microstructures and environmental conditions.

The bacterial attachment to surfaces is the first step of biofilm formation. This attachment is governed by adhesion forces which act between the bacterium and the substrate. Such forces can be measured by single cell force spectroscopy, where a single bacterium is attached to a cantilever of a scanning force microscope, and force-distance curves are measured. For the productive sea-water bacterium Paracoccus seriniphilus, pH dependent measurements reveal the highest adhesion forces at pH 4. Adhesion forces measured at salinities between 0% and 4.5% NaCl are in general higher for higher salinity. However, there is an exception for 0.9% where a higher adhesion force was measured than expected. These results are in line with zeta potential measurements of the bacterium, which also show an exceptionally low zeta potential at 0.9% NaCl. In the absence of macromolecular interactions, the adhesion forces are thus governed by (unspecific) electrostatic interactions, which can be adjusted by pH and ionic strength. It is further shown that microstructures on the titanium surface increase the adhesion force. Growth medium reduces the interaction forces dramatically, most probably through macromolecular bridging.

Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation.

Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (p)ppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of incubation. Analysis of some mutants demonstrated the interest of this proteomic approach in identifying genes involved in the early phase of adhesion to a surface.

Substrate effects on cell-envelope deformation during early-stage Staphylococcus aureus biofilm formation.

Bacterial adhesion to a surface is the first step in biofilm formation, and adhesive forces between the surface and a bacterium are believed to give rise to planktonic-to-biofilm phenotypic changes. Here we use Focused-Ion-Beam (FIB) tomography with backscattered scanning electron microscopy (SEM) to image Staphyolococcus aureus (S. aureus) biofilms grown on Au-coated polystyrene (PS) and Au-coated PS modified by mixed thiols of triethylene glycol mono-11-mercaptoundecyl ether (EG3) and 1-dodecanethiol (CH3). The FIB-SEM technique enables a direct measurement of the contact area between individual bacteria and the substrate. The area of adhesion is effectively zero on the EG3 substrate. It is nonzero on all of the other substrates and increases with increasing hydrophobicity. The fact that the contact area is highest on the unmodified gold, however, indicates that other forces beyond hydrophobicity are significant. The magnitude of bacterial deformation suggests that the adhesive forces are on the order of a few nN, consistent with AFM force measurements reported in the literature. The resolution afforded by electron microscopy furthermore enables us to probe changes in the cell-envelope thickness, which decreases within and near the contact area relative to other parts of the same bacterium. This finding supports the idea that mechanosensing due to stress-induced membrane thinning plays a role in the planktonic-to-biofilm transition associated with bacterial adhesion.