Listeria monocytogenes LO28: surface physicochemical properties and ability to form biofilms at different temperatures and growth phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20 degrees C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37 degrees C. At 8 degrees C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

Different, overlapping mechanisms for colonization of abiotic and plant surfaces by Pseudomonas putida

Mechanisms governing biofilm formation have generated considerable interest in recent years, yet comparative analyses of processes for bacterial establishment on abiotic and biotic surfaces are still limited. In this report we have expanded previous information on the genetic determinants required for colonization of plant surfaces by Pseudomonas putida populations and analyzed their correlation with biofilm formation processes on abiotic surfaces. Insertional mutations affecting flagellar genes or the synthesis and transport of the large adhesin LapA lead to decreased adhesion to seeds and biofilm formation on abiotic surfaces. The latter also causes reduced fitness in the rhizosphere. Decreased seed adhesion and altered biofilm formation kinetics are observed in mutants affected in heme biosynthesis and a gene that might participate in oxidative stress responses, whereas a mutant in a gene involved in cytochrome oxidase assembly is affected in the bacterium-plant interaction but not in bacterial establishment on abiotic surfaces. Finally, a mutant altered in lipopolysaccharide biosynthesis is impaired in seed and root colonization but seems to initiate attachment to plastic faster than the wild type. This variety of phenotypes reflects the complexity of bacterial adaptation to sessile life, and the partial overlap between mechanisms leading to biofilm formation on abiotic and biotic surfaces.     

Listeria monocytogenes LO28: Surface Physicochemical Properties and Ability To Form Biofilms at Different Temperatures and Growth Phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20°C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37°C. At 8°C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

Bacterial adhesion: From mechanism to control

Bacterial adhesion is the initial step in colonization and biofilm formation. Biofilms can, on the one hand, be detrimental to both human life and industrial processes, for example, causing infection, pathogen contamination, and slime formation, while on the other hand, be beneficial in environmental technologies and bioprocesses. For control and utilization of bacterial adhesion and biofilms, adhesion mechanisms must be elucidated. Conventional physicochemical approaches based on Lifshitz-van der Waals, electrostatic and acid–base interactions provide important models of bacterial adhesion but have a limited capacity to provide a complete understanding of the complex adhesion process of real bacterial cells. In conventional approaches, bacterial cells, whose surfaces are structurally and chemically heterogeneous, are often described from the viewpoint of their overall cellular properties. Cell appendages such as polysaccharide chains and proteinous nanofibers have an important function bridging between cells and the substratum in conventional adhesion models, but sometimes cause deviation from the models of cell adhesion. In reality, cell appendages are responsible for specific and nonspecific cell adhesion to biotic and abiotic surfaces. This paper reviews conventional physicochemical models and cell appendage-mediated cell adhesion. State-of-the-art technologies for controlling microbial adhesion and biofilm formation are also described. These technologies are based on the adhesion mechanisms.     

Fluorescence-Based Quasicontinuous and In Situ Monitoring of Biofilm Formation Dynamics in Natural Marine Environments

Analyzing the dynamics of biofilm formation helps to deepen our understanding of surface colonization in natural environments. While methods for screening biofilm formation in the laboratory are well established, studies in marine environments have so far been based upon destructive analysis of individual samples and provide only discontinuous snapshots of biofilm establishment. In order to explore the development of biofilm over time and under various biotic and abiotic conditions, we applied a recently developed optical biofilm sensor to quasicontinuously analyze marine biofilm dynamics in situ. Using this technique in combination with microscope-assisted imaging, we investigated biofilm formation from its beginning to mature multispecies biofilms. In contrast to laboratory studies on biofilm formation, a smooth transition from initial attachment to colony formation and exponential growth could not be observed in the marine environment. Instead, initial attachment was followed by an adaptation phase of low growth and homogeneously distributed solitary bacterial cells. Moreover, we observed a diurnal variation of biofilm signal intensity, suggesting a transient state of biofilm formation of bacteria. Overall, the biofilm formation dynamics could be modeled by three consecutive development stages attributed to initial bacterial attachment, bacterial growth, and attachment and growth of unicellular eukaryotic microorganisms. Additional experiments showed that the presence of seaweed considerably shortened the adaptation phase in comparison with that on control surfaces but yielded similar growth rates. The outlined examples highlight the advantages of a quasicontinuous in situ detection that enabled, for the first time, the exploration of the initial attachment phase and the diurnal variation during biofilm formation in natural ecosystems.     

Blocking of bacterial biofilm formation by a fish protein coating

Bacterial biofilm formation on inert surfaces is a significant health and economic problem in a wide range of environmental, industrial, and medical areas. Bacterial adhesion is generally a prerequisite for this colonization process and, thus, represents an attractive target for the development of biofilm-preventive measures. We have previously found that the preconditioning of several different inert materials with an aqueous fish muscle extract, composed primarily of fish muscle alpha-tropomyosin, significantly discourages bacterial attachment and adhesion to these surfaces. Here, this proteinaceous coating is characterized with regards to its biofilm-reducing properties by using a range of urinary tract infectious isolates with various pathogenic and adhesive properties. The antiadhesive coating significantly reduced or delayed biofilm formation by all these isolates under every condition examined. The biofilm-reducing activity did, however, vary depending on the substratum physicochemical characteristics and the environmental conditions studied. These data illustrate the importance of protein conditioning layers with respect to bacterial biofilm formation and suggest that antiadhesive proteins may offer an attractive measure for reducing or delaying biofilm-associated infections.     

Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture

Although exopolysaccharides (EPSs) are a large component of bacterial biofilms, their contribution to biofilm structure and function has been examined for only a few organisms. In each of these cases EPS has been shown to be required for cellular attachment to abiotic surfaces. Here, we undertook a genetic approach to examine the potential role of colanic acid, an EPS of Escherichia coli K-12, in biofilm formation. Strains either proficient or deficient in colanic acid production were grown and allowed to adhere to abiotic surfaces and were then examined both macroscopically and microscopically. Surprisingly, we found that colanic acid production is not required for surface attachment. Rather, colanic acid is critical for the formation of the complex three-dimensional structure and depth of E. coli biofilms.     

Vancomycin-Eluting Niosomes: A New Approach to the Inhibition of Staphylococcal Biofilm on Abiotic Surfaces

A new vancomycin (VCM)-eluting mixed bilayer niosome formulation was evaluated for the control of staphylococcal colonization and biofilm formation on abiotic surfaces, a niosome application not explored to date. Cosurfactant niosomes were prepared using a Span 60/Tween 40/cholesterol blend (1: 1: 2). Tween 40, a polyethoxylated amphiphile, was included to enhance VCM entrapment and confer niosomal surface properties precluding bacterial adhesion. VCM-eluting niosomes showed good quality attributes including relatively high entrapment efficiency (～50%), association of Tween 40 with vesicles in a constant proportion (～87%), biphasic release profile suitable for inhibiting early bacterial colonization, and long-term stability at 4°C for a 12-month study period. Niosomes significantly enhanced VCM activity against planktonic bacteria of nine staphylococcal strains. Using microtiter plates as abiotic surface, VCM-eluting niosomes proved superior to VCM in inhibiting biofilm formation, eradicating surface-borne biofilms, inhibiting biofilm growth, and interfering with biofilm induction by VCM subminimal inhibitory concentrations. Data suggest dual functionality of cosurfactant VCM-eluting niosomes as passive colonization inhibiting barrier and active antimicrobial-controlled delivery system, two functions recognized in infection control of abiotic surfaces and medical devices.     

The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharides

The ability to form biofilm is seen as an increasingly important colonization strategy among both pathogenic and environmental Klebsiella pneumoniae strains. The aim of the present study was to identify abiotic surface colonization factors of K. pneumoniae using different models at different phases of biofilm development. A 2200 K. pneumoniae mutant library previously obtained by signature-tagged mutagenesis was screened in static and dynamic culture models to detect clones impaired at early and/or mature stages of biofilm formation. A total of 28 mutants were affected during late phases of biofilm formation, whereas 16 mutants displayed early adhesion defect. These mutants corresponded to genes involved in potential cellular and DNA metabolism pathways and to membrane transport functions. Eight mutants were deficient in capsule or LPS production. Gene disruption and microscopic analyses showed that LPS is involved in initial adhesion on both glass and polyvinyl-chloride and the capsule required for the appropriate initial coverage of substratum and the construction of mature biofilm architecture. These results give new insight into the bacterial factors sequentially associated with the ability to colonize an abiotic surface and reveal the dual roles played by surface exopolysaccharides during K. pneumoniae biofilm formation.     

Identification of Tn10 insertions in the dsbA gene affecting Escherichia coli biofilm formation

Escherichia coli was used as model to study initial adhesion and early biofilm development to an abiotic surface. Tn10 insertion mutants with reduced attachment to a polystyrene surface were isolated. Three adhesion mutants harbored the transposon in the dsbA gene, whose product, DsbA, catalyses folding of numerous extracytoplasmic disulfide bond-containing proteins. All three mutants were weakly adherent and grew poorly. Cell surface structure analysis showed that motility. type 1 fimbriation and lipopolysaccharide structure were affected in these mutants. The pleiotropic effect of the dsbA mutations on biofilm formation is discussed.     

Developmental pathway for biofilm formation in curli-producing Escherichia coli strains: role of flagella, curli and colanic acid

This work was performed to establish a model describing bacterial surface structures involved in biofilm development, in curli-overproducing Escherichia coli K-12 strains, at 30°C, and in minimal growth medium. Using a genetic approach, in association with observations of sessile communities by light and electron microscopic techniques, the role of protein surface structures, such as flagella and curli, and saccharidic surface components, such as the E. coli exopolysaccharide, colanic acid, was determined. We show that, in the context of adherent ompR234 strains, (i) flagellar motility is not required for initial adhesion and biofilm development; (ii) both primary adhesion to inert surfaces and development of multilayered cell clusters require curli synthesis; (iii) curli display direct interactions with the substratum and form interbacterial bundles, allowing a cohesive and stable association of cells; and (iv) colanic acid does not appear critical for bacterial adhesion and further biofilm development but contributes to the biofilm architecture and allows for the formation of voluminous biofilms.     

Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri

The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.     

Hydrophobin coating prevents Staphylococcus epidermidis biofilm formation on different surfaces

Staphylococcus epidermidis is a significant nosocomial pathogen in predisposed hosts because of its capability of forming a biofilm on indwelling medical devices. The initial stage of biofilm formation has a key role in S. epidermidis abiotic surface colonization. Recently, many strategies have been developed to create new anti-biofilm surfaces able to control bacterial adhesion mechanisms. In this work, the self-assembled amphiphilic layers formed by two fungal hydrophobins (Vmh2 and Pac3) have proven to be able to reduce the biofilm formed by different strains of S. epidermidis on polystyrene surfaces. The reduction in the biofilm thickness on the coated surfaces and the preservation of cell vitality have been demonstrated through confocal laser scanning microscope analysis. Moreover, the anti-biofilm efficiency of the self-assembled layers on different medically relevant materials has also been demonstrated using a CDC biofilm reactor.     

Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili

We have used Escherichia coli as a model system to investigate the initiation of biofilm formation. Here, we demonstrate that E. coli forms biofilms on multiple abiotic surfaces in a nutrient-dependent fashion. In addition, we have isolated insertion mutations that render this organism defective in biofilm formation. One-half of these mutations was found to perturb normal flagellar function. Using defined fli , flh , mot and che alleles, we show that motility, but not chemotaxis, is critical for normal biofilm formation. Microscopic analyses of these mutants suggest that motility is important for both initial interaction with the surface and for movement along the surface. In addition, we present evidence that type I pili (harbouring the mannose-specific adhesin, FimH) are required for initial surface attachment and that mannose inhibits normal attachment. In light of the observations presented here, a working model is discussed that describes the roles of both motility and type I pili in biofilm development.     

Substrata mechanical stiffness can regulate adhesion of viable bacteria

The competing mechanisms that regulate adhesion of bacteria to surfaces and subsequent biofilm formation remain unclear, though nearly all studies have focused on the role of physical and chemical properties of the material surface. Given the large monetary and health costs of medical-device colonization and hospital-acquired infections due to bacteria, there is considerable interest in better understanding of material properties that can limit bacterial adhesion and viability. Here we employ weak polyelectrolyte multilayer (PEM) thin films comprised of poly(allylamine) hydrochloride (PAH) and poly(acrylic acid) (PAA), assembled over a range of conditions, to explore the physicochemical and mechanical characteristics of material surfaces controlling adhesion of Staphylococcus epidermidis bacteria and subsequent colony growth. Although it is increasingly appreciated that eukaryotic cells possess subcellular structures and biomolecular pathways to sense and respond to local chemomechanical environments, much less is known about mechanoselective adhesion of prokaryotes such as these bacteria. We find that adhesion of viable S. epidermidis correlates positively with the stiffness of these polymeric substrata, independently of the roughness, interaction energy, and charge density of these materials. Quantitatively similar trends observed for wild-type and actin analogue mutant Escherichia coli suggest that these results are not confined to only specific bacterial strains, shapes, or cell envelope types. These results indicate the plausibility of mechanoselective adhesion mechanisms in prokaryotes and suggest that mechanical stiffness of substrata materials represents an additional parameter that can regulate adhesion of and subsequent colonization by viable bacteria.     

Bacterial cell attachment, the beginning of a biofilm

The ability of bacteria to attach to surfaces and develop into a biofilm has been of considerable interest to many groups in numerous industries, including the medical and food industry. However, little is understood in the critical initial step seen in all biofilm development, the initial bacterial cell attachment to a surface. This initial attachment is critical for the formation of a bacterial biofilm, as all other cells within a biofilm structure rely on the interaction between surface and bacterial cell for their survival. This review examines what are believed to be some of the most important aspects involved in bacterial attachment to a surface.     

The role of initial spore adhesion in pellet and biofilm formation in Aspergillus niger

Fungi grow on a great variety of organic and inorganic materials. Colony establishment and growth on solid surfaces require adhesion of spores and hyphae to the substrate, while cell-to-cell interactions among spores and/or hyphae are a prerequisite for the development of three-dimensional mycelial structures such as pellets or biofilms. Surface adherence has been described as a two-step process, comprised of the initial attachment of ungerminated conidia followed by further adhesion of the forming germ tubes and growing hyphae. In the present study, we analyzed the contribution of adhesion of ungerminated spores to pellet and biofilm formation in Aspergillus niger. Mutants deficient in melanin biosynthesis were constructed by the deletion of the alb1 gene, encoding a polyketide synthase essential for pigment biosynthesis. Δalb1 conidia have an altered surface structure and changed physicochemical surface properties. Spore aggregation in liquid culture as well as spore surface attachment differ between the wild type and the mutant in a pH-dependent manner. In liquid culture further pellet formation is unaffected by altered spore-spore interactions, indicating that germ tube and hyphal adherence can compensate for deficiencies in the initial step of spore attachment. In contrast, under conditions promoting adhesion of Δalb1 conidia to polymer surfaces the mutant forms more stable biofilms than the wild type, suggesting that initial spore adhesion supports sessile growth.     

Initial Phases of biofilm formation in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative Fe(III)- and Mn(IV)-reducing microorganism and serves as a model for studying microbially induced dissolution of Fe or Mn oxide minerals as well as biogeochemical cycles. In soil and sediment environments, S. oneidensis biofilms form on mineral surfaces and are critical for mediating the metabolic interaction between this microbe and insoluble metal oxide phases. In order to develop an understanding of the molecular basis of biofilm formation, we investigated S. oneidensis biofilms developing on glass surfaces in a hydrodynamic flow chamber system. After initial attachment, growth of microcolonies and lateral spreading of biofilm cells on the surface occurred simultaneously within the first 24 h. Once surface coverage was almost complete, biofilm development proceeded with extensive vertical growth, resulting in formation of towering structures giving rise to pronounced three-dimensional architecture. Biofilm development was found to be dependent on the nutrient conditions, suggesting a metabolic control. In global transposon mutagenesis, 173 insertion mutants out of 15,000 mutants screened were identified carrying defects in initial attachment and/or early stages in biofilm formation. Seventy-one of those mutants exhibited a nonswimming phenotype, suggesting a role of swimming motility or motility elements in biofilm formation. Disruption mutations in motility genes (flhB, fliK, and pomA), however, did not affect initial attachment but affected progression of biofilm development into pronounced three-dimensional architecture. In contrast, mutants defective in mannose-sensitive hemagglutinin type IV pilus biosynthesis and in pilus retraction (pilT) showed severe defects in adhesion to abiotic surfaces and biofilm formation, respectively. The results provide a basis for understanding microbe-mineral interactions in natural environments.     

Quantitative characterization of the influence of the nanoscale morphology of nanostructured surfaces on bacterial adhesion and biofilm formation

Bacterial infection of implants and prosthetic devices is one of the most common causes of implant failure. The nanostructured surface of biocompatible materials strongly influences the adhesion and proliferation of mammalian cells on solid substrates. The observation of this phenomenon has led to an increased effort to develop new strategies to prevent bacterial adhesion and biofilm formation, primarily through nanoengineering the topology of the materials used in implantable devices. While several studies have demonstrated the influence of nanoscale surface morphology on prokaryotic cell attachment, none have provided a quantitative understanding of this phenomenon. Using supersonic cluster beam deposition, we produced nanostructured titania thin films with controlled and reproducible nanoscale morphology respectively. We characterized the surface morphology; composition and wettability by means of atomic force microscopy, X-ray photoemission spectroscopy and contact angle measurements. We studied how protein adsorption is influenced by the physico-chemical surface parameters. Lastly, we characterized Escherichia coli and Staphylococcus aureus adhesion on nanostructured titania surfaces. Our results show that the increase in surface pore aspect ratio and volume, related to the increase of surface roughness, improves protein adsorption, which in turn downplays bacterial adhesion and biofilm formation. As roughness increases up to about 20 nm, bacterial adhesion and biofilm formation are enhanced; the further increase of roughness causes a significant decrease of bacterial adhesion and inhibits biofilm formation. We interpret the observed trend in bacterial adhesion as the combined effect of passivation and flattening effects induced by morphology-dependent protein adsorption. Our findings demonstrate that bacterial adhesion and biofilm formation on nanostructured titanium oxide surfaces are significantly influenced by nanoscale morphological features. The quantitative information, provided by this study about the relation between surface nanoscale morphology and bacterial adhesion points towards the rational design of implant surfaces that control or inhibit bacterial adhesion and biofilm formation.     

Carbohydrate Coating Reduces Adhesion of Biofilm-Forming Bacillus subtilis to Gold Surfaces

The growth of bacterial biofilms in pipes and food tanks causes severe problems in industry. Biofilms growing on medical implants or catheters are of great concern, as they can cause serious infections and decrease the functionality of the medical device. The prevention of bacterial adhesion—the first step in colonization and biofilm formation—is therefore very important. Current research comprises alterations in surface properties, the prevention of adhesin biosynthesis, inhibition with receptor analogs, or the development of anti-adhesive vaccines. We present a new approach that allows us to study bacterial adhesion with high sensitivity in real-time while testing several different surfaces in parallel. Using the cantilever-array technique we demonstrate that coating of gold surfaces with mono- or disaccharides results in a reduction of the bacterial adhesion of the biofilm-forming bacterium Bacillus subtilis NCIB 3610 to these gold surfaces. This reduction in bacterial adhesion is independent of the studied carbohydrate. Using several mutant strains, we investigate the underlying molecular interactions, and our results suggest that adhesion to gold surfaces is mediated by thiol groups present in proteins of the bacterial cell membrane or biofilm matrix proteins expressed at low levels by the wild-type strain. Furthermore, our data indicate that the adhesion of B. subtilis NCIB 3610 to carbohydrate-coated gold surfaces is facilitated by interactions between carbohydrates installed on the cantilever gold surface and an exopolysaccharide expressed by this strain. Understanding general and specific contributions of molecular interactions mediating bacterial adhesion will enable its prevention in the future.