The effect of mutations in the synthesis of lipopolysaccharides and calcofluor-binding polysaccharides on biofilm formation by Azospirillum brasilense

The thickness and antigenic properties of biofilms produced by Azospirillum brasilense Sp245 and its mutants deficient in the synthesis of lipopolysaccharides (Lps) and calcofluor-binding polysaccharides (CBPS) at the interface between water and hydrophilic or hydrophobic solid surfaces were compared. The mutants deficient in acidic LpsI synthesis produce thicker biofilms on hydrophilic surfaces. Biofilms produced on hydrophobic surfaces by bacteria that are unable to synthesize CBPS are less pronounced. Defects in CBPS production in Azospirillum mutants with impaired flagellar motility can cause adverse effects on the cell ability to attach to hydrophobic and hydrophilic surfaces. The loss of the neutral LpsII antigen by the mutants capable of producing CBPS does not affect their behavior on hydrophobic surfaces, which is probably due to the compensatory increase in the total polysaccharide production. The fundamental change in the Lps structure correlates with the activation of biofilm formation by the relevant mutants on hydrophilic and hydrophobic surfaces.     

The effect of mutations affecting synthesis of lipopolysaccharides and calcofluor-binding polysaccharides on biofilm formation by <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

The thickness and antigenic properties of biofilms produced by Azospirillum brasilense Sp245 and its mutants deficient in the synthesis of lipopolysaccharides (Lps) and calcofluor-binding polysaccharides (CBPS) at the interface between water and hydrophilic or hydrophobic solid surfaces were compared. The mutants deficient in acidic LpsI synthesis produce thicker biofilms on hydrophilic surfaces. Biofilms produced on hydrophobic surfaces by bacteria that are unable to synthesize CBPS are less pronounced. Defects in CBPS production in Azospirillum mutants with impaired flagellar motility can cause adverse effects on the cell ability to attach to hydrophobic and hydrophilic surfaces. The loss of the neutral LpsII antigen by the mutants capable of producing CBPS does not affect their behavior on hydrophobic surfaces, which is probably due to the compensatory increase in the total polysaccharide production. The fundamental change in the Lps structure correlates with the activation of biofilm formation by the relevant mutants on hydrophilic and hydrophobic 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 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.     

Comparison of the fouling release properties of hydrophobic fluorinated and hydrophilic PEGylated block copolymer surfaces: attachment strength of the diatom Navicula and the green alga Ulva

To understand the role of surface wettability in adhesion of cells, the attachment of two different marine algae was studied on hydrophobic and hydrophilic polymer surfaces. Adhesion of cells of the diatom Navicula and sporelings (young plants) of the green macroalga Ulva to an underwater surface is mainly by interactions between the surface and the adhesive exopolymers, which the cells secrete upon settlement and during subsequent colonization and growth. Two types of block copolymers, one with poly(ethylene glycol) side-chains and the other with liquid crystalline, fluorinated side-chains, were used to prepare the hydrophilic and hydrophobic surfaces, respectively. The formation of a liquid crystalline smectic phase in the latter inhibited molecular reorganization at the surface, which is generally an issue when a highly hydrophobic surface is in contact with water. The adhesion strength was assessed by the fraction of settled cells (Navicula) or biomass (Ulva) that detached from the surface in a water flow channel with a wall shear stress of 53 Pa. The two species exhibited opposite adhesion behavior on the same sets of surfaces. While Navicula cells released more easily from hydrophilic surfaces, Ulva sporelings showed higher removal from hydrophobic surfaces. This highlights the importance of differences in cell-surface interactions in determining the strength of adhesion of cells to substrates.     

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.     

Diversity in surface colonization behavior in marine bacteria

Using laminar flow chambers and time-lapse video imaging, colonization of surfaces by four marine bacteria revealed a diverse range of morphological characteristics and cell-cell interactions. The strain SW5 formed a compact, multilayered single- and double-cell biofilm on hydrophobic surfaces but developed long multicellular chains on hydrophilic surfaces. The morphologically similar SW8 showed unusual proximal vertical packing of cells on both substrata.Vibrio sp strain S14 exhibited cyclical colonization-detachment events on both substrata.Pseudomonas sp strain S9 initially displayed reversible and then irreversible adhesion apparently triggered by a cell density phenomenon that led to the development of regular microcolonies on both substrata with individual cells translocating between the colonies. The length of time bacteria were exposed to and their density at a surface influenced behavioral traits, with diverse and distinctive species-specific behavioral events.     

Role of hydrophobicity in bacterial adherence to carbon nanostructures and biofilm formation

The role of cell and surface hydrophobicity in the adherence of the waterborne bacterium Mycobacterium smegmatis to nanostructures and biofilm formation was investigated. Carbon nanostructures (CNs) were synthesized using a flame reactor and deposited on stainless steel grids and foils, and on silicon wafers that had different initial surface hydrophobicities. Surface hydrophobicity was measured as the contact angle of water droplets. The surfaces were incubated in suspensions of isogenic hydrophobic and hydrophilic strains of M. smegmatis and temporal measurements of the numbers of adherent cells were made. The hydrophobic, rough mutant of M. smegmatis adhered more readily and formed denser biofilms on all surfaces compared to its hydrophilic, smooth parent. Biofilm formation led to alterations in the hydrophobicity of the substratum surfaces, demonstrating that bacterial cells attached to CNs are capable of modifying the surface characteristics.     

Development of pure culture biofilms of P. putida on solid supports

Pseudomonas putida biofilms were developed on and biofilm accumulation rate data were obtained for the following two classes of support materials: charged surfaces and noncharged hydrophobic and hydrophilic surfaces. The effects of surface roughness and porosity on the rate of microbial attachment were also examined.Materials bearing a net positive or negative surface charge supported the greatest biofilm accumulation and the highest biofilm accumulation rate. Uncharged hydrophobic materials achieved the next greatest biofilm accumulation, averaging approximately 50% of the total biomass which was accumulated on the charged surface materials after 16 days. Uncharged hydrophilic materials supported very little biofilm development. In general, biofilm accumulation increased with decreased surface roughness. The effect of pore size on biofilm accumulation was not conclusive.The biofilm accumulation kinetics showed an exponential accumulation rate for the charged surfaces and an approximately linear accumulation rate for the hydrophobic materials. This difference in accumulation kinetics is consistent with proposed differences in the physicochemical mechanism governing attachment to these two types of surface materials.     

Efficient rhizosphere colonization by Pseudomonas fluorescens f113 mutants unable to form biofilms on abiotic surfaces

Motility is a key trait for rhizosphere colonization by Pseudomonas fluorescens. Mutants with reduced motility are poor competitors, and hypermotile, more competitive phenotypic variants are selected in the rhizosphere. Flagellar motility is a feature associated to planktonic, free‐living single cells, and although it is necessary for the initial steps of biofilm formation, bacteria in biofilm lack flagella. To test the correlation between biofilm formation and rhizosphere colonization, we have used P. fluorescens F113 hypermotile derivatives and mutants affected in regulatory genes which in other bacteria modulate biofilm development, namely gacS (G), sadB (S) and wspR (W). Mutants affected in these three genes and a hypermotile variant (V35) isolated from the rhizosphere were impaired in biofilm formation on abiotic surfaces, but colonized the alfalfa root apex as efficiently as the wild‐type strain, indicating that biofilm formation on abiotic surfaces and rhizosphere colonization follow different regulatory pathways in P. fluorescens. Furthermore, a triple mutant gacSsadBwspR (GSW) and V35 were more competitive than the wild‐type strain for root‐tip colonization, suggesting that motility is more relevant in this environment than the ability to form biofilms on abiotic surfaces. Microscopy showed the same root colonization pattern for P. fluorescens F113 and all the derivatives: extensive microcolonies, apparently held to the rhizoplane by a mucigel that seems to be plant produced. Therefore, the ability to form biofilms on abiotic surfaces does not necessarily correlates with efficient rhizosphere colonization or competitive colonization.     

Plasmid Transfer between Marine Bacteria in the Aqueous Phase and Biofilms in Reactor Microcosms

Plasmid transfer of broad-host-range plasmid RP1 from marine Vibrio sp. strain S14 to marine strain SW5 under optimum conditions on the surface of nutrient plates was improved 2 orders of magnitude by using the plasmid transfer process to select an SW5 recipient more efficient than the wild type in receiving and/or maintaining the plasmid. This recipient strain, SW5H, was used to form biofilms under flow conditions on the surfaces of glass beads in reactors. The S142(RP1) donor strain was introduced to the reactors after either 48 or 170 h of biofilm formation, and production of transconjugants in the aqueous phases and biofilms without selection pressure was assessed. Plasmid transfer to the recipient cells in the biofilm was detected for biofilms formed for 170 h but not in those formed for 48 h. The plasmid transfer frequency was significantly higher (P < 0.05) among cells attached to the bead surfaces in the biofilm than among cells in the aqueous phase.     

Comparison of the Phenotypes and Genotypes of Biofilm and Solitary Epiphytic Bacterial Populations on Broad-Leaved Endive

The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety. Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor. However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies. Broad-leaved endive (Cichorium endivia var. latifolia) harbors biofilms containing fluorescent pseudomonads. These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads. To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive. Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation. The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains. However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms. We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states. This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate.     

Plastics select for distinct early colonizing microbial populations with reproducible traits across environmental gradients

Little is known about early plastic biofilm assemblage dynamics and successional changes over time. By incubating virgin microplastics along oceanic transects and comparing adhered microbial communities with those of naturally occurring plastic litter at the same locations, we constructed gene catalogues to contrast the metabolic differences between early and mature biofilm communities. Early colonization incubations were reproducibly dominated by Alteromonadaceae and harboured significantly higher proportions of genes associated with adhesion, biofilm formation, chemotaxis, hydrocarbon degradation and motility. Comparative genomic analyses among the Alteromonadaceae metagenome assembled genomes (MAGs) highlighted the importance of the mannose-sensitive hemagglutinin (MSHA) operon, recognized as a key factor for intestinal colonization, for early colonization of hydrophobic plastic surfaces. Synteny alignments of MSHA also demonstrated positive selection for mshA alleles across all MAGs, suggesting that mshA provides a competitive advantage for surface colonization and nutrient acquisition. Large-scale genomic characteristics of early colonizers varied little, despite environmental variability. Mature plastic biofilms were composed of predominantly Rhodobacteraceae and displayed significantly higher proportions of carbohydrate hydrolysis enzymes and genes for photosynthesis and secondary metabolism. Our metagenomic analyses provide insight into early biofilm formation on plastics in the ocean and how early colonizers self-assemble, compared to mature, phylogenetically and metabolically diverse biofilms.     

Chemical defenses of seaweeds against microbial colonization

Any living or non-living surface immersed in seawaterrapidly acquires a bacterial biofilm. For living marineorganisms, biofilm formation can result in the death ofthe host, and thus there is strong evolutionary pressure formarine eukaryotes to evolve mechanisms which inhibit orcontrol the development of biofilms on their surfaces.Some marine eukaryotes are indeed successful incontrolling biofilms on their surfaces, and in manyinstances this control is achieved by the production ofinhibitory chemicals which act at or near the surface ofthe organism. In some cases these natural inhibitors aresimply toxic to bacteria. However, increasingly it appearsthat at least some of these compounds act by interferingspecifically with bacterial characteristics which effect theability of bacteria to colonize their hosts, such asattachment, surface spreading, or the production ofextracellular macromolecules. As an example, theAustralian seaweed Delisea pulchra appears tocontrol bacterial colonization by interfering with abacterial regulatory system (the acylated homoserinelactone system) that regulates several colonizationrelevant bacterial traits. Understanding how marineorganisms control specific bacterial colonization traitsshould provide us with insights into new technologies forthe control of biofilms on artificial surfaces.     

IR study of self-assembly of capsular exopolymers from Pseudomonas sp. NCIMB 2021 on hydrophilic and hydrophobic surfaces

Capsular exopolymers (EPS) of the bacterium Pseudomonas sp. NCIMB 2021 are allowed to self-assemble on hydrophilic and hydrophobic gold surfaces. Tapping mode atomic force microscopy confirms the differences in the surface topography between EPS adsorbed on both surfaces. Fourier-transform IR spectroscopy indicates that the EPS surface coverage is much greater on the hydrophobic surface. Furthermore, an increased contribution is observed from hydrophobic (i.e., methyl and tyrosyl residues) and electrostatic (i.e., carboxylate residues) groups at the hydrophobic surface, but there is relatively less neutral polymer compared to the hydrophilic surface. The behavior of this EPS is in agreement with the behavior of cells of Pseudomonas sp. NCIMB 2021 at hydrophilic and hydrophobic surfaces.     

An in vitro study of initial adsorption from human parotid and submandibular/sublingual resting saliva at solid/liquid interfaces

The influence of saliva concentration, saliva total protein content and the wetting characteristics of exposed solids on in vitro film formation was studied by the technique of in situ ellipsometry. The rates and plateau values of adsorption (45 min) at solid/liquid interfaces (hydrophilic silica and hydrophobic methylated silica surfaces) were determinated for human parotid (HPS) and submandibular/sublingual (HSMSLS) resting saliva solutions (0.1 and 1.0%, (v/v), saliva in phosphate buffered saline). Adsorption rates were related to a model assuming mass transport through an unstirred layer adjacent to the surface. The results showed that the adsorption was rapid, concentration dependent and higher on hydrophobic than on hydrophilic surfaces. Analysis of the influence of protein concentration on the adsorbed amounts demonstrated an interaction between protein concentration and the two surfaces for HPS and HSMSLS, respectively. This may indicate differences in binding mode. Inter‐individual differences were found not to be significant at the 1% level of probability. Comparison of the observed adsorption and calculated diffusion rates suggest that on hydrophilic surfaces initial adsorption of proteins diffusing at rates corresponding to those of statherin and aPRPs takes place, whereas on hydrophobic surfaces lower molecular mass compounds appear to be involved.     

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.     

Potential of antimicrobial treatment of linear low-density polyethylene with poly((tert-butyl-amino)-methyl-styrene) to reduce biofilm formation in the food industry

Antimicrobial surfaces are one approach to prevent biofilms in the food industry. The aim of this study was to investigate the effect of poly((tert-butyl-amino)-methyl-styrene) (poly(TBAMS)) incorporated into linear low-density polyethylene (LLDPE) on the formation of mono- and mixed-species biofilms. The biofilm on untreated and treated LLDPE was determined after 48 and 168 h. The comparison of the results indicated that the ability of Listeria monocytogenes to form biofilms was completely suppressed by poly(TBAMS) (Δ_168 h 3.2 log₁₀ cfu cm^?2) and colonization of Staphylococcus aureus and Escherichia coli was significantly delayed, but no effect on Pseudomonas fluorescens was observed. The results of dual-species biofilms showed complex interactions between the microorganisms, but comparable effects on the individual bacteria by poly(TBAMS) were identified. Antimicrobial treatment with poly(TBAMS) shows great potential to prevent biofilms on polymeric surfaces. However, a further development of the material is necessary to reduce the colonization of strong biofilm formers.     

Biofilm formation in environmental bacteria is influenced by different macromolecules depending on genus and species

The formation of biofilms by diverse bacteria isolated from contaminated soil and groundwater on model substrata with different surface properties was assessed in a multifactorial screen. Diverse attachment phenotypes were observed as measured by crystal violet dye retention and confocal laser scanning microscopy (CLSM). Bulk measurements of cell hydrophobicity had little predictive ability in determining whether biofilms would develop on hydrophobic or hydrophilic substrata. Therefore selected pairs of bacteria from the genera Rhodococcus, Pseudomonas and Sphingomonas that exhibited different attachment phenotypes were examined in more detail using CLSM and the lipophilic dye, Nile Red. The association of Rhodococcus sp. cell membranes with lipids was shown to influence the attachment properties of these cells, but this approach was not informative for Pseudomonas and Sphingomonas sp. Confocal Raman Microspectroscopy of Rhodococcus biofilms confirmed the importance of lipids in their formation and indicated that in Pseudomonas and Sphingomonas biofilms, nucleic acids and proteins, respectively, were important in identifying the differences in attachment phenotypes of the selected strains. Treatment of biofilms with DNase I confirmed a determining role for nucleic acids as predicted for Pseudomonas. This work demonstrates that the attachment phenotypes of microbes from environmental samples to different substrata varies markedly, a diverse range of macromolecules may be involved and that these differ significantly between genera. A combination of CLSM and Raman spectroscopy distinguished between phenotypes and could be used to identify the key macromolecules involved in cell attachment to surfaces for the specific cases studied.     

Retention of bacteria on a substratum surface with micro-patterned hydrophobicity

Bacteria adhere to almost any surface, despite continuing arguments about the importance of physico-chemical properties of substratum surfaces, such as hydrophobicity and charge in biofilm formation. Nevertheless, in vivo biofilm formation on teeth and also on voice prostheses in laryngectomized patients is less on hydrophobic than on hydrophilic surfaces. With the aid of micro-patterned surfaces consisting of 10-microm wide hydrophobic lines separated by 20-microm wide hydrophilic spacings, we demonstrate here, for the first time in one and the same experiment, that bacteria do not have a strong preference for adhesion to hydrophobic or hydrophilic surfaces. Upon challenging the adhering bacteria, after deposition in a parallel plate flow chamber, with a high detachment force, however, bacteria were easily wiped-off hydrophobic lines, most notably when these lines were oriented parallel to the direction of flow. Adhering bacteria detached slightly less from the hydrophilic spacings in between, but preferentially accumulated adhering on the hydrophilic regions close to the interface between the hydrophilic spacings and hydrophobic lines. It is concluded that substratum hydrophobicity is a major determinant of bacterial retention while it hardly influences bacterial adhesion.     

Proteomic studies highlight outer‐membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer‐membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer‐membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB‐dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT‐PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.