New Device for High-Throughput Viability Screening of Flow Biofilms

Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.Bacterial biofilms are surface-attached communities that are encased in a polymeric matrix, which exhibit a high degree of resistance to antimicrobial agents and the host immune system (12, 16). This makes them medically important; diseases with a biofilm component are chronic and difficult to eradicate. Examples of such diseases are cystitis (1), endocarditis (31), cystic fibrosis (35), and middle-ear (17) and indwelling medical device-associated (20) infections. Biofilms also play important environmental roles in, for example, wastewater treatment (38), bioremediation (29, 30), biofouling (7), and biocorrosion (2). Better control of biofilms requires elucidation of the molecular basis of their superior resistance (by identifying resistance-compromised mutants) and identification of compounds with antibiofilm activity. While our understanding of these aspects of biofilms has increased (11, 15, 25-27, 36), further work, including development of accurate high-throughput (HTP) methods for screening biofilm viability, is needed.Two major biofilm models are studied in the laboratory, biofilms grown without a continuous flow of fresh medium and biofilms grown with a continuous flow of fresh medium; examples of these two models are microtiter well biofilms and flow cell biofilms, respectively. Methods have been developed for HTP screening of the viability of static biofilms (6, 28, 32, 33), but there are no methods for HTP screening of flow biofilms. The latter biofilms are typically grown in flow cells, which have to be examined individually to determine viability and thus cannot be used for rapid screening. An HTP screening method for flow biofilms is desirable, as these biofilms more closely approximate natural biofilms and can differ from static biofilms evidently due to hydrodynamic influences on cell signaling (22, 34). For example, the ability of rpoS-deficient Escherichia coli (lacking σ^S) to form flow biofilms is impaired, but its capacity to form biofilms under static conditions is enhanced (18).We describe here a new application of a recently developed device (8-10, 13), the “BioFlux” device consisting of microfluidic channels for biofilm growth. Other microfluidic devices have recently been used for biofilm formation (14, 19, 21, 23), but none of them has been used for HTP screening. The BioFlux device permits rapid measurement of the fluorescence of flow biofilms with a plate reader, which permits initial HTP screening of the viability of such biofilms.     

Increased Antibiotic Resistance of Escherichia coli in Mature Biofilms

Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 μg/ml), kanamycin (25 μg/ml), and ofloxacin (10 μg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.Reduced susceptibility of biofilm bacteria to antimicrobial agents is a crucial problem for treatment of chronic infections (11, 29, 48). It has been estimated that 65% of microbial infections are associated with biofilms (11, 29, 37), and biofilm cells are 100 to 1,000 times more resistant to antimicrobial agents than planktonic bacterial cells (11, 29, 32).The molecular nature of this apparent resistance has not been elucidated well, and a number of mechanisms have been proposed to explain the reduced susceptibility, such as restricted antibiotic penetration (47), decreased growth rates and metabolism (7, 52), quorum sensing and induction of a biofilm-specific phenotype (8, 29, 35, 39, 49), stress response activation (7, 52), and an increase in expression of efflux pumps (14). Biofilm resistance has generally been assumed to be due to the fact that the cells in the deeper layers of thick biofilms, which grow more slowly, have less access to antibiotics and nutrients. However, this is not the only reason in many cases. Familiar mechanisms of antibiotic resistance, such as modifying enzymes and target mutations, do not seem to be responsible for the biofilm resistance. Even sensitive bacteria that do not have a known genetic basis for resistance can exhibit profoundly reduced susceptibility when they form biofilms (48).It was reported previously that changes in gene expression induced a biofilm-specific phenotype (5, 13, 22, 35, 41, 42). Several genes have been proposed to be particularly important for biofilm formation, and the importance of the rpoS gene in Escherichia coli biofilm formation was suggested recently (1, 10, 22, 42). It has been suggested that induction of an rpoS-mediated stress response results in physiological changes that could contribute to antibiotic resistance (29). Although several mechanisms and genes have been proposed to explain biofilm resistance to antibiotics, this resistance is not still fully understood because these mechanisms seem to work together within a biofilm community. In addition, the physiology of biofilm cells is remarkably heterogeneous and varies according to the location of individual cells within biofilms (33, 34, 46).In this study, susceptibility of E. coli cells in biofilms to antibiotics was investigated. The E. coli cells in the deeper layers of mature biofilms were directly treated with three antibiotics with different molecular targets, the β-lactam ampicillin, the aminoglycoside kanamycin, and the fluoroquinolone ofloxacin. The biofilm biomass was removed before antibiotic treatment, and only the cells located in the deeper layers of the mature biofilms were directly exposed to antibiotics; thus, the effects of restricted antibiotic and nutrient penetration, as well as heterogeneous physiological states in biofilms, were reduced. Although ofloxacin and kanamycin effectively killed the biofilm cells, ampicillin could not kill the cells, which led to regrowth of biofilms. However, the cells in young colony biofilms were completely killed by ampicillin. Therefore, to determine which genes are induced in the mature biofilm cells, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. Based on the experimental data obtained, possible mechanisms of the increased biofilm resistance to ampicillin are discussed below.     

Application of Paramagnetically Tagged Molecules for Magnetic Resonance Imaging of Biofilm Mass Transport Processes

Molecules become readily visible by magnetic resonance imaging (MRI) when labeled with a paramagnetic tag. Consequently, MRI can be used to image their transport through porous media. In this study, we demonstrated that this method could be applied to image mass transport processes in biofilms. The transport of a complex of gadolinium and diethylenetriamine pentaacetic acid (Gd-DTPA), a commercially available paramagnetic molecule, was imaged both in agar (as a homogeneous test system) and in a phototrophic biofilm. The images collected were T₁ weighted, where T₁ is an MRI property of the biofilm and is dependent on Gd-DTPA concentration. A calibration protocol was applied to convert T₁ parameter maps into concentration maps, thus revealing the spatially resolved concentrations of this tracer at different time intervals. Comparing the data obtained from the agar experiment with data from a one-dimensional diffusion model revealed that transport of Gd-DTPA in agar was purely via diffusion, with a diffusion coefficient of 7.2 × 10⁻¹⁰ m² s⁻¹. In contrast, comparison of data from the phototrophic biofilm experiment with data from a two-dimensional diffusion model revealed that transport of Gd-DTPA inside the biofilm was by both diffusion and advection, equivalent to a diffusion coefficient of 1.04 × 10⁻⁹ m² s⁻¹. This technology can be used to further explore mass transport processes in biofilms, either by using the wide range of commercially available paramagnetically tagged molecules and nanoparticles or by using bespoke tagged molecules.Biofilms are utilized in a wide range of biotechnological processes, such as cleansing municipal and industrial wastewater, bioremediation of hazardous waste sites, biofuel production, and the generation of electricity in microbial fuel cells (20, 31, 35). They also play an important role in mediating the geochemistry of the natural environment (35). Critically, our growing understanding of the biology, physics, and chemistry of biofilms is allowing us to manipulate biofilms and enhance their performance in a variety of biotechnologies (33). The optimization of biofilm processes is, however, hindered when a lack of quantitative measurements of critical biofilm parameters exists.For the biofilm to function, the relevant substrates must be transported through the biofilm matrix, where they are metabolized. The rate at which these metabolites are transported through the biofilm can be critical in controlling the performance of the biofilm (5, 8, 13, 31). Equally, the rate at which the biofilm can sequester nonmetabolizable pollutants, such as nonmetabolizable heavy metals and recalcitrant organics, is also mediated by the transport rate (9, 28). Previous studies of mass transport inside biofilms show that transport occurs not only by diffusion but also by advection if the biofilm contains interconnected channels (5, 9, 13, 19, 39, 40, 45). When transported by diffusion, the mass of the diffusing solute plays a key role in mediating the transport rate. That is, the higher the molecular mass of the solute, the lower its diffusion coefficient (7, 39). Moreover, the molecular masses and diffusion rates of these solutes vary considerably, ranging from low-mass, fast-diffusing metabolites, such as H₂ and O₂, to large, slowly diffusing organic macromolecules tens to hundreds of kDa in size. Indeed, high-molecular-mass molecules and nanoparticles are an important part of the substrate and pollutant load in both wastewater treatment and natural aquatic systems (21). At a certain size, large macromolecules and nanoparticles become too large to diffuse into the dense extracellular polymeric substance (EPS) matrix, although they still can be transported deep into the biofilm along open channels (9, 39).Moreover, due to the heterogeneous nature of biofilms, substrates can also display significant spatial variation in mass transport rates, such as a decrease in transport rate with biofilm depth (4). As attempts to understand biofilm function or enhance biofilm performance are dependent upon accurate mass transport data sets, quantifying the transport behaviors of different-molecular-mass molecules in different biofilms is key to allowing us to model real biofilm systems more accurately.Recognizing the importance of mass transport, researchers have already used a variety of methods, such as microelectrodes, confocal laser scanning microscopy (CLSM), fluorescence recovery after photobleaching (FRAP), and two-photon excitation microscopy to obtain mass transport data from biofilms (7, 11, 12). These approaches have provided invaluable data on mass transport within biofilms. However, as with any method, each has certain limitations. For example, microelectrodes are used to measure the mass transport of low-molecular-mass molecules; particulates and high-molecular-mass molecules are undetectable by this method. Moreover, the insertion of a probe is invasive and thus has potential to disrupt the surrounding material, altering results. This could be problematic when numerous insertions must be made, such as during spatial mapping of diffusion coefficients in heterogeneous biofilms. Conversely, CLSM is noninvasive. However, small molecules such as H₂ or O₂ cannot be labeled with the fluorescent probe, and thus only the transport of higher-molecular-weight compounds can be determined. This method, which relies on photons penetrating the biofilm, is limited both to biofilm thickness (<100 μm) and to its density due to optical scattering effects (26, 43). Although the two-photon excitation method can overcome the depth penetration limitation of CLSM by approximately four times (26), it is not suitable where biofilms exceed these thicknesses. FRAP also suffers similar thickness limitations and light-scattering effects. However, the capacity of magnetic resonance imaging (MRI) for completely noninvasive measurement of the transport of both low- and high-molecular-mass compounds and its ability to image inside hydrated biological matrices (1, 30), no matter what thickness, means that it has significant potential for mass transport analysis of biofilms and can thus be an invaluable additional tool in this research field.Researchers have already used MRI to examine flow dynamics over biofilm surfaces (22, 37), metabolite consumption and production (23), the flux of heavy metals in metal-immobilizing bioreactors (15, 25), water diffusion in biofilms (28, 44), and the transport and fate of metals both in natural and artificial biofilms (28, 29) and in real methanogenic granules which are employed in anaerobic wastewater treatment (2).     

Antibody-Mediated Immobilization of Cryptococcus neoformans Promotes Biofilm Formation

Most microbes, including the fungal pathogen Cryptococcus neoformans, can grow as biofilms. Biofilms confer upon microbes a range of characteristics, including an ability to colonize materials such as shunts and catheters and increased resistance to antibiotics. Here, we provide evidence that coating surfaces with a monoclonal antibody to glucuronoxylomannan, the major component of the fungal capsular polysaccharide, immobilizes cryptococcal cells to a surface support and, subsequently, promotes biofilm formation. We used time-lapse microscopy to visualize the growth of cryptococcal biofilms, generating the first movies of fungal biofilm growth. We show that when fungal cells are immobilized using surface-attached specific antibody to the capsule, the initial stages of biofilm formation are significantly faster than those on surfaces with no antibody coating or surfaces coated with unspecific monoclonal antibody. Time-lapse microscopy revealed that biofilm growth was a dynamic process in which cells shuffled position during budding and was accompanied by emergence of planktonic variant cells that left the attached biofilm community. The planktonic variant cells exhibited mobility, presumably by Brownian motion. Our results indicate that microbial immobilization by antibody capture hastens biofilm formation and suggest that antibody coating of medical devices with immunoglobulins must exclude binding to common pathogenic microbes and the possibility that this effect could be exploited in industrial microbiology.Cryptococcus neoformans is a fungal pathogen that is ubiquitous in the environment and enters the body via the inhalation of airborne particles. The C. neoformans cell is surrounded by a layer of polysaccharide that consists predominantly of glucuronoxylomannan (GXM), which forms a protective capsule around the microbe. The capsule has been shown to be essential for virulence in murine models of infection (5-7) and, thus, is considered a key virulence factor. C. neoformans is the causative agent of cryptococcosis, a disease that primarily affects individuals with impaired immune systems, and is a significant problem in AIDS patients (21, 31). The most common manifestation of cryptococcosis is meningoencephalitis.Biofilms are communities of microbes that are attached to surfaces and held together by an extracellular matrix, often consisting predominantly of polysaccharides (8, 10). A great deal is known about bacterial biofilms (3, 9, 24, 30), but fungal biofilm formation is much less studied. Candida albicans is known to synthesize biofilms (11, 28, 29), as is C. neoformans. Biofilm-like structures consisting of innumerable cryptococcal cells encased in a polysaccharide matrix have been reported in human cases of cryptococcosis (32). Biofilm formation confers upon the microbe the capacity for drug resistance, and microbial cells in biofilms are less susceptible to host defense mechanisms (2, 4, 9, 12). In this regard, cells within C. neoformans biofilms are significantly less susceptible to caspofungin and amphotericin B than are planktonic cells (19). The cells within the biofilm are also resistant to the actions of fluconazole and voriconazole and various microbial oxidants and peptides (17, 19).Bacterial and fungal biofilms form readily on prosthetic materials, which poses a tremendous risk of chronic infection (10, 13, 15, 27). C. neoformans biofilms can form on a range of surfaces, including glass, polystyrene, and polyvinyl, and material devices, such as catheters (16). C. neoformans can form biofilms on the ventriculoatrial shunts used to decompress intracerebral pressure in patients with cryptococcal meningoencephalitis (32).The polysaccharide capsule of C. neoformans is essential for biofilm formation (18), and biofilm formation involves the shedding and accumulation of large amounts of GXM into the biofilm extracellular matrix (16). Previously, we reported that antibody to GXM in solution could inhibit biofilm formation through a process that presumably involves interference with polysaccharide shedding (18, 20). However, the effect of antibody-mediated immobilization of C. neoformans cells on cryptococcal biofilm formation has not been explored. In this paper, we report that the monoclonal antibody (MAb) 18B7, which is specific for the capsular polysaccharide GXM, can capture and immobilize C. neoformans to surfaces, a process that promotes biofilm formation. Interestingly, we identified planktonic variant C. neoformans cells that appeared to escape from the biofilm, but whose functions are not known. The results provide new insights on biofilm formation.     

Poly-N-Acetylglucosamine Matrix Polysaccharide Impedes Fluid Convection and Transport of the Cationic Surfactant Cetylpyridinium Chloride through Bacterial Biofilms

Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix. Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG) is a major biofilm matrix component in phylogenetically diverse bacteria. In this study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced in vitro by the gram-negative porcine respiratory pathogen Actinobacillus pleuropneumoniae and the gram-positive device-associated pathogen Staphylococcus epidermidis. The effect of PNAG on bulk fluid flow was determined by measuring the rate of fluid convection through biofilms cultured in centrifugal filter devices. The rate of fluid convection was significantly higher in biofilms cultured in the presence of the PNAG-degrading enzyme dispersin B than in biofilms cultured without the enzyme, indicating that PNAG decreases bulk fluid flow. PNAG also blocked transport of the quaternary ammonium compound cetylpyridinium chloride (CPC) through the biofilms. Binding of CPC to biofilms further impeded fluid convection and blocked transport of the azo dye Allura red. Bioactive CPC was efficiently eluted from biofilms by treatment with 1 M sodium chloride. Taken together, these findings suggest that CPC reacts directly with the PNAG matrix and alters its physical and chemical properties. Our results indicate that PNAG plays an important role in controlling the physiological state of biofilms and may contribute to additional biofilm-associated processes such as biocide resistance.Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix (7). The main function of the biofilm matrix is to provide a structural framework that holds the cells together in a mass and firmly attaches the bacterial mass to the underlying surface. In addition to having a structural role, the matrix provides biofilm cells with a protected microenvironment containing dissolved nutrients, secreted enzymes, DNA, and phages. The matrix may also contribute to the increased antimicrobial resistance exhibited by biofilm cells, either by providing a diffusion barrier or by directly binding to antimicrobial agents and preventing their penetration into the biofilm (19).Polysaccharides are a major matrix component in most bacterial biofilms (26). Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm cohesion in numerous gram-negative members of the Proteobacteria family, including Escherichia coli, Yersinia pestis, Pseudomonas fluorescens, Bordetella spp., Xenorhabdus nematophila, Aggregatibacter actinomycetemcomitans, and Actinobacillus pleuropneumoniae (4, 8, 15, 22), and in the gram-positive species Staphylococcus aureus and Staphylococcus epidermidis (3, 17). Specific biofilm-related functions ascribed to PNAG include abiotic surface attachment (1), epithelial cell attachment (23, 28), intercellular adhesion (15, 17), and resistance to killing by antibiotics, detergents, antimicrobial peptides, and mammalian phagocytic cells (9, 10, 16, 27, 29).In the present study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced by the porcine respiratory pathogen A. pleuropneumoniae and the device-associated pathogen S. epidermidis. By using a novel centrifugal filter device assay, we obtained evidence that PNAG significantly inhibits fluid convection and solute transport through A. pleuropneumoniae and S. epidermidis biofilms.     

Role of Extracellular DNA during Biofilm Formation by Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen that is capable of living in harsh environments. It is believed to do this by forming biofilms, which are surface-associated multicellular structures encased in a self-produced matrix. In this paper we show that in L. monocytogenes extracellular DNA (eDNA) may be the only central component of the biofilm matrix and that it is necessary for both initial attachment and early biofilm formation for 41 L. monocytogenes strains that were tested. DNase I treatment resulted in dispersal of biofilms, not only in microtiter tray assays but also in flow cell biofilm assays. However, it was also demonstrated that in a culture without eDNA, neither Listeria genomic DNA nor salmon sperm DNA by itself could restore the capacity to adhere. A search for additional necessary components revealed that peptidoglycan (PG), specifically N-acetylglucosamine (NAG), interacted with the DNA in a manner which restored adhesion. If a short DNA fragment (less than approximately 500 bp long) was added to an eDNA-free culture prior to addition of genomic or salmon sperm DNA, adhesion was prevented, indicating that high-molecular-weight DNA is required for adhesion and that the number of attachment sites on the cell surface can be saturated.The food-borne pathogen Listeria monocytogenes is known to persist in food processing plants (28, 48), and it has been reported that some strains of this species are capable of forming biofilms (2, 16). The mechanisms of biofilm formation have not been elucidated, but this process seems to depend on factors such as temperature and inducing compounds (14). One inducing compound is NaCl (22), but ethanol, isopropanol (14), quorum sensing (36), and an increasing temperature (8, 14, 38) also seem to enhance attachment and biofilm formation, whereas an acidic pH reduces adhesion (17, 38, 43). Furthermore, at 30°C flagellum-based motility seems to be a specific determinant for the initial adhesion (23, 42) and biofilm formation (23); however, it has recently been reported that in time nonflagellated mutants can produce hyperbiofilms (42).Since bacteria adhering to surfaces, both in biofilms and as single cells, exhibit increased resistance to sanitizers and antimicrobial agents (10, 41), examining the essential steps in adhesion and biofilm formation is important in order to develop new and improved sanitation processes.Extracellular DNA (eDNA) is a ubiquitous component of the organic matter pool in soil, marine, and freshwater habitats (26), but it is also found in environments as diverse as tissue cultures and the blood of mammals (11, 25). The presence of eDNA in the matrix of multicellular structures has recently been reported to influence the initial attachment and/or biofilm structure of Pseudomonas (1, 47), Streptococcus (29), and Staphylococcus (21, 33, 34) species.The prevalence of eDNA in nature appears to be associated with both lysis of cells and active secretion. The concentrations of eDNA released can be up to 2 μg g⁻¹ soil (30) and up to 0.5 g (m²)⁻¹ in the top few centimeters of deep-sea sediment (where more than 90% of the DNA is extracellular) (5). In the deep sea eDNA plays a key role in the ecosystem, functioning as a nitrogen and phosphorus reservoir (5). At present, there are different theories concerning both the function and the release of eDNA in multicellular structures. The presence of eDNA could be a result of either cell lysis (33, 34) or vesicle release (47), whereas active transport is a more speculative explanation. The role of eDNA in biofilm structure has not been revealed yet, but various functions, including a role as a structural component, an energy and nutrition source, or a gene pool for horizontal gene transfer (HGT) in naturally competent bacteria, can be envisaged.Until now there have been no studies of L. monocytogenes eDNA as a possible matrix component in relation to adhesion and biofilm development. In this study, we determined for the first time the presence of L. monocytogenes eDNA, its origin, and its role as a matrix component for both single-cell adhesion and biofilm formation using static assays, as well as flow cell systems. Furthermore, we showed that an additional component is necessary for eDNA-mediated adhesion.     

Flagellated but Not Hyperfimbriated Salmonella enterica Serovar Typhimurium Attaches to and Forms Biofilms on Cholesterol-Coated Surfaces

The asymptomatic, chronic carrier state of Salmonella enterica serovar Typhi occurs in the bile-rich gallbladder and is frequently associated with the presence of cholesterol gallstones. We have previously demonstrated that salmonellae form biofilms on human gallstones and cholesterol-coated surfaces in vitro and that bile-induced biofilm formation on cholesterol gallstones promotes gallbladder colonization and maintenance of the carrier state. Random transposon mutants of S. enterica serovar Typhimurium were screened for impaired adherence to and biofilm formation on cholesterol-coated Eppendorf tubes but not on glass and plastic surfaces. We identified 49 mutants with this phenotype. The results indicate that genes involved in flagellum biosynthesis and structure primarily mediated attachment to cholesterol. Subsequent analysis suggested that the presence of the flagellar filament enhanced binding and biofilm formation in the presence of bile, while flagellar motility and expression of type 1 fimbriae were unimportant. Purified Salmonella flagellar proteins used in a modified enzyme-linked immunosorbent assay (ELISA) showed that FliC was the critical subunit mediating binding to cholesterol. These studies provide a better understanding of early events during biofilm development, specifically how salmonellae bind to cholesterol, and suggest a target for therapies that may alleviate biofilm formation on cholesterol gallstones and the chronic carrier state.The serovars of Salmonella enterica are diverse, infect a broad array of hosts, and cause significant morbidity and mortality in impoverished and industrialized nations worldwide. S. enterica serovar Typhi is the etiologic agent of typhoid fever, a severe illness characterized by sustained bacteremia and a delayed onset of symptoms that afflicts approximately 20 million people each year (14, 19). Serovar Typhi can establish a chronic infection of the human gallbladder, suggesting that this bacterium utilizes novel mechanisms to mediate enhanced colonization and persistence in a bile-rich environment.There is a strong correlation between gallbladder abnormalities, particularly gallstones, and development of the asymptomatic Salmonella carrier state (47). Antibiotic regimens are typically ineffective in carriers with gallstones (47), and these patients have an 8.47-fold-higher risk of developing hepatobiliary carcinomas (28, 46, 91). Elimination of chronic infections usually requires gallbladder removal (47), but surgical intervention is cost-prohibitive in developing countries where serovar Typhi is prevalent. Thus, understanding the progression of infection to the carrier state and developing alternative treatment options are of critical importance to human health.The formation of biofilms on gallstones has been hypothesized to facilitate enhanced colonization of and persistence in the gallbladder. Over the past 2 decades, bacterial biofilms have been increasingly implicated as burdens for food and public safety worldwide, and they are broadly defined as heterogeneous communities of microorganisms that adhere to each other and to inert or live surfaces (17, 22, 67, 89, 102). A sessile environment provides selective advantages in natural, medical, and industrial ecosystems for diverse species of commensal and pathogenic bacteria, including Streptococcus mutans (40, 92, 104), Staphylococcus aureus (15, 35, 100), Escherichia coli (21, 74), Vibrio cholerae (39, 52, 107), and Pseudomonas aeruginosa (23, 58, 73, 105). Bacterial biofilms are increasingly associated with many chronic infections in humans and exhibit heightened resistance to commonly administered antibiotics and to engulfment by professional phagocytes (54, 55, 59). The bacterial gene expression profiles for planktonic and biofilm phenotypes differ (42, 90), and the changes are likely regulated by external stimuli, including nutrient availability, the presence of antimicrobials, and the composition of the binding substrate.Biofilm formation occurs in sequential, highly ordered stages and begins with attachment of free-swimming, planktonic bacteria to a surface. Subsequent biofilm maturation is characterized by the production of a self-initiated extracellular matrix (ECM) composed of nucleic acid, proteins, or exopolysaccharides (EPS) that encase the community of microorganisms. Planktonic cells are continuously shed from the sessile, matrix-bound population, which can result in reattachment and fortification of the biofilm or systemic infection and release of the organism into the environment. Shedding of serovar Typhi by asymptomatic carriers can contaminate food and water and account for much of the person-to-person transmission in underdeveloped countries.Our laboratory has previously reported that bile is required for formation of mature biofilms with characteristic EPS production by S. enterica serovars Typhimurium, Enteritidis, and Typhi on human gallstones and cholesterol-coated Eppendorf tubes (18, 78). Cholesterol is the primary constituent of human cholesterol gallstones, and use of cholesterol-coated tubes creates an in vitro uniform surface that mimics human gallstones (18). It was also demonstrated that Salmonella biofilms that formed on different surfaces had unique phenotypes and required expression of specific EPS (18, 77), yet the factors mediating Salmonella binding to gallstones and cholesterol-coated surfaces during the initiation of biofilm formation remain unknown. Here, we show that the presence of serovar Typhimurium flagella promotes binding specifically to cholesterol in the early stages of biofilm development and that the FliC subunit is a critical component. Bound salmonellae expressing intact flagella provided a scaffold for other cells to bind to during later stages of biofilm growth. Elucidation of key mechanisms that mediate adherence to cholesterol during Salmonella bile-induced biofilm formation on gallstone surfaces promises to reveal novel drug targets for alleviating biofilm formation in chronic cases.     

Evolution of Diversity in Spatially Structured Escherichia coli Populations

The stochastic Ricker population model was used to investigate the generation and maintenance of genetic diversity in a bacterial population grown in a spatially structured environment. In particular, we showed that Escherichia coli undergoes dramatic genetic diversification when grown as a biofilm. Using a novel biofilm entrapment method, we retrieved 64 clones from each of six different depths of a mature biofilm, and after subculturing for ∼30 generations, we measured their growth kinetics in three different media. We fit a stochastic Ricker population growth model to the recorded growth curves. The growth kinetics of clonal lineages descendant from cells sampled at different biofilm depths varied as a function of both the depth in the biofilm and the growth medium used. We concluded that differences in the growth dynamics of clones were heritable and arose during adaptive evolution under local conditions in a spatially heterogeneous environment. We postulate that under nutrient-limited conditions, selective sweeps would be protracted and would be insufficient to purge less-fit variants, a phenomenon that would allow the coexistence of genetically distinct clones. These findings contribute to the current understanding of biofilm ecology and complement current hypotheses for the maintenance and generation of microbial diversity in spatially structured environments.The mechanisms that lead to the genesis and maintenance of diversity in communities have intrigued geneticists and ecologists alike for decades (6, 17, 27, 33, 39, 49). This is particularly challenging for microbial communities, in which ecological and evolutionary processes occur on roughly the same time scale (3, 16, 38) and where the outcome of these processes may be affected by the spatial structure in which these communities grow.Bacterial biofilms are examples of spatially structured communities that have been the subject of intense research in medical and engineering contexts in recent years (3, 8, 20, 48, 56). Previous work has shown that the phenotypic characteristics of bacterial populations in biofilms are distinct from those of their free-swimming counterparts (8). These bacterial assemblages form physically and chemically heterogeneous structures (20) whose complex architecture strongly influences mass transfer (56). This results in the formation of steep gradients of nutrients, waste products, pH, redox potential, and electron acceptors, which results in the creation of distinct and perhaps unique niches on a microscale. This places selective pressure on variants that have enhanced fitness and are well adapted to local conditions. From a theoretical perspective, this would be expected to increase genetic diversity within a population by precluding competitive exclusion, yet this has not previously been demonstrated empirically.The degree of diversification that occurs within populations growing in biofilms is not well understood, nor are the spatial and temporal dynamics of bacterial species succession in biofilms. However, it is known that the physical and chemical heterogeneity of microbial biofilms has profound effects on microbial growth and activity. Most bacterial cells in biofilms are not highly active and grow slowly if at all. For example, active protein synthesis occurs only in the uppermost zone (32 ± 3 μm) of Pseudomonas aeruginosa biofilms (4). Likewise, in Klebsiella pneumoniae biofilms, fast growth occurs near the interface of the biofilm and bulk fluid, and cells inside the biofilm show little growth (55). The near absence of growth in interior regions of biofilms may lead to an increased tempo of diversification, since numerous studies have shown that mutation frequencies are elevated in slowly growing cells (28). If this occurs within a biofilm, then clones might exhibit a high genotypic variability that could have significant practical implications in terms of yielding spontaneous mutants that are resistant to antimicrobial agents.Experimental evolution has contributed greatly to our understanding of the causes and consequences of genetic diversity in populations (reviewed in references 23, 29, and 42). Initially, research focused on characterizing diversity within populations that evolved in spatially homogenous environments (e.g., chemostat and batch systems) (13, 15, 19, 30-32, 45, 47, 50-53). Several studies have highlighted a role for spatial heterogeneity in the emergence and maintenance of genetic diversity (25, 26, 43). Korona and colleagues (25, 26) compared populations that evolved in batch cultures to populations that evolved with a spatial structure and demonstrated that phenotypic diversity was greatest with spatial structure. In other work, Rainey and Travisano (43) showed that populations of Pseudomonas grown in static broth microcosms diversified so that some ecotypes occupied a floating biofilm on the surface of the broth while others occupied the liquid phase or glass surface of the culture. Boles et al. (2, 3) investigated the extent of diversification of Pseudomonas using biofilms that evolved in flow-cell systems. They reported that genetic changes produced by a recA-dependent mechanism affected multiple traits, with some biofilm-derived variants being better able to disseminate while others were better able to form biofilms (3). Further study showed that in some cells, endogenous oxidative stress caused double-stranded DNA breaks that when repaired by recombinatorial DNA repair genes gave rise to mutations (2). These previous studies demonstrate the pivotal role of spatial structure in the generation and maintenance of diversity in evolving bacterial populations.In this study, we extended this work by using novel techniques to characterize diversity in Escherichia coli biofilms that allowed us to recover clones from specific depths within a biofilm. The growth kinetics of clones from six different biofilm depths were measured and modeled using an analysis-of-variance formulation of the stochastic Ricker model of population dynamics with environmental noise (11, 40). Rigorous statistical methods were used to show that after 1 month of cultivation, the extant diversity in E. coli biofilms was extraordinarily high and varied with depth.     

Organoselenium Coating on Cellulose Inhibits the Formation of Biofilms by Pseudomonas aeruginosa and Staphylococcus aureus

Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims, patients with traumatic wounds, necrotic lesions in people with diabetes, and patients with surgical wounds. Within a wound, infecting bacteria frequently develop biofilms. Many current wound dressings are impregnated with antimicrobial agents, such as silver or antibiotics. Diffusion of the agent(s) from the dressing may damage or destroy nearby healthy tissue as well as compromise the effectiveness of the dressing. In contrast, the antimicrobial agent selenium can be covalently attached to the surfaces of a dressing, prolonging its effectiveness. We examined the effectiveness of an organoselenium coating on cellulose discs in inhibiting Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation. Colony biofilm assays revealed that cellulose discs coated with organoselenium completely inhibited P. aeruginosa and S. aureus biofilm formation. Scanning electron microscopy of the cellulose discs confirmed these results. Additionally, the coating on the cellulose discs was stable and effective after a week of incubation in phosphate-buffered saline. These results demonstrate that 0.2% selenium in a coating on cellulose discs effectively inhibits bacterial attachment and biofilm formation and that, unlike other antimicrobial agents, longer periods of exposure to an aqueous environment do not compromise the effectiveness of the coating.Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims (10), patients with traumatic wounds (33), people with diabetes (27), and patients with surgical wounds (29, 31). Two of the more common causative agents of wound infections are Staphylococcus aureus and Pseudomonas aeruginosa (10, 27, 29, 31, 33). Such infections often lead to fatality; the mortality rate among patients infected with P. aeruginosa ranges from 26% to 55% (9, 49), while mortality from S. aureus infection ranges from 19% to 38% (28, 46, 50). As opportunistic pathogens, S. aureus and P. aeruginosa cause few infections in healthy individuals but readily cause infection once host defenses are compromised, such as with the removal of skin from burns (10). S. aureus infection originates from the normal flora of either the patient or health care workers (48), while P. aeruginosa is acquired from the environment surrounding the patient (41). Once established on the skin, S. aureus and P. aeruginosa are then able to colonize the wound. Infection results if the organisms proliferate in the wound environment.Both P. aeruginosa and S. aureus often exist within burn wounds as biofilms (43, 47). A biofilm is presently defined as a sessile microbial community characterized by cells that are irreversibly attached either to a substratum or to each other (16). Biofilms, which can attain over 100 μm in thickness, are made up of multiple layers of bacteria in an exopolysaccharide matrix (12, 16, 42). Sauer et al. showed that P. aeruginosa biofilms form in distinct developmental stages: reversible attachment, irreversible attachment, two stages of maturation, and a dispersion phase (42). Clinically, biofilms present serious medical management problems through their association with different chronic infections (37). During vascular catheter-related infections and sepsis, biofilms serve as a reservoir of bacteria from which planktonic cells detach and spread throughout the tissue and/or enter the circulatory system, resulting in bacteremia or septicemia (15). Factors specific to the bacterium may influence the formation of bacterial biofilms at different infection sites or surfaces. For example, during the initial attachment stage the flagellum, lipopolysaccharide, and possibly outer membrane proteins play a major role in bringing P. aeruginosa into proximity with the surface as well as mediating the interaction with the substratum (12). Using the murine model of thermal injury, we recently showed that P. aeruginosa forms a biofilm within the thermally injured tissues (43). Clinically, the surgeons debride the infected or dead tissues; however, a few microorganisms may remain on the tissue surface and reinitiate biofilm formation.Antibiotics, silver, or chitosan, attached to or embedded in gauze, have been shown to be efficacious in preventing wound infection (21, 24, 26, 36). However, the resistance of P. aeruginosa and S. aureus to available antibiotics severely limits the choices for antibiotic treatment (13, 40). Additionally, silver compounds, such as silver nitrate and silver sulfadiazine, leaching from dressings are toxic to human fibroblasts even at low concentrations (20, 25). Thus, effective alternative antimicrobial agents that contact the thermally injured/infected tissues and prevent the development of bacterial biofilms are required. Previous studies have shown that selenium (Se) can be covalently bound to a solid matrix and retain its ability to catalyze the formation of superoxide radicals (O₂^·−) (30). These superoxide radicals inhibit bacterial attachment to the solid surface (30). In this study, we examined the ability of a newly synthesized organoselenium-methacrylate polymer (Se-MAP) to block biofilm formation by both S. aureus and P. aeruginosa. These bacteria were chosen since they cause a major share of wound infections and because drug-resistant forms of these bacteria have become a serious problem in the treatment and management of these wound infections (6, 13, 17, 18, 38). Results of the study show that 0.2% (wt/wt) Se in Se-MAP covalently attached to cellulose discs inhibited P. aeruginosa and S. aureus biofilm formation. This could lead to the development of a selenium-based antimicrobial coating for cotton materials that will prevent the bacterial attachment and colonization that can ultimately lead to bacterial biofilm formation during chronic infections.     

Evaluation of Different Methods for Extracting Extracellular DNA from the Biofilm Matrix

The occurrence of high concentrations of extracellular DNA (eDNA) in the extracellular matrices of biofilms plays an important role in biofilm formation and development and possibly in horizontal gene transfer through natural transformation. Studies have been conducted to characterize the nature of eDNA and its potential function in biofilm development, but it is difficult to extract eDNA from the extracellular matrices of biofilms without any contamination from genomic DNA released by cell lysis during the extraction process. In this report, we compared several different extraction methods in order to obtain highly pure eDNA from different biofilm samples. After different extraction methods were explored, it was concluded that using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method. There was no detectable cell lysis when the enzymatic treatment methods were used, but substantial cell lysis was observed when the chemical treatment methods were used. These data suggest that eDNA may bind to other extracellular polymers in the biofilm matrix and that enzymatic treatment methods are effective and favorable for extracting eDNA from biofilm samples. Moreover, randomly amplified polymorphic DNA analysis of eDNA in Acinetobacter sp. biofilms and Acinetobacter sp. genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA.A biofilm is a well-organized community of microorganisms that adheres to surfaces and is embedded in the slimy extracellular polymeric substances (EPSs). EPSs are a complex mixture composed of high-molecular-mass polymers (>10,000 Da) generated by the bacterial cells, cell lysis and hydrolysis products, and organic matter adsorbed from the substrate. EPSs are involved in the establishment of stable arrangements of microorganisms in biofilms (40), and it recently was found that extracellular DNA (eDNA) is one of the major components of EPSs (7, 31). eDNA plays a very important role in biofilm development (39), and it is believed to be involved in providing substrates for sibling cells, maintaining the three-dimensional structure of biofilms, and enhancing the exchange of genetic materials (18, 31). eDNA has also been found to be accumulated in cultures of several bacterial species and has been postulated as being released by bacterial cells (11, 15, 21, 30). Although it is commonly accepted that eDNA is released mainly from cell lysis (11, 23, 24, 28, 34, 41), several studies have revealed that some other active secretion mechanisms may exist (1, 6, 11, 27). Recent evidence, however, indicates the possibility that eDNA is secreted actively via transport vesicles for the purpose of creating the biofilm matrix (39). Bockelmann et al. found that eDNA formed a defined, network-like spatial structure in the biofilm of an aquatic bacterium and identified that eDNA was not completely identical to genomic DNA by using randomly amplified polymorphic DNA (RAPD) and restriction endonuclease analyses (3). By using RAPD analysis, principal-components analysis, and terminal restriction fragment length polymorphism analysis, Steinberger and Holden (33) also characterized eDNA in single- and multiple-species unsaturated biofilm and found that it was different from genomic DNA. However, research is still needed to elucidate the role of eDNA in biofilm structures and in the development and origins of eDNA. In order to further investigate these questions, it is important to extract most of the eDNA of high purity in the biofilm matrix and separate eDNA from other components in the EPSs and from the genomic DNA released during the extraction process. Several methods, such as high-speed centrifugation (2, 33) and membrane filtration (3), have been used to isolate eDNA from biofilm samples. However, these methods may isolate only a portion of the eDNA from biofilm samples.EPSs are composed mainly of high-molecular-weight compounds, including polysaccharides, proteins, and amphiphilic polymers (19, 20), that are secreted by microorganisms into their environment (32). The majority of proteins in the EPSs are bridged by divalent ions, including Ca²⁺ and Mg²⁺, and a small fraction of carbohydrates and nucleic acids are linked to these divalent ions. Under neutral conditions, the carboxyl of protein would become ionized and negative. Through ion interaction, the divalent ions bridge the protein and the cells. In addition, eDNA may be physically or chemically associated with extracellular proteins, polysaccharides, and other polymers in the EPS matrix. The structural assemblage of proteins and polysaccharides in the complex matrix of the EPS might hinder the liberating eDNA from the EPS matrix. Therefore, it is difficult to release eDNA and other materials from the EPS matrix by only vortexing or homogenizing. Additionally, it is necessary to degrade certain components of EPSs in the biofilm matrix in order to release eDNAs that may bind to these compounds.In this study, the following extractants were chosen to treat biofilm samples for isolation of eDNA from Acinetobacter sp. strain AC811 biofilm: EDTA and cation-exchange resin (CER) (16), which both have the ability to remove cations from the EPS matrix; sodium dodecyl sulfate (SDS) and NaOH, which are strong denaturants and are used frequently for EPS extraction from various pure and mixed cultures (17, 29); and N-glycanase (glycoprotein degradation hydrolase) (35), dispersin B (biofilm-dispersing glycoside hydrolase) (25), and proteinase K (protein hydrolase). We evaluated the efficiencies of these treatments and their impacts on the quantity and quality of eDNA extracted, and we propose that eDNA may bind to other extracellular polymers in the Acinetobacter biofilm matrix, based on the release of eDNA from the biofilm matrix after such treatments.     

Competition of Listeria monocytogenes Serotype 1/2a and 4b Strains in Mixed-Culture Biofilms

The majority of Listeria monocytogenes isolates recovered from foods and the environment are strains of serogroup 1/2, especially serotypes 1/2a and 1/2b. However, serotype 4b strains cause the majority of human listeriosis outbreaks. Our investigation of L. monocytogenes biofilms used a simulated food-processing system that consisted of repeated cycles of growth, sanitation treatment, and starvation to determine the competitive fitness of strains of serotypes 1/2a and 4b in pure and mixed-culture biofilms. Selective enumeration of strains of a certain serotype in mixed-culture biofilms on stainless steel coupons was accomplished by using serotype-specific quantitative PCR and propidium monoazide treatment to prevent amplification of extracellular DNA or DNA from dead cells. The results showed that the serotype 1/2a strains tested were generally more efficient at forming biofilms and predominated in the mixed-culture biofilms. The growth and survival of strains of one serotype were not inhibited by strains of the other serotype in mixed-culture biofilms. However, we found that a cocktail of serotype 4b strains survived and grew significantly better in mixed-culture biofilms containing a specific strain of serotype 1/2a (strain SK1387), with final cell densities averaging 0.5 log₁₀ CFU/cm² higher than without the serotype 1/2a strain. The methodology used in this study contributed to our understanding of how environmental stresses and microbial competition influence the survival and growth of L. monocytogenes in pure and mixed-culture biofilms.A prominent food-borne pathogen, Listeria monocytogenes can cause severe infections in humans, primarily in high-risk populations, though the disease (listeriosis) is relatively rare (11, 30, 43). Outbreaks of listeriosis have resulted from the contamination of a variety of foods by L. monocytogenes, especially meat and dairy products (27). L. monocytogenes is ubiquitous in the environment, able to grow at refrigeration temperature, and tolerant of the low pHs (3 to 4) typical of acidified foods (28, 32, 44). The capacity to produce biofilms confers protection against stresses common in the food-processing environment (13, 33).Biofilms are characterized by dense clusters of bacterial cells embedded in extracellular polymeric substances which are secreted by cells to aid in adhesion to surfaces and to other cells (4, 5). Strains of L. monocytogenes have been known to persist for years in food-processing environments, presumably in biofilms. Of the 13 known serotypes of L. monocytogenes, three (1/2a, 1/2b, and 4b) account for >95% of the isolates from human illness (21). Serotype 1/2a accounts for >50% of the L. monocytogenes isolates recovered from foods and the environment, while most major outbreaks of human listeriosis have been caused by serotype 4b strains (1, 3, 14, 15, 17, 22, 29, 31, 41, 47, 49,). No correlation between L. monocytogenes strain fitness and serotype has been identified (16, 19). Some studies have reported that strains repeatedly isolated from food and environmental samples (defined as persistent strains) had a higher adherence capacity than strains that were sporadically isolated (2, 36), while this phenomenon was not observed by others (7). Serotype 4b strains exhibited a higher capacity for biofilm formation than did serotype 1/2a strains (36), whereas this was not observed by Di Bonaventura and colleagues (6). It has been suggested that serotype 1/2a strains could be more robust than serotype 4b strains in biofilm formation under a variety of environmental conditions. Furthermore, strains of these serotypes differ in terms of the medium that promotes biofilm formation. Biofilm formation by serotype 4b strains was higher in full-strength tryptic soy broth than in diluted medium, whereas the opposite was observed with serotype 1/2a strains, which produced more biofilm in diluted medium (12).There is limited information on microbial competition between strains of different serotypes in biofilms or on how the environmental stresses present in food-processing environments may affect the biofilm formation and survival of L. monocytogenes of different serotypes. In food-processing plants, the environmental stresses encountered by bacteria are more complex and variable than most laboratory systems used for microbial ecology and biofilm studies. A simulated food-processing (SFP) system has been developed to address this issue (38). The SFP system incorporates several stresses that may affect bacteria in biofilms in the food-processing environment, including exposure to sanitizing agents, dehydration, and starvation. When biofilms were subjected to the SFP regimen over a period of several weeks, the cell numbers of L. monocytogenes strains in the biofilms initially were reduced and then increased as the culture adapted (38). The development of resistance to sanitizing agents was specific to the biofilm-associated cells and was not apparent in the detached cells (38). This suggested that extracellular polymeric substances present in the biofilm matrix were responsible for the resistance to sanitizing agents. It was subsequently found that real-time PCR, in combination with propidium monoazide (PMA) treatment of samples prior to DNA isolation, was an effective method for enumerating viable cells in biofilms (37).The objective of this study was to determine if strains of serotype 1/2a or 4b have a selective advantage under stress conditions. We investigated and compared the initial attachment and biofilm formation capabilities of L. monocytogenes strains of these two serotypes and analyzed the survival and growth of bacteria of each serotype in mixed-serotype biofilms in the SFP system by using PMA with quantitative PCR.     

Effects of Trp- and Arg-Containing Antimicrobial-Peptide Structure on Inhibition of Escherichia coli Planktonic Growth and Biofilm Formation

Biofilms are sessile microbial communities that cause serious chronic infections with high morbidity and mortality. In order to develop more effective approaches for biofilm control, a series of linear cationic antimicrobial peptides (AMPs) with various arginine (Arg or R) and tryptophan (Trp or W) repeats [(RW)_n-NH₂, where n = 2, 3, or 4] were rigorously compared to correlate their structures with antimicrobial activities affecting the planktonic growth and biofilm formation of Escherichia coli. The chain length of AMPs appears to be important for inhibition of bacterial planktonic growth, since the hexameric and octameric peptides significantly inhibited E. coli growth, while tetrameric peptide did not cause noticeable inhibition. In addition, all AMPs except the tetrameric peptide significantly reduced E. coli biofilm surface coverage and the viability of biofilm cells, when added at inoculation. In addition to inhibition of biofilm formation, significant killing of biofilm cells was observed after a 3-hour treatment of preformed biofilms with hexameric peptide. Interestingly, treatment with the octameric peptide caused significant biofilm dispersion without apparent killing of biofilm cells that remained on the surface; e.g., the surface coverage was reduced by 91.5 ± 3.5% by 200 μM octameric peptide. The detached biofilm cells, however, were effectively killed by this peptide. Overall, these results suggest that hexameric and octameric peptides are potent inhibitors of both bacterial planktonic growth and biofilm formation, while the octameric peptide can also disperse existing biofilms and kill the detached cells. These results are helpful for designing novel biofilm inhibitors and developing more effective therapeutic methods.Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics (5). Native AMPs are part of the host defense in organisms ranging from bacteria to insects, plants, and animals (14). They are capable of eliminating a broad spectrum of microorganisms, including viruses, bacteria, and fungi (4, 14). Compared with widespread antibiotic resistance (38), resistance to AMPs is rare, possibly because AMPs directly target cell membranes that are essential to microbes (14, 29). In addition, no cross-resistance has been observed in clinic due to the diversity of peptide sequences (42). Thus, native and synthetic AMPs offer potential alternatives to antibiotics for treating drug-resistant infections (3, 26, 27).In mammalian innate immune systems, some AMPs are produced constitutively, while others are inducible within hours after detection of invading microorganisms (4, 13). Although the detailed mechanism of AMPs'' activities remains elusive (5), AMPs are known to disrupt cell membranes of microbes, interfere with metabolism, and/or target cytoplasmic components (41). Most known AMPs are cationic and amphiphilic (29). It is hypothesized that the initial interaction occurs via an electrostatic attraction between the AMP molecule and microbial membrane. Cationic AMPs can cover bacterial membranes, disrupt the membrane potential, create pores across the membrane, and consequently cause the leak of cell contents and cell death (27, 41). AMPs are relatively selective in targeting microbes rather than mammalian cells, most likely because of the fundamental differences between microbial and host membranes (41), e.g., a higher abundance of negatively charged phospholipids and an absence of cholesterol in microbial membranes.Known AMPs vary dramatically in sequence, size (from 12 to 50 amino acids), and structure (α-helices or β-sheets) (23). However, most AMPs have two types of side chains with relatively conservative sequences: positively charged basic residues, containing arginine (R), lysine (K), and/or histidine (H), that presumably mediate the interaction with the negatively charged microbial membrane, and bulky hydrophobic residues, rich in tryptophan (W), proline (P), and/or phenylalanine (F), that facilitate permeabilization and membrane disruption (26).Although AMPs are promising agents for antimicrobial therapies (15), only a few have made it to clinical trials and applications, with varied success (15, 42). There are several issues that need further development. First, the MICs of AMPs are relatively high compared to those of conventional antibiotics. Recent studies suggest that the peptide/lipid (P/L) ratio needs to be higher than a threshold to allow the AMPs to be oriented perpendicular to the membrane so that pores can be created to kill bacteria (22, 30). Thus, an optimization of peptide structure and size may improve their antimicrobial activities. In addition to the high MICs, the wide application of AMPs is also hindered by their high manufacturing costs and the cytotoxicity of some AMPs.Given the limit of currently available AMPs, it is important to develop more effective AMPs with reduced manufacturing cost and enhanced activity (17, 26, 28, 39). Strøm et al. (39) chemically synthesized a series of short cationic AMPs containing repeating R and W residues in order to identify the minimal pharmacophore with high antimicrobial activities. The data suggest that tetrapeptides or capped tripeptides are effective and there is no correlation between the order of amino acids and antimicrobial activity. Liu et al. (26) analyzed the effects of chain length on the activities of AMPs with repeating pharmacophore sequences (RW)_n-NH₂ (n = 1, 2, 3, 4, or 5). The tests of antimicrobial activities and the hemolysis of red blood cells suggest that (RW)₃-NH₂ has the optimal chain length. Although longer chains are more potent antimicrobials, they can stimulate hemolysis.Most of the AMP studies to date are focused on planktonic bacteria. However, the majority of pathogenic bacteria tend to adhere to surfaces and form sessile microbial communities with highly hydrated structures of secreted polysaccharide matrix, collectively known as biofilms (9). Biofilms can tolerate up to 1,000 times more antibiotics and disinfectants than their planktonic counterparts (2, 7, 8). For example, Folkesson et al. (12) reported that biofilm formation of E. coli K-12 increases its tolerance to polymyxin E, a polypeptide antibiotic that kills Gram-negative bacteria by disrupting membranes (34, 40). Since biofilms are involved in 80% of human bacterial infections (1), it is necessary to study biofilm inhibition and dispersion by AMPs.In this study, a series of linear peptides (RW)_n-NH₂ (where n = 2, 3, or 4) were studied for the effects of their activities on planktonic cells and biofilms of E. coli to understand the structural effects on the antimicrobial activities of AMPs. We chose E. coli RP437 in this study because it is one of the model strains for biofilm research and allows us to compare the data with those of our previous studies (6, 16, 19, 20).     

Biofilm Formation by Campylobacter jejuni Is Increased under Aerobic Conditions

The microaerophilic human pathogen Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that C. jejuni NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O₂) than under microaerobic conditions (5% O₂, 10% CO₂), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in C. jejuni. Moreover, preformed biofilms shed high numbers of viable C. jejuni cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions, C. jejuni adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of C. jejuni.Infection with Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world and is often associated with the consumption of undercooked poultry products (19). The United Kingdom Health Protection Agency reported more than 45,000 laboratory-confirmed cases for England and Wales in 2006 alone, although this is thought to be a 5- to 10-fold underestimation of the total number of community incidents (20, 43). The symptoms associated with C. jejuni infection usually last between 2 and 5 days and include diarrhea, vomiting, and stomach pains. Sequelae of C. jejuni infection include more-serious autoimmune diseases, such as Guillain-Barré syndrome, Miller-Fisher syndrome (18), and reactive arthritis (15).Poultry represents a major natural reservoir for C. jejuni, since the organism is usually considered to be a commensal and can reach densities as high as 1 × 10⁸ CFU g of cecal contents⁻¹ (35). As a result, large numbers of bacteria are shed via feces into the environment, and consequently, C. jejuni can spread rapidly through a flock of birds in a broiler house (1). While well adapted to life in the avian host, C. jejuni must survive during transit between hosts and on food products under stressful storage conditions, including high and low temperatures and atmospheric oxygen levels. The organism must therefore have mechanisms to protect itself from unfavorable conditions.Biofilm formation is a well-characterized bacterial mode of growth and survival, where the surface-attached and matrix-encased bacteria are protected from stressful environmental conditions, such as UV radiation, predation, and desiccation (7, 8, 28). Bacteria in biofilms are also known to be >1,000-fold more resistant to disinfectants and antimicrobials than their planktonic counterparts (11). Several reports have now shown that Campylobacter species are capable of forming a monospecies biofilm (21, 22) and can colonize a preexisting biofilm (14). Biofilm formation can be demonstrated under laboratory conditions, and environmental biofilms, from poultry-rearing facilities, have been shown to contain Campylobacter (5, 32, 44). Campylobacter biofilms allow the organism to survive up to twice as long under atmospheric conditions (2, 21) and in water systems (27).Molecular understanding of biofilm formation by Campylobacter is still in its infancy, although there is evidence for the role of flagella and gene regulation in biofilm formation. Indeed, a flaAB mutant shows reduced biofilm formation (34); mutants defective in flagellar modification (cj1337) and assembly (fliS) are defective in adhering to glass surfaces (21); and a proteomic study of biofilm-grown cells shows increased levels of motility-associated proteins, including FlaA, FlaB, FliD, FlgG, and FlgG2 (22). Flagella are also implicated in adhesion and in biofilm formation and development in other bacterial species, including Aeromonas, Vibrio, Yersinia, and Pseudomonas species (3, 23, 24, 31, 42).Previous studies of Campylobacter biofilms have focused mostly on biofilm formation under standard microaerobic laboratory conditions. In this work we have examined the formation of biofilms by motile and nonmotile C. jejuni strains under atmospheric conditions that are relevant to the survival of this organism in a commercial context of environmental and food-based transmission.     

Coaggregation by the Freshwater Bacterium Sphingomonas natatoria Alters Dual-Species Biofilm Formation

Coaggregation is hypothesized to enhance freshwater biofilm development. To investigate this hypothesis, the ability of the coaggregating bacterium Sphingomonas natatoria to form single- and dual-species biofilms was studied and compared to that of a naturally occurring spontaneous coaggregation-deficient variant. Attachment assays using metabolically inactive cells were performed using epifluorescence and confocal laser scanning microscopy. Under static and flowing conditions, coaggregating S. natatoria 2.1gfp cells adhered to glass surfaces to form diaphanous single-species biofilms. When glass surfaces were precoated with coaggregation partner Micrococcus luteus 2.13 cells, S. natatoria 2.1gfp cells formed densely packed dual-species biofilms. The addition of 80 mM galactosamine, which reverses coaggregation, mildly reduced adhesion to glass but inhibited the interaction and attachment to glass-surface-attached M. luteus 2.13 cells. As opposed to wild-type coaggregating cells, coaggregation-deficient S. natatoria 2.1COGgfp variant cells were retarded in colonizing glass and did not interact with glass-surface-attached M. luteus 2.13 cells. To determine if coaggregation enhances biofilm growth and expansion, viable coaggregating S. natatoria 2.1gfp cells or the coaggregation-deficient variant S. natatoria 2.1COGgfp cells were coinoculated in flow cells with viable M. luteus 2.13 cells and allowed to grow together for 96 h. Coaggregating S. natatoria 2.1gfp cells outcompeted M. luteus 2.13 cells, and 96-h biofilms were composed predominantly of S. natatoria 2.1gfp cells. Conversely, when coaggregation-deficient S. natatoria 2.1COGgfp cells were coinoculated with M. luteus 2.13 cells, the 96-h biofilm contained few coaggregation-deficient S. natatoria 2.1 cells. Thus, coaggregation promotes biofilm integration by facilitating attachment to partner species and likely contributes to the expansion of coaggregating S. natatoria 2.1 populations in dual-species biofilms through competitive interactions.In nature, most biofilms are not composed of one bacterial species but instead contain multiple species (24). These multispecies communities can be responsible for the fouling of ships (9, 44), the corrosion of liquid-carrying vessels (3, 14), and chronic infections in higher organisms (41, 42, 57). Recent research has demonstrated that in order for multispecies biofilm communities to develop, interbacterial communication is often essential (62) and facilitates the coordination of bacterial activities to promote the formation and to maintain the integrity of multispecies biofilm communities (28, 32, 60). Interspecies communication can be mediated by chemical or physical means. Mechanisms for chemical communication between different species include the secretion and uptake of metabolic by-products (11, 19), the exchange of genetic material (40), and the production and recognition of interspecies signal molecules such as short peptides (36) and autoinducer-2 (10). Mechanisms for interspecies physical communication can involve cell surface structures such as flagella or fimbriae (31, 48) and also include nonspecific adhesion between bacterial species (5) as well as highly specific coaggregations mediated by lectin-saccharide interactions (48).Coaggregation, the highly specific recognition and adhesion of different bacterial species to one another, was first discovered to occur between human oral bacteria in 1970 (23). Since then, research has shown that coaggregation occurs between specific bacterial species in environments other than the human oral cavity (48). Coaggregation interactions have been detected between bacteria isolated from canine dental plaque (21), the crop of chickens (61), the human female urogenital tract (30), the human intestine (34), and wastewater and freshwater biofilms (27, 37, 53). In particular, Buswell et al. (8) first demonstrated that coaggregation occurred between 19 freshwater strains that were isolated from a drinking water biofilm. Further studies by Rickard et al. demonstrated that coaggregation between these 19 strains was mediated by growth-phase-dependent lectin-saccharide interactions (49, 50) and occurred at the interspecies and intraspecies levels for nine different genera (50). From this aquatic biofilm consortium, coaggregation between the gram-negative bacterium Sphingomonas (Blastomonas) natatoria 2.1 and the gram-positive bacterium Micrococcus luteus 2.13 have been studied further. Coaggregation between this pair is mediated by the growth-phase-dependent expression of a lectin-like adhesin(s) on S. natatoria 2.1 and a complementary polysaccharide-containing receptor(s) on the cell surface of M. luteus 2.13 (47, 49). The addition of millimolar concentrations of galactosamine resulted in the dispersion of the coaggregates (47, 49). Coaggregation between this pair also occurs after growth in artificial biofilm constructs composed of poloxamer (47). These findings suggested that coaggregation may contribute to the integration of S. natatoria 2.1 into freshwater biofilms through specific adhesive interactions with M. luteus 2.13. Indeed, while coaggregation is hypothesized to contribute to the integration of species into freshwater biofilms (31, 32, 48), no direct evidence has yet been presented. If coaggregation promotes the integration of species into a freshwater biofilm, it may contribute to the retention of pathogens in drinking water pipelines (7) as well as the maintenance of the species diversity of aquatic biofilms that are exposed to shear stress (52, 53).S. natatoria and M. luteus are commonly isolated from moist environments. M. luteus is environmentally ubiquitous and is found in biofilms of aquatic ecosystems (8, 35), in soil (54), and on human and animal skin (17, 29). Cells of M. luteus are gram positive, coccus shaped, arranged in clusters of tetrads, and nonmotile. S. natatoria is indigenous to freshwater environments (55) and has been isolated from swimming pools, deep-ice boreholes, and drinking water systems (1, 50, 56). Cells are gram negative, are rod shaped, and have the propensity to form rosettes containing 4 to 14 cells (55). Each rosette-forming cell has a polar tuft of fimbriae at its nonreproductive pole by which it attaches to other S. natatoria cells and, possibly, solid surfaces (46, 55). Reproduction occurs by asymmetric division (budding) to produce an ovoid daughter cell, which is highly motile, with a single polar flagellum. These ovoid daughter cells do not coaggregate, and only mature cells within rosettes can attach to other species of bacteria. Previous studies indicated that while coaggregation between S. natatoria 2.1 and M. luteus 2.13 is inhibited by the addition of galactosamine, the propensity of S. natatoria 2.1 to form rosettes was unaffected (46, 49).The aim of this work was to determine if coaggregation enhances the attachment of planktonic S. natatoria 2.1 cells to clean glass surfaces as well as glass surfaces precoated with M. luteus 2.13 cells under static and flowing conditions. This study also aimed to provide insight into whether coaggregation contributes to the expansion of S. natatoria 2.1 populations within dual-species biofilms containing M. luteus 2.13. Epifluorescence microscopy and confocal laser scanning microscopy (CLSM) coupled with three different computer-based analysis programs were used throughout this study. Attachment assays were performed using metabolically inactive planktonic coaggregating or coaggregation-deficient variants of S. natatoria 2.1 that were suspended over or that were flowed across metabolically inactive glass-surface-attached M. luteus 2.13 cells. The potential role of coaggregation in promoting the expansion of S. natatoria 2.1 populations within biofilms containing M. luteus 2.13 was investigated by inoculating flow cells with viable cells and monitoring spatiotemporal development. By achieving these two aims, this work demonstrates that coaggregation contributes to biofilm integration and indicates that there is a possible role for coaggregation interactions in the establishment and expansion of S. natatoria populations in freshwater biofilms.     

Interactions of DNA with Biofilm-Derived Membrane Vesicles

The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs) and DNA, both matrix components, demonstrated concentration-, pH-, and cation-dependent interactions. Furthermore, MV-DNA association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction of matrix polymers and other constituents.The biofilm matrix contributes to the chemistry, structure, and function of biofilms and is crucial for the development of fundamental biofilm properties (46, 47). Early studies defined polysaccharides as the matrix component, but proteins, lipids, and nucleic acids are all now acknowledged as important contributors (7, 15). Indeed, DNA has emerged as a vital participant, fulfilling structural and functional roles (1, 5, 6, 19, 31, 34, 36, 41, 43, 44). The phosphodiester bond of DNA renders this polyanionic at a physiological pH, undoubtedly contributing to interactions with cations, humic substances, fine-dispersed minerals, and matrix entities (25, 41, 49).In addition to particulates such as flagella and pili, membrane vesicles (MVs) are also found within the matrices of gram-negative and mixed biofilms (3, 16, 40). MVs are multifunctional bilayered structures that bleb from the outer membranes of gram-negative bacteria (reviewed in references 4, 24, 27, 28, and 30) and are chemically heterogeneous, combining the known chemistries of the biofilm matrix. Examination of biofilm samples by transmission electron microscopy (TEM) has suggested that matrix material interacts with MVs (Fig. ​(Fig.1).1). Since MVs produced in planktonic culture have associated DNA (11, 12, 13, 20, 21, 30, 39, 48), could biofilm-derived MVs incorporate DNA (1, 39, 40, 44)?Open in a separate windowFIG. 1.Possible interactions between matrix polymers and particulate structures. Shown is an electron micrograph of a thin section through a P. aeruginosa PAO1 biofilm. During processing, some dehydration occurred, resulting in collapse of matrix material into fibrillate arrangements (black filled arrows). There is a suggestion of interactions occurring with particulate structures such as MVs (hollow white arrow) and flagella (filled white arrows) (identified by the appearance and cross-dimension of these highly ordered structures when viewed at high magnification), which was consistently observed with other embedded samples and also with whole-mount preparations of gently disrupted biofilms (data not shown). The scale bar represents 200 nm.     

Real-Time Microsensor Measurement of Local Metabolic Activities in Ex Vivo Dental Biofilms Exposed to Sucrose and Treated with Chlorhexidine

Dental biofilms are characterized by structural and functional heterogeneity. Due to bacterial metabolism, gradients develop and diverse ecological microniches exist. The aims of this study were (i) to determine the metabolic activity of microorganisms in naturally grown dental biofilms ex vivo by measuring dissolved oxygen (DO) and pH profiles with microelectrodes with high spatial resolution and (ii) to analyze the impact of an antimicrobial chlorhexidine (CHX) treatment on microbial physiology during stimulation by sucrose in real time. Biofilms were cultivated on standardized human enamel surfaces in vivo. DO and pH profiles were measured in a flow cell system in sterile human saliva, after sucrose addition (10%), again after alternative treatment of the sucrose exposed biofilms with CHX (0.2%) for 1 or 10 min or after being killed with paraformaldehyde (4%). Biofilm structure was visualized by vitality staining with confocal microscopy. With saliva as the sole nutrient source oxygen consumption was high within the superficial biofilm layers rendering deeper layers (>220 μm) anoxic. Sucrose addition induced the thickness of the anaerobic zone to increase with a concurrent decrease in pH (7.1 to 4.4). CHX exposure reduced metabolic activity and microbial viability at the biofilm surface and drove metabolic activity deeper into the biofilm. CHX treatment led to a reduced viability at the biofilm surface with minor influence on overall biofilm physiology after 1 min; even after 10 min there was measurable respiration and fermentation inside the biofilm. However, the local microenvironment was more aerated, less acidogenic, and presumably less pathogenic.Biofilms are complex, surface-associated, microbiological communities (7) that are characterized by microscale spatial, structural, and functional heterogeneity (40). The biofilm consists of microorganisms that are embedded in an extracellular slime matrix consisting of biopolymers of microbial origin such as polysaccharides, proteins, and DNA (16). This extracellular polymeric slime is highly hydrated and influences both the structure and the diffusion behavior within the biofilm (39). Bacterial metabolism results in the development of chemical and physiologic/metabolic gradients within the biofilm (17). Due to different concentrations of oxygen, nutrients, and microbial metabolic by-products, local microecological niches are created, allowing the coexistence of microorganisms with different growth requirements in close proximity (30). For example, the growth of anaerobic microorganisms within a generally aerobic environment within the oral cavity is possible. Carbohydrates and sugar are the most important energy sources for microorganisms in dental plaque (23) and, in the case of a lacking external substrate supply, they are able to metabolize salivary glycoproteins (5). Nutrient depletion causes the microorganisms to either grow very slowly or to stop growing completely, entering a dormant-like state.Changes in the ecologic balance of the oral microflora and in dental biofilms are a causative factor for the development of dental caries (43), gingivitis, and periodontitis (1); thus, these diseases can be considered as biofilm mediated. Fundamental factors that may lead to a shift in the microflora and the predominance of pathogens are the local pH value, the redox potential, and the availability of nutrients and/or carbohydrates (30). Caries, for instance, is a multifactorial disease. However, its main cause is the bacterial carbohydrate catabolism and the release of organic acids by acidogenic bacteria in the biofilm. This promotes the predominance of cariogenic pathogens such as Streptococcus mutans, Streptococcus sobrinus, and other acidogenic microorganisms (28, 43). Consequently, this results in further acid production and a decreasing pH. Associated with this is the demineralization and lesion development of dental hard substance (54).Next to individual improvement of mechanical oral hygiene (i.e., mechanical and manual brushing, as well as flossing), prevention and therapy of oral disease is achieved by adjunctive oral hygiene products containing antimicrobial agents (29, 47). A concentration of 0.2% chlorhexidine (CHX) in oral mouth rinses showed the best efficacy in clinical studies and is still considered a “gold standard” (45). The antiplaque effect of CHX is based on a broad antibacterial spectrum. During application it immediately shows bactericidal effect and continuous bacteriostatic effect due to its high substantivity in the oral cavity (20). Electron microscopic examinations showed that CHX binds to and damages bacterial cell membranes and leads to structural changes and leakage of cytoplasm (3). Furthermore, contraction of in vitro grown biofilms after CHX exposure was shown in addition to cell damage (19, 44), which could cause changes in the diffusion behavior by changes to extracellular polymeric slime density. However, in deep layers of oral biofilms not all bacteria were reached (49). Direct visualization by fluorescence microscopy of the CHX effect was described by Takenaka et al. (44) for a three-species oral biofilm grown in vitro. Cell damage started from the periphery of bacterial aggregates and slowly continued into the depths. Other authors have reported the inability of CHX to completely kill all bacteria in different in vitro biofilm systems (15, 19, 32) and in vivo studies (48, 55) when a normally used clinical concentration was applied at usual exposure times. The killing efficacy was dependent on the age of the biofilm and thus on its thickness and composition.Detection of concentration gradients in oxygen, pH, and metabolites in undisturbed biofilms in situ requires a microsensor technique. Microelectrodes with a tip diameter of a few micrometers offer precisely localized measurements on the microscale in three dimensions under real-time conditions (9, 35). Thus far, microelectrodes have been applied for measuring plaque pH in dental research in a groove model ex vivo (53, 54) and in in vitro-grown S. mutans biofilms (13, 42). However, there is no information on direct measurements of oxygen distribution and consumption in dental biofilms grown in vivo in the human oral cavity. In the field of environmental microbiology, analysis of the microenvironment, local activities, and gradients by microelectrodes is correlated with microscopic examination of the biofilm structure, thus allowing a relationship to be made between biofilm physiology and structure (18, 37). We decided to take a similar approach to characterize the effects of sucrose and CHX on ex vivo plaque biofilm physiology. The goal of the present study was to examine the metabolic activity (oxygen consumption and acid formation) and viability and the effect of antimicrobial CHX treatment on the physiology of the dental biofilms during exposure to sucrose. The spatial distribution of live and dead biofilm cells was examined by confocal laser scanning microscopy (CLSM), as well as by microbiological culture.     

Genetic Features of Resident Biofilms Determine Attachment of Listeria monocytogenes

Planktonic Listeria monocytogenes cells in food-processing environments tend most frequently to adhere to solid surfaces. Under these conditions, they are likely to encounter resident biofilms rather than a raw solid surface. Although metabolic interactions between L. monocytogenes and resident microflora have been widely studied, little is known about the biofilm properties that influence the initial fixation of L. monocytogenes to the biofilm interface. To study these properties, we created a set of model resident Lactococcus lactis biofilms with various architectures, types of matrices, and individual cell surface properties. This was achieved using cell wall mutants that affect bacterial chain formation, exopolysaccharide (EPS) synthesis and surface hydrophobicity. The dynamics of the formation of these biofilm structures were analyzed in flow cell chambers using in situ time course confocal laser scanning microscopy imaging. All the L. lactis biofilms tested reduced the initial immobilization of L. monocytogenes compared to the glass substratum of the flow cell. Significant differences were seen in L. monocytogenes settlement as a function of the genetic background of resident lactococcal biofilm cells. In particular, biofilms of the L. lactis chain-forming mutant resulted in a marked increase in L. monocytogenes settlement, while biofilms of the EPS-secreting mutant efficiently prevented pathogen fixation. These results offer new insights into the role of resident biofilms in governing the settlement of pathogens on food chain surfaces and could be of relevance in the field of food safety controls.Listeria monocytogenes is a food pathogen that has been implicated in numerous food-borne disease outbreaks (5, 58). This organism is found not only in food products but also on surfaces in food-processing plants (18). It is well documented that L. monocytogenes is able to adhere and form persistent biofilms on a variety of solid materials, such as stainless steel, glass, or polymers (18, 48, 51, 52). However, in food-manufacturing plants (and particularly in fermented-food-processing environments), it is most likely that the first contact between a pathogen and a surface will concern a resident microbial biofilm covering the solid surface (10, 35, 46). In this context, such a resident biofilm may be regarded as a “conditioning film” that modifies the topographic and physicochemical characteristics of the surface and hence the adhesion capability of planktonic microorganisms coming into contact with this substratum (6).Once the pathogens are immobilized on the surface, interactions between the pathogens and their environment (physiological interactions with resident flora, nutrient availability, pH, water activity, temperature, and cleaning and disinfection procedures) govern the long-term settlement and persistence of the pathogens on the surface. Various studies have demonstrated the inhibition of L. monocytogenes development by natural “protective” biofilms (10, 66). Competition for nutrients has been demonstrated as a major mechanism underlying the inhibition of pathogen development (25, 27). The production of antimicrobial agents (bacteriocins, acids, and hydrogen peroxide) has also been reported as being of importance to such interactions (13, 20, 36). For example, Lactococcus lactis has been described as being exceptionally efficient in controlling the development of L. monocytogenes on food-processing surfaces by means of competitive exclusion (66) or bacteriocin production (35). It has been reported that treating a surface with a bacterial polysaccharide prevented the adhesion of different nosocomial pathogens (60). Furthermore, alginate-overexpressing Pseudomonas aeruginosa biofilms reduced the retention of Cryptosporidium parvum oocysts (54). Other recent studies have shown that the composition and quantity of specific exopolysaccharides (EPS) in Pseudomonas biofilms can inhibit the fixation of Escherichia coli or Erwinia chrysanthemi planktonic cells in porous media (37, 38).The present study investigated those properties of resident biofilms that could affect the settlement of L. monocytogenes. L. lactis was used as a model resident biofilm strain, as this is widely used in dairy fermentations and its cell wall properties have been the subject of considerable study (22, 23). Cell wall mutants of L. lactis MG1363 were used to create a set of model biofilms that differed in terms of their architecture, EPS synthesis, and cell surface hydrophobicity. These biofilms were used to evaluate the attachment of fluorescent inert polystyrene microbeads and of two reference strains of L. monocytogenes (LO28 and EGDe) using in situ confocal fluorescence imaging.