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George A Otoole - One of the best experts on this subject based on the ideXlab platform.
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microtiter dish Biofilm Formation assay
Journal of Visualized Experiments, 2011Co-Authors: George A OtooleAbstract:Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a Biofilm probably represents the predominate mode of growth for microbes in most environments. Mature Biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a Biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics. The microtiter dish assay is an important tool for the study of the early stages in Biofilm Formation, and has been applied primarily for the study of bacterial Biofilms, although this assay has also been used to study fungal Biofilm Formation. Because this assay uses static, batch-growth conditions, it does not allow for the Formation of the mature Biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of Biofilm Formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that Biofilms grown in microtiter dishes do develop some properties of mature Biofilms, such a antibiotic tolerance and resistance to immune system effectors. This simple microtiter dish assay allows for the Formation of a Biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing Biofilm Formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early Biofilm Formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi. In the protocol described here, we will focus on the use of this assay to study Biofilm Formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of Biofilm Formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of Biofilm Formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of Biofilms.
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catheter lock solutions influence staphylococcal Biofilm Formation on abiotic surfaces
Nephrology Dialysis Transplantation, 2006Co-Authors: Robert M Q Shanks, Martha L Graber, Jennifer L Sargent, Raquel M Martinez, George A OtooleAbstract:Background. Microbial Biofilms form on central venous catheters and may be associated with systemic infections as well as decreased dialysis efficiency due to catheter thrombosis. The most widely used anticoagulant catheter lock solution in the US is sodium heparin. We have previously shown that sodium heparin in clinically relevant concentrations enhances Staphylococcus aureus Biofilm Formation. In the present study, we examine the effect of several alternative catheter lock solutions on in vitro Biofilm Formation by laboratory and clinical isolates of S. aureus and coagulase-negative staphylococci (CNS). Methods. Lepirudin, low molecular weight heparin, tissue plasminogen activator, sodium citrate, sodium citrate with gentamicin and sodium ethylene diamine tetra-acetic acid (EDTA) were assessed for their effect on Biofilm Formation on polystyrene, polyurethane and silicon elastomer. Results. Sodium citrate at concentrations above 0.5% efficiently inhibits Biofilm Formation and cell growth of S. aureus and Staphylococcus epidermidis. Subinhibitory concentrations of sodium citrate significantly stimulate Biofilm Formation in most tested S. aureus strains, but not in CNS strains. Sodium EDTA was effective in prevention of Biofilm Formation as was a combination of sodium citrate and gentamicin. Low molecular weight heparin stimulated Biofilm Formation of S. aureus, while lepirudin and tissue plasminogen activator had little effect on S. aureus Biofilm Formation. Conclusions. This in vitro study demonstrates that heparin alternatives, sodium citrate and sodium EDTA, can prevent the Formation of S. aureus Biofilms, suggesting that they may reduce the risk of Biofilm-associated complications in indwelling catheters. This finding suggests a biological mechanism for the observed improvement in catheter-related outcomes in recent clinical comparisons of heparin and trisodium citrate as catheter locking solutions. A novel and potential clinically relevant finding of the present study is the observation that citrate at low levels strongly stimulates Biofilm Formation by S. aureus.
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heparin stimulates staphylococcus aureus Biofilm Formation
Infection and Immunity, 2005Co-Authors: Robert M Q Shanks, Niles P Donegan, Martha L Graber, Sarah E Buckingham, Michael E Zegans, Ambrose L Cheung, George A OtooleAbstract:Heparin, known for its anticoagulant activity, is commonly used in catheter locks. Staphylococcus aureus, a versatile human and animal pathogen, is commonly associated with catheter-related bloodstream infections and has evolved a number of mechanisms through which it adheres to biotic and abiotic surfaces. We demonstrate that heparin increased Biofilm Formation by several S. aureus strains. Surface coverage and the kinetics of Biofilm Formation were stimulated, but primary attachment to the surface was not affected. Heparin increased S. aureus cell-cell interactions in a protein synthesis-dependent manner. The addition of heparin rescued Biofilm Formation of hla, ica, and sarA mutants. Our data further suggest that heparin stimulation of Biofilm Formation occurs neither through an increase in sigB activity nor through an increase in polysaccharide intracellular adhesin levels. These finding suggests that heparin stimulates S. aureus Biofilm Formation via a novel pathway.
Robert M Q Shanks - One of the best experts on this subject based on the ideXlab platform.
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catheter lock solutions influence staphylococcal Biofilm Formation on abiotic surfaces
Nephrology Dialysis Transplantation, 2006Co-Authors: Robert M Q Shanks, Martha L Graber, Jennifer L Sargent, Raquel M Martinez, George A OtooleAbstract:Background. Microbial Biofilms form on central venous catheters and may be associated with systemic infections as well as decreased dialysis efficiency due to catheter thrombosis. The most widely used anticoagulant catheter lock solution in the US is sodium heparin. We have previously shown that sodium heparin in clinically relevant concentrations enhances Staphylococcus aureus Biofilm Formation. In the present study, we examine the effect of several alternative catheter lock solutions on in vitro Biofilm Formation by laboratory and clinical isolates of S. aureus and coagulase-negative staphylococci (CNS). Methods. Lepirudin, low molecular weight heparin, tissue plasminogen activator, sodium citrate, sodium citrate with gentamicin and sodium ethylene diamine tetra-acetic acid (EDTA) were assessed for their effect on Biofilm Formation on polystyrene, polyurethane and silicon elastomer. Results. Sodium citrate at concentrations above 0.5% efficiently inhibits Biofilm Formation and cell growth of S. aureus and Staphylococcus epidermidis. Subinhibitory concentrations of sodium citrate significantly stimulate Biofilm Formation in most tested S. aureus strains, but not in CNS strains. Sodium EDTA was effective in prevention of Biofilm Formation as was a combination of sodium citrate and gentamicin. Low molecular weight heparin stimulated Biofilm Formation of S. aureus, while lepirudin and tissue plasminogen activator had little effect on S. aureus Biofilm Formation. Conclusions. This in vitro study demonstrates that heparin alternatives, sodium citrate and sodium EDTA, can prevent the Formation of S. aureus Biofilms, suggesting that they may reduce the risk of Biofilm-associated complications in indwelling catheters. This finding suggests a biological mechanism for the observed improvement in catheter-related outcomes in recent clinical comparisons of heparin and trisodium citrate as catheter locking solutions. A novel and potential clinically relevant finding of the present study is the observation that citrate at low levels strongly stimulates Biofilm Formation by S. aureus.
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heparin stimulates staphylococcus aureus Biofilm Formation
Infection and Immunity, 2005Co-Authors: Robert M Q Shanks, Niles P Donegan, Martha L Graber, Sarah E Buckingham, Michael E Zegans, Ambrose L Cheung, George A OtooleAbstract:Heparin, known for its anticoagulant activity, is commonly used in catheter locks. Staphylococcus aureus, a versatile human and animal pathogen, is commonly associated with catheter-related bloodstream infections and has evolved a number of mechanisms through which it adheres to biotic and abiotic surfaces. We demonstrate that heparin increased Biofilm Formation by several S. aureus strains. Surface coverage and the kinetics of Biofilm Formation were stimulated, but primary attachment to the surface was not affected. Heparin increased S. aureus cell-cell interactions in a protein synthesis-dependent manner. The addition of heparin rescued Biofilm Formation of hla, ica, and sarA mutants. Our data further suggest that heparin stimulation of Biofilm Formation occurs neither through an increase in sigB activity nor through an increase in polysaccharide intracellular adhesin levels. These finding suggests that heparin stimulates S. aureus Biofilm Formation via a novel pathway.
Roberto Kolter - One of the best experts on this subject based on the ideXlab platform.
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Biofilm Formation as a response to ecological competition
PLOS Biology, 2015Co-Authors: Nuno Oliveira, Esteban Martinezgarcia, Joao B Xavier, William M Durham, Roberto Kolter, Kevin R FosterAbstract:Bacteria form dense surface-associated communities known as Biofilms that are central to their persistence and how they affect us. Biofilm Formation is commonly viewed as a cooperative enterprise, where strains and species work together for a common goal. Here we explore an alternative model: Biofilm Formation is a response to ecological competition. We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on Biofilm Formation. We show that strain mixing reliably increases Biofilm Formation compared to unmixed conditions. Importantly, strain mixing leads to strong competition: one strain dominates and largely excludes the other from the Biofilm. Furthermore, we show that pyocins, narrow-spectrum antibiotics made by other P. aeruginosa strains, can stimulate Biofilm Formation by increasing the attachment of cells. Side-by-side comparisons using microfluidic assays suggest that the increase in Biofilm occurs due to a general response to cellular damage: a comparable Biofilm response occurs for pyocins that disrupt membranes as for commercial antibiotics that damage DNA, inhibit protein synthesis or transcription. Our data show that bacteria increase Biofilm Formation in response to ecological competition that is detected by antibiotic stress. This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded. Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.
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flagellar motility is critical for listeria monocytogenes Biofilm Formation
Journal of Bacteriology, 2007Co-Authors: Katherine P Lemon, Darren E Higgins, Roberto KolterAbstract:The food-borne pathogen Listeria monocytogenes attaches to environmental surfaces and forms Biofilms that can be a source of food contamination, yet little is known about the molecular mechanisms of its Biofilm development. We observed that nonmotile mutants were defective in Biofilm Formation. To investigate how flagella might function during Biofilm Formation, we compared the wild type with flagellum-minus and paralyzed-flagellum mutants. Both nonmotile mutants were defective in Biofilm development, presumably at an early stage, as they were also defective in attachment to glass during the first few hours of surface exposure. This attachment defect could be significantly overcome by providing exogenous movement toward the surface via centrifugation. However, this centrifugation did not restore mature Biofilm Formation. Our results indicate that it is flagellum-mediated motility that is critical for both initial surface attachment and subsequent Biofilm Formation. Also, any role for L. monocytogenes flagella as adhesins on abiotic surfaces appears to be either minimal or motility dependent under the conditions we examined.
Douglas R Call - One of the best experts on this subject based on the ideXlab platform.
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variation in Biofilm Formation among strains of listeria monocytogenes
Applied and Environmental Microbiology, 2003Co-Authors: Monica K Borucki, Jason D Peppin, David C White, Frank J Loge, Douglas R CallAbstract:Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular Biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between Biofilm Formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for Biofilm Formation to determine if there is a robust relationship between Biofilm Formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased Biofilm Formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in Biofilm Formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased Biofilm Formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-Biofilm-producing strains. Scanning electron microscopy showed that a high-Biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-Biofilm-forming strain produced a thin, patchy Biofilm. These data are consistent with data on persistent strains forming Biofilms but do not support a consistent relationship between enhanced Biofilm Formation and disease incidence.
Martha L Graber - One of the best experts on this subject based on the ideXlab platform.
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catheter lock solutions influence staphylococcal Biofilm Formation on abiotic surfaces
Nephrology Dialysis Transplantation, 2006Co-Authors: Robert M Q Shanks, Martha L Graber, Jennifer L Sargent, Raquel M Martinez, George A OtooleAbstract:Background. Microbial Biofilms form on central venous catheters and may be associated with systemic infections as well as decreased dialysis efficiency due to catheter thrombosis. The most widely used anticoagulant catheter lock solution in the US is sodium heparin. We have previously shown that sodium heparin in clinically relevant concentrations enhances Staphylococcus aureus Biofilm Formation. In the present study, we examine the effect of several alternative catheter lock solutions on in vitro Biofilm Formation by laboratory and clinical isolates of S. aureus and coagulase-negative staphylococci (CNS). Methods. Lepirudin, low molecular weight heparin, tissue plasminogen activator, sodium citrate, sodium citrate with gentamicin and sodium ethylene diamine tetra-acetic acid (EDTA) were assessed for their effect on Biofilm Formation on polystyrene, polyurethane and silicon elastomer. Results. Sodium citrate at concentrations above 0.5% efficiently inhibits Biofilm Formation and cell growth of S. aureus and Staphylococcus epidermidis. Subinhibitory concentrations of sodium citrate significantly stimulate Biofilm Formation in most tested S. aureus strains, but not in CNS strains. Sodium EDTA was effective in prevention of Biofilm Formation as was a combination of sodium citrate and gentamicin. Low molecular weight heparin stimulated Biofilm Formation of S. aureus, while lepirudin and tissue plasminogen activator had little effect on S. aureus Biofilm Formation. Conclusions. This in vitro study demonstrates that heparin alternatives, sodium citrate and sodium EDTA, can prevent the Formation of S. aureus Biofilms, suggesting that they may reduce the risk of Biofilm-associated complications in indwelling catheters. This finding suggests a biological mechanism for the observed improvement in catheter-related outcomes in recent clinical comparisons of heparin and trisodium citrate as catheter locking solutions. A novel and potential clinically relevant finding of the present study is the observation that citrate at low levels strongly stimulates Biofilm Formation by S. aureus.
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heparin stimulates staphylococcus aureus Biofilm Formation
Infection and Immunity, 2005Co-Authors: Robert M Q Shanks, Niles P Donegan, Martha L Graber, Sarah E Buckingham, Michael E Zegans, Ambrose L Cheung, George A OtooleAbstract:Heparin, known for its anticoagulant activity, is commonly used in catheter locks. Staphylococcus aureus, a versatile human and animal pathogen, is commonly associated with catheter-related bloodstream infections and has evolved a number of mechanisms through which it adheres to biotic and abiotic surfaces. We demonstrate that heparin increased Biofilm Formation by several S. aureus strains. Surface coverage and the kinetics of Biofilm Formation were stimulated, but primary attachment to the surface was not affected. Heparin increased S. aureus cell-cell interactions in a protein synthesis-dependent manner. The addition of heparin rescued Biofilm Formation of hla, ica, and sarA mutants. Our data further suggest that heparin stimulation of Biofilm Formation occurs neither through an increase in sigB activity nor through an increase in polysaccharide intracellular adhesin levels. These finding suggests that heparin stimulates S. aureus Biofilm Formation via a novel pathway.
