Wednesday AM, November 29, 2006
Room 205 (Hynes)
9:30 AM - **H1.1
(Genome) Size Matters.
Steven Projan 1 Show Abstract
1 Biological Technologies, Wyeth, Cambridge, Massachusetts, United States
It is a commonly held view that all bacteria will eventually become resistant to any antibiotic given enough time and exposure. However not all bacteria develop resistance to antibiotics at the same rate (and perhaps some species of bacteria may never develop resistance). And some bacteria that have been labeled “resistant” are not, in any clinically meaningful way, actually resistant. Another piece of dubious conventional wisdom is that “Bacterial resistance to drug therapy was first discovered in the 1940s, following the introduction of penicillin.” The fact is that the first description of a beta-lactamase producing (and therefore penicillin resistant) bacterial strain was published on Dec. 28, 1940 by Abraham and Chain while the first successful therapeutic use of penicillin was in April of 1942. Rather than blaming the profligate use of antibiotics on the emergence of resistant strains it should be understood that resistance to antibiotics is a natural thing, at least for bacteria that are freely living in the environment. Third generation cephalosporin antibiotics, when used clinically, appear to rapidly select for resistant strains, especially MRSA, while penicillins do not appear to exert the same degree of selective pressure. This is despite both being members of the same of class of antibiotics and sharing a similar spectrum of antibacterial activity. None of our current resistance dogma can account for this observation. Are there differential affects on commensal microflora at work here? If so this may mean that we grossly underestimate the importance of commensal microflora and how they are be affected by antibiotic use or even protect their human hosts from infection by pathogenic bacteria. It certainly demonstrates that all antibiotics, even within the same class, are not created equal. It has not previously been noted that the ability of a given bacterium to evolve towards a multidrug resistance phenotype is a function of genome size, however this observation appears to hold true across bacterial species. By way of example infectious with Streptococcus pyogenes (a small genome bacterium at 1.6 mB) have been successfully treated with penicillin for over sixty years, yet there has not emerged a single penicillin resistant strain which Pseudomonas aeruginosa (a genetic behemoth at 6.3 mB) eventually develops clinical resistance to all antimicrobial agents. In other words the larger the genome the greater the propensity of a bacterium to display multidrug resistant phenotypes and that the smaller the genome the less likely antibacterial resistance will emerge and disseminate within that species. Until we develop a more sophisticated (and accurate) understanding of bacterial resistance to antimicrobial agents how can we ever control resistance?
10:00 AM - H1.2
Antibiotic Resistance: There Is No Turning Back!
Barry Kreiswirth 1 Show Abstract
1 PHRI TB Center, Public Health Research Institute, Newark, New Jersey, United States
Antibiotic resistance has been the major side effect of the antibiotic era; a 60 year experiment that has produced highly drug resistant bacteria that have spread in hospitals, in the environment, and in the beef and poultry industries. The development of new classes of effective antibiotics is limited and the research dollars in the major pharmaceutical companies are being directed toward life style drugs and against chronic diseases. The obvious consequence and it is already occurring in New York City hospitals, is the spread of nosocomial strains that are refractive to available antibiotics. The recent identification of vancomycin resistant S. aureus strains is ominous and analogous to the early MRSA identified in the 1960’s; the lessen is that these resistant strains will spread aggressively and most significantly, obviate the most effect hospital treatment for MRSA infections. The rise in resistant strains and limited number of lead compounds in the pharmaceutical pipeline is further confounded by the realization that hospital patients are highly susceptible to infections as a result of immunosuppression brought on cancer therapy, transplant procedures and the spread of HIV disease. The need for new therapies and strategies to lessen the future rise in bacterial resistance is paramount to controlling the spread of infectious diseases.
10:15 AM - **H1.3
Prevention of Catheter-related Bloodstream Infections.
Stephen Heard 1 Show Abstract
1 Anesthesiology, UMass Medical School, Worcester, Massachusetts, United States
More than 80,000 cases of catheter-related bloodstream infections (CRBSI) occur annually in the intensive care unit (ICU). The majority of these infections are cause by Gram positive organisms: S. aureus, S. epidermidis and enterococci. Most infections are caused by propagation of bacteria along the subcutaneous catheter tract or by colonization of the catheter lumen(s) when medications are administered or blood is withdrawn for lab testing. Strategies to reduce CRBSI include educational programs, use of maximum barrier precautions during catheter insertion (including skin preparation with a chlorhexidine solution), use of the subclavian vein insertion site, dressing the insertion site with a chlorhexedine impregnated sponge and use of catheters that are impregated with antiseptics or antibiotics. These special catheters have combinations of chlorhexidine and silver sulfadiazine; silver, carbon and platinum; or minocycline and rifampin. Clinical studies have demonstrated the utility of these catheters in reducing catheter colonization and CRBSI. Current research is focused on coatings that reduce further the incidence of CRBSI and increase the duration of antimicrobial activity.
10:45 AM - H1.4
Infection in Internal Fracture Fixation.
Geoff Richards 1 Show Abstract
1 AO Foundation, AO Research Institute, Davos Switzerland
H2: Biofilm Development I
Wednesday AM, November 29, 2006
Room 205 (Hynes)
11:30 AM - **H2.1
Differences in Gene Expression and Biofilm Development.
Karin Sauer 1 Show Abstract
1 Biological Sciences, Binghamton University, Binghamton, New York, United States
12:00 PM - **H2.2
Staphylococcus Biofilms: Mechanisms of Increased Biofilm Formation and Strategies for Blocking Biofilm Formation.
George O'Toole 1 , M. Grinstaff 2 , D. Kenan 3 Show Abstract
1 Department of Microbiology & Immunology, Dartmouth Medical School, Hanover, New Hampshire, United States, 2 , Boston University, Boston, Massachusetts, United States, 3 , Duke University, Durham, Massachusetts, United States
12:30 PM - H2.3
Direct Measurements of Bacterial Adhesion using Atomic Force Microscopy.
Terri Camesano 1 Show Abstract
1 Chemical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts, United States
While bacterial adhesion and biofilm formation affect a wide range of problems from catheter-related bloodstream infections to urinary tract infections and dental plaque, a clear relationship between bacterial surface properties and the adhesion of these microbes to surfaces does not exist. The inability to perform molecular-scale characterizations of bacterial surface macromolecules has rendered scientists and engineers unable to explain bacterial adhesion based on a first-principles approach. Our methodology is to characterize bacterial surfaces at the single-molecule level using atomic force microscopy, and to relate the physical and chemical properties of bacterial surface macromolecules to bacterial adhesion. In this presentation, we will highlight recent work on two medically-relevant bacterial systems: 1) Quantifying the interaction forces between Escherichia coli and uroepithelial cells, which is related to the development of urinary tract infections; and 2) Measuring the efficacy of self-assembled monolayer-based surfaces for preventing the adhesion and impairing the viability of Staphyloccocus epidermidis, a model organism in the consideration of catheter-related bloodstream infections. The results of our work will be instrumental to practitioners and clinicians seeking to prevent pathogenic bacterial infections.
12:45 PM - H2.4
Biofilm Formation and Electrostatic Force Characteristics of Escherichia coli O157:H7 Observed by Atomic Force Microscopy
YooJin Oh 1 , W. Jo 1 , Y. Yang 2 , S. Park 2 Show Abstract
1 Physics, Ewha womans univ., seoul Korea (the Republic of), 2 Nano Sciences, Ewha womans univ., seoul Korea (the Republic of)
Biofilms are complex microbial communities that are resistant against attacks by bacteriophages and removal by drugs and chemicals. Initial interactions between bacterial cells and the substrate are governed in part by electrostatic interactions. The charge and hydrophobicity of the cell surface can change with respect to environmental factors such as ion availability and pH. The actual mechanism that allows a bacterium to sense or perceive another surface is the physical force that exists in the nanometer scale interface between a cell and that other surface. Bacterial surface charge can also play a role in bacterial interaction with solid surfaces. In this study, biofilms of Escherichia coli O157:H7, a bacterial pathogen, were investigated using atomic force microscopy (AFM) in terms of the dynamic transition of morphology and surface properties of bacterial cells over the development of biofilms. Moreover, the electrical properties of biofilms were studied by electrostatic force microscopy (EFM). The determination of surface charge properties of bacterial cells is of paramount importance for modeling cell function and behavior in various environmental conditions. Although zeta potential method exists for indirectly determining surface charge, a direct determination of the EFM has proven to be alternative. Of the various analytical methods that have been developed for estimating surface charge, the technique of EFM offers distinct advantages with regard to accuracy, measurement time and ease of use. Application of high-resolution techniques such as AFM in conjunction with conventional electrical measurements provides a unique opportunity to achieve microscopic insight into the physical processes occurring on cell surface. As a result, traditionally EPS was generally thought to carry a net negative charge, however this studies reveal that the bacterial EPS composition is heterogeneous both chemically and spatially. EPS regions contained both positive and negative charges, suggesting the existence of specific functional regions within complex biofilms communities. As the biofilm is getting matured, the magnitude of the alteration seems to be smaller, indicating that electrostatic force is a sign of extra-cellular matrix of the bacteria and a measure of biofilms formation. Cell surface electrostatic force increases concomitantly with increasing time.
H3: Microfluidic and Microfabricated Technology Platforms
Wednesday PM, November 29, 2006
Room 205 (Hynes)
2:30 PM - **H3.1
Integrated Lab-on-a-Chip Devices for Complex Fluid and BioFilm Analysis
John T. McDevitt 1 Show Abstract
1 Chemistry and Biochemistry, University of Texas. Austin, Austin, Texas, United States
Despite remarkable advances in development of miniaturized sensing systems, the ability to assemble and interface individual components in order to achieve a high level of integration in complete working systems continues to pose daunting challenges for the scientific community. Inspired by nature’s taste sensation, the McDevitt group has launched a research program directed towards development of fully integrated lab-on-a-chip based methodologies that are suitable for the rapid analysis of complex fluids and biofilms. Because sample preparation and analyte detection is completed within the confines of miniaturized reaction vessels created within nano-bio-chip structures, multiple tests can be performed simultaneously. These nano-bio-chips can be used to identify and quantify analytes in the solution-phase via colorimetric and fluorescence changes to receptor and indicator molecules that are covalently attached to the polymer microspheres. The optical response of each microsphere is monitored using a charged coupled device (CCD), allowing for the near-real-time analysis of complex fluids. From these activities a number of powerful miniaturized sensor concepts suitable for important application areas such as clinical, environmental, bioterrorism, humanitarian and saliva-based diagnostic tools have been developed. Microfabrication methods are used here to generate these nano-bio-chip sensor systems. This lab-on-a-chip approach is extremely versatile, making it suitable for the measurement of electrolytes, protein antigens, antibodies, whole bacteria, cells and DNA/RNA. While these systems exhibit impressive analytical and diagnostic capabilities as compared with gold standards (such as pH meters for acidity, ELISA for protein analysis, FDA approved automated instruments for cardiac risk factors and planar DNA chips for nucleotide detection), their compact design and low cost also allows for their use in numerous important applications which require point-of-need capabilities. Furthermore, the nano-bio-chip system has been adapted recently for cellular analyses. Here immune function monitoring microchip systems have been fashioned suitable for HIV testing. This promising new system has been tested and validated in US hospitals as well as in a HIV reference laboratory in Botswana, Africa. The miniaturized platform also compares quite favorably with the gold standard method of flow cytometry.
3:00 PM - **H3.2
Microreactors as New Translation Tools for Controlled Complexity in Biofilm-Material Interactions.
Woo Lee 1 , Joung Hyun Lee 1 Show Abstract
1 Chemical Engineering, Stevens Institute of Technology, Hoboken, New Jersey, United States
In many infections, bacteria attach to the surfaces of medical devices or compromised tissue and subsequently develop into highly cooperative microenvironments known as biofilms. These microbiological environments interact in very complex and dynamic manners with device surfaces, host cells, and therapeutic interventions. From a perspective of rapidly and effectively translating newly discovered therapeutic concepts into clinical practice, this degree of complexity imposes a far greater number of independent variables than can be practically evaluated through animal and clinical studies. At the other extreme, the well-controlled environment of in vitro studies often reduces that level of complexity to a point where the resulting research is too far removed from clinical relevance. Recent advances in microreactor technology provide a timely opportunity to introduce controlled complexity into in vitro studies. We will present our initial microreactor-based catheter and orthopaedic implant infection models in the context of evaluating bacteria repulsive surface coating concepts. Through these examples, we will also address the issues and challenges associated with systematically and quantitatively elevating the level of in vitro complexity for physiologically relevant emulations of biofilm-associated infectious diseases.
3:30 PM - H3.3
Polyethylene Glycol Deposition Techniques for Antifouling Surfaces: Using Antistiction to Conserve Bioparticles for Recovery and Analysis.
Norine Chang 1 , Meng Lean 1 , Scott Limb 1 Show Abstract
1 Hardware Systems Laboratory, Palo Alto Research Center, Inc., Palo Alto, California, United States
3:45 PM - H3.4
Use of a Novel Fluidic Microplotter in Macroelectronics, Photonics, and Biodetection.
Brad Larson 1 , Douglas Lagally 1 , Paul Rugheimer 2 , Boy Tanto 2 , Padma Gopalan 2 , Max Lagally 2 1 Show Abstract
1 , SonoPlot, Inc., Madison, Wisconsin, United States, 2 , University of Wisconsin-Madison, Madison, Wisconsin, United States
Many future applications in electronics, bioassays, drug development, and the integration of these technologies will focus on broad uses such as fast flexible electronics, imaging and displays, biodefense, environmental and health monitoring, and drug screening. One potential overarching need is the deposition of a wide range of materials in the fluid state. We describe a new fluidic microplotter that enables the deposition of a very wide range of materials at lateral scales down to one micrometer. The dispensing depends on a novel axial ultrasonic resonance of a fluid micropipette that allows a gentle noncontact deposition of spots, lines, curves, and 3D objects with high precision and very good CV values. We will demonstrate some of the capabilities of the plotter in 1) creating highly precise spots for DNA and protein microarrays, 2) writing patterns on MEMS membranes, 3) writing a polymer LED, 4) writing parallel lines separated by 1 μm, 5) writing polymer waveguides, and 6) writing on Si nanomembranes that serve as the basis for very fast flexible electronics. The physical basis for the unique dispensing action in this device, as well as broader features of the plotter will be described. Potential additional applications will be discussed.Support: NSF, DOE
H4: Bacterial Interactive Surfaces
Wednesday PM, November 29, 2006
Room 205 (Hynes)
4:30 PM - H4.1
Antimicrobial Properties of Nanostructured Materials
Roger Narayan 1 , Topher Berry 2 , Robin Brigmon 2 Show Abstract
1 Biomedical Engineering, University of North Carolina, Chapel Hill, North Carolina, United States, 2 , Washington Savannah River Company, Aiken, South Carolina, United States
A major concern in the treatment of hospitalized or chronically ill individuals is medical device infection. Infection of catheters is especially troublesome, because these infections may quickly progress from the device site to involve other organs (e.g., endocarditis) or the entire body (e.g., septicemia). We have examined the antimicrobial activity of nanocrystalline diamond thin films, nanotube composite thin films, and other surfaces using in vitro studies and CDC biofilm reactor studies. Colonization of Staphylococcus aureus, Staphylococcus warneri, and Pseudomonas fluorescens was examined at several time intervals using epifluorescent microscopy and laser scanning confocal microscopy. These novel materials may be useful for inhibiting microorganism attachment and biofilm formation in hemodialysis catheters and other medical devices.
4:45 PM - H4.2
Surfaces That Resist Bacterial Adhesion: Novel Nonfouling Polymers.
Rupert Konradi 1 , Bidhari Pidhatika 1 , Marcus Textor 1 , Jens Möller 2 , Viola Vogel 2 Show Abstract
1 Dept. of Materials, Lab. for Surface Science and Technology, ETH Zurich, Zurich Switzerland, 2 Dept. of Materials, Lab. for Biologically Oriented Materials, ETH Zurich, Zurich Switzerland
Bacterial infections are usually established through several steps including attachment of the pathogen to the biomaterial surface, multiplication and biofilm formation. Antibiotics usually prevent the multiplication of bacteria either by active killing or by slowing down their growth such that the host defense mechanism can clear the infection. Recently, attempts have been made to impart resistance to bacterial infection by addressing the first step in this cascade of events, i.e. to inhibit the bacterial adhesion to surfaces. By far most frequently, this has been done by coating the inanimate surface with the nonfouling polymer poly(ethylene glycol) (PEG). Depending on the grafting chemistry and the bacterial adhesion assay the surfaces showed a reduction in the adhesion of different pathogens including S. epidermidis, S. aureus and P. aeruginosa of up to 90% for a typical incubation time of approximately 1 day. Despite the well documented advantages of PEG, there are also drawbacks. One major concern is the tendency of PEG to undergo oxidative degradation leading not only to a partial loss in its function but also to the generation of hazardous (hydro)peroxides and/or radicals. In the present study we explore the properties of poly(2-methyl-2-oxazoline) (PMOXA) based coatings as an alternative. This polymer is uncharged, hydrophilic and has a backbone structure similar to that of PEG with a heteroatom (nitrogen) separated by an ethylene unit. We have grafted carboxy-functional PMOXA to poly(L-lysine) to obtain graft-copolymers (PLL-g-PMOXA) with a structure very similar to that of the respective PEG analog (PLL-g-PEG). PLL-g-PEG sponateously adsorbs onto oppositely charged metal oxide surfaces, renders them highly protein-resistant and strongly reduces bacterial adhesion.[1, 3] The very similar architecture of the two copolymers allows us to carry out a systematic and detailed comparison of the two nonfouling polymers. Since unspecific protein adhesion is thought to occur as the first step in the cascade of biological events promoting surface colonization by bacteria, we first examine the interaction of PLL-g-PMOXA coated surfaces with full human serum using highly sensitive in situ techniques (optical waveguide lightmode spectroscopy OWLS and quarz crystal microbalance QCM). We then study and compare the adhesion of bacteria to surfaces modified with either of the two polymers by light microscopy as a function of the graft-copolymer architecture. We find that PLL-g-PMOXA is equally efficient in both, protein and bacterial repellence as compared to PLL-g-PEG.Harris, L. G.; Tosatti, S.; Wieland, M.; Textor, M.; Richards, R. G. Biomaterials 2004, 25, 4135.Harris, J. M.; Zalipsky, S.; Editors, Polyethylene glycol: Chemistry and Biological Applications. In: ACS Symp. Ser. 1997, 680.Pasche, S.; De Paul, S. M.; Voeroes, J.; Spencer, N. D.; Textor, M. Langmuir 2003, 19, 9216.
5:00 PM - H4.3
Plasma-enhanced Generation of Anti-Bacterial Surfaces.
Soujanya Jampala 1 2 , S. Manolache 2 , A. Wong 1 , K. Leonas 3 , F. Denes 1 2 4 Show Abstract
1 Materials Science Program, University of Wisconsin-Madison, Madison , Wisconsin, United States, 2 Center for Plasma-Aided Manufacturing, University of Wisconsin-Madison, Madison, Wisconsin, United States, 3 Textile Science, University of Georgia, Athens, Georgia, United States, 4 Department of Biological Systems Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
The permanent need to combat microbial infections in all biomedical devices drives interest in developing surfaces with antibacterial properties that inhibit the growth of attached bacteria, decrease the potential for biofilm development, and minimize the opportunity for contamination of biomaterials. Covalent implantation of polycationic structures onto organic and inorganic material surfaces is an attractive approach to develop stable antimicrobial surfaces with no residual toxicity. The cationic surface-active agents containing quaternary ammonium groups with appropriate alkyl substituents fatally disrupt the bacterial cell membrane/wall. The surfaces coated (covalently attached) with bactericidal quaternary ammonium thin layer macromolecular structures are generated using low pressure, non-equilibrium plasma (LP-NEP)-enhanced synthesis onto stainless steel (SS) and cellulose-based substrates. Ethylene Diamine (ED) plasma is used to deposit films of high concentrations of reactive nitrogen functionalities (around 35% nitrogen surface concentration) consisting of amine and imine groups. The inert SS substrate is pretreated with O2 and hexamethyldisiloxane plasmas to form an intermediate layer that stabilizes the top ED-plasma-deposited structure. These films are covalently attached and do not delaminate during washing with water or acetone in an ultrasonicator. The paper substrates are coated with ED plasma without any prior functionalization. The quaternization of plasma-deposited surface amines are done by a subsequent ex-situ reaction with hexyl bromide, and further methylated with methyl iodide. X-ray Photoelectron Spectroscopy (XPS) and Fourier Transform Infrared Spectroscopy (FTIR) are used to determine and confirm the surface chemistry and the nature of functional groups at all steps in the process. The evaluation of antifouling ability of the functionalized surfaces is carried out using standard colony counting procedures. Compared to unmodified SS and paper, quaternized substrates showed about 99% decrease in Staphylococcus aureus attachment at 24 h contact time. This cold plasma mediated deposition of quaternary ammonium groups provides a promising technique to synthesize surfaces that kill bacteria on contact.
5:15 PM - H4.4
Preliminary Studies of Attachment, Survival and Growth of Bone Marrow Stromal Cells on Nanocrystalline Ultra-Hydrophilic Hard Adherent Ceramic Coatings.
Fereydoon Namavar 1 , John Jackson 2 , J. Sharp 3 , Shailaja Varma 1 , Hani Haider 1 , Connie Feschuk 1 , Kevin Garvin 1 Show Abstract
1 Orthopaedics & Rehabilitation, University of Nebraska Medical Center, Omaha, Nebraska, United States, 2 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, United States, 3 Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, United States
There is a great need to develop methods to regulate cellular growth in order to enhance or prevent cell proliferation as needed, to either improve health or prevent disease. The present studies were devised to evaluate the adhesion, survival and growth of cells on the surface of new engineered nano-crystal films of pure cubic zirconia (with a hardness of 16 GPa), titanium, tantalum, cerium oxides, as well as silver. In vivo, much of the proliferative activity in bone cell development is associated with mesenchymal precursors. However, in vitro, osteoblast cell lines often have characteristics resembling tumor cancer cells, including dysregulated cell proliferation. Consequently, their growth on surface coatings may not be typical of normal cells. Because of these concerns over the use of osteoblast cell lines, the current studies were performed using a cloned bone marrow stromal cell line from C57Bl mice termed OMA-AD cells. This is a spontaneously immortalized undifferentiated stromal cell population that resembles multipotential mesenchymal stromal cells (MMSC). OMA-AD cell line duplicates, in vitro, all of the characteristics of primary mesenchymal stem cells and is a valid experimental model to probe the impact of nanocrystalline hard ceramic coatings on the attachment, survival and growth of bone marrow stromal cells. The engineered nano-crystal films with ultra-hydrophilic properties are produced by employing an ion beam assisted deposition (IBAD) technique. IBAD combines physical vapor deposition with concurrent ion beam bombardment (ionic hammer), in a high vacuum environment, to produce films (with 7 to 70 nm grain size) with superior properties. These films are “stitched” to the orthopaedic artificial implant materials with characteristics that affect the wettability and mechanical properties of the coating. Because of the opacity of substrates of our preliminary samples, the OMA-AD cells on these surfaces had to be viewed in incident light. Morphologically, there were different frequencies of cells attached to the different surfaces. For example, preparations of zirconium oxide had the highest frequency and silver the lowest frequency of cells. Also morphologically, the cells attached to some surfaces, for example tantalum oxide, showed much greater spreading with occasional large “blanket” cells. Based on cell counts, silver supported the lowest growth (about 1x103 cells/cm2), tantalum and titanium oxide and some preparations of zirconium oxide were intermediate (3-6x103 cells/cm2, but note, some of these cells were very large, and one surface nanostructure of cubic zirconium oxide supported approximately 8x103 cells/cm2). Physical, chemical and nanoscale properties of surface influence attachment, survival and growth of OMA-AD cells. The biophysics of these differences and the impact on the differentiation of OMA-AD cells is under ongoing investigation.
H5: Poster Session: Biofilm-Material Interactions
Wednesday PM, November 29, 2006
Exhibition Hall D (Hynes)
9:00 PM - H5.1
Bacterial Adhesion to PLL-g-PEG Modified Surfaces.
Raj Maddikeri 1 2 3 , Samuelle Tosatti 2 , Marcus Textor 2 , Julie Gold 3 , Geoff Richards 1 , Llinos Harris 1 Show Abstract
1 AO Research Institute, AO Foundation, Davos Switzerland, 2 Lab for Surface Science & Technology, ETH , Zürich Switzerland, 3 Chalmers University of technology , Göteborg University, Göteborg Sweden
9:00 PM - H5.10
Fractal Analysis of Xylella fastidiosa biofilm surface evolution
Alberto Luis Moreau 1 , Gabriela Lorite 1 , Antônio Borges 3 , Enilza Espreafico 3 , Marco Takita 2 , Alessandra de Souza 2 , Mônica Cotta 1 Show Abstract
1 Departamento de Física Aplicada, Instituto de Física Gleb Wataghin - UNICAMP, Campinas, São Paulo, Brazil, 3 Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto - USP, Ribeirão Preto, São Paulo, Brazil, 2 Centro APTA Citros Sylvio Moreira, Instituto Agronômico de Campinas, Cordeirópolis, São Paulo, Brazil
Xylella fastidiosa is a phytopathogenic bacteria that colonizes the xilema vessels of a large number of plants (vine, coffee, citrus, etc) and has affected crops in Brazil and around the world. The main pathogenic mechanism of X. fastidiosa is the formation of biofilm that induces the improper functioning of the water conducting system of the plant. In recent years, scanning probe microscopies – Atomic Force Microscopy (AFM) in particular – have become an important tool for the biomaterial study, essentially analyzing surfaces. In particular, AFM can be used for a more detailed structural analysis of the biofilm formation, providing information on the assembling mechanisms involved. In this work we have studied Xylella fastidiosa bacterial colonies cultivated on glass by AFM. The samples were metallized with a ~50nm-thick gold film, which allowed their in-air AFM imaging. We were thus able to obtain critical exponents and the fractal dimension for the topographic structures, since scaling laws were observed for the roughness dependence with system size. Moreover, different colonies in the same sample presented similar fractal behavior, making it possible a comparative analysis for samples with different growth times. The analysis of the roughness exponent and correlation length for different samples suggested the existence of different aggregation mechanisms during the evolution of biofilm formation.
9:00 PM - H5.11
Design and Fabrication of Nano to Microscale Porous Carbon Films for Biosensor Application.
Peter Feng 1 , Luis M Colon 1 , Boqian Yang 1 , Hongxin Zhang 1 , Xinpeng Wang 1 Show Abstract
1 Physics, UPR, San Juan, Puerto Rico, United States
9:00 PM - H5.13
Design of Fluorescent Sensing Interfaces for Detection of Catecholamines Based on Fluorogenic Derivatization
Nobuko Fukuda 1 , Kazuma Tsuboi 1 , Hirobumi Ushijima 1 Show Abstract
1 Photonics Research Institute, AIST, Tsukuba Japan
A fluorescent sensing interface based on fluorogenic derivatization was designed for detection of catecholamines, dopamine, epinephrine and norepinephrine on solid substrates. The advantages of fluorogenic derivatization on solid substrates are label-free detection and high throughput. In addition, this sensing surface can combine with surface plasmon resonance (SPR) and waveguide optical systems for high sensitive fluorescent detection by field enhancement of irradiation light. The sensing interface was fabricated by immobilization of fluorogenic derivatization reagents onto a glass substrate. Catecholamines as analytes in a buffer solution chemically reacted with fluorogenic derivatization molecules mixed into a polymer films on the glass substrates. When 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)  was immobilized as a derivatization reagent into a poly(methylmethacrylate-co-styrene) film, NBD-F in the polymer film reacted with dopamine in the mixture solution of 50 mM borate buffer (pH 8.0) and acetonitrile (volume ratio = 1:1) within 5 min at room temperature. The fluorescent spectrum of the film after reaction showed the peak at 510 nm by excitation of 442 nm. The change in the fluorescent intensity at 510 nm was observed with the change in the concentration of dopamine. The high sensitive detection of catecholamines using waveguide optical system is also investigated. This work is supported by Industrial Technology Research Grant Program in ’05 from New Energy and Industrial Technology Development Organization (NEDO) of Japan.  X. Zhu, P. N. Shaw, D. A. Barrett, Anal. Chim. Acta 478 (2003) 259.
9:00 PM - H5.15
Drop-by-drop Polymer Deposition by Acoustic Picoliter Droplet Generators for Applications in Semiconductor Industry and Biotechnology.
Utkan Demirci 1 Show Abstract
1 Surgery, Harvard Medical School, Charlestown, Massachusetts, United States
9:00 PM - H5.16
Electrophoresis of DNA and Conformation Studies on Microporous Membranes.
Eli Hoory 1 2 , Andrew Dubitsky 2 , John Frenna 2 , Michael Ding 1 , Miriam Rafailovich 1 , Jonathan Sokolov 1 Show Abstract
1 Materials Science, SUNY @ Stony Brook, Stony Brook, New York, United States, 2 , Pall Corporation, Port Washington, New York, United States
The ability to separate DNA via electrophoresis on solid surfaces has been demonstrated on insulating and semiconducting substrates. Due to an array of surface-DNA interactions and surface morphologies, preliminary studies of DNA surface electrophoresis on microporous membranes are currently in progress to determine the feasibility of DNA separation in comparison with solid surfaces previously analyzed. DNA length and conformation will also be analyzed to determine if DNA molecules remain in tact while moving along the microporous surface. Separation of Genomic DNA molecules on solid surfaces can potentially be used for genomic research and diagnostic applications. Utilization of sputter coating and other surface modifications of best candidate membranes will be explored to optimize efficiency of separation.
9:00 PM - H5.2
Microreactor Design and Fabrication for Studying Staphylococcus epidermidis – Implant Coating Interactions.
Joung Hyun Lee 1 , Woo Lee 1 Show Abstract
1 CBME, Stevens Institute of Technology, Hoboken, New Jersey, United States
9:00 PM - H5.3
Nanopatterned Hydrogels for Rapid Detection of Staphylococcus Aureus
Ishtiaq Saaem 1 , Barry Kreiswirth 2 , Matthew Libera 1 Show Abstract
1 Dept. of Chemical Engineering, Stevens Institute of Technology, Hoboken, New Jersey, United States, 2 , Public Health Research Institute, Newark, New Jersey, United States
Staphylococcus aureus can cause nosocomial and community-acquired infections ranging from mild conditions, such as skin and soft tissue infections, to severe, life-threatening sepsis. The rapid determination that a particular infection is due to S. aureus is important in order to quickly begin the administration of the most effective type of antibiotic. Conventional identification methods are time consuming, and traditional cell-plating or bottle-incubation techniques can take as long as 2-3 days to reach a definitive identification. As part of an effort to develop a rapid S. aureus detection method, we have been exploring how to functionalize surfaces to preferentially enhance S. aureus binding at specific surface locations. We use focused electron beams to create surface-patterned hydrogels by radiation crosslinking of amine-terminated poly(ethylene glycol) [PEG] thin films. The swelling properties can be controlled by the electron dose. These hydrogels can be patterned on glass or silicon at submicron spacings, and we can pattern ~7500 nanohydrogels in a 100 micron diameter area in ~10 seconds. After patterning, we treat any exposed substrate surface with an antifouling Pluronic triblock (PEO-PPO-PEO) copolymer. We find that high-swelling e-beam patterned gels resist nonspecific adhesion of both proteins and bacteria. These gels can be made adhesive to S. aureus by covalently binding IgG to the gel amine groups. We find that wild-type S. aureus binds to these immobilized IgG molecules, and the surface-bound S. aureus cells can be detected by fluorescence optical microscopy using anti-protein antibodies. As a control, we demonstrate that a protein A-knockout strain of S. aureus does not bind to the immobilized IgG. Our ongoing work addresses issues associated with the limits of sensitivity of this approach.
9:00 PM - H5.4
An NMR Metabolomic Study of Differences between Planktonic and Biofilm Modes of Growth
Erica Gjersing 1 , Julie Herberg 1 , Joanne Horn 1 , Charlene Schaldach 1 , Robert Maxwell 1 Show Abstract
1 , LLNL, Livermore, California, United States
Bacteria often reside in communities where the cells have secreted sticky, polymeric compounds, which allow them to attach to surfaces. This sessile lifestyle, referred to as a biofilm, affords the cells within these communities a tolerance of antibiotics and antimicrobial treatments. Biofilms of the bacterium Pseudomonas aeruginosa have been implicated in cystic fibrous and are capable of colonizing medical implant devices, such as heart valves and catheters, where treatment of the infection often requires the removal of the infected device. This mode of growth is in stark contrast to planktonic, free floating cells which are more easily eradicated with antibiotics. The mechanisms contributing to a biofilm’s tenacity and a planktonic cell’s susceptibility are just beginning to be explored. In this study, we have used a metabolomic approach employing NMR techniques to study the metabolic distinctions between these two modes of growth in P. aeruginosa. One-dimensional proton spectra of fresh growth media were compared with spent media supernatants from planktonic and biofilm cultures. In addition, high resolution magic angle spinning (HRMAS) techniques were employed to collect proton NMR spectra of the corresponding cells. Principal component analysis and spectral comparisons revealed that, judging from the spent media samples, the overall metabolism of planktonic and biofilm modes of growth appeared similar. However, when cells themselves were analyzed they display marked differences relative to each other.Work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under contract number W-7405-ENG-48. The project (05-ERD-026) was funded by the Laboratory Directed Research and Development Program at LLNL.
9:00 PM - H5.5
Early-Stage Staphylococcus Epidermidis Biofilm Development on Solid Surfaces.
Hongyi Mu 1 , Peter Krsko 1 , Jeffery Kaplan 2 , Matthew Libera 1 Show Abstract
1 Dept. of Chemical Engineering, Stevens Institute of Technology, Hoboken, New Jersey, United States, 2 Dept. of Oral Biology, New Jersey Dental School, Newark, New Jersey, United States
Staphylococcus epidermidis (S. epi) is one of the main pathogenic species associated with the infection of percutaneous and indwelling biomedical devices such as catheters and orthopedic implants. Infection of such devices can substantially amplify the cost of a given procedure as well as dramatically affect patient comfort and well being, particularly in the case of indwelling orthopedic implants such as replacement knees and hips. Despite its importance, however, relatively little is known about the interaction between S. epi and synthetic surfaces. We have been working to quantify the early-stage growth of S. epi on a number of synthetic materials including glass and poly(dimethyl siloxane) [PDMS]. To standardize the inoculation conditions, we used an inoculum prepared by culturing a loopful of S. epi cells in a polystyrene dish at 37 oC for 18 h in 20 ml of tryptic soy broth. The thick biofilm that formed was scraped into 3 ml of fresh medium. Clumps of cells were disrupted by high-speed vortex agitation for 30 sec and then passed through a 5 micron sterile syringe filter. Imaging by optical microscopy indicated that the resulting inoculum contained either single cells or pairs of cells, and standard culture techniques measured a concentration of 108 cfu/ml in typical inocula. Larger clusters were not observed. Glass microscope slides cleaned by UV and oxygen-plasma exposure were inoculated for 1 min, then placed in 3 ml of fresh tryptic soy broth and cultured for various times at 37 oC in air. We monitored the cell coverage of the slides by imaging at 100x followed by digital image processing. After a lag phase of approximately 5 hours, the cultures entered an exponential growth period, and the surface was fully covered after 10 hours. Significantly, higher-magnification imaging by scanning electron microscopy shows that the growth remained almost exclusively two dimensional until the area fraction covered reached approximately 60% after 7 hours of culture. Beyond that time point, the cells began to proliferate into multilayered colonies. Similar experiments using PDMS substrates showed that fewer cells initially adhered to the PDMS surface than the glass. These were mostly localized at defects in the PDMS surface. The lag phase was longer on the PDMS, and after 8 hours less than 5% of the slide surface was covered by bacteria. These results indicate that the nature of a synthetic surface can substantially affect the early stages of S. epi biofilm formation, and they raise interesting questions concerning how surfaces may be modified not simply to repel bacterial adhesion altogether but rather to control the development of colonies and biofilms once bacteria have adhered to a surface.
9:00 PM - H5.6
Contribution of Biofilms on Corrosion of AISI 316 Stainless Steel
Julia Claudia Mirza Rosca 1 , Daniel Mareci 2 Show Abstract
1 Mechanical Engineering, Las Palmas de Gran Canaria University, Las Palmas de GC Spain, 2 , Technical University Gh. Asachi, Iasi Romania
9:00 PM - H5.7
Inhibiting the Attachment of Staphylococcus epidermidis Using Self-assembled Monolayers.
Joshua Strauss 1 , Yatao Liu 1 , Eftim Milkani 2 , W. Grant McGimpsey 2 3 , Terri Camesano 1 Show Abstract
1 Chemical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts, United States, 2 Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts, United States, 3 Bioengineering Institute, Worcester Polytechnic Institute, Worcester, Massachusetts, United States
Catheters are important to treating patients suffering from many disorders including dehydration, organ failure, clogging arteries, and build-up of internal fluids. In the USA, catheter related blood stream infections (CRBSIs) are one of the most common nosocomial infections, with estimated annual costs of over $4.5 billion, or $10K/patient. Our work focuses on using a series of self-assembled monolayers (SAMs) to inhibit the attachment of clinically-isolated Staphylococcus epidermidis to a surface. Bacterial retention to SAM-coated slides and bacterial viability were quantified using a dual staining technique (live/dead kit), which consists of propidium iodide and Syto 9. Model proteins such as fetal bovine serum (FBS) and fibronectin were first adsorbed onto the SAMs to more closely mimic bacterial retention under in vivo conditions. The most promising coating to date is SAM with a terminal isopthalic acid group, complexed with silver ions. This molecule resulted in the least amount of retention of S. epidermidis to the slide, even when serum proteins were also present.
9:00 PM - H5.8
Study of Initial Steps of Biofilm Formation: Surface Characteristics and Interaction Forces Between Staphylococcus epidermidis and Serum Protein-Coated Substrata
Yatao Liu 1 , Joshua Strauss 1 , Terri Camesano 1 Show Abstract
1 Chemical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts, United States
With the great need for and rapid development of implanted biomedical devices, the issue of biofilm infection on biomaterials is of increasing importance. Microorganisms grow in a matrix of extracellular polymeric substances that they secrete, which can resist the host immune system and antimicrobial therapy. Hence, surgical excision of the infected device is often the only solution. Biofilm formation is preceded by two steps. Serum proteins in the blood are adsorbed onto the implant surface, followed by bacteria retention on the protein-coated substratum. Physicochemical properties, such as hydrophobicity, surface charge, and surface free energy, of both protein-coated biomaterial surfaces and the microorganisms, influence the initial attachment step. Atomic force microscopy (AFM) was used to directly measure the interaction forces between the microorganisms and the proteins-coated substrata, with force measurements being conducted as a function of contact time. Clinically-isolated Staphylococcus epidermidis was investigated for its interactions with model proteins, such as fetal bovine serum, human fibronectin, and fibrin. Nanoscopic AFM force measurements were correlated to the physicochemical properties from bulk measurements. This research is aimed to provide a fundamental understanding of the initial steps of bacterial adhesion to a biomedical device.
9:00 PM - H5.9
Antimicrobial Properties of Diamondlike Carbon-Metal Films
Roger Narayan 1 , Topher Berry 2 , Mark Morrison 3 , Robin Brigmon 2 Show Abstract
1 Biomedical Engineering, University of North Carolina, Chapel Hill, North Carolina, United States, 2 , Washington Savannah River Company, Aiken, South Carolina, United States, 3 , Smith & Nephew, Inc, Memphis, Tennessee, United States
We have deposited diamondlike carbon-silver, diamondlike carbon-platinum, and diamondlike carbon-silver-platinum thin films using pulsed laser deposition. Transmission electron microscopy of the DLC-silver and DLC-platinum composite films revealed that the silver and platinum form nanoparticle arrays within the diamondlike carbon matrix. The diamondlike carbon-metal composite films exhibited passive behavior at open-circuit potentials. In addition, low corrosion rates were observed during testing in a phosphate-buffered saline electrolyte. The diamondlike carbon-metal composite films were found to be immune to localized corrosion below 1000 mV (SCE). DLC-silver-platinum films demonstrated antimicrobial properties against Staphylococcus bacteria. It is believed that a galvanic couple forms between platinum and silver, which accelerates silver ion release and leads to improved antimicrobial properties. Diamondlike carbon-biofunctional metal nanocomposite films have a variety of potential medical and antimicrobial applications.
H6: Engineering Biofilm-Material Interactions I
Thursday AM, November 30, 2006
Room 205 (Hynes)
9:30 AM - **H6.1
A Combinatorial Approach for the Development of Polymer Surfaces Exhibiting Cell-Specific Adhesion.
Paul Holmes 1 , Patrick Johnson 1 , Larisa Sheihet 1 , Joachim Kohn 1 Show Abstract
1 New Jersey Center for Biomaterials, Rutgers University, Piscataway, New Jersey, United States
A significant drawback in the application of biomedical materials is the occurrence of biomaterial-centred infections. In this work, the development of a rapid-screening assay for the characterization of initial bacterial adhesion on polymer surfaces is presented. By solvent casting films in a 384-well plate, a facile method is afforded employing a fluorescent plate reader to quantify microbial adhesion of stained cells. Data are presented for four species commonly associated with biomaterial centred infections, Staphylococcus epidermidis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. A library of ~40 tyrosine-derived polycarbonate surfaces chemically modified with iodine, poly(ethylene glycol) (PEG) and desaminotyrosyl tyrosine (DT), a negatively charged carboxylic acid group, allowed us to study the complex interplay between these components and their effect on bacterial adhesion.Validation data for the fluorescence-based rapid-screening assay are presented by comparing quantitative data from polymer films solvent cast on a photo-cured 2D grid format. The initial adhesion of all species is shown to be reduced by the progressive incorporation of PEG. However, upon iodination of the tyrosine ring, the PEG effect was significantly reduced. The effect of copolymerization with DT is found to only suppress initial bacterial adhesion at intermediate concentrations (~ 25% DT). Finally, data will be presented on the development of surfaces capable of selectively promoting the adhesion of mammalian cells vs. bacterial cells and the effect on adhesion by pre-adsorbing surfaces with extracellular matrix proteins.
10:00 AM - **H6.2
New Tools to Control the Interactiveness of Surfaces with Proteins, Bacteria and Cells.
Marcus Textor 1 Show Abstract
1 Department of Materials, ETH Zurich, Zurich Switzerland
10:30 AM - H6.3
Novel Engineered Substratum Topographies to Control Bacterial Biofilm Formation.
Kenneth Chung 1 , James Schumacher 2 , Richard Cannon 3 , Patrick Antonelli 3 , Anthony Brennan 1 2 Show Abstract
1 Department of Materials Science & Engineering, University of Florida, Gainesville, Florida, United States, 2 J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, Florida, United States, 3 Department of Otolaryngology, University of Florida, Gainesville, Florida, United States
The irreversible process of biological adhesion on synthetic surfaces is of great concern in the successful development of biomaterials, ultrafiltration, and underwater vessels. Theories on the mechanisms and thermodynamics for bioadhesion are as plentiful as they are diverse. The general consensus for micro and macrofouling on substratum topography has been that organisms preferentially attach to randomly roughened surfaces. However, previously we have successfully demonstrated engineered micro-topographies in polydimethylsiloxane elastomer (PDMSe) that inhibit settlement of algae spores and various larvae (barnacles and tubeworms) of marine organisms. These topographies differ in the sense that they have clearly defined surface structures that are tailored to the critical dimensions of the fouling organism. Our most successful topography, a biomimetic structure based on the skin of fast-moving sharks (Sharklet AF™), has been evaluated for its ability to disrupt and prohibit biofilm formation. In the preliminary study, the engineered topography was designed and fabricated at a 2µm critical dimension and created on the surface of PDMSe (Silastic® T2). Three types of surfaces were used: glass, smooth (unmodified) PDMSe, and Sharklet AF™ PDMSe. Samples were statically exposed to 107 CFU/mL of Staphylococcus aureus in growth medium for up to 12 days to promote biofilm formation. Light and scanning electron micrographs showed abundant biofilm on glass and slightly less on the smooth PDMSe, but no evidence of biofilm on the engineered micro-topography. These results suggest a correlation between the defined microscale topography and the bacteria’s ability to settle and form subsequent biofilm colonies on the surface.
10:45 AM - H6.4
A Novel Polymerizable Antibacterial: Surface Modification of Ti-6Al-4V Alloy and Biofilm Inhibition.
McKinley Lawson 1 2 , Kristi Anseth 1 3 , Christopher Bowman 1 Show Abstract
1 Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado, United States, 2 Medical Scientist Training Program, University of Colorado School of Medicine, Denver, Colorado, United States, 3 Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, United States
Infection following the implantation of orthopaedic hardware remains a serious and expensive complication. Though modern surgical practice has reduced the incidence of infection following total hip arthroplasty, for example, to approximately 1-2%, those patients with periprosthetic joint infection must undergo lengthy antibiotic therapy and quite often surgical revision. The physiologic location of these infections make them inherently difficult to treat due to poor antibiotic penetration into bone and joint spaces and the formation of bacterial biofilms on implant surfaces. Problems including loss of viable bone stock, recurrent infection, and less than ideal treatment options present definite challenges to both surgeon and patient.
We have developed a new antibacterial monomer (a modified form of vancomycin termed VPA) that can be photochemically polymerized and covalently attached to implant grade Ti-6Al-4V orthopaedic alloy. The newly synthesized monomer has been purified by gel filtration chromatography, and its chemical structure has been characterized by 1H NMR spectroscopy. It has been found to maintain biological activity similar to its parent antibacterial agent against Staphylococcus epidermidis ATCC 12228 both in terms of minimum inhibitory concentration and minimum bactericidal concentration.
Following polymerization to appropriately silanized Ti-6Al-4V alloy, a stable, delamination-resistant polymer coating is formed that imparts surface-contact-mediated bacteriostatic/bactericidal action against S. epidermidis ATCC 12228. X-ray photoelectron spectroscopy has been used to characterize alloy surfaces before and after functional modification, and standard microbiological culture techniques have been used to elucidate the surface-contact mode of antibacterial action. In addition, polymer-coated surfaces have been found resistant to biofilm formation when challenged with S. epidermidis ATCC 35984 (prototypical biofilm producer) in an improvised flow chamber. Experiments are currently underway to elucidate the mechanism of biofilm inhibition and to extend the anti-biofilm results to poly(methyl methacrylate) bone cement loaded with VPA.
The coating briefly described here has bacteriostatic/bactericidal properties that do not require release of an active agent. In general, the material offers a large increase in available antibiotic when compared to monolayer coatings, carries an antibacterial that does not require internalization and that is well characterized clinically, and provides multiple layers of antibacterial activity should the outermost layers be removed. Moreover, the polymerization technique described can be well-controlled to yield complex, biologically active co-polymer constructs. It is anticipated that this approach will be useful in the treatment of periprosthetic infections and osteomyelitis and potentially for other medical applications.
H7: Engineering Biofilm-Material Interactions II
Thursday AM, November 30, 2006
Room 205 (Hynes)
11:30 AM - **H7.1
Controlling Biofilm Formation on Surfaces.
Henny van der Mei 1 , Henk Busscher 1 Show Abstract
1 Biomedical Engineering, University of Groningen, Groningen Netherlands
12:00 PM - **H7.2
Microscale Tools for Building and Studying Biofilms
Milan Mrksich 1 Show Abstract
1 , University of Chicago, Chicago, Illinois, United States
This presentation will describe recent developments in analytical methods that can be applied to studying biofilms. Methods include the use of patterned and dynamic self-assembled monolayers to organize cells into complex architectures, mass spectrometry methods to analyze protein and enzyme activities in biofilms and microfluidic systems for controlling the environments of biofilms.
12:30 PM - H7.3
Spatially Controlled Bacterial Aadhesion to Nanopatterned PEG Hydrogels.
Peter Krsko 1 , Jeffery Kaplan 2 , Matthew Libera 1 Show Abstract
1 Dept. of Chemical Engineering, Stevens Institute of Technology, Hoboken, New Jersey, United States, 2 Dept. of Oral Biology, New Jersey Dental School, Newark, New Jersey, United States
12:45 PM - H7.4
Multifunctional Antibacterial Coatings.
Zhi Li 1 , Daeyeon Lee 2 , Xiaoxia Sheng 1 , Robert Cohen 2 , Michael Rubner 1 Show Abstract
1 Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Department of Chemical Enigneering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
H8: Biofilm Development II
Thursday PM, November 30, 2006
Room 205 (Hynes)
2:30 PM - **H8.1
Inter-species Signaling Role in Biofilm Community Development.
Paul Kolenbrander 1 , Alexander Rickard 1 , Robert Palmer 1 Show Abstract
1 Oral Infection and Immunity Branch, National Institutes of Health/NIDCR, Bethesda, Maryland, United States
In nature, bacteria exist on surfaces within multispecies communities called biofilms. Dental plaque is one example of such a biofilm community and harbors over 500 species of bacteria that live in close association with one another, and all oral bacteria coaggregate (physically interact) with a set of specific partner species. This specificity in cell-cell networking contributes towards organized spatiotemporal colonization of biofilms on enamel. Such organization, by definition, requires communication among the participants. Small extracellular signal molecules are the currency of bacterial communication. Intimate cell-cell contact enhances communication by small diffusible signal molecules among oral bacteria in flowing saliva-fed biofilm systems. Communication among genetically distinct bacteria coordinates group behavior and community building. On some complex nutritional sources, mutualistic interactions between two or more species are required to obtain bacterial growth. Individually, the species cannot grow on the complex nutrient. Mutualistic interactions are evident as enhanced growth and interdigitation of mixed-species within communities.We hypothesized that mutualistic growth on saliva was mediated by small molecule signals. One of these signals is AI-2, a product of the LuxS enzyme and which has been proposed to be a universal signal molecule mediating inter-species communication among bacteria. To examine the role of AI-2 in inter-species signaling, we used chemically synthesized DPD which spontaneously cyclizes to form AI-2. DPD was added to dual-species biofilms containing the S. oralis 34 luxS mutant, which cannot synthesize AI-2, and A. naeslundii T14V. Ten-fold incremental concentrations of synthetic DPD were added to saliva reservoirs that fed developing biofilms. In S. oralis 34 luxS mutant-A. naeslundii T14V biofilms, mutualistic interdigitated growth and biovolume were dependent upon DPD concentration. The optimal concentration of DPD lies between 0.08 nM and 0.8 nM. Significantly, in this natural two-species system, the optimal DPD concentration is 100- to 1000-fold lower than the detection limit of the currently accepted AI-2 bioassay, indicating that only a very low concentration of AI-2 is required for these organisms to conduct AI-2-signaled inter-species communication under natural conditions. In context, attached-biofilm cells that are in intimate contact through cell-cell interactions, such as coaggregation, will be exposed to summed concentrations of AI-2. Thus, in a flowing environment, close proximity may be essential for AI-2 mediated mutualism. These results indicate that picomolar concentrations of AI-2 mediate mutualistic interactions among members of a natural dual-species community. Thus, AI-2 is a bona-fide inter-species signal, and its concentration is critical for mutualism between two species of oral bacteria grown under conditions that are representative of the human oral cavity.
3:00 PM - **H8.2
Mechanisms of Antibiotic Tolerance in Staphylococcal Biofilms.
Phil Stewart 1 Show Abstract
1 Center for Biofilm Engineering, Montana State University, Bozeman, Montana, United States
Staphylococcal bacteria that adhere to implanted medical devices or damaged tissue can become the nidus of persistent infections. Key to understanding the recalcitrance of these infections is the protection from killing by antimicrobial agents afforded to microorganisms when growing in biofilms. Three aspects of biofilm tolerance of antimicrobial challenge will be explored in this presentation and illustrated with experimental data from staphylococcal biofilms. A critical question is whether the antimicrobial penetrates the biofilm. Time lapse microscopy was applied to directly observe the diffusive access of antibiotic-sized fluorescent dyes into the center of biofilm cell clusters. Such dyes penetrated large staphylococcal cell clusters within a few minutes. A second mechanism of reduced biofilm susceptibility to antibiotics hinges on physiological differences between attached and suspended microorganisms. Spatial patterns of cellular replication and protein synthetic activity were visualized by pulse labeling DNA with bromodeoxyuridine followed by immunofluorescent detection of brominated DNA, and by using a strain containing an inducible green fluorescent protein, respectively. These techniques revealed regions of active anabolism and also regions of inactivity within staphylococcal biofilms. Biofilms were stained with a fluorogenic esterase substrate. Cells were thereby loaded with an unbound green fluorescent dye that remains trapped inside the cell as long as the cell membrane is intact. If membrane integrity is compromised, for example by an antimicrobial agent, the dye leaks out and the cell becomes dark. This method was used to prepare time-lapse movies of the action of antimicrobial agents on biofilms. These latter data suggest that phenotypic variation from cell to cell may be an important component of the biofilm defense.
3:30 PM - H8.3
Vacuum Ultraviolet Postionization for Mass Spectrometry of Small Molecule Analytes in Bacterial Biofilms
Praneeth Edirisinghe 1 , Luke Hanley 1 , Manshui Zhou 1 , Kelly Skinner-Nemec 2 , Carol Giometti 2 , Jerry Moore 3 2 , Jerry Hunt 2 , Michael Pellin 2 Show Abstract
1 Chemistry, MC 111, University of Illinois at Chicago, Chicago, Illinois, United States, 2 , Argonne National Laboratory, Argonne, Illinois, United States, 3 , MassThink, Inc., Naperville, Illinois, United States
Mass spectrometric analysis and imaging of intact microbial biofilms are difficult with established methods. A new experimental strategy is discussed for analyzing small molecule analytes within intact biofioms: laser desorption followed by postionization with 7.87 eV radiation of molecular analytes whose ionization potentials have been lowered by chemical derivatization with an aromatic tag [1-3]. Postionization mass spectrometry with derivatization is developed on small peptides with aromatic or native tags such as a tryptophan residue. The new method is then applied to the detection of a quorum sensing peptide in a Bacillus subtilis bacterial biofilm. Finally, detection of an antibiotic is demonstrated by direct 7.87 eV postionization, without derivatization. These mass spectrometric methods show promise for the study of antibiotic resistance in microbial biofilms as well as other studies of small molecule analytes within complex biological matrices.1. P.D. Edirisinghe et al., Anal. Chem. 76 (2004) 4267.2. L. Hanley et al., Appl. Surf. Sci. (2006), in press.3. P.D. Edirisinghe et al., Anal. Chem. (2006) in press.
3:45 PM - H8.4
Spectral Self-interference 4Pi Microscopy
Mehmet Dogan 1 , Bennett Goldberg 1 2 , Anna Swan 2 , M. Selim Unlu 2 1 Show Abstract
1 Physics Department, Boston University, Boston, Massachusetts, United States, 2 ECE Department, Boston University, Boston, Massachusetts, United States
In the past decade there has been a great advancement in the fluorescence microscopy with the development of 4Pi confocal microscopy [1,2] that increases the axial resolution of standard confocal microscopes 3- to 7-fold depending on the mode of operation. Spectral Self-interference Fluorescence Microscopy (SSFM)  has also been introduced to determine the location of fluorescent molecules above a reflecting surface with nanometer precision. SSFM has been used to determine the position of fluorescent markers attached to sub-cellular structures such as lipid bilayer membranes and DNA strands revealing conformational information .Despite the unprecedented axial precision capability, SSFM lacks high lateral resolution for planar substrates when emission is collected from one side of the sample. We have developed a 4Pi microscope with a spectroscopic detection system that combines the two technologies, 4Pi and SSFM, to localize fluorescent emitters axially with nanometer precision without sacrificing the lateral resolution. In this configuration the emitters are excited through two opposing high numerical aperture objectives; emitted signal is coherently collected by the same objectives and coupled to a spectroscopy system. The common foci of the two objectives allow the use of high NA objectives without phase averaging for interference measurements. The path length difference of the two interference arms is adjusted to several tens of micrometers to induce modulations in the spectrum of the emitter. In order to test the axial position localization, a monolayer of Alexa Fluor 488 dye deposited on a glass cover slip was placed in the common foci of the objectives of the 4Pi microscope and spectrum of the signal was measured for two different axial positions of the monolayer 5 nm apart. The spectral response clearly showed a shift in spectral fringes corresponding to the change in the axial position of the fluorophores.With the fluorescent localization technology we have developed, we are able to measure nanometer scale changes in the axial position of fluorescent emitters. Spatial orientation of proteins, organelles and other type of structures can also be studied with this high localization capability.  S. Hell , E. H. K. Stelzer, “Properties of a 4Pi confocal fluorescence microscope”, JOSA A, 9, 12 , pp .2159-2166 (1992) A. Egner, S. Verrier, A. Goroshkov, H.D. Soling, S.W. Hell, “4Pi Microscopy of Golgi apparatus in live mammalian cells”,J. Str. Biol., Vol. 147, pp .70-76 (2004) L.A. Moiseev, C.R. Cantor, I. Aksun, M. Dogan, B.B. Goldberg, A.K. Swan and M. S. Ünlü, “Spectral self-interference fluorescence microscopy”, Journal of Applied Physics, Vol.96, pp.5311-5315 (2004) Lev Moiseev, Anna K. Swan, M. Selim Ünlü, Bennett B. Goldberg, Charles R. Cantor, “DNA Conformation on Surfaces Measured by Fluorescence Self-Interference”, PNAS, Vol. 103, No. 8, pp. 2623-2628 (February 21, 2006)