Nehal Abu-Lail Washington State University
Wendy Goodson Air Force Research Laboratory
BrianH. Lower Ohio State University
Mark Fornalik Industrial Biofouling Science, LLC
Roberto Lins Federal University of Pernambuco
Air Force Research Laboratory
Office of Naval Research
KK5: Poster Session: Microbial Life on Surfaces
Tuesday PM, April 26, 2011
Exhibition Hall (Moscone West)
KK1: Nanoscale Investigations of Microbial Properties and Interactions
Tuesday PM, April 26, 2011
Nob Hill CD (Marriott)
9:45 AM - **KK1.1
Physico-chemical Mechanisms of Initial Microbial Adhesion to Surfaces and Bond-maturation.
Henny Van der Mei 1 Show Abstract
1 Biomedical Engineering, University Medical Center Groningen, Groningen Netherlands
Upon initial microbial adhesion to a surface, multiple events occur that are unlikely to be captured in a single mechanism. Measurements of residence-time dependent desorption in flow displacement systems have shown that the desorption probabilities of initially adhering organisms strongly decrease within seconds to minutes after first contact. Interaction force measurements using Atomic Force Microscopy (AFM) have supported that the bond strength between adhering organisms and substratum surfaces increases within that time span. Surface thermodynamic analyses, application of DLVO-theories and Poisson analysis of retract force-distant curves from AFM indicated that this bond-maturation is due to the progressive involvement of acid-base interactions. Acid-base interactions require close approach between the interacting surfaces, which is firstly achieved by attractive, long-range Lifshitz-Van der Waals forces. Once brought in the close vicinity of a surface, bond-maturation follows as a result of interfacial re-arrangements. Interfacial re-arrangements in the region between an adhering organism and a surface are often associated with changes in the rigidity of the coupling. Quartz Crystal Microbalance with Dissipation (QCM-D) allows monitoring of these interfacial re-arrangements or changes in coupling-rigidity from a change in its dissipation signal, but has been little applied yet to study this aspect of microbial adhesion. Application of physico-chemical mechanisms to explain microbial adhesion to surfaces requires better knowledge of the interfacial re-arrangement occurring immediately after adhesion than hitherto available.
10:15 AM - KK1.2
Using Atomic Force Microscopy to Map the Distribution of Protein Molecules on the Surface of Live Microorganisms.
Brian Lower 1 Show Abstract
1 School of Environment & Natural Resources, The Ohio State University, Columbus, Ohio, United States
Antibody-recognition force microscopy (Ig-RFM) is a relatively new technique that uses atomic force microscopy (AFM) to study antibody-antigen interactions, identify proteins, and map the nanoscale distribution of protein molecules in complex biological structures. This is a powerful technique because it permits the study of live cells or isolated biomolecules (e.g., protein) under physiological conditions. Here we describe the use Ig-RFM to probe the cell surface of live bacterial cells using AFM tips that were functionalized with protein-specific antibodies. In doing so we were able to identify specific proteins that were targeted to the external cell surface. We were also able to map the distribution of protein molecules on the cell surface and relative to the substrate on which the bacteria were growing.
10:30 AM - KK1.3
Effects of the Temperature and Ionic Strength of Growth Conditions on the Nanoscale Adhesion of L. monocytogenes EGDe to Silicon Nitride.
Pinar Gordesli 1 , Nehal Abu-Lail 1 Show Abstract
1 Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington, United States
The food-borne pathogen Listeria monocytogenes is a Gram-positive, facultatively anaerobic and rapidly growing bacterium, with the ability to form persistent biofilms. It is transmitted to animals and humans by contaminated food and can cause listeriosis, a severe disease with high hospitalization and fatality rates. L. monocytogenes can adapt to survive and grow in a wide range of environmental conditions allowing this pathogen to overcome the safety barriers in food processing and storage. In this study, the nanoscale adhesion forces of L. monocytogenes EGDe to a model surface of silicon nitride were quantified by using atomic force microscopy (AFM) for bacterial cells grown under five different temperatures (10, 20, 30, 37 and 40oC) and five different ionic strengths (0.005, 0.05, 0.1, 0.3 and 0.5M NaCl). Our findings for the cells grown under different temperatures show that the adhesion ability of L. monocytogenes EGDe change due to the growth temperature. It was observed that L. monocytogenes EGDe adhesion affinity to model inert surfaces achieved its highest values at 30oC followed by those quantified at 37oC. Our nanoscale measurements agree well with studies in the literature that quantified the effects of growth temperature on the ability of the same bacteria to form biofilms. The adhesion affinity of L. monocytogenes to silicon nitride surface for cells grown under various ionic strength conditions are currently ongoing. Our results will be used to elucidate some of the fundamental aspects of the survival mechanisms of L. monocytogenes EGDe under physical conditions of stress.
10:45 AM - KK1: Nanoscale
11:15 AM - **KK1.4
Mineral Surfaces, Amino Acids, and the Origins of Life.
Robert Hazen 1 , Dimitri Sverjensky 2 1 , Kateryna Klochko 1 , Adrian Villegas-Jimenez 1 , Namhey Lee 2 1 , Charlene Estrada 2 1 Show Abstract
1 Geophysical Laboratory, Carnegie Institution, Washington, District of Columbia, United States, 2 Earth & Planetary Sciences, Johns Hopkins University, Baltimore, Maryland, United States
The chemical origins of life occurred in several steps, each of which increased molecular complexity and patterning of Earth’s near-surface environment. The first step, abiotic synthesis of amino acids, sugars, lipids and other essential molecular bio-building blocks, has been well documented through experiments that mimic environments on Earth and in space. However, the second step, which includes selection, concentration and assembly of those molecules into the functional membranes and polymers of life, is less well understood. Our research team investigates how mineral surfaces might have played a role in the critical transition from a dilute, indiscriminate prebiotic soup to micro-environments that were concentrated in molecules poised to foster life. Studies on adsorption of biomolecules onto common mineral surfaces, including competitive molecular adsorption, batch adsorption, molecular stability and decomposition, and potentiometric titration experiments, coupled with extended triple-layer surface complexation and density functional theory modeling, point to at least four plausible roles that such interactions may have played in life’s origins. (1) Minerals are able to concentrate molecules from dilute solutions by factors of 1000 or more, thus potentially overcoming the problem of a dilute prebiotic soup. (2) Molecules bound to mineral surfaces may be much more stable than those in solution, thus countering a strong objection to the hypothesis that life’s origins occurred at or near a hydrothermal system. (3) Some minerals are able to select and concentrate specific molecules, notably chiral (right- versus left-handed) amino acids, thus providing a possible mechanism for the origins of biological handedness. (4) Finally, mineral surfaces may juxtapose and align molecules to facilitate polymerization and other modes of biomolecular assembly. Our work also underscores the importance of including realistic prebiotic contributions in any origin of life scenario. Any geochemical model for life’s origins must thus incorporate such physico-chemical complexities as cycles, gradients, fluxes, and interfaces.
11:45 AM - **KK1.5
Molecular Interactions of Staphylococcus aureus and Implanted Biomedical Devices.
Nadia Casillas-Ituarte 1 , Supaporn Lamlertthon 2 , Eric Taylor 1 , Alex DiBartola 1 , Vance Fowler 2 , Steven Lower 1 Show Abstract
1 School of Earth Sciences, Ohio State University, Columbus, Ohio, United States, 2 Duke Clinical Research Institute, Duke University, Durham, North Carolina, United States
Staphylococcus aureus is responsible for a large percentage of infections associated with implanted biomedical devices. The molecular interactions of this bacterium with a fibronectin-coated probe (model of an implanted device) were analyzed with atomic force microscopy. A group of 100 different isolates of this bacterium were obtained from either patients with an infected cardiac device (invasive group) or healthy carriers (control group). The average binding-force frequency is statistically different (p = 0.003) between the two populations, suggesting that a microorganism’s “force taxonomy” may provide a fundamental and practical indicator of the pathogen related risk that infections pose to patients with implanted medical devices.
12:15 PM - KK1.6
Extracellular DNA Enhances Bacterial Adhesion and Aggregation by Influencing Acid – Base Interactions.
Theerthankar Das 1 , Prashant Sharma 1 , Bastiaan Krom 1 , Henk Busscher 1 , Henny van der Mei 1 Show Abstract
1 Biomedical Engineering, W.J. Kolff Institute, University Medical Center Groningen and University of Groningen, Groningen Netherlands
Significance and objectives: Bacteria in nature attach to nearly all surfaces and form biofilms with the help of self produced extracellular polymeric substances (EPS). Extracellular DNA (eDNA) present in EPS acts as an adhesive and strengthens the biofilm. In this study we investigated the effect of naturally occurring eDNA on adhesion and aggregation of several bacterial strains and there mechanism of interaction.Methods: Initial bacterial adhesion of Staphylococcus epidermidis 1457, S. epidermidis 1457 ΔatlE and Streptococcus mutans LT11 to hydrophilic and hydrophobic substrata and surface aggregation in presence and absence of eDNA were studied using a parallel plate flow chamber. Physico-chemical surface characteristics of S. epidermidis 1457 and its mutant ΔatlE were determined by contact angle and zeta potential measurements. Adhesion force and bond formation between S. mutans LT11 and substratum or between two S. mutans LT11 in the presence and absence of eDNA was measured by Atomic force microscopy (AFM). Extended DLVO theory was used to calculate total interaction energies of staphylococcal adhesion to the substrata and bacteria in aggregates. All experiments were done in phosphate buffer saline.Results: In the presence of eDNA all bacterial strains showed a higher initial deposition rate and adhered in higher numbers after 60 min to both hydrophilic and hydrophobic substrata when compared to adhesion in the absence of eDNA. On hydrophilic surfaces in the presence of eDNA an increase in the percentage of bacteria in large aggregates was observed compared to aggregates on hydrophobic surfaces. Physico-chemical surface characterization showed that removal of eDNA, from S. epidermidis 1457 surface by DNaseI treatment decreased its hydrophobicity similar to the hydrophobicity of its mutant strain ΔatlE, which lacks production of eDNA. Whereas the zeta potential of the staphylococcal cell surfaces became less negative upon removal of eDNA. Accordingly, favourable total interaction energies in the presence of eDNA became unfavourable in the absence of eDNA due to changes in acid-base interaction energies. However Lifshitz-Van der Waals and electrostatic interaction energies remains attractive and repulsive respectively regardless of eDNA presence. AFM measurements showed significant increases in adhesion force and bond formation between S. mutans LT11 and substratum or between two S. mutans LT11 in presence of eDNA compared to the absence of eDNA. Presence of eDNA molecules on bacterial cell surfaces enhance exchange of electrons between interacting substratum or bacteria and thus increases bond formation.Conclusions: The presence of eDNA on bacterial cell surfaces enhances adhesion kinetics, aggregation, forces of interaction and bond formation due to the involvement of acid-base interactions.
KK5: Poster Session: Microbial Life on Surfaces
Tuesday PM, April 26, 2011
Exhibition Hall (Moscone West)
6:00 PM - KK5.1
The Effects of Sodium and Calcium Binding on the Structure of the LPS Membrane of Pseudomonas Aeruginosa.
Agrinaldo Nascimento 1 , Roberto Lins 1 Show Abstract
1 Química Fundamental, Universidade Federal de Pernmbuco - UFPE, Recife , Pernambuco, Brazil
Bacterial Lipopolysaccharide (LPS) molecules consist of a lipid A - an endotoxin, a nonrepeating “core”oligosaccharide, and the O-antigen, a long variable polysaccharide chain. LPS is credited as the major factor of virulence in humans and other mammals. It acts as a weak non-specific antigen, which is poorly neutralized by antibodies. Unlike its planctonic counterpart, Gram-negative bacteria when forming biolfims can cause sceptic shock, fever and even lead to death. These microorganisms have a great metail ions sorption capacity in their cell walls. Such characteristic is very important to explain phenotypical variation, mobility and bioavailability of metals in environment. Metal ions and their complexes have been reported to bind to the negatively charged phosphoryl and carboxyl groups in the LPS. The availability of metal ions is highly dependent on the local environment. In turn, ionic coordination number, solvation shell and net charge are expected to influence the packing, stability, adhesion and permeability properties of these membranes. In addition, the pH has been reported to have a significant impact the adhesion of LPS to different materials. Therefore, the elucidation of the interactions between LPS and different metal ions is expected to shed light into problems such as antibiotic resistance and material adhesion. In this work, we have performed quantum calculations and molecular dynamics simulations of the LPS membrane of Pseudomonas aeruginosa in the presence of several concentrations of Na+ and Ca2+ ions. While both ions are abundant ions in physiological media, Ca2+ ions are commonly found in the LPS of soil-living bacteria. On the other hand, these microorganisms are exposed to high concentration of Na+ ions in infecting tissues. Changes in the pKa values for the phosphoryl and carboxyl groups were monitored as a function of the ratio Ca2+/Na+ ions in the LPS membrane. Differences in the LPS lateral diffusion and acyl chain order parameters suggest that metal ion and pH can dramatically affect membrane dynamics, surface charge and stability.
6:00 PM - KK5.10
Interaction of Alcohols and Ions with Biofilms Using Microrheology.
Anderson Sunda Meya 1 , Jasmine Jones 1 , Kamirah Demouchet 1 , Fook Cheong 2 , Simone Duarte 3 , David Grier 2 Show Abstract
1 Department of Physics , Xavier University of Louisiana, New Orleans, Louisiana, United States, 2 Department of Physics & Center for Soft Matter Research, New York University, New York, New York, United States, 3 Department of Basic Science and Craniofacial , New York University College of Dentistry, New York, New York, United States
Dental Biofilms, the extracellular matrix formed by the growth of polysaccharides from sucrose, provide the optimal environment enabling the pathogenesis of the bacteria, Streptococcus mutans (S. mutans). The presence of S. mutans is primarily associated with dental caries or tooth decay, the most virulent disease today. The objective of this research is to understand the effects of various ions and alcohols on the viscoelastic properties, the mechanical properties, and the intermolecular interactions of the dental biofilms. A holographic microrheological study allows a non-invasive method to accurately measure the physical properties of the dental biofilm in three-dimensions with nanometer resolution by measuring precisely probes particles’ three-dimensional trajectories. Applying this technique to biofilms, in particular, shows promise for high-throughput combinatorial screening of candidate therapeutic or remedial agents.
6:00 PM - KK5.11
Use of Antimicrobial Peptides and Proteins for Prevention of Biofilm Formation.
Caitlin Knight 2 1 , Matthew Dickerson 2 1 , Lawrence Brott 1 , Wendy Goodson 1 Show Abstract
2 , UES Inc., Dayton, Ohio, United States, 1 Nanostructured and Biological Materials Branch, Materials and Manufacturing Directorate, Air Force Research Laboratory, Air Force Research Laboratory, Wright-Patterson AFB, Ohio, United States
Biofilms are communities of bacteria that colonize surfaces. They are problematic in both medical and industrial settings where biofilms cause chronic infections on medical implants and foul pipelines and storage tanks. Their ability to colonize many different substrates and to resist to biocidal treatment makes freeing biofilms from surfaces a challenging and costly task. Regular maintenance intervals require pipelines to be shut down and application of biocidal agents may be costly or have negative environmental impacts. Thus, preventing initial biofilm formation is a much more economical solution. We propose developing coatings that will help prevent initial attachment and maturation of biofilms by covalently attaching AMPs (antimicrobial peptides) and enzymes, such as DNase, to surfaces. Part of the innate host defense system, antimicrobial peptides have evolved over millions of years to provide protection against a wide range of microbes, including Gram positive and Gram negative bacteria, and fungi. AMPs are small (1-10 kDa), cationic, amphipathic peptides that are resistant to degradation. Unlike many antibiotics whose efficacy requires cells to be metabolically active, AMPs act by forming pores in the cell membrane, causing leakage of cytosol. Using silane chemistry and the heterobifunctional crosslinker PMPI (N-[p-Maleimidophenyl]isocyanate) the antimicrobial peptide Cecropin A was attached to glass surfaces and successfully decreased the number of cells attached to the surface after 24 hours.
6:00 PM - KK5.12
Bacterial Effects on CaCO3 Crystallization.
Jenny Cappuccio 2 , Veronica Pillar 1 , Caroline Ajo-Franklin 1 Show Abstract
2 Earth Sciences Division, Lawrence Berkeley National Lab, Berkeley, California, United States, 1 Materials Science Division, Lawrence Berkeley National Lab, Berkeley, California, United States
Geologic carbon dioxide sequestration, the underground storage of carbon dioxide, will be an essential component of global climate change mitigation. Carbonate minerals are a promising form of stable CO2 storage, but their formation occurs on a geologic timescale, not than human timescale. Many microbes can influence the precipitation of carbonate minerals; however the mechanisms of such mineralization are largely unknown. Hypothesized mechanisms include metabolic processes altering pH and supersaturation, as well as interactions with cell surface molecule , i.e. extracellular polymeric substances (EPS), cell membrane, and protein surface layers (S-layers), that may alter mineral nucleation. This work investigates these mechanisms by allowing calcium carbonate (CaCO3) to form in abiotic or microbial solutions of Escherichia coli (E. coli) or Synechocystis sp. PCC 6803 (Syn. sp. 6803) with varying calcium ion concentrations, via the ammonium carbonate diffusion method. Both the resulting CaCO3 and bacteria was imaged using optical microscopy. Surprisingly, formation of crystalline CaCO3 was accelerated in the presence of both species. This rate acceleration also occurred for metabolically inactive bacteria, suggesting metabolic change was not the operating mechanism under these conditions. Calcium carbonate crystals increased in number as cell density increased. Scanning electron microscopy and fluorescent microscopy show that both species of bacteria cluster on the edges and crevices of the crystals, further supporting this idea. Bacterial surface charge was assessed using zeta potential measurements and correlated to biomineralization experiments. From these results, we postulate that the charged bacterial surfaces attract Ca2+ ions, serving as nucleation sites for CaCO3, thereby accelerating crystal formation. These observations provide substantive evidence for a non-specific nucleation mechanism, and stress the importance of microbes, even dead ones, on the rate of formation of carbonate minerals. This work also indicates that additional microbial engineering could optimize these interactions and be used to implement the sub-surface sequestration of CO2 as stable mineral carbonates on an accelerated timescale.
6:00 PM - KK5.3
Biodegradable Star Polymers for Antimicrobial Applications.
Daniel Coady 1 , James Hedrick 1 , Kazuki Fukushima 1 , Yi-Yan Yang 2 Show Abstract
1 , IBM, San Jose , California, United States, 2 , IBN, The Nanos Singapore
Biocompatible and biodegradable antimicrobial materials are becoming increasingly important due to the rise in antibiotic resistant bacteria. Previously, our efforts have utilized the self-assembly and aggregation of amphiphilic poly(carbonate) block copolymers with pendent tetraalkylammonium groups for such applications. In an effort to simplify the self-assembly process we have synthesized analogous amphiphilic block-star polymers to eliminate the need for aggregation and create more consistent size distributions. These advancements are envisioned to mimic natural antibiotic proteins for potential use as polymeric drugs.
6:00 PM - KK5.4
Rechargeable Antimicrobial and Biofilm-controlling Biomaterials.
Yuyu Sun 1 Show Abstract
1 Biomedical Engineering, The University of South Dakota, Sioux Falls, South Dakota, United States
Despite major medical advances, infectious diseases continue to be the third leading cause of death in the United States and the leading cause worldwide. The use of antimicrobial devices can be a potentially effective approach to reduce such risks. However, most of the currently available antimicrobial devices are only effective for a short period of time (days), and are not suitable for long-term applications. Novel rechargeable antimicrobial and biofilm-controlling biomaterials are developed to fight disease. The new biomaterials act as “rechargeable batteries” that bind and then slowly release various antimicrobial agents to prevent microbial colonization and biofilm formation. Extended use consumes antimicrobials and reduces disease-fighting activities. However, the consumed antimicrobials can be repeatedly recharged to extend antimicrobial duration for long-term use. In recharging, antimicrobials can be changed/rotated to enhance antimicrobial potency and reduce the risk of microbial resistance. At the time when the infections are cleared, the remaining drugs in the biomaterials can be “quenched” to stop the therapy when no further drug release is need. If needed, the biomaterials can be recharged again to re-initiate drug release. The new biomaterials are attractive novel drug carriers for multiple medical/dental applications in which the devices are readily accessible for recharging. Representative examples include long-term central venous catheters, dentures, tubing in dental unit waterlines and ventilators, etc. The new biomaterials can also be used for the antimicrobial treatment of high-touch, high-risk surfaces in healthcare settings and other related fields to reduce the risk of cross-contamination and cross-infection.
6:00 PM - KK5.5
Potential of Amoxicillin based Chitosan Nanoparticle against Escherichia Coli Biofilm.
Vivek Pandey 1 , Sandeep Singh 1 , Preetam Varma 1 2 , Himanshu Pandey 2 , Vikas Pruthi 3 , Ravi Tewari 1 , Vishnu Agarwal 1 Show Abstract
1 , Motilal Nehru National Institute of Technology, Allahabad, India, Allahabad India, 2 , Sam Higginbottom Institutes of Agriculture, Technology & Sciences, Allahabad, India., Allahabad India, 3 Department of Biotechnology, Indian Institute of Technology, Roorkee, India, Roorkee India
Escherichia coli is a gram negative bacilli generally reside in lower intestine of endotherms. The harmless strains are part of the normal flora of the human gut, and can benefit their hosts by producing vitamin K2 and by preventing the establishment of pathogenic bacteria within the intestine. The pathogenic form of E. coli strains can cause serious food poisoning in humans including gastroenteritis, urinary tract infections, and neonatal meningitis. In some cases, virulent strains can cause for haemolytic-uremic syndrome (HUS), peritonitis, mastitis, septicemia and gram-negative pneumonia. E. coli causes infections mostly in its biofilm mode of growth which is characterized by production of exopolysaccharide (EPS) and enhanced antibiotic resistance. The resistance of biofilm residing cell against the drug generally developed due to penetration barrier, expression of drug resistant phenotypes or reaction of drug with EPS components. To check the efficacy of commonly used antibiotic amoxicillin against E. coli in absence of reaction of drug molecule with EPS components, in the present work we analyzed effect of chitosan based nanoparticle mediated drug delivery system as a potent biofilm inhibitor. The E. coli biofilm was developed on polypropylene pieces at 37C for 48h using CDC biofilm reactor. The chitosan nanoparticles were synthesized by ionotropic gelation method. Amoxicillin with minimum inhibitory concentration (MIC90) of 2.5 µg/ml was used to inhibit the biofilms either directly or encapsulated within chitosan nanoparticle. It was found that the biofilm inhibition was about 35% more in case of drug releasing chitosan nanoparticle. The study demonstrated that a significant amount of drug reacts with EPS components and hence the effective dosage available to sessile community is less than the amount added.
6:00 PM - KK5.6
Effect of Nano-and-Micro Crystalline Diamond Surfaces in the Size of Bacteria.
Adriana Collazo 2 , Olga Medina 1 , Jose Nocua 1 , Ramon Gomez-Moreno 5 , Daniel Montano 1 , Javier Avalos 3 4 , Concepcion Rodriguez 5 , Gerardo Morell 1 3 Show Abstract
2 Department of Biology, University of Puerto Rico, San Juan, Puerto Rico, United States, 1 Department of Physics, University of Puerto Rico, San Juan, Puerto Rico, United States, 5 Department of Biology, University of Puerto Rico at Bayamón, Bayamón, Puerto Rico, United States, 3 , Institute for Functional Nanomaterials, University of Puerto Rico, San Juan, Puerto Rico, United States, 4 Department of Physics, University of Puerto Rico at Bayamón, Bayamón, Puerto Rico, United States
The following work shows the changes in the bacterial division of the P. aeruginosa on nanocrystalline diamond (NCD) surfaces and compares it with microcrystalline diamond (MCD), stainless steel AISI 304 (SS), silver (Ag), polyethylene (Poly) and copper (Cu), with the purpose of comparing their antibacterial efficiency with NCD's. The results show that the inhibitory properties of NCD become perceivable just after 13 hours of bacterial transference. NCD was shown to be a good bactericidal surface, overmatched only by copper. The polyethylene, silver, stainless steel and MCD were found to be less inhibiting than NCD. Valuable properties of NCD as the high resistance to oxidation and corrosion, the extreme mechanical hardness and the biological compatibility with blood and tissue makes it more useful than copper. In order to study the bactericidal properties of each surface and their effect on the bacterial size, different characterization techniques were employed, such as scanning electron microscopy (SEM), atomic force microscopy (AFM), the measurement of the contact angle and the evaluation of the colonization factors via a statistical analysis of the bacterial count. These techniques helped to establish a correlation between the bacteria size (NCD: 1.82μm y MCD: 3.02μm), the roughness, the hydrophobicity/hydrophilicity and the colonization susceptibility of the given materials.
6:00 PM - KK5.7
Functionalized Single Wall Carbon Nanotubes as an Antimicrobial Agent for Pseudomonas Aeruginosa and Staphylococcus Aureus.
D. Hernandez-Lugo 1 2 3 , O. Medina 3 4 , J. Nocua 3 4 , M. Rivera 4 , A. Colon 6 , A. Collazo 6 , D. Montano 4 , A. Borrero 7 , R. Rivera 7 , J. Avalos 3 5 , G. Morell 3 4 , B. Weiner 1 3 Show Abstract
1 Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, San Juan United States, 2 Center for Advance Nanoscale Materials, NASA, University of Puerto Rico, Rio Piedras Campus, San Juan United States, 3 Institute of Functional Nanomaterials, University of Puerto Rico, Rio Piedras Campus, San Juan United States, 4 Department of Physics, University of Puerto Rico, Rio Piedras Campus, San Juan United States, 6 Department of Biology, University of Puerto Rico, Rio Piedras Campus, San Juan United States, 7 Department of Biology, University of Puerto Rico, Bayamon Campus, Bayamon United States, 5 Department of Physics, University of Puerto Rico, Bayamon Campus, Bayamon United States
Carbon nanotubes (CNTs) have emerged as a novel and promising class of nanomaterials with unique optical, electrical, mechanical, and thermal properties. Several studies have demonstrated that single-walled CNTs (SWCNTs) in suspensions have strong antimicrobial activities to bacterial cells. As part of this study we analyze the antimicrobial activity of these CNTs in relation to their surface group. Functionalized SWCNTs (-COOH) and NO-functionalized SWCNTs have been used in order to determine their inhibitory efficiency. As part of this study we found that functionalized SWCNTs have a higher antimicrobial activity when compared to NO-functionalized SWCNTs. The antimicrobial activity of CNTs was examined by looking at the growth curve using 640nm wavelength. Functionalized and NO-functionalized single-wall carbon nanotubes were characterized by using scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Raman Spectroscopy, X-ray Photoelectron Spectroscopy (XPS) and IR.
6:00 PM - KK5.9
Biofilm Elimination and Detachment Using Photocatalytic TiO2 Surfaces.
Yanling Cai 1 , Hakan Engqvist 2 , Maria Stroemme 1 , Ken Welch 1 Show Abstract
1 Nanotechnology and Functional Materials, Uppsala University, Uppsala Sweden, 2 Division for Materials Science, Uppsala University, Uppsala Sweden
Biofilms are a prevalent mode of microbial life found in nature. Bacteria in biofilms are 10-1000 times more resistant to antibiotics than when in planktonic form, and in many cases are developing resistances to existing antibiotics; as such, there is a growing requirement for new strategies in biofilm elimination. Dental plaque is an example of a biofilm that often results in dental diseases such as caries. Furthermore, dental plaque is often associated with restorative dentistry materials, which often enhance and increase the accumulation of bacteria. The aim of the present work was to perform an in vitro evaluation a novel dental adhesive containing photocatalytic TiO2 nanoparticles for on-demand biofilm elimination and detachment through ultraviolet (UV-A) irradiation. The dental adhesive was prepared by adding 20 wt% TiO2 nanoparticles to a light cured resin matrix of HEMA and bis-GMA polymers. Spontaneous hydroxylapatite formation on the surface of the adhesive samples upon storage in simulated body fluid indicated good bioactive properties, and suggests that the material should better integrate with the adjacent tooth tissue. The nanoparticle-containing adhesive was shown to be photocatalytic by the degradation of rhodamine-B dye under UV-A irradiation. Biofilm elimination and detachment testing was accomplished by irradiating the biofilm-coated surface of the adhesive with UV-A light. Detachment of the biofilm was assessed after the UV treatment by measuring the amount of biofilm remaining on the surface after the samples were subjected to an ultrasound treatment. It was found that UV-A irradiation led to a significant increase in detachment of biofilm from the adhesive surface compared to the non-irradiated adhesive surface. Results also showed that a dose of approximately 6 J/cm2 led to a 1 log reduction in the concentration of viable bacteria in a biofilm that was grown on the surface of the adhesives. As much as 7 log reduction in bacteria was achieved with a total UV-A dose of 45 J/cm2.
Nehal Abu-Lail Washington State University
Wendy Goodson Air Force Research Laboratory
BrianH. Lower Ohio State University
Mark Fornalik Industrial Biofouling Science, LLC
Roberto Lins Federal University of Pernambuco
Air Force Research Laboratory
Office of Naval Research
KK6: Antimicrobials and Antifouling Coatings
Wednesday AM, April 27, 2011
Nob Hill CD (Marriott)
9:15 AM - **KK6.1
Starved Bacterial Biofilms and the Possible Origin of Life between Mica Sheets.
Helen Greenwood Hansma 1 Show Abstract
1 Department of Physics, University of California at Santa Barbara, Santa Barbara, California, United States
Biofilms of Pseudomonas aeruginosa bacteria respond differently to nutrient limitation than bacteria grown in liquid culture. While bacteria in liquid culture become round when starved, P. aeruginosa in biofilms become elongated when starved (Steinberger, et al., 2002, Microbial Ecol. 43:416). In both cases, the response to starvation serves to maximize the bacterial surface area that is available for nutrient uptake. This research on biofilms was done by Atomic Force Microscopy, which also led indirectly to the hypothesis that life might have originated between mica sheets (Hansma, 2010, J. Theor. Biol. 266:175). The spaces between mica sheets may have served as cells or compartments within which life could originate and evolve before free-living cells existed. The mica lattice spacing of 0.5 nm is comparable to the periodicities of biological macromolecules such as single-stranded nucleic acids, carbohydrates, and proteins. The potassium ions that hold mica sheets together may be the original source of the high potassium ion concentration in the cytoplasms of cells. Error tolerance is also extremely high in the Mica Hypothesis for the origin of life. Error tolerance is a major requirement for the origin of life, because almost everything is likely to go wrong. With a million mica sheets per millimeter of thickness, mica provides the potential for a huge redundancy in prebiotic molecules of all types.
9:45 AM - **KK6.2
Hilary Lappin-Scott 1 , Sara Burton 2 Show Abstract
1 Centre for Nanohealth, Swansea University, Swansea, Wales, United Kingdom, 2 Biosciences, University of Exeter, Exeter, Devon, United Kingdom
Various nanomaterials (both manufactured and naturally produced) reach natural environments, for example from waste waters containing healthcare products and pharmaceuticals or be taken into the human body as nanomaterials used for targeted drug delivery. Consequently, they will be in contact with interacting microbial communities carrying out essential functions in these habitats. Given this, little is known of the ecotoxicity of various nanomaterials on microorganisms and specifically whether they disrupt such processes. However there is some evidence that nanoparticles disrupt lipid bi-layers and affect some genetic and transcriptional processes. Our work includes how to effectively monitor nanoparticle-microbial interactions, including the importance of standardisation of methodologies; and to understand nanoparticle- microbial uptake and toxicity and the control of deleterious microbial growth on surfaces using nanomaterials. The novel characteristics of nanoparticles within newly developed materials will be explored.
10:15 AM - KK6.3
Bio-inspired Bactericidal Macromolecular Coatings.
Thomas Blin 2 , Viswas Purohit 2 , Xavier Laloyaux 1 , Alain Jonas 1 , Karine Glinel 1 Show Abstract
2 Laboratoire Polymères, Biopolymères, Surfaces, CNRS - Université de Rouen, Mont Saint Aignan France, 1 Institute of Condensed Matter and Nanosciences (Bio- & Soft-Matter) , Université catholique de Louvain, Louvain-la-Neuve Belgium
The formation of biofilms on material surfaces is a persisting problem inducing many damages in industrial equipments such as the clogging and the corrosion of pipelines or the reduction of heat transfer. More dramatically, the biofilms serve as a reservoir for the development of pathogens. Therefore, there is a great interest to fabricate materials preventing the bacterial attachment which is the first step of the biofilm formation. The most efficient approach to prevent bacterial adhesion is to immobilize a bactericidal substance on material surface. Different routes based on silver derivatives, antibiotics or poly(ammonium) salts have been developed in this way. However, they are not completely satisfying regarding their efficiency, their environmental impact or their role in the emergence of multi-resisting pathogens.Beside these synthetic approaches, there is a fascinating strategy developed by living organisms such as frogs which secrete a thin skin mucus containing antibacterial peptides to protect themselves against bacterial attachment. Compared to conventional bactericidal substances, these peptides offer the advantages to act at very low concentrations, to have a broad spectrum of antibacterial activities and to have a very low propensity to promote pathogen resistance.Here we explore the fabrication of coatings inspired from the frog skin and based on biocompatible macromolecular layers functionalized by an antibacterial peptide. For this, polysaccharide layers or poly(ethylene glycol) derived brushes were grafted onto substrates by “grafting to” and “grafting from” techniques, respectively. Then magainin-I peptide produced by a claw frog was immobilized by one of its extremities onto the hydroxyl groups of the polymer layers through a heterolinker, keeping its accessibility and its activity against bacteria. The antibacterial properties of the coatings were evidenced against various gram+ and gram- micro-organisms such as L. ivanovii, P. aeruginosa and E. coli. This strategy was also adapted to coat superparamagnetic microparticles in order to prepare killing magnetic particles which can be used to disinfect sensitive aqueous solutions or to achieve localized antibacterial action. Moreover, smart coatings switching their surface properties from bactericidal to cell-repellent with temperature were prepared by grafting magainin-I peptide onto temperature responsive brushes showing a collapse temperature in the physiological range. These non conventional approaches could be advantageously adapted to coat various materials or items used in medicine or food industries.References:(1) X. Laloyaux et al. Adv. Mater. 2010, in press.(2) K. Glinel et al. Bioconj. Chem. 2009, 20, 71.(3) A. M Jonas et al. Macromolecules 2007, 40, 4403.
10:30 AM - KK6.4
The Role of the pH Conditions of Growth on the Bioadhesion of Individual and Lawns of Pathogenic L. monocytogenes Cells.
Nehal Abu-Lail 1 , Bong-Jae Park 1 Show Abstract
1 Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington, United States
The work of adhesion that governs the interactions between pathogenic Listeria monocytogenes and silicon nitride in water was probed for individual cells using atomic force microscopy and for lawns of cells using contact angle measurements combined with a thermodynamic-based harmonic mean model. The work of adhesion was probed for cells cultured under variable pH conditions of growth that ranged from pH 5 to pH 9. Our results indicated that L. monocytogenes cells survived and adapted well to the chemical stresses applied. For all pH conditions investigated, a transition was observed in the generation time, physiochemical properties, biopolymer grafting density and bioadhesion for cells cultured in media adjusted to pH 7 of growth. In media with pH 7, the generation time for the bacterial cells was lowest, the specific growth rate constant was highest, the cells were the most polar, cells displayed the highest grafting density of surface biopolymers and the highest bioadhesion to silicon nitride in water represented in terms of the work of adhesion. When compared, the work of adhesion values quantified between silicon nitride and lawns of L. monocytogenes cells were linearly correlated with the work of adhesion values quantified between silicon nitride and individual L. monocytogenes cells.
10:45 AM - KK6.5
Nanostructured Mesoporous Silicon as an Effective Carrier For Extended Antibacterial Therapy.
Mengjia Wang 1 , Phil Hartman 2 , Armando Loni 3 , Leigh Canham 3 , Jeffery Coffer 1 Show Abstract
1 Chemistry, Texas Christian University, Fort Worth, Texas, United States, 2 Biology, Texas Christian University, Fort Worth, Texas, United States, 3 , Intrinsiq Materials Ltd, Malvern, Wors, United Kingdom
Surface modification of devices to permit incorporation and subsequent sustained release of antimicrobials is an active strategy in biofilm control. One nanostructured candidate capable of acting as a time-release surface is mesoporous silicon (PSi), a porous form of the key elemental semiconductor. Porous silicon possesses the useful properties of a simple fabrication procedure, significant control over pore size and surface chemistry, a large surface area, and in vivo biocompatibility. For drug delivery, the porous character of the matrix offers the potential to ideally improve the delivery of poorly soluble agents for an extended duration in a tailored manner. In this work, nanostructured particles of porous silicon are demonstrated to act as an effective carrier for the sustained delivery of antibacterial agents with an enhanced inhibitory activity. Methods are described for the incorporation of significant amounts of the established antibacterial compound triclosan (Irgasan) into mesoporous silicon of varying porosities. Such materials were characterized by a combination of scanning electron microscopy (SEM), energy dispersive x-ray analysis (EDX), x-ray diffraction (XRD), thermal gravimetric analysis (TGA), and antimicrobial assays. Assessment of antibacterial activity was carried out versus the bacterium Staphylococcus aureus as a function of time with concomitant assessment of triclosan release; significant, sustained inhibition of bacterial growth was demonstrated in the triclosan-containing porous Si for time intervals greater than 100 days. Significantly, enhanced dissolution (relative to room temperature equilibrium solubility) of the triclosan was observed for the initial 15 days of drug release, inferring some amorphatization or nanostructuring by the porous Si matrix.
11:00 AM - KK6: Anti
11:30 AM - KK6.6
Assessment of Marine Biofilm Attachment and Growth for Antifouling Surfaces under Static and Controlled Hydrodynamic Conditions.
Maria Salta 1 , Julian Wharton 1 , Paul Stoodley 1 , Simon Dennington 1 , Robert Wood 1 , Keith Stokes 2 1 Show Abstract
1 nCATS, School of Engineering Sciences, University of Southampton, Southampton, Hampshire, United Kingdom, 2 Physical Sciences Department, Dstl, Salisbury, Wiltshire, United Kingdom
Marine biofouling is the accumulation of organisms on underwater surfaces, causing increased ship hydrodynamic drag, which results in higher fuel consumption and decreased speed and range. Biofilms constitute a major component of the overall biofouling and may lead to a 14 % increase in ship fuel costs. Past solutions to antifouling (AF) have used toxic coatings which have subsequently been shown to severely affect marine life. The prohibited use of these antifoulants has led to the search for bio-inspired AF strategies. Current approaches towards the production of alternative coatings include the incorporation of natural AF compounds into paints. Significant effort is being directed towards more environmentally benign strategies, however, ultimately we believe that a combination of surface texturing and chemistry will lead to the most effective antifouling performance.Screening assays for novel AF compounds are often separated into two categories; toxicity and AF assays. Increasingly there is evidence that active compounds affect organisms at non-toxic concentrations, hence, the necessity for more insightful AF testing directly on surfaces for both static and hydrodynamic conditions. Our study assessed natural product (NP) antifouling performance of an isolated compound from a terrestrial source (a derivative of quinone) against biofilm organisms which included the marine bacteria, Cobetia marina, Marinobacter hydrocarbonoclasticus and the bioluminescent bacterium Vibrio harveyi and the diatom Amphora coffeaeformis. Novel bioassay protocols were developed to test the in-situ AF efficacy of the NP on coated surfaces. This was assessed by quantifying biofilm growth and adhesion kinetics using a multidetection microplate reader utilising viability staining and natural bioluminescence. Additionally, flow cells and microfluidic channels have been uniquely adapted permitting the AF performance of coatings and NPs to be explored in terms of biofilm attachment and growth for controlled hydrodynamic regimes. These bioassays were corroborated using a suit of microscopy techniques, including atomic force and confocal laser scanning microscopy, in order to compare biofilm structures in the presence and absence of the NP. The NP showed a marked AF efficacy against C. marina and M. hydrocarbonoclasticus attachment at very low concentrations (10 μg mL–1) with a clear impact on biofilm morphology on NP-containing surfaces. By directly assessing the surface AF effect on biofilm formation, greater insights on NP activity have be obtained (i.e. toxicity, microtopography and/or contact effects), as well as better understanding of the NP kinetics within the coating system and how its interaction with the biofilm.
11:45 AM - KK6.7
Preparation, Characterization and In Vitro Antibacterial Activity of Fluoridated Hydroxyapatite Nanothick Coatings for Biomedical Applications.
Xiang Ge 1 , Yang Leng 1 , Chongyun Bao 2 , Sherry Xu 3 , Renke Wang 2 , Fuzeng Ren 1 Show Abstract
1 Department of Mechanical Engineering, The Hong Kong University of Science and Technology, Hong Kong Hong Kong, 2 West China College of Stomatology, Sichuan University, Chengdu China, 3 Department of Biology, The Hong Kong University of Science and Technology, Hong Kong Hong Kong
Introduction: Percutaneous type of orthopedic and dental implants requires not only a good adhesion with bone, but also the ability to form good attachment and seal with connective tissues and skins. Currently, the skin-seal of such implants still remains as a problem to be resolved. Electrochemical deposition method can be used to modify the surfaces of metallic implants with coatings in order to improve the antibacterial activity and skin seal of the implants. With a carefully control of electrochemical parameters, we successfully deposited a nanothick and dense coating of fluoridated calcium phosphate on titanium substrate. After heat treatment, the fluoridated calcium phosphate transformed to fluoridated hydroxyapatite (FHA). The FHA nanothick coating was systematically characterized by various techniques to obtain comprehensive properties of the coating. The in vitro antibacterial activity evaluation of samples was conducted with a film attachment method against S.aureus, E.coli and P.gingivalis.Materials and Methods: Titanium plates were used as substrates. The electrolyte was mixed with three kinds of aqueous solutions (0.042 M Ca(NO3)2, 0.025 M NH4H2PO4 and 0.01M NaF) sequentially. The titanium plates were cathodically treated in an electrochemical cell which contained three electrodes: a titanium plate as the cathode, a platinum plate as the anode and a saturated calomel electrode as the reference electrode. The electrochemical deposition process was conducted at a constant current density within acidic environment at room temperature for 6 minutes. Then, the specimens were thermally treated at 600°C for 3 hours in a humid air atmosphere. Dissolution behavior of coatings was examined by immersing each type of specimens into an solution (mixing 0.1M Tris and HCl, 30mL, pH=7.3) at 37°C. Nanoscratching tests were conducted by a nanoindentation system. The zeta potential of coatings was tested with an electro kinetic analyzer. The in vitro antibacterial activity of FHA and HA coating was tested against S.aureus, E.coli and P.gingivalis; while the acid etched pure titanium plate was selected as control.Results and Conclusions: A nanothick (~200 nm) coating of FHA was deposited on acid etched pure titanium substrates with an electrochemical deposition method followed by a heat treatment. The dissolution test results indicate the importance of crystalline structure on chemical stability and also the positive role of F¬- ions in apatite structure stability. The Lc of the FHA coating was 147% higher than that of the HA coating with the same thickness level. The zeta potential of FHA is 13.9% less negative than that of HA. The survivability of bacteria on the FHA coating was much less than that on HA coating and pure titanium substrates, which indicates that FHA nanothick coating has potential clinical applications for inhibiting percutaneous orthopedic and dental implants associated bacterial infections.
12:00 PM - KK6.8
Anti-biofilm Betaine Medical Device Surfaces after 90 Day Serum Exposure.
Sarah Guedez 1 , Heather Lapp 1 , Raisa Fabre 1 , Victoria Wagner 1 , Christopher Loose 1 Show Abstract
1 , Semprus BioSciences, Cambridge, Massachusetts, United States
Indwelling catheters put patients at risk for infections which often result in significant morbidity and mortality. Biofilms associated with such device infections are often recalcitrant to currently available therapeutics. Traditional prevention strategies have largely focused on applying leaching antimicrobial coatings to devices with variable clinical success, and drawbacks include short-term duration, limited spectrum of activity, potential toxicity and generation of drug-resistant strains. We examined the performance of a potentially superior approach by using highly water-coordinating, nonfouling betaine polymers as inert coatings to prevent bacterial attachment and subsequent biofilm formation in a blood product environment. This study demonstrates the long-term antimicrobial activity of betaine-modified Carbothane®/ BaSO4 using a modified flow biofilm reactor system (mCDC). Polyurethane catheter substrates (Carbothane®/ BaSO4, 14-French rods) were modified using betaine, zwitterionic structures. To mimic the clinical setting, we subjected betaine-modified materials to serum, a complex media, for periods up to 90 days prior to biofilm challenge. Escherichia coli ATCC 25922 was used as the challenge microbe. Briefly, samples of control and betaine-modified rods exposed to 50% fetal bovine serum for 1, 30, 60, or 90 days were tested for antimicrobial/antibiofilm activity using the mCDC system. Samples were incubated with a bacterial suspension of 1e6 cfu/ml in 1xPBS in the mCDC reactor (batch mode) for 2 hours at 37°C with agitation. Thereafter, the rods were transferred to a fresh reactor and exposed to modified M63 media under flow at 8 ml/min. Biofilm growth was monitored by plate counts and macroscopic visualization of biofilm surface coverage after 24 hours. Log reduction (LR) differences were calculated on surface modified rods and polyurethane controls. Betaine surface modified rods maintained performance over the 90 days of serum exposure with a mean LR of 1.94 (p<0.0001). Previous work has demonstrated that such betaine structures show superior resistance to thrombus formation in blood flow-loop studies after serum exposure, giving the potential for dual antimicrobial and antithrombotic performance.
12:15 PM - KK6.9
Structural Dynamics of Pseudomonas Aeruginosa Lipid A as a Function of the Number of Acyl Chains.
Frederico de Santana Pontes 1 , Thereza Soares 1 , Roberto Lins 1 Show Abstract
1 , UFPE, Recife Brazil
Lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacteria, perform an important role in the structural integrity of the microbe as well as protect the membrane from certain kinds of chemical attack through complexation, uptake of ions and efflux mechanisms. LPS are comprised of three parts: the O antigen, the oligosaccharide core and the lipid A. The latter exhibits long fatty acid chains, which binds the sugar moiety into the bacterial membrane. The lipid A domain is the main responsible for the toxicity of Gram-negative bacteria. Changes in the number of acyl chains affects directly the toxicity levels, adhesion and permeability of the LPS. The understanding at molecular level of these processes are indispensable for future applications of Gram-negative bacteria in relevant tasks such as decontamination of soil and water, adhesion to solid surfaces and antibiotic resistance, for example. Previously, an atomistic model for LPS membranes of Pseudomonas aeruginosa was developed (Lins and Straatsma, Biophys. J., 2001), validated (Soares and Straatsma, Mol. Simulation, 2008) and successfully applied to structural studies (Soares et al, J. Braz. Chem. Soc., 2008) and metal uptake processes (Lins et al, Biomacromolecules, 2008). In the present work, we have used computer simulations to investigate the influence of phenotypical variations of the acyl chains on the flexibility, electrostatic potential and charge distribution of the lipid A of P. aeruginosa. Dependence of lipid A lateral diffusion, order parameter, molecular shape and diglucosamine tilt angle for symmetrical tetraacyl, pentaacyl and symmetrical hexaacyl lipid A membranes are characterized. These results are compared against experimental data and insights into the relationship between the number of acyl chains and endotoxicity are drawn.