Article pubs.acs.org/Langmuir
Effect of Particulate Contaminants on the Development of Biofilms at Air/Water Interfaces Zhenhuan Zhang and Gordon Christopher* Department of Mechanical Engineering, Texas Tech University, Lubbock, Texas 79409-1035, United States ABSTRACT: The development of biofilms at air/water or oil/water interfaces has important ramifications on several applications, but it has received less attention than biofilm formation on solid surfaces. A key difference between the growth of biofilms on solid surfaces versus liquid interfaces is the range of complicated boundary conditions the liquid interface can create that may affect bacteria, as they adsorb onto and grow on the interface. This situation is exacerbated by the existence of complex interfaces in which interfacially adsorbed components can even more greatly affect interfacial boundary conditions. In this work, we present evidence as to how particle-laden interfaces impact biofilm growth at an air/water interface. We find that particles can enhance the rate of growth and final strength of biofilms at liquid interfaces by providing sites of increased adhesive strength for bacteria. The increased adhesion stems from creating localized areas of hydrophobicity that protrude in the water phase and provide sites where bacteria preferentially adhere. This mechanism is found to be primarily controlled by particle composition, with particle size providing a secondary effect. This increased adhesion through interfacial conditions creates biofilms with properties similar to those observed when adhesion is increased through biological means. Because of the generally understood ubiquity of increased bacteria attachment to hydrophobic surfaces, this result has general applicability to pellicle formation for many pellicle-forming bacteria.
1. INTRODUCTION Free swimming bacteria can attach to surfaces through processes extending from their surfaces. When surface-attached bacteria reach a critical concentration, they form a biofilm, which consists of a community of microorganisms embedded in a matrix of self-secreted extracellular polymeric substances (EPS). The biofilm creates advantageous circumstances for bacteria growth and survival by producing favorable environmental conditions and defending against antimicrobial agents.1,2 Because of their ability to enhance bacteria survivability, biofilms are found extensively in nature; many serious and persistent infections occur due to biofilm formation on human tissue or medical devices. 3,4 In industrial applications, biofilms on solid surfaces form in industrial pipelines, water treatment systems, paper manufacturing plants, and in food production facilities, causing blockage, corrosion, and contamination.5−7 Because of their ubiquity and negative outcomes, there have been many studies of biofilm formation, mechanical properties, and prevention on solid surfaces, of which a few relevant reviews are given.8−12 Biofilm formation is a complex process, but it can be broken down into three primary stages: initial adsorption of bacteria close to a surface, adhesion of bacteria to the surface, and, finally, growth of the biofilm.13,14 These stages are affected by the environmental conditions of surrounding bulk liquids, including nutrient supply, pH, ionic strength, and more.15−18 Bacteria have biologically adapted to ensure successful colonization using motility, adjusting their surface properties, or changing the content of their EPS. Because of their importance, these area impacts on biofilm formation are still actively studied, as current reviews show.19−28 © XXXX American Chemical Society
Biofilms can also form at liquid/liquid interfaces. These biofilms, which are referenced as pellicles, can be found in both nature and industrial applications. Pellicles can create infections29,30 and are important to the development of cystic fibrosis.31 Pellicles can be beneficial in the process of microbial hydrocarbon degradation during carbon sequestration, because of their ability to stabilize emulsions.32,33 Industrially, pellicles reduce the efficacy of microbial enhanced oil recovery34−38 and negatively impact oil processing.39−42 Pellicle formation has the same basic steps as solid surface biofilm formation. However, pellicle formation adds another variable to every step: the properties of the interface, which are complicated and vary widely. Most liquid interfaces typical of the aforementioned applications are complex and have multiple adsorbed species affecting interfacial properties. Complex interfaces such as these will have varying interfacial rheology and chemistry, which will affect the hydrodynamic, steric/physical, and physio-chemical interfacial boundary conditions, and bacteria that are confronted during biofilm development.43−45 Although not widely studied, the effects of changing these interfacial properties and boundary conditions on pellicle development have been examined for some specific situations. During adsorption, surface viscosity magnitude affects the rotation direction of adsorbing bacteria.44,46,47 Adhesion can be impacted by modifying surface tension and charge through addition of interfacially adsorbed components.48,49 Growth and Received: January 14, 2016 Revised: March 3, 2016
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Langmuir final pellicle strength can be affected by the addition of surfactant immediately prior to biofilm formation, interrupting curli fiber formation and, hence, preventing cellular aggregates.50 The addition of a nonionic surfactant after biofilm formation begins will also reduce viscoelasticity.51 Although there have been some studies, the role of complex interfaces on the development and mechanical properties of biofilms is not well understood for most complex interfaces. There are still many questions regarding how various interface types affect pellicle formation. In this paper, the role of particulate contaminants on pellicles is studied. Many applications present interfaces contaminated by particulates; however, the effect of particle-laden interfaces on the ability of bacteria to form pellicles is unknown. The purpose of this paper is to explore the influence of interfacially adsorbed particles on pellicle formation, and determine what mechanisms cause any changes in the known behavior of pellicle formation.
2. MATERIALS AND EXPERIMENTAL METHODS
Figure 1. (a) Photograph of interfacial rheometer geometry and modified base used to perform experiments described in this work. (b) Schematic of interfacial rheometer base modified for biofilm growth. Units are given in millimeters (mm).
2.1. Materials. Uropathogenic Escherichia coli (E. coli) UTI89 stock culture was provided by Dr. Scott Hultgren from University of Washington at St. Louis. E. coli are rod-shaped bacteria that are ∼2 μm long and range in width from 0.25 μm to 1 μm.52 The starting culture was grown from frozen stock by inoculating the strain into 4 mL of LB broth, prepared with deionized water, at 37 °C for 20 h, shaking at 150 rpm. After 20 h, the planktonic cell concentration of working culture was measured by cell culture optical density at 600 nm. A density of 3.2 A was found. Three types of particles were used in the study: 3 μm (Life Technology, No. S37223) and 30 nm (Life Technology, No. A37351) hydrophobic-coated sulfate latex polystyrene particles. In addition, 20 nm silver particles (Sigma−Aldrich, No. 730793) were used. 2.2. Pellicle and Interface Formation. Pellicles were formed by inoculating 0.75 mL of working culture into 150 mL of broth. In all experiments, YESCA broth (10 g/L Casamino acids, 0.5 g/L yeast extract) or YESCA broth with 4% dimethyl sulfoxide (DMSO) was used. YESCA was prepared with deionized (DI) water and sterilized by using an autoclave. Samples of the bacteria broth were added to the rheometer base (described below), creating an air/water interface upon which the biofilm can grow. This broth was kept at a constant temperature of 25 °C and continuously resupplied. In clean interface experiments (i.e., those without added particles), the interfacial rheometer geometry was immediately brought into contact with the liquid interface and the experiment was started. For experiments with particulate containments, particles were added directly to the interface after bacterial broth was introduced. This was done by dispensing a mixture of particles in water and isopropanol to the air/water interface directly and letting the isopropanol on the interface evaporate. To control surface concentration, the particle solution was added with a specified volume at a known bulk particle concentration. Using these values and the total interfacial area, the surface concentration can be controlled. Surface concentration was then verified using a microscope system attached to the rheometer, as described in a previous work.53 Particle surface concentrations of 10% and 20% were used in experiments. The particles distribute across the interface with some aggregation (observed optically). 2.3. Interfacial Rheology. Interfacial rheology has been found to track well with pellicle growth.50,51,54−56 All interfacial rheology measurements were done using a double-wall ring geometry attached to an AR-G2 stress-controlled rheometer (TA Instruments). This interfacial geometry provides high sensitivity measurements by creating a surface Couette flow on either side of the ring. Because of the high torque sensitivity of the AR-G2, interfacial viscoelastic moduli can be measured at a wide range of values.57 The base of the double-wall ring system has been modified significantly to allow the growth of biofilms, as shown in Figure 1. This design is based on those used by Hollenbeck and co-workers for a Du Noüy ring interfacial geometry.56 The system has expanded reservoirs that are connected to
syringe pumps that allow continually replenishment of broth and ensure that the level of the interface does not change over the long time scales of the experiments. A heater allows the temperature of the broth to be controlled in each reservoir. The interface was shielded from the air to avoid any contamination. Furthermore, the trough was designed to allow the use of an existing interface microscope on the rheometer, as detailed in earlier work.53 The position of the doublewall ring attached in the interface can be monitored by using microscopy underneath the trough. Each experiment was run for 72 h, during which the biofilm was allowed to grow and surface viscoelastic moduli were taken for 10 min at the end of each hour with an angular frequency of 0.5 rad/s and strain of 1%. During experiments, temperature was kept at 25 °C and an infusion rate of 0.003 mL/min was used for the broth. When working with biological materials, there is typically variability in the material properties. Therefore, each experiment is carried out twice and average moduli are reported. Measurements of viscoelastic moduli were corrected using a code that accounts for bulk shear effects that may occur at small Boussinesq numbers.57 It is possible for interfacially adsorbed proteins to create significant interfacial viscosity.58 Therefore, we tested the interfacial rheological properties of pure YESCA and YESCA+DMSO broths over 72 h in a manner identical to the experiments described above. Interfacial storage moduli with values growing from 10−5 to 10−4 over the course of 72 h were observed. These values were below measured biofilm values by at least an order of magnitude (see below). Therefore, the proteins do not contribute to observed behaviors. It is possible that bacteria or EPS also interacts with the adsorbed proteins from the broth. This effect would be incredibly difficult to assess using current experimental methods, and is only important if multiple broth types were used. However, since only one bacteria and one broth were used, these effects will be consistent across all tests and, therefore, can be ignored. Particle-laden interfaces made on top of YESCA broth did not exhibit measurable interfacial viscoelasticity within the sensitivity limits of our interfacial rhometer. 2.4. Scanning Electron Microscopy (SEM). The biofilms floating on the air/liquid interface are collected on glass slides and submerged in liquid nitrogen for fast freezing at the end of 72 h of growth. These frozen sample slides are then kept in a freeze dryer at −25 °C for 3 days. Before imaging, these slides are sputter-coated with a thin layer of Au/Pd alloy to improve the sample conductivity. Those samples are imaged by a Hitachi, Model SE/N 4300 scanning electron microscopy (SEM) system. B
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3. RESULTS 3.1. Pellicle Formation Relation to Interfacial Viscoelasticity. Using interfacial rheology to probe the timedependent properties of an interface, three distinct regions in the interfacial viscoelastic moduli are observed (Figure 2a). At
Figure 3. Effects of microparticles and nanoparticles on biofilm surface elastic modulus over time. The solid line () represents the biofilm on a clean interface with YESCA broth; open circle symbols (○) represent data for the interface with 20% micropolystyrene particles, and the cross symbols (+) represent data for the interface with 20% nanopolystyrene particles. Figure 2. Typical interfacial surface moduli for a biofilm grown in a YESCA and DMSO broth over 72 h. The solid line () represents the surface elastic modulus (G′), and the dashed line (- - -) represents the surface loss modulus (G″).
behavior of the interface during the growth stage of pellicle formation. The interfaces with microparticles quickly grew at the 30−40 h period, showing an ∼2 orders of magnitude increase in moduli. The nanoparticle interface and the clean interface began to exhibit growth at a later time and growth is much more gradual. Toward the end of the 72 h experiment, the nanoparticle interface began to plateau. However, the clean interface does not appear to be reaching a plateau, and was still slowly growing. However, within experimental error, the nanoscopic polystyrene particles and the clean interfaces showed no differences in behavior over the course of 72 h. At the end of the experiments, both the clean and nanoparticle interface had weaker elasticity, by ∼1 order of magnitude, in comparison to the interface with the microparticles. In order to further examine the effects of particles on the interface, the final microstructure of the biofilms at the 72 h mark is shown in Figure 4. When examining the clean interface, the biofilm is, for lack of a better term, patchy. Dense raftlike structures were observed in the biofilm. Holes and cracks are discernible between the rafts. Finally, the biofilm appears to be nonuniform in thickness and density, indicating uneven growth. The interface with nanoparticles is similar to the clean interface; however, at higher magnifications (as shown in Figure 4b), clusters of aggregated nanoparticles embedded in the biofilm rafts were observed. The microstructure of the microparticles biofilm interface was clearly different than the clean and nanoparticle biofilms. The microparticles were distributed evenly throughout the biofilm. There was some aggregation, but no dense clusters as observed. The biofilm was much more uniform and resembles a dense network of fibers. The thickness and density of the biofilm appear consistent, and there are no holes in the structure. 3.3. Effect of Particle Composition on Growth. Based on results with polystyrene, the effects of particle composition on biofilm growth were studied. Figure 5 displays the results comparing biofilm growth on a clean interface to one with 20% surface coverage of nanopolystyrene and one with 10% surface coverage of nanosilver particles. As seen previously, the nanopolystyrene gave nearly identical (within error of the measurements) interfacial rheology results in comparison to the clean interface. The nanosilver interface shows similar initial results with some degree of adhesion and adsorption in the first 10 h. In comparison to the other two interfaces, nanosilver did not exhibit any further growth after these stages. 3.4. Effect of Growth Medium in Comparison to Particle Contamination. It has been previously established
the beginning of an experiment, the G′ and G″ values of the interface are too low to be measured, because the interface is clean and bacteria have not been adsorbed close to the interface. During this phase, in the first 10 h, for the system in Figure 2, the storage and loss moduli grow from nothing to small but measurable values. After this initial adsorption phase, adhesion occurs over the next 15 h for the system shown in Figure 2. During this phase, interfacial moduli slowly grow as bacteria adhere to the interface. The bacteria act like distinct particles, creating interfacial viscoelasticity from their interaction with each in a manner similar to that of interfacially adsorbed particles. After enough bacteria are on the interface to initiate quorum sensing, the bacteria increase EPS production to form a biofilm; there is a large increase in the storage and loss moduli. Over the course of 10 h for the system in Figure 2, the biofilm fills the entire testing area and storage and loss moduli values reach a plateau. After the biofilm has fully covered the entire area of the interfacial trough, the moduli slightly increase over time as the biofilm becomes denser. All results in this work exhibited G′ and G″ with similar trends as that depicted in Figure 2. Depending on the specific system, different timing for the various stages and different overall moduli magnitude were observed. However, the pellicles were always dominated by interfacial elasticity, and the loss moduli followed similar trends to the surface storage modulus. Therefore, in the following results, only the storage modulus (G′) is reported versus time. 3.2. Effect of Particle Size on Pellicle Growth. In order to gauge the effect of particulate contaminants, the effect of particle diameter on the growth of biofilms over time was characterized (see Figure 3). When polystyrene particles 3 μm and 30 nm in diameter were added to the air/water interface at a surface coverage of 20%, little difference was observed in the rheological behavior of the three systems over the first few hours of the experiment, corresponding to the adsorption phase. Around 10 h, both the microparticles and nanoparticles exhibit slightly increased moduli, in comparison to the clean interface; this increase is within the variability seen between experiments for identical system compositions. A significant difference in the interfacial rheology was observed in terms of the length of the adhesion stage and the C
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grown in DMSO and YESCA with microparticles, and finally one grown in YESCA only.
Figure 6. Combined effects of DMSO and particles on biofilm surface elastic modulus over time. The solid line () represents data for the biofilm on a clean interface with YESCA broth, and the dashed line (- - -) represents data for the biofilm on a clean interface with YESCA and DMSO broth; the inverted triangle symbol (▽) interface with 20% micropolystyrene particles and YESCA and DMSO broth.
As can be seen, the rate of growth and overall viscoelastic moduli for a clean interface of YESCA + DMSO results in faster adhesion and growth, in comparison to an interface with particles on the interface. When the SEM images of the biofilms after 72 h of growth are examined (Figure 7), the DMSO interface and interfaces with particles in either broth share
Figure 4. Scanning electron microscopy (SEM) images of pellicles grown in a YESCA broth with (a) 20% micropolystyrene particles, (b) 30 nm polystyrene particles, and (c) a clean interface.
Figure 5. Effects of nanopolystyrene and nanosilver particles on biofilm surface elastic modulus over time. The solid line () represents the biofilm on a clean interface with YESCA broth; the cross symbols (+) represent data for the interface with 20% nanopolystyrene particles, and the times sign symbols (×) represent data for the interface with 10% nanosilver particles.
that the addition of DMSO to the growth medium enhances the production of amyloid curli fiber on the bacteria surface. The resulting biofilms were stronger, with greater elasticity, in comparison to those without DMSO. This was theorized to be caused by the increase of curli, allowing the bacteria to more quickly adhere and organize on the air/water interface.55 These results are similar to what was observed for the 20% surface coverage of micropolystyrene. In Figure 6, results from three biofilms are shown: one grown in DMSO and YESCA, one
Figure 7. SEM images of pellicles grown on (a) a clean interface with YESCA and DMSO broth, (b) interface with 20% micropolystyrene particles and YESCA and DMSO broth, and (c) a clean interface with YESCA broth. D
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Langmuir similar microstructures. In both cases, a dense network of fibers is formed to create the biofilm. Interfaces with particles see those particles embedded into the biofilm. The clean interface, in comparison, is more varied in thickness, exhibiting holes in the film, and generally appears to be less thoroughly developed.
to the nanoparticles or natural biological variability. However, it is important to note that the nanopolystyrene particles have no negative effect on the biofilm, and in no way appear to act in any antimicrobial fashion. This is seen in both the identical interfacial rheological behavior and the similar microstructures observed in SEM images of the pellicles. Interestingly, the structure of the biofilm is affected by enhanced adhesion. The structure of biofilms with DMSO is sufficiently different from those of interfaces formed in the pure YESCA broth. In the SEM micrographs shown in Figure 7, the YESCA+DMSO formed a more percolated, dense network, in comparison to the sparser, inconsistent structures formed at the clean interface. Interestingly, similar structures to the DMSO biofilms are seen when examining the SEM for both microparticle systems (pure YESCA and YESCA+DMSO). This indicates that the mechanism for this structure of biofilm relies on a biological response to increased adhesion, but the mechanisms of that increased adhesion do not matter. 4.2. Effect of Particle Composition on Growth. Not surprisingly, the particle composition matters very much. When switching to a low concentration of nanosilver particles, bacteria still adsorbed and adhered to the interface, but never went to the growth phase. Silver is a known antimicrobial agent,61 so it is not surprising to find that it inhibits the growth of the biofilm. In this case, the size of the particle has nothing to do with the change in the interface; the known antimicrobial properties of the silver at the interface itself are simply enough to stop any further growth after adhesion.
4. DISCUSSION 4.1. Effect of Enhanced Adhesion on Biofilm Formation. Previous work with DMSO showed that amyloid curli fiber increased the adhesion of bacteria at an air/water interface, which, in turn, increased their overall viscoelasticity and rate of growth of the pellicle. This result was attributed to the ability of the fibers to enhance adhesion.55 In this work, we believe enhanced adhesion also describes the mechanism for increased viscoelasticity at the interface; however, in this case, the adhesion is caused by particulates at an interface. The particles used in this study are coated, such that they are hydrophobic. E. coli are known to preferentially adhere to hydrophobic surfaces.59 In fact, it can be generally stated, that most bacteria preferentially attach to a hydrophobic surface.60 Therefore, the interfacially adsorbed hydrophobic particles are creating cites of increased adhesive strength and probability for individual bacteria approaching the interface. As adsorbing bacteria approach the interface, the protruding hydrophobic particles provide a closer and more energetically favorable place to which bacteria can adhere, in comparison to the clean interface, and, hence, enhance the overall adhesion rate. This enhanced adhesion increases the number of bacteria on the interface, resulting in faster growth rate of bacteria on the interface to provide the quorum sensing to begin biofilm formation. This mechanism is essentially identical to the amyloid curli fibers created by DMSO; however, the mechanism in this case is the physio-chemical properties of the interface itself, rather than changes to the biological functions of the bacteria. When both DMSO and particles are used, results that lie somewhere between the clean interface and pure DMSO are seen. This could indicate that the biological mechanism (i.e., curli) are better suited at increasing adhesion of the bacteria. However, it is also possible that changing particle types by changing particle surface wettability or roughness could improve the physiochemical mechanism observed with the particles. What can be concluded, at least from this work, is that particles that promote bacteria adhesion should create biofilms faster and with greater viscoelastic moduli in a manner similar to changing the surface properties of the bacteria through biological manipulation. This mechanism works for microsized particles but not nanosized ones, because the bacteria are of similar size to the microparticles. This similar size makes adsorption to the physical surface of the particles possible. The smaller nanoparticles, although of similar surface coverage, do not individually provide enough surface area for bacteria adhesion. Even in their clumped state, the particles do not provide sufficient area for bacteria attachment. Hence, they do not affect the development of the biofilm sufficiently. Although some increase in the strength of these networks is observed, the overall behavior of the nanopolystyrene systems was very similar to that of the clean interface. The increased strength observed in these systems may have something to do with nanoparticles filling gaps in the biofilm structure of clean interfaces. However, because of the variability of these interfaces, it is difficult to say if the observed results are due
5. CONCLUSIONS The development of biofilms at air/water or oil/water interfaces has applications in which either prevention (such as disease or oil processing) or promotion (such as carbon sequestration) are important. Therefore, looking at the effects of interfacial properties on biofilm growth should provide new tools with which to manipulate these systems and create desired outcomes. Previous work has begun this examination by looking at the role of surfactants on pellicle formation. In this work, the role of particulates on interfaces and their effect on pellicle growth has been characterized using interfacial rheology. In particular, the addition of hydrophobic microparticles provides enhanced adhesion of bacteria, which allows faster and more robust biofilm development. However, both composition and size of particulate containments affect this process. Interestingly, this mechanism works through the physical mechanism as described in this work, but also through biological mechanisms such as enhanced adhesion due to the production of curli through gene regulation. The degree to which this mechanism is effective will be dependent both on particle type and specific bacteria surface properties and biology. However, since the principle itself is due to physical adsorption driven by known preferences of bacteria to hydrophobic surfaces, the general conclusions drawn here can be applied to other systems. That being said, we have found that particle composition can negate this effect by changing particle properties away from hydrophobic and to known antimicrobial conditions. Furthermore, particle size seems to also matter in comparison to bacteria size. However, in lieu of an antimicrobial coating, particles do not appear to be antimicrobial on their own, as seen by the nanopolystyrene. Overall, this work points to the importance in considering the condition of the interface on the formation of pellicle E
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developing antibiofilm agents. Future Med. Chem. 2015, 7 (4), 493− 512. (13) Macedo, A. J.; Kuhlicke, U.; Neu, T. R.; Timmis, K. N.; Abraham, W.-R. Three stages of a biofilm community developing at the liquid−liquid interface between polychlorinated biphenyls and water. Appl. Environ. Microbiol. 2005, 71 (11), 7301−7309. (14) Hobley, L.; Harkins, C.; MacPhee, C. E.; Stanley-Wall, N. R. Giving structure to the biofilm matrix: An overview of individual strategies and emerging common themes. FEMS Microbiol. Rev. 2015, 39 (5), 649−669. (15) Toyofuku, M.; Inaba, T.; Kiyokawa, T.; Obana, N.; Yawata, Y.; Nomura, N. Environmental factors that shape biofilm formation. Biosci., Biotechnol., Biochem. 2016, 80 (1), 7−12. (16) Renner, L. D.; Weibel, D. B. Physicochemical regulation of biofilm formation. MRS Bull. 2011, 36 (5), 347−355. (17) Sabater, S.; Guasch, H.; Ricart, M.; Romanı ́, A.; Vidal, G.; Klunder, C.; Schmitt-Jansen, M. Monitoring the effect of chemicals on biological communities. The biofilm as an interface. Anal. Bioanal. Chem. 2007, 387 (4), 1425−1434. (18) Pavlovsky, L.; Sturtevant, R. A.; Younger, J. G.; Solomon, M. J. Effects of Temperature on the Morphological, Polymeric, and Mechanical Properties of Staphylococcus epidermidis Bacterial Biofilms. Langmuir 2015, 31 (6), 2036−2042. (19) Rusconi, R.; Stocker, R. Microbes in flow. Curr. Opin. Microbiol. 2015, 25, 1−8. (20) Cunliffe, M.; Upstill-Goddard, R. C.; Murrell, J. C. Microbiology of aquatic surface microlayers. FEMS Microbiol. Rev. 2011, 35 (2), 233−246. (21) Guttenplan, S. B.; Kearns, D. B. Regulation of flagellar motility during biofilm formation. FEMS Microbiol. Rev. 2013, 37 (6), 849− 871. (22) Wei, Q.; Ma, L. Z. Biofilm Matrix and Its Regulation in Pseudomonas aeruginosa. Int. J. Mol. Sci. 2013, 14 (10), 20983−21005. (23) Wozniak, D. J.; Parsek, M. R. Surface-associated microbes continue to surprise us in their sophisticated strategies for assembling biofilm communities. F1000Prime Rep. 2014, 6, 26−26. (24) Hung, C.; Zhou, Y. Z.; Pinkner, J. S.; Dodson, K. W.; Crowley, J. R.; Heuser, J.; Chapman, M. R.; Hadjifrangiskou, M.; Henderson, J. P.; Hultgren, S. J. Escherichia coli Biofilms Have an Organized and Complex Extracellular Matrix Structure. mBio 2013, 4 (5), e00645-13. (25) Romanı ́, A. M.; Amalfitano, S.; Artigas, J.; Fazi, S.; Sabater, S.; Timoner, X.; Ylla, I.; Zoppini, A. Microbial biofilm structure and organic matter use in Mediterranean streams. Hydrobiologia 2013, 719 (1), 43−58. (26) Dourou, D.; Beauchamp, C. S.; Yoon, Y.; Geornaras, I.; Belk, K. E.; Smith, G. C.; Nychas, G. J. E.; Sofos, J. N. Attachment and biofilm formation by Escherichia coli O157:H7 at different temperatures, on various food-contact surfaces encountered in beef processing. Int. J. Food Microbiol. 2011, 149 (3), 262−268. (27) Rickard, A. H.; McBain, A. J.; Stead, A. T.; Gilbert, P. Shear rate moderates community diversity in freshwater biofilms. Appl. Environ. Microbiol. 2004, 70 (12), 7426−7435. (28) Ylla, I.; Borrego, C.; Romanı ́, A. M.; Sabater, S. Availability of glucose and light modulates the structure and function of a microbial biofilm. FEMS Microbiol. Ecol. 2009, 69 (1), 27−42. (29) Flores-Mireles, A. L.; Walker, J. N.; Caparon, M.; Hultgren, S. J. Urinary tract infections: Epidemiology, mechanisms of infection and treatment options. Nat. Rev. Microbiol. 2015, 13 (5), 269−284. (30) Xu, Z.-G.; Gao, Y.; He, J.-G.; Xu, W.-F.; Jiang, M.; Jin, H.-S. Effects of azithromycin on Pseudomonas aeruginosa isolates. from catheter-associated urinary tract infection. Exp. Ther. Med. 2014, 9 (2), 569−572. (31) Nazzari, E.; Torretta, S.; Pignataro, L.; Marchisio, P.; Esposito, S. Role of biofilm in children with recurrent upper respiratory tract infections. Eur. J. Clin. Microbiol. Infect. Dis. 2015, 34 (3), 421−429. (32) Bajaj, S.; Singh, D. K. Biodegradation of persistent organic pollutants in soil, water and pristine sites by cold-adapted microorganisms: Mini review. Int. Biodeterior. Biodegrad. 2015, 100, 98−105.
biofilms and how that interface may affect the desired outcomes in a range of applications. What remains unclear is the impact of other interfacial physiochemical boundary conditions on pellicle growth, in comparison to biological or environmental changes. It would appear from the results in this work that biological changes alone provide a stronger effect than the inclusion of hydrophobic particles at the interface. However, it is possible that a more robust study looking at a wider range of particle sizes, wettability, and roughness may find that certain particles provide even greater increases in bacteria interfacial adhesion and improved pellicle strength.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to acknowledge Dr. Gerry Fuller (Stanford University) and Emily Hollenbeck (Stanford University), for their help in designing the rheometer base used for characterizing biofilms interfacial properties, Dr. Michael San Francisco (Texas Tech University), for his assistance in growing and measuring bacteria cultures, and finally Dr. Scott Hultgren, for providing the bacteria stock used in this study.
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REFERENCES
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DOI: 10.1021/acs.langmuir.6b00143 Langmuir XXXX, XXX, XXX−XXX
