Scholarly Work - Center for Biofilm Engineering
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Item Assessing biofouling on polyamide reverse osmosis (RO) membrane surfaces in a laboratory system(2010-04) Khan, Mohiuddin M. T.; Stewart, Philip S.; Moll, D. J.; Mickols, W. E.; Burr, Mark D.; Nelson, Sara E.; Camper, Anne K.Biofouling of reverse osmosis (RO) membranes is a major impediment in both wastewater reuse and desalination of sea/brackish waters. A benefit to the industry would be a simple screening approach to evaluate biofouling resistant RO membranes for their propensity to biofoulants. To observe the relationship between initial membrane productivity and control of biofilm formation governed by surface modification to the aromatic polyamide thin-film composite RO membranes, three different RO membranes developed by the FilmTec Corporation including FilmTec’s commercial membrane BW30 (RO#1) and two experimental membranes (RO #2 and #3) were used. RO #2 and RO #3 were modified with a proprietary aliphatic group and with an extra proprietary aromatic group, respectively. Membrane swatches were fixed on coupons in rotating disk reactor systems without filtration and exposed to water with indigenous organisms supplemented with 1.5 mg/L organic carbon under continuous flow. After biofouling had developed, the membranes were sacrificed and subjected to several analyses. Staining and epifluorescence microscopy revealed more cells on RO #2 and #3 compared to RO #1. Based on image analysis of 5-µmthick stained biofoulant cryo-sections, the accumulation of hydrated biofoulants on RO #1 and #3 were from 0.87 to 1.26µm/day, which was lower than that on RO#2 (2.19µm/day). Biofoulants increased the hydrophobicity of RO #2 to the greatest amount, up to 32°, as determined by contact angle. In addition, a wide range of changes of the chemical elements of the RO surfaces was observed with X-ray photoelectron spectroscopy analysis. RO #2 with the highest initial membrane productivity showed the poorest biofouling resistance. A combination of these novel approaches showed good agreement and suggested that membrane productivity, heterogeneity of anti-biofouling agents on membrane surface, stability of surface chemical elements and the role of virgin RO surface hydrophobicity should be jointly considered during the development of anti-biofouling polyamide thin-film RO surfaces.Item Robustness analysis of culturing perturbations on Escherichia coli colony biofilm beta-lactam and aminoglycoside antibiotic tolerance(2010-07) Zuroff, Trevor R.; Bernstein, Hans C.; Lloyd-Randolfi, J.; Jimenez-Taracido, L.; Stewart, Philip S.; Carlson, Ross P.BACKGROUND: Biofilms are ubiquitous. For instance, the majority of medical infections are thought to involve biofilms. However even after decades of investigation, the in vivo efficacy of many antimicrobial strategies is still debated, suggesting there is a need for better understanding of biofilm antimicrobial tolerances. The current study's goal is to characterize the robustness of biofilm antibiotic tolerance to medically and industrially relevant culturing perturbations. By definition, robust systems will return similar, predictable responses when perturbed, while non-robust systems will return very different, and potentially unpredictable, responses. The predictability of an antibiotic tolerance response is essential to developing, testing, and employing antimicrobial strategies. RESULTS: The antibiotic tolerance of Escherichia coli colony biofilms was tested against beta-lactam and aminoglycoside class antibiotics. Control scenario tolerances were compared to tolerances under culturing perturbations including 1) different nutritional environments 2) different temperatures 3) interruption of cellular quorum sensing and 4) different biofilm culture ages. Here, antibiotic tolerance was defined in terms of culturable biofilm cells recovered after a twenty four hour antibiotic treatment.Colony biofilm antibiotic tolerances were not robust to perturbations. Altering basic culturing parameters like nutritional environment or temperature resulted in very different, non-intuitive antibiotic tolerance responses. Some minor perturbations—like increasing the glucose concentration from 0.1 to 1 g/L—caused a ten-million fold difference in culturable cells over a twenty four hour antibiotic treatment. CONCLUSIONS: The current study presents a basis for robustness analysis of biofilm antibiotic tolerance. Biofilm antibiotic tolerance can vary in unpredictable manners based on modest changes in culturing conditions. Common antimicrobial testing methods, which only consider a single culturing condition, are not desirable since slight culturing variations can lead to very different outcomes. The presented data suggest it is essential to test antimicrobial strategies over a range of culturing perturbations relevant to the targeted application. In addition, the highly dynamic antibiotic tolerance responses observed here may explain why some current antimicrobial strategies occasionally fail.Item Spatial and temporal patterns of biocide action against Staphylococcus epidermidis biofilms(2010-05) Davison, William M.; Pitts, Betsey; Stewart, Philip S.The dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of Staphylococcus epidermidis was investigated using time-lapse confocal scanning laser microscopy. The technique allowed for the simultaneous imaging of changes in biofilm structure and disruption of cellular membrane integrity through the loss of an unbound fluorophore loaded into bacterial cells prior to antimicrobial challenge. Each of the four antimicrobial agents produced distinct spatial and temporal patterns of fluorescence loss. The antimicrobial action of chlorine was localized around the periphery of biofilm cell clusters. Chlorine was the only antimicrobial agent that caused any biofilm removal. Treatment with the quaternary ammonium compound caused membrane permeabilization that started at the periphery of cell clusters, then migrated steadily inward. A secondary pattern superimposed on the penetration dynamic suggested a subpopulation of less-susceptible cells. These bacteria lost fluorescence much more slowly than the majority of the population. Nisin caused a rapid and uniform loss of green fluorescence from all parts of the biofilm without any removal of biofilm. Glutaraldehyde caused no biofilm removal and also no loss of membrane integrity. Measurements of biocide penetration and action time at the center of cell clusters yielded 46 min for 10 mg liter-1 chlorine, 21 min for 50 mg liter-1 chlorine, 25 min for the quaternary ammonium compound, and 4 min for nisin. These results underscore the distinction between biofilm removal and killing and reinforce the critical role of biocide reactivity in determining the rate of biofilm penetration.Item Biofilm maturity studies indicate sharp debridement opens a time-dependent therapeutic window(2010-08) Wolcott, Randall D.; Rumbaugh, Kendra P.; James, Garth A.; Schultz, Gregory; Phillips, P.; Yang, Q.; Watters, C.; Stewart, Philip S.; Dowd, Scot E.Objective: To investigate the hypothesis that newly formed wound biofilms (or bioburdens) are more susceptible to antimicrobial treatment.Method: Four separate and distinct models were performed by four separate biofilm research laboratories to evaluate the resistance of biofilms to antimicrobial treatments over time. These included a drip-flow biofilm model along with a hydrodebridement study, a porcine skin punch biopsy ex vivo model, a mouse chronic wound model and clinical longitudinal debridement study.Results: All four models showed that, within the first 24 hours, the biofilm community was more susceptible to the selected antibiotics, and after maturing for up to 48 hours became increasingly tolerant. In each model, there was at least a 24-hour period in which the biofilms were more resistant to antibiotics. Each of the models utilised showed a significant decrease in the resistance of the biofilm/ burden to gentamicin for up to 24 hours with a confidence interval of at least 95%. The resistance increased in each of the models by 48 hours and reached original resistance levels by 72 hours.Conclusion: These data suggest the principles of biofilm-based wound care, along with the use of serial debridement to continually remove mature biofilm, followed by biofilm wound management strategies, including topical antibiotics while the bioburden is still immature and more susceptible, are valid.Conflict of interest: SED is director of Research and Testing Laboratory, a commercial laboratory that develops molecular methods for diagnosis of wounds and infections and CEO of Pathogenius Laboratories, which is a molecular pathogen diagnostic company with a focus on chronic wounds. RDW is medical director of Southwest Regional Wound Care Center and inventor of biofilm-based wound care principles.Item An in vitro model for the growth and analysis of chronic wound MRSA biofilms(2011-09) Agostinho, Alessandra; Hartman, A.; Lipp, C.; Parker, Albert E.; Stewart, Philip S.; James, Garth A.Aims: To develop an in vitro model (Colony/drip-flow reactor – C/DFR) for the growth and analysis of methicillin-resistant Staphylococcus aureus (MRSA) biofilms. Methods and Results: Using the C/DFR model, biofilms were grown on the top of polycarbonate filter membranes inoculated with a clinical isolate of MRSA, placed on absorbent pads in the DFR and harvested after 72 h. The biofilms varied from 256 to 308 µm in thickness with a repeatability standard deviation of 0·22. Testing of antimicrobial agents was also performed where C/DFR biofilms were grown in parallel with conventional colony biofilms. A saline solution (control), 1% silver sulfadiazine solution, and 0·25% Dakin’s solution were used to treat the biofilms for 15 min. Microscopic evaluation of biofilm morphology and thickness was conducted. The Dakins solution in both models produced statistically significantly higher log reductions than silver sulfadiazine treatment. Conclusions: The C/DFR biofilms were thick and repeatable and exhibited higher resistance to Dakins solution than the treated colony biofilms. Significance and Impact of the Study: The C/DFR can be used as a tool for examining complex biofilm physiology as well as for performing comparative experiments that test wound care products and novel antimicrobials.Item Antimicrobial penetration and efficacy in an in vitro oral biofilm model(2011-05) Corbin, A.; Pitts, Betsey; Parker, Albert E.; Stewart, Philip S.The penetration and overall efficacy of six mouthrinse actives was evaluated by using an in vitro flow cell oral biofilm model. The technique involved preloading biofilm cells with a green fluorescent dye that leaked out as the cells were permeabilized by a treatment. The loss of green color, and of biomass, was observed by time-lapse microscopy during 60 min of treatment under continuous flow conditions. The six actives analyzed were ethanol, sodium lauryl sulfate, triclosan, chlorhexidine digluconate (CHX), cetylpyridinium chloride, and nisin. Each of these agents effected loss of green fluorescence throughout biofilm cell clusters, with faster action at the edge of a cell cluster and slower action in the cluster center. The time to reach half of the initial fluorescent intensity at the center of a cell cluster, which can be viewed as a combined penetration and biological action time, ranged from 0.6 to 19 min for the various agents. These times are much longer than the predicted penetration time based on diffusion alone, suggesting that anti-biofilm action was controlled more by the biological action time than by the penetration time of the active. None of the agents tested caused any removal of the biofilm. The extent of fluorescence loss after 1 h of exposure to an active ranged from 87 to 99.5%, with CHX being the most effective. The extent of fluorescence loss in vitro, but not penetration and action time, correlated well with the relative efficacy data from published clinical trials.Item Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or DispersinB(2011-07) Brindle, Eric R.; Miller, David A.; Stewart, Philip S.The force-deflection and removal characteristics of bacterial biofilm were measured by two different techniques before and after chemical, or enzymatic, treatment. The first technique involved time lapse imaging of a biofilm grown in a capillary flow cell and subjected to a brief shear stress challenge imparted through increased fluid flow. Biofilm removal was determined by calculating the reduction in biofilm area from quantitative analysis of transmission images. The second technique was based on microindentation using an atomic force microscope. In both cases, biofilms formed by Staphylococcus epidermidis were exposed to buffer (untreated control), urea, chlorhexidine, iron chloride, or DispersinB.In control experiments, the biofilm exhibited force-deflection responses that were similar before and after the same treatment. The biofilm structure was stable during the post-treatment shear challenge (1% loss). Biofilms treated with chlorhexidine became less deformable after treatment and no increase in biomass removal was seen during the post-treatment shear challenge (2% loss). In contrast, biofilms treated with urea or DispersinB became more deformable and exhibited significant biofilm loss during the post-treatment flow challenge (71% and 40%, respectively). During the treatment soak phase, biofilms exposed to urea swelled. Biofilms exposed to iron chloride showed little difference from the control other than slight contraction during the treatment soak. These observations suggest the following interpretations: (1) chemical or enzymatic treatments, including those that are not frankly antimicrobial, can alter the cohesion of bacterial biofilm; (2) biocidal treatments (e.g., chlorhexidine) do not necessarily weaken the biofilm; and (3) biofilm removal following treatment with agents that make the biofilm more deformable (e.g., urea, DispersinB) depend on interaction between the moving fluid and the biofilm structure. Measurements such as those reported here open the door to development of new technologies for controlling detrimental biofilms by targeting biofilm cohesion rather than killing microorganisms.Item In vitro efficacy of bismuth thiols against biofilms formed by bacteria isolated from human chronic wounds(2011-08) Folsom, James P.; Baker, B.; Stewart, Philip S.Aims: The purpose of this study was to evaluate the antimicrobial efficacy of thirteen bismuth thiol preparations for bactericidal activity against established biofilms formed by two bacteria isolated from human chronic wounds.Methods: Single species biofilms of a Pseudomonas aeruginosa or a methicillin resistant Staphylococcus aureus (MRSA) were grown in either colony biofilm or drip-flow reactors systems. Biofilms were challenged with bismuth thiols, antibiotics or silver sulfadiazine, and log reductions were determined by plating for colony formation.Conclusions: Antibiotics were ineffective or inconsistent against biofilms of both bacterial species tested. None of the antibiotics tested was able to achieve >2 log reductions in both biofilm models. The 13 different bismuth thiols tested in this investigation achieved widely varying degrees of killing, even against the same microorganism in the same biofilm model. For each microorganism, the best bismuth thiol easily outperformed the best conventional antibiotic. Against P. aeruginosa biofilms, bismuth-2,3-dimercaptopropanol (BisBAL) at 40–80 µg ml-1 achieved >7.7 mean log reduction for the two biofilm models. Against MRSA biofilms, bismuth-1,3-propanedithiolâ „bismuth-2-mercaptopyridine N-oxide (BisBDTâ „PYR) achieved a 4.9 log reduction.Significance and Impact of the Study: Bismuth thiols are effective antimicrobial agents against biofilms formed by wound bacteria and merit further development as topical antiseptics for the suppression of biofilms in chronicwounds.Item Efficacy of zosteric acid sodium salt on the yeast biofilm model Candida albicans(2011-05) Villa, Federica; Stewart, Philip S.; Giussani, Barbara; Roncoroni, S.; Albanese, Domenico; Giordano, C.; Tunesi, M.; Cappitelli, FrancescaCandida albicans is the most notorious and the most widely studied yeast biofilm former. Design of experiments (DoE) showed that 10 mg/L zosteric acid sodium salt reduced C. albicans adhesion and the subsequent biofilm formation by at least 70%, on both hydrophilic and hydrophobic surfaces of 96-well plates. Indeed, biofilm imaging revealed the dramatic impact of zosteric acid sodium salt on biofilm thickness and morphology, due to the inability of the cells to form filamentous structures while remaining metabolically active. In the same way, 10 mg/L zosteric acid sodium salt inhibited C. albicans biofilm formation when added after the adhesion phase. Contrary to zosteric acid sodium salt, methyl zosterate did not affect yeast biofilm. In addition, zosteric acid sodium salt enhanced sensitivity to chlorhexidine, chlorine, hydrogen peroxide, and cis-2-decenoic acid, with a reduction of 0.5 to 8 log units. Preliminary in vitro studies using suitable primary cell based models revealed that zosteric acid sodium salt did not compromise the cellular activity, adhesion, proliferation or morphology of either the murine fibroblast line L929 or the human osteosarcoma line MG-63. Thus the use of zosteric acid sodium salt could provide a suitable, innovative, preventive, and integrative approach to preventing yeast biofilm formation.Item Chemical and antimicrobial treatments change the viscoelastic properties of bacterial biofilms(2011-01) Jones, Warren L.; Sutton, Michael P.; McKittrick, Ladean R.; Stewart, Philip S.Changes in the viscoelastic material properties of bacterial biofilms resulting from chemical and antimicrobial treatments were measured by rheometry. Colony biofilms of Staphylococcus epidermidis or a mucoid Pseudomonas aeruginosa were subjected to a classical creep test performed using a parallel plate rheometer. Data were fit to the 4-parameter Burger model to quantify the material properties. Biofilms were exposed to the chloride salts of several common mono-, di-, and tri- valent cations, and to urea, industrial biocides, and antibiotics. Many of these treatments resulted in statistically significant alterations in the material properties of the biofilm. Multivalent cations stiffened the P. aeruginosa biofilm, while ciprofloxacin and glutaraldehyde weakened it. Urea, rifampin, and a quaternary ammonium biocide weakened the S. epidermidis biofilm. In general, there was no correspondence between the responses of the two different types of biofilms to a particular treatment. These results underscore the distinction between the killing power of an antimicrobial agent and its ability to alter biofilm mechanical properties and thereby influence biofilm removal. Understanding biofilm rheology and how it is affected by chemical treatment could lead to improvements in biofilm control.
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