Center for Biofilm Engineering (CBE)
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At the Center for Biofilm Engineering (CBE), multidisciplinary research teams develop beneficial uses for microbial biofilms and find solutions to industrially relevant biofilm problems. The CBE was established at Montana State University, Bozeman, in 1990 as a National Science Foundation Engineering Research Center. As part of the MSU College of Engineering, the CBE gives students a chance to get a head start on their careers by working on research teams led by world-recognized leaders in the biofilm field.
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Item Analysis of Clostridium difficile biofilms: imaging and antimicrobial treatment(2018-01) James, Garth A.; Chesnel, L.; Boegli, Laura; Pulcini, Elinor D.; Fisher, Steve T.; Stewart, Philip S.BACKGROUND: Clostridium difficile, a spore-forming Gram-positive anaerobic bacillus, is the most common causative agent of healthcare-associated diarrhoea. Formation of biofilms may protect C. difficile against antibiotics, potentially leading to treatment failure. Furthermore, bacterial spores or vegetative cells may linger in biofilms in the gut causing C. difficile infection recurrence. OBJECTIVES: In this study, we evaluated and compared the efficacy of four antibiotics (fidaxomicin, surotomycin, vancomycin and metronidazole) in penetrating C. difficile biofilms and killing vegetative cells. METHODS: C. difficile biofilms grown initially for 48 or 72 h using the colony biofilm model were then treated with antibiotics at a concentration of 25 × MIC for 24 h. Vegetative cells and spores were enumerated. The effect of treatment on biofilm structure was studied by scanning electron microscopy (SEM). The ability of fidaxomicin and surotomycin to penetrate biofilms was studied using fluorescently tagged antibiotics. RESULTS: Both surotomycin and fidaxomicin were significantly more effective than vancomycin or metronidazole (P < 0.001) at killing vegetative cells in established biofilms. Fidaxomicin was more effective than metronidazole at reducing viable spore counts in biofilms (P < 0.05). Fluorescently labelled surotomycin and fidaxomicin penetrated C. difficile biofilms in < 1 h. After 24 h of treatment, SEM demonstrated that both fidaxomicin and surotomycin disrupted the biofilm structure, while metronidazole had no observable effect. CONCLUSIONS: Fidaxomicin is effective in disrupting C. difficile biofilms, killing vegetative cells and decreasing spore counts.Item Paired methods to measure biofilm killing and removal: a case study with Penicillin G treatment of Staphylococcus aureus biofilm(2018-03) Ausbacher, D.; Lorenz, Lindsey A.; Pitts, Betsey; Stewart, Philip S.; Goeres, Darla M.Biofilms are microbial aggregates that show high tolerance to antibiotic treatments in vitro and in vivo. Killing and removal are both important in biofilm control, therefore methods that measure these two mechanisms were evaluated in a parallel experimental design. Kill was measured using the single tube method (ASTM method E2871) and removal was determined by video microscopy and image analysis using a new treatment flow cell. The advantage of the parallel test design is that both methods used biofilm covered coupons harvested from a CDC biofilm reactor, a well-established and standardized biofilm growth method. The control Staphylococcus aureus biofilms treated with growth medium increased by 0 6 logs during a 3-h contact time. Efficacy testing showed biofilms exposed to 400 lmol l1 penicillin G decreased by only 0 3 logs. Interestingly, time-lapse confocal scanning laser microscopy revealed that penicillin G treatment dispersed the biofilm despite being an ineffective killing agent. In addition, no biofilm removal was detected when assays were performed in 96-well plates. These results illustrate that biofilm behaviour and impact of treatments can vary substantially when assayed by different methods. Measuring both killing and removal with well-characterized methods will be crucial for the discovery of new anti-biofilm strategies.Item Bacterial biofilms: A common cause of persistent infections(1999-05) Costerton, J. William; Stewart, Philip S.; Greenberg, E. P.Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.Item Electrolytic generation of oxygen partially explains electrical enhancement of tobramycin efficacy against pseudomonas aeruginosa biofilm(1999-02) Stewart, Philip S.; Wattanakaroon, Wanida; Goodrum, L.; Fortun, Susana M.; McLeod, Bruce R.The role of electrolysis products, including protons, hydroxyl ions, reactive oxygen intermediates, oxygen, hydrogen, and heat, in mediating electrical enhancement of killing of Pseudomonas aeruginosa biofilms by tobramycin (the bioelectric effect) was investigated. The log reduction in biofilm viable cell numbers compared to the numbers for the untreated positive control effected by antibiotic increased from 2.88 in the absence of electric current to 5.58 in the presence of electric current. No enhancement of antibiotic efficacy was observed when the buffer composition was changed to simulate the reduced pH that prevails during electrolysis. Neither did stabilization of the pH during electrical treatment by increasing the buffer strength eliminate the bioelectric effect. The temperature increase measured in our experiments, less than 0.2°C, was far too small to account for the greatly enhanced antibiotic efficacy. The addition of sodium thiosulfate, an agent capable of rapidly neutralizing reactive oxygen intermediates, did not abolish electrical enhancement of killing. The bioelectric effect persisted when all of the ionic constituents of the medium except the two phosphate buffer components were omitted. This renders the possibility of electrochemical generation of an inhibitory ion, such as nitrite from nitrate, an unlikely explanation for electrical enhancement. The one plausible explanation for the bioelectric effect revealed by this study was the increased delivery of oxygen to the biofilm due to electrolysis. When gaseous oxygen was bubbled into the treatment chamber during exposure to tobramycin (without electric current), a 1.8-log enhancement of killing resulted. The enhancement of antibiotic killing by oxygen was not due simply to physical disturbances caused by sparging the gas because similar delivery of gaseous hydrogen caused no enhancement whatsoever.Item Color measurement as a means of quantifying surface biofouling(1998-11) Pitts, Betsey; Hamilton, Martin A.; McFeters, Gordon A.; Stewart, Philip S.; Willse, Alan Ray; Zelver, NickLaboratory reactors fitted with removable ceramic porcelain growth surfaces were inoculated with a consortium of biofilm forming environmental isolates. A Minolta colorimeter CR-200 (Minolta Camera Co., Ltd, Ramsey, NJ) was used in conjunction with a specially designed adapter to evaluate the reflective color of the porcelain disks as biofilm accumulated on them. Areal viable cell counts were monitored over a period of eleven days in two separate experiments and direct color measurements of the untreated, microbially fouled test surfaces were collected. This colorimetric assay was both non-destructive and immediate. A strong linear relationship between log cell density and log color change was observed. The Pearson product moment correlation coefficient for all 45 observations combined was r=0.95. Separate regression lines for each experiment were not significantly different (P=0.19). When adjusted for time, the (partial) correlation coefficient between log cell density and log color change was r=0.87, which suggests that the relationship between the two measures can not be explained by their mutual dependence on time. Reflective color measurement provided a rapid, non-destructive and quantitative measure of biofilm accumulation.Item Bacterial characterization of toilet bowl biofilms(1998-08) Pitts, Betsey; Stewart, Philip S.; McFeters, Gordon A.; Hamilton, Martin A.; Willse, Alan Ray; Zelver, NickMethods have been developed and applied for sampling, characterizing and quantifying naturally occurring toilet bowl biofilms. Ceramic porcelain disks mounted in neoprene rubber strips were sealed in place in toilet bowls in three residences in Bozeman, Montana. In each bowl, duplicate strips were placed above, at and below the water level. In 7 consecutive weeks, duplicate disks from each zone in each bowl were removed. Surface biofouling was measured by viable cell areal density. Specific fouling rates were calculated and variability among toilet bowls and water levels was assessed. Specific fouling rates ranged from 0.0 to 0.46d‐1. Average areal cell densities at the end of 7 weeks ranged from 103 to 107cfu cm‐2. The extent of fouling was highest below the water line. Neutralization of the chlorine residual (typically 0.9 mg l‐1) in one toilet did not increase the extent of fouling compared to the controls. Biofilm areal viable cell densities and bowl water viable counts were positively correlated (r = 0.78). The visual threshold for detection of toilet bowl biofilm by the naked eye was approximately 105 cfu cm‐2. In a heavily fouled toilet bowl, the biofilm was up to 20 μm thick. Microorganisms were isolated from the biofilm and identified. Of the 32 organisms that were further characterized, 10 were identified as Pseudomonas, Sphingomonas or Chryseomonas species.Item Analysis of biocide transport limitation in an artificial biofilm system(1998-09) Stewart, Philip S.; Grab, L.; Diemer, J. A.An alginate gel bead artificial biofilm system was used to assay biofilm susceptibility to four biocides and to analyse the extent to which each agent penetrated the biofilm. Chlorine, glutaraldehyde, an isothiazolone, and a quaternary ammonium compound were tested on alginate-entrapped Enterobacter aerogenes in gel beads ranging from 1·8 to 6 mm in diameter. Gel-entrapped bacteria were less susceptible to all four antimicrobial agents than were planktonic micro-organisms. The degree of kill measured in artificial biofilm gel beads depended on the size of the gel bead and the cell density at which it was loaded. Disinfection efficacy decreased as gel bead radius or cell density increased. The manifest dependence of biofilm disinfection efficacy on the physical properties of the artificial biofilm (radius and cell density) suggests the impingement of transport limitation of biocide transport into the biofilm. A previously developed theory of biocide reaction and diffusion in biofilm was tested by calculating an appropriate Thiele modulus. In accordance with the theory, the efficacy of all four biocides decreased, albeit noisily, as the Thiele modulus exceeded 1. This result demonstrates that transport limitation can impact antimicrobial performance against biofilms not only of oxidizing biocides but also of non-oxidizing agents.Item Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability(1998-10) Xu, Karen D.; Stewart, Philip S.; Xia, Fuhu; Huang, Ching-Tsan; McFeters, Gordon A.Item A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms(1998-08) Stewart, Philip S.Experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms are reviewed. Effective diffusive permeabilities, the parameter appropriate to the analysis of reaction-diffusion interactions, depend on solute type and biofilm density. Three categories of solute physical chemistry with distinct diffusive properties were distinguished by the present analysis. In order of descending mean relative effective diffusive permeability (De/Daq) these were inorganic anions or cations (0.56), nonpolar solutes with molecular weights of 44 or less (0.43), and organic solutes of molecular weight greater than 44 (0.29). Effective diffusive permeabilities decrease sharply with increasing biomass volume fraction suggesting a serial resistance model of diffusion in biofilms as proposed by Hinson and Kocher (1996). A conceptual model of biofilm structure is proposed in which each cell is surrounded by a restricted permeability envelope. Effective diffusion coefficients, which are appropriate to the analysis of transient penetration of nonreactive solutes, are generally similar to effective diffusive permeabilities in biofilms of similar composition. In three studies that examine diffusion of very large molecular weight solutes ( > 5000) in biofilms, the average ratio of the relative effective diffusion coefficient of the large solute to the relative effective diffusion coefficient of either sucrose or fluorescein was 0.64, 0.61, and 0.36. It is proposed that large solutes are effectively excluded from microbial cells, that small solutes partition into and diffuse within cells, and that ionic solutes are excluded from cells but exhibit increased diffusive permeability (but decreased effective diffusion coefficients) due to sorption to the biofilm matrix. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:261–272, 1998.Item The study of microbial biofilms by classical fluorescence microscopy(1998) Huang, Ching-Tsan; Stewart, Philip S.; McFeters, Gordon A.