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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.Item Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation(1998-04) Huang, Ching-Tsan; Xu, Karen D.; McFeters, Gordon A.; Stewart, Philip S.The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 μmol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor.Item Enhanced bacterial biofilm control using electromagnetic fields in combination with antibiotics(1999) McLeod, Bruce R.; Fortun, Susana M.; Costerton, J. William; Stewart, Philip S.