Scholarly Work - Center for Biofilm Engineering
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Item Modeling biocide action against biofilms(2000-03) Stewart, Philip S.; Hamilton, Martin A.; Goldstein, B. R.; Schneider, B. T.A phenomenological model of biocide action against microbial biofilms was derived. Processes incorporated in the model include bulk flow in and out of a well-mixed reactor, transport of dissolved species into the biofilm, substrate consumption by bacterial metabolism, bacterial growth, advection of cell mass within the biofilm, cell detachment from the biofilm, cell death, and biocide concentration-dependent disinfection. Simulations were performed to analyze the general behavior of the model and to perform preliminary sensitivity analysis to identify key input parameters. The model captured several general features of antimicrobial agent action against biofilms that have been observed widely by experimenters and practitioners. These included (1) rapid disinfection followed by biofilm regrowth, (2) slower detachment than disinfection, and (3) reduced susceptibility of microorganisms in biofilms. The results support the plausibility of a mechanism of biofilm resistance in which the biocide is neutralized by reaction with biofilm constituents, leading to a reduction in the bulk biocide concentration and, more significantly, biocide concentration gradients within the biofilm. Sensitivity experiments and analyses identified which input parameters influence key response variables. Each of three response variables was sensitive to each of the five input parameters, but they were most sensitive to the initial biofilm thickness and next most sensitive to the biocide disinfection rate coefficient. Statistical regression modeling produced simple equations for approximating the response variables for situations within the range of conditions covered by the sensitivity experiment. The model should be useful as a tool for studying alternative biocide control strategies. For example, the simulations suggested that a good interval between pulses of biocide is the time to minimum thickness.Item Confocal laser microscopy on biofilms: Successes and limitations(2008-07) Pitts, Betsey; Stewart, Philip S.Imaging of bacterial biofilms with microscopes has been an essential and transformative method in biofilm research. Fluorescence microscopy can elucidate specific biofilm components and cellular activities that cannot be separated otherwise. In particular, confocal fluorescence microscopy extends that examination through the thickness of a fully hydrated, in-situ biofilm, affording the potential for 3D, non-invasive, time-lapse imaging. This article discusses some striking examples of the insight provided by confocal fluorescence microscopy into biofilm structure, composition, and heterogeneity, and will also enumerate some limitations of this imaging process.Item Battling biofilms(2001-07) Costerton, J. William; Stewart, Philip S.Item Gene expression and protein levels of the stationary phase sigma factors, RpoS, in continuously-fed Pseudomonas aeruginosa biofilms(2001-05) Xu, Karen D.; Franklin, Michael J.; Park, C-H; McFeters, Gordon A.; Stewart, Philip S.Bacteria growing in biofilms experience gradients of environmental conditions, including varying levels of nutrients and oxygen. Therefore, bacteria within biofilms may enter distinct physiological states, depending on the surrounding conditions. In this study, rpoS expression and RpoS levels were measured as indicators of stationary phase growth within thick continuously-fed Pseudomonas aeruginosa biofilms. The level of rpoS expression in a 3-day-old biofilm was found to be three-fold higher than the average expression in stationary phase planktonic cultures. In planktonic cultures, oxygen limitation did not lead to increased levels of RpoS, suggesting that oxygen limitation was not the environmental signal causing increased expression of rpoS. These results suggest that bacteria within P. aeruginosa biofilms may exhibit stationary phase characteristics even when cultured in flow conditions that continually replenish nutrients.Item Multicellular resistance: Biofilms(2001-05) Stewart, Philip S.Introduction: In 1987, Bill Costerton and colleagues published a landmark review article that was the first to articulate the general phenomenon of biofilm resistance to antimicrobial agents 1. Fourteen years later, the question of how bacteria in biofilms manage to evade killing by antiseptics, antibiotics and antimicrobial components of the host defenses remains unanswered. Recently, Mah and O'Toole provided an excellent overview of the current hypotheses and recent data on mechanisms of biofilm resistance 2. Recognition of the significance of this problem is bound to grow as the role of biofilms in chronic infections becomes increasingly clear 3 and 4.Why has the underlying basis of reduced susceptibility of bacteria in biofilms proven to be such a difficult nut to crack? Mah and O'Toole register the possibility that multiple resistance mechanisms operate in concert within a single biofilm community. I would like to offer two further perceptions, both admittedly speculative, about the general nature of the resistance of bacterial biofilms to antimicrobial agents. Although these ideas are probably only partially correct, perhaps they can guide some fresh approaches to solving the problem of biofilm recalcitrance.Item Biofilm removal caused by chemical treatments(2000-12) Chen, Xiao; Stewart, Philip S.Biofilm protein removal by a variety of chemical treatments was investigated. Binary population biofilms of P. aeruginosa and K. pneumoniae were grown in continuous flow annular reactors for 7–9 days prior to a 1-h treatment period. Treatments that caused removal of more than 25% of the biomass (as total protein) included NaCl and CaCl2, two chelating agents (EDTA and Dequest 2006), surfactants (SDS, Tween 20, and Triton X-100), a pH increase, lysozyme, hypochlorite, monochloramine, and concentrated urea. Treatments that caused little removal (less than 25%) included a control, MgCl2, sucrose, nutrient upshifts and downshifts, and a pH decrease. The amount of biofilm protein removal and the reduction in viable cell numbers in the biofilm were not correlated. Some treatments caused significant killing but not much removal while other treatments caused removal with little killing. These results underscore the fact that biofilm removal and killing are distinct processes. The chemical diversity of agents that bring about biofilm removal suggests that multiple interactive forces contribute to biofilm cohesion. No pattern of differential removal of the two microbial species could be discerned.Item Mechanisms of antibiotic resistance in bacterial biofilms(2003-01) Stewart, Philip S.Bacteria that attach to a surface and grow as a biofilm are protected from antibiotic killing. Reduced antibiotic susceptibility contributes to the persistence of biofilm infections such as those associated with implanted devices. The protective mechanisms at work in biofilms appear to be distinct from those that are responsible for conventional antibiotic resistance. In biofilms, poor antibiotic penetration, nutrient limitation, slow growth, adaptive stress responses, and formation of persister cells are hypothesized to constitute a multi-layered defense. The genetic and biochemical details of these biofilm defenses are only now beginning to emerge. Each gene and gene product contributing to this resistance may be a target for the development of new chemotherapeutic agents. Disabling biofilm resistance may enhance the ability of existing antibiotics to clear infections involving biofilms that are refractory to current treatments.Item Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates(2001-09) Stewart, Philip S.; Rayner, Joanna; Roe, Frank L.; Ree, Wayne M.AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two-species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l-1. Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into sim 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as the penetration time for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l-1 alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport was clearly retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing, despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm, demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.Item Role of dose concentration in biocide efficacy against pseudomonas aeruginosa biofilms(2002-07) Grobe, Katherine Jean; Zahller, Jeff; Stewart, Philip S.Pseudomonas aeruginosa entrapped in alginate gel beads to form artificial biofilms resisted killing by chlorine, glutaraldehyde, 2,2-dibromo-3-nitrilopropionamide (DBNPA), and an alkyl dimethyl benzyl ammonium compound (ADBAC). The degree of resistance was quantified by a resistance factor that compared killing times for biofilm and planktonic cells in response to the same concentration of antimicrobial agent. Resistance factors averaged 120 for chlorine, 34 for glutaraldehyde, 29 for DBNPA, and 1900 for ADBAC. In every case resistance factors decreased with increasing concentration of the antimicrobial agent. An independent analysis of the concentration dependence of the apparent rates of killing of planktonic and biofilm bacteria showed that elevating the treatment concentration increased bacterial killing more in the biofilm than it did in a suspension culture. Calculation of a transport modulus comparing the rates of biocide reaction and diffusion suggested that at least part of the biofilm resistance to chlorine, glutaraldehdye, and DBNPA could be attributed to incomplete or slow penetration of these agents into the biofilm. Time-kill curves were nonlinear for biofilm bacteria in some cases. The shapes of these curves implicated retarded antimicrobial penetration for chlorine and glutaraldehyde and the presence of a tolerant subpopulation for DBNPA and ADBAC. The results indicate that treating biofilms with a concentrated dose of biocide is more effective than using prolonged doses of a lower concentration.Item Transmission electron microscopic study of antibiotic action on klebsiella pneumoniae biofilm(2002-08) Zahller, Jeff; Stewart, Philip S.The penetration of ampicillin and ciprofloxacin through biofilms formed by Klebsiella pneumoniae was confirmed by transmission electron microscopic observation of antibiotic-affected cells at the distal edge of the biofilm. Because the bacteria nevertheless survived antibiotic treatment, some protective mechanism other than inadequate penetration must have been at work in the biofilm.