Scholarly Work - Center for Biofilm Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/9335
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Item Nisin penetration and efficacy against Staphylococcus aureus biofilms under continuous-flow conditions(2019-07) Godoy-Santos, Fernanda; Pitts, Betsey; Stewart, Philip S.; Mantovani, Hilario C.Biofilms may enhance the tolerance of bacterial pathogens to disinfectants, biocides and other stressors by restricting the penetration of antimicrobials into the matrix-enclosed cell aggregates, which contributes to the recalcitrance of biofilm-associated infections. In this work, we performed real-time monitoring of the penetration of nisin into the interior of Staphylococcus aureus biofilms under continuous flow and compared the efficacy of this lantibiotic against planktonic and sessile cells of S. aureus . Biofilms were grown in Center for Disease Control (CDC) reactors and the spatial and temporal effects of nisin action on S. aureus cells were monitored by real-time confocal microscopy. Under continuous flow, nisin caused loss of membrane integrity of sessile cells and reached the bottom of the biofilms within ~20 min of exposure. Viability analysis using propidium iodide staining indicated that nisin was bactericidal against S. aureus biofilm cells. Time-kill assays showed that S. aureus viability reduced 6.71 and 1.64 log c.f.u. ml-1 for homogenized planktonic cells in exponential and stationary phase, respectively. For the homogenized and intact S. aureus CDC biofilms, mean viability decreased 1.25 and 0.50 log c.f.u. ml-1, respectively. Our results demonstrate the kinetics of biofilm killing by nisin under continuous-flow conditions, and shows that alterations in the physiology of S. aureus cells contribute to variations in sensitivity to the lantibiotic. The approach developed here could be useful to evaluate the antibiofilm efficacy of other bacteriocins either independently or in combination with other antimicrobials.Item A permeability-increasing drug synergizes with bacterial efflux pump inhibitors and restores susceptibility to antibiotics in multi-drug resistant Pseudomonas aeruginosa strains(2019-03) Ferrer-Espada, Raquel; Shahrour, Hawraa; Pitts, Betsey; Stewart, Philip S.; Sánchez-Gómez, Susana; Martínez-de-Tejada, GuillermoResistance to antibiotics poses a major global threat according to the World Health Organization. Restoring the activity of existing drugs is an attractive alternative to address this challenge. One of the most efficient mechanisms of bacterial resistance involves the expression of efflux pump systems capable of expelling antibiotics from the cell. Although there are efflux pump inhibitors (EPIs) available, these molecules are toxic for humans. We hypothesized that permeability-increasing antimicrobial peptides (AMPs) could lower the amount of EPI necessary to sensitize bacteria to antibiotics that are efflux substrates. To test this hypothesis, we measured the ability of polymyxin B nonapeptide (PMBN), to synergize with antibiotics in the presence of EPIs. Assays were performed using planktonic and biofilm-forming cells of Pseudomonas aeruginosa strains overexpressing the MexAB-OprM efflux system. Synergy between PMBN and EPIs boosted azithromycin activity by a factor of 2,133 and sensitized P. aeruginosa to all tested antibiotics. This reduced several orders of magnitude the amount of inhibitor needed for antibiotic sensitization. The selected antibiotic-EPI-PMBN combination caused a 10 million-fold reduction in the viability of biofilm forming cells. We proved that AMPs can synergize with EPIs and that this phenomenon can be exploited to sensitize bacteria to antibiotics.Item Polynomial accelerated solutions to a LARGE Gaussian model for imaging biofilms: in theory and finite precision(2018-06) Parker, Albert E.; Pitts, Betsey; Lorenz, Lindsey A.; Stewart, Philip S.Three-dimensional confocal scanning laser microscope images offer dramatic visualizations of living biofilms before and after interventions. Here, we use confocal microscopy to study the effect of a treatment over time that causes a biofilm to swell and contract due to osmotic pressure changes. From these data (the video is provided in the supplementary materials), our goal is to reconstruct biofilm surfaces, to estimate the effect of the treatment on the biofilm’s volume, and to quantify the related uncertainties. We formulate the associated massive linear Bayesian inverse problem and then solve it using iterative samplers from large multivariate Gaussians that exploit well-established polynomial acceleration techniques from numerical linear algebra. Because of a general equivalence with linear solvers, these polynomial accelerated iterative samplers have known convergence rates, stopping criteria, and perform well in finite precision. An explicit algorithm is provided, for the first time, for an iterative sampler that is accelerated by the synergistic implementation of preconditioned conjugate gradient and Chebyshev polynomials.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 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 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 A microtiter-plate screening method for biofilm disinfection and removal(2003-08) Pitts, Betsey; Hamilton, Martin A.; Zelver, Nick; Stewart, Philip S.A quantitative spectrophotometric method was developed to measure the removal and killing efficacy of antibiofilm agents. Biofilms of Pseudomonas aeruginosa or Staphylococcus epidermidis were grown in 96-well plates, treated with an agent, then stained with either the biomass indicator crystal violet or the respiratory indicator 5-cyano-2,3-ditolyl tetrazolium chloride. This rapid screening method is sensitive enough to elucidate concentration–response relationships as well as differences between species responses to treatments. Using these assays, agents can be ranked by their ability to remove or kill biofilm.Item A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance(2003-11) Mah, Thien-Fah; Pitts, Betsey; Pellock, Brett; Walker, Graham C.; Stewart, Philip S.; O'Toole, George A.Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.Item Stratified growth in Pseudomonas aeruginosa biofilms(2004-10) Werner, Erin M.; Roe, Frank L.; Bugnicourt, Amandine; Franklin, Michael J.; Heydorn, Arne; Molin, Søren; Pitts, Betsey; Stewart, Philip S.In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp1 promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 µm wide in colony biofilms and 30 µm wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 µm into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.
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