Browsing by Author "Lorenz, Lindsey A."

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Development, standardization, and validation of a biofilm efficacy test: The single tube method
(2019-10) Goeres, Darla M.; Walker, Diane K.; Buckingham-Meyer, Kelli; Lorenz, Lindsey A.; Summers, Jennifer; Fritz, Blaine; Goveia, Danielle; Dickerman, Grace; Schultz, Johanna M.; Parker, Albert E.
Methods validated by a standard setting organization enable public, industry and regulatory stakeholders to make decisions on the acceptability of products, devices and processes. This is because standard methods are demonstrably reproducible when performed in different laboratories by different researchers, responsive to different products, and rugged when small (usually inadvertent) variations from the standard procedure occur. The Single Tube Method (ASTM E2871) is a standard method that measures the efficacy of antimicrobials against biofilm bacteria that has been shown to be reproducible, responsive and rugged. In support of the reproducibility assessment, a six-laboratory study was performed using three antimicrobials: a sodium hypochlorite, a phenolic and a quaternary/alcohol blend, each tested at low and high efficacy levels. The mean log reduction in viable bacteria in this study ranged from 2.32 to 4.58 and the associated reproducibility standard deviations ranged from 0.89 to 1.67. Independent follow-up testing showed that the method was rugged with respect to deviations in sonication duration and sonication power but slightly sensitive to sonicator reservoir degassing and tube location within the sonicator bath. It was also demonstrated that when a coupon was dropped into a test tube, bacteria can splash out of reach of the applied antimicrobials, resulting in substantial bias when estimating log reductions for the products tested. Bias can also result when testing products that hinder the harvesting of microbes from test surfaces. The culmination of this work provided recommended changes to the early version of the standard method E2871-13 (ASTM, 2013b) including use of splashguards and microscopy checks. These changes have been incorporated into a revised ASTM method E2871-19 (ASTM 2019) that is the basis for the first regulatory method (ATMP-MB-20) to substantiate “kills biofilm” claims for antimicrobials registered and sold in the US.
Drip flow reactor method exhibits excellent reproducibility based on a 10-laboratory collaborative study
(Elsevier BV, 2020) Goeres, Darla M.; Parker, Albert E.; Walker, Diane K.; Meier, Kelsey; Lorenz, Lindsey A.; Buckingham-Meyer, Kelli
A standard method for growing Pseudomonas aeruginosa biofilm in the Drip Flow Biofilm Reactor was assessed in a 10-laboratory study. The mean log density was 9.29 Log10(CFU/cm2). The repeatability and reproducibility SDs were equal to 0.22 and 0.24, respectively, providing statistical confidence in data generated by the method.
Evaluation and remediation of bulk soap dispensers for biofilm
(2012-01) Lorenz, Lindsey A.; Ramsay, Bradley D.; Goeres, Darla M.; Fields, Matthew W.; Zapka, Carrie A.; Macinga, David R.
Recent studies evaluating bulk soap in public restroom soap dispensers have demonstrated up to 25% of open refillable bulk-soap dispensers were contaminated with ~6 log10(CFU ml-1) heterotrophic bacteria. In this study, plastic counter-mounted, plastic wall-mounted and stainless steel wall-mounted dispensers were analyzed for suspended and biofilm bacteria using total cell and viable plate counts. Independent of dispenser type or construction material, the bulk soap was contaminated with 4â€“7 log10(CFU ml-1) bacteria, while 4â€“6 log10(CFU cm-2) biofilm bacteria were isolated from the inside surfaces of the dispensers (n=6). Dispenser remediation studies, including a 10 min soak with 5000 mg 1-1 sodium hypochlorite, were then conducted to determine the efficacy of cleaning and disinfectant procedures against established biofilms. The testing showed that contamination of the bulk soap returned to pre-test levels within 7â€“14 days. These results demonstrate biofilm is present in contaminated bulk-soap dispensers and remediation studies to clean and sanitize the dispensers are temporary.
Holistic Management of Textile Odor Using Novel Silver-Polymeric Complexes
(2018-08) Frattarelli, Dave; Powers, Lisa; Doshi, Deepack; Vargo, Kevin; Patel, Bhavin; Liboon, Jennifer; Gallagher, Michelle; Monticello, Robert; Goeres, Darla M.; Lorenz, Lindsey A.; Buckingham-Meyer, Kelli
Odor poses a growing concern in clothing and apparel applications due to laundering limitations at managing odor-causing microorganisms. Herein, a novel silver-polymer complex was applied to textile materials and studied using quantitative antimicrobial assays, gas chromatography techniques, and odor panel sensory tests to ascertain odor control function and effectiveness. A known chemical odor pathway involving leucine conversion to isovaleric acid was studied and found to be disrupted in silver-treated fabrics. Furthermore, its odor absorption function was confirmed with up to 90% retention of select thiol and fatty acid odors at body temperature in a model odor bouquet. Lastly, human sensory studies were used to support laboratory odor measurements using seven-day wear trials and milk odor generation techniques after 50 launderings.
A method for growing a biofilm under low shear at the air–liquid interface using the drip flow biofilm reactor
(2009-04) Goeres, Darla M.; Hamilton, Martin A.; Beck, Nicholas A.; Buckingham-Meyer, Kelli; Hilyard, Jackie D.; Loetterle, Linda R.; Lorenz, Lindsey A.; Walker, Diane K.; Stewart, Philip S.
This protocol describes how to grow a Pseudomonas aeruginosa biofilm under low fluid shear close to the airâ€“liquid interface using the drip flow reactor (DFR). The DFR can model environments such as food-processing conveyor belts, catheters, lungs with cystic fibrosis and the oral cavity. The biofilm is established by operating the reactor in batch mode for 6 h. A mature biofilm forms as the reactor operates for an additional 48 h with a continuous flow of nutrients. During continuous flow, the biofilm experiences a low shear as the media drips onto a surface set at a 101 angle. At the end of 54 h, biofilm accumulation is quantified by removing coupons from the reactor channels, rinsing the coupons to remove planktonic cells, scraping the biofilm from the coupon surface, disaggregating the clumps, then diluting and plating for viable cell enumeration. The entire procedure takes 13 h of active time that is distributed over 5 d.
Optimal surface estimation and thresholding of confocal microscope images of biofilms using Beer's Law
(2020-05) Parker, Albert E.; Christen, J. A.; Lorenz, Lindsey A.; Smith, Heidi J.
Beer's Law explains how light attenuates into thick specimens, including thick biofilms. We use a Bayesian optimality criterion, the maximum of the posterior probability distribution, and computationally efficiently fit Beer's Law to the 3D intensity data collected from thick living biofilms by a confocal scanning laser microscope. Using this approach the top surface of the biofilm and an optimal image threshold can be estimated. Biofilm characteristics, such as bio-volumes, can be calculated from this surface. Results from the Bayesian approach are compared to other approaches including the method of maximum likelihood or simply counting bright pixels. Uncertainty quantification (i.e., error bars) can be provided for the parameters of interest. This approach is applied to confocal images of stained biofilms of a common lab strain of Pseudomonas aeruginosa, stained biofilms of Janthinobacterium isolated from the Antarctic, and biofilms of Staphylococcus aureus that have been genetically modified to fluoresce green.
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.
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.