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Item Novel phenolic antimicrobials enhanced activity of iminodiacetate prodrugs against biofilm and planktonic bacteria(Wiley, 2020-09) Walsh, Danica J.; Livinghouse, Tom; Durling, Greg M.; Arnold, Adrienne D.; Brasier, Whitney; Berry, Luke; Goeres, Darla M.; Stewart, Philip S.Prodrugs are pharmacologically attenuated derivatives of drugs that undergo bioconversion into the active compound once reaching the targeted site, thereby maximizing their efficiency. This strategy has been implemented in pharmaceuticals to overcome obstacles related to absorption, distribution, and metabolism, as well as with intracellular dyes to ensure concentration within cells. In this study, we provide the first examples of a prodrug strategy that can be applied to simple phenolic antimicrobials to increase their potency against mature biofilms. The addition of (acetoxy)methyl iminodiacetate groups increases the otherwise modest potency of simple phenols. Biofilm-forming bacteria exhibit a heightened tolerance toward antimicrobial agents, thereby accentuating the need for new antibiotics as well as those, which incorporate novel delivery strategies to enhance activity toward biofilms.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 Measuring antimicrobial effects on biofilm bacteria: From laboratory to field(1999) Zelver, Nick; Hamilton, Martin A.; Pitts, Betsey; Goeres, Darla M.; Walker, Diane K.; Sturman, Paul J.; Heersink, JoannaItem Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications(2017-09) Gomes, I. B.; Meireles, A.; Goncalves, A. L.; Goeres, Darla M.; Sjollema, J.; Simoes, L. C.; Simoes, M.Biofilms can cause severe problems to human health due to the high tolerance to antimicrobials; consequently, biofilm science and technology constitutes an important research field. Growing a relevant biofilm in the laboratory provides insights into the basic understanding of the biofilm life cycle including responses to antibiotic therapies. Therefore, the selection of an appropriate biofilm reactor is a critical decision, necessary to obtain reproducible and reliable in vitro results. A reactor should be chosen based upon the study goals and a balance between the pros and cons associated with its use and operational conditions that are as similar as possible to the clinical setting. However, standardization in biofilm studies is rare. This review will focus on the four reactors (Calgary biofilm device, Center for Disease Control biofilm reactor, drip flow biofilm reactor, and rotating disk reactor) approved by a standard setting organization (ASTM International) for biofilm experiments and how researchers have modified these standardized reactors and associated protocols to improve the study and understanding of medical biofilms.Item Evaluation of disinfectant efficacy against biofilm and suspended bacteria in a laboratory swimming pool model(2004-07) Goeres, Darla M.; Palys, T.; Sandel, B. B.; Geiger, J.Laboratory reactor systems designed to model specific environments enable researchers to explore environmental dynamics in a more controlled manner. This paper describes the design and operation of a reactor system built to model a swimming pool in the laboratory. The model included relevant engineering parameters such as filter loading and turn-overs per day. The water chemistry in the system's bulk water was balanced according to standard recommendations and the system was challenged with a bacterial load and synthetic bather insult, formulated to represent urine and perspiration. The laboratory model was then used to evaluate the efficacy of six chemical treatments against biofilm and planktonic bacteria. Results showed that the biofilm was able to accumulate on coupons and in the filter systems of reactors treated with either 1-3 mg/L free chlorine or 10 mg/L polyhexamethylene biguanide (PHMB). All the treatments tested resulted in at least a 4-log reduction in biofilm density when compared to the control, but shock treatments were the most effective at controlling biofilm accumulation. A once-weekly shock dose of 10 mg/L free chlorine resulted in the greatest log reduction in biofilm density. The research demonstrated the importance of studying a biofilm in addition to the planktonic bacteria to assess the microbial dynamics that exist in a swimming pool model.Item Statistical assessment of a laboratory method for growing biofilms(2005-03) Goeres, Darla M.; Loetterle, Linda R.; Hamilton, Martin A.; Murga, Ricardo; Kirby, D. W.; Donlan, R. M.Microbial biofilms have been grown in laboratories using a variety of different approaches. A laboratory biofilm reactor system, called the CDC biofilm reactor (CBR) system, has been devised for growing biofilms under moderate to high fluid shear stress. The reactor incorporates 24 removable biofilm growth surfaces (coupons) for sampling and analysing the biofilm. Following preliminary experiments to verify the utility of the CBR system for growing biofilms of several clinically relevant organisms, a standard operating procedure for growing a Pseudomonas aeruginosa biofilm was created. This paper presents the results of a rigorous, intra-laboratory, statistical evaluation of the repeatability and ruggedness of that procedure as well as the results of the experiments with clinically relevant organisms. For the statistical evaluations, the outcome of interest was the density (c.f.u. cm-2) of viable P. aeruginosa. Replicate experiments were conducted to assess the repeatability of the log density outcome. The mean P. aeruginosa log10 density was 7·1, independent of the coupon position within the reactor. The repeatability standard deviation of the log density based on one coupon per experiment was 0·59. Analysis of variance showed that the variability of the log density was 53% attributable to within-experiment sources and 47% attributable to between-experiments sources. The ruggedness evaluation applied response-surface design and regression analysis techniques, similar to those often used for sensitivity analyses in other fields of science and engineering. This approach provided a quantitative description of ruggedness; specifically, the amount the log density was altered by small adjustments to four key operational factors – time allowed for initial surface colonization, temperature, nutrient concentration, and fluid shear stress on the biofilm. The small size of the regression coefficient associated with each operational factor showed that the method was rugged; that is, relatively insensitive to minor perturbations of the four factors. These results demonstrate that the CBR system is a reliable experimental tool for growing a standard biofilm in the laboratory and that it can be adapted to study several different micro-organisms.Item A laboratory hot tub model for disinfectant efficacy evaluation(2007-01) Goeres, Darla M.; Loetterle, Linda R.; Hamilton, Martin A.This paper describes a novel laboratory hot tub (LHT) apparatus and associated standard operating procedure (SOP) designed to reproduce the key biological, chemical, and engineering parameters associated with recreational and therapeutic hot tubs. Efficacy, as measured quantitatively by log reduction values, was determined against both biofilm and planktonic bacteria. When the LHT was run according to the SOP, with no antimicrobial treatment, a consistent level of bacterial contamination occurred. The means of log10 viable cell densities (± the repeatability standard deviation of log densities) were 7.2 (± 0.31) for the bulk water (density in units of cfu ml− 1), 5.3 (± 0.56) for the coupons (density in units of cfu cm− 2), and 6.6 (± 0.50) for the filters (density in units of cfu cm− 2). When control and chlorine treated LHTs were run in parallel, the log reduction increased significantly with chlorine concentration for samples of planktonic bacteria in the bulk water (p = 0.016), biofilm bacteria on the coupons (p = 0.09) and biofilm bacteria on the filter (p = 0.005), indicating that the method was sensitive to chlorine concentration. The method also displayed sensitivity by differentiating between chlorine and bromine treatments; in every case, chlorine produced a greater log reduction than did the same concentration of bromine. The model and SOP were shown to be rugged with respect to slight changes in fluid mixing intensity, water chemistry (saturation index), inoculum size, and organic loading. The LHT and associated SOP provide a reliable second tier in a three-tiered testing process, in which the first tier is a suspension test and the final tier is a field test.Item Comparative evaluation of biofilm disinfectant efficacy tests(2007-08) Buckingham-Meyer, Kelli; Goeres, Darla M.; Hamilton, Martin A.Regulatory agencies are receiving registration applications for unprecedented, antibiofilm label claims for disinfectants. Reliable, practical, and relevant laboratory biofilm test methods are required to support such claims. This investigation describes the influence of fluid dynamics on the relevancy of a laboratory test. Several disinfectant formulations were tested using three different biofilm testing systems run side-by-side: the CDC biofilm reactor system that created turbulent flow (Reynolds number between 800 and 1850), the drip flow biofilm reactor system that created slow laminar flow (Reynolds number between 12 and 20), and the static biofilm system that involved no fluid flow. Each comparative experiment also included a dried surface carrier test and a dried biofilm test. All five disinfectant tests used glass coupons and followed the same steps for treatment, neutralization, viable cell counting, and calculating the log reduction (LR). Three different disinfectants, chlorine, a quaternary ammonium compound, and a phenolic, were each applied at two concentrations. Experiments were conducted separately with Pseudomonas aeruginosa and Staphylococcus aureus and every experiment was independently repeated. The results showed that biofilm grown in the CDC reactor produced the smallest LR, the static biofilm produced the largest LR, and biofilm grown in the drip flow reactor produced an intermediate LR. The differences were large enough to be of practical importance. The dried surface test often produced a significantly higher LR than the tests against hydrated or dried biofilm. The dried biofilm test produced LR values similar to those for the corresponding hydrated biofilm test. These results show that the efficacy of a disinfectant must be measured by using a laboratory method where biofilm is grown under fluid flow conditions similar to the environment where the disinfectant will be applied.Item 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 air–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.Item Understanding the importance of biofilm growth in hot tubs(2009-10) Goeres, Darla M.Introduction: Microorganisms attach to surfaces and form biofilms (Figure 5.1). From an historical perspecitve, biofilms first bacame an accepted entity as well as an accepted problem in environmental settings, particularly in industry. The Center for Biofilm Engineering at Montana State University-Bozeman in 1990, funded through the National Science Foundation's Engineering Research Center Program, was established primarily to study environmental biofilms. The fact that biofilms were growing and clogging water pipes and cooling towers was easily accepted because the effects of the biofilms were readily apparent in the reduction and eventual loss of flow through a system.