Browsing by Author "Walker, Diane K."

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Design and fabrication of biofilm reactors
(2020) Goeres, Darla M.; Pedersen, Stephen; Warwood, B. K.; Walker, Diane K.; Parker, Albert E.; Mettler, Madelyn; Sturman, Paul J.
Laboratory biofilm reactors are tools that researchers use to grow biofilms that exhibit characteristics sufficiently similar to the environment of interest. Numerous biofilm reactors that model various fluid dynamics are described in scientific literature, each with its associated list of advantages and limitations. This chapter focuses on the process used to design and fabricate biofilm reactors with the stated goal of generating a commercial product. The process begins with identifying the environment of interest and key attributes the reactor should include or model. A prototype is then designed, built, and tested in the laboratory. Modifications are made based upon laboratory performance until a design is achieved that is affordable, practical, operationally simple, and relevant and that provides repeatable, convincing results. This process was used to design the industrial surfaces biofilm reactor, developed to model cooling tower biofilms but suitable to study biofilms grown under low shear, high gas transfer, and intermittently wet conditions.
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 of Petrifilm™ Aerobic Count Plates as an Equivalent Alternative to Drop Plating on R2A Agar Plates in a Biofilm Disinfectant Efficacy Test
(2014-12) Fritz, Blaine; Walker, Diane K.; Goveia, Dani; Parker, Albert E.; Goeres, Darla M.
This paper compares Petrifilm™ aerobic count (AC) plates to drop plating on R2A agar plates as an alternative method for biofilm bacteria enumeration after application of a disinfectant. A Pseudomonas aeruginosa biofilm was grown in a Centers for Disease Control and Prevention biofilm reactor (ASTM E2562) and treated with 123 ppm sodium hypochlorite (as free chlorine) according to the Single Tube Method (ASTM E2871). Aliquots from the same dilution tubes were plated on Petrifilm™ AC plates and drop plated on R2A agar plates. The Petrifilm™ AC and R2A plates were incubated for 48 and 24 h, respectively, at 36 ± 1 °C. After nine experimental runs performed by two technicians, the mean difference in biofilm log densities [log biofilm density (LD) = log10(CFU/cm2)] between the two methods for control coupons, treated coupons, and log reduction (LR) was 0.052 (p = 0.451), −0.102 (p = 0.303), and 0.152 (p = 0.313). Equivalence testing was used to assess equivalence of the two plating methods. The 90 % confidence intervals for the difference in control and treated mean LDs between methods were (−0.065, 0.170) and (−0.270, 0.064), both of which fall within a (−0.5, +0.5) equivalence criterion. The 90 % confidence interval for the mean LR difference (−0.113, 0.420) also falls within this equivalence criterion. Thus, Petrifilm™ AC plates were shown to be statistically equivalent to drop plating on R2A agar for the determination of control LDs, treated LDs, and LR values in an anti-biofilm efficacy test. These are the first published results that establish equivalency to a traditional plate counting technique for biofilms and for a disinfectant assay.
Hand hygiene product use by food employees in casual dining and quick-service restaurants
(Elsevier BV, 2023-02) Manuel, Clyde S.; Robbins, Greg; Slater, Jason; Walker, Diane K.; Parker, Albert; Arbogast, James W.
Hand hygiene product usage characteristics by food employees when hand sanitizers are made available are not well understood. To investigate hand hygiene product usage in casual dining and quick-service restaurants, we placed automated monitoring soap and sanitizer dispensers side-by-side at handwash sinks used by food employees in seven restaurants. Dispenses were monitored, and multiple dispenses that occurred within 60 s of each other were considered a single hand hygiene event. This resulted in 186,998 events during the study (149,779 soap only, 21 985 sanitizer only, and 15,234 regimen [defined as soap followed by sanitizer at the same sink within 60 s]) over 15,447 days of use. Soap was the most frequently used hand hygiene method by food employees in both restaurant types. Regimen use, despite being the preferred hand hygiene method by both restaurant chains, was the least used hand hygiene method. When pooled over restaurant types, the median daily usage for soap was statistically significantly highest of all methods at 23.5 dispenses per sink per day (p < 0.0001), the sanitizer median daily usage was 4.27 dispenses per sink per day, and regimen use was statistically significantly lowest of all methods at 4.02 dispenses per sink per day (p < 0.0001). When hand hygiene event types were pooled, casual dining restaurants had similar median hand hygiene event rates (11.4 dispenses per sink per day) compared to quick-service restaurants (11.9 dispenses per sink per day; p = 0.890). The number of events by sink location varied, with sinks located at a warewash station having the highest number of events (19.3 dispenses per sink per day; p < 0.0001), while sinks located by a ready-to-eat food preparation area had the lowest number of events (6.8 dispenses per sink per day; p < 0.0001). These data provide robust baseline benchmarks for future hand hygiene intervention studies in these settings.
Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research
(MyJove Corporation, 2022-04) Buckingham-Meyer, Kelli; Miller, Lindsey A.; Parker, Albert E.; Walker, Diane K.; Sturman, Paul; Novak, Ian; Goeres, Darla M.
Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.
Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research
(MyJove Corporation, 2022-04) Buckingham-Meyer, Kelli; Miller, Lindsey A.; Parker, Albert E.; Walker, Diane K.; Sturman, Paul; Novak, Ian; Goeres, Darla M.
Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.
Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research
(MyJove Corporation, 2022-04) Buckingham-Meyer, Kelli; Miller, Lindsey A.; Parker, Albert E.; Walker, Diane K.; Sturman, Paul; Novak, Ian; Goeres, Darla M.
Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.
Inactivation of Pseudomonas aeruginosa biofilms formed under high shear stress on various hydrophilic and hydrophobic surfaces by a continuous flow of ozonated water
(2018-10) Shelobolina, Evgenya S.; Walker, Diane K.; Parker, Albert E.; Lust, Dorian V.; Schultz, Johanna M.; Dickerman, Grace E.
The inactivation of Pseudomonas aeruginosa biofilms grown on glass under high shear stress and exposed to a range of dissolved ozone concentrations (2, 5 and 7 ppm) at 10 and 20 min was investigated. The regression equation, log reduction (biofilm) = 0.64 + 0.59×(C – 2) + 0.33×(T – 10), described the dependence of biofilm inactivation on the dissolved ozone concentration (C, ppm) and contact time (T, min). The predicted D-values were 11.1, 5.7 and 2.2 min at 2, 5 and 7 ppm, respectively. Inactivation of biofilms grown on various surfaces was tested at a single dissolved ozone concentration of 5 ppm and a single exposure time of 20 min. Biofilms grown on plastic materials showed inactivation results similar to that of biofilms on glass, while biofilms grown on ceramics were statistically significantly more difficult to inactivate, suggesting the importance of utilizing non-porous materials in industrial and clinical settings.
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, Joanna
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.
Predictive modeling for hot water inactivation of planktonic and biofilm-associatedSphingomonas parapaucimobilis to support hot water sanitization programs
(2016-06) Kaatz Wahlen, L.; Parker, Albert E.; Walker, Diane K.; Pasmore, M.; Sturman, Paul J.
Hot water sanitization is a common means to maintain microbial control in process equipment for industries where microorganisms can degrade product or cause safety issues. This study compared the hot water inactivation kinetics of planktonic and biofilm-associated Sphingomonas parapaucimobilis at temperatures relevant to sanitization processes used in the pharmaceutical industry, viz. 65, 70, 75, and 80°C. Biofilms exhibited greater resistance to hot water than the planktonic cells. Both linear and nonlinear statistical models were developed to predict the log reduction as a function of temperature and time. Nonlinear Michaelis-Menten modeling provided the best fit for the inactivation data. Using the model, predictions were calculated to determine the times at which specific log reductions are achieved. While ≥80°C is the most commonly cited temperature for hot water sanitization, the predictive modeling suggests that temperatures ≥75°C are also effective at inactivating planktonic and biofilm bacteria in timeframes appropriate for the pharmaceutical industry.
Ruggedness and reproducibility of the MBEC biofilm disinfectant efficacy test
(2014-07) Parker, Albert E.; Walker, Diane K.; Goeres, Darla M.; Allan, N.; Olson, M. E.; Omar, A.
The MBEC™ Physiology & Genetics Assay recently became the first approved ASTM standardized biofilm disinfectant efficacy test method. This report summarizes the results of the standardization process using Pseudomonas aeruginosa biofilms. Initial ruggedness testing of the MBEC method suggests that the assay is rugged (i.e., insensitive) to small changes to the protocol with respect to 4 factors: incubation time of the bacteria (when varied from 16 to 18 h), treatment temperature (20â€“24 Â°C), sonication duration (25â€“35 min), and sonication power (130â€“480 W). In order to assess the repeatability of MBEC results across multiple tests in the same laboratory and the reproducibility across multiple labs, an 8-lab study was conducted in which 8 concentrations of each of 3 disinfectants (a non-chlorine oxidizer, a phenolic, and a quaternary ammonium compound) were applied to biofilms using the MBEC method. The repeatability and reproducibility of the untreated control biofilms were acceptable, as indicated by small repeatability and reproducibility standard deviations (SD) (0.33 and 0.67 log10(CFU/mm2), respectively). The repeatability SDs of the biofilm log reductions after application of the 24 concentration and disinfectant combinations ranged from 0.22 to 1.61, and the reproducibility SDs ranged from 0.27 to 1.70. In addition, for each of the 3 disinfectant types considered, the assay was statistically significantly responsive to the increasing treatment concentrations.
The use of a CDC biofilm reactor to grow multi-strain Listeria monocytogenes biofilm
(Elsevier BV, 2020-12) Mendez, Ellen; Walker, Diane K.; Vipham, Jessie; Trinetta, Valentina
Listeria monocytogenes is one of the most concerning pathogens for the food industry due to its ability to form biofilms, particularly in difficult-to-clean sites of processing facilities. There is a current industry-wide lack of data to refer to when selecting a strategy to control L. monocytogenes biofilms in the food premises. Many strategies have been developed to study biofilm formation of bacteria; however, few have targeted L. monocytogenes biofilms under dynamic conditions. This study addresses the biofilm formation ability of L. monocytogenes on stainless steel and polycarbonate under dynamic conditions using TSBYE or BHI as media culture at 30 °C or 37 °C. Higher cell counts were recovered at 30 °C in TSBYE on polycarbonate while lower counts were obtained at 37 °C in BHI on stainless steel (P < 0.05). Nonetheless, all factors (temperature, media and material) were statistically significant (P < 0.05) and an interaction between temperature and media was observed (P < 0.05). To our knowledge, this work represents an initial framework to develop L. monocytogenes biofilms under different dynamic conditions. The use of CDC Biofilm Reactor is not widely used yet in the food industry and represent a novel approach to help sanitary control strategies implementation.