Browsing by Author "Goeres, Darla M."

Now showing 1 - 20 of 44

Antimicrobial activity of naturally occurring phenols and derivatives against biofilm and planktonic bacteria
(2019-10) Walsh, Danica J.; Livinghouse, Tom; Goeres, Darla M.; Mettler, Madelyn; Stewart, Philip S.
Biofilm-forming bacteria present formidable challenges across diverse settings, and there is a need for new antimicrobial agents that are both environmentally acceptable and relatively potent against microorganisms in the biofilm state. The antimicrobial activity of three naturally occurring, low molecular weight, phenols, and their derivatives were evaluated against planktonic and biofilm Staphylococcus epidermidis and Pseudomonas aeruginosa. The structure activity relationships of eugenol, thymol, carvacrol, and their corresponding 2- and 4-allyl, 2-methallyl, and 2- and 4-n-propyl derivatives were evaluated. Allyl derivatives showed a consistent increased potency with both killing and inhibiting planktonic cells but they exhibited a decrease in potency against biofilms. This result underscores the importance of using biofilm assays to develop structure-activity relationships when the end target is biofilm.
Approaches to biofilm-associated infections: the need for standardized and relevant biofilm methods for clinical applications
(2017-02) Malone, Matthew; Goeres, Darla M.; Gosbell, Iain; Vickery, Karen; Jensen, Slade; Stoodley, Paul
Introduction: The concept of biofilms in human health and disease is now widely accepted as cause of chronic infection. Typically, biofilms show remarkable tolerance to many forms of treatments and the host immune response. This has led to vast increase in research to identify new (and sometimes old) anti-biofilm strategies that demonstrate effectiveness against these tolerant phenotypes. Areas covered: Unfortunately, a standardized methodological approach of biofilm models has not been adopted leading to a large disparity between testing conditions. This has made it almost impossible to compare data across multiple laboratories, leaving large gaps in the evidence. Furthermore, many biofilm models testing anti-biofilm strategies aimed at the medical arena have not considered the matter of relevance to an intended application. This may explain why some in vitro models based on methodological designs that do not consider relevance to an intended application fail when applied in vivo at the clinical level. Expert commentary: This review will explore the issues that need to be considered in developing performance standards for anti-biofilm therapeutics and provide a rationale for the need to standardize models/methods that are clinically relevant. We also provide some rational as to why no standards currently exist.
The biofilm life cycle: expanding the conceptual model of biofilm formation
(Springer Science and Business Media LLC, 2022-10) Sauer, Karin; Stoodley, Paul; Goeres, Darla M.; Hall-Stoodley, Luanne; Burmølle, Mette; Stewart, Philip S.; Bjarnsholt, Thomas
Bacterial biofilms are often defined as communities of surface-attached bacteria and are typically depicted with a classic mushroom-shaped structure characteristic of Pseudomonas aeruginosa. However, it has become evident that this is not how all biofilms develop, especially in vivo, in clinical and industrial settings, and in the environment, where biofilms often are observed as non-surface-attached aggregates. In this Review, we describe the origin of the current five-step biofilm development model and why it fails to capture many aspects of bacterial biofilm physiology. We aim to present a simplistic developmental model for biofilm formation that is flexible enough to include all the diverse scenarios and microenvironments where biofilms are formed. With this new expanded, inclusive model, we hereby introduce a common platform for developing an understanding of biofilms and anti-biofilm strategies that can be tailored to the microenvironment under investigation.
Biofilms vs. cities and humans vs. aliens – a tale of reproducibility in biofilms
(Elsevier BV, 2021-06) Azevedo, Nuno F.; Allkja, Jontana; Goeres, Darla M.
In recent decades the scientific community has started to appreciate that most microorganisms live in complex 3D structures composed of cells, polysaccharides, and other components such as proteins, extracellular (e)DNA, and lipids. These structures are commonly designated 'biofilms'. Similar to other areas of research, biofilm studies have been affected by a lack of reproducibility. In this article, we propose a new scheme on how to classify the level of reproducibility in biofilms. This consists of four different levels: level 1, no reproducibility; level 2, standard reproducibility; level 3, potential standard reproducibility; and level 4, total reproducibility. Some methods aim to improve reproducibility by focusing on biofilm growth reactors, while others focus on biofilm characterization methods. Moreover, initiatives such as minimum information guidelines and biofilm-centered databases offer alternative strategies to tackle the reproducibility problem. The path to total reproducibility is certainly complex, but novel experimental and computational strategies are bringing us closer to achieving this goal.
Checking the validity of the harvesting and disaggregating steps in laboratory tests of surface disinfectants
(2009-11) Hamilton, Martin A.; Buckingham-Meyer, Kelli; Goeres, Darla M.
A chemical disinfectant against surface-associated bacteria typically uses carriers (e.g., glass disks)that are purposely contaminated with bacteria prior to disinfection. After disinfection, the bacteria are harvested by mechanically separating them from the carrier surface to form a suspension of cells in a dilution tube. Bacterial clumps in the tube are disaggregated using mechanical or chemical techniques, thereby creating a well-mixed suspension of single cells suitable for enumeration. Efficacy is quantified by comparing the viable cell count for a disinfected carrier to the viable cell count for sham-disinfected (control) carrier. A test is said to be biased (invalid) if the observed efficacy measure is systematically higher or lower than the true efficacy. It is shown here for the first time that the bias attributable to the harvesting and disaggregating steps of a disinfectant test can be measured. For some conventional biofilm harvesting and disaggregating techniques, laboratory checks showed either negligible bias or important bias, depending on the disinfectant. Quantitative bias checks on the harvesting and disaggregating steps are prudent for each combination of carrier material, microorganism, and disinfectant. The quantitative results should be augmented by microscopic examination of harvested disinfected and control carriers and of the disaggregated suspensions.
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.
Coupon position does not affect Pseudomonas aeruginosa and Staphylococcus aureus biofilm densities in the CDC biofilm reactor
(Elsevier BV, 2024-08) Buckner, Elizabeth; Buckingham-Meyer, Kelli; Miller, Lindsey A.; Parker, Albert E.; Jones, Christopher J.; Goeres, Darla M.
The CDC Biofilm Reactor method is the standard biofilm growth protocol for the validation of US Environmental Protection Agency biofilm label claims. However, no studies have determined the effect of coupon orientation within the reactor on biofilm growth. If positional effects have a statistically significant impact on biofilm density, they should be accounted for in the experimental design. Here, we isolate and quantify biofilms from each possible coupon surface in the reactor to quantitatively determine the positional effects in the CDC Biofilm Reactor. The results showed no statistically significant differences in viable cell density across different orientations and vertical positions in the reactor. Pseudomonas aeruginosa log densities were statistically equivalent among all coupon heights and orientations. While the Staphylococcus aureus cell growth showed no statistically significant differences, the densities were not statistically equivalent among all coupon heights and orientations due to the variability in the data. Structural differences were observed between biofilms on the high-shear baffle side of the reactor compared to the lower shear glass side of the reactor. Further studies are required to determine whether biofilm susceptibility to antimicrobials differs based on structural differences attributed to orientation.
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.
Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle
(Elsevier BV, 2024-06) Barbosa, Ana; Azevedo, Nuno F.; Goeres, Darla M.; Cerqueira, Laura
There has been considerable discussion regarding the environmental life cycle of Legionella pneumophila and its virulence potential in natural and man-made water systems. On the other hand, the bacterium's morphogenetic mechanisms within host cells (amoeba and macrophages) have been well documented and are linked to its ability to transition from a non-virulent, replicative state to an infectious, transmissive state. Although the morphogenetic mechanisms associated with the formation and detachment of the L. pneumophila biofilm have also been described, the capacity of the bacteria to multiply extracellularly is not generally accepted. However, several studies have shown genetic pathways within the biofilm that resemble intracellular mechanisms. Understanding the functionality of L. pneumophila cells within a biofilm is fundamental for assessing the ecology and evaluating how the biofilm architecture influences L. pneumophila survival and persistence in water systems. This manuscript provides an overview of the biphasic cycle of L. pneumophila and its implications in associated intracellular mechanisms in amoeba. It also examines the molecular pathways and gene regulation involved in L. pneumophila biofilm formation and dissemination. A holistic analysis of the transcriptional activities in L. pneumophila biofilms is provided, combining the information of intracellular mechanisms in a comprehensive outline. Furthermore, this review discusses the techniques that can be used to study the morphogenetic states of the bacteria within biofilms, at the single cell and population levels.
Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle
(Elsevier BV, 2024-06) Barbosa, Ana; Azevedo, Nuno F.; Goeres, Darla M.; Cerqueira, Laura
There has been considerable discussion regarding the environmental life cycle of Legionella pneumophila and its virulence potential in natural and man-made water systems. On the other hand, the bacterium's morphogenetic mechanisms within host cells (amoeba and macrophages) have been well documented and are linked to its ability to transition from a non-virulent, replicative state to an infectious, transmissive state. Although the morphogenetic mechanisms associated with the formation and detachment of the L. pneumophila biofilm have also been described, the capacity of the bacteria to multiply extracellularly is not generally accepted. However, several studies have shown genetic pathways within the biofilm that resemble intracellular mechanisms. Understanding the functionality of L. pneumophila cells within a biofilm is fundamental for assessing the ecology and evaluating how the biofilm architecture influences L. pneumophila survival and persistence in water systems. This manuscript provides an overview of the biphasic cycle of L. pneumophila and its implications in associated intracellular mechanisms in amoeba. It also examines the molecular pathways and gene regulation involved in L. pneumophila biofilm formation and dissemination. A holistic analysis of the transcriptional activities in L. pneumophila biofilms is provided, combining the information of intracellular mechanisms in a comprehensive outline. Furthermore, this review discusses the techniques that can be used to study the morphogenetic states of the bacteria within biofilms, at the single cell and population levels.
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.
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.
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.
Guidelines for the statistical analysis of a collaborative study of a laboratory method for testing disinfectant product performance
(2013-09) Hamilton, Martin A.; Hamilton, G. C.; Goeres, Darla M.; Parker, Albert E.
This paper presents statistical techniques suitable for analyzing a collaborative study (multilaboratory study or ring trial) of a laboratory disinfectant product performance test (DPPT) method. Emphasis is on the assessment of the repeatability, reproducibility, resemblance, and responsiveness of the DPPT method. The suggested statistical techniques are easily modified for application to a single laboratory study. The presentation includes descriptions of the plots and tables that should be constructed during initial examination of the data, including a discussion of outliers and QA checks. The statistical recommendations deal with evaluations of prevailing types of DPPTs, including both quantitative and semiquantitative tests. The presentation emphasizes tests in which the disinfectant treatment is applied to surface-associated microbes and the outcome is a viable cell count; however, the statistical guidelines are appropriate for suspension tests and other test systems. The recommendations also are suitable for disinfectant tests using any microbe (vegetative bacteria, virus, spores, etc.) or any disinfectant treatment. The descriptions of the statistical techniques include either examples of calculations based on published data or citations to published calculations. Computer code is provided in an appendix.
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.
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.