College of Engineering
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The College of Engineering at Montana State University will serve the State of Montana and the nation by fostering lifelong learning, integrating learning and discovery, developing and sharing technical expertise, and empowering students to be tomorrow's leaders.
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Item Comparison of quantification methods for an endoscope lumen biofilm model(Elsevier BV, 2023-12) Haas, Bruno; James, Sarah; Parker, Albert E.; Gagnon, Marie-Claude; Goulet, Noémie; Labrie, PhilippeBiofilm has been implicated in multi-drug resistant organism outbreaks following endoscopic procedures. Automated Endoscope Reprocessors (AER) are devices validated to clean and disinfect endoscopes per applicable standards. The ISO 15883 part 4 standard guides performance testing validation of AERs, including cleaning performance using a biofilm test soil. The standard recommends assessment of biofilm reduction using protein or carbohydrate quantification methods. The aim of this study was to assess the suitability of various quantification methods using the ISO biofilm model. The ISO 15883 part 5 biofilm test soil method was used to grow biofilm within lumens representative of endoscopes channels. The biofilm was then quantified using five methods: Crystal Violet (CV), Colony Forming Units (CFU), Total Organic Carbon (TOC), protein assay with Orthophtalaldehyde (OPA), and protein assay by micro bicinchoninic acid (μBCA). The five methods were statistically analyzed for their ability to assess biofilm reduction on samples accurately and precisely. In addition, the quantification methods were compared to demonstrate statistical equivalency, and thus their suitability for assessing biofilm cleaning performance testing of AERs.Item Seven genome sequences of bacterial, environmental isolates from Pony Lake, Antarctica(American Society for Microbiology, 2023-12) Foreman, Christine M.; Smith, Heidi J.; Dieser, MarkusDissolved organic matter (DOM) in Antarctic inland waters is unique in that its precursor molecules are microbially derived and lack the chemical signature of higher plants. Here, we report the genomic sequences of seven environmental, bacterial isolates from Pony Lake, Antarctica, to explore the genetic potential linked to DOM processing.Item Detection of Microbes in Ice Using Microfabricated Impedance Spectroscopy Sensors(The Electrochemical Society, 2023-12) Kaiser-Jackson, Lauren B.; Dieser, Markus; McGlennen, Matthew; Parker, Albert E.; Foreman, Christine M.; Warnat, StephanDuring the growth of a polycrystalline ice lattice, microorganisms partition into veins, forming an ice vein network highly concentrated in salts and microbial cells. We used microfabricated electrochemical impedance spectroscopy (EIS) sensors to determine the effect of microorganisms on the electrochemical properties of ice. Solutions analyzed consisted of a 176 μS cm−1 conductivity solution, fluorescent beads, and Escherichia coli HB101-GFP to model biotic organisms. Impedance spectroscopy data were collected at −10 °C, −20 °C, and −25 °C within either ice veins or ice grains (i.e., no veins) spanning the sensors. After freezing, the fluorescent beads and E. coli were partitioned into the ice veins. The corresponding impedance data were discernibly different in the presence of ice veins and microbial impurities. The presence of microbial cells in ice veins was evident by decreased electrical characteristics (electrode polarization between electrode and ice matrix) relative to solid ice grains. Further, this electrochemical behavior was reversed in all bead-doped solutions, indicating that microbial processes influence sensor response. Linear mixed-effects models empirically corroborated the differences in polarization associated with the presence and absence of microbial cells in ice. We show that EIS has the potential to detect microbes in ice and differentiate between veins and solid grains.Item Sample sizes for estimating the sensitivity of a monitoring system that generates repeated binary outcomes with autocorrelation(Sage Publications, 2023-11) Parker, Albert E.; Arbogast, James W.Sample size formulas are provided to determine how many events and how many patient care units are needed to estimate the sensitivity of a monitoring system. The monitoring systems we consider generate time series binary data that are autocorrelated and clustered by patient care units. Our application of interest is an automated hand hygiene monitoring system that assesses whether healthcare workers perform hand hygiene when they should. We apply an autoregressive order 1 mixed effects logistic regression model to determine sample sizes that allow the sensitivity of the monitoring system to be estimated at a specified confidence level and margin of error. This model overcomes a major limitation of simpler approaches that fail to provide confidence intervals with the specified levels of confidence when the sensitivity of the monitoring system is above 90%.Item Development of Martian saline seep models and their implications for planetary protection(Elsevier BV, 2023-12) Mettler, Madelyn K.; Goemann, Hannah M.; Mueller, Rebecca C.; Vanegas, Oscar A.; Lopez, Gabriela; Singh, Nitin; Venkateswaran, Kasthuri; Peyton, Brent M.While life on Mars has not been found, Earth-based microorganisms may contaminate the Red Planet during rover expeditions and human exploration. Due to the survival advantages conferred by the biofilm morphology to microorganisms, such as resistance to UV and osmotic stress, biofilms are particularly concerning from a planetary protection perspective. Modeling and data from the NASA Phoenix mission indicate that temporary liquid water might exist on Mars in the form of high salinity brines. These brines could provide colonization opportunities for terrestrial microorganisms brought by spacecraft or humans. To begin testing for potential establishment of microbes, results are presented from a simplified laboratory model of a Martian saline seep inoculated with sediment from Hailstone Basin, a terrestrial saline seep in Montana (USA). The seep was modeled as a sand-packed drip flow reactor at room temperature fed media with either 1 M MgSO4 or 1 M NaCl. Biofilms were established within the first sampling point of each experiment. Endpoint 16S rRNA gene community analysis showed significant selection of halophilic microorganisms by the media. Additionally, we detected 16S rRNA gene sequences highly similar to microorganisms previously detected in two spacecraft assembly cleanrooms. These experimental models provide an important foundation for identifying microbes that could hitch-hike on spacecraft and may be able to colonize Martian saline seeps. Future model optimization will be vital to informing cleanroom sterilization procedures.Item Α,α-disubstituted β-amino amides eliminate Staphylococcus aureus biofilms by membrane disruption and biomass removal(Elsevier BV, 2023-12) Ausbacher, Dominik; Miller, Lindsey A.; Goeres, Darla M.; Stewart, Philip S.; Strøm, Morten B.; Fallarero, AdyaryBacterial biofilms account for up to 80% of all infections and complicate successful therapies due to their intrinsic tolerance to antibiotics. Biofilms also cause serious problems in the industrial sectors, for instance due to the deterioration of metals or microbial contamination of products. Efforts are put in finding novel strategies in both avoiding and fighting biofilms. Biofilm control is achieved by killing and/or removing biofilm or preventing transition to the biofilm lifestyle. Previous research reported on the anti-biofilm potency of α,α-disubstituted β-amino amides A1, A2 and A3, which are small antimicrobial peptidomimetics with a molecular weight below 500 Da. In the current study it was investigated if these derivatives cause a fast disintegration of biofilm bacteria and removal of Staphylococcus aureus biofilms. One hour incubation of biofilms with all three derivatives resulted in reduced metabolic activity and membrane permeabilization in S. aureus (ATCC 25923) biofilms. Bactericidal properties of these derivatives were attributed to a direct effect on membranes of biofilm bacteria. The green fluorescence protein expressing Staphylococcus aureus strain AH2547 was cultivated in a CDC biofilm reactor and utilized for disinfectant efficacy testing of A3, following the single tube method (American Society for Testing and Materials designation number E2871). A3 at a concentration of 90 μM acted as fast as 100 μM chlorhexidine and was equally effective. Confocal laser scanning microscopy studies showed that chlorhexidine treatment lead to fluorescence fading indicating membrane permeabilization but did not cause biomass removal. In contrast, A3 treatment caused a simultaneous biofilm fluorescence loss and biomass removal. These dual anti-biofilm properties make α,α-disubstituted β-amino amides promising scaffolds in finding new control strategies against recalcitrant biofilms.Item Monitoring biofilm growth and dispersal in real-time with impedance biosensors(Oxford University Press, 2023-02) McGlennen, Matthew; Dieser, Markus; Foreman, Christine M; Warnat, StephanMicrobial biofilm contamination is a widespread problem that requires precise and prompt detection techniques to effectively control its growth. Microfabricated electrochemical impedance spectroscopy (EIS) biosensors offer promise as a tool for early biofilm detection and monitoring of elimination. This study utilized a custom flow cell system with integrated sensors to make real-time impedance measurements of biofilm growth under flow conditions, which were correlated with confocal laser scanning microscopy (CLSM) imaging. Biofilm growth on EIS biosensors in basic aqueous growth media (tryptic soy broth, TSB) and an oil–water emulsion (metalworking fluid, MWF) attenuated in a sigmoidal decay pattern, which lead to an ∼22–25% decrease in impedance after 24 Hrs. Subsequent treatment of established biofilms increased the impedance by ∼14% and ∼41% in TSB and MWF, respectively. In the presence of furanone C-30, a quorum-sensing inhibitor (QSI), impedance remained unchanged from the initial time point for 18 Hrs in TSB and 72 Hrs in MWF. Biofilm changes enumerated from CLSM imaging corroborated impedance measurements, with treatment significantly reducing biofilm. Overall, these results support the application of microfabricated EIS biosensors for evaluating the growth and dispersal of biofilm in situ and demonstrate potential for use in industrial settings.Item Open-source pneumatic pressure pump for drop-based microfluidic flow controls(IOP Publishing, 2023-07) Sanchez, Humberto S.; Chang, Connie B.An open-source pneumatic pressure pump is engineered for driving fluid flow in a microfluidic device. It is designed to be a cost-effective and customizable alternative to commercial systems. The pneumatic pressure pump utilizes a single open-source microcontroller to control four dual-valve pressure regulators. The control scheme is written in the Arduino development environment and the user interface is written in Python. The pump was used to pressurize water and a fluorinated oil that have similar viscosities. The pump can accurately control pressures to a resolution of less than 0.02 psig with rapid response times of less than one second, overshoot of desired pressures by less than 30%, and setting response times of less than two seconds. The pump was also validated in its ability to produce water-in-oil drops using a drop-making microfluidic device. The resultant drop size scaled as expected with the pressures applied to the emulsion phases. The pump is the first custom-made dual-valve regulator that is used to precisely control fluid flow in a microfluidic device. The presented design is an advancement towards making more fully open-source pneumatic pressure pumps for controlling flow in microfluidic devices.Item 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.Item Bacterial transfer and biofilm formation in needleless connectors in a clinically simulated in vitro catheter model(Cambridge University Press, 2023-04) Ryder, Marcia; deLancey-Pulcini, Elinor; Parker, Albert E.; James, Garth A.Objective: Although needleless connectors (NCs) are widely used in clinical practice, they carry significant risk of bloodstream infection (BSI). In this study, we quantified differences in bacterial transfer and biofilm formation between various NCs. Design: Prospective, clinically simulated in vitro experimental study. Methods: We tested 20 NCs in a 5-day clinical simulation of Staphylococcus aureus inoculations onto NC septum surfaces, which were then flushed with saline and cultured for bacterial transfer. Biofilm formation was measured through destructive sampling of the connector-catheter system. Moreover, 8 NC design factors were evaluated for their influence on bacterial transfer and biofilm formation. This study was designed without a disinfection protocol to ascertain the intrinsic risk of each NC. Results: Clave Neutron and MicroClave had the lowest overall mean log density of bacteria in the flush compared to other NCs (P < .05), except there were no statistically significant differences between Clave Neutron, Microclave, SafeTouch, and SafeAccess (P ≥ .05). The amount of biofilm in the NC was positively associated with bacteria in the flush (P < .0005). Among 8 design factors, flow path was most important, with the internal cannula associated with a statistically significant 1 log reduction (LR) in bacteria in the flush (R2 = 49%) and 0.5–2 LR in the connector (R2 = 34%). All factors together best explained bacteria in the flush (R2 = 65%) and biofilm in the connector (R2 = 48%). Conclusions: Bacterial transfer and biofilm formation in the connector-catheter system varied statistically significantly between the 20 NCs, suggesting that NC choice can lower the risk of developing catheter-related BSIs.