Scholarly Work - Center for Biofilm Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/9335
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Item A Novel Irrigant to Eliminate Planktonic Bacteria and Eradicate Biofilm Superstructure With Persistent Effect During Total Hip Arthroplasty(Elsevier BV, 2022-02) Bashyal, Ravi K.; Mathew, Matt; Bowen, Edward; James, Garth A.; Stulberg, S. DavidBackground Numerous studies have examined the use of topical and irrigation-related adjuvants to decrease the risk of periprosthetic joint infection (PJI) after total hip arthroplasty. Many issues related to their use remain to be investigated. These include cost, antibiotic stewardship, bactericidal effect on planktonic bacteria, host cytotoxicity, necessity to irrigate/dilute potentially cytotoxic agents after their application, and impact on biofilm. Methods Bacterial strains of microorganisms were grown in optimal medium. After the growth phase, the organisms were exposed to the novel irrigation solution (XPerience) or phosphate buffer solution (PBS) for 5 minutes before a neutralizing broth was added. The colony-forming units per milliliter and the log reduction in colony-forming units in the treated sample vs the control were then determined. Subsequently, biofilms of microorganisms were grown on hydroxyapatite-coated glass slides. Each slide was then exposed to irrigation solutions for various contact times. Biofilm quantification was performed and the log10 density of each organism was obtained. Results In vitro testing of the irrigant demonstrated 6-log reductions in planktonic bacteria in 5 minutes, and 4-log to 8-log reductions in biofilms. Laboratory tissue testing has demonstrated minimal cytotoxic effects to host tissue allowing for solution to remain in contact with the host without need for subsequent irrigation, creating a barrier to biofilm for up to 5 hours after its application. Conclusion This novel irrigant demonstrates high efficacy against both planktonic bacteria and bacterial biofilms in laboratory testing. Large series in vivo data are necessary to further establish its efficacy in reducing primary and recurrent surgical site infections.Item Screening of Additive Formulations Enables Off-Chip Drop Reverse Transcription Quantitative Polymerase Chain Reaction of Single Influenza A Virus Genomes(American Chemical Society, 2021-03) Loveday, Emma Kate; Zath, Geoffrey K.; Bikos, Dimitri A.; Jay, Zackary J.; Chang, Connie B.The miniaturization of polymerase chain reaction (PCR) using drop-based microfluidics allows for amplification of single nucleic acids in aqueous picoliter-sized drops. Accurate data collection during PCR requires that drops remain stable to coalescence during thermocycling and drop contents are retained. Following systematic testing of known PCR additives, we identified an optimized formulation of 1% w/v Tween-20, 0.8 μg/μL bovine serum albumin, 1 M betaine in the aqueous phase, and 3 wt % (w/w) of the polyethylene glycol-perfluoropolyether2 surfactant in the oil phase of 50 μm diameter drops that maintains drop stability and prevents dye transport. This formulation enables a method we call off-chip drop reverse transcription quantitative PCR (OCD RT-qPCR) in which drops are thermocycled in a qPCR machine and sampled at various cycle numbers “off-chip”, or outside of a microfluidic chip. qPCR amplification curves constructed from hundreds of individual drops using OCD RT-qPCR and imaged using epifluorescence microscopy correlate with amplification curves of ≈300,000 drops thermocycled using a qPCR machine. To demonstrate the utility of OCD RT-qPCR, influenza A virus (IAV) RNA was detected down to a single viral genome copy per drop, or 0.320 cpd. This work was extended to perform multiplexed detection of IAV M gene RNA and cellular β-actin DNA in drops, and direct amplification of IAV genomes from infected cells without a separate RNA extraction step. The optimized additive formulation and the OCD-qPCR method allow for drop-based RT-qPCR without complex devices and demonstrate the ability to quantify individual or rare nucleic acid species within drops with minimal processing.Item An inexpensive, versatile, compact, programmable temperature controller and thermocycler for simultaneous analysis and visualization within a microscope(Springer Science and Business Media LLC, 2021-05) Cruz, Pablo Martínez; Wood, Mikayla A.; Abbasi, Reha; LeFevre, Thomas B.; McCalla, Stephanie E.Microfluidic Lab on a Chip (LOC) devices are key enabling technologies for research and industry due to their compact size, which increases the number of integrated operations while decreasing reagent use. Common operations within these devices such as chemical and biological reactions, cell growth, or kinetic measurements often require temperature control. Commercial temperature controllers are constrained by cost, complexity, size, and especially versatility for use in a broad range of applications. Small companies and research groups need temperature control systems that are more accessible, which have a wide applicability. This work describes the fabrication and validation of an inexpensive, modular, compact, and user-friendly temperature control system that functions within a microscope. This system provides precise temperature acquisition and control during imaging of any arbitrary sample which complies with the size of a microscope slide. The system includes two parts. The first part is a compact and washable Device Holder that is fabricated from high temperature resistant material and can fit securely inside a microscope stage. The second part is a robust Control Device that incorporates all the necessary components to program the temperature settings on the device and to output temperature data. The system can achieve heating and cooling times between 50°C and 100°C of 32 seconds and 101 seconds, respectively. A Bluetooth enabled smartphone application has been developed for real-time data visualization. The utility of the temperature control system was shown by monitoring rhodamine B fluorescence in a microfluidic device over a range of temperatures, and by performing a polymerase chain reaction (PCR) within a microscope. This temperature control system could potentially impact a broad scope of applications that require simultaneous imaging and temperature control.Item Subsurface hydrocarbon degradation strategies in low- and high-sulfate coal seam communities identified with activity-based metagenomics(Springer Science and Business Media LLC, 2022-02) Schweitzer, Hannah D.; Smith, Heidi J.; Barnhart, Elliott P.; McKay, Luke J.; Gerlach, Robin; Cunningham, Alfred B.; Malmstrom, Rex R.; Goudeau, Danielle; Fields, Matthew W.Environmentally relevant metagenomes and BONCAT-FACS derived translationally active metagenomes from Powder River Basin coal seams were investigated to elucidate potential genes and functional groups involved in hydrocarbon degradation to methane in coal seams with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the establishment of coal-associated microbial communities under in situ conditions for metagenomic analyses from environmental and translationally active populations. Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and anaerobic phenol degradation pathways were present in active populations across the sampled coal seams. In particular, results suggested the importance of anaerobic degradation pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low-sulfate conditions, a mixture of both aerobic and anaerobic pathways was observed but with a predominance of aerobic dioxygenases. The putative low-molecular-weight biosurfactant, lichysein, appeared to play a more important role compared to rhamnolipids. The methods used in this study—subsurface environmental samplers in combination with metagenomic sequencing of both total and translationally active metagenomes—offer a deeper and environmentally relevant perspective on community genetic potential from coal seams poised at different redox conditions broadening the understanding of degradation strategies for subsurface carbon.Item In Situ Enhancement and Isotopic Labeling of Biogenic Coalbed Methane(American Chemical Society, 2022-02) Barnhart, Elliott P.; Ruppert, Leslie; Hiebert, Randy; Smith, Heidi J.; Schweitzer, Hannah D.; Clark, Arthur C.; Weeks, Edwin P.; Orem, William H.; Varonka, Matthew S.; Platt, George; Shelton, Jenna L.; Davis, Katherine J.; Hyatt, Robert J.; McIntosh, Jennifer C.; Ashley, Kilian; Ono, Shuhei; Martini, Anna M.; Hackley, Keith C.; Gerlach, Robin; Spangler, Lee; Phillips, Adrienne J.; Barry, Mark; Cunningham, Alfred B.; Fields, Matthew W.Subsurface microbial (biogenic) methane production is an important part of the global carbon cycle that has resulted in natural gas accumulations in many coal beds worldwide. Laboratory studies suggest that complex carbon-containing nutrients (e.g., yeast or algae extract) can stimulate methane production, yet the effectiveness of these nutrients within coal beds is unknown. Here, we use downhole monitoring methods in combination with deuterated water (D2O) and a 200-liter injection of 0.1% yeast extract (YE) to stimulate and isotopically label newly generated methane. A total dissolved gas pressure sensor enabled real time gas measurements (641 days preinjection and for 478 days postinjection). Downhole samples, collected with subsurface environmental samplers, indicate that methane increased 132% above preinjection levels based on isotopic labeling from D2O, 108% based on pressure readings, and 183% based on methane measurements 266 days postinjection. Demonstrating that YE enhances biogenic coalbed methane production in situ using multiple novel measurement methods has immediate implications for other field-scale biogenic methane investigations, including in situ methods to detect and track microbial activities related to the methanogenic turnover of recalcitrant carbon in the subsurface.Item Experimental Designs to Study the Aggregation and Colonization of Biofilms by Video Microscopy With Statistical Confidenc(Frontiers Media SA, 2022-01) Pettygrove, Brian A.; Smith, Heidi J.; Pallister, Kyler B.; Voyich, Jovanka M.; Stewart, Philip S.; Parker, Albert E.The goal of this study was to quantify the variability of confocal laser scanning microscopy (CLSM) time-lapse images of early colonizing biofilms to aid in the design of future imaging experiments. To accomplish this a large imaging dataset consisting of 16 independent CLSM microscopy experiments was leveraged. These experiments were designed to study interactions between human neutrophils and single cells or aggregates of Staphylococcus aureus (S. aureus) during the initial stages of biofilm formation. Results suggest that in untreated control experiments, variability differed substantially between growth phases (i.e., lag or exponential). When studying the effect of an antimicrobial treatment (in this case, neutrophil challenge), regardless of the inoculation level or of growth phase, variability changed as a frown-shaped function of treatment efficacy (i.e., the reduction in biofilm surface coverage). These findings were used to predict the best experimental designs for future imaging studies of early biofilms by considering differing (i) numbers of independent experiments; (ii) numbers of fields of view (FOV) per experiment; and (iii) frame capture rates per hour. A spreadsheet capable of assessing any user-specified design is included that requires the expected mean log reduction and variance components from user-generated experimental results. The methodology outlined in this study can assist researchers in designing their CLSM studies of antimicrobial treatments with a high level of statistical confidence.Item Innovating carbon-capture biotechnologies through ecosystem-inspired solutions(Elsevier BV, 2021-01) Schweitzer, Hannah; Aalto, Nerea J.; Busch, Wolfgang; Chan, Dennis Tin Chat; Chiesa, Matteo; Elvevoll, Edel O.; Gerlach, Robin; Krause, Kristen; Mocaer, Karel; Moran, James J.; Noel, Joseph P.; Patil, Shalaka Kiran; Schwab, Yannick; Wijffels, René H.; Wulff, Angela; Øvreås, Lise; Bernstein, Hans C.Rising atmospheric carbon concentrations affect global health, the economy, and overall quality of life. We are fast approaching climate tipping points that must be addressed, not only by reducing emissions but also through new innovation and action toward carbon capture for sequestration and utilization (CCSU). In this perspective, we delineate next-generation biotechnologies for CCSU supported by engineering design principles derived from ecological processes inspired by three major biomes (plant-soil, deep biosphere, and marine). These are to interface with existing industrial infrastructure and, in some cases, tap into the carbon sink potential of nature. To develop ecosystem-inspired biotechnology, it is important to identify accessible control points of CO2 and CH4 within a given system as well as value-chain opportunities that drive innovation. In essence, we must supplement natural biogeochemical carbon sinks with new bioengineering solutions.Item Pseudomonas aeruginosa reverse diauxie is a multidimensional, optimized, resource utilization strategy(Springer Science and Business Media LLC, 2021-01) McGill, S. Lee; Yung, Yeni; Hunt, Kristopher A.; Henson, Michael A.; Hanley, Luke; Carlson, Ross P.Pseudomonas aeruginosa is a globally-distributed bacterium often found in medical infections. The opportunistic pathogen uses a different, carbon catabolite repression (CCR) strategy than many, model microorganisms. It does not utilize a classic diauxie phenotype, nor does it follow common systems biology assumptions including preferential consumption of glucose with an ‘overflow’ metabolism. Despite these contradictions, P. aeruginosa is competitive in many, disparate environments underscoring knowledge gaps in microbial ecology and systems biology. Physiological, omics, and in silico analyses were used to quantify the P. aeruginosa CCR strategy known as ‘reverse diauxie’. An ecological basis of reverse diauxie was identified using a genome-scale, metabolic model interrogated with in vitro omics data. Reverse diauxie preference for lower energy, nonfermentable carbon sources, such as acetate or succinate over glucose, was predicted using a multidimensional strategy which minimized resource investment into central metabolism while completely oxidizing substrates. Application of a common, in silico optimization criterion, which maximizes growth rate, did not predict the reverse diauxie phenotypes. This study quantifies P. aeruginosa metabolic strategies foundational to its wide distribution and virulence including its potentially, mutualistic interactions with microorganisms found commonly in the environment and in medical infections.Item Symmetry breaking propulsion of magnetic microspheres in nonlinearly viscoelastic fluids(Springer Nature, 2021-02) Rogowski, Louis William; Ali, Jamel; Zhang, Xiao; Wilking, James N.; Fu, Henry C.; Kim, Min JunMicroscale propulsion impacts a diverse array of fields ranging from biology and ecology to health applications, such as infection, fertility, drug delivery, and microsurgery. However, propulsion in such viscous drag-dominated fluid environments is highly constrained, with time-reversal and geometric symmetries ruling out entire classes of propulsion. Here, we report the spontaneous symmetry-breaking propulsion of rotating spherical microparticles within non-Newtonian fluids. While symmetry analysis suggests that propulsion is not possible along the fore-aft directions, we demonstrate the existence of two equal and opposite propulsion states along the sphere’s rotation axis. We propose and experimentally corroborate a propulsion mechanism for these spherical microparticles, the simplest microswimmers to date, arising from nonlinear viscoelastic effects in rotating flows similar to the rod-climbing effect. Similar possibilities of spontaneous symmetry-breaking could be used to circumvent other restrictions on propulsion, revising notions of microrobotic design and control, drug delivery, microscale pumping, and locomotion of microorganisms.Item In Silico Analysis of Functionalized Hydrocarbon Production Using Ehrlich Pathway and Fatty Acid Derivatives in an Endophytic Fungus(MDPI, 2021-05) Hunt, Kristopher A.; Mallette, Natasha D.; Peyton, Brent M.; Carlson, Ross P.Functionalized hydrocarbons have various ecological and industrial uses, from signaling molecules and antifungal/antibacterial agents to fuels and specialty chemicals. The potential to produce functionalized hydrocarbons using the cellulolytic, endophytic fungus, Ascocoryne sarcoides, was quantified using genome-enabled, stoichiometric modeling. In silico analysis identified available routes to produce these hydrocarbons, including both anabolic- and catabolic-associated strategies, and determined correlations between the type and size of the hydrocarbons and culturing conditions. The analysis quantified the limits of the wild-type metabolic network to produce functionalized hydrocarbons from cellulose-based substrates and identified metabolic engineering targets, including cellobiose phosphorylase (CP) and cytosolic pyruvate dehydrogenase complex (PDHcyt). CP and PDHcyt activity increased the theoretical production limits under anoxic conditions where less energy was extracted from the substrate. The incorporation of both engineering targets resulted in near-complete conservation of substrate electrons in functionalized hydrocarbons. The in silico framework was integrated with in vitro fungal batch growth experiments to support O2 limitation and functionalized hydrocarbon production predictions. The metabolic reconstruction of this endo-phytic filamentous fungus describes pathways for both specific and general production strategies of 161 functionalized hydrocarbons applicable to many eukaryotic hosts.