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Item How long is enough? Identification of product dry-time as a primary driver of alcohol-based hand rub efficacy(2018-05) Suchomel, Miranda; Leslie, Rachel; Parker, Albert E.; Macinga, David R.Background The World Health Organization has called for the development of improved methodologies to evaluate alcohol-based handrub (ABHR) efficacy, including evaluation at “short application times and volumes that reflect actual use in healthcare facilities”. The objective of this study was to investigate variables influencing ABHR efficacy, under test conditions reflective of clinical use. Methods The test product (60% V/V 2-propanol) was evaluated according to a modified EN 1500 methodology, where application volumes of 1 mL, 2 mL, and 3 mL were rubbed until dry. Statistical analyses were performed to investigate the relative influences of product volume, hand size, and product dry-time on efficacy, and hand size and hand contamination on product dry-time. Results Mean log10 reduction factors (SD) were 1.99 (0.66), 2.96 (0.84) and 3.28 (0.96); and mean dry-times (SD) were 24 s (7 s), 50 s (14 s), and 67 s (20 s) at application volumes of 1 mL, 2 mL, and 3 mL, respectively (p ≤ 0.030). When data were examined at the individual volunteer level, there was a statistically significant correlation between dry-time and log reduction factor (p < 0.0001), independent of application volume. There was also a statistically significant correlation between hand surface area and dry-times (p = 0.047), but no correlation between hand surface area and efficacy (p = 0.698). Conclusions When keeping other variables such as alcohol type and concentration constant, product dry-time appears to be the primary driver of ABHR efficacy suggesting that dosing should be customized to each individual and focus on achieving a product dry-time delivering adequate efficacy.Item Assessment of Nannochloropsis gaditana growth and lipid accumulation with increased inorganic carbon delivery(2018-04) Pedersen, Todd C.; Gardner, Robert D.; Peyton, Brent M.Algal biomass refineries for sustainable transportation fuels, in particular biodiesel, will benefit from algal strain enhancements to improve biomass and lipid productivity. Specifically, the supply of inorganic carbon to microalgal cultures represents an area of great interest due to the potential for improved growth of microalgae and the possibility for incorporation with CO2 mitigation processes. Combinations of bicarbonate (HCO3−) salt addition and application of CO2 to control pH have shown compelling increases in growth rate and lipid productivity of fresh water algae. Here, focus was placed on the marine organism, Nannochloropsis gaditana, to investigate growth and lipid accumulation under various strategies of enhanced inorganic carbon supply. Three gas application strategies were investigated: continuous sparging of atmospheric air, continuous sparging of 5% CO2 during light hours until nitrogen depletion, and continuous sparging of atmospheric air supplemented with 5% CO2 for pH control between 8.0 and 8.3. These gas sparging schemes were combined with addition of low concentrations (5 mM) of sodium bicarbonate at inoculation and high concentration (50 mM) of sodium bicarbonate amendments just prior to nitrogen depletion. The optimum scenario observed for growth of N. gaditana under these inorganic carbon conditions was controlling pH with 5% CO2 on demand, which increased both growth rate and lipid accumulation. Fatty acid methyl esters were primarily comprised of C16:0 (palmitic) and C16:1 (palmitoleic) aliphatic chains. Additionally, the use of high concentration (50 mM) of bicarbonate amendments further improved lipid content (up to 48.6%) under nitrogen deplete conditions when paired with pH-controlled strategies.Item Low-Field Borehole NMR Applications in the Near-Surface Environment(2018-01) Kirkland, Catherine M.; Codd, Sarah L.The inherent heterogeneity of the near subsurface (<200 m below the ground surface) presents challenges for agricultural water management, hydrogeologic characterization, and engineering, among other fields. Borehole nuclear magnetic resonance (NMR) has the potential not only to describe this heterogeneity in space nondestructively but also to monitor physical and chemical changes in the subsurface with time. Nuclear magnetic resonance is sensitive to parameters of interest like porosity and permeability, saturation, fluid viscosity, and formation mineralogy. Borehole NMR tools have been used to measure soil moisture in model soils, and recent advances in lowfield borehole NMR instrumentation allow estimation of hydraulic properties of unconsolidated aquifers. We also demonstrate the potential for low-field borehole NMR tools to monitor field-relevant biogeochemical processes like biofilm accumulation and microbially induced calcite precipitation at laboratory and field scales. Finally, we address some remaining challenges and areas of future research, as well as other possible applications where borehole. NMR could provide valuable complementary data.Item Challenges and Opportunities for Tribal Waters: Addressing Disparities in Safe Public Drinking Water on the Crow Reservation in Montana, US(2018-03) Doyle, John T.; Kindness, L.; Real Bird, James; Eggers, Margaret J.; Camper, Anne K.Disparities in access to safe public drinking water are increasingly being recognized as contributing to health disparities and environmental injustice for vulnerable communities in the United States. As the Co-Directors of the Apsaálooke Water and Wastewater Authority (AWWWA) for the Crow Tribe, with our academic partners, we present here the multiple and complex challenges we have addressed in improving and maintaining tribal water and wastewater infrastructure, including the identification of diverse funding sources for infrastructure construction, the need for many kinds of specialized expertise and long-term stability of project personnel, ratepayer difficulty in paying for services, an ongoing legacy of inadequate infrastructure planning, and lack of water quality research capacity. As a tribal entity, the AWWWA faces additional challenges, including the complex jurisdictional issues affecting all phases of our work, lack of authority to create water districts, and additional legal and regulatory gaps—especially with regards to environmental protection. Despite these obstacles, the AWWWA and Crow Tribe have successfully upgraded much of the local water and wastewater infrastructure. We find that ensuring safe public drinking water for tribal and other disadvantaged U.S. communities will require comprehensive, community-engaged approaches across a broad range of stakeholders to successfully address these complex legal, regulatory, policy, community capacity, and financial challenges.Item Cr(VI) reduction and physiological toxicity are impacted by resource ratio in Desulfovibrio vulgaris(2018-03) Franco, Lauren C.; Steinbeisser, Sadie; Zane, Grant M.; Wall, Judy D.; Fields, Matthew W.Desulfovibrio spp. are capable of heavy metal reduction and are well-studied systems for understanding metal fate and transport in anaerobic environments. Desulfovibrio vulgaris Hildenborough was grown under environmentally relevant conditions (i.e., temperature, nutrient limitation) to elucidate the impacts on Cr(VI) reduction on cellular physiology. Growth at 20 °C was slower than 30 °C and the presence of 50 μM Cr(VI) caused extended lag times for all conditions, but once growth resumed the growth rate was similar to that without Cr(VI). Cr(VI) reduction rates were greatly diminished at 20 °C for both 50 and 100 μM Cr(VI), particularly for the electron acceptor limited (EAL) condition in which Cr(VI) reduction was much slower, the growth lag much longer (200 h), and viability decreased compared to balanced (BAL) and electron donor limited (EDL) conditions. When sulfate levels were increased in the presence of Cr(VI), cellular responses improved via a shorter lag time to growth. Similar results were observed between the different resource (donor/acceptor) ratio conditions when the sulfate levels were normalized (10 mM), and these results indicated that resource ratio (donor/acceptor) impacted D. vulgaris response to Cr(VI) and not merely sulfate limitation. The results suggest that temperature and resource ratios greatly impacted the extent of Cr(VI) toxicity, Cr(VI) reduction, and the subsequent cellular health via Cr(VI) influx and overall metabolic rate. The results also emphasized the need to perform experiments at lower temperatures with nutrient limitation to make accurate predictions of heavy metal reduction rates as well as physiological states in the environment.Item Analysis of Clostridium difficile biofilms: imaging and antimicrobial treatment(2018-01) James, Garth A.; Chesnel, L.; Boegli, Laura; Pulcini, Elinor D.; Fisher, Steve T.; Stewart, Philip S.BACKGROUND: Clostridium difficile, a spore-forming Gram-positive anaerobic bacillus, is the most common causative agent of healthcare-associated diarrhoea. Formation of biofilms may protect C. difficile against antibiotics, potentially leading to treatment failure. Furthermore, bacterial spores or vegetative cells may linger in biofilms in the gut causing C. difficile infection recurrence. OBJECTIVES: In this study, we evaluated and compared the efficacy of four antibiotics (fidaxomicin, surotomycin, vancomycin and metronidazole) in penetrating C. difficile biofilms and killing vegetative cells. METHODS: C. difficile biofilms grown initially for 48 or 72 h using the colony biofilm model were then treated with antibiotics at a concentration of 25 × MIC for 24 h. Vegetative cells and spores were enumerated. The effect of treatment on biofilm structure was studied by scanning electron microscopy (SEM). The ability of fidaxomicin and surotomycin to penetrate biofilms was studied using fluorescently tagged antibiotics. RESULTS: Both surotomycin and fidaxomicin were significantly more effective than vancomycin or metronidazole (P < 0.001) at killing vegetative cells in established biofilms. Fidaxomicin was more effective than metronidazole at reducing viable spore counts in biofilms (P < 0.05). Fluorescently labelled surotomycin and fidaxomicin penetrated C. difficile biofilms in < 1 h. After 24 h of treatment, SEM demonstrated that both fidaxomicin and surotomycin disrupted the biofilm structure, while metronidazole had no observable effect. CONCLUSIONS: Fidaxomicin is effective in disrupting C. difficile biofilms, killing vegetative cells and decreasing spore counts.Item Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation and fusion events(2018-02) Sebrell, T. Andrew; Sidar, Barkan; Bruns, Rachel; Wilkinson, Royce A.; Wiedenheft, Blake A.; Taylor, Brian A.; Samuelson, Linda C.; Wilking, James N.; Bimczok, DianeThree-dimensional cultures of primary epithelial cells including organoids, enteroids and epithelial spheroids have become increasingly popular for studies of gastrointestinal development, mucosal immunology and epithelial infection. However, little is known about the behavior of these complex cultures in their three-dimensional culture matrix. Therefore, we performed extended time-lapse imaging analysis (up to 4 days) of human gastric epithelial spheroids generated from adult tissue samples in order to visualize the dynamics of the spheroids in detail. Human gastric epithelial spheroids cultured in our laboratory grew to an average diameter of 443.9 ± 34.6 μm after 12 days, with the largest spheroids reaching diameters of >1000 μm. Live imaging analysis revealed that spheroid growth was associated with cyclic rupture of the epithelial shell at a frequency of 0.32 ± 0.1/day, which led to the release of luminal contents. Spheroid rupture usually resulted in an initial collapse, followed by spontaneous re-formation of the spheres. Moreover, spheroids frequently rotated around their axes within the Matrigel matrix, possibly propelled by basolateral pseudopodia-like formations of the epithelial cells. Interestingly, adjacent spheroids occasionally underwent luminal fusion, as visualized by injection of individual spheroids with FITC–Dextran (4 kDa). In summary, our analysis revealed unexpected dynamics in human gastric spheroids that challenge our current view of cultured epithelia as static entities and that may need to be considered when performing spheroid infection experiments.Item Quantifying NMR relaxation correlation and exchange in articular cartilage with time domain analysis(2018-02) Mailhiot, Sarah E.; Zong, Fangrong; Maneval, James E.; Galvosas, Petrik; Seymour, Joseph D.Measured nuclear magnetic resonance (NMR) transverse relaxation data in articular cartilage has been shown to be multi-exponential and correlated to the health of the tissue. The observed relaxation rates are dependent on experimental parameters such as solvent, data acquisition methods, data analysis methods, and alignment to the magnetic field. In this study, we show that diffusive exchange occurs in porcine articular cartilage and impacts the observed relaxation rates in T1-T2 correlation experiments. By using time domain analysis of T2-T2 exchange spectroscopy, the diffusive exchange time can be quantified by measurements that use a single mixing time. Measured characteristic times for exchange are commensurate with T1 in this material and so impacts the observed T1 behavior. The approach used here allows for reliable quantification of NMR relaxation behavior in cartilage in the presence of diffusive fluid exchange between two environments.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 Detection of Pseudomonas aeruginosa biomarkers from thermally injured mice in situ using imaging mass spectrometry(2017-12) Hamerly, Timothy; Everett, Jake A.; Paris, Nina; Fisher, Steve T.; Karunamurthy, Arivarasan; James, Garth A.; Rumbaugh, Kendra P.; Rhoads, Daniel D.; Bothner, BrianMonitoring patients with burn wounds for infection is standard practice because failure to rapidly and specifically identify a pathogen can result in poor clinical outcomes, including death. Therefore, a method that facilitates detection and identification of pathogens in situ within minutes of biopsy would be a significant benefit to clinicians. Mass spectrometry is rapidly becoming a standard tool in clinical settings, capable of identifying specific pathogens from complex samples. Imaging mass spectrometry (IMS) expands the information content by enabling spatial resolution of biomarkers in tissue samples as in histology, without the need for specific stains/antibodies. Herein, a murine model of thermal injury was used to study infection of burn tissue by Pseudomonas aeruginosa. This is the first use of IMS to detect P. aeruginosa infection in situ from thermally injured tissue. Multiple molecular features could be spatially resolved to infected or uninfected tissue. This demonstrates the potential use of IMS in a clinical setting to aid doctors in identifying both presence and species of pathogens in tissue.