Center for Biofilm Engineering (CBE)
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/9334
At the Center for Biofilm Engineering (CBE), multidisciplinary research teams develop beneficial uses for microbial biofilms and find solutions to industrially relevant biofilm problems. The CBE was established at Montana State University, Bozeman, in 1990 as a National Science Foundation Engineering Research Center. As part of the MSU College of Engineering, the CBE gives students a chance to get a head start on their careers by working on research teams led by world-recognized leaders in the biofilm field.
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Item Pseudomonad reverse carbon catabolite repression, interspecies metabolite exchange, and consortial division of labor(Springer Science and Business Media LLC, 2019-11) Park, Heejoon; McGill, S. Lee; Arnold, Adrienne D.; Carlson, Ross P.Microorganisms acquire energy and nutrients from dynamic environments, where substrates vary in both type and abundance. The regulatory system responsible for prioritizing preferred substrates is known as carbon catabolite repression (CCR). Two broad classes of CCR have been documented in the literature. The best described CCR strategy, referred to here as classic CCR (cCCR), has been experimentally and theoretically studied using model organisms such as Escherichia coli. cCCR phenotypes are often used to generalize universal strategies for fitness, sometimes incorrectly. For instance, extremely competitive microorganisms, such as Pseudomonads, which arguably have broader global distributions than E. coli, have achieved their success using metabolic strategies that are nearly opposite of cCCR. These organisms utilize a CCR strategy termed ‘reverse CCR’ (rCCR), because the order of preferred substrates is nearly reverse that of cCCR. rCCR phenotypes prefer organic acids over glucose, may or may not select preferred substrates to optimize growth rates, and do not allocate intracellular resources in a manner that produces an overflow metabolism. cCCR and rCCR have traditionally been interpreted from the perspective of monocultures, even though most microorganisms live in consortia. Here, we review the basic tenets of the two CCR strategies and consider these phenotypes from the perspective of resource acquisition in consortia, a scenario that surely influenced the evolution of cCCR and rCCR. For instance, cCCR and rCCR metabolism are near mirror images of each other; when considered from a consortium basis, the complementary properties of the two strategies can mitigate direct competition for energy and nutrients and instead establish cooperative division of labor.Item Nisin penetration and efficacy against Staphylococcus aureus biofilms under continuous-flow conditions(2019-07) Godoy-Santos, Fernanda; Pitts, Betsey; Stewart, Philip S.; Mantovani, Hilario C.Biofilms may enhance the tolerance of bacterial pathogens to disinfectants, biocides and other stressors by restricting the penetration of antimicrobials into the matrix-enclosed cell aggregates, which contributes to the recalcitrance of biofilm-associated infections. In this work, we performed real-time monitoring of the penetration of nisin into the interior of Staphylococcus aureus biofilms under continuous flow and compared the efficacy of this lantibiotic against planktonic and sessile cells of S. aureus . Biofilms were grown in Center for Disease Control (CDC) reactors and the spatial and temporal effects of nisin action on S. aureus cells were monitored by real-time confocal microscopy. Under continuous flow, nisin caused loss of membrane integrity of sessile cells and reached the bottom of the biofilms within ~20 min of exposure. Viability analysis using propidium iodide staining indicated that nisin was bactericidal against S. aureus biofilm cells. Time-kill assays showed that S. aureus viability reduced 6.71 and 1.64 log c.f.u. ml-1 for homogenized planktonic cells in exponential and stationary phase, respectively. For the homogenized and intact S. aureus CDC biofilms, mean viability decreased 1.25 and 0.50 log c.f.u. ml-1, respectively. Our results demonstrate the kinetics of biofilm killing by nisin under continuous-flow conditions, and shows that alterations in the physiology of S. aureus cells contribute to variations in sensitivity to the lantibiotic. The approach developed here could be useful to evaluate the antibiofilm efficacy of other bacteriocins either independently or in combination with other antimicrobials.Item DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology(2019-09) Pratt, Shawna L.; Zath, Geoffrey K.; Williamson, Kelly S.; Franklin, Michael J.; Chang, Connie B.The physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic approaches now provide a means to study microbial physiology and bacterial heterogeneity at the single cell level, improving our ability to isolate and examine these subpopulations. Drop-based microfluidics provides a high-throughput approach to study individual cell physiology within bacterial populations. Using this approach, single cells are isolated from the population and encapsulated in growth medium dispersed in oil using a 15 μm diameter drop making microfluidic device. The drops are arranged as a packed monolayer inside a polydimethylsiloxane (PDMS) microfluidic device. Growth of thousands of individual cells in identical microenvironments can then be imaged using confocal laser scanning microscopy (CLSM). A challenge for this approach has been the maintenance of drop stability during extended time-lapse imaging. In particular, the drops do not maintain their volume over time during incubation in PDMS devices, due to fluid transport into the porous PDMS surroundings. Here, we present a strategy for PDMS device preparation that stabilizes drop position and volume within a drop array on a microfluidic chip for over 20 h. The stability of water-in-oil drops is maintained by soaking the device in a reservoir containing both water and oil in thermodynamic equilibrium. This ensures that phase equilibrium of the drop emulsion fluids within the porous PDMS material is maintained during drop incubation and imaging. We demonstrate the utility of this approach, which we label DropSOAC (DropStabilization On AChip), for time-lapse studies of bacterial growth. We characterize growth of Pseudomonas aeruginosa and its Δhpf mutant derivative during resuscitation and growth following starvation. We demonstrate that growth rate and lag time heterogeneity of hundreds of individual bacterial cells can be determined starting from single isolated cells. The results show that the DropSOAC capsule provides a high-throughput approach toward studies of microbial physiology at the single cell level, and can be used to characterize physiological differences of cells from within a larger population.Item Janthinobacterium CG23_2: comparative genome analysis reveals enhanced environmental sensing and transcriptional regulation for adaptation to life in an Antarctic supraglacial stream(2019-10) Dieser, Markus; Smith, Heidi J.; Ramaraj, Thiruvarangan; Foreman, Christine M.As many bacteria detected in Antarctic environments are neither true psychrophiles nor endemic species, their proliferation in spite of environmental extremes gives rise to genome adaptations. Janthinobacterium sp. CG23_2 is a bacterial isolate from the Cotton Glacier stream, Antarctica. To understand how Janthinobacterium sp. CG23_2 has adapted to its environment, we investigated its genomic traits in comparison to genomes of 35 published Janthinobacterium species. While we hypothesized that genome shrinkage and specialization to narrow ecological niches would be energetically favorable for dwelling in an ephemeral Antarctic stream, the genome of Janthinobacterium sp. CG23_2 was on average 1.7 ± 0.6 Mb larger and predicted 1411 ± 499 more coding sequences compared to the other Janthinobacterium spp. Putatively identified horizontal gene transfer events contributed 0.92 Mb to the genome size expansion of Janthinobacterium sp. CG23_2. Genes with high copy numbers in the species-specific accessory genome of Janthinobacterium sp. CG23_2 were associated with environmental sensing, locomotion, response and transcriptional regulation, stress response, and mobile elements—functional categories which also showed molecular adaptation to cold. Our data suggest that genome plasticity and the abundant complementary genes for sensing and responding to the extracellular environment supported the adaptation of Janthinobacterium sp. CG23_2 to this extreme environment.Item Long-Term Flow through Human Intestinal Organoids with the Gut Organoid Flow Chip (GOFlowChip)(2019-09) Sidar, Barkan; Jenkins, Brittany R.; Huang, Sha; Spence, Jason R.; Walk, Seth T.Human intestinal organoids (HIOs) are millimeter-scale models of the human intestinal epithelium and hold tremendous potential for advancing fundamental and applied biomedical research. HIOs resemble the native gut in that they consist of a fluid-filled lumen surrounded by a polarized epithelium and associated mesenchyme; however, their topologically-closed, spherical shape prevents flow through the interior luminal space, making the system less physiological and leading to the buildup of cellular and metabolic waste. These factors ultimately limit experimentation inside the HIOs. Here, we present a millifluidic device called the gut organoid flow chip (GOFlowChip), which we use to “port” HIOs and establish steady-state liquid flow through the lumen for multiple days. This long-term flow is enabled by the use of laser-cut silicone gaskets, which allow liquid in the device to be slightly pressurized, suppressing bubble formation. To demonstrate the utility of the device, we establish separate luminal and extraluminal flow and use luminal flow to remove accumulated waste. This represents the first demonstration of established liquid flow through the luminal space of a gastrointestinal organoid over physiologically relevant time scales. Flow cytometry results reveal that HIO cell viability is unaffected by long-term porting and luminal flow. We expect the real-time, long-term control over luminal and extraluminal contents provided by the GOFlowChip will enable a wide variety of studies including intestinal secretion, absorption, transport, and co-culture with intestinal microorganisms.Item Facultative and anaerobic consortia of haloalkaliphilic ureolytic micro-organisms capable of precipitating calcium carbonate(Wiley, 2019-08) Skorupa, Dana J.; Akyel, Arda; Fields, Matthew W.; Gerlach, RobinAims Development of biomineralization technologies has largely focused on microbially induced carbonate precipitation (MICP) via Sporosarcina pasteurii ureolysis; however, as an obligate aerobe, the general utility of this organism is limited. Here, facultative and anaerobic haloalkaliphiles capable of ureolysis were enriched, identified and then compared to S. pasteurii regarding biomineralization activities. Methods and Results Anaerobic and facultative enrichments for haloalkaliphilic and ureolytic micro‐organisms were established from sediment slurries collected at Soap Lake (WA). Optimal pH, temperature and salinity were determined for highly ureolytic enrichments, with dominant populations identified via a combination of high‐throughput SSU rRNA gene sequencing, clone libraries and Sanger sequencing of isolates. The enrichment cultures consisted primarily of Sporosarcina‐ and Clostridium‐like organisms. Ureolysis rates and direct cell counts in the enrichment cultures were comparable to the S. pasteurii (strain ATCC 11859) type strain. Conclusions Ureolysis rates from both facultatively and anaerobically enriched haloalkaliphiles were either not statistically significantly different to, or statistically significantly higher than, the S. pasteurii (strain ATCC 11859) rates. Work here concludes that extreme environments can harbour highly ureolytic active bacteria with potential advantages for large scale applications, such as environments devoid of oxygen. Significance and Impact of the Study The bacterial consortia and isolates obtained add to the possible suite of organisms available for MICP implementation, therefore potentially improving the economics and efficiency of commercial biomineralization.Item A Novel Gastric Spheroid Co-culture Model Reveals Chemokine-Dependent Recruitment of Human Dendritic Cells to the Gastric Epithelium(2019-03) Sebrell, Thomas A.; Hashimi, Marziah; Sidar, Barkan; Wilkinson, Royce A.; Kirpotina, Liliya; Quinn, Mark T.; Malkoc, Zeynep; Taylor, Paul J.; Wilking, James N.; Bimczok, DianeBackground & Aims Gastric dendritic cells (DCs) control the adaptive response to infection with Helicobacter pylori, a major risk factor for peptic ulcer disease and gastric cancer. We hypothesize that DC interactions with the gastric epithelium position gastric DCs for uptake of luminal H pylori and promote DC responses to epithelial-derived mediators. The aim of this study was to determine whether the gastric epithelium actively recruits DCs using a novel co-culture model of human gastric epithelial spheroids and monocyte-derived DCs. Methods Spheroid cultures of primary gastric epithelial cells were infected with H pylori by microinjection. Co-cultures were established by adding human monocyte-derived DCs to the spheroid cultures and were analyzed for DC recruitment and antigen uptake by confocal microscopy. Protein array, gene expression polymerase chain reaction array, and chemotaxis assays were used to identify epithelial-derived chemotactic factors that attract DCs. Data from the co-culture model were confirmed using human gastric tissue samples. Results Human monocyte-derived DCs co-cultured with gastric spheroids spontaneously migrated to the gastric epithelium, established tight interactions with the epithelial cells, and phagocytosed luminally applied H pylori. DC recruitment was increased upon H pylori infection of the spheroids and involved the activity of multiple chemokines including CXCL1, CXCL16, CXCL17, and CCL20. Enhanced chemokine expression and DC recruitment to the gastric epithelium also was observed in H pylori–infected human gastric tissue samples. Conclusions Our results indicate that the gastric epithelium actively recruits DCs for immunosurveillance and pathogen sampling through chemokine-dependent mechanisms, with increased recruitment upon active H pylori infection.Item Kinetics of Calcite Precipitation by Ureolytic Bacteria under Aerobic and Anaerobic Conditions(2019-05) Mitchell, Andrew C.; Espinosa-Ortiz, Erika J.; Parks, Stacy L.; Phillips, Adrienne J.; Cunningham, Alfred B.; Gerlach, RobinThe kinetics of urea hydrolysis (ureolysis) and induced calcium carbonate (CaCO3) precipitation for engineering use in the subsurface was investigated under aerobic conditions using Sporosarcina pasteurii (ATCC strain 11859) as well as Bacillus sphaericus strains 21776 and 21787. All bacterial strains showed ureolytic activity inducing CaCO3 precipitation aerobically. Rate constants not normalized to biomass demonstrated slightly higher-rate coefficients for both ureolysis (kurea) and CaCO3 precipitation (kprecip) for B. sphaericus 21776 (kurea=0.10±0.03 h−1, kprecip=0.60±0.34 h−1) compared to S. pasteurii (kurea=0.07±0.02 h−1, kprecip=0.25±0.02 h−1), though these differences were not statistically significantly different. B. sphaericus 21787 showed little ureolytic activity but was still capable of inducing some CaCO3 precipitation. Cell growth appeared to be inhibited during the period of CaCO3 precipitation. Transmission electron microscopy (TEM) images suggest this is due to the encasement of cells and was reflected in lower kurea values observed in the presence of dissolved Ca. However, biomass regrowth could be observed after CaCO3 precipitation ceased, which suggests that ureolysis-induced CaCO3 precipitation is not necessarily lethal for the entire population. The kinetics of ureolysis and CaCO3 precipitation with S. pasteurii was further analyzed under anaerobic conditions. Rate coefficients obtained in anaerobic environments were comparable to those under aerobic conditions; however, no cell growth was observed under anaerobic conditions with NO−3, SO2−4 or Fe3+ as potential terminal electron acceptors. These data suggest that the initial rates of ureolysis and ureolysis-induced CaCO3 precipitation are not significantly affected by the absence of oxygen but that long-term ureolytic activity might require the addition of suitable electron acceptors. Variations in the ureolytic capabilities and associated rates of CaCO3 precipitation between strains must be fully considered in subsurface engineering strategies that utilize microbial amendments.Item Quorum sensing inhibition as a promising method to control biofilm growth in metalworking fluids(2019-04) Ozcan, Safiye S.; Dieser, Markus; Parker, Albert E.; Balasubramanian, Narayanaganesh; Foreman, Christine M.Microbial contamination in metalworking systems is a critical problem. This study determined the microbial communities in metalworking fluids (MWFs) from two machining shops and investigated the effect of quorum sensing inhibition (QSI) on biofilm growth. In both operations, biofilm-associated and planktonic microbial communities were dominated by Pseudomonadales (60.2–99.7%). Rapid recolonization was observed even after dumping spent MWFs and meticulous cleaning. Using Pseudomonas aeruginosa PAO1 as a model biofilm organism, patulin (40 µM) and furanone C-30 (75 µM) were identified as effective QSI agents. Both agents had a substantially higher efficacy compared to α-amylase (extracellular polymeric substance degrading enzyme) and reduced biofilm formation by 63% and 76%, respectively, in MWF when compared to untreated controls. Reduced production of putatively identified homoserine lactones and quinoline in MWF treated with QS inhibitors support the effect of QSI on biofilm formation. The results highlight the effectiveness of QSI as a potential strategy to eradicate biofilms in MWFs.Item Multiscale analysis of autotroph-heterotroph interactions in a high-temperature microbial community(2018-09) Hunt, Kristopher A.; Jennings, Ryan deM.; Inskeep, William P.; Carlson, Ross P.Interactions among microbial community members can lead to emergent properties, such as enhanced productivity, stability, and robustness. Iron-oxide mats in acidic (pH 2–4), high-temperature (> 65 °C) springs of Yellowstone National Park contain relatively simple microbial communities and are well-characterized geochemically. Consequently, these communities are excellent model systems for studying the metabolic activity of individual populations and key microbial interactions. The primary goals of the current study were to integrate data collected in situ with in silico calculations across process-scales encompassing enzymatic activity, cellular metabolism, community interactions, and ecosystem biogeochemistry, as well as to predict and quantify the functional limits of autotroph-heterotroph interactions. Metagenomic and transcriptomic data were used to reconstruct carbon and energy metabolisms of an important autotroph (Metallosphaera yellowstonensis) and heterotroph (Geoarchaeum sp. OSPB) from the studied Fe(III)-oxide mat communities. Standard and hybrid elementary flux mode and flux balance analyses of metabolic models predicted cellular- and community-level metabolic acclimations to simulated environmental stresses, respectively. In situ geochemical analyses, including oxygen depth-profiles, Fe(III)-oxide deposition rates, stable carbon isotopes and mat biomass concentrations, were combined with cellular models to explore autotroph-heterotroph interactions important to community structure-function. Integration of metabolic modeling with in situ measurements, including the relative population abundance of autotrophs to heterotrophs, demonstrated that Fe(III)-oxide mat communities operate at their maximum total community growth rate (i.e. sum of autotroph and heterotroph growth rates), as opposed to net community growth rate (i.e. total community growth rate subtracting autotroph consumed by heterotroph), as predicted from the maximum power principle. Integration of multiscale data with ecological theory provides a basis for predicting autotroph-heterotroph interactions and community-level cellular organization.