Scholarly Work - Center for Biofilm Engineering
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Item Optimal surface estimation and thresholding of confocal microscope images of biofilms using Beer's Law(2020-05) Parker, Albert E.; Christen, J. A.; Lorenz, Lindsey A.; Smith, Heidi J.Beer's Law explains how light attenuates into thick specimens, including thick biofilms. We use a Bayesian optimality criterion, the maximum of the posterior probability distribution, and computationally efficiently fit Beer's Law to the 3D intensity data collected from thick living biofilms by a confocal scanning laser microscope. Using this approach the top surface of the biofilm and an optimal image threshold can be estimated. Biofilm characteristics, such as bio-volumes, can be calculated from this surface. Results from the Bayesian approach are compared to other approaches including the method of maximum likelihood or simply counting bright pixels. Uncertainty quantification (i.e., error bars) can be provided for the parameters of interest. This approach is applied to confocal images of stained biofilms of a common lab strain of Pseudomonas aeruginosa, stained biofilms of Janthinobacterium isolated from the Antarctic, and biofilms of Staphylococcus aureus that have been genetically modified to fluoresce green.Item Contribution of wild foods to diet, food security, and cultural values amidst climate change(2019-11) Smith, Erin; Ahmed, Selena; Running Crane, MaryAnn; Eggers, Margaret J.; Pierre, Mike; Flagg, Kenneth A.; Byker Shanks, CarmenWild foods are recognized to contribute to diet and food security through enhancing the availability of local, diverse, and nonmarket food sources. We investigated the contribution of wild foods to diet, food security, and cultural identity in a Native American[1] community in the context of climate change. Structured interviews were conducted with low-income residents of the Flathead Indian Reservation[2] in Northwestern Montana who participate in the federal Food Distribution Program on Indian Reservations, also known by participants as ‘Commodities.’ Responses to structured questions were analyzed for frequency, and open-ended responses were coded and analyzed to identify prevalent themes. Our analysis indicated that half of participants were food insecure. Approximately 28% of participants engaged in at least one wild food procurement activity, including hunting, fishing, and harvesting. On average, participants who engaged in one or more wild food procurement activities were more food secure than those who did not. Results highlight the multidimensional valuation of wild foods by participants including taste, freshness, nutritional quality, being a traditional community practice, and providing a sense of self-sufficiency. Climate change is perceived by participants to be adversely impacting wild food systems due to increased variability in seasonality and precipitation and increased incidences of wild fire. Findings point to the need for community-based strategies to strengthen wild food knowledge toward enhancing food sovereignty in Native American communities, in the context of climate change. [1] The term ‘Native American’ was determined to be the preferred term for referencing the Native American community in this study, based on consultation from our community advisory board. [2] The term ‘Flathead Indian Reservation’ was determined to be the preferred term for referencing the location in which this study was held, based on consultation from our community advisory board.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 Metagenomic Profiling of Microbial Pathogens in the Little Bighorn River, Montana(2019-03) Hamner, Steve; Brown, Bonnie L.; Hasan, Nur A.; Franklin, Michael J.; Doyle, John T.; Eggers, Margaret J.; Colwell, Rita R.; Ford, Tim E.The Little Bighorn River is the primary source of water for water treatment plants serving the local Crow Agency population, and has special significance in the spiritual and ceremonial life of the Crow tribe. Unfortunately, the watershed suffers from impaired water quality, with high counts of fecal coliform bacteria routinely measured during run-off events. A metagenomic analysis was carried out to identify potential pathogens in the river water. The Oxford Nanopore MinION platform was used to sequence DNA in near real time to identify both uncultured and a coliform-enriched culture of microbes collected from a popular summer swimming area of the Little Bighorn River. Sequences were analyzed using CosmosID bioinformatics and, in agreement with previous studies, enterohemorrhagic and enteropathogenic Escherichia coli and other E. coli pathotypes were identified. Noteworthy was detection and identification of enteroaggregative E. coli O104:H4 and Vibrio cholerae serotype O1 El Tor, however, cholera toxin genes were not identified. Other pathogenic microbes, as well as virulence genes and antimicrobial resistance markers, were also identified and characterized by metagenomic analyses. It is concluded that metagenomics provides a useful and potentially routine tool for identifying in an in-depth manner microbial contamination of waterways and, thereby, protecting public health.Item Microbial community changes during a toxic cyanobacterial bloom in an alkaline Hungarian lake(2018-08) Bell, Tisza A. S.; Feldoldi, Tamas; Sen-Kilic, Emel; Vasas, Gabor; Fields, Matthew W.; Peyton, Brent M.The Carpathian Basin is a lowland plain located mainly in Hungary. Due to the nature of the bedrock, alluvial deposits, and a bowl shape, many lakes and ponds of the area are characterized by high alkalinity. In this study, we characterized temporal changes in eukaryal and bacterial community dynamics with high throughput sequencing and relate the changes to environmental conditions in Lake Velence located in Fejer county, Hungary. The sampled Lake Velence microbial populations (algal and bacterial) were analyzed to identify potential correlations with other community members and environmental parameters at six timepoints over 6weeks in the Spring of 2012. Correlations between community members suggest a positive relationship between certain algal and bacterial populations (e.g. Chlamydomondaceae with Actinobacteria and Acidobacteria), while other correlations allude to changes in these relationships over time. During the study, high nitrogen availability may have favored non-nitrogen fixing cyanobacteria, such as the toxin-producing Microcystis aeruginosa, and the eutrophic effect may have been exacerbated by high phosphorus availability as well as the high calcium and magnesium content of the Carpathian Basin bedrock, potentially fostering exopolymer production and cell aggregation. Cyanobacterial bloom formation could have a negative environmental impact on other community members and potentially affect overall water quality as well as recreational activities. To our knowledge, this is the first prediction for relationships between photoautotrophic eukaryotes and bacteria from an alkaline, Hungarian lake.Item Impact of hydrologic boundaries on microbial planktonic and biofilm communities in shallow terrestrial subsurface environments(2018-09) Smith, Heidi J.; Zelaya, Anna J.; De León, Kara B.; Chakraborty, R.; Elias, Dwayne A.; Hazen, Terry C.; Arkin, Adam P.; Cunningham, Alfred B.; Fields, Matthew W.Subsurface environments contain a large proportion of planetary microbial biomass and harbor diverse communities responsible for mediating biogeochemical cycles important to groundwater used by human society for consumption, irrigation, agriculture and industry. Within the saturated zone, capillary fringe and vadose zones, microorganisms can reside in two distinct phases (planktonic or biofilm), and significant differences in community composition, structure and activity between free-living and attached communities are commonly accepted. However, largely due to sampling constraints and the challenges of working with solid substrata, the contribution of each phase to subsurface processes is largely unresolved. Here, we synthesize current information on the diversity and activity of shallow freshwater subsurface habitats, discuss the challenges associated with sampling planktonic and biofilm communities across spatial, temporal and geological gradients, and discuss how biofilms may be constrained within shallow terrestrial subsurface aquifers. We suggest that merging traditional activity measurements and sequencing/-omics technologies with hydrological parameters important to sediment biofilm assembly and stability will help delineate key system parameters. Ultimately, integration will enhance our understanding of shallow subsurface ecophysiology in terms of bulk-flow through porous media and distinguish the respective activities of sessile microbial communities from more transient planktonic communities to ecosystem service and maintenance.