Microbiology & Cell Biology
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Item Comparison of Enrichment and Plating Media for Recovery of Virulent Strains of Yersinia enterocolitica from Inoculated Beef Stew(Elsevier BV, 1983-11) Schiemann, D.A.Five plating agar media were evaluated for their ability to recover pure cultures of virulent strains of Yersinia enterocolitica serotypes O:3, O:8 and O:5,27. Cellobiose-arginine-lysine and bismuth sulfite agars were unproductive at 32°C but gave quantitative recovery with 48 h of incubation at 22°C. Adjustment of the pH of bismuth sulfite agar to 7.4 made the medium inhibitory. MacConkey, DHL and cefsulodin-irgasan-novobiocin agars gave quantitative recovery with 24 h of incubation at 32°C. Four preenrichment media incubated at four different temperatures, three selective enrichment media incubated at 22°C, and the five plating media were evaluated for their ability to recovery Y. enterocolitica from beef stew seeded with a background of ten other gram-negative bacteria. None of the plating media was superior for recovery; however, cefsulodin-irgasan-novobiocin agar showed the highest confirmation rate for presumptive colonies. Buffered-sorbitol-bile broth was inferior to richer media such as trypticase soy broth for preenrichment. Of the three selective enrichment media examined, only bile-oxalate-sorbose broth was found useful, especially for strains of serotype O:8 which could be recovered after 1 d of preenrichment and 3 d of selective enrichment at 22°C. Strains of serotypes O:8 and O:3 were recovered when two cells with 107 cells of ten other gram-negative bacteria were added to 10 g of beef stew following preenrichment in trypticase soy broth at 2°C for 7 d and selective enrichment in bile oxalate-sorbose broth at 22°C for 3 to 5 d. Strains of serotype O:5,27 were more difficult to recover even with longer enrichment times. These studies indicated that the most comprehensive enrichment system for recovery of Y. enterocolitica from foods is preenrichment in trypticase soy broth at 22°C for 1 d and 2 to 4°C for 4 to 7 d followed by selective enrichment in bile-oxalate-sorbose broth at 22°C for 3 to 5 d and isolation on cefsulodin-irgasan-novobiocin agar.Item Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group AStreptococcusIs Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but Not in the Skin(2019-09) Lei, Benfang; Minor, Dylan; Feng, Wenchao; Jerome, Maria; Quinn, Mark T.; Jutila, Mark A.; Liu, MengyaoGroup A Streptococcus (GAS) commonly causes pharyngitis and skin infections. Little is known why streptococcal pharyngitis usually does not lead to pneumonia and why the skin is a favorite niche for GAS. To partially address these questions, the effectiveness of neutrophils in clearing wild-type (wt) M1T1 GAS strain MGAS2221 from the lung and from the skin was examined in murine models of intratracheal pneumonia and subcutaneous infection. Ninety-nine point seven percent of the MGAS2221 inoculum was cleared from the lungs of C57BL/6J mice at 24 h after inoculation, while there was no MGAS2221 clearance from skin infection sites. The bronchial termini had robust neutrophil infiltration, and depletion of neutrophils abolished MGAS2221 clearance from the lung. Phagocyte NADPH oxidase but not myeloperoxidase was required for MGAS2221 clearance. Thus, wt M1T1 GAS can be cleared by neutrophils using an NADPH oxidase-dependent mechanism in the lung. MGAS2221 induced robust neutrophil infiltration at the edge of skin infection sites and throughout infection sites at 24 h and 48 h after inoculation, respectively. Neutrophils within MGAS2221 infection sites had no nuclear staining. Skin infection sites of streptolysin S-deficient MGAS2221 ΔsagA were full of neutrophils with nuclear staining, whereas MGAS2221 ΔsagA infection was not cleared. Gp91phox knockout (KO) and control mice had similar GAS numbers at skin infection sites and similar abilities to select SpeB activity-negative (SpeBA-) variants. These results indicate that phagocyte NADPH oxidase-mediated GAS killing is compromised in the skin. Our findings support a model for GAS skin tropism in which GAS generates an anoxic niche to evade phagocyte NADPH oxidase-mediated clearance.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 Type I-F CRISPR-Cas provides protection from DNA, but not RNA phages(2019-10) Buyukyoruk, Murat; Wiedenheft, Blake A.The type I-F CRISPR–Cas system from Pseudomonas aeruginosa (PA14) provides sequence-specific elimination of invading DNA1,2. Recently, a paper published in Cell Research reported that this system targets mRNA through a non-canonical mechanism3. Here, we implement the proposed design rules for generating CRISPR-RNA (crRNA)-guides that target RNA and test whether the proposed RNA-targeting activity provides immunity against RNA phage infection. Our experiments reveal that the type I-F CRISPR system provides protection from DNA, but not RNA phages.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 Protective Effects of a New C-Jun N-terminal Kinase Inhibitor in the Model of Global Cerebral Ischemia in Rats(2019-05-19) Plotnikov, Mark B.; Chernysheva, Galina A.; Aliev, Oleg I.; Smol'iakova, Vera I.; Fomina, Tatiana I.; Osipenko, Anton N.; Rydchenko, Victoria S.; Anfinogenova, Yana J.; Khlebnikov, Andrei I.; Schepetkin, Igor A.; Atochin, Dmitriy N.c-Jun N-terminal kinase (JNK) is activated by various brain insults and is implicated in neuronal injury triggered by reperfusion-induced oxidative stress. Some JNK inhibitors demonstrated neuroprotective potential in various models, including cerebral ischemia/reperfusion injury. The objective of the present work was to study the neuroprotective activity of a new specific JNK inhibitor, IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime sodium salt), in the model of global cerebral ischemia (GCI) in rats compared with citicoline (cytidine-5′-diphosphocholine), a drug approved for the treatment of acute ischemic stroke and to search for pleiotropic mechanisms of neuroprotective effects of IQ-1S. The experiments were performed in a rat model of ischemic stroke with three-vessel occlusion (model of 3VO) affecting the brachiocephalic artery, the left subclavian artery, and the left common carotid artery. After 7-min episode of GCI in rats, 25% of animals died, whereas survived animals had severe neurological deficit at days 1, 3, and 5 after GCI. At day 5 after GCI, we observing massive loss of pyramidal neurons in the hippocampal CA1 area, increase in lipid peroxidation products in the brain tissue, and decrease in local cerebral blood flow (LCBF) in the parietal cortex. Moreover, blood hyperviscosity syndrome and endothelial dysfunction were found after GCI. Administration of IQ-1S (intragastrically at a dose 50 mg/kg daily for 5 days) was associated with neuroprotective effect comparable with the effect of citicoline (intraperitoneal at a dose of 500 mg/kg, daily for 5 days).The neuroprotective effect was accompanied by a decrease in the number of animals with severe neurological deficit, an increase in the number of animals with moderate degree of neurological deficit compared with control GCI group, and an increase in the number of unaltered neurons in the hippocampal CA1 area along with a significant decrease in the number of neurons with irreversible morphological damage. In rats with IQ-1S administration, the LCBF was significantly higher (by 60%) compared with that in the GCI control. Treatment with IQ-1S also decreases blood viscosity and endothelial dysfunction. A concentration-dependent decrease (IC50 = 0.8 ± 0.3 μM) of tone in isolated carotid arterial rings constricted with phenylephrine was observed after IQ-1S application in vitro. We also found that IQ-1S decreased the intensity of the lipid peroxidation in the brain tissue in rats with GCI. 2.2-Diphenyl-1-picrylhydrazyl scavenging for IQ-1S in acetonitrile and acetone exceeded the corresponding values for ionol, a known antioxidant. Overall, these results suggest that the neuroprotective properties of IQ-1S may be mediated by improvement of cerebral microcirculation due to the enhanced vasorelaxation, beneficial effects on blood viscosity, attenuation of the endothelial dysfunction, and antioxidant/antiradical IQ-1S activity.Item Neutrophil Immunomodulatory Activity of Natural Organosulfur Compounds(2019-05-19) Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrie I.; Balasubramanian, Ganesh; Quinn, Mark T.Organosulfur compounds are bioactive components of garlic essential oil (EO), mustard oil, Ferula EOs, asafoetida, and other plant and food extracts. Traditionally, garlic (Allium sativum) is used to boost the immune system; however, the mechanisms involved in the putative immunomodulatory effects of garlic are unknown. We investigated the effects of garlic EO and 22 organosulfur compounds on human neutrophil responses. Garlic EO, allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC) directly activated Ca2+ flux in neutrophils, with the most potent being AITC. Although 1,3-dithiane did not activate neutrophil Ca2+ flux, this minor constituent of garlic EO stimulated neutrophil reactive oxygen species (ROS) production. In contrast, a close analog (1,4-dithiane) was unable to activate neutrophil ROS production. Although 1,3-dithiane-1-oxide also stimulated neutrophil ROS production, only traces of this oxidation product were generated after a 5 h treatment of HL60 cells with 1,3-dithiane. Evaluation of several phosphatidylinositol-3 kinase (PI3K) inhibitors with different subtype specificities (A-66, TGX 221, AS605240, and PI 3065) showed that the PI3K p110δ inhibitor PI 3065 was the most potent inhibitor of 1,3-dithiane-induced neutrophil ROS production. Furthermore, 1,3-dithiane enhanced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), glycogen synthase kinase 3 α/β (GSK-3α/β), and cAMP response element binding (CREB) protein in differentiated neutrophil-like HL60 cells. Density functional theory (DFT) calculations confirmed the reactivity of 1,3-dithiane vs. 1,4-dithiane, based on the frontier molecular orbital analysis. Our results demonstrate that certain organosulfur compounds can activate neutrophil functional activity and may serve as biological response modifiers by augmenting phagocyte functions.Item Efficacy and Safety of Immuno-Magnetically Sorted Smooth Muscle Progenitor Cells Derived from Human-Induced Pluripotent Stem Cells for Restoring Urethral Sphincter Function(2017-04) Li, Yanhui; Green, Morgaine; Wen, Yan; Wei, Yi; Wani, Prachi; Wang, Zhe; Reijo Pera, Renee A.; Chen, BerthaHuman-induced pluripotent stem cells (hiPSCs)-based cell therapy holds promise for treating stress urinary incontinence (SUI). However, safety concerns, especially tumorgenic potential of residual undifferentiated cells in hiPSC derivatives, are major barriers for its clinical translation. An efficient, fast and clinical-scale strategy for purifying committed cells is also required. Our previous studies demonstrated the regenerative effects of hiPSC-derived smooth muscle progenitor cells (pSMCs) on the injured urethral sphincter in SUI, but the differentiation protocol required fluorescence-activated cell sorting (FACS) which is not practical for autologous clinical applications. In this study, we examined the efficacy and safety of hiPSC-derived pSMC populations sorted by FDA-approved magnetic-activated cell sorting (MACS) using cell-surface marker CD34 for restoring urethral sphincter function. Although the heterogeneity of MACS-sorted pSMCs was higher than that of FACS-sorted pSMCs, the percentage of undifferentiated cells dramatically decreased after directed differentiation in vitro. In vivo studies demonstrated long-term cell integration and no tumor formation of MACS-sorted pSMCs after transplantation. Furthermore, transplantation of MACS-sorted pSMCs into immunodeficient SUI rats was comparable to transplantation with FACS-sorted pSMCs for restoration of the extracellular matrix metabolism and function of the urethral sphincter. In summary, purification of hiPSC derivatives using MACS sorting for CD34 expression represent an efficient approach for production of clinical-scale pSMCs for autologous stem cell therapy for regeneration of smooth muscle tissues.