The role of competition versus cooperation in microbial community coalescence

New microbial communities often arise through the mixing of two or more separately assembled parent communities, a phenomenon that has been termed “community coalescence”. Understanding how the interaction structures of complex parent communities determine the outcomes of coalescence events is an important challenge. While recent work has begun to elucidate the role of competition in coalescence, that of cooperation, a key interaction type commonly seen in microbial communities, is still largely unknown. Here, using a general consumer-resource model, we study the combined effects of competitive and cooperative interactions on the outcomes of coalescence events. To do so, we simulate coalescence events between pairs of communities with different degrees of competition for shared carbon resources and cooperation through cross-feeding on leaked metabolic by-products (facilitation). We also study how structural and functional properties of post-coalescence communities evolve when they are subjected to repeated coalescence events. We find that in coalescence events, the less competitive and more cooperative parent communities contribute a higher proportion of species to the new community because of their superior ability to deplete resources and resist invasions. Consequently, when a community is subjected to repeated coalescence events, it gradually evolves towards being less competitive and more cooperative, as well as more speciose, robust and efficient in resource use. Encounters between microbial communities are becoming increasingly frequent as a result of anthropogenic environmental change, and there is great interest in how the coalescence of microbial communities affects environmental and human health. Our study provides new insights into the mechanisms behind microbial community coalescence, and a framework to predict outcomes based on the interaction structures of parent communities.     

Stomach-Specific Controlled Release Gellan Beads of Acid-Soluble Drug Prepared by Ionotropic Gelation Method

The purpose of the present work was the development and evaluation of stomach-specific controlled release mucoadhesive drug delivery system prepared by ionotropic gelation of gellan beads, containing acid-soluble drug amoxicillin trihydrate, using 3² factorial design with concentration of gellan gum and quantity of drug as variables. The study showed that beads prepared in alkaline cross-linking medium have higher entrapment efficiency than the acidic cross-linking medium. The entrapment efficiency was in the range of 32% to 46% w/w in acidic medium, which increased up to 60% to 90% w/w in alkaline medium. Batches with lowest, medium, and highest drug entrapment were subjected to chitosan coating to form a polyelectrolyte complex film. As polymer concentration increases, entrapment efficiency and particle size increases. Scanning electron microscopy revealed spherical but rough surface due to leaching of drug in acidic cross-linking solution, dense spherical structure in alkaline cross-linking solution, and rough surface of chitosan-coated beads with minor wrinkles. The in vitro drug release up to 7 h in a controlled manner following the Peppas model (r = 0.9998). In vitro and in vivo mucoadhesivity study showed that beads have good mucoadhesivity and more than 85% beads remained adhered to stomach mucosa of albino rat even after 7 h. In vitro growth inhibition study showed complete eradication of Helicobacter pylori. These results indicate that stomach-specific controlled release mucoadhesive system of amoxicillin gellan beads may be useful in H. pylori treatment.     

Importance of flagella in acute and chronic Pseudomonas aeruginosa infections

Pseudomonas aeruginosa is an environmental microorganism and a causative agent of diverse acute and chronic, biofilm-associated infections. Advancing research-based knowledge on its adaptation to conditions within the human host is bound to reveal novel strategies and targets for therapeutic intervention. Here, we investigated the traits that P. aeruginosa PA14 as well as a virulence attenuated ΔlasR mutant need to survive in selected murine infection models. Experimentally, the genetic programs that the bacteria use to adapt to biofilm-associated versus acute infections were dissected by passaging transposon mutant libraries through mouse lungs (acute) or mouse tumours (biofilm-infection). Adaptive metabolic changes of P. aeruginosa were generally required during both infection processes. Counter-selection against flagella expression was observed during acute lung infections. Obviously, avoidance of flagella-mediated activation of host immunity is advantageous for the wildtype bacteria. For the ΔlasR mutant, loss of flagella did not confer a selective advantage. Apparently, other pathogenesis mechanisms are active in this virulence attenuated strain. In contrast, the infective process of P. aeruginosa in the chronic biofilm model apparently required expression of flagellin. Together, our findings imply that the host immune reactions against the infectious agent are very decisive for acuteness and duration of the infectious disease. They direct disease outcome.     

Targeting bioenergetics is key to counteracting the drug-tolerant state of biofilm-grown bacteria

Embedded in an extracellular matrix, biofilm-residing bacteria are protected from diverse physicochemical insults. In accordance, in the human host the general recalcitrance of biofilm-grown bacteria hinders successful eradication of chronic, biofilm-associated infections. In this study, we demonstrate that upon addition of promethazine, an FDA approved drug, antibiotic tolerance of in vitro biofilm-grown bacteria can be abolished. We show that following the addition of promethazine, diverse antibiotics are capable of efficiently killing biofilm-residing cells at minimal inhibitory concentrations. Synergistic effects could also be observed in a murine in vivo model system. PMZ was shown to increase membrane potential and interfere with bacterial respiration. Of note, antibiotic killing activity was elevated when PMZ was added to cells grown under environmental conditions that induce low intracellular proton levels. Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. Abrogation of antibiotic tolerance by interfering with the cell’s bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. Repurposing promethazine as a biofilm-sensitizing drug has the potential to accelerate the introduction of new treatments for recalcitrant, biofilm-associated infections into the clinic.     

Use of Hydrogenophaga pseudoflava Penetration To Quantitatively Assess the Impact of Filtration Parameters for 0.2-Micrometer-Pore-Size Filters

Filters rated as having a 0.2-μm pore size (0.2-μm-rated filters) are used in laboratory and manufacturing settings for diverse applications of bacterial and particle removal from process fluids, analytical test articles, and gasses. Using Hydrogenophaga pseudoflava, a diminutive bacterium with an unusual geometry (i.e., it is very thin), we evaluated passage through 0.2-μm-rated filters and the impact of filtration process parameters and bacterial challenge density. We show that consistent H. pseudoflava passage occurs through 0.2-μm-rated filters. This is in contrast to an absence of significant passage of nutritionally challenged bacteria that are of similar size (i.e., hydrodynamic diameter) but dissimilar geometry.The 0.2-μm-pore-size filter class (0.2-μm-rated filters) includes a large and diverse set of products (22). They include air filters, particle reduction filters, filters used for bioburden reduction, lab-grade filters, and “sterilizing-grade” filters used in sterile-dosage-form manufacture. ASTM F 838-05, the Brevundimonas diminuta challenge test, is a standard for the “sterilizing-grade” filters (4), a subset of the 0.2-μm-rated filters. The “0.2-μm” designation is applied to the larger and more diverse set of products. This designation is based on physical measurements (e.g., the bubble point, the force necessary to extrude air through the capillary network of a wet filter) and mathematical extrapolations (5, 14, 29).The current filter validation approach for parenteral pharmaceuticals involves a demonstration of removal of 7 log₁₀ CFU/cm² of nutritionally starved B. diminuta from bulk drug product liquids (4, 8, 11, 29). B. diminuta can penetrate 0.2-μm-rated filters, but only sporadically and at low levels (12, 21). Larger bacteria (Listeria monocytogenes) have been demonstrated to be able to penetrate 0.2-μm filters after long-term exposure (27). Recently, a species of small waterborne bacteria, Hydrogenophaga pseudoflava, has been shown to penetrate 0.2-μm-rated filters (31-36) to a greater extent than the above-described bacteria. None of these bacteria are actually physically smaller than 0.2 μm, even H. pseudoflava (25, 37, 38).Because H. pseudoflava penetrates 0.2-μm-rated filters in a potentially quantifiable manner, it can be used to study filtration efficiency. In this report, we evaluate the impact of filtration process parameters and bacterial challenge density on passage. We benchmark H. pseudoflava passage against that of nutritionally challenged bacteria which are of similar size (i.e., hydrodynamic diameter) but dissimilar geometry.     

Intramolecular reductive cyclization strategy to the synthesis of (-)-6-methyl-3-hydroxy-piperidine-2-carboxylic acid, (+)-6-methyl-(2-hydroxymethyl)-piperidine-3-ol and their glycosidase inhibitory activity

The first stereoselective synthesis of (2S,3R,6S)-6-methyl-3-hydroxy-piperidine-2-carboxylic acid (-)-6 and (2R,3R,6S)-6-methyl-(2-hydroxymethyl)-piperidine-3-ol (+)-7 was achieved starting from readily available d-glucose in 14 steps with 17% overall yield for both the compounds. The key feature of the present strategy includes the Wittig-olefination for the preparation of required conjugated keto-azide 9 and construction of 2,3,6-trisubstituted piperidine skeleton 11 by applying intramolecular reductive cyclization of conjugated keto-azide intermediate. The glycosidase inhibitory activity of compounds 6 and 7 towards several glycosidases has been evaluated.     

Search for novel antifungal agents by monitoring fungal metabolites in presence of synthetically designed fluconazole derivatives using NMR spectroscopy

Resistance to currently available antifungal drugs necessitates development of new drugs using rapid, robust and automated methods to test a large number of newly synthesized drugs in less time. We have compared the effect of ketoconazole, fluconazole and its synthesized analogues on Candida albicans ATCC 10231. A metabolic profile of C.albicans ATCC 10231 in presence of drugs has been compared using ¹H NMR. Signals from metabolites have been monitored with time. MIC determined using conventional methods has been compared with Metabolic End Point (MEP) obtained from NMR spectroscopy. Results indicate that the activity of the fluconazole derivatives is in the order fluconazole p-methoxybenzoate > fluconazole = fluconazole benzoate > fluconazole toluate > fluconazole p-nitrobenzoate.     

Larvicidal activity of bis(2-ethylhexyl) benzene-1,2-dicarboxylate from Sterculia guttata seeds against two mosquito species

The larvicidal activities of various fractions of the hexane extract of the seeds of Sterculia guttata against larvae of Aedes aegypti and Culex quinquefasciatus were determined. Bis(2-ethylhexyl) benzene-1,2-dicarboxylate (1) was identified as one of the active principles, displaying chronic toxicity against both types of larvae, with LD50 values of 79 and 64 ppm, respectively.     

Calmodulin binding to the Fas-mediated death-inducing signaling complex in cholangiocarcinoma cells

We have previously demonstrated that the antagonists of calmodulin (CaM) induce apoptosis of cholangiocarcinoma cells partially through Fas-mediated apoptosis pathways. Recently, CaM has been shown to bind to Fas, which is regulated during Fas or CaM antagonist-mediated apoptosis in Jurkat cells and osteoclasts. Accordingly, the present studies were designed to determine whether Fas interacts with CaM in cholangiocarcinoma cells and to elucidate its role in regulating Fas-mediated apoptosis. CaM bound to Fas in cholangiocarcinoma cells. CaM was identified in the Fas-mediated death inducing signaling complex (DISC). The amount of CaM recruited into the DISC was increased after Fas-stimulation, a finding confirmed by immunofluorescent analysis that demonstrated increased membrane co-localization of CaM and Fas upon Fas-stimulation. Consistently, increased Fas microaggregates in response to Fas-stimulation were found to bind to CaM. Fas-induced recruitment of CaM into the DISC was inhibited by the Ca(2+) chelator, EGTA, and the CaM antagonist, trifluoperazine (TFP). TFP decreased DISC-induced cleavage of caspase-8. Further, inhibition of actin polymerization, which has been demonstrated to abolish DISC formation, inhibited the recruitment of CaM into the DISC. These results suggest an important role of CaM in mediating DISC formation, thus regulating Fas-mediated apoptosis in cholangiocarcinoma cells. Characterization of the role of CaM in Fas-mediated DISC formation and apoptosis signaling may provide important insights in the development of novel therapeutic targets for cholangiocarcinoma.