Development and application of aerobic, chemically defined media for Dysgonomonas.

Members of Dysgonomonas are Gram-stain-negative, non-motile, facultatively anaerobic coccobacilli originally described in relation to their isolation from stool and wounds of human patients (CDC group DF-3). More recently, Dysgonomonas have been found to be widely distributed in terrestrial environments and are particularly enriched in insect systems. Their prevalence in xylophagous insects such as termites and wood-feeding cockroaches, as well as in soil-fed microbial fuel cells, elicit interest in lignocellulose degradation and biofuel production, respectively. Their occurrence in mosquito and fruit fly have implications relating to symbiosis, host immunology and developmental biology. Additionally, their presence in termite, mosquito and nematode present novel opportunities for pest and vector control. Currently, the absolute growth requirements of Dysgonomonas are unknown, and they are commonly cultured under anaerobic conditions on complex media containing blood, peptones, tryptones, and yeast, plant or meat extracts. Restrictive and undefined culturing conditions preclude physiological and genetic studies, and thus further understanding of their metabolic potential. Here we describe the requirements for growth of termite-derived Dysgonomonas isolates and create parallel complex, defined and minimal media that permit vigorous and reliable aerobic growth. Furthermore, we show that these media can be used to easily enrich for Dysgonomonas isolates from densely-colonized and microbially-diverse environmental samples.

Pickering emulsions stabilized nanocellulosic-based nanoparticles for coumarin and curcumin nanoencapsulations: In vitro release, anticancer and antimicrobial activities.

Coumarin and curcumin have a wide spectrum of biological and pharmacological activities including antioxidant, anti-inflammatory, antimicrobial and anticancer but hindered therapeutic applications due to low stability and poor solubility in water. The main objective of the current study was to overcome these drawbacks via improved bioavailability by nanoencapsulated emulsions. Pickering emulsion (PE) via oil-in-water approach were stabilized by aminated nanocellulose (ANC) particles through application of a full factorial optimization design for nanoemulsions containing different composition of oil phase with medium chain triglyceride (MCT) and Tween 80. The fabricated nanoemulsions and PEs with average particle sizes (≤150 nm) were obtained. Influencing factors such as ANC concentration, storage time and pH on the stability of emulsions were examined alongside zeta potentials. Encapsulation efficiency (EE) of coumarin and curcumin were determined as >90%. Release kinetic profiles for encapsulated PEs displayed sustained release with supposed increase bioavailability. Higher release percent were detected for curcumin encapsulated PE in contrast to coumarin. In vitro cytotoxicity evaluation for coumarin and curcumin loaded PEs were further investigated for anticancer and antimicrobial activities using human cell lines (L929 and MCF-7) and different microorganisms (Gram (+), Gram (-) and fungi), respectively. The results clearly demonstrated PE coumarin and curcumin as promising candidates to inhibit microbial growth and to prevent preferential killing of cancer cells compared to normal cells.

Non-covalent interaction between CA-TAT and calf thymus DNA: Deciphering the binding mode by in vitro studies.

CA-TAT, a novel peptide analog, was modified at the N-terminus of TAT (47-57), the cell-penetrating peptide transacting activator of transcription, by attaching cecropin A (1-7). CA-TAT, TAT (47-57), and cecropin A (1-7) were synthesized using standard Fmoc solid-phase peptide synthesis procedures, purified using reversed-phase high performance liquid chromatography (RP-HPLC), and characterized using ESI-MS. CA-TAT demonstrated antibacterial activities against bacteria with low hemolysis (MHC > 128 µM). The minimum inhibitory concentration (MIC) values of CA-TAT were in the range of 1-16 µM, which completely inhibited both gram-positive and gram-negative bacteria. The interactions between CA-TAT or TAT (47-57) and calf thymus DNA (ct-DNA) were investigated using multi-spectroscopic techniques and viscometry. The results showed that both CA-TAT and TAT (47-57) can interact with DNA via the minor groove-binding mode, and binding constant was calculated to be 2.83 × 105 L mol-1 at 310 K, which is lower than that with the classical intercalation binder ethidium bromide (EB). Compared with TAT (47-57) or cecropin A (1-7), CA-TAT combined with DNA much closer. The study results suggest that CA-TAT can be used as a novel antibacterial peptide in the development of new antibiotics because of its antibacterial activity that targets intracellular DNA.

Synthesis and antibacterial activity of silver@carbon nanocomposites.

In this work, hollow multiple-Ag-nanoclustes- C-shell nanocomposites (Ag@C) were synthesized by using silane coupling agent to graft carbon dots (CDs) with silver nanoparticles (AgNPs). CDs act as coating and stabilizing agent, protecting AgNPs from aggregation and oxidation. The resulting Ag@C nanocomposites demonstrate strong bactericidal effect against both gram-negative and gram-positive bacteria in the disk diffusion test. Cellular toxicity evaluation was performed using MTT assay. Meanwhile, the as-prepared Ag@C nanocomposites show a good biocompatibility.

Agricultural anaerobic digestion power plants in Ireland and Germany: policy and practice.

The process of anaerobic digestion (AD) is valued as a carbon-neutral energy source, while simultaneously treating organic waste, making it safer for disposal or use as a fertilizer on agricultural land. The AD process in many European nations, such as Germany, has grown from use of small, localized digesters to the operation of large-scale treatment facilities, which contribute significantly to national renewable energy quotas. However, these large AD plants are costly to run and demand intensive farming of energy crops for feedstock. Current policy in Germany has transitioned to support funding for smaller digesters, while also limiting the use of energy crops. AD within Ireland, as a new technology, is affected by ambiguous governmental policies concerning waste and energy. A clear governmental strategy supporting on-site AD processing of agricultural waste will significantly reduce Ireland's carbon footprint, improve the safety and bioavailability of agricultural waste, and provide an indigenous renewable energy source. © 2016 Society of Chemical Industry.

In-vitro activity of solithromycin against anaerobic bacteria from the normal intestinal microbiota.

Solithromycin is a novel fluoroketolide with high activity against bacteria associated with community-acquired respiratory tract infections as well as gonorrhea. However, data on the activity of solithromycin against anaerobic bacteria from the normal intestinal microbiota are scarce. In this study, 1024 Gram-positive and Gram-negative anaerobic isolates from the normal intestinal microbiota were analyzed for in-vitro susceptibility against solithromycin and compared to azithromycin, amoxicillin/clavulanic acid, ceftriaxone, metronidazole and levofloxacin by determining the minimum inhibitory concentration (MIC). Solithromycin was active against Bifidobacteria (MIC50, 0.008 mg/L) and Lactobacilli (MIC50, 0.008 mg/L). The MIC50 for Clostridia, Bacteroides, Prevotella and Veillonella were 0.5, 0.5, 0.125 and 0.016 mg/L, respectively. Gram-positive anaerobes were more susceptible to solithromycin as compared to the other antimicrobials tested. The activity of solithromycin against Gram-negative anaerobes was equal or higher as compared to other tested agents.

Microbial community structure and dynamics in two-stage vs single-stage thermophilic anaerobic digestion of mixed swine slurry and market bio-waste.

The microbial community of a thermophilic two-stage process was monitored during two-months operation and compared to a conventional single-stage process. Qualitative and quantitative microbial dynamics were analysed by Denaturing Gradient Gel Electrophoresis (DGGE) and real-time PCR techniques, respectively. The bacterial community was dominated by heat-shock resistant, spore-forming clostridia in the two-stage process, whereas a more diverse and dynamic community (Firmicutes, Bacteroidetes, Synergistes) was observed in the single-stage process. A significant evolution of bacterial community occurred over time in the acidogenic phase of the two-phase process with the selection of few dominant species associated to stable hydrogen production. The archaeal community, dominated by the acetoclastic Methanosarcinales in both methanogen reactors, showed a significant diversity change in the single-stage process after a period of adaptation to the feeding conditions, compared to a constant stability in the methanogenic reactor of the two-stage process. The more diverse and dynamic bacterial and archaeal community of single-stage process compared to the two-stage process accounted for the best degradation activity, and consequently the best performance, in this reactor. The microbiological perspective proved a useful tool for a better understanding and comparison of anaerobic digestion processes.

Biological sulfate reduction in the acidogenic phase of anaerobic digestion under dissimilatory Fe (III)--reducing conditions.

In this study, a novel approach was developed for sulfate - containing wastewater treatment via dosing Fe2O3 in a two - stage anaerobic reactor (A1, S1). The addition of Fe2O3 in its second stage i.e. acidogenic sulfate-reducing reactor (S1) resulted in microbial reduction of Fe (III), which significantly enhanced the biological sulfate reduction. In reactor S1, increasing influent sulfate concentration to 1400 mg/L resulted in a higher COD removal (27.3%) and sulfate reduction (57.9%). In the reference reactor without using Fe2O3 (S2), the COD and sulfate removal were 15.6% and 29%, respectively. The combined performance of the two-stage anaerobic reactor (A1, S1) also showed a higher COD removal of 74.2%. Denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis showed that the dominant bacteria with high similarity to IRB species as well as sulfate reducer Desulfovibrio and acidogenic bacteria (AB) were enriched in S1. Quantitative Polymerase Chain Reaction (qPCR) analysis presented a higher proportion of sulfate reducer Desulfovibrio marrakechensis and Fe (III) reducer Iron-reducing bacteria HN54 in S1.

Occurrence and fate of volatile siloxanes in a municipal wastewater treatment plant of Beijing, China.

The occurrence and fate of four cyclic and two linear volatile siloxanes were studied in a municipal Wastewater Treatment Plant (WWTP), Beijing City, China. Aqueous and sludge samples were analyzed by solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS). In the studied WWTP, four cyclic analogs (D3-D6) had significantly higher concentrations and frequencies than the two linear analogs [Octamethyltrisiloxane (L3) and Decamethyltetrasiloxane (L4)], with inputs into the WWTP ranging from 78.2 to 387.7 kg/year. Removal efficiencies of volatile cyclic siloxanes in two parallel secondary treatment processes ranged from 59.3 to 92.7%. For volatile cyclic siloxanes, relative fractions of mass loss by adsorption to sludge ranged from 8.3 to 53.0%, and their adsorption capacities were significantly affected by the dissolved organic matter. Besides adsorption, they were eliminated mainly in anaerobic units (44.4-84.3%). Through in vitro biodegradation experiments, we concluded that in the anaerobic compartments, Hexamethylcyclotrisiloxane (D3) and Dodecamethylcyclohexasiloxane (D6) were eliminated mostly by volatilization, while Octamethylcyclotetrasiloxane (D4) and Decamethylcyclopentasiloxane (D5) may be eliminated by both volatilization and degradation. Furthermore, microbe catalysis hydrolysis was identified as one of the main degradation pathways for D4 and D5 in anaerobic compartments.

Chicken egg yolk antibody (IgY) controls Solobacterium moorei under in vitro and in vivo conditions.

Solobacterium moorei is a causative agent in diseases such as oral halitosis, bacteremia, and necrobacillosis-associated thrombophlebitis. The objective of this study was to determine the effectiveness of chicken egg yolk antibody (IgY) in controlling S. moorei. Intact S. moorei cells were used as an immunogen to immunize four White Leghorn laying hens. IgY, extracted from egg yolks obtained from these immunized hens, was purified using water dilution, two-step salt precipitation, and ultrafiltration. The purity of the IgY obtained was approximately 87.3 %. The antibody titer of the IgY was determined by enzyme-linked immunosorbent assay. The antibody titer peaked at 10,000 following the third immunization. In order to evaluate the inhibitory effects of the specific IgY, the growth of S. moorei in liquid media was measured every 12 h using a microplate reader at 600 nm. Biofilm formation of S. moorei was quantified by staining with crystal violet. The specific binding ability of IgY was further confirmed by the use of immunofluorescence and immunoelectron microscopy. Growth and biofilm formation of S. moorei were significantly (P<0.05) inhibited by 20 and 40 mg/ml specific IgY compared with the control. The specific IgY also decreased the bacterial level in the oral cavity of mice after infection with S. moorei. This study demonstrates that the growth and biofilm formation of S. moorei can be effectively inhibited by specific IgY. As a result, IgY technology may have application in the control of diseases caused by S. moorei.

Oral colonization of Gram-negative anaerobes as a risk factor for preterm delivery.

BACKGROUND: Preterm birth significantly impacts on neonatal morbidity and mortality and is apparently increasing world-wide. Several studies have attempted to define a causative role for periodontal disease in adverse pregnancy outcomes but few have focused on the microbiology of periodontal disease in relation to these outcomes. RESULTS: The evidence for a positive correlation is strong, supported by microbiological and immunological findings. Conflicting results are often associated with uncontrolled confounding factors. MATERIALS AND METHODS: A literature search was conducted in order to establish whether or not a role exists for oral Gram-negative bacteria in adverse pregnancy outcomes. Association and intervention studies are summarized along with pathogenic potential of the Gram-negative bacteria most frequently implicated in periodontal disease.

Isolation and characterization of Thermanaerothrix daxensis gen. nov., sp. nov., a thermophilic anaerobic bacterium pertaining to the phylum "Chloroflexi", isolated from a deep hot aquifer in the Aquitaine Basin.

A new strictly anaerobic thermophilic multicellular filamentous bacterium (0.2-0.3µm×>100µm), designated GNS-1(T), was isolated from a deep hot aquifer in France. It was non-motile, and stained Gram-negative. Optimal growth was observed at 65°C, pH 7.0, and 2gL(-1) of NaCl. Strain GNS-1(T) was chemoorganotrophic fermenting ribose, glucose, galactose, arabinose, fructose, mannose, maltose, sucrose, xylose, raffinose, pyruvate, and xylan. Yeast extract was required for growth. The end products of glucose fermentation were lactate, acetate, CO(2), and H(2). The G+C content of the DNA was 57.6mol%. Its closest phylogenetic relative was Bellilinea caldifistulae with 92.5% similarity. Based on phylogenetic, genotypic and phenotypic characteristics, strain GNS-1(T) (DSM 23592(T), JCM 16980(T)) is proposed to be assigned to a novel species of a novel genus within the class Anaerolineae (subphylum I), phylum "Chloroflexi", Thermanaerothrix daxensis gen. nov., sp. nov. The GenBank accession number is HM596746.

Bacterial lifestyle in a deep-sea hydrothermal vent chimney revealed by the genome sequence of the thermophilic bacterium Deferribacter desulfuricans SSM1.

The complete genome sequence of the thermophilic sulphur-reducing bacterium, Deferribacter desulfuricans SMM1, isolated from a hydrothermal vent chimney has been determined. The genome comprises a single circular chromosome of 2,234,389 bp and a megaplasmid of 308,544 bp. Many genes encoded in the genome are most similar to the genes of sulphur- or sulphate-reducing bacterial species within Deltaproteobacteria. The reconstructed central metabolisms showed a heterotrophic lifestyle primarily driven by C1 to C3 organics, e.g. formate, acetate, and pyruvate, and also suggested that the inability of autotrophy via a reductive tricarboxylic acid cycle may be due to the lack of ATP-dependent citrate lyase. In addition, the genome encodes numerous genes for chemoreceptors, chemotaxis-like systems, and signal transduction machineries. These signalling networks may be linked to this bacterium's versatile energy metabolisms and may provide ecophysiological advantages for D. desulfuricans SSM1 thriving in the physically and chemically fluctuating environments near hydrothermal vents. This is the first genome sequence from the phylum Deferribacteres.

[Metabolism of the thermophilic bacterium Oceanithermus profundus].

The metabolism of the novel facultatively anaerobic thermophilic bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurs via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.

In vitro antimicrobial susceptibility of anaerobic bacteria isolated from caprine footrot.

The in vitro antimicrobial susceptibility of Gram-negative anaerobic bacilli commonly isolated from footrot in goats was studied. A total of 97 isolates belonging to the genera Dichelobacter, Fusobacterium, Prevotella, Porphyromonas and Bacteroides, obtained from clinical cases of footrot in south-western Spain between March 2000 and May 2001, were tested against 25 antimicrobial agents comprising beta-lactams, aminoglycosides, macrolides, chloramphenicol, quinolones, lincosamides, sulphonamides and tetracyclines in order to optimise antibiotic treatment of this disease in goats. beta-lactams, tetracyclines and metronidazole displayed the highest in vitro efficacy against the species involved in the pathogenesis of footrot.

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium Thermotoga hypogea.

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90 degrees C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO(2), H(2), L-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 +/- 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 +/- 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe(2+). The enzyme was thermostable with a half-life of about 10 h at 70 degrees C, and its catalytic activity increased along with the rise of temperature up to 95 degrees C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K (m) values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.

[Symbiotic microflora in fishes of different ecological groups].

Published data on the taxonomic composition of the intestinal bacterial flora in fishes living under different conditions (marine and freshwater fishes) and differing in the type of feeding are reviewed. The results confirm the conclusion that the composition of intestinal microflora differs depending on fish ecology. A limited number of bacterial taxa found in the intestines of some fish species may be evidence not only for a low diversity of these bacteria but also for insufficient knowledge about them. Considerable differences in the composition of intestinal microflora in marine and freshwater fishes are described.

Effect of fatty acids on growth of conjugated-linoleic-acids-producing bacteria in rumen.

Microorganisms with high activity of linoleic acid delta12-cis,delta11-trans-isomerase were isolated from the digestive tract of ruminants and characterized. The isolate with the highest isomerase activity was identified as Pseudobutyrivibrio ruminis. The susceptibility of this strain to 3 fatty acids added to the grow medium was determined. A significant inhibition of bacterial growth (during a 3-d period) by linoleic acid (0.1 %) and oleic acid (5 ppm) was observed; no inhibition was found in the presence of stearic acid.

Growth and pectate-lyase activity of the ruminal bacterium Lachnospira multiparus in the presence of short-chain organic acids.

AIMS: Acetic, propionic, butyric and lactic acids are end products of feed fermentation by rumen microbes. The effects of these short chain acids on growth and pectate-lyase (PL) activity of Lachnospira multiparus were studied. METHODS AND RESULTS: The bacterial strain used was L. multiparus D32. Acids were tested between 50 and 300 mmol l(-1). Growth and PL activity were measured by the increase in total protein content and by the increase in absorbance at 235 nm in the reaction medium respectively. With the exception of lactic acid, all acids decreased bacterial growth rates; generally, these effects were more pronounced at higher concentrations and with acids of longer chains. PL activity was inhibited by all the acids except by butyric acid at 50 and 100 mmol l(-1). Enzyme inhibition increased with the concentrations of the acids and lactic acid was the most inhibitory. CONCLUSIONS: High concentrations of short chain acids can differentially inhibit the growth rate and the PL activity of L. multiparus. SIGNIFICANCE AND IMPACT OF THE STUDY: Products of fermentation generated by the ruminal microbiota could modify the degradation of pectic substances by this bacterium.

Syntrophomonas erecta sp. nov., a novel anaerobe that syntrophically degrades short-chain fatty acids.

Two novel anaerobes, strains GB4-38(T) and SB9-1, were isolated from an upflow anaerobic sludge blanket reactor for treating bean-curd farm wastewater and lotus field mud, respectively. The strains degraded straight-chain fatty acids with 4-8 carbon atoms in syntrophic association with methanogens and converted 1 mol butyrate into about 2 mol acetate and presumably 2 mol H(2). None of the branched-chain fatty acids tested could be degraded. Benzoate was not degraded. Fumarate, sulfate, thiosulfate, sulfur and nitrate did not serve as electron acceptors for butyrate degradation. In the absence of a methanogen partner, strain GB4-38(T) grew on crotonate in pure culture; the generation time was about 5 h at 37 degrees C. However, strain SB9-1 grew on butyrate plus pentenoate, but not crotonate, in pure culture and the generation time was 18 h at 37 degrees C. Cells of GB4-38(T) and SB9-1 were straight rods and stained Gram-negative. The major cellular fatty acids of GB4-38(T) were C(14 : 0) (29.74 %), C(16 : 0) (17.00 %), C(16 : 1)omega5c (16.63 %) and isoC(17 : 1) I (15.34 %). ll-Diaminopimelic acid existed in the cellular peptidoglycan. The genomic DNA G+C content of strain GB4-38(T) was 43.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences supported clustering of the two strains with syntrophic bacterial species of the genus Syntrophomonas (89.6-92.4 % sequence similarity), but phenotypic, chemotaxonomic and genetic characters differentiated the two strains from members of this genus. Therefore, it is proposed that the two strains are representatives of a novel species, Syntrophomonas erecta sp. nov. The type strain is GB4-38(T) (=CGMCC 1.5013(T)=DSM 16215(T)).