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  • Open access
  • 108 Reads
Concentration of short chain fatty acids produced by gut microbiota are related with cognitive dysfunction in a murine model of Alzheimer´s disease

Introduction. Alzheimer´s disease (AD) is the most common dementia in aged population and is characterized by cognitive impairment and dysbiosis, not to mention amyloid plaques, neurofibrillary tangles and reactive gliosis as the main hallmarks in the brain. Currently, there is not an effective treatment against this condition. Recent reports have suggested an elevated abundance of pro-inflammatory bacteria in the gut microbiota (GM) of transgenic (Tg) AD models compared to wild type (WT) animals. Moreover, the short chain fatty acids (SCFAs) propionate and butyrate, produced by GM, have been reported as pro- and anti-inflammatory species respectively. Methods. In order to determine changes in GM diversity and SCFAs concentration related with cognitive impairment in AD pathology, we used 6 months-old WT and AD Tg male mice to evaluate working and spatial memory status utilizing T- and water-maze paradigms respectively, SCFAs (acetate, propionate and butyrate) concentration in feces using high performance liquid chromatography (HPLC), and GM diversity, enrichment and functional predictive metabolism by DNA sequencing. Results. We observed increases in the proportion of lactic acid bacterial taxa, and also in the concentration, proportion and predictive metabolism of propionate for Tg compared to WT mice. Acetate and butyrate fecal amounts, in addition to working and spatial memory scores, were decreased in Tg animals. Conclusions. Our data suggests lactic acid bacterial increases, acetate and butyrate reductions, and an enhanced propionate metabolism and production could be related with AD pathology. Further studies are need in order to attribute cognitive dysfunctions to these changes.

  • Open access
  • 42 Reads
Subtypes of NanS-p sialate O-acetylesterase encoded by Stx2a bacteriophages
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Shiga toxin (Stx)-producing Escherichia coli strains are foodborne pathogens that can cause severe human diseases, such as haemorrhagic colitis and haemolytic-uraemic syndrome. Stxs are encoded by bacteriophages (Stx phages) which show remarkable variations in genome composition and harbour several genes of unknown function. Recently, a gene encoding a sialate O-acetylesterase (NanS-p) was identified in some relevant Stx2a phages and it was suggested that it could provide advantages for bacterial growth in the gut. The aim of this study was to analyze the presence and sequence of nanS-p genes in available Stx2a genomes. A total of 59 DNA sequences of Stx2a phages were extracted from NCBI GenBank database with the BLASTN program using the stx2a sequence from the phage 933W as query sequence, either as complete phage genomes (45) or from bacterial genomes by subsequent analysis with PHASTER web server (14). Comparative analysis revealed that nanS-p was located downsteam stx2a in all genomes. Twenty different amino acid sequences of NanS-p were identified among the 59 Stx2a phages. Specifically, catalytic esterase domains were clustered in 11 groups, with differences mainly observed in nine amino acid positions. Sequences corresponding to the N-terminal domain (DUF1737) clustered into three groups, two of them closely related, while C-terminal domain was highly variable giving place to four groups. Since sialate O-acetylesterase activity has been determined from particular Stx2a phages, new studies are necessary to evaluate if the NanS-p subtypes identified in the present study also differ in their biological activity.

  • Open access
  • 56 Reads
Structural studies of a fungal polyphenol oxidase with application to bioremediation of contaminated water

Polyphenol oxidases (PPOs) are a group of Cu-containing enzymes distributed from bacteria to humans, exhibiting two activities, catechol oxidase and tyrosinase. However, their precise mechanism of action and the structural elements that determine the distinction between the two activities are yet to be fully understood. In nature, PPOs catalyse the oxidation of several phenols to o-quinones, considerably affecting colour, flavour, nutritional properties, shelf life and therefore market values of numerous vegetables and fruits. On the other hand, PPOs have been widely employed as biocatalysts in many reactions, with a vast spectrum of applications in food, pharmaceutical, and cosmetic industries. One of the most important although least studied application of PPOs is as sensitive detectors of phenol derivatives in polluted waters as well as efficient tools towards biodegradation of these substances.

In a previous work, the use of a PPO from the thermophilic fungus Thermothelomyces thermophila (TtPPO), for the degradation of chlorophenols (CPs), contagious by-products of various pesticides, was presented and evaluated. Based on a homology model and available literature on PPO structure-function relations, point mutations were designed, that led to TtPPO variants with altered activity. In order to shed light on the structure-function relations of TtPPO, we solved the structure of specific TtPPO mutant (G292N) (PDB code 6Z1S). Unfortunately, subsequent efforts to determine TtPPO structure in complex with various ligands or substrate analogues have not been yet successful.

The present work attempts to shed light on the structural determinants of TtPPO function, by performing protein-ligand docking experiments via YASARA software. The docking results are compared with the biochemical data, and the role of specific aminoacids in TtPPO function is discussed. Observations concerning the binding of the different substrates to the active site of the enzyme, i.e the identification of the amino acids involved in this process, extracted form high-resolution structural models, would allow for structure-based design and production of a more potent biocatalyst for the bioremediation of CPs, providing an economic, effective and sustainable tool for wastewater treatment.

  • Open access
  • 88 Reads
Lactococcus lactis RBT18: from the rainbow trout farm to the lab, the tale of a nisin Z producer

Infectious diseases, such as lactococcosis caused by Lactococcus garvieae, are portrayed as critical limiting factors in aquaculture. The antimicrobial properties of Lactic Acid Bacteria (LAB), mainly the production of organic acids and bacteriocins (e. g., the lanthionine containing nisins A and Z; NisA and NisZ, respectively), led to propose LAB as probiotics to be used as an alternative and/or complementary strategy to vaccination and chemotherapy in aquaculture. L. lactis RBT18, isolated from cultured rainbow trout, exerts a strong direct and extracellular antimicrobial activity against L. garvieae and other ichthyopathogens, being the latter heat-resistant (100ºC, 10 min), and thus suggesting the involvement of a thermostable antimicrobial compound (i. e., bacteriocin). Cross-immunity tests using the agar-well-diffusion test (ADT) and PCR assays suggested that NisA/Z is the bacteriocin responsible for the extracellular antimicrobial activity exerted by L. lactis RBT18. To demonstrate this hypothesis, the bacteriocin was purified to homogeneity by two multi-chromatographic procedures. MALDI TOF-MS analyses of purified samples after the last reverse-phase chromatography step identified the presence of NisZ (3,330 Da), and its oxidized form (3,346 Da), derived from the oxidation of a lanthionine ring. The oxidized NisZ showed a diminished antimicrobial activity which would increase the chances of bacterial pathogens to evade its antimicrobial activity. Further experiments are necessary to assess the in vitro and in vivo safety and efficiency of L. lactis RBT18 as probiotic in aquaculture, but also to optimize the environmental conditions to reduce bacteriocin oxidation and thus bacterial pathogen resistance.

  • Open access
  • 46 Reads
Characterization of Methanosarcina mazei JL01 isolated from Holocene Arctic Permafrost and study of the archaeon cooperation with bacterium Sphaerochaeta associata GLS2T
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Methanogens are the unique and only group of microorganisms responsible for the generation of more than 90% of Earth`s methane. In terms of the necessary of the biological processes, these archaea use very small range of substrates but they are so adoptable that can be found in various anoxic habitats, even in extreme environments. Research of Arctic permafrost revealed the presence of viable methanogenic archaea, what may lead to the increase of methane flux in permafrost in the future. Several investigations on the CH4 emissions and the methanogenic community of Arctic permanently frozen grounds of different ages have been conducted previously (Rivkina et al., 2007; Wagner et al., 2013; Shcherbakova et al., 2016). The use of cultivated methods allowed to identify and describe new methanogenic species of the genera Methanosarcina and Methanobacterium in the Pliocene and Pleistocene permafrost, which are responsible for the formation of methane under extreme subzero conditions (Rivkina et al., 2004; Rivkina et al., 2007; Krivushin et al., 2010; Shcherbakova et al., 2011).

Tied methane-producing binary culture JL01 was isolated from 2.0-m-deep Holocene permafrost (Kolyma lowland, Russia) after long-term cultivation at 15°C. Using molecular techniques and repeated transfers in the presence of various antibiotics the binary culture was divided into archaeal (JL01) and bacterial (GLS2) strains. Later the bacterium was described as a novel spherical spirochaete Sphaerochaeta associata GLS2T (Troshina et al., 2015).

Preliminary studies of strain JL01 have shown that it was strictly anaerobic, nonmotile methanogenic аrchaeon which cells were organized into aggregates and had a typical appearance for Methanosarcina species. Strain JL01 cell wall corresponded to the Gram-positive type. Methane production was observed under the temperature from 10 to 37oC, pH 5.5-9.0 and NaCl concentration from 0.9 to 10 g L-1. The most suitable substrates for strain JL01 growth were methanol and trimethylamine. The genome sequence was determined in the Centre for Genomic Regulation (Barcelona, Spain). Genome was assembled from 323 contigs. The sequence total and ungapped lengths were 4,186,733 bp and 4,127,022 bp, respectively. GC content was 41.59%. A comparison of the genome sequences of our strain and Methanosarcina mazei S-6T (ANI 98.5%) showed that the Arctic isolate belonged to the species M. mazei despite significant phenotypic differences between strain JL01 and its closest relative M. mazei S-6T.

This study comprises the characterization of methanogenic archaeon strain JL01 and the data of the long-term cooperation with saccharolytic bacterial partner S. associata GLS2T. Supported by genomic data this finding confirm a possibility of methane production in this cooperation without any additional carbon and energy sources.

The genome analysis of S. associata GLS2T shows a particular adaptation of this bacterium towards methanochondroitin matrix of M. mazei JL01 due to the presence of necessary genes responsible for various carbohydrates. It is possible that such cooperation between the methanogen and the bacterium, that we observed in our experiments, allows the participants to carry out a continuous process of decomposition of methanochondroitin of dead methanogen cells to form acetate (Troshina et al., 2015), the substrate for methanogenesis. The analysis of genomes of Sphaerochaeta spp. showed the absence of several genes coding for vitamin B12 synthesis. Methane producing archaea can produce its precursors (Zhang et al., 2008, Caro-Quintero et al., 2012) and can supply them to the bacterium.

Thus, we hypothesize that such interaction allows partners to stay viable and active in the permafrost condition.

  • Open access
  • 89 Reads
New bacterial strains that destroy aromatic compounds act as antagonists of highly active phytopathogens

The growth of the world's population sets the task of providing people with quality food. Getting a high yield of plants is impossible without the use of fertilizers and plant protection products. However, the use of a large number of chemical compounds leads to their excessive accumulation in plants and can have a negative impact on human health. On the other hand, compounds used for multidirectional treatment of plants partially enter the soil, penetrate into the groundwater, which ultimately affects the quality of natural waters and bottom sediments. Therefore, the primary task is to intensify agricultural production without causing additional damage to the environment. This problem can be partially solved using microorganisms with target properties. For example, microorganisms that increase the bioavailability of nutrients that have phytostimulating properties, have antifungal, insecticidal, acaricidal effects and, at the same time, decompose pesticide residues. Microorganisms that combine several useful traits are especially valuable.

Thphe aim of this work was to search for new microbial strains that are applicable for agricultural production and possess a complex technologically significant potential.

30 strains of soil organisms were isolated from soil samples of the central chernozem region of Russia. The primary selection of strains was performed by direct plating on an agar mineral medium containing sodium benzoate as the only source of carbon and energy. We proceeded from the assumption that the ability to degrade benzoate can serve as a criterion for the presence of a minimum biodegradative potential for two reasons. Despite the relatively simple structure of the benzoate molecule, the biodegradation of this compound presupposes the presence of specialized biodegradation pathways and genes. Bacterial strains capable of degrading benzoate are, to varying degrees, capable of degrading other pollutants. It is important to emphasize that the isolation of soil microorganisms on a mineral medium with benzoate made it possible to quickly assess the presence of one of two biodegradative pathways - ortho- or meta-cleavage of catechol. The data obtained did not allow us to detect strains decomposing benzoate via the pathway of meta-cleavage, since there was no yellow coloration of the medium, characteristic of 2-hydroxymuconic semialdehyde formed as a result of meta-cleavage of catechol, a product of benzoate biodegradation. Thus, it was shown that the metabolism of benzoate in the isolated strains occurs with the induction of catechol 1,2-dioxygenase.

Some of the isolated strains showed high antagonistic activity against fungi-phytopathogens. The new Pseudomonas strain showed high activity. This strain completely inhibited the growth of the fungus Fusarium graminearum, preventing the phytopathogen from developing within a month of co-cultivation. The ability of this strain to inhibit the growth of the bacterial phytopathogen Ralstonia sp. 7-1 should be specially noted. Thus, a number of bacterial strains have been identified that are promising for use in plant protection technologies.

Among the highlighted ones, a new bacterial strain Bacillus sp. This strain is a spore-forming culture and is distinguished by an unusual morphology and development cycle of gram-positive cells. During growth, the strain forms chains of cells of irregular shape, which on the first day of growth on rich nutrient media are filled with multiple lipid granules of unknown nature. Of particular interest is the ability of one of the cells in the chain to divide, forming a helical cell form, which is further split into multiple small irregular cell forms.

Thus, the study of the microbial diversity of chernozems made it possible to identify strains potentially significant for biotechnology, combining such important properties as activity against phytopathogens and decomposition of pollutants, which makes it possible to develop biopreparations for multipurpose purposes. In addition, the isolation of new, previously undescribed bacteria significantly expands the understanding of microbial biodiversity.

The reported study was funded by RFBR according to the research project № 19-54-80003.

  • Open access
  • 363 Reads
Test for the production and assay of the enzymatic activity of bacterial and Archean halophilic proteolytic strains isolated from Algerian hypersaline environments

The present work was carried out on 133 halophilic strains, isolated on MGM medium at 12 and 23% (w / v) of salt. A screening of the extracellular proteolytic activities, carried out on the same medium supplemented with casein or gelatin at 1% (w / v), allowed us to select 24 bacterial strains and 21 Archean strains presenting a precipitate around the colonies for casein and / or a translucent halo (after addition of Frazier's reagent) for the gelatin. The enzymatic test was done on liquid medium in micro-culture on a 2 mL Eppendorf tube. The assay of the proteolytic activity using Azocasein as substrate, following 2 protocols the first with PBS and the second with tris HCl, with positive and negative controls, demonstrated interesting results for 10 strains among the 45 tested including 5 bacteria and 5 archaea. These have undergone morphological, physiological and molecular characterization based on amplification and sequencing of the 16S ribosomal RNA gene.

  • Open access
  • 117 Reads
Antibacterial Properties of Cymbopogon martinii essential Oil against Bacillus subtillis food industry pathogen
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Essential oils have great potential in the field of food industry as they effectively prevent the presence of several bacterial and fungal pathogens. Essential oils are complex volatile compounds, synthesized naturally in different plant parts during the process of secondary metabolism. The main goal of this work was to made a qualitative evaluation antibacterial properties of 24 chemotyped essential oils against the growth of Bacillus subtillis. This gram positive bacteria is responsible for "rope" disease in bread preservation processes.

The study was carried out using disk-diffusion in agar method. Biological activity is observed in five essential oils from Cymbopogon Martinii var. motia, Thymus vulgaris QT Linanol, Thymus satureioides, Mentha piperita and Eugenia caryophyllus. The first three have in common some terpenic derivatives: Geraniol, Linalool and Carvacrol.

The Cymbopogon Martinii essential oil, is one of the botanicals with the highest geraniol content (up to 93%), showed more activity antimicrobial.

A contributing role of this knowledge could be the design of Cymbopogon Martinii essential oil formula, can be used in bakery industry as preservative, such as nano-encapsulation for bakery doughs, active packaging of baked products or surface disinfectants.

  • Open access
  • 123 Reads
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Background: Probiotics are a live microbial supplement which improves hosts health by maintaining intestinal microbiota. Lactic acid bacteria strains have been extensively used as probiotics. The health benefits it provides are prevention of gastrointestinal problems, improving host immunity, reducing blood cholesterol level, lowering blood pressure, suppressing pathogenic microorganisms etc. This evidence suggests that probiotics can be used as a therapeutic strategy to improve overall digestive health.

Method: The aim of the present study was to isolate lactic acid bacteria with probiotic potential from food samples. Probiotic properties such as tolerance to low pH, bile, sodium chloride, lysozyme, antibiotic susceptibility, cell surface hydrophobicity and antimicrobial activity were determined.

Results: Ten different isolates were examined to study their probiotic potential. In this study, Lactobacillus brevis was isolated, it showed most of the probiotic properties like 10% sodium chloride tolerance, 1% bile tolerance, growth in pH 2 and antimicrobial activity against E. coli, S. aureus, K. pneumoniae, and P. aeruginosa. Formation of biofilm by Klebsiella pneumoniae, and Pseudomonas aeruginosa was also inhibited by cell free extracts of L. brevis which reveals its therapeutic relevance. Also, it was found to be stable at low temperature (4°C).

Conclusion: Based on the above-mentioned results of L. brevis it suggests that it has promising potential to be considered as ‘Probiotic’. Further in vivo assessments could be carried out which will provide its dual role of prevention as well as use in therapy.

  • Open access
  • 171 Reads
Allium cepa L. seed inoculation with a consortium of plant growth-promoting bacteria: effects on plant growth and development and soil fertility status and microbial community

The present work was aimed at investigating the effects of a four strains consortium – Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, and Burkholderia ambifaria – on crops of Allium cepa L. and soil health. The bacterial consortium was inoculated on onion seeds of two different varieties; inoculated seeds and control ones (treated with autoclaved inoculum) were sown in open-field and followed until harvest. Plant growth development parameters, as well as soil chemical and molecular profiles (DNA extraction and 16S community sequencing on the Mi-Seq Illumina platform), were investigated. The results showed a positive influence of bacterial application on plant growth, with increased plant height (+ 18%), total chlorophylls (+ 42%), crop yields (+ 13 %) and bulbs dry matter (+ 3%) than the control. The differences between control and treated experimental conditions were also underlined in the bulb extracts in terms of total phenolic contents (+25%) and antioxidant activities (+20%). Soil fertility and microbial community structure and diversity were also positively affected by bacterial presence. At harvest, the soil with the presence of bacterial consortium showed increased total organic carbon, organic matter and available P and higher concentrations of nutrients than control. The ecological indexes calculated on molecular profiles showed that community diversity was positively affected by the bacterial treatment. The present work allowed to remark the effective use of plant growth-promoting bacteria as valid fertilization strategy to improve yield in productive landscapes, whilst safeguarding soil biodiversity.

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