Effect of the gradual increase of salt on stability and microbial diversity of granular sludge and ammonia removal.

Two sequential batch reactors were operated, aiming at forming aerobic granular sludge and studying the effects of the gradual increase of the NaCl concentration on the granule. structure and microbial diversity, and on the efficiency of ammonia removal. The reactors were fed with ammonia-enriched synthetic effluent and 5 g L-1 of NaCl per week were applied. A decrease in the size of the granules was observed until they were completely disintegrated as the salt concentration increased up to 10 g L-1. However, the ammonia removal efficiency remained high in all the salinities applied. By sequencing the 16S rRNA amplicon gene, the microbial community structure allowed the verification of the presence of several genera affiliated with the bacteria that perform both heterotrophic nitrification and aerobic denitrification, besides those involved in the conventional nitrification and denitrification and the ANAMMOX process. Salinity affected the microbial population related to the formation and stability of the granules.

Ammonium removal from high-salinity oilfield-produced water: assessing the microbial community dynamics at increasing salt concentrations.

Water generated during oil exploration is chemically complex and contains high concentrations of ammonium and, in some cases, high salinity. The most common way to remove ammonium from effluent is a biological process, which can be performed by different routes and different groups of microorganisms. However, the presence of salts in the effluents could be an inhibiting factor for biological processes, interfering directly with treatment. This study aimed to evaluate changes in the profile of a microbial community involved in the process of ammonium removal when subjected to a gradual increase of salt (NaCl), in which the complete inhibition of the ammonium removal process occurred at 125 g L-1 NaCl. During the sludge acclimatization process, samples were collected and submitted to denaturing gradient gel electrophoresis (DGGE) and massive sequencing of the 16S ribosomal RNA (rRNA) genes. As the salt concentration increased in the reactor, a change in the microbial community was observed by the DGGE band profiles. As a result, there was a reduction in the presence of bacterial populations, and an increase in archaeal populations was found. The sequencing data suggested that ammonium removal in the reactor was carried out by different metabolic routes by autotrophic nitrifying bacteria, such as Nitrosococcus, Nitrosomonas, Nitrosovibrio, Nitrospira, and Nitrococcus; ammonium-oxidizing archaea Candidatus nitrosoarchaeum; ANAMMOX microorganisms, such as Candidatus brocadia, Candidatus kuenenia, and Candidatus scalindua; and microorganisms with the potential to be heterotrophic nitrifying, such as Paracoccus spp., Pseudomonas spp., Bacillus spp., Marinobacter sp., and Alcaligenes spp.

Applying functional metagenomics to search for novel lignocellulosic enzymes in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure.

Environments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes ß-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three ß-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.

Endophytic and mycorrhizal fungi associated with roots of endangered native orchids from the Atlantic Forest, Brazil.

The composition and diversity of fungal communities associated with three endangered orchid species, Hadrolaelia jongheana, Hoffmannseggella caulescens, and Hoffmannseggella cinnabarina, found in different vegetation formations of the Atlantic Forest were determined by constructing clone libraries and by applying diversity and richness indices. Our results demonstrated the presence of Basidiomycetes. Sebacinales (81.61%) and Cantharellales (12.10%) were the dominant orders and are potential candidates for orchid mycorrhizal fungi. The Ascomycetes identified included the Helotiales (29.31%), Capnodiales (18.10%), and Sordariales (10.34%), among others. These orders may represent potentially endophytic fungi. A Shannon-Wiener diversity index (H') analysis showed a relatively high fungal community diversity associated with these tropical orchids. This diversity may offer greater flexibility in terms of the adaptation of the plants to changing environmental conditions and the potential facilitation of reintroduction programs. The Simpson diversity index values showed that all of the libraries included dominant species, and a LIBSHUFF analysis showed that the fungal communities were structurally different from each other, suggesting an influence of local factors on this diversity. This study offers important information for the development of conservation strategies for threatened and endemic species of Brazilian flora in an important and threatened hotspot.

Natural products as source of potential dengue antivirals.

Dengue is a neglected disease responsible for 22,000 deaths each year in areas where it is endemic. To date, there is no clinically approved dengue vaccine or antiviral for human beings, even though there have been great efforts to accomplish these goals. Several approaches have been used in the search for dengue antivirals such as screening of compounds against dengue virus enzymes and structure-based computational discovery. During the last decades, researchers have turned their attention to nature, trying to identify compounds that can be used as dengue antivirals. Nature represents a vast reservoir of substances that can be explored with the aim of discovering new leads that can be either used directly as pharmaceuticals or can serve as lead structures that can be optimized towards the development of new antiviral agents against dengue. In this review we describe an assortment of natural products that have been reported as possessing dengue antiviral activity. The natural products are organized into classes of substances. When appropriate, structure-activity relationships are outlined. The biological assays used to assess antiviral activity are briefly described.

Melipona stingless bees and honey microbiota reveal the diversity, composition, and modes of symbionts transmission.

The Melipona gut microbiota differs from other social bees, being characterized by the absence of crucial corbiculate core gut symbionts and a high occurrence of environmental strains. We studied the microbial diversity and composition of three Melipona species and their honey to understand which strains are obtained by horizontal transmission (HT) from the pollination environment, represent symbionts with HT from the hive/food stores or social transmission (ST) between nestmates. Bees harbored higher microbial alpha diversity and a different and more species-specific bacterial composition than honey. The fungal communities of bee and honey samples are also different but less dissimilar. As expected, the eusocial corbiculate core symbionts Snodgrassella and Gilliamella were absent in bees that had a prevalence of Lactobacillaceae - including Lactobacillus (formerly known as Firm-5), Bifidobacteriaceae, Acetobacteraceae, and Streptococcaceae - mainly strains close to Floricoccus, a putative novel symbiont acquired from flowers. They might have co-evolved with these bees via ST, and along with environmental Lactobacillaceae and Pectinatus (Veillonellaceae) strains obtained by HT, and Metschnikowia and Saccharomycetales yeasts acquired by HT from honey or the pollination environment, including plants/flowers, possibly compose the Melipona core microbiota. This work contributes to the understanding of Melipona symbionts and their modes of transmission.

Extinction of anciently associated gut bacterial symbionts in a clade of stingless bees.

Animal-microbe symbioses are often stable for millions of years. An example is the clade consisting of social corbiculate bees-honeybees, bumblebees, and stingless bees-in which a shared ancestor acquired specialized gut bacteria that subsequently diversified with hosts. This model may be incomplete, however, as few microbiomes have been characterized for stingless bees, which are diverse and ecologically dominant pollinators in the tropics. We surveyed gut microbiomes of Brazilian stingless bees, focusing on the genus Melipona, for which we sampled multiple species and biomes. Strikingly, Melipona lacks Snodgrassella and Gilliamella, bacterial symbionts ubiquitous in other social corbiculate bees. Instead, Melipona species harbor more environmental bacteria and bee-specific Starmerella yeasts. Loss of Snodgrassella and Gilliamella may stem from ecological shifts in Melipona or the acquisition of new symbionts as functional replacements. Our findings demonstrate the value of broadly sampling microbiome biodiversity and show that even ancient symbioses can be lost.

Efficient Plant Production of Recombinant NS1 Protein for Diagnosis of Dengue.

Dengue fever is endemic in more than 120 countries, which account for 3.9 billion people at risk of infection worldwide. The absence of a vaccine with effective protection against the four serotypes of this virus makes differential molecular diagnosis the key step for the correct treatment of the disease. Rapid and efficient diagnosis prevents progression to a more severe stage of this disease. Currently, the limiting factor in the manufacture of dengue (DENV) diagnostic kits is the lack of large-scale production of the non-structural 1 (NS1) protein (antigen) to be used in the capture of antibodies from the blood serum of infected patients. In this work, we use plant biotechnology and genetic engineering as tools for the study of protein production for research and commercial purposes. Gene transfer, integration and expression in plants is a valid strategy for obtaining large-scale and low-cost heterologous protein production. The authors produced NS1 protein of the dengue virus serotype 2 (NS1DENV2) in the Arabidopsis thaliana plant. Transgenic plants obtained by genetic transformation expressed the recombinant protein that was purified and characterized for diagnostic use. The yield was 203 µg of the recombinant protein per gram of fresh leaf. By in situ immunolocalization, transgenic protein was observed within the plant tissue, located in aggregates bodies. These antigens showed high sensitivity and specificity to both IgM (84.29% and 91.43%, respectively) and IgG (83.08% and 87.69%, respectively). The study goes a step further to validate the use of plants as a strategy for obtaining large-scale and efficient protein production to be used in dengue virus diagnostic tests.

Milk microbial composition of Brazilian dairy cows entering the dry period and genomic comparison between Staphylococcus aureus strains susceptible to the bacteriophage vB_SauM-UFV_DC4.

Brazil has the second-largest dairy cattle herd in the world, and bovine mastitis still can cause significant losses for dairy farmers. Despite this fact, little information is available about milk microbial composition of Brazilian dairy cows, as well as the potential use of bacteriophages in the control of S. aureus. Here, we investigated milk bacterial composition of 28 Holstein Fresian cows (109 teats), selected in the dry-off period, using 16S rRNA analysis. Furthermore, a representative S. aureus strain (UFV2030RH1) was obtained at drying-off for isolation of a bacteriophage (vB_SauM-UFV_DC4, UFV_DC4) and bacterial genomic comparison purposes. Our outcomes revealed that Staphylococcus was the third most prevalent genus and positively correlated with subclinical mastitis events. As a major finding, genomic analyses showed the presence of adhesive matrix molecules that recognize microbial surface components (MSCRAMM) in UFV2030RH1 and might indicate great biofilm formation capability. A minimum inhibitory concentration (MIC) assay showed that resistance to ampicillin was the highest among the antibiotic tested in S. aureus 3059 and UFV2030RH1, displaying values four and sixteen times greater than MIC resistance breakpoint, respectively. Together, our results suggest that Staphylococcus is highly prevalent in dairy cows at drying-off and the use of the phage UFV_DC4 as a biocontrol agent must be investigated in future studies.

Desulfovibrio alaskensis prophages and their possible involvement in the horizontal transfer of genes by outer membrane vesicles.

Desulfovibrio alaskensis is a Gram-negative bacterial species that belongs to the group of Sulphate Reducing Bacteria (SRB) and presents prophages in genomes, a common characteristic of the genus Desulfovibrio. Genetic material can be transported by outer membrane vesicles, however, no data regarding the production of these vesicles has been reported for D. alaskensis. To verify the expression of D. alaskensis prophages and their involvement with outer membrane vesicles, the DSM16109 strain was used. The DSM16109 strain had three prophages and presented reduced growth after mitomycin C addition when compared to the control culture. This reduction was accompanied by the presence of virus-like particles (VLPs), indicating mitomycin C dependent prophage induction. The increase in the number of cap gene copies and transcriptions of the three prophages was verified in the control sample, however, without the formation of VLPs. Prophage genes were identified in outer membrane vesicles from cultures treated and not treated with mitomycin C. DSM16109 prophages are expressed spontaneously but only in the presence of mitomycin C was it possible to observe VLP formation. Due to the genetic material detection from the prophages within outer membrane vesicles, this property may be related to the horizontal transfer of viral genes.

Animal model of arthritis and myositis induced by the Mayaro virus.

BACKGROUND: The Mayaro virus (MAYV) is an endemic arbovirus in South American countries, where it is responsible for sporadic outbreaks of Mayaro fever. Clinical manifestations include fever, headache, ocular pain, rash, myalgia, and debilitating and persistent polyarthralgia. Understanding the mechanisms associated with MAYV-induced arthritis is of great importance due to the potential for its emergence, urbanization and dispersion to other regions. METHODS: 15-day old Balb/c mice were infected by two distinct pathways, below the forelimb and in the rear footpad. Animals were observed for a period of 21 days. During this time, they were monitored every 24 hours for disease signs, such as weight loss and muscle weakness. Histological damage in the muscles and joints was evaluated 3, 7, 10, 15 and 20 days post-infection. The cytokine profile in serum and muscles during MAYV infection was evaluated by flow cytometry at different post-infection times. For pain analysis, the animals were submitted to the von Frey test and titre in different organs was evaluated throughout the study to obtain viral kinetics. FINDINGS: Infection by two distinct pathways, below the forelimb and in the rear footpad, resulted in a homogeneous viral spread and the development of acute disease in animals. Clinical signs were observed such as ruffled fur, hunched posture, eye irritation and slight gait alteration. In the physical test, both groups presented loss of resistance, which was associated with histopathological damage, including myositis, arthritis, tenosynovitis and periostitis. The immune response was characterized by a strong inflammatory response mediated by the cytokines TNF-α, IL-6 and INF-γ and chemokine MCP-1, followed by the action of IL-10 and IL-4 cytokines. INTERPRETATION: The results showed that Balb/c mice represent a promising model to study mechanisms involved in MAYV pathogenesis and for future antiviral testing.

Screening and characterization of prophages in Desulfovibrio genomes.

Bacteria of the genus Desulfovibrio belong to the group of Sulphate Reducing Bacteria (SRB). SRB generate significant liabilities in the petroleum industry, mainly due to their ability to microbiologically induce corrosion, biofilm formation and H2S production. Bacteriophages are an alternative control method for SRB, whose information for this group of bacteria however, is scarce. The present study developed a workflow for the identification of complete prophages in Desulfovibrio. Poly-lysogenesis was shown to be common in Desulfovibrio. In the 47 genomes analyzed 53 complete prophages were identified. These were classified within the order Caudovirales, with 69.82% belonging to the Myoviridade family. More than half the prophages identified have genes coding for lysozyme or holin. Four of the analyzed bacterial genomes present prophages with identity above 50% in the same strain, whose comparative analysis demonstrated the existence of colinearity between the sequences. Of the 17 closed bacterial genomes analyzed, 6 have the CRISPR-Cas system classified as inactive. The identification of bacterial poly-lysogeny, the proximity between the complete prophages and the possible inactivity of the CRISPR-Cas in closed bacterial genomes analyzed allowed the choice of poly-lysogenic strains with prophages belonging to the Myoviridae family for the isolation of prophages and testing of related strains for subsequent studies.

A T4virus prevents biofilm formation by Trueperella pyogenes.

Trueperella pyogenes is an opportunistic pathogen of many animal species. It causes economic losses worldwide, through mastitis, metritis and mainly endometritis in dairy cows. The ability of this bacterium to form biofilms is implicated in chronic infections through hampering immune system recognition and antibiotic penetration. Since it is difficult to eradicate T. pyogenes infections with antibiotics, phage therapy presents itself as a non-toxic, effective and economically viable alternative. The present study evaluated the use of the bacteriophage vB_EcoM-UFV13 (UFV13) in the prevention of T. pyogenes biofilm development. Based upon two different approaches (crystal violet and sessile cell counting) we observed that only a multiplicity of infection (MOI) of 10 showed a statistically significant reduction in biofilm formation. Although the exact mechanisms of biofilm disruption and cell-adhesion inhibition have not been determined, genome sequence analysis of the Escherichia phage UFV13 revealed a repertoire of virion-associated peptidoglycan hydrolases (VAPGHs). The present study presents new findings regarding the disruption of biofilm formation of a Gram-positive bacterium. Subsequent transcriptomic and proteomic research will help us to understand the exact interaction mechanisms between UFV13 and T. pyogenes.

Genomic analysis and immune response in a murine mastitis model of vB_EcoM-UFV13, a potential biocontrol agent for use in dairy cows.

Bovine mastitis remains the main cause of economic losses for dairy farmers. Mammary pathogenic Escherichia coli (MPEC) is related to an acute mastitis and its treatment is still based on the use of antibiotics. In the era of antimicrobial resistance (AMR), bacterial viruses (bacteriophages) present as an efficient treatment or prophylactic option. However, this makes it essential that its genetic structure, stability and interaction with the host immune system be thoroughly characterized. The present study analyzed a novel, broad host-range anti-mastitis agent, the T4virus vB_EcoM-UFV13 in genomic terms, and its activity against a MPEC strain in an experimental E. coli-induced mastitis mouse model. 4,975 Single Nucleotide Polymorphisms (SNPs) were assigned between vB_EcoM-UFV13 and E. coli phage T4 genomes with high impact on coding sequences (CDS) (37.60%) for virion proteins. Phylogenetic trees and genome analysis supported a recent infection mix between vB_EcoM-UFV13 and Shigella phage Shfl2. After a viral stability evaluation (e.g pH and temperature), intramammary administration (MOI 10) resulted in a 10-fold reduction in bacterial load. Furthermore, pro-inflammatory cytokines, such as IL-6 and TNF-α, were observed after viral treatment. This work brings the whole characterization and immune response to vB_EcoM-UFV13, a biocontrol candidate for bovine mastitis.

Prokaryotic Diversity in the Rhizosphere of Organic, Intensive, and Transitional Coffee Farms in Brazil.

Despite a continuous rise in consumption of coffee over the past 60 years and recent studies showing positive benefits linked to human health, intensive coffee farming practices have been associated with environmental damage, risks to human health, and reductions in biodiversity. In contrast, organic farming has become an increasingly popular alternative, with both environmental and health benefits. This study aimed to characterize and determine the differences in the prokaryotic soil microbiology of three Brazilian coffee farms: one practicing intensive farming, one practicing organic farming, and one undergoing a transition from intensive to organic practices. Soil samples were collected from 20 coffee plant rhizospheres (soil directly influenced by the plant root exudates) and 10 control sites (soil 5 m away from the coffee plantation) at each of the three farms for a total of 90 samples. Profiling of 16S rRNA gene V4 regions revealed high levels of prokaryotic diversity in all three farms, with thousands of species level operational taxonomic units identified in each farm. Additionally, a statistically significant difference was found between each farm's coffee rhizosphere microbiome, as well as between coffee rhizosphere soils and control soils. Two groups of prokaryotes associated with the nitrogen cycle, the archaeal genus Candidatus Nitrososphaera and the bacterial order Rhizobiales were found to be abundant and statistically different in composition between the three farms and in inverse relationship to each other. Many of the nitrogen-fixing genera known to enhance plant growth were found in low numbers (e.g. Rhizobium, Agrobacter, Acetobacter, Rhodospirillum, Azospirillum), but the families in which they belong had some of the highest relative abundance in the dataset, suggesting many new groups may exist in these samples that can be further studied as potential plant growth-promoting bacteria to improve coffee production while diminishing negative environmental impacts.

Diversity of endophytic fungi in Glycine max.

Endophytic fungi are microorganisms that live within plant tissues without causing disease during part of their life cycle. With the isolation and identification of these fungi, new species are being discovered, and ecological relationships with their hosts have also been studied. In Glycine max, limited studies have investigated the isolation and distribution of endophytic fungi throughout leaves and roots. The distribution of these fungi in various plant organs differs in diversity and abundance, even when analyzed using molecular techniques that can evaluate fungal communities in different parts of the plants, such as denaturing gradient gel electrophoresis (DGGE). Our results show there is greater species richness of culturable endophytic filamentous fungi in the leaves G. max as compared to roots. Additionally, the leaves had high values for diversity indices, i.e. Simpsons, Shannon and Equitability. Conversely, dominance index was higher in roots as compared to leaves. The fungi Ampelomyces sp., Cladosporium cladosporioides, Colletotrichum gloeosporioides, Diaporthe helianthi, Guignardia mangiferae and Phoma sp. were more frequently isolated from the leaves, whereas the fungi Fusarium oxysporum, Fusarium solani and Fusarium sp. were prevalent in the roots. However, by evaluating the two communities by DGGE, we concluded that the species richness was higher in the roots than in the leaves. UPGMA analysis showed consistent clustering of isolates; however, the fungus Leptospora rubella, which belongs to the order Dothideales, was grouped among species of the order Pleosporales. The presence of endophytic Fusarium species in G. max roots is unsurprising, since Fusarium spp. isolates have been previously described as endophyte in other reports. However, it remains to be determined whether the G. max Fusarium endophytes are latent pathogens or non-pathogenic forms that benefit the plant. This study provides a broader knowledge of the distribution of the fungal community in G. max leaves and roots, and identifies the genetic relationships among the isolated species.