Effects of Starch Overload and Cecal Buffering on Fecal Microbiota of Horses.

Starch overload in horses causes gastrointestinal and metabolic disorders that are associated with microbiota changes. Therefore, we identified the fecal microbiota and hypothesized that intracecal injection of alkaline solution (buffer; Mg(OH)2 + Al(OH)3) could stabilize these microbiota and clinical changes in horses submitted to corn starch overload. Ten crossbred horses (females and geldings) were allocated to group I (water−saline and starch−buffer treatments) and group II (water−buffer and starch−saline treatments). Clinical signs, gross analysis of the feces, and fecal microbiota were evaluated through 72 h (T0; T8; T12; T24; T48; T72). Corn starch or water were administrated by nasogastric tube at T0, and the buffer injected into the cecum at T8 in starch−buffer and water−buffer treatments. Starch overload reduced the richness (p < 0.001) and diversity (p = 0.001) of the fecal microbiota. However, the starch−buffer treatment showed greater increase in amylolytic bacteria (Bifidobacterium 0.0% to 5.6%; Lactobacillus 0.1% to 7.4%; p < 0.05) and decrease in fibrolytic bacteria (Lachnospiraceae 10.2% to 5.0%; Ruminococcaceae 11.7% to 4.2%; p < 0.05) than starch−saline treatment. Additionally, animals that received starch−buffer treatment showed more signs of abdominal discomfort and lameness associated with dysbiosis (amylolytic r > 0.5; fribolytic r < 0.1; p < 0.05), showing that cecal infusion of buffer did not prevent, but intensified intestinal disturbances and the risk of laminitis.

Interrelationship between the Microbial Communities of the Root Canals and Periodontal Pockets in Combined Endodontic-Periodontal Diseases.

Periodontal and Endodontic diseases are biofilm-related diseases. The presence of microorganisms in root canals (RCs) and the complex microbiota of periodontal pockets (PPs) contribute to the development of endodontic-periodontal diseases. This study performed a systemic analysis using state-of-the-art sequence data to assess the microbial composition of infected RCs and PPs to further assess the microbiota and verify the possibility of cross-infection between these sites. The microbiomes of these combined diseases were examined with a focus on the V3-V4 hypervariable region of the 16S rRNA gene. The number of species in PP was higher than in RC, and there was a predominance of obligate anaerobes and gram-negative bacteria. In the RCs, the genera Enterococcus, Parvimonas, Stomatobaculum predominated, in contrast, the PPs revealed a predominance of Enterococcus, Parvimonas, Stomatobaculum, Peptostreptococcus and Mogibacterium. The RC and PP microbiome was not similar with regards to the sharing of OTUs for phyla and genera (8 and 67, respectively). The evaluation of molecular markers revealed a large number of markers for resistance to antibiotics of the carbapenem and beta-lactam type (broad spectrum). Another relevant finding of this study was the markers related to systemic diseases related to cardiac muscle and rheumatology, among others. In conclusion, the RC microbiota was less complex and diverse than PP. Interactions between microbial communities were present. The shared genus can signal communication between the endodontic and periodontal microbiomes.

Microbial Community Profiling in Intensive Care Units Expose Limitations in Current Sanitary Standards.

Hospital-associated infections (HAIs) are a leading cause of morbidity and mortality in intensive care units (ICUs) and neonatal intensive care units (NICUs). Organisms causing these infections are often present on surfaces around the patient. Given that microbiota may vary across different ICUs, the HAI-related microbial signatures within these units remain underexplored. In this study, we use deep-sequencing analyses to explore and compare the structure of bacterial communities at inanimate surfaces of the ICU and NICU wards of The Medical School Clinics Hospital (Brazil). The data revealed that NICU presents higher biodiversity than ICU and surfaces closest to the patient showed a peculiar microbiota, distinguishing one unit from the other. Several facultative anaerobes or obligate anaerobes HAI-related genera were classified as biomarkers for the NICU, whereas Pseudomonas was the main biomarker for ICU. Correlation analyses revealed a distinct pattern of microbe-microbe interactions for each unit, including bacteria able to form multi-genera biofilms. Furthermore, we evaluated the effect of concurrent cleaning over the ICU bacterial community. The results showed that, although some bacterial populations decreased after cleaning, various HAI-related genera were quite stable following sanitization, suggesting being well-adapted to the ICU environment. Overall, these results enabled identification of discrete ICU and NICU reservoirs of potentially pathogenic bacteria and provided evidence for the presence of a set of biomarkers genera that distinguish these units. Moreover, the study exposed the inconsistencies of the routine cleaning to minimize HAI-related genera contamination.

Draft genome of Thermomonospora sp. CIT 1 (Thermomonosporaceae) and in silico evidence of its functional role in filter cake biomass deconstruction.

The filter cake from sugar cane processing is rich in organic matter and nutrients, which favors the proliferation of microorganisms with potential to deconstruct plant biomass. From the metagenomic data of this material, we assembled a draft genome that was phylogenetically related to Thermomonospora curvata DSM 43183, which shows the functional and ecological importance of this bacterium in the filter cake. Thermomonospora is a gram-positive bacterium that produces cellulases in compost, and it can survive temperatures of 60 ºC. We identified a complete set of biomass depolymerizing enzymes in the draft genome of Thermomonospora sp. CIT 1, such as α-amylase, catalase-peroxidases, ß-mannanase, and arabinanase, demonstrating the potential of this bacterium to deconstruct the components of starch, lignin, and hemicellulose. In addition, the draft genome of Thermomonospora sp. CIT 1 contains 18 genes that do not share identity with five other species of Thermomonospora, suggesting that this bacterium has different genetic characteristics than those present in genomes reported so far for this genus. These findings add a new dimension to the current understanding of the functional profile of this microorganism that inhabits agro-industrial waste, which may boost new gene discoveries and be of importance for application in the production of bioethanol.

Comparison of two DNA extraction methods widely used in aquatic microbial ecology.

In recent years, the rapid advances of culture-independent methods and new molecular tools have revolutionized our understanding of microbial biodiversity and ecological functions. DNA extraction from microbial communities is a critical step in this process and several methods have been proposed and used, but the influence of the extraction method on the outcome and ultimately on ecological inferences from the results is not yet precisely determined. Here, we compared two of the most commonly used extraction methods in aquatic microbial ecology, and investigated whether the two methods yielded comparable results for community ecology analyses. We extracted DNA from 15 different shallow lakes with phenol:chloroform, a classical and widely used extraction method, and with the PowerSoil DNA isolation Kit, often suggested as the standard DNA extraction method, with some adaptations for aquatic environments. We found that although only 5% of all OTUs showed significant differences in pairwise comparisons (using the 15 lakes as replicates), these OTUs accounted for >35% (on average) of the relative abundance. Diversity and richness did not differ significantly between the two extraction methods, but the beta-dispersion of the communities indicated that the organic extraction yielded more homogeneous communities, while the kit extraction generated variability. Consequently, we conclude that despite the small number of OTUs with significant differences, their impact on the community composition obtained was not negligible, and therefore the results from these two extraction methods were not comparable.

Draft Genome Sequence of Bradyrhizobium elkanii TnphoA 33, a Producer of Polyhydroxyalkanoates.

The genus Bradyrhizobium comprises bacteria with the ability to form nitrogen-fixing symbioses with legumes. They are of great interest in agriculture, as well as for the production of biopolymers such as polyhydroxyalkanoates. Here, we report the draft genome assembly of Bradyrhizobium elkanii TnphoA 33 comprising 9 Mb, 1,124 contigs, and 9,418 open reading frames.

Draft Genome Sequence of a Chitinophaga Strain Isolated from a Lignocellulose Biomass-Degrading Consortium.

Chitinophaga comprises microorganisms capable of degrading plant-derived carbohydrates, serving as a source of new tools for the characterization and degradation of plant biomass. Here, we report the draft genome assembly of a Chitinophaga strain with 8.2 Mbp and 7,173 open reading frames (ORFs), isolated from a bacterial consortium that is able to degrade lignocellulose.

Metagenomic Assembly and Draft Genome Sequence of an Uncharacterized Prevotella sp. from Nelore Rumen.

Prevotella is one of the most abundant genera in bovine rumen, although no genome has yet been assembled by a metagenomics approach applied to Brazilian Nelore. We report the draft genome sequence of Prevotella sp., comprising 2,971,040 bp, obtained using the Illumina sequencing platform. This genome includes 127 contigs and presents a low 48% GC.

A novel ß-fructofuranosidase in Coleoptera: Characterization of a ß-fructofuranosidase from the sugarcane weevil, Sphenophorus levis.

ß-fructofuranosidases or invertases (EC 3.2.1.26) catalyze the hydrolysis of sucrose into fructose and glucose. ß-fructofuranosidases have been widely described in microorganisms, but were not known in the animal kingdom until very recently. There are studies reporting lepidopteran ß-fructofuranosidases, but no ß-fructofuranosidase gene sequence or encoding transcript has previously been identified in beetles. Considering the scarcity of functional studies on insect ß-fructofuranosidases and their apparent non-occurrence among coleopterans, the aim of the present study was to investigate the occurrence and characterize a ß-fructofuranosidase transcript identified in a cDNA library from the sugarcane weevil, Sphenophorus levis (Curculionidae). To validate that the ß-fructofuranosidase sequence (herein denominated Sl-ß-fruct) is indeed encoded by the S. levis genome, PCRs were performed using genomic DNA extracted from the larval fat body as well as DNA from the midgut with microbial content. Amplification of Sl-ß-fruct gene using larval fat body DNA indicated its presence in the insect's genomic DNA. The Sl-ß-fruct gene was cloned in Pichia pastoris to produce the recombinant enzyme (rSl-ß-fruct). Molecular weight of the recombinant protein was about 64 kDa, indicating possible glycosylation, since the theoretical weight was 54.8 kDa. The substrate specificity test revealed that rSl-ß-fruct hydrolyzes sucrose and raffinose, but not melibiose or maltose, thereby confirming invertase activity. The pH curve revealed greatest activity at pH 5.0, demonstrating rSl-ß-fruct to be an acidic ß-fructofuranosidase. Quantitative PCR (qRT-PCR) analyses indicated that the production of mRNA only occurs in the midgut and reaches the greatest expression level in 30-day-old larvae, which is the expected pattern for digestive enzymes. Chromatography of glycosidases from S. levis midguts showed two enzymes acting as ß-fructofuranosidase, indicating the presence of a Sl-ß-fruct isoform or a ß-fructofuranosidase from insect intestinal microbiota. Moreover, it was found that α-glucosidases do not act on sucrose hydrolysis. Phylogenetic analyses indicated this enzyme to be similar to enzymes found in other coleopteran and lepidopteran ß-fructofuranosidases, but also closely similar to bacterial enzymes, suggesting potential horizontal gene transfer. Despite this, the enzyme seems to be restricted to different groups of bacteria, which suggests distinct origin events. The present study expands the concept of the occurrence of ß-fructofuranosidase in insects. Despite the few descriptions of this gene in the animal kingdom, it is possible to state that ß-fructofuranosidase is crucial to the establishment of some insects throughout their evolutionary history, especially members of the Lepidoptera and Coleoptera clades.

RNA-Seq analysis of Citrus reticulata in the early stages of Xylella fastidiosa infection reveals auxin-related genes as a defense response.

BACKGROUND: Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is one the most important citrus diseases, and affects all varieties of sweet orange (Citrus sinensis L. Osb). On the other hand, among the Citrus genus there are different sources of resistance against X. fastidiosa. For these species identifying these defense genes could be an important step towards obtaining sweet orange resistant varieties through breeding or genetic engineering. To assess these genes we made use of mandarin (C. reticulata Blanco) that is known to be resistant to CVC and shares agronomical characteristics with sweet orange. Thus, we investigated the gene expression in Ponkan mandarin at one day after infection with X. fastidiosa, using RNA-seq. A set of genes considered key elements in the resistance was used to confirm its regulation in mandarin compared with the susceptible sweet orange. RESULTS: Gene expression analysis of mock inoculated and infected tissues of Ponkan mandarin identified 667 transcripts repressed and 724 significantly induced in the later. Among the induced transcripts, we identified genes encoding proteins similar to Pattern Recognition Receptors. Furthermore, many genes involved in secondary metabolism, biosynthesis and cell wall modification were upregulated as well as in synthesis of abscisic acid, jasmonic acid and auxin. CONCLUSIONS: This work demonstrated that the defense response to the perception of bacteria involves cell wall modification and activation of hormone pathways, which probably lead to the induction of other defense-related genes. We also hypothesized the induction of auxin-related genes indicates that resistant plants initially recognize X. fastidiosa as a necrotrophic pathogen.

Global expression profile of biofilm resistance to antimicrobial compounds in the plant-pathogenic bacterium Xylella fastidiosa reveals evidence of persister cells.

Investigations of biofilm resistance response rarely focus on plant-pathogenic bacteria. Since Xylella fastidiosa is a multihost plant-pathogenic bacterium that forms biofilm in the xylem, the behavior of its biofilm in response to antimicrobial compounds needs to be better investigated. We analyzed here the transcriptional profile of X. fastidiosa subsp. pauca in response to inhibitory and subinhibitory concentrations of copper and tetracycline. Copper-based products are routinely used to control citrus diseases in the field, while antibiotics are more widely used for bacterial control in mammals. The use of antimicrobial compounds triggers specific responses to each compound, such as biofilm formation and phage activity for copper. Common changes in expression responses comprise the repression of genes associated with metabolic functions and movement and the induction of toxin-antitoxin systems, which have been associated with the formation of persister cells. Our results also show that these cells were found in the population at a ca. 0.05% density under inhibitory conditions for both antimicrobial compounds and that pretreatment with subinhibitory concentration of copper increases this number. No previous report has detected the presence of these cells in X. fastidiosa population, suggesting that this could lead to a multidrug tolerance response in the biofilm under a stressed environment. This is a mechanism that has recently become the focus of studies on resistance of human-pathogenic bacteria to antibiotics and, based on our data, it seems to be more broadly applicable.

Global gene expression of Poncirus trifoliata, Citrus sunki and their hybrids under infection of Phytophthora parasitica.

BACKGROUND: Gummosis and root rot caused by Phytophthora are among the most economically important diseases in citrus. Four F1 resistant hybrids (Pool R), and four F1 susceptible hybrids (Pool S) to P. parasitica, were selected from a cross between susceptible Citrus sunki and resistant Poncirus trifoliata cv. Rubidoux. We investigated gene expression in pools of four resistant and four susceptible hybrids in comparison with their parents 48 hours after P. parasitica inoculation. We proposed that genes differentially expressed between resistant and susceptible parents and between their resistant and susceptible hybrids provide promising candidates for identifying transcripts involved in disease resistance. A microarray containing 62,876 UniGene transcripts selected from the CitEST database and prepared by NimbleGen Systems was used for analyzing global gene expression 48 hours after infection with P. parasitica. RESULTS: Three pairs of data comparisons (P. trifoliata/C. sunki, Pool R/C. sunki and Pool R/Pool S) were performed. With a filter of false-discovery rate less than 0.05 and fold change greater than 3.0, 21 UniGene transcripts common to the three pairwise comparative were found to be up-regulated, and 3 UniGene transcripts were down-regulated. Among them, our results indicated that the selected transcripts were probably involved in the whole process of plant defense responses to pathogen attack, including transcriptional regulation, signaling, activation of defense genes participating in HR, single dominant genes (R gene) such as TIR-NBS-LRR and RPS4 and switch of defense-related metabolism pathway. Differentially expressed genes were validated by RT-qPCR in susceptible and resistant plants and between inoculated and uninoculated control plants CONCLUSIONS: Twenty four UniGene transcripts were identified as candidate genes for Citrus response to P. parasitica. UniGene transcripts were likely to be involved in disease resistance, such as genes potentially involved in secondary metabolite synthesis, intracellular osmotic adjustment, signal transduction pathways of cell death, oxidative burst and defense gene expression. Furthermore, our microarray data suggest another type of resistance in Citrus-Phytophthora interaction conferred by single dominant genes (R gene) since we encountered two previously reported R genes (TIR-NBS-LRR and RPS4) upregulated in the resistant genotypes relative to susceptible. We identified 7 transcripts with homology in other plants but yet unclear functional characterization which are an interesting pool for further analyses and 3 transcripts where no significant similarity was found. This is the first microarray study addressing an evaluation of transcriptional changes in response to P. parasitica in Citrus.