Insights into multiple use water enterobacteria from the Brazilian semi-arid: the first approach by pyrosequencing

Thousands of people living in semi-arid regions face problems of drought and loss of water quality. In addition, high incidence of acute diarrheal diseases related to water consumption has been responsible for a high number of deaths and high economic costs for human health. Many of the diseases can be caused by the presence of enterobacteria in reservoirs that serve for multiple purposes. This study aimed to confirm the presence of potentially harmful bacteria, which was highlighted in other articles, and to reveal non-identified genera by culture-dependent methods and pyrosequencing. Twenty-three genera of the Enterobacteriaceae family were detected, with emphasis on Escherichia genus and confirmation of the presence of species such as Salmonella enterica and Enterobacter cloacae. The abundance of heterotrophic prokaryotes and the physical and chemical data show an expected average for this type of environment due to the numbers historically presented in previous articles. The unprecedented detection of the presence of some potentially pathogenic species can alert and raise awareness of the populations that use stored water in the semi-arid regions. Consequently, as a result of the peculiar characteristics of reservoirs under this climate influence, there is a cosmopolitanism of enterobacteria that may be related to the alarming numbers of infections from Waterborne Diseases.

Insights into multiple use water enterobacteria from the Brazilian semi-arid: the first approach by pyrosequencing

Thousands of people living in semi-arid regions face problems of drought and loss of water quality. In addition, high incidence of acute diarrheal diseases related to water consumption has been responsible for a high number of deaths and high economic costs for human health. Many of the diseases can be caused by the presence of enterobacteria in reservoirs that serve for multiple purposes. This study aimed to confirm the presence of potentially harmful bacteria, which was highlighted in other articles, and to reveal non-identified genera by culture-dependent methods and pyrosequencing. Twenty-three genera of the Enterobacteriaceae family were detected, with emphasis on Escherichia genus and confirmation of the presence of species such as Salmonella enterica and Enterobacter cloacae. The abundance of heterotrophic prokaryotes and the physical and chemical data show an expected average for this type of environment due to the numbers historically presented in previous articles. The unprecedented detection of the presence of some potentially pathogenic species can alert and raise awareness of the populations that use stored water in the semi-arid regions. Consequently, as a result of the peculiar characteristics of reservoirs under this climate influence, there is a cosmopolitanism of enterobacteria that may be related to the alarming numbers of infections from Waterborne Diseases.(AU)

Outstanding impact of soil tillage on the abundance of soil hydrolases revealed by a metagenomic approach

The soil represents the main source of novel biocatalysts and biomolecules of industrial relevance. We searched for hydrolases in silico in four shotgun metagenomes (4,079,223 sequences) obtained in a 13-year field trial carried out in southern Brazil, under the no-tillage (NT), or conventional tillage (CT) managements, with crop succession (CS, soybean/wheat), or crop rotation (CR, soybean/maize/wheat/lupine/oat). We identified 42,631 hydrolases belonging to five classes by comparing with the KEGG database, and 44,928 sequences by comparing with the NCBI-NR database. The abundance followed the order: lipases > laccases > cellulases > proteases > amylases > pectinases. Statistically significant differences were attributed to the tillage system, with the NT showing about five times more hydrolases than the CT system. The outstanding differences can be attributed to the management of crop residues, left on the soil surface in the NT, and mechanically broken and incorporated into the soil in the CT. Differences between the CS and the CR were slighter, 10% higher for the CS, but not statistically different. Most of the sequences belonged to fungi (Verticillium, and Colletotrichum for lipases and laccases, and Aspergillus for proteases), and to the archaea Sulfolobus acidocaldarius for amylases. Our results indicate that agricultural soils under conservative managements may represent a hotspot for bioprospection of hydrolases.(AU)

Metagenômica ruminal de cabras leiteiras alimentadas com dietas adicionadas ou não de tanino

A caprinocultura é uma importante fonte de renda e alimento em ambientes extremos (altas temperaturas e baixa umidade), como o semiárido nordestino brasileiro, com curtos período chuvosos. No período da chuva, a vegetação abundante e de qualidade. Porém, na maior parte do ano, no período seco, esses alimentos são escassos e de qualidade nutricional reduzida, e apresentam altos índices de compostos secundários, como os taninos. Esses compostos apresentam características físico-químicas que lhe concedem diversas aplicações, desde controle de endoparasitas e modulação da microbiota ruminal. Diante de tais qualidades, a sua utilização tem sido bastante estudada nos mais diversos contextos produtivos e biotecnológicos. Até o presente momento, poucos trabalhos estudaram como os taninos modulam a microbiota ruminal nas espécies domésticas de ruminantes. Sendo assim, objetivou-se com este trabalho caracterizar os perfis funcionais de caprinos nativos submetidos a dietas com e sem adição de tanino condensados, utilizando a metagenômica total do liquido ruminal, com o intuito de melhor entender como esse microbioma se comporta frente a esse composto, e quais vias metabólicas, genes e funções possuem suas abundâncias modificadas. Doze caprinos nativos das raças Canindé e Repartida foram submetidos a dietas com e sem adição de tanino (5%). Esses animais foram distribuídos em quatro grupos de três animais cada, em um esquema fatorial 2x2, onde se avaliou os efeitos dos fatores dieta e raça. Ao final do período experimental foi coletado líquido ruminal de cada animal, de onde o DNA total foi extraído e utilizado para sequenciamento de metagenoma total. O controle de qualidade das sequencias foi feita no Trimmomatic, os metagenomas foram montados e avaliados os genes foram identificados e anotados utilizando o MegaHit e Metaquast, MetaProdigal, InterproScan respetivamente, e as análises estatísticas para identificação dos genes diferencialmente presentes no edgeR. Verificou-se que o tanino na dieta produziu alterações funcionais no rúmen dos animais desafiados. Na raça Canindé, a concentração testada produziu alterações mais intensas do que nos animais da raça Repartida. Nos animais desafiados foram identificados genes envolvidos com e metabolismo de carboidratos e peptidoglicano aumentados e, diminuição de genes de enzimas de hidrólise de ligações glicosídicas e biossíntese microbiana de cobalamina, bem como diminuição na contagem de sequências pertencentes a endoparasitas.

Goat farming is an important source of income and food in extreme environments (high temperatures and low humidity), such as the Brazilian northeastern semiarid region, which has a low concentration of rainfall, with short rainy periods. During this period of rain, there is abundant vegetation and excellent quality for the animals' diet. However, for most of the year, when the period is dry, these foods are scarce and of reduced nutritional quality, and still have high levels of secondary compounds, among which tannins stand out. This group of compounds has physicochemical characteristics that grant it several applications, such as endoparasite control and modulation of the ruminal microbiota. Given these qualities, its use has been extensively studied in the most diverse productive and biotechnological contexts. So far, few studies have studied how tannins modulate the ruminal microbiota in domestic ruminant species. Thus, the objective of this work was to characterize the functional profiles of native goats fed diets with and without the addition of commercial condensed tannin, using the total metagenomics of the rumen fluid, to better understand how this microbiome behaves against this compound, and which metabolic pathways, genes and functions have their abundances modified. Twelve native goats of the Canindé and Repartida breeds were fed diets with and without addition of tannin (5%). These animals were distributed into four groups of three animals each, in a 2x2 factorial scheme, where the effects of diet and race factors were evaluated. At the end of the experimental period, ruminal fluid was collected from each animal, from which the total DNA was extracted and used for metagenoma sequencing, The quality control of the sequences was carried out in Trimmomatic, the metagenomes were assembled and evaluated, the genes were identified and annotated using MegaHit and Metaquast, MetaProdigal, InterproScan respectively, and statistical analysis to identify genes differentially present in edgeR. It was found that tannin in the diet produced functional changes in the rumen of challenged animals. In the Canindé breed, the concentration tested produced more intense changes than in the Repartida breed. In challenged animals, genes involved with increased carbohydrate and peptidoglycan metabolism and decreased glycosidic bond hydrolysis enzyme genes and microbial cobalamin biosynthesis, as well as decreased sequence count belonging to endoparasites were identified.

DETECÇÃO E CARACTERIZAÇÃO DO MICROBIOMA DA CONJUNTIVA OCULAR DE CÃES SEM AFECÇÕES OFTALMOLÓGICAS

A conjuntiva ocular desempenha um papel importante na função lacrimal, na proteção imunológica do olho, nos movimentos oculares e na regeneração corneana, agindo como uma barreira à entrada de microrganismos. Infecções conjuntivais em cães são comuns e resultam tanto da invasão de microrganismos patogênicos, quanto do crescimento descontrolado da microbiota previamente existente. O presente estudo analisou a comunidade microbiana presente na conjuntiva ocular de uma população heterogênea de cães sem afecções oftalmológicas por meio de técnicas de Sequenciamento de Última Geração (NGS). Suabes conjuntivais foram coletados de ambos os olhos de 30 cães que, após o processamento, foram submetidos ao sequenciamento metagenômico através da plataforma Illumina. Foi identificada a predominância do filo Proteobacteria e dos gêneros Ralstonia e Burkholderia, além de uma minoria de fungos, enquanto vírus não foram encontrados. Por meio de sequenciamento de DNA microbiano total, o presente estudo trouxe novos dados a respeito deste tema, demonstrando a presença de organismos não-cultiváveis e previamente desconhecidos como parte do ambiente ocular. Como a conjuntivite é uma afecção comum que afeta o bem-estar e a qualidade de vida dos cães, portanto, é importante conhecer a microbiota conjuntival para entender os processos infecciosos que afetam este sítio e assim direcionar os tratamentos empregados de forma racional.

The conjunctiva plays essential role in the lacrimal function, immune protection of the eye, eye mobility and corneal regeneration, acting as a barrier to the entry of microorganisms. Conjunctival infections are common in dogs and result from both the invasion of pathogenic microorganisms and the uncontrolled growth of the existing microbiota. The present study analyzed the microbial community present on the ocular conjunctivae of a heterogeneous dog population without ophthalmological disorders by New Generation Sequencing (NGS) techniques. Conjunctival swabs were collected from both eyes of 30 dogs, which after processing were subjected to NGS sequencing through the Illumina platform. A predominance of the phylum Proteobacteria and the genera Ralstonia and Burkholderia were identified along with a minority of fungi, while viruses were not found. Through total microbial DNA sequencing, the present study have brought new data on this subject showing the presence of non-cultivable organisms that were previously unknown as part of the ocular environment. As conjunctivitis is a common condition that affects welfare and life quality of dogs, so it is important to know the microbiota of the conjunctiva in order to understand the infectious processes that affect it and to direct the treatments employed rationally.

Molecular characterization of bacterial populations of different soils

Until recently, few studies were carried out in Brazil about diversity of bacterial soil communities. Aiming to characterize the bacterial population in the soil through 16S rRNA analysis, two types of soil have been analyzed: one of them characterized by intensive use where tomato, beans and corn were cultivated (CS); the other analyzed soil was under forest (FS), unchanged by man; both located in Guaíra, São Paulo State, Brazil. Using specific primers, 16S rRNA genes from metagenomic DNA in both soils were amplified by PCR, amplicons were cloned and 139 clones from two libraries were partially sequenced. The use of 16S rRNA analysis allowed identification of several bacterial populations in the soil belonging to the following phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria Verrucomicrobia in addition to the others that were not classified, beyond Archaea domain. Differences between FS and CS libraries were observed in size phyla. A larger number of phyla and, consequently, a greater bacterial diversity were found in the under-forest soil. These data were confirmed by the analyses of genetic diversity that have been carried out. The characterization of bacterial communities of soil has made its contribution by providing facts for further studies on the dynamics of bacterial populations in different soil conditions in Brazil.

Até o momento poucos estudos foram realizados no Brasil a respeito da diversidade de comunidades bacterianas no solo. Com o objetivo de caracterizar as populações bacterianas presentes no solo através da análise do gene 16S rRNA, foram analisados dois solos: um caracterizado pelo uso intensivo, principalmente para a produção de tomate, feijão e milho (CS); e outro sob floresta (FS), não modificado pelo homem, ambos do município de Guaíra, no estado de São Paulo, Brasil. Usando oligonucleotídeos específicos, de genes 16S rRNA do DNA metagenomico de ambos os solos foram amplificados por PCR, amplicons foram clonados e 139 clones de duas bibliotecas foram seqüenciados. O uso da técnica de 16S rRNA, gerou a identificação de diferentes populações de bactérias de solo pertencentes aos filos Acidobacteria Actinobacteria Bacteroidetes Firmicutes Proteobacteria Verrucomicrobia, Archaea, além das não classificadas. Diferenças entre as bibliotecas FS e CS foram observadas no tamanho dos filos. Um grande número de filos e, consequentemente, uma grande diversidade bacteriana foi observada no solo sob floresta. Estes dados foram confirmados pela análise de diversidade genética realizada. A caracterização de comunidades do solo apresentada neste trabalho contribuiu fornecendo dados para estudos posteriores sobre a dinâmica das populações bacterianas em solos de diferentes condições no Brasil.