Detection and evaluation of volatile and non-volatile antifungal compounds produced by Bacillus spp. strains.

The identification of antifungal compounds produced by microorganisms is crucial in the context of sustainable agriculture. Bacteria of the genus Bacillus have a broad spectrum of action that can influence plant growth and control pests, vectors of public health relevance and phytopathogens. Lipopeptides are the main compounds related to the biological control of several pathogen species. Strains with biotechnological potential are identified by means of in vitro bioassays and molecular tests. In this study, strains from the Bacillus Bank of Brazilian Agricultural Research Corporation (EMBRAPA/DF/Brazil) were selected to control the fungal pathogens Sclerotinia sclerotiorum and Fusarium oxysporum by pairing assays. The detection of genes for biosynthesis of antifungal compounds from strains with high pathogen-inhibition capacity was correlated with peptide synthesis, such as bacillomycin D, fengycin d, bacilysin and surfactin. Their gene expression in contact with the pathogen was analyzed by Real-Time PCR. The volatile organic compounds produced by selected Bacillus strains were identified and quantified. In co-culture assays, the inhibition zone between Bacillus strains and Sclerotinia sclerotiorum was evaluated by scanning electron microscopy. Thirteen potentially anti-pathogenic strains were selected. Genes related to the synthesis of antifungal peptides were detected in 11 of them. In five strains, all tested genes were detected. Bacillomycin was the most frequently found lipopeptide gene. The fungus-bacteria interaction potentiated the production of volatiles. Several ketones and other volatile compounds with antifungal activity were identified. Relevant morphological changes in the fungus were observed when paired with bacteria. The study demonstrated the efficacy of the selected strains with regard to the biological control of phytopathogens and their biotechnological potential.

Distribution and Genetic Diversity of Genes from Brazilian Bacillus thuringiensis Strains Toxic to Agricultural Insect Pests Revealed by Real-Time PCR.

Bacillus thuringiensis is a Gram-positive aerobic bacterium and the most used biopesticide worldwide. Given the importance of B. thuringiensis strain characterization for the development of new bioinsecticides or transgenic events and the identification and classification of new B. thuringiensis genes and strains to understand its distribution and diversity, this work is aimed at creating a gene identification system based on qPCR reactions utilizing core B. thuringiensis genes cry1, cry2, cry3, cry4, cry5, app6, cry7, cry8, cry9, cry10, cry11, vpb1, vpa2, vip3, cyt1, and cyt2 for the characterization of 257 strains of B. thuringiensis. This system was based on the Invertebrate Bacteria Collection from Embrapa Genetic Resources and Biotechnology and analyzed (a) the degree of correlation between the distribution of these strains and the origin of the substrate from which the strain was isolated and (b) between its distribution and geoclimatic conditions. This study made it possible to observe that the cry1, cry2, and vip3A/B genes occur homogeneously in the Brazilian territory, and some genes are found in specific regions. The biggest reservoir of variability is within B. thuringiensis strains in each region, and it is suggested that both geoclimatic conditions and regional crops interfere with the genetic diversity of the B. thuringiensis strains present in the region, and B. thuringiensis strains can constantly exchange genetic information.

Biocontrol potential of bacteria belonging to the Bacillus subtilis group against pests and diseases of agricultural interest through genome exploration.

The usage of microorganisms as biocontrol agents and biofertilizers has been recommended and recognized as an ecologically correct alternative to maintaining the productivity and safety of crops. Thus, the objectives of this work were to characterize twelve strains belonging to Invertebrate Bacteria Collection of Embrapa Genetic Resources and Biotechnology by molecular, morphological, and biochemical methods and to evaluate the pathogenicity of these strains against pests and diseases of agricultural interest. The morphological characteristic of the strains was performed according to the principles of Bergy's Manual of Systematic Bacteriology. The genomes of the 12 strains were sequenced in Macrogen, Inc. (Seoul, Korea) using the HiSeq2000 and GS-FLX Plus high-performance platforms. In the determination of antibiotic sensibility profiles, disc-diffusion methods (Cefar Diagnótica Ltda) were adopted©. Selective bioassays were carried out with insects of the Lepidoptera (Spodoptera frugiperda, Helicoverpa armigera, and Chrysodeixis includens), Coleoptera (Anthonomus grandis), Diptera (Aedes aegypti) and Hemiptera (Euschistus heros) orders, and with the nematode Caenorhabditis elegans. In addition, the antagonistic action of the phytopathogens Fusarium oxysporum f. sp. vasinfectum and Sclerotinia sclerotiorum against the strains under study, and in vitro assays of phosphate solubilization were also performed. Sequencing of the complete genome of the 12 strains determined that all of them belonged to the Bacillus subtilis sensu lato group. In the strains genome were detected genic clusters responsible for encoding secondary metabolites such as surfactin, iturin, fengycins/plipastatin, bacillomycin, bacillisin, and siderophores. Due to the production of these compounds, there was a survival reduction of the Lepidoptera order insects and a reduction in the phytopathogens mycelial growth. These results show that the species of group B. subtilis s.l. can become promising microbiological alternatives to pest and disease control.

Identification of cry genes in Bacillus thuringiensis by multiplex real-time PCR.

Bacillus thuringiensis is an important bacterium of the group Bacillus cereus sensu lato due to its insecticidal properties. This microorganism has high genetic variability and its strains produce different Cry toxins, known as δ-endotoxins, which are mainly responsible for its toxic effect on insects that are agricultural pests or vector human diseases. Each strain can express a variety of cry genes, out of a total of 789 cry genes described so far. The detection of these genes is very important to characterize strains, as they may indicate their toxic potential. Several methods have been used to characterize B. thuringiensis strains, but one of the most common techniques is Polymerase Chain Reaction (PCR) from primers that detect the presence of cry genes. This technique has been optimized to make real-time multiplex quantitative PCR (qPCR) assays faster, more efficient, and safer, because the presence of three genes can be detected in a single reaction. In this work, a multiplex assay was developed to identify the presence of genes from the cry1A, cry1C, and cry1F families whose respective toxins are present in both bioinsecticides, and commercial transgenic plants used to control caterpillars. Specific primers were designed to identify the families of the cited genes and the system was validated with samples that were sequenced by next-generation sequencing (NGS). The system was implemented and used to characterize 214 strains. Of these, eight were submitted to conventional PCR, and the results matched, again validating the system. Thus, the application of the proposed technique allows the reliable evaluation through this system to detect the presence of the genes of the families cry1A, cry1C, and cry1F in samples of B. thuringiensis.

Factors Associated with Suicidal Behavior in Farmers: A Systematic Review.

This review aimed to investigate the factors associated with suicidal behavior in farmers in the scientific literature. Two researchers participated independently in searching databases, specifically PubMed/MEDLINE, LILACS, Web of Science, Scopus, PsycINFO, and SciELO. Only observational studies were included. The quality of the selected studies was assessed with a critical assessment checklist for cross-sectional analytical and case-control studies, prepared by the Joanna Briggs Institute. Data related to the publication were collected (author and year; city/country); methodological design; sample/population (gender; average age), outcome, measuring instrument and factors associated with suicidal behavior. A total of 14 studies were included in the systematic review, and factors associated with farmers' behavior in mental health (depression), seasonal impacts (drought), and work exposures (herbicides and insecticides) were identified. However, heterogeneity was found in terms of the method, measurement of suicidal behavior, and associated factors, which indicates the need for further studies.

Notifiable Diseases Information System and human poisoning by pesticides in Brazil. / Sistema de Informação de Agravos de Notificação e as intoxicações humanas por agrotóxicos no Brasil.

OBJECTIVE: To analyze trends in pesticide poisoning incidence rates in Brazilian regions, according to sex and circumstances of poisoning, between 2001 and 2014. METHOD: Ecological time-series study, with data from the national Notifiable Diseases Information System (SINAN). The incidence rates were calculated by dividing the number of confirmed new cases of pesticide poisoning by the total resident population in the same period and location. Both Polynomial regression analysis and Mann-Whitney and Kruskal-Wallis tests were performed. When significant differences were found, these tests were followed by the Bonferroni penalty, in order to identify the difference more precisely. RESULTS: In Brazil, 80,069 notified poisoning cases were recorded from 2001 to 2014. There was a steadily increasing growth of pesticide poisoning in this population, whose growth trend was 0,377 for 100,000 inhabitants per year. The highest incidence of poisoning occurred in the South and Midest Regions. Regarding sex, no significant differences were found (p < 0,347), and attempted suicide was the most significant circumstance of poisoning (p < 0,001). CONCLUSION: The incidence of pesticide poisoning in Brazil has been continuously increasing in the twenty-first century.

OBJETIVO: Analisar as tendências das taxas de incidência da intoxicação por agrotóxicos nas regiões brasileiras, de acordo com sexo e circunstância da intoxicação, no período de 2001 a 2014. MÉTODO: Estudo de séries temporais, com dados do Sistema de Informação de Agravos de Notificação (SINAN). As taxas de incidência foram calculadas por meio da razão entre o número de casos novos confirmados de intoxicações por agrotóxicos e a população residente no mesmo período e local. Foram realizados análise de regressão polinomial e testes de Mann-Whitney e Kruskal-Wallis. Quando encontradas diferenças significativas, os testes foram seguidos pela penalização de Bonferroni com a finalidade de identificar onde residia a diferença. RESULTADOS: No Brasil, foram registrados 80.069 casos de intoxicação nesse período. Houve um crescimento linear de notificações de intoxicação por agrotóxico, cuja taxa de tendência de crescimento encontrada foi de 0,377 por 100 mil habitantes/ano. As regiões Sul e Centro-Oeste apresentam as maiores taxas de intoxicação. Em relação ao sexo, não foram encontradas diferenças significativas (p < 0,347), sendo a tentativa de suicídio a circunstância de intoxicação mais significativa (p < 0,001). CONCLUSÃO: A incidência de intoxicação por agrotóxico no Brasil segue em aumento no século XXI.

Sistema de Informação de Agravos de Notificação e as intoxicações humanas por agrotóxicos no Brasil / Notifiable Diseases Information System and human poisoning by pesticides in Brazil

RESUMO: Objetivo: Analisar as tendências das taxas de incidência da intoxicação por agrotóxicos nas regiões brasileiras, de acordo com sexo e circunstância da intoxicação, no período de 2001 a 2014. Método: Estudo de séries temporais, com dados do Sistema de Informação de Agravos de Notificação (SINAN). As taxas de incidência foram calculadas por meio da razão entre o número de casos novos confirmados de intoxicações por agrotóxicos e a população residente no mesmo período e local. Foram realizados análise de regressão polinomial e testes de Mann-Whitney e Kruskal-Wallis. Quando encontradas diferenças significativas, os testes foram seguidos pela penalização de Bonferroni com a finalidade de identificar onde residia a diferença. Resultados: No Brasil, foram registrados 80.069 casos de intoxicação nesse período. Houve um crescimento linear de notificações de intoxicação por agrotóxico, cuja taxa de tendência de crescimento encontrada foi de 0,377 por 100 mil habitantes/ano. As regiões Sul e Centro-Oeste apresentam as maiores taxas de intoxicação. Em relação ao sexo, não foram encontradas diferenças significativas (p < 0,347), sendo a tentativa de suicídio a circunstância de intoxicação mais significativa (p < 0,001). Conclusão: A incidência de intoxicação por agrotóxico no Brasil segue em aumento no século XXI.

ABSTRACT: Objective: To analyze trends in pesticide poisoning incidence rates in Brazilian regions, according to sex and circumstances of poisoning, between 2001 and 2014. Method: Ecological time-series study, with data from the national Notifiable Diseases Information System (SINAN). The incidence rates were calculated by dividing the number of confirmed new cases of pesticide poisoning by the total resident population in the same period and location. Both Polynomial regression analysis and Mann-Whitney and Kruskal-Wallis tests were performed. When significant differences were found, these tests were followed by the Bonferroni penalty, in order to identify the difference more precisely. Results: In Brazil, 80,069 notified poisoning cases were recorded from 2001 to 2014. There was a steadily increasing growth of pesticide poisoning in this population, whose growth trend was 0,377 for 100,000 inhabitants per year. The highest incidence of poisoning occurred in the South and Midest Regions. Regarding sex, no significant differences were found (p < 0,347), and attempted suicide was the most significant circumstance of poisoning (p < 0,001). Conclusion: The incidence of pesticide poisoning in Brazil has been continuously increasing in the twenty-first century.

Purified citritin in combination with vancomycin inhibits VRE in vitro and in vivo.

Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) have been frequently associated with bacterial resistance mechanisms. These mechanisms, in turn, restrict a range of therapeutic opportunities for the treatment of infections caused by these micro-organisms. Faced with this problem, the present study aims to isolate and characterize molecules with antimicrobial activity derived from the fungus Penicillium citrinum isolated from Cerrado soil. Furthermore, we also tested possible antibacterial potential alone and in combination with commercial antimicrobial agents. In this context, citrinin was isolated and characterized by nuclear magnetic resonance and electrospray ionization. Functional analyses showed MIC of 128 µg ml-1 against S. aureus ATCC 25923, E. faecalis ATCC 29212 and a clinical isolate of vancomycin-resistant E. faecium (VRE01). However, for a clinical strain of methicillin-resistant S. aureus (MRSA01), the MIC was 256 µg ml-1. In order to avoid such high concentrations and reduce the collateral effects, additive effects were evidenced by combining citrinin with cefoxitin against MRSA01 (FIC index=0.5) and also citrinin with vancomycin toward VRE01 (FIC index=0.5). In vivo studies with BALB/c-tipe mice (MRSA assay) demonstrated a clinical ineffectiveness of cefoxitin associated with citrinin (9.8 mg kg-1 of cefoxitin +0.2 mg kg-1 of citrinin), with this combination being inefficient to increase animal survival. However, the combination used in the treatment of VRE (23.5 mg kg-1 of citrinin +1.5 mg kg-1 of vancomycin) sepsis model was extremely promising, leading to an animal survival rate of 80 percent. In summary, our data show, for the first time, the possible successful use of citrinin associated with vancomycin for pathogenic bacteria control.

Prevalência e fatores associados a queimaduras de terceiro grau no município de Natal, RN - Brasil / Prevalence and factors associated with third degree burns in the city of Natal, RN- Brazil

Introdução: A queimadura é a lesão dos tecidos decorrente de um trauma causado por um agente térmico. Pode ser provocada por chama, escaldadura, eletricidade e substâncias químicas ácidas ou álcalis, dentre outros. Objetivo: Este estudo teve como objetivo identificar a prevalência e os fatores associados da queimadura de 3º grau das vítimas internadas com o diagnóstico de queimadura no Hospital Monsenhor Walfredo Gurgel, Natal/RN. Método: Realizou-se um estudo retrospectivo, no qual foram analisados os prontuários de 164 pacientes entre janeiro e dezembro de 2009 quanto às seguintes variáveis: sexo, idade, renda familiar, escolaridade, local geográfico do trauma, zona geográfica acometida, local físico do trauma, agente agressor, região do corpo afetada, profundidade da lesão e mês do trauma. Resultados: A prevalência da queimadura de 3º grau (23,4%), embora tenha sido inferior quando comparada as de 1º e 2º graus, foi considerada elevada. Dos fatores associados à queimadura de 3º grau, a idade, renda familiar, local físico do trauma e o agente agressor formam os que apresentaram significância estatística. Conclusão: O estudo demonstrou uma alta prevalência de queimadura de 3º grau. Ressaltase, ainda, a importância dos estudos epidemiológicos e de políticas de educação continuada visando à prevenção.

Introduction: Burning is the injury of fabrics decurrent of a trauma caused for a thermal agent. It can be provoked by flame, scalding, electricity, and acid or alkalis chemical substances, amongst others. Objective: This study it had as objective to identify to the prevalence and the factors associates of the third degree burn, of the victims interned with the diagnosis of burning in the Hospital Monsenhor Walfredo Gurgel, Natal/RN. Methods: A retrospective study was become fulfilled where had been analyzed the handbooks of 164 patients between January and December of 2009 how much to the following variable: sex, age, familiar income, instruction level, geographic place of the trauma, geographic zone attack, physical place of the trauma, aggressive agent, region of the affected, depth of the injury and month of the trauma. Results: The prevalence of the burning of 3º degree (23.4%), even so has been inferior when compared of 1º and 2º degrees, were considered raised.Of the factors associates to the burning of 3º degree, the age, familiar income, local physicist of the trauma and the aggressive agent presented the statistical significance. Conclusion: The study demonstrated high prevalence of burning of 3º degree. It is noteworthy the importance of the studies epidemiologists and education policies continued aiming at to the prevention.

Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putative receptors for the Cry1B protein of Bacillus thuringiensis.

Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. They interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in midgut epithelial cells lysis. In this work we had cloned, sequenced and expressed a cry1Ba toxin gene from the B thuringiensis S601 strain which was previously shown to be toxic to Anthonomus grandis, a cotton pest. The Cry1Ba6 protein expressed in an acrystaliferous B. thuringiensis strain was toxic to A. grandis in bioassays. The binding of Cry1Ba6 toxin to proteins located in the midgut brush border membrane of A. grandis was analyzed and we found that Cry1Ba6 binds to two proteins (62 and 65kDa) that showed alkaline phosphatase (ALP) activity. This work is the first report that shows the localization of Cry toxin receptors in the midgut cells of A. grandis.