The microbiological signature of human cutaneous leishmaniasis lesions exhibits restricted bacterial diversity compared to healthy skin

Localised cutaneous leishmaniasis (LCL) is the most common form of cutaneous leishmaniasis characterised by single or multiple painless chronic ulcers, which commonly presents with secondary bacterial infection. Previous culture-based studies have found staphylococci, streptococci, and opportunistic pathogenic bacteria in LCL lesions, but there have been no comparisons to normal skin. In addition, this approach has strong bias for determining bacterial composition. The present study tested the hypothesis that bacterial communities in LCL lesions differ from those found on healthy skin (HS). Using a high throughput amplicon sequencing approach, which allows for better populational evaluation due to greater depth coverage and the Quantitative Insights Into Microbial Ecology pipeline, we compared the microbiological signature of LCL lesions with that of contralateral HS from the same individuals.Streptococcus, Staphylococcus,Fusobacterium and other strict or facultative anaerobic bacteria composed the LCL microbiome. Aerobic and facultative anaerobic bacteria found in HS, including environmental bacteria, were significantly decreased in LCL lesions (p < 0.01). This paper presents the first comprehensive microbiome identification from LCL lesions with next generation sequence methodology and shows a marked reduction of bacterial diversity in the lesions.

Land-use systems affect Archaeal community structure and functional diversity in western Amazon soils

The study of the ecology of soil microbial communities at relevant spatial scales is primordial in the wide Amazon region due to the current land use changes. In this study, the diversity of the Archaea domain (community structure) and ammonia-oxidizing Archaea (richness and community composition) were investigated using molecular biology-based techniques in different land-use systems in western Amazonia, Brazil. Soil samples were collected in two periods with high precipitation (March 2008 and January 2009) from Inceptisols under primary tropical rainforest, secondary forest (5-20 year old), agricultural systems of indigenous people and cattle pasture. Denaturing gradient gel electrophoresis of polymerase chain reaction-amplified DNA (PCR-DGGE) using the 16S rRNA gene as a biomarker showed that archaeal community structures in crops and pasture soils are different from those in primary forest soil, which is more similar to the community structure in secondary forest soil. Sequence analysis of excised DGGE bands indicated the presence of crenarchaeal and euryarchaeal organisms. Based on clone library analysis of the gene coding the subunit of the enzyme ammonia monooxygenase (amoA) of Archaea (306 sequences), the Shannon-Wiener function and Simpson's index showed a greater ammonia-oxidizing archaeal diversity in primary forest soils (H' = 2.1486; D = 0.1366), followed by a lower diversity in soils under pasture (H' = 1.9629; D = 0.1715), crops (H' = 1.4613; D = 0.3309) and secondary forest (H' = 0.8633; D = 0.5405). All cloned inserts were similar to the Crenarchaeota amoA gene clones (identity > 95 %) previously found in soils and sediments and distributed primarily in three major phylogenetic clusters. The findings indicate that agricultural systems of indigenous people and cattle pasture affect the archaeal community structure and diversity of ammonia-oxidizing Archaea in western Amazon soils.

Polyethylene glycol and polyvinylpirrolidone effect on bacterial rRNA extraction and hybridization from cells exposed to tannins

In order to detect fluctuations in ruminal microbial populations due to forage tannins using 16S ribosomal RNA (rRNA) probes, recovery of intact rRNA is required. The objective of this work was to evaluate the effect of polyethylene glycol (PEG) and polyvinylpirrolidone (PVP) on extraction of bacterial rRNA, in the presence of tannins from tropical legume forages and other sources, that hybridize with oligonucleotide probes. Ruminococcus albus 8 cells were exposed to 8 g/L tannic acid or 1 g/L condensed tannins extracted from Acacia angustissima, banana (Musa sp.) skin, Desmodium ovalifolium, red grape (Vitis vinifera) skin and Inga edulis, or no tannins. Cells were rinsed with Tris buffer pH 7 containing either 8% PEG or 6% PVP prior to cell lysis. Total RNA samples rinsed with either PEG or PVP migrated through denaturing agarose gels. The 16S rRNA bands successfully hybridized with a R. albus species-specific oligonucleotide probe, regardless of tannin source. The effect of rinsing buffers on the density of 16S rRNA bands, as well as on the hybridization signals was compared. There were significant effects (P<0.01) when the controls were compared to either buffer treatments due to tannin type, buffer used and the interaction of tannin type and buffer. The significant interaction indicates the influence of tannin type on the parameters evaluated.

Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA) and an eucalyptus arboretum (EAA). PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.

Functional diversity of bacterial genes associated with aromatic hydrocarbon degradation in anthropogenic dark earth of Amazonia

The objective of this work was to evaluate the catabolic gene diversity for the bacterial degradation of aromatic hydrocarbons in anthropogenic dark earth of Amazonia (ADE) and their biochar (BC). Functional diversity analyses in ADE soils can provide information on how adaptive microorganisms may influence the fertility of soils and what is their involvement in biogeochemical cycles. For this, clone libraries containing the gene encoding for the alpha subunit of aromatic ring-hydroxylating dioxygenases (α-ARHD bacterial gene) were constructed, totaling 800 clones. These libraries were prepared from samples of an ADE soil under two different land uses, located at the Caldeirão Experimental Station - secondary forest (SF) and agriculture (AG) -, and the biochar (SF_BC and AG_BC, respectively). Heterogeneity estimates indicated greater diversity in BC libraries; and Venn diagrams showed more unique operational protein clusters (OPC) in the SF_BC library than the ADE soil, which indicates that specific metabolic processes may occur in biochar. Phylogenetic analysis showed unidentified dioxygenases in ADE soils. Libraries containing functional gene encoding for the alpha subunit of the aromatic ring-hydroxylating dioxygenases (ARHD) gene from biochar show higher diversity indices than those of ADE under secondary forest and agriculture.

Explorando produtos naturais microbianos nas fronteiras da Química e da Biologia

The chemistry of natural products has been remarkably growing in the past few decades in Brazil. Aspects related to the isolation and identification of new natural products, as well as their biological activities, have been achieved in different laboratories working on this subject in the country. More recently, the introduction of new molecular biology tools has strongly influenced the research on natural products, mainly those produced by microorganisms, creating new possibilities to assess the chemical diversity of secondary metabolites. This paper describes some ideas on how the research on natural products can have a considerable input from molecular biology in the generation of chemical diversity. We also explore the role of microbial natural products in mediating interspecific interactions and their relevance to ecological studies. Examples of the generation of chemical diversity are highlighted by using genome mining, mutasynthesis, combinatorial biosynthesis, metagenomics, and synthetic biology, while some aspects of microbial ecology are also discussed. The idea to bring up this topic is linked to the remarkable development of molecular biology techniques to generate useful chemicals from different organisms. Here, we focus mainly on microorganisms, even though similar approaches have also been applied to the study of plants and other organisms. Investigations in the frontier of chemistry and biology require interactions between different areas, characterizing the interdisciplinarity of this research field. The necessity of a real integration of chemistry and biology is pivotal to finding correct answers to a number of biological phenomena. The use of molecular biology tools to generate chemical diversity and control biosynthetic pathways is largely explored in the production of important biologically active compounds. Finally, we briefly comment on the Brazilian organization of research in this area, the necessity of new strategies for the graduation programs, and the establishment of networks as a way of organization to overcome some of the problems faced in the area of natural products.

Bacterial ecology of tilapia fresh fillets and some factors that can influence their microbial quality

The purpose of research was to investigate the bacterial ecology of tilapia (Oreochromis niloticus) fresh fillets and some factors that can influence its microbial quality. Samples of fish cultivation water (n = 20), tilapia tegument and gut (n = 20) and fresh fillets (n = 20) were collected in an experimental tilapia aquaculture located in the city of Lavras, Minas Gerais, Brazil. Staphylococcus spp., Aeromonas spp., Enterococcus spp. and Enterobacteriaceae were quantified using selective plating. For the enumeration of Pseudomonas spp., the most probable number technique (MPN) was utilized. Bacterial colonies (n = 198) were identified by Gram strain and biochemical tests. Aeromonas spp., Pseudomonas spp., Enterococcus spp. and Enterobacteriaceae were found in the cultivation water (water from a fishpond cultivation), tegument, gut, and fresh fillets. Staphylococcus spp. was not isolated in the cultivation water. Salmonella spp. was not detected. The count variable of 10 to 10³ CFU or MPN.(g or mL)-1. Associated to freshwater tilapia fillet processing, there is a large variety of microorganisms related to foodborne illnesses and fish products deterioration.

Dinámica microbial del suelo asociada a diferentes estrategias de manejo de Phytophthora cinnamomi Rands en aguacate

La marchitez del aguacate es la enfermedad más limitante de este cultivo, cuyo agente causal más relevante es el oomycete Phytophthora cinnamomi Rands. Es por esto que se han desarrollado diferentes estrategias para su manejo integrado, pero aún prevalece el uso de productos químicos, como única medida de manejo, generando impactos negativos en el ambiente y la salud. Uno de los efectos perjudiciales que se ocasiona es la alteración de las poblaciones microbianas en el suelo. Este trabajo estuvo encaminado a conocer la dinámica microbiana del suelo, bajo diferentes estrategias de manejo de esta enfermedad, para lo cual se midió su dinamismo mediante unidades formadoras de colonias (UFC), para hongos, bacterias y actinomicetos, a partir de muestras de suelo y rizósfera de la raíz, bajo incubación en condiciones de anaerobiosis y aerobiosis, además se midió la actividad microbiana total, en condiciones de laboratorio, como complemento se cuantificaron microorganismos como: Trichiderma spp, bacterias formadoras de endosporas (BAFE), celulolíticos, proteolíticos, amilolíticos, solubilizadores de fosfato, fijadores asimbióticos de nitrógeno y promotores del crecimiento, como Pseudomonas spp., fluorescentes. Los resultados encontrados en esta investigación, sugieren que el uso individual y combinado de mantillo orgánico, material compostado de estiércol bovino, enmienda mineral y cascarilla de arroz y la propuesta de integración; incrementan significativamente la población y actividad microbiana aerobia, en la cual se identificaron microorganismos antagonistas como, Trichiderma spp., celulolíticos, Pseudomonas spp. fluorescentes y BAFE.

The ecology of Triatoma sordida in natural environments in two different regions of the state of Minas Gerais, Brazil

A study was undertaken about T. sordida in the natural environment in two different regions of the state of Minas Gerais: Itapagipe (Triângulo), an area of cerrado modified by the formation of fields of pasture and agriculture, and Mato Verde (north) an area of transition between caatinga and cerrado with profound deforestation in the last years due to the expansion of cotton cultivation. In both regions the principal ecotopes identified were hollow trees and the bark of live or dead trees, where the occurrence of a food source is not frequent. In this environment, the triatomines utilize various food sources; opposums appear to represent an important source of infection. In the north of Minas, a greater concentration of reservoirs and vectors was observed than in the Triángulo which could explain the higher level of infection of the triatomines in the north. Close attention to the process of domiciliation of T. sordida in the north of Minas is recommended where an extensive intervention by man in the natural environment has occurred and where a rise in the population of triatomines in the peridomestic environment has been observed in recent years.

New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

ASPECTS OF THE ECOLOGY OF PHLEBOTOMINES (Diptera: Psychodidae: Phlebotominae) IN AN AREA OF CUTANEOUS LEISHMANIASIS OCCURRENCE, MUNICIPALITY OF ANGRA DOS REIS, COAST OF RIO DE JANEIRO STATE, BRAZIL

Over a complete two-year period, phlebotomine specimens were caught in an area of cutaneous leishmaniasis occurrence in the municipality of Angra dos Reis. A manual suction tube was used to catch phlebotomines on house walls, and also light traps in domestic and peridomestic settings and in the forest. This yielded 14,170 specimens of 13 species: two in the genus Brumptomyia and eleven in the genus Lutzomyia. L. intermedia predominantly in domestic and peridomestic settings, with little presence in the forest, with the same trend being found in relation to L. migonei, thus proving that these species have adapted to the human environment. L. fischeri appeared to be eclectic regarding location, but was seen to be proportionally more endophilic. L. intermedia and L. migonei were more numerous in peridomestic settings, throughout the year, while L. fischeri was more numerous in domestic settings except in March, April, May and September. From the prevalence of L. intermedia, its proven anthropophily and findings of this species naturally infected with Leishmania(Viannia) braziliensis, it can be incriminated as the main vector for this agent of cutaneous leishmaniasis in the study area, especially in the peridomestic environment. L. fischeri may be a coadjuvant in carrying the parasite.

Ecology of Phlebotomine sandflies (Diptera, Psychodidae) in Brazilian Atlantic Forest

The phlebotomine sandfly fauna of a primary forest reserve at Morretes (eastern Paraná State) was studied, using CDC-like light traps, one night per month, at canopy and ground level, between April 1995 and March 1996. A total of 3,106 insects were collected, identified as belonging to nine species. Lutzomyia ayrozai and Lu.geniculata were predominant, seven other species also being present. Monthly mean temperature, rainfall and the temperature of the collection night significantly influenced the numbers of Lu. ayrozai while the two first factors influenced the numbers of Lu. geniculata, besides the collected quantities of females of the two species. The influence of the factors on Lu. ayrozai numbers was more immediate than in those of Lu. geniculata. Numbers of both species and of the females of Lu. geniculata collected in different seasons, but not at the different heights, varied significantly. Differences between the behaviour of Lu. ayrozai in Morretes and in other regions could be attributed to environmental differences and/or to regional variations in the species, which could constitute species complexes. Hourly variations of collections were different in the species and seasons.