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.

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

Lankesterella alencari n. sp., a toxoplasma-like organism in the central nervous system of Amphibia (Protozoa, Sporozoa)

Lankesterella alencari n. sp. a Sporozoa that occur in the blood and CNS of the South American frog Leptodactylus acellatus is described. Since the tissue forms of this parasite have been previously reported as belonging to the genus Toxoplasma, we attempted in fection of 2 species of amphibia (Bufo marinus an dLeptodactylus ocellatus) with a Toxoplasma strain of human origen; inoculation was by intraperitoneal injection of parasite-containing ascitic fluid from infected mice. Attempt of experimental inoculation of the parasite found in the CNS of L. ocellatus in a highly susceptible host (mice) was unsuccessful. These results suggest that Toxoplasma does not occur naturally in the amphibia; be related to Toxoplasma is excluded. The following genera of haematozoa found in brazilian amphibia have been considered briedfly: Haemobartonella, Cytamoeba, Dactylosoma, Hepatozoon and Trypanosoma.

Immunomodulation by microbial ribosomes

Over the past twenty years, many authors have reported evidence of the immunoprotective capacity of ribosomes isolated from bacteria, fungi and parasites. Since 1971 we have explored the protective capacity of ribosomes isolated from a large variety of microorganisms responsible for human and animal diseases. More recently, using monoclonal antibodies raised against ribosomes and then selected for their ability to confer passive immunity to mice, we have studied the mechanism of the protection induced by ribosomes. These studies, in parallel with the development of a technology for the large scale production of ribosomes, have allowed us to achieve a new regard for ribosomal vaccines for use in human. The general concept of ribosomal vaccines in presented and examples of two such vaccines are described with data on the specific protection that they induce in mice against experimental infections with Klebsiella peneumoniae, Streptococcus pneumoniae, S. pyogenes and Haemophilus influenzae for the first one, and against Candida albicans type A and type B for the second one. Because of their high immunogenicity and their innocuity these vaccines represent a decisive improvement over classical microbial vaccines.

Biological Parameters and Molecular Markers of Clone CL Brener - The Reference Organism of the Trypanosoma cruzi Genome Project

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. Some biological parameters of CL Brener were determined: (a) the doubling time of epimastigote forms cultured in liver infusion-tryptose (LIT) medium at 28oC is 58±13 hr; (b) differentiation of epimastigotes to metacyclic trypomastigotes is obtained by incubation in LIT-20% Grace´s medium; (c) trypomastigotes infect mammalian cultured cells and perform the complete intracellular cycle at 33 and 37oC; (d) blood forms are highly infective to mice; (e) blood forms are susceptible to nifurtimox and benznidazole. The molecular typing of CL Brener has been determined: (a) isoenzymatic profiles are characteristic of zymodeme ZB; (b) PCR amplification of a 24Sa ribosomal RNA sequence indicates it belongs to T. cruzi lineage 1; (c) schizodeme, randomly amplified polymorphic DNA (RAPD) and DNA fingerprinting analyses were performed

Microbial flora variations in the respiratory tract of mice

A stable microbial system in the respiratory tract acts as an important defense mechanism against pathogenic microorganisms. Perturbations in this system may allow pathogens to establish. In an ecological environment such as the respiratory tract, there are many diverse factors that play a role in the establishment of the indigenous flora. In the present work we studied the normal microbial flora of different areas of the respiratory tract of mice and their evolution from the time the mice were born. Our interest was to know which were the dominant groups of microorganisms in each area, which were the first capable of colonizing and which dominated over time to be used as probiotic microorganisms. Our results show that Gram negative facultatively anaerobic bacilli and strict anaerobic microorganisms were the last ones to appear in the bronchia, while aerobic and Gram positive cocci were present in all the areas of the respiratory tract. The number of facultative aerobes and strict anaerobes were similar in the nasal passage, pharynx instilled and trachea, but lower in bronchia. The dominant species were Streptococcus viridans and Staphylococcus saprophyticcus, followed by S. epidermidis, Lactobacilli and S. cohnii I which were present on every studied days but at different proportions. This paper is the first part of a research topic investigating the protective effect of the indigenous flora against pathogens using the mice as an experimental model.

Coevolutionary networks: a novel approach to understanding the relationships of humans with the infectious agents

Human organism is interpenetrated by the world of microorganisms, from the conception until the death. This interpenetration involves different levels of interactions between the partners including trophic exchanges, bi-directional cell signaling and gene activation, besides genetic and epigenetic phenomena, and tends towards mutual adaptation and coevolution. Since these processes are critical for the survival of individuals and species, they rely on the existence of a complex organization of adaptive systems aiming at two apparently conflicting purposes: the maintenance of the internal coherence of each partner, and a mutually advantageous coexistence and progressive adaptation between them. Humans possess three adaptive systems: the nervous, the endocrine and the immune system, each internally organized into subsystems functionally connected by intraconnections, to maintain the internal coherence of the system. The three adaptive systems aim at the maintenance of the internal coherence of the organism and are functionally linked by interconnections, in such way that what happens to one is immediately sensed by the others. The different communities of infectious agents that live within the organism are also organized into functional networks. The members of each community are linked by intraconnections, represented by the mutual trophic, metabolic and other influences, while the different infectious communities affect each other through interconnections. Furthermore, by means of its adaptive systems, the organism influences and is influenced by the microbial communities through the existence of transconnections. It is proposed that these highly complex and dynamic networks, involving gene exchange and epigenetic phenomena, represent major coevolutionary forces for humans and microorganisms.

Bacterial and fungal colonization of burn wounds

A prospective study of fungal and bacterial flora of burn wounds was carried out from February 2004 to February 2005 at the Burns Unit of Hospital Regional da Asa Norte, Brasília, Brazil. During the period of the study, 203 patients were treated at the Burns Unit. Wound swab cultures were assessed at weekly intervals for four weeks. Three hundred and fifty four sampling procedures (surface swabs) were performed from the burn wounds. The study revealed that bacterial colonization reached 86.6% within the first week. Although the gram-negative organisms, as a group, were more predominant, Staphylococcus aureus (28.4%) was the most prevalent organism in the first week. It was however surpassed by Pseudomonas aeruginosa form third week onwards. For S. aureus and P. aeruginosa vancomycin and polymyxin were found to be the most effective drugs. Most of the isolates showed high level resistance to antimicrobial agents. Fungi were found to colonize the burn wound late during the second week postburn, with a peak incidence during the third and fourth weeks. Species identification of fungi revealed that Candida tropicalis was the most predominant, followed by Candida parapsilosis. It is crucial for every burn institution to determine the specific pattern of burn wound microbial colonization, the time-related changes in the dominant flora, and the antimicrobial sensitivity profiles. This would enable early treatment of imminent septic episodes with proper empirical systemic antibiotics, without waiting for culture results, thus improving the overall infection-related morbidity and mortality.

In vitro anti-microbial activity of the Cuban medicinal plants Simarouba glauca DC, Melaleuca leucadendron L and Artemisia absinthium L

In the present study, an extensive in vitro antimicrobial profiling was performed for three medicinal plants grown in Cuba, namely Simarouba glauca, Melaleuca leucadendron and Artemisia absinthium. Ethanol extracts were tested for their antiprotozoal potential against Trypanosoma b. brucei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum. Antifungal activities were evaluated against Microsporum canis and Candida albicans whereas Escherichia coli and Staphylococcus aureus were used as test organisms for antibacterial activity. Cytotoxicity was assessed against human MRC-5 cells. Only M. leucadendron extract showed selective activity against microorganisms tested. Although S. glauca exhibited strong activity against all protozoa, it must be considered non-specific. The value of integrated evaluation of extracts with particular reference to selectivity is discussed.

Microbial contamination of procedure gloves after opening the container and during exposure in the environment

The objective of this study was to quantify the colony forming units (cfu) on latex procedure gloves in the beginning, middle, and end of the containers in real (professional) and controlled (researcher) gloving situations; evaluate the microbial load of the gloves, considering the time of exposure in the environment. This comparative prospective study was conducted at an intensive care unit of a teaching hospital. The microbiological data was collected from the gloves using digital-pressure. Microbiological evaluations were performed on 186 pairs of gloves: 93 in the control group and 93 in real gloving situations. In the control group, the average cfu was 4.7 against 6.2 in the real gloving situation. Hence, no statistically significant difference was found (p=.601). In addition, the cfu values of gloves in the beginning, middle and end of the containers also did not show any significant differences (p>.05). The most common strain was Staphylococcus spp. The time of exposure in the environment did not increase the cfu value of the latex gloves.

Nitrogen fractions in the microbial biomass in soils of southern Brazil

The reaction of nitrogen compounds with ninhydrin can be used as an indicator of cytoplasmic materials released from microbial cells killed by fumigation. Total-N, ninhydrin-reactive-N (NR-N), ammonium-N (A-N), and α-amino-N in the microbial biomass of soils from the State of Rio Grande do Sul, Brazil, were determined, in 1996, in 0.5 mol L-1 K2SO4 extracts of fumigated and non-fumigated soils. Total-N varied from 20.3 to 104.4 mg kg-1 and the ninhydrin-reactive-N corresponded, in average, to 27% of this. The ninhydrin-reactive-N was made up of 67% ammonium-N and 33% aminoacids with the amino group at the α-carbon position. It was concluded that colorimetric analysis of NR-N and A-N may be used as a direct measure of microbial N in soil. This simple and rapid procedure is adequate for routine analyses.

Biomassa microbiana e produção de C-CO2 e N mineral de um podzólico vermelho-escuro submetido a diferentes sistemas de manejo: Microbial biomass and C-CO2 and mineral nitrogen production in paleudult soil cultivated under different management systems

Os sistemas de manejo, com diferenças no revolvimento do solo e na composição dos resíduos vegetais, alteram as propriedades biológicas do solo, com reflexos na qualidade do solo e na produtividade das culturas. Com vistas em medir estas alterações nas propriedades biológicas do solo, a biomassa e a atividade microbiana foram avaliadas em um Podzólico Vermelho-Escuro, em Eldorado do Sul (RS), utilizando diferentes preparos (convencional, reduzido e plantio direto) e dois sistemas de sucessões de culturas (aveia preta + vica/milho + caupi e aveia/milho). As avaliações foram realizadas em quatro épocas, durante 12 meses, e em duas profundidades (0-5 e 5-15 cm). O carbono da biomassa microbiana foi analisado pelo método de fumigação-incubação, e a atividade microbiana, pela produção de C-CO2 e N mineral, após 60 dias de incubação. As diferenças na biomassa e na atividade microbiana, entre os sistemas de manejo, foram mais pronunciadas na camada de 0-5 cm. Nesta camada de solo, observaram-se os maiores valores de biomassa e de atividade nos preparos conservacionistas e no sistema aveia + vica/milho + caupi. Dentre as variáveis estudadas, a mineralização de N mostrou-se a mais sensível aos manejos, à profundidade e à época de amostragem.

Bacteria diversity and microbial biomass in forest, pasture and fallow soils in the southwestern Amazon basin

It is well-known that Amazon tropical forest soils contain high microbial biodiversity. However, anthropogenic actions of slash and burn, mainly for pasture establishment, induce profound changes in the well-balanced biogeochemical cycles. After a few years the grass yield usually declines, the pasture is abandoned and is transformed into a secondary vegetation called "capoeira" or fallow. The aim of this study was to examine how the clearing of Amazon rainforest for pasture affects: (1) the diversity of the Bacteria domain evaluated by Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE), (2) microbial biomass and some soil chemical properties (pH, moisture, P, K, Ca, Mg, Al, H + Al, and BS), and (3) the influence of environmental variables on the genetic structure of bacterial community. In the pasture soil, total carbon (C) was between 30 to 42 % higher than in the fallow, and almost 47 % higher than in the forest soil over a year. The same pattern was observed for N. Microbial biomass in the pasture was about 38 and 26 % higher than at fallow and forest sites, respectively, in the rainy season. DGGE profiling revealed a lower number of bands per area in the dry season, but differences in the structure of bacterial communities among sites were better defined than in the wet season. The bacterial DNA fingerprints in the forest were stronger related to Al content and the Cmic:Ctot and Nmic:Ntot ratios. For pasture and fallow sites, the structure of the Bacteria domain was more associated with pH, sum of bases, moisture, total C and N and the microbial biomass. In general microbial biomass in the soils was influenced by total C and N, which were associated with the Bacteria domain, since the bacterial community is a component and active fraction of the microbial biomass. Results show that the genetic composition of bacterial communities in Amazonian soils changed along the sequence forest-pasture-fallow.