Assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1H NMR spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism. In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for competitive fitness against bacteria and yeasts

When analysing the secretome of the plant pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, we identified hemolysin co-regulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the Pst DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of hcp genes and tested the fitness of hcp knock-out mutants in host plant colonization and in inter-microbial competition. We found that the hcp2 gene is expressed, most actively at the stationary growth phase, and that the Hcp2 protein is secreted via T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and it does not contribute to virulence or colonisation in tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition against yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive in conditions where it has to compete with other micro-organisms for resources.

The evolutionary basis for differences between the immune systems of man, mouse, pig and ruminants

Studying the pathogenesis of an infectious disease like colibacillosis requires an understanding of the responses of target hosts to the organism both as a pathogen and as a commensal. The mucosal immune system constitutes the primary line of defence against luminal micro-organisms. The immunoglobulin-superfamily-based adaptive immune system evolved in the earliest jawed vertebrates, and the adaptive and innate immune system of humans, mice, pigs and ruminants co-evolved in common ancestors for approximately 300 million years. The divergence occurred only 100 mya and, as a consequence, most of the fundamental immunological mechanisms are very similar. However, since pressure on the immune system comes from rapidly evolving pathogens, immune systems must also evolve rapidly to maintain the ability of the host to survive and reproduce. As a consequence, there are a number of areas of detail where mammalian immune systems have diverged markedly from each other, such that results obtained in one species are not always immediately transferable to another. Thus, animal models of specific diseases need to be selected carefully, and the results interpreted with caution. Selection is made simpler where specific host species like cattle and pigs can be both target species and reservoirs for human disease, as in infections with Escherichia coli.

Rooting theories of plant community ecology in microbial interactions

Predominant frameworks for understanding plant ecology have an aboveground bias that neglects soil micro-organisms. This is inconsistent with recent work illustrating the importance of soil microbes in terrestrial ecology. Microbial effects have been incorporated into plant community dynamics using ideas of niche modification and plant–soil community feedbacks. Here, we expand and integrate qualitative conceptual models of plant niche and feedback to explore implications of microbial interactions for understanding plant community ecology. At the same time we review the empirical evidence for these processes. We also consider common mycorrhizal networks, and propose that these are best interpreted within the feedback framework. Finally, we apply our integrated model of niche and feedback to understanding plant coexistence, monodominance and invasion ecology.

Hematologically important mutations: X-linked chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations. (C) 2010 Elsevier Inc. All rights reserved.

Quantitative detection of periodontopathic bacteria in atherosclerotic plaques from coronary arteries

Oral pathogens, including periodontopathic bacteria, are thought to be aetiological factors in the development of cardiovascular disease. In this study, the presence of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum-periodonticum-simiae group, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythia in atheromatous plaques from coronary arteries was determined by real-time PCR. Forty-four patients displaying cardiovascular disease were submitted to periodontal examination and endarterectomy of coronary arteries. Approximately 60-100 mg atherosclerotic tissue was removed surgically and DNA was obtained. Quantitative detection of periodontopathic bacteria was performed using universal and species-specific TaqMan probe/primer sets. Total bacterial and periodontopathic bacterial DNA were found in 94.9 and 92.3 %, respectively, of the atheromatous plaques from periodontitis patients, and in 80.0 and 20.0%, respectively, of atherosclerotic tissues from periodontally healthy subjects. All periodontal bacteria except for the F. nucleatum-periodonticum-simiae group were detected, and their DNA represented 47.3 % of the total bacterial DNA obtained from periodontitis; patients. Porphyromonas gingivalis, A. actinomycetemcomitans and Prevotella intermedia were detected most often. The presence of two or more periodontal species could be observed in 64.1 % of the samples. In addition, even in samples in which a single periodontal species was detected, additional unidentified microbial DNA could be observed. The significant number of periodontopathic bacterial DNA species in atherosclerotic tissue samples from patients with periodontitis suggests that the presence of these micro-organisms in coronary lesions is not coincidental and that they may in fact contribute to the development of vascular diseases.

Effects of Aspergillus spp. exogenous fibrolytic enzymes on in vitro fermentation of tropical forages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of a gene encoding adaptin-like protein in the Paracoccidioides brasiliensis genome by random amplified polymorphic DNA analysis

Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pbl 8) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pbl Ba by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein beta-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.

Curcumin modulates the immune response associated with LPS-induced periodontal disease in rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Jejum Pré-abate em Frangos de Corte

As condições ideais dos frangos de corte no momento do abate devem ser conhecidas a fim de possibilitar a produção de carne de excelente qualidade, uma vez que diversos fatores pré e pós-abate estão envolvidos na qualidade final. em condições normais de abate e processamento, a retirada de ração é feita de 6 a 8 horas antes da apanha das aves, resultando em um período total de jejum de 8 a 12 horas antes do abate, para esvaziar o intestino e com isso minimizar a contaminação no abatedouro. A escalda, depena e evisceração são pontos importantes de contaminação cruzada no abatedouro devido à grande quantidade de microorganismos aderidos às penas, pele e patas das aves e ao rompimento das vísceras durante a evisceração. Entretanto, a desidratação da carcaça começa imediatamente após o início do jejum. Períodos prolongados de jejum podem afetar o pH das diversas partes do intestino, aumentando a presença de Salmonella e outros microorganismos patogênicos. Além disso, determinam uma maior contaminação pela bile, e são, subjetivamente, associados à fragilidade dos intestinos durante a evisceração mecânica. Portanto, os esquemas de processamento devem ser estabelecidos levando-se em conta a integridade e o esvaziamento do intestino e da vesícula biliar, bem como a desidratação e os seus efeitos sobre o bem estar das aves, contaminação da carcaça e qualidade da carne. Como alguns efeitos do jejum ainda não são bem conhecidos, sugerem-se pesquisas nas seguintes áreas: definir o tempo ótimo de jejum para atender o bem estar das aves, minimizar a contaminação e otimizar os parâmetros de qualidade de carcaça; estudar os efeitos de períodos prolongados de jejum sobre o pH e a colonização do papo, pró-ventrículo, moela, intestino delgado, intestino grosso e cecos por enterobactérias, como Salmonella, por exemplo; efeito do jejum sobre o tamanho e cor do fígado. O resultado esperado é um aumento na qualidade final dos produtos aliado a uma redução nas perdas e no custo de produção.

An infection control protocol: effectiveness of immersion solutions to reduce the microbial growth on dental prostheses

This investigation evaluated the effectiveness of an infection control protocol for cleansing and disinfecting removable dental prostheses. Sixty-four dentures were rubbed with sterile cotton swab immediately after they had been taken from patients' mouths. Samples were individually placed in the culture medium and immediately incubated at 37 +/- 2 degreesC. The dentures were scrubbed for 1 min with 4% chlorhexidine, rinsed for 1 min in sterile water and placed for 10 min in one of the following immersion solutions: 4% chlorhexidine gluconate, 1% sodium hypochlorite, Biocide (iodophors) and Amosan (alkaline peroxide). After the disinfection procedures, the dentures were immersed in sterile water for 3 min, reswabbed and the samples were incubated. All samples obtained in the initial culture were contaminated with micro-organisms. All the lower dentures immersed in Biocide showed positive growth, and the upper dentures were positive for growth in six of eight dentures. The 4% chlorhexidine gluconate, 1% sodium hypochlorite and Amosan solutions have been proved effective to reduce the growth of the micro-organisms in the 10 min immersion period. The protocol evaluated in this study seems to be a viable method to prevent cross-contamination between dental personnel and patients.

Skin and pulmonary models using coated bentonite particles for the study of the inflammation evoked by Paracoccidioides brasiliensis antigens in previously immunized mice

Bentonite particles coated with polysaccharide antigen or crude soluble antigen of Paracoccidioides brasiliensis were injected intradermally or intravenously in mice. In control animals that were not pre-immunized with P. brasiliensis antigens, coated and uncoated bentonite caused minimal and nonspecific inflammation around the cutaneous injection site or around the bentonite thrombi in small lung vessels after intravenous injection. However, in mice previously immunized with P. brasiliensis antigens, the coated bentonite particles boosted the humoral and cellular immune responses to P. brasiliensis and evoked intense inflammatory reactions. Twelve days after intradermal injection, the inflammatory reaction around the bentonite was rich in neutrophils, macrophages, lymphocytes and plasma cells associated with young granulation tissue. In intravenously injected mice, the pulmonary inflammation was maximal at day 2, and was characterized by a florid neutrophilic and macrophagic cellular infiltration around bentonite thrombi; in some foci, there was incipient organization to mature granuloma. However, in both models, there was no formation of epithelioid granulomata, demonstrating that in paracoccidioidomycosis cellular immunity alone, without the presence of intact micro-organisms, may not be enough for the development of this type of granuloma.

Cumulative effects of vinasse on the characteristics of red-yellow latosols under cerrado vegetation

Vinasse, a liquor effluent from the alcohol and sugar making industry, was applied annually for twelve years to medium-textured red-yellow latosols under cerrado vegetation sensu stricto, to study the environmental impacts on the biotic and abiotic factors. Four plots were established of which two acted as control and the other two received annual doses of vinasse. The studies were begun in 1980 when the first annual dose of 20 L m-2 year-1 was added to the soil without removing the top scrub layer. Theses doses were added to the soil until 1983, but in 1984 the doses were increased to 50 L m-2 year-1 and used until 1991. Soil samples were taken at a depth of 15 cm every three months from 1987 to 1991. Twenty seven environmental variables in the vinasse-treated and untreated plots were studied. These factors consisted of different enzymatic activities, a number of filamentous fungi, bacteria, actinomycetes and other micro-organisms, nutrients and some micro-climatic factors. The results obtained were statistically analyzed using the Tukey test, Pearson correlation and variance test methods with replicates and three factors. Matrices were determined using the correlation coefficient method and were compared with those of earlier published studies in the same area. The comparison of the results helped characterize changes in the environmental factors studied and in the correlation between them, after using annual cumulative doses of vinasse. Positive effects were observed only for the first six years of this application but vinasse had negative effects after the seventh year. It is concluded that medium-textured red-yellow latosols cannot be treated with vinasse for proloned periods.

Microbiological quality of ice used to refrigerate foods

Ice used for human consumption or to refrigerate foods can be contaminated with pathogenic microorganisms and may become a vehicle for human infection. To evaluate the microbiological content of commercial ice and ice used to refrigerate fish and seafood, 60 ice samples collected at six different retail points in the city of Araraquara, SP, Brazil, were studied. The following parameters were determined: total plate counts (37° C and 4° C), most probable number (MPN) for total coliforms, fecal coliforms and Escherichia coli, presence of Salmonella spp., Shigella spp., Yersinia spp., E. coli, Vibrio cholerae and Aeromonas spp.. Results suggested poor hygienic conditions of ice production due to the presence of indicator micro-organisms. Fifty strains of E. coli of different serotypes, as well as one Y. enterocolitica biotype 1, serogroup 0:5, 27 and phage type Xz (Ye 1/05,27/Xz) and one Salmonella Enteritidis phage type 1 (PT1) were isolated. Aeromonas spp., Shigella spp. and V. cholerae were not detected. The presence of high numbers of coliforms, heterotrophic indicator micro-organisms and pathogenic strains suggested that commercial ice and ice used to refrigerate fish and seafood may rep resent a potential hazard to the consumer in our community. © 2002 Elsevier Science Ltd. All rights reserved.