Comparison of different protocols for the extraction of microbial DNA from reef corals

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Microbial Translocation Is Associated with Extensive Immune Activation in Dengue Virus Infected Patients with Severe Disease

Background: Severe dengue virus (DENV) disease is associated with extensive immune activation, characterized by a cytokine storm. Previously, elevated lipopolysaccharide (LPS) levels in dengue were found to correlate with clinical disease severity. In the present cross-sectional study we identified markers of microbial translocation and immune activation, which are associated with severe manifestations of DENV infection. Methods: Serum samples from DENV-infected patients were collected during the outbreak in 2010 in the State of Sa˜o Paulo, Brazil. Levels of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and IgM and IgG endotoxin core antibodies were determined by ELISA. Thirty cytokines were quantified using a multiplex luminex system. Patients were classified according to the 2009 WHO classification and the occurrence of plasma leakage/shock and hemorrhage. Moreover, a (non-supervised) cluster analysis based on the expression of the quantified cytokines was applied to identify groups of patients with similar cytokine profiles. Markers of microbial translocation were linked to groups with similar clinical disease severity and clusters with similar cytokine profiles. Results: Cluster analysis indicated that LPS levels were significantly increased in patients with a profound pro-inflammatory cytokine profile. LBP and sCD14 showed significantly increased levels in patients with severe disease in the clinical classification and in patients with severe inflammation in the cluster analysis. With both the clinical classification and the cluster analysis, levels of IL-6, IL-8, sIL-2R, MCP-1, RANTES, HGF, G-CSF and EGF were associated with severe disease. Conclusions: The present study provides evidence that both microbial translocation and extensive immune activation occur during severe DENV infection and may play an important role in the pathogenesis.

Is there a seamount effect on microbial community structure and biomass?: the case study of Seine and Sedlo seamounts (Northeast Atlantic)

[EN] Seamounts are considered to be ??hotspots?? of marine life but, their role in oceans primary productivity is still under discussion. We have studied the microbial community structure and biomass of the epipelagic zone (0?150 m) at two northeast Atlantic seamounts (Seine and Sedlo) and compared those with the surrounding ocean. Results from two cruises to Sedlo and three to Seine are presented. Main results show large temporal and spatial microbial community variability on both seamounts. Both Seine and Sedlo heterotrophic community (abundance and biomass) dominate during winter and summer months, representing 75% (Sedlo, July) to 86% (Seine, November) of the total plankton biomass. In Seine, during springtime the contribution to total plankton biomass is similar (47% autotrophic and 53% heterotrophic). Both seamounts present an autotrophic community structure dominated by small cells (nano and picophytoplankton). It is also during spring that a relatively important contribution (26%) of large cells to total autotrophic biomass is found. In some cases, a ??seamount effect?? is observed on Seine and Sedlo microbial community structure and biomass. In Seine this is only observed during spring through enhancement of large autotrophic cells at the summit and seamount stations. In Sedlo, and despite the observed low biomasses, some clear peaks of picoplankton at the summit or at stations within the seamount area are also observed during summer. Our results suggest that the dominance of heterotrophs is presumably related to the trapping effect of organic matter by seamounts. Nevertheless, the complex circulation around both seamounts with the presence of different sources of mesoscale variability (e.g. presence of meddies, intrusion of African upwelling water) may have contributed to the different patterns of distribution, abundances and also changes observed in the microbial community.

Patterns in marine microbial community structure

Programa en Oceanografía

Krill excretion boosts microbial activity in the Southern Ocean

[EN]Antarctic krill are known to release large amounts of inorganic and organic nutrients to the water column. Here we test the role of krill excretion of dissolved products in stimulating heterotrophic bacteria on the basis of three experiments where ammonium and organic excretory products released by krill were added to bacterial assemblages, free of grazers. Our results demonstrate that the addition of krill excretion products (but not of ammonium alone), at levels expected in krill swarms, greatly stimulates bacteria resulting in an order-of-magnitude increase in growth and production. Furthermore, they suggest that bacterial growth rate in the Southern Ocean is suppressed well below their potential by resource limitation. Enhanced bacterial activity in the presence of krill, which are major sources of DOC in the Southern Ocean, would further increase recycling processes associated with krill activity, resulting in highly efficient krill-bacterial recycling that should be conducive to stimulating periods of high primary productivity in the Southern Ocean.

Abundance and diversity of marine microbial eukaryotes

[EN]Microeukaryotes are important ecological players in any kind of ecosystem, most notably in the ocean, and it is therefore essential to collect information about their abundance and diversity. To achieve this general goal this thesis was structured in two parts. The first part represents an effort to define our “diversity unit” from studies based on the well-known cloning and Sanger sequencing approach. Basically, we wanted to establish a solid baseline for the second part of the thesis. We started with data from one cruise (Chapter 1) and then continued with the analysis of the complete dataset of 18S DNA sequences available at that time (Chapter 2).

Changes in plant metabolism induced by dioxygenase inhibitors and their effect on the epiphytic microbial community and fire blight (Erwinia amylovora) control

Fire blight, caused by the gram negative bacterium Erwinia amylovora, is one of the most destructive bacterial diseases of Pomaceous plants. Therefore, the development of reliable methods to control this disease is desperately needed. This research investigated the possibility to interfere, by altering plant metabolism, on the interactions occurring between Erwinia amylovora, the host plant and the epiphytic microbial community in order to obtain a more effective control of fire blight. Prohexadione-calcium and trinexapac-ethyl, two dioxygenase inhibitors, were chosen as a chemical tool to influence plant metabolism. These compounds inhibit the 2-oxoglutarate-dependent dioxygenases and, therefore, they greatly influence plant metabolism. Moreover, dioxygenase inhibitors were found to enhance plant resistance to a wide range of pathogens. In particular, dioxygenase inhibitors application seems a promising method to control fire blight. From cited literature, it is assumed that these compounds increase plant defence mainly by a transient alteration of flavonoids metabolism. We tried to demonstrate, that the reduction of susceptibility to disease could be partially due to an indirect influence on the microbial community established on plant surface. The possibility to influence the interactions occurring in the epiphytic microbial community is particularly interesting, in fact, the relationships among different bacterial populations on plant surface is a key factor for a more effective biological control of plant diseases. Furthermore, we evaluated the possibility to combine the application of dioxygenase inhibitors with biological control in order to develop an integrate strategy for control of fire blight. The first step for this study was the isolation of a pathogenic strain of E. amylovora. In addition, we isolated different epiphytic bacteria, which respond to general requirements for biological control agents. Successively, the effect of dioxygenase inhibitors treatment on microbial community was investigated on different plant organs (stigmas, nectaries and leaves). An increase in epiphytic microbial population was found. Further experiments were performed with aim to explain this effect. In particular, changes in sugar content of nectar were observed. These changes, decreasing the osmotic potential of nectar, might allow a more consistent growth of epiphytic bacteria on blossoms. On leaves were found similar differences as well. As far as the interactions between E. amylovora and host plant, they were deeply investigated by advanced microscopical analysis. The influence of dioxygenase inhibitors and SAR inducers application on the infection process and migration of pathogen inside different plant tissues was studied. These microscopical techniques, combined with the use of gpf-labelled E. amylovora, allowed the development of a bioassay method for resistance inducers efficacy screening. The final part of the work demonstrated that the reduction of disease susceptibility observed in plants treated with prohexadione-calcium is mainly due to the accumulation of a novel phytoalexins: luteoforol. This 3-deoxyflavonoid was proven to have a strong antimicrobial activity.

Changes in the microbial community based on seawater pH variations

Ocean acidification is an effect of the rise in atmospheric CO2, which causes a reduction in the pH of the ocean and generates a number of changes in seawater chemistry and consequently potentially impacts seawater life. The effect of ocean acidification on metabolic processes (such as net community production and community respiration and on particulate organic carbon (POC) concentrations was investigated in summer 2012 at Cap de la Revellata in Corsica (Calvi, France). Coastal surface water was enclosed in 9 mesocosms and subjected to 6 pCO2 levels (3 replicated controls and 6 perturbations) for approximately one month. No trend was found in response to increasing pCO2 in any of the biological and particulate analyses. Community respiration was relatively stable throughout the experiment in all mesocosms, and net community production was most of the time close to zero. Similarly, POC concentrations were not affected by acidification during the whole experimental period. Such as the global ocean, the Mediterranean Sea has an oligotrophic nature. Based on present results, it seems likely that seawater acidification will not have significant effects on photosynthetic rates, microbial metabolism and carbon transport.

Characterization and exploiment of Microbial Strains to be used in Meat Processing and Fermentation

The principal aim of this research project has been the evaluation of the specific role of yeasts in ripening processes of dry-cured meat products, i.e. speck and in salami produced by adding Lactobacilli starter cultures, i.e. L. sakei, L. casei, L. fermentum, L. rhamnosus, L.sakei + S.xylosus. In particular the contribution of the predominant yeasts to the hydrolytic patterns of meat proteins has been studied both in model system and in real products. In fact, although several papers have been published on the microbial, enzymatic, aromatic and chemical characterization of dry-cured meat e.g. ham over ripening, the specific role of yeasts has been often underestimated. Therefore this research work has been focused on the following aspects: 1. Characterization of the yeasts and lactic acid bacteria in samples of speck produced by different farms and analyzed during the various production and ripening phases 2. Characterization of the superficial or internal yeasts population in salami produced with or without the use of lactobacilli as starter cultures 3. Molecular characterization of different strains of yeasts and detection of the dominant biotypes able to survive despite environmental stress factors (such as smoke, salt) 4. Study of the proteolytic profiles of speck and salami during the ripening process and comparison with the proteolytic profiles produced in meat model systems by a relevant number of yeasts isolated from speck and salami 5. Study of the proteolytic profiles of Lactobacilli starter cultures in meat model systems 6. Comparative statistical analysis of the proteolytic profiles to find possible relationships between specific bands and peptides and specific microorganisms 7. Evaluation of the aromatic characteristics of speck and salami to assess relationships among the metabolites released by the starter cultures or the dominant microflora

Evaluation of Microbial Contamination in Bivalve Mollusks: Epidemiology and Diagnosis

Shellfish are filter-feeding organisms that can accumulate many bacteria and viruses. Considering that depuration procedures are not effective in removal of certain microorganisms, shellfish-borne diseases are frequent in many parts of the world, and their control must rely primarily on investigation of prevalence of human pathogens in shellfish and water environment. However, the diffusion of enteric viruses and Vibrio bacteria is not known in many geographical areas, for example in Sardinia, Italy. A survey aimed at investigating the prevalence of Norovirus (NoV), hepatitis A virus (HAV), V. parahaemolyticus, V. cholerae and V. vulnificus was carried out, analyzing both local and imported purified, non-purified and retail shellfish from North Italy and Sardinia. Shellfish from both areas were found contaminated by NoVs, HAV and Vibrio, including retail and purified animals. Molecular analysis evidenced different NoV genogroups and genotypes, including bovine NoVs, as well as pathogenic Vibrio strains, underlining the risk for shellfish consumers. However, also other approaches are needed to control the diffusion of shellfish-borne diseases. It was originally thought that enteric viruses are passively accumulated by shellfish. Recently, it was proven that NoVs bind to specific carbohydrate ligands in oysters, and various NoV strains are characterized by a different bioaccumulation pattern. To deepen the knowledge on this argument, a study was carried out, analyzing bioaccumulation of up to 8 different NoV strains in four different species of shellfish. Different bioaccumulation patterns were observed for each shellfish species and NoV strain used, potentially important in setting up effective shellfish purification protocols. Finally, a novel study of evaluation of viral contamination in shellfish from the French Atlantic coast was carried out following the passage of Xynthia tempest over Western Europe which caused massive destruction. Different enteric viruses were found over a one month period, evidencing the potential of these events of contaminating shellfish.

Diversity of microbial communities at shallow-hydrothermal vents off Dominica, Lesser Antilles

Hydrothermal vents are often compared to desert oases, because of the presence of highly diverse and abundant biotic communities inhabiting these extreme environments. Nevertheless, the microbial communities associated with shallow-hydrothermal systems have been poorly studied. Hydrothermal activity at Dominica Island is quite well known under the geological and geochemical aspects, but no previous information existed about the microbial communities associated to this area. This thesis is therefore targeting the microbiology of hydrothermal sediments combining geochemical and molecular biological investigations, focusing on differences between hydrothermal vents and background (i.e. control) areas, and between hydrothermal sites. It was also intended to assess relationship between geochemical parameters and microbial diversity at the two hydrothermally impacted sites. Two shallow-sea hydrothermal vents located south-west off Dominica Island (Lesser Antilles) have been investigated in this study: Champagne Hot Springs and Soufriere Bay offshore vent. During this study, sediments for geochemical and molecular analyses were collected every 2 cm from the two impacted areas and from two control sites not associated with hydrothermal activity; in situ temperatures measurements were also taken every 5 cm deep in the sediment for all the sites. A geochemical characterization of the sediment porewater was performed through the analysis of several elements’ concentrations (i.e. H2S, Cl-, Br-, SO42-, Fe2+, Na+, K+, B+, Si+). Microbial communities at the different sites were studied by Automated Intergenic Spacer Analysis (ARISA). Inspection of the operational taxonomic units (OTUs) distribution was performed, as well as statistical analyses for communities’ structure and composition differences, and for changes of β-diversity along with sediment geochemistry. Data suggested that mixing between hydrothermal fluids and seawater results in distinct different environmental gradients and potential ecological niches between the two investigated hydrothermal vents, reflecting a difference in microbial community structures between them.