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To evaluate the taxonomic profile of the gut microbiota using metagenomics and the association with diet-dependent childhood obesity.A cross-sectional study of a subsample of 46 children was conducted. The children were classified as normal-weight, overweight, and obese according to their age and sex and the World Health Organization (WHO) guidelines. Dietary patterns were determined through principal component analysis. The profile of the human gut microbiota was determined by bioinformatic analysis using whole metagenome shotgun sequencing. The association of gut microbiota and z-BMI, waist circumference and hip circumference, and the possible modifying effect of diet were analyzed using multiple regression models.Children with an abundance of Holdemania spp. and high protein and complex carbohydrate consumption had a lower z-BMI (β -19.06, p = 0.011), waist circumference (β -171.92, p = 0.003), and hip circumference (β -157.57, p = 0.004). In contrast, observed a positive association between Coprococcus catus and the low intake of this dietary pattern with hip circumference (β 147.87, p = 0.025). Furthermore, the presence of Bilophila spp. and Paraprevotella xylaniphila with high saturated fat and simple carbohydrate consumption we observed a positive association between z-BMI (β 47.5, p = 0.002), hip circumference (β 44.54, p = 0.025), and waist circumference (β 44.34, p = 0.004).We suggest that the synergism between diet and the profile of children's gut microbiota can be a factor that could be associated with the development of obesity and its complications in childhood.

In the present study, we evaluated the biogas production from the anaerobic co-digestion of raw glycerol (RG) in combination with swine manure (SM) and the interaction of the microbial community during anaerobic co-digestion. In the same way, different RG and SM concentrations were examined in the range of 6 to 15 g L−1 and 7.5 to 22.5 g L−1, respectively. According to the results, the highest values for biogas yield and methane yield were observed in test 1(6 g RG/7.5 g SM) with 340 mL g−1 COD, test 2 (6 g RG/22.5 g SM) with 330 mL g−1 COD, test 4 (15 g RG/22.5 g SM) with 344 mL g−1 COD, and test 5 (3.75 g RG/15 g SM) with 328 mL g−1 COD. Although the concentration of substrates increases, glycerol remains between 20 and 21% of RG for both tests (2 and 5). An increase in the concentration of RG (> 21% w/w) generated inhibitory effects for the co-digestion process since there was an overload of organic acids, which increase the impurity concentration and lower the biogas yield. Thus, the microbial consortium is a function of the organic load of the RG/SM ratio. The greater the concentration of RG, the higher the population of clostridium and low biogas yield in anaerobic co-digestion.

Catechol 1,2 dioxygenases (C12DOs) have been studied for its ability to cleavage the benzene ring of catechol, the main intermediate in the degradation of aromatic compounds derived from aerobic degradation of hydrocarbons. Here we report the genome sequence of the marine bacterium Pseudomonas stutzeri GOM2, isolated from the southwestern Gulf of Mexico, and the biochemical characterization of its C12DO (PsC12DO). The catA gene, encoding PsC12DO of 312 amino acid residues, was cloned and expressed in Escherichia coli. Many C12DOs have been described as dimeric enzymes including those present in Pseudomonas species. The purified PsC12DO enzyme was found as an active trimer, with a molecular mass of 107 kDa. Increasing NaCl concentration in the enzyme reaction gradually reduced activity; in high salt concentrations (0.7 M NaCl) quaternary structural analysis determined that the enzyme changes to a dimeric arrangement and causes a 51% decrease in specific activity on catechol substrate. In comparison with other C12DOs, our enzyme showed a broad range of action for PsC12DO in solutions with pH values ranging from neutral to alkaline (70%). The enzyme is still active after incubation at 50°C for 30 min and in low temperatures to long term storage after 6 weeks at 4°C (61%). EDTA or Ca2+ inhibitors cause no drastic changes on residual activity; nevertheless, the activity of the enzyme was affected by metal ions Fe3+, Zn2+ and was completely inhibited by Hg2+. Under optimal conditions the k cat and K m values were 16.13 s-1 and 13.2 μM, respectively. To our knowledge, this is the first report describing the characterization of a marine C12DOs from P. stutzeri isolated from the Gulf of Mexico that is active in a trimeric state. We consider that our enzyme has important features to be used in environments in presence of EDTA, metals and salinity conditions.

Metagenomics research has recently thrived due to DNA sequencing technologies improvement, driving the emergence of new analysis tools and the growth of taxonomic databases. However, there is no all-purpose strategy that can guarantee the best result for a given project and there are several combinations of software, parameters and databases that can be tested. Therefore, we performed an impartial comparison, using statistical measures of classification for eight bioinformatic tools and four taxonomic databases, defining a benchmark framework to evaluate each tool in a standardized context. Using in silico simulated data for 16S rRNA amplicons and whole metagenome shotgun data, we compared the results from different software and database combinations to detect biases related to algorithms or database annotation. Using our benchmark framework, researchers can define cut-off values to evaluate the expected error rate and coverage for their results, regardless the score used by each software. A quick guide to select the best tool, all datasets and scripts to reproduce our results and benchmark any new method are available at https://github.com/Ales-ibt/Metagenomic-benchmark . Finally, we stress out the importance of gold standards, database curation and manual inspection of taxonomic profiling results, for a better and more accurate microbial diversity description.

Indigenous bacterial populations play an important role in the restoration of crude oil-polluted marine environments. The identification and characterization of these bacteria are key in defining bioremediation strategies for the mitigation of possible future oil spills. In this work, we characterized Pseudomonas aeruginosa strain GOM1, which was isolated from the water column in the southwestern Gulf of Mexico. Phylogenetic analysis revealed that GOM1 strain was most closely related to P. aeruginosa WC55, a strain isolated from the northern Gulf of Mexico after the Deepwater Horizon oil spill. The hydrocarbon-degrading capacity of P. aeruginosa GOM1 was investigated using various approaches. This strain degraded 96% of the aliphatic fraction (C12–C38) of crude oil during a 30-day incubation period, exhibiting a high activity on long-chain alkanes, and expressing alkane hydroxylases AlkB1, AlkB2 and AlmA. Addition of nitrogen and phosphate to seawater culture medium enhanced hexadecane degradation by GOM1. Additionally, the strain exhibited high surfactant/rhamnolipid production and emulsifying activity when grown in a complex medium in the presence of hexadecane. Comparisons of growth kinetics, hydrocarbon degradation and gene expression between GOM1 and the closely related P. aeruginosa laboratory strain PAO1 revealed that the marine isolate is better adapted to degrade alkanes. Taken together, our results place P. aeruginosa GOM1 as a potentially effective candidate to be included in a consortium for use in the bioremediation of oil-polluted sites.

Marine sediments are an example of one of the most complex microbial habitats. These bacterial communities play an important role in several biogeochemical cycles in the marine ecosystem. In particular, the Gulf of Mexico has a ubiquitous concentration of hydrocarbons in its sediments, representing a very interesting niche to explore. Additionally, the Mexican government has opened its oil industry, offering several exploration and production blocks in shallow and deep water in the southwestern Gulf of Mexico (swGoM), from which there are no public results of conducted studies. Given the higher risk of large-scale oil spills, the design of contingency plans and mitigation activities before oil exploitation is of growing concern. Therefore, a bacterial taxonomic baseline profile is crucial to understanding the impact of any eventual oil spill. Here, we show a genus level taxonomic profile to elucidate the bacterial baseline, pointing out richness and relative abundance, as well as relationships with 79 abiotic parameters, in an area encompassing ∼150,000 km2, including a region where the exploitation of new oil wells has already been authorized. Our results describe for the first time the bacterial landscape of the swGoM, establishing a bacterial baseline “core” of 450 genera for marine sediments in this region. We can also differentiate bacterial populations from shallow and deep zones of the swGoM based on their community structure. Shallow sediments have been chronically exposed to aromatic hydrocarbons, unlike deep zones. Our results reveal that the bacterial community structure is particularly enriched with hydrocarbon-degrading bacteria in the shallow zone, where a greater aromatic hydrocarbon concentration was determined. Differences in the bacterial communities in the swGoM were also observed through a comprehensive comparative analysis relative to various marine sediment sequencing projects, including sampled sites from the Deep Water Horizon oil spill. This study in the swGoM provides clues to the bacterial population adaptation to the ubiquitous presence of hydrocarbons and reveals organisms such as Thioprofundum bacteria with potential applications in ecological surveillance. This resource will allow us to differentiate between natural conditions and alterations generated by oil extraction activities, which, in turn, enables us to assess the environmental impact of such activities.

Metagenomics research has recently thrived due to DNA sequencing technologies improvement, driving the emergence of new analysis tools and the growth of taxonomic databases. However, there is no all-purpose strategy that can guarantee the best result for a given project and there are several combinations of software, parameters and databases that can be tested. Therefore, we performed an impartial comparison, using statistical measures of classification for eight bioinformatic tools and four taxonomic databases, defining a benchmark framework to evaluate each tool in a standardized context. Using in silico simulated data for 16S rRNA amplicons and whole metagenome shotgun data, we compared the results from different software and database combinations to detect biases related to algorithms or database annotation. Using our benchmark framework, researchers can define cut-off values to evaluate the expected error rate and coverage for their results, regardless the score used by each software. A quick guide to select the best tool, all datasets and scripts to reproduce our results and benchmark any new method are available at https://github.com/Ales-ibt/Metagenomic-benchmark. Finally, we stress out the importance of gold standards, database curation and manual inspection of taxonomic profiling results, for a better and more accurate microbial diversity description.

Summary Objectives To determine the contribution of influenza and respiratory syncytial virus (RSV) as the cause of lower respiratory tract infection (LRTI) associated hospitalizations during the first year of the influenza A(H1N1) 2009 pandemic and to assess the severity of illness during the second pandemic wave. Methods Patients admitted with LRTI from April 2009 through March 2010 were assessed for the presence of influenza and RSV. Pandemic influenza virus was detected by means of a nested RT-PCR assay and/or the CDC's real time-PCR protocol. RSV was detected using a one-step RT-PCR assay. The characteristics of patients admitted during the first and second pandemic outbreaks were compared. Results 657 patients with LRTI were admitted during the study period. Pandemic influenza virus was detected in 180 and RSV in 133. Influenza was the most common cause of infection in adults, while RSV was more common in children. There were no differences in disease severity between the first and second pandemic outbreaks. Conclusions Pandemic influenza virus was associated to increased numbers of hospitalizations and deaths; particularly in adults. The severity of the first and second pandemic outbreaks was similar. RSV continues to be the main pathogen responsible for hospitalizations in young children.

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

Background Despite the potential to produce antibodies that can neutralize different virus (heterotypic neutralization), there is no knowledge of why vaccination against influenza induces protection predominantly against the utilized viral strains (homotypic response). Identification of structural patterns of the B cell repertoire associated to heterotypic neutralization may contribute to identify relevant epitopes for a universal vaccine against influenza. Methods Blood samples were collected from volunteers immunized with 2008/2009 trivalent inactivated vaccine (TIV), pandemic H1N1 (pdmH1N1) monovalent inactivated vaccine (MIV) and the 2014/2015 TIV. Neutralization was assessed by hemagglutination and microneutralization test. IgG VH amplicons derived from peripheral blood RNA from pre-immune and 7 days post vaccination were subjected to 454-Roche sequencing. Full reconstruction of the sampled repertoires was done with ImmunediveRsity. Results The TIV induced a predominantly homotypic neutralizing serologic response, while the 09 MIV induced a heterotypic neutralizing seroconversion in 17 % of the individuals. Both the 08/09 and the 14/15 TIV were associated with a reduction in clonotypic diversity, whereas 09 MIV was the opposite. Moreover, TIV and MIV induced distinctive patterns of IGHV segment use that are consistent with B cell selection by conserved antigenic determinants shared by the pre-pandemic and the pandemic strains. However, low somatic hypermutation rates in IgG after 09 MIV immunization, but not after 08/09 and 14/15 TIV immunization were observed. Furthermore, no evidence of the original antigenic sin was found in the same individuals after vaccination with the three vaccines. Conclusions Immunization with a new influenza virus strain (2009 pdmH1N1) induced unique effects in the peripheral B cell repertoire clonal structure, a stereotyped response involving distinctive IGHV segment use and low somatic hypermutation levels. These parameters were contrastingly different to those observed in response to pre-pandemic and post-pandemic vaccination, and may be the result of clonal selection of common antigenic determinants, as well as germinal center-independent responses that wane as the pandemic strain becomes seasonal. Our findings may contribute in the understanding of the structural and cellular basis required to develop a universal influenza vaccine. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0239-y) contains supplementary material, which is available to authorized users.

Background The study of human B cell response to dengue virus (DENV) infection is critical to understand serotype-specific protection and the cross-reactive sub-neutralizing response. Whereas the first is beneficial and thus represents the ultimate goal of vaccination, the latter has been implicated in the development of severe disease, which occurs in a small, albeit significant, fraction of secondary DENV infections. Both primary and secondary infections are associated with the production of poly-reactive and cross-reactive IgG antibodies. Methods To gain insight into the effect of DENV infection on the B cell repertoire, we used VH region high-throughput cDNA sequencing of the peripheral blood IgG B cell compartment of 19 individuals during the acute phase of infection. For 11 individuals, a second sample obtained 6 months later was analyzed for comparison. Probabilities of sequencing antibody secreting cells or memory B cells were estimated using second-order Monte Carlo simulation. Results We found that in acute disease there is an increase in IgG B cell diversity and changes in the relative use of segments IGHV1-2, IGHV1-18, and IGHV1-69. Somewhat unexpectedly, an overall low proportion of somatic hypermutated antibody genes was observed during the acute phase plasmablasts, particularly in secondary infections and those cases with more severe disease. Conclusions Our data are consistent with an innate-like antiviral recognition system mediated by B cells using defined germ-line coded B cell receptors, which could provide a rapid germinal center-independent antibody response during the early phase of infection. A model describing concurrent T-dependent and T-independent B cell responses in the context of DENV infection is proposed, which incorporates the selection of B cells using hypomutated IGHV segments and their potential role in poly/cross-reactivity. Its formal demonstration could lead to a definition of its potential implication in antibody-dependent enhancement, and may contribute to rational vaccine development efforts. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0276-1) contains supplementary material, which is available to authorized users.