The effect of pH on Marinobacter hydrocarbonoclasticus denitrification pathway and nitrous oxide reductase.

Increasing atmospheric concentration of N2O has been a concern, as it is a potent greenhouse gas and promotes ozone layer destruction. In the N-cycle, release of N2O is boosted upon a drop of pH in the environment. Here, Marinobacter hydrocarbonoclasticus was grown in batch mode in the presence of nitrate, to study the effect of pH in the denitrification pathway by gene expression profiling, quantification of nitrate and nitrite, and evaluating the ability of whole cells to reduce NO and N2O. At pH 6.5, accumulation of nitrite in the medium occurs and the cells were unable to reduce N2O. In addition, the biochemical properties of N2O reductase isolated from cells grown at pH 6.5, 7.5 and 8.5 were compared for the first time. The amount of this enzyme at acidic pH was lower than that at pH 7.5 and 8.5, pinpointing to a post-transcriptional regulation, though pH did not affect gene expression of N2O reductase accessory genes. N2O reductase isolated from cells grown at pH 6.5 has its catalytic center mainly as CuZ(4Cu1S), while that from cells grown at pH 7.5 or 8.5 has it as CuZ(4Cu2S). This study evidences that an in vivo secondary level of regulation is required to maintain N2O reductase in an active state.

Genomic organization, gene expression and activity profile of Marinobacter hydrocarbonoclasticus denitrification enzymes.

BACKGROUND: Denitrification is one of the main pathways of the N-cycle, during which nitrate is converted to dinitrogen gas, in four consecutive reactions that are each catalyzed by a different metalloenzyme. One of the intermediate metabolites is nitrous oxide, which has a global warming impact greater then carbon dioxide and which atmospheric concentration has been increasing in the last years. The four denitrification enzymes have been isolated and biochemically characterized from Marinobacter hydrocarbonoclasticus in our lab. METHODS: Bioinformatic analysis of the M. hydrocarbonoclasticus genome to identify the genes involved in the denitrification pathway. The relative gene expression of the gene encoding the catalytic subunits of those enzymes was analyzed during the growth under microoxic conditions. The consumption of nitrate and nitrite, and the reduction of nitric oxide and nitrous oxide by whole-cells was monitored during anoxic and microoxic growth in the presence of 10 mM sodium nitrate at pH 7.5. RESULTS: The bioinformatic analysis shows that genes encoding the enzymes and accessory factors required for each step of the denitrification pathway are clustered together. An unusual feature is the co-existence of genes encoding a q- and a c-type nitric oxide reductase, with only the latter being transcribed at similar levels as the ones encoding the catalytic subunits of the other denitrifying enzymes, when cells are grown in the presence of nitrate under microoxic conditions. Using either a batch- or a closed system, nitrate is completely consumed in the beginning of the growth, with transient formation of nitrite, and whole-cells can reduce nitric oxide and nitrous oxide from mid-exponential phase until being collected (time-point 50 h). DISCUSSION: M. hydrocarbonoclasticus cells can reduce nitric and nitrous oxide in vivo, indicating that the four denitrification steps are active. Gene expression profile together with promoter regions analysis indicates the involvement of a cascade regulatory mechanism triggered by FNR-type in response to low oxygen tension, with nitric oxide and nitrate as secondary effectors, through DNR and NarXL, respectively. This global characterization of the denitrification pathway of a strict marine bacterium, contributes to the understanding of the N-cycle and nitrous oxide release in marine environments.

Global Assessment of the Impact of Type 2 Diabetes on Sleep through Specific Questionnaires. A Case-Control Study.

ABSTRACT: Type 2 diabetes (T2D) is an independent risk factor for sleep breathing disorders. However, it is unknown whether T2D affects daily somnolence and quality of sleep independently of the impairment of polysomnographic parameters. MATERIAL AND METHODS: A case-control study including 413 patients with T2D and 413 non-diabetic subjects, matched by age, gender, BMI, and waist and neck circumferences. A polysomnography was performed and daytime sleepiness was evaluated using the Epworth Sleepiness Scale (ESS). In addition, 135 subjects with T2D and 45 controls matched by the same previous parameters were also evaluated through the Pittsburgh Sleep Quality Index (PSQI) to calculate sleep quality. RESULTS: Daytime sleepiness was higher in T2D than in control subjects (p = 0.003), with 23.9% of subjects presenting an excessive daytime sleepiness (ESS>10). Patients with fasting plasma glucose (FPG ≥13.1 mmol/l) were identified as the group with a higher risk associated with an ESS>10 (OR 3.9, 95% CI 1.8-7.9, p = 0.0003). A stepwise regression analyses showed that the presence of T2D, baseline glucose levels and gender but not polysomnographic parameters (i.e apnea-hyoapnea index or sleeping time spent with oxigen saturation lower than 90%) independently predicted the ESS score. In addition, subjects with T2D showed higher sleep disturbances [PSQI: 7.0 (1.0-18.0) vs. 4 (0.0-12.0), p<0.001]. CONCLUSION: The presence of T2D and high levels of FPG are independent risk factors for daytime sleepiness and adversely affect sleep quality. Prospective studies addressed to demonstrate whether glycemia optimization could improve the sleep quality in T2D patients seem warranted.

High throughput phenotypic selection of Mycobacterium tuberculosis mutants with impaired resistance to reactive oxygen species identifies genes important for intracellular growth.

Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS). Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.

Phylogeny of Mycobacterium tuberculosis Beijing strains constructed from polymorphisms in genes involved in DNA replication, recombination and repair.

BACKGROUND: The Beijing family is a successful group of M. tuberculosis strains, often associated with drug resistance and widely distributed throughout the world. Polymorphic genetic markers have been used to type particular M. tuberculosis strains. We recently identified a group of polymorphic DNA repair replication and recombination (3R) genes. It was shown that evolution of M. tuberculosis complex strains can be studied using 3R SNPs and a high-resolution tool for strain discrimination was developed. Here we investigated the genetic diversity and propose a phylogeny for Beijing strains by analyzing polymorphisms in 3R genes. METHODOLOGY/PRINCIPAL FINDINGS: A group of 3R genes was sequenced in a collection of Beijing strains from different geographic origins. Sequence analysis and comparison with the ones of non-Beijing strains identified several SNPs. These SNPs were used to type a larger collection of Beijing strains and allowed identification of 26 different sequence types for which a phylogeny was constructed. Phylogenetic relationships established by sequence types were in agreement with evolutionary pathways suggested by other genetic markers, such as Large Sequence Polymorphisms (LSPs). A recent Beijing genotype (Bmyc10), which included 60% of strains from distinct parts of the world, appeared to be predominant. CONCLUSIONS/SIGNIFICANCE: We found SNPs in 3R genes associated with the Beijing family, which enabled discrimination of different groups and the proposal of a phylogeny. The Beijing family can be divided into different groups characterized by particular genetic polymorphisms that may reflect pathogenic features. These SNPs are new, potential genetic markers that may contribute to better understand the success of the Beijing family.

Innate immune response to Mycobacterium tuberculosis Beijing and other genotypes.

BACKGROUND: As a species, Mycobacterium tuberculosis is more diverse than previously thought. In particular, the Beijing family of M. tuberculosis strains is spreading and evaluating throughout the world and this is giving rise to public health concerns. Genetic diversity within this family has recently been delineated further and a specific genotype, called Bmyc10, has been shown to represent over 60% of all Beijing clinical isolates in several parts of the world. How the host immune system senses and responds to various M. tuberculosis strains may profoundly influence clinical outcome and the relative epidemiological success of the different mycobacterial lineages. We hypothesised that the success of the Bmyc10 group may, at least in part, rely upon its ability to alter innate immune responses and the secretion of cytokines and chemokines by host phagocytes. METHODOLOGY/PRINCIPAL FINDINGS: We infected human macrophages and dendritic cells with a collection of genetically well-defined M. tuberculosis clinical isolates belonging to various mycobacterial families, including Beijing. We analyzed cytokine and chemokine secretion on a semi-global level using antibody arrays allowing the detection of sixty-five immunity-related soluble molecules. Our data indicate that Beijing strains induce significantly less interleukin (IL)-6, tumor necrosis factor (TNF), IL-10 and GRO-α than the H37Rv reference strain, a feature that is variously shared by other modern and ancient M. tuberculosis families and which constitutes a signature of the Beijing family as a whole. However, Beijing strains did not differ relative to each other in their ability to modulate cytokine secretion. CONCLUSIONS/SIGNIFICANCE: Our results confirm and expand upon previous reports showing that M. tuberculosis Beijing strains in general are poor in vitro cytokine inducers in human phagocytes. The results suggest that the epidemiological success of the Beijing Bmyc10 is unlikely to rely upon any specific ability of this group of strains to impair anti-mycobacterial innate immunity.

DNA repair in Mycobacterium tuberculosis revisited.

Our understanding of Mycobacterium tuberculosis DNA repair mechanisms is still poor compared with that of other bacterial organisms. However, the publication of the first complete M. tuberculosis genome sequence 10 years ago boosted the study of DNA repair systems in this organism. A first step in the elucidation of M. tuberculosis DNA repair mechanisms was taken by Mizrahi and Andersen, who identified homologs of genes involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes required for nucleotide excision repair, base excision repair, recombination, and SOS repair and mutagenesis were identified. Notably, no homologs of genes involved in mismatch repair were identified. Novel characteristics of the M. tuberculosis DNA repair machinery have been found over the last decade, such as nonhomologous end joining, the presence of Mpg, ERCC3 and Hlr - proteins previously presumed to be produced exclusively in mammalian cells - and the recently discovered bifunctional dCTP deaminase:dUTPase. The study of these systems is important to develop therapeutic agents that can counteract M. tuberculosis evolutionary changes and to prevent adaptive events resulting in antibiotic resistance. This review summarizes our current understanding of the M. tuberculosis DNA repair system.

Evolution and diversity of clonal bacteria: the paradigm of Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis complex species display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task. PRINCIPAL FINDINGS: We found that single-nucleotide polymorphism (SNP) analysis of DNA repair, recombination and replication (3R) genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study. Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets of genes that may lead to suboptimal 3R system activity. In turn, the relaxed fidelity of 3R genes may allow the occurrence of adaptive variants, some of which will survive. Furthermore, 3R-based phylogenetic trees are a new tool for distinguishing between M. tuberculosis complex strains. CONCLUSIONS/SIGNIFICANCE: This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis. They also facilitate further epidemiological studies of these bacteria, through the development of high-resolution tools. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria.

[Detection and handling of alcohol problems in primary care in Catalonia]. / Detección y abordaje de los problemas de alcohol en la atención primaria de Cataluña.

OBJECTIVE: To gain information on how well strategies for alcohol problem detection and interventions are being implemented in primary care in Catalonia, Spain. DESIGN: Longitudinal pre/post study to evaluate the impact of the distribution to primary care professionals of a training programme for detecting alcohol problems and intervening in them. Descriptive analysis of the basic situation, using interviews with patients and professionals and examination of clinical histories (CH). SETTING: Health districts that existed in Catalonia in 2001. PARTICIPANTS: Twenty eight health districts, 973 patients, 80 professionals, and 852 clinical histories examined. MAIN MEASUREMENTS: Data were collected, by means of questionnaires for professionals and patients and of ad-hoc instruments for examining clinical histories, on the levels of primary care screening for alcohol consumption and of intervention. RESULTS: There was a major disparity in the data between the 3 sources. Thus the screening of consumption and counselling was recorded much less in the CHs than amounts that patients said they received and that professionals said they performed. In addition, most of the at-risk drinkers that were seen in PC were not detected. On comparing them with non-risk drinkers we found significant differences in sex, job, familiarity with the centre and having been asked or not about their alcohol consumption. CONCLUSIONS: Given the deficiencies found in PC preventive activity on alcohol consumption and as alcohol consumption has such huge social and health repercussions on the general population, we think it is fully justified to introduce into PC specific training programmes on screening and brief intervention techniques for alcohol problems.

Detección y abordaje de los problemas de alcohol en la atención primaria de Cataluña

Objetivo. Conocer el grado de implementación de las estrategias de detección e intervención en los problemas de alcohol en la atención primaria de Cataluña. Diseño. Estudio de evaluación del impacto de la diseminación de un programa de formación de los profesionales de atención primaria (AP) en la detección e intervención en los problemas de alcohol. Análisis descriptivo de la situación basal a partir de las entrevistas a pacientes, profesionales y el vaciado de las historias clínicas. Emplazamiento. Áreas básicas de salud (ABS) de Cataluña en el 2001. Participantes. Participaron 28 ABS seleccionadas aleatoriamente, 973 pacientes, 80 profesionales y 852 historias clínicas auditadas. Mediciones principales. Se recogieron datos sobre el nivel de cribado e intervención en el consumo de alcohol en la atención primaria a través de cuestionarios a profesionales y a pacientes y la auditoria de las historias clínicas. Resultados. Se observa una importante disparidad en los datos obtenidos a partir de las 3 fuentes. Así, el cribado de consumo y el consejo se registran mucho menos en la historia clínica de lo que los pacientes dicen recibirlo y de lo que los profesionales dicen hacerlo. Además, la mayor parte de los bebedores de riesgo atendidos en AP no son detectados y presentan diferencias significativas en cuanto al sexo, la profesión, la familiaridad con el centro y el hecho de haberles preguntado o no sobre su consumo de alcohol, en relación con los no bebedores de riesgo. Conclusiones. Dadas las deficiencias detectadas en la actividad preventiva de AP en relación con el consumo de alcohol y las enormes repercusiones sociales y sanitarias que ocasiona el consumo de alcohol en la población general, creemos que está ampliamente justificado impulsar la implementación en AP de programas específicos de formación sobre técnicas de cribado e intervención breve en los problemas de alcohol.(AU)

Detección y abordaje de los problemas de alcohol en la atención primaria de Cataluña / Detection and handling of alcohol problems in primary care in Catalonia

Objetivo. Conocer el grado de implementación de las estrategias de detección e intervención en los problemas de alcohol en la atención primaria de Cataluña. Diseño. Estudio de evaluación del impacto de la diseminación de un programa de formación de los profesionales de atención primaria (AP) en la detección e intervención en los problemas de alcohol. Análisis descriptivo de la situación basal a partir de las entrevistas a pacientes, profesionales y el vaciado de las historias clínicas. Emplazamiento. Áreas básicas de salud (ABS) de Cataluña en el 2001. Participantes. Participaron 28 ABS seleccionadas aleatoriamente, 973 pacientes, 80 profesionales y 852 historias clínicas auditadas. Mediciones principales. Se recogieron datos sobre el nivel de cribado e intervención en el consumo de alcohol en la atención primaria a través de cuestionarios a profesionales y a pacientes y la auditoría de las historias clínicas. Resultados. Se observa una importante disparidad en los datos obtenidos a partir de las 3 fuentes. Así, el cribado de consumo y el consejo se registran mucho menos en la historia clínica de lo que los pacientes dicen recibirlo y de lo que los profesionales dicen hacerlo. Además, la mayor parte de los bebedores de riesgo atendidos en AP no son detectados y presentan diferencias significativas en cuanto al sexo, la profesión, la familiaridad con el centro y el hecho de haberles preguntado o no sobre su consumo de alcohol, en relación con los no bebedores de riesgo. Conclusiones. Dadas las deficiencias detectadas en la actividad preventiva de AP en relación con el consumo de alcohol y las enormes repercusiones sociales y sanitarias que ocasiona el consumo de alcohol en la población general, creemos que está ampliamente justificado impulsar la implementación en AP de programas específicos de formación sobre técnicas de cribado e intervención breve en los problemas de alcohol

Objective. To gain information on how well strategies for alcohol problem detection and interventions are being implemented in primary care in Catalonia, Spain. Design. Longitudinal pre/post study to evaluate the impact of the distribution to primary care professionals of a training programme for detecting alcohol problems and intervening in them. Descriptive analysis of the basic situation, using interviews with patients and professionals and examination of clinical histories (CH). Setting. Health districts that existed in Catalonia in 2001. Participants. Twenty eight health districts, 973 patients, 80 professionals, and 852 clinical histories examined. Main measurements. Data were collected, by means of questionnaires for professionals and patients and of ad-hoc instruments for examining clinical histories, on the levels of primary care screening for alcohol consumption and of intervention. Results. There was a major disparity in the data between the 3 sources. Thus the screening of consumption and counselling was recorded much less in the CHs than amounts that patients said they received and that professionals said they performed. In addition, most of the at-risk drinkers that were seen in PC were not detected. On comparing them with non-risk drinkers we found significant differences in sex, job, familiarity with the centre and having been asked or not about their alcohol consumption. Conclusions. Given the deficiencies found in PC preventive activity on alcohol consumption and as alcohol consumption has such huge social and health repercussions on the general population, we think it is fully justified to introduce into PC specific training programmes on screening and brief intervention techniques for alcohol problems