RESUMO
Mycobacterium tuberculosis (MTB) lineage 2/Beijing is associated with high virulence and drug resistance worldwide. In Colombia, the Beijing genotype has circulated since 1997, predominantly on the pacific coast, with the Beijing-Like SIT-190 being more prevalent. This genotype conforms to a drug-resistant cluster and shows a fatal outcome in patients. To better understand virulence determinants, we performed a transcriptomic analysis with a Beijing-Like SIT-190 isolate (BL-323), and Beijing-Classic SIT-1 isolate (BC-391) in progressive tuberculosis (TB) murine model. Bacterial RNA was extracted from mice lungs on days 3, 14, 28, and 60. On average, 0.6% of the total reads mapped against MTB genomes and of those, 90% against coding genes. The strains were independently associated as determined by hierarchical cluster and multidimensional scaling analysis. Gene ontology showed that in strain BL-323 enriched functions were related to host immune response and hypoxia, while proteolysis and protein folding were enriched in the BC-391 strain. Altogether, our results suggested a differential bacterial transcriptional program when evaluating these two closely related strains. The data presented here could potentially impact the control of this emerging, highly virulent, and drug-resistant genotype.
Assuntos
Doenças dos Animais , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Animais , Pequim , Progressão da Doença , Resistência a Medicamentos , Genótipo , Humanos , Camundongos , Transcriptoma , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Bioelectrochemical sensing of Mycobacterium tuberculosis through electro-immunosensors is a promising technique to detect relevant analytes. In general, immunosensors require the formation of organic assemblies by the adsorption of molecular constituents. Moreover, they depend on the correct immobilization of the bio-recognition element in the biosensor. These procedures cannot be easily monitored without the use of invasive methods. In this work, an impedance analysis technique was used, as a non-invasive method, to measure and differentiate the manufacturing stages of the sensors. Biomicrosystems were fabricated through physical vapor deposition (PVD) of 80 nm Au nanolayers on 35 µm copper surfaces. Later, the surface was modified through thiolation methods generating a self-assembled-monolayer (SAM) with 20 mM 4-aminothiophenol (4-ATP) on which a polyclonal antibody (pAb) was covalently attached. Using impedance analysis, every step of the electro-immunosensor fabrication protocol was characterized using 40 independent replicas. Results showed that, compared to the negative controls, distilled water, and 0.5 µg/mL HSA, a maximum variation of 171% between each replica was achieved when compared to samples containing 0.5 µg/mL of ESAT-6 M. tuberculosis immunodominant protein. Therefore, this development validates a non-invasive method to electrically monitor the assembly process of electro-immunosensors and a tool for its further measure for detection of relevant antigens.
Assuntos
Anticorpos Antibacterianos/análise , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Microtecnologia/métodos , Mycobacterium tuberculosis/isolamento & purificação , Compostos de Anilina/química , Anticorpos Antibacterianos/metabolismo , Compostos de Sulfidrila/químicaRESUMO
BACKGROUND: The chemical treatment of Plasmodium falciparum for human infections is losing efficacy each year due to the rise of resistance. One possible strategy to find novel anti-malarial drugs is to access the largest reservoir of genomic biodiversity source on earth present in metagenomes of environmental microbial communities. METHODS: A bioluminescent P. falciparum parasite was used to quickly detect shifts in viability of microcultures grown in 96-well plates. A synthetic gene encoding the Dermaseptin 4 peptide was designed and cloned under tight transcriptional control in a large metagenomic insert context (30 kb) to serve as proof-of-principle for the screening platform. RESULTS: Decrease in parasite viability consistently correlated with bioluminescence emitted from parasite microcultures, after their exposure to bacterial extracts containing a plasmid or fosmid engineered to encode the Dermaseptin 4 anti-malarial peptide. CONCLUSIONS: Here, a new technical platform to access the anti-malarial potential in microbial environmental metagenomes has been developed.
Assuntos
Antimaláricos/farmacologia , Biblioteca Genômica , Testes de Sensibilidade Parasitária , Extratos Vegetais/farmacologia , Plantas/química , Plasmodium falciparum/efeitos dos fármacos , Biodiversidade , Malária Falciparum/tratamento farmacológico , Metagenoma , Plasmodium falciparum/genéticaRESUMO
Salmonellosis outbreaks in Europe, the United States, and Latin America have been associated with contaminated food derivatives including meat from the poultry industry. Salmonella grown under iron-limiting conditions has the capability to increase concentration of several iron-regulated outer-membrane proteins to augment the acquisition of the metal. These proteins have been proved to have immunogenic properties. Our aim was to increase the relative expression of iroN, fepA, and cirA in Salmonella Enteritidis domestic strain. Furthermore, we proposed a 3-dimensional structure model for each protein to predict and locate antigenic peptides. Our eventual objective is to produce an effective vaccine against regional avian salmonellosis. Two simple factorial designs were carried out to discriminate between 2 nitrogen sources and determine chelating-agent addition timing to augment relative gene expression. Two antigenic peptides located at the external face of each protein and 2 typical domains of iron-regulated outer-membrane proteins, plug and TonB-dep-Rec, were identified from the 3-dimensional models. Tryptone was selected as the best nitrogen source based on growth rate (µx = 0.36 h(-1)) and biomass productivity (Px = 0.9 gâ¢h(-1)â¢L(-1)) as determined by a general factorial design. Optimum timing for chelating agent addition was in the middle of the log phase, which allowed relative expressions at 4 h of culture. Increase in iroN, fepA, and cirA relative expression was favored by the length of log phase and the addition of chelating agent, which decreased chelating toxicity and enhanced cell growth rate.
Assuntos
Proteínas de Bactérias/metabolismo , Simulação por Computador , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ferro/farmacologia , Salmonella enteritidis/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Quelantes/farmacologia , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Nitrogênio/metabolismo , Conformação Proteica , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/genéticaRESUMO
As a follow-up of the "spoligoriftyping" development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.
Assuntos
Farmacorresistência Bacteriana Múltipla , Tipagem Molecular/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Monitoramento Epidemiológico , Humanos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana/métodos , Epidemiologia Molecular/métodos , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/genética , Medicina de Precisão/métodos , Rifampina/farmacologia , Sensibilidade e EspecificidadeRESUMO
The Mycobacterium tuberculosis insertion sequence IS6110, besides being a very useful tool in molecular epidemiology, seems to have an impact on the biology of bacilli. In the present work, we mapped the 12 points of insertion of IS6110 in the genome of a successful strain named M. tuberculosis Zaragoza (which has been referred to as the MTZ strain). This strain, belonging to principal genetic group 3, caused a large unsuspected tuberculosis outbreak involving 85 patients in Zaragoza, Spain, in 2001 to 2004. The mapping of the points of insertion of IS6110 in the genome of the Zaragoza strain offers clues for a better understanding of the adaptability and virulence of M. tuberculosis. Surprisingly, the presence of one copy of IS6110 was found in Rv2286c, as was recently described for a successful Beijing sublineage. As a result of this analysis, a rapid method for detecting this particular M. tuberculosis strain has been designed.
Assuntos
Elementos de DNA Transponíveis , Surtos de Doenças , Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Tuberculose/microbiologia , Técnicas Bacteriológicas/métodos , DNA Bacteriano/genética , Genoma Bacteriano , Humanos , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Espanha/epidemiologia , Tuberculose/diagnósticoRESUMO
BACKGROUND: The insertion element IS6110 is one of the main sources of genomic variability in Mycobacterium tuberculosis, the etiological agent of human tuberculosis. Although IS 6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment. RESULTS: We identified a total of 6,976 IS6110 flanking regions on the different isolates. When validated using reference strains, the method had 100% specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed. CONCLUSIONS: This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in M. tuberculosis strains.
Assuntos
Elementos de DNA Transponíveis/genética , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Biblioteca Gênica , Humanos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/microbiologia , Virulência/genéticaRESUMO
[This corrects the article DOI: 10.3389/fmicb.2022.956602.].
RESUMO
The establishment of Mycobacterium tuberculosis (Mtb) long-term infection in vivo depends on several factors, one of which is the availability of key nutrients such as iron (Fe). The relation between Fe deprivation inside and outside the granuloma, and the capacity of Mtb to accumulate lipids and persist in the absence of growth is not well understood. In this context, current knowledge of how Mtb modifies its lipid composition in response to growth arrest, depending on iron availability, is scarce. To shed light on these matters, in this work we compare genome-wide transcriptomic and lipidomic profiles of Mtb at exponential and stationary growth phases using cultures with glycerol as a carbon source, in the presence or absence of iron. As a result, we found that transcriptomic responses to growth arrest, considered as the transition from exponential to stationary phase, are iron dependent for as many as 714 genes (iron-growth interaction contrast, FDR <0.05), and that, in a majority of these genes, iron deprivation enhances the magnitude of the transcriptional responses to growth arrest in either direction. On the one hand, genes whose upregulation upon growth arrest is enhanced by iron deprivation were enriched in functional terms related to homeostasis of ion metals, and responses to several stressful cues considered cardinal features of the intracellular environment. On the other hand, genes showing negative responses to growth arrest that are stronger in iron-poor medium were enriched in energy production processes (TCA cycle, NADH dehydrogenation and cellular respiration), and key controllers of ribosomal activity shut-down, such as the T/A system mazE6/F6. Despite of these findings, a main component of the cell envelope, lipid phthiocerol dimycocerosate (PDIM), was not detected in the stationary phase regardless of iron availability, suggesting that lipid changes during Mtb adaptation to non-dividing phenotypes appear to be iron-independent. Taken together, our results indicate that environmental iron levels act as a key modulator of the intensity of the transcriptional adaptations that take place in the bacterium upon its transition between dividing and dormant-like phenotypes in vitro.
RESUMO
Mycobacteria, like other microorganisms, survive under different environmental variations by expressing an efficient adaptive response, oriented by regulatory elements, such as transcriptional repressors of the TetR family. These repressors in mycobacteria also appear to be related to cholesterol metabolism. In this study, we have evaluated the effect of a fatty acid (oleic-palmitic-stearic)/cholesterol mixture on some phenotypic and genotypic characteristics of a tetR-mutant strain (BCG_2177c mutated gene) of M. bovis BCG, a homologous of Rv2160A of M. tuberculosis. In order to accomplish this, we have analyzed the global gene expression of this strain by RNA-seq and evaluated its neutral-lipid storage capacity and potential to infect macrophages. We have also determined the macrophage response by measuring some pro- and anti-inflammatory cytokine expressions. In comparison with wild-type microorganisms, we showed that the mutation in the BCG_2177c gene did not affect the growth of M. bovis BCG in the presence of lipids but it probably modified the structure/composition of its cell envelope. Compared to with dextrose, an overexpression of the transcriptome of the wild-type and mutant strains was observed when these mycobacteria were cultured in lipids, mainly at the exponential phase. Twelve putative intracellular redox balance maintenance genes and four others coding for putative transcriptional factors (including WhiB6 and three TetR-like) were the main elements repeatedly overexpressed when cultured in the presence of lipids. These genes belonged to the central part of what we called the "genetic lipid signature" for M. bovis BCG. We have also found that all these mycobacteria genotypic changes affected the outcome of BCG-infected macrophages, being the mutant strain most adapted to persist longer inside the host. This high persistence result was also confirmed when mutant-infected macrophages showed overexpression of the anti-inflammatory cytokine TGF-ß versus pro-inflammatory cytokines. In summary, the lack of this TetR-like repressor expression, within a lipid environment, may help mycobacteria overcome intracellular redox stress and survive longer inside their host.
Assuntos
Infecções por Mycobacterium , Mycobacterium bovis , Mycobacterium tuberculosis , Vacina BCG , Colesterol/metabolismo , Citocinas/metabolismo , Humanos , Macrófagos/microbiologia , OxirreduçãoRESUMO
OBJECTIVES: To perform a multicentre study to evaluate the performance of the colorimetric redox indicator (CRI) assay and to establish the MICs and critical concentrations of rifampicin, isoniazid, ofloxacin, kanamycin and capreomycin. METHODS: The study was carried out in two phases. Phase I determined the MIC of each drug. Phase II established critical concentrations for the five drugs tested by the CRI assay compared with the conventional proportion method. RESULTS: Phase I: a strain was considered resistant by the CRI assay if the MIC was ≥0.5 mg/L for rifampicin, ≥0.25 mg/L for isoniazid, ≥4.0 mg/L for ofloxacin and ≥5.0 mg/L for kanamycin and capreomycin. Sensitivity was 99.1% for isoniazid and 100% for the other drugs and specificity was 97.9% for capreomycin and 100% for the other drugs. Phase II: the critical concentration was 0.5 mg/L for rifampicin, 0.25 mg/L for isoniazid, 2.0 mg/L for ofloxacin and 2.5 mg/L for kanamycin and capreomycin giving an overall accuracy of 98.4%, 96.6%, 96.7%, 98.3% and 90%, respectively. CONCLUSIONS: Results demonstrate that the CRI assay is an accurate method for the rapid detection of XDR Mycobacterium tuberculosis. The CRI assay is faster than the conventional drug susceptibility testing method using solid medium, has the same turnaround time as the BACTEC MGIT 960 system, but is less expensive, and could be an adequate method for low-income countries.
Assuntos
Antituberculosos/farmacologia , Colorimetria/métodos , Farmacorresistência Bacteriana Múltipla , Tuberculose Extensivamente Resistente a Medicamentos/diagnóstico , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Humanos , Indicadores e Reagentes/metabolismo , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/isolamento & purificação , OxirreduçãoRESUMO
The global demand for fine-flavour cocoa has increased worldwide during the last years. Fine-flavour cocoa offers exceptional quality and unique fruity and floral flavour attributes of high demand by the world's elite chocolatiers. Several studies have highlighted the relevance of cocoa fermentation to produce such attributes. Nevertheless, little is known regarding the microbial interactions and biochemistry that lead to the production of these attributes on farms of industrial relevance, where traditional fermentation methods have been pre-standardized and scaled up. In this study, we have used metagenomic approaches to dissect on-farm industrial fermentations of fine-flavour cocoa. Our results revealed the presence of a shared core of nine dominant microorganisms (i.e. Limosilactobacillus fermentum, Saccharomyces cerevisiae, Pestalotiopsis rhododendri, Acetobacter aceti group, Bacillus subtilis group, Weissella ghanensis group, Lactobacillus_uc, Malassezia restricta and Malassezia globosa) between two farms located at completely different agro-ecological zones. Moreover, a community metabolic model was reconstructed and proposed as a tool to further elucidate the interactions among microorganisms and flavour biochemistry. Our work is the first to reveal a core of microorganisms shared among industrial farms, which is an essential step to process engineering aimed to design starter cultures, reducing fermentation times, and controlling the expression of undesirable phenotypes.
Assuntos
Cacau/química , Cacau/microbiologia , Fermentação/genética , Metagenoma/genética , Chocolate/microbiologia , Aromatizantes/química , Microbiologia de Alimentos/métodosRESUMO
A reverse line blot DNA hybridization format for rapid detection of multidrug-resistant tuberculosis was developed. Simultaneous detection of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and specificity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identified. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrug-resistant tuberculosis but that cannot afford expensive commercial tests because of limited resources.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla , Isoniazida/farmacologia , Mycobacterium tuberculosis/genética , Oligonucleotídeos , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Proteínas de Bactérias/genética , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/normas , Catalase/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Hibridização de Ácido Nucleico/métodos , Oxirredutases/genética , Sensibilidade e Especificidade , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Tuberculosis is the world's leading cause of death due to a single infectious agent, and efforts aimed at its control require a better understanding of host, environmental, and bacterial factors that govern disease outcome. Growing evidence indicates that certain Mycobacterium tuberculosis strains of distinct phylogeographic lineages elicit unique immunopathological events. However, identifying the genetic basis of these phenotypic peculiarities has proven difficult. Here we report the presence of six large sequence polymorphisms which, together with two single-nucleotide changes previously described by our group, consistently differentiate Haarlem strains from the remaining M. tuberculosis lineages. The six newly found Haarlem-specific genetic events are four deletions, which altogether involve more than 13 kb, and two intragenic insertions of the element IS6110. The absence of the genes involved in these polymorphisms could have an important physiological impact on Haarlem strains, i.e., by affecting key genes, such as Rv1354c and cyp121, which have been recently proposed as plausible drug targets. These lineage-specific polymorphisms can serve as genetic markers for the rapid PCR identification of Haarlem strains, providing a useful tool for strain surveillance and molecular epidemiology studies. Strain variability such as that described here underscores the need for the definition of a core set of essential genes in M. tuberculosis that are ubiquitously present in all circulating lineages, as a requirement in the development of effective antituberculosis drugs and vaccines.
Assuntos
Antituberculosos/farmacologia , DNA Bacteriano/genética , Mycobacterium tuberculosis/genética , Polimorfismo Genético , Vacinas contra a Tuberculose/imunologia , Tuberculose/microbiologia , Elementos de DNA Transponíveis , Marcadores Genéticos , Humanos , Mutagênese Insercional , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Reação em Cadeia da Polimerase/métodos , Deleção de SequênciaRESUMO
Fusarium spp. have frequently been isolated from patients with onychomycosis. In Colombia, several studies have shown that Fusarium is the most common non-dermatophyte mould causing onychomycosis and its spread has increased in the past years. In this study, samples were collected in 2003 and 2004 from 137 patients who were diagnosed with onychomycosis caused by Fusarium spp. Three species of Fusarium were identified: Fusarium solani (64.9%), Fusarium oxysporum (32.8%) and Fusarium verticillioides (2.3%). The diseases were more common in women (73%) than in men (27%) and occurred mainly among adults between 31 and 40 years old. The percentage of patients who had received previous treatments was 63.5%. In the last years, new and improved antifungal agents like echinocandins or new triazoles like voriconazole have been developed. For this reason, susceptibility testing using voriconazole was performed, by broth microdilution and disk diffusion. The results showed that F. solani had the highest minimum inhibitory concentration. Using the disk diffusion test, many of the isolates showed variable susceptibility. Genetic diversity of F. oxysporum isolates was determined by random amplified polymorphic DNA. Twenty isolates belonging to different haplotypes were selected for PCR amplification of a region of the gene encoding α-l-arabinofuranosidase B, a specific test to determine if the isolates were F. oxysporum f. sp. dianthi. On the basis of these PCR results, we found that five out of the 20 F. oxysporum isolates corresponded to f. sp. dianthi.
Assuntos
Fusarium/isolamento & purificação , Onicomicose/microbiologia , Adulto , Colômbia/epidemiologia , Feminino , Proteínas Fúngicas/genética , Fusarium/classificação , Fusarium/enzimologia , Fusarium/genética , Glicosídeo Hidrolases/genética , Humanos , Masculino , Pessoa de Meia-Idade , Unhas/microbiologia , Onicomicose/epidemiologia , Adulto JovemRESUMO
The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/genética , Enzimas/genética , Biblioteca Gênica , Metagenômica/métodos , Solo/química , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Enzimas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Microbiologia do SoloRESUMO
OBJECTIVES: The purpose of this study was to develop the nitrate reductase assay (NRA) for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide resistance as a marker of pyrazinamide resistance in Löwenstein-Jensen (LJ) medium at neutral pH. METHODS: We tested 68 M. tuberculosis isolates using nicotinamide at three different concentrations (1000, 500 and 250 mg/L) by the NRA in LJ medium and compared the results with those obtained with the BACTEC 460-TB or the BACTEC MGIT 960 as reference standard methods. Mutations in the pncA gene were detected by DNA sequencing of the pyrazinamide-resistant isolates. RESULTS: Out of 34 M. tuberculosis pyrazinamide-resistant isolates, 31 were found to be resistant to 1000 and 500 mg/L nicotinamide giving sensitivity and specificity of 91% and 94%, respectively. At 250 mg/L nicotinamide, the sensitivity and specificity decreased to 91% and 71%, respectively. Results were obtained in an average of 10 days. Based on these results, a tentative breakpoint concentration of 500 mg/L nicotinamide was defined. DNA sequencing of the pncA gene detected mutations in 26 out of 34 M. tuberculosis isolates resistant to pyrazinamide. CONCLUSIONS: The NRA using nicotinamide to detect resistance to pyrazinamide in LJ medium is a rapid and accurate method that could be useful in limited-resource countries where the BACTEC 460-TB or the BACTEC MGIT 960 system is not available.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Niacinamida/farmacologia , Nitrato Redutase/metabolismo , Pirazinamida/farmacologia , Sequência de Bases , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos , Humanos , Testes de Sensibilidade Microbiana/métodos , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Fatores de Tempo , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
DevR is a transcriptional regulator that mediates the genetic response of Mycobacterium tuberculosis and Mycobacterium bovis to oxygen limitation and nitric oxide exposure. devR is part of an operon that includes the genes devS and Rv3134c, which encode an oxygen sensor protein and a protein that contains a universal stress protein domain, respectively. Here, we report the transcriptional analysis and quantitative expression of Rv3134c/devR/devS under in vitro stress conditions including oxygen limitation, low nutrients and ex vivo macrophage infection. At least three different promoters were found to control Rv3134c/devR/devS expression under the stresses tested. Two promoters were identified upstream of devR, one was active under hypoxia and the other under nutrient starvation. A single promoter was identified upstream of Rv3134c, and transcripts from this promoter were detected only under hypoxia. Rv3134c to devR were found to be co-transcribed only under hypoxia, whereas devR/devS were co-transcribed both in aerobiosis and starvation. RT-qPCR showed an increase in the ratio hypoxia/aerobiosis and in starvation/nutrients in all genes. devR/devS showed transient expression in the first days of macrophage infection. Our results indicate that Rv3134c/devR/devS of M. bovis BCG constitutes an operon with complex regulation that participates in bacterial response against a wide range of stresses.
Assuntos
Proteínas de Bactérias/genética , Mycobacterium bovis/genética , Óperon/genética , Protamina Quinase/genética , Estresse Fisiológico/fisiologia , Transcrição Gênica/genética , Animais , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Macrófagos/microbiologia , Camundongos , Mycobacterium bovis/metabolismo , Óxido Nítrico , Oxigênio , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Protamina Quinase/metabolismoRESUMO
The capacity of Mycobacterium tuberculosis (Mtb) to sense, respond and adapt to a variable and hostile environment within the host makes it one of the most successful human pathogens. During different stages of infection, Mtb is surrounded by a plethora of lipid molecules and current evidence points out the relevance of fatty acids during the infectious process. In this study, we have compared the transcriptional response of Mtb to hypoxia in cultures supplemented with a mix of even long-chain fatty acids or dextrose as main carbon sources. Using RNA sequencing, we have identified differential expressed genes in early and late hypoxia, defined according to the in vitro Wayne and Hayes model, and compared the results with the exponential phase of growth in both carbon sources. We show that the number of genes over-expressed in the lipid medium was quite low in both, early and late hypoxia, relative to conditions including dextrose, with the exception of transcripts of stable and non-coding RNAs, which were more expressed in the fatty acid medium. We found that sigB and sigE were over-expressed in the early phase of hypoxia, confirming their pivotal role in early adaptation to low oxygen concentration independently of the carbon source. A drastic contrast was found with the transcriptional regulatory factors at early hypoxia. Only 2 transcriptional factors were over-expressed in early hypoxia in the lipid medium compared to 37 that were over-expressed in the dextrose medium. Instead of Rv0081, known to be the central regulator of hypoxia in dextrose, Rv2745c (ClgR), seems to play a main role in hypoxia in the fatty acid medium. The low level of genes associated to the stress-response during their adaptation to hypoxia in fatty acids, suggests that this lipid environment makes hypoxia a less stressful condition for the tubercle bacilli. Taken all together, these results indicate that the presence of lipid molecules shapes the metabolic response of Mtb to an adaptive state for different stresses within the host, including hypoxia. This fact could explain the success of Mtb to establish long-term survival during latent infection.
Assuntos
Anaerobiose , Exposição Ambiental , Ácidos Graxos/metabolismo , Mycobacterium tuberculosis/fisiologia , Estresse Fisiológico , Adaptação Fisiológica , Carbono/metabolismo , Meios de Cultura/química , Perfilação da Expressão Gênica , Glucose/metabolismo , Mycobacterium tuberculosis/genética , Análise de Sequência de RNARESUMO
The analysis of the DNA repair genes ogt and ung was carried out in 117 Mycobacterium tuberculosis clinical isolates from Argentina and Colombia in order to explore correlation between mutations in these genes and multi-drug resistance. With the exception of two Beijing family isolates, the rest of the strains harbored either two wild-type or two mutant alleles with identical single nucleotide polymorphisms (SNPs) in each gene (ogt44 and ung501). These ogt44 and ung501 mutations were not associated with multi-drug resistance and occurred simultaneously in circulating Haarlem genotype M. tuberculosis strains. We therefore propose the use of these markers as tools in phylogenetic and epidemiologic studies.