Rapid identification of lineage and drug resistance in clinical samples of Mycobacterium Tuberculosis

Background: Mycobacterium tuberculosis is a slow-growing bacterium, which could delay its diagnosis and, therefore, promote the spread of the disease. Whole-genome sequencing allows us to obtain the complete drug-resistance profile of the strain; however, bacterial cultivation of clinical samples, along with complex processing, is required. Methods: In this work, we explore AmpliSeq, an amplicon-based enrichment method for preparing libraries for targeted next-generation sequencing, to identify lineage and drug resistance directly from clinical samples. Results: In our study, 111 clinical samples were tested. The lineage was identified in 100% of the culture-derived samples (52/52), in 95% of the smear (BK)-positive clinical samples (38/40) and in 42.1% of the BK-negative clinical samples (8/19). The drug-resistance profile was accurately identified in all but 11 samples, in which some phenotypic and genotypic discrepancies were found. In this respect, our panels were not exact in the detection of streptomycin resistance for isolates derived from clinical samples, as an extremely high number of SNPs in the rrs and rrl genes were detected due to cross-contamination. Conclusion: This technique has demonstrated high sensitivity in obtaining the drug-resistance profile of the isolates, as even those samples with DNA concentrations below the detection limit of Qubit produced a result. AmpliSeq technology is cheaper than whole-genome sequencing, easy to perform by laboratory technicians and applicable to any microorganism using the Ion Torrent platform

A combination of transposable elements and magnetic cell sorting provides a very efficient transgenesis system for chicken primary erythroid progenitors

<p>Abstract</p> <p>Background</p> <p>Stable transgenesis is an undeniable key to understanding any genetic system. Retrovirus-based insertional strategies, which feature several technical challenges when they are used, are often limited to one particular species, and even sometimes to a particular cell type as the infection depends on certain cellular receptors. A universal-like system, which would allow both stable transgene expression independent of the cell type and an efficient sorting of transfected cells, is required when handling cellular models that are incompatible with retroviral strategies.</p> <p>Results</p> <p>We report here on the combination of a stable insertional transgenesis technique, based on the Tol2 transposon system together with the magnetic cell sorting (MACS) technique, which allows specific selection of cells carrying the transgene in an efficient, reliable and rapid way.</p> <p>Conclusion</p> <p>This new Tol2/MACS system leads to stable expression in a culture of primary chicken erythroid cells highly enriched in cells expressing the transgene of interest. This system could be used in a wide variety of vertebrate species.</p

Analysis of the twenty-six largest outbreaks of tuberculosis in Aragon using whole-genome sequencing for surveillance purposes

The incidence of tuberculosis in Aragon, Spain, is around ten cases per 100,000 inhabitants. Since 2004, a molecular surveillance protocol has been carried out; therefore, all M. tuberculosis strains are genotyped. Recently, whole-genome sequencing has been implemented for relevant isolates. The aim of this work is to characterise at the molecular level the causative strains of the 26 largest outbreaks of the community (including ten or more cases), genotyped by IS6110-RFLP and causing 26% of tuberculosis cases. To achieve this objective, two or three isolates of each IS6110-cluster belonging to different years were selected for sequencing. We found that strains of lineages L4.8, L4.3 and L4.1.2 were the most frequent. The threshold of 12 SNPs as the maximum distance for confirming the belonging to an outbreak was met for 18 of the 26 IS6110-clusters. Four pairs of isolates with more than 90 SNPs were identified as not belonging to the same strain, and four other pairs were kept in doubt as the number of SNPs was close to 12, between 14 and 35. The study of Regions of Difference revealed that they are lineage conserved. Moreover, we could analyse the IS6110 locations for all genome-sequenced isolates, finding some frequent locations in isolates belonging to the same lineage and certain IS6110 movements between the paired isolates. In the vast majority, these movements were not captured by the IS6110-RFLP pattern. After classifying the genes containing SNP by their functional category, we could confirm that the number of SNPs detected in genes considered as virulence factors and the number of cases the strain produced were not related, suggesting that a particular SNP is more relevant than the number. The characteristics found in the most successful strains in our community could be useful for other researchers in epidemiology, virulence and pathogenesis

Conservation of the links between gene transcription and chromosomal organization in the highly reduced genome of Buchnera aphidicola

<p>Abstract</p> <p>Background</p> <p>Genomic studies on bacteria have clearly shown the existence of chromosomal organization as regards, for example, to gene localization, order and orientation. Moreover, transcriptomic analyses have demonstrated that, in free-living bacteria, gene transcription levels and chromosomal organization are mutually influenced. We have explored the possible conservation of relationships between mRNA abundances and chromosomal organization in the highly reduced genome of <it>Buchnera aphidicola</it>, the primary endosymbiont of the aphids, and a close relative to <it>Escherichia coli</it>.</p> <p>Results</p> <p>Using an oligonucleotide-based microarray, we normalized the transcriptomic data by genomic DNA signals in order to have access to inter-gene comparison data. Our analysis showed that mRNA abundances, gene organization (operon) and gene essentiality are correlated in <it>Buchnera </it>(i.e., the most expressed genes are essential genes organized in operons) whereas no link between mRNA abundances and gene strand bias was found. The effect of <it>Buchnera </it>genome evolution on gene expression levels has also been analysed in order to assess the constraints imposed by the obligate symbiosis with aphids, underlining the importance of some gene sets for the survival of the two partners. Finally, our results show the existence of spatial periodic transcriptional patterns in the genome of <it>Buchnera</it>.</p> <p>Conclusion</p> <p>Despite an important reduction in its genome size and an apparent decay of its capacity for regulating transcription, this work reveals a significant correlation between mRNA abundances and chromosomal organization of the aphid-symbiont <it>Buchnera</it>.</p

Estimation of the mutation rate of Mycobacterium tuberculosis in cases with recurrent tuberculosis using whole genome sequencing

The study of tuberculosis latency is problematic due to the difficulty of isolating the bacteria in the dormancy state. Despite this, several in vivo approaches have been taken to mimic the latency process. Our group has studied the evolution of the bacteria in 18 cases of recurrent tuberculosis. We found that HIV positive patients develop recurrent tuberculosis earlier, generally in the first two years (p value = 0.041). The genome of the 36 Mycobacterium tuberculosis paired isolates (first and relapsed isolates) showed that none of the SNPs found within each pair was observed more than once, indicating that they were not directly related to the recurrence process. Moreover, some IS6110 movements were found in the paired isolates, indicating the presence of different clones within the patient. Finally, our results suggest that the mutation rate remains constant during all the period as no correlation was found between the number of SNPs and the time to relapse

The MtZ Strain: Molecular Characteristics and Outbreak Investigation of the Most Successful Mycobacterium tuberculosis Strain in Aragon Using Whole-Genome Sequencing

Since 2004, a tuberculosis surveillance protocol has been carried out in Aragon, thereby managing to detect all tuberculosis outbreaks that take place in the community. The largest outbreak was caused by a strain named Mycobacterium tuberculosis Zaragoza (MtZ), causing 242 cases as of 2020. The main objective of this work was to analyze this outbreak and the molecular characteristics of this successful strain that could be related to its greater transmission. To do this, we first applied whole-genome sequencing to 57 of the isolates. This revealed two principal transmission clusters and six subclusters arising from them. The MtZ strain belongs to L4.8 and had eight specific single nucleotide polymorphisms (SNPs) in genes considered to be virulence factors [ptpA, mc3D, mc3F, VapB41, pks15 (two SNPs), virS, and VapC50]. Second, a transcriptomic study was carried out to better understand the multiple IS6110 copies present in its genome. This allowed us to observe three effects of IS6110: the disruption of the gene in which the IS6110 is inserted (desA3), the overexpression of a gene (ppe38), and the absence of transcription of genes (cut1:Rv1765c) due to the recombination of two IS6110 copies. Finally, because of the disruption of ppe38 and ppe71 genes by an IS6110, a study of PE_PGRS secretion was carried out, showing that MtZ secretes these factors in higher amounts than the reference strain, thereby differing from the hypervirulent phenotype described for the Beijing strains. In conclusion, MtZ consists of several SNPs in genes related to virulence, pathogenesis, and survival, as well as other genomic polymorphisms, which may be implicated in its success among our population

Inclusión. Aula Estable con alumnos/as con discapacidad intelectual: perfil y desarrollo de los alumnos. Una propuesta para alumnos/as del Grado de Pedagogía

Depto. de Investigación y Psicología en EducaciónFac. de EducaciónFALSEsubmitte

Caractérisation des capacités de régulation génétique de la bactérie Buchnera aphidicola en liaison avec sa fonction symbiotique chez le puceron Acyrthosiphon pisum

L\u27association entre la bactérie Buchnera aphidicola et les pucerons est l\u27un des modèles de symbiose les plus étudiés aux plans évolutif, biochimique et plus récemment moléculaire. Il est admis que Buchnera fournit les acides aminés essentiels aux pucerons, qui se nourrissent de la sève phloémienne des plantes, aliment particulièrement pauvre en apports protéiques. La question des mécanismes de régulation du symbiote en réponse aux exigences nutritionnelles de son hôte reste cependant ouverte. Pour répondre à cette question, dans la première partie de cette thèse, nous avons analysé les relations entre l\u27expressivité des gènes et l\u27organisation du chromosome de Buchnera et montré qu\u27il existe une influence réciproque entre ces deux paramètres. Dans la deuxième partie de ce travail nous avons mis en évidence que la bactérie est capable d\u27ajuster la néosynthèse de leucine en réponse à des concentrations changeantes de cet acide aminé dans l\u27alimentation de son hôte. Les mécanismes moléculaires sous-jacents sont constitués par une réponse transcriptionnelle rapide complétée, plus tardivement, par une modification du nombre de copies du plasmide pLeu, portant les gènes de biosynthèse de la leucine. L\u27ensemble de ces résultats suggère que Buchnera a conservé, malgré la réduction de son génome, des capacités régulatrices essentielles pour l\u27adaptation du puceron aux contraintes de son environnement

Towards experimental manipulation of stochasticity in gene expression.

International audienceFor decades, most of molecular biology was driven by the "central dogma" in which the phenotype is defined by the genotype following a fully deterministic point of view. However, during the last 10 years, a wealth of studies has demonstrated that a given genotype can generate multiple phenotypes in identical environmental conditions, mainly because of the inherently probabilistic nature of the transcription process. It has also been shown that cells can tune this variability at the molecular level. Although previously described as a useless "noise", stochastic gene expression has now been shown by many authors to be an essential part of diverse biological processes. Chromatin dynamics having a central role in higher eukaryotes, we decided to investigate its involvement in the generation and control of stochasticity in gene expression (SGE). Our experiments reveal that the chromatin environment of a gene plays an important role in regulating SGE. Indeed, we find that histone acetylation and DNA methylation significantly affect SGE, suggesting that cells are able to adjust the variability of the expression of their genes through modification of chromatin marks. Given that the alteration of chromatin marks is itself subject to the expression of chromatin modifiers, our results shed light on a complex circular causality with on the one hand, the effect of gene expression on chromatin and on the other hand, the influence of the local chromatin environment of a gene on the dynamics of its expression