1,721,064 research outputs found
Genetic diversity and phylogeny of Mycobacterium avium
No full textMycobacterium avium, one of the species of the M. avium complex (MAC), includes 4 subspecies, i.e., M. avium subsp. hominissuis (MAH), M. avium subsp. avium (MAA), M. avium subsp. silvaticum (MAS) and M. avium subsp. paratuberculosis (MAP), in turn classified into the S (sheep) and C (cattle) types. These subspecies, although closely related, represent distinct organisms, each endowed with specific pathogenetic and host range characteristics, ranging from environmental opportunistic bacteria that cause infections in swine and immunocompromised patients to pathogens of birds and ruminants. The present review summarizes the basic epidemiological and pathological features of the M. avium subspecies, describes the major genomic events responsible of M. avium subspecies diversity (insertion sequences, sequence variations in specific chromosome loci or genes, deletions, duplications and insertions of large genomic regions) and then reconstructs the phylogenetic relationships among the M. avium subspecies
Intracellular cytokine detection by fluorescence-activated flow cytometry: Basic principles and recent advances
No full textIntracellular cytokine staining is a flow cytometric technique consisting of culturing stimulated cytokine-producing cells in the presence of a protein secretion inhibitor, followed by fixation, permeabilization and staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies. Up to 18 different colors can be detected by modern flow cytometers, making it the only immunological technique allowing simultaneous determination of antigen-specific T cell function and phenotype. In addition, cell proliferation and viability can be also measured. For this reason, it is probably the most popular method to measure antigenicity during vaccine trials and in the study of infectious diseases, along with ELISPOT. In this review, we will summarize its features, provide the protocol used by most laboratories and review its most recent applications
Geni di virulenza di Mycobacterium tuberculosis
No full textLa tubercolosi rappresenta la principale causa di morte nel mondo causata da un singolo agente patogeno. Nonostante la sua importanza, le basi genetiche della patogenicità di Mycobacterium tuberculosis non sono ancora state chiarite per la mancanza di validi metodi di studio. Recentemente, sono stati ottenuti notevoli progressi nello studio della genetica micobatterica. In primo luogo, è stata resa nota la sequenza completa del genoma di M. tuberculosis; inoltre, sono state sviluppate nuove efficienti strategie molecolari, incluso lo sviluppo di metodiche mirate alla inattivazione di geni. Tali tecnologie hanno permesso di analizzare l’espressione di geni micobatterici in vivo e hanno portato alla identificazione di una serie di geni coinvolti nella virulenza di M. tuberculosis. Alcuni di questi geni sono implicati nella sintesi e nel trasporto dello ftiocerolo dimicocerosato, un lipide presente unicamente nei micobatteri patogeni, necessario per la crescita di M. tuberculosis nel polmone. Inoltre, mutazioni in geni che codificano enzimi correlati, enzimi coinvolti nel metabolismo dei lipidi, e altre strutture della parete batterica riducono la crescita in vivo del bacillo tubercolare, dimostrando l’importanza dei componenti della parete nella virulenza. E’ stato dimostrato che numerosi processi fisologici svolgono un ruolo significativo per la sopravvivenza di M. tuberculosis nell’ospite. Questi includono la biosintesi di purine, che presumibilmente sono assenti nell’ambiente intracellulare, e la risposta allo stress ambientale. Inoltre, la regolazione della trascrizione genica rappresenta un rilevante processo coinvolto nella virulenza di M. tuberculosis, e di particolare interesse è la funzione dei geni che codificano per i fattori sigma e il repressore ferro-dipendente. In futuro, alcuni di questi geni e le proteine da essi codificate, insieme ad eventuali nuovi fattori, potranno fornire nuovi bersagli batterici da poter impiegare per l’allestimento di farmaci e vaccini
Prevalence of Epstein-Barr virus type 1 and 2 in oropharyngeal fluids of HIV-infected patients
No full textGenomic diversity and molecular epidemiology of Mycobacterium tuberculosis strains of Beijing genotype isolated in Tuscany, Italy
No full textIntroduction: The Beijing genotype of Mycobacterium tuberculosis (MTB) is cause of global concern as it is rapidly spreading worldwide, is considered hypervirulent, and is most often associated to MDR/XDR tuberculosis, although these properties have not been confirmed for all strains and in all geographic settings. In this study we report the molecular characterization of a collection of Beijing strains isolated in Tuscany, Italy, a region where the ethnic diversity of patients provides an opportunity to study a global sample of Beijing strains and gain new insights into the heterogeneity and phylogeny of the Beijing genotype.
Materials and Methods: A total of 80 MTB Beijing strains isolated in the years 2002-2010 from 18 Italian- and 62 foreign-born patients were studied. Spoligotype patterns, deletions of large genomic sequences RD105, RD181, RD150 and RD142, missense mutations in putative DNA repair genes mutT4 and mutT2, and MIRU-VNTR profiles, based on 11 discriminative loci, were determined according to standard procedures.
Results: Molecular analysis of MTB Beijing isolates revealed 56 MIRU-VNTR profiles, 6 spoligotype patterns, 3 deletions of large genomic sequences and polymorphic codons in mutT genes. Based on these polymorphisms, a phylogenetic reconstruction of the Beijing lineage was drawn. A minimun spanning tree (MST), constructed by the MIRU-VNTR profiles, showed that strains with deletions RD105 and RD181 and mutated mutT4/mutT2 genes form a large clonal complex of strains linked together, representing the prevalent expanding strain population and that RD105/RD181-deleted strains with mutated mutT4 and wild-type mutT2 form two star-like clonal complexes typical of expanding strain populations. Moreover, MIRU-VNTR analysis revealed 47 unique profiles and 9 clusters including 33 (41%) isolates; active transmission rate of Beijing strains (30.0%) was two-fold higher than non-Beijing strains previously reported in our setting.
Conclusions: Our study shows a considerable genomic heterogeneity of MTB Beijing isolates, yielding both ancient and recent phylogenetic sublineages, and confirms the high transmissibility of Beijing strains also in our setting. Notably, as the prevalent Beijing sublineages harbour missense mutations in one or both putative DNA repair mutT genes, it can be speculated that a defective DNA repair system may confer selective advantages and contribute to the successful spreading of the Beijing family
Genes of Mycobacterium tuberculosis H37Rv downregulated in the attenuated strain H37Ra are restricted to M. tuberculosis complex species
No full textBy comparing gene expression of virulent Mycobacterium tuberculosis H37Rv and attenuated strain H37Ra, we previously detected six genes that appear to be markedly downregulated in the attenuated strain compared with the virulent one. Three of these genes, i.e. Rv1345, Rv2770c, and Rv0288, code for proteins that can be predictively associated to immunological or pathogenetic aspects of M. tuberculosis infection; the other genes, i.e. Rv2336, Rv1320c, and Rv2819c, code for proteins with unknown functions (Rindi et al., 1999). In this paper we searched for the above mentioned genes in Pvu II-digested genomic DNA of a number of mycobacterial species by southern blot analysis employing PCR-generated probes in high-stringency conditions. Hybridization signals were only found in species belonging to the M. tuberculosis complex, i.e., M. tuberculosis, M. bovis, including the BCG strain, and M. microti, but not in other mycobacterial species, including M. avium, M. intracellulare, M. malmoense, M. xenopi, M. kansasii, M. simiae, M. marinum, M. scrofulaceum, M. gordonae, M. fortuitum, and M. smegmantis. These results indicate that genes Rv1345, Rv2770c, Rv0288, Rv2336, Rv1320c, and Rv2819c are associated with the most virulent mycobacteria and further support their potential role in M. tuberculosis virulence
Identification of one insertion site of IS6110 in Mycobacterium tuberculosis H37Ra and analysis of the RvD2 deletion in M. tuberculosis clinical isolates
No full textMycobacterium tuberculosis H37Rv and the attenuated strain H37Ra were used as a model to investigate the virulence properties of M. tuberculosis at the genetic level. To test whether transposition of the insertion element IS6110 might be involved in the loss of virulence of strain H37Ra, the nucleotide sequence of a differential IS6110-positive restriction fragment detected in strain H37Ra, but not in strain H37Rv, was determined. The region flanking the 3' end of the IS6110 element showed partial sequence homology with internal sequences of M. tuberculosis H37Rv genes plcA, plcB and plcC, each one coding for phospholipase C, a well-known bacterial virulence factor. A 100% homology was found between the IS6110-flanking region and an internal sequence of M. bovis plcD, a further phospholipase C gene that is truncated and partly lost in strain H37Rv in the so-called RvD2 deletion. This result indicates that the differential restriction fragment of strain H37Ra originally stems from the plcD gene interrupted by the insertion of the IS6110 element. The occurrence of the RvD2 deletion was then investigated in 45 clinical isolates of M. tuberculosis by Southern blot. The deletion was demonstrated in 15 isolates; the entire RvD2 region (including the undisrupted plcD gene) was detected in 29 isolates, whereas only one isolate showed the RvD2 region in which the plcD gene was interrupted by an IS6110 insertion. It is concluded that disruption of the plcD gene and deletion of the RvD2 region by IS6110 insertion have no consequence for the virulence of M. tuberculosis, although the role of phospholipase C as a virulence factor of M. tuberculosis remains debatable
Increase in non-tuberculous mycobacteria isolated from humans in Tuscany, Italy, from 2004 to 2016
No full textIntroduction: Non-tuberculous mycobacteria (NTM) include all Mycobacterium species other than Mycobacterium tuberculosis complex and Mycobacterium leprae. NTM are a group of over 150 environmental species; they are generally endowed with low pathogenicity to human, however some species are associated with a variety of human diseases: respiratory tract infection are the most frequent, followed by lymphadenitis in children, disseminated infections in severely immunocompromised patients and skin infections. In Italy, the prevalence of NTM in human infections is largely unknown. The aim of the present survey is to report the epidemiology and recent trend of NTM infections in a region of central Italy, Tuscany, over the last 13 years, and provide a review of the recent literature on NTM isolation rates in different geographic regions.
Materials and Methods: The complete collection of NTM strains isolated from 50.150 clinical specimens at the Laboratory of Clinical Mycobacteriology of Pisa University Hospital, Italy, from 1 January 2004 to 31 December 2016 was included.
Results: In our setting, in the period 2004-2016 a total of 215 patients had cultures positive for NTM. The number of NTM isolates increased considerably from 5 isolates in 2004 to 36 in 2016; a sharp increase occurred in the last 5 years. Overall, 18 NTM species were isolated; the most common were M. avium, M. intracellulare and M. gordonae detected in respectively in 37.7%, 15.8% and 13.5% of NTM patients. Notably, M. kansasii, a pulmonary pathogen not reported before 2012, was repeatedly isolated in the last 5 years, representing 7,1% of total NTM isolates. In general, NTM isolates were largely prevalent in people older than 60 (59.5%); patients aged 1-10 year-old almost exclusively yielded M. avium and M. intracellulare. Of the 215 NTM clinical isolates, 77.2% were from respiratory specimens, 10.7% from lymph nodes, 2.3% from blood (yielding exclusively M. avium), and the remaining 9.8% from other clinical specimens.
Conclusions: The present study provides a snapshot of the prevalent NTM species in our setting and shows an increase in NTM isolation rate, which is in keeping with the general increase in NTM infections reported worldwide in the past two decades, although the distribution of the NTM prevalent species differs by geographic region
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