High genetic heterogeneity of Mycobacterium intracellulare isolated from respiratory specimens.

Background: M. intracellulare is a frequent causative pathogen of nontuberculous mycobacteria infection that causes infections in the respiratory tract, whose incidence is increasing in many countries. This study aimed at determining the VNTR-based genetic diversity of a collection of 39 M.

Effect of efflux pump inhibitors on the susceptibility of Mycobacterium avium complex to clarithromycin.

In this study we aimed to evaluate the effect of the combination of clarithromycin and four inhibitors of efflux pumps (EPIs), including berberine (BER), carbonyl cyanide m-chlorophenylhydrazone (CCCP), piperine (PIP) and tetrandrine (TET), against 12 Mycobacterium avium complex clinical isolates. The minimum inhibitory concentration (MIC) of clarithromycin showed at least a fourfold reduction in presence of BER (83% of total isolates), CCCP (67%), PIP (25%) and TET (75%). Our results showed that the EPIs tested are active against both clarithromycin susceptible and resistant isolates.

Genotyping and clarithromycin susceptibility testing of Mycobacterium avium subsp. hominissuis isolated in Tuscany, Italy.

Mycobacterium avium subsp. hominissuis (MAH) is a major cause of nontuberculous mycobacteria infection and the incidence of MAH infections is increasing in many countries. This study aimed at determining the VNTR-based genetic diversity and the susceptibility to clarithromycin of a collection of 71 MAH human strains isolated in the last seven years.

Virulence of Subsp. Human Isolates in an Macrophage Infection Model.

Background: Mycobacterium avium subsp. hominissuis (MAH) is an environmental opportunistic pathogen for humans and swine worldwide; in humans, the vast majority of MAH infections is due to strains belonging to specific genotypes, such as the internal transcribed spacer (ITS)-sequevars Mav-A and Mav-B that mostly cause pulmonary infections in elderly patients and severe disseminated infections in acquired immunodeficiency syndrome patients, respectively. To test whether the different types of infections in distinct patients' populations might reflect a different virulence of the infecting genotypes, MAH human isolates, genotyped by ITS sequencing and MIRU-VNTR minisatellite analysis, were studied for the capacity to infect and replicate in human macrophages in vitro.

Genomic diversity of Mycobacterium tuberculosis Beijing strains isolated in Tuscany, Italy, based on large sequence deletions, SNPs in putative DNA repair genes and MIRU-VNTR polymorphisms.

The Beijing genotype of Mycobacterium tuberculosis is cause of global concern as it is rapidly spreading worldwide, is considered hypervirulent, and is most often associated to massive spread of MDR/XDR TB, although these epidemiological or pathological properties have not been confirmed for all strains and in all geographic settings. In this paper, to gain new insights into the biogeographical heterogeneity of the Beijing family, we investigated a global sample of Beijing strains (22% from Italian-born, 78% from foreign-born patients) by determining large sequence polymorphism of regions RD105, RD181, RD150 and RD142, single nucleotide polymorphism of putative DNA repair genes mutT4 and mutT2 and MIRU-VNTR profiles based on 11 discriminative loci. We found that, although our sample of Beijing strains showed a considerable genomic heterogeneity, yielding both ancient and recent phylogenetic strains, the prevalent successful Beijing subsets were characterized by deletions of RD105 and RD181 and by one nucleotide substitution in one or both mutT genes.

Genomic variability of Mycobacterium tuberculosis strains of the Euro-American lineage based on large sequence deletions and 15-locus MIRU-VNTR polymorphism.

A sample of 260 Mycobacterium tuberculosis strains assigned to the Euro-American family was studied to identify phylogenetically informative genomic regions of difference (RD). Mutually exclusive deletions of regions RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 and RD761 were found in 202 strains; the RD(Rio) deletion was detected exclusively among the RD174-deleted strains. Although certain deletions were found more frequently in certain spoligotype families (i.

Large Sequence Polymorphisms of the Euro-American lineage of Mycobacterium tuberculosis: a phylogenetic reconstruction and evidence for convergent evolution in the DR locus.

The Euro-American lineage of the Mycobacterium tuberculosis complex consists of 10 sublineages, each defined by a deletion of a large genomic region (RD, region of difference); by spoligotyping, that probes the polymorphism of the Direct Repeat (DR) locus, the Euro-American strains are classified into 5 lineages (T, Haarlem, LAM, S and X) and 34 sublineages, but the relationships between the RD-defined sublineages and the spoligotype groupings are largely unclear. By testing a global sample of 158 Euro-American strains, mutually exclusive deletions of RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 or RD761 were found in 122 strains; deletion of RD724, typical of strains from Central Africa, was not found. The RD-defined sublineages, tested for katG463/gyrA95 polymorphism, belonged to Principal Genotypic Group (PGG) 2, with the exception of RD219 sublineage belonging to PGG3; the 36 strains with no deletion were of either PGG2 or 3.

Genetic diversity of human isolates of Mycobacterium bovis assessed by spoligotyping and Variable Number Tandem Repeat genotyping.

A collection of clinical isolates including 9 Mycobacterium bovis bacille Calmette-Guérin (BCG), 37 M. bovis and 1 isolate identified as M. bovis/caprae intermediate, recovered from humans in Tuscany, Italy, from 1990 to 2009, was genotyped by spoligotyping and Variable Number Tandem Repeat (VNTR) typing.

A duplex real-time PCR assay for detection of mutT4 and mutT2 mutations in Mycobacterium tuberculosis of Beijing genotype.

To determine the polymorphism of mutT genes of Mycobacterium tuberculosis of Beijing genotype, we developed a duplex real-time PCR assay based on hybridization probes for the Roche LightCycler instrument. The assay rapidly detects mutations at codons 48 and 58 of genes mutT4 and at mutT2, respectively.

Evolutionary pathway of the Beijing lineage of Mycobacterium tuberculosis based on genomic deletions and mutT genes polymorphisms.

Among the genotypes that prevail in the modern spectrum of Mycobacterium tuberculosis strains, the Beijing genotype is the one that causes major concern, as it is geographically widespread and it is considered hypervirulent. Comparative genomic studies have shown that Beijing strains have principally evolved through mechanisms of deletion of chromosomal regions, designated regions of difference (RD), and mutations. In this paper, we aimed to determine the evolutionary history of Beijing strains through the analysis of polymorphisms generated by deletions of large specific sequences, i.

Variation of the expression of Mycobacterium tuberculosis ppe44 gene among clinical isolates.

PPE44 is a member of the Mycobacterium tuberculosis PPE proteins, a polymorphic family of 69 glycine-rich proteins that predictively represent a source of antigenic variation. The genetic diversity of gene ppe44 among clinical isolates has been studied. No genomic polymorphism of ppe44 was found by a PCR-restriction fragment length polymorphism assay using three restriction enzymes.

Three-year longitudinal study of genotypes of Mycobacterium tuberculosis isolates in Tuscany, Italy.

The genetic diversity of 829 strains of Mycobacterium tuberculosis isolated during a 3-year period in Tuscany, Italy, a country with a low prevalence of tuberculosis, from 480 Italian-born and 349 foreign-born patients was determined by spoligotyping. The predominant spoligotype families were T (30.2% of isolates), Haarlem (19.

Molecular analysis of clinical isolates of Mycobacterium bovis recovered from humans in Italy.

In order to achieve a better knowledge of Mycobacterium bovis epidemiology in Italy, 42 clinical isolates from humans were genotyped. Predominant molecular patterns were found in one cluster of 15 isolates sharing spoligotype (ST482), variable-number tandem repeat (VNTR), and IS6110-based restriction fragment length polymorphism (one 1.9-kb band) profiles and in two clusters of 6 and 3 Mycobacterium bovis BCG isolates differing by one VNTR character.

Mutations in mutT genes of Mycobacterium tuberculosis isolates of Beijing genotype.

Missense alterations in genes mutT4 and mutT2, which encode DNA repair enzymes, were sequenced from 30 clinical isolates of Mycobacterium tuberculosis of Beijing genotype, mostly from patients with primary tuberculosis, to evaluate their contribution to anti-mycobacterial drug resistance. The mutation Arg to Gly at codon position 48 (CGG to GGG) of mutT4 was found in 21 isolates; of these, 16 isolates also harboured the mutation Gly to Arg at position 58 (GGA to CGA) of mutT2. No statistically significant association was found between mutT4 and mutT2 mutations, and drug resistance.

Immunogenicity of mycobacterial PPE44 (Rv2770c) in Mycobacterium bovis BCG-infected mice.

The Pro-Pro-Glu (PPE) protein family of Mycobacterium tuberculosis includes 69 glycine-rich proteins with a conserved N-terminal domain. Their role in tuberculosis is unknown, but it has been speculated that they may have an important immunological significance. In this investigation, the immunogenicity of the ppe44 (Rv2770c) gene product in BALB/c mice infected subcutaneously or intravenously with Mycobacterium bovis bacille Calmette-Guerin (BCG) was evaluated.

Genetic diversity, determined on the basis of katG463 and gyrA95 polymorphisms, Spoligotyping, and IS6110 typing, of Mycobacterium tuberculosis complex isolates from Italy.

Mycobacterium tuberculosis complex isolates (n = 248) collected during a 1-year period in Tuscany, Italy, were genotyped for the katG463 and gyrA95 polymorphisms and by standard spacer oligonucleotide typing (spoligotyping) and IS6110 restriction fragment length polymorphism (RFLP) assays. Most of the isolates (n = 212; 85.5%) belonged to genotypic groups 2 and 3, which included most isolates from Italian-born patients.

Detection of Mycobacterium tuberculosis genotypic groups by a duplex real-time PCR targeting the katG and gyrA genes.

A duplex real-time PCR assay was developed for the assignment of Mycobacterium tuberculosis isolates to the three genotypic groups based on the katG463/gyrA95 polymorphism. The assay was as sensitive and specific as nucleotide sequencing and proved also able to detect unambiguously the isolate genotype in clinical specimens.

Proposal to elevate the genetic variant MAC-A, included in the Mycobacterium avium complex, to species rank as Mycobacterium chimaera sp. nov.

The possibility that the strains included within the Mycobacterium avium complex (MAC), but not belonging either to M. avium or to Mycobacterium intracellulare, may be members of undescribed taxa, has already been questioned by several taxonomists. A very homogeneous cluster of 12 strains characterized by identical nucleotide sequences both in the 16S rDNA and in the 16S-23S internal transcribed spacer was investigated.

Most human isolates of Mycobacterium avium Mav-A and Mav-B are strong producers of hemolysin, a putative virulence factor.

Hemolysin was quantified in 58 isolates of Mycobacterium avium from human, animal, and environmental sources. Human Mav-A and Mav-B isolates were the strongest producers; in contrast, animal and environmental Mav-A isolates and human, animal, and environmental Mav-C organisms were low-level producers. Hemolysin production was not restricted to isolates causing invasive infections.

A real-time PCR assay for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates.

A real-time PCR genotypic assay was developed for the detection of isoniazid (INH) resistance in Mycobacterium tuberculosis. The assay detects mutations C(-15)T and, possibly, G(-24)T in the regulatory region of the inhA gene and proved as sensitive and specific as nucleotide sequencing in all the clinical isolates tested. Our assays mapped the mutations efficiently in 10 out of 35 resistant isolates, thereby covering 29% of all resistant strains.

Identification of one insertion site of IS6110 in Mycobacterium tuberculosis H37Ra and analysis of the RvD2 deletion in M. tuberculosis clinical isolates.

Mycobacterium 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.

Molecular typing of Mycobacterium avium isolates by sequencing of the 16S-23S rDNA internal transcribed spacer and comparison with IS1245-based fingerprinting.

The nucleotide sequence of the variable 16S-23S rDNA internal transcribed spacer (ITS) was determined in 32 strains of Mycobacterium avium, including 29 clinical isolates, two environmental isolates and the reference strain M. avium ATCC 35712. The results were compared with those obtained by the IS1245-based restriction fragment length polymorphism (RFLP) assay.