Nicoletta Lari

Association of Mycobacterium tuberculosis complex isolates of BOVIS and Central Asian (CAS) genotypic lineages with extrapulmonary disease

The association between isolate genotype, defined as in the international spoligotype database SpolDB4, and extrapulmonary tuberculosis was determined among 1009 patients in a population-based, 4-year survey performed in Tuscany, Italy. Extrapulmonary disease occurred in 24.2% of patients. A statistically significant association with extrapulmonary disease was found for the BOVIS (adjusted OR 3.2; 95% CI 1.2-8.1) and for the Central Asian (CAS) lineages (adjusted OR 2.3; 95% CI 1.0-5.1). These findings support the view that Mycobacterium tuberculosis strains within individual genotypic lineages might have evolved unique pathogenic characteristics that are capable of influencing the clinical outcome of the infection.

Genetic Diversity, Determined on the Basis of katG463 and gyrA95 Polymorphisms, Spoligotyping, and IS6110 Typing, of Mycobacterium tuberculosis Complex Isolates from Italy

ABSTRACT 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. The remaining isolates were genotypic group 1 organisms, which were prevalent among foreign-born patients (29 of 36; 80.6%). Spoligotype analysis detected 116 unique patterns and 34 clusters including 166 isolates. The combination of spoligotyping and IS6110 RFLP analyses yielded 28 distinct clusters including 65 identical isolates (26.2%)—22 clusters with 2 isolates, 4 clusters with 3 isolates, 1 cluster with 4 isolates, and 1 cluster with 5 isolates—thus proving a low transmission rate in the community. Predominant spoligotypes representing 50% of clustered isolates were found in six clusters that included widespread type ST53 (clade T1) with 29 isolates (11.7% of total isolates); types ST50 and ST47 (Haarlem family) with 18 isolates (7.3%) and 8 isolates (3.2%), respectively; type ST42 (Latino-American and Mediterranean clade) with 13 isolates (5.2%); new type ST1737 (named “Tuscany”) with 8 isolates (3.2%); and type ST1 (W-Beijing family) with 7 isolates (2.8%). Other spoligotype families, such as the Mycobacterium africanum, East African-Indian (EAI2/Manila), and central Asia 1 (CAS1/Delhi) families (all including organisms of genotypic group 1) and the Cameroun family (genotypic group 2), were detected especially among immigrant patients. The occurrence of genotypes originally found in distant geographic areas with a high prevalence of tuberculosis may represent a hallmark for changes in the dynamics of transmission of tuberculosis in the region in the near future.

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.e., RD105, RD181, RD150, and RD142, and by single nucleotide substitutions in genes mutT4 and mutT2, coding for DNA repair enzymes. Based on the molecular characteristics of a collection of Beijing strains recently isolated in Tuscany, Italy, we propose a phylogenetic reconstruction of the Beijing family. According to our model, the Beijing family evolved from a M. tuberculosis progenitor following deletion of the RD207 region, an event responsible for the loss of spacers 1-34 in the direct repeat (DR) locus. The major lineages of the Beijing family then evolved via subsequent deletions of regions RD105, RD181 and RD150. In the most ancient evolutionary lineages genes mutT4 and mutT2 were in wild type configuration; the mutT4 mutation was acquired subsequent to the RD181 deletion in a progenitor strain that, in turn, gave rise to a sublineage bearing the mutT2 mutation. Within the major branches of the Beijing family, deletion of additional spacers in the DR locus led to evolution of sublineages characterized by different spoligotypes. Our evolutionary model of the Beijing family provides a deeper framework than previously proposed for epidemiologic and phylogenetic studies of circulating M. tuberculosis Beijing strains, thus allowing a more systematic and comprehensive evaluation of the relevance of Beijing strain variability.

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

ABSTRACT 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. The remaining 18 isolates yielded unique profiles. Our results confirm the potential utility of spoligotyping and VNTR typing as a major typing system of M. bovis isolates.

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. The strains belonged to three distinct ITS sequevars, Mav-A, Mav-B and Mav-C. Sixteen of 17 isolates of the Mav-B sequevar were from HIV-positive patients; the Mav-A sequevar included six and five isolates from HIV-positive and HIV-negative individuals, respectively, as well as the two environmental isolates and the M. avium reference strain ATCC 35712; only one isolate, from a HIV-infected patient, belonged to the Mav-C sequevar. IS1245-RFLP analysis of M. avium isolates of sequevars Mav-A and Mav-B showed that isolates occurring in clusters of identical or highly related RFLP patterns generally belong to the same sequevar, and that M. avium strains belonging to the same sequevar may present distinct and unrelated IS1245-RFLP patterns. The question of the molecular markers specific for M. avium clones pathogenic for man is discussed.

Comparison of three restriction endonucleases in IS1245-based RFLP typing of Mycobacterium avium

IS1245-based restriction fragment length polymorphism (RFLP) analysis has been proposed recently for molecular typing of Mycobacterium avium isolates. As there is no standardised method with respect to the optimal restriction enzyme, three restriction endonucleases were tested for analysis of 17 human isolates. The restriction endonucleases, selected on the basis of the physical maps of IS1245 and of the highly homologous IS1311, were BsaAI, that cleaves IS1245, PvuII, that cleaves IS1311, and NruI, that cleaves both IS1245 and IS1311. All the restriction endonucleases yielded polymorphic and complex RFLP patterns. However, BsaAI- and NruI-generated bands were more evenly distributed and easier to detect than PvuII-generated bands, most of which clustered in a narrow zone of the fingerprint. In some cases, DNA digestion with BsaAI or NruI yielded probe-specific restriction fragments of molecular size lower than expected. Moreover, digestion with NruI, which was expected to generate the highest numbers of bands in all the isolates, yielded fewer bands than were obtained with BsaAI or PvuII in 14 and 5 isolates, respectively. These findings might suggest the existence of unidentified IS1245-related insertion element(s) in M. avium isolates. Computer analysis of the IS1245-based RFLP patterns of M. avium isolates showed that the restriction endonucleases were capable, although with minor differences, of defining distinct banding patterns and clusters of identical or highly related isolates, thus confirming IS1245-based RFLP analysis as a useful technique for epidemiological studies.

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.

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. Based on these polymorphisms, a phylogenetic reconstruction of the Euro-American lineage, that integrates the previously reported phylogeny, is proposed. Although certain deletions were found to be associated to certain spoligotype lineages (i.e., deletion RD115 to T and LAM, RD174 to LAM, RD182 to Haarlem, RD219 to T), our analysis indicates a general lack of concordance between RD-defined sublineages and spoligotype groupings. Moreover, of the 42 spoligotypes detected among the study strains, sixteen were shared by strains belonging to different RD sublineages. IS6110-RFLP analysis of strains sharing spoligotypes confirmed a poor genetic relatedness between strains of different RD sublineages. These findings provide evidence for the occurrence of a high degree of homoplasy in the DR locus leading to convergent evolution to identical spoligotypes. The incongruence between Large Sequence Polymorphism and spoligotype polymorphism argues against the use of spoligotyping for establishing phylogenetic relationships within the Euro-American lineage.

Beijing/W Mycobacterium tuberculosis in Italy.

To the Editor: Molecular typing of Mycobacterium tuberculosis strains isolated in several countries in recent years has shown that a group of strains known as “Beijing” is widespread around the world (1). The Beijing group of M. tuberculosis has been associated with drug resistance; one multidrug-resistant strain, designated “W,” was found in New York City in the early 1990s and caused large institutional outbreaks of tuberculosis (TB) in the United States (2). M. tuberculosis strains of Beijing/W genotype are mostly prevalent in Asia (1), but recent data suggest that they have been spreading in Indochina and are prevalent among younger persons in Vietnam (3). Beijing/W strains are also widespread in Eastern Europe (1); during the last decade, the Beijing/W genotype of M. tuberculosis, with more prevalent drug-resistant mutations than non-Beijing strains, has been identified in 40% to 50% of clinical isolates studied in Russia (4). We studied a total of 245 M. tuberculosis strains collected during a 1-year period, from January to December 2002, from the same number of TB patients hospitalized in Tuscany, Italy. All the isolates were typed by the standardized IS6110 restriction fragment length polymorphism (RFLP) and the spoligotyping (spacer oligonucleotide typing) techniques. A total of 216 distinct IS6110 RFLP patterns were found among the 245 isolates; 51 isolates (20.8%) occurred in 23 clusters, each constituting strains with an identical IS6110 RFLP and spoligotype pattern; 19 clusters contained two isolates each, 3 contained three isolates, and 1 contained four isolates. Spoligotype analysis showed seven isolates with the typical Beijing/W pattern of probe hybridization only to spacer sequences 35–43. The Beijing/W isolates yielded distinct IS6110 RFLP profiles with similarity coefficient >57.8%. Characteristics of the Beijing/W strains and respective patients, obtained from clinical records, are reported in the Table. Although the overall prevalence of Beijing/W strains was low (7/245, 2.9%), five of the seven strains were from recent immigrants to Italy from China who live in the same area; the other two strains were from Italian citizens also living in that area. Recent immigration from high-prevalence areas is therefore likely to be associated with the occurrence of the Beijing/W genotype in Italy. None of the Beijing/W strains was associated with TB outbreaks; nonetheless, infection of Italian residents with Beijing strains suggests that spread of this genotype is ongoing. Table Characteristics of Mycobacterium tuberculosis strains of Beijing/W genotype isolated in 2002 in Tuscany, Italya Beijing/W strains have been strongly associated with drug resistance in a number of countries (2,4–6), but elsewhere the association was weak or absent. In our survey, no substantial drug resistance was observed; all Beijing/W strains isolated in Tuscany were susceptible to rifampin, ethambutol, pirazinamide, and streptomycin (tested only in two strains), and all but one were susceptible to isoniazid. Although we detected only a few cases, our data do not show a trend of Beijing/W strains’ being associated with infection in young people, as has been observed in other settings (3). The age of immigrants with Beijing/W TB (mean 33.2 years, standard deviation [SD] 8.2 years) did not significantly differ from that of immigrants infected with non-Beijing/W strains (30.7 years, SD 7.4 years), a find that indicates that, at least in our setting, immigrant status, rather than M. tuberculosis genotype, is associated with infection in young people. The few cases of Beijing/W infections in Italian-born patients do not allow us to draw conclusions regarding nonimmigrant patients. In conclusion, M. tuberculosis strains of Beijing/W genotype are becoming widespread worldwide, including in countries with a low prevalence of TB. Their association with drug resistance and infection in young people, clearly shown in certain settings, remains to be defined. Further molecular epidemiologic surveillance is needed to monitor trends in prevalence and spread of these strains.

Most Human Isolates of Mycobacterium avium Mav-A and Mav-B Are Strong Producers of Hemolysin, a Putative Virulence Factor

ABSTRACT 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.