Genetic Diversity, Determined on the Basis of katG463 and gyrA95 Polymorphisms, Spoligotyping, and IS 6110 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.Since the discovery of polymorphic DNA in Mycobacterium tuberculosis, molecular typing of strains has become a valuable tool in the study of the epidemiology of tuberculosis (TB) by allowing investigators to track epidemics, detect new outbreaks, and achieve better knowledge of moving and expanding strains (1).Analysis of restriction fragment length polymorphisms (RFLPs), based on the variability of the chromosomal location and copy number of insertion sequence IS6110, is considered the standard approach for the genotyping of M. tuberculosis isolates and has allowed the construction of large databases of IS6110-based genotypes (24). IS6110 fingerprinting has been shown to have great potential for the clonal differentiation of M. tuberculosis isolates (11); however, IS6110 RFLP analysis is a labor-intensive and time-consuming methodology, and its use is hampered for strains with fewer than six IS6110 insertions that have a limited degree of IS6110 polymorphisms or when rapid results are needed, such as in cases of nosocomial transmission of TB or laboratory cross-contaminations. In this context, PCR-based spacer oligonucleotide...

Global Phylogeny ofMycobacterium tuberculosisBased on Single Nucleotide Polymorphism (SNP) Analysis: Insights into Tuberculosis Evolution, Phylogenetic Accuracy of Other DNA Fingerprinting Systems, and Recommendations for a Minimal Standard SNP Set

“…Our results showed that the T clade comprised the majority of SCG-6a and -6b, but it could also be found in SCG-3abc, -4, and -5, suggesting that the T clade classification in fact includes a number of phylogenetically distinct subgroups. The Haarlem (H) clade is another spoligotype group that occurs at high frequency in human tuberculosis studies (20,44). Our results showed that the H clade could be found in two distinct genetic clusters, SCG-3b and SCG-5, although neither of these SCGs contained only H clade isolates.…”

Three-Year Longitudinal Study of Genotypes of Mycobacterium tuberculosis Isolates in Tuscany, Italy

“…Among these, the predominant spoligotypes, shown in Table 2, include (i) ST53, with a total of 98 isolates, a ubiquitous spoligotype belonging to clade T1; (ii) ST1, with 48 isolates, which represents the predominant spoligotype of the Beijing family; (iii) ST50, ST47, and ST62, belonging to the Haarlem family, with 43, 30, and 22 isolates, respectively; and (iv) ST42 of the LAM9 clade, with 35 isolates. Among the other clustered major spoligotypes, it is worthwhile to mention the occurrence of spoligotype ST1737, clade T1 Tuscany, with 10 isolates which likely represent autochthonous spoligotypes, as previously reported (20), or of spoligotypes which are endemic in distant geographical areas, such as ST26 of the CAS1_Delhi clade, with 11 isolates; ST19 of the EAI2_Manilla clade, with 10 isolates; and ST181 of the Africanum type 1 clade, with 5 isolates. Among the remaining 213 (25.7%) unclustered isolates, 99 (11.9%) were not yet reported to the SpolDB4 database and represented the true orphan isolates, indicated with ST0; the remaining 114 (13.8%) unclustered isolates, though unique, were already reported in SpolDB4, which allowed the attribution of an ST number.…”

Distribution of Strain Families ofMycobacterium tuberculosisCausing Pulmonary and Extrapulmonary Disease in Hospitalized Children in Cape Town, South Africa

“…Spoligotyping identified 11 genotype families, namely, LAM3/F11 (n ϭ 86), W-Beijing (n ϭ 70), X (European low-copy-number family by IS6110-based restriction fragment length polymorphism analysis; n ϭ 30), S/F28 (n ϭ 21), Zimbabwean (n ϭ 7), other LAM (n ϭ 8), Tuscany and Russian (n ϭ 12) (Brudley et al, unpublished), Haarlem (n ϭ 11), T clade (n ϭ 20), CAS (n ϭ 5) and "unknown" clades (n ϭ 15) (4,8).…”