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PubMed2006

rpoB Gene mutations and molecular characterization of rifampin-resistant Mycobacterium tuberculosis isolates from Shandong Province, China.

Sixty rifampin (RIF)-resistant and 75 RIF-susceptible Mycobacterium tuberculosis isolates from Shandong Province, China, were analyzed for rpoB gene mutations and genotyped. Mycobacterial interspersed repetitive unit (MIRU) genotype 223325173533 was overrepresented among RIF-resistant isolates. MIRU combined with IS6110 restriction fragment length polymorphism analysis as the second-line genotyping method may reflect epidemiologic links more reliably than each method alone.

Antibiotics, Antitubercular↗
PubMed2006

Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis.

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%--and by 23% in combination with spoligotyping--among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold--by threefold in combination with spoligotyping--under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.

Bacterial Typing Techniques↗
PubMed2006

Assessment of an optimized mycobacterial interspersed repetitive- unit-variable-number tandem-repeat typing system combined with spoligotyping for population-based molecular epidemiology studies of tuberculosis.

An optimized set of 24 mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) loci, including a discriminatory subset of 15 loci, has recently been defined for the typing of Mycobacterium tuberculosis. Here, we evaluated the performances of this MIRU-VNTR typing system in combination with spoligotyping for the detection of transmission chains in a population-based study comprising 91% of culture-confirmed tuberculosis patients reported in 2003 in Hamburg, Germany. Of the 154 isolates investigated, more than 90% had high IS6110 copy numbers (>/=6). IS6110 restriction fragment length polymorphism (RFLP) typing resulted in 13 clusters, 5 of which had a confirmed epidemiological link. All five, as well as six of the eight IS6110 clusters with no identified epidemiological link, were perfectly matched by MIRU-VNTR typing with the 24 loci. Two IS6110 clusters were split by differences into 6 to 12 MIRU-VNTR loci, clearly supporting the absence of a link, as judged by contact tracing data. In contrast, only one MIRU-VNTR cluster, grouping what were probably epidemiologically unlinked isolates, was split by IS6110 RFLP. However, these isolates were also distinguished by spoligotyping. Both the optimized 24-locus and 15-locus sets thus showed a comparable to slightly better predictive value, especially when combined with spoligotyping, than the current gold standard IS6110 RFLP for the study of tuberculosis transmission in Hamburg. Because the epidemiological characteristics of this setting are similar to those of many developed countries, these results support the wide applicability of this real-time genotyping approach for population-based studies of M. tuberculosis transmission.

Bacterial Typing Techniques↗
PubMed2006

Progression toward an improved DNA amplification-based typing technique in the study of Mycobacterium tuberculosis epidemiology.

While high-copy-number IS6110-based restriction fragment length polymorphism (HCN-RFLP) is the gold standard for typing most Mycobacterium tuberculosis strains, the time taken for culturing and low throughput make it impractical for large-scale prospective typing of large numbers of isolates. The development of a new method, mycobacterial interspersed repetitive units (MIRU), a variation of the original variable-number tandem repeat (VNTR) technique, may provide a viable alternative. Panels based on the original 12-loci MIRU (12MIRU), a combination of 12MIRU and remaining ETR loci (15MIRU-VNTR), and an extended panel with an additional 10 novel regions (25VNTR) were used to study three populations with varying degrees of epidemiological data. MIRU discrimination increased with panel size and the addition of spoligotyping. Combining these two techniques enabled a reduction in the panel size from 25 to 14 loci without a significant loss in discrimination. However, 25VNTR alone or in combination with spoligotyping still possessed weaker discrimination than RFLP for high-copy-number isolates.

Bacterial Typing Techniques↗
PubMed2004

Application of mycobacterial interspersed repetitive unit typing to Manitoba tuberculosis cases: can restriction fragment length polymorphism be forgotten?

Since 1993, all Mycobacterium tuberculosis isolates recovered in the province of Manitoba, Canada, have been genotyped by the standard IS6110-restriction fragment length polymorphism (RFLP) method for routine surveillance, prevention, and control purposes. To date, our laboratory has collected 1,290 isolates, from which we have identified approximately 390 unique fingerprint patterns or "types." Although the standard method is well known for being a lengthy and labor-intensive procedure, a more efficient alternative for typing tuberculosis isolates, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method, has recently gained acceptance. Consequently, all isolates acquired in 2003 (n = 126) were typed by both methods in order to determine the utility of replacing the RFLP method with MIRU typing for all future isolates. Application of Hunter's discriminatory index to the available study population showed that the MIRU method was close in discriminatory power (D) to the RFLP method (D(MIRU) = 0.831 to 0.984 versus D(RFLP) = 0.821 to 0.997). Clustering of isolates by using MIRU data correlated with RFLP-derived clustering, lending useful information for either an investigation or confirmation of an incidence of recent transmission. In addition, it was determined that each predominant RFLP type in Manitoba had a corresponding, recognizable MIRU type. It is conceivable that in the future RFLP typing can be replaced with MIRU for real-time, ongoing tuberculosis surveillance in the province.

Bacterial Typing Techniques↗
PubMed2005

Utility of mycobacterial interspersed repetitive unit typing for differentiating multidrug-resistant Mycobacterium tuberculosis isolates of the Beijing family.

Mycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping of Mycobacterium tuberculosis and may represent a feasible approach to study global M. tuberculosis molecular epidemiology. To evaluate the use of MIRU typing in discriminating drug-resistant M. tuberculosis strains of the Beijing genotype family, 102 multidrug-resistant (MDR) clinical isolates and 253 randomly selected non-MDR isolates collected from 2000 to 2003 in Hong Kong were subjected to 12-locus MIRU typing, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. Spoligotyping showed that 243 (68.5%) of 355 isolates belonged to Beijing family genotype. MIRU typing showed lower discrimination in differentiating between the Beijing family strains (Hunter-Gaston discriminative index [HGI] of 0.8827) compared with the IS6110 RFLP method (HGI = 0.9979). For non-Beijing strains, MIRU typing provided discrimination (HGI = 0.9929) comparable to that of the RFLP method (HGI = 0.9961). There was no remarkable difference in discrimination power between the two methods in differentiating both within and between MDR and non-MDR strains of M. tuberculosis. Dendrograms constructed with the MIRU typing data showed a clear segregation between the Beijing and non-Beijing genotype. Addition of RFLP to MIRU typing offered a higher discrimination ability (92.6%) than did addition of MIRU typing to RFLP (40.0%). This supported the potential use of this method to analyze the global genetic diversity of MDR M. tuberculosis strains that may be at different levels of evolutionary divergence.

Bacterial Typing Techniques↗
PubMed2005

Sensitivities and specificities of spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing methods for studying molecular epidemiology of tuberculosis.

The development of PCR-based genotyping modalities (spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat [MIRU-VNTR] typing) offers promise for real-time molecular epidemiological studies of tuberculosis (TB). However, the utility of these methods depends on their capacity to appropriately classify isolates. To determine the operating parameters of spoligotyping and MIRU-VNTR typing, we have compared results generated by these newer tests to the standard typing method, IS6110 restriction fragment length polymorphism, in analyses restricted to high-copy-number IS6110 isolates. Sensitivities of the newer tests were estimated as the percentages of isolates with identical IS6110 fingerprints that had identical spoligotypes and MIRU-VNTR types. The specificities of these tests were estimated as the percentages of isolates with unique IS6110 fingerprints that had unique spoligotypes and MIRU-VNTR types. The sensitivity of MIRU-VNTR typing was 52% (95% confidence interval [CI], 31 to 72%), and the sensitivity of spoligotyping was 83% (95% CI, 63 to 95%). The specificity of MIRU-VNTR typing was 56% (95% CI, 51 to 62%), and the specificity of spoligotyping was 40% (95% CI, 35 to 46%). The proportion of isolates estimated to be due to recent transmission was 4% by identical IS6110 patterns, 19% by near-identical IS6110 patterns, 33% by MIRU-VNTR typing, and 53% by spoligotyping. The low calculated specificities of spoligotyping and MIRU-VNTR typing led to misclassification of cases, inflated estimates of TB transmission, and low positive predictive values, suggesting that these techniques have unsuitable operating parameters for population-based molecular epidemiology studies.

Bacterial Typing Techniques↗
PubMed2005

Characterization of Mycobacterium caprae isolates from Europe by mycobacterial interspersed repetitive unit genotyping.

Mycobacterium caprae, a recently defined member of the Mycobacterium tuberculosis complex, causes tuberculosis among animals and, to a limited extent, in humans in several European countries. To characterize M. caprae in comparison with other Mycobacterium tuberculosis complex members and to evaluate genotyping methods for this species, we analyzed 232 M. caprae isolates by mycobacterial interspersed repetitive unit (MIRU) genotyping and by spoligotyping. The isolates originated from 128 distinct epidemiological settings in 10 countries, spanning a period of 25 years. We found 78 different MIRU patterns (53 unique types and 25 clusters with group sizes from 2 to 9) but only 17 spoligotypes, giving Hunter-Gaston discriminatory indices of 0.941 (MIRU typing) and 0.665 (spoligotyping). For a subset of 103 M. caprae isolates derived from outbreaks or endemic foci, MIRU genotyping and IS 6110 restriction fragment length polymorphism were compared and shown to provide similar results. MIRU loci 4, 26, and 31 were most discriminant in M. caprae, followed by loci 10 and 16, a combination which is different than those reported to discriminate M. bovis best. M. caprae MIRU patterns together with published data were used for phylogenetic inference analysis employing the neighbor-joining method. M. caprae isolates were grouped together, closely related to the branches of classical M. bovis, M. pinnipedii, M. microti, and ancestral M. tuberculosis, but apart from modern M. tuberculosis. The analysis did not reflect geographic patterns indicative of origin or spread of M. caprae. Altogether, our data confirm M. caprae as a distinct phylogenetic lineage within the Mycobacterium tuberculosis complex.

Animals↗
PubMed2005

Evaluation of a two-step approach for large-scale, prospective genotyping of Mycobacterium tuberculosis isolates in the United States.

Genotyping of Mycobacterium tuberculosis isolates is useful in tuberculosis control for confirming suspected transmission links, identifying unsuspected transmission, and detecting or confirming possible false-positive cultures. The value is greatly increased by reducing the turnaround time from positive culture to genotyping result and by increasing the proportion of cases for which results are available. Although IS6110 fingerprinting provides the highest discrimination, amplification-based methods allow rapid, high-throughput processing and yield digital results that can be readily analyzed and thus are better suited for large-scale genotyping. M. tuberculosis isolates (n = 259) representing 99% of culture-positive cases of tuberculosis diagnosed in Wisconsin in the years 2000 to 2003 were genotyped by using spoligotyping, mycobacterial interspersed repetitive unit (MIRU) typing, and IS6110 fingerprinting. Spoligotyping clustered 64.1% of the isolates, MIRU typing clustered 46.7% of the isolates, and IS6110 fingerprinting clustered 29.7% of the isolates. The combination of spoligotyping and MIRU typing yielded 184 unique isolates and 26 clusters containing 75 isolates (29.0%). The addition of IS6110 fingerprinting reduced the number of clustered isolates to 30 (11.6%) if an exact pattern match was required or to 44 (17.0%) if the definition of a matching IS6110 fingerprint was expanded to include patterns that differed by the addition of a single band. Regardless of the genotyping method chosen, the addition of a second or third method decreased clustering. Our results indicate that using spoligotyping and MIRU typing together provides adequate discrimination in most cases. IS6110 fingerprinting can then be used as a secondary typing method to type the clustered isolates when additional discrimination is needed.

Bacterial Typing Techniques↗
PubMed2005

Analysis of Mycobacterium tuberculosis genotypes in Madrid and identification of two new families specific to Spain-related settings.

In Spain, tuberculosis (TB) patterns are changing because of the recent increase in the number of cases among immigrants. To establish the composition of circulating Mycobacterium tuberculosis strains before the effects of foreign strains appear, this study focused on molecular characterization of 233 patient isolates using spoligotyping. The spoligotyping data were further analyzed using an international database, SpolDB4. The results obtained showed that the general features of the M. tuberculosis population in Spain are coherent with those of other European countries, with the Latin American and Mediterranean group, and with the Haarlem 3 and T1 families as the most prevalent genotypes. The Spanish isolates clustered mostly with genotypes which had previously been isolated in countries linked with Spain. We also describe and fully characterize two novel M. tuberculosis families, Madrid1 and Madrid2, which are specific to Spain-related settings. The data reported here provide a solid reference when monitoring changes in the composition of the M. tuberculosis population in Spain as a consequence of the increasing rate of TB in the foreign population.

Adult↗
PubMed2005

Molecular typing of Mycobacterium tuberculosis by mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis, a more accurate method for identifying epidemiological links between patients with tuberculosis.

IS6110 fingerprinting of Mycobacterium tuberculosis is the standard identification method in studies on transmission of tuberculosis. However, intensive epidemiological investigation may fail to confirm transmission links between patients clustered by IS6110-restriction fragment length polymorphism (RFLP) typing. We applied typing based on variable numbers of tandem repeats (VNTRs) of mycobacterial interspersed repetitive units (MIRUs) to isolates from 125 patients in 42 IS6110 clusters, for which thorough epidemiological data were available, to investigate the potential of this method in distinguishing epidemiologically linked from nonlinked patients. Of seven IS6110 clusters without epidemiological links, five were split by MIRU-VNTR typing, while nearly all IS6110 clusters with proven or likely links displayed conserved MIRU-VNTR types. These results provide molecular evidence that not all clusters determined on the basis of multibanded IS6110 RFLP patterns necessarily reflect transmission of tuberculosis. They support the use of MIRU-VNTR typing as a more reliable and faster method for transmission analysis.

Bacterial Typing Techniques↗
PubMed2006

Rapid identification of Mycobacterium tuberculosis Beijing genotypes on the basis of the mycobacterial interspersed repetitive unit locus 26 signature.

Mycobacterium tuberculosis Beijing strains are prevalent in many parts of the world and often give rise to large institutional outbreaks. Such highly transmissible strains, often associated with multidrug resistance, are likely underrepresented in outbreaks reported from developing countries, mainly due to nonavailability of fast detection methods suitable in epidemiological surveillance studies. We evaluated a PCR assay based on amplification of mycobacterial interspersed repetitive unit locus 26 as a stand-alone method for unambiguous identification of Beijing strains. The method was used on blinded samples from 10 standard strains whose Beijing status was already confirmed by spoligotyping. All 10 strains were accurately identified, and their profiles were corroborated successfully with spoligotypes. The method was also applied to 70 different non-Beijing clinical isolates from different countries to allow discrimination of isolates. Owing to its accuracy, simplicity, and rapidity, the assay can be employed in laboratory-level testing of isolates linked to certain outbreaks. The test can also be adopted for direct application on clinical samples to save time on culturing bacilli for genotyping.

Bacterial Typing Techniques↗
PubMed2026

Ecological and evolutionary implications of a mobile genetic element-rich haloarchaeon with unique osmotic resilience.

We isolated a novel halophilic archaeon, strain DSL9, representing the proposed new species Haloliberatus hailidukes gen. nov., sp. nov., from Dishui Lake, China. Unlike most obligate halophiles, DSL9 survives in low salinity, even distilled water, without lysis. Genomic analysis revealed dual salinity adaptation strategies: salt-in and compatible solutes, including a complete trehalose biosynthesis pathway. The strain harbors multiple plasmids, notably a 111,311 bp large plasmid (pHdsl9-3) encoding replication (Orc1/Cdc6, SSB), transcription (TFIIB), transmission (T4SS cluster, ArdC-like protein), and recombination (XerA) modules. pHdsl9-3 provides auxiliary functions such as defense, genome diversification, ion detoxification, and suggests active horizontal gene transfer. Similar elements are widespread in Halobacteriales, highlighting their role in haloarchaeal genetic diversity and plasticity. The encoded XerA hinted at a function beyond DNA dimer resolution, suggesting it may have been adapted by other archaeal mobile genetic elements. These findings underscore the need to investigate plasmid-driven evolution and environmental adaptation mechanisms in haloarchaea.IMPORTANCEThis study reports the isolation and characterization of DSL9, a novel halophilic archaeon from a freshwater lake. Remarkably, DSL9 defies the typical obligate halophilic lifestyle by surviving in low-salinity environments, including distilled water, without cell lysis. A key discovery is the identification of a 111,311 bp large plasmid harboring essential modules for replication, transcription, transmission, and integration. Widespread distribution of similar elements across Halobacteriales suggests their crucial role in haloarchaeal genetic diversity and plasticity, warranting further study of plasmid-mediated evolution and adaptation strategies.

Lakes↗
PubMed2005

Mobile genetic elements and sexual reproduction.

Transposable elements (TE) are prominent components of most eukaryotic genomes. In addition to their possible participation in the origin of sexual reproduction in eukaryotes, they may be also involved in its maintenance as important contributors to the deleterious mutation load. Comparative analyses of transposon content in the genomes of sexually reproducing and anciently asexual species may help to understand the contribution of different TE classes to the deleterious load. The apparent absence of deleterious retrotransposons from the genomes of ancient asexuals is in agreement with the hypothesis that they may play a special role in the maintenance of sexual reproduction and in early extinction for which most species are destined upon the abandonment of sex.

Animals↗
PubMed1996

A somatic cell hybrid panel for pig regional gene mapping characterized by molecular cytogenetics.

A panel of 27 pig x rodent somatic cell hybrids was produced and characterized cytogenetically. The first step of this study consisted of hybridizing a SINE probe to GTG-banded metaphases of each hybrid clone in order to count and identify the normal pig chromosomes and to detect rearranged ones. The second step consisted of using the DNA of each clone as a probe after pIRS-PCR (porcine interspersed repetitive sequence-polymerase chain reaction) amplification to highly enrich it in pig sequences. These probes, hybridized to normal pig metaphase chromosomes, enabled the identification of the complete porcine complement in the hybrid lines. Whole chromosomes and fragments were characterized quickly and precisely, and results were compared. In addition to this cytogenetic characterization, molecular verification was also carried out by using primers specific to six microsatellites and to one gene previously mapped to pig chromosomes. The results obtained allow us to conclude that we have produced a panel that is informative for all porcine chromosomes. This panel constitutes a highly efficient tool to establish not only assignments of genes and markers but also regional localizations on pig chromosomes.

Animals↗