Polymorphism of variable-number tandem repeats at multiple loci in Mycobacterium tuberculosis

Genotyping based on variable-number tandem repeats (VNTR) is currently a very promising tool for studying the molecular epidemiology and phylogeny of Mycobacterium tuberculosis. Here we investigate the polymorphisms of 48 loci of direct or tandem repeats in M. tuberculosis previously identified by our group. Thirty-nine loci, including nine novel ones, were polymorphic. Ten VNTR loci had high allelic diversity (Nei's diversity indices >or= 0.6) and subsequently were used as the representative VNTR typing set for comparison to IS 6110-based restriction fragment length polymorphism (RFLP) typing. The 10-locus VNTR set, potentially providing >2 x 10(9) allele combinations, obviously showed discriminating capacity over the IS 6110 RFLP method for M. tuberculosis isolates with fewer than six IS 6110-hybridized bands, whereas it had a slightly better resolution than IS 6110 RFLP for the isolates having more than five IS 6110-hybridized bands. Allelic diversity of many VNTR loci varied in each IS 6110 RFLP type. Genetic relationships inferred from the 10-VNTR set supported the notion that M. tuberculosis may have evolved from two different lineages (high and low IS 6110 copy number). In addition, we found that the lengths of many VNTR loci had statistically significant relationships to each other. These relationships could cause a restriction of the VNTR typing discriminating capability to some extent. Our results suggest that VNTR-PCR typing is practically useful for application to molecular epidemiological and phylogenetic studies of M. tuberculosis. The discriminating power of the VNTR typing system can still be enhanced by the supplementation of more VNTR loci.     

Stability of variable-number tandem repeats of mycobacterial interspersed repetitive units from 12 loci in serial isolates of Mycobacterium tuberculosis

Variable number tandem repeats (VNTRs) of elements named mycobacterial interspersed repetitive units (MIRUs) have previously been identified in 12 minisatellite loci of the Mycobacterium tuberculosis genome. These markers allow reliable high-throughput genotyping of M. tuberculosis and represent a portable approach to global molecular epidemiology of M. tuberculosis. To assess their temporal stability, we genotyped 123 serial isolates, separated by up to 6 years and belonging to a variety of distinct IS6110 restriction fragment length polymorphism (RFLP) families, from 56 patients who had positive sputum cultures. All 12 MIRU VNTR loci were completely identical within the groups of serial isolates in 55 out of 56 groups (98.2%), although 11 pairs of isolates from the same patients with conserved MIRU VNTRs displayed slightly different IS6110 RFLP profiles. In a single case, serial isolates with an unchanged IS6110 RFLP profile showed a change in 1 out of 12 MIRU VNTR loci. These results indicate that MIRU VNTRs are stable over time and therefore are suitable for reliable follow-up of patients chronically infected with tuberculosis over long periods. Moreover, they support MIRU VNTR genotyping as a powerful first-line method followed by subtyping by IS6110 RFLP to define ongoing transmission clusters.     

Molecular typing of Mycobacterium tuberculosis by using nine novel variable-number tandem repeats across the Beijing family and low-copy-number IS6110 isolates

Molecular epidemiological tools for genotyping clinical isolates of Mycobacterium tuberculosis have been developed and used to help track and contain transmission of tuberculosis. We identified 87 short sequence repeat loci within the genome of the M. tuberculosis H37Rv strain. Nine tandem repeats were found to be variable (variable-number tandem repeats [VNTRs]) in a set of 91 isolates. Fifty-seven of the isolates had only four IS6110 bands. The other 34 isolates were members of the Beijing strain family. The number of alleles of each these nine VNTRs was determined by examining each isolate. Six of the loci (Mtb-v1, -v4, -v10, -v15, -v18, and -v20) were able to differentiate the Beijing spoligotype identical isolates into seven distinct genotypes. Five of the loci (Mtb-v3, -v5, -v6, -v10, and -v15) were informative in discriminating the four-band IS6110 restriction fragment length polymorphism isolates from each other. The Nei's diversity values of each marker ranged from 0.02 to 0.59, with the number of alleles ranging from two to eight across the entire strain set. These nine loci provide a useful, discriminatory extension of VNTR typing methods for application to molecular epidemiologic studies of M. tuberculosis.     

Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units

A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.     

7个VNTRs用于65株中国分离的结核分枝杆菌基因多态性研究

目的　应用数目可变串联重复位点 (variablenumberoftandemrepeats ,VNTRs)分子分型技术 ,初步探讨我国结核分枝杆菌的基因多态性特征。方法　采用PCR和琼脂糖凝胶电泳技术 ,对结核分枝杆菌的 11个数目可变重复位点进行检测 ,并应用Gel Proanalyzer 3.1软件和BioNumerics(Ver sion 3.0 )软件进行结果分析 ,分析结核分枝杆菌DNA多态性特征。结果　选取结核分枝杆菌基因组中的 7个 (ETR A、ETR B、ETR C、ETR D、ETR E、ETR F、MPTR A)具有明显多态性特征的串联重复位点 ,检测分析了 6 5株来自安徽、湖南和江苏省的结核分枝杆菌 ,结果显示被测菌株可分为 10个不同的基因型 ,其中以 2个型别为主 ,占总菌株数的 6 9.2 % ,其他菌株呈散在分布。结论　采用VNTR分型技术初步分析表明 ,中国结核分枝杆菌具有明显的基因多态性 ,且存在主要流行菌群。     

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.     

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.     

Phenotypic and Genotypic Characterization of Mycobacterium africanum Isolates from West Africa

The Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, M. africanum, and M. microti. Most clinical isolates are M. tuberculosis or M. bovis. These species can be distinguished by phenotypes and genotypes. However, there is no simple definition of M. africanum, and some authors question the validity of this species. We analyzed 17 human isolates from Sierra Leone, identified as M. africanum by biochemical and growth characteristics. We sequenced polymorphic genes and intergenic regions. We amplified DNA from six loci with variable numbers of tandem repeats (VNTRs) and determined the exact number of repeats at each locus in each strain. All M. africanum isolates had the ancestral CTG Leu at katG codon 463. Drug-resistant M. africanum isolates had katG and rpoB mutations similar to those found in drug-resistant M. bovis and M. tuberculosis. Fourteen Sierra Leone M. africanum isolates (designated group A) had katG codon 203 ACC Thr, also found in M. africanumT (the T indicates type strain) from Senegal. Group A isolates clustered with M. africanumT by VNTR analysis. Three M. africanum isolates (group B) had katG codon 203 ACT Thr, found in M. tuberculosisT, and clustered with M. tuberculosisT by VNTR analysis. Phenotypic identification of M. africanum yielded a heterogeneous collection of strains. Genotypic analyses identified a cluster (M. africanum group A) which included M. africanumT and was distinct from the rest of the M. tuberculosis complex. Future studies of M. africanum should include both phenotypic and genotypic analyses.     

Multiple polymorphic loci for molecular typing of strains of Mycobacterium leprae

The need for molecular tools for the differentiation of isolates of Mycobacterium leprae, the organism that causes leprosy, is urgent in view of the continuing high levels of new case detection, despite years of aggressive chemotherapy and the consequent reduction in the prevalence of leprosy. The slow onset of leprosy and the reliance on physical examination for detection of disease have restricted the epidemiological tracking necessary to understand and control transmission. Two genetic loci in several isolates of M. leprae have previously been demonstrated to contain variable-number tandem repeats (VNTRs). On the basis of these reports and the availability of the full genome sequence, multiple-locus VNTR analysis for strain typing has been undertaken. A panel of 11 short tandem repeat (STR) loci with repeat units of 1, 2, 3, 6, 12, 18, 21, and 27 bp from four clinical isolates of M. leprae propagated in armadillo hosts were screened by PCR. Fragment length polymorphisms were detected at 9 of the 11 loci by agarose gel electrophoresis. Sequencing of representative DNA products confirmed the presence of VNTRs between isolates. The application of nine new polymorphic STRs in conjunction with automated methods for electrophoresis and size determination allows greater discrimination between isolates of M. leprae and enhances the potential of this technique to track the transmission of leprosy.     

Stability of multiple-locus variable-number tandem repeats in Salmonella enterica serovar typhimurium

Variable-number tandem repeats (VNTRs) may evolve so rapidly that multiple profiles emerge during an outbreak. A total of 190 isolates from eight Salmonella enterica serovar Typhimurium outbreaks and 15 isolates from seven patients were analyzed by pulsed-field gel electrophoresis and VNTR typing. Small changes in loci were noted; otherwise, the VNTR profiles were stable during the course of the outbreaks.     

Genetic diversity of Mycobacterium tuberculosis in Sicily based on spoligotyping and variable number of tandem DNA repeats and comparison with a spoligotyping database for population-based analysis

In a previous study, we proposed to associate spoligotyping and typing with the variable number of tandem DNA repeats (VNTR) as an alternative strategy to IS6110-restriction fragment length polymorphism (RFLP) for molecular epidemiological studies on tuberculosis. The aim of the present study was to further evaluate this PCR-based typing strategy and to describe the population structure of Mycobacterium tuberculosis in another insular setting, Sicily. A collection of 106 DNA samples from M. tuberculosis patient isolates was characterized by spoligotyping and VNTR typing. All isolates were independently genotyped by the standard IS6110-RFLP method, and clustering results between the three methods were compared. The totals for the clustered isolates were, respectively, 15, 60, and 82% by IS6110-RFLP, spoligotyping, and VNTR typing. The most frequent spoligotype included type 42 that missed spacers 21 to 24 and spacers 33 to 36 and derived types 33, 213, and 273 that, together represented as much as 26% of all isolates, whereas the Haarlem clade of strains (types 47 and 50, VNTR allele 32333) accounted for 9% of the total strains. The combination of spoligotyping and VNTR typing results reduced the number of clusters to 43% but remained superior to the level of IS6110-RFLP clustering (ca. 15%). All but one IS6110-defined cluster were identified by the combination of spoligotyping and VNTR clustering results, whereas 9 of 15 spoligotyping-defined clusters could be further subdivided by IS6110-RFLP. Reinterpretation of previous IS6110-RFLP results in the light of spoligotyping-VNTR typing results allowed us to detect an additional cluster that was previously missed. Although less discriminative than IS6110-RFLP, our results suggest that the use of the combination of spoligotyping and VNTR typing is a good screening strategy for detecting epidemiological links for the study of tuberculosis epidemiology at the molecular level.     

Mycobacterial interspersed repetitive unit typing of Mycobacterium tuberculosis compared to IS6110-based restriction fragment length polymorphism analysis for investigation of apparently clustered cases of tuberculosis

An evaluation of the utility of IS6110-based restriction fragment length polymorphism (RFLP) typing compared to a combination of variable number tandem repeat (VNTR) typing and mycobacterial interspersed repetitive unit (MIRU) typing was undertaken. A total of 53 patient isolates of Mycobacterium tuberculosis from four presumed episodes of cross-infection were examined. Genomic DNA was extracted from the isolates by a cetyl trimethylammonium bromide method. The number of copies of tandem repeats of the five loci ETR(A) to ETR(E) and 12 MIRU loci was determined by PCR amplification and agarose gel electrophoresis of the amplicons. VNTR typing identified the major clusters of strains in the three investigations in which they occurred (each representing a different evolutionary clade: 32333, 42235, and 32433). The majority of unrelated isolates (by epidemiology and RFLP typing) were also identified by VNTR typing. The concordance between the RFLP and MIRU typing was complete, with the exception of two isolates with RFLP patterns that differed by one band each from the rest of the major epidemiologically linked groups of isolates in investigation A. All of these isolates had identical MIRU and VNTR types. A further pair of isolates differed in the number of tandem repeat copies at two MIRU alleles but had identical RFLP patterns. The speed of the combined VNTR and MIRU typing approach enabled results for some of the investigations to be supplied in "real time," influencing choices in contact tracing. The ease of comparison of results of MIRU and VNTR typing, which are recorded as single multidigit numbers, was also found to greatly facilitate investigation management and the communication of results to health care professionals.     

Use of variable-number tandem-repeat typing to differentiate Mycobacterium tuberculosis Beijing family isolates from Hong Kong and comparison with IS6110 restriction fragment length polymorphism typing and spoligotyping

The Mycobacterium tuberculosis Beijing family isolates may cause more than a quarter of all tuberculosis cases worldwide, are emerging in some areas, and are often associated with drug resistance. Early recognition of transmission of this genotype is therefore important. To evaluate the usefulness of variable-number tandem-repeat (VNTR) typing to discriminate and recognize strains of the Beijing family, M. tuberculosis isolates from Hong Kong were subjected to VNTR analysis, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. The allelic diversity of the 14 VNTR loci included in the analysis varied from 0 to 0.618 among Beijing strains. The discriminatory power of VNTR analysis was slightly lower than that of IS6110 RFLP. Our analysis shows that VNTR typing, which has many practical advantages over RFLP typing, can be used for epidemiological studies of Beijing strains. However, VNTR-defined clusters should be subtyped with IS6110 RFLP for maximal resolution.     

Use of VNTR typing to test genetic diversity of <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> isolates

Variable number tandem repeats (VNTRs) have been identified in silico by the present study in the Neisseria gonorrhoeae genome. Using a collection of 48 N. gonorrhoeae isolates derived from patients with gonorrhea in Almaty (Kazakhstan), the identified VNTR loci have been studied for the possibility of applying them for VNTR typing and compared to opa typing. The Hunter-Gaston discrimination index (HGDI) for VNTR typing on two loci (VNTR2048 and NMTR-12) was 0.734, which is lower than HGDI for opa typing (0.959). The VNTR1192-locus has a complex structure based on repeated elements, which created difficulties for its electrophoretic analysis. However, the analysis of the nucleotide sequence of this locus allows one to differentiate between all 48 isolates that participate in the study. The proposed VNTR loci can be used to analyze gonorrhea transmission through sexual contact and solve other molecular and epidemiological problems.     

Evaluation of Mycobacterium tuberculosis typing methods in a 4-year study in Schleswig-Holstein, Northern Germany

In order to evaluate the discriminatory power of different methods for genotyping of Mycobacterium tuberculosis complex (MTBC) isolates, we compared the performance of (i) IS6110 DNA fingerprint typing, (ii) spoligotyping, and (iii) 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in a long-term study on the epidemiology of tuberculosis (TB) in Schleswig-Holstein, the northernmost federal state of Germany. In total, we analyzed 277 MTBC isolates collected from patients between the years 2006 and 2010. The collection comprised a broad spectrum of 13 different genotypes, among which strains of the Haarlem genotype (31%) were most prominent, followed by strains belonging to the Delhi and Beijing lineages (7% and 6%, respectively). On the basis of IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping analyses, 211 isolates had unique patterns (76%) and 66 isolates (24%) were in 20 clusters. MIRU-VNTR combined with spoligotyping analyses revealed 202 isolates with unique patterns (73%) and 75 isolates in 18 clusters (27%). Overall, there was 93.1% concordance between the typing results obtained; 198 strains were identified as unique, and 60 isolates were clustered by both typing combinations (including all 31 isolates with confirmed epidemiological links). Of the remaining 19 isolates with discrepant results, 15 were falsely clustered by MIRU-VNTR (six Beijing genotype strains) and four were clustered by IS6110 RFLP (low IS6110 copy number) only. In conclusion, in the study population investigated, a minority of isolates, especially of the Beijing genotype, clustered by standard 24-loci MIRU-VNTR and without an obvious epidemiological link may require second-line typing by IS6110 RFLP or hypervariable MIRU-VNTR loci.     

Molecular epidemiology of disease due to Mycobacterium bovis in humans in the United Kingdom

Mycobacterium bovis is the causative agent of bovine tuberculosis, with a wide host range. Fifty human M. bovis isolates were typed using spoligotyping and variable number tandem repeats (VNTR). Fifteen of these spoligotypes have not yet been recorded in cattle. The predominant spoligotype in humans and cattle was subdivided by VNTR.     

Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units

Large-scale genotyping of Mycobacterium tuberculosis is especially challenging, as the current typing methods are labor-intensive and the results are difficult to compare among laboratories. Here, automated typing based on variable-number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 mammalian minisatellite-like loci of M. tuberculosis is presented. This system combines analysis of multiplex PCRs on a fluorescence-based DNA analyzer with computerized automation of the genotyping. Analysis of a blinded reference set of 90 strains from 38 countries (K. Kremer et al., J. Clin. Microbiol. 37:2607-2618, 1999) demonstrated that it is 100% reproducible, sensitive, and specific for M. tuberculosis complex isolates, a performance that has not been achieved by any other typing method tested in the same conditions. MIRU-VNTRs can be used for analysis of the global genetic diversity of M. tuberculosis complex strains at different levels of evolutionary divergence. To fully exploit the portability of this typing system, a website was set up for the analysis of M. tuberculosis MIRU-VNTR genotypes via the Internet. This opens the way for global epidemiological surveillance of tuberculosis and should lead to novel insights into the evolutionary and population genetics of this major pathogen.     

The discovery,function and development of the variable number tandem repeats in different Mycobacterium species

Abstract

The method of genotyping by variable number tandem repeats (VNTRs) facilitates the epidemiological studies of different Mycobacterium species worldwide. Until now, the VNTR method is not fully understood, for example, its discovery, function and classification. The inconsistent nomenclature and terminology of VNTR is especially confusing. In this review, we first describe in detail the VNTRs in Mycobacterium tuberculosis (M. tuberculosis), as this pathogen resulted in more deaths than any other microbial pathogen as well as for which extensive studies of VNTRs were carried out, and then we outline the recent progress of the VNTR-related epidemiological research in several other Mycobacterium species, such as M. abscessus, M. africanum, M. avium, M. bovis, M. canettii, M. caprae, M. intracellulare, M. leprae, M. marinum, M. microti, M. pinnipedii and M. ulcerans from different countries and regions. This article is aimed mainly at the practical notes of VNTR to help the scientists in better understanding and performing this method.     

Mixed tuberculosis infections in rural South Vietnam

Tuberculosis patients may be infected with or have disease caused by more than one Mycobacterium tuberculosis strain, usually referred to as "mixed infections." These have mainly been observed in settings with a very high tuberculosis incidence and/or high HIV prevalence. We assessed the rate of mixed infections in a population-based study in rural Vietnam, where the prevalences of both HIV and tuberculosis are substantially lower than those in previous studies looking at mixed infections. In total, 1,248 M. tuberculosis isolates from the same number of patients were subjected to IS6110 restriction fragment length polymorphism (RFLP) typing, spoligotyping, and variable-number-tandem-repeat (VNTR) typing. We compared mixed infections identified by the presence of (i) discrepant RFLP and spoligotype patterns in isolates from the same patient and (ii) double alleles at ≥ 2 loci by VNTR typing and assessed epidemiological characteristics of these infections. RFLP/spoligotyping and VNTR typing identified 39 (3.1%) and 60 (4.8%) mixed infections, respectively (Cohen's kappa statistic, 0.57). The number of loci with double alleles in the VNTR pattern was strongly associated with the proportion of isolates with mixed infections according to RFLP/spoligotyping (P < 0.001). Mixed infections occurred more frequently in newly treated than in previously treated patients, were significantly associated with minor X-ray abnormalities, and were almost significantly associated with lower sputum smear grades. Although the infection pressure in our study area is lower than that in previously studied populations, mixed M. tuberculosis infections do occur in rural South Vietnam in at least 3.1% of cases.     

Differentiation of clinical Mycobacterium tuberculosis complex isolates by gyrB DNA sequence polymorphism analysis

The discriminatory power of gyrB DNA sequence polymorphisms for differentiation of the species of the Mycobacterium tuberculosis complex (MTBC) was evaluated by sequencing and restriction fragment length polymorphism (RFLP) analysis of a 1,020-bp fragment amplified from clinical isolates of M. tuberculosis, Mycobacterium bovis (pyrazinamide [PZA] resistant as well as PZA susceptible), Mycobacterium africanum subtypes I and II, and Mycobacterium microti types vole and llama. We found sequence polymorphisms in four regions described previously and at one additional position. These differences in the gyrB sequences allow an accurate discrimination of M. bovis, M. microti, and M. africanum subtype I. The PZA-susceptible subtypes of M. bovis shared the M. bovis-specific substitution at position 756 with the PZA-resistant strains, but can be unambiguously differentiated by a characteristic substitution at position 1311. As a drawback, M. tuberculosis and M. africanum subtype II showed an identical gyrB sequence that facilitates discrimination from the other species, but not from each other. A PCR-RFLP technique applying three restriction enzymes could be shown to be a rapid and easy-to-perform tool for the differentiation of the members of the MTBC. Based on these results, we present a clear diagnostic algorithm for the differentiation of species of the MTBC.