Evaluation of Four DNA Typing Techniques in Epidemiological Investigations of Bovine Tuberculosis

DNA fingerprinting techniques were used to type 273 isolates of Mycobacterium bovis from Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS6110, the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of Mycobacterium tuberculosis (J. Kamerbeek et al., J. Clin. Microbiol. 35:907–914, 1997). Eighty-five percent of the isolates harbored a single copy of IS6110, and 81.5% of these carried IS6110 on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS6110 identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS6110 was sufficiently sensitive for the typing of isolates with more than three copies of IS6110, but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS6110. Spoligotyping may have advantages for the rapid typing of M. bovis, but it needs to be made more sensitive.     

Study of Restriction Fragment Length Polymorphism Analysis and Spoligotyping for Epidemiological Investigation of Mycobacterium bovis Infection

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS6110, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type Mycobacterium bovis isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS6110 or DR probes. The majority of isolates from all species carried a single IS6110 copy. In four RFLP types IS6110 polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of M. bovis infection in Ireland.     

Evaluation of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Analysis and Spoligotyping for Genotyping of Mycobacterium bovis Isolates and a Comparison with Restriction Fragment Length Polymorphism Typing

Common strain typing methods for differentiation of Mycobacterium bovis isolates include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. MIRU-VNTR typing and spoligotyping were evaluated in this study, and these typing methods were compared with RFLP typing. A total of 386 M. bovis isolates from cattle, badgers, and deer in the Republic of Ireland that had previously been typed by IS6110, polymorphic GC-rich sequence (PGRS), and direct-repeat (DR) RFLP were included in the study. Spoligotyping and analysis of six VNTR loci (QUB 11a, QUB 11b, ETR A, 4052, MIRU 26, and 1895) were performed on the samples. RFLP analysis was the method that gave the greatest differentiation of strains, with a Hunter-Gaston discriminatory index (HGDI) of 0.927; the HGDI recorded for MIRU-VNTR typing was marginally lower at 0.918, and spoligotyping was the least discriminatory method, with an HGDI of 0.7. Spoligotype SB0140 represented approximately 50% of the isolates. Within the group of isolates represented by SB0140, there was a much lower level of concordance between RFLP and MIRU-VNTR typing than for groups represented by other spoligotypes. A combination of spoligotyping and MIRU-VNTR typing offered advantages over MIRU-VNTR typing alone. In a combined spoligotyping and MIRU-VNTR typing protocol, the number of VNTR loci could be reduced to four (QUB 11a, QUB 11b, ETR A, and 4052) while maintaining a high level of strain differentiation.The development of molecular techniques for differentiation of Mycobacterium bovis isolates has been of considerable benefit in epidemiological studies. Typing methods that have been commonly used include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing (7, 19).RFLP analysis of M. bovis isolates has commonly utilized polymorphism of the insertion sequence IS6110 and repetitive DNA elements such as the polymorphic GC-rich sequence (PGRS) and the direct-repeat (DR) region. Analysis of polymorphism of IS6110, the PGRS, and the DR region in combination has provided a high level of discrimination between strains (7, 19). REA has been widely used in New Zealand and has also given excellent resolution of strains (4). However, both RFLP analysis and REA require relatively large quantities of DNA and are laborious and time-consuming procedures. Complex banding patterns make analysis and interlaboratory comparisons difficult. Spoligotyping is a PCR-based typing method that reveals the presence or absence of unique spacer sequences located between the direct-repeat sequences of the DR region (12). It is a relatively easy procedure to perform, and the results can be expressed in a digital format. However, spoligotyping does not differentiate M. bovis strains to the same extent as RFLP analysis or REA (7, 19). Minisatellite-like loci in the Mycobacterium tuberculosis complex genome, described as mycobacterial interspersed repetitive units, may show polymorphism of the number of tandem repeats. A wide range of M. tuberculosis complex MIRU-VNTR loci have been evaluated, and loci which are informative for M. bovis isolates have been identified (8, 16, 17, 20, 23). Similar to spoligotyping, MIRU-VNTR typing has the advantages of ease of procedure and the generation of results in a digital format.In recent years, genotyping by IS6110, PGRS, and DR RFLP has been used in epidemiological studies of M. bovis infection in the Republic of Ireland (5, 6, 14). While RFLP analysis has given a high level of strain differentiation, its replacement by MIRU-VNTR typing or by a combination of MIRU-VNTR typing and spoligotyping offers potential advantages. The objective of this study was to evaluate MIRU-VNTR typing or a combination of MIRU-VNTR typing and spoligotyping for discrimination of M. bovis strains, to compare the discriminatory powers of the two methods against RFLP analysis, and to investigate the level of concordance between the three typing systems.     

Improvement of differentiation and interpretability of spoligotyping for Mycobacterium tuberculosis complex isolates by introduction of new spacer oligonucleotides

The direct repeat (DR) region in Mycobacterium tuberculosis complex strains is composed of multiple well-conserved 36-bp DRs interspersed with nonrepetitive DNA spacer sequences of similar size. Clinical isolates show extensive polymorphism in this DR region, and this has led to the development of a 43-spacer reversed line blot methodology: spoligotyping. Although this method has contributed significantly to the molecular epidemiology of tuberculosis in the last decade, the discriminatory power and the readability of this method were not found to be optimal. In order to improve the discriminatory power, the usefulness of 43 redesigned oligonucleotides and the usefulness of 51 new spacer oligonucleotides were evaluated. For 314 M. tuberculosis complex strains isolated in the central part of The Netherlands over a 5-year period, 264 different IS6110 RFLP types could be distinguished, and 160 different spoligotype patterns were identified by traditional spoligotyping. After the introduction of 51 new spacer oligonucleotides, 14 additional spoligotypes were recognized. This enabled us to split 11 clusters of isolates identified by the traditional spoligotyping. Furthermore, on the basis of the new spacer oligonucleotides a dichotomy was found among the Beijing genotype isolates. Among 76 Mycobacterium bovis strains, 20 patterns were found by traditional spoligotyping and 30 patterns were found by novel probe spoligotyping, respectively. Nine M. bovis subsp. caprae isolates yielded six patterns by traditional spoligotyping and eight patterns by novel probe spoligotyping. A part of the redesigned oligonucleotides slightly improved the reading of spoligotype patterns. The reproducibility of spoligotyping, based on internal control probes, invariably yielded a high score; only 4 (1%) of the 314 patient isolates gave discrepant results. Analysis of a set of 31 duplicate M. tuberculosis complex strains demonstrated a 10% error rate for the identification of blinded duplicate samples. In a redundancy analysis, 40 essential spacer oligonucleotides of the 94-spacer sequences were selected, yielding the same number of spoligotype patterns. We propose to leave the traditional commercialized first-generation membrane for spoligotyping unchanged for current applications and to introduce a second-generation spoligotyping membrane whenever extended discrimination is required, e.g., for low-copy-number IS6110 strains or for phylogenetic studies of Beijing genotype strains.     

Molecular fingerprinting of Mycobacterium bovis subsp. caprae isolates from central Europe

To study the dissemination of Mycobacterium bovis subsp. caprae, 79 European isolates from cattle, humans, and other hosts were examined by spoligotyping and IS6110 restriction fragment length polymorphism (RFLP) analysis. Among a total of 11 different spoligotypes identified, type C1 proved to be predominant (n = 62). Five of the spoligotypes are described for the first time. A total of 43 different RFLP types were identified, thus allowing further differentiation for epidemiological tracking. Isolates from a series of outbreaks in one village proved to be of the same spoligotype and of identical or closely related RFLP types.     

Characterization of the Mycobacterium bovis restriction fragment length polymorphism DNA probe pUCD and performance comparison with standard methods

In this study, the newly described Mycobacterium bovis restriction fragment length polymorphism (RFLP) typing probe pUCD was characterized by sequence analysis and the previously observed polymorphic banding pattern was reproduced with a combination of three oligonucleotide probes in a single, mixed hybridization. In addition, the ability of pUCD to distinguish between 299 M. bovis isolates from the Republic of Ireland was assessed in relation to established methods and a statistical function for objective comparison of RFLP probes was derived. It was found that typing with pUCD alone produced greater discrimination between M. bovis isolates than typing with the commonly used mycobacterial DNA probes IS6110, PGRS, and DR and also by the spoligotyping technique. pUCD and DR in combination produced the highest level of discrimination while maintaining a high level of concordance with known epidemiological data relating to the samples. The reduction of pUCD to the level of oligonucleotides should in future allow pUCD and DR to be included together in a mixed hybridization, thus producing a high level of M. bovis strain type discrimination from a single round of RFLP analysis.     

Characterization of Finnish Mycobacterium tuberculosis isolates by spoligotyping

The molecular epidemiology of tuberculosis (TB) in Finland was studied by spoligotyping 380 Mycobacterium tuberculosis isolates. The isolates were obtained during a 1-year study period from July 2000 to June 2001 and represented 90% of new M. tuberculosis findings by culture in the whole country during the study period. The spoligotyping results were compared to the World Spoligotyping Database of the Institut Pasteur de Guadeloupe, which contains data from >14,000 M. tuberculosis isolates obtained worldwide. A total of 138 different spoligotypes were identified among the 380 M. tuberculosis isolates. Thirty-eight (10%) isolates had unique spoligotypes, while 342 (90%) isolates belonged to 100 shared types. The four most common spoligotypes caused approximately one-third of the Finnish TB cases. Forty-seven of the 138 (34.1%) spoligotypes and 61 (16.1%) of the 380 M. tuberculosis isolates had spoligotypes that had not been previously reported. Only four (1.1%) patients were infected with an isolate belonging to the Beijing genotype. The characterization of Finnish M. tuberculosis isolates by spoligotyping shows that ubiquitous spoligotypes were common, but many spoligotypes specific to Finland were also found. However, Beijing family isolates were rarely encountered, although this spoligotype is predominant in our eastern and southern neighbors.     

Detection and Molecular Characterization of Mycobacterium microti Isolates in Wild Boar from Northern Italy

Approximately 23,000 hunter-harvested wild boars from the pre-Alpine area of northern Italy were examined for tuberculosis over a 9-year period (2003 to 2011). Retropharyngeal and mandibular lymph nodes from the wild boars were examined grossly, and 1,151 of the lymph nodes were analyzed in our laboratory by histology (728 samples) and culture isolation (819 samples). Mycobacterium tuberculosis complex (MTBC)-specific PCR (1,142 samples) was used for molecular-level detection in tissue samples, as was a gyrB restriction fragment length polymorphism (RFLP) assay (322 samples). Lesions compatible with tuberculosis and indistinguishable from those described in cases of Mycobacterium bovis infection had been observed since 2003. Mycobacterium microti was identified directly in 256 tissue samples by the adopted molecular approaches. However, only 26 M. microti strains were obtained by culture isolation due to the well-known difficulties in isolating this slow-growing mycobacterium. During 2006, a prevalence study was performed in two provinces of the area, and the diffusion of M. microti was calculated to be 5.8% (95% confidence intervals surrounding the estimated prevalences [CI_P95%], 3.94 to 7.68%). Over the following years (2007 to 2011), the presence of M. microti appeared to be stable. All isolates were genotyped by spoligotyping and exact tandem repeat analysis (ETR types A to F). In addition to the typical vole type (SB0118), a new spoligotype lacking the 43 spacers was found. Spoligotyping was also applied directly to tissue samples, and a geographical cluster distribution of the two spoligotypes was observed. This is the first report studying the diffusion and genetic variability of M. microti in wild boar.     

Identification of a novel DNA probe for strain typing Mycobacterium bovis by restriction fragment length polymorphism analysis

Bovine tuberculosis caused by Mycobacterium bovis remains a significant disease of farmed cattle in many countries despite ongoing tuberculosis eradication programs. Molecular typing methods such as restriction fragment length polymorphism (RFLP) analysis and spoligotyping have been used to identify related herd breakdowns in an attempt to identify more precisely the route of infection into cattle herds and to trace the transmission of bovine tuberculosis. A recent geographical survey of Irish M. bovis isolates demonstrated that a significant proportion of isolates ( approximately 20%) exhibit a common strain type, limiting the value of current strain typing methods as an epidemiological tool. We have identified and cloned a region of the M. bovis genome, pUCD, which generates a clear, highly polymorphic banding pattern when used as an RFLP probe on AluI restriction-digested M. bovis genomic DNA and which effectively subdivides this common strain type. When used to type 60 Irish M. bovis isolates, pUCD exhibited greater discriminatory power than the commonly used mycobacterial RFLP probes IS6110, PGRS, and DR and detected an equivalent number of strain types to a combination of these three probes. pUCD also detected significantly more strain types than the spoligotyping technique, while maintaining a high level of concordance between epidemiologically related and unrelated herd breakdowns. The polymorphic element within pUCD remains to be fully characterized, however the potential for this probe to greatly decrease the workload necessary to genotype M. bovis by RFLP analysis is compelling.     

Differentiation of Mycobacterium bovis isolates from animals by DNA typing.

The insertion sequence IS6110 and the direct repeat (DR) specific to tuberculosis complex mycobacteria and the highly repeated DNA sequence, the polymorphic GC-rich repeat sequence (PGRS), were systematically used to identify restriction fragment length polymorphisms (RFLPs) within 210 isolates of Mycobacterium bovis. The isolates were primarily of bovine origin, but isolates from badgers, feral deer, sheep, humans, and a pig were included. The RFLP probes IS6110, DR, and PGRS individually identified 17, 18, and 18 different RFLP types, respectively, but in combination these probes identified a total of 39 different M. bovis RFLP types. The recommendations (J. D. A. van Embden, M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. W. M. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small, J. Clin. Microbiol. 31:406-409, 1993) for a standardized RFLP analysis for M. tuberculosis were adapted to facilitate gel documentation, image analysis, and construction of a database of RFLP types. In the present study the same M. bovis RFLP types were evident in the various animal species included, indicating that the strains were not host restricted. Application of these techniques to defined field studies should help elucidate more accurately aspects of the epidemiology of bovine tuberculosis in different countries.     

Spoligotyping Followed by Double-Repetitive-Element PCR as Rapid Alternative to IS6110 Fingerprinting for Epidemiological Studies of Tuberculosis

A total of 129 clinical isolates of Mycobacterium tuberculosis representing 91 patients were typed by a combination of direct-repeat (DR)-based spoligotyping and an inter-IS6110–PGRS (polymorphic GC-rich region)–PCR, also designated double-repetitive-element PCR (DRE-PCR). During the first phase of this investigation, 72 clinical strains representing 52 patients were initially typed by IS6110-restriction fragment length polymorphism (RFLP) and DR-RFLP, followed by spoligotyping and DRE-PCR. In the second phase of this investigation, the discriminating ability of spoligotyping plus DRE-PCR was studied for 57 isolates from 39 patients who were suspected to be epidemiologically linked, and the typing results were later confirmed by IS6110-RFLP and DR-RFLP analyses. The molecular clustering of the isolates remained identical irrespective of the methods used. These results show that the association of two PCR-based fingerprinting techniques for molecular epidemiology of tuberculosis has a discriminating ability similar to the IS6110-RFLP reference method.     

The IS6110 Restriction Fragment Length Polymorphism in Particular Multidrug-Resistant Mycobacterium tuberculosis Strains May Evolve Too Fast for Reliable Use in Outbreak Investigation

To study possible nosocomial transmission of multidrug-resistant (MDR) Mycobacterium tuberculosis, strain types and other information on 24, mostly human immunodeficiency virus-positive patients, were collected. Isolates from 11 patients had identical IS6110 restriction fragment length polymorphism (RFLP) patterns as well as spoligotype patterns and resistance profiles. Noticeably, nine other isolates from related cases also exhibited identical spoligotypes but slightly different RFLP patterns. These results indicate that for some MDR strains, the evolutionary clock of IS6110 RFLP may run too fast for reliable interpretation of strain typing results over a period of a few years.     

Molecular characterization of Mycobacterium tuberculosis isolates from different regions of Bulgaria

Mycobacterium tuberculosis isolates from different regions of Bulgaria were studied by a variety of molecular typing tools. Based on spacer oligonucleotide typing (spoligotyping), the 113 strains were subdivided into 35 spoligotypes: 5 unique profiles and 15 profiles shared by two to 29 strains; the Hunter-Gaston diversity index (HGI) was 0.9. Comparison with the international database SITVIT2 at the Institut Pasteur de Guadeloupe showed the presence of two globally distributed shared types, ST53 (25.7%) and ST47 (6.2%). Nineteen (16.8%) and six (5.3%) strains belonged to the ST125 (LAM/S subfamily) and ST41 (LAM7_TUR subfamily) types described in SITVIT2 as ubiquitous/rare and ubiquitous/common types, respectively. Seven spoligoprofiles (12 strains) were not found in the database; two of them constituted new shared types. The Beijing genotype strains were not found in the studied collection in spite of close contacts with Russia in the recent and historical past. Additional subtyping by IS6110-restriction fragment length polymorphism (RFLP) and 12-locus mycobacterial interspersed repetitive unit (MIRU)-variable number of tandem repeat analyses were performed within selected spoligotypes. In particular, MIRU typing showed better discrimination within ST125 than IS6110-RFLP typing (HGI = 0.83 versus 0.39). A high gradient for ST125 in Bulgaria compared to its negligible presence in the global database and neighboring countries leads us to suggest a Bulgarian phylogeographic specificity of this spoligotype. To conclude, this first study of the Bulgarian M. tuberculosis population demonstrated its heterogeneity and predominance of several worldwide-distributed and Balkan-specific spoligotypes.     

Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China

A total of 2,346 Mycobacterium tuberculosis isolates from 13 provinces in China were genotyped by spoligotyping. Two hundred seventy-eight spoligotypes were identified: 2,153 isolates were grouped into 85 clusters, and the remaining 193 isolates were orphans. Comparison with the SpolDB4.0 database revealed that 118 spoligotypes had shared international type numbers in the database and the other 160 were novel. These 160 novel spoligotypes were assigned to families and subfamilies using the SpotClust program. The most prevalent family was the Beijing family (74.08%), followed by the T family (14.11%). CAS family strains were found only in the Xinjiang and Tibet regions, while EAI family strains were found only in Fujian Province. In conclusion, the present study of the M. tuberculosis population in China demonstrated that Beijing family isolates are the most prevalent strains in China and that they exhibit geographical variation. Furthermore, many new spoligotypes were found in this study.Tuberculosis (TB) continues to be a major public health problem in China. Based on the data from a nationwide random survey of the epidemiology of TB in China in 2000, there were probably 4.51 million active-TB patients in the country, including 1.50 million smear-positive cases, which were the infectious sources (16). From 2006 to 2009, more than 1 million new TB cases emerged each year. Consequently, the task of controlling TB in China remains difficult.The genotyping of Mycobacterium tuberculosis strains is important for TB control because it allows the detection of suspected outbreaks and the tracing of transmission chains. It is also important to monitor species diversity, as well as to identify secondary infections (4, 7, 13, 19). Insertion sequence (IS) 6110 restriction fragment length polymorphism (IS6110 RFLP) is thought of as the gold standard genotyping method for M. tuberculosis strain genotype identification (6, 13, 21). However, the method is time-consuming, labor-intensive, and costly. Furthermore, it is difficult to compare results between laboratories. Spacer oligonucleotide typing (spoligotyping), which is based on the analysis of polymorphisms of direct-repeat (DR) regions comprised of 36-bp DRs interspersed with 35- to 41-bp unique spacer sequences, is a good alternative to traditional IS6110 RFLP fingerprinting because of its simplicity, speed, and reliability (9, 11). Spoligotyping is useful for classifying M. tuberculosis strains into spoligotype families and subfamilies according to the presence or absence of spacer regions (24). Brosch et al. reported that M. tuberculosis can be divided into ancestral or modern strains based on M. tuberculosis-specific deletion 1 (TbD1) region analysis. The TbD1 region is present in ancestral M. tuberculosis strains but is absent from modern ones. These ancestral strains predominantly originated from endemic foci, whereas modern M. tuberculosis strains represent epidemic strains that were introduced into the same geographical regions more recently as a consequence of the worldwide spread of the tuberculosis epidemic (4).Presently, an international spoligotype database, SpolDB4.0, has been established. Although the updated SpolDB4.0 version reflects the global distribution of M. tuberculosis spoligotypes, it contains little information regarding M. tuberculosis strains in China (5). In this study, we typed 2,346 M. tuberculosis clinical isolates from 13 different provinces across China between 2005 and 2007 using spoligotyping to study M. tuberculosis diversity in China.     

Molecular typing of Mycobacterium bovis isolates from Cameroon

In order to gain a better understanding of the molecular epidemiology of Mycobacterium bovis isolates in Cameroon, 75 isolates of M. bovis collected in three provinces of northern Cameroon were studied by spoligotyping. For 65 of these isolates, typing was also carried out by pulsed-field gel electrophoresis (PFGE) with DraI, and 18 of the isolates were also typed by restriction fragment length polymorphism (RFLP) analysis with probe IS6110-RHS. Molecular typing of the isolates by these techniques revealed a high degree of homogeneity, with 10 spoligotypes for 75 isolates, four PFGE profiles for 65 isolates, and three RFLP types for 18 isolates. Some types were present in the three different provinces, while some were confined to one or two areas. These results suggest that geographical mapping of M. bovis strains could be helpful for the control of bovine tuberculosis at the regional level. An interesting feature of all the spoligotypes was the absence of spacer 30, suggesting a common origin for all of the Cameroon isolates tested; an evolutionary scenario for the isolates is discussed. In addition, a comparison of the three techniques showed that for M. bovis strain differentiation in Cameroon and in surrounding countries, spoligotyping would be a more discriminating and practical tool for molecular typing than the other two techniques used in this study.     

Characterization of Mycobacterium tuberculosis isolates from patients in Houston, Texas, by spoligotyping

Mycobacterium tuberculosis isolates (n = 1,429) from 1,283 patients collected as part of an ongoing population-based tuberculosis epidemiology study in Houston, Texas, were analyzed by spoligotyping and IS6110 profiling. The isolates were also assigned to one of three major genetic groups on the basis of nucleotide polymorphisms located at codons 463 and 95 in the genes (katG and gyrA) encoding catalase-peroxidase and the A subunit of DNA gyrase, respectively. A total of 225 spoligotypes were identified in the 1,429 isolates. There were 54 spoligotypes identified among 713 isolates (n = 623 patients) assigned to 73 IS6110 clusters. In addition, among 716 isolates (n = 660 patients) with unique IS6110 profiles, 200 spoligotypes were identified. No changes were observed either in the IS6110 profile or in the spoligotype for the 281 isolates collected sequentially from 133 patients. Five instances in which isolates with slightly different spoligotypes had the same IS6110 profile were identified, suggesting that in rare cases isolates with different spoligotypes can be clonally related. Spoligotypes correlated extremely well with major genetic group designations. Only three very similar spoligotypes were shared by isolates from genetic groups 2 and 3, and none was shared by group 1 and group 2 organisms or by group 1 and group 3 organisms. All organisms belonging to genetic groups 2 and 3 failed to hybridize with spacer probes 33 to 36. Taken together, the results support the existence of three distinct genetic groups of M. tuberculosis organisms and provide new information about the relationship between IS6110 profiles, spoligotypes, and major genetic groups of M. tuberculosis.     

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.     

Microevolution of the direct repeat region of Mycobacterium tuberculosis: implications for interpretation of spoligotyping data

The direct repeat (DR) region has been determined to be an important chromosomal domain for studying the evolution of Mycobacterium tuberculosis. Despite this, very little is known about microevolutionary events associated with clonal expansion and how such events influence the interpretation of both restriction fragment length polymorphism (RFLP) and spoligotype data. This study examined the structure of the DR region in three independently evolving lineages of M. tuberculosis with a combination of DR-RFLP, spoligotyping, and partial DNA sequencing. The results show that the duplication of direct variable repeat (DVR) sequences and single-nucleotide polymorphisms is rare; conversely, the deletion of DVR sequences and IS6110-mediated mutation is observed frequently. Deletion of either single or contiguous DVR sequences was observed. The deletion of adjacent DVR sequences occurred in a dependent manner rather than as an accumulation of independent events. Insertion of IS6110 into either the direct repeat or spacer sequences influenced the spoligotype pattern, resulting in apparent deletion of DVR sequences. Homologous recombination between adjacent IS6110 elements led to extensive deletion in the DR region, again demonstrating a dependent evolutionary mechanism. Different isolates from the same strain family and isolates from different strain families were observed to converge to the same spoligotype pattern. In conclusion, the binary data of the spoligotype are unable to provide sufficient information to accurately establish genotypic relationships between certain clinical isolates of M. tuberculosis. This has important implications for molecular epidemiologic strain tracking and for the application of spoligotype data to phylogenetic analysis of M. tuberculosis isolates.     

Genetic diversity of Mycobacterium tuberculosis in Mbarara, South Western Uganda

Background

We determined the genetic diversity of mycobacteria isolated from tuberculosis patients in Mbarara Uganda, using region of difference (RD) analysis and spacer oligonucleotide typing (spoligotyping).

Methods

Sputum samples were cultured on Lowenstein Jensen media. The isolates were characterized using RD analysis and spoligotyping.

Results

The majority (92.8%) of the patients were new cases, 60% were males and 44% were HIV positive with a mean age of 33.7 years. All the 125 isolates were identified as M.tuberculosis sensu stricto. Most (92.8%) of the isolates were modern strains. Spoligotyping revealed 79 spoligotype patterns, with an overall diversity of 63.2%. Sixty (48%) isolates formed 16 clusters each consisting of 2–15 isolates. Mst (59.2 %) of the isolates were Uganda genotype strains. The major shared spoligotypes in our sample were SIT 135 (T2-Uganda) with 12 isolates and SIT 128 (T2) with 5 isolates. Sixty nine (87%) patterns had not yet been defined in the SpolDB4.0.database.

Conclusion

The TB epidemic in Mbarara is caused mainly by modern M.tuberculosis strains of the Uganda genotype. The wide diversity of strains may indicate that the majority of the TB cases are reactivation rather than re-infection. However this needs to be ascertained with more discriminative finger printing techniques.     

Splitting of a Prevalent Mycobacterium bovis Spoligotype by Variable-Number Tandem-Repeat Typing Reveals High Heterogeneity in an Evolving Clonal Group

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.