Mycobacterium tuberculosis in Ontario,Canada: Insights from IS6110 Restriction Fragment Length Polymorphism and Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Genotyping

A collection of 1,308 clinical Mycobacterium tuberculosis isolates from Ontario, Canada, was genotyped by IS6110 restriction fragment length polymorphism (RFLP) and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. RFLP or >12 MIRU-VNTR loci were necessary for resolution of Indo-Oceanic strains. The low clustering rate and high strain diversity indicate that, in Ontario, most tuberculosis results from reactivation of latent infections.Tuberculosis (TB), which is caused by pathogens of the Mycobacterium tuberculosis complex (MTBC), remains a global scourge (19). In Canada, the average incidence rate is 5.0 cases/100,000 people, but the burden of disease varies across the country. In 2007, the four Atlantic provinces accounted for only ∼1% of TB cases, whereas ∼42% of new cases occurred in the province of Ontario (11). The Public Health Laboratories (PHL) of the Ontario Agency for Health Protection and Promotion provide diagnostic testing for TB in Ontario. The TB and Mycobacteriology Laboratory at PHL-Toronto is the largest facility of its kind in Canada, processing 50,000 patient samples plus 2,000 referred acid-fast positive cultures, with 600 to 650 new cases of TB detected annually (8, 11). Historically, the PHL has employed IS6110 restriction fragment length polymorphism (RFLP) for genotyping. Despite its utility, IS6110 RFLP is labor-intensive and only performed upon request. The current turnaround time of 21 days also makes the method incompatible with the PHL goal of universal, real-time MTBC genotyping. More recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing has been introduced (6, 7, 9, 15). The current PHL strategy relies upon agarose gel electrophoresis for comparison of PCR products. This method is low throughput, and gel-to-gel variability confounds comparison of samples processed at different times. Here, we describe implementation and validation of an improved MIRU-VNTR strategy and its utility for analysis of a large clinical strain collection.The semiautomated MIRU-VNTR strategy was derived from the 12-loci method of Cowan et al. (6, 7). Briefly, multiplex PCR was performed with dye-labeled primers in 96-well plates. For each reaction, 5 ng of template DNA was combined with 11 μl of a master mix (Red Taq; Sigma-Aldrich, Oakville, Canada) containing three primer pairs. PCR conditions were 95°C for 10 min and 34 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 60 s, with a final extension at 72°C for 7 min. PCR amplification was confirmed by electrophoresis on 1% Tris-borate-EDTA agarose gels. Samples were diluted 1:20 in sequence loading solution (Beckman Coulter, Mississauga, Canada) containing a 600-bp sequencing standard (Beckman Coulter) and then subjected to fragment analysis on a CEQ8800 genetic analysis system (Beckman Coulter). To reduce the manual labor and potential for human error associated with extensive pipetting, a Biomek NX Span-8 automation workstation (Beckman Coulter) was programmed to set up the initial PCRs and dilute PCR products for fragment analysis.To validate the method, a blinded set of 99 DNA samples was provided by the Public Health Research Institute (NJ). Strain identities and MIRU-VNTR patterns were unblinded only after complete 12-digit patterns were generated for all 99 DNA samples. Concordance between PHL and Public Health Research Institute results was 100%.To evaluate the utility of MIRU-VNTR in Ontario, typing was performed on 1,308 clinical samples from the PHL strain collection for which IS6110 RFLP profiles were also available. Strains were identified as MTBC by using DNA probes (AccuProbe; Gen-Probe, San Diego, CA) and were originally isolated during 1999 to 2001. Strains identified as Mycobacterium bovis or M. bovis BCG and samples containing multiple Mycobacterium species were excluded from analysis. For cases with multiple cultures, only the first isolate was used. Genomic DNA was extracted according to standard protocols (17) in a dedicated biosafety containment facility.MIRU-VNTR and IS6110 RFLP data were analyzed with BioNumerics 5.0 (Applied Maths, St-Martin Latem, Belgium). RFLP patterns were compared using band-based Dice statistics with 1% position tolerance such that clustered strains exhibited bands of identical number and position. Strains clustered by MIRU-VNTR were identical at all 12 loci. Analysis by MIRU-VNTR plus IS6110 RFLP employed the unweighted-pair group method with arithmetic mean. MTBC lineages were assigned by comparison to the MIRU-VNTR reference strain database (1).Independently, both methods revealed a large number of unique profiles and some clustered isolates (Table ​(Table1).1). Maximum strain resolution was achieved when both methods were combined. In general, RFLP was superior for multiband IS6110 strains, whereas resolution of single-band IS6110 isolates required MIRU-VNTR. Initial typing at 12 MIRU-VNTR loci generated two large “pseudoclusters.” One contained East Asian lineage strains (pattern 223325173533; n = 110), which are known to be poorly resolved by these 12 loci (12, 16). The second was comprised of strains from the Indo-Oceanic lineage (pattern 254326223432; n = 80). This group was typed at 12 additional loci (15). Even though four of the new loci were invariant, extended typing generated 47 new patterns (Fig. ​(Fig.1).1). Thirty-five Indo-Oceanic strains had unique profiles, whereas the remaining isolates formed 12 MIRU-VNTR-defined clusters (6 clusters with 2 isolates in each cluster, 3 with 3 each, 1 with 4, and 2 with 10 each). However, 11 of these could be resolved further by RFLP.Open in a separate windowFIG. 1.Improved typing of the Indo-Oceanic pseudocluster. Whereas all strains (n = 80) shared the same, original 12-loci pattern (254326223432), typing at 12 additional loci produced 47 distinct patterns, including 35 unique profiles. The remaining strains formed 12 clusters (6× n = 2; 3× n = 3; 1× n = 4; 2× n = 10), most of which could be further resolved by RFLP. However, one pair (cluster A) was identical by both methods, and two (clusters D and G) exhibited single band shifts. Conversely, one RFLP-defined pair (cluster M) exhibited differences at two loci upon extended MIRU-VNTR typing. For each strain, IS6110 RFLP profiles and repeat values at the extended MIRU-VNTR loci are shown. Relationships between strains are indicated by the phylogenetic (unweighted-pair group method with arithmetic mean) tree, and strains in MIRU-VNTR-defined clusters are labeled (clusters A to L).

TABLE 1.

Clustering results from MIRU-VNTR and IS6110 RFLP genotyping

Comparison of Variable Number Tandem Repeat and IS6110-Restriction Fragment Length Polymorphism Analyses for Discrimination of High- and Low-Copy-Number IS6110 Mycobacterium tuberculosis Isolates

The present study was designed to evaluate the use of variable number tandem repeat (VNTR) and IS6110-restriction fragment length polymorphism (RFLP) analyses in combination as a two-step strategy for discrimination (as measured by the Hunter-Gaston Discrimination Index [HGDI]) of both high- and low-copy-number IS6110 Mycobacterium tuberculosis isolates compared to IS6110-RFLP alone with an unselected collection of isolates. Individually, IS6110-RFLP fingerprinting produced six clusters that accounted for 69% of the low-copy-number IS6110 isolates (five clusters) and 5% of the high-copy-number IS6110 isolates (one cluster). A total of 39% of all the isolates were clustered (HGDI = 0.97). VNTR analysis generated a total of 35 different VNTR allele profile sets from 93 isolates (HGDI = 0.938). Combining IS6110-RFLP analysis with VNTR analysis reduced the overall percentage of clustered isolates to 29% (HGDI = 0.988) and discriminated a further 27% of low-copy-number isolates that would have been clustered by IS6110-RFLP alone. The use of VNTR analysis as an initial typing strategy facilitates further analysis by IS6110-RFLP, and more importantly, VNTR analysis subdivides some IS6110-RFLP-defined clusters containing low- and single-copy IS6110 isolates.     

Stability of Mycobacterium tuberculosis IS6110 Restriction Fragment Length Polymorphism Patterns and Spoligotypes Determined by Analyzing Serial Isolates from Patients with Drug-Resistant Tuberculosis

The stability of Mycobacterium tuberculosis IS6110 fingerprint patterns and spoligotypes has been assessed by analyzing serial isolates from patients with drug-resistant tuberculosis. Altogether, 165 M. tuberculosis isolates obtained from 56 patients have been analyzed. The time spans between the first and the last or a changed isolate from one patient ranged from 1 to 772 days. Among the 56 patients, 5 (9%) were infected with isolates with changes in their IS6110 fingerprint patterns. According to the total number of strains analyzed, 5% of the subsequent isolates showed variations in their IS6110 restriction fragment length polymorphism patterns compared to the pattern of the first isolates. Up to 10 isolates from one patient sampled at time intervals of up to 772 days with no changes in their IS6110 patterns have been analyzed. A statistically significant correlation could be found between changes in insertion sequence (IS) patterns and the increased time intervals over which the isolates were obtained, whereas changes in IS patterns are not correlated to changes in the drug resistance of the isolates. In contrast to the observed variations in IS6110 fingerprint patterns, no changes in the spoligotypes of the isolates analyzed could be found. In conclusion, our results confirm that the IS6110 fingerprint patterns of M. tuberculosis isolates have high degrees of stability. Compared to IS6110, the direct repeat (DR) region, which is the basis for spoligotyping, has a lower rate of change. Partial deletions, e.g., deletions induced by homologous recombination between the repetitive DR elements, could not be detected in this study.     

Molecular Fingerprinting of Mycobacterium tuberculosis Isolates Obtained in Havana, Cuba, by IS6110 Restriction Fragment Length Polymorphism Analysis and by the Double-Repetitive-Element PCR Method

Mycobacterium tuberculosis sputum isolates from 38 patients, obtained in the first 6 months of 1997 in Havana, Cuba, were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis and the double-repetitive-element PCR (DRE-PCR) method. Among 41 strains from 38 patients, 24 and 25 unique patterns, and 5 and 4 cluster patterns, were found by the RFLP and DRE-PCR methods, respectively. Patients within two of these clusters were found to be epidemiologically related, while no relation was observed in patients in the other clusters. The DRE-PCR method is rapid, and it was as discriminating as IS6110 RFLP analysis in identifying an epidemiological association. Its simplicity makes the technique accessible for subtyping of M. tuberculosis strains in laboratories not equipped to perform RFLP analysis.     

IS1245 Restriction Fragment Length Polymorphism Typing of Mycobacterium avium Isolates: Proposal for Standardization

Mycobacterium avium has become a major human pathogen, primarily due to the emergence of the AIDS epidemic. Restriction fragment length polymorphism (RFLP) typing, using insertion sequence IS1245 as a probe, provides a powerful tool in the molecular epidemiology of M. avium-related infections and will facilitate well-founded studies into the sources of M. avium infections in animal and environmental reservoirs. The standardization of this technique allows computerization of IS1245 RFLP patterns for comparison on a local level and the establishment of M. avium DNA fingerprint databases for interlaboratory comparison. Moreover, by combining international DNA typing results of M. avium complex isolates from a broad spectrum of sources, long-lasting questions on the epidemiology of this major agent of mycobacterial infections will be answered.     

Comparative Study of IS6110 Restriction Fragment Length Polymorphism and Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Isolates in the Netherlands,Based on a 5-Year Nationwide Survey

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% (n = 3,123). Of the remaining 855 cases, 12% (n = 479) of the cases were clustered only by VNTR, 7.7% (n = 305) only by RFLP typing, and 1.8% (n = 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% (n = 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLP-clustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems.     

Typing of Human Mycobacterium avium Isolates in Italy by IS1245-Based Restriction Fragment Length Polymorphism Analysis

All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern.     

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 the Mycobacterium bovis Restriction Fragment Length Polymorphism Probe pUCD, in Combination with the Direct Repeat Probe, for Molecular Typing of Mycobacterium tuberculosis Strains in Ireland

A mycobacterial restriction fragment length polymorphism probe, pUCD, has recently been described which represents an effective tool for the strain typing of Mycobacterium bovis. The present study evaluated this probe, in combination with the direct repeat probe (DR), for the molecular typing of 90 strains of Mycobacterium tuberculosis from 87 patients, looking at a group (62 isolates) of nonselected samples to assess pUCD combined with DR as a general tool and a subset of 32 isolates with a common specific IS6110 strain type in Ireland. Within the group of 62 isolates, pUCD-DR identified 42 strains and was comparable to both IS6110 (41 strains) and polymorphic guanine-cytosine-rich sequence (PGRS) (37 strains) analysis. pUCD-DR was found to be comparable to IS6110 and PGRS in identifying four separate clusters of isolates which were confirmed to be clinically related. pUCD-DR divided the common IS6110 isolates into six distinct types and was comparable to PGRS (seven strain types). The usefulness of this probe as an epidemiological tool is discussed.     

Usefulness of Spoligotyping To Discriminate IS6110 Low-Copy-Number Mycobacterium tuberculosis Complex Strains Cultured in Denmark

Mycobacterium tuberculosis complex strains cultured in Denmark have been analyzed by IS6110 restriction fragment length polymorphism (RFLP) on a routine basis from 1992 and onwards. Due to the influx of immigrants with tuberculosis, the number of strains harboring only one to five copies of IS6110 has increased steadily. Since the discriminatory power of IS6110 fingerprinting for such strains is poor, we have performed additional genotyping of all low-copy-number strains by the recently described PCR-based method known as spoligotyping. A total of 311 clinical strains were typed: 14 Mycobacterium bovis BCG, 48 M. bovis, and 249 M. tuberculosis strains. Spoligotyping correctly differentiated M. bovis and M. bovis BCG from M. tuberculosis strains, but it did not differentiate M. bovis from M. bovis BCG. All M. bovis BCG strains exhibited identical spoligotype patterns. The discriminatory power of spoligotyping of low-copy-number M. tuberculosis strains was higher than that of IS6110 fingerprinting. Based on RFLP typing solely, 83% of the low-copy-number M. tuberculosis strains were found to form part of a cluster, and 75% were found to form a cluster on the basis of spoligotyping. When the two techniques were combined, the amount of clustering decreased to 55%. The combination of these two techniques might be valuable in studying the epidemiology of M. tuberculosis strains harboring few copies of the IS6110 element.     

Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR.

Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues.     

Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR.

Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues.     

Fast ligation-mediated PCR, a fast and reliable method for IS6110-based typing of Mycobacterium tuberculosis complex

IS6110 restriction fragment length polymorphism (RFLP) analysis is the most widely applied method for strain differentiation of Mycobacterium tuberculosis complex. We have previously described mixed-linker PCR, an IS6110-based PCR method that favorably compared with other typing methods for M. tuberculosis complex according to reproducibility and ability to differentiate between strains. Here we report the further development of this method, called fast ligation-mediated PCR (FLiP), which allows analysis of strains within one working day and starting from less than 1 ng of mycobacterial DNA or a crude cell lysate. Blinded analysis of a standard set of 131 M. tuberculosis complex and nontuberculous isolates showed the ability to differentiate 81 types among 90 M. tuberculosis complex isolates with 84 different IS6110 RFLP fingerprint patterns and detected 97% of the 31 duplicate samples. We suggest that FLiP can serve to rapidly detect chains of transmission prior to starting high-throughput analysis or standard IS6110 RFLP. It may as well serve as a secondary typing technique for other, non-IS6110-based methods.     

Use of a PCR method based on IS6110 polymorphism for typing Mycobacterium tuberculosis strains from BACTEC cultures.

Two PCR typing methods, based on polymorphism of the insertion sequence IS6110, were compared with Mycobacterium tuberculosis strains by using a single primer complementary to the inverted repeats of IS6110. Total M. tuberculosis DNA either was amplified directly (IS6110-PCR) or was amplified following digestion and ligation (IS6110-inverse-PCR). Both PCR techniques showed a similar degree of discrimination. Because of its simplicity, IS6110-PCR was chosen to confirm that a single M. tuberculosis strain was responsible for an outbreak of tuberculosis in a secondary school. IS6110-PCR was used to study the degree of differentiation in 85 clinical M. tuberculosis isolates from BACTEC 12B broth cultures. Results were consistent with those of the standardized IS6110 restriction fragment length polymorphism (RFLP) analysis method, showing identical PCR types for identical RFLPs, although the degree of discrimination was greater by RFLP analysis. The study concludes that due to its simplicity, IS6110-PCR is a good screening method when quick differentiation between M. tuberculosis strains is needed because BACTEC cultures may be used directly.     

Use of spoligotyping to study the evolution of the direct repeat locus by IS6110 transposition in Mycobacterium tuberculosis

Based on the variability of 43 spacers within the direct repeat (DR) locus of Mycobacterium tuberculosis complex organisms, spoligotyping is a rapid method that aids in the study of the epidemiology of tuberculosis. It was recently hypothesized that despite its presence in the DR locus, spacer 31 could not be amplified in M. tuberculosis clinical isolates belonging to spoligotype 50 due to the insertion of an extra copy of IS6110 between spacers 31 and 32 that could lead to an asymmetrical split of the primer targets (I. Filliol, C. Sola, and N. Rastogi, J. Clin. Microbiol. 38:1231--1234, 2000). In the present investigation, previous observations were extended to 25 clinical isolates of type 50 showing that the primer set IS6-DRb that selectively amplified the left and central DR regions was indeed able to demonstrate the presence of spacer 31. IS6110-restriction fragment length polymorphism (RFLP) and DR-RFLP showed that type 50 isolates were characterized by the presence of two copies of IS6110 associated with the DR locus and an additional double IS6110 band of 1.4 kb. The primer set IS3-IS6 was then used to selectively amplify a 750-bp inter-IS6110 fragment within the DR locus. The sequencing of the central DR region corroborated our previous findings and showed that the absence of spacer 31 among the type 50 isolates was due to the asymmetric insertion of an extra copy of IS6110 between spacers 31 and 32, leading to an unequal split of the DRa-DRb target into two portions, of 6 and 30 bp, respectively. These results show that the DR locus constitutes an ideal IS6110 preferential locus (ipl), permitting the insertion of two or more copies of IS6110, and provide new clues for epidemiological and phylogenetic interpretation of changes in IS6110-RFLP and spoligotyping profiles.     

Determination of Genotypic Diversity of Mycobacterium avium Subspecies from Human and Animal Origins by Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat and IS1311 Restriction Fragment Length Polymorphism Typing Methods

Members of the Mycobacterium avium complex (MAC) are ubiquitous bacteria that can be found in water, food, and other environmental samples and are considered opportunistic pathogens for numerous animal species, mainly birds and pigs, as well as for humans. We have recently demonstrated the usefulness of a PCR-based mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing for the molecular characterization of M. avium subsp. paratuberculosis and M. avium strains exclusively isolated from AIDS patients. In the present study we extended our analysis, based on eight MIRU-VNTR markers, to a strain collection comprehensively comprising the other M. avium subspecies, including M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. silvaticum, isolated from numerous animal species, HIV-positive and HIV-negative humans, and environmental sources. All strains were fully typeable, with the discriminatory index being 0.885, which is almost equal to that obtained by IS1311 restriction fragment length polymorphism (RFLP) typing as a reference. In contrast to IS1311 RFLP typing, MIRU-VNTR typing was able to further discriminate M. avium subsp. avium strains. MIRU-VNTR alleles strongly associated with or specific for M. avium subspecies were detected in several markers. Moreover, the MIRU-VNTR typing-based results were consistent with a scenario of the independent evolution of M. avium subsp. avium/M. avium subsp. silvaticum and M. avium subsp. paratuberculosis from M. avium subsp. hominissuis, previously proposed on the basis of multilocus sequence analysis. MIRU-VNTR typing therefore appears to be a convenient typing method capable of distinguishing the three main subspecies and strains of the complex and providing new epidemiological knowledge on MAC.The most frequent agents of nontuberculous mycobacterioses belong to the Mycobacterium avium complex (MAC); in particular, M. avium subsp. hominissuis is a frequent agent of human mycobacterioses (12, 25). Members of this subspecies are also frequent infectious agents for pigs, leading to significant economic losses in pig farming, albeit that subspecies produces very low rates of morbidity in this animal species (23, 24). Two other MAC members, M. avium subsp. avium and M. avium subsp. paratuberculosis, are the causative agents of two other important, often fatal (2) animal pathologies, avian tuberculosis (40) and ruminant paratuberculosis (Johne''s disease) (6), respectively. Like other opportunistic agents, M. avium subsp. avium and M. avium subsp. hominissuis are also capable of infecting a wide range of animal species, including cattle, deer, wild boars, goats, and horses (40). In contrast, M. avium subsp. silvaticum is taxonomically very close to M. avium subsp. avium but almost exclusively infects wood pigeons (41).In the particular case of M. avium subsp. hominissuis, strains with similar or identical genotypes are usually found in common between pigs and human patients (26), which does not permit the potential zoonotic risk of this subspecies to be discarded. Moreover, these mycobacteria can be found in environmental sources such as water, biofilms, soil, aerosols, and phagocytic protozoa and amoebae (11), all of which can act as common sources of infection for animals and humans.For epidemiological investigations of MAC, the current reference molecular typing technique is restriction fragment length polymorphism analysis (RFLP) based on the IS1245 (47) and IS1311 (19, 20) insertion sequences. Whereas IS1311 RFLP usually generates clear hybridization patterns, IS1245 RFLP yields complex multiband patterns which are difficult to compare among different experiments and laboratories, mainly because of the heterogeneity in the intensities of the hybridization bands (19, 20, 42). Recently, an even simpler PCR-based molecular typing method, multilocus variable-number tandem-repeat analysis (MLVA), which is based on mycobacterial repetitive elements called mycobacterial interspersed repetitive-unit-variable-number tandem repeats (MIRU-VNTRs) (14, 34, 36, 37), has been described for M. avium subsp. paratuberculosis (38). This method presented better results for the differentiation of strains of this subspecies than those obtained by the standard IS900 RFLP method (38) and showed a promisingly good discrimination index (DI) with a panel of M. avium strains isolated from human AIDS patients (38).In the study described here, we extended that initial study by applying MIRU-VNTR typing to a large strain panel set comprising M. avium subsp. hominissuis, M. avium subsp. avium, and M. avium subsp. silvaticum strains isolated from diverse animal and human sources. Our aim was to further analyze the power of MIRU-VNTR typing to discriminate isolates within these subspecies and to identify possible specific signatures within the complex for better characterization and detection of interspecies transmission patterns.     

Analysis of the regions responsible for IS6110 RFLP in a single Mycobacterium tuberculosis strain.

A high degree of IS6110 restriction fragment length polymorphism (RFLP) is observed amongst the different strains of the Mycobacterium tuberculosis complex. The sequences of the IS6110 flanking regions from a M. tuberculosis strain harbouring four IS6110 copies were determined. Duplication of 3-4 nucleotides was found at the extremities of the four IS6110 copies, suggesting that IS6110 RFLP is due to transposition of the IS element. One of the copies of IS6110 analysed in the study was shown to be located at the same site in the genome of M. tuberculosis as the single copy present in an M. bovis BCG strain.     

Patients with Multidrug-Resistant Tuberculosis Display Impaired Th1 Responses and Enhanced Regulatory T-Cell Levels in Response to an Outbreak of Multidrug-Resistant Mycobacterium tuberculosis M and Ra Strains

In Argentina, multidrug-resistant tuberculosis (MDR-TB) outbreaks emerged among hospitalized patients with AIDS in the early 1990s and thereafter disseminated to the immunocompetent community. Epidemiological, bacteriological, and genotyping data allowed the identification of certain MDR Mycobacterium tuberculosis outbreak strains, such as the so-called strain M of the Haarlem lineage and strain Ra of the Latin America and Mediterranean lineage. In the current study, we evaluated the immune responses induced by strains M and Ra in peripheral blood mononuclear cells from patients with active MDR-TB or fully drug-susceptible tuberculosis (S-TB) and in purified protein derivative-positive healthy controls (group N). Our results demonstrated that strain M was a weaker gamma interferon (IFN-γ) inducer than H37Rv for group N. Strain M induced the highest interleukin-4 expression in CD4⁺ and CD8⁺ T cells from MDR- and S-TB patients, along with the lowest cytotoxic T-lymphocyte (CTL) activity in patients and controls. Hence, impairment of CTL activity is a hallmark of strain M and could be an evasion mechanism employed by this strain to avoid the killing of macrophages by M-specific CTL effectors. In addition, MDR-TB patients had an increased proportion of circulating regulatory T cells (Treg cells), and these cells were further expanded upon in vitro M. tuberculosis stimulation. Experimental Treg cell depletion increased IFN-γ expression and CTL activity in TB patients, with M- and Ra-induced CTL responses remaining low in MDR-TB patients. Altogether, these results suggest that immunity to MDR strains might depend upon a balance between the individual host response and the ability of different M. tuberculosis genotypes to drive Th1 or Th2 profiles.Human interventions, namely, mistreatment of tuberculosis (TB) and poor patient compliance, selectively favor the multiplication of drug-resistant Mycobacterium tuberculosis mutants over drug-susceptible bacilli in tuberculous lesions. M. tuberculosis isolates are considered to be multidrug resistant (MDR) when showing resistance to isoniazid and rifampin (rifampicin), the most effective drugs for TB treatment; they become extensively drug resistant when showing additional resistance to key second-line drugs (32, 36). MDR-TB and extensively drug-resistant TB are very difficult to treat, their prognosis is somber, and mortality is high (14, 27).In Argentina, a total of 11,464 new cases of TB were reported in 2006, with an incidence of 29.1 per 100,000 inhabitants. MDR-TB occurred in 4.5% of the cases. MDR-TB outbreaks emerged in Argentina among hospitalized patients with AIDS in the early 1990s (1, 45) and thereafter disseminated to immunocompetent individuals (37-39). Epidemiological, bacteriological, and genotyping data allowed the identification of certain MDR M. tuberculosis outbreak strains, such as the so-called strain M of the Haarlem family and strain Ra of the Latin America and Mediterranean (LAM) family. Each of these two strains managed to perpetuate in its geographical niche, i.e., Buenos Aires and the Rosario City area, respectively. In particular, strain M appears to be highly prosperous in the country and is able to build up further drug resistance without impairing its ability to spread (29).TB development depends not only on the host immune response and on its natural resistance/susceptibility to M. tuberculosis infection but also on differences in transmissibility, virulence, and immunogenicity among M. tuberculosis strains, determined by the genetic background of the organisms. It is becoming evident that certain strains of M. tuberculosis with special transmission potential are able to manipulate host immunity by inducing Th1 or Th2 responses which could impact disease outcome and/or evolution (5, 28, 30, 31, 40-42, 51). Protective immunity against TB is mediated by a Th1-type immune response characterized by high levels of interleukin-12 (IL-12) from infected macrophages and gamma interferon (IFN-γ) from antigen-specific T cells, which control and contain infection in the lungs (24). Peripheral blood mononuclear cells (PBMC) from MDR-TB patients have been shown to poorly respond in vitro to H37Ra whole bacilli, purified protein derivative (PPD), and specific antigens, such as ESAT-6 and the 30-kDa protein (16, 25, 26, 33). Furthermore, increased IL-4 secretion by CD4⁺ T cells after H37Rv total lipid stimulation was observed in MDR-TB patients (50).Although a Th1 profile is necessary for a protective response, it may also cause immunopathologic damage; for this reason, either regulatory T cells (Treg cells) or a Th2 response might play important regulatory functions protecting the patient from collateral host tissue damage. Nevertheless, an excessive Th1 downregulation might favor disease progression. In this context, increased levels of CD4⁺ CD25^high Foxp3⁺ Treg cells have been detected in PBMC from drug-susceptible tuberculosis (S-TB) patients compared to those from PPD-positive (PPD⁺) healthy donors (18-20, 46). In addition, the results of studies involving in vitro depletion of CD4⁺ CD25^high T lymphocytes suggest a role of Treg cells in TB pathogenesis (18-20, 43).In the current study, we examined immune profiles induced by two MDR M. tuberculosis strains disseminated in Argentina, namely, strains M and Ra. Our results demonstrate that strain M is a weak inducer of IFN-γ and elicits a remarkably low cytotoxic T-cell (CTL) activity. Also, in vitro expansion of Treg cells in PBMC from TB patients is not M. tuberculosis strain dependent and efficiently suppresses antigen-induced IFN-γ and CTL responses.