Beijing family Mycobacterium tuberculosis strains differ in their intracellular growth in THP-1 macrophages.

SETTING: Previous studies have shown that isolates from cases in IS6110 restriction fragment length polymorphism (RFLP) clusters that have persisted over several years and are widely distributed grow significantly faster in macrophages than isolates from cases with unique RFLP patterns. As members of the Beijing family of Mycobacterium tuberculosis are widely distributed and have been responsible for several large outbreaks, it has been suggested that this genotype may have a selective advantage over other strains. OBJECTIVE: To determine whether rapid growth in macrophages is a common characteristic of Beijing family strains. DESIGN: T-helper precursor-1 human macrophages were infected with various Beijing family strains, and intracellular growth and tumor necrosis factor alpha (TNF-alpha) secretion were assessed. Strains differed in their genotype, with IS6110 copy number ranging from 9 to 22. RESULTS: Strains demonstrated a range of growth phenotypes over the 7-day infection period. Three grew significantly more slowly than the other strains, whereas the fastest growth was observed consistently with isolates of strain 210. CONCLUSION: Rapid growth in macrophages is not a common characteristic of all Beijing strains. Few Beijing strains are as virulent as strain 210. The growth advantage is consistent with strain 210 having persisted many years in different locations and having caused many outbreaks.

Epidemiologic import of tuberculosis cases whose isolates have similar but not identical IS6110 restriction fragment length polymorphism patterns.

Isolates of Mycobacterium tuberculosis from patients with epidemiologic links frequently demonstrate identical IS6110 restriction fragment length polymorphism (RFLP) patterns (i.e., RFLP clustering) because they are infected with the same strain. Uncertainty arises with isolates that differ from one another by a few IS6110 hybridizing bands. During the period from 1 January 1996 to 31 December 1999, isolates from 585 tuberculosis (TB) cases were analyzed by RFLP, representing 98.2% of the 596 culture-positive TB cases reported in Arkansas during the study period. Of the 585 cases for which RFLP was available, 419 (71.6%) had an RFLP pattern with more than five copies of IS6110. Of the total 74 clusters, 48 comprised isolates with more than five copies of IS6110 and included 164 cases. Sixty-nine isolates with more than five copies of IS6110 comprising 16 clusters and 60 unique isolates were found to be similar to at least 1 other isolate (differing from it by one or two hybridizing bands). Among the 129 cases whose isolates were similar to other clustered or unique isolates, 16 cases were discovered with epidemiologic links: 14 (15.2%) were among the 92 cases with IS6110 RFLP patterns similar to those in clusters, and 2 (5.2%) were among the 37 unique cases that were similar to another unique case. The isolates from the epidemiologically linked patients shared common spoligotypes; all except one case shared common polymorphic GC-rich sequence (PGRS) patterns. Of the 129 patients whose isolates differed from another by one or two hybridizing IS6110 bands, 101 (78.3%) shared common spoligotypes and 87 (67.4%) shared common PGRS RFLP patterns.

Differences in the prevalence of IS6110 insertion sites in Mycobacterium tuberculosis strains: low and high copy number of IS6110.

SETTING: Mycobacterium tuberculosis (M. tuberculosis) isolates from various parts of the USA which have few copies of the insertion sequence IS6110. OBJECTIVES: To characterize the sites of insertion of IS6110 among M. tuberculosis isolates that have one to six copies of the insertion sequence. DESIGN: The mixed-linker polymerase chain reaction (ML-PCR) procedure was used to amplify the terminal repeats on the ends of IS6110 and adjacent flanking sequences. From the ML-PCR products, sequences flanking 14 copies of IS6110 in strains containing less than seven copies of the insertion were determined. Sequence information from the flanking deoxyribonucleic acid was used to construct flanking primers that can be used to indicate the presence of IS6110 at a particular site when paired with outbound IS6110 primers in a PCR. Over 200 strains of diverse origin were screened for the insertion of IS6110 at several distinct sites using this procedure. RESULTS: The direct repeat (DR) locus has been described as a highly preferred site for insertion of IS6110 in strains of M. tuberculosis. Another highly preferred site of insertion of IS6100, DK1, is herein described. Insertions at DK1 are highly prevalent in M. tuberculosis strains harboring two to six copies of IS6110. The prevalence of insertions at this site decreases in strains with more than six copies of IS6110, even though the sequence itself is present in strains lacking a copy of IS6110 at this site. CONCLUSION: In addition to the DR locus there are other conserved sites of insertion among M. tuberculosis strains. The data further suggest a separate lineage for the high copy and the low copy strains, and a possible sequential insertion of IS6110 in strains of M. tuberculosis with less than seven copies.

Restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in northern Tanzania.

Isolates of Mycobacterium tuberculosis from 88 patients with pulmonary tuberculosis in northern Tanzania were subjected to IS6110 restriction fragment length polymorphism (RFLP) analysis. Of 88 isolates, 73 fell into 11 groups of which 9 contained 2-5 isolates. Of 2 large homology groups one, group H (20 isolates), was isolated only from patients resident in the Kilimanjaro region, whereas 79% of isolates from other groups came from this region. A significant association (P = 0.023) was found between another group, M (24 isolates) and isolation from patients of the Masai tribe. The data from this pilot study support the idea that IS6110 RFLP analysis provides information which may be of value in the control of tuberculosis in Africa.

Insertion sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110.

DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.

Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium.

A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20') were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited in M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.

Transpositional activity of IS986 in Mycobacterium smegmatis.

The insertion sequence IS986 (also known as IS6110) has been widely used for typing Mycobacterium tuberculosis isolates, due to the extensive multiple polymorphism shown using this probe. Although this polymorphism is presumed to be due to the mobility of IS986, transposition of this element has not previously been demonstrated in the laboratory. Using artificial composite transposons constructed in a vector unable to replicate in mycobacteria, we have succeeded in demonstrating the mobility of IS986 in M. smegmatis, apparently through cointegrate formation and transposition. The apparently random nature of IS986 insertions in M. tuberculosis is advantageous for transposon mutagenesis, although its efficient use will require more effective delivery systems.

Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains.

Gene probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.