Genetic markers for the epidemiology of tuberculosis.
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AIDS-Related Opportunistic Infections↗
Biomedical subjects
D van Soolingen
Publications and source records attributed to D van Soolingen.
Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES.
In contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene. The complete DNA sequence of this groESL operon was determined. The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85-90% and 59-61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins. Southern blotting analyses with M. leprae groES- and groEL-specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis. This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene. Using five T-cell clones from two leprosy patients as probes, expression of the M. leprae GroES protein in Escherichia coli after heat shock was demonstrated. Four of these clones recognized the same M. leprae-specific GroES-derived peptide in a DR2-restricted fashion. No expression of the groEL gene from this operon was detected in E. coli after heat shock, as tested with a panel of T-cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria.
Amino Acid Sequence↗
Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansasii and Mycobacterium gordonae.
In this study, the occurrence of repeated DNA sequences in the chromosome of Mycobacterium tuberculosis was investigated systematically. By screening a M. tuberculosis lambda gt-11 gene library with labeled total chromosomal DNA, five strongly hybridizing recombinants were selected, and these contained DNA sequences that were present in multiple copies in the chromosome of M. tuberculosis. These recombinants all contained repeated sequences belonging to a single family of repetitive DNA, which shares homology with a previously described repeated sequence present in recombinant pPH7301. Sequences analysis of pPH7301 showed the presence of a 10-bp sequence that was tandemly repeated and invariably separated by 5-bp unique spacer sequences. Southern blot analysis revealed that the majority of the repeated DNA in M. tuberculosis is composed of this family of repetitive DNA. Because the 10-bp repeats are slightly heterogeneous in sequence, we designated this DNA as a major polymorphic tandem repeat, MPTR. The presence of this repeated sequence in various other mycobacterial species was investigated. Among the MPTR-containing mycobacterial species the chromosomal location of the repetitive DNA is highly variable. The potential use of this polymorphism in the epidemiology of mycobacterioses is discussed.
Amino Acid Sequence↗
Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG.
Recently, the insertion element IS1081 from Mycobacterium bovis was identified. In this study, the usefulness of IS1081 in the epidemiology of tuberculosis was investigated. The host range of this insertion sequence was found to be restricted exclusively to the group of Mycobacterium tuberculosis complex bacteria, whereas none of the 10 mycobacterial species which do not belong to the M. tuberculosis complex contained IS1081-homologous DNA. All 99 M. tuberculosis complex strains investigated carried five or six copies of IS1081, and very limited IS1081-associated restriction fragment length polymorphisms were observed among the strains. Seven different IS1081-containing bands were distinguished in each strain, and the patterns differed only in one or two insertion sequence-containing bands. The banding pattern of M. bovis BCG differed in the presence of a 8.0-kb IS1081-containing PvuII fragment which was absent from all other M. tuberculosis complex strains.
Base Sequence↗
Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains.
Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like element, and these strains are highly polymorphic with regard to the site of integration in the chromosome. In contrast, Mycobacterium bovis BCG contains a single copy of the insertion element, and in all strains this copy is integrated at the same site in the chromosome. In this study, we determined the sequence of the single-copy insertion element from M. bovis BCG, IS987, and its flanking regions. The analysis of IS987 revealed that this element was virtually identical to the sequence of IS986 from M. tuberculosis. IS987 is located in a region containing direct repeats (DRs). The cloned flanking regions contained 20 virtually identical DRs of 36 bp, each separated by 35 to 41 bp of spacer DNA. Analysis of chromosomal DNA by the polymerase chain reaction revealed the presence of a cluster of 49 DRs, and IS987 is inserted in the 30th DR. Furthermore, the DR sequences were found to occur only in species of the M. tuberculosis complex and not in nine other mycobacterial species tested. Analysis of 14 M. tuberculosis strains revealed the presence of one insertion sequence element in the DR-containing region of eight strains, two insertion sequence elements were located in the DR region of five strains, and one strain did not contain an insertion sequence element in this region. Additionally, the DR-containing regions of these 14 M. tuberculosis strains were polymorphic in length and composition. We conclude that the DR cluster is a specific, hot-spot region for integration of insertion elements in the chromosome of M. tuberculosis complex strains.
Amino Acid Sequence↗
Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis.
In this study we established the usefulness of DNA fingerprinting for the epidemiology of tuberculosis on the basis of the DNA polymorphism generated by the insertion sequence (IS) IS986. Although clinical isolates of Mycobacterium tuberculosis displayed a remarkably high degree of restriction fragment length polymorphism, we showed that transposition of this IS element is an extremely rare event in M. tuberculosis complex strains grown either in vitro or in vivo for long periods of time. The M. tuberculosis and Mycobacterium africanum strains tested in this study contained 6 to 17 IS copies. In the Mycobacterium bovis strains, the copy numbers ranged between 1 and 5, and all 27 M. bovis BCG strains investigated invariably contained a single IS copy. This copy was located at a unique chromosomal position, reinforcing the idea that the frequency of IS transposition is very low in M. tuberculosis complex strains. Various microepidemics are described in which each microepidemic corresponds to a particular fingerprint type. The extent of similarity between Dutch and African strains was quantitatively assessed by computer-assisted analysis of DNA fingerprints. The results indicate that M. tuberculosis strains from regions in central Africa, where tuberculosis is highly prevalent, are generally more related to each other than isolates from the Netherlands, where the transmission rate is low and where the majority of the tuberculosis cases are presumed to be the result of reactivation of previously contracted M. tuberculosis infections.
Africa↗
Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family.
A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).
Amino Acid Sequence↗
Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis.
IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it has previously been shown to be present in multiple copies in the chromosome of M. tuberculosis. In this study we investigated the value of a IS986-based DNA probe in the diagnosis and epidemiology of tuberculosis. IS986 was found only in species belonging to the M. tuberculosis complex. Independent isolates of M. tuberculosis complex strains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. In contrast, Mycobacterium bovis BCG vaccine strains as well as clinical isolates of M. bovis BCG contained one copy of IS986, which was present at the same location in the chromosome. Different M. tuberculosis isolates from a recent M. tuberculosis outbreak showed an identical banding pattern. We concluded that IS986 is an extremely suitable tool for the diagnosis and epidemiology of tuberculosis.
Animals↗
The impact of nontuberculous mycobacteria on management of presumed pulmonary tuberculosis.
BACKGROUND: The presence of nontuberculous mycobacteria (NTM) in sputum or bronchial washings may cause diagnostic problems which affect clinical management. PATIENTS AND METHODS: In a retrospective analysis of 135 patients in a Dutch tuberculosis center, patients with NTM isolates were thoroughly investigated. Colonization or contamination by NTM was differentiated from true lung disease. RESULTS: 25 HIV-seronegative and two HIV-seropositive patients with NTM were identified. NTM were a likely cause of disease in only 14 (52%) patients. In 15 (55%), their presence led to preliminary diagnosis and treatment of tuberculosis. Unnecessary or inappropriate treatment was instituted in 17 (63%) patients with NTM. In two patients, detection of NTM in sputum also led to delay in diagnosing malignant disease. CONCLUSION: In this series, NTM in sputum or bronchial washings poorly reflected disease and often led to diagnostic and therapeutic errors. Although it is common knowledge that the presence of NTM should be considered in smear-positive patients, this apparently is a diagnostic pitfall in clinical practice. Reliable DNA-based techniques and better communication between physicians and microbiologists may improve management of suspected mycobacterial infections.
Adult↗
Mycobacterium canettii, the smooth variant of M. tuberculosis, isolated from a Swiss patient exposed in Africa.
An exceptionally smooth and glossy morphotype of Mycobacterium tuberculosis complex was isolated from a 56-year-old Swiss patient with mesenteric tuberculosis. Direct 16S rRNA sequence analysis of the hypervariable signature gene regions revealed a 100% homology to the specific M. tuberculosis complex sequence. Spoligotyping and restriction fragment length polymorphism analyses using the insertion sequences IS6110 and IS1081 and the polymorphic GC-rich sequence as additional genetic markers identified the isolate as the novel taxon M. canettii. Like a Somali child with a similar case, this patient probably contracted the infection in Africa, which raises questions about the geographic distribution of M. canettii.
Genetic Variation↗
Mycobacterium tuberculosis Beijing genotype emerging in Vietnam.
To assess whether the Mycobacterium tuberculosis Beijing genotype is emerging in Vietnam, we analyzed 563 isolates from new cases by spoligotyping and examined the association between the genotype and age, resistance, and BCG vaccination status. Three hundred one (54%) patients were infected with Beijing genotype strains. The genotype was associated with younger age (and hence with active transmission) and with isoniazid and streptomycin resistance, but not with BCG vaccination.
Adult↗
Mycobacterium tuberculosis Beijing genotype, Thailand--reply to Dr. Prodinger.
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China↗
Mycobacterium tuberculosis Beijing genotype strains associated with febrile response to treatment.
DNA fingerprinting has demonstrated predominance of the Beijing genotype among Mycobacterium tuberculosis strains isolated in Southeast Asia. We prospectively examined the occurrence of Beijing genotype strains in tuberculosis patients in Indonesia. Early in treatment, patients infected with Beijing genotype strains more often had fever unrelated to disease severity, toxicity, or drug resistance, indicating that Beijing genotype strains may have specific pathogenic properties.
Adolescent↗