Africanum Strains Research Articles

The Mycobacterium bovis homologous protein of the Mycobacterium tuberculosis serine/threonine protein kinase Mbk (PknD) is truncated.

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.

A novel polymorphic genetic locus in members of the Mycobacterium tuberculosis complex.

It has previously been shown that the PAN promoter from Mycobacterium paratuberculosis can be used as a DNA probe to identify an RFLP between wild-type Mycobacterium bovis and the vaccine strain Mycobacterium bovis BCG. To investigate the genetic basis of this phenomenon, DNA fragments from a New Zealand M. bovis cattle strain and M. bovis BCG Pasteur, containing the PAN-binding region, were isolated from gene libraries, sequenced and characterized. Sequence analysis and comparison with database sequences showed that the PAN region in M. bovis, M. bovis BCG and Mycobacterium tuberculosis is identical and shares 70% similarity to the PAN sequence from M. paratuberculosis. The Shine-Dalgarno sequence and the -10 and -35 promoter regions are conserved between the different species. Analysis of the flanking sequences of the PAN region revealed that less than 1 kb downstream of PAN is a 2405 bp fragment that is present in M. bovis BCG but absent in the M. bovis wild-type strain. The distribution of the 2405 bp fragment in members of the M. tuberculosis complex was investigated and found to be present in 70 out of 70 M. tuberculosis strains, and 7 out of 7 M. bovis BCG daughter strains, 2 out of 2 Mycobacterium africanum strains, 2 out of 2 Mycobacterium microti strains and 7 out of 25 M. bovis strains. This is the first report of a genetic region of M. bovis BCG that is not universally present in M. bovis strains. The fragment does not appear to be present in any mycobacterial species outside the M. tuberculosis complex. It does not possess any characteristics of known transposable elements and the flanking sequences do not have any obvious features to suggest a deletion mechanism. The genetic location of this region is close to the 3' end of the RD1 region of M. bovis and M. tuberculosis. The polymorphic nature of this locus in M. bovis will provide an additional genetic marker for strain differentiation.

Rapid differentiation of bovine and human tubercle bacilli based on a characteristic mutation in the bovine pyrazinamidase gene.

Bovine tuberculosis (TB) caused by Mycobacterium bovis is an important veterinary disease that can also afflict humans. Although M. bovis shares an almost identical genome with M. tuberculosis, subtle differences in host specificity and several biochemical parameters can be used to distinguish the two closely related species. The current method for distinguishing M. bovis from M. tuberculosis relies on tedious testing of biochemical parameters, including natural resistance to pyrazinamide and defective pyrazinamidase (PZase) activity of M. bovis strains. In this study, we report the development of a rapid PCR-single-strand conformation polymorphism (SSCP) assay to differentiate M. bovis from M. tuberculosis strains, based on the detection of a single characteristic point mutation in the PZase gene (pncA) of M. bovis. Eighty-seven of 89 M. bovis strains could be distinguished from M. tuberculosis strains. Surprisingly, two animal isolates which were initially identified as M. bovis were shown to be M. africanum because they had a wild-type pncA sequence with positive PZase. These two M. africanum strains contain multiple (three and six) copies of insertion sequence IS6110, a feature they have in common with M. tuberculosis. The implication of this finding for the taxonomy of M. tuberculosis complex is discussed in relation to host preference and epidemiology. The development of a rapid PCR-SSCP test for distinguishing M. bovis from M. tuberculosis will be useful for monitoring the spread of bovine TB to humans in areas where bovine TB is endemic and for directing the treatment of human TB caused by M. bovis.

Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex.

It is important to correctly identify species within the Mycobacterium tuberculosis complex because of the zoonotic implications of bovine tuberculosis, especially in developing countries. We assessed the use of various genetic markers for species-specific identification of mycobacteria from the M. tuberculosis complex. A multiplex PCR designed for detection of the mtp40 and IS1081 elements was optimized and evaluated in 339 mycobacterial strains from different animal and geographic origins. The host range of the IS6110, MPB70, and 16S rRNA genes was also studied by PCR in all the strains. Finally, the usefulness of the genetic markers was compared by an immunoperoxidase test for specific identification of Mycobacterium bovis strains. The mtp40 sequence was detected in 87 of the 91 strains of M. tuberculosis and in 9 of the 11 Mycobacterium africanum strains but not in any of the M. bovis or Mycobacterium microti strains, indicating that the mtp40 element was also found in all of the M. tuberculosis complex strains isolated from seals. This organism is considered to be a true seal pathogen, but its origin is essentially unknown. The finding of the mtp40 element in the strains from seals suggests a closer relationship of these strains with a human origin than to an animal origin. The mtp40 element was not found in any other mycobacterial species included in the study. As a result of this study, we suggest that biochemical tests or alternate genetic markers are still needed to differentiate M. tuberculosis from M. africanum when these species coexist as causative agents of tuberculosis. The immunoperoxidase test worked well for the identification of M. bovis strains. We also report, for the first time, PCR amplification of the repetitive element IS6110 in an isolate of Mycobacterium ulcerans and an isolate of Mycobacterium gilvum, which emphasizes the need for further investigation of the host range of this sequence.

Drug resistance of M. tuberculosis and M. africanum strains isolated in Sierra Leone, West Africa

Drug resistance of M. tuberculosis and M. africanum strains isolated in Sierra Leone, West Africa

M. africanum strains isolated in East-Africa, Uganda

M. africanum strains isolated in East-Africa, Uganda

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.

In vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae to ticarcillin in combination with clavulanic acid.

The in vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae (M. chelonei) to ticarcillin in combination with calvulanic acid (CA) was studied by the agar dilution method. All the M. tuberculosis, M. bovis, and M. africanum strains were inhibited at a ticarcillin concentration of 32 micrograms/ml or lower in combination with 5 micrograms of CA. M. chelonae and M. avium strains proved resistant to more than 128 micrograms of ticarcillin plus 5 micrograms of CA per ml. M. fortuitum strains needed 128 micrograms of ticarcillin plus 5 micrograms of CA to inhibit approximately 30% of the isolates.

DNA restriction endonuclease analysis of Mycobacterium bovis and other members of the tuberculosis complex.

DNA preparations from 24 New Zealand isolates, two reference strains of Mycobacterium bovis, and one reference strain each of Mycobacterium microti, Mycobacterium africanum, and Mycobacterium tuberculosis were characterized by restriction endonuclease analysis. Twenty-five restriction enzymes were investigated. The clearest differences in M. bovis patterns were obtained with the enzymes BstEII and BclI. These produced four and five different patterns, respectively, for the 24 local isolates. When the results from both enzymes were considered, seven different combinations were obtained. The patterns produced for the two reference strains of M. bovis could be distinguished from each other and also from the patterns produced for the local isolates. All patterns were reproducible and are now being used for typing M. bovis isolates. With either enzyme, the patterns produced for the M. tuberculosis, M. bovis, and M. africanum strains had many features in common, but all the M. bovis patterns were clearly more similar to each other than to the M. tuberculosis patterns. The patterns produced for the M. microti strain were markedly different from those produced for the other species. Restriction endonuclease analysis is clearly a useful method for inter- and intraspecific classifications of the tuberculosis complex.

Numerical Taxonomy Analysis of Mycobacterium africanum

A numerical taxonomy analysis of Mycobacterium africanum strains from Rwanda (6 strains), Burundi (5 strains), Yaoundá (23 strains), Dakar (17 strains), and Mauretania (9 strains) indicated that these bacteria did not cluster separate from the M. tuberculosis or the M. bovis clusters, nor did they eliminate the phenetic discontinuity between the latter. However, the African strains introduced considerable heterogeneity in the usually homogeneous M. tuberculosis and M. bovis clusters. Judging from the data, it did not seem justifiable to create a new species M. africanum. Preliminary genetic analysis did not support the view expressed earlier that these bacteria mutated at high frequencies, and further genetic analysis of these bacteria seemed necessary.

Thermal and ultraviolet light inactivation of Mycobacterium africanum

Mycobacterium africanum Yaoundé and Rwanda were more heat-resistant than the tubercle bacilli. The ultraviolet susceptibilities of M. africanum strains were within the range usually found in the mycobacteria (e −1 doses), however marked differences were found in the intersept numbers of the Yaounde and the Dakar types.