The Global Burden of Tuberculosis, Leprosy, and Other Mycobacterioses

Diseases Caused by Mycobacterium tuberculosis Complex

Mycobacterium tuberculosis complex (MTBC) includes closely related bacteria showing higher than 99% similarity at the DNA level. The group comprises several main members, such as M. tuberculosis, responsible for most human tuberculosis cases, Mycobacterium bovis, the agent of bovine tuberculosis, which can also infect other animals as well as humans, Mycobacterium africanum, a prevalent cause of human tuberculosis on the African continent, and the vole bacillus Mycobacterium microti (1) (Genus Mycobacterium). It also contains the vaccine strain Bacille Calmette-Guérin (BCG). Other recently proposed members of the MTBC are Mycobacterium caprae, primarily infecting goats in Spain (2, 3), and Mycobacterium pinnipedii, infecting seals in Australia and Argentina (4). A variant human isolate of M. tuberculosis, the canetti strain, was also described recently and has been associated with the “ancient” tuberculosis lineages (5). Despite this close relationship, they show a large variability in their phenotypic properties, epidemiology, and incidence in human tuberculosis, and for this and other historical reasons, they are still considered different species (6). However, the extent of MTBC speciation is not yet resolved (7).

According to WHO reports, one-third of the world’s population is currently infected with the tuberculosis bacillus, and each year, about two million people die of tuberculosis (WHO Tuberculosis). Lack of control of tuberculosis is largely attributable to delayed or under diagnosis, non-efficient protection by BCG vaccination, the use of multiple drugs and prolonged treatment regimens, which leads to the existence and spread of multidrug-resistant strains, and association with HIV infection. Other social factors also have strong influence, such as increase in migration and poverty. The evolution of human tuberculosis incidence since 1990 is depicted in the maps in Figure 1 and Figure 2.

Figure 1

TB incidence, all forms (per 100,000 population per year), By Country, Total, 1990. From: WHO|Global TB database (http://www.who.int/tb/country/global_tb_database/en/index1.html).

Figure 2

TB incidence, all forms (per 100,000 population per year), By Country, Total, 2004. From: WHO|Global TB database (http://www.who.int/tb/country/global_tb_database/en/index1.html).

Bovine tuberculosis is a chronic zoonotic disease whose etiological agent is M. bovis. It constitutes a serious animal health problem, causing economic losses due to decreased meat and milk production and to low exportation of cattle products (TB in cattle). Although the main host of M. bovis is cattle, other animals, including humans, may be affected. A study performed in the main milk production region of Argentina showed that in the period 1984 to 1989, M. bovis was responsible for 2.4 to 6.2% of tuberculosis cases in human beings, of which 64% were rural and meat workers (8). The AIDS epidemic has also increased the risk of transmission of M. bovis to humans. Nosocomial transmission of bovine tuberculosis produced by multidrug-resistant M. bovis strains among HIV-positive individuals was described recently in Spain (9).

Leprosy: A Public Health Problem

Leprosy remains a public health problem, mainly in developing countries, because in spite of the actions promoted by WHO, prevalence has been reduced, but the number of new cases has remained constant, with more than 690,000 cases reported (WHO; New Case Detection). The discovery of the leprosy bacillus by Hansen in 1873 constituted the first demonstration of a clear association of a microorganism to human disease. Mycobacterium leprae, the etiological agent of leprosy, is still unable to be grown as axenic culture and has an extremely slow doubling time in tissues (approximately 14 days). Large quantities of bacilli could be isolated for biochemical and genetic studies using the nine-banded armadillo as a surrogate host. The complete genome sequence of the armadillo-derived Indian isolate, the TN strain, was performed (10).

Leprosy is considered a low transmissible disease, and the means of transmission are uncertain. However, M. leprae infection is thought to spread by the respiratory route, because bacilli could be isolated from nasal swabs of patients. Leprosy develops very slowly in the infected host (2 to 10 years); there are two polar, stable forms based on skin smears (named multibacillary (MB) and paucibacillary (PB) leprosy, respectively) and a complex spectrum of unstable forms (Classification).

Using comparative genomics, seven strains of the leprosy bacilli from separate geographic origins were recently analyzed, showing a surprisingly stable genome. Authors concluded that cases of leprosy around the world could be attributable to a single clone, providing a general evolutionary scheme for M. leprae on the basis of single nucleotide polymorphism (SNP) data (11) (Figure 3). More recently, the computer analysis of substitutions in the pseudogenes, and its comparison with the functional orthologs of closely related genomes, has allowed the reconstruction of the gene content of the common ancestor of M. leprae and mycobacteria (12).

Figure 3

From Leprae Monot, Science 308:1040-42, 2005.

Other Mycobacterioses: Neglected Diseases

Buruli Ulcer

Mycobacterium ulcerans is an emerging human pathogen responsible for Buruli ulcer, a necrotizing skin disease found most commonly in West Africa (13), but outbreaks have also been reported in the Americas, Australia, and Asia (WHO Buruli Ulcer). The first detailed clinical description of ulcers caused by M. ulcerans has been attributed to Dr. Albert Cook, a missionary physician who worked in Uganda in the late 1800s (Buruli ulcer). Since the 1980s, Buruli ulcer has emerged as a serious public health problem in an increasing number of countries. In terms of numbers of cases, it is probably the third most common mycobacterial disease in humans, after tuberculosis and leprosy (Figure 4).

Figure 4

Buruli ulcer: global situation.

Knowledge of M. ulcerans at the molecular level has come only in the last 5 years, primarily from research focused on developing tools for either rapid diagnosis or molecular epidemiological investigation. M. ulcerans has an unexpectedly close genetic relationship with Mycobacterium marinum. Analysis of the 16S rRNA gene of both species revealed greater than 99.8% sequence identity. Biochemically, M. ulcerans has also been found to resemble M. marinum. However, there are also substantial phenotypic differences and dramatic differences in the pathologies and treatment of the diseases caused by each of these organisms. Also at a genetic level, multi-copy insertion sequences, IS2404 and IS2606, are present in the genome of M. ulcerans and absent from M. marinum (14).

This close relationship has been exploited in comparative genetic analysis by multi-locus sequencing (15). The results of this analysis suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA driven mainly by the activity of mobile DNA elements. M. ulcerans, but not M. marinum, expresses a plasmid-encoded immunomodulatory macrolide toxin, mycolactone, which plays an important role in virulence and pathology (16). Recently, it was suggested that the acquisition of this plasmid and the subsequent ability to produce mycolactone appears to be a key issue in the evolution of M. ulcerans from a common M. marinum progenitor (17)

Johne's Disease

Johne’s disease or paratuberculosis is a chronic disease produced by Mycobacterium avium subsp. paratuberculosis (MAP) that causes chronic enteritis in bovine, ovine, and other small ruminants, thus leading to production losses because of deficient food conversion. The disease has also been diagnosed in primates, deer, rabbits, foxes, and camels, as well as in a wide variety of wild animals (Johne´s disease, paratuberculosis). It is currently associated with Crohn’s disease in humans; therefore, it is considered a zoonotic disease (18).

MAP belongs to the M. avium complex (MAC), which comprises a group of opportunistic pathogenic mycobacteria able to infect humans and other animals and is also widely distributed in the environment. The group includes four species: M. avium, Mycobacterium intracellulare, and the recently described Mycobacterium chimaera (19) and Mycobacterium colombiense (20). M. avium has now been divided into four subspecies: M. avium subsp. paratuberculosis, M. avium subsp. silvaticum, M. avium subsp. avium, and M. avium subsp. hominisuis (21). The subspecies paratuberculosis presents higher than 95% sequence similarity with M. avium subsp. avium but is unique in the fact that the majority of its isolates require the iron-chelating agent mycobactin for growth.

Diseases Caused by Rapidly Growing Mycobacteria

Infections caused by rapidly growing mycobacteria (RGM) are not reportable diseases, and consequently their incidence and prevalence are largely unknown. Nevertheless, an increasing number of outbreaks and clusters of infections caused by RGM have been reported worldwide in the last years (22-27). RGM species are widely distributed in the environment and have been isolated from potable water, wastewater, soil, and inanimate surfaces (28).

Mycobacterial taxonomy is in constant evolution, and genotypic studies led to recent splitting and redefinition of species defined previously by phenotype-based methods (29). The rationale of genotypic taxonomy consists in the identification of highly conserved genomic regions harboring hypervariable sequences that show species-specific deletions, insertions, or replacements of single nucleotides. The 16S rRNA gene was, for many years, the primary target of molecular taxonomic studies, combined with analysis of unique mycobacterial cell wall lipids, the mycolic acids, and led to classification of RGM in three main groups: the Mycobacterium chelonae-abscessus group, the M. fortuitum group, and the M. smegmatis group.

RGM species are closely related to each other, and the indication that bacterial strains belong to the same species if they have fewer than 5- to 15-base differences in their 16S rRNA gene sequences is not applicable to RGM (30). Therefore, genes other than 16S rRNA have recently been included to assess phylogenetic relationships and to help clarify the taxonomy of RGM (31-34). Phylogenetic trees based upon individual sequences of five genes—16S rRNA, recA, rpoB, sodA, and hsp65—of 19 RGM species were compared with trees based on the combined dataset of the five genes and combined datasets of recA and rpoB. Six phylogenetic groups were recognized: the M. chelonae-abscessus group, M. mucogenicum group, M. fortuitum group, M. mageritense group, M. wolinsky group, and M. smegmatis group (35).

Information derived from the ribosomal RNA has been applied for further recognition of species within RGM. Mycobacteria have a minimum number of ribosomal operons, one or two copies per genome. Typical RGM have two copies. It was demonstrated that the chromosomal location of the two rrn operons in RGM is conserved within each species, as indicated by the Restriction Fragment Length Polymorphism (16S-RFLP)-derived patterns (36). Since the first description in 1994 (37), this approach has been demonstrated to complement the DNA-DNA hybridization data in the delimitation of closely related RGM species (38, 39).

The near completion of the genomes of M. smegmatis, M. chelonae, and M. abscessus will certainly further improve taxonomy studies and our understanding of these emerging pathogens.

Mycobacteria and AIDS

Soon after the beginning of AIDS pandemy, it was evident that mycobacterial infections, not only tuberculosis but also opportunistic infections, were a major cause of morbidity and mortality among patients living with HIV/AIDS worldwide. MAC members are responsible for most opportunistic bacterial infections in late stages of the disease, and other non-tuberculous mycobacteria can also be involved. The panorama dramatically changed in countries where highly active antiretroviral treatment (HAART) became widely available, with the beneficial effect of restoration of pathogen-specific immune responses. Thus, the incidence of mycobacterial infections, including tuberculosis, decreased in treated cohorts living in high- and low-income countries (40).

MAC bacilli are acquired from the environment (person-to-person transmission had not been demonstrated), enter the host by the gastrointestinal mucosa, and cause disseminated disease with fatal consequences in patients with AIDS. Epidemiological evidence indicates that most AIDS patients with disseminated MAC infections have recently acquired these organisms, suggesting that infection was not due to previously viable MAC bacilli that could reactivate; therefore, MAC bacteria seem not to be able to establish latent infection, compared with that of M. tuberculosis (41).

Association of HIV infection and tuberculosis is the major adult infectious killer in the developing world, and approximately 10% of all global tuberculosis cases are attributable to HIV. About 13 million people are infected with both causative organisms. The biggest impact has been described in the sub-Saharan Africa: tuberculosis notifications have tripled since the mid-1980s, and death rates are fourth times higher compared with countries with good tuberculosis-control programs. Probably because of its association with HIV, Africa is the only continent where tuberculosis incidence is rising, causing a global increase of about 1% per year (42) (Figure 5).

Figure 5

From Nunn et al. (42).

Molecular epidemiology studies on MTBC strains isolated from AIDS patients showed that both re-infection and new infection occur in these patients. It has been speculated that HIV/AIDS patients could be an ecological niche for M. tuberculosis low virulent clones without the selective pressure provided by the immunocompetent patient (43). As a consequence of the increase in the number of tuberculosis cases due to HIV, the performance of tuberculosis-control programs has deteriorated. The lack of human resources is the major bottleneck for implementation of services in developing countries. Global development efforts have been established until 2015 within the Millenium development goals by the United Nations with prominent features addressed against Tuberculosis and HIV/AIDS. The achievements will depend on progress in Africa.

Mycobacterial Genomes

Several mycobacterial genomes have been completed or are near completion. The updated list of mycobacterial genome projects can be viewed at Mycobacterial genome-sequencing projects and GOLD: Genomes OnLine Database Homepage

Impact of Genome Projects on Mycobacterial Research

The recent determination of genomic nucleotide sequences of M. tuberculosis H37Rv and CDC 1551, M. bovis, M. leprae, and MAP makes Mycobacterium one of the most sequenced bacterial genera (57). This wealth of comparative genome sequence information provides unique opportunities for new insights into the biology of these globally important pathogens.

Comparative Genomics

Comparison of mycobacterial genomes can be performed by a set of different approaches. One of them, the array technology, easily identify deletion events but cannot readily detect insertions or duplications. Other powerful method includes whole-genome sequence comparison that covers from SNP to gene rearrangements (80) and allows detection of large sequence polymorphisms (LSP), considered the major contributor to genetic diversity within members of the genus. Several of these methodologies have been used to compare members of MTBC, and more rarely were applied to other members of the genus.

Comparative Genomics between Members of MTBC

Whole genome comparisons of M. tuberculosis H37Rv and M. bovis BCG Pasteur were undertaken using two different approaches. Using BAC-arrays and direct comparison of canonical BACs from ordered libraries, several polymorphic genomic regions were uncovered (48). By performing comparative hybridization experiments on a DNA microarray comprising 4896 spots, representing 3902 ORFs of M. tuberculosis H37Rv, 16 regions of difference (RD1-16) were shown to be absent from some M. bovis BCG relative to M. tuberculosis H37Rv (81). In parallel, five regions deleted in H37Rv were described (RvD)1-5 (82) compared with genomes of other members of the complex. The combined findings of these studies can be found at UnitéGénétique Moléculaire Bactérienne.

Recently, a M. tuberculosis-specific deletion (TbD1) was identified. This deletion is characterized by the absence of a 2,153-bp fragment truncating genes mmpS6 and mmpL6 in “modern” strains of M. tuberculosis; however, this region is present in all other members of the M. tuberculosis complex (6).

In another study (83), M. tuberculosis H37Rv and M. bovis AF2122/97 genomic sequences were compared using blastn and MSPcrunch (84) software and visualized with the Artemis Comparison Tool (ACT) version 4.0 for Apple. The criteria used to select loci for further studies were that they were not IS6110 insertions, PE-PPE family genes, single-nucleotide polymorphisms, or previously published regions of difference. Four polymorphisms were found: 1) a deletion of Rv3479 specific to M. bovis; 2) that the rpfA gene is shortened to various extents in M. bovis; 3) an insertion in Rv0648 in M. bovis,; and 4) a duplication of lppA/B in M. bovis. Interestingly, lppA is also duplicated in M. tuberculosis CDC 1551.

Comparative genomics has given also some clues to understand the evolution of members of the complex. Thus, the analysis of the distribution of variable regions indicates that, contrary to the initial proposed scheme, M. tuberculosis did not descend from M. bovis; instead, both bacteria descended from a single common ancestor named “ancient” TB, which appears to be closely related to M. canetti, a member of the complex isolated from humans described recently. Both M. bovis and “modern” M. tuberculosis evolved from that common ancestor, as well as all other components (82) (Figure 6).

Figure 6

From Brosch et al. (82).

Identification of the different members of MTBC in the laboratory is difficult using standard methodologies; however, their correct differentiation has important influence in the treatment to be chosen and the consequent management of the patients. The development of comparative genomics has allowed the description of PCR-based schemes for such purposes. On the basis of variable distribution of insertion and deletions in the genomes of the several members, some flow charts were described for M. tuberculosis complex differentiation (80, 85).

Comparative Genomics between Members of MAC

Some few studies have been performed mainly focused on the detection of specific targets for identification of MAP, by comparison of its genome with that of other related members of MAC. The annotated sequence of M. avium subsp. avium strain 104 (provided by TIGR) was used initially to assemble a whole-genome DNA microarray representing the predicted coding sequences. Seventy-base-pair-long oligonucleotide probes were designed and synthesized for 4,158 of 4,480 predicted open reading frames (ORFs). Each probe was printed in duplicate onto microarray slides to permit genomic DNA comparison of M. avium subsp. avium strain 104 and the following strains: 1) M. avium subsp. paratuberculosis K10 (cow strain); 2) M. avium subsp. paratuberculosis LN20 (sheep strain); and 3) M. avium subsp. silvaticum 49884 (ATCC strain). Microarray comparison revealed 14 LSP regions that distinguish a single strain of M. avium subsp. avium (MAA) from other strains of MAP (86). These LSP regions encompass 572 genes, more than 700 Kb, that represents 13.5% of the MAA genome. Remarkably higher diversity was demonstrated in this complex compared with the genomic variability described among M. tuberculosis complex isolates (87). This could be in agreement with consideration of MAP and MAA as different subspecies within the species M. avium.

As part of this ongoing work to characterize M. avium complex organisms, sequenced genome of M. avium subsp. avium strain 104 (TIGR) and M. avium subsp. paratuberculosis strain K10 (GenBank accession number NC_002944) were compared using Artemis software for visualization of the M. avium subsp. paratuberculosis strain K10 genome and annotation of M. avium subsp. avium strain 104. Genome sequence comparison, via visual inspection in Artemis, revealed two types of LSPs: those present in the former but missing in the latter (LSP^As), and those only present in the latter (LSP^Ps). Three LSP^As and 17 LSP^Ps were found. The distribution of these LSPs were examined across a panel of M. avium complex isolates, revealing 1 LSP^A absent in all MAP isolates tested; then this absence was described as 100% specific of the MAP genome, and 7 LPS^Ps genomic regions present only in the analyzed MAP strains, and described as highly specific of MAP isolates; other 10 LPS^P regions were not specific of the MAP genome (86).

The analysis of a larger number of MAP isolates showed that these isolates have a high degree of genetic conservation, with no differences with the reference strain K10. Deletions detected by comparison with other members of the MAC are, as expected, associated with the presence of mobile genetic elements (88).

Comparative Genomics as a Tool for the Analysis of Mycobacterial Speciation

The pool of different mycobacterial genome projects currently in progress opens new possibilities for comparison between members of different species in the genus, a task still to be undertaken. LSPs, based on insertion/deletions and rearrangements of the genomes, are being considered the main mechanism that occurs during the evolution of the bacterial genomes. These changes are considered important in the separation of isolates in different genomic species and could putatively be studied by determining a gene’s location and its organization in the chromosome.

Recently, a new website (Microbesonline) has been described and organized for quick comparison of bacterial genomes (89). Only five mycobacterial genomes are included in that site thus far, three of them members of MTBC, plus MAP and M. leprae.

Using the facilities offered at that website (MicrobesOnline Comparative Genome Browser), a multispecies comparison of the location and gene organization of the available mycobacterial genomes was performed. An expected result was found when the M. leprae genome was analyzed; this genome showed major differences from all other mycobacterial genomes, differences that were scattered along the chromosome. As an exception, only short regions showed conserved gene organization in the M. leprae genome when compared with the equivalent region in other mycobacterial genomes. For example, the region corresponding to recN (ML1360 and Rv1696) shows gene conservation in about a 10-Kb length among all five compared mycobacterial genomes; and the region corresponding to dnaA (ML0001 and Rv0001) is also conserved along 12 Kb in all five genomes.

The genome of M. tuberculosis H37Rv was considered as reference for organization of the contained ORFs (from Rv0001 to Rv3924) and was theoretically divided into eight regions (containing approximately 500 ORFs each), taking the following ORFs as references: Rv0001 (dnaA); Rv0500 (proC); Rv1001 (arcA); Rv1522 (mmpL12); Rv2031 (acr/hspX); Rv2500 (fadE19); Rv3065 (emr); and Rv3504 (fadE26). Approximately 50 Kb were checked upstream and downstream from each of the previous genes. A genome multispecies browser was applied to each of the selected regions, and as a result, the eight genomic regions could be divided into three groups.

Group I regions were conserved in members of the MTBC as well as in MAP. This includes Rv0001, where genes related to chromosome replication and partitioning are located. Group II regions were conserved in members of the MTBC but were different than the corresponding region in MAP. This includes Rv0500 (this region encloses the icl gene, which has been related to dormancy in M. tuberculosis) and Rv3065 (this region includes components of the cluster nrd involved in DNA replication). Group III variable regions were conserved in all four mycobacteria, including members of the MTBC. This includes four of the remaining regions, such as Rv1522 (the mmpL12 gene thought to be involved in the fatty acid transport), Rv2031 (gene coding for the stress protein α-crystallin), Rv2500 (belonging to a family of genes associated with fatty acid degradation and whose components have more or less scattered distribution along the chromosome), and Rv3504 (several genes coding for members of the PE-PGRS family of proteins, which have been related to the pathogenesis of the genus, are located within this region). A curious result was that the region defined by the gene Rv1001 divided two genomic zones: one belongs to Group II, located in the corresponding downstream region, and the other to Group III, located in the corresponding upstream region.

The previous data are deeply biased because of the small number of species compared (actually only three clearly different species); however, as more mycobacterial genomes were included in this site, a similar approach can be used to help in the detection of genomic regions that putatively would participate in mycobacterial speciation.

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