Mycobacterium bovis BCG, the only presently available vaccine against tuberculosis, was obtained from virulent M. bovis after serial passages in vitro. The vaccine strain retained at least some of its original virulence, as it persists in immune-competent hosts and occasionally may cause fatal disease in immunedeficient hosts. Mycobacterial persistence in vivo is thought to depend on anaerobic metabolism, an apparent paradox since all mycobacteria are obligate aerobes. Here we report that M. bovis BCG lacking anaerobic nitrate reductase (NarGHJI), an enzyme essential for nitrate respiration, failed to persist in the lungs, liver, and kidneys of immune-competent (BALB/c) mice. In immune-deficient (SCID) mice, however, bacilli caused chronic infection despite disruption of narG, even if growth of the mutant was severely impaired in lungs, liver, and kidneys. Persistence and growth of BCG in the spleens of either mouse strain appeared largely unaffected by lack of anaerobic nitrate reductase, indicating that the role of the enzyme in pathogenesis is tissue specific. These data suggest first that anaerobic nitrate reduction is essential for metabolism of M. bovis BCG in immune-competent but not immune-deficient mice and second that its role in mycobacterial disease is tissue specific, both of which are observations with important implications for pathogenesis of mycobacteria and vaccine development.Mycobacterium tuberculosis claims more human lives each year than any other bacterial pathogen. Mycobacterium bovis BCG, the only presently available vaccine against tuberculosis, belongs phylogenetically to the M. tuberculosis complex. In humans, M. bovis BCG, like M. tuberculosis, forms granulomas and abscesses in various tissues. Following vaccination in immune-competent individuals, M. bovis BCG may persist for extended periods (34). In immune-compromised individuals the vaccine strain may even lead to fatal disease (2,3,10,11,14,21,27,30,36,39).Mycobacteria become firmly established within host tissues, adapting their metabolism to the available source of carbohydrates, nitrogen, and energy (4). Although the acquisition of essential nutrients by mycobacteria is an area of considerable interest, our knowledge of bacterial metabolism throughout the course of infection remains rudimentary. A recent study revealed that metabolism of fatty acids serves as a source of carbohydrates and is required for persistence of M. tuberculosis in mice and activated macrophages (25). Nitrate, through nitrate respiration, could provide energy for bacterial metabolism in an anaerobic environment, because anaerobic nitrate reductase (NarGHJI) couples the reduction of nitrate (NO 3 ) to the generation of ATP by replacing oxygen as a terminal electron acceptor (29). Anaerobic nitrate reductase coding sequences (narGHJI) have been identified in both obligate aerobes such as Bacillus and Pseudomonas and facultative anaerobes such as Escherichia coli (5, 17, 28). However, a role of this enzyme in virulence was not established. In mycobacteria,...

In this study we used LightCycler PCR amplification and product detection by fluorescence resonance energy transfer probes to identify mycobacteria and differentiate between Mycobacterium tuberculosis complex, Mycobacterium avium, and other nontuberculous mycobacteria. Targeting the 16S rRNA gene, three different probes specific for mycobacteria, M. tuberculosis complex, and M. avium were constructed. As few as five genome copies of target nucleic acid were detected by the probes, illustrating the high sensitivity of the system. All 33 mycobacterial species tested but none of the closely related actinomycetes and other bacteria produced a specific fluorescence signal. A specificity of 100% was also demonstrated for the M. tuberculosis complex-specific probe and the M. avium-specific probe. Within 45 min, the LightCycler method correctly detected mycobacteria and specifically identified M. tuberculosis complex and M. avium without any post-PCR sample manipulation. In view of future clinical studies, we also constructed and tested an internal control which could be used to assure successful amplification and detection of mycobacteria. Monitoring of PCR inhibition will be essential for evaluation of this system for direct detection of mycobacteria in clinical specimens. Finally, we tested our system on sputum seeded with mycobacteria and were able to detect as few as 10 organisms. At present, this system is the fastest available method for identification and differentiation of mycobacteria from culturepositive specimens and offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnostic mycobacterial laboratory.

Mycobacterium tuberculosis, the major pathogen of human tuberculosis, predominantly affects the respiratory tract, whereas M. bovis, the major pathogen of tuberculosis in cattle, is typically found in extrapulmonary tuberculosis (5, 32). Attenuated M. bovis BCG, the only currently available vaccine against tuberculosis, has been administered to more than 3 billion people worldwide (17). It may be isolated from immunocompromised individuals, who might develop disseminated disease with the vaccine strain after vaccination with M. bovis BCG. M. africanum and M. microti are rarely encountered members of the M. tuberculosis complex (MTBC) (11,18,22). M. africanum causes human tuberculosis in certain regions of tropical Africa. M. microti causes naturally acquired generalized tuberculosis in voles and produces local lesions in guinea pigs, rabbits, and calves and has been described very occasionally as a cause of infection in human immunodeficiency virus-positive patients.Nitrate reductase activity is a widely used phenotypic trait to differentiate between M. tuberculosis, which rapidly accumulates nitrite from nitrate, and other members of the MTBC (18). In other pathogenic and environmental bacteria, at least three different nitrate reductases have been found, one of which is the respiratory nitrate reductase encoded by narGHJI (19). The membrane-bound complex consists of NarG, -H, and -I, with NarG being the catalytic subunit, whereas NarJ is required for the assembly of the enzyme. Expression of narGHJI is typically induced under anaerobic conditions (20). Previous studies on mycobacterial nitrate reduction were limited to its role in classification and identification of the genus Mycobacterium (2, 6, 28, 29). However, recent reports revived interest in a possible role for enzymes involved in nitrate metabolism during infection with M. tuberculosis or M. bovis BCG (4, 12, 30). We provided evidence that M. bovis BCG weakly accumulates nitrite from nitrate under strictly anaerobic conditions, that this activity is mediated by narGHJI, and that a mutant of M. bovis BCG with a partial deletion of the narG gene was attenuated in mice, linking anaerobic nitrate reduction to mycobacterial pathogenesis (7,31).In the present study, we generated targeted deletion of narG to compare the role of narGHJI in nitrite accumulation by M. tuberculosis, M. bovis (non-BCG [referred to here as simply "M. bovis"]), and M. bovis BCG. Analysis of the promoter region of narGHJI from either species revealed a single nucleotide polymorphism that separated M. tuberculosis from the bovine mycobacteria. We tested a variety of different strains within the MTBC by using LightCycler technology and showed that this single nucleotide polymorphism was specific for M. tuberculosis, thus allowing rapid identification of M. tuberculosis. For further differentiation within the MTBC based on LightCycler technology, we adapted analysis of the oxyR polymorphism, which is specific for the bovine mycobacteria, and the region of differences 1 (RD1) polymorphi...

The recent publication of the genome sequence of Mycobacterium bovis showed >99.95% identity to M. tuberculosis. No genes unique to M. bovis were found. Instead numerous single-nucleotide polymorphisms (SNPs) were identified. This has led to the hypothesis that differential gene expression due to SNPs might explain the differences between the human and bovine tubercle bacilli. One phenotypic distinction between M. tuberculosis and M. bovis is nitrate reduction, which not only is an essential diagnostic tool but also contributes to mycobacterial pathogenesis. We previously showed that narGHJI encodes a nitrate reductase in both M. tuberculosis and M. bovis and that NarGHJI-mediated nitrate reductase activity was substantially higher in the human tubercle bacillus. In the present study we used a genetic approach to demonstrate that an SNP within the promoter of the nitrate reductase gene cluster narGHJI is responsible for the different nitrate reductase activity of M. tuberculosis and M. bovis. This is the first example of an SNP that leads to differential gene expression between the human and bovine tubercle bacilli.Mycobacterium tuberculosis infects one-third of the world's population, with a fatality rate higher than that for any other bacterial organism. Mycobacterium bovis, which causes tuberculosis in a range of animal species, generates worldwide annual losses to agriculture of $3 billion. Both fall within the M. tuberculosis complex (TBC), a group of closely related mycobacteria including M. bovis BCG, a live attenuated vaccine strain against tuberculosis, M. africanum, and M. microti (14).Comparative genome analyses has shown that M. bovis does not have any unique genes compared to M. tuberculosis. Instead, there are numerous single-nucleotide polymorphisms SNPs that distinguish M. bovis from M. tuberculosis (11). Therefore, it has been suggested that SNPs are responsible for the differences between the human and the bovine tubercle bacilli. One such difference is nitrate reduction leading to accumulation of nitrite. M. tuberculosis rapidly reduces nitrate (23). We provided evidence that M. bovis also accumulates nitrite but at a much lower rate. We also found that nitrate reduction leading to accumulation of nitrite in M. tuberculosis and M. bovis is mediated by the same narGHJI-encoded nitrate reductase and asked why nitrite accumulation in M. tuberculosis was so much stronger than in the bovine mycobacteria (21).Therefore, we compared the narGHJI promoter of M. tuberculosis and M. bovis in both laboratory and clinical strains and found that M. tuberculosis carries a thymine residue at nucleotide Ϫ215 prior to the start codon of narG whereas M. bovis carries a cytosine residue at this position. Interestingly, M. bovis BCG and other members of the TBC such as M. africanum and M. microti also contained a cytosine residue at this position (21). Comparing the available genome sequences of M. tuberculosis and M. bovis, we found additional SNPs within the coding sequences of narGHJI, which could account for t...

LytR-cpsA-Psr (LCP) domain containing proteins fulfil important functions in bacterial cell wall synthesis. In Mycobacterium tuberculosis complex (Mtbc) strains, the causative agents of tuberculosis (TB), the genes Rv3484 and Rv3267 encode for LCP proteins which are putatively involved in arabinogalactan transfer to peptidoglycan. To evaluate the significance of Rv3484 for Mtbc virulence, we generated a deletion mutant in the Mtbc strain H37Rv and studied its survival in mice upon aerosol infection. The deletion mutant failed to establish infection demonstrating that Rv3484 is essential for growth in mice. Following an initial phase of marginal replication in the lungs until day 21, the Rv3484 deletion mutant was almost eliminated by day 180 post-infectionem. Interestingly, the mutant also showed higher levels of resistance to meropenem/clavulanate and lysozyme, both targeting peptidoglycan structure. We conclude that Rv3484 is essential for Mtbc virulence in vivo where its loss of function cannot be compensated by Rv3267.