Rapid Diagnosis of Tuberculosis by Real-Time High-Resolution Imaging of Mycobacterium tuberculosis Colonies.

Culture remains the cornerstone of diagnosis for pulmonary tuberculosis, but the fastidiousness of Mycobacterium tuberculosis may delay culture-based diagnosis for weeks. We evaluated the performance of real-time high-resolution imaging for the rapid detection of M. tuberculosis colonies growing on a solid medium. A total of 50 clinical specimens, including 42 sputum specimens, 4 stool specimens, 2 bronchoalveolar lavage fluid specimens, and 2 bronchial aspirate fluid specimens were prospectively inoculated into (i) a commercially available Middlebrook broth and evaluated for mycobacterial growth indirectly detected by measuring oxygen consumption (standard protocol) and (ii) a home-made solid medium incubated in an incubator featuring real-time high-resolution imaging of colonies (real-time protocol). Isolates were identified by Ziehl-Neelsen staining and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Use of the standard protocol yielded 14/50 (28%) M. tuberculosis isolates, which is not significantly different from the 13/50 (26%) M. tuberculosis isolates found using the real-time protocol (P = 1.00 by Fisher's exact test), and the contamination rate of 1/50 (2%) was not significantly different from the contamination rate of 2/50 (4%) using the real-time protocol (P = 1.00). The real-time imaging protocol showed a 4.4-fold reduction in time to detection, 82 ± 54 h versus 360 ± 142 h (P < 0.05). These preliminary data give the proof of concept that real-time high-resolution imaging of M. tuberculosis colonies is a new technology that shortens the time to growth detection and the laboratory diagnosis of pulmonary tuberculosis.

Long-term survival of tuberculosis complex mycobacteria in soil.

While there is evidence for the persistence of Mycobacterium bovis in soil, there are no reports for the other Mycobacterium tuberculosis complex (MTC) mycobacteria. Here, soil was inoculated with 10(8) c.f.u. g(-1) M. tuberculosis, M. bovis and M. canettii and subcultured monthly for 12 months. The pathogenicity of mycobacterial colonies, identified by using matrix-assisted laser desorption/ionization time of flight mass spectrometry, was assessed in a mouse model. Moreover, mice were fed with food that contained 16.7% M. tuberculosis-contaminated soil. The three tested MTC species survived in soil for 12 months with a final inoculum of 2 × 10(3) c.f.u. g(-1) for M. tuberculosis, 150 c.f.u. g(-1) for M. bovis and 2 × 10(4) c.f.u. g(-1) for M. canettii. In an experiment that included negative controls, all (5/5) mice inoculated with such M. tuberculosis and M. canettii developed 0.03-0.3 granulomas mm(-2) in their lungs and spleen and grew mycobacteria; five mice that were inoculated with M. bovis from soil did not develop granulomas but grew mycobacteria. Furthermore, 0.2-0.4 granulomas mm(-2) were observed in the lungs and spleen of 3/5 mice fed with M. tuberculosis-contaminated soil in the presence of two negative control mice. M. tuberculosis grew in the stomach, intestine, spleen and lung in 5/5 challenged mice, whereas the negative controls remained M. tuberculosis-free (P = 0.008, Fisher exact test). This study provides clear evidence that MTC mycobacteria survive in soil, and that M. tuberculosis remains virulent while in the soil, outside its hosts, for extended periods of time.

Magnetic bead protocol for culturing Mycobacterium tuberculosis from sputum specimens.

A magnetic bead protocol and a standard centrifugation protocol yielded Mycobacterium tuberculosis in 40/50 sputum specimens in 12 ± 1 days and 11 ± 2 days, respectively (P > 0.05 by Student's t test). Manipulation took 35 ± 5 min and 45 ± 10 min, respectively (P < 0.05). The magnetic bead protocol could advantageously replace centrifugation for culturing M. tuberculosis from sputum.

Dramatic reduction of culture time of Mycobacterium tuberculosis.

Mycobacterium tuberculosis culture, a critical technique for routine diagnosis of tuberculosis, takes more than two weeks. Here, step-by-step improvements in the protocol including a new medium, microaerophlic atmosphere or ascorbic-acid supplement and autofluorescence detection dramatically shortened this delay. In the best case, primary culture and rifampicin susceptibility testing were achieved in 72 hours when specimens were inoculated directly on the medium supplemented by antibiotic at the beginning of the culture.

Non-human sources of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a successful pathogen responsible for the vast majority of deadly tuberculosis cases in humans. It rests in a dormant form in contaminated people who constitute the reservoir with airborne interhuman transmission during pulmonary tuberculosis. M. tuberculosis is therefore regarded majoritary as a human pathogen. Here, we review the evidence for anthroponotic M. tuberculosis infection in non-human primates, other mammals and psittacines. Some infected animals may be sources for zoonotic tuberculosis caused by M. tuberculosis, with wild life trade and zoos being amplifying factors. Moreover, living animals and cadavers can scatter M. tuberculosis in the environment where it could survive for extended periods of time in soil where amoebae could play a role. Although marginal in the epidemiology of human tuberculosis, these data indicate that M. tuberculosis is not uniquely adapted to humans.

Cultivation of the first mesophilic representative ("mesotoga") within the order Thermotogales.

Cultivated members of the order Thermotogales comprise only thermophilic to hyperthermophilic anaerobic microorganisms. However, based on molecular studies, the existence of mesophilic members ("mesotoga") within this order has been postulated but has not been demonstrated by cultural approaches so far. A "mesotoga" (strain PhosAc3) that belonged to an uncultivated lineage distantly related to the thermophilic Kosmotoga genus has now been cultivated in axenic culture. It grew between 30°C and 50°C (optimum 40°C) and oxidized lactate using elemental sulphur as a terminal electron acceptor. Further genomic and physiological characterization of strain PhosAc3 will be important not only for understanding bacterial adaptation to high and moderate temperatures at small evolutionary scales, but also because "mesotoga" might play a crucial ecological role in ecosystems polluted by aromatic compounds.