Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB , katG , embB , rrs , gyrA , and the promoter regions of inhA and eis . The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis . In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis . The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis .

BackgroundThe P27-P55 (lprG-Rv1410c) operon is crucial for the survival of Mycobacterium tuberculosis, the causative agent of human tuberculosis, during infection in mice. P55 encodes an efflux pump that has been shown to provide Mycobacterium smegmatis and Mycobacterium bovis BCG with resistance to several drugs, while P27 encodes a mannosylated glycoprotein previously described as an antigen that modulates the immune response against mycobacteria. The objective of this study was to determine the individual contribution of the proteins encoded in the P27-P55 operon to the resistance to toxic compounds and to the cell wall integrity of M. tuberculosis.MethodIn order to test the susceptibility of a mutant of M. tuberculosis H37Rv in the P27-P55 operon to malachite green, sodium dodecyl sulfate, ethidium bromide, and first-line antituberculosis drugs, this strain together with the wild type strain and a set of complemented strains were cultivated in the presence and in the absence of these drugs. In addition, the malachite green decolorization rate of each strain was obtained from decolorization curves of malachite green in PBS containing bacterial suspensions.ResultsThe mutant strain decolorized malachite green faster than the wild type strain and was hypersensitive to both malachite green and ethidium bromide, and more susceptible to the first-line antituberculosis drugs: isoniazid and ethambutol. The pump inhibitor reserpine reversed M. tuberculosis resistance to ethidium bromide. These results suggest that P27-P55 functions through an efflux-pump like mechanism. In addition, deletion of the P27-P55 operon made M. tuberculosis susceptible to sodium dodecyl sulfate, suggesting that the lack of both proteins causes alterations in the cell wall permeability of the bacterium. Importantly, both P27 and P55 are required to restore the wild type phenotypes in the mutant.ConclusionsThe results clearly indicate that P27 and P55 are functionally connected in processes that involve the preservation of the cell wall and the transport of toxic compounds away from the cells.

Bovine tuberculosis is caused by Mycobacterium bovis, a mycobacterium highly similar to M. tuberculosis that belongs to the M. tuberculosis complex. The main host of M. bovis is cattle but it also affects many other mammalians including humans. Tuberculosis in humans caused by either M. bovis or M. tuberculosis is clinically hard to distinguish. During 2004-2005, samples from 448 patients with diagnosis of TB were collected from different regions of Argentina. The PRA technique identified 400 isolates with representative patterns of mycobacterium. The predominant ones were the M. tuberculosis complex, the M. avium-M. intracellulare complex and M. gordonae. Samples with M. tuberculosis complex PRA restriction profiles were analyzed with a multiplex PCR to differentiate between M. tuberculosis and M. bovis. Multiplex PCR identified nine M. bovis. The results allowed the possibility to establish that 2% of pulmonary tuberculosis was due to M. bovis. Isolates of M. bovis from humans were examined using spoligotyping. These isolates presented five different spoligotypes. The main spoligotype was also the most frequently one found in cattle. The remaining human spoligotypes (grouped in clusters) are occasionally found in cattle. Variable number tandem repeat (VNTR) analysis identified five different patterns. By combining the results of spoligotyping and VNTR analysis, we were able to differentiate seven M. bovis isolates. The remaining two M. bovis samples showed the same spoligotype and VNTR profile and belonged to household contacts. An MDR-M. bovis was isolated from the samples of these household contacts. The identification of two epidemiologically linked cases of human M. bovis infection suggests person-to-person transmission of an MDR-M. bovis.

Results demonstrate that the CRI assay is an accurate method for the rapid detection of XDR Mycobacterium tuberculosis. The CRI assay is faster than the conventional drug susceptibility testing method using solid medium, has the same turnaround time as the BACTEC MGIT 960 system, but is less expensive, and could be an adequate method for low-income countries.

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