Molecular characterisation of antibiotic resistance in Mycobacterium tuberculosis

Abstract: To counteract the emergence of multi-drug resistant tuberculosis (MDR-TB) a number of approaches have been proposed, amongst which are the sound usage of the present anti-TB drugs and the development of prompt and specific tools for the diagnosis of resistance. To examine the role of drugs in the treatment of resistant TB, we investigated the general crossresistance between kanamycin and amikacin, which has been reported in numerous studies. Forty clinical isolates were shown to be low-to intermediate resistant to kanamycin but fully sensitive to amikacin. Sequencing of the 16S rRNA gene, rrs, revealed no specific genotype correlating to this. We could however confirm the previously reported correlation between mutations at position 1400 and dually highly resistant strains. Since cross-resistance is not present in all strains, caution should thus be taken when extrapolating the results of susceptibility testing between the closely related drugs kanamycin and amikacin. A rapid detection of MDR-TB infections makes it possible to promptly change to a more effective treatment of the patient, which could shorten the patient s period of infectiousness and thus reduce the risk for additional new cases. We evaluated the usefulness of the commercially available hybridisation Line Probe Assay (INNO-LiPATM Rif.TB). We also developed an assay based on Pyrosequencing technology, for the identification of rpoB mutations, and thus the rapid detection of rifampicin resistance. Both methods detected mutations in all rifampicin resistant strains. Among the susceptible strains, the Pyrosequencing assay detected additional mutations whereas these could not be discriminated by LiPA. Since the M. tuberculosis Beijing family has been associated with major outbreaks and MDR-TB, we investigated whether rifampicin resistance-levels and rpoB mutations could be strain dependent. We studied 189 in vitro generated rifampicin resistant mutants, of which approximately half were of the Beijing family. There was no general difference in resistance-level or mutations between the two subsets of mutants. The two most common mutations found, irrespective of strain origin, were the Ser531Leu and His526Tyr, which reflect what is found among clinical isolates. Thus, the predominance of Beijing strains in terms of resistance and high prevalence in certain geographical areas are likely due to other factors than mutations in the rpoB gene. Lastly, we investigated the use of a sequencing assay directed to the pncA gene and sequences in its proximity, for the detection of pyrazinamide resistance. The phenotypic drug-susceptibility tests for the first-line agent pyrazinamide are cumbersome to use and known to be difficult to reproduce. Not only would a sequencing assay circumvent the obstacles associated with the phenotypic methods but also provide a shorter turnaround time. We identified mutations in all but one resistant strain. Among the susceptible strains, only a few mutations were found, and these were silent mutations. Thus, pncA sequencing seems to offer an attractive alternative to phenotypic tests. In the present studies, we have shown that DNA-based methods directed to rpoB and pncA correlate well to the phenotypic methods for the detection of rifampicin and pyrazinamide resistance. A hybridisation-based method would be of second choice as there is a risk for false positive readings. Thus, we recommend the use of sequencing assays that will detect and directly define mutations. Unfortunately, for aminoglycosidic resistance there is yet no clear correlation between mutations and resistance, and only highly resistant strains could be identified using our sequencing assay. More importantly however, in contrast to what has earlier been described, we showed that cross-resistance between amikacin and kanamycin is not a general rule. Therefore we propose that, if these two drugs are being considered for treatment, drug-susceptibility testing on both drugs should be conducted simultaneously. These studies show that molecular techniques not only offer tools for a rapid detection of drug resistance, but also increase our understanding of how resistance is developed.