Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India.

Background & objectives: Drug resistance surveillance offers useful information on trends of drug resistance and the efficacy of control measures. Studies and reports of drug-resistant mutations and phenotypic assays thus become important. This study was conducted to investigate the molecular characteristics of ofloxacin (OFX)-resistant, multidrug-resistant tuberculosis (MDR-TB) isolates from different geographical regions of India and their association with strains of different genotypes. Further, the nitrate reductase assay (NRA) was tested against Mycobacteria Growth Indicator Tube(MGIT) for the determination of OFX resistance as an alternative and cost-effective method. Methods: A total of 116 Mycobacterium tuberculosis isolates were used to assess the mutations in the gyrA, gyrB genes and resistance levels to OFX. Mutational analysis in gyrA and gyrB genes and genotype analysis of M. tuberculosis isolates was done by gene-specific polymerase chain reaction (PCR) followed by DNA sequencing and spoligotyping, respectively. Results: Three (6.25%), 12 (44.44%) and 12 (29.27%) MDR-TB isolates from western, northern and southern India, respectively, were found to be OFX-resistant MDR-TB isolates. OFX resistance was observed to be significantly higher in MDR-TB cases for all study regions. Beijing genotypes from northern India were observed to be associated with OFX-resistant MDR-TB cases (P <0.05). Among 35 (30.15%) phenotypically OFX-resistant isolates, 22 (62.86%) had mutations in the gyrA gene and two (5.71%) isolates had mutations in the gyrB gene. Interpretation & conclusions: These results caution against the PCR-based prediction of OFX resistance patterns and highlight the need for searching other genetic loci for the detection of mutations conferring resistance to OFX in M. tuberculosis. Our study also showed the usefulness of NRA as an alternative method to detect OFX resistance.

PknB remains an essential and a conserved target for drug development in susceptible and MDR strains of M. Tuberculosis.

BACKGROUND: The Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India. METHODS: A total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations. RESULTS: The sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding. CONCLUSIONS: Hence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study says that it has no significant effect on the binding and hence the activity of the protein.