Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations

Abstract

The emergence of multidrug-resistant strains ofMycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions ofgyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons ofgyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = 14) for which the ciprofloxacin MIC was >2 ,ug/ml. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of 1 in 107 to 108 at ciprofloxacin concentrations of 2 ,ug/ml, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.The resurgence of tuberculosis and its incidence in human immunodeficiency virus-positive populations in both developing countries and the industrialized world have been accompanied by the alarming emergence of virulent multidrugresistant tuberculosis (MDR-TB) strains in North American cities (7). Many of these strains have acquired resistance to almost all first-and second-line antituberculosis agents. For this reason, there is an increasing interest in the antimycobacterial actions of the fluoroquinolones (FQs). Against Mycobacterium tuberculosis, the FQs show moderate in vitro activity (4), with sparfloxacin (MIC, 0.25 to 0.5 ,ug/ml) perhaps being the most effective compound (17). The principal target of the quinolones is the DNA gyrase, a type II DNA topoisomerase that is composed of two A and two B subunits (30) encoded by gyrA and gyrB, respectively. Mutations in the putative FQbinding region of the A subunit have been found to confer high-level FQ resistance in several bacterial species (8,19,22,31,33). Other mutations that confer resistance to quinolones have been found in gyrB, in genes that lower the intracellular concentration of the drug (although these tend to confer lower-level resistance than do the gyrA mutations [32,34]), or

Detection of Multidrug Resistance in Mycobacterium tuberculosis

“…Some researchers have described mutations that caused amino acid substitutions but not drug resistance (30,33,39). In the present study, we identified several novel mutations that cause amino acid substitutions but do not confer drug resistance.…”

“…Mutations in the FQ resistancedetermining region (QRDR) in gyrA are responsible for resistance in at least 96% of FQ-resistant M. tuberculosis isolates (17,30,39). In the present study, we found one novel mutation, E21Q, in gyrA, and all isolates tested, except the H37Rv strain, contained this mutation.…”

“…We also identified a polymorphism, S95T, that is not associated with FQ resistance. The G88C, D89G, S91P, and D94A, -N, -H, or -Y mutations in gyrA have also been found in FQ-resistant isolates (4,30). These mutations are presumed to be located in the FQ-binding region (4,30).…”

“…Of STR-resistant isolates, 52% to 59% and 8% to 21% have mutations in rpsL, which encodes ribosomal protein S12, and rrs, which encodes 16S rRNA, respectively (17,19,39). Of FQ-resistant isolates, 75% to 94% have mutations in gyrA, which encodes the A subunit of DNA gyrase (17,30,39). …”

Rapid Diagnosis of Extensively Drug-Resistant Tuberculosis by Use of a Reverse Line Blot Hybridization Assay

“…Resistance to fluoroquinolones and second-line aminoglycosides is most frequently associated with mutations in gyrA and gyrB and in rrs, respectively. Studies have demonstrated that by targeting mutations in codons 90, 91, and 94 in gyrA, approximately 70 to 90% of all fluoroquinolone resistance can be accurately predicted (2,5,17). Similarly, mutations in the rrs gene, particularly the hotspot region from nucleotides 1401 to 1484, can accurately predict resistance to second-line aminoglycosides (10,16).…”