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

Due to the resurgence of tuberculosis and the emergence of multidrug-resistant strains, fluoroquinolones (FQ) are being used in selected tuberculosis patients, but FQ-resistant strains of Mycobacterium tuberculosis have rapidly begun to appear. The mechanisms involved in FQ resistance need to be elucidated if the effectiveness of this class of antibiotics is to be improved and prolonged. By using the rapid-growing Mycobacterium smegmatis as a model genetic system, a gene was selected that confers low-level FQ resistance when present on a multicopy plasmid. This gene, lfrA, encodes a putative membrane efflux pump of the major facilitator family, which appears to recognize the hydrophilic FQ, ethidium bromide, acridine, and some quaternary ammonium compounds. It is homologous to qacA from Staphylococcus aureus, tcmA, ofStreptomyces glaucescens, and actII and mmr, both from Streptomyces coelicoler. Increased expression of lfrA augments the appearance of subsequent mutations to higher-level FQ resistance.The worldwide reemergence of tuberculosis as a major public health problem has been accompanied by an ominous increase in multidrug resistant strains (1). This increase has stimulated an intense search for new antimycobacterial agents, but at present only one additional class of drugs, the fluoroquinolones (FQ), has been added to the traditional anti-tuberculosis armamentarium (2). Introduced into clinical practice in the 1980s, the FQ were initially active against many pathogens (3), but their use has been limited by the rapid appearance of resistance in a large percentage of clinical isolates, especially in Staphylococcus aureus and Pseudomonas aeruginosa (41,42). The therapeutic use of the FQ in tuberculosis began only within the past 3-4 years, and they are generally reserved for infections resistant to other agents. However, most FQ are only moderately active against the mycobacteria, and unfortunately, FQ-resistant (FQr) clinical isolates of Mycobacterium tuberculosis have already appeared (4,5). If more can be learned about what determines the effectiveness of a particular FQ against the mycobacteria and the mechanisms by which resistance develops, new agents or strategies may be designed that can prevent or circumvent this resistance.The principal targets of the FQ are bacterial type II topoisomerases, including both the bacterial DNA gyrase, an essential type II topoisomerase that introduces supercoils into the DNA chromosome (6), and the highly homologous topoisomerase IV, which deconcatenates the chromosome after DNA replication (7,8). Mutations in a particular region of gyrA, which encodes the gyrase A subunit, have been associated with moderate-to-high level [>5x minimal inhibitory concentration (MIC)] FQ resistance in many species of bacteria, including M. tuberculosis (5), and similar mutations have been found in the homologous region of topoisomerase IV (9, 10). Mutations conferring low-level resistance have also beenThe publication costs of this article were defrayed in part by page charge p...

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The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.