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

Fluoroquinolones are gaining increasing importance in the treatment of tuberculosis. The expression of MfpA, a member of the pentapeptide repeat family of proteins from Mycobacterium tuberculosis, causes resistance to ciprofloxacin and sparfloxacin. This protein binds to DNA gyrase and inhibits its activity. Its three-dimensional structure reveals a fold, which we have named the right-handed quadrilateral beta helix, that exhibits size, shape, and electrostatic similarity to B-form DNA. This represents a form of DNA mimicry and explains both its inhibitory effect on DNA gyrase and fluoroquinolone resistance resulting from the protein's expression in vivo.

The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United