Isoniazid (INH) is one of the primary chemotherapeutic and prophylactic drugs used against Mycobacterium tuberculosis, the causative agent of tuberculosis, which remains the leading single cause of death due to an infectious agent throughout the world. Recent studies indicate that the median rate of primary resistance to INH is 7.3% (range, 1.5 to 32%) and that the rates of acquired resistance range from 5.3 to 70% globally (9, 45). The overall rate of resistance to INH is 8.4% in the United States and has remained relatively stable in the last decade (23). Global reports of clusters of tuberculosis cases caused by drug-resistant strains together with the emergence and dissemination of multidrug-resistant tuberculosis have underscored the need for research into the mechanisms of drug resistance and the design of more effective antituberculous agents. Despite the use of INH for several decades, the molecular basis for its bactericidal action and the mechanisms by which INH resistance evolves in M. tuberculosis are only beginning to be understood.INH has a simple chemical structure consisting of a hydrazide group attached to a pyridine ring, but its mode of action is very complex (8). It is proposed that INH enters M. tuberculosis as a prodrug by passive diffusion and is activated by catalase-peroxidase, encoded by katG, to generate free radicals, which then attack multiple targets in the cells (6). Recent studies have shown that an NADH-dependent enoyl acyl carrier protein (ACP) reductase, encoded by inhA, and a ␤-ketoacyl ACP synthase, encoded by kasA, are two potential intracellular enzymatic targets for activated INH; and both of these enzymes are involved in the biosynthesis of mycolic acids (4,19,20). Resistance-associated amino acid substitutions have been identified in the katG, inhA, and kasA genes of INHresistant clinical isolates of M. tuberculosis (7,20,24,26,29). In addition, mutations in the oxyR-ahpC intergenic region have been identified in INH-resistant isolates (36). Additional genetic and biochemical studies have shown that certain promoter mutations of alkylhydroperoxide reductase, encoded by ahpC, in INH-resistant isolates result in overexpression of ahpC as a compensatory mechanism for the loss of catalase activity due to katG mutations (15, 32). Recently, missense mutations were identified in ndh, a gene encoding NADH dehydrogenase, which is an essential respiratory chain enzyme that regulates the NADH/NAD ϩ ratio in cells (18,22). The molecular mechanism by which mutations in ndh confer INH resistance in M. tuberculosis is poorly understood. In addition, low-level INH resistance in mycobacteria has been shown to be

Ethambutol (EMB) is a central component of drug regimens used worldwide for the treatment of tuberculosis. To gain insight into the molecular genetic basis of EMB resistance, approximately 2 Mb of five chromosomal regions with 12 genes in 75 epidemiologically unassociated EMB-resistant and 33 EMB-susceptibleMycobacterium tuberculosis strains isolated from human patients were sequenced. Seventy-six percent of EMBresistant organisms had an amino acid replacement or other molecular change not found in EMB-susceptible strains. Thirty-eight (51%) EMB-resistant isolates had a resistance-associated mutation in only 1 of the 12 genes sequenced. Nineteen EMB-resistant isolates had resistance-associated nucleotide changes that conferred amino acid replacements or upstream potential regulatory region mutations in two or more genes. Most isolates (68%) with resistance-associated mutations in a single gene had nucleotide changes in embB, a gene encoding an arabinosyltransferase involved in cell wall biosynthesis. The majority of these mutations resulted in amino acid replacements at position 306 or 406 of EmbB. Resistance-associated mutations were also identified in several genes recently shown to be upregulated in response to exposure of M. tuberculosis to EMB in vitro, including genes in the iniA operon. Approximately one-fourth of the organisms studied lacked mutations inferred to participate in EMB resistance, a result indicating that one or more genes that mediate resistance to this drug remain to be discovered. Taken together, the results indicate that there are multiple molecular pathways to the EMB resistance phenotype. Ethambutol [EMB; (S, S)-2,2Ј-(ethylenediimino)di-1-buta-nol] is used worldwide as one of the primary antituberculosis agents. The mechanism of action and the molecular genetic basis of resistance to EMB are not fully understood. Only the dextro isomer of EMB is biologically active, an observation consistent with the idea that the drug binds to a specific cellular target (7, 37). Several studies have implicated membrane-associated arabinosyltransferases as targets for EMB (1,5,20,22). These enzymes are well conserved in mycobacteria and are involved in the biosynthesis of arabinan, a component of arabinogalactan present in cell walls (6,8,17,33,34,39). Inhibition of arabinan synthesis leads to accumulation of mycolic acids and eventually to cell death.Three contiguous genes encoding arabinosyltransferases and designated embC, embA, and embB have been identified in Mycobacterium tuberculosis (35). The proteins encoded by these genes are about 65% identical to each other. Previous studies based on limited sequencing of the 10-kb region containing the embCAB genes have identified mutations that result in replacement of amino acid residues and are found only in EMB-resistant organisms cultured from humans. The most commonly affected amino acid was Met306 of EmbB. For example, Sreevatsan et al. (31) identified five distinct mutant codons that resulted in replacement of wild-type Met306 with Ile, Leu, or Va...

To test this idea, a new nonpolar mutagenesis method employing a spectinomycin resistance cassette was used to inactivate the sic gene in an M1 GAS strain. The isogenic Sic-negative mutant strain was significantly (P < 0.019) impaired in ability to colonize the mouse mucosal surface after intranasal infection. These results support the hypothesis that the predominance of M1 strains in human infections is related, in part, to a Sic-mediated enhanced colonization ability.

Serotype M1 group A Streptococcus strains cause epidemic waves of human infections long thought to be mono- or pauciclonal. The gene encoding an extracellular group A Streptococcus protein (streptococcal inhibitor of complement) that inhibits human complement was sequenced in 1,132 M1 strains recovered from population-based surveillance of infections in Canada, Finland and the United States. Epidemic waves are composed of strains expressing a remarkably heterogeneous array of variants of streptococcal inhibitor of complement that arise very rapidly by natural selection on mucosal surfaces. Thus, our results enhance the understanding of pathogen population dynamics in epidemic waves and infectious disease reemergence.

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