We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of the M. tuberculosis samples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting that M. tuberculosis first arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction of M. tuberculosis into these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured by G ST . Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences of M. tuberculosis.Compared to many bacterial species, Mycobacterium tuberculosis harbors relatively little genetic diversity (21, 34, 37); however, there is increasing evidence that the interstrain variation that exists is biologically significant. Clinical M. tuberculosis isolates have variable gene expression profiles (25) and have different numbers of genes deleted from their chromosome (32). In animal models, M. tuberculosis appears to engender a range of immune responses and variable degrees of virulence depending on the infecting strain (5,7,47,55). In human infections, molecular epidemiological studies have suggested that certain M. tuberculosis types, identified by DNA fingerprinting, can be especially prone to drug resistance acquisition (17, 59, 65) or to global dissemination (3, 9, 27, 40, 66,

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drugresistant phenotypes.Isoniazid (INH) is one of the most effective and specific agents for the treatment of infections with Mycobacterium tuberculosis. INH is the cornerstone of treatment for drug-susceptible tuberculosis, and it is also widely used to treat latent M. tuberculosis infections. Recent increases in INH-resistant (INH r ) and multidrug-resistant (MDR) tuberculosis are jeopardizing the continued utility of this drug (13, 61). Furthermore, the development of INH resistance is a common first step in the evolution to MDR (11). Thus, there has been considerable interest in identifying the molecular basis of INH resistance in clinical M. tuberculosis isolates.INH is a prodrug that requires activation by the catalaseperoxidase enzyme encoded by the katG gene (65). Activated INH appears to disrupt the synthesis of essential mycolic acids by inhibiting the NADH-dependent enoyl-ACP reductase enzyme encoded by inhA (45). INH resistance is likely to arise

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Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P ‫؍‬ 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P ‫؍‬ 0.008) and katG315 (odds ratio, 1.99; P ‫؍‬ 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.The antibiotic ethambutol (EMB) appears to inhibit the growth of both Mycobacterium tuberculosis and Mycobacterium smegmatis by blocking the synthesis of arabinogalactan. Arabinogalactan biosynthesis is dependent on the activity of the embABC gene cluster, which encodes the arabinotransferases that mediate the polymerization of arabinose into arabinan. Several lines of evidence suggest that EMB exerts its toxic effect on mycobacteria by inhibiting the embABC-encoded proteins (26,27,34,35,46), and mutations in embABC also appear to play a key role in the development of EMB resistance in both M. tuberculosis and M. smegmatis (34). Associations between EMB resistance and mutations in embA, embB, and embC have been reported in clinical strains of M. tuberculosis (41), and mutations in codon 306 of the embB gene (embB306) in M. tuberculosis are seen in approximately 50% of all EMB-* Corresponding author. Mailing address: