Generalist and specialist species differ in the breadth of their ecological niche. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis Lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that Lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that while the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of Lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZAr). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZAr strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZAr strains, (iii) mutations with an unclear role found in less than 70% of PZAr strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZAr; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%.

Mutations at codon 315 of the katG gene were detected in 312 of 364 (85.7%) isoniazid-resistant Mycobacterium tuberculosis isolates. Seven of 52 (13.5%) isoniazid-resistant isolates with the wild-type Ser315 codon and 10 of 52 (19.2%) isoniazid-resistant isolates with a mutated katG allele had mutation ؊15C3T in the promoter of the mabA-inhA operon.Isoniazid (INH) together with rifampin (RIF) form the cornerstone of a short chemotherapy course for tuberculosis, and resistance to either drug hampers seriously the complete cure of patients (17). Mutations at several chromosome loci of Mycobacterium tuberculosis (fur-katG, mabA-inhA, oxyR-aphC, kasA, and ndh) are associated with resistance to INH (6,7,11,13). Mutations both at the Ser315 codon of katG, encoding catalase-peroxidase, the enzyme oxidizing INH (4), and at the regulatory region of the mabA-inhA operon, encoding a target of activated prodrug, enoyl-acyl carrier protein reductase (2), occur most frequently in INH-resistant isolates (9,11,13). In this study we searched for the most common mutations in katG and the mabA-inhA regulon of INH-resistant isolates to evaluate their significance for rapid drug resistance prediction in Lithuania.In total, 364 INH-resistant and 78 INH-susceptible M. tuberculosis isolates collected from different patients (93 female, 349 male; age range, 19 to 94 years) in the National Tuberculosis Reference Laboratory from 1998 to May 2002 were studied. New and previously treated tuberculosis cases were defined according to World Health Organization recommendations (19). Among INH-resistant isolates 157 were from new cases and 207 were from previously treated cases. Among INH-susceptible isolates 66 and 12 were from new and previously treated cases, respectively. Analysis of M. tuberculosis was carried out by standard procedures (5, 18). Drug susceptibility testing was done either by the proportion method on Löwenstein-Jensen medium or with BACTEC 460 (Becton Dickinson, Sparks, Md.). Critical drug concentrations were 0.2 g/ml for INH, 4 g/ml for streptomycin (STR), 40 g/ml for RIF, and 2 g/ml for ethambutol (EMB) in Löwenstein-Jensen medium and 0.1, 2, 2, and 2.5 g/ml for INH, STR, RIF, and EMB, respectively, with the BACTEC 460. DNA from M. tuberculosis was extracted as described elsewhere (15). The 264-bp katG fragment targeting codon 315 and the 248-bp fragment spanning the mabA-inhA regulon were amplified by using primers 5Ј-TGGAGCAGATGGGCTTGG and 5Ј-CAG TGGCCAGCATCGTCG (12) and primers 5Ј-CCTCGCTGC CCAGAAAGGGA and 5Ј-ATCCCCCGGTTTCCTCCGG (14), respectively. PCR products purified with silica powder were subjected to manual sequencing by using the CycleReader DNA sequencing kit (Fermentas, Vilnius, Lithuania). For the restriction fragment length polymorphism (RFLP) assay, katG-specific PCR products were digested with TauI or SatI (Fermentas) and analyzed in a 1.5% agarose gel. Both endonucleases possess the overlapping GC sequence at a single recognition site of the 315 triplet AGC, and mutations at these positions eliminate cle...