We suggest that the evolution of the population structure of microbial pathogens is influenced by that of modern humans. Consequently, the timing of hallmark changes in bacterial genomes within the last 100,000 yr may be attempted by comparison with relevant human migrations. Here, we used a lineage within Mycobacterium tuberculosis, a Beijing genotype, as a model and compared its phylogeography with human demography and Y chromosome-based phylogeography. We hypothesize that two key events shaped the early history of the Beijing genotype: (1) its Upper Palaeolithic origin in the Homo sapiens sapiens K-M9 cluster in Central Asia, and (2) primary Neolithic dispersal of the secondary Beijing NTFϻIS6110 lineage by Proto-Sino-Tibetan farmers within east Asia (human O-M214/M122 haplogroup). The independent introductions of the Beijing strains from east Asia to northern Eurasia and South Africa were likely historically recent, whereas their differential dissemination within these areas has been influenced by demographic and climatic factors.[Supplemental material is available online at www.genome.org.]Intriguing clues about the history of a biological species can be derived from the study of the geographical distribution of the phylogenetic/genealogical lineages, in the approach known as "phylogeography" (Avise et al. 1987). The underlying assumption of human phylogeography is that there is a correspondence between the overall distribution of haplotypes and haplogroups and past human movements. The uniparentally inherited nonrecombining haploid mtDNA and the Y chromosome loci are particularly sensitive to the influences of drift, especially founder effect. Consequently these loci are suitable for assessing the origins of contemporary population diversity and provide context for paleontological hypothesis testing (Foley 1998). The mutation rate of the maternally transmitted mitochondrial genome is ∼10 times higher than that of nuclear DNA, which provides abundance of polymorphic sites but creates difficulties in reconstructing genealogies owing to repeated and reverse mutations. By contrast, the mutation rate of the paternally inherited nonrecombining portion of the Y chromosome (NRY) is comparable to that of nuclear DNA, which means that polymorphisms are more difficult to find but genealogies are easier to reconstruct. In addition, the greater length of the NRY DNA compared with mtDNA compensates in data analysis for its lower mutation rate (Cavalli-Sforza and Feldman 2003).The rarity of back and recurrent mutations in NRY contributes to the property of displaying the strongest geographic correlation and greatest diversity among, rather than within, populations. To date, NRY binary polymorphisms have been widely used to trace the origin and migration events of modern humans (Underhill et al. 2001). Here, we propose the hypothesis that NRY-based phylogeography of H. sapiens sapiens, offers a convenient spatiotemporal framework for inferring early primary dispersals of those human pathogens that are essentially (1) devo...

A total of 204 isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different patients in the northwestern region of Russia from 1996 to 2001 were screened by a PCR-restriction fragment length polymorphism (RFLP) assay. This assay uses HapII cleavage of an amplified fragment of the katG gene to detect the transversion 315AGC→ACC (Ser→Thr), which is associated with INH resistance. This analysis revealed a 93.6% prevalence of the katG S315T mutation in strains from patients with both newly and previously diagnosed cases of tuberculosis (TB). This mutation was not found in any of 57 INH-susceptible isolates included in the study. The specificity of the assay was 100%; all isolates that contained the S315T mutation were classified as resistant by a culture-based susceptibility testing method. The Beijing genotype, defined by IS6110-RFLP analysis and the spacer oligonucleotide typing (spoligotyping) method, was found in 60.3% of the INH-resistant strains studied. The katG S315T shift was more prevalent among Beijing genotype strains than among non-Beijing genotype strains: 97.8 versus 84.6%, respectively, for all isolates, including those from patients with new and previously diagnosed cases, isolated from 1999 to 2001 and 100.0 versus 86.5%, respectively, for isolates from patients with new cases isolated from 1996 to 2001. The design of this PCR-RFLP assay allows the rapid and unambiguous identification of the katG 315ACC mutant allele. The simplicity of the assay permits its implementation into routine practice in clinical microbiology laboratories in regions with a high incidence of TB where this mutation is predominant, including northwestern Russia

In this work, we studied the variation in the gyrA and gyrB genes in ofloxacin-and multidrug-resistant Mycobacterium tuberculosis strains circulating in northwest Russia. Comparison with spoligotyping data suggested that similar to the spread of multidrug-resistant tuberculosis, the spread of fluoroquinolone-resistant tuberculosis in Russia may be due, at least partly, to the prevalence of the Beijing genotype in a local population of M. tuberculosis.Fluoroquinolones (FQ) are potent second-line drugs recommended to treat multidrug-resistant tuberculosis. The main target of FQ, in Mycobacterium tuberculosis and other pathogens, is DNA gyrase, a type II topoisomerase composed of two A and two B subunits encoded by the gyrA and gyrB genes, respectively; mutations in the quinolone resistance-determining regions (QRDR) in these genes serve as a primary mechanism of the development of FQ resistance (1,7,10,20).FQ susceptibility testing is not performed in all regional laboratories in Russia, and data on FQ resistance rates are limited. In 2006, the rate of ofloxacin (OFL)-resistant M. tuberculosis strains was in the range of 1.1 to 1.6% for patients newly diagnosed with tuberculosis and 4.1 to 10.3% for previously treated patients in different regions of northwest Russia. Here, we report the results of the first population-based study of the molecular basis of FQ resistance in M. tuberculosis strains currently circulating in Russia.The bacteriology laboratory in the St. Petersburg Research Institute of Phthisiopulmonology serves as a reference center for northwest Russia. In the present study, we included all OFL-resistant strains isolated from March to November 2006 (see the table in the supplemental material); these strains were recovered from 48 patients among the total of 261 patients screened. The patients were epidemiologically unlinked and originated from different regions of the Russian Federation.Drug susceptibility testing was done by the absolute concentration method according to the guidelines of the Russian Ministry of Health (order no. 109 of 21 March 2003) by using recommended MIC breakpoints (in particular, 2 g/ml for OFL). All 48 strains were multidrug resistant (resistant to at least rifampin [RIF] and isoniazid [INH]), while 45 of them were resistant to at least INH, RIF, OFL, and kanamycin (see the table in the supplemental material) and were classified as extensively drug resistant (22).DNA extraction from Löwenstein-Jensen cultures, spoligotyping, and variable-number tandem repeat (VNTR) typing (using a 24-locus format and three hypervariable loci, QUB-3232, VNTR-3820, and VNTR-4120) were done as described previously (8,9,19). The gyrA QRDR was amplified and sequenced using the forward primer 5Ј-ACCGCAGCCACGCC AAGTCG and the reverse primer 5Ј-CCTGGCGAGCCGAA GTTGCC. The gyrB QRDR was amplified and sequenced using primers described by Takiff et al. (20). Mutations in the rpoB hot-spot region (codons 507 to 533) and katG S315T (AGCϾACC) were detected as described previously (12, 15). To minimize th...

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We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB.The spread of multidrug-resistant tuberculosis (MDR-TB) has increased worldwide and reached epidemic proportions in many countries. MDR Mycobacterium tuberculosis strains are considered those resistant to at least rifampin (RIF) and isoniazid. RIF is a key component of the World Health Organization DOTS (directly observed therapy, short course) regimen: because RIF monoresistance is extremely rare and development of isoniazid resistance usually precedes that to RIF, resistance to the latter is considered to be the MDR marker (8). It has been shown in many studies that RIF resistance in up to 95 to 98% of resistant strains is caused by mutations in the rpoB gene encoding the RNA polymerase ␤-subunit (20, 21). These mutations are generally described in the short 81-bp region in rpoB (RIF resistance-determining region: codons 507 to 533 [21]). In addition, RIF resistance may be caused by mutations in other parts of rpoB outside the hot spot region, such as codon 176 (146 [21]) in the N-terminal part (9) and codons 541 and 553 (18). Methods used so far to detect rpoB mutations associated with RIF-resistance were direct DNA sequencing, PCR-single-strand conformation polymorphism, heteroduplex mobility, dot spot, RNA-RNA mismatch, and some other assays (reviewed in references 4 and 25). Previously, we have described a multiplex allele-specific PCR (MAS-PCR) assay based on standard PCR and agarose gel electrophoresis to detect mutations in katG315 (15) and embB306 (14) in M. tuberculosis strains. A similar assay to detect rpsL43, rpoB531, and katG315 mutations was very recently published by Victor et al. (24). The MAS-PCR method in our design (14, 15) ...

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Parts of katG and rpoB from 27 RussianMycobacterium tuberculosis isolates were sequenced to detect mutations causing resistance to isoniazid (INH) and rifampin (RMP), respectively. All 24 INH-resistant isolates had a mutatedkatG, and 22 of them (91.7%) carried a mutation coding for a Ser315Thr shift. An rpoB mutation was noted for each of the 21 RMP-resistant isolates, with Ser531Leu being the most prevalent change encoded. Only two isolates had identical IS6110fingerprints.