Takayuki Wada

Dual-Probe Assay for Rapid Detection of Drug-Resistant Mycobacterium tuberculosis by Real-Time PCR

ABSTRACT Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.

Genetic diversity of the Mycobacterium tuberculosis Beijing family in East Asia revealed through refined population structure analysis

The Beijing/W family is the endemic lineage of Mycobacterium tuberculosis in East Asia: it has disseminated worldwide. To elucidate its genetic diversity in Japan, phylogenetic reconstruction was performed using 403 M. tuberculosis Beijing family clinical isolates. Variable number of tandem repeats analysis revealed the strains from Japan to be dispersed mainly among five subgroups in a phylogenetic tree. Interestingly, the genotypes of the strains from China and Mongolia were restricted mainly to a single branch; they exhibited high clonality. IS6110 insertion in the NTF region was also analyzed. The majority (78.6%) of Japanese isolates belonged to the ancient sublineage. The modern Beijing strains were observed to correspond to the branch containing the foreign strains, although the ancient Beijing strains were dispersed among the trees other branches. Our results reflect the singular genetic diversity and the epidemiological pattern of Beijing M. tuberculosis in Japan.

Population Structure Analysis of the Mycobacterium tuberculosis Beijing Family Indicates an Association between Certain Sublineages and Multidrug Resistance

ABSTRACT Our population-based study of the Mycobacterium tuberculosis Beijing family examined the frequency of occurrence of each sublineage of this family, classified by using 10 synonymous single-nucleotide polymorphisms. The results revealed the overabundance of two evolutionary sublineages in a population of multidrug-resistant and extensively drug-resistant tuberculosis bacteria.

Genetic Diversity and Transmission Characteristics of Beijing Family Strains of Mycobacterium tuberculosis in Peru

Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs). They were also subjected to variable number tandem-repeat (VNTR) typing using an international standard set of 15 loci (15-MIRU-VNTR) plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10), which belongs to a modern subfamily of Beijing strains (178/198, 89.9%). Twelve strains belonged to the ancient subfamily (ST3 [n = 3], ST25 [n = 1], ST19 [n = 8]). Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3%) and a high recent transmission index (RTIn−1 = 0.707). These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999.

Population Structure Dynamics of Mycobacterium tuberculosis Beijing Strains during Past Decades in Japan

ABSTRACT We used 909 strains to compare the population structures of the Mycobacterium tuberculosis Beijing family between different birth-year cohorts in Japan. The results revealed that the spread of a modern sublineage that has high transmissibility is currently increasing, while the spread of an ancient sublineage, STK, has significantly decreased in younger generations.

High transmissibility of the modern Beijing Mycobacterium tuberculosis in homeless patients of Japan

A population-based study of Mycobacterium tuberculosis isolated from homeless tuberculosis patients was performed during 2002-2004 in Osaka City, Japan. The data show that the ancient Beijing subfamily was predominant, whereas clustered isolates based on refined variable number of tandem repeats genotyping (19 loci) mainly belonged to the modern Beijing subfamily, suggesting its increased transmissibility.

Phylogeographical particularity of the Mycobacterium tuberculosis Beijing family in South Korea based on international comparison with surrounding countries

To understand the domestic population structure of Mycobacterium tuberculosis clinical isolates in the Republic of Korea, we genotypically analysed 80 isolates obtained from various geographical origins in the country. Of these, 64 (80.0 %) isolates were identified as Beijing family strains. It is particularly interesting that their phylogenetic classification, based on the ancient/modern separation and the presence/absence of the genomic region RD181, revealed a majority of the ancient (RD181+) subfamily in the population. The 15 loci of variable number of tandem repeat(s) of mycobacterial interspersed repetitive units (15-MIRU-VNTR) were also analysed. Combination with the previous VNTR data reported from surrounding countries revealed that the topology of the minimum spanning tree was linked tightly not to the geographical origins of the patients but to the phylogenetic characteristics of the isolates. These results show that the phylogeographical distribution of the M. tuberculosis Beijing family around far-eastern Asia could be estimated using international accumulation and comparison of VNTR genotyping data.

Allelic diversity of variable number of tandem repeats provides phylogenetic clues regarding the Mycobacterium tuberculosis Beijing family

The Beijing family is the putative hypervirulent lineage of Mycobacterium tuberculosis that has been endemic in East Asia and has disseminated worldwide. The genetic population structure of Beijing family strains with regard to Japan is notable in its high diversity and dominance of the ancestral sublineage, in contrast to the modern sublineage found worldwide. Therefore, it is expected to be a suitable population model for investigating the microevolutionary process of the lineage. Variable number of tandem repeats (VNTR) has become a reliable genotyping method for M. tuberculosis, but its dynamics in the phylogenetic process remains unclear. Using 355 clinical Beijing family isolates in Japan, genetic traits, including VNTR, were analyzed and subjected to minimum spanning tree (MST) reconstruction. In the results, the topology of the tree was tightly related to other genotypic characters. We also found that some VNTR alleles were specific to each sublineage and provided clues for reconstructing a valid MST topology for the population. It is suggested that VNTR typing can elucidate genetic markers representing the phylogenetic classification in the lineage.

Evolutionary robust SNPs reveal the misclassification of Mycobacterium tuberculosis Beijing family strains into sublineages.

Genotypic classification in Mycobacterium tuberculosis has greatly contributed to the comprehension of phylogenetic and population genetic relationships. It is, therefore, necessary to verify the robustness of the genetic markers for phylogenetic classification. In this study, we report some examples of homoplasy for two molecular markers, the IS6110 insertion at the NTF region, and a single nucleotide polymorphism (SNP) at locus 909166, through genotyping of 1054 Beijing family strains. Our data revealed that a small fraction of strains traditionally classified into modern sublineages by IS6110 insertion at NTF actually belong to an ancient sublineage. We also proved that the robustness of branches in the evolutionary tree established using the putative homoplasious SNP 909166 is relatively low. Our findings highlight the importance of validating genetic markers used to establish phylogeny, evolution, and phenotypic characteristics.

Multi-locus variable number tandem repeat analysis for Clostridium botulinum type B isolates in Japan: Comparison with other isolates and genotyping methods

Clostridium botulinum produces botulinum neurotoxin (BoNT) and causes botulism in humans and animals. Recently, 15-loci multi-locus variable number tandem repeat analysis (MLVA) for C. botulinum was developed for high-resolution and inter-lab comparative genotyping. This study examines the relation between MLVA and other genotyping methods such as pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), BoNT/B subtyping and bont/b gene location to evaluate MLVA as a method applicable to the genetic markers for C. botulinum type B. Japanese isolates were genotyped using MLVA and were compared with strains from other sources reported previously. Results show that the discriminatory power of MLVA was comparable to that of PFGE and higher than that of MLST. The topology of the minimum spanning tree (MST) constructed using MLVA data was very consistent with the phylogenetic classifications of PFGE and MLST. The MST topology also represented genetic diversity between the strains possessing bont/b gene on chromosomes and plasmids. Some Japanese isolates including those associated with infant botulism were inferred to be related to isolates of Europe origin from MLVA genotyping results. The MLVA scheme used for this study is apparently useful not only for high-resolution molecular typing, but also for phylogenetic characterization of C. botulinum type B.