High-resolution MIRU-VNTRs typing reveals the unique nature of Mycobacterium tuberculosis Beijing genotype in Okinawa, Japan

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Abstract

Mycobacterium tuberculosis Beijing lineage is highly prevalent in Japan. The aim of the present study was to describe the population structure of the Beijing lineage in this country based on 12-, 15-, and 21-loci MIRU-VNTR genotyping schemes. The MIRU-VNTR patterns of Beijing strains from Okinawa, Ryukyu Islands were compared to those recently published from the Osaka-Kobe megalopolis of the main island of Japan, Honshu (Wada et al., 2009). We also compared proportions of “modern/typical” vs. “ancient/atypical” Beijing strains as defined by structure of the NTF locus. Contrarily to the 12-loci Minimum Spanning Tree (MST), the 15- and 21-loci trees allowed the distinction of two groups of strains in Okinawa. A 12-loci MIRU-VNTR pattern (223325173533) corresponding to MIRU international type MIT17 was identified as the most prevalent Beijing genotype in Japan. In the SITVIT2 database, this pattern was found to be disseminated worldwide and corresponded to the most widely distributed Beijing profile in East Asia and former USSR countries. A comparison of 15- and 21-loci MIRU-VNTR patterns showed that two loci (QUB-4156 and Mtub21) were most polymorphic in our study, and could be potential candidates to distinguish between NTF locus based subclassification of Beijing strains. High-resolution VNTR typing using 15- and 21-loci underlined an evolutionarily distinct “ancient/atypical” subpopulation of the Beijing genotype in Okinawa as well as a subgroup of strains closely related to “modern/typical” Beijing strains observed in Osaka/Kobe.

Introduction

Mycobacterium tuberculosis Beijing genotype is a well-characterized genetic lineage of this important human pathogen. This genotype is endemically prevalent in East Asia and the countries of the former Soviet Union; recent decade witnessed a rapid dissemination of these strains to all continents, albeit in different extent, fueled by human migrations (Mokrousov, 2008, Wirth et al., 2008). Genetically, the Beijing family strains were long believed to be homogeneous as best exemplified by their highly conserved spoligotyping profile (absence of spacers 1–34, plus presence of at least three spacers among the nine remaining; although most of the Beijing strains are positive for spacers 35–43; van Soolingen et al., 1995, Kremer et al., 2004). Whereas IS6110-RFLP has long remained a method of choice for fine epidemiological subtyping of the Beijing strains, the recent years have seen multiple attempts to find new typing schemes based on different combinations of the MIRU-VNTR loci (Yokoyama et al., 2007, Millet et al., 2007, Mokrousov et al., 2008, Kam et al., 2006, Murase et al., 2008, Surikova et al., 2005). In Japan, where the Beijing lineage have been reported to represent 71.3–79.4% of M. tuberculosis strains (Millet et al., 2007, Iwamoto et al., 2007, Wada et al., 2007), the studies conducted have used various molecular markers such as: IS6110-RFLP (Ohata and Tada, 2004, Fujikane et al., 2004, Ano et al., 2006), MIRU-VNTRs (Murase et al., 2008, Wada et al., 2007) as well as less discriminatory NTF and regions of deletion (RD) (Wada et al., 2009, Maeda et al., 2010).

Based on a number of evolutionary markers such as IS6110, IS1547 and Rv3135, a rough subdivision of the Beijing genotype into two large sublineages of ancient (atypical) vs. modern (typical) strains was first proposed by Mokrousov et al. (2002). The NTF locus (Plikaytis et al., 1994, Kurepina et al., 1998) was further helpful to update this concept since these sublineages could be distinguished on the basis of the presence or absence of insertion sequence IS6110 in the NTF region (Mokrousov et al., 2005). Firstly, intact NTF locus is a feature of the “ancient/atypical” sublineage within Beijing genotype. Secondly, most Beijing strains worldwide harbor only one IS6110 insertion and were defined as NTF::IS6110 branch (Mokrousov et al., 2005). The W-branch prevalent in the USA harbors two head-to-tail IS6110 insertions separated by a 556-bp non-coding spacer (Kurepina et al., 1998); it was suggested to be the youngest Beijing sublineage likely originated in situ from the main Beijing NTF::IS6110 lineage imported to the modern USA with immigrants, perhaps from Russia or East Asia (Mokrousov et al. 2005). These sublineages have been suggested to differ in terms of their association with drug resistance (Mokrousov et al., 2006) and ability to escape BCG-vaccination induced immunity by typical Beijing strains (Kremer et al., 2009).

The aim of the present study was to describe the population structure of isolates of the Beijing lineage from Japan based on 12-, 15-, and 21-loci MIRU-VNTR genotyping schemes and to compare patterns of strains isolated in Okinawa of the Ryukyu Islands to those recently published for strains of the Beijing lineage from Osaka and Kobe located on the main island of Japan, Honshu (Wada et al., 2009). We have also compared proportions of modern/typical and ancient/atypical Beijing strains from Okinawa and the Osaka-Kobe megalopolis.

Section snippets

Set of strains

This study describes MIRU-VNTRs profiles of a total of 419 M. tuberculosis Beijing genotype strains isolated from patients (1 isolate per patient) residing in various parts of Japan as follows: Okinawa, Ryukyu Islands (n = 70), Osaka (n = 174), and Kobe (n = 175) located on the main island of Japan, Honshu. All the Beijing isolates were selected on the basis of their spoligotype patterns; the strains from Okinawa were from our previous study describing supposedly epi-unlinked patients (Millet et al.,

Genotyping profiles of the Beijing strains and discriminatory power of typing schemes

The 70 M. tuberculosis Beijing strains from Okinawa have been previously genotyped using eight selected loci out of the 12 of the “classical” MIRU-VNTR typing scheme (loci numbered 10, 16, 23, 26, 27, 31, 39, 40) as well as seven QUB markers (Millet et al., 2007). In the present study, complete 12-, 15-, 21-, and 24-loci MIRU-VNTR typing profiles were obtained for the 70 isolates (Supplemental Table S1) and further compared to the 12-, 15-, and 21-loci patterns of the Beijing isolates from

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