We analysed the genotypes of 325 Mycobacterium tuberculosis clinical isolates obtained during 2002 throughout Japan. The genotyping methods included insertion sequence IS6110 RFLP, spoligotyping and variable number of tandem repeat (VNTR) analyses. Clustered isolates revealed by IS6110 RFLP analysis accounted for 18.5 % (60/325) of the isolates. Beijing genotype tuberculosis (TB) accounted for 73.8 % (240/325) of the isolates. Using VNTR, we analysed 35 loci, including 12 standard mycobacterial interspersed repetitive units and 4 exact tandem repeats. The discriminatory power of these 16 loci was low. Using VNTR analyses of the 35 loci, 12 loci (VNTRs 0424, 0960, 1955(VNTRs 0424, 0960, , 2074(VNTRs 0424, 0960, , 2163b(VNTRs 0424, 0960, , 2372(VNTRs 0424, 0960, , 2996 were selected for the genotyping of Beijing genotype strains. Comparison of the discriminatory power of the 12-locus VNTR [Japan Anti-Tuberculosis Association (JATA)] to that of the 15-locus and 24-locus VNTRs proposed by Supply et al. (2006) showed that our established VNTR system was superior to the reported 15-locus VNTR and had almost equal discriminatory power to the 24-locus VNTR. This 12-locus VNTR (JATA) can therefore be used for TB genotyping in areas where Beijing family strains are dominant.

Whole-genome sequencing (WGS) with next-generation DNA sequencing (NGS) is an increasingly accessible and affordable method for genotyping hundreds of Mycobacterium tuberculosis (Mtb) isolates, leading to more effective epidemiological studies involving single nucleotide variations (SNVs) in core genomic sequences based on molecular evolution. We developed an all-in-one web-based tool for genotyping Mtb, referred to as the Total Genotyping Solution for TB (TGS-TB), to facilitate multiple genotyping platforms using NGS for spoligotyping and the detection of phylogenies with core genomic SNVs, IS6110 insertion sites, and 43 customized loci for variable number tandem repeat (VNTR) through a user-friendly, simple click interface. This methodology is implemented with a KvarQ script to predict MTBC lineages/sublineages and potential antimicrobial resistance. Seven Mtb isolates (JP01 to JP07) in this study showing the same VNTR profile were accurately discriminated through median-joining network analysis using SNVs unique to those isolates. An additional IS6110 insertion was detected in one of those isolates as supportive genetic information in addition to core genomic SNVs. The results of in silico analyses using TGS-TB are consistent with those obtained using conventional molecular genotyping methods, suggesting that NGS short reads could provide multiple genotypes to discriminate multiple strains of Mtb, although longer NGS reads (≥300-mer) will be required for full genotyping on the TGS-TB web site. Most available short reads (~100-mer) can be utilized to discriminate the isolates based on the core genome phylogeny. TGS-TB provides a more accurate and discriminative strain typing for clinical and epidemiological investigations; NGS strain typing offers a total genotyping solution for Mtb outbreak and surveillance. TGS-TB web site: https://gph.niid.go.jp/tgs-tb/.

Tuberculosis is the leading cause of death due to a single infectious agent in the world and the emergence of multidrug-resistant strains prompted us to develop new drugs with novel targets and mechanism. Here, we screened a natural antibiotics library with Mycobacterium smegmatis membrane-bound dehydrogenases and identified polymyxin B (cationic decapeptide) and nanaomycin A (naphtoquinone derivative) as inhibitors of alternative NADH dehydrogenase [50% inhibitory concentration (IC(50)) values of 1.6 and 31 microg/ml, respectively] and malate: quinone oxidoreductase (IC(50) values of 4.2 and 49 microg/ml, respectively). Kinetic analysis on inhibition by polymyxin B showed that the primary site of action was the quinone-binding site. Because of the similarity in K(m) value for ubiquinone-1 and inhibitor sensitivity, we examined amino acid sequences of actinobacterial enzymes and found possible binding sites for L-malate and quinones. Proposed mechanisms of polymyxin B and nanaomycin A for the bacteriocidal activity were the destruction of bacterial membranes and production of reactive oxygen species, respectively, while this study revealed their inhibitory activity on bacterial membrane-bound dehydrogenases. Screening of the library with bacterial respiratory enzymes resulted in unprecedented findings, so we are hoping that continuing efforts could identify lead compounds for new drugs targeting to mycobacterial respiratory enzymes.

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The Philippines has a high incidence of tuberculosis disease (TB), with an increasing prevalence of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strains making its control difficult. Although the M . tuberculosis “Manila” ancient lineage 1 strain-type is thought to be prevalent in the country, with evidence of export to others, little is known about the genetic diversity of circulating strains. By whole genome sequencing (WGS) 178 isolates from the Philippines National Drug Resistance Survey, we found the majority (143/178; 80.3%) belonged to the lineage 1 Manila clade, with the minority belonging to lineages 4 (European-American; n = 33) and 2 (East Asian; n = 2). A high proportion were found to be multidrug-resistant (34/178; 19.1%), established through highly concordant laboratory drug susceptibility testing and in silico prediction methods. Some MDR-TB isolates had near identical genomic variation, providing potential evidence of transmission. By placing the Philippine isolates within a phylogeny of global M . tuberculosis (n > 17,000), we established that they are genetically similar to those observed outside the country, including a clade of Manila-like strain-types in Thailand. An analysis of the phylogeny revealed a set of ~200 SNPs that are specific for the Manila strain-type, and a subset can be used within a molecular barcode. Sixty-eight mutations known to be associated with 10 anti-TB drug resistance were identified in the Philippine strains, and all have been observed in other populations. Whilst nine putative streptomycin resistance conferring markers in gid (8) and rrs (1) genes appear to be novel and with functional consequences. Overall, this study provides an important baseline characterisation of M . tuberculosis genetic diversity for the Philippines, and will fill a gap in global datasets and aid the development of a nation-wide database for epidemiological studies and clinical decision making. Further, by establishing a molecular barcode for detecting Manila strains it will assist with the design of diagnostic tools for disease control activities.

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. INTRODUCTIONThe Beijing family, a lineage of Mycobacterium tuberculosis, is well known for its highly endemic prevalence around East Asian countries and as a causative agent of tuberculosis (TB) (van Soolingen et al., 1995). In the Republic of Korea (South Korea), TB is still a major public health concern, with 34 157 (70.3 per 100 000) registered new TB patients in 2008 and 2376 deaths attributable to TB in 2007 (Korea Centers for Disease Control & Prevention, 2008). More than 70 % of M. tuberculosis strains isolated from Korean pulmonary TB patients belong to the Beijing family. 'K strain' (Park et al., 2000), one of the Beijing family strains, has been reported as the cause of a severe outbreak of TB in South Korea (Kim et al., 2001). Although their phylogenetic position in the Beijing family lineage has been unclear, Shamputa et al. (2010) reported recently that K strains show genetic diversity by some genotyping methods, even in isolates obtained from a single hospital.The detailed phylogenetic variation of the Beijing family has been unveiled by various genetic markers such as single nucleotide polymorphisms, regions of difference and variable number of tandem repeat (VNTR) loci (Filliol et al., 2006;Tsolaki et al., 2005;. A notable characteristic of the lineage is the insertion of IS6110 in a genomic region named NTF (Mokrousov et al., 2005;Plikaytis et al., 1994). This phylogenetic marker can classify the Beijing family into two distinct sublineages: ancient and modern. The modern Beijing sublineage has been more predominant than the ancient sublineage throughout the world, including countries surrounding South Korea (Bifani et al., 2002;Dou et al., 2008; Kremer et al., 2009; Mokrousov et al., 2005Mokrousov et al., , 2006 van Soolingen & Kremer, 2009). However, reported that the ancient Beijing sublineage has been observed to be exceptionally predominant in Japan.Abbreviations: MIRU-VNTR, ...

Strain clustering suggests community transmission plays a critical role in incidence.

In bacterial membranes and plant, fungus and protist mitochondria, NADH dehydrogenase (NDH-II) serves as an alternative NADH : quinone reductase, a non-proton-pumping single-subunit enzyme bound to the membrane surface. Because NDH-II is absent in mammalian mitochondria, it is a promising target for new antibiotics. However, inhibitors for NDH-II are rare and unspecific. Taking advantage of the simple organization of the respiratory chain in Gluconobacter oxydans, we carried out screening of natural compounds and identified scopafungin and gramicidin S as inhibitors for G. oxydans NDH-II. Further, we examined their effects on Mycobacterium smegmatis and Plasmodium yoelii NDH-II as model pathogen enzymes.