Whole genome sequencing reveals candidate genes involving in PAS resistance in M. Tuberculosis isolated from patients in Thailand.

Para-amino salicylic acid (PAS) was first reported by Lehmann in 1946 and used for tuberculosis treatment. However, due to its adverse effects, it is now used only as a second line anti-tuberculosis drug for treatment of multidrug resistant or extensively drug resistant M. tuberculosis. The structure of PAS is similar to para-amino benzoic acid (pABA), an intermediate metabolite in the folate synthesis pathway. The study has identified mutations in genes in folate pathway and their intergenic regions for their possibilities in responsible for PAS resistance. Genomic DNA from 120 PAS-resistant and 49 PAS-sensitive M. tuberculosis isolated from tuberculosis patients in Thailand were studied by whole genome sequencing. Twelve genes in the folate synthesis pathway were investigated for variants associated with PAS resistance. Fifty-one SNVs were found in nine genes and their intergenic regions (pabC, pabB, folC, ribD, thyX, dfrA, thyA, folK, folP). Functional correlation test confirmed mutations in RibD, ThyX, and ThyA are responsible for PAS resistance. Detection of mutation in thyA, folC, intergenic regions of thyX, ribD, and double deletion of thyA dfrA are proposed for determination of PAS resistant M. tuberculosis.

In Vitro Activity and MIC of Sitafloxacin against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis Isolated in Thailand.

New fluoroquinolones (FQs) have been shown to be more active against drug-resistant Mycobacterium tuberculosis strains than early FQs, such as ofloxacin. Sitafloxacin (STFX) is a new fluoroquinolone with in vitro activity against a broad range of bacteria, including M. tuberculosis This study aimed to determine the in vitro activity of STFX against all groups of drug-resistant strains, including multidrug-resistant M. tuberculosis (MDR M. tuberculosis), MDR M. tuberculosis with quinolone resistance (pre-XDR), and extensively drug-resistant (XDR) strains. A total of 374 drug-resistant M. tuberculosis strains were tested for drug susceptibility by the conventional proportion method, and 95 strains were randomly submitted for MIC determination using the microplate alamarBlue assay (MABA). The results revealed that all the drug-resistant strains were susceptible to STFX at a critical concentration of 2 µg/ml. Determination of the MIC90s of the strains showed different MIC levels; MDR M. tuberculosis strains had a MIC90 of 0.0625 µg/ml, whereas pre-XDR and XDR M. tuberculosis strains had identical MIC90s of 0.5 µg/ml. Common mutations within the quinolone resistance-determining region (QRDR) of gyrA and/or gyrB did not confer resistance to STFX, except that double mutations of GyrA at Ala90Val and Asp94Ala were found in strains with a MIC of 1.0 µg/ml. The results indicated that STFX had potent in vitro activity against all the groups of drug-resistant M. tuberculosis strains and should be considered a new repurposed drug for treatment of multidrug-resistant and extensively drug-resistant TB.

In Vitro Activities of Enantiopure and Racemic 1'-Acetoxychavicol Acetate against Clinical Isolates of Mycobacterium tuberculosis.

In the process of evaluating the effect of several plant extracts against Mycobacterium tuberculosis using the Microplate Alamar Blue Assay (MABA), an extract of Thai herb Alpinia galanga rhizome and its major component, 1'-acetoxychavicol acetate (ACA), exhibited marked anti-tuberculosis activity. The minimal inhibition concentrations (MICs) of the S-enantiomer of ACA (S-ACA) against M. tuberculosis H37Ra ATCC 25177 and H37Rv ATCC 27294 strains were 0.2 µg/mL and 0.7 µg/mL, respectively. More than 95% of 100 drug-sensitive and 50 drug-resistant mycobacterial clinical isolates were inhibited by extracted S-ACA at 1.0 µg/mL. All of the remaining isolates were inhibited at 2.0 µg/mL. In contrast to the S-enantiomer, synthetic racemic 1'-R,S-ACA (rac-ACA) showed MICs of 0.5 µg/mL and 2.7 µg/mL for M. tuberculosis H37Ra ATCC 25177 and H37Rv ATCC 27294, respectively, suggesting that the anti-tuberculosis effect might be primarily due to the S-form. These observations were in line with the MICs of rac-ACA against 98% of 93 drug-resistant clinical isolates, which showed the effective inhibitory dose at 2.0 µg/mL. After exposure to 2.7 µg/mL of rac-ACA for at least 3 h, the tubercle bacilli were completely killed. These demonstrated that ACA had potent anti-TB activity.

Mutations in rrs, rpsL and gidB in streptomycin-resistant Mycobacterium tuberculosis isolates from Thailand.

The objectives of this study were to characterise mutations in rrs, rpsL and gidB genes in Mycobacterium tuberculosis isolates from Thailand and to examine possible associations between mutations and strain genotypes. In total, 110 streptomycin (STR)-resistant M. tuberculosis isolates and 51 STR-susceptible isolates obtained from a sample collection in Thailand during 1999-2011 were sequenced for mutation analysis in rrs, rpsL and gidB. Genotypes of the isolates were identified using spoligotyping and large sequence polymorphisms. Mutations at codons 43 and 88 in rpsL represented 63.6% of the STR-resistant isolates and were mostly associated with Beijing strains. Mutations in rrs existed in 17.3% of the STR-resistant isolates; only 8.2% harboured resistance-associated mutations. Twenty-five different mutations were found in gidB, twelve of which are new. Eight gidB mutations were likely to contribute to STR resistance in ca. 14% of the resistant isolates; about one-half of the isolates also had a mutation in rrs or rpsL. Nearly all of the double mutants belonged to Beijing strains, whereas isolates carrying only STR-associated gidB mutation were non-Beijing strains. Three different alleles in gidB were also found, each specific to Beijing, East-African Indian and Euro-American lineages, respectively. Most of the STR-resistant isolates (80.9%) carried putative resistance-associated mutations in the analysed genes. Beijing strains were related not only to single resistance-associated mutations in rpsL or rrs but usually harboured a second mutation in gidB. Strains harbouring resistance-associated gidB mutations without rrs or rpsL mutations were more associated with non-Beijing isolates. Certain gidB mutations were also potential lineage markers.

Different behaviours of promoters in Mycobacterium tuberculosis H37Rv and H37Ra.

Mycobacterium tuberculosis H37Rv and H37Ra are two closely-related bacterial strains in which the former is virulent whereas the latter is not. Although the genetic differences between these strains are known, our understanding of how they control the difference in virulence characteristics is incomplete. In this work, we tested the activities of different mycobacterium gene promoters in the two strains using a gfp reporter gene. The promoter activities were compared between growth in vitro and growth of bacteria residing in U937 cells (a-macrophage-like cell line). The promoters tested included M. tuberculosis isocitrate lyase (icl), alpha crystalline homolog (hspX) and moeZ, and the M. avium macrophage-induced gene (mig) promoter. Two hspX constructs with different lengths of the promoter sequence were tested. All promoters except the shorter hspX construct were active in the H37Ra strain in both liquid culture and in U937 cells. In the H37Rv strain, the shorter hspX and icl constructs were induced in infected U937 cells relative to liquid culture, whereas the mig construct was active in both conditions. In conclusion, the inducible properties of the shorter hspX and the icl promoters evident in H37Rv appeared to be lost in the H37Ra strain. Furthermore, sequence further upstream of the hspX gene appears to modulate the inducible property of the hspX promoter in a strain-dependent manner.

Genetic characterisation of a whiB7 mutant of a Mycobacterium tuberculosis clinical strain.

Mycobacterium tuberculosis is naturally resistant to clarithromycin (CLR). The genes Rv3197A (whiB7) and Rv1988 (ermMT) have been shown to be involved in the resistant phenotype. In this study, a CLR-susceptible M. tuberculosis clinical strain was identified, designated as DS3214, and the nucleotide sequences and expression profiles of whiB7 and ermMT were investigated. The results revealed that strain DS3214 contained a one nucleotide deletion in whiB7, leading to a truncated peptide. Expression of whiB7 was low, whereas comparable expression of ermMT was determined compared with the reference strain M. tuberculosis H37Rv. Overexpression of the mutant whiB7 in M. tuberculosis H37Ra did not increase the minimum inhibitory concentration (MIC) to CLR or kanamycin, indicating the defect of the mutant WhiB7. The CLR-susceptible M. tuberculosis clinical strain, whose whiB7 is naturally mutated, was first described in this study and whiB7 has been shown to play a role in the CLR-susceptible phenotype.

A role for 16S rRNA dimethyltransferase (ksgA) in intrinsic clarithromycin resistance in Mycobacterium tuberculosis.

The emergence of extensively drug-resistant tuberculosis (XDR-TB) makes the control of tuberculosis (TB) difficult. As a result, there is an urgent need to develop new anti-TB drugs. Alternatively, drugs that have already been used in humans as anti-infectives and later found to have antitubercular activity might be useful as anti-TB drugs, particularly against drug-resistant TB. Clarithromycin (CLR), a 14-membered macrolide and protein synthesis inhibitor, has potent activity against most mycobacterial infections, except Mycobacterium tuberculosis. Mycobacterium tuberculosis is naturally resistant to CLR [minimum inhibitory concentration (MIC) of 8-16 µg/mL] owing to the presence of inducible erm methylase (ErmMT). With a view to gaining further insight into the mechanisms of innate CLR resistance in M. tuberculosis, CLR-susceptible M. tuberculosis H37Rv mutants were generated by transposon mutagenesis. One mutant, designated as Tn-196, was further investigated and it was found that ksgA (Rv1010) was inactivated by the transposon. The ksgA gene encodes a 16S rRNA adenine dimethyltransferase that methylates A1518 and A1519 (Escherichia coli numbering) of 16S rRNA and plays an important role in ribosome biogenesis. Complementation of the Tn-196 mutant with a wild-type ksgA gene restored the resistant phenotype (MIC of 8-16 µg/mL), corroborating the association of ksgA with intrinsic CLR resistance in M. tuberculosis.

Variable-number tandem repeats typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 in Thailand.

Spoligotyping and variable-number tandem repeats (VNTR) typing have been increasingly used for differentiating Mycobacterium tuberculosis strains with low copy numbers of IS6110. However, there are few studies comparing their potential to type the strains originating from South and Southeast Asia where many of the isolates have only a few copies, or even single copy, of IS6110. Here, we evaluated the genotyping of 187M. tuberculosis isolates harboring 1-6 copies of IS6110, available from a population-based study in Chiangrai, northern Thailand during 1998-2000, using spoligotyping and VNTR typing. The low-copy-number isolates constituted about 34% of all M. tuberculosis isolated in the province. Discriminating capacities and cluster identification by the two methods were compared with each other and to those obtained by the standard IS6110-restriction fragment length polymorphism (RFLP) method. We found that VNTR typing based on the studied 10-loci set generated more distinct patterns (151 patterns) than spoligotyping (54 patterns) and IS6110-RFLP (65 patterns). Most of the RFLP- or spoligotyping-defined clusters were subdivided by VNTR typing. Combining IS6110-RFLP with VNTR typing produced 164 distinct patterns and 21.9% of clustered isolates whereas the combination of IS6110-RFLP and spoligotyping gave 103 different patterns and 59.4% of clustered isolates. Our results confirm the utility of VNTR typing as the secondary method of choice for investigating the epidemiology of M. tuberculosis with low copy numbers of IS6110.

Evolution of some variable-number tandem repeat loci among a group of Beijing strains of Mycobacterium tuberculosis.

The patterns of variable-number tandem repeats (VNTR) genotypes of a clonal group of Mycobacterium tuberculosis Beijing isolates with very similar IS6110-restriction fragment length polymorphism (RFLP) patterns were studied. Differences between VNTR were mostly by a single repeat unit. However, a multiple-unit change also occurred. This suggests that a mechanism other than the slipped-strand mispairing might be responsible for the instability of VNTR in M. tuberculosis as well. This finding is useful for inferring phylogenetics of M. tuberculosis based on the VNTR genotypes.

Polymorphism of variable-number tandem repeats at multiple loci in Mycobacterium tuberculosis.

Genotyping based on variable-number tandem repeats (VNTR) is currently a very promising tool for studying the molecular epidemiology and phylogeny of Mycobacterium tuberculosis. Here we investigate the polymorphisms of 48 loci of direct or tandem repeats in M. tuberculosis previously identified by our group. Thirty-nine loci, including nine novel ones, were polymorphic. Ten VNTR loci had high allelic diversity (Nei's diversity indices >or= 0.6) and subsequently were used as the representative VNTR typing set for comparison to IS 6110-based restriction fragment length polymorphism (RFLP) typing. The 10-locus VNTR set, potentially providing >2 x 10(9) allele combinations, obviously showed discriminating capacity over the IS 6110 RFLP method for M. tuberculosis isolates with fewer than six IS 6110-hybridized bands, whereas it had a slightly better resolution than IS 6110 RFLP for the isolates having more than five IS 6110-hybridized bands. Allelic diversity of many VNTR loci varied in each IS 6110 RFLP type. Genetic relationships inferred from the 10-VNTR set supported the notion that M. tuberculosis may have evolved from two different lineages (high and low IS 6110 copy number). In addition, we found that the lengths of many VNTR loci had statistically significant relationships to each other. These relationships could cause a restriction of the VNTR typing discriminating capability to some extent. Our results suggest that VNTR-PCR typing is practically useful for application to molecular epidemiological and phylogenetic studies of M. tuberculosis. The discriminating power of the VNTR typing system can still be enhanced by the supplementation of more VNTR loci.