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    Overexpression of eis without a mutation in promoter region of amikacin- and kanamycin-resistant Mycobacterium tuberculosis clinical strain
    (2018-07-16)
    Sowajassatakul, Angkanang
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    Prammananan, Therdsak
    ;
    Chaiprasert, Angkana
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    Background: Aminoglycosides such as amikacin and kanamycin are effective injectable second-line drugs for treatment of multidrug-resistant tuberculosis. Molecular mechanisms underlying aminoglycoside resistance are not well understood. We have previously identified the amikacin- and kanamycin-resistant M. tuberculosis MT433 clinical strain, of which all known mutations related to resistance have not been found. Drug efflux pump is one of reported resistance mechanisms that might play a role in aminoglycoside resistance. Methods: The expression levels of sixteen putative efflux pump genes, including eis and one regulator gene, whiB7, of MT433 in the presence of kanamycin were determined using the reverse transcription-quantitative PCR method. The effects of upregulated genes on amikacin and kanamycin resistance were investigated by overexpression in M. tuberculosis H37Ra strain. Results: Upon kanamycin exposure, other than whiB7 and eis that were found extremely overexpressed, two drug efflux pump genes, namely Rv1877 and Rv2846c, showed specifically high-level of expression in M. tuberculosis MT433 strain. However, direct effect of overexpressed Rv1877 and Rv2846c on amikacin and kanamycin resistance could not be demonstrated in M. tuberculosis H37Ra overexpressed strain. Conclusions: Our finding demonstrated that overexpression of eis could occur without any mutations in the promoter region and be detectable in clinical isolate. This might be a consequence of overexpressed whiB7, resulting in amikacin and kanamycin resistance in M. tuberculosis MT433 strain.
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    Genetic characterisation of a whiB7 mutant of a Mycobacterium tuberculosis clinical strain
    (2015-12-01)
    Warit, Saradee
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    ;
    Jityam, Chaitas
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    Jaitrong, Sarinya
    ;
    Billamas, Pamaree
    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.
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    Draft genome sequence of amikacin- and kanamycin-resistant Mycobacterium tuberculosis MT433 without rrs and eis mutations
    (2015-01-01)
    Sowajassatakul, Angkanang
    ;
    Coker, Olabisi O.
    ;
    Prammananan, Therdsak
    ;
    Chaiprasert, Angkana
    ;
    We announce the draft genome sequence of amikacin- and kanamycin-resistant Mycobacterium tuberculosis MT433, which has been previously described as the strain carrying an unknown resistance mechanism.
      2
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    Molecular characterization of amikacin, kanamycin and capreomycin resistance in M/XDR-TB strains isolated in Thailand
    (2014-06-22)
    Sowajassatakul, Angkanang
    ;
    Prammananan, Therdsak
    ;
    Chaiprasert, Angkana
    ;
    Background: The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) makes the treatment and control of tuberculosis difficult. Rapid detection of drug-resistant strains is important for the successful treatment of drug-resistant tuberculosis; however, not all resistance mechanisms to the injectable second-line drugs such as amikacin (AK), kanamycin (KM), and capreomycin (CAP) are well understood. This study aims to validate the mechanisms associated with AK, KM, and CAP resistance in M. tuberculosis clinical strains isolated in Thailand. Results: A total of 15,124 M. tuberculosis clinical strains were isolated from 23,693 smear-positive sputum samples sent from 288 hospitals in 46 of 77 provinces of Thailand. Phenotypic analysis identified 1,294 strains as MDR-TB and second-line drugs susceptibility was performed in all MDR-TB strains and revealed 58 XDR-TB strains. Twenty-nine KM-resistant strains (26 XDR-TB and 3 MDR-TB) could be retrieved and their genes associated with AK, KM, and CAP resistance were investigated compared with 27 KM-susceptible strains. Mutation of the rrs (A1401G) was found in 21 out of 29 KM-resistant strains whereas mutations of eis either at C-14 T or at G-37 T were found in 5 strains. Three remaining KM-resistant strains did not contain any known mutations. Capreomycin resistance was determined in 28 of 29 KM-resistant strains. Analysis of tlyA revealed that the A33G mutation was found in all CAP-resistant strains and also in susceptible strains. In contrast, the recently identified tlyA mutation T539G and the novel Ins49GC were found in two and one CAP-resistant strains, respectively. In addition, our finding demonstrated the insertion of cytosine at position 581 of the tap, a putative drug efflux encoding gene, in both KM-resistant and KM-susceptible strains. Conclusions: Our finding demonstrated that the majority of KM resistance mechanism in Thai M. tuberculosis clinical strains was rrs mutation at A1401G. Mutations of the eis promoter region either at C-14 T or G-37 T was found in 5 of 29 strains whereas three strains did not contain any known mutations. For CAP resistance, 3 of 28 CAP-resistant strains contained either T539G or Ins49GC mutations at tlyA that might be associated with the resistant phenotype. © 2014 Sowajassatakul et al.; licensee BioMed Central Ltd.
      2
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    Distribution of hsp65 PCR-restriction enzyme analysis patterns among Mycobacterium avium complex isolates in Thailand
    (2006-10-01)
    Prammananan, Therdsak
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    Tingtoy, Nipa
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    Srimuang, Somboon
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    Chaiprasert, Angkana
    A total of 227 clinical Mycobacterium avium complex isolates from Thailand were differentiated into species and types by using PCR-restriction enzyme analysis of hsp65. The distribution of types showed the predominance of M. avium I (77%) in blood specimens, whereas M. intracellulare I was more commonly found in pulmonary specimens (44.2%). In addition, infections with M. avium were more likely to be found in younger adults (20 to 39 years old), while infections with M. intracellulare were more likely to be found in older adults (≥60 years old). Our results provide the useful epidemiological information that some particular types have more invasive and virulent characters than others. Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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