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Genotypic susceptibility testing of Mycobacterium tuberculosis isolates for amikacin and kanamycin resistance by use of a rapid sloppy molecular beacon-based assay identifies more cases of low-level drug resistance than phenotypic Lowenstein-Jensen testing.

ABSTRACT: Resistance to amikacin (AMK) and kanamycin (KAN) in clinical Mycobacterium tuberculosis strains is largely determined by specific mutations in the rrs gene and eis gene promoter. We developed a rapid, multiplexed sloppy molecular beacon (SMB) assay to identify these mutations and then evaluated assay performance on 603 clinical M. tuberculosis DNA samples collected in South Korea. Assay performance was compared to gold-standard phenotypic drug susceptibility tests, including Lowenstein-Jensen (LJ) absolute concentration, mycobacterial growth indicator tubes (MGIT), and TREK Sensititre MycoTB MIC plate (MycoTB) methods. Target amplicons were also tested for mutations by Sanger sequencing. The SMB assay correctly detected 115/116 mutant and mixed sequences and 487/487 wild-type sequences (sensitivity and specificity of 99.1 and 100%, respectively). Using the LJ method as the reference, sensitivity and specificity for AMK resistance were 92.2% and 100%, respectively, and sensitivity and specificity for KAN resistance were 87.7% and 95.6%, respectively. Mutations in the rrs gene were unequivocally associated with high-level cross-resistance to AMK and KAN in all three conventional drug susceptibility testing methods. However, eis promoter mutations were associated with KAN resistance using the MGIT or MycoTB methods but not the LJ method. No testing method associated eis promoter mutations with AMK resistance. Among the discordant samples with AMK and/or KAN resistance but wild-type sequence at the target genes, we discovered four new mutations in the whiB7 5' untranslated region (UTR) in 6/22 samples. All six samples were resistant only to KAN, suggesting the possible role of these whiB7 5' UTR mutations in KAN resistance.

SUBMITTER: Chakravorty S

PROVIDER: S-EPMC4290905 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Genotypic susceptibility testing of Mycobacterium tuberculosis isolates for amikacin and kanamycin resistance by use of a rapid sloppy molecular beacon-based assay identifies more cases of low-level drug resistance than phenotypic Lowenstein-Jensen testing.

Chakravorty Soumitesh S Lee Jong Seok JS Cho Eun Jin EJ Roh Sandy S SS Smith Laura E LE Lee Jiim J Kim Cheon Tae CT Via Laura E LE Cho Sang-Nae SN Barry Clifton E CE Alland David D

Journal of clinical microbiology 20141022 1

Resistance to amikacin (AMK) and kanamycin (KAN) in clinical Mycobacterium tuberculosis strains is largely determined by specific mutations in the rrs gene and eis gene promoter. We developed a rapid, multiplexed sloppy molecular beacon (SMB) assay to identify these mutations and then evaluated assay performance on 603 clinical M. tuberculosis DNA samples collected in South Korea. Assay performance was compared to gold-standard phenotypic drug susceptibility tests, including Lowenstein-Jensen (L ...[more]

PMID: 25339395

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Project description: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.

Dataset Information

Genotypic susceptibility testing of Mycobacterium tuberculosis isolates for amikacin and kanamycin resistance by use of a rapid sloppy molecular beacon-based assay identifies more cases of low-level drug resistance than phenotypic Lowenstein-Jensen testing.

Publications

Genotypic susceptibility testing of Mycobacterium tuberculosis isolates for amikacin and kanamycin resistance by use of a rapid sloppy molecular beacon-based assay identifies more cases of low-level drug resistance than phenotypic Lowenstein-Jensen testing.

Similar Datasets

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