Molecular characterization of amikacin, kanamycin and capreomycin resistance in M/XDR-TB strains isolated in Thailand

Abstract

BackgroundThe 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.ResultsA 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.ConclusionsOur 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.

Evaluation of Pyrosequencing for Detecting Extensively Drug-Resistant Mycobacterium tuberculosis among Clinical Isolates from Four High-Burden Countries

“…Upon completion of the present study, Rodwell et al determined through Sanger sequencing that the majority of the KAN isolates examined in their study that had no rrs 1401 SNPs had SNPs in the eis promoter (18). Undetected eis promoter mutations, along with recently reported mutations in whiB7 gene regions (42)(43)(44), might be responsible for the observed KAN-resistant phenotypes that were not detected by our PSQ assay. Adding the eis promoter region as a PSQ target in a future version of the PSQ assay should considerably improve its sensitivity for detecting KAN resistance in any location where these SNPs are prevalent.…”

“…In addition, as discussed in Sirgel et al (40), clinical isolates from South Africa with the 1401 A-G mutation in the rrs gene showed decreased phenotypic susceptibility to CAP. Furthermore, while the rrs 1401 A-G mutation is implicated in cross-resistance to all three injectable drugs, some studies have shown that mutations outside our considered genomic regions, most notably mutations in eis promoter and tlyA, may independently confer resistance to KAN and CAP, respectively (41)(42)(43)(44). The sensitivity and specificity of PSQ for detecting phenotypic CAP and KAN resistance are likely to remain at Ͻ100% unless the CAP critical concentration issue is addressed independently of the PSQ platform, and other gene regions are added to the PSQ assay.…”

Performance of REBA MTB-XDR to detect extensively drug-resistant tuberculosis in an intermediate-burden country

“…Recently, it was reported that the overexpression of the eis gene, which encodes an aminoglycoside acetyltransferase, is associated with low-level kanamycin resistance [38e40]. Mutations within the eis promoter region were reported in 26e80% of TB patients with kanamycin resistance [38]. In Korea, eis gene mutations were found in 13.7% of TB patients with kanamycin resistance, and those mutations demonstrated crossresistance with other injectable drugs (e.g.…”

Mutations in Streptomycin Resistance Genes and Their Relationship to Streptomycin Resistance and Lineage ofMycobacterium tuberculosisThai Isolates

“…Oligonucleotide primers used for polymerase chain reaction (PCR) amplification and sequencing of rpsL , rrs , gidB , and whiB7 were based on previous studies or newly designed in the present study (Table 2)1718192021. Amplifications were carried out using Pfu DNA polymerase (Bioneer Corp., Daegeon, Korea).…”