Mutations In Rrs Genes Research Articles

Assessment of the GenoType MTBDRsl VER 2.0 compared to the phenotypic drug susceptibility testing and whole genome sequencing for the rapid detection of resistance to fluoroquinolone and second-line injectable drugs among rifampicin-resistant Mycobacterium tuberculosis isolates.

Molecular techniques have considerable advantages for rapid detection, a reduction of infectiousness, prevention of further resistance development and surveillance of drug-resistant TB. MTBDRsl VER 2.0 was used to detect resistance to second-line anti-tuberculosis drugs on 35 rifampicin-resistant M. tuberculosis (RR-MTB) isolates compared to the minimum inhibitory concentrations (MICs) and whole genome sequencing (WGS). The MTBDRsl VER 2.0 (Hain Life Science, Nehren, Germany) and WGS (San Diego, CA, USA) were performed for tracing mutations in resistant-related genes involved in resistance to fluoroquinolone (FLQ) and second-line injectable drugs. The broth microdilution method using 7H9 Middlebrook media supplemented with OADC was used to determine the MICs. The MTBDRsl VER 2.0 correctly detected 5/6 (83.3%) of FLQ-resistant strains. The MUT1 A1401G (seven strains) and MUT2 G1484T (one strain) mutations in rrs gene were detected in eight AMK/KAN/CAP-resistant strains. Four low-level KAN-resistant strains with the G-10A/C-12T (three strains) and eis C-14T (one strain) mutations in eis gene was diagnosed using MTBDRsl VER 2.0. Five errors were found in detecting resistance to kanamycin and capreomycin compared to the phenotypic drug susceptibility testing and WGS. Failling wild-type bands without improved mutant bands did not indicate a reliable resistance. WGS could efficiently resolve the discrepancies of the results. MTBDRsl showed better performance in detecting XDR strains than pre-XDR.

Assessment of line probe assay for rapid detection of extensively drug-resistant Mycobacterium tuberculosis isolates

Aims and objectives: Molecular techniques have considerable advantages for rapid detection, a reduction of infectiousness, prevention of further resistance development and surveillance of drug-resistant TB. MTBDRsl VER 2.0 was used to detect resistance to second-line anti-tuberculosis drugs on rifampicin-resistant M. tuberculosis (RR-MTB) isolates compared to the minimum inhibitory concentrations (MICs) and whole genome sequencing (WGS). Methods: The MTBDRsl VER 2.0 (Hain Life Science, Nehren, Germany) and WGS (San Diego, CA, USA) was performed for tracing mutations in resistant-related genes involved in resistance to fluoroquinolone (FLQs) and second-line injectable drugs (SLIDs). The broth microdilution method using 7H9 Middlebrook media supplemented with OADC was used to determine the MICs. Results: The MTBDRsl VER 2.0 correctly detected 5/6 (83.3%) of FLQ-resistant strains having gyrA mutations. The MUT1 A1401G (7 strains) and MUT2 G1484T (1 strain) mutations in rrs gene were detected in 8 AMK/KAN/CAP-resistant strains. Four low-level KAN-resistant strains with the G-10A/C-12T (3 strains) and C-14T (1 strain) mutations in eis gene was diagnosed using MTBDRsl VER 2.0. Conclusions: Five error results were found in detecting resistance to kanamycin and capreomycin compared to the phenotypic drug susceptibility testing and WGS. Failing wild type-bands without improved mutant-bands did not indicate a reliable resistance. WGS could efficiently resolve the discrepancies of the results. MTBDRsl showed better performance in detecting extensively drug-resistant strains.

RETROSPECTIVE ANALYSIS OF SLOVENIAN MYCOBACTERIUM AVIUM COMPLEX AND MYCOBACTERIUM ABSCESSUS COMPLEX ISOLATES AND MOLECULAR RESISTANCE PROFILE

Abstract. Mycobacteria belonging to Mycobacterium (M.) avium complex (MAC) and M. abscessus complex (MABSC) are the most frequent causes of mycobacteriosis in the world. In the last few years MAC and MABSC taxonomy was rapidly changing due to new molecular methods conveying the possibility to differentiate between species. New techniques are able to identify M. chimaera that was previously recognized as M. intracellulare and also differentiate subspecies of MABSC. Due to their natural habitat, non-tuberculous mycobacteria (NTM) are constantly exposed to various concentrations of antimicrobial drugs and other chemicals and consequently they had developed different mechanisms of resistance. Macrolides and aminoglycosides are frequently used drugs to treat MAC and MABSC infections. The aim of our nation-wide survey was to obtain information about MABSC subspecies prevalence in Slovenia and to assess the percentage of misidentifications of M. chimaera isolates as M. intracellulare in the past. Moreover, the purpose of our study was to reveal, which of the two species M. intracellulare or M. chimaera is clinically more relevant in Slovenia. Further, the aim of the study was to detect mutations in erm(41), rrl and rrs genes, which are known to convey macrolide resistance (erm(41) and rrl) and aminoglycoside resistance (rrs). One hundred and thirty-two Slovenian mycobacterial isolates obtained from the National Mycobacterial Collection that belong to MAC and MABSC were analysed. GenoType NTM-DR was used to differentiate M. intracellulare from M. chimaera and subspecies of MABSC. Our results showed that 48% of previously identified M. intracellulare isolates were actually M. chimaera isolates and that M. abscessus subsp. abscessus was the most frequent subspecies of MABSC. Most of the MABSC isolates carried the inducible macrolide resistance genes (erm(41) and rrl), however none of the isolates of MAC and MABSC had mutations in rrs genes for aminoglycoside resistance.

Analysis of rrs gene mutations in amikacin resistant clinical isolates of Mycobacterium tuberculosis from Khyber Pakhtunkhwa, Pakistan.

Tuberculosis is a major infectious disease caused by Mycobacterium tuberculosis complex. Antimicrobial drugs are used to control TB infections. Molecular mechanisms controlling resistance to second-line drugs are not completely understood and no endogenous information is available regarding these mechanisms. The present study reports mutational analysis of rrs gene in Mycobacterium tuberculosis isolates collected from Khyber Pakhtunkhwa province of Pakistan. A total of 499 Mycobacterium tuberculosis isolates were analyzed for resistance against amikacin. Thirty resistant isolates were selected for mutational analysis in rrs gene. Among the 30 amikacin resistant isolates of Mycobacterium tuberculosis, 9 (30%) had mutation in the hotspot region of rrs gene. The predominant mutation was 1401A>G which was observed in 5 isolates. Maximum number of mutations was observed in isolate 6 and isolate 16 with six different mutations each. Mutations in isolate 6 included 1260G>A, 1278A>T, 1278_1279insT, 1300C>T, 1321G>A and 1445C>T. Mutation in isolate 16 included 1255_1256insA, 1364_1365insG, 1384_1385insA, 1880_1881insT, 1487G>A, and 1493delA. The mutation 1263G>A was observed in isolate 1. Isolate 2 had the 1484G>T mutation. The findings could be used as reference for future endures. It was evident from the results that mutations in rrs gene do not always contribute to amikacin resistance; hence, traditional drug susceptibility testing is still helpful for evaluation of such samples.

Novel mutations conferring resistance to kanamycin in Mycobacterium tuberculosis clinical isolates from Northern India

Twenty-nine Kanamycin resistant clinical isolates of Mycobacterium tuberculosis from Northern India were screened to evaluate genetic mutations in rrs gene, eis gene with its promoter, and whiB7 gene along with its 5'UTR. 14 strains (~48.0%) collectively exhibited mutations in rrs, eis or whiB7 target regions. While the highest frequency of mutations was found in rrs gene, eis and whiB7 loci displayed novel mutations. The novel mutations displayed by eis and whiB7 loci were found to be associated specifically with the Kanamycin resistance as none of the twenty nine Kanamycin sensitive strains harbor them. The inclusion of novel mutations of eis and whiB7 loci will be useful in improving the specificity of future diagnostics.