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RpoB mutations in multidrug-resistant strains of Mycobacterium tuberculosis isolated in Italy.

ABSTRACT: Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different rpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser-->Leu) and 11 (29.7%) had GAC in position 526 (His-->Asp).

SUBMITTER: Pozzi G

PROVIDER: S-EPMC88675 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

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rpoB mutations in multidrug-resistant strains of Mycobacterium tuberculosis isolated in Italy.

Pozzi G G Meloni M M Iona E E Orrù G G Thoresen O F OF Ricci M L ML Oggioni M R MR Fattorini L L Orefici G G

Journal of clinical microbiology 19990401 4

Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different rpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from diffe ...[more]

PMID: 10074552

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Project description:INTRODUCTION:Tuberculosis remains the leading causes of death worldwide with frequencies of mutations in rifampicin and isoniazid resistant Mycobacterium tuberculosis isolates varying according to geographical location. There is limited information in Zimbabwe on specific antibiotic resistance gene mutation patterns in MTB and hence, increased rate of discordant results and mortality due to inappropriate antibiotic prescriptions. The rpoB and katG genes molecular markers are used for detecting rifampicin and isoniazid resistance respectively. Some mutations within these gene sequences are associated with drug resistance as they directly alter gene function. The objectives of this research was to determine the drug resistance profiles in M. tuberculosis isolates that are phenotypically resistant but not detected by the GeneXpert and MTBDRplus kit and also to detect mutations in the rpoB and katG genes which are not detected by the Hain Genotype MTBDRplus kit and GeneXpert diagnosis. METHODS:PCR was used for the amplification of the rpoB and katG genes from MTB isolates collected from human clinical samples between 2008 and 2015. The genes were sequenced and compared to the wild type MTB H37Rv rpoB (accession number L27989) and kat G genes (KP46920), respectively. Sequence analysis results were compared to genotyping results obtained from molecular assays and culture results of all isolates. RESULTS:The most frequent mutation responsible for rifampicin resistance was (25/92) S531L that was detected by using all molecular assays. Some inconsistencies were observed between phenotypic and genotypic assay results for both katG and rpoB genes in 30 strains. For these, eight codons; G507S, T508A, L511V, del513-526, P520P, L524L, R528H, R529Q and S531F were novel mutations. In addition, the I572P/F, E562Q, P564S, and Q490Y mutations were identified as novel mutations outside the rifampicin resistance determining region. In katG gene, amino acid changes to threonine, asparagine and isoleucine exhibited high degrees of polymorphism such as V473N, D311N, and L427I. The R463L (20/92) amino acid substitution was most common but was not associated with isoniazid resistance. CONCLUSION:These finding indicate that molecular assay kit diagnosis that is based on the rpoB and katG genes should be improved to cater for the genetic variations associated with the geographic specificity of the target genes and be able to detect most prevalent mutations in different areas.

Project description:BackgroundEthiopia is one of the high multidrug-resistant tuberculosis (MDR-TB) burden countries. However, phenotypic drug susceptibility testing can take several weeks due to the slow growth of Mycobacterium tuberculosis complex (MTBC) strains. In this study, we assessed the performance of a Sanger sequencing approach to predict resistance against five anti-tuberculosis drugs and the pattern of resistance mediating mutations.MethodsWe enrolled 226 MTBC culture-positive MDR-TB suspects and collected sputum specimens and socio-demographic and TB related data from each suspect between June 2015 and December 2016 in Addis Ababa, Ethiopia. Phenotypic drug susceptibility testing (pDST) for rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin using BACTEC MGIT 960 was compared with the results of a Sanger sequencing analysis of seven resistance determining regions in the genes rpoB, katG, fabG-inhA, pncA, embB, rpsL, and rrs.ResultDNA isolation for Sanger sequencing was successfully extracted from 92.5% (209/226) of the MTBC positive cultures, and the remaining 7.5% (17/226) strains were excluded from the final analysis. Based on pDST results, drug resistance proportions were as follows: isoniazid: 109/209 (52.2%), streptomycin: 93/209 (44.5%), rifampicin: 88/209 (42.1%), ethambutol: 74/209 (35.4%), and pyrazinamide: 69/209 (33.0%). Resistance against isoniazid was mainly mediated by the mutation katG S315T (97/209, 46.4%) and resistance against rifampicin by rpoB S531L (58/209, 27.8%). The dominating resistance-conferring mutations for ethambutol, streptomycin, and pyrazinamide affected codon 306 in embB (48/209, 21.1%), codon 88 in rpsL (43/209, 20.6%), and codon 65 in pncA (19/209, 9.1%), respectively. We observed a high agreement between phenotypic and genotypic DST, such as 89.9% (at 95% confidence interval [CI], 84.2%-95.8%) for isoniazid, 95.5% (95% CI, 91.2%-99.8%) for rifampicin, 98.6% (95% CI, 95.9-100%) for ethambutol, 91.3% (95% CI, 84.6-98.1%) for pyrazinamide and 57.0% (95% CI, 46.9%-67.1%) for streptomycin.ConclusionWe detected canonical mutations implicated in resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin. High agreement with phenotypic DST results for all drugs renders Sanger sequencing promising to be performed as a complementary measure to routine phenotypic DST in Ethiopia. Sanger sequencing directly from sputum may accelerate accurate clinical decision-making in the future.

Dataset Information

RpoB mutations in multidrug-resistant strains of Mycobacterium tuberculosis isolated in Italy.

Publications

rpoB mutations in multidrug-resistant strains of Mycobacterium tuberculosis isolated in Italy.

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