Characterization of IS6110 restriction fragment length polymorphism patterns and mechanisms of antimicrobial resistance for multidrug-resistant isolates of Mycobacterium tuberculosis from a major reference hospital in Assiut, Egypt.
ABSTRACT: We evaluated 25 Mycobacterium tuberculosis isolates from patients at a major Egyptian reference hospital in Assiut, Egypt, who had been treated for at least 1 year for tuberculosis. Typing patterns (IS6110) were diverse, and multidrug resistance was found among 11 (44%) of the isolates. Mutations associated with antimicrobial drug resistance were found in rpoB, katG, rpsL, and embB in the resistant isolates.
Project description:BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem. Early diagnosis of MDR-TB patients is essential for minimizing the risk of Mycobacterium tuberculosis (MTB) transmission. The conventional drug susceptibility testing (DST) methods for detection of drug-resistant M. tuberculosis are laborious and cannot provide the rapid detection for clinical practice. METHODS: The aim of this study was to develop a pyrosequencing approach for the simultaneous detection of resistance to rifampin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (SM), ofloxacin (OFL) and amikacin (AMK) in M. tuberculosis clinical isolates and sputum samples from re-treatment pulmonary tuberculosis (PTB) patients. We identified the optimum conditions for detection mutation of rpoB, katG, rpsl, embB, gyrA and rrs gene by pyrosequencing. Then this approach was applied to detect 205 clinical isolates and 24 sputum samples of M. tuberculosis from re-treatment PTB patients. RESULTS: The mutations of rpoB and gyrA gene were detected by pyrosequencig with the SQA mode, and the mutations of katG, rpsl, embB, gyrA and rrs gene were detected by pyrosequencing with SNP mode. Compared with the Bactec MGIT 960 mycobacterial detection system, the accuracy of pyrosequencing for the detection of RIF, INH, EMB, SM, AMK and OFL resistance in clinical isolates was 95.0%, 79.2%, 70.3%, 84.5%, 96.5% and 91.1%, respectively. In sputum samples the accuracy was 83.3%, 83.3%, 60.9%, 83.3%, 87.5% and 91.7%, respectively. CONCLUSIONS: The newly established pyrosequencing assay is a rapid and high-throughput method for the detection of resistance to RIF, INH, SM, EMB, OFL and AMK in M. tuberculosis. Pyrosequencing can be used as a practical molecular diagnostic tool for screening and predicting the resistance of re-treatment pulmonary tuberculosis patients.
Project description:Development of modern genomics provides us an effective method to understand the molecular mechanism of drug resistance and diagnose drug-resistant Mycobacterium tuberculosis. In this study, mutations in 18 genes or intergenic regions acquired by whole-genome sequencing (WGS) of 183 clinical M. tuberculosis strains, including 137 multidrug-resistant and 46 pan-susceptible isolates from China, were identified and used to analyze their associations with resistance of isoniazid, rifampin, ethambutol, and streptomycin. Using the proportional method as the gold standard method, the accuracy values of WGS to predict resistance were calculated. The association between synonymous or lineage definition mutations with different genotypes were also analyzed. The results show that, compared to the phenotypic proportional method, the sensitivity and specificity of WGS for resistance detection were 94.2 and 100.0% for rifampicin (based on mutations in rpoB), 90.5 and 97.8% for isoniazid (katG), 83.0 and 97.8% for streptomycin (rpsL combined with rrs 530 loop and 912 loop), and 90.9 and 65.1% for ethambutol (embB), respectively. WGS data also showed that mutations in the inhA promoter increased only 2.2% sensitivity for INH based on mutations in katG. Synonymous mutation rpoB A1075A was confirmed to be associated with the Beijing genotype. This study confirmed that mutations in rpoB, katG, rrs 530 loop and 912 loop, and rpsL were excellent biomarkers for predicting rifampicin, isoniazid, and streptomycin resistance, respectively, and provided clues in clarifying the drug-resistance mechanism of M. tuberculosis isolates from China.
Project description:To determine the prevalence and molecular characteristics of drug-resistant tuberculosis in Hunan province, drug susceptibility testing and spoligotyping methods were performed among 171 M. tuberculosis isolates. In addition, the mutated characteristics of 12 loci, including katG, inhA, rpoB, rpsL, nucleotides 388 to 1084 of the rrs gene [rrs(388-1084)], embB, pncA, tlyA, eis, nucleotides 1158 to 1674 of the rrs gene [rrs(1158-1674)], gyrA, and gyrB, among drug-resistant isolates were also analyzed by DNA sequencing. Our results indicated that the prevalences of isoniazid (INH), rifampin (RIF), streptomycin (SM), ethambutol (EMB), pyrazinamide (PZA), capreomycin (CAP), kanamycin (KAN), amikacin (AKM), and ofloxacin (OFX) resistance in Hunan province were 35.7%, 26.9%, 20.5%, 9.9% 15.2%, 2.3%, 1.8%, 1.2%, and 10.5%, respectively. The previously treated patients presented significantly increased risks for developing drug resistance. The majority of M. tuberculosis isolates belonged to the Beijing family. Almost all the drug resistance results demonstrated no association with genotype. The most frequent mutations of drug-resistant isolates were katG codon 315 (katG315), inhA15, rpoB531, rpoB526, rpoB516, rpsL43, rrs514, embB306, pncA96, rrs1401, gyrA94, and gyrA90. These results contribute to the knowledge of the prevalence of drug resistance in Hunan province and also expand the molecular characteristics of drug resistance in China.
Project description:<h4>Background</h4>Increasing incidence of multidrug-resistant Mycobacterium tuberculosis infections is hampering global tuberculosis control efforts. Kuwait is a low-tuberculosis-incidence country, and ~?1% of M. tuberculosis strains are resistant to rifampicin and isoniazid (MDR-TB). This study detected mutations in seven genes predicting resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin in MDR-TB strains. Sequence data were combined with spoligotypes for detecting local transmission of MDR-TB in Kuwait.<h4>Methods</h4>Ninety-three MDR-TB strains isolated from 12 Kuwaiti and 81 expatriate patients and 50 pansusceptible strains were used. Phenotypic drug susceptibility was determined by MGIT 460 TB/960 system. Mutations conferring resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin were detected by genotype MTBDRplus assay and/or PCR sequencing of three rpoB regions, katG codon 315 (katG315)?+?inhA regulatory region, pncA, three embB regions and rpsL?+?rrs-500-900 regions. Spoligotyping kit was used, spoligotypes were identified by SITVIT2, and phylogenetic tree was constructed by using MIRU-VNTRplus software. Phylogenetic tree was also constructed from concatenated sequences by MEGA7 software. Additional PCR sequencing of gidB and rpsA was performed for cluster isolates.<h4>Results</h4>Pansusceptible isolates contained wild-type sequences. Mutations in rpoB and katG and/or inhA were detected in 93/93 and 92/93 MDR-TB strains, respectively. Mutations were also detected for pyrazinamide resistance, ethambutol resistance and streptomycin resistance in MDR-TB isolates in pncA, embB and rpsL?+?rrs, respectively. Spoligotyping identified 35 patterns with 18 isolates exhibiting unique patterns while 75 isolates grouped in 17 patterns. Beijing genotype was most common (32/93), and 11 isolates showed nine orphan patterns. Phylogenetic analysis of concatenated sequences showed unique patterns for 51 isolates while 42 isolates grouped in 16 clusters. Interestingly, 22 isolates in eight clusters by both methods were isolated from TB patients typically within a span of 2 years. Five of eight clusters were confirmed by additional gidB and rpsA sequence data.<h4>Conclusions</h4>Our study provides the first insight into molecular epidemiology of MDR-TB in Kuwait and identified several potential clusters of local transmission of MDR-TB involving 2-6 subjects which had escaped detection by routine surveillance studies. Prospective detection of resistance-conferring mutations can identify possible cases of local transmission of MDR-TB in low MDR-TB settings.
Project description:We examined the feasibility of a full-length gene analysis for the drug resistance-related genes inhA, katG, rpoB, pncA, rpsL, embB, eis, and gyrA using ion semiconductor next-generation sequencing (NGS) and compared the results with those obtained from conventional phenotypic drug susceptibility testing (DST) in multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates.We extracted genomic DNA from 30 pure MDR-TB isolates with antibiotic susceptibility profiles confirmed by phenotypic DST for isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA), amikacin (AMK), kanamycin (KM), streptomycin (SM), and fluoroquinolones (FQs) including ofloxacin, moxifloxacin, and levofloxacin. Enriched ion spheres were loaded onto Ion PI Chip v3, with 30 samples on a chip per sequencing run, and Ion Torrent sequencing was conducted using the Ion AmpliSeq TB panel (Life Technologies, USA).The genotypic DST results revealed good agreement with the phenotypic DST results for EMB (Kappa 0.8), PZA (0.734), SM (0.769), and FQ (0.783). Agreements for INH, RIF, and AMK+KM were not estimated because all isolates were phenotypically resistant to INH and RIF, and all isolates were phenotypically and genotypically susceptible to AMK+KM. Moreover, 17 novel variants were identified: six (p.Gly169Ser, p.Ala256Thr, p.Ser383Pro, p.Gln439Arg, p.Tyr597Cys, p.Thr625Ala) in katG, one (p.Tyr113Phe) in inhA, five (p.Val170Phe, p.Thr400Ala, p.Met434Val, p.Glu812Gly, p.Phe971Leu) in rpoB, two (p.Tyr319Asp and p.His1002Arg) in embB, and three (p.Cys14Gly, p.Asp63Ala, p.Gly162Ser) in pncA.Ion semiconductor NGS could detect reported and novel amino acid changes in full coding regions of eight drug resistance-related genes. However, genotypic DST should be complemented and validated by phenotypic DSTs.
Project description:BACKGROUND: Drug resistance displays a problem for the therapy of Mycobacterium tuberculosis infections. For molecular resistance testing, it is essential to have precise knowledge on genomic variations involved in resistance development. However, data from high-incidence settings are only sparely available. Therefore we performed a systematic approach and analyzed a total of 97 M. tuberculosis strains from previously treated patients in Sierra Leone for mutations in katG, rpoB, rrs, rpsL, gidB, embB, pncA and where applicable in inhA and ahpC. Of the strains investigated 50 were either mono- or poly-resistant to isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide or MDR and 47 fully susceptible strains served as controls. RESULTS: The majority of isoniazid and rifampin resistant strains had mutations in katG315 (71.9%) and rpoB531 (50%). However, rpoB mutations in codons 511, 516 and 533 were also detected in five rifampin susceptible strains. MIC determinations revealed low-level rifampin resistance for those strains. Thus, the sensitivity and specificity of sequencing of katG for detection of drug resistance were 86.7% and 100% and for sequencing of rpoB 100% and 93.8%, respectively.Strikingly, none of the streptomycin resistant strains had mutations in rrs, but 47.5% harboured mutations in rpsL. Further changes were detected in gidB. Among ethambutol resistant strains 46.7% had mutations at embB306. Pyrazinamide resistant strains displayed a variety of mutations throughout pncA. The specificities of sequencing of rpsL, embB and pncA for resistance detection were high (96-100%), whereas sensitivities were lower (48.8%, 73.3%, 70%). CONCLUSIONS: Our study reveals a good correlation between data from molecular and phenotypic resistance testing in this high-incidence setting. However, the fact that particular mutations in rpoB are not linked to high-level resistance is challenging and demonstrates that careful interpretation of molecular resistance assays is mandatory. In addition, certain variations, especially in gidB, appear to be phylogenetically informative polymorphisms rather than markers for drug resistance.
Project description:Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance.To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods.The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites.Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance mutations to improve the design of tuberculosis control measures, such as diagnostics, and inform patient management.
Project description:The insertion sequence IS6110 has an important role in diagnostic PCR and typing of Mycobacterium tuberculosis. We have evaluated a one-tube nested PCR which detects IS6110. Positive results were obtained with DNAs from four of four M. tuberculosis isolates, seven of eight M. fortuitum isolates, seven of seven M. avium-M. intracellulare complex isolates, four of five M. kansasii isolates, four of five M. xenopi isolates, two of four M. malmoense isolates, and one of two M. chelonei isolates. These results were confirmed by hybridization of genomic DNA from bp 505 to 685 of the IS6110 from M. tuberculosis H37Rv. Dot blot hybridization of genomic DNAs from these isolates with the same probe cinfirmed the presence of a homologous sequence in these mycobacterial species. These data suggest that false-positive results may be obtained for clinical samples when some methods based on IS6110 are used [corrected].
Project description:Insertion sequence (IS) 6110 is found at multiple sites in the Mycobacterium tuberculosis genome and displays a high degree of polymorphism with respect to copy number and insertion sites. Therefore, IS6110 is considered to be a useful molecular marker for diagnosis and strain typing of M. tuberculosis. Generally IS6110 elements are identified using experimental methods, useful for analysis of a limited number of isolates. Since short read genome sequences generated using next-generation sequencing (NGS) platforms are available for a large number of isolates, a computational pipeline for identification of IS6110 elements from these datasets was developed. This study shows results from analysis of NGS data of 1377 M. tuberculosis isolates. These isolates represent all seven major global lineages of M. tuberculosis. Lineage specific copy number patterns and preferential insertion regions were observed. Intra-lineage differences were further analyzed for identifying spoligotype specific variations. Copy number distribution and preferential locations of IS6110 in different lineages imply independent evolution of IS6110, governed mainly through ancestral insertion, fitness (gene truncation, promoter activity) and recombinational loss of some copies. A phylogenetic tree based on IS6110 insertion data of different isolates was constructed in order to understand genome level variations of different markers across different lineages.
Project description:A 267-nucleotide Mycobacterium tuberculosis genomic sequence (ipl, the IS6110 preferential locus) which can harbor the insertion sequence IS6110 at six alternative locations has been identified in some three-quarters of the isolates tested. Only one IS6110 copy was observed at this locus in the ipl::IS6110(+)-containing isolates tested, and all insertions had the same orientation. The implications of this finding for IS6110 fingerprint typing methods is discussed in this work.