Molecular characterization of ofloxacin-resistant Mycobacterium tuberculosis strains from Russia.
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ABSTRACT:
SUBMITTER: Mokrousov I
PROVIDER: S-EPMC2493099 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tuberculosis caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) strains is a growing problem in many countries. The availability of the complete nucleotide sequences of several MTB genomes allows to use the comparative genomics as a tool to study the relationships of strains and differences in their evolutionary history including acquisition of drug-resistance. In our work, we sequenced three genomes of Russian MTB strains of different phenotypes--drug susceptible, MDR and XDR. Of them, MDR and XDR strains were collected in Tomsk (Siberia, Russia) during the local TB outbreak in 1998-1999 and belonged to rare KQ and KY families in accordance with IS6110 typing, which are considered endemic for Russia. Based on phylogenetic analysis, our isolates belonged to different genetic families, Beijing, Ural and LAM, which made the direct comparison of their genomes impossible. For this reason we performed their comparison in the broader context of all M. tuberculosis genomes available in GenBank. The list of unique individual non-synonymous SNPs for each sequenced isolate was formed by comparison with all SNPs detected within the same phylogenetic group. For further functional analysis, all proteins with unique SNPs were ascribed to 20 different functional classes based on Clusters of Orthologous Groups (COG). We have confirmed drug resistant status of our isolates that harbored almost all known drug-resistance associated mutations. Unique SNPs of an XDR isolate CTRI-4(XDR), belonging to a Beijing family were compared in more detail with SNPs of additional 14 Russian XDR strains of the same family. Only type specific mutations in genes of repair, replication and recombination system (COG category L) were found common within this group. Probably the other unique SNPs discovered in CTRI-4(XDR) may have an important role in adaptation of this microorganism to its surrounding and in escape from antituberculosis drugs treatment.
| S-EPMC3577857 | biostudies-literature
Project description:Ofloxacin (OFX) resistant Mycobacterium tuberculosis (MTB) isolates have been increasingly observed and are a major concern in recent years. This study investigated the genetic mutations associated with OFX resistance among clinical OFX mono-resistant MTB isolates from new and previously treated tuberculosis patients. A total of 50 unrelated OFX mono-resistant MTB isolates were analyzed. For all isolates, the quinolone resistance determining regions of gyrA and gyrB were PCR amplified and sequenced. Single mutations in the quinolone resistance determining regions of gyrA (positions D94A, G, N, and Y; A90V; and S91P) and gyrB (positions T539A and E540D) were observed in 62% (31/50) and 4% (2/50) of all OFX mono-resistant isolates, respectively. No differences were detected between the proportions of isolates with mutations in gyrA/gyrB from new and previously treated tuberculosis patients (P=.820). Although mutations in gyrB were rare, they were as important as mutations in gyrA in predicting OFX resistance in MTB in Tianjin, China.
| S-EPMC6817024 | biostudies-literature
Project description:A total of 105 rifampin (RMP)- and/or isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different parts of Poland in 2000 were screened for mutations associated with resistance to these drugs by two molecular methods, namely sequence analysis and real-time PCR technology. Three loci associated with drug resistance were selected for characterization: they were rpoB (RMP), katG, and the regulatory region of inhA (INH). Nineteen different mutations were identified in 64 RMP-resistant strains, and five new alleles were described. The most common point mutations were in codons 531 (41%), 516 (16%), and 526 (9%) of the rpoB gene. Mutations were not found in two (3%) of the isolates. In the case of resistance to INH, six different mutations in the katG gene of 83 resistant strains were detected. Fifty-seven (69%) isolates exhibited nucleotide substitutions at codon 315. One strain harbored a mutation affecting codon 279 (Gly279Thr). Twelve of 26 INH-resistant strains with the wild-type codon 315 (14.5% of all strains tested) had the mutation -15C-->T in the regulatory region of inhA. A full correlation between the DNA sequence analysis and real-time PCR data was obtained. We conclude that the real-time PCR method is fast and reliable for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.
| S-EPMC427864 | biostudies-literature
Project description:In this study, a total of 77 multidrug-resistant and extensively drug-resistant (MDR and XDR, respectively) isolates of Mycobacterium tuberculosis were characterized among samples from patients living in Jiangxi province, China. The following two approaches were used: (i) genotyping all drug-resistant isolates by the 15-locus MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) method and identifying the Beijing family genotype using the RD105 deletion targeted multiplex PCR and (ii) determining the mutation profiles associated with the resistance to the first-line antituberculous drugs rifampin (RIF) and isoniazid (INH) and the second-line drugs ofloxacin (OFX), kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) with DNA sequencing. Six loci were examined: rpoB (for resistance to RIF), katG and mabA-inhA (INH), gyrA and gyrB (OFX), and rrs (KAN, AMK, and CAP). It is shown that the Beijing genotype was predominant (80.5%) among these strains and that the selected drug-resistant strains were genetically diverse, suggesting that they probably had independently acquired drug resistance. In comparison to the phenotypic data, the sensitivities for the detection of RIF, INH, OFX, and KAN/AMK/CAP resistance by DNA sequencing were 94.8, 80.5, 84.6, and 78.9%, respectively. The most prevalent mutations involved in RIF, INH, OFX, and KAN/AMK/CAP resistance were Ser531Leu in rpoB (44.2%), Ser315Thr in katG (55.8%) and C-15T in mabA-inhA (11.7%), Asp94Gly in gyrA (48.7%), and A1401G in rrs (73.7%), respectively. Five novel katG mutants (Trp191Stop, Thr271Pro, Trp328Phe, Leu546Pro, and Asp695Gly) and six new alleles (Ile569Val, Ile572Met, Phe584Ser, Val615Met, Asp626Glu, and Lys972Thr) in the rpoB gene were identified.
| S-EPMC3405621 | biostudies-literature
Project description:RationaleCentral dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations.ObjectiveTo determine whether rifampicin induces efflux pumps activation in rifampicin resistant M. tuberculosis strains thereby defining rifampicin resistance levels and reducing ofloxacin susceptibility.MethodsRifampicin and/or ofloxacin minimum inhibitory concentrations (MICs) were determined in rifampicin resistant strains by culture in BACTEC 12B medium. Verapamil and reserpine were included to determine their effect on rifampicin and ofloxacin susceptibility. RT-qPCR was applied to assess expression of efflux pump/transporter genes after rifampicin exposure. To determine whether verapamil could restore susceptibility to first-line drugs, BALB/c mice were infected with a MDR-TB strain and treated with first-line drugs with/without verapamil.Measurements and main findingsRifampicin MICs varied independently of rpoB mutation and genetic background. Addition reserpine and verapamil significantly restored rifampicin susceptibility (p = 0.0000). RT-qPCR demonstrated that rifampicin induced differential expression of efflux/transporter genes in MDR-TB isolates. Incubation of rifampicin mono-resistant strains in rifampicin (2 μg/ml) for 7 days induced ofloxacin resistance (MIC > 2 μg/ml) in strains with an rpoB531 mutation. Ofloxacin susceptibility was restored by exposure to efflux pump inhibitors. Studies in BALB/c mice showed that verapamil in combination with first-line drugs significantly reduced pulmonary CFUs after 1 and 2 months treatment (p < 0.05).ConclusionExposure of rifampicin resistant M. tuberculosis strains to rifampicin can potentially compromise the efficacy of the second-line treatment regimens containing ofloxacin, thereby emphasising the need for rapid diagnostics to guide treatment. Efflux pump inhibitors have the potential to improve the efficacy of anti-tuberculosis drug treatment.
| S-EPMC3698754 | biostudies-literature
Project description:To investigate the molecular characterization of multidrug-resistant tuberculosis (MDR-TB) isolates from China and the association of specific mutations conferring drug resistance with strains of different genotypes, we performed spoligotyping and sequenced nine loci (katG, inhA, the oxyR-ahpC intergenic region, rpoB, tlyA, eis, rrs, gyrA, and gyrB) for 128 MDR-TB isolates. Our results showed that 108 isolates (84.4%) were Beijing family strains, 64 (59.3%) of which were identified as modern Beijing strains. Compared with the phenotypic data, the sensitivity and specificity of DNA sequencing were 89.1% and 100.0%, respectively, for isoniazid (INH) resistance, 93.8% and 100.0% for rifampin (RIF) resistance, 60.0% and 99.4% for capreomycin (CAP) resistance, 84.6% and 99.4% for kanamycin (KAN) resistance, and 90.0% and 100.0% for ofloxacin (OFX) resistance. The most prevalent mutations among the MDR-TB isolates were katG315, inhA15, rpoB531, -526, and -516, rrs1401, eis-10, and gyrA94, -90, and -91. Furthermore, there was no association between specific resistance-conferring mutations and the strain genotype. These findings will be helpful for the establishment of rapid molecular diagnostic methods to be implemented in China.
| S-EPMC4023728 | biostudies-literature
Project description:To develop a better understanding of the epidemiology and molecular biology of rifampin-resistant Mycobacterium tuberculosis strains in Australia, 50 clinical isolates (33 rifampin-resistant and 17 rifampin-sensitive strains) cultured between 1990 and 1997 were analyzed by a number of bacteriological and molecular techniques. Examination of the drug resistance profiles of the 33 rifampin-resistant isolates revealed that 91% were resistant to rifampin in combination with resistance to isoniazid, 88% were resistant to rifampin on first isolation, and 81% showed cross-resistance with rifabutin. On the basis of the demographic data provided for the patients infected with the rifampin-resistant strains, 90% of the patients were born overseas. Of these patients, 64% developed clinical symptoms within 5 years of residence in Australia. On a molecular level, analysis of the rpoB gene revealed that 97% of the rifampin-resistant isolates had missense mutations within a conserved region of the gene, and eight types of missense mutations were detected. Of the 31 rifampin-resistant isolates that were typed by restriction fragment length polymorphism (RFLP) analysis, 28 distinct patterns were obtained by RFLP analysis with IS6110, and three clusters of genetically related isolates were identified. All isolates within the clusters were from patients who were born overseas and who had the same country of origin. The results from this study provide an overview of the current situation of rifampin resistance in Australia and can serve as a basis for continued monitoring of drug-resistant M. tuberculosis strains isolated within the country.
| S-EPMC85826 | biostudies-literature
Project description:China is one of the countries with the highest burdens of multidrug-resistant (MDR) and fluoroquinolone (FQ)-resistant tuberculosis (TB) globally. Nevertheless, knowledge about the prevalence and molecular characterization of FQ-resistant Mycobacterium tuberculosis isolates from this region remains scant. In this study, 138 M. tuberculosis isolates determined by the agar proportion susceptibility method to be resistant to ofloxacin (OFX) were enrolled from a national drug resistance survey of China. All these strains were tested for susceptibility to ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, and sparfloxacin using liquid Middlebrook 7H9 medium. The entire gyrA and gyrB genes conferring FQ resistance were sequenced, and spoligotyping was performed to distinguish different genotypes. Overall, the prevalence of resistance in China was highest for ofloxacin (3.76%), intermediate for levofloxacin (3.18%) and moxifloxacin (3.12%), and lowest for sparfloxacin (1.91%) and gatifloxacin (1.33%). Mutations in the gyrA gene were observed in 89 (64.5%) out of the 138 OFX-resistant M. tuberculosis strains. Positions 94 and 90 were the most frequent sites of mutation conferring FQ resistance on these strains, accounting for high-level FQ resistance. Furthermore, the Beijing genotype showed no association with high-level FQ resistance or distribution in hot spots in the quinolone resistance-determining region (QRDR) of gyrA. Our findings provide essential implications for the feasibility of genotypic tests relying on detection of mutations in the QRDR of gyrA and the shorter first-line treatment regimens based on FQs in China.
| S-EPMC3910797 | biostudies-other
Project description:Prothionamide (PTH) has been widely used in the treatment of tuberculosis (TB), especially multidrug resistant tuberculosis (MDR-TB), while data regarding prevalence of resistance-causing mutation is limited. In this study, we aimed to investigate the molecular characteristics of PTH-resistant MTB isolates, and also analyzed the risk factors for PTH resistance among Mycobacterium tuberculosis (MTB) isolates in southern China. A total of 282 MTB isolates were enrolled in from Guangzhou Chest Hospital. Among these isolates, 46 (16.3%) were resistant to PTH. Statistical analysis revealed that PTH resistance was more likely to be associated with resistance to levofloxacin (LFX; OR: 2.18, 95% CI: 1.02-4.63; P = 0.04). Of the 46 PTH-resistant MTB isolates, 37 (80.4%) isolates harbored 19 different mutation types, including 10 (21.7%) isolates with double nucleotide substitutions and 27 (58.7%) with single nucleotide substitution. The mutations in ethA (51.4%, 19/37) were most frequently observed among PTH-resistant isolates, followed by 16 (43.2%) in the promoter of inhA and 6 (16.2%) in inhA. In addition, no significant difference was found in the distribution of isolates with different mutation types between Beijing and non-Beijing genotypes (P > 0.05). In conclusion, our data demonstrate that high diversity of genetic mutations conferring PTH resistance is identified among MTB isolates from southern China. Mutations in inhA, ethA, mshA, and ndh genes confer increased resistance of MTB to PTH. Ancient Beijing genotype strains have higher proportion of drug resistance compared with modern Beijing strains. In addition, PTH resistance is more likely to be observed in the LFX-resistant MTB isolates.
| S-EPMC5714880 | biostudies-literature
Project description:Multidrug- (MDR) and extensively drug-resistant tuberculosis (XDR TB) are commonly associated with Beijing strains. However, in KwaZulu-Natal, South Africa, which has among the highest incidence and mortality for MDR and XDR TB, data suggest that non-Beijing strains are driving the epidemic. We conducted a retrospective study to characterize the strain prevalence among drug-susceptible, MDR, and XDR TB cases and determine associations between strain type and survival. Among 297 isolates from 2005-2006, 49 spoligotype patterns were found. Predominant strains were Beijing (ST1) among drug-susceptible isolates (27%), S/Quebec (ST34) in MDR TB (34%) and LAM4/KZN (ST60) in XDR TB (89%). More than 90% of patients were HIV co-infected. MDR TB and XDR TB were independently associated with mortality, but TB strain type was not. We conclude that, although Beijing strain was common among drug-susceptible TB, other strains predominated among MDR TB and XDR TB cases. Drug-resistance was a stronger predictor of survival than strain type.
| S-EPMC3944869 | biostudies-literature