Background Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB), is characterized by low sequence diversity making this bacterium one of the classical examples of a genetically monomorphic pathogen. Because of this limited DNA sequence variation, routine genotyping of clinical MTBC isolates for epidemiological purposes relies on highly discriminatory DNA fingerprinting methods based on mobile and repetitive genetic elements. According to the standard view, isolates exhibiting the same fingerprinting pattern are considered direct progeny of the same bacterial clone, and most likely reflect ongoing transmission or disease relapse within individual patients.Methodology/Principal FindingsHere we further investigated this assumption and used massively parallel whole-genome sequencing to compare one drug-susceptible (K-1) and one multidrug resistant (MDR) isolate (K-2) of a rapidly spreading M. tuberculosis Beijing genotype clone from a high incidence region (Karakalpakstan, Uzbekistan). Both isolates shared the same IS6110 RFLP pattern and the same allele at 23 out of 24 MIRU-VNTR loci.We generated 23.9 million (K-1) and 33.0 million (K-2) paired 50 bp purity filtered reads corresponding to a mean coverage of 483.5 fold and 656.1 fold respectively. Compared with the laboratory strain H37Rv both Beijing isolates shared 1,209 SNPs. The two Beijing isolates differed by 130 SNPs and one large deletion. The susceptible isolate had 55 specific SNPs, while the MDR variant had 75 specific SNPs, including the five known resistance-conferring mutations.ConclusionsOur results suggest that M. tuberculosis isolates exhibiting identical DNA fingerprinting patterns can harbour substantial genomic diversity. Because this heterogeneity is not captured by traditional genotyping of MTBC, some aspects of the transmission dynamics of tuberculosis could be missed or misinterpreted. Furthermore, a valid differentiation between disease relapse and exogenous reinfection might be impossible using standard genotyping tools if the overall diversity of circulating clones is limited. These findings have important implications for clinical trials of new anti-tuberculosis drugs.
Besides mutations in embB306, mutations in embB406 and embB497 were confirmed as hot spots for genomic variation in ethambutol-resistant clinical isolates. Of all resistant strains 70.6% carry a mutation in a relatively short region in embB, which therefore represents a promising target for inclusion in molecular assays for rapid detection of ethambutol resistance.
We analyzed 159 Mycobacterium tuberculosis isolates (101 ethambutol [EMB]-resistant strains, 33 multidrugresistant but not EMB-resistant strains, and 25 fully susceptible strains) for the presence of mutations in embB codon 306 (embB306). Mutations were detected only in EMB-resistant strains (n ؍ 69; 68%), thus confirming the significance of embB306 mutations for the prediction of resistance to EMB.Drug-resistant tuberculosis (TB) has become a major public health problem in several regions around the world (14). The third report of the WHO/IUATLD Global Project on AntiTuberculosis Drug Resistance confirmed the serious magnitude and widespread occurrence of drug resistance. Documented rates of drug-resistant TB have reached tremendous levels of up to 57% among new cases (14).The rapid determination of drug resistance is the prerequisite for the initiation of effective chemotherapy to ensure successful treatment of the patient and to prevent the further spread of drug-resistant isolates (7). Based on the knowledge that the development of drug resistance in Mycobacterium tuberculosis complex isolates is the result of random genetic mutations in particular genes conferring resistance (15), molecular assays which allow the prediction of drug resistance in clinical isolates within 1 working day have been established. Thus, these assays are potentially the most rapid methods for the detection of drug resistance (6).In the case of ethambutol (EMB), which, in combination with isoniazid, rifampin, and pyrazinamide, is a key component of the first-line anti-TB treatment regimen, resistance was most frequently associated with mutations in the embCAB operon and particularly with mutations in embB codon 306 (embB306) (15). Overall, approximately 60% of EMB-resistant M. tuberculosis isolates carry a mutation in embB306 (15). The determination of alterations of this codon was suggested as a rapid screening method for the detection of EMB resistance in clinical isolates (3,8,13).More recently, however, discrepancies between the results of genotypic and phenotypic EMB resistance testing have raised concerns about the accuracy of molecular assays based on the detection of point mutations in embB306 for the prediction of EMB resistance (2, 4, 5, 11). Discordant results were especially reported for multidrug-resistant (MDR) strains phenotypically susceptible to EMB (5), and in a recent paper, Hazbón and colleagues described for embB codon 306 mutations "a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance" (2).To further investigate this question, we analyzed a large collection of 159 M. tuberculosis strains isolated in Germany in the year 2001 for the presence of mutations in embB306 and the association with phenotypic resistance to EMB. Resistance to the key antimycobacterial drugs was determined at the Supranational Reference Laboratory (SRL) in Borstel, Germany, by using the proportion method on Löwenstein-Jensen medium (critical concentration, 2.0 g/ml for EMB) (1) and/or the ...
s u m m a r y embB306 mutations are potential markers for detecting ethambutol resistance in clinical Mycobacterium tuberculosis isolates. However, more recently, embB306 mutations have been found in ethambutol susceptible isolates and an association with broad drug resistance rather than ethambutol resistance has been reported.To further investigate this question, we analyzed the association between embB306 mutations and phenotypic ethambutol resistance among 197 isolates from a drug resistance survey performed in Karakalpakstan, Uzbekistan.39 strains had an embB306 mutation, out of which seven were ethambutol susceptible, thus, displaying discrepant test results. After re-analysis, the seven isolates were tested ethambutol resistant. All of these strains had an increased ethambutol MIC, however, three strains showed no or weak growth on the critical concentration of 2 mg/ml on Lö wenstein-Jensen. In three strains we confirmed the presence of heteroresistant mixed populations which might influence conventional ethambutol testing. Final concordance between molecular and phenotypic EMB testing was high with a sensitivity of 78% and a specificity of 100%.Our results confirm that embB306 mutations are useful markers for predicting ethambutol resistance. Discrepancies between molecular and phenotypic ethambutol resistance test results are most likely caused by problems with conventional susceptibility testing.
Ethambutol (EMB) is a major component of the first-line therapy of tuberculosis. Mutations in codon 306of embB (embB306) were suggested as a major resistance mechanism in clinical isolates. To directly analyze the impact of individual embB306 mutations on EMB resistance, we used allelic exchange experiments to generate embB306 mutants of M. tuberculosis H37Rv. The level of EMB resistance conferred by particular mutations was measured in vitro and in vivo after EMB therapy by daily gavage in a mouse model of aerogenic tuberculosis. The wild-type embB306 ATG codon was replaced by embB306 ATC, ATA, or GTG, respectively. All of the obtained embB306 mutants exhibited a 2-to 4-fold increase in EMB MIC compared to the wild-type H37Rv. In vivo, the one selected embB306 GTG mutant required a higher dose of ethambutol to restrict its growth in the lung compared to wild-type H37Rv. These experiments demonstrate that embB306 point mutations enhance the EMB MIC in vitro to a moderate, but significant extent, and reduce the efficacy of EMB treatment in the animal model. We propose that conventional EMB susceptibility testing, in combination with embB306 genotyping, may guide dose adjustment to avoid clinical treatment failure in these low-level resistant strains.
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