RESUMEN

Here we described phenotypical, molecular and epidemiological features of a highly rifampicin-resistant Mycobacterium tuberculosis strain emerging in Southern Brazil, that carries an uncommon insertion of 12 nucleotides at the codon 435 in the rpoB gene. Employing a whole-genome sequencing-based study on drug-resistant Mycobacterium tuberculosis strains, we identified this emergent strain in 16 (9.19%) from 174 rifampicin-resistant clinical strains, all of them belonging to LAM RD115 sublineage. Nine of these 16 strains were available to minimum inhibitory concentration determination and for all of them was found a high rifampicin-resistance level (≥to 32 mg/L). This high resistance level could be explained by structural changes into the RIF binding site of RNA polymerase caused by the insertions, and consequent low-affinity interaction with rifampicin complex confirmed through protein modeling and molecular docking simulations. Epidemiological investigation showed that most of the individuals (56.25%) infected by the studied strains were prison inmate individuals or that spent some time in prison. The phylogenomic approach revealed that strains carrying on insertion belonged to same genomic cluster, evidencing a communal transmission chain involving inmate individuals and community. We stress the importance of tuberculosis genomic surveillance and introduction of measures to interrupt Mycobacterium tuberculosis transmission chain in this region.

Asunto(s)

ADN Bacteriano/genética , Mutación , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Brasil/epidemiología , Análisis Mutacional de ADN , Humanos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Estudios Retrospectivos , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología

RESUMEN

Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.

RESUMEN

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

Asunto(s)

Proteínas Bacterianas/genética , Catalasa/genética , Farmacorresistencia Bacteriana Múltiple/genética , Mutación/genética , Mycobacterium tuberculosis/genética , Oxidorreductasas/genética , Colorimetría , ADN Bacteriano/genética , Técnicas de Genotipaje , Isoniazida/farmacología , Reacción en Cadena de la Polimerasa Multiplex , Mycobacterium tuberculosis/efectos de los fármacos , Hibridación de Ácido Nucleico , Rifampin/farmacología

RESUMEN

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

Asunto(s)

Proteínas Bacterianas/genética , Catalasa/genética , Farmacorresistencia Bacteriana Múltiple/genética , Mutación/genética , Mycobacterium tuberculosis/genética , Oxidorreductasas/genética , Colorimetría , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN , Técnicas de Genotipaje , Isoniazida/farmacología , Reacción en Cadena de la Polimerasa Multiplex , Mycobacterium tuberculosis/efectos de los fármacos , Hibridación de Ácido Nucleico , Rifampin/farmacología

RESUMEN

Rapid identification of drug resistance in clinical isolates of Mycobacterium tuberculosis is important in determining treatment for tuberculosis. The aim of this work was evaluate the performance of the GenoType MDRTBplus assay directly on sputum of patients who had treatment failure or relapse in a routine outpatient setting in southern Brazil.

Asunto(s)

Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Esputo/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antibióticos Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Secuencia de Bases , Brasil , Catalasa/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Isoniazida/uso terapéutico , Pruebas de Sensibilidad Microbiana , Técnicas de Diagnóstico Molecular , Mutación , Mycobacterium tuberculosis/genética , Oxidorreductasas/genética , Recurrencia , Rifampin/uso terapéutico , Análisis de Secuencia de ADN , Insuficiencia del Tratamiento , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico

RESUMEN

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3% and 98.1%, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6% and 100%, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100% consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

Asunto(s)

Antibióticos Antituberculosos/farmacología , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Mutación/genética , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Southern Blotting , ARN Polimerasas Dirigidas por ADN , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad

RESUMEN

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3 percent and 98.1 percent, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6 percent and 100 percent, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100 percent consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

Asunto(s)

Humanos , Antibióticos Antituberculosos , Proteínas Bacterianas , ADN Bacteriano , Farmacorresistencia Bacteriana , Mutación , Mycobacterium tuberculosis , Rifampin , Southern Blotting , Genotipo , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad

RESUMEN

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC-->ACC (Ser-->Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100% of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.

Asunto(s)

Antituberculosos/farmacología , Proteínas Bacterianas/genética , Catalasa/genética , Farmacorresistencia Bacteriana/genética , Isoniazida/farmacología , Mutación/genética , Mycobacterium tuberculosis , Colorimetría/métodos , ADN Bacteriano/análisis , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa

RESUMEN

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100 percent of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.

Asunto(s)

Antituberculosos/farmacología , Proteínas Bacterianas/genética , Catalasa/genética , Farmacorresistencia Bacteriana/genética , Isoniazida/farmacología , Mycobacterium tuberculosis , Mutación/genética , Colorimetría/métodos , ADN Bacteriano/análisis , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa