RESUMO
According to studies carried out, approximately 10 million people developed tuberculosis in 2018. Of this total, 1.5 million people died from the disease. To study the behavior of the genome sequences of Mycobacterium tuberculosis (MTB), the bacterium responsible for the development of tuberculosis (TB), an analysis was performed using k-mers (DNA word frequency). The k values ranged from 1 to 10, because the analysis was performed on the full length of the sequences, where each sequence is composed of approximately 4 million base pairs, k values above 10, the analysis is interrupted, as consequence of the program's capacity. The aim of this work was to verify the formation of the phylogenetic tree in each k-mer analyzed. The results showed the formation of distinct groups in some k-mers analyzed, taking into account the threshold line. However, in all groups, the multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains remained together and separated from the other strains.
Assuntos
Tuberculose Extensivamente Resistente a Medicamentos , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Análise por Conglomerados , Farmacorresistência Bacteriana Múltipla/genética , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Tuberculose Extensivamente Resistente a Medicamentos/genética , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Filogenia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Whole genome sequencing (WGS) has been proposed as a tool for the diagnosis of drug resistance in tuberculosis (TB); however, there have been few studies on its effectiveness in countries with significantly high drug resistance rates. This study therefore aimed to evaluate the effectiveness of WGS to identify mutations related to drug resistance in TB isolates from an endemic region of Mexico. The results showed that, of 35 multidrug-resistant isolates analyzed, the values of congruence found between the phenotypic drug susceptibility testing and polymorphisms were 94% for isoniazid, 97% for rifampicin, 90% for ethambutol, and 82% for pyrazinamide. It was also possible to identify eight isolates as potential pre-extensive drug resistant (XDR) and one as XDR. Twenty nine isolates were classified within L4 and two transmission clusters were identified. The results show the potential utility of WGS for predicting resistance against first- and second-line drugs, as well as providing a phylogenetic characterization of TB drug-resistant isolates circulating in Mexico.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/genética , Tuberculose Extensivamente Resistente a Medicamentos/genética , Humanos , México/epidemiologia , Testes de Sensibilidade Microbiana , Fenótipo , Polimorfismo Genético , Sequenciamento Completo do GenomaRESUMO
Tuberculosis (TB) is still considered a major global public health problem in the world and there is a concern about the worldwide increase of drug-resistance (DR). This paper describes the analysis of three Mycobacterium tuberculosis isolates from a single patient collected over a long treatment period of time. DR was initially investigated through phenotypic testing, followed by line probe assays (LPAs) and whole genome sequencing (WGS). It presents an intriguing situation where a multidrug-resistant (MDR-) TB case was diagnosed and treated based only on late phenotypic drug susceptibility testing of isolate 1. During the treatment, another two isolates were cultivated: isolate 2, nine months after starting MDR-TB treatment; and isolate 3, cultivated five months later, during regular use of anti-TB drugs. These two isolates were evaluated using molecular LPA and WGS, retrospectively. All mutations detected by LPA were also detected in the WGS, including conversion from fluoroquinolones susceptibility to resistance from isolate 2 to isolate 3. WGS showed additional mutations, including some which may confer resistance to other drugs not tested (terizidone/cycloserine) and mutations with no correspondent resistance in drug susceptibility testing (streptomycin and second-line injectable drugs).
Assuntos
Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Resistência a Medicamentos , Tuberculose Extensivamente Resistente a Medicamentos/genética , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/genética , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Fatores de TempoRESUMO
Resistance phenomenon in M tuberculosis is mainly based on decreased permeability of the bacterial envelope and function of effluent pumps. The regulatory gene of the whiB7 transcription determines drug resistance in these bacteria. Increases in WhiB7 protein activity induce transcription of resistance genes leading to intrinsic multidrug resistance. The aim of this work was to evaluate the whiB7 gene sequence in susceptible, MDR and XDR clinical isolates of M tuberculosis in order to further design an inhibitor. Thirty-three clinical isolates of MTB identified as susceptible, MDR and XDR-TB were investigated by PCRfor sequencing of the entire promoter (429 bp), structural gene (279 bp) and the end of the upstream gene uvrD (265 bp). No differences were detected in the sequences of the structural gene in susceptible and MDR with XDR isolates and all of them terminated at TGA as stop codon. Examination of sequence profiles of the promoter part of whiB7 by several sets ofprimers proved that there were no differences between sequence ofsusceptible, MDR and XDR isolates by type strain (H37Rr). Furthermore, the structure of WhiB7 protein was studied in achieved sequences from clinical isolates. We found that the promoter and structural gene of whiB7 are highly conservative in clinical susceptible and resistant isolates. It is a key finding that would assist in the design ofan inhibitor for the WhiB7 protein in all clinical forms in further studies.
El fenómeno de resistencia en M tuberculosis se basa principalmente en la disminución de la permeabilidad de la envoltura bacterial y la función de las bombas efluentes. El gene regulador de la trascripción de whiB7 determina la resistencia al medicamento en estas bacterias. Los aumentos en la actividad de proteína de WhiB7 inducen la trascripción de genes de resistencia que llevan a la resistencia intrínseca de multimedicamentos. El objetivo de este trabajo fue evaluar la secuencia de genes de whiB7 en aislados clínicos susceptibles MDR y XDR de M tuberculosis para mejorar el diseño de un inhibidor. Treinta y tres aislados clínicos de MTB identificados como MDR y XDR-TB susceptibles, fueron investigados por PCR para la secuenciación del promotor entero (429 bp), el gene estructural (279 bp) y el extremo del uvrD gen arriba (265 bp). No se detectó diferencia alguna en las secuencias del gene estructural en aislados susceptibles, MDR y XDR, terminando todos ellos en TGA como codón de terminación. El examen de perfiles de la secuencia de la parte de promotor de whiB7 por varios conjuntos de iniciadores (primers), demostró que no había ninguna diferencia entre la secuencia de aislados susceptibles MDR y XDR por tipo de cepa (H37Rv). Además, la estructura de la proteína de WhiB7 se estudió en secuencias logradas de aislados clínicos. Se encontró que el promotor y el gene estructural whiB7 son muy conservadores en aislados clínicos susceptibles y resistentes. Se trata de un hallazgo clave que ayudaría a designar un inhibidor para la proteína WhiB7 en todas las formas de este patógeno en estudios ulteriores.
Assuntos
Humanos , Proteínas de Bactérias/genética , Tuberculose Extensivamente Resistente a Medicamentos/genética , Genes Bacterianos , Genes Reguladores , Mycobacterium tuberculosis/genética , Fatores de Transcrição/genética , Tuberculose Resistente a Múltiplos Medicamentos/genética , Predisposição Genética para Doença , Mycobacterium tuberculosis/isolamento & purificação , Análise de Sequência de DNA , Escarro/microbiologiaRESUMO
Resistance phenomenon in M tuberculosis is mainly based on decreased permeability of the bacterial envelope and function of effluent pumps. The regulatory gene of the whiB7 transcription determines drug resistance in these bacteria. Increases in WhiB7 protein activity induce transcription of resistance genes leading to intrinsic multidrug resistance. The aim of this work was to evaluate the whiB7 gene sequence in susceptible, MDR and XDR clinical isolates of M tuberculosis in order to further design an inhibitor. Thirty-three clinical isolates of MTB identified as susceptible, MDR and XDR-TB were investigated by PCR for sequencing of the entire promoter (429 bp), structural gene (279 bp) and the end of the upstream gene uvrD (265 bp). No differences were detected in the sequences of the structural gene in susceptible and MDR with XDR isolates and all of them terminated at TGA as stop codon. Examination of sequence profiles of the promoter part of whiB7 by several sets of primers proved that there were no differences between sequence of susceptible, MDR and XDR isolates by type strain (H37Rr). Furthermore, the structure of WhiB7 protein was studied in achieved sequences from clinical isolates. We found that the promoter and structural gene of whiB7 are highly conservative in clinical susceptible and resistant isolates. It is a key finding that would assist in the design of an inhibitor for the WhiB7 protein in all clinical forms in further studies.
Assuntos
Proteínas de Bactérias/genética , Tuberculose Extensivamente Resistente a Medicamentos/genética , Genes Bacterianos , Genes Reguladores , Mycobacterium tuberculosis/genética , Fatores de Transcrição/genética , Tuberculose Resistente a Múltiplos Medicamentos/genética , Predisposição Genética para Doença , Humanos , Mycobacterium tuberculosis/isolamento & purificação , Análise de Sequência de DNA , Escarro/microbiologiaRESUMO
Como entre 25%-50% dos isolados de Mycobacterium tuberculosis resistentes à INH não apresentam mutações nos genes katG, inhA, ahpC e kasA que possam justificar sua resistência, foi proposta a influência de mutações específicas no gene nat nos mecanismos de resistência e atividade da NAT. Todos os isolados obtidos (n=125) foram identificados e caracterizados através da amplificação pela PCR da IS6110 e por MIRU-VNTR, respectivamente. A determinação da concentração inibitória mínima (CIM) foi realizada pelo método REMA. Após triagem de mutações nos genes caracteristicamente envolvidos com resistência pela PCR-SSCP, seguida de seqüenciamento de DNA, foram selecionados 45 isolados para o estudo de mutações específicas (pela PCR e sequenciamento) e expressão gênica do mRNA do gene nat através da RT-PCR em tempo real. Confirmou-se que mutação no gene katG é a mais correlacionada com a resistência à INH, pois 68,4% das cepas resistentes apresentaram mutação neste gene. Mutações na região promotora do gene inhA, na região intergênica oxyR-ahpC e no gene kasA foram encontradas em 8,8%, 5,6% e 21,6% dos isolados, respectivamente...