RESUMO
The present study aimed to determine the genetic diversity of isolates of Mycobacterium tuberculosis (Mtb) from presumed drug-resistant tuberculosis patients from several states of Brazil. The isolates had been submitted to conventional drug susceptibility testing for first- and second-line drugs. Multidrug-resistant (MDR-TB) (54.8%) was the most frequent phenotypic resistance profile, in addition to an important high frequency of pre-extensive resistance (p-XDR-TB) (9.2%). Using whole-genome sequencing (WGS), we characterized 298 Mtb isolates from Brazil. Besides the analysis of genotype distribution and possible correlations between molecular and clinical data, we determined the performance of an in-house WGS pipeline with other online pipelines for Mtb lineages and drug resistance profile definitions. Sub-lineage 4.3 (52%) was the most frequent genotype, and the genomic approach revealed a p-XDR-TB level of 22.5%. We detected twenty novel mutations in three resistance genes, and six of these were observed in eight phenotypically resistant isolates. A cluster analysis of 170 isolates showed that 43.5% of the TB patients belonged to 24 genomic clusters, suggesting considerable ongoing transmission of DR-TB, including two interstate transmissions. The in-house WGS pipeline showed the best overall performance in drug resistance prediction, presenting the best accuracy values for five of the nine drugs tested. Significant associations were observed between suffering from fatal disease and genotypic p-XDR-TB (p = 0.03) and either phenotypic (p = 0.006) or genotypic (p = 0.0007) ethambutol resistance. The use of WGS analysis improved our understanding of the population structure of MTBC in Brazil and the genetic and clinical data correlations and demonstrated its utility for surveillance efforts regarding the spread of DR-TB, hopefully helping to avoid the emergence of even more resistant strains and to reduce TB incidence and mortality rates.
RESUMO
Tuberculosis patients taking second line drugs such as ethionamide (ETH) have often experienced previous treatment failure and usually have a complex history of disease and treatment that can span decades. Mutations in the ETH activating enzyme, EthA, confer resistance through undescribed mechanisms. To explore the impact of EthA mutations on ETH resistance, data from a total of 160 ETHR isolates was analysed. The most frequently mutated positions are within regions that display sequence conservation with the active site of OTEMO, another FAD-containing NADH-binding Baeyer-Villiger monooxygenase (BVMO), or with the sugar binding site of galectin-4N. Additionally, to look at a possible role of EthR on ETH resistance we purified an EthR mutant identified in a clinical isolate, F110L, and found it to bind the ethA-ethR intergenic region with higher affinity than the wild type regulator in gel shift assays. The ability of cyclic di-GMP to enhance DNA binding is maintained in the EthR mutant. To our knowledge, this is the first ETH resistance study that combines sequence and resistance data of clinical isolates with functional and structural information.
Assuntos
Antituberculosos/uso terapêutico , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Etionamida/uso terapêutico , Loci Gênicos , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Sítios de Ligação , DNA Bacteriano/isolamento & purificação , Genótipo , Humanos , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/isolamento & purificação , Oxirredutases/genética , Fenótipo , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/genética , Relação Estrutura-Atividade , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológicoRESUMO
Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.
Assuntos
Genoma Bacteriano , Genótipo , Pneumopatias/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium kansasii/genética , Brasil , Gráficos por Computador , DNA Bacteriano , Genômica , Humanos , Mycobacterium kansasii/isolamento & purificação , Polimorfismo de Fragmento de RestriçãoRESUMO
Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.
Assuntos
DNA Bacteriano , Genoma Bacteriano/genética , Mycobacterium kansasii/genética , Gráficos por ComputadorRESUMO
Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as "LAM-like", 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.
Assuntos
Repetições Minissatélites/genética , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleotídeo Único , Tuberculose/microbiologia , Alelos , Proteínas de Bactérias/genética , Brasil , Frequência do Gene , Genótipo , Humanos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , FilogeniaRESUMO
This work comprises 9 pulmonary nontuberculous mycobateria isolates obtained from sputum of 4 different patients from Brazil. The sequencing and phylogenetic analysis allowed their accurate identification as Mycobacterium intracellulare. We report a mutation at position 453 creating a new HaeIII cutting site and, therefore, a new PRA-hsp65 M. intracellulare profile.
Assuntos
Proteínas de Bactérias/genética , Chaperonina 60/genética , Tipagem Molecular , Complexo Mycobacterium avium/classificação , Complexo Mycobacterium avium/genética , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Brasil , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Humanos , Dados de Sequência Molecular , Complexo Mycobacterium avium/isolamento & purificação , Infecção por Mycobacterium avium-intracellulare/microbiologia , Filogenia , Pneumonia/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Escarro/microbiologiaAssuntos
Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium/efeitos dos fármacos , Pneumonia Bacteriana/microbiologia , Antituberculosos/farmacologia , Genes Bacterianos , Heterogeneidade Genética , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Tipagem Molecular , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Pneumonia Bacteriana/diagnóstico , RNA Ribossômico 16S/genética , Estudos RetrospectivosRESUMO
Os testes bioquímicos realizados, o seqüenciamento de diferentes alvos genéticos e a construção de uma árvore concatenada, construída a través do método Neighbor-Joining, permitiram a identificação das cepas brasileiras como M. kyorinense.
Assuntos
Brasil , Micobactérias não Tuberculosas/isolamento & purificação , Micobactérias não Tuberculosas/virologia , Mycobacterium/isolamento & purificação , Mycobacterium/citologia , Mycobacterium/virologiaRESUMO
Three isolates of a slow-growing, non-chromogenic mycobacterium were grown from three sputum samples of a patient from the north-eastern Ceará state in Brazil. Identification at species level could not be obtained with PCR restriction analysis of the hsp65 gene. In order to characterize the isolates we carried out phenotypic and genotypic tests. We sequenced the nearly complete 16S rRNA gene and obtained partial sequences of the hsp65 (encoding the hypervariable region of the 65 kDa heat-shock protein) and rpoB (encoding the beta-subunit of RNA polymerase) genes. The three isolates turned out to be identical and most closely related to the species Mycobacterium celatum and Mycobacterium kyorinense. The results, however, showed significant differences between these species and the isolates studied, which led us to consider them members of a novel species for which we propose the name Mycobacterium fragae. The type strain is HF8705(T) ( = Fiocruz-INCQS/CMRVS P4051(T) = DSM 45731(T)).
Assuntos
Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , Filogenia , Escarro/microbiologia , Proteínas de Bactérias/genética , Brasil , Chaperonina 60/genética , DNA Bacteriano/genética , Genes Bacterianos , Humanos , Pneumopatias/microbiologia , Dados de Sequência Molecular , Mycobacterium/genética , Mycobacterium/isolamento & purificação , RNA Ribossômico 16S/genéticaRESUMO
A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.
Assuntos
Humanos , Infecções por Mycobacterium/microbiologia , Mycobacterium/genética , Infecção da Ferida Cirúrgica/microbiologia , Sequência de Bases , Brasil , Técnicas de Tipagem Bacteriana/métodos , DNA Bacteriano/análise , DNA Ribossômico/análise , Eletroforese em Gel de Campo Pulsado , Infecções por Mycobacterium/epidemiologia , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Análise de Sequência de DNA , Infecção da Ferida Cirúrgica/epidemiologiaRESUMO
In this article, the first isolation of Mycobacterium kyorinense specimens in Brazil is described. M. kyorinense is a recently identified species, with a few strains reported only in Japan. The Brazilian isolates were initially identified as Mycobacterium celatum by PCR restriction enzyme pattern analysis (PRA) with hsp65. However, biochemical tests indicated the same profile of M. kyorinense and distinguished them from M. celatum and Mycobacterium branderi. The sequencing of the hsp65, rpoB, and 16S rRNA genes allowed the accurate identification of isolates as M. kyorinense.
Assuntos
Infecções por Mycobacterium/diagnóstico , Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Proteínas de Bactérias/genética , Brasil , Chaperonina 60/genética , DNA Bacteriano/química , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Humanos , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.
Assuntos
Infecções por Mycobacterium/microbiologia , Mycobacterium/genética , Infecção da Ferida Cirúrgica/microbiologia , Técnicas de Tipagem Bacteriana/métodos , Sequência de Bases , Brasil , DNA Bacteriano/análise , DNA Ribossômico/análise , Eletroforese em Gel de Campo Pulsado , Humanos , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Infecções por Mycobacterium/epidemiologia , Análise de Sequência de DNA , Infecção da Ferida Cirúrgica/epidemiologiaRESUMO
In order to evaluate the Organon Teknika MB/BacT system used for testing indirect susceptibility to the alternative drugs ofloxacin (OFLO), amikacin (AMI), and rifabutin (RIF), and to the usual drugs of standard treatment regimes such as rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB), and ethionamide (ETH), cultures of clinical specimens from 117 patients with pulmonary tuberculosis under multidrug-resistant investigation, admitted sequentially for examination from 2001 to 2002, were studied. Fifty of the Mycobacterium tuberculosis cultures were inoculated into the gold-standard BACTEC 460 TB (Becton Dickinson) for studying resistance to AMI, RIF, and OFLO, and the remaining 67 were inoculated into Lowenstein Jensen (LJ) medium (the gold standard currently used in Brazil) for studying resistance to RMP, INH, PZA, SM, EMB, and ETH. We observed 100 percent sensitivity for AMI (80.8-100), RIF (80.8-100), and OFLO (78.1-100); and 100 percent specificity for AMI (85.4-100), RIF (85.4-100), and OFLO (86.7-100) compared to the BACTEC system. Comparing the results obtained in LJ we observed 100 percent sensitivity for RMP (80-100), followed by INH - 95 percent (81.8-99.1), EMB - 94.7 percent (71.9-99.7), and 100 percent specificity for all drugs tested except for PZA - 98.3 (89.5-99.9) at 95 percent confidence interval. The results showed a high level of accuracy and demonstrated that the fully automated, non-radiometric MB/BacT system is indicated for routine use in susceptibility testing in public health laboratories
Assuntos
Humanos , Antituberculosos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose Pulmonar , Intervalos de Confiança , Estudo de Avaliação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tuberculose Resistente a Múltiplos MedicamentosRESUMO
In order to evaluate the direct-method test of sensitivity to drugs used in the principal tuberculosis treatment regimes, in the Organon Teknika MB/BacT system, we tested 50 sputum samples positive to microscopy taken from patients with pulmonary tuberculosis and with clinical indications for an antibiogram, admitted sequentially for examination during the routine of the reference laboratory. The material was treated v/v with 23% trisodium phosphate solution, incubated for 24 h at 35 degrees C, and neutralized v/v with 20% monosodium phosphate solution. The material was then centrifuged and the sediment inoculated into flasks containing Rifampin - 2 micro g/ml, Isoniazid - 0.2 micro g/ml, Pyrazinamide - 100 micro g/ml, Ethambutol - 2.5 micro g/ml, Ethionamide - 1.25 micro g/ml, and Streptomycin - 2 micro g/ml. The tests were evaluated using the indirect method in the BACTEC 460 TB (Becton Dickinson) system as the gold standard. The results showed that the Rifampin test performed best, i.e., 100% sensitivity at 95% Confidence Interval (82.2-100) and 100% specificity at 95% Confidence Interval (84.5-100), followed by Isoniazid and Pyrazinamide. In this experiment, 92% of the materials showed a final reading in 30 days; this period represents the time for primary isolation as well as the results of the sensitivity profile, and is within Centers for Disease Control and Prevention recommendations regarding time for performance of the antibiogram. The inoculated flasks showed no contamination during the experiment. The MB/BacT is shown to be a reliable, rapid, fully automated nonradiometric system for the tuberculosis antibiogram.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Kit de Reagentes para Diagnóstico , Intervalos de Confiança , Humanos , Testes de Sensibilidade Microbiana/métodos , Sensibilidade e EspecificidadeRESUMO
In order to evaluate the direct-method test of sensitivity to drugs used in the principal tuberculosis treatment regimes, in the Organon Teknika MB/BacT system, we tested 50 sputum samples positive to microscopy taken from patients with pulmonary tuberculosis and with clinical indications for an antibiogram, admitted sequentially for examination during the routine of the reference laboratory. The material was treated v/v with 23 percent trisodium phosphate solution, incubated for 24 h at 35ºC, and neutralized v/v with 20 percent monosodium phosphate solution. The material was then centrifuged and the sediment inoculated into flasks containing Rifampin - 2 æg/ml, Isoniazid - 0.2 æg/ml, Pyrazinamide - 100 æg/ml, Ethambutol - 2.5 æg/ml, Ethionamide - 1.25 æg/ml, and Streptomycin - 2 æg/ml. The tests were evaluated using the indirect method in the BACTEC 460 TB (Becton Dickinson) system as the gold standard. The results showed that the Rifampin test performed best, i.e., 100 percent sensitivity at 95 percent Confidence Interval (82.2-100) and 100 percent specificity at 95 percent Confidence Interval (84.5-100), followed by Isoniazid and Pyrazinamide. In this experiment, 92 percent of the materials showed a final reading in 30 days; this period represents the time for primary isolation as well as the results of the sensitivity profile, and is within Centers for Disease Control and Prevention recommendations regarding time for performance of the antibiogram. The inoculated flasks showed no contamination during the experiment. The MB/BacT is shown to be a reliable, rapid, fully automated nonradiometric system for the tuberculosis antibiogram