Integrated analysis of ethionamide resistance loci in Mycobacterium tuberculosis clinical isolates.

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.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity.

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.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity

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.

Strain classification of Mycobacterium tuberculosis isolates in Brazil based on genotypes obtained by spoligotyping, mycobacterial interspersed repetitive unit typing and the presence of large sequence and single nucleotide polymorphism.

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.

Description of a new Mycobacterium intracellulare pattern of PCR restriction enzyme analysis of hsp65 gene.

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.

Primeiro isolamento de Mycobacterium kyorinense em espécime clínico no Brasil / First isolation of Mycobacterium kyorinense in a clinical specimen in Brazil

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.

Primeiro isolamento de Mycobacterium kyorinense em espécime clínico no Brasil / First isolation of Mycobacterium kyorinense in a clinical specimen in Brazil

Os testes bioquímicos realizados, o seq³enciamento de diferentes alvos genéticos e a construþÒo de uma árvore concatenada, construída a través do método Neighbor-Joining, permitiram a identificaþÒo das cepas brasileiras como M. kyorinense. (AU)

Mycobacterium fragae sp. nov., a non-chromogenic species isolated from human respiratory specimens.

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)).

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil

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.

First isolation of Mycobacterium kyorinense from clinical specimens in Brazil.

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.

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil.

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.

Análise da confiabilidade dos testetes de sensibilidade a Mycobacterium tuberculosis no Brasil no período de 2000 a 2004

O teste de sensibilidade às drogas utilizadas no tratamento da tuberculose, isoniazida (INH), rifampicina (RMP), estreptomicina (SM), etambutol (EMB), realizado pela grande maioria dos Laboratórios Centrais (LACENs) nas capitais dos estados de Alagoas, Amapá, Amazonas, Bahia, Ceará, Distrito Federal, Espírito Santo, Goiás, Maranhão, Mato Grosso do Sul, Minas Gerais, Paraíba, Paraná, Pernambuco, Rio Grande do Sul, Rio Grande do Norte, Rio de Janeiro, Piauí e Santa Catarina, foram avaliados quanto à confiabilidade no período de 2000 a 2004. As 659 culturas analisadas mostraram, pelo coeficiente de Kappa, concordância máxima de 83% referentes à RMP e à INH. Já o EMB e a SM produziram resultados concordantes moderados, na ordem de 48% e de 55%, respectivamente. A dificuldade de adesão dos estados ao projeto possibilitou a analise de aproximadamente 2% dos exames realizados no período. Apesar disto, esta ação permitiu a melhoria do desempenho de alguns laboratórios, assim como a implementação de outros para a participação efetiva na vigilância epidemiológica da resistência em tuberculose

Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system

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

Direct sensitivity test of the MB/BacT system.

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.

Direct sensitivity test of the MB/BacT system

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