RESUMEN
Mycobacterium tuberculosis K, a member of the Beijing family, was first identified in 1999 as the most prevalent genotype in South Korea among clinical isolates of M. tuberculosis from high school outbreaks. M. tuberculosis K is an aerobic, non-motile, Gram-positive, and non-spore-forming rod-shaped bacillus. A transmission electron microscopy analysis displayed an abundance of lipid bodies in the cytosol. The genome of the M. tuberculosis K strain was sequenced using two independent sequencing methods (Sanger and Illumina). Here, we present the genomic features of the 4,385,518-bp-long complete genome sequence of M. tuberculosis K (one chromosome, no plasmid, and 65.59 % G + C content) and its annotation, which consists of 4194 genes (3447 genes with predicted functions), 48 RNA genes (3 rRNA and 45 tRNA) and 261 genes with peptide signals.
RESUMEN
Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.
Asunto(s)
Antígenos Bacterianos/química , Proteínas Bacterianas/química , Mycobacterium tuberculosis/química , Proteoma/química , Tuberculina/química , Animales , Antígenos Bacterianos/análisis , Antígenos Bacterianos/clasificación , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/análisis , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/inmunología , Cobayas , Mycobacterium tuberculosis/inmunología , Proteoma/análisis , Tuberculina/análisis , Tuberculina/inmunología , Tuberculosis/inmunología , Tuberculosis/microbiologíaRESUMEN
Among 53,974 cases of measles that occurred during the 2000-2001 outbreak in Korea, the incidence of tuberculosis following measles was 47 cases per 214,949.6 person-years, which was significantly lower than that in the general population (standardized incidence ratio, 0.73; 95% confidence interval, 0.54-0.96). In conclusion, we did not find a positive relationship between measles and tuberculosis.
Asunto(s)
Brotes de Enfermedades , Sarampión/epidemiología , Tuberculosis/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Notificación de Enfermedades/estadística & datos numéricos , Femenino , Humanos , Incidencia , Lactante , Corea (Geográfico)/epidemiología , Masculino , Sarampión/inmunología , Persona de Mediana EdadRESUMEN
As the incidence of nontuberculous mycobacterial infection has been increasing recently in Korea, the importance of drug susceptibility test for clinical isolates of mycobacteria has become larger. In this study we determined the antimicrobial susceptibility patterns of clinical isolates of M. fortuitum and M. abscessus in Korea, and evaluated the efficacy of a modified broth microdilution method using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC), in terms of its ability to provide accurate and easy-to-read minimal inhibitory concentration (MIC) endpoints for the susceptibility testing of rapidly growing mycobacteria. Most isolates of M. fortuitum and M. abscessus in Korea are susceptible or intermediately susceptible to amikacin, cefoxitin, ciprofloxacin, and clarithromycin. Many isolates of M. fortuitum are susceptible to doxycycline, sulfamethoxazole, and imipenem, while many M. abscessus isolates are resistant to these drugs. In the present study, the modified broth microdilution method using STC was found to be reliable, easy to read, and inexpensive for M. fortuitum and M. abscessus susceptibility testing. The modified colorimetric MIC testing method using STC was proven to be a useful surrogate for RGM antibiotic susceptibility testing.
Asunto(s)
Pruebas de Sensibilidad Microbiana , Mycobacterium fortuitum/metabolismo , Mycobacterium/metabolismo , Sales de Tetrazolio/farmacología , Antibacterianos/farmacología , Cefoxitina/farmacología , Química Farmacéutica/métodos , Ciprofloxacina/farmacología , Claritromicina/farmacología , Colorimetría/métodos , Farmacorresistencia Bacteriana , Corea (Geográfico)RESUMEN
Mycobacterium xenopi is a nontuberculous mycobacterium (NTM) that rarely causes pulmonary disease in Asia. Here we describe the first case of M. xenopi pulmonary disease in Korea. A 66-year-old man was admitted to our hospital with a 2-month history of productive cough and hemoptysis. His past medical history included pulmonary tuberculosis 44 years earlier, leading to a right upper lobectomy. Chest X-ray upon admission revealed cavitary consolidation involving the entire right lung. Numerous acid-fast bacilli were seen in his initial sputum, and M. xenopi was subsequently identified in more than five sputum cultures, using molecular methods. Despite treatment with clarithromycin, rifampicin, ethambutol, and streptomycin, the infiltrative shadow revealed on chest X-ray increased in size. The patient's condition worsened, and a right completion pneumonectomy was performed. The patient consequently died of respiratory failure on postoperative day 47, secondary to the development of a late bronchopleural fistula. This case serves as a reminder to clinicians that the incidence of NTM infection is increasing in Korea and that unusual NTM are capable of causing disease in non-immunocompromised patients.
Asunto(s)
Enfermedades Pulmonares/diagnóstico , Enfermedades Pulmonares/microbiología , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Mycobacterium xenopi/aislamiento & purificación , Anciano , Proteínas Bacterianas/genética , Proteínas de Choque Térmico/genética , Humanos , Corea (Geográfico) , Enfermedades Pulmonares/diagnóstico por imagen , Masculino , Infecciones por Mycobacterium no Tuberculosas/diagnóstico por imagen , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium xenopi/clasificación , Mycobacterium xenopi/genética , Filogenia , Radiografía , Análisis de Secuencia de ADNRESUMEN
A proficiency review of antituberculous drug susceptibility testing (DST) was undertaken by the regional tuberculosis reference laboratories of the Western Pacific Region of WHO to evaluate the performance of national reference laboratories (NRLs) and to ensure that the results from the participating laboratories are reliable and similar. A panel of 30 Mycobacterium tuberculosis strains with various patterns of resistance to isoniazid, rifampin, ethambutol, and streptomycin was sent to the NRLs, and their DST results were analyzed by comparing them with the judicial results. The efficiency scores for each drug were 90 to 99% (mean, 95%) for isoniazid, 77 to 100% (mean, 94%) for rifampin, 82 to 97% (mean, 90%) for ethambutol, and 82 to 98% (mean, 89%) for streptomycin. Significant changes over time in the rates of accordance with the judicial results were observed for rifampin (P < 0.0001) and streptomycin (P = 0.0002), whereas no changes were observed for ethambutol (P = 0.0880). The efficiency score for isoniazid was consistently good throughout the nine rounds. As a whole, NRL02 showed the highest score (95%) in accordance rates for all drugs, while NRL03 (86%) and NRL04 (88%) ranked lowest. Continued proficiency testing with subsequent technical assistance improved the DST quality of participating laboratories, demonstrating the importance of the current WHO/IUATLD external quality assurance program for DST proficiency testing.
Asunto(s)
Antituberculosos/farmacología , Pruebas de Sensibilidad Microbiana/normas , Mycobacterium tuberculosis/efectos de los fármacos , Humanos , Laboratorios , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
In Korea, the Mycobacterium tuberculosis K-strain is the most prevalent clinical isolates and belongs to the Beijing family. In this study, we conducted comparative porteomics of expressed proteins of clinical isolates of the K-strain with H37Rv, H37Ra as well as the vaccine strain of Mycobacterium bovis BCG following phagocytosis by the human monocytic cell line U-937. Proteins were analyzed by 2-D PAGE and MALDITOF-MS. Two proteins, Mb1363 (probable glycogen phosphorylase GlgP) and MT2656 (Haloalkane dehalogenase LinB) were most abundant after phagocytosis of M. tuberculosis K-strain. This approach provides a method to determine specific proteins that may have critical roles in tuberculosis pathogenesis.
Asunto(s)
Macrófagos/microbiología , Mycobacterium bovis/química , Mycobacterium tuberculosis/química , Proteoma/análisis , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Humanos , Corea (Geográfico) , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Mycobacterium tuberculosis strains that are resistant to an increasing number of second-line drugs used to treat multidrug-resistant tuberculosis (MDR TB) are becoming a threat to public health worldwide. We surveyed the Network of Supranational Reference Laboratories for M. tuberculosis isolates that were resistant to second-line anti-TB drugs during 2000-2004. We defined extensively drug-resistant TB (XDR TB) as MDR TB with further resistance to > or = 3 of the 6 classes of second-line drugs. Of 23 eligible laboratories, 14 (61%) contributed data on 17,690 isolates, which reflected drug susceptibility results from 48 countries. Of 3,520 (19.9%) MDR TB isolates, 347 (9.9%) met criteria for XDR TB. Further investigation of population-based trends and expanded efforts to prevent drug resistance and effectively treat patients with MDR TB are crucial for protection of public health and control of TB.
Asunto(s)
Antituberculosos/farmacología , Salud Global , Mycobacterium tuberculosis/efectos de los fármacos , Vigilancia de Guardia , Tuberculosis/prevención & control , Control de Enfermedades Transmisibles , Humanos , Laboratorios , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Tuberculosis Resistente a Múltiples MedicamentosRESUMEN
Although mycobacterial culture and the subsequent drug-susceptibility test (DST) for anti-tuberculosis (TB) drugs take several months to complete using solid media, there are no reports on the turnaround times of these tests under clinical conditions. The aim of this study was to determine the interval between initiation of anti-TB treatment and receipt of DST requested at an outpatient clinic. We prospectively enrolled patients with culture-positive pulmonary TB at Seoul National University Hospital from September 2002 to December 2004. Patients were followed up monthly. Mycobacterial cultures were done using Ogawa media at Seoul National University Hospital. DST were performed at the Korean Institute of Tuberculosis. Of the 104 patients enrolled, 54 were male. The median age was 41 yr. The median interval from initiation of anti-TB treatment to receipt of mycobacterial culture results by clinicians was 37 days (range, 0-89 days). The median interval from initiation of treatment to confirmation of DST by requesting clinicians was 80.5 days (range, 28-145 days). Clinicians only received the results of DST more than two months after initiation of treatment when they followed up patients monthly and mycobacterial culture was performed using solid media.
Asunto(s)
Antituberculosos/uso terapéutico , Pruebas de Sensibilidad Microbiana , Tuberculosis Pulmonar/tratamiento farmacológico , Adolescente , Adulto , Anciano , Antituberculosos/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de TiempoRESUMEN
The clinical efficacy of a spoligotyping method for discriminating Mycobacterium tuberculosis strains was evaluated. Among the strains other than Beijing or Beijing like family, 30 different spoligotypes out of 39 strains were produced, which included 4 strains not having IS6110 sequence. The oligonucleotide chip-based spoligotyping technique is useful for early screening and detection of clonal proximity of M. tuberculosis isolates.
Asunto(s)
Dermatoglifia del ADN/métodos , Mycobacterium tuberculosis/clasificación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Tuberculosis/microbiología , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Intergénico/química , ADN Intergénico/genética , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificaciónRESUMEN
Here we describe a novel duplex PCR method which can differentiate Mycobacterium tuberculosis and nontuberculosis mycobacteria (NTM) strains by amplifying hsp65 DNAs of different sizes (195 and 515 bp, respectively). The devised technique was applied to 54 reference and 170 clinical isolates and differentiated all strains into their respective groups with 100% sensitivity and specificity. Furthermore, a duplex PCR-restriction analysis (duplex PRA) and a direct sequencing protocol were developed to differentiate NTM strains at the species and subspecies levels based on previously reported hsp65 DNA sequences (H. Kim et al., Int. J. Syst. Evol. Microbiol. 55:1649-1656, 2005) and then applied to 105 NTM clinical isolates. All NTM isolates were clearly differentiated at the species and subspecies levels by subsequent procedures (PRA or direct sequencing) targeting 515-bp NTM duplex PCR amplicons. Our results suggest that novel duplex PCR-based methods are sensitive and specific for identifying mycobacterial culture isolates at the species level.
Asunto(s)
Proteínas Bacterianas/genética , Chaperoninas/genética , Mycobacterium tuberculosis/clasificación , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN , Chaperonina 60 , Humanos , Mycobacterium tuberculosis/genética , Sensibilidad y EspecificidadRESUMEN
An oligonucleotide chip (Combichip Mycobacteria chip) detecting specific mutations in the rpoB, katG, and inhA genes of Mycobacterium tuberculosis was compared with conventional antimicrobial susceptibility results. The probes detecting drug resistance were as follows: 7 wild-type and 13 mutant probes for rifampin and 2 wild-type and 3 mutant probes for isoniazid. Target DNA of M. tuberculosis was amplified by PCR, followed by hybridization and scanning. Direct sequencing was performed to verify the results of the oligonucleotide chip. One-hundred seven of 115 rifampin-resistant strains (93%) had mutations in the rpoB gene. Eighty-five of 119 isoniazid-resistant strains (71%) had mutations in the katG gene or inhA gene. The diagnostic oligonucleotide chip with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of resistance against rifampin and isoniazid in M. tuberculosis isolates.
Asunto(s)
Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Antibióticos Antituberculosos/farmacología , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Secuencia de Bases , Catalasa/genética , Sondas de ADN/genética , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN , Farmacorresistencia Bacteriana/genética , Genes Bacterianos , Humanos , Isoniazida/farmacología , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Oxidorreductasas/genética , Reacción en Cadena de la Polimerasa/métodos , Rifampin/farmacologíaRESUMEN
STUDY OBJECTIVES: Precise epidemiologic data regarding nontuberculous mycobacterial (NTM) lung infection in many Asian countries have been relatively unavailable. In order to determine the clinical significance of NTM isolated from respiratory specimens, we reviewed medical records from all patients from whom NTM isolates were recovered within a 2-year period. MATERIALS AND METHODS: We identified all NTM isolates from respiratory specimens at the Samsung Medical Center (Seoul, South Korea) obtained from January 2002 to December 2003. We then reviewed the clinical and radiologic characteristics of the patients from whom NTM was isolated. Patients were classified as having either definite, probable, or unlikely NTM lung disease, as defined by the guidelines of both the American Thoracic Society and the British Thoracic Society. RESULTS: During the study period, 1,548 NTM isolates were recovered from 794 patients. Of these 794 patients, 131 patients (17%) were determined to have definite NTM lung disease, and 64 patients (8%) were designated as probable disease candidates. The most commonly involved organisms in the definite and probable NTM lung disease cases were Mycobacterium avium complex (n = 94, 48%) and Mycobacterium abscessus (n = 64, 33%). In 195 patients with NTM lung disease, 82 patients (42%) manifested the upper lobe cavitary form, 101 patients (52%) exhibited the nodular bronchiectatic form, and 12 patients (6%) exhibited the unclassifiable form. CONCLUSIONS: About one fourth of the patients in whom NTM was isolated from respiratory specimens were found to have clinically significant NTM lung infections. The spectrum of organisms responsible for the NTM lung disease in these Korean patients differed from those reported in other regions of the world. However, the estimates of clinical significance in this study may be underestimates due to the retrospective analysis. Some of the patients might have "true" NTM lung disease that could be diagnosed with continued evaluation and follow-up.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Micobacterias no Tuberculosas/aislamiento & purificación , Tuberculosis Pulmonar/diagnóstico , Adulto , Anciano , Bronquios/microbiología , Femenino , Humanos , Corea (Geográfico)/epidemiología , Masculino , Persona de Mediana Edad , Infecciones por Mycobacterium no Tuberculosas/diagnóstico por imagen , Infecciones por Mycobacterium no Tuberculosas/epidemiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Radiografía , Esputo/microbiología , Tuberculosis Pulmonar/diagnóstico por imagen , Tuberculosis Pulmonar/epidemiología , Tuberculosis Pulmonar/microbiologíaRESUMEN
Mycobacterium kansasii is one of the most common cause of pulmonary diseases due to nontuberculous mycobacteria. We investigated the changing in the number of isolation of M. kansasii and the clinical characteristics of M. kansasii pulmonary disease in Korea. Through searching the database of the Korean Institute of Tuberculosis, we identified the cases of isolated M. kansasii from 1992 to 2002. The number of M. kansasii isolation had increased from once in 1992 to 62 in 2002. Fifteen patients with M. kansasii pulmonary disease were identified during the period January 1997 to December 2002. Twelve patients (80%) were male and fourteen (93%) were from highly industrialized areas. The most common symptom was a cough. Seven patients (47%) had a cavitary lesion and right upper lobe was most commonly involved. Patients responded well to isoniazid and rifampicin based regimens both bacteriologically and radiographically. In conclusion, M. kansasii isolation has increased, especially in highly industrialized areas, as well as other nontuberculous mycobacteria in Korea.
Asunto(s)
Enfermedades Pulmonares/epidemiología , Infecciones por Mycobacterium no Tuberculosas/epidemiología , Mycobacterium kansasii , Adulto , Anciano , Antibacterianos/uso terapéutico , Femenino , Humanos , Corea (Geográfico)/epidemiología , Enfermedades Pulmonares/diagnóstico , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/microbiología , Masculino , Persona de Mediana Edad , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium kansasii/aislamiento & purificaciónRESUMEN
The nucleotide sequences (604 bp) of partial heat-shock protein genes (hsp65) from 161 Mycobacterium strains containing 56 reference Mycobacterium species and 105 clinical isolates were determined and compared. hsp65 sequence analysis showed a higher degree of divergence between Mycobacterium species than did 16S rRNA gene analysis. Generally, the topology of the phylogenetic tree based on the hsp65 DNA sequences was similar to that of the 16S rRNA gene, thus revealing natural relationships among Mycobacterium species. When a direct sequencing protocol targeting 422 bp sequences was applied to 70 non-tuberculous mycobacterium (NTM) clinical isolates, all NTMs were clearly identified. In addition, an XhoI PCR restriction fragment length polymorphism analysis method for the differentiation of Mycobacterium tuberculosis complex from NTM strains was developed during this study. The results obtained suggest that 604 bp hsp65 sequences are useful for the phylogenetic analysis and species identification of mycobacteria.
Asunto(s)
Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Chaperoninas/genética , Mycobacterium/clasificación , Chaperonina 60 , ADN Bacteriano/análisis , ADN Ribosómico/análisis , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Humanos , Datos de Secuencia Molecular , Mycobacterium/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
A method based on PCR-restriction fragment length polymorphism analysis (PRA) using a novel region of the hsp65 gene was developed for the rapid and exact identification of mycobacteria to the species level. A 644 bp region of hsp65 in 62 mycobacteria reference strains, and 4 related bacterial strains were amplified, and the amplified DNAs were subsequently digested with restriction enzymes, namely, AvaII, HphI, and HpaII. Most of the mycobacteria species were easily differentiated at the species level by the developed method. In particular, the method enabled the separation of M. avium, M. intracellulare and M. tuberculosis to the species level by AvaII digestion alone. An algorithm was constructed based on the results and a blind test was successfully performed on 251 clinical isolates, which had been characterized by conventional biochemical testing. Our results suggest that this novel PRA offers a simple, rapid, and accurate method for the identification of mycobacteria culture isolates at the species level.
Asunto(s)
Proteínas Bacterianas/genética , Chaperoninas/genética , Mycobacterium/clasificación , Reacción en Cadena de la Polimerasa/métodos , Algoritmos , Proteínas Bacterianas/química , Chaperonina 60 , Chaperoninas/química , ADN Bacteriano/química , ADN Bacteriano/genética , Humanos , Mycobacterium/genética , Mycobacterium/aislamiento & purificación , Infecciones por Mycobacterium/microbiología , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADNRESUMEN
The frequency of resistance genotypes among Beijing and non-Beijing strains was compared using a reverse blot hybridization assay to detect mutations within genes associated with rifampicin (rpoB) and isoniazid (katG, inhA, and ahpC) resistance. Of the 743 Mycobacterium tuberculosis isolates, 569 (77%) belonged to Beijing family. The proportion of Beijing strains was significantly higher among MDR-TB isolates than among drug-susceptible strains (82% vs. 72%, p<0.01). Genotype analysis of the rpoB gene revealed significantly lower rates of the Ser531Leu mutation rate among Beijing vs. non-Beijing MDR-TB strains (41% vs. 66%, p<0.005). While the mutation for Ser315Thr in the katG gene was more common among Beijing vs. non-Beijing family strains (65% vs. 50%, p<0.01), the mutation rate of promoter region of the inhA gene was lower among Beijing strains compared with non-Beijing strains (14% vs. 25%, p<0.05). Reverse hybridization successfully detected over 80% of isoniazid-resistant strains and over 92% of rifampicin-resistant strains among Korean isolates. Significant differences in mutation rates in the rpoB, katG, and inhA genes between Beijing strains and non-Beijing strains could explain discrepancies in mutation rates of genotypes in different countries. Reverse hybridization was useful for rapid detection of isoniazid and rifampicin resistant strains.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Tuberculosis Pulmonar/epidemiología , Proteínas Bacterianas/genética , China , Genotipo , Humanos , Corea (Geográfico)/epidemiología , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , Prevalencia , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Pulmonar/microbiologíaRESUMEN
The species identification within Mycobacterium terrae complex has been known to be very difficult. In this study, the genomic diversity of M. terrae complex with eighteen clinical isolates, which were initially identified as M. terrae complex by phenotypic method, was investigated, including that of three type strains (M. terrae, M. nonchromogenicum, and M. triviale ). 16S rRNA and 65-kDa heat shock protein (hsp 65) gene sequences of mycobacteria were determined and aligned with eleven other references for the comparison using similarity search against the GenBank and Ribosomal Database Project II (RDP) databases. 16S rRNA and hsp 65 genes of M. terrae complex showed genomic heterogeneity. Amongst the eighteen clinical isolates, nine were identified as M. nonchromogenicum, eight as M. terrae, one as M. mucogenicum with the molecular characteristic of rapid growth. M. nonchromogenicum could be subdivided into three subgroups, while M. terrae could be subdivided into two subgroups using a 5 bp criterion (>1% difference). Seven isolates in two subgroups of M. nonchromogenicum were Mycobacterium sp. strain MCRO 6, which was closely related to M. nonchromogenicum. The hsp 65 gene could not differentiate one M. nonchromogenicum from M. avium or one M. terrae from M. intracellulare. The nucleotide sequence analysis of 16S rRNA and hsp 65 genes was shown to be useful in identifying the M. terrae complex, but hsp 65 was less discriminating than 16S rRNA.
Asunto(s)
Proteínas Bacterianas/genética , Chaperoninas/genética , Micobacterias no Tuberculosas/genética , ARN Ribosómico 16S/genética , Secuencia de Bases , Chaperonina 60 , ADN Bacteriano/análisis , ADN Ribosómico/análisis , Bases de Datos Factuales , Variación Genética , Genoma Bacteriano , Datos de Secuencia Molecular , Micobacterias no Tuberculosas/química , Micobacterias no Tuberculosas/clasificación , Filogenia , ARN Ribosómico 16S/análisisRESUMEN
Sequence analysis of a specific region of the mycobacterium rpoB gene in 35 mycobacterial strains representing 26 different mycobacterial species of clinical importance showed that there exists a highly polymorphic region. Based on the sequences of the polymorphic region, the oligonucleotide probes of 14 mycobacterial species with relatively high clinical importance were designed and shown to be specific to their corresponding mycobacterial species by dot blot hybridization. The results showed that the probes designed in this study are highly specific to each mycobacterial species, which suggests that these sequences may be useful for the species identification of mycobacteria.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Genes Bacterianos , Mycobacterium/genética , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Sondas de ADN/genética , ADN Bacteriano/genética , Humanos , Datos de Secuencia Molecular , Mycobacterium/clasificación , Mycobacterium/enzimología , Polimorfismo de Longitud del Fragmento de Restricción , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Resistance of Mycobacterium tuberculosis to ethambutol (EMB) has been assigned to an operon, embCAB, which has been proposed to be a structural gene for mycobacterial arabinosyl transferases. Recently, genetic events resulting in structural mutations at embB have been proposed as major contributors to the EMB-resistance of isolates whose minimum inhibitory concentration (MIC) level is higher than 20 microgram/ml. On the contrary, isolates with a MIC level lower than 20 microgram/ml do not seem to contain any sequence alterations. In this study, in an effort to understand the role of embB mutations at a low-level of EMB resistance, we investigated the sequence polymorphisms of clinical isolates whose MIC levels are lower than 10 microgram/ml. Accordingly, the sequence alterations of a 312-bp region of the embB gene containing the 306th codon, which has been assigned as a hot-spot for EMB-resistance related mutations, were determined for 21 EMB-resistant and 5 EMB-susceptible clinical isolates. In brief, among 21 EMB- resistant isolates examined, 12 (57.1%) contained mutations in embB (10 at the 306th codon and 2 at other sites), and the remaining isolates 9 contained no mutations in any region of embB. The observed mutations included M306V, M306I, and M306L substitutions that have been reported previously. However, 3 were novel types, which included M306T, A313G and Y319C, D328Y [corrected] double substitutions. On the other hand, all of the EMB-susceptible isolates were found to be free of mutations. In conclusion, our findings suggest that sequence polymorphism of embB may play a pivotal role in the EMB- resistance of M. tuberculosis.