Our study was to investigate the epidemiological characteristics of M.tuberculosis from a national tuberculosis referral center in China. All strains isolated from TB patients, were genotyped by the RD105 deletion, 8 and 51 SNP loci and VNTR. The high differentiation SNPs of modern Beijing strains were analyzed for protein function and structure. 413 M. tuberculosis were included. Of 379 Beijing lineage M. tuberculosis, 'modern' and 'ancient' strains respectively represented 85.5% (324/379) and 14.5% (55/379). Rv2494 (V48A) and Rv0245 (S103F) were confirmed as high differentiation SNPs associated with modern strains. In a word, Modern Beijing lineage M.tuberculosis was dominant and the structural models suggested that modern sub-lineage may more easily survive in 'extreme' host condition.
China ; epidemiology ; DNA, Bacterial ; genetics ; Genome, Bacterial ; Hospitals, Chronic Disease ; Humans ; Molecular Epidemiology ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Phylogeny ; Phylogeography ; Polymorphism, Single Nucleotide ; Tuberculosis ; epidemiology ; microbiology

No abstract available.
Aged ; Bronchiectasis/*diagnosis/microbiology ; Humans ; Male ; Mycobacterium/classification/*genetics/isolation & purification ; Mycobacterium Infections/*diagnosis/microbiology ; Phylogeny ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; Republic of Korea ; Sequence Analysis, DNA

Mycobacterium longobardum is a slow-growing, nontuberculous mycobacterium that was first characterized from the M. terrae complex in 2012. We report a case of M. longobardum induced chronic osteomyelitis. A 71-yr-old man presented with inflammation in the left elbow and he underwent a surgery under the suspicion of tuberculous osteomyelitis. The pathologic tissue culture grew M. longobardum which was identified by analysis of the 65-kDa heat shock protein and full-length 16S rRNA genes. The patient was cured with the medication of clarithromycin and ethambutol without further complications. To the best of our knowledge, this is the first report of a M. longobardum infection worldwide.
Aged ; Anti-Bacterial Agents/therapeutic use ; Bacterial Proteins/genetics ; Chaperonin 60/genetics ; Clarithromycin/therapeutic use ; Elbow/pathology ; Ethambutol/therapeutic use ; Humans ; Male ; Mycobacterium Infections, Nontuberculous/*microbiology ; Nontuberculous Mycobacteria/classification/genetics/*isolation & purification ; Osteomyelitis/diagnosis/drug therapy/*microbiology/pathology ; RNA, Ribosomal, 16S/genetics ; Treatment Outcome

OBJECTIVETo identify the novel species 'Mycobacterium fukienense' sp. nov of Mycobacterium chelonae/abscessus complex from tuberculosis patients in Fujian Province, China.

METHODSFive of 27 clinical Mycobacterium isolates (Cls) were previously identified as M. chelonae/abscessus complex by sequencing the hsp65, rpoB, 16S-23S rRNA internal transcribed spacer region (its), recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobacterium. Clinical Mycobacterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed.

RESULTSThe 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 16S-23S rRNA internal transcribed spacer region (its), sodA, and recA genes as compared with the M. abscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences.

CONCLUSIONThe novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. chelonea/abscessus complex.

Bacterial Proteins ; genetics ; Base Sequence ; China ; epidemiology ; Cluster Analysis ; DNA, Bacterial ; genetics ; Humans ; Molecular Sequence Data ; Mycobacterium ; classification ; genetics ; isolation & purification ; Mycobacterium Infections, Nontuberculous ; epidemiology ; microbiology ; Mycobacterium chelonae ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Tuberculosis ; epidemiology ; microbiology

PURPOSE: The Mycobacterium tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis bacillus Calmette-Guerin (BCG) and M. africanum, and causes tuberculosis in humans and animals. Identification of Mycobacterium spp. and M. tuberculosis complex to the species level is important for practical use in microbiological laboratories, in addition to optimal treatment and public health. MATERIALS AND METHODS: A novel multiplex PCR assay targeting a conserved rpoB sequence in Mycobacteria spp., as well as regions of difference (RD) 1 and RD8, was developed and evaluated using 37 reference strains and 178 clinical isolates. RESULTS: All mycobacterial strains produced a 518-bp product (rpoB), while other bacteria produced no product. Virulent M. tuberculosis complex strains, M. tuberculosis, M. bovis and M. africanum, produced a 254-bp product (RD1), while M. bovis BCG, M. microti and nontuberculous mycobacteria produced no RD1 region product. Additionally, M. tuberculosis and M. africanum produced a 150-bp product (RD8), while M. bovis and M. bovis BCG produced a 360-bp product (deleted form of RD8). M. microti and nontuberculous mycobacteria produced no RD8 region product. This assay identified all Mycobacterium spp. and all M. tuberculosis complex strains to the species level. CONCLUSION: The multiplex PCR assay of the present study could be implemented as a routine test in microbiology laboratories, and may contribute to more effective treatment and surveillance of tuberculosis stemming from the M. tuberculosis complex.
Animals ; Cattle ; Classification/methods ; DNA Primers ; Genes, Bacterial ; Humans ; Multiplex Polymerase Chain Reaction/*methods ; Mycobacterium/classification/genetics/*isolation & purification ; Mycobacterium tuberculosis/classification/genetics/*isolation & purification ; Species Specificity

We herein report a case in which the recently characterized species Mycobacterium monacense was isolated from the sputum of an Iranian patient. This case represents the first isolation of M. monacense from Iran. The isolate was identified by conventional and molecular techniques. Our findings show that M. monacense infection is not restricted to developed countries.
Bacterial Proteins/genetics ; Chaperonin 60/genetics ; Chronic Disease ; Female ; Humans ; Iran ; Lung Diseases/diagnosis/*microbiology ; Middle Aged ; Mycobacterium/classification/*genetics/isolation & purification ; Mycobacterium Infections/*microbiology/pathology ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/genetics ; Sputum/microbiology

A slowly growing, non-chromogenic mycobacterial strain was isolated from sputum and bronchial lavage fluid samples of a patient presenting with productive cough, blood-tinged sputum, low-grade fever, and weakness. A positive acid-fast bacilli sputum smear result prompted the initiation of an anti-tuberculosis regimen. Multiplex real-time PCR showed a negative result for Mycobacterium tuberculosis complex and a positive result for nontuberculous mycobacteria. The DNA chip test confirmed this organism as a member of the genus Mycobacterium, but could not specify the species. Interestingly, the mycolic acid patterns obtained by HPLC nearly overlapped with those of M. simulans. The sequences of the Mycobacterium 16S rRNA gene and 16S-23S internal transcribed spacer region were unique and were found to have 100% similarity with those of M. riyadhense. After a review of the literature, we report this case as the first Korean case of M. riyadhense lung infection.
Adult ; Antitubercular Agents/pharmacology ; Chromatography, High Pressure Liquid ; Female ; Humans ; Lung Diseases/*microbiology ; Microbial Sensitivity Tests ; Mycobacterium/classification/drug effects/*isolation & purification ; Mycobacterium Infections/microbiology ; Mycobacterium tuberculosis/genetics/isolation & purification ; Mycolic Acids/analysis ; Oligonucleotide Array Sequence Analysis ; Phylogeny ; RNA, Ribosomal, 16S/chemistry/genetics ; RNA, Ribosomal, 23S/chemistry/genetics ; Republic of Korea ; Sequence Analysis, DNA

BACKGROUND: Single-nucleotide polymorphism (SNP) analysis is a powerful strategy for large-scale molecular population studies examining phylogenetic relationships among bacterial strains. Mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) can be easily digitized to share data among laboratories. This study applied SNP and MIRU-VNTR analyses for molecular strain typing of Mycobacterium tuberculosis isolates collected throughout Korea. METHODS: We studied 102 clinical M. tuberculosis isolates, including 6 paired strains, collected from 11 university hospitals in Korea in 2008 and 2009. SNPs were detected using hairpin primer assays, and then, MIRU-VNTR analysis was performed. RESULTS: Thirty-five SNPs contained polymorphisms that helped differentiate the 96 tested isolates. The isolates were classified into 15 clusters. The Beijing family strains were distributed within closely related clusters in the SNP dendrogram. For MIRU-VNTR analysis, the 96 isolates were divided into 12 groups. The discriminatory index in 8 of these groups (MIRU-10, -23, -26, and -31; ETR-A, -B, -C, and -F) was high (Hunter-Gaston diversity index > 0.6). Unlike the SNP method, MIRU-VNTR analysis did not identify any notable localizations of Beijing or non-Beijing family isolates in specific clusters. CONCLUSIONS: SNP and MIRU-VNTR analyses are surrogate molecular strain-typing methods for M. tuberculosis in Korea where Beijing family isolates are predominant.
Cluster Analysis ; DNA Primers/chemistry ; Interspersed Repetitive Sequences ; *Minisatellite Repeats ; Mycobacterium tuberculosis/*classification/genetics/isolation & purification ; Phylogeny ; *Polymorphism, Single Nucleotide ; Republic of Korea

Tuberculosis remains a severe public health problem worldwide. Presently, genotyping is used for conducting epidemiologic and clinical studies on tuberculosis cases. We evaluated the efficacy of the repetitive sequence-based PCR (rep-PCR)-based DiversiLab(TM) system (bioMerieux, France) over the IS6110-restriction fragment length polymorphism analysis for detecting Mycobacterium tuberculosis. In all, 89 clinical M. tuberculosis isolates collected nationwide from Korea were used. The DiversiLab system allocated the 89 isolates to 8 groups with 1 unique isolate when a similarity level of 95% was applied. Seventy-six isolates of the Beijing family and 13 isolates of non-Beijing family strains were irregularly distributed regardless of rep-PCR groups. The DiversiLab system generated a rapid, sensitive, and standardized result. It can be used to conduct molecular epidemiologic studies to identify clinical M. tuberculosis isolates in Korea.
Automation ; *Bacterial Typing Techniques ; *Epidemiologic Methods ; Genotype ; Humans ; Mycobacterium tuberculosis/*classification/genetics/isolation & purification ; *Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Reagent Kits, Diagnostic ; Repetitive Sequences, Nucleic Acid ; Republic of Korea/epidemiology ; Tuberculosis/diagnosis/*epidemiology/microbiology

The Beijing family of Mycobacterium tuberculosis has been emerging in the world. However, there are few nationwide data of genotypic distribution in Korea. This study aimed to identify the genotypic diversity of clinical isolates of M. tuberculosis and to demonstrate the population of Beijing family in Korea. We collected 96 clinical M. tuberculosis isolates from 11 university hospitals nationwide in Korea from 2008 to 2009. We observed 24 clusters in IS6110-RFLP analysis and 19 patterns in spoligotyping. Seventy-five isolates were confirmed to be Beijing family. Two isolates of the K strain and 12 isolates of the K family strain were also found. We found that drug resistance phenotypes were more strongly associated with Beijing family than non-Beijing family (P=0.003). This study gives an overview of the distribution of genotypes of M. tuberculosis in Korea. These findings indicate that we have to pay more attention to control of M. tuberculosis strains associated with the Beijing family.
Drug Resistance, Bacterial ; Genotype ; Humans ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis/*classification/genetics/isolation & purification ; Phenotype ; Polymorphism, Restriction Fragment Length ; Republic of Korea ; Tuberculosis/*epidemiology/genetics/microbiology