OBJECTIVEWe determined the genetic diversity of Mycobacterium tuberculosis (MTB) in a remote mountainous area of southwest China and evaluated the resolving ability of single nucleotide polymorphism (SNP) genotyping combined with variable number tandem repeat (VNTR) genotyping for Beijing family strains in association with drug resistance status.

METHODSThree hundred thirty-one MTB strains were isolated from patients living in mountainous regions of southwest China, and 8-loci SNP, VNTR-15 genotyping assays, and drug susceptibility testing of 9 drugs were performed.

RESULTSA total of 183 [55.29% (183/331)] strains were classified into the Beijing family. Of the 183 strains, 111 (60.66%) were defined as modern Beijing strains. The most predominant modern Beijing sub-lineage and ancient Beijing sub-lineage were Bmyc10 [39.34% (72/183)] and Bmyc25 [20.77% (38/183)], respectively. Of the isolates, 19.64% (65/331) were resistant to at least 1 of the 9 anti-TB drugs and 17 [4.98% (17/331)] MTB isolates were multi-drug resistant tuberculosis (MDR-TB). Two hundred sixty-one isolates showed a clustering rate of 14.18% (37/261) and a discriminatory index of 0.9990. The Beijing lineage exhibited a significantly higher prevalence of MDR-TB, as well as resistance to isoniazid (INH), rifampin (RIF), and para-aminosalicylic acid (PAS) when analyzed independently (P = 0.005, P = 0.017, P = 0.014, and P = 0.006 respectively). The Beijing lineage was not associated with genetic clustering or resistance to any drug. In addition, genetic clustering was not associated with drug resistance.

CONCLUSIONMTB strains demonstrate high genetic diversity in remote mountainous areas of southwest China. Beijing strains, especially modern Beijing strains, are predominant in remote mountainous area of China. The combination of 8-loci SNPs and VNTR-15 genotyping is a useful tool to study the molecular epidemiology of MTB strains in this area.

Antitubercular Agents ; pharmacology ; China ; epidemiology ; Cluster Analysis ; Drug Resistance, Bacterial ; Genotype ; Humans ; Mycobacterium tuberculosis ; drug effects ; genetics ; Phylogeny ; Polymorphism, Single Nucleotide ; Tuberculosis ; epidemiology ; microbiology

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

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

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

Bovine tuberculosis (TB) is a major zoonosis that's caused by Mycobacterium bovis (M. bovis). Being able to detect M. bovis is important to control bovine TB. We applied a molecular technique, the variable number tandem repeat (VNTR) typing method, to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea. From 2003 to 2004, 59 M. bovis clinical strains were isolated from dairy cattle in Gyeonggi-do, Korea, and these cattle had tuberculosis-like lesions. Twenty-four published MIRUVNTR markers were applied to the M. bovis isolates and ten of them showed allelic diversity. The most discriminatory locus for the M. bovis isolates in Korea was QUB 3336 (h = 0.64). QUB 26 and MIRU 31 also showed high discriminative power (h = 0.35). The allelic diversity by the combination of all VNTR loci was 0.86. Six loci (MIRU 31, ETR-A and QUB-18, -26, -3232, -3336) displayed valuable allelic diversity. Twelve genotypes were identified from the 59 M. bovis isolates that originated from 20 cattle farms that were dispersed throughout the region of Gyenggi-do. Two genotypes [designation index (d.i.) = e, g] showed the highest prevalence (20% of the total farms). For the multiple outbreaks on three farms, two successive outbreaks were caused by the same genotype at two farms. Interestingly, the third outbreak at one farm was caused by both a new genotype and a previous genotype. In conclusion, this study suggests that MIRU-VNTR typing is useful to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea.
Animals ; Cattle ; DNA Primers/genetics ; *Genetic Variation ; Genotype ; Korea/epidemiology ; Minisatellite Repeats/*genetics ; Mycobacterium bovis/*genetics ; Prevalence ; Tuberculosis, Bovine/*epidemiology/*microbiology

OBJECTIVETo assess the application of IS6110-restriction fragment length polymorphism (RFLP), Spoligotyping and mycobacterial interspersed repetitive unit (MIRU) in epidemiological studies of tuberculosis and to discuss the characteristics of Mycobacterium tuberculosis strains in different regions in China.

METHODSMycobacterium tuberculosis strains, with a total number of 158 isolates, were subjected to IS6110-RFLP, Spoligotyping and MIRU.

RESULTSThe numbers of patterns produced by IS6110-RFLP, Spoligotyping, and MIRU typing were 118, 20 and 105 respectively. The discriminatory power of IS6110-RFLP was higher than that of Spoligotyping. However, when the copies of IS6110 were lower than 10, the discriminatory power of Spoligotyping improved obviously. The discriminatory power of MIRU typing was close to that of IS6110-RFLP for typing of Mycobacterium tuberculosis. In MIRU loci, there were four loci (loci 4, 10, 26, 40) with higher diversity. Significant differences among the Mycobacterium tuberculosis between Guangdong and other regions in clustered rate and the proportion of Beijing genotype (P < 0.05) were found. The clustered rates and the proportion of Beijing genotype in Guangdong were lower than that in other regions.

CONCLUSIONThe results of this study indicated that either IS6110-RFLP, Spoligotyping or MIRU technique was useful for epidemiological studies on tuberculosis in China and the strains in different regions had different characterishes in China.

China ; epidemiology ; DNA Fingerprinting ; methods ; DNA, Bacterial ; genetics ; Humans ; Molecular Epidemiology ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length ; Tuberculosis ; epidemiology ; microbiology

OBJECTIVETo study the correlation between DNA fingerprinting of Mycobacterium tuberculosis (MTB) stains isolated from the Chinese army in the south and from local residents, and to investigate the molecular epidemiological characteristics of tuberculosis (TB) in the army, for the sake of TB prevention in the army.

METHODSMTB DNA was digested with restriction endonuclease PvuII and electrophoresed in agarose gel, after Southern Blotting, the membrane was hybridized with a 245 bp fragment of IS6110 which labeled [alpha(32)P]-dCTP as probe. Finally, a restriction fragment length polymorphism (RFLP) patterns was shown, and analyzed logestic with epidemiological data from the patients.

RESULTSA total number of 185 TB strains were detected and the IS6110 copy numbers ranged from 1 - 22. No significant difference was found in the IS6110 copy numbers between patients from army and local patients. IS6110 copy numbers of TB strains in army patients were centered in 6 - 20, however, with 7 - 20 copies in local TB patients. The TB strains were dispersed into 8 groups and the majority of TB strains in both army and local patients was centered in groups I, II, III. The distribution of DNA fingerprint for drug resistance TB strains was significantly different from those for sensitive strains. No different distribution of among groups was found regarding BCG history.

CONCLUSIONSThe genetics of TB stains were roughly the same between the army patients and local ones, but there was a strong correlation in the gene levels. Data suggested that a close connection should be considered on TB prevention and treatment for TB patients in the army and local residents.

China ; epidemiology ; DNA Fingerprinting ; DNA, Bacterial ; analysis ; genetics ; Humans ; Military Personnel ; Molecular Epidemiology ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Polymorphism, Restriction Fragment Length ; Tuberculosis ; epidemiology ; genetics ; microbiology

OBJECTIVETo evaluate the gene-chip detecting rifaman-resistance Mycobacterium tuberculosis applied in TB diagnosis and drug-resistant detection.

METHODSMycobacterium tuberculosis and rifaman-resistant strains among 35 rifaman-resistance isolated strains and 102 sputa specimens from TB patients, 27 sputa specimens from other patients were examined the gene-chips. Results obtained were compared with sputum examination, bacteriological culture and standard drug susceptibility test of Mycobacterium tuberculosis.

RESULTSThirty-five rifaman-resistance strains were detected by gene-chips and 33 were identified as rifaman-resistance strains and the concordance with the traditional drug susceptibility test of Mycobacterium tuberculosis was 94.29%. Twenty-seven sputa specimens from other patients were examined Mycobacterium tuberculosis by the gene-chips, 2 were positive, the detection specialty was 92.59%. Using three methods detecting Mycobacterium tuberculosis among 102 sputa specimens the positive rate respectively was, sputum examination 35.29% (36/102), bacteriological culture 28.43% (29/102), gene-chip 77.45% (79/102). Among 102 sputa specimens only 29 examined Mycobacterium tuberculosis by the traditional drug susceptibility test and 8 were rifaman-resistant strains. While using gene-chip, there were 20 among 102 sputa specimens identified as rifaman-resistance strains. Among total 55 rifaman-resistance strains detected by the gene-chips, the most frequent mutations were those associated with codon 531 (23 of 55; 41.8%), 526 (15 of 55; 27.27%) and 516 (9 of 55; 16.36%).

CONCLUSIONResults showed that this was a rapid, simple and highly specific method when using gene-chip to detect Mycobacterium tuberculosis and rifaman-resistant strains.

China ; epidemiology ; DNA, Bacterial ; genetics ; Drug Resistance, Bacterial ; genetics ; Female ; Humans ; Male ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes ; Point Mutation ; Rifampin ; pharmacology ; Sensitivity and Specificity ; Sputum ; microbiology ; Tuberculosis, Multidrug-Resistant ; epidemiology ; microbiology ; Tuberculosis, Pulmonary ; epidemiology ; microbiology

OBJECTIVETo explore the epidemic distribution of Mycobacterium tuberculosis isolates from Beijing, Guangdong and Ningxia, and to determine M. tuberculosis strains of the "Beijing Family".

METHODSTwo hundred and six IS6110 DNA fingerprinting patterns of M. tuberculosis strains from three provinces (city) were transferred to digital data, compared with the world M. tuberculosis DNA fingerprinting database, and then clustered by Gel compare 4.1 software. The clustering values in different patients with tuberculosis were compared by chi(2) test. Risk factors for recent transmission were calculated using odd ratios.

RESULTSNo M. tuberculosis strains were found the same as those of DNA fingerprint database. 56.8% (117/206) fingerprinting patterns of M. tuberculosis shared by least two-thirds of the IS6110 fragments and their Spoligotyping fingerprinting patterns were consistent with those of M.tuberculosis strains of the "Beijing Family". There were significant differences between female and male, different age groups (< 42 years old) and older (>or= 42 years old) (P < 0.05). Odd ratio was 5.06 in the group younger than 42 years old (95% CI: 1.00 - 34.34) and was 4.43 (95% CI: 0.94 - 28.76) in males.

CONCLUSIONM. tuberculosis strains of "Beijing Family" were popular in Beijing, Guangdong and Ningxia. Men and younger age group (< 42) were shown to be infected by identical strains more often than women and older aged which might play an important role in the recent transmission of tuberculosis in these areas. IS6110 DNA fingerprinting of M. tuberculosis could be used to trace the source of tuberculosis infection.

Adult ; Age Factors ; China ; epidemiology ; Cluster Analysis ; DNA Fingerprinting ; methods ; DNA, Bacterial ; genetics ; Databases, Nucleic Acid ; Female ; Humans ; Male ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Odds Ratio ; Polymerase Chain Reaction ; methods ; Polymorphism, Genetic ; Sex Factors ; Tuberculosis ; epidemiology ; microbiology ; transmission