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

OBJECTIVETo evaluate the application of different variable number tandem repeats (VNTR) locus in genotyping of Mycobacterium tuberculosis (M.tuberculosis) strains isolated from eight provinces in China, and to find the suitable locus-set of VNTR for epidemical strains in China.

METHODSAll 140 M.tuberculosis strains were randomly selected from 2800 M.tuberculosis strains isolated from eight provinces in China, 27 VNTR loci were used for typing all isolates. Discriminatory power (Hunter-Gaston Index, HGI) of every locus and different locus-set were analyzed by BioNumerics software. Meanwhile, Spoligotyping was used to identify Beijing family and non-Beijing family. Then the HGI of different locus-sets in two families was also evaluated.

RESULTSAll 140 isolates were clustered into Beijing kindred (112 strains, 80%) and non-Beijing kindred (28 strains, 20%) by Spoligotyping. The discriminatory power of Spoligotyping in 140 isolates was 0.4589. Every locus showed different polymorphism and HGI were from 0 to 0.809. The number of VNTR loci with HGI higher than 0.5 in all strains, Beijing family and non-Beijing family was 8, 7 and 14 respectively. 27 loci were combined into four groups which included 8, 12, 15 and 24 VNTR loci respectively. Four locus-sets showed different polymorphism, HGI of eight-locus, 12-locus, 15-locus, and 24-locus set in 140 strains was 0.9991, 0.9882, 0.9980 and 0.9986, and their discriminatory power were calculated in Beijing kindred (HGI: 0.9987, 0.9318, 0.9969 and 0.9975) and non-Beijing kindred (HGI: 1, 0.9894, 1 and 1).

CONCLUSIONDifferent VNTR locus and locus-set showed different discriminatory power in the selected M.tuberculosis strains isolated from China. Eight-locus set can be used in molecular epidemiological study of M.tuberculosis in China after standardization.

Bacterial Typing Techniques ; DNA, Bacterial ; genetics ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Tandem Repeat Sequences

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

Bacterial Typing Techniques ; China ; Humans ; Interspersed Repetitive Sequences ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Ribotyping

OBJECTIVETo access the application of spacer oligotyping (Spoligotyping) and Multiple Locus VNTR(MLVA) in epidemiological studies of Mycobacterium tuberculosis.

METHODS224 clinical isolates of M. tuberculosis were collected and typed by Spoligotyping and MLVA respectively, to compare the results of both methods and to access their application in epidemiological studies of M. tuberculosis.

RESULTSData from Spoligotyping showed that 224 strains presented 55 kinds of genotypes. Of these, 39 were represented by a unique isolate, with the remaining 185 isolates being grouped in 16 clusters whereas the result of MLVA showed that 224 strains presenting 160 kinds of genotypes. Of these, 132 were represented by a unique isolate, with the remaining 92 isolates being grouped in 28 groups. Data from the combination of Spoligotyping and VNTR showed that 224 strains presenting 179 kinds of genotypes. Of these, 159 were represented by a unique isolate, with the remaining 65 isolates being grouped in 20 groups. There was significant difference noticed among M. tuberculosis between Hunan and Anhui in the proportion of Beijing family (P < 0.001). The proportion of Beijing family in Anhui was higher than that in Hunan.

CONCLUSIONResults from this direct comparison studies demonstrated that MLVA analysis was more effective than Spoligotyping in discriminating individual M. tuberculosis isolates. However, Spoligotyping had an advantage over MLVA in identifying Beijing family strains and M. bovis. Taking Spoligotyping as a first-line typing technique and VNTR as second-line typing technique, the arrangement would improve the effectiveness of epidemiological investigation and pathological inspection of tuberculosis. The strains in different regions seemed to have had different characteristics.

China ; epidemiology ; Genotype ; Humans ; Mycobacterium bovis ; genetics ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Oligonucleotide Array Sequence Analysis ; Tandem Repeat Sequences ; Tuberculosis ; epidemiology

Forty mycobacterial strains comprising clinical Indian isolates of Mycobacterium tuberculosis (28 field isolates +1H37 Rv) and Mycobacterium bovis (10 field isolates +1 AN5) were subjected to restriction fragment length polymorphism analysis (RFLP) using IS6110 and IS1081 probes. Most of these strains originated from dairy cattle herd and human patients from Indian Veterinary research Institute (IVRI) campus isolated from the period of 1986 to 2000. Our study showed presence of 8 copies of IS6110 in most of the M.tuberculosis (96.6%) strains irrespective of their origin with the exception of one M.tuberculosis strain with presence of an extra copy (3.4%). All M.bovis strains showed a single copy of IS6110 on the characteristic 1.9kb restriction fragment. RFLP analysis with IS1081 invariably showed the presence of 5 copies in all isolates of M.bovis and M.tuberculosis at the same chromosomal location. Similarity of IS6110 RFLP fingerprints of M.tuberculosis strains from animals and human suggested the possibility of dissemination of single M.tuberculosis strain among animals as well as human. It was not possible to discriminate within the isolates of either M.tuberculosis or M.bovis, when IS1081 was used as target sequence. The IS6110 RFLP is a valuable tool for disclosing transmission chain of M. tuberculosis and M. bovis among humans as well as animals
Animals ; Bacterial Typing Techniques ; Cattle ; DNA Fingerprinting/*veterinary ; DNA, Bacterial/*genetics ; Deer ; Humans ; India/epidemiology ; Mycobacterium bovis/classification/*genetics/isolation&purification ; Mycobacterium tuberculosis/classification/*genetics/isolation & purification ; Polymerase Chain Reaction/veterinary ; Polymorphism, Restriction Fragment Length ; Zoonoses/epidemiology

Infrequent restriction site amplification (IRS-PCR) is a method of amplifying DNA sequences, which flank an infrequent restriction site, and produces a strain-specific electrophoretic pattern. We studied the use of IRS-PCR to characterize Mycobacterium tuberculosis and non-tuberculous mycobactria (NTM). One-hundred and sixteen M. tuberculosis and nine NTM isolated at Hanyang University Hospital in Seoul, Korea were used in this study. IRS-PCR using AH1 and PX-G primers produced unique patterns for reference strains, M. tuberculosis H37Rv, M. bovis BCG, M. kansasii, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulae, M. fortuitum, and M. chelonae, respectively. Reference strains M. tuberculosis H37Rv, M. bovis, M. africanum, and all isolates of M. tuberculosis showed similar IRS-PCR patterns. The IRS-PCR patterns generated with multiple isolates of M. tuberculosis from the same patients were essentially identical. IRS-PCR revealed the greatest difference between electrophoretic DNA patterns from M. avium, M. intracellulae, and M. fortuitum that differed from each other and from the reference strains. We concluded that IRS-PCR is a useful tool for strain typing of NTM, but not for M. tuberculosis.
Bacterial Typing Techniques/methods ; Base Sequence ; DNA Fingerprinting ; DNA Primers/genetics ; DNA, Bacterial/genetics/isolation & purification ; Genotype ; Humans ; Korea ; Mycobacterium/classification/*genetics/isolation & purification ; Mycobacterium Infections/microbiology ; Mycobacterium tuberculosis/classification/*genetics/isolation & purification ; Polymerase Chain Reaction/*methods ; Species Specificity ; Tuberculosis, Pulmonary/microbiology

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

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

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