Bacterial Typing Techniques ; Molecular Biology ; Mycobacterium tuberculosis ; classification ; genetics

Bacterial Typing Techniques ; Mycobacterium tuberculosis ; classification ; genetics ; pathogenicity

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

BACKGROUNDCurrently, migration has become one of the risk factors of high burden of tuberculosis in China. This study was to explore the influence of mass migration on the dynamics of Mycobacterium (M.) tuberculosis in Beijing, the capital and an urban area of China.

METHODSThree hundred and thirty-six M. tuberculosis strains from the Changping district, where the problem of urban migrants was more pronounced than in other Beijing regions, were genotyped by Spoligotyping, large sequence polymorphisms (LSPs 105 and 181), and variable number tandem repeat (VNTR) typing. Based on the genotype data, the phylogeny of the isolates was studied.

RESULTSIn Changping district, the proportion of Beijing lineage M. tuberculosis isolates amounted to 89.0% (299/336), among which 86.6 % (252) belonged to the modern lineage. The frequency of modern Beijing lineage strains is so high (around 75% (252/336)) that associated risk factors affecting the tuberculosis epidemic cannot be determined. The time to the most recent common ancestor (TMRCA) of the Beijing lineage strains was estimated to be 5073 (95% CI: 4000-6200) years. There was no significant difference in the genetic variation of Beijing isolates from urban migrants and local residents.

CONCLUSIONSThe clone of modern Beijing lineage M. tuberculosis, which is dominant in the Beijing area, most likely started to expand with the five thousand-year-old Chinese civilization. In the future, with the urbanization in the whole of China, modern Beijing lineage M. tuberculosis may gain the larger geographical spread.

China ; Genetics, Population ; Genotype ; Humans ; Mycobacterium tuberculosis ; classification ; genetics ; Phylogeny ; Transients and Migrants

Antitubercular Agents ; pharmacology ; Drug Resistance, Bacterial ; genetics ; Genotype ; Mycobacterium tuberculosis ; classification ; drug effects ; genetics

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

OBJECTIVEMultiple locus variable number-tandem repeat (VNTR) analysis (MLVA) had been proposed as a means of strain typing for tracking of source and studying the transmission chain of pathogens. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy.

METHODSMLVA on 7 VNTR loci was applied to the strain typing on prevalent Mycobacterium leprae isolates collected from Qiubei county, Yunnan province during 2002-2006 in the study on the relationship between geographic distribution and genotypes of M. leprae. The strain typing, combined with conventional epidemiological investigation was performed to trace the transmission chain.

RESULTS(1) Phylogenetic analyses through application of PAUP 4.0, The M. leprae were grouped into A, B, C, D and E strains according to the allelic range 9, 11-13, 15-26 and > 26 on the GTA9 locus. The strains with 9 copies on GTA9 locus, was named A. (2) Genotypes of strains from the five multi-case families located at North and North-West parts were similar and belonged to A strains. VNTR patterns of intra-family were identical or similar but not identical inter-family. (3) Not only A cluster appeared higher proportion in total isolates but also distributes cluster, indicating ongoing transmission from recent findings.

CONCLUSIONVNTR strain typing was suitable to trace the short chain of transmission in both small area and intra-families. Multi-case families might constitute epidemic foci and source of M. leprae in villages, causing the predominant strain or cluster which tends to be those identified in multi-case families and resulted in the spreading of leprosy. A long-term study was underway to reveal whether A strain was predominant strain and to observe the evolution of M. leprae in this spatially and temporally defined endemic population.

Female ; Genotype ; Humans ; Leprosy ; microbiology ; Male ; Minisatellite Repeats ; genetics ; Molecular Epidemiology ; Mycobacterium leprae ; classification ; genetics ; Phylogeny ; Polymerase Chain Reaction