ABSTRACT
To evaluate the relationship between mutations in rpsL or rrs genes and streptomycin (SM) resistance, we compared four molecular methods for their clinical value in the detection of SM resistance. Genotypic analysis of SM resistance in 167 M. tuberculosis clinical strains isolated from Chinese patients was performed by direct DNA sequencing, SSCP, RFLP, and reverse dot-blot hybridization (RDBH) assays. Of the 98 SM-resistant isolates, 78 (79.6%) had missense mutations in codon 43 or 88 of rpsL resulting in a Lys to Arg substitution, 6 (6.1%) had mutations of the rrs gene at positions 513 A to C or T or 516 C to T, and 14 (14.3%) had the wild-type sequence. None of the 69 SM-susceptible isolates examined had alterations in rpsL or rrs. The results of the SSCP, RFLP, and RDBH analyses for these mutations and wild-type sequences were completely consistent with DNA sequencing data. Five distinct single-nucleotide substitutions in codon 43 or 88 of rpsL gene or in position 513 or 516 of rrs gene were correctly identified in 84 of 98 (85.7%) phenotypically SM-resistant isolates by RDBH assay. Molecular analyses of the rpsL and rrs genes are useful for rapid prediction of SM resistance in most clinical strains of M. tuberculosis. Reverse dot-blot hybridization assay is a rapid, simple, and reliable method for the detection of drug resistance.
Subject(s)
Antibiotics, Antitubercular/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Mycobacterium tuberculosis/drug effects , Streptomycin/pharmacology , China , Humans , Microbial Sensitivity Tests/methods , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Polymorphism, Single-Stranded ConformationalABSTRACT
BACKGROUND: Nontuberculous mycobacterium (NTM) had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacterium marinum (M. marinum) are mostly seen to cause skin infection. It is therefore important to establish a rapid approach for detection and identification of M. marinum from lesions of patients with suspected M. marinum infections. METHODS: Specimens were obtained from 5 patients with swimming pool granuloma. DNA was extracted and polymerase chain reaction (PCR) was performed. PCR products were digested with Hae III and BstE II, then analysed by pattern restriction analysis to detect heat shock protein (hsp) 65 kD gene. RESULTS: The 65 kD hsp gene was found in all specimens from patients with swimming pool granuloma. PCR restriction analysis (PRA) identified all 5 samples to be M. marinum infections, and the result was consistent with that of routine bacteriological identification. The lesions subsided or markedly improved upon treatment. CONCLUSIONS: PRA is a sensitive, specific and rapid method in identification of mycobacteria. Application of this method will be helpful for early diagnosis of mycobacterial skin infections.
Subject(s)
Bacterial Proteins/genetics , Chaperonins/genetics , Granuloma/microbiology , Mycobacterium marinum/genetics , Polymerase Chain Reaction/methods , Skin Diseases, Bacterial/diagnosis , Adolescent , Adult , Chaperonin 60 , Female , Humans , Male , Mycobacterium marinum/isolation & purification , Staining and Labeling , Swimming PoolsABSTRACT
OBJECTIVE: To explore the epidemical distribution characteristics of Mycobacterium tuberculosis isolated from China. METHODS: The M. tuberculosis strains were gained by multi-stratified grouping random sampling method from the nationwide random survey for the epidemiology of tuberculosis in China, 2000, and analyzed by IS6110-based RFLP and DR-based Spoligotyping DNA fingerprinting. These fingerprinting patterns were transferred into digital data and compared each other, and clustered by Gel compar4.1 Software. The clustering values in different TB patients were compared by chi(2) test and the risk factors for recent transmission were calculated by Odd Ratios. RESULTS: Two hundreds and four of 402 M. tuberculosis strains from this survey were determined to be "Beijing Genotype" M. Tuberculosis strain by DNA fingerprinting analysis. Three clusters M. tuberculosis strains sharing identical fingerprint pattern were found, including one cluster belong to a same family. There were significant difference between female and male, and younger (< 40 years old) and elder (>or= 40 years old) (P < 0.05). Odds Ratio 1 showed youth [1.68 95% CI (1.01 - 2.80)] and male [2.04 95% CI (1.07 - 3.96)], but Odds Ratio 2 showed youth [0.3 95% CI (0.2 - 0.44)] and male [4.96 95% CI (2.77 - 9.00)]. CONCLUSION: M. tuberculosis strains of Beijing Genotype are prevailing in China at present. Male and elder were shown to be significant risk factors for recent transmission. The infection source of M. tuberculosis could be traced by DNA fingerprinting.
