Molecular characterization of environmental non-Tuberculous mycobacteria using PCR- RFLP analysis of 441 Bp heat shock protein 65 fragments

Non- Tuberculous Mycobacteria are environmental opportunistic pathogens that can be found in various terrestrial and aquatic habitats. There are an epidemiological links between species isolated in tap water and those isolated from patients. hsp65 gene has more variability in its sequences, compared to the some more conserved genes in NTM, for identification of mycobacteria to species level. In this study, the prevalence of NTM in Isfahan City water samples was determined using culture, biochemical tests and PCR-RFLP analyses of hsp65 gene. Eighty-five water samples were collected and cultured. The mycobacterial isolates were identified by conventional biochemical tests. A 441 bp fragment of hsp65 genes was amplified and digested by two restriction enzymes, BstEII and HaeII. Digested products were analyzed using polyacrilamid gel electrophoresis [PAGE]. 25.9% of the water samples contained different species of NTM. Dominant isolates were M. fortuitum [26.7%], M. chelonae like organism [13.3%] and M. mucogenicum [13.3%]. Nineteen isolates of Mycobacteria were differentiated using hsp65 genes PCR-RFLP. Three isolates could not be identified at the species level because their RFLP patterns were different from other known PCR-RFLP profiles. There were different hsp65 gene PCR-RFLP profiles produced by digestion with BstEII and HaeIII. This study showed that PCR-RFLP of hsp65 gene in mycobacteria is more reliable method for identification of NTM at the specie level than conventional phenotypic methods [P<0.05]. In comparing of RFLP patterns of this study to other investigation, some minor differences were negligible

[Detection of mutation in codon 315 katG gene as a gene marker associated with isoniazid resistance, in mycobacterium tuberculosis strains isolated from patients in Isfahan and Tehran by PCR-RFLP method]

Drug resistance to tuberculosis is continuously increasing and is a significant threat to tuberculosis control programs because afew effective drugs are present against Mycobacterium tuberculosis. Although isoniazid [INH] is the most effective drug against tuberculosis, resistance to this drug also develops readily. Mutations in katG, specially the Ser315Thr substitution, are responsible for isoniazid resistance in a large proportion of patients with tuberculosis. However, the frequency of the katG Ser315Thr substitution varies among population samples. This study provided molecular characterization of isoniazid resistance of M. tuberculosis strains and extended our knowledge about molecular basis of M. tuberculosis drug resistance that is widely applicable for rapid drug resistance detection. Using 1% proportional method, the sensitivity of 126 strains isolated from patients in Isfahan and Tehran to isoniazid was determined. The katG mutations in codon 315 associated with isoniazid resistance among isoniazid resistant isolates was determined by PCR-RFLP. In this way, 355 bp PCR products were digested by MspI. Out of 126 isolates of M. tuberculosis, 32 [25.4%] strains were determined as INH resistant. Resistance rate was 22.6% [19 strains] in Isfahan and 31% [13 strains] in Tehran. Overall, 72% of isoniazidresistant isolates could be identified by analysis of just katG 315 loci. The PCR-RFLP using MspI restriction enzyme that detects katG Ser315Thr substitution could be identified in 72% of isoniazid-resistant strains. Elucidation of the molecular characterization of isoniazid resistance in M. tuberculosis has led to the development of different genotypic approaches to the rapid detection of isoniazid resistant in clinical isolates

Detection of s315t mutation in the katg gene associated with isoniazid resistance in mycobacterium tuberculosis isolated from isfahan and tehran by PCR-RFLP

Drug resistance to tuberculosis is increasing continuously and is a significant threat to tuberculosis control programs because there arc few drugs effective against Mycobacterium tuberculosis. Although isoniazid is most efficient in killing the tuberculosis bacilli, resistance to this drug also develops most readily. Mutations in katG, in particular the Ser 315 Thr substitution, are responsible for isoniazid resistance in a large proportion of tuberculosis cases. However, the frequency of the katG Ser 315 Thr substitution varies with population samples. This study provided the first molecular characterization of isoniazid resistance of M. tuberculosis strains and extended our knowledge of molecular basis of M. tuberculosis drug resistance that are widely applicable for rapid drug resistance detection. Using 1% proportional method, the sensitivity of 126 strains collected from Isfahan and Tehran to isoniazid was determined. The katG mutations in codon 315 associated with isoniazid resistance among isoniazid resistant isolates were determined by PCR-RFLP. In this way, 355 bp PCR products were digested by Mspl of 126 isolates of M. tuberculosis, 32 [25.4%] strains were determined as INH resistant. Resistance rate was 22.6% [19 strains] in Isfahan and 31% [13 strains] in Tehran. In total, 72% of isoniazid-resistant isolates could be identified by analysis of just katG 315 loci. The PCR-RFLP with Mspl that detect katG Ser315Thr substitution identified more isoniazid-resistant strains with mutations at codon 315 in the katG. Elucidation of the molecular basis of isoniazid resistance in M. tuberculosis has led to the development of different genotypic approaches for the rapid detection of isoniazid resistance in clinical isolates