[Comparative study of protein profile in Mycobacterium tuberculosis and Mycobacterium bovis with SDS-PAGE method]

Mycobacterium bovine, as the etiology of bovine TB, involves human in some cases and with Mycobacterium tuberculosis are regarded as global health problems. The aim of this study was to extract and compare protein profile of these strains in order to achieve effective biomarkers for diagnosis and the vaccine components. First, the clinical samples were cultured on Lowenstein-Jensen [LJ] medium by the N-acetyl-L-cysteine-sodium hydroxide method and the biochemical tests and antibiotic susceptibility were used. Colonies were grown in the Middlebrook 7H9 medium and, upon harvesting the new colonies, secretory and membrane proteins were extracted by sonication and ammonium sulfate and alcohol precipitation. Concentration was determined by Bradford method and finally the comparison was made through one dimensional electrophoresis. The major discrepancy between two strains of Mycobacterium tuberculosis and Mycobacterium bovis was 45 and 60 KDa bands as well as the zone between 14 and 45 KDa bands of secretory proteins, in the banding separation membrane proteins. Discrepancy in the protein bands of sensitive strains and Mycobacterium bovis seems to be used as an effective protein marker or even a biomarker in distinguishing the sensitive and bovis strains; also similarities can be used for immunization purposes

Mutations in rpoB gene and genotypes of rifampin resistant Mycobacterium tuberculosis isolates in Iran

Prevention and treatment of drug-resistant clones is important in guiding TB control strategies. The simultaneous rapid detection of the type of mutation conferring resistance and the genotype reflect the extent of drug resistant TB transmission within the communities.Mutations conferring resistance to rifampin in rifampin-resistant clinical Mycobacterium tuberculosis isolates occur mostly in the 81 bp rifampin-resistance-determining region [RRDR] of the rpoB gene.Spoligotyping, IS6110- restriction fragment length polymorphism [RFLP] typing and sequencing of the rpoB gene were performed for 30 rifampin resistant M. tuberculosis isolates from patients referred to "Iranian National TB Laboratory" from 2006 to 2007 Mutations in the RRDR of the rpoB gene were identified in 96.6% of rifampin-resistant isolates. The spoligotyping analysis identified one [3.3%] East African-Indian [EAI] family, 7 [23.3%] Haarlem family, 9 [30.0%] Beijing family and 12 [40.0%] Central Asia [CAS] family isolates. Sixty- six percent of CAS isolates carried a mutation in codon 516, 37% of Beijing isolates carried a mutation in codon 531 and 33% of Haarlem isolates carried a mutation in codon 526 Overall, there appeared to be a correlation between the genotype and specific mutations conferring resistance to rifampin in the Beijing and Haarlem families

Mutations in codon 315 of the katG gene associated with high-level resistance to isoniazid

The aim of this study was to investigate the significance of mutation in codon 315 of katG gene and its correlation with high-level of resistance to isoniazid, nuclotide and amino acid changes in mycobacterium tuberculosis [MTB] isolates randomly collected from sputums of 42 patients with active pulmonary tuberculosis in different regions of Belarus. Drug susceptibility testing was determined using the CDC standard conventional proportional method. DNA Extraction, katG gene amplification, and DNA sequencing analysis were performed. Six isolates [14%] bearing multi-mutations in three codons [309,315 and 316], 26 Isolates [61.9%] demonstrated multi-mutations in all or two of the above codons, and 8 [19%] were found to have a single mutation in 315. Four types of mutations were identified in codons 315: AGC-ACC [n=36]85%, AGC-AGG [n=1] 2.3%, AGC-AAC [n=2] 4.7%, AGC-GGC [n=1] 2.3%, one type of mutation in 316: GGC-AGC [n=18]41.4%, and four types of mutations in 309: GGT-GGT [n=7]16.1%, GGT-GCT [n=4]9.2%, GGT-GTC [n=3]6.9%, GGT-GGG [n=1]2.7%. In 2 [4.7%] isolates mutations were identified in codons 463, 357, and in codons 454, 357 respectively. MTB in patients from Belarus were found to have high-level resistance to isoniazid in the isolates with mutations in codon 315 [> 10 micro g/mL]

KatG mutation of isoniazid-resistant isolated from tuberculosis patients

The emergence of drug-resistant strains of Mycobacterium tuberculosis [MTB] is an increasing problem in developed and developing countries. The aims of the present study were to identify various types of mutations in katG region from 28 MDR strains isolated from sputum of tuberculosis patients. Twenty-eight rifampin-resistant strains isolated from sputum of patients with active pulmonary tuberculosis were obtained from various geographic regions of Iran. Drug susceptibility was determined by using the BACTEC system. DNA extraction, standard PCR identification, katG gene amplification, DNA sequencing and analysis were done. There was no mutation in 2 strains. In 20 strains, mutation was shown to be in codon 315. Three types of mutations were detected consisting of AGC-ACC [Ser-Thr] [80%], AGC-AGG [Ser-Arg] [5%] and AGC-AAC[Ser-Asn] [15%].Furthermore, one type of mutation was seen in codons 311, 299, and 323. Twelve strains showed one mutation in codon 315 [46%], 7 strains 2 mutations [27%], 5 isolate 3 mutations [19%] and in 2 strains 4 mutations [8%] were observed in different codons. Nine silent mutations was demonstrated in codon 311[GAC1TAC]. This research demonstrated that mutations were mostly seen in codons 315 and 299 indicating resistance to isoniazide

Mycobacterial antibodies against BCG and M.tuberculosis H37RA do not recognize pathogenic M.tuberculosis cells but do recognize their cytoplasmic constituents

The humoral immune response against mycobacterial pathogens and BCG vaccine is still poorly understood. It is not known if antibodies raised against BCG react with all pathogenic M. tuberculosis strains. The question arose during the development of a tuberculosis detection system based on antibodies. It was found that antibodies raised against cells of avirulent TB strain H37Ra and against whole sonicates of BCG do not react consistently with whole cells of pathogenic TB strains and very poorly with whole BCG cells. These antibodies; however, react very well with the internal components of the BCG cell [i.e. A 60 and cytoplasmic constituents]