Spinal Streptomycin Resistant Strains Of Mycobacterium Tuberculosis Screening And Its Resistance Mechanism Preliminarily Study

Objective:Explore the spinal tuberculous mycobacteria streptomycin resistance gene of therpsL,gidB and rrs mutations in its new resistance mechanisms.To provide a basis forfurther in-depth exploration of spinal tuberculosis resistance mechanisms and ascientific theoretical and experimental basis for drug research and development of anti-spine tuberculous, to propose new therapeutic strategies and ideas. Ultimately curb thespread of spinal tuberculosis and tuberculosis in the world and deterioration.Materials and methods:Select from November2010to December2012,19patients were treated in our hospitalpatients with spinal tuberculosis. After surgery revealed tuberculous lesions, charged thecaseous necrosis matter, sequestrum and other diseased tissue, with a specimen bagsealed submission culture.1. The BACTECTMMGITTM960fully automated mycobacterial quick culture systemfor mycobacterial culture (increased within MGIT PANTA bacteriostatic agentsinhibit pollution and bacteria) and susceptibility testing of absolute concentrationmethod: The specimens which had been treated in conventional way were inoculatedto MGIT culture tubes,that were set The Fully Automatic mycobacteria inBACTECTMMGITTM960rapid culture system for culture; After cultivate apositive result, susceptibility testing of absolute concentration method (includingisoniazid, rifampicin, ethambutol, streptomycin), while traditional strainidentification.2. Target gene extraction (rpsL, gidB, rrs), electrophoresis and DNA sequencing:culturing the obtained colonies after the conventional pre-treatment, after thegenomic extraction, PCR amplification and agarose gel electrophoresis, to obtain thedesired DNA was separated, and finally by DNA sequencing. Sequencing results obtained with H37Rv DNA (from Gene bank) to do a comparative study. Analysisof gene mutation and drug resistance of the correlation.Result：1. BACTECTMMGITTM960fully automated rapid culture of Mycobacteriuminstrument training and absolute concentration method drug susceptibility testing:1.1BACTECTMMGITTM960fully automated rapid culture of Mycobacteriuminstrument culture results:19cases of co-cultured specimens culture positive in16cases, negative in threecases. The positive rate of84.2%.1.2Absolute concentration method susceptibility test resultsA total of16cases were positive strains susceptibility testing, three cases thepresence of resistance, the resistance rate was18.7%(3/16).1.3Strain identification results:16specimens culture results were positive, TCH (thiophene-2-carboxylic acidhydrazine), PNB (nitrobenzoic acid) and LJ (Lowenstein-Jensen medium)identify the species. Identification results:16cases were identified as MTB, andare humanoid.2. Strain identification of gene chips and DNA sequencing2.1The gene chip Mycobacterium strain identification results16cases with positive isolates were cultured fully automaticBACTECTMMGITTM960Mycobacterium Quick Culture Instrument cultureconduct strain identification of gene chip, identification results all are humanoid,BACTECTMMGITTM960system culture results fully in line with strainidentification results of gene chip2.2DNA sequencing resultsA, B, C resistant strains in gidB gene sequencing, were found a new mutation sites,the A strains:615(205alanine-> alanine),299(serine100-> Phe),276(glutamicacid92-> aspartic acid) mutation, wherein615is a new mutation point. B strains:615 (205alanine-> alanine),299(serine100-> Phe),276(92glutamate-> asparticacid) mutation,224frameshift (deletion C670), wherein615,670new mutation point.C strains:615(205alanine-> alanine),299(100serine-> Phe),276(92glutamicacid-> aspartic acid),102(34glycine-> glycine) mutation, wherein205,34for a newmutation point. Three strains in the A, B, C363(121lysine-> lysine) weresynonymous mutations in rpsL gene; were not found in the rrs gene mutation.Conclusions:3、 The experiment found in the spinal Mycobacterium tuberculosis streptomycinresistant strains gidB gene of some new mutation sites: C102T, T615C,224frameshift (deletion C670).4、 The experiment preliminary obtain that T276G, G299A double-digit pointsmutation may lead to spinal Mycobacterium tuberculosis SM high level ofresistance, while224frameshift (deletionC670) may also be one of the reasons inrrs, rpsL the genes not occurred of mutations and gidB gene C102T,T615C aresynonymous mutations.