Species Identification And Drug Resistance Analysis Of Non-Tuberculosis Mycobacterium Clinical Isolates In Dali Region

Objective:The purpose of this thesis is to identify the clinical isolates strains of non-tuberculous mycobacteria?NTM?in Dali area.The drug resistance test was used to detect the drug resistance of the NTM,analysis of gene mutations in rpoB and inhA related to rifampicin and isoniazid drug resistance in NTM resistant strains,preliminary study on the drug resistance mechanism of NTM.Methods:1.Species identification:Preliminary identification of 18 strains suspected NTM clinical strains by using PNB/TCH identification medium,and polymerase chain reaction was performed to the DNA of the initially identified clinical isolate of NTM by using designed 16SrRNA primers.The gene fragments of the recovered strain were sent to the company for sequencing,then the strain identification results were obtained by perfoming homology analysis through submitting the sequencing results to the NCBI-BLAST website to compare with the standard sequences in GenBank.Using hsp65 and rpoB primers,the DNA of two strains of Mycobacterium abscessus was amplified by PCR,the target gene was sent to the company for sequencing,and after BLAST comparison,the Mycobacterium abscessus were eventually identified as differently subspecies.2.Drug resistance analysis of Non-Tuberculosis Mycobacterium.?1?Drug sensitivity test:In order to analyze the resistance of NTM,four drugs including Isoniazid?INH?,Rifampicin?RFP?,Ethambutol?EMB?,and Kanamycin?KM?were applied to all NTM to perform drug sensitivity test based on proportional method.Besides,the drug sensitivity test for Mycobacterium aviumadded Moxafloxacin?MFX?,and the Mycobacterium abscessus added Amikacin?AK?.?2?Resistance-related gene analysis:1)Primer inhA was designed and the PCR technology was perfomed to amplify the inhA gene of Mycobacterium abscessus.After sequencing,the sequence of the target inhA genes was compared with the sequence of theinhA gene of Mycobacterium tuberculosis sensitive strain?H37RV?and the Mycobacteriumabscessus standard strain?ATCC19977?to count the genetic differences between them.Thus,the relationship between thegene mutation of inhA and the resistance to isoniazid of Mycobacterium abscessus was analyzed.2)Primer rpoB₂ was designed,and the rpoB₂ gene of Rifampicin-resistant Mycobacterium abscess clinical strain was amplified by performing the PCR technology,the sequence of the amplified target gene was compared with the rpoB₂ gene sequence of the Mycobacterium abscessus standard strain?ATCC19977?,finally,look for differences.3)Primer rpoB₁ was designed,the 81bp core region of rpoB₁gene of Rifampicin-resistant Mycobacterium avium was amplified by performing PCR technology.Then the fragments of target gene of rpoB₁ was extracted and sent to company for sequencing.The sequencing results were compared with the gene sequence of rpoB₁ of Mycobacterium avium standard strain?ATCC25291?,and finally the genetic differences in this region between the Rifampicin-resistant Mycobacterium avium and the standard strain?ATCC25291?were found.Results:1.Species identification:The 18 suspected NTM clinical strains were initially identified as 14 strains of NTM and 4 strains of MTB using PNB/TCH identification medium,then by amplifying the 16S rRNA gene,the 14 clinical strain were finally accurately identified as 12 strain of NTM,1 strain of Tsukamurella and 1 strain of Gordonia.Among the identified NTM,by using 16S rRNA primers,10 strains?83%?is identified as mycobacterium avium which was a kind of slowly growing mycobacterium?SGM?and 2 strains is mycobacteria abscess of rapidly growing mycobacterium?RGM?.The mycobacteria abscess were further identified as Bolletii subspecies and Abscessussubspecies by using primers hsp65and rpoB,In the results of this experiment,there are includes one Bollettii subspecies and one Abscessussubspecies.2.Drug resistance analysis of the NTM:?1?Drug sensitivity test:The results showed thatin the drug susceptibility test of the 10 strainsmycobacterium avium,the10 strains mycobacterium avium were all resistant to isoniazid and kanamycin;9 strains were resistant to rifampicin,and 1 strain is sensitive;7 strains resistant to ethambutol and moxifloxacin,3 strains sensitive.The 2 strains of Mycobacterium abscessus were all resistant to 5 anti-tuberculosis drugs.?2?Resistance-related gene analysis:1)Three bands inhA gene fragments of Mycobacterium abscess with length of 810 bp were amplified by PCR technology.and the length is identical to the corresponding gene in Genebank;Comparison of two clinical isolates of Mycobacterium abscessus with standard strains showed that there were2 nucleotide differential expression sites in No.1 clinical strain and 7 nucleotide differential expression sites in No.5 clinical strain.Through sequencing results of the 3 strains of Mycobacterium abscessus and H37Rv showed thatthere were 607 differential expression locus of No.1 strain,581 of No.5 strain,and 567 of standard strain.In inhA gene,the amino acid expressed by codon 94 of the H37Rv was S?serine?,while 2 clinical strains of Mycobacterium abscessus were expressed as T?threonine?.2)Using rpoB₂ gene,the 2 strains of Mycobacterium abscessus were amplified to a DNA fragment with length of 1300 bp fragment by PCR technology.Comparing the Mycobacterium abscessus standard strain and Mycobacterium tuberculosis standard strain?H37Rv?,Mycobacterium abscessus standard strain and rifampin-resistant Mycobacterium abscessus clinical strain in 146 codon,in gene mutation assembly?region,and in gene mutation assembly?region,there were no positive mutations found,and they all encoded the same amino acid.3)5 strains of rifampicin-resistant Mycobacterium avium were amplified to a target gene fragment with length of 1400 bp by using rpoB₁ gene.Detection in the 81 bp core region of the rpoB₁ gene of rifampicin-resistant mycobacterium avium showed that there were no mutation occurred in this region.Conclusion:1.Using 16S rRNA primers,NTM clinical isolates can be further identified as different strains.The gene sequencing method of hsp65 and rpoB can be used to identify Mycobacterium abscessus as a different subspecies,and the results of the two methods are consistent.2.Mycobacterium avium is highly resistant to anti-tuberculosis drugs.Mycobacterium abscessus is not only resistant to tuberculosis drugs,but also shows multidrug resistance.Mycobacterium abscessus clinical strain and mycobacterium tuberculosis standard strain?H37Rv?had different sites on the inhA gene,the differences in nucleotides may be responsible for the natural resistance of Mycobacterium abscessus to isoniazid.The resistance mechanism of mycobacteria to isoniazid is not due to the substitution of serine?Ser?at the94th codon by alanine?Ala?,it also proved that there are other reasons for mycobacterium abscessus resistance to isoniazid.The mycobacterium abscessus,which is resistant to rifampicin does not undergo a sense mutation in the high-risk mutation region of rpoB₂ gene,it indicated that the drug resistance mechanism of mycobacterium abscessus to rifampicin was different from mycobacterium tuberculosis.There were no mutation occurred in the 81bp core region ofrpoB₁gene of rifampicin-resistant mycobacteria.The results suggested that the rifampicin-resistant mechanism of mycobacterium avium was different from mycobacterium tuberculosis.