Study On The Correlation Between PAS-risistant Genotype And Phenotype Of Mycobacterium Tuberculosis

Objective: To understand the mutation frequency of para aminosalicylic acid(PAS)related genes in clinical isolates of Mycobacterium tuberculosis and its correlation with the drug susceptibility phenotype of Mycobacterium tuberculosis,and to provide laboratory data for molecular detection of PASrelated genes.And provide a reference for the treatment of drug-resistant tuberculosis.Methods:The clinical specimens of tuberculosis patients from March 2015 to March2018 were collected for acid-fast staining,Mycobacterium tuberculosis culture and drug susceptibility testing.The results of 226 clinical isolates of Mycobacterium tuberculosis with susceptibility results were obtained,of which 120 cases were sensitive and 106 cases were drug resistant.The DNA of the above strains was extracted by CTAB method,and the PAS-related genes thyA,folC and ribD were sequenced by PCR and gene sequencing,and compared with the Mycobacterium tuberculosis standard strain H37 Rv sequence to obtain the mutation position of PAS resistance-related genes,and use of the findings to compare with PAS susceptibility phenotype.Results:In this study,PCR amplification and sequencing analysis of PAS-related genes thyA,folC and ribD were performed.Among the 226 clinical isolates,the thyA gene analysis of 215 clinical isolates,111 cases were sensitive strains,104 cases were resistant strains,212 cases were PAS phenotype sensitive strains,and 3 cases were PAS phenotype resistant strains.4 of 215 cases had thyA gene mutations.The total mutation rate was 1.9%(4/215),3 of them were resistant strains,the mutation rate was 2.9%(3/104),and had a mutant form of ACC22GCC(Thr22Ala),CAC75AAC(His75Asn)and CAA97CGA(Gln97Arg),ACC22 GCC was found to be a new mutation,which has been submited to the GenBank,the numbers is MK674466;1 case was a sensitive strain with a mutation rate of0.9%(1/111)and had a mutant form of TAC164AAC(Tyr164Asn).The mutation rate in the PAS phenotype sensitive strains was 1.4%(3/212),and the mutation rate in the PAS phenotype resistant strain was 33.3%(1/3).Among the 226 clinical isolates,the folC gene analysis of 216 clinical isolates,118 cases were sensitive strains,98 cases were resistant strains,212 cases were PAS phenotype sensitive strains,and 4 cases were PAS phenotype resistant strains.2 of 206 cases had folC gene mutations.The total mutation rate was 0.9%(2/216),the two cases ware resistant strains with a mutation rate of 2.0%(2/98),and mutant form was GGT206AGT(Gly206Ser)and GCC325GTC(Ala325Val),the GGT206 AGT was a new mutations,and has been submited to the GenBank,the numbers is MK674466;The mutation rate in PAS phenotype sensitive strains was 0.9%(2/212),no mutation was detected in the PAS phenotype resistant strains.Among the 226 clinical isolates,the ribD gene analysis of 206 clinical isolates,111 cases were sensitive,95 cases were resistant,203 cases were PAS phenotype sensitive,and3 cases were PAS phenotype resistant.Only 1 in 206 cases had ribD mutation with a total mutation rate of 0.5%(1/206),the mutated starin was resistant case with a mutation rate of 1.1%(1/95),and the mutant form was ACC245GCC(Thr245Ala).The mutation rate in the PAS phenotype sensitive strain was 0.9%(1/203),and no mutation was detected in the PAS phenotype resistant strain.Conclusion:This study showed that mutations of ACC22GCC(Thr22Ala),CAC75AAC(His75Asn)and CAA97CGA(Gln97Arg)in thyA,GGT206AGT(Gly206Ser)and GCC325GTC(Ala325Val)in folC and ACC245GCC(Thr245Ala)in ribD were closely related to isoniazid resistance and multidrug resistance,and it suggests that these detected mutaions may have a cross-resistant with isoniazid.The mutation of ACC22 GCC in thyAgene was associated with PAS resistance,and the remaining mutations were not associated with PAS phenotype resistance.The study also found two new mutation sites of ACC22GCC(Thr22Ala)in thyA gene and GGT206AGT(Gly206Ser)in folC gene.