Capreomycin Resistance Related Genes In Clinical Isolated Strains Of Mycobacterium Tuberculosis

Objective:To understand the circumstances of capreomycin resistance related genes in clinical isolated strains of Mycobacterium tuberculosis and its correlation with capreomycin drug susceptibility phenotype,and provide reference for the clinical application of capreomycin.Methods:Clinical sputum isolates from tuberculosis patients in our hospital from 2017 to2018 were collected for acid-fast staining and culture of Mycobacterium tuberculosis,and clinical isolated strains of Mycobacterium tuberculosis were obtained.The cultured clinical isolates were extracted by CTAB method,and the whole genome was sequenced,the sequences were compared with the standard Mycobacterium tuberculosis H37 Rv sequence,to understand the mutations of genes related to capreomycin resistance rrs,tlyA,eis,rpsL,Rv1258 C and Rv0194;and to perform clinical susceptibility testing of anti-tuberculous first-line and second-line drugs in clinical isolates of Mycobacterium tuberculosis with positive sputum culture,the relationship between mutations in capreomycin resistance related genes and drug-susceptible phenotypes was analyzed and compared,and the correlation between capreomycin and rifampicin resistance.Results:In this study,124 strains of clinical isolates of Mycobacterium tuberculosis were extracted by CTAB method and the whole genome were sequenced,123 strains were successfully obtained.6 genes of capreomycin resistance related rrs,tlyA,eis,rpsL,Rv1258 C and Rv0194 mutations are as follows: rrs appeared a total of 6 mutation points,respectively A1401 G,A1356G,G5 T,A514C,A908 C and C1236 T,the mutation rate was10.6 %(13/123),the gene does not encode a protein;tlyA has 3 mutation points,of which 2mutation points A33 G and C582 G are synonymous mutations,C86 T mutation points are non-synonymous mutations,and the rate of non-synonymous mutations is 0.8%(1/123);eis has 3 mutation points,of which 2 mutation points G11 A and T501 G are non-synonymous mutations,C1122 T mutation point is synonymous mutation,non-synonymous mutation rate is 1.6 %(2/123);There are 3 mutation points in rpsL,the three mutation points A128 G,A263G and A263 C are non-synonymous mutations,and the mutation rate is 22.0%(27/123);there are 6 mutation points in Rv1258 C,among them,4mutation points C55 T,C633T,G1240 A and C322 T are non-synonymous mutations,2mutation points C1065 T and C57 T are synonymous mutations,and the non-synonymous mutation rate is 3.3%(4/123);Rv0194 had 16 mutation points,13 of which were T221 C,C3293T,A1384 G,C292G,T2575 C,C1810T,A571 G,C633G,T3395 A,C752A,A3133 G,C661A and C1985 T are non-synonymous mutations,The three mutation points G2814 A,C2308T and C93 T are synonymous mutations,and the non-synonymous mutation rate is100%(123/123).The susceptibility to capreomycin of 123 clinical isolates of Mycobacterium tuberculosis showed 113 strains of sensitivity,4 strains of resistance,1strain of contamination,and 5 strains of aseptic growth.In 4 strains of capreomycin phenotype-resistant strains,4 non-synonymous mutation points of 3 genes appeared:Rv0194 genes C3293 T and T221 C,rpsL gene A128 G,and rrs gene A1401 G.4 strains of capreomycin phenotype-resistant strains had T221 C and C3293 T mutations in Rv0194 gene,3 of them also had A128 G mutation in rpsL gene,and 2 of these 3 strains had A1401 G mutation in rrs gene.Rifampicin phenotype susceptibility of 123 clinical isolates of Mycobacterium tuberculosis showed that 71 strains were sensitive,52 strains resistant,and 4 strains of capreomycin phenotype-resistant strains appeared in rifampicin phenotype-resistant.Conclusion:In this study,23 non-synonymous mutations were found in the genes rrs,tlyA,eis,rpsL,Rv1258 C and Rv01946 related to capreomycin resistance.Mutations of rrs genes A1401 G,Rv0194 genes C3293 T and rpsL genes A128 G may be related to phenotype resistance of capreomycin,rrs genes A1356 G,G5T,A514 C and C1236 T,tlyA genes A33 G,C582G and C86 T,eis genes G11 A,T501G and C1122 T,rpsL genes A263 G,Rv1258C genes C55 T,C633T,G1240 A,C322T,C1065 T and C57 T,Rv0194 genes T221 C,A1384G,C292 G,T2575C,A571 G,C633G,T3395 A,C752A,A3133 G,C661A,C1985 T,G2814A,C2308 T and C93 T mutations have no significant relationship with capreomycin resistance.Among the 23 non-synonymous mutations detected,eis genes G11 A and T501 G,Rv1258C genes C55 T,C633T and C322 T,Rv01946 genes C292 G,T2575C,C1810 T,A571G,C633 G,T3395A,C752 A,A3133G,C661 A and C1985 T,a total of 15 mutation points and mutations in the rrs genes A1356 G,G5T and C1236 T mutation points,a total of 18 mutations have not been reported in the literature.In addition,there was a correlation between resistance to capreomycin and resistance to rifampicin.It is suggested that tuberculosis patients have the risk of resistance to capreomycin,screening for resistance tocapreomycin should be conducted before clinical administration to guide clinical medication.