Characterization On Genotypes,Drug Susceptibility Phenotypes And Gene Mutations Associated With Drug Resistance Of Mycobacterium Tuberculosis Clinical Isolates From Hebei

Objective: To explore the role of Mycobacterial interspersed repetitive units-variable number tandem repeats(MIRU-VNTR)and spacer oligonucleotide typing(Spoligotyping)in molecular epidemiology study of Mycobacterium tuberculosis(MTB)and to identify the genotypic characterization and distribution of MTB strains in Hebei province.Drug susceptibility testing and gene sequencing were used to identify the drug resistance profiles of MTB strains in this area.The associations between drug resistance phenotypes and gene mutations associated with drug resistance,drug resistance phenotypes and genotypes,and genotypes and gene mutations associated with drug resistance were investigated.Furthermore,the clinical predict value of gene mutations associated with drug resistance in drug resistance phenotypes was evaluated,which may provide basic science evidence for the development of rapid detection technology of drug resistance phenotypes.Methods: A total of 1017 MTB clinical isolates from Hebei province and the corresponding clinical information of the patients were collected during 2014.Differential PNB/TCH medium and multi-locus PCR were performed to the identification of MTB strains.First-line anti-TB drug susceptibility testing(streptomycin,isoniazid,rifampin and ethambutol)was performed using Lowenstein-Jensen(L-J)proportion method.Fifteen-locus MIRU-VNTR and spoligotyping were conducted to identify the genotypes of strains.Seven target genes(rpsL,rrs,katG,inhA,oxyR-ahpC intergenic region,rpoB and embB)were sequenced and aligned.Clustering analysis was done by BioNumerics software 5.0 and statistical analysis was done by GraphPad Prism 5.0.Results:1 The total drug resistance rate of MTB in Hebei was 34.1% and multi-drug resistance(MDR)rate was 13.2%.The initial drug resistance rate and initial MDR rate were 26.6% and 6.1%,respectively;the acquired drug resistance rate and acquired MDR rate were 59.7% and 37.6%,respectively.The acquired drug resistance rate was significantly higher than the initial drug resistance rate(P<0.0001)and the acquired MDR rate was significantly higher than the initial MDR rate(P<0.0001).2 The MTB strains in Hebei exhibited high genetic diversity.Spoligotyping showed that 90.5% of all strains belonged to the Beijing family and 9.5% to the non-Beijing family.The predominant genotype of the non-Beijing family was T genotype,but LAM,MANU,H and U genotypes were also found.Fifteen-locus MIRU-VNTR showed that 1017 MTB strains with 846 VNTR genotypes were clustered into 16 genetic groups.The HGDI value of spoligotyping and 15-locus MIRU-VNTR was 0.279 and 0.999,respectively.3 Distribution of gender,age,treatment history and drug resistance phenotypes in the Beijing family strains and the non-Beijing family strains had no significant difference.However,the clustering rate of the Beijing family strains was significantly higher than that of the non-Beijing family strains(P<0.0001).Compared to the non-Beijing family strains,the Beijing family strains had significantly higher mutation rates of rpsL and emb B(P =0.0051;0.0180).4 The mutation rate of rpsL43 was 56.0%,rrs was 17.0%,kat G315 was 62.3%,inhA(-15)was 14.1%,oxyR-ahpC intergenic region was 10.5%,rpo B531 was 53.7% and embB306 was 56.5%.5 The mutation rates of rpsL43,katG315,rpoB526/531 and embB306 increased with the rising numbers of drug resistance(P value for trend <0.0001).The mutation rates of rpsL43,katG315,rpoB526/531 and embB306 in MDR strains were significantly higher than those in non-MDR strains(P=0.0136,<0.0001,<0.0001 and <0.0001,respectively).The sensitivity and specificity of cotesting rpoB526/531 and embB306 for detection of MDR phenotypes was 83.2% and 80.8%,respectively.Conclusions:1 The total first-line anti-TB drug resistance rate and MDR rate of the MTB isolates from Hebei are similar to the nation baseline level.The occurrence of MTB drug resistance is associated with anti-TB chemotherapy treatment.2 The MTB isolates from Hebei exhibit high genetic diversity and the Beijing family is the predominant lineage.Fifteen-locus MIRU-VNTR shows a higher discriminatory power than spoligotyping.Cotesting of 15-locus MIRU-VNTR and spoligotyping is suitable for genotyping MTB strains in Hebei.3 The Beijing family strains have stronger recent transmission ability than the non-Beijing family strains in Hebei.Mutations of rpsL and emb B are associated with the Beijing family strains,suggesting that the clinical effects of streptomycin and ethambutol for the Beijing family strains may be less satisfactory and this fitness cost makes the Beijing family strains better adapt to the external condition,which genetically explains the widespread of the Beijing family strains in this area.4 The mutation rates of rpsL43,katG315,rpoB526/531 and embB306 increase with the rising numbers of drug resistance.Cotesting of rpoB526/531 and embB306 is suitable for detection of MDR phenotypes.