Identification And Drug Resistance Analysis Of Mycobacterium Isolated From Cattle In Parts Of Northwestern China

Tuberculosis is a type of zoonotic infectious disease that is prevalent throughout the world and poses a serious threat to human and animal health.As an important source of human production and living materials,cattle plays an important role in the spread of tuberculosis.In this study,the suspected pathogens of PPD-positive cows in large-scale dairy farms in the northwestern part of Ningxia and Xinjiang were isolated and identified by multi-site PCR.The genotype of bovine isolates was explored using the MIRU-VNTR genotyping technique.The drug sensitivity test was used to understand the phenotypic resistance of bovine isolates.Through the sequencing of drug resistance genes,the correlation between drug resistance gene mutations and phenotypic resistance and genotypes of resistant strains was studied.Using gene sequencing technology to explore the types of nontuberculosis mycobacterium(NTM)in the five northwestern provinces provides a reliable theoretical basis for controlling bovine tuberculosis in the northwest.82 frozen strains of tuberculosis suspected pathogenic bacteria were reconstituted and subcultured by Lowenstein-Jensen,and 80 strains were successfully regenerated.According to the preliminary identification of 16SrRNA,80 clinical isolates belong to the genus Mycobacterium.Thirty MTBCs were identified by MPT64 gene and multi-site PCR,one of which was Ningxia strain,and the other 29 isolates were Xinjiang strains.Sensitivity test of first-line anti-tuberculosis drugs(isoniazid,rifampicin,streptomycin and ethambutol)by proportional method,20 strains(20/30,66.7%)of Mycobacterium tuberculosis against four first-line anti-tuberculosis drugs All were sensitive,10 strains(10/30,33.3%)were resistant to at least one drug,and 9(30.0%)were multidrug-resistant(MDR)strains.The RIF single resistance rate was 3.3%(1/30),and the other three first-line anti-tuberculosis drugs(STR,INH,and EMB)had a single resistance rate of 0%.The total resistance rates of STR,INH,RIF and EMB were 30.0%(9/30),30.0%(9/30),33.3%(10/30)and 6.7%(2/30).The first-line anti-tuberculosis drug-resistant gene sequencing results showed that the mutation rate of rpoB1333 site was 10.0%in 10 strains of drug-resistant.tuberculosis;the mutation rate of katG1388 site was 90.0%;the mutation rate of inhA 1812 site was 70.0%.The mutation rate of the 1714 locus was 10.0%;the mutation rate of the rrs 932 locus was 10.0%;the mutation rate of the embB1333 locus was 90.0%;and the oxy R-ahp C and rps L loci were not.Cluster analysis of the genotyping results of 15 MIRU-VNTR loci,30 strains of M.tuberculosis clustered into four gene groups,showing 18 VNTR genotypes,14 of which were single genotypes,ie one strain represented One genotype,accounting for 46.7%of all strains;16 strains(53.3%)aggregated into four gene clusters,namely C1,C2,C3,and C4,each containing 3,2,5,and 6 isolates,respectively.The clustering rate of the strain was 46.7%.The largest gene group(both Xinjiang strains)contains C3 and C4 gene clusters,and the strains contained in this gene group may be dominant strains in Xinjiang.50 non-tuberculous mycobacterial hsp65 sequencing sequences were submitted to the NCBI website for homology comparison to obtain 6 NTMs.32 strains of mycobacterium nonchromogenicum(64.0%,32/50),4 strains of rare mycobacterium peregrinum(8.0%,4/50),and 8 strains of mycobacterium alvei(16.0%,8/50),3 strains of mycobacterium engbaekii(6.0%,3/50),2 strains of mycobacterium fortuitum(4.0%,2/50),mycobacterium arupense 1 strain(2.0%,1/50).The main non-tuberculous mycobacteria prevalent in the herd may be Mycobacterium mutans.