Isolation And Identification Of Bovine Mycobacterium Tuberculosis Complexand Its Analysis Of Molecular Evolution

Mycobacterium tuberculosis（M.tb） and Mycobacterium bovis（M.bovis） are the main members of Mycobacterium tuberculosis complex, and the causative agents of tuberculosis（TB）. M.tb mainly infects humans, while M.bovis infects bovines, and other livesotcks, and a broad range of wild animals, as well as humans. In recent years, that M. tb infected cattle and was transmitted among the herd was confirmed. However, whether M.tb is pathogenic to cattle has been controversial for a long time. In addition, how serious the M.tb infection is in bovine herds remains to be demonstrated.As is known, the strains of M.tb complex share high similarity at genome level witha very low level of DNA sequence variation. The development of genotyping, including region of difference（RD） based PCR and direct repeat region based Spoligotyping, make it possible to classify the clinic isolates. With the application of high-throughput sequencing technology, genomic sequences reveal enriched gene information at genomic level to analyze the different phenotypes of strains. In addition, the data would help develop novel drugs and vaccines.In this study, we isolated the clinical strains from nasal swabs and O-P liquid of the dairy cattle positive to the PPD skin test. Meanwhile, we completed the whole genome sequencing of and comparativing analysis of a M.tb Beijing type isolate 1458, and comparative genomic analysis. The main findings are listed as follows:1. We obtained 5 mycobacteria isolates from 497 nasal swab and O-P liquid of cattle positive to PPD skin test.2. We finished identifying 24 clinic isolates from human TB patients. PCR identification determined 23 isolates to be M.tb strains. Furthermore, Spoligotyping demonstrated 22 isolates to be M.tb, including 19 M.tb Beijing type, 86.4% of the total. By referring to Spol DB4 database, 17 M.tb Beijing type strains（000000000003731） belong to ST1 type, 2 M.tb Beijing type strains（000000000003731） belong to ST190type, the remaining 3 strains（775767577740771） don’t get a match. The results show that M.tb Beijing type is the dominant type that causes human TB in China.3. Whole genome sequencing and annotation of M.tb 1458 strain isolated from cattle positive to PPD skin testIn this study, the whole genome of M.tb 1458 strain was sequenced by 454 GS-FLX technology platform. Moreover, the gaps were filled by manual PCR, all sequences were jointed into a circular chromosome with 29-fold coverage and 99.99% accuracy of whole genome. The genome of 1458 strain is 4402033 bp in size and 65.6% G+C content on average.The annotation of 1458 strain resulted in total 4349 open reading frames, 2898 genes could be classified into Clusters of Orthologous Groups（COG） families. Biological functions were assigned to 3130 genes, and the other 1219 genes encoded hypothetical proteins. Insertion elements were predicted to be 57 trusted insertion sequence including 21 of IS6110. The polymorphism of copy numbers and insertion location of IS6110 in genome might be important in the pathogenicity of 1458 strain.4. Phylogenetic analysisOrthologs of known M.tb, M.bovis and BCG genomes were obtained from the NCBI database. The 1458 genome phylogenetic position was determined based on ortholog proteins. Concatenated protein sequences of orthologous Mycobacterium species proteins were aligned, finally the phylogenetic tree was generated. The phylogenetic tree shows that 1458 strain is close to CCDC5079 and CCDC5180, which are both M.tb Beijing type isolates from China.5. Comparative genomic analysisWe finished pan-genome analysis of M.tb 1458, M.tb H37 Rv and M.bovis AF2122₉7. The core-genome of three has 3562 genes which covers all necessary genes for M.tb. Through the KEGG pathway enrichment analysis of the non-synonymous SNPs, the affected genes found that ace Aa is specific for H37 Rv due to a single nucleotide deletion of ace A. Isocitrate lyase, which is encoded by ace A,involvs in lipid metabolism and mediates multi-drug resistant, implicated that be a latent factor implicating M.tb transmission over cattles.We analyzed mutations affected genes in 1458 strain and four Mycobacterium tuberculosis M.tb Beijing genotype strains. The results show that NC₀21054, NC₀17523 and 1458 are likely to present a larger number of mutations than NC₀17522 and NC₀21251. Besides, NC₀21054 contains far more individual SNPs than others. This results are in accordance with the phylogenetic analysis.