The Origin, Evolution Of Mycobacterium Tuberculosis Beijing Family Strains And Its Relationship With Modern Human Migration

China is among the22highest TB burden countries with a second largest total TB cases after India. TB in China is mainly caused by Mycobacterium tuberculosis Beijing family strains, which is highly prevalent in East Asia and transmits worldwide. Beijing strains are usually thought to be highly virulent and resistant. However, until now the phylogeny and population structure of these strains remains largely unclear. It is of significant importance to study the origin, evolution and transmission history of Beijing strains which will shed light on its success of worldwide distribution and further exploring its resistance and virulence.By sequencing223R genes of58Beijing strains collected worldwide, we have constructed a high resolution phylogeny of this family. Then, by typing305Beijing strains with SNPs in3R genes, we have further explored the population structure of Beijing strains worldwide. Surprisingly, we found that about60%Beijing strains around the world belong to a more recently evolved sublineage Bmyc10. The highly similarity of VNTR profiles of these strains further indicated its wide distribution might be the result of recent population expansion.To further explore the origin, evolution of Beijing strains and its relationship with human migration, we genotyped more than1500Beijing strains collected from11provinces across China. Furthermore, we sequenced the whole genomes of39representative strains of different Beijing sublineages. Combining with other22MTBC genomes represented different lineages worldwide, we reconstructed the phylogeny of MTBC and dated the divergence time of different lineage or Beijing sublineages. According to the whole genome based phylogeny, we found the "proto-Beijing" strains collected in Southeast Asia represented the most basal branch among the East Asia lineage. Our genotyping results indicated the "proto-Beijing" strains are rare in China and only eight isolates were indentified in Guangxi, Fujian, Zhejiang and Sichuan, which all located in Southern China. The Bayesian evolutionary analysis indicated the ancestor of "proto-Beijing" and Beijing strains originated around20-30kyr ago, which is highly accordant with the origin time of human people in East Asia. Take together, we conclude that Beijing strains are most likely originated in somewhere between Southeast Asia and Southern China. According to the distribution of Beijing strains in11provinces, the early diverged "ancient" Beijing strains were widely distributed across China. Furthermore, results from Xizang, Japan and Korea showed these "ancient" Beijing strains were dominated in those areas. Combine with the peopling of East Asia, we speculated that after the origin in Southeast Asia, Beijing strains transmitted to other areas of East Asia along with the South-to-North human migrations around10-30kyr ago and reached to Korea and Japan. Except in Xizang,"modern" Beijing strains predominated in other10studied areas. The prevalence of "modern" strains is especially high in Han populations. Furthermore, the prevalence in Northern Han populations is significantly higher than in Southern Han populations. Thus, we speculated "modern" Beijing might originate in the ancestral Han populations in Northern China. Whole genome based dating indicated "modern" Beijing originated around6000years ago and had most likely undergone a population expansion during that date. This date,6000years ago, is perfect matched the date of human population explosion of Yangshao society, the ancestor of Northern Han, during the Neolithic Age after the origin of agriculture in East Asia. The Northern Han population underwent several large scaled migrations during recent2000years, which finally enabled Han peopled reached most areas of East Asia. The wide distribution of Beijing strains could only been associated with these migrations. This speculation could be further evidenced by the lower prevalence of "modern" Beijing strains in the minority areas and in Korea and Japan, where the ratio Han people are relatively low.The early expansion of "modern" Beijing strains in ancestral populations of Chinese, its dominance in other areas of East Asia and its further worldwide transmissions, all together, indicate a higher virulence of these strains. We believed that, by more comprehensive whole genome comparison, it is possible to find out the genetic bases for the success of "modern" Beijing strains. On the other hand, the predominance of these highly transmissible "modern" strains in China highlights the importance of comprehensive case finding and early diagnosis to prevent its further transmission and expansions.The large population size and high genetic similarity of "modern" Beijing strains in China makes tracing transmissions of these trains difficult. The clustered cases defined by standard15or24loci VNTR usually have no epidemic links. By typing a population based collection of191Beijing strains with15loci VNTR and8SNPs, we found5 VNTR-15defined clusters contain strains from different "modern" SNP sublineages respectively. By combine the SNP phylogeny and MSTs of each sublineage, we proved these failures of VNTR-15are caused, on one hand, by low discriminative power. On the other hand, the highly similar VNTR-15genotypes of "modern" strains are prone to homoplasy by parallel or convergent evolutions. Thus, we recommend the combination of SNP and VNTR typing for studying the epidemiology in areas dominated by Beijing strains. However, utill now a uniformed VNTR typing loci set is still lacked for the molecular epidemiology studies in China. Due to the large number of low discriminatory loci, the standard VNTR-15and VNTR-24typing sets are not optimized for resourse limited countries, such as China. By evaluating the dricriminatory power of selected24VNTR loci in six population-based collections of1375M. tuberculosis isolates around China, we further selected an optimized10loci (MIRU10,26,31,40; QUB11b,18,26; Mtub04,21and ETR-A) as the first-line typing methods in China. These10loci combined with3hypervariable loci and QUB11a provided a robut typing assy to define potencial recently transmissions.The whole genome comparison indicate Beijing strains are much diverse than we previously thought and contains several genetically distantly related sublineages. We speculate the genetic difference would lead to phenotypic difference among Beijing sublineages. By sequencing the RIF and INH resistance related genes of242MDR strains, we found the frequency of rpoB531mutations is higher in "modern" Beijing strains than that in other sublineages (OR=1.8, P=0.04). Furthermore, in the population of less popular "ancient" sublineages, most of the rpoB mutations located in codons other than526and531(OR=3.16, P<0.01). Mutations in rpoB526and rpoB531are well known as low cost RIF resistance conferring mutations. The higher prevalence of these mutations in "modern" Beijing strains may indicate a higher bacterial load within patients which enable the selection of low cost mutations. Based on the mutation profiles of242MDR strains, we also selected11maximum parsimonious mutation loci which can detect93.8%RIF resistance,89.3%INH resistance and86.8%MDR. We further developed a multiplex Real-time PCR assay to detect these mutations, which is sample, cost effective and can be applied to most Real-time PCR detection systems.In summary, by studying the origin and evolution of Beijing strains, we have further uncovered the paralleled relationship between the transmissions of MTB with human demographics. Our finding provided insights about the virulence and resistance of Beijing strains. At the same time, we provided reliable molecular typing assay for both studying the molecular epidemiology and rapid detecting antibiotic resistance in areas predominated by Beijing strains.