Analysis Of A Beijing Genotype Rifampin-enhanced Mycobacterium Tuberculosis Strain

Mycobacterium tuberculosis is one of the most prevalent pathogens in the world. Tub erculosis（TB） is an epidemic disease which threatens people’s health severely. Rifampin （RI F） is an important first-line anti-TB drug. In recent years, the emergence of drug-resistant tuberculosis, multi-drug resistant tuberculosis （MDR-TB） and extensively drug-resistant tub erculosis （XDR-TB）, especially rifampin resistant tuberculosis, has posed serious threats to public health worldwide. On the basis of rifampin resistance, Zhong Min et al reported rif ampin-dependent or enhanced （RIF-D/E） phenomenon. To shed more light on the mechanis m of RIF-D/E, we carried out the research on rifampin enhanced mechanism. We isolated one Beijing family M. tuberculosis strain1034from Lanzhou Pulmonary Hospital. Strain1034was not only resistant to RIF, but also its growth was enhanced by RIF. To study th e stability of rifampin enhanced phenomenon, we serially subcultured strain1034and foun d that this RIF-enhanced（RIF-E） phenomenon is still present up to the49th generation at t he time of this manuscript preparation. In order to investigate the relevant genetic characte ristics between strain1034and RIF-enhancement, we sequenced strain1034using whole-g enome sequencing （WGS） method at Beijing Genomics Institute. This study identified spec ific mutations in strain1034, which do not exist in the reference sequence NC₀00962（vi rulent strain H37Rv） and NC₀02755（clinical strain CDC1551）. A total of371mutations are found among1034, H37Rv and CDC1551, and63of them are non-synonymous SNP s（nsSNPs） in protein coding sequences compared with H37Rv and CDC1551, including68nsSNP sites. For further study, the63nsSNPs in protein coding sequences were examine d in three other rifampin enhanced strains（859,1216,1353） to shed light on the mechanism of RIF-D/E. Among these63nsSNPs,31nsSNPs（32nsSNP sites） were shared by four rif ampin enhanced strains （1034,859,1216,1353）, but the31nsSNPs were also present in rifa mpin resistant and sensitive strains. In addition, we found three mutations of RpoB gene i n strain1034, including one non-synonymous mutation H526R（A76867G）, and two synony mous mutations K1115（G78635A） and A1156（A78758G）. The non-synonymous mutation H526R exists in two rifampin enhanced strains（1034and1353）, and one synonymous mutatio n A1156exists in four rifampin enhanced strains（1034,859,1216,1353）. Our research sh ows that the rifampin enhanced Mycobacterium tuberculosis exists in clinical patients, but seems to be somewhat unstable. It is of interest to note that the RIF-D/E strain harbors a H526R mutation in RpoB. The role of this RpoB mutation in facilitating the phenomenon of RIF-D/E remains to be confirmed Further studies are needed to understand the mecha-nism of the rifampin-enhanced phenomenon. Timely surveillance of the rifampin-enhanced strain may have significance for tuberculosis treatment. The Beijing genotype Mycobacterium tuberculosis is apparently the most prevalent genotype in China, and the second most prevalent genotype worldwide[1]. Whether Beijing genotype strain has a selective advantage over other genotypes in acquired resistance and higher pathogenicity remain to be answered. Mycobacterium tuberculosis Beijing genotype strains is an aggressively expanding genotype that has been identified in different populations around the world. It can be divided further with genotyping methods, such as mycobacterial interspersed repetitive unit （MIRU） typing. MIRU223325173533type is the most prevalent Beijing genotype in Russia, China and the rest of Asia. In earlier stage, our lab isolated one Beijing genotype M. tuberculosis strain1034with MIRU223325173533type. We sequenced this Beijing strain1034, using whole-genome sequencing （WGS） method at Beijing Genomics Institute. To look for the specific and common mutated genes in highly prevalent Beijing strains, we analysed the specific mutated genes of1034compared with H37Rv and a clinical isolate CDC1551. Then we analysed the common mutated genes between1034and other Beijing strains. A total of631034-specific non-synonymous SNPs（nsSNPs） that are all different among1034, H37Rv and CDC1551and23common mutated nsSNPs in7Beijing genotype Mycobacterium tuberculosis strains were identified, using whole-genome sequencing and bioinformatics method. Missense mutations of individual genes may affect the phenotype of strains, improve their ability to adapt to the environment like immune escape and etc. Investigation of the difference between Beijing strains at gene level may contribute to the reason for the high prevalence of the Beijing genotype strains. Analysing the specific nsSNPs of Beijing type in the genetic coding region will help to address the role of these nsSNP loci in the prevalence of Beijing strains.