Inner Mongolia Tuberculosis Clinical Isolates Molecular Typing Of Mycobacteria

Objectives:In this study, we researched the genotyping of the clinical Mycobacteriumtuberculosis (M. tuberculosis) strains in Inner Mongolia, in order to understand thegenotypes composition, prevalent genotype and the related epidemic characteristics,providing certain scientific basis for tuberculosis (TB) control and prevention.Methods:1. We collected the clinical mycobacteriun strains including thebackgroundinformation of the patients clinically diagnosed with tuberculosis from January2011to June2012in Inner Mongolia Institute for Tuberculosis Control and Prevention, andthe standard strain H37Rv（ATCC27294）. These strains were provided for this studyby the Laboratory ofTuberculosis Research, National Institute for CommunicableDisease Control and Prevention, Chinese Center for Disease Control and Prevention.2. The bacteria were cultured with conventional LJ media methods, and collected,form which the genome DNA samples were extracted for the species identificationand genotyping.3. The Mycobacterium speices were identified with multi-locus PCR.4. The clinical strains identified as M. tuberculosis were genotyped by MLVAwith21MIRU-VNTR loci and traditional43spaces Spoligotyping. The results of theDNA fingerprinting were analyzed with BioNumerics5.0software, and thespoligotypes were compared with SpolD4.0database. In addition, Nucleartranscription factors or neuronot (NTF) and large sequence polymorphism (LSP) wereused to explore the characteristics of the M. tuberculosis Beijing family strains.5. By means of the SPSS13.0software, χ2test was used to analyze the statisticrelationship between M. tuberculosis Beijing family strains and major nationalities(Mongolian and Han), ages, genders, TB clinical types and case types, in order tolearn the prevalence and transmission mechanism of M. tuberculosis Beijing familystrains, in which P≤0.05shows that there is significant difference. Results:1. We collected a total of500clinical mycobacterium strains isolated from thesputum samples of the patients clinically diagnosed with tuberculosis. Withmulti-locus PCR these strains were confirmed to482M. tuberculosis strains whichwere used for the next genntyping research, and18non-tuberculosis mycobacterium.2. Of all the482M. tuberculosis strains tested by MLVA with21MIRU-VNTRloci and clustered with BioNumerics5.0software, we have obtained349genotypes,in which279types represented the single isolate and203strains belonged to70genotypes which had2or more strains respectively. The clustering rate was27.59%.3. Of all the482M. tuberculosis strains tested with Spoligotyping and clusteredwith BioNumerics5.0software and referring to SpolDB4.0database, we haveobtained52genotypes, in which35types represented the single isolate and447strains belonged to17genotypes which had2or more strains respectivly. Amongthese trains, M. tuberculosis Beijing genotype strains accounting for86.72%（418/482）is the most prevalent family in Inner Mongolia.4. The NTF analysis of the Beijing family strains showed that69.62%(291/418)strains which had one or two IS6110insertions in the NTF region were designated as“modern” sublineage. The remaining strains30.38%(127/418) which had an intactNTF region were designated as “ancient” sublineage.5. Of all the482strains analysed with LSP, the results of RD105analysis werethe same as the results of Spoligotyping. The strains lacking RD105represent thepositive3to9hybrid spots from35to43spaces by Spoligotyping, meaning Beijingfamily strains. The results of analysis on RD181showed that6.46%（27/418）strainshad an intact RD181, while93.54%（391/418）strains had RD181deletion. The27(21.26%，27/127) Beijing family strains that had an intact RD181were all included inthe “ancient” sublineage determined by NTF analysis, but the other100“ancient”sublineage strains had RD181deletion.6. During the comparison between MLVA and Spologotyping, the discriminationof the former is higher than the latter obviously. The identification of the Beijingfamily by Spoligotyping is conducive to the strain level analysis via MLVA. 7. The statistic analysis on relationship between Beijing family strainsand majornationalities (Mongolian and Han), ages, genders, TB clinical types and case typesshowed that there was no significant difference between them (all of P＞0.05).Conclusions:1. There is significant genotypic diversity of clinical M. tuberculosis strains andBeijing genotype is the most prevalent family in Inner Mongolia.2. Identification of the Beijing family with the RD105analysis and theSpoligotyping are consistent effects. Most of the Beijing family strains belong to the“modern” group, and the RD181deletion happened before the insertion of IS6110inthe NTF region.3. The results of these researches showed that there was no statistic relationshipbetween Beijing family strains and major nationalities (Mongolian and Han), ages,genders, TB clinical types and case types. Therefor, we could not consider thecorrelation between the popular transmission of Beijing family and above factors.4. By the comparison of MLVA and Spoligotyping methods, we found that thecombination of these two genotyping methods can improve more comprehensivestrain level identification.