Phenotypic And Genotypic Variation Of Rifampin-dependent Mycobacterium Tuberculosis And Molecular Epidemiology Of Mycobacterium Tuberculosis

Objective:Mycobacterial interspersed repetitive units-variable-number tandem repeat (MIRU-VNTR) and Beijing family typing based on detecting the deletion of RD105 sequence are two common genotyping methods to study the molecular epidemiologic characteristics of Mycobacterium tuberculosis (M. tuberculosis), in order to identify dominant strains and evaluate value of the genotyping methods for molecular epidemiology of Mycobacterium tuberculosis.Methods:Center For Disease Prevention and Control(CDC) of GanSu Province is in charge of monitoring and collecting M.tuberculosis strains and correlated data of case from Linxia Muslim Minority Autonomous Region that from August 2004 to June 2006.318 M.tuberculosis strains were collected, and 218 strains that culture-succeeding and identifying as M. tuberculosis according to the PNB and TCH method, respectively, were analyzed by mycobacterial interspersed repetitive units (MIRU) genotyping method and Beijing family genotyping. All 218 of isolates were derived from smear-positive sputum samples and cultured in Lowenstein-Jensen (L-J) medium for Drug susceptibility test by the absolute concentration method.DNA of M.tuberculosis strains were extracted by hand-made with chloroform and alcohol. Polymerase chain reaction was carried out for in vitro amplification of 12 known loci. The result is analyzed by gel documentation.We can decided copies of 12 MIRU loci according to the size of amplified products. Typing results of this method are expressed as numerical codes. Each number of the code represents the number of tandem repeats at a particular repeat locus. We did the clustering analysis for the digitalized result of genotyping using the UPGMA method. We calculated Hunter-Caston Discrimination Index (HGDI) and polymorphism of allel (h) using Microsoft Excel software.We also analysed the epidemic and transmission characteristic by combining the epidemiology date.All isolates were identified for Beijing family genotype with detection the deletion of RD105 by Deletion-Targeted Multiplex PCR (DTM-PCR) method as previously described. Briefly, PCR primers PI, P2, P3 were previously described. D2000 Marker was used as size marker. H37Rv and distilled water were positive and negative control. The amplified product was a 761bp sequence for Beijing family strains or 1600bp sequence for non-Beijing family strains.Results:218 M.tuberculosis strains isolated from distinct patients,_including 144 men(66%) and 74 women(34%) with the mean age 45.4 years old(the range is from 14 to 79 years). There were 54 (24.77%) isolates were at least resistant to one drug, among them,31(14.22%) strains were resistant to rifampin and isoniazid, which are multidrug-resistant (MDR) isolates. And the remainder strains were drug susceptible strains.The MIRU typing yielded 115 distinct genotypes, including 98 unique isolates and 17 different clusters containing 120 isolates (55.05%), the cluster rate was 47.25%, the number of MIRU patterns varied from 2 to 16, and the largest cluster contained 58 strains with MIRU genotype of 223325173533. By detecting the deletion of RD105 sequence,188 of 218 (86.23%) isolates belonged to Beijing family, and 30 (13.77%) isolates belonged to non-Beijing family. Combination of Beijing family typing and MIRU typing yielded 118 distinct patterns, including 101 unique isolates and 17 clusters containing 117 isolates (54.13%). The largest cluster contained 58 strains with MIRU genotype of 223325173533 which contained 50 strains belonging to Beijing family and 8 strains belonging to non-Beijing family. Twelve MIRU loci showed different allelic diversity within all of strains, Beijing strains and non-Beijing strains. The calculation of the discriminative power by the Hunter-Caston Discrimination Index (HGDI) for MIRU typing gave the values was 0.93, while the HGDI for combining MIRU genotyping and Beijing family genotyping was 0.94.Conclusions:Our results indicate that identification the Beijing family strains is more important in epidemiology, and combining detecting the deletion of RD105 and MIRU typing together to discover that the MIRU genotype 223325173533 is dominant strain in Linxia, Gansu, moreover Beijing family strains occupied a large proportion in these strains. Combining these two methods a simple, fast and effective method with low cost, which might be practical and suitable for M. tuberculosis genotyping in China. Objective:Screening rifampin-dependent Mycobacterium tuberculosis (MTB) from patients'sputum specimens in Lanzhou Pulmonary Hospital, Gansu province, analysising phenotypic and genotypic variation of the rifampin-dependent MTB preliminarily, and researching the pathogenic mechanism in order to provide a new idea for the diagnosis and prevention and cure of the rifampin-dependent MTB.Methods:6 rifampin-dependent (RIF-D) strains that from Lanzhou pulmonary hospital of Gansu province, then inoculated 0.1 ml and cultured stationarily L-J media with or without rifamcin, respectively, subculturing for ascertaimmenting the rifampin-dependent strains and observationing the phenotypic variation of bacteria. Then the growth of colonies was observed, then done acid-fast staining for strains by Ziehl-Neelsen method, and observed the morphology of the bacteria. Extraction DNA from bacteria that were_rifampin-dependent strains which were the most obviously strains and genotyped by MIRU genotyping and Beijing genotyping. Then sequenced the whole DNA and RNA sequencing of the rifampin-dependent Mycobacterium tuberculosis.Results:In six rifampin-dependent TB strains, there have four RIF-D TB strains were the most obviously strains. Among the four patients infected with RIF-D MTB. The result of L-J media subculturing revealed that the bacterial colony were bigger and more in the with rifamcin group than in the without rifamcin group, the dependent phenomenon continuing until the sixteenth generation. Whereas, the thallus were stronger in the media with RIF than in the media without RIF by acid fast staining in anterior generations. After subculturing 6-7 generations, the phenomenon was disappeared. With MIRU genotyping, among 4 RIF-D strains, the MIRU genotype of 223325173533 which contained 3 strains and the remain 1 strain genotype was 222325173533, they are belonging to Beijing family. We have extracted DNA and RNA from the rifampin-dependent Mycobacterium tuberculosis, and send to Huada gene company for the whole sequence sequencing.Conclusions:The rifampin-dependent Mycobacterium tuberculosis existed in clinical patients, but unstabilized. Both the dependent phenomenon is acquired in the in vitro subculturing or inherent. Surveillance the rifampin-dependent strain have a significance for tuberculosis therapy.