Large Sequence Polymorphisms Of Mycobacterium Tuberculosis From Plateau And Their Relations With Drug Resistance

Objectives To study the drug resistance of mycobacterium tuberculosis(MTB)in Qinghai province and to explore the relationships between patients' basic information and drug resistance.To detect genotypes with large sequence polymorphisms(LSP;Regions of differences,RD)and polymorphisms of phospholipase C genes(plc)of these MTB strains,and to observe their relations with drug resistance and linkages between RD and plc genes.Methods(1)Sputum samples of tuberculosis patients in Qinghai province were collected.After inoculated and identified,MTB colonies were prepared for proportional drug susceptibility test to detect drug resistance of anti-tuberculosis drugs.(2)The DNA of MTB strains were extracted by Boiling.RD genes were amplified by polymerase chain reaction(PCR)for genotyping.The sequencing of some strains' rpoB/katG was detected,and the relationships between these drug-resistance genes and their RD genotypes were analyzed.(3)The polymorphisms of plc genes were also obtained by PCR.Then the relations between mutations of plc and RD genotypes,and the linkages between plc and drug-resistant phenotypes were studied.Results 249 strains of mycobacterium tuberculosis were identified.The findings of this study were listed as follows.(1)The results of proportional drug susceptibility test: Of 249 strains,108 were isoniazid resistant,93 were resistant to rifampin,97 were streptomycin resistant,70 were ethambutol resistant,ofloxacin resistance were 34 and 10 kanamycin resistance.Among 249 isolates,79 weremultidrug-resistant tuberculosis(MDR TB)and 3 were extensively drug-resistant tuberculosis(XDR TB).(2)RD genotyping: Of these 249 MTB strains,190 were identified as Beijing family strains,including 172 modern Beijing family strains and18 ancient Beijing family strains.Another 59 MTB were identified as non-Beijing family strains,5 of which were found RD239 gene deleted.Most patients were infected with modern Beijing family strains which RD150 and RD142 genes were both existed.The infections of Beijing/non-Beijing family strains in this region have nothing to do with the drug susceptibility results or the mutations of katG/rpoB,while the modern Beijing family strains were more likely to obtain the resistance to at least one first-line drugs than that of ancient Beijing family strains.(3)The polymorphisms of plcABCD: 249 MTB isolates were tested for plc polymorphisms,of which the plcD mutation rate was as high as 97.19%,and the plcABCD of most strains were +++-(+:wild type;-:mutant type),which was consistent with the reference strain H37 Rv.According to RD genotyping results,Beijing family strains were found associated with plc ABC mutations,but no association were found between plcD and Beijing family strains.For drug resistance results,plc ABCD mutations were not observed relations with any four first-line drugs or MDR TB,besides the association between plcC gene mutations and the resistance of EMB.Conclusions The main epidemic strains of mycobacterium tuberculosis in Qinghai province are the modern Beijing family strains,and the situations of drug resistance are severe.The genotypes of Beijing/non-Beijing family strains based on RD typing are not correlated with the phenotypes of drug resistance and the mutations of hot spot resistance genes(rpoB/katG).This research reveals plc genes are polymorphic,which is not correlated to drug resistance.This study suggests Beijing family strains are associated with the mutations of plcABC,excepting plcD.Therefore,it can be speculated that the virulence of Beijing family strains might be potentially associated with plcABC gene mutations which need to be further studied.