Construction Of The In Vitro Evaluation Strain Panel For Anti-tuberculosis Drugs

Tuberculosis(TB)is the top tenth cause of death in global scale.China is a high burden country of TB.In recent years,with the increasing prevalence of multidrugresistant tuberculosis(MDR-TB)and extensively drug-resistant tuberculosis(XDRTB),the trend of TB has become more severe.Therefore,it's imperative to develop new anti-TB drugs under this urgent current.It is indispensible that the systematic strain panel containing different resistant levels and mechanisms for screening and evaluation of anti-tuberculosis drugs.At the same time,the proportion of non-tuberculous mycobacteria(NTM)diseases have constantly increased among TB cases,which made the prevention and control of tuberculosis become more complicated and also posed a double threat to the human health.There were two conspicuous factors which induce this situation.Firstly,compared with TB,which has a systematic monitoring mechanism,it was not mandatory to report NTM cases.As a result,there was not enough systematic routine surveillance data for NTM.Secondly,the complex methods of NTM isolation and identification,high technical requirements and limited research resources were all led NTM to a stagnation stage.And also these three factors prompted the comprehensive data on NTM prevalence and drug resistance only relied on meta-analysis.On the basis of real need,this study has established two contents.1)this project aimed to establish a more systematic system panel that included diverse drug-resistant sites and strains of different drug-resistant phenotypes.According to the distribution of tasks,this study undertook the proportion of rifampicin and ethambutol,then used this system to evaluate the anti-tuberculousis activity of 71 candidate drugs.2)A systematic review and meta-analysis of the trend of NTM was concluded in mainland China about the past 20 years.In the first chapter,the first step was to retrieve studies containing resistant mechanisms of two first-line anti-tuberculosis drugs which were rifampicin and ethambutol to carry out the confirmed resistance-related mutation sites verified by multiple drug sensitivity tests.Secondly,strains with different drug resistance levels were selected to subculture according to the results of drug sensitivity tests.After the detection and review of drug resistance sites,monocolonial cultivation was performed.Next,the drug sensitivity tests(DST)were verified again.Finally,the chosen strains were amplified and preserved to establish a systematic strain combination including different drug resistance levels and sites.The collected papers showed that the confirmed mutation sites of rifampicin were mainly in rpo B gene including 511(CTG511CGG/CCG),513(CAA513AAA/CCA/GAA/CTA),516(GAC516TTC/TAC/GTC/GGC/GCC),526(CA526CCCG/TGC/AAC/CTC/CGC/GAC/TAC/TTT/TGG/TTG),531(TCG531TTG/TGG/TTT)et.al.The counterpart of ethambutol sites were distributed in emb B,emb A,emb C and ubi A four genes,they mainly occurred in emb B.In emb B gene,they were 306(ATG306CTG/ATA/ATC/GTG),319(TAT319TCT),334(TAC334CAC),354(GAC354GCC/AAC),361(TGC361TAC),406(GGC406GAC/AGC/GCC)and 497(CAG497CAT/AAG).428 strains with different drug resistance levels were selected from the bacteria base.77 strains of 511,513,516,526,531,533 and 572 sites were obtained in rop B of rifampicin.And 38 strains of 306 and 497 of emb B,-16,-12 and-11 of emb A,270 of emb C and 174 of ubi A sites were acquired in ethambutol by amplification and sequecing analysis.Using this established system,drug sensitivity tests were performed on 71 candidate compounds with potential antituberculosis activity.The experimental results showed that the compounds L11,L5,L6,L2,L8,L9,L1,Z3,Z10,Z16,Z38 and Z46,especially L11,had good inhibitory effects on Mycobacterium tuberculosis and fastgrowing nontuberculous mycobacteria.The component 1 and 2 isolated from Fissistigma cavaleriei root also had good inhibitory effects on mono-resistant strains(isoniazid and rifampicin resistance)and MDR strains,which suggested that this crude drug deserved further development under the increasing emergency of drug-resistant strains,MDR and XDR strains in TB cases.In the second chapter,primary research reports of NTM clinical specimens from mainland China published between January 1,2000 and May 31,2019 were retrieved from four online resources(BIOSIS,Embase,Pub Med and Web of Science)and three Chinese medical databases(CNKI,Wanfang and Vip).In total,339 publications were acquired.129 were classified in drug susceptibility analysis and 95 were used in isolation meta-analysis.The statistical results showed that Lowenstein-Jensen(L-J)slants combined with P-nitrobenzene acid(PNB)and thiophene-2-carboxylic acid hydrazine(TCH)medium were the most commonly method for isolation,identification and drug susceptibility testing of NTM in China.The crude isolation rate of NTM was 4.66-5.78%,while the isolation rate of NTM in Mycobacterium was 11.57%.Mycobacteroides abscessus(M.abscessus)and Mycobacterium avium(M.avium)were the most common clinical NTM species.In mainland of China,NTM morbidity showed a downward trend after 2015.And also Mycobacterium intracellulare(M.intracellulare)replaced M.abscessus as the dominant strain.At the same time,the geographical diversity of the mainland exsited in it which costal region was higher than inland.Environmental and economic differences also had a significant impact on the distribution of NTM strains.Finally,we discovered that NTM species only showed general sensitivity to Ethambutol,Linezolid,Clofazimine,Amikacin,Tobramycin,and Clarithromycin.In summary,the mututation sites database and systematic strain panel established in this study will provide a technical platform for the development of anti-tuberculosis drugs,and the comprehensive data analysis of NTM can be used as a reference for clinical treatment and guidance for the adjustment of TB prevention and control strategies.