结核病药效评价用结核分枝杆菌参考菌株的筛选

Screening of national reference strains for evaluation of protective effect of drugs against tuberculosis

目的筛选结核病(tuberculosis,TB)药效评价用结核分枝杆菌(Mycobacterium tuberculosis,MTB)国家参考菌株。方法取169株MTB临床分离株,采用结核杆菌生长指示管(mycobacterial growth indicator tube,MGIT)960液体培养基,对4种一线抗TB药物[链霉素(streptomycin,STR)、异烟肼(isoniazid,INH)、利福平(rifampin,RFP)和乙胺丁醇(ethambutol,EMB)]进行药敏试验,并对耐INH和/或耐RFP的菌株的相关耐药基因位点进行测序,确认突变位点;用间隔区寡核苷酸分型法(Spoligotyping)对所有菌株进行基因分型,多位点可变数量串联重复序列分型方法(mutiple loci VNTR analysis,MLVA)对北京家族MTB临床分离株进行基因分型。结果 169株MTB临床分离株中耐STR 118株,INH 143株,RFP 129株,EMB 95株,耐多药(至少对NIH和RFP耐药)120株,单耐INH 5株,单耐RFP7株,四种抗TB药均敏感共11株。143株INH耐药菌株中,kat G基因突变菌株占76.9%,inh A基因突变菌株占27.3%,aph C基因突变菌株占25.2%,至少2个基因发生突变的菌株有31株;129株RFP耐药菌株中,rpo B基因531位点发生突变,占67.4%,526位发生突变,占17.8%,516位点发生突变,占16.3%,至少2个位点发生突变的菌株有10株。169株MTB临床分离株分为北京家族和非北京家族,北京家族111株;非北京家族58株,主要有CAS家族2株,EAI家族7株,H家族11株,LAM家族6株,MANU家族6株,T家族7株,U家族1株,X3家族3株,还有15株在数据库中未表现的新基因型。111株北京家族MTB临床分离株分为4大群,其中Ⅱ群分布最广,占68.5%,代表性最好。筛选出耐多药菌株27株,敏感菌株5株,单耐异烟肼2株,单耐利福平3株。结论成功筛选出敏感菌株、单耐药菌株、MDR菌株作为候选菌株。

Objective To screen the national reference Mycobacterium tuberculosis（MTB） strains for evaluation of protective effect of drugs against tuberculosis（TB）. Methods A total of 169 M. tuberculosis clinical isolates were tested for sensitivities to streptomycin（STR）, isoniazid（INH）, rifampin（RFP） and ethambutol（EMB） by using mycobacterial growth indicator tuber（MGIT）960 system. The resistance-associated mutant sites of isolates resistant to INH and / or RFP were sequenced. All the 169 isolates were genotyped by Spoligotyping, and those belonging to Beijing MTB family were further genotyped by multiple loci VNTR analysis（MLVA）. Results Of the 169 clinical isolates, 118 were resistant to STR, while 143 to INH, 129 to RFP, 95 to EMB, 120 to multiple drugs（at least NIH and RFP）, 5 to INH alone, and 7 to RFP alone. However, 11 isolates were sensitive to all the four drugs. The mutations of kat G gene were observed in 76. 9%,while those of inh A gene in 27. 3%, and those of aph C gene in 25. 2% of the 143 isolates resistant to INH, while muta-tions of at least two genes in 31 isolates. However, the mutations of rpo B gene at sites 531, 526 and 516 were observed in67. 4%, 17. 8% and 16. 3% of 129 isolates resistant to RFP, respectively, while the mutation in at least two sites in 10 isolates. Of the 169 isolates, 111 were of Beijing genotype, and 58 were of non-Beijing genotypes, including CAS（2 strains）, EAI（7 strains）, H（11 strains）, LAM（6 strains）, MANU（6 strains）, T（7 strains）, U（1 strain）and X（3strains） lineages, as well as 15 new genotypes not included in the Spol DB4. 0 database. All the 111 isolates of Beijing genotype were divided into four groups by MLVA, among which group Ⅱ accounted for 68. 5% and was the most representative one. Twenty-seven multiple drug-resistant（MDR） and 5 sensitive isolates, as well as 2 isolates resistant to INH alone and 3 isolates resistant to RFP alone were screened. Conclusion Sensitive and MDR isolates as well as those resistant to a single drug were successfully screened.