深圳市耐多药结核分枝杆菌耐药基因突变特征分析

目的 利用全基因组序列分析方法研究深圳市耐多药结核分枝杆菌(MDR-MTB)的耐药基因突变类型特点,为耐药结核病的快速分子检测提供依据。方法 对2013—2017年深圳市慢性病防治中心及各区慢性病防治院门诊收集的所有449株MDR-MTB临床分离株进行全基因组测序,经与结核分枝杆菌标准菌株(H37Rv)基因组模板序列比对后获得447株满足分析要求的全基因组测序菌株。每株菌株经单核苷酸多态性(SNP)鉴定后与H37Rv各基因型和亚型的特异性突变和对11种抗结核药品已知的耐药基因突变进行比对,获得各菌株独属的基因型或亚型和耐药基因突变,并分析不同药品耐药基因中的主要耐药突变类型不同基因型或亚型间的相关性。结果 447株MDR-MTB中全基因组测序结果显示,432株(96.6%)发生基因突变,MTB对11种药品主要的耐药突变类型分别为katG-315-S/TINH, 81.7%(353/432)]、rpoB-450-S/LRFP, 59.7%(258/432)]、rpsL-43-K/RSm, 66.0% (198/300)]、embB-306-M/VEMB, 35.5% (94/265)]、pncA启动子-11-T/CPZA, 8.9% (11/123)]、gyrA-90-A/V Ofx, 31.5% (39/124)]、rrs-1401-A/GAm,48.1% (25/52)]、rrs-1401-A/G Cm, 100.0% (27/27)]、rrs-1401-A/G Km, 84.4% (27/32)]、inhA-15-C/TEto, 84.2% (48/57)]、thyA-75-H/NPAS, 31.3% (10/32)],且发现存在2种及2种以上联合突变者有对INH、RFP、EMB和Sm耐药的菌株。深圳市MDR-MTB菌株的3个基因型为L10.4%(2/447)]、L284.6% (378/447)]和L415.0%(67/447)]型,其中L2型又分为3个亚型L2.1型:1.9%(7/378)、L2.2型: 37.0%(140/378)、L2.3型: 61.1%(231/378)]。突变类型为katG-315-S/T、rpsL-43-K/R和embB-306-M/V在L2型菌株中的突变率分别为79.6%(301/378)、50.5%(191/378)、23.0%(87/378)]明显高于L4型菌株分别为61.2%(41/67)、10.4% (7/67)、10.4% (7/67)](χ ²值分别为10.874、37.021、5.396,P值分别为0.001、0.000、0.020),且PAS耐药突变类型folC-43-I/T和thyA-75-H/N仅出现在L2型菌株中;其中katG-315-S/T在L2.2型菌株中突变频率88.6%(124/140)]高于L2.3型74.5%(172/231)](χ ²=10.764,P=0.000),而inhA-15-C/T(INH)、rpoB-450-S/L、rpsL-43-K/R和inhA-15-C/T(Eto) 在L2.3型菌株中的突变频率分别为8.2% (19/231)、61.9%(143/231)、 59.3% (137/231)、15.6% (36/231)]明显高于L2.2型分别为2.9% (4/140)、49.3% (69/140)、37.9% (53/140)、3.6% (5/140);χ ²值分别为4.319、5.668、16.053、12.797,P值分别为0.038、0.017、0.000、0.000)]。结论 深圳市MDR-MTB菌株中11种药品耐药基因主要突变类型分别为katG-315-S/T、rpoB-450-S/L、rpsL-43-K/R、embB-306-M/V、pncA启动子-11-T/C、gyrA-90-A/V、rrs-1401-A/G、inhA-15-C/T、thyA-75-H/N;基因型为L1、L2和L4型,以L2.2型、L2.3型和L4型为主。

Analysis of characteristic of resistant gene mutations among multidrug-resistant Mycobacterium tuberculosis in Shenzhen

Objective To analyze the characteristics of gene mutations of MDR-MTB in Shenzhen using the whole-genome sequencing (WGS) data, providing basement for rapid molecular detection of drug-resistant tuberculosis. Methods WGS was performed on 449 MDR-MTB clinical isolates collected from Shenzhen Center for Chronic Disease Control and Chronic Disease Control of districts of Shenzhen among 2013-2017. Among them, 447 strains were enrolled after sequence alignment with the standard strain of Mycobacterium tuberculosis (H37RV) genome template sequence. After being identified by single nucleotide polymorphism (SNP), each strain was aligned with the specific mutation of genotype or subtype of H37Rv and the drug-resistant gene mutation of 11 anti-TB drugs, to explore the genotype or subtype and drug-resistant gene mutation of each strain, and analyze the correlation of main mutation types from different drugs resistance genes in genotype or subtype. Results The WGS results of 447 MDR-MTB showed that 432 strains (96.6%) had gene mutation. The major resistant mutation types to 11 drugs were katG-315-S/T (INH,81.7% (353/432)), rpoB-450-S/L (RFP, 59.7% (258/432)), rpsL-43-K/R (Sm, 66.0% (198/300)), embB-306-M/V (EMB, 35.5% (94/265)), pncA promoter -11-T/C (PZA,8.9% (11/123)), gyrA-90-A/V (Ofx,31.5% (39/124)), rrs-1401-A/G (Am,48.1% (25/52)), rrs-1401-A/G (Cm, 100.0% (27/27)), rrs-1401-A/G (Km, 84.4% (27/32)), inhA-15-C/T (Eto, 84.2% (48/57)), and thyA-75-H/N (PAS, 31.3% (10/32)). Among mutation types, two or more mutations were found in INH, RFP, EMB and Sm. The 3 genotypes of MDR strains in Shenzhen were lineage 1 (L1) (0.4% (2/447)), lineage 2 (L2) (84.6% (378/447)) and lineage 4 (L4) (15.0% (67/447)) in Shenzhen. The L2 was further divided into three subtypes: L2.1 (1.9% (7/378)), L2.2 (37.0% (140/378)) and L2.3 (61.1% (231/378)). katG-315-S/T, rpsL-43-K/R and embB-306-M/V in L2 (79.6% (301/378), 50.5%(191/378), 23.0%(87/378)) were significantly higher than those in L4 (61.2% (41/67), 10.4% (7/67), 10.4% (7/67)) (χ ² were 10.874, 37.021 and 5.396, respectively; P values were 0.001, 0.000, and 0.020, respectively). However, the PAS resistant mutations, folC-43-I/T and thyA-75-H/N, were only found in L2. The mutation frequency of katG-315-S/T in L2.2 (88.6% (124/140)) was significantly higher than in L2.3 (74.5%(172/231)) (χ ²=10.764, P=0.000), while the mutation frequencies of inhA-15-C/T (INH), rpoB-450-S/L, rpsL-43-K/R and inhA-15-C/T (Eto) in L2.3 (8.2% (19/231), 61.9% (143/231), 59.3% (137/231), 15.6% (36/231)) were higher than those in L2.2 (2.9% (4/140), 49.3% (69/140), 37.9% (53/140), 3.6% (5/140)(χ ² were 4.319, 5.668, 16.053 and 12.797, respectively;P values were 0.038, 0.017, 0.000 and 0.000, respectively). Conclusion The major resistant mutation types of 11 drugs in MDR-MTB of Shenzhen were katG-315-S/T, rpoB-450-S/L, rpsL-43-K/R, embB-306-M/V, pncA promoter -11-T/C, gyrA-90-A/V, rrs-1401-A/G, inhA-15-C/T and thyA-75-H/N; and the genotypes were L1, L2 and L4, of which the main types were L2.2, L2.3 and L4.