关键词: 结核, 抗多种药物性, 基因组, 细菌, 高通量核苷酸测序, 基因型, 寡核苷酸序列分析, 深圳

Abstract:

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.

Key words: Tuberculosis, multidrug-resistant, Genome, bacterial, High-throughput nucleotide sequencing, Genotype, Point mutation, Oligonucleotide array sequence analysis, Shenzhen