关键词: 分枝杆菌, 结核, 利福平, 突变, 基因组, 微生物敏感性试验

Abstract:

Objective：To learn about the mutation characteristics of the rpoB gene in Mycobacterium tuberculosis （MTB） in Shanghai，and explore the correlation between rpoB gene mutation and rifampin resistance level. Methods：Strains of pulmonary tuberculosis patients who were newly registered and culture-positive in Shanghai from 2020 to 2022 were selected using a simple random sampling. Minimal inhibitory concentration （MIC） drug susceptibility test was performed on 2766 MTB strains. After excluding duplicate samples and those with failed tests，valid phenotypic drug susceptibility results were obtained from 2734 strains. Whole genome sequencing （WGS） was performed on 462 strains from these patients （176 retreatment and 286 primary patients）. After excluding 12 non-compliant samples，450 MTB strains were analyzed for rifampicin resistance gene mutations，then the relationship between rpoB mutation type and rifampicin MIC were analyzed with Wilcoxon rank sum test. Results：Among the 450 strains of clinical MTB，139 strains （including 8 RFP sensitive strains，18 strains with low-level RFP resistant and 113 strains with high-level RFP resistant） had RFP resistance gene mutations，among which 95.68% （133/139） of the mutations occurred in the rifampicin rpoB RRDR region. The most frequent rpoB gene mutations were observed at codons 450，445 and 435，which had mutated frequencies of 60.43% （84/139），21.58% （30/139） and 7.91% （11/139），respectively. The Wilcoxon rank sum test showed that rpoB_p.Ser450Leu，rpoB_p.Ser450Trp，rpoB_p.His445Asp，rpoB_p.His445Tyr，rpoB_p.Asp435Gly，rpoB_p.Asp435Val，rpoB_p.Asp435Tyr and rpoB_p.Leu452Pro were significantly associated with RFP resistance （Ps<0.05）. Among them，stains with these five mutations （rpoB_p.Ser450Leu，rpoB_p.Ser450Trp，rpoB_p.His445Asp，rpoB_p.His445Tyr and rpoB_p.Asp435Val） had a median MIC over 16 μg/ml，showing high level resistance. The median MIC values for rpoB_p.Asp435Gly，rpoB_p.Asp435Tyr and rpoB_p.Leu452Pro mutant strains were 4 μg/ml，1 μg/ml and 4 μg/ml，respectively，showing low-level resistance. Among the RFP-resistant strains with multicodon mutations，70.59% （12/17） of the rpoB RRDR gene mutation were accompanied by compensatory mutations of rpoA and rpoC or rpoB non-RRDR. All multicodon mutant strains showed high level resistance （MIC>16 μg/ml）. Lineage analysis of 450 MTB strains revealed that the L2 lineage predominated at 88.44% （398/450），the L4 lineage accounted for 10.90% （49/450），while the L1 lineage accounted for the least，only 0.44% （2/450）. One strain （0.22%） was identified as a mixed strain of both L2 and L4 lineages. Among RFP-resistant strains，the L2 lineage showed a higher proportion at 92.09% （128/139），while the L4 lineage accounted for 7.19% （10/139），and no L1 lineage resistant strains were detected. Analysis of sublineage distribution revealed that the L2 lineage was dominated by the L2.2.1 sublineage （55.78%，251/450），while the L4 lineage was primarily composed of the L4.4.2 and L4.5 sublineages，accounted for 4.00% （18/450） and 3.56% （16/450），respectively. Conclusion：Different rifampicin rpoB mutation types in Shanghai can cause different levels of RFP resistance. The mutation of rpoB_p.Ser450Leu，rpoB_p.Ser450Trp，rpoB_p.His445Asp，rpoB_p.His445Tyr and rpoB_p.Asp435Val are associated with high-level resistance of RFP. The mutation of rpoB_p.Asp435Gly，rpoB_p.Asp435Tyr and rpoB_p.Leu452Pro were associated with low-level resistance of RFP. Multicodon mutations，especially those carrying compensatory mutations，may synergistically enhance RFP resistance level.

Key words: Mycobacterium tuberculosis, Rifampicin, Mutation, Genome, Microbial sensitivity tests