Department of Bacteriology and Immunology, Beijing Key Laboratory On Drug-Resistant Tuberculosis Research, Beijing Tuberculosis and Thoracic Tumor Research Institute/Beijing Chest Hospital, Capital Medical University, Beijing, 101149, People's Republic of China.
Department of Tuberculosis, Beijing Tuberculosis and Thoracic Tumor Research Institute/Beijing Chest Hospital, Capital Medical University, Beijing, 101149, People's Republic of China.
Ann Clin Microbiol Antimicrob. 2022 Nov 5;21(1):48. doi: 10.1186/s12941-022-00537-z.
BACKGROUND & OBJECTIVES: Accurate determination of antimicrobial resistance profiles is of great importance to formulate optimal regimens against multidrug-resistant tuberculosis (MDR-TB). Although para-aminosalicylic acid (PAS) has been widely used clinically, the reliable testing methods for PAS susceptibility were not established. Herein, we aimed to establish critical test concentration for PAS on the Mycobacterial Growth Indicator Tube (MGIT) 960 in our laboratory settings.
A total of 102 clinical isolates were included in this study, including 82 wild-type and 20 resistotype isolates. Minimum inhibitory concentration (MIC) was determined by MGIT 960. Whole-genome sequencing was used to identify the mutation patterns potentially conferring PAS resistance. Sequence alignment and structure modelling were carried out to analyze potential drug-resistant mechanism of folC mutant.
Overall, the Minimum inhibitory concentration (MIC) distribution demonstrated excellent separation between wild-type and resistotype isolates. The wild-type population were all at least 1 dilution below 4 μg/ml, and the resistotype population were no lower than 4 μg/ml, indicating that 4 μg/ml was appropriate critical concentration to separate these two populations. Of 20 mutant isolates, 12 (60.0%) harbored thyA mutations, 2 (10%) had a mutation on upstream of dfrA, and the remaining isolates had folC mutations. Overall, thyA and folC mutations were scattered throughout the whole gene without any one mutation predominating. All mutations within thyA resulted in high-level resistance to PAS (MIC > 32 μg/ml); whereas the MICs of isolates with folC mutations exhibited great diversity, ranged from 4 to > 32 μg/ml, and sequence and structure analysis partially provided the possible reasons for this diversity.
We propose 4 μg/ml as tentative critical concentration for MGIT 960. The major mechanism of PAS resistance is mutations within thyA and folC in MTB isolations. The whole-gene deletion of thyA locus confers high-level resistance to PAS. The diversity of many distinct mutations scattered throughout the full-length folC gene challenges the PCR-based mutation analysis for PAS susceptibility.
准确测定抗菌药物耐药谱对于制定针对耐多药结核分枝杆菌（MDR-TB）的最佳方案非常重要。虽然对氨基水杨酸（PAS）在临床上得到了广泛应用，但尚未建立可靠的 PAS 药敏检测方法。本研究旨在确定本实验室分枝杆菌液体培养药敏检测管（MGIT960）中 PAS 的临界测试浓度。
共纳入 102 株临床分离株，包括 82 株野生型和 20 株耐药型分离株。采用 MGIT960 测定最小抑菌浓度（MIC）。全基因组测序用于鉴定可能导致 PAS 耐药的突变模式。序列比对和结构建模用于分析 folC 突变体的潜在耐药机制。
总体而言，MIC 分布在野生型和耐药型分离株之间表现出良好的分离。野生型菌株均至少稀释 1 倍，MIC 值<4μg/ml，耐药型菌株 MIC 值均不低于 4μg/ml，表明 4μg/ml 是分离这两种菌株的合适临界浓度。20 株突变株中，thyA 突变 12 株（60.0%），dfrA 上游突变 2 株（10%），其余菌株 folC 突变。总体而言，thyA 和 folC 突变在整个基因中散布，没有一个突变占主导地位。thyA 内的所有突变均导致 PAS 高水平耐药（MIC>32μg/ml）；而 folC 突变株的 MIC 值表现出很大的多样性，范围为 4 至>32μg/ml，序列和结构分析部分提供了这种多样性的可能原因。
我们建议将 4μg/ml 作为 MGIT960 的暂定临界浓度。PAS 耐药的主要机制是 MTB 分离株中 thyA 和 folC 内的突变。thyA 基因座的完全缺失导致 PAS 高水平耐药。folC 全长内散布着许多不同的突变，这给基于 PCR 的 PAS 药敏突变分析带来了挑战。
Zotero 插件