Clinical Laboratory' Affiliated Dongguan People's Hospital, Southern Medical University, No.3 Xinguchong Wandao South Road, Wangjiang District, Dongguan, 523059, Guangdong, China.
Curr Microbiol. 2020 Nov;77(11):3264-3269. doi: 10.1007/s00284-020-02148-3. Epub 2020 Aug 12.
The purpose of this study was to analyze the metallo-β-lactamases (MBLs) genotype and oprD mutations of the β-lactam antibiotic-resistant Pseudomonas aeruginosa (PA) strains isolated from southern China. We collected 110 strains of β-lactam antibiotic-resistant PA from 2 hospitals during January 2016-December 2017 from Dongguan, South China. MBLs were detected, amplified, and typed using EDTA disc synergy test, PCR, and Sanger gene sequencing. The mutations and expression levels of oprD were detected using Sanger gene sequencing and qPCR. A total of 16.36% (18/110) β-lactam antibiotic-resistant PA strains produced MBLs, and the main genotypes of MBLs were IMP-25, VIM-2, and SIM-2. Sanger gene sequencing results showed that 107 of the 110 strains harbored mutations in oprD sequence, while 3 strains were negative for oprD amplification (2.73%). Among the 107 strains with positive amplification (97.27%), the rate of intentional mutations (including deletions, insertions, and premature stop codons) was 93.46% (100/107) and that of no disrupted mutation was 6.54% (7/107). qPCR analysis confirmed that the expression level of the OprD protein in the 7 strains of no disrupted mutation was significantly reduced. Among the β-lactam antibiotic-resistant PA strains in southern China, 16.36% were positive for MBLs. The loss rate of oprD was 2.73%, and almost all PA strains showed oprD amplification variation or transcription downregulation. Thus, impaired oprD expression and MBLs production may be some of the mechanisms of β-lactam antibiotic-resistance of PA strains in southern China.
本研究旨在分析来自中国南方的耐β-内酰胺抗生素铜绿假单胞菌（PA）分离株的金属β-内酰胺酶（MBLs）基因型和 oprD 突变。我们收集了 2016 年 1 月至 2017 年 12 月期间来自中国南方东莞的 2 家医院的 110 株耐β-内酰胺抗生素 PA 株。使用 EDTA 圆盘协同试验、PCR 和 Sanger 基因测序检测 MBLs，扩增和分型。使用 Sanger 基因测序和 qPCR 检测 oprD 的突变和表达水平。共有 16.36%（18/110）的耐β-内酰胺抗生素 PA 株产生 MBLs，MBLs 的主要基因型为 IMP-25、VIM-2 和 SIM-2。Sanger 基因测序结果显示，110 株中有 107 株 oprD 序列发生突变，而 3 株 oprD 扩增阴性（2.73%）。在 107 株扩增阳性的菌株中（97.27%），有意突变（包括缺失、插入和提前终止密码子）的发生率为 93.46%（100/107），无破坏突变的发生率为 6.54%（7/107）。qPCR 分析证实，7 株无破坏突变的 oprD 蛋白表达水平显著降低。在中国南方的耐β-内酰胺抗生素 PA 株中，16.36%为 MBLs 阳性。oprD 的缺失率为 2.73%，几乎所有的 PA 株都表现出 oprD 扩增变异或转录下调。因此，oprD 表达受损和 MBLs 产生可能是中国南方 PA 株耐β-内酰胺抗生素的部分机制。
