Wang Yan, Yang Haitao, Wang Kun, Zhu Wenjun, Wang Xizhen
Department of Hospital Infection Control, The Affiliated Lianyungang Hospital of Bengbu Medical University, The Second People's Hospital of Lianyungang, Lianyungang, 222000, China.
Department of Medicine Laboratory, The Second People's Hospital of Lianyungang Affiliated with Kangda College of Nanjing Medical University, The Second People's Hospital of Lianyungang City, Lianyungang, 222000, China.
Arch Microbiol. 2025 Jun 18;207(8):179. doi: 10.1007/s00203-025-04373-5.
Pseudomonas aeruginosa (PA), which is a common Gram-negative bacterium, can become carbapenem-resistant Pseudomonas aeruginosa (CRPA) upon the development of antibiotic resistance, making clinical treatment difficult. CRPA with antibiotic resistance genes (ARGs), such as bla_NDM and bla_KPC, is common in China. The development of tests for ARGs would facilitate the more rapid identification of CRPA in China. Isothermal amplification research has improved, but limitations remain, including a lack of specialized equipment, the difficulty of developing sophisticated primers, and aerosol pollution. Thus, clinical examination methods must improve. We successfully integrated RPA with CRISPR/Cas12a, and we identified bla_NDM as our institution's primary CRPA. RPA-CRISPR/Cas12a could accurately detect target DNA within 40 min without cross-reacting with other bacteria. The results showed high concordance with conventional culture-based methods, including 100% agreement in clinical sample validation. The method reliably identified standard PA strains and 29 clinical isolates, detecting PA at concentrations as low as 10 CFU. In addition, the closed-tube format effectively minimized the risk of aerosol contamination. This platform offers a rapid and user-friendly tool for identifying bla_NDM-positive CRPA, and this tool is particularly suitable for early screening and clinical management in resource-limited settings. The simplicity and accuracy of this approach make it a promising option for infection control and public health surveillance.
铜绿假单胞菌（PA）是一种常见的革兰氏阴性菌，在产生抗生素耐药性后可成为耐碳青霉烯铜绿假单胞菌（CRPA），这使得临床治疗变得困难。携带抗生素耐药基因（ARGs），如bla_NDM和bla_KPC的CRPA在中国很常见。针对ARGs的检测方法的开发将有助于在中国更快速地鉴定CRPA。等温扩增研究有所改进，但仍存在局限性，包括缺乏专门设备、难以设计复杂的引物以及气溶胶污染等问题。因此，临床检测方法必须改进。我们成功地将重组酶聚合酶扩增（RPA）与CRISPR/Cas12a整合，并将bla_NDM鉴定为我们机构主要的CRPA。RPA-CRISPR/Cas12a能够在40分钟内准确检测目标DNA，且不会与其他细菌发生交叉反应。结果显示与传统的基于培养的方法高度一致，在临床样本验证中一致性达100%。该方法能够可靠地鉴定标准PA菌株和29株临床分离株，检测低至10 CFU浓度的PA。此外，闭管形式有效降低了气溶胶污染的风险。该平台为鉴定bla_NDM阳性CRPA提供了一种快速且用户友好的工具，尤其适用于资源有限环境下的早期筛查和临床管理。这种方法的简单性和准确性使其成为感染控制和公共卫生监测的一个有前景的选择。
