Department of Pharmacy, Singapore General Hospital, Singapore, Singapore.
SingHealth-Duke-NUS Academic Clinical Programme, Singapore, Singapore.
PLoS One. 2024 Jun 24;19(6):e0305939. doi: 10.1371/journal.pone.0305939. eCollection 2024.
Current antimicrobial susceptibility testing (AST) requires 16-24 hours, delaying initiation of appropriate antibiotics. Hence, there is a need for rapid AST. This study aims to develop and evaluate the feasibility of a rapid flow cytometric AST assay to determine minimum inhibitory concentration (MIC) for carbapenem-resistant Acinetobacter baumannii (CRAB). Antibiotic exposure causes increased intracellular reactive oxygen species (ROS) in bacteria. We hypothesized that ROS can be used as a marker to determine MIC. We assessed three CRAB clinical isolates across fifteen antibiotics at various concentrations in a customized 96-well microtiter plate. The antibiotics assessed include amikacin, beta-lactams (ampicillin/sulbactam, aztreonam, cefepime, ceftolozane/tazobactam, doripenem, imipenem, meropenem, and piperacillin/tazobactam), levofloxacin, polymyxin B, rifampicin, trimethoprim/sulfamethoxazole, and tetracyclines (tigecycline and minocycline). These clinical CRAB isolates were assessed for ROS after antibiotic treatment. Increased ROS levels indicated by increased RedoxSensorTM Green (RSG) fluorescence intensity was assessed using flow cytometry (FCM). MIC was set as the lowest antibiotic concentration that gives a ≥1.5-fold increase in mode RSG fluorescence intensity (MICRSG). Accuracy of MICRSG was determined by comparing against microtiter broth dilution method performed under CLSI guidelines. ROS was deemed accurate in determining the MICs for β-lactams (83.3% accuracy) and trimethoprim/sulfamethoxazole (100% accuracy). In contrast, ROS is less accurate in determining MICs for levofloxacin (33.3% accuracy), rifampicin (0% accuracy), amikacin (33.3% accuracy), and tetracyclines (33.3% accuracy). Collectively, this study described an FCM-AST assay to determine antibiotic susceptibility of CRAB isolates within 5 hours, reducing turnaround time up to 19 hours.
目前的抗菌药物敏感性测试（AST）需要 16-24 小时，从而延迟了适当抗生素的使用。因此，需要快速 AST。本研究旨在开发和评估一种快速流式细胞术 AST 测定法，以确定耐碳青霉烯鲍曼不动杆菌（CRAB）的最小抑菌浓度（MIC）。抗生素暴露会导致细菌内活性氧物种（ROS）增加。我们假设 ROS 可以用作确定 MIC 的标志物。我们评估了三种 CRAB 临床分离株，在定制的 96 孔微量滴定板中，在十五种不同浓度的抗生素下进行了测试。评估的抗生素包括阿米卡星、β-内酰胺类（氨苄西林/舒巴坦、氨曲南、头孢吡肟、头孢他啶/他唑巴坦、多利培南、亚胺培南、美罗培南和哌拉西林/他唑巴坦）、左氧氟沙星、多粘菌素 B、利福平、复方磺胺甲噁唑和四环素（替加环素和米诺环素）。这些临床 CRAB 分离株在抗生素治疗后进行 ROS 评估。使用流式细胞术（FCM）通过增加的 RedoxSensorTM Green（RSG）荧光强度来评估 ROS 水平的增加。MIC 被设定为导致模式 RSG 荧光强度增加≥1.5 倍的最低抗生素浓度（MICRSG）。通过与根据 CLSI 指南进行的微量肉汤稀释法比较来确定 MICRSG 的准确性。ROS 在确定β-内酰胺类（83.3%的准确性）和复方磺胺甲噁唑（100%的准确性）的 MIC 方面是准确的。相比之下，ROS 在确定左氧氟沙星（33.3%的准确性）、利福平（0%的准确性）、阿米卡星（33.3%的准确性）和四环素（33.3%的准确性）的 MIC 方面准确性较低。总的来说，这项研究描述了一种 FCM-AST 测定法，可在 5 小时内确定 CRAB 分离株的抗生素敏感性，将周转时间缩短至 19 小时。
