School of Public Health, University of Alberta, Edmonton, Alberta, Canada.
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
Cytometry A. 2019 May;95(5):555-564. doi: 10.1002/cyto.a.23768. Epub 2019 Apr 15.
Pseudomonas aeruginosa is a Gram-negative bacterium that is abundant in the environment and water systems, with strains that cause serious infections, especially in patients with compromised immune systems. In times of stress or as part of its natural life cycle, P. aeruginosa can adopt a viable but not culturable (VBNC) state, which renders it undetectable by current conventional food and water testing methods and makes it highly resistant to antibiotic treatment. Specific conditions can resuscitate these coccoid VBNC P. aeruginosa cells, which returns them to their active, virulent rod-shaped form. Underreporting the VBNC cells of P. aeruginosa by standard culture-based methods in water distribution systems may therefore pose serious risks to public health. As such, being able to accurately detect and quantify the presence of VBNC P. aeruginosa, especially in a hospital setting, is of critical importance. Herein, we describe a method to analyze VBNC P. aeruginosa using imaging flow cytometry. With this technique, we can accurately distinguish between active and VBNC forms. We also show here that association of VBNC P. aeruginosa with Acanthamoeba polyphaga results in resuscitation of P. aeruginosa to an active form within 2 h. Our approach could provide an alternative, reliable detection method of VBNC P. aeruginosa when coupled with species-specific staining. Most importantly, our experiments demonstrate that the coculture with amoebae can lead to a resuscitation of P. aeruginosa of culturable morphology after only 2 h, indicating that VBNC P. aeruginosa could potentially resuscitate in piped water (healthcare) environments colonized with amoebae. © 2019 International Society for Advancement of Cytometry.
铜绿假单胞菌是一种革兰氏阴性细菌，在环境和水系统中大量存在，其菌株可引起严重感染，尤其是在免疫系统受损的患者中。在压力下或作为其自然生命周期的一部分，铜绿假单胞菌可以采用一种可行但不可培养（VBNC）的状态，这使得它无法被当前常规的食品和水检测方法检测到，并使其对抗生素治疗具有高度抗性。特定条件可以使这些球菌状 VBNC 铜绿假单胞菌细胞复苏，使其恢复到活跃的、有毒的杆状形式。因此，在水分配系统中，基于标准培养的方法对铜绿假单胞菌的 VBNC 细胞进行低报可能对公共健康构成严重威胁。因此，能够准确检测和定量 VBNC 铜绿假单胞菌的存在，特别是在医院环境中，至关重要。在此，我们描述了一种使用成像流式细胞术分析 VBNC 铜绿假单胞菌的方法。通过这项技术，我们可以准确地区分活性和 VBNC 形式。我们还在这里表明，VBNC 铜绿假单胞菌与棘阿米巴属聚生体的关联会导致铜绿假单胞菌在 2 小时内复苏为活跃形式。我们的方法可以提供一种替代的、可靠的 VBNC 铜绿假单胞菌检测方法，当与种特异性染色结合使用时。最重要的是，我们的实验表明，与阿米巴共生仅 2 小时后，就可以使可培养形态的铜绿假单胞菌复苏，这表明 VBNC 铜绿假单胞菌可能会在被阿米巴属定植的管道水（医疗保健）环境中复苏。
