Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom.
Shell International Exploration & Production Inc., Westhollow Technology Center, Houston, TX, United States.
Front Cell Infect Microbiol. 2021 Jul 21;11:716592. doi: 10.3389/fcimb.2021.716592. eCollection 2021.
Bacteria modify their morphology in response to various factors including growth stage, nutrient availability, predation, motility and long-term survival strategies. Morphological changes may also be associated with specific physiological phenotypes such as the formation of dormant or persister cells in a "viable but non-culturable" (VBNC) state which frequently display different shapes and size compared to their active counterparts. Such dormancy phenotypes can display various degrees of tolerance to antibiotics and therefore a detailed understanding of these phenotypes is crucial for combatting chronic infections and associated diseases. Cell shape and size are therefore more than simple phenotypic characteristics; they are important physiological properties for understanding bacterial life-strategies and pathologies. However, quantitative studies on the changes to cell morphologies during bacterial growth, persister cell formation and the VBNC state are few and severely constrained by current limitations in the most used investigative techniques of flow cytometry (FC) and light or electron microscopy. In this study, we applied high-throughput Imaging Flow Cytometry (IFC) to characterise and quantify, at single-cell level and over time, the phenotypic heterogeneity and morphological changes in cultured populations of four bacterial species, and Morphologies in relation to growth stage and stress responses, cell integrity and metabolic activity were analysed. Additionally, we were able to identify and morphologically classify dormant cell phenotypes such as VBNC cells and monitor the resuscitation of persister cells in following antibiotic treatment. We therefore demonstrate that IFC, with its high-throughput data collection and image capture capabilities, provides a platform by which a detailed understanding of changes in bacterial phenotypes and their physiological implications may be accurately monitored and quantified, leading to a better understanding of the role of phenotypic heterogeneity in the dynamic microbiome.
细菌会根据各种因素改变其形态，包括生长阶段、营养物质可用性、捕食、运动和长期生存策略。形态变化也可能与特定的生理表型相关，例如在“存活但不可培养”（VBNC）状态下形成休眠或持久细胞，与活跃状态下的细胞相比，其形状和大小通常会有所不同。这些休眠表型可能对抗生素表现出不同程度的耐受性，因此，详细了解这些表型对于对抗慢性感染和相关疾病至关重要。因此，细胞形状和大小不仅仅是简单的表型特征，它们还是理解细菌生活策略和病理学的重要生理特性。然而，关于细菌生长过程、持久细胞形成和 VBNC 状态下细胞形态变化的定量研究很少，并且受到当前最常用的流式细胞术（FC）和光或电子显微镜调查技术的限制。在这项研究中，我们应用高通量成像流式细胞术（IFC）来表征和定量分析四种细菌培养物群体的单细胞水平和随时间变化的表型异质性和形态变化，以及与生长阶段和应激反应、细胞完整性和代谢活性相关的形态变化。此外，我们能够识别和形态分类休眠细胞表型，如 VBNC 细胞，并监测抗生素治疗后 中持久细胞的复苏。因此，我们证明 IFC 凭借其高通量数据收集和图像捕获功能，为准确监测和量化细菌表型变化及其生理意义提供了一个平台，从而更好地理解表型异质性在动态微生物组中的作用。
