Bacterial Zombies and Ghosts: Production of Inactivated Gram-Positive and Gram-Negative Species with Preserved Cellular Morphology and Cytoplasmic Content

ABSTRACT There are many approaches available to inactivate bacteria, each with a different efficacy, impact on cell integrity, and potential for application in high-throughput. The aim of this study was to compare these approaches and develop a standardized protocol for generation of intact Gram-positive as well as on Gram-negative “bacterial zombies”, i.e. cells that are metabolically dead with retained cellular integrity. Here, we introduce the term “bacterial zombies” in addition to “bacterial ghosts” to differentiate inactivated bacteria with preserved cellular integrity from those with perforated membranes, where DNA and cytoplasmic contents have been released. This differentiation of inactivated bacteria is important if the cell content is the subject of study, or if cell contents in the media may cause unwanted effects in downstream applications. We inactivated eight different bacterial species by treatment with beta-propiolactone, ethanol, formalin, sodium hydroxide, and pasteurization. Inactivation efficacy was determined by culturing, and cell wall integrity assessed by quantifying released DNA and visualization by scanning electron microscopy. Based on these results, we discuss the choice of bacterial inactivation methods, and conclude that beta-propiolactone and ethanol are the most promising approaches for standardized generation of bacterial zombies. IMPORTANCE For applications such as vaccination or analyses that are sensitive to bacterial growth, inactivated bacteria are preferred because they simplify the analyses and the interpretation of results. This study compared various bacterial inactivation treatments that maintain cell integrity and may be used in high-throughput. Our results demonstrated that beta-propiolactone and 70% ethanol were the best techniques to achieve these goals.