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

Abstract

25There are many approaches available to inactivate bacteria, each with a different efficacy, 26 impact on cell integrity, and potential for application in high-throughput. The aim of this 27 study was to compare these approaches and develop a standardized protocol for generation of 28 intact Gram-positive as well as on Gram-negative "bacterial zombies", i.e. cells that are 29 metabolically dead with retained cellular integrity. Here, we introduce the term "bacterial 30 zombies" in addition to "bacterial ghosts" to differentiate inactivated bacteria with preserved 31 cellular integrity from those with perforated membranes, where DNA and cytoplasmic 32 contents have been released. This differentiation of inactivated bacteria is important if the cell 33 content is the subject of study, or if cell contents in the media may cause unwanted effects in 34 downstream applications. We inactivated eight different bacterial species by treatment with 35 beta-propiolactone, ethanol, formalin, sodium hydroxide, and pasteurization. Inactivation 36 efficacy was determined by culturing, and cell wall integrity assessed by quantifying released 37 DNA and visualization by scanning electron microscopy. Based on these results, we discuss 38 the choice of bacterial inactivation methods, and conclude that beta-propiolactone and ethanol 39 are the most promising approaches for standardized generation of bacterial zombies. 40 41 3 IMPORTANCE 42For applications such as vaccination or analyses that are sensitive to bacterial growth, 43 inactivated bacteria are preferred because they simplify the analyses and the interpretation of 44 results. This study compared various bacterial inactivation treatments that maintain cell 45 integrity and may be used in high-throughput. Our results demonstrated that beta-46 propiolactone and 70% ethanol were the best techniques to achieve these goals. 48Bacterial inactivation refers to bactericidal methods that kill bacteria by damaging DNA or 49 protein synthesis, resulting in termination of growth. While several techniques are available to 50 achieve bacterial inactivation, most studies focus on single bacteria and there is no protocol 51 for standardized and high-throughput inactivation of bacteria in general. Experiments that are 52 sensitive to host-microbe interaction often involve inactivation of the bacteria before they are 53 applied. Examples include immunization of humans and animals (vaccinations), analysis of 54 cell response to bacterial outer-membrane structures, and the use of inactivated bacteria as 55 carriers for drugs or antigens [1]. 56 One challenge for a standardized protocol for bacterial inactivation lies in the diversity of the 57 targeted bacteria. For example, the structurally different walls of Gram-positive and Gram-58 negative bacteria may inhibit certain inactivation treatments. One example is the production 59 of bacterial ghosts by using a plasmid with an E gene insert derived from bacteriophage 60 ФX174, that lyses Gram-negative bacteria by forming a lysis tunnel across...

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