INTRODUCTION

Spores are dormant bodies formed by spore-producing bacteria under nutrient-poor conditions [1]. Spore formation is an extremely complex process involving multiple changes in morphology, structure, chemical composition, and other aspects. During sporulation, trophoblast cells divide asymmetrically to form the prespore and mother cell compartments, where the smaller prespore compartment is used for spore formation and the larger mother cell compartment is used to cultivate the spores and synthesize the external proteolipid layer structure. Until the prespore is fully developed, the mother cell compartment engulfs and fuses with the prespore compartment and forms a multilayered protective layer around the prespore; when the spore is formed, the mother cell ruptures to release the spore into the environment [2]. The spore contains the complete genome of the species and is capable of resuming growth to form a trophoblast under nutritive conditions. In addition, spores have a special multi-layered structure and extremely low water content, giving them the ability to survive for long periods of time, even in extreme environments, making them extremely resilient. For example, spores can survive in low and high temperatures, including space environments, and can be exposed to strong acids and bases, ethanol, etc [3-7]. The resilience of spores is mainly dependent on two types of wall structures, an external protein wall, the spore shell, and a peptidoglycan wall, the spore cortex. The spore shell is resistant to a wide range of chemical and enzymatic treatments and acts as a permeable barrier, limiting the entry of macromolecules into the spore. The cortex, on the other hand, is a special peptidoglycan-rich structure deposited between the inner and outer membranes of the spore, responsible for maintaining the core in a highly dehydrated state, thus contributing to the extreme dormancy and heat resistance [2]. In addition, maintaining the dehydration of the spore core is dependent on the inner membrane, which is relatively permeable and limits the entry and exit of small molecules and chemicals [6]. Degradation of a spore’s cortex and damage to the inner membrane can result in the rapid rehydration of the spore core and consequent loss of resistance [8]. Notably, a specific molecule within the spore core, pyridine-2,6-dicarboxylic acid (DPA), chelated 1:1 with Ca2+ (CaDPA), plays an important role in spore resistance and stability [9]. Although the exact function of CaDPA in spores is not yet known, a lack of, or low levels of, CaDPA can result in very unstable spores that germinate spontaneously [10].
Currently, chlorination is a popular method for sterilization and sodium hypochlorite (bleach) is widely used by virtue of its cheapness and availability [11]. Many studies have evaluated the effects of sodium hypochlorite on spores in terms of (i) spore viability, (ii) release of budding molecules and (iii) electron microscopic capture of budding structures. These studies have shown that at suitable concentrations and exposure times, sodium hypochlorite is able to kill spores by damaging their shells, germination proteins and DNA [12, 13]. However, there is controversy as to whether CaDPA is released from spores after sodium hypochlorite treatment. It has been shown that sodium hypochlorite destroys the outer shell of spores and damages proteins on the inner membrane, thus disrupting the permeability barrier, and leading to the release of CaDPA from the core and DNA denaturation [14]. However, in another study, spores did not release CaDPA after hypochlorite treatment but released CaDPA at germination and were unable to grow further [15]. The ability of hypochlorite to inactivate spores is not disputed; however, its mechanism of action is not yet fully understood. There are still many unresolved issues, including (i) whether sodium hypochlorite-treated spores release substances, such as DNA, at the time of CaDPA release and (ii) whether the degradation of spore shells by sodium hypochlorite is localized or global. In addition, methods used in previous studies, such as transmission electron microscopy and scanning electron microscopy, require the special treatment of individual organisms, which may cover up some information about treated spores [16]. It should also be noted that the overuse of chemicals to treat spores results in a series of problems, such as strain resistance and contamination of the environment [11, 17].
In the present study, we comprehensively evaluated the response of spores to sodium hypochlorite by using single-cell techniques. We used laser tweezer Raman spectroscopy (LTRS) to detect molecular changes in spores after sodium hypochlorite treatment, evaluated spore morphology before and after treatment using atomic force microscopy (AFM), and observed the germination and growth process of single spores before and after treatment using live-cell dynamic imaging. The results of this study deepen our understanding of the response of spores to sodium hypochlorite, including internal molecular and morphological structural changes, and provide new insights into the effects of sodium hypochlorite on spores at the single-cell level.