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.