Analysis of injury and growth behaviors of stressed bacillus subtilis spores using the double subculture method

Tuyển tập báo cáo Hội nghị Khoa học và Công nghệ hạt nhân toàn quốc lần thứ 14
Proceedings of Vietnam conference on nuclear science and technology VINANST-14

ANALYSIS OF INJURY AND GROWTH BEHAVIORS OF STRESSED BACILLUS
SUBTILIS SPORES USING THE DOUBLE SUBCULTURE METHOD
R. ASADA1, 2*, S. HORIKIRI2, H. DEN1, J.J. SAKAMOTO2, T. TSUCHIDO2, M. FURUTA1, 2
1
2

Graduate School of Engineering, Osaka Prefecture University, Sakai, Japan

Research Center of Microorganism Control, Organization of Research Promotion,
Osaka Prefecture University, Sakai, Japan
*E-mail:

Abstract: The control of bacterial spores resistant to sterilization is important for the microbiological safety and integrity
of foods. Spores are considered sub-lethally injured during the sterilization process but remain alive and can germinate and
grow under non-stress conditions during storage and distribution. In this study, we aimed to establish a method to control
such injured bacterial spores, the survival of injured Bacillus subtilis spores after heat or gamma-ray irradiation treatment
was investigated. The numbers of injured spores were determined using the double subculture (DS) method, estimating the
injured population via the differential between the traditional plate count survival rate and the integrated viability (IV)
using growth delay analysis. The spores of Bacillus subtilis 168 wild-type and deficient strains that lacked either of the
small acid-soluble protein genes (△sspA△sspB) were heated and gamma-irradiated; then their homologous recombination
repair gene (△recA), or a non-homologous end binding repair gene (△ykoUV) were determined with the DS method. Using
the △sspA△sspB strain, we confirmed that DNA protection was involved in heat and gamma-ray irradiation resistance. In
addition, evaluation of the sterilization stress-treated △ykoVU and △recA strains indicated that ykoUV, in particular,
functioned to repair DNA injury, thus leading to normal post-germination growth after gamma-ray irradiation.
Keywords: gamma-ray irradiation, Bacillus subtilis spores, DNA repair, DNA protection

1. INTRODUCTION
The microbiological safety and wholesomeness of food products are achieved by strict microbial

control throughout the food chain, from the initial acquisition of raw materials, through pretreatment and
processing, to the distribution and consumption of the final product. The possible occurrence of
sterilization-resistant bacterial spores is problematic in various fields, including food and pharmaceuticals.
The resistance of bacterial spores to bactericidal stress is believed to be due to their unique layered
structure and intracellular environment. The outermost layer of the spore is surrounded by a spore coat
consisting of highly cross-linked coat proteins [1], while the inner cortex is composed of peptidoglycans
[2] that protect the core, which contains the spore’s DNA and multiple enzymes [3]. In addition, the DNA
in the core is protected by intracellular substances such as dipicolinic acid (DPA) [4], which chelates with
Ca2+, and α/β-type small acid-soluble spore proteins (SASPs) [5,6]. Furthermore, the core is dehydrated [7].
Overall, these factors contribute to the high tolerance of spores to bactericidal stress.
Many of the spores damaged by heat sterilization exist in a sub-lethal state and recover during
incubation under non-stress conditions, such as storage and distribution. The actual condition of such
injured spores remains unknown. Moreover, the use of conventional sterilization technology alone requires
high temperature or long treatment time, resulting in the inevitable deterioration of product quality.
Sterilization conditions have been relaxed in recent years due to consumer preference for high quality; thus,
the potentials for injured spores to recover and countermeasures against this occurrence are of increasing
interest. Furthermore, the colony count unit (CFU) method using agar plates is usually employed as an
indicator of bacterial spore kill, but it may not accurately determine the number of injured bacteria because
bacterial damage includes factors such as delayed growth initiation and viability. It is impossible to
distinguish between healthy bacteria and bacteria in a state of recovery, leading to ambiguity when
assessing spore control measures.
In this study, to address these problems, Bacillus subtilis spores were subjected to heat sterilization
and γ-ray irradiation treatment, which are commonly used in the food industry. The goal of the study was
to estimate the injured population based on the difference between the survival rate determined using CFU
data obtained by the plate count method and the converted survival rate obtained using the growth delay
analysis method [8,9]. Furthermore, we aimed to determine the relationships between DNA damage and
repair mechanisms, cell damage, and recovery mechanisms by comparing injured populations of strains
deficient in several stress-response control genes. The study findings provide basic knowledge to develop
effective countermeasures against spore resistance and recovery in food products.
485

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Section D3-D4: Application of nuclear techniques in agriculture, radiation technology application

2. METHODOLOGY
2.1. Strains and spore preparation method
B. subtilis 168 wild-type (WT) strain, SASP-deficient strain (△sspA△sspB), homologous
recombination (HR)-deficient strain (△recA), and non-homologous end joining (NHEJ)-deficient strain
(△ykoVU) were cultured on Schaefer's sporulation agar at 37 °C to generate spores.

2.2. Heat and γ-ray sterilization
Heat sterilization treatments of B. subtilis spores were carried out at 95 °C for 10, 15, 20, 30, and 40
min. A 10-fold dilution series of unheated or heat-treated spores for each treatment time was prepared
using 50 mM potassium phosphate buffer (KPB, pH 7.0) containing 0.1% Tween 80.
γ-ray sterilization treatment of B. subtilis spores was carried out at 0, 2, 4, 6, 8, and 10 kGy, with the
same dose rate of approximately 1.87 kGy/h. The spore solution was diluted 10-fold with 50 mM KPB in a
brown test tube with a stopper, and subsequently, γ-ray irradiated in the 60Co gamma irradiation pool at the
Radiation Research Center of the Research Promotion Organization of Osaka Prefecture University. A
dilution series of the treated spore solutions was also prepared as described above for heat sterilization
treatment.
2.3. Determination of the number of viable bacteria and converted viability
The growth delay analysis method [8] estimates the number of viable bacteria based on the delay of
turbidity change in liquid culture (cultured in LB medium at 37 °C, absorbance measured using a
microplate reader at OD 650 nm). The number of surviving spores is denoted by ν [8]. When the number of
spores is 1/10 the difference in the delay time is expressed as G10. The delay time in the growth of spores
when exposed to various stresses compared to that of the untreated spores is defined as τ. The converted
viability (IV) is expressed as the ratio of τ to G10 and is calculated using equation 1 mentioned below.
IV: − log 𝜈 = ( 𝜏⁄G10 ) (1)
The differential viabilities between solid and liquid media (DiVSal) method or double subculture

(DS) method [9,10] compares the number of viable bacteria counted using the growth delay analysis
method with the number of viable bacteria obtained using the plate count method (cultured on LB agar
medium at 37 °C for 48 h). The number of damaged bacteria was obtained from the difference between the
two methods.
2.4. Evaluation of the number of injured spores after sterilization treatment
After heat treatment and γ-ray irradiation of various B. subtilis spore solutions, susceptibility to
sterilization treatment and the number of injured bacteria were evaluated using the plate count, growth
delay analysis, and DS methods.

3. RESULTS
3.1. Quantification of surviving spores
The killing effects of γ-ray irradiation and 95 °C heat treatments on B. subtilis spores (WT) were
evaluated using the plate count method. In the case of γ-ray irradiation, the spores lost their ability to form
colonies exponentially with the increasing dose (Fig. 1A). However, in the case of 95 °C heat treatment, a
shoulder was observed at 10 min after which the spores lost their ability to form colonies exponentially
with the increasing heating time (Fig. 2B). The D10 values were 1.6 kGy for gamma irradiation and 21.5
min for heating.

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Proceedings of Vietnam conference on nuclear science and technology VINANST-14

Figure 1. Survival curve of sterilization stress-treated B. subtilis spores (WT)
(A) γ-ray irradiation (B) 95 °C heat treatment.

3.2. Analysis of developmental dynamics
Using the plate count method, cells treated with and without sterilization stress were cultured on LB
agar, and the resulting colonies were counted and compared to evaluate the killing effect. However, it is

impossible to evaluate the number of injured spores using this method unless they form colonies. Therefore,
the initial and treated spores were cultured in a liquid medium, and their OD650 values were measured with
time to investigate the developmental dynamics of the injured spores. For γ-ray irradiated spores, the time
between germination and growth and the increase in OD650 due to nutrient growth was delayed in a dosedependent manner. After a certain time lag, the spores grew and reached the logarithmic growth phase like
non-treated spores. If spores are damaged during sterilization treatment, they can only germinate and
proliferate after successfully recovering through the spore germination process. This recovery time also
contributes to the extended delay. Therefore, the extended delay time should be taken into account when
calculating the converted survival rate.

Figure 2. Growth curves of sterilization stress-treated B. subtilis spores (WT). (A) γ-ray irradiation (B)
95 °C heat treatment.

487


Tiểu ban D3-D4: Ứng dụng kỹ thuật hạt nhân trong nông nghiệp, ứng dụng công nghệ bức xạ
Section D3-D4: Application of nuclear techniques in agriculture, radiation technology application

3.3. Quantification of injured spores by heat treatment and γ-ray irradiation
The developmental behavior of B. subtilis spores damaged by heat and irradiation and the
applicability of the DS method to assess the number of injured spores were investigated. The delay of the
growth curve is fundamental in the evaluation of damaged bacteria. Therefore, we evaluated the growth
curve delay against the dilution series of untreated spores and found that it showed linearity, indicating that

Figure 3. Applicability of the growth delay analysis method by confirming the correlation between
dilution rate and delay time in B. subtilis spores (WT).

the number of viable spores could be estimated from the delay time (Fig. 3).
The delay time (G10 value) for each 10-fold dilution was estimated to be 59.6 min. The plate count
method displayed a large shoulder, while the growth delay analysis revealed a large decay, indicating a

difference between the number of spores calculated using each method (Fig. 4). Radiation-induced injury
depended on the radiation dose, and heat-induced injury depended on heating time. These results confirmed the
applicability of the DS method for evaluating injured spores and showed that more injured spores were

generated by heat treatment than by γ-ray irradiation.
Figure 4. Analysis of injured B. subtilis spores (WT) spores after (A) heat treatment and (B) γray irradiation by double subculture method. ND: number of dead spores; NI: number of injured
spores; NL: number of live spores.

3.4. Quantification of injured spores and growth behavior of various gene-deficient strains
damaged by heat treatment and γ-ray irradiation
This study evaluated the effects of heat and γ-ray irradiation treatment on spore damage, including
germination growth, post-germination growth, and nutrient growth. The number of injured spores was
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Proceedings of Vietnam conference on nuclear science and technology VINANST-14

compared between the WT strain and the strain deficient in a SASP gene (ΔsspA ΔsspB), which has a
DNA-protective effect. The growth delay analysis method was used to compare the killing effect of heat
treatment on spores of the WT and △sspA△ sspB strains (Fig. 5). Heat treatment at 95 °C for 10 and 20
min did not kill WT spores by one order of magnitude, while three orders of magnitude of death were
observed in the △sspA △sspB strain after 10 min and four orders of magnitude after 20 min heating. Both
strains showed a lower conversion survival rate in the growth delay analysis method compared to the plate
count method, which may be due to a delay in growth initiation caused by damage to the functions
involved in germination and post-germination growth. These results indicated the involvement of DNA
damage in the development of injured spores, which may be strongly influenced by DNA stability.
Therefore, we assessed the damage recovery system, employing HR-deficient (△recA ) and NHEJdeficient (△ykoVU) strains to evaluate the relationship between damage caused by γ-ray irradiation and
DNA repair. The slopes of the survival curves of the HR- and NHEJ-deficient strains were higher than that
of the WT strain (Fig. 6). The WT strain displayed a slightly lower survival rate using the growth delay

analysis method than the plate count method, while the △recA strain demonstrated almost the same
survival rates. Comparatively, the △ykoVU strain showed a tendency towards a slightly lower decay of the
converted survival rate than that obtained using the plate count method.

Figure 5. Change in the viability of the ΔsspAΔsspB strain in response to 95 °C
heat treatment.

Figure 6. Survival curves and converted viability curves of spores after gamma-ray irradiation in B. subtilis mutant

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Section D3-D4: Application of nuclear techniques in agriculture, radiation technology application

strains. (A) ΔsspAΔsspB, (B) △recA, (C) △ykoVU strains.

The killing effects of 95 °C heat treatment and 60Co -ray irradiation on various deficient B. subtilis
strains were evaluated using the plate count method. The times to reach 90% reduction, determined from
these survival curves, are listed in Table 1.
Table 1. D10 values calculated from survival curves

Strain
WT
△sspA △sspB
△recA
△ykoVU

D10 [min]
21.5

3.06
22.0
20.1

D10 [kGy]
1.60
0.92
1.66
0.84

3. DISCUSSION
The growth kinetics of injured B. subtilis spores after γ-ray irradiation or heat treatment at 95 °C
showed different levels of damage may due to different causes. In the case of γ-ray irradiation, the time
from post-germination to increase in OD600 value due to nutrient growth was delayed in a dose-dependent
manner. It suggests that γ-ray irradiated spores repaired the damage during germination or postgermination growth and then switched to the nutrient growth cycle [11,12].
DNA damage is believed to be the main effect of irradiation on spores. When DNA damage occurs in
spores, their metabolism becomes dormant, and DNA damage repair occurs when they germinate and
resume metabolic activities during post-germination growth [13]. Spores have a variety of mechanisms that
can be activated to repair damaged DNA, but HR and NHEJ are particularly responsible for repairing
double-strand breaks (DSBs), which are thought to be directly related to cell lethality. These two
mechanisms are important for spore survival [14,15]. The killing effect of -ray irradiation on B. subtilis
spores determined by the plate count method was 1.60 kGy for the WT strain, 0.92 kGy for the △sspA△
sspB strain, 1.66 kGy for the △recA strain, and 0.84 kGy for the △ykoVU strain. This value is the D10
value, and a smaller D10 value indicates a higher susceptibility. In particular, △sspA△sspB strain lacking
DNA-protecting SASPs and △ykoVU strain lacking NHEJ repair ability showed altered sensitivity. SASPs
that bind to spore DNA to protect the DNA backbone from chemical and enzymatic cleavage are degraded
within the first few minutes of germination, providing amino acids for both new protein synthesis and
metabolism. Therefore, it is likely that SASPs contribute to the γ-ray resistance of spores, both in terms of
their protective effect and as a nutrient source for subsequent metabolism. NHEJ repair is supposedly
involved in DNA repair during spore germination [13], and the difference between NHEJ and HR repair,

which is also a DSB repair mechanism, suggests that NHEJ repair does not require homologous DNA,
which may be advantageous over HR repair.
Interestingly, the SASP-deficient strain displayed the largest difference between the survival rate by
plate count method and the converted survival rate by growth delay analysis, suggesting this strain was
more susceptible to damage than the other strains. Therefore, it is very likely that SASPs protect against or
repair DNA damage induced by -ray irradiation. Moreover, membrane damage and protein denaturation
are generally considered important in heat treatment; we also confirmed that DNA stability is important in
evaluating sterilization treatments. In the present study, we confirmed that the DNA protection function
directly contributed to the heat sensitivity of the ΔsspAΔsspB strain via the degree of damage generated. We
also confirmed the role of ykoVU in the repair of DNA damage caused by γ-ray irradiation. Since the type
of spore injury depends on the sterilization method, the findings establish a comprehensive analysis and are
expected to contribute to a controlled method based on the individual evaluation of the occurrence and
repair of each type of damage.
4. CONCLUSION
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Proceedings of Vietnam conference on nuclear science and technology VINANST-14

In this study, we compared the damage mechanisms of spores subjected to heat treatment and -ray
irradiation and the developmental dynamics of the injured spores. This study found that more injured
spores were generated by heat treatment than by γ-ray irradiation. The results with the SASP-deficient
strain suggested that SASPs may help protect against or repair DNA damage induced by -ray irradiation.
The results with the NHEJ-deficient strain suggested that NHEJ repair is involved in DNA repair during
spore germination, and NHEJ repair may be advantageous over HR repair. The present study provides
useful insights into the damage, developmental dynamics, and stress response caused by -ray irradiation.
The findings are expected to contribute to the future application of -ray irradiation as a non-thermal and
gentle approach for spore control, which has been industrially commercialized for some food applications.

ACKNOWLEDGMENTS
This research was partially supported by the Osaka Prefecture University Female Researcher Support
Program.

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