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

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.

ANALYSIS OF INJURY AND GROWTH BEHAVIORS OF STRESSED BACILLUS

SUBTILIS SPORES USING THE DOUBLE SUBCULTURE METHOD

R ASADA1, 2*, S HORIKIRI 2 , H DEN 1 , J.J SAKAMOTO 2 , T TSUCHIDO 2 , M FURUTA 1, 2

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

2 Research Center of Microorganism Control, Organization of Research Promotion,

Osaka Prefecture University, Sakai, Japan

*E-mail: asada@riast.osakafu-u.ac.jp

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

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

2 METHODOLOGY

2.1 Strains and spore preparation method

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

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)

(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

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 dose-dependent 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

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

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

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

Figure 3 Applicability of the growth delay analysis method by confirming the correlation between

dilution rate and delay time in B subtilis spores (WT)

Figure 4 Analysis of injured B subtilis spores (WT) spores after (A) heat treatment and (B)

γ-ray irradiation by double subculture method N D : number of dead spores; N I : number of injured

spores; N L : number of live spores.

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

min did not kill WT spores by one order of magnitude, while three orders of magnitude of death were

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 NHEJ-deficient (△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

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

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

heat treatment

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

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. D 10 values calculated from survival curves

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 post-germination 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△

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

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

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

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

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

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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