Effects of sterilization methods on the survival of pathogenic bacteria in potting soil stored at various temperatures

Fresh food products can be contaminated with pathogenic bacteria in various agricultural environments. Potting soil is sterilized by heat sterilization and then reused. This study evaluated the effects of three sterilization methods (non-sterilized, pasteurized, and sterilized) on the survival of pathogenic bacteria in potting soil during storage for 60 days at 5, 15, 25, and 35 °C. The reduction in Escherichia coli O157:H7, Salmonella Typhimurium, and Staphylococcus aureus in potting soil was higher at higher temperatures (25 and 35 °C) than at lower temperatures (5 and 15 °C). The population of pathogenic bacteria in pasteurized and sterilized potting soil was reduced below the detectable levels within 30 days at 35 °C. In contrast, the population of Bacillus cereus did not change in potting soil during storage for 60 days at all temperatures. These results indicate that sterilization and storage temperature of potting soil are critical factors influencing the survival of pathogenic bacteria.


Introduction
Fresh, nutritious, and healthy foods such as vegetable sprouts, which are usually consumed raw, can be contaminated by various microorganisms. From 2005 to 2017, Escherichia coli O157:H7 and Salmonella spp. were the major pathogenic bacteria contributing to outbreaks in alfalfa sprouts (Centers for Disease Control and Prevention, 2016), clover sprouts (CDC, 2017), and mung bean sprouts (Rohekar et al., 2008). In 2011, a large outbreak of E. coli O104:H4, which was associated with the consumption of raw vegetable sprouts, occurred in Europe (European Food Safety Authority, 2011). In general, vegetable sprouts can be contaminated with pathogenic bacteria in various agricultural environments during harvesting. Possible sources of contamination in agricultural products are soil, raw manure, compost, irrigation water, and human handling (Olaimat and Holley, 2012). Although soil provides essential nutrients for agricultural product growth, improper management can be attributed to a major source of contamination during seed germination (Steele and Odumeru, 2004).
Ginseng spouts (Panax ginseng sprouts) are grown in greenhouses using soil-based or hydroponic systems at 23-24 °C and harvested after approximately 25-40 days (Chang et al., 2020). Among the types of soil, potting soil, also known as growing media or cultural media, is an active substance that supplies nutrients and moisture required for growth in ginseng sprouts. Most of potting soil is reused without sterilization or disinfection until an injury occurs by the continuous cropping of ginseng sprouts. Hyun et al. (2022) reported populations of Bacillus cereus ranged from 1 3 1.00 to 4.84 log CFU/g on 20 ginseng sprouts distributed in Korea. A previous study showed that the levels of total aerobic bacteria differed depending on the site of ginseng sprouts, with average populations of 5. 69, 5.17, and 7.15 log CFU/g in the leaves, stems, and roots of ginseng sprouts, respectively (unpublished data). These results indicated a higher level of contamination in the roots than in the leaves and stems. The survival and growth of pathogenic bacteria in soil are determined by various factors, such as light, pH, moisture content, temperature, and soil component (Alegbeleye et al., 2018). Several studies have reported that E. coli O157:H7 and Salmonella spp. can survive in the soil from 7 up to 25 weeks, depending on the temperature, moisture content, and type of soil (Guo et al., 2002;Lang and Smith, 2007;Nicholson et al., 2005;Zhang et al., 2009). Solomon et al. (2002) also demonstrated that E. coli O157:H7 was detected in lettuce cultivated on soil using manure or irrigation water contaminated with E. coli O157:H7. According to these results, pathogenic bacteria can enter the roots of agricultural products and thus increase contamination levels of pathogenic bacteria in agricultural products. Thus, there is a need to develop efficient technologies to control pathogenic bacteria and improve soil safety. Various sterilization technologies, including heat treatment using autoclaving, γ-radiation, and ethylene oxide, have been used to change the organic matter in the soil (Berns et al., 2008;McNamara et al., 2003;Nègre et al., 1995). However, the effect of sterilization methods on the survival of pathogenic bacteria in potting soils has not been intensively studied. Therefore, this study aimed to investigate the survival of pathogenic bacteria in potting soil using different sterilization methods (non-sterilized, pasteurized, and sterilized) during storage at various temperatures (5, 15, 25, and 35 °C).

Bacterial strain and culture conditions
Three strains of E. coli O157:H7 (ATCC 13890, 43889, and 43894), Salmonella Typhimurium (ATCC 19586, 43174, and DT104), Staphylococcus aureus (ATCC 13565, 23235, and 25923), and Bacillus cereus (ATCC 13061, KACC 1092, and KACC 13066) were obtained from the Bacterial Culture Collection of the National Institute of Agricultural Sciences (Wanju-gun, Jeollabuk-do, Korea). Each strain was cultured in tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA) at 37 °C for 24 h before use. Antibioticmarked strains were used because microorganisms are naturally present in the soil. The rifampicin-resistant strain was prepared as previously described (Choi et al., 2017) with some modifications. A stock solution of 1 mg/mL (w/v) rifampicin (Gold Biotechnology, St. Louis, MO, USA) was prepared in dimethyl sulfoxide (Biosesang, Seongnam-si, Gyeonggi-do, Korea). The solutions were filtered through a 0.45 μm syringe filter (Taeshin Bio Science, Namyangjusi, Gyeonggi-do, Korea). Each strain was inoculated with TSB containing 50 μg/mL rifampicin and incubated at 37 °C for 24 h. Subsequently, each strain was spread on tryptic soy agar (TSA; Difco) containing 50 μg/mL rifampicin. All rifampicin-resistant strains were cultured in TSB containing 50 μg/mL rifampicin at 37 °C for 24 h, harvested by centrifugation at 6000 × g for 5 min at 4 °C, and washed twice with sterilized 0.1 M phosphate-buffered saline (PBS, Biosesang). The cell pellets were resuspended in 0.1 M PBS to prepare a bacterial culture with a concentration of approximately 10 7 -10 8 CFU/mL. For cocktail preparation, equal volumes of the three strains of pathogenic bacteria were mixed (1:1:1, v/v/v), and the cell suspension was used as an inoculum.

Preparation of potting soil and inoculation
The potting soil for ginseng sprouts was purchased from Nongkyung (Jincheon-gun, Korea). The soil was composed of peat moss (70%, w/w) and perlite (30%, w/w). The potting soil treatment process is shown in Fig. 1. Potting soil (ca. 200 g) was placed in plastic beaker and treated using three different sterilization methods (non-sterilized, pasteurized at 60 °C for 30 min, and sterilized at 121 °C for 15 min) by autoclave (SJ-220A80, Sejong Technology Co. Ltd., Bucheon-si, Gyeonggi-do, Korea). Non-sterilized potting soil was used as the control. For potting soil inoculation, 20 mL suspensions of each strain were inoculated on potting soil (200 g) to reach a final concentration of ca. 10 6 -10 7 CFU/g. The inoculated potting soil was shaken in a sterile plastic bag (Microgiene Co., Ltd, Suwon-si, Gyeonggi-do, Korea) for 10 min to mix it well. After mixing, potting soil (ca. 200 g) was packaged in a sterile plastic bag (Microgiene Co., Ltd). The packaged potting soil was stored in an incubator at 5, 15, 25, and 35 °C for 0, 3, 7, 10, 15, 30, and 60 days. These temperatures were chosen based on the recommended cultivation temperature of ginseng sprouts (25 °C), and low (5 and 15 °C) or high temperatures (35 °C) were chosen for accelerated testing.

Measurement of pH and moisture content (%)
The pH level was measured using a pH meter (SevenEasy S20, Mettler Toledo, GmbH, Switzerland). The treated potting soil (1 g) was homogenized thoroughly with distilled water (20 mL). To determine weight loss, the samples were weighed using a digital balance (BCE224i-1SKR, Sartorius, Göttingen, Germany). The moisture content (%) of the potting soil was determined using the gravimetric method (Magdić et al., 2022). Moisture content (%) was calculated by the weight loss of the sample (50 g) maintained in a dry oven (DS-80-1, Dasol Scientific Co. Ltd., Hwaseong-si, Korea) at 105 °C until a constant weight was reached. The sample was allowed to cool in a desiccator before the potting soil weight was recorded.

Statistical analysis
All experiments were repeated three times with duplicate samples. Before analysis, the means of the plate counts from three replicates were converted to log CFU/g. Analysis of variance (ANOVA) for a completely randomized design was performed using SPSS (version 25, IBM Corporation, Endicott, NY, USA). Means were separated using Duncan's multiple range test when there were significant differences (p < 0.05) among treatments.

Results and discussion pH and moisture content (%) in potting soil
The changes in pH and moisture content (%) in the potting soil by the three different sterilization methods are shown in Table 1. The pH levels of the non-sterilized, pasteurized, and sterilized potting soils were 5.76, 5.39, and 5.01,  respectively. pH levels slightly decreased in the potting soil depending on the sterilization method. Tanaka et al. (2003) reported that the physicochemical properties of soil, such as pH, change after autoclaving. This change may be due to the release of organic acids from organic matter in the soil (Berns et al., 2008;Skipper and Westermann, 1973). However, it seems that pH levels of samples did not affect the populations of pathogenic bacteria. For example, nonsterilized potting soil was effective at decreasing levels of Staphylococcus aureus however, its pH level (5.76 ± 0.05) was higher than that of the pasteurized potting soil that were less effective. The moisture content (%) of potting soil did not significantly change with different sterilization methods (p > 0.05).

Survival characteristics of pathogenic bacteria in potting soil by three different sterilization methods
The survival characteristics of E. coli O157:H7, S. Typhimurium, S. aureus, and B. cereus in potting soil treated using different sterilization methods during storage at various temperatures (5, 15, 25, and 35 °C) are shown in Figs. 2, 3, 4, and 5, respectively. The initial populations of E. coli O157:H7 in non-sterilized potting soil were 6.76 and 7.13 log CFU/g on selective (CT-SMAC) and non-selective agar (TSA), respectively ( Fig. 2A, B). The population of E. coli O157:H7 in non-sterilized potting soil stored at 5 °C for 60 days was 4.00 log CFU/g ( Fig. 2A). However, E. coli O157:H7 counts were reduced below detectable levels (≤ 2.00 log CFU/g) in non-sterilized potting soil after 60 days of storage at 15, 25, and 35 °C. In contrast, those stored at 5, 15, 25, and 35 °C were 4.89, 5.74, 5.30, and 5.21 log CFU/g, respectively, after 60 days when enumerated on TSA (Fig. 2B). These results indicate that the sublethal injury cell induces in non-sterilized potting soil. During 60 days of storage, the reduction in the population of E. coli O157:H7 in potting soil was higher at 35 °C than at 5 °C. In particular, the population of E. coli O157:H7 was reduced below detectable levels after storage for 30 days at 35 °C. In contrast, the population of E. coli O157:H7 was reduced to 2.87-3.11 log CFU/g at 5, 15, and 25 °C when enumerated on CT-SMAC (Fig. 2C). In addition, the population of E. coli O157:H7 was reduced to below detectable levels in sterilized potting soil after storage for 3 days at 35 °C (Fig. 2E). There were no appreciable differences between survived levels enumerated on CT-SMAC and TSA. According to these results, sterilized potting soil effectively reduced levels of E. coli O157:H7 without producing sublethally injured cells.
The survival curve of S. Typhimurium in potting soil treated with the three different sterilization methods is shown in Fig. 3. The initial populations of S. Typhimurium in non-sterilized potting soil were 5.80 and 6.52 log CFU/g as determined on selective (XLT4) and non-selective agar (TSA), respectively (Fig. 3A, B). Overall, the growth of S. Typhimurium was rapidly reduced compared to that of E. coli O157:H7 during storage for 60 days. At the end of the storage period, the populations of S. Typhimurium in pasteurized potting soil were reduced below detectable levels at 5, 25, and 35 °C (Fig. 3C). In contrast, S. Typhimurium in pasteurized potting soil was maintained at approximately 4.00 log CFU/g after storage at 15 °C for 60 days when enumerated on XLT4. Similar to E. coli O157:H7, S. Typhimurium was not detected in sterilized potting soil after storage for 3 days at 35 °C (Fig. 3E, F). There were no differences between survived levels enumerated on XLT4 and TSA. These results reveal that S. Typhimurium inactivated in sterilized potting soil without producing sublethally injured cells. In addition, populations of S. Typhimurium were not detected in sterilized potting soil after 7 days of storage at 5 and 15 °C. On the other hand, the population of S. Typhimurium decreased and then slightly increased in sterilized potting soil at 25 °C after 7 days. This phenomenon is probably a difference between survival mechanisms of bacteria in potting soil treated with non-sterilization and sterilization. Figure 4 shows the populations of S. aureus in potting soil stored at various temperatures using three different sterilization methods. The initial populations of S. aureus in nonsterilized potting soil were 7.83 and 7.92 log CFU/g enumerated on selective (BPA) and non-selective agar (TSA), respectively (Fig. 4A, B). The initial population of S. aureus was maintained in three types of potting soil enumerated on TSA (Fig. 4B, D, F). On the other hand, the populations of S. aureus were reduced according to the storage temperature in the three types of potting soil during storage for 60 days at all temperatures, indicating that sublethal injury cell produced (Fig. 4A, C, E). Among the storage temperature, S. aureus was more effective at inhibiting at higher temperatures (25 and 35 °C) than at lower temperatures (5 and 15 °C). Similar to E. coli O157:H7 and S. Typhimurium, storage at 35 °C in pasteurized and sterilized potting soil was the most effective at reducing S. aureus after storage for 3 and 7 days, respectively. These results indicated that the survival of E. coli O157:H7, S. Typhimurium, and S. aureus depends on the storage temperature of the potting soil.
Among the pathogenic bacteria tested, B. cereus was the most resistant in the soil (Fig. 5). The initial populations of B. cereus in non-sterilized potting soil were 5.29 and 5.22 log CFU/g enumerated on selective (MYP) and non-selective agar (TSA), respectively (Fig. 5A, B). The initial population of B. cereus in non-sterilized potting soil did not change during storage for 60 days at all temperatures when enumerated on MYP (Fig. 5A), whereas the growth of B. cereus stored at 35 °C slightly increased on TSA (Fig. 5B). The results showed that the initial levels of B. cereus were maintained in non-sterilized potting soil during 60 days of storage at all temperatures when enumerated on MYP (Fig. 5A). On the other hand, the number of B. cereus in three types of potting soil grown on both TSA and MYP were almost similar, indicating that there was no injury produced.
The survival and growth of microorganisms in soil differ depending on various factors, including pH, temperature, moisture content, nutrient availability, and soil type (Alegbeleye et al., 2018). The storage temperature is important  (Arrus et al., 2006). In this study, storage at 35 °C was effective in reducing the levels of pathogenic bacteria in pasteurized and sterilized potting soils during storage.
Several studies have demonstrated that higher temperatures may be more effective in inhibiting pathogenic bacteria than lower temperatures. Cools et al. (2001) observed that E. coli populations in sandy soil were reduced below detectable  agar (B, D, F). Sterilization methods using non-sterilized (A) and (B); pasteurized (C) and (D); and sterilized (E) and (F). Data represent mean ± standard deviations of three measurements levels after 68 days at 5 °C, whereas populations of E. coli were reduced below detectable levels after 26 days of storage at 25 °C. García et al. (2010) showed that the survival of S. Typhimurium in the top soil was maintained at 5 °C compared to 25 °C. Similar studies reported by Danyluk et al. (2007) showed that the population of Salmonella spp. was reduced in soil from almond orchards by approximately 1.00 and 5.00 log CFU/g at 20 °C and 35 °C after 30 days of storage, respectively. This was probably due to the differences in soil moisture levels according to the storage temperature. A few studies have demonstrated that the populations of bacteria or viruses are reduced in dry environments rather than in cool and moist environments (Ghorbani et al., 2008;Santamaria and Toranzos, 2003). In addition, the population of pathogenic bacteria was more effective at inhibiting in sterilized potting soil than in non-sterilized and pasteurized ; pasteurized (C) and (D); and sterilized (E) and (F). Data represent mean ± standard deviations of three measurements potting soil. This was probably due to the absence of naturally resident microbiomes in the soil after sterilization. In other words, the growth of pathogenic bacteria increased without competitive organisms than that with competitive organisms. This phenomenon is consistent with the results of Choi et al. (2010), who reported that growth of S. aureus was more active without competitive organisms compared to with competitive organisms in bean sprouts. These results indicate the difference between survival mechanisms of bacteria in potting soil treated with non-sterilization and sterilization. In this study, the growth of E. coli O157:H7 and B. cereus tended to be maintained in non-sterilized and pasteurized potting soil at after 60 days of storage at 5 °C. Several studies have shown that populations of pathogenic bacteria are maintained in the soil during plant cultivation. Zhang et al. (2009) demonstrated that E. coli O157:H7 survived in soil for at least 60 days during storage at 23 °C for 12 h (day) and 7 °C for 12 h (night). Jechalke et al. (2019) reported that the average population of S. Typhimurium persisted at approximately 2.50 log CFU/g in the soil during the growth period of lettuce (44 days). Another study showed that the population of Salmonella spp. increased by 2.50 log CFU/ tomato on tomatoes in contact with soil during storage at 20 °C for 4 days and remained constant for an additional 10 days (Guo et al., 2002). These microorganisms may cause the soil on which plants grow is a potential source of contamination. Soil inoculated with E. coli was detected on the surface of spinach after cultivation for 42 days at 20-26 °C (Warriner et al., 2003). Ingham et al. (2004) showed that E. coli was detected in lettuce grown in soil in the presence of bovine manure contaminated with E. coli, indicating that E. coli in soil was transferred to lettuce. According to this result, the survival of B. cereus was not influenced by the storage temperature or sterilization method. B. cereus is widely distributed in natural environments, including soil and aquatic habitats, from which it can be transferred to plants and food products (Bartoszewicz and Czyżewska, 2017). In particular, the soil is the primary source of contamination in raw vegetables contaminated with B. cereus (Kotiranta et al., 2000). In our previous study, B. cereus was frequently detected in raw materials (ginseng sprouts, ginseng seedlings, potting soil, and green moss) and working tools (gloves, working table, dishcloth, scissors, boots, and knob of greenhouses) in ginseng sprout farms (data not shown). These results indicated that B. cereus contaminated with soil could be easily transferred to various raw materials and working tools on farms. Therefore, appropriate technologies to control the contamination of B. cereus in potting soil need to be developed. Furthermore, further study is needed to understand the difference in inactivation susceptibility according to bacterial strains.
These results could provide valuable information for improving the microbial safety of reused soil using appropriate sterilization methods and storage temperatures. However, limited studies have investigated the effects of sterilization methods and storage temperature on the survival of pathogenic bacteria in the soil. Our results showed that storage temperature is one of the most important factors for reusing potting soil. Moreover, sterilization methods, such as pasteurization or sterilization in soil, are effective treatments for reducing the survival of pathogenic bacteria. Therefore, storage at high temperatures after pasteurization or sterilization of potting soil can be used to effectively control the presence of pathogenic bacteria. Further studies should be conducted to investigate the appropriate conditions, such as sterilization temperature and time, to control the microbial safety and physiochemical characteristics of potting soil.