Food Microbiology 46 (2015) 234e238
Contents lists available at ScienceDirect
Food Microbiology journal homepage: www.elsevier.com/locate/fm
Short communication
Effect of quantity of food residues on resistance to desiccation of foodrelated pathogens adhered to a stainless steel surface Takashi Kuda*, Gensui Shibata, Hajime Takahashi, Bon Kimura Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo 108-8477, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 April 2014 Received in revised form 21 June 2014 Accepted 17 August 2014 Available online 27 August 2014
In order to study the effect of food residues on the survival of food-borne pathogens, Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes were subjected to drying conditions in the presence of small amounts of food such as carrot juice, aqueous solution of nori, milk, and soy-milk. After drying for 2 h at room temperature in the absence of food residue, cell counts of S. Typhimurium, S. aureus, and L. monocytogenes decreased from 8 to 3, 6, and 5 log cfu/dish, respectively. Five milligrams of fresh carrot, 0.05 mg dried nori, and 100 nL milk or soy milk per 10 mm 4 surface were sufficient to demonstrate a protective effect on the adhered pathogens, as confirmed by atomic force microscopy. Results from this study suggest that small sediments of food, not only protein rich but also carbohydrate rich, increase the resistance of surface-adherent bacteria to desiccation, rendering sanitization processes ineffective and encouraging cross contamination. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Surface contamination Food residue Salmonella Typhimurium Staphylococcus aureus Listeria monocytogenes
1. Introduction Microbial adhesion to surfaces is a potential route of transmission of pathogens in the food-processing industry (Giauris and ~ es et al., 2010) and in the domestic environment Nychas, 2006; Simo (Humphrey et al., 2001; Hayson and Sharp, 2005). Microorganisms within and/or on wet surfaces of utensils and medical equipment often form a biofilm, which exhibits resistance to various kinds of stress conditions (Boles and Singh, 2008; Finn et al., 2013). In particular biofilms formed by Pseudomonas aeruginosa (Baird et al., 2012; Murphy et al., 2014), Staphylococcus aureus (Chen et al., 2012; Kuda et al., 2011), Listeria monocytogenes (Chaturongkasumrit et al., 2011; Lourenço et al., 2013), and Salmonella Typhimurium (Nguyen and Yuk, 2013; Kuda et al., 2012) serious threats because of their strong resistance to disinfectants and their role in nosocomial infections. We have previously reported that when food poisoning agents such as Salmonella Typhimurium and S. aureus were dried and adhered onto stainless steel or glass surfaces in the presence of nutrient-rich food residue such as milk, meat, and egg, they showed resistance to desiccation, surfactant disinfectants such as benzalkonium chloride, as well as 254-nm ultraviolet (UV)-C irradiation (Kuda et al., 2008, 2011, 2012; Li et al., 2014). This indicated that
* Corresponding author. Tel./fax: þ81 3 5463 0602. E-mail address: [email protected] (T. Kuda). http://dx.doi.org/10.1016/j.fm.2014.08.014 0740-0020/© 2014 Elsevier Ltd. All rights reserved.
protein-, lipid-, and/or carbohydrate-rich food residue could protect pathogens from stress conditions. The gram-positive rod L. monocytogenes, regarded as tolerant to desiccation, is similarly protected by food residue (Takahashi et al., 2011). These studies indicate that washing and rinsing prior to sterilization are essential to meet the strictly sterile requirements for food safety. However, the quantum of food residue that is sufficient to protect pathogens from stress such as desiccation, disinfectants, or UV-C remains unclear. In this study, we investigated the quantity-dependent effects of milk, soy milk, carrot, and laver nori residue on survival rates of Salmonella Typhimurium, S. aureus, and L. monocytogenes adhered to and desiccated on a stainless steel surface. The cells adhered in the presence of food residue were observed using atomic force microscopy. 2. Materials and methods 2.1. Bacterial culture and food material Salmonella enterica subsp. enterica serotype Typhimurium NBRC 13245, S. aureus NBRC 12732, and L. monocytogenes Scott A were adhered onto utensil surfaces. To produce cultures, the bacterial cells were inoculated into 10 mL trypticase soy broth (TSB; Becton, Dickinson and Co.; Sparks, MD) and incubated at 37 C for 20 h. Under these conditions, the culture reached stationary phase (Kuda et al., 2013).
T. Kuda et al. / Food Microbiology 46 (2015) 234e238
Fresh carrot, sundried sheets of laver Porphyra sp. (called nori in Japanese), milk, and soy milk were purchased from a retail shop in Tokyo. The carrot was minced with an equal amount of distilled water (DW) for 60 s in a blender (16-Speed Blender; Osaka Chemical; Osaka, Japan) and shaken for 60 min at room temperature. The carrot extract was collected by centrifugation (2500 g for 10 min) and sterilized by filtration with a 0.22-mm-pore filter. The dried nori was milled in the blender and autoclaved (121 C for 15 min) with 40 time volumes of DW. After centrifugation, the supernatant was used as nori extract. The carrot extract, nori extract, milk, and soy milk samples were diluted 10-fold in DW to obtain 104 (0e10,000-fold diluted) solutions. 2.2. Adhesion of food pathogens to surfaces Fifty-millimeter-4 stainless steel dishes were purchased from As One Co. (Osaka, Japan) and used as the experimental surface (Kuda et al., 2008). Prior to use, in order to equalize the effect of the surface conditions on the survival cell count (Bohinc et al., 2014), the steel dishes were ultra-sonicated twice for 15 min, brushed for 60 s, and autoclaved at 121 C for 15 min. The bacterial cells were placed in the dish and attached as previously reported (Kuda et al., 2011), with slight modifications. Briefly, bacterial cells in the TSB culture were washed by centrifugation at 2000 g for 10 min at 4 C and re-suspended in phosphate-buffered saline (PBS; Nissui Pharmaceutical Co.; Tokyo, Japan); this process was repeated twice. The cells were finally re-suspended in 10 mL DW or the diluted food samples such that the final cell concentration was approximately 8e9 log cfu/mL. A bacterial suspension (0.1 mL) was placed in about 10 mm-4 of the center of the dish (n ¼ 3) and dried for 120 min at room temperature (20e24 C) in a bio-safety cabinet (Class IIA; Airtech Japan Co.; Tokyo, Japan) with ventilation. After drying, the adhered cells were detached by rubbing for 60 s using a sterile cotton swab and re-suspended in 5 mL TSB (Nissui Pharmaceutical). The detached cell suspension (0.1 mL) was immediately diluted in PBS, spread on trypticase soy agar (TSA; Becton, Dickinson and Co.) and incubated at 37 C for 24 h. 2.3. Microscopic observations To observe the bacterial cells, 0.01 ml of the cell suspension, prepared as same as above, was adhered by drying on a cover slip and the cells were observed using an atomic force microscope (AFM; Naio AFM; Nanosurf AG; Liestal, Switzerland) in dynamic force mode. 2.4. Statistical analysis Bacterial cell viability was expressed in terms of mean and standard deviation of log cfu/dish (n ¼ 3). Statistical analysis was performed using EXCEL Statistic 5.0 software (Esumi Co., Ltd.; Tokyo, Japan). One-way ANOVA was used to assess differences among groups, and individual means were compared by Tukey's multiple-range test. Differences were considered significant at p < 0.05. 3. Results and discussion 3.1. Effect of drying on survival of bacterial cells on the surface In our previous study, the sensitivity of logarithmic or early stationary growth phase cells to drying was higher than that of the stationary growth phase cells (Kuda et al., 2008). It has been reported that sigma factors produced in the stationary phase lead to
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the generation of self-defense proteins that protect the cells against acidic conditions, desiccation, low temperature, and reactive oxidants (Spector and Kenyon, 2012). Therefore, we used stationaryphase bacterial cells in previous studies (Kuda et al., 2011, 2012; 2013) as well as in this study. The viable count of the bacterial cells before and after drying on the stainless steel surface is summarized in Fig. 1. During the 120min ventilation period at room temperature, viable cells of Salmonella Typhimurium in DW were dried thoroughly and the cell count decreased from approximately 8.1 log cfu/dish to 3.4 log cfu/dish on the stainless steel surface (Fig. 1A). On the other hand, S. aureus and L. monocytogenes showed resistance to desiccation in contrast to Salmonella Typhimurium (Fig. 1B and C). The food samples of 50% carrot juice and 5% nori solution, milk, and soy milk clearly protected the cells from desiccation. The images of the dried and adhered cells on the cover slip observed by the AFM in dynamic force mode are shown in Fig. 2AeC. In this study, the gram-negative Salmonella Typhimurium cells were flattened by desiccation (Fig. 2A); however, the gram-positive S. aureus and L. monocytogenes cells retained their cell shape (Fig. 2A and B). This observation correlates with our and other previous reports regarding survival rates of gram-positive rez-Rodríguez and -negative pathogens (Kuda et al., 2011; Pe et al., 2013). 3.2. Protective effect of decimal diluted food residues on the adhered pathogens In Fig. 3, the horizontal lines marked as “DW” shows the survival cell counts of pathogens adhered with DW. Although the 100-fold diluted nori solution did not affect the survival rate of Salmonella Typhimurium, the 1000-fold diluted carrot juice, milk, and soy milk samples provided protection to Salmonella Typhimurium (Fig. 3A). S. aureus and L. monocytogenes were protected by the 100-fold diluted carrot juice and nori samples (Fig. 3B and C) as well as the 1000-fold diluted milk and soy milk samples. Of particular note is the finding that only 100 nL (103 of 0.1 mL) milk and soy milk could protect 10% of the gram-positive bacterial cells from desiccation. In the case of S. aureus, the protection capacity of the 100-fold diluted nori sample was higher than that of carrot juice. This finding was not in agreement with the results from Salmonella Typhimurium. In the case of L. monocytogenes, the protective effect from carrot juice and nori samples was similar. In the AFM observation, the adhered and dried pathogenic bacterial cells were not detected with the 50% carrot and 5% nori solutions, undiluted milk, and undiluted soy milk (Fig. 2DeG). Furthermore, 10% milk and 10% soy milk also covered the pathogenic cells (image not shown). Although the cells adhered with 10fold diluted carrot and nori samples as well as 100-fold diluted milk and soy milk samples were detected, the cells were covered with food residue (Fig. 2HeK). Interestingly, L. monocytogenes showed an aggregation of food residue particles around the cells (Fig. 2H,K, and L). Salmonella Typhimurium cells adhered with 1000-fold diluted milk and soy milk samples were not flattened (Fig. 2MeO). AFM is used to observe the effect of disinfectants such as quaternary ammonium compounds on pathogens (Crismaru et al., 2011), as this is considered a powerful tool for nano-level analysis of the utensil surface-adhered microorganisms. In addition, observations could be made in real time, without pretreatments such as fixation, dehydration, or metal-coating, as are required for scanning electron microscopy. In this study, cover slips were used for AFM observation instead of stainless steel. In future, we would like to kinetic study with the actual stainless surface. Milk and soy milk are rich in protein (approximately 33 mg/g and 38 mg/g, respectively), lipids (38 mg/g and 20 mg/g,
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Fig. 1. Survival rates in terms of cell counts of Salmonella Typhimurium (A), Staphylococcus aureus (B), and Listeria monocytogenes (C) on stainless steel dishes before (BF) and after 2 h drying with distilled water (DW), 50% carrot juice (CJ), 5% dried nori solution (NR), milk, or soy milk (SM). Values are expressed as mean and SD (n ¼ 3). a-d Different letters indicate significant differences (p < 0.05) between groups.
respectively), as well as carbohydrates (48 mg/g and 31 mg/g, respectively) (Standard Tables of Food Composition in Japan, 2005). These compounds may protect the cells from desiccation. Carrots are rich in carotenoids (94 mg/g) and carbohydrates (90 mg/g).
Although dried nori is a popular algal food, it is not only rich in carbohydrates (417 mg/g) but also in proteins (221 mg/g). These differences probably affected the protective activity, as shown in Fig. 3. Water-soluble polysaccharides such as porphyran and other
Fig. 2. Atomic force microscopy images of Salmonella Typhimurium (ST), Staphylococcus aureus (Sa), and Listeria monocytogenes (Lm) on cover slips after drying with distilled water (AeC), and food residues (DeO). DeG are images of undiluted food residue solutions. H and I are images of Lm with 10-fold diluted food residue solutions. JeM are images of pathogens with the 100-fold dilutions. N and O are images of ST with1000-fold diluted milk and soy milk solution.
T. Kuda et al. / Food Microbiology 46 (2015) 234e238
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Fig. 3. Survival number of Salmonella Typhimurium (A), Staphylococcus aureus (B), and Listeria monocytogenes (C) adhered and desiccated on stainless steel dishes with decimal dilutions of 50% carrot juice (circles), 5% dried nori solution (open triangles), milk (closed triangles), or soy milk (squares). Values are expressed as mean and SD (n ¼ 3). The horizontal line (DW) corresponds to survival cell counts without food residue. aed Different letters indicate significant differences (p < 0.05) between groups.
low-molecular weight compounds were probably extracted in the boiled nori. Porphyra sp. has been reported to exhibit antioxidant and anti-inflammatory activity, in addition to providing protection from UV radiation (Kuda et al., 2005; Kazłowska et al., 2010; Zhang et al., 2010). The activity of these compounds may have affected the survival rate of pathogenic cells in this study. Thus, bacteria drying with food residue on utensil surfaces can occur in both industrial and domestic environments (Giauris and ~es et al., 2010; Humphrey et al., 2001; Hayson Nychas, 2006; Simo and Sharp, 2005). This can lead to cross contamination, inactivation of sterilization techniques that use disinfectants or UV-C, and contribute to biofilm formation (Kuda et al., 2011, 2012). Several reports regarding biofilms and methods for bacterial detachment and disinfection have been published (Chaturongkasumrit et al., ~es et al., 2010), which demonstrate that once biofilms 2011; Simo are formed, bacterial detachment and sterilization are challenging. In the present study, we showed that a minimal amount of food residue can protect bacterial cells. Therefore, adequate and regular washing to remove all traces of food residue is essential. Further study considering with temperature, humidity and other surface materials in food processing environment is also needed. 4. Conclusions In conclusion, after drying for 2 h at room temperature in the absence of food residue, cell counts of Salmonella Typhimurium, S. aureus, and L. monocytogenes decreased from 8 to 3, 6, and 5 log cfu/dish, respectively. It was observed that 5 mg fresh carrot, 0.05 mg dried nori, and 100 nL milk or soy milk per 10 mm 4 surface were sufficient to demonstrate a protective effect on the adhered pathogens, as confirmed by atomic force microscopy. Results from this study suggest that small sediments of food, not only protein rich but also carbohydrate rich, increase the resistance of surfaceadherent bacteria to desiccation, rendering sanitization processes ineffective and encouraging cross contamination and some food poisonings. Acknowledgment This work was supported by the Nisshin Seifun Foundation ( 2012-8) (Tokyo, Japan). References Baird, F.J., Wadsworth, M.P., Hill, J.E., 2012. Evaluation and optimization of multiple fluorophore analysis of a Pseudomonas aeruginosa biofilm. J. Microbiol. Methods 90, 192e196.
Bohinc, K., Dra zi c, G., Fink, R., Oder, M., Jevsnik, M., Nipi c, D., Godi c-Torkar, K., Raspor, P., 2014. Available surface dictates microbial adhesion capacity, 50, 265e272. Boles, B.R., Singh, P.K., 2008. Endogenous oxidative stress produces diversity and adaptability in biofilm communities. Proc. Natl. Acad. Sci. U. S A. 105, 12503e12508. Chaturongkasumrit, Y., Takahashi, H., Keeratipibul, S., Kuda, T., Kimura, B., 2011. The effect of polyesterurethane belt surface roughness on Listeria monocytogenes biofilm formation and its cleaning efficiency. Food Control 22, 1893e1899. Chen, P., Abercrobie, J.J., Jeffrey, N.R., Leung, K.P., 2012. An improved medium for growing Staphylococcus aureus biofilm. J. Microbiol. Methods 90, 115e118. Crismaru, M., Asri, L.A.T.W., Loontjens, T.J.A., Krom, B.P., de Vries, J., van der Mei, H.C., Busscher, H.J., 2011. Survival of adhering Staphylococci during exposure to a quaternary ammonium compound evaluated by using atomic force microscopy imaging. Antimicrob. Agents Chemother. 55, 5010e5017. €ndler, K., Condell, O., Colgan, A., Cooney, S., McClure, P., Ame zquita, A., Finn, S., Ha Hinton, J.C.D., Fanning, S., 2013. ProP is required for the survival of desiccated Salmonella enterica serovar Typhimurium cells on a stainless steel surface. Appl. Environ. Microbiol. 79, 4376e4384. Giauris, E.D., Nychas, G.J.E., 2006. The adherence of Salmonella Enteritidis PT4 to stainless steel: the importance of the aireliquid interface and nutrient availability. Food Microbiol. 23, 747e752. Hayson, I.W., Sharp, A.K., 2005. Bacterial contamination of domestic kitchens over a 24-hour period. Br. Food J. 107, 453e466. Humphrey, T.J., Martin, K.W., Slader, J., Durham, K., 2001. Campylobacter spp. in the kitchen: spread and persistence. J. Appl. Microbiol. 90, 115Se120S. Kazłowska, K., Hsu, T., Hou, C.C., Yang, W.C., Tsai, G.J., 2010. Anti-inflammatory properties of phenolic compounds and crude extract from Porphyra dentate. J. Ethnopharmacol. 128, 123e130. Kuda, T., Tsunekawa, M., Goto, H., Araki, Y., 2005. Antioxidant properties of four edible algae harvested in the Noto Peninsula, Japan. J. Food Compos. Analysis 18, 625e633. Kuda, T., Yano, T., Kuda, M.T., 2008. Resistances to benzalkonium chloride of bacteria dried with food elements on stainless steel surface. LWT-Food Sci. Technol. 41, 988e993. Kuda, T., Iwase, T., Chaturongkasumrit, Y., Takahashi, H., Koyanagi, T., Kimura, B., 2011. Surfactant-disinfectant resistance of Salmonella and Staphylococcus adhered and dried on surfaces with egg compounds. Food Microbiol. 28, 920e925. Kuda, T., Iwase, T., Chaturongkasumrit, Y., Takahashi, H., Koyanagi, T., Kimura, B., 2012. Resistances to UV-C irradiation of Salmonella Typhimurium and Staphylococcus aureus in wet and dried suspensions on surface with egg residues. Food Control 23, 485e490. Kuda, T., Yazaki, T., Takahashi, H., Kimura, B., 2013. Effect of dried and vinegar flavored squid products on acid resistance of Salmonella Typhimurium and Staphylococcus aureus. Food Control 30, 569e574. Li, R., Kuda, T., Yano, T., 2014. Effect of food residues on efficiency of surfactant disinfectants against food related pathogens adhered on polystyrene and ceramic surfaces. LWT-Food Sci. Technol. 57, 200e206. Lourenço, A., de Las Heras, A., Scortti, M., Vazquez-Boland, J., Frank, J.F., Brito, L., 2013. Comparison of Listeria monocytogenes exoproteomes from biofilm and planktonic state: Lmo2504, a protein associated with biofilms. Appl. Environ. Microbiol. 79, 6075e6082. Murphy, K., Park, A.J., Hao, Y., Brewer, D., Lam, J.S., Khursigara, M., 2014. Influence of O polysaccharides on biofilm development and outer membrane vesicle biogenesis in Pseudomonas aeruginosa PAO1. J. Bacteriol. 196, 1306e1317. Nguyen, H.D.N., Yuk, H.G., 2013. Changes in resistance of Salmonella Typhimurium biofilms formed under various conditions to industrial sanitizers. Food Control 29, 236e240. rez-Rodríguez, F., Posada-Izquierdo, G.D., Valero, A., García-Gimeno, R.M., Zur, G., Pe 2013. Modelling survival kinetics of Staphylococcus aureus and Escherichia coli
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O157:H7 on stainless steel surfaces soiled with different substrates under static conditions of temperature and relative humidity. Food Microbiol. 33, 197e204. ~es, M., Simo ~es, L.C., Vieirab, M.J., 2010. A review of current and emergent Simo biofilm control strategies. LWT-Food Sci. Technol. 43, 573e583. Standard Tables of Food Composition in Japan, 2005. Ministry of Education, Culture, Sports, Science and Technology. http://www.mext.go.jp/b_menu/shingi/ gijyutu/gijyutu3/toushin/05031802/002.htm.
Takahashi, H., Kuramoto, S., Miya, S., Kimura, B., 2011. Desiccation survival of Listeria monocytogenes and other potential foodborne pathogens on stainless steel surfaces is affected by different food soils. Food Control 22, 633e637. Zhang, Z., Zhang, Q., Wang, J., Song, H., Zhang, H., Niu, X., 2010. Chemical modification and influence of function groups on the in vitro-antioxidant activities of porphyran from Porphyra. Carbohydr. Polym. 79, 290e295.
Contents lists available at ScienceDirect
Food Microbiology journal homepage: www.elsevier.com/locate/fm
Short communication
Effect of quantity of food residues on resistance to desiccation of foodrelated pathogens adhered to a stainless steel surface Takashi Kuda*, Gensui Shibata, Hajime Takahashi, Bon Kimura Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo 108-8477, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 April 2014 Received in revised form 21 June 2014 Accepted 17 August 2014 Available online 27 August 2014
In order to study the effect of food residues on the survival of food-borne pathogens, Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes were subjected to drying conditions in the presence of small amounts of food such as carrot juice, aqueous solution of nori, milk, and soy-milk. After drying for 2 h at room temperature in the absence of food residue, cell counts of S. Typhimurium, S. aureus, and L. monocytogenes decreased from 8 to 3, 6, and 5 log cfu/dish, respectively. Five milligrams of fresh carrot, 0.05 mg dried nori, and 100 nL milk or soy milk per 10 mm 4 surface were sufficient to demonstrate a protective effect on the adhered pathogens, as confirmed by atomic force microscopy. Results from this study suggest that small sediments of food, not only protein rich but also carbohydrate rich, increase the resistance of surface-adherent bacteria to desiccation, rendering sanitization processes ineffective and encouraging cross contamination. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Surface contamination Food residue Salmonella Typhimurium Staphylococcus aureus Listeria monocytogenes
1. Introduction Microbial adhesion to surfaces is a potential route of transmission of pathogens in the food-processing industry (Giauris and ~ es et al., 2010) and in the domestic environment Nychas, 2006; Simo (Humphrey et al., 2001; Hayson and Sharp, 2005). Microorganisms within and/or on wet surfaces of utensils and medical equipment often form a biofilm, which exhibits resistance to various kinds of stress conditions (Boles and Singh, 2008; Finn et al., 2013). In particular biofilms formed by Pseudomonas aeruginosa (Baird et al., 2012; Murphy et al., 2014), Staphylococcus aureus (Chen et al., 2012; Kuda et al., 2011), Listeria monocytogenes (Chaturongkasumrit et al., 2011; Lourenço et al., 2013), and Salmonella Typhimurium (Nguyen and Yuk, 2013; Kuda et al., 2012) serious threats because of their strong resistance to disinfectants and their role in nosocomial infections. We have previously reported that when food poisoning agents such as Salmonella Typhimurium and S. aureus were dried and adhered onto stainless steel or glass surfaces in the presence of nutrient-rich food residue such as milk, meat, and egg, they showed resistance to desiccation, surfactant disinfectants such as benzalkonium chloride, as well as 254-nm ultraviolet (UV)-C irradiation (Kuda et al., 2008, 2011, 2012; Li et al., 2014). This indicated that
* Corresponding author. Tel./fax: þ81 3 5463 0602. E-mail address: [email protected] (T. Kuda). http://dx.doi.org/10.1016/j.fm.2014.08.014 0740-0020/© 2014 Elsevier Ltd. All rights reserved.
protein-, lipid-, and/or carbohydrate-rich food residue could protect pathogens from stress conditions. The gram-positive rod L. monocytogenes, regarded as tolerant to desiccation, is similarly protected by food residue (Takahashi et al., 2011). These studies indicate that washing and rinsing prior to sterilization are essential to meet the strictly sterile requirements for food safety. However, the quantum of food residue that is sufficient to protect pathogens from stress such as desiccation, disinfectants, or UV-C remains unclear. In this study, we investigated the quantity-dependent effects of milk, soy milk, carrot, and laver nori residue on survival rates of Salmonella Typhimurium, S. aureus, and L. monocytogenes adhered to and desiccated on a stainless steel surface. The cells adhered in the presence of food residue were observed using atomic force microscopy. 2. Materials and methods 2.1. Bacterial culture and food material Salmonella enterica subsp. enterica serotype Typhimurium NBRC 13245, S. aureus NBRC 12732, and L. monocytogenes Scott A were adhered onto utensil surfaces. To produce cultures, the bacterial cells were inoculated into 10 mL trypticase soy broth (TSB; Becton, Dickinson and Co.; Sparks, MD) and incubated at 37 C for 20 h. Under these conditions, the culture reached stationary phase (Kuda et al., 2013).
T. Kuda et al. / Food Microbiology 46 (2015) 234e238
Fresh carrot, sundried sheets of laver Porphyra sp. (called nori in Japanese), milk, and soy milk were purchased from a retail shop in Tokyo. The carrot was minced with an equal amount of distilled water (DW) for 60 s in a blender (16-Speed Blender; Osaka Chemical; Osaka, Japan) and shaken for 60 min at room temperature. The carrot extract was collected by centrifugation (2500 g for 10 min) and sterilized by filtration with a 0.22-mm-pore filter. The dried nori was milled in the blender and autoclaved (121 C for 15 min) with 40 time volumes of DW. After centrifugation, the supernatant was used as nori extract. The carrot extract, nori extract, milk, and soy milk samples were diluted 10-fold in DW to obtain 104 (0e10,000-fold diluted) solutions. 2.2. Adhesion of food pathogens to surfaces Fifty-millimeter-4 stainless steel dishes were purchased from As One Co. (Osaka, Japan) and used as the experimental surface (Kuda et al., 2008). Prior to use, in order to equalize the effect of the surface conditions on the survival cell count (Bohinc et al., 2014), the steel dishes were ultra-sonicated twice for 15 min, brushed for 60 s, and autoclaved at 121 C for 15 min. The bacterial cells were placed in the dish and attached as previously reported (Kuda et al., 2011), with slight modifications. Briefly, bacterial cells in the TSB culture were washed by centrifugation at 2000 g for 10 min at 4 C and re-suspended in phosphate-buffered saline (PBS; Nissui Pharmaceutical Co.; Tokyo, Japan); this process was repeated twice. The cells were finally re-suspended in 10 mL DW or the diluted food samples such that the final cell concentration was approximately 8e9 log cfu/mL. A bacterial suspension (0.1 mL) was placed in about 10 mm-4 of the center of the dish (n ¼ 3) and dried for 120 min at room temperature (20e24 C) in a bio-safety cabinet (Class IIA; Airtech Japan Co.; Tokyo, Japan) with ventilation. After drying, the adhered cells were detached by rubbing for 60 s using a sterile cotton swab and re-suspended in 5 mL TSB (Nissui Pharmaceutical). The detached cell suspension (0.1 mL) was immediately diluted in PBS, spread on trypticase soy agar (TSA; Becton, Dickinson and Co.) and incubated at 37 C for 24 h. 2.3. Microscopic observations To observe the bacterial cells, 0.01 ml of the cell suspension, prepared as same as above, was adhered by drying on a cover slip and the cells were observed using an atomic force microscope (AFM; Naio AFM; Nanosurf AG; Liestal, Switzerland) in dynamic force mode. 2.4. Statistical analysis Bacterial cell viability was expressed in terms of mean and standard deviation of log cfu/dish (n ¼ 3). Statistical analysis was performed using EXCEL Statistic 5.0 software (Esumi Co., Ltd.; Tokyo, Japan). One-way ANOVA was used to assess differences among groups, and individual means were compared by Tukey's multiple-range test. Differences were considered significant at p < 0.05. 3. Results and discussion 3.1. Effect of drying on survival of bacterial cells on the surface In our previous study, the sensitivity of logarithmic or early stationary growth phase cells to drying was higher than that of the stationary growth phase cells (Kuda et al., 2008). It has been reported that sigma factors produced in the stationary phase lead to
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the generation of self-defense proteins that protect the cells against acidic conditions, desiccation, low temperature, and reactive oxidants (Spector and Kenyon, 2012). Therefore, we used stationaryphase bacterial cells in previous studies (Kuda et al., 2011, 2012; 2013) as well as in this study. The viable count of the bacterial cells before and after drying on the stainless steel surface is summarized in Fig. 1. During the 120min ventilation period at room temperature, viable cells of Salmonella Typhimurium in DW were dried thoroughly and the cell count decreased from approximately 8.1 log cfu/dish to 3.4 log cfu/dish on the stainless steel surface (Fig. 1A). On the other hand, S. aureus and L. monocytogenes showed resistance to desiccation in contrast to Salmonella Typhimurium (Fig. 1B and C). The food samples of 50% carrot juice and 5% nori solution, milk, and soy milk clearly protected the cells from desiccation. The images of the dried and adhered cells on the cover slip observed by the AFM in dynamic force mode are shown in Fig. 2AeC. In this study, the gram-negative Salmonella Typhimurium cells were flattened by desiccation (Fig. 2A); however, the gram-positive S. aureus and L. monocytogenes cells retained their cell shape (Fig. 2A and B). This observation correlates with our and other previous reports regarding survival rates of gram-positive rez-Rodríguez and -negative pathogens (Kuda et al., 2011; Pe et al., 2013). 3.2. Protective effect of decimal diluted food residues on the adhered pathogens In Fig. 3, the horizontal lines marked as “DW” shows the survival cell counts of pathogens adhered with DW. Although the 100-fold diluted nori solution did not affect the survival rate of Salmonella Typhimurium, the 1000-fold diluted carrot juice, milk, and soy milk samples provided protection to Salmonella Typhimurium (Fig. 3A). S. aureus and L. monocytogenes were protected by the 100-fold diluted carrot juice and nori samples (Fig. 3B and C) as well as the 1000-fold diluted milk and soy milk samples. Of particular note is the finding that only 100 nL (103 of 0.1 mL) milk and soy milk could protect 10% of the gram-positive bacterial cells from desiccation. In the case of S. aureus, the protection capacity of the 100-fold diluted nori sample was higher than that of carrot juice. This finding was not in agreement with the results from Salmonella Typhimurium. In the case of L. monocytogenes, the protective effect from carrot juice and nori samples was similar. In the AFM observation, the adhered and dried pathogenic bacterial cells were not detected with the 50% carrot and 5% nori solutions, undiluted milk, and undiluted soy milk (Fig. 2DeG). Furthermore, 10% milk and 10% soy milk also covered the pathogenic cells (image not shown). Although the cells adhered with 10fold diluted carrot and nori samples as well as 100-fold diluted milk and soy milk samples were detected, the cells were covered with food residue (Fig. 2HeK). Interestingly, L. monocytogenes showed an aggregation of food residue particles around the cells (Fig. 2H,K, and L). Salmonella Typhimurium cells adhered with 1000-fold diluted milk and soy milk samples were not flattened (Fig. 2MeO). AFM is used to observe the effect of disinfectants such as quaternary ammonium compounds on pathogens (Crismaru et al., 2011), as this is considered a powerful tool for nano-level analysis of the utensil surface-adhered microorganisms. In addition, observations could be made in real time, without pretreatments such as fixation, dehydration, or metal-coating, as are required for scanning electron microscopy. In this study, cover slips were used for AFM observation instead of stainless steel. In future, we would like to kinetic study with the actual stainless surface. Milk and soy milk are rich in protein (approximately 33 mg/g and 38 mg/g, respectively), lipids (38 mg/g and 20 mg/g,
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Fig. 1. Survival rates in terms of cell counts of Salmonella Typhimurium (A), Staphylococcus aureus (B), and Listeria monocytogenes (C) on stainless steel dishes before (BF) and after 2 h drying with distilled water (DW), 50% carrot juice (CJ), 5% dried nori solution (NR), milk, or soy milk (SM). Values are expressed as mean and SD (n ¼ 3). a-d Different letters indicate significant differences (p < 0.05) between groups.
respectively), as well as carbohydrates (48 mg/g and 31 mg/g, respectively) (Standard Tables of Food Composition in Japan, 2005). These compounds may protect the cells from desiccation. Carrots are rich in carotenoids (94 mg/g) and carbohydrates (90 mg/g).
Although dried nori is a popular algal food, it is not only rich in carbohydrates (417 mg/g) but also in proteins (221 mg/g). These differences probably affected the protective activity, as shown in Fig. 3. Water-soluble polysaccharides such as porphyran and other
Fig. 2. Atomic force microscopy images of Salmonella Typhimurium (ST), Staphylococcus aureus (Sa), and Listeria monocytogenes (Lm) on cover slips after drying with distilled water (AeC), and food residues (DeO). DeG are images of undiluted food residue solutions. H and I are images of Lm with 10-fold diluted food residue solutions. JeM are images of pathogens with the 100-fold dilutions. N and O are images of ST with1000-fold diluted milk and soy milk solution.
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Fig. 3. Survival number of Salmonella Typhimurium (A), Staphylococcus aureus (B), and Listeria monocytogenes (C) adhered and desiccated on stainless steel dishes with decimal dilutions of 50% carrot juice (circles), 5% dried nori solution (open triangles), milk (closed triangles), or soy milk (squares). Values are expressed as mean and SD (n ¼ 3). The horizontal line (DW) corresponds to survival cell counts without food residue. aed Different letters indicate significant differences (p < 0.05) between groups.
low-molecular weight compounds were probably extracted in the boiled nori. Porphyra sp. has been reported to exhibit antioxidant and anti-inflammatory activity, in addition to providing protection from UV radiation (Kuda et al., 2005; Kazłowska et al., 2010; Zhang et al., 2010). The activity of these compounds may have affected the survival rate of pathogenic cells in this study. Thus, bacteria drying with food residue on utensil surfaces can occur in both industrial and domestic environments (Giauris and ~es et al., 2010; Humphrey et al., 2001; Hayson Nychas, 2006; Simo and Sharp, 2005). This can lead to cross contamination, inactivation of sterilization techniques that use disinfectants or UV-C, and contribute to biofilm formation (Kuda et al., 2011, 2012). Several reports regarding biofilms and methods for bacterial detachment and disinfection have been published (Chaturongkasumrit et al., ~es et al., 2010), which demonstrate that once biofilms 2011; Simo are formed, bacterial detachment and sterilization are challenging. In the present study, we showed that a minimal amount of food residue can protect bacterial cells. Therefore, adequate and regular washing to remove all traces of food residue is essential. Further study considering with temperature, humidity and other surface materials in food processing environment is also needed. 4. Conclusions In conclusion, after drying for 2 h at room temperature in the absence of food residue, cell counts of Salmonella Typhimurium, S. aureus, and L. monocytogenes decreased from 8 to 3, 6, and 5 log cfu/dish, respectively. It was observed that 5 mg fresh carrot, 0.05 mg dried nori, and 100 nL milk or soy milk per 10 mm 4 surface were sufficient to demonstrate a protective effect on the adhered pathogens, as confirmed by atomic force microscopy. Results from this study suggest that small sediments of food, not only protein rich but also carbohydrate rich, increase the resistance of surfaceadherent bacteria to desiccation, rendering sanitization processes ineffective and encouraging cross contamination and some food poisonings. Acknowledgment This work was supported by the Nisshin Seifun Foundation ( 2012-8) (Tokyo, Japan). References Baird, F.J., Wadsworth, M.P., Hill, J.E., 2012. Evaluation and optimization of multiple fluorophore analysis of a Pseudomonas aeruginosa biofilm. J. Microbiol. Methods 90, 192e196.
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