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Effect of heat shock on the fatty acid and protein profiles of Cronobacter sakazakii BCRC 13988 as well as its growth and survival in the presence of various carbon, nitrogen sources and disinfectants
Food Microbiology 36 (2013) 142e148
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Effect of heat shock on the fatty acid and protein profiles of Cronobacter sakazakii BCRC 13988 as well as its growth and survival in the presence of various carbon, nitrogen sources and disinfectants Po-Ting Li, Wan-Ling Hsiao, Roch-Chui Yu, Cheng-Chun Chou* Graduate Institute of Food Science and Technology, National Taiwan University, 59, Lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2012 Received in revised form 14 June 2012 Accepted 28 April 2013 Available online 15 May 2013
In the present study, Cronobacter sakazakii, a foodborne pathogen, was first subjected to heat shock at 47 C for 15 min. Effect of heat shock on the fatty acid and protein profiles, carbon and nitrogen source requirements as well as the susceptibilities of C. sakazakii to Clidox-S, a chlorine-containing disinfectant and Quatricide, a quaternary ammonium compound were investigated. Results revealed that heat shock increased the proportion of myristic acid (14:0), palmitic acid (16:0) and the ratio of saturated fatty acid to unsaturated fatty acid, while reducing the proportion of palmitoleic acid (16:1) and cis-vacceric acid (18:1). In addition, eleven proteins showed enhanced expression, while one protein showed decreased expression in the heat-shocked compared to the non-heat-shocked cells. Non-heat-shocked cells in the medium supplemented with beef extract exhibited the highest maximum population. On the contrary, the highest maximum population of heat-shocked C. sakazakii was noted in the medium having either tryptone or yeast extract as the nitrogen source. Among the various carbon sources examined, the growth of the test organism, regardless of heat shock, was greatest in the medium having glucose as the carbon source. Furthermore, heat shock enhanced the resistance of C. sakazakii to Clidox-S or Quatricide. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Cronobacter sakazakii Heat shock Fatty acid and protein profiles Nitrogen and carbon sources Disinfectant
1. Introduction Cronobacter sakazakii, formerly Enterobacter sakazakii, is an emerging foodborne pathogen that has been implicated as a cause of meningitis and enterocolitis in premature infants and has resulted in high mortality rates in those affected (Van Acker et al., 2001; Iversen et al., 2008). This pathogen has also been reported to be the cause of death of the elderly people and immunocompromised adults (Lehner et al., 2005). It appears to be ubiquitous in nature and has been isolated from various food sources (Iversen and Forsythe, 2003; Beuchat et al., 2009). However, it is its presence in dried infant formula milk that has been linked to the severe systemic neonatal infections (Clark et al., 1990; Van Acker et al., 2001; Himelright et al., 2002). The International Commission of Microbiological Specifications for Foods has ranked this pathogen as a “severe hazard for restricted population, life threatening or substantial chronic sequelae or long duration” (ICMSF, 2002). Thus, this pathogen has garnered the increased interest and concern of
* Corresponding author. Tel.: þ886 2 3366 4111; fax: þ886 2 2362 0849. E-mail addresses: [email protected], [email protected] (C.-C. Chou). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.04.018
regulatory agencies, health care providers, the food industry and the scientific community. Various degrees of heat treatment are commonly employed in the food industry and in households when foods and food products are prepared or processed. These treatments may inactivate, reduce and prevent the proliferation of pathogenic and spoilage microorganisms that are present in the food. This treatment extends the shelf-life of prepared food and increases its safety. Nevertheless, sub-lethal heat treatment (heat shock) might induce heat shock response on microorganisms. Cells of microorganisms exposed to temperatures a few degrees centigrade above their optimal growth temperature may develop enhanced tolerance to more adverse heat stress or other stresses otherwise lethal to them (Bunning et al., 1990; Linton et al., 1992; Murano and Pierson, 1993). This phenomenon of heat shock response has been observed in various foodborne pathogens and has important implications in food safety (Bunning et al., 1990; Farber and Brown, 1990; Mackey and Derrick, 1990; Chang et al., 2004; Lin and Chou, 2004). These implications are more pressing because currently employed control measures were developed based on data collected with non-heat-shocked microorganisms, and therefore may be inadequate in controlling the proliferation of the more resilient heat-shocked microorganisms.
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In the past few years, a series of experiments concerning the heat shock response of C. sakazakii has been conducted in our laboratory to obtain the data necessary for developing adequate control measures and risk assessments for C. sakazakii. We found that heat-shocked C. sakazakii exhibited enhanced survival when exposed to 51 C and other lethal stress conditions such as ethanol (15%, v/v), acidity (pH 3.3) (Chang et al., 2009, 2010) and simulated gastric juice (Hsiao et al., 2010a). In addition, heat shock enhanced the survival of C. sakazakii during the lactic fermentation of milk, after freeze-drying or spray-drying of milk (Hsiao et al., 2010b). In this study, we further investigated the effect of heat shock on the fatty acid and protein profile of C. sakazakii to elucidate the heat response of this organism. The susceptibility of the heat-shocked and non-shocked cells of C. sakazakii to disinfectants was also examined. Furthermore, considering the possible effect of nutrient availability on the growth of heat shocked and non-heat shocked microorganisms as observed for Listeria monocytogenes and Vibrio parahaemolyticus (Busch and Donnelly, 1992; Chiang and Chou, 2008), growth pattern of heat-shocked and non-shocked C. sakazakii in presence of various nitrogen and carbon sources was examined and compared in the present study. 2. Materials and methods 2.1. C. sakazakii and preparation of the heat-shocked cells In the present study, C. sakazakii BCRC 13988 (ATCC 29544), obtained from the Bioresources Collection and Research Center in Hsinchu, Taiwan, was used as the test organism. It is a clinical strain originally isolated from throat of an infant patient (Farmer et al., 1980). Procedures as described previously (Chang et al., 2009) were followed to prepare the heat-shocked cells of C. sakazakii. Briefly, the activated cultures obtained by two successive transfers of the test organism in Tryptic soy broth (TSB) (Acumedia Manufactures, Lansing, MI, USA) at 37 C for 24 h, were grown in TSB (pH 7.2) at 37 C for 6 h to reach late exponential growth. The culture was centrifuged at 8000 g for 10 min. After re-suspending the pellet in fresh TSB which was pre-warmed at 47 C, the cultures were subjected to heating at 47 C for 15 min to obtain the heat-shocked cells of C. sakazakii. Cultures not exposed to the heat shock treatment were included as control. 2.2. Growth study with various carbon and nitrogen sources In this study, the prepared heat-shocked and control were first centrifuged at 4000 g for 15 min at 4 C. Cells were washed and re-suspended in phosphate buffer solution (PBS, pH 7.0) to give a population of ca. 107 cfu/ml. The nitrogen sources tested were N-Z amine (Sigma, St. Louis, MO, USA), beef extract (Difco), casamino acid (Difco), malt extract (Difco), peptone (Difco), soytone (Difco), tryptone (Difco), tryptone and yeast extract (Difco). On the other hand, fructose (Sigma), galactose (Sigma), glucose (Sigma), lactose (Sigma), maltose (Sigma), mannitol (Sigma), sorbitol (Sigma) and sucrose (Sigma) were tested as carbon sources. To study the effects of nitrogen sources, 2% (w/v) of each nitrogen source was added to a basal medium containing 0.25% glucose, 0.5% NaCl and 0.25% K2HPO4, pH 7.0. To examine the effects of carbon sources, a basal medium containing 2% tryptone, 0.5% NaCl and 0.25% K2HPO4, pH 7.0 was supplemented with 0.25% of each carbon source. After inoculation with the heat-shocked or non-shocked cells at 106 cfu/ ml, the media were incubated at 37 C for 7 h. Growth was determined from increases of the absorbance at 600 nm, measured using a spectrophotometer (Helios Alpha, Spectronic Unicam, Cambridge, UK).
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2.3. Study on the susceptibility of heat-shocked and control C. sakazakii to disinfectants In the present study, the susceptibility of the test organism to disinfectants including Clidox-S, a chlorine base disinfectant, and Quatricide, a quaternary ammonium compound (QACs) was investigated. Both disinfectants are products of Pharmacal Research Labs, Inc. (Naugatuck, CT, USA) Formulation of Clidox-S solution was performed by mixing Clidox-S, distilled water and Clidox-S activator at a ratio of 1:18:1 (v:v:v) while ca. 59.1 ml (two ounces) of Quatricide was mixed with 3785.4 ml (one gallon) of distilled water (w/v) according to the manufacture’s instructions. To examine the susceptibility of heat-shocked and control C. sakazakii to disinfectants, the procedures of Kim et al. (2007) were followed. Essentially, one ml of C. sakazakii cells was first mixed with 9.0 ml of reconstituted infant formula (Wyeth Nutritionals Inc., Singapore) to give a population of ca. 7.0 log cfu/ml. The reconstituted infant formula was prepared by dissolving 8.5 g S-26 powder infant formula in 60 ml distilled water and autoclaving at 121 for 15 min. Five milliliters of cell suspension were then mixed with 5 ml Clidox-S or 15 ml Quatricide and incubated at 25 or 40 C for 300 s. At specific time intervals during incubation, one ml of sample was withdrawn and combined with one ml of Dey-Engley (DE) neutralizing broth (Sigma) to neutralize those compounds that might be lethal to the test organism (Sutton et al., 1991). Then the viability of test organism was determined. 2.4. Enumeration of C. sakazakii To determine the viable population of C. sakazakii, samples were serially diluted in PBS containing 0.1% peptone. The viable counts were then made by spread plating 0.1 ml on Tryptic soy agar (Acumedia Manufactures). Colonies were counted after 18 h of incubation at 37 C. 2.5. The preparation of cell extract and protein determination The method described by Wouters et al. (1999) was followed to prepare the protein extract, cells of C. sakazakii were collected by centrifugation at 3000 g for 10 min and were suspended in 50 mM phosphate buffer (pH 7.0) containing 0.1 M NaCl, 0.1 mM EDTA. The cell suspension, maintained in an ice bath, was then sonicated with a sonicator (Model 3000, Misonix, Farmingdale, NY, USA) for sixteen 30-sec intervals. Cell debris was removed by centrifugation at 10,000 g for 20 min. The supernatant extracted from cells served as the cellular proteins. The content of protein in the cell lysate was measured by the colorimetric method of Bradford (1976), with bovine serum albumin (BSA) (Sigma) as the standard. SDS-PAGE was performed according to the method of Laemmli et al. (1970) using a separating gel of 12.5% acrylamide. The cell extract was mixed with an equal volume of sample buffer (250 mM Tris, 4 mM EDTA-2Na, 4% SDS, 10% b-mercaptoethanol) and then a small amount of bromophenol blue (5 ml) was added, after which it was heated in a water bath at 100 C for 5 min. After cooling to room temperature, the same quantity of extracted protein (10 mg) was loaded onto a gel and separated with a mini-vertical electrophoresis unit (Model SE260, Hoefer, San Francisco, CA, USA) at 150 V for 1.5 h. The standard marker proteins (11e98 KDa) (Bio-Rad Laboratories, Inc., Hercules, California, USA) were used to run concurrently as a size marker. The gels were stained with colloidal Coomassie blue (2 mM Coomassie Brilliant R-250, 45% methanol, 10% acetic acid for 1 h and destained with a solution containing 20% methanol and 10% acetic acid. The images of the gels were scanned and analyzed using Multi-Gauge (Fuji Photo Film Co., Tokyo, Japan).
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2.6. Analysis of fatty acids To determine the acid profile of C. sakazakii, cultures containing control or heat-shocked cells were first centrifuged (3000 g, 10 min) and twice washed with phosphate buffer solution (pH 7.0, PBS). Transesterification of fatty acids as described by Lepage and Roy (1986) was then followed to prepare the methylesters of cellular fatty acids. The prepared methylated fatty acids were dissolved in n-hexane and subjected to GC analysis. Chromatography was conducted on a 30-m fused silica column with an internal diameter of 0.32 mm. The column was wall-coated with 0.2 mm RTXÒ-2330 (Restekcorp Co., PA, USA). Analysis was performed on a Hewlett Packard Model 5890 SERIES II gas chromatograph equipped with a flame ionization detector (Hewlett Packard, Palo Alto, CA, USA). Hydrogen was used as a carrier gas and nitrogen as a make-up gas. The split ratio was 1:25 (column flow:split vent flow). The injection port temperature was 230 C and the detector temperature was 250 C. The column temperature was held at 130 C for 2 min, and then the temperature was increased to 180 C by 3 C per min and held for 1 min. After that, the temperature was increased again to 195 C by 2 C per min and applied for 0.2 min. A final increase in temperature to 205 C by 3 C per min was then held for 28 min. Commercial standards of fatty acid methylesters (Sigma) were run under identical conditions and the chromatograms were evaluated with reference to the retention time standards. 2.7. Statistical analysis Mean values and standard deviations were calculated from the data obtained from the three separate experiments. Data were analyzed using an unpaired two-tailed Student’s t-test. Statistical significance was set at p < 0.05 (SAS, 2001). 3. Results and discussion 3.1. The effect of heat shock on fatty acid composition Table 1 shows the fatty acid composition of the heat shocked and control cells of C. sakazakii. It was noted that palmitic acid (16:0) and cis-vaccenic acid (18:1) was, respectively, the major saturated and unsaturated fatty acid noted in the non heat-shocked cells of C. sakazakii. Heat shock resulted in an increase in the content of myristic acid (14:0) and palmitic acid (16:0) in the cells of C. sakazakii. On the other hand, palmitoleic acid (16:1) and cis-vaccenic acid (18:1) content reduced from 15.94% to 25.79%, respectively, to 10.01 and 13.15% (heat-shocked cells) after heat shock. Compared with non-shocked cells, the proportion of unsaturated fatty acid (USFA) Table 1 Fatty acid composition of heat-shocked and non- heat-shocked C. sakazakii cells. Fatty acida
Fatty acid proportion (%)b Non-heat-shocked
12:0 14:0 16:0 16:1 18:1 SFA USFA SFA/USFA
1.68 5.82 50.84 15.94 25.79 58.34 41.66 1.44
0.16 0.28 3.97 2.39 1.72 0.56 1.99 0.06
Ac B B A A B A B
Heat-shocked 1.54 12.52 62.78 10.01 13.15 76.84 23.16 3.31
0.14 0.98 2.21 1.22 1.25 0.67 1.23 0.07
A A A B B A B A
a 12:0, Lauric acid; 14:0 Myristic acid: 16:0, Palmitic acid; 16:1 Palmitoleic acid; 18:1 cis-vaccenic acid; SFA, saturated fatty acid; USFA, unsaturated fatty acid. b Values are proportion of total fatty acid (%). c Values in the same row with different upper case letters (A, B) are significantly different (p < 0.05).
decreased, while content of saturated fatty acids (SFA) and the proportion of SFA to USFA increased in the heat-shocked cells of C. sakazakii. A similar phenomenon has also been observed on Escherichia coli (Sinensky, 1974), V. parahaemolyticus (Chiang et al., 2005) and Pediococcus sp. (Annous et al., 1999). The relative proportions of SFA and USFA affected the fluidity and permeability of cell membranes (Sinensky, 1974; Mejia et al., 1995). Hansen (1971) and Chang et al. (2004), respectively, reported that there was a relationship between increased SFA/USFA ratios in lipids and the enhanced heat resistance of E. coli and V. parahaemolyticus 690 incubated at elevated temperatures. Previously, it was noted that the induced formation of the heat shock protein Ibp B, which both facilitates the fouling of protein and reduces the heat coagulation of protein, might lead to the increased thermal resistance (Pérez et al., 2007). The alteration of fatty acid profile observed in the present study might also contribute to the increased thermal resistance and the alteration of the cell membrane permeability of the heat-shocked cells of C. sakazakii as observed by Chang et al. (2009). 3.2. The effect of heat shock on protein profile Thermal tolerance has been identified as an important factor relevant to the pathogenicity of C sakazakii. Previously, Williams et al. (2005) reported that thermotolerant Cronobacter strains contained a protein, Mfla_1165, which is a homologue of a hypothetical protein from the thermotolerant bacterium Methylobacillus flagellatus KT.126. Besides, Gajdosova et al. (2011) showed that the gene encoding this homologue, orfI, is associated with an 18 kbp region containing 22 open reading frames which were upregulated under heat adaptation conditions. The major feature of the region is a cluster of conserved genes, most of them having significant homologies with known bacterial proteins involved in some type of stress response, including heat, oxidation and acid stress. Furthermore, heat shock response has been proposed as one of the adaptive responses with its induction of heat-shocked proteins (HpsP). Analysis with SDS-PAGE has shown an alteration in the expression of proteins due to sublethal heat shock and has been observed on various microorganisms (Sahu et al., 1994; Jeevanjyot and Ghosh, 1995; Chiang et al., 2008). The elevated levels of some proteins in microbial cells are believed to be connected with an increase in thermal as well as other forms of stress. Fig. 1 shows the SDS-PAGE pattern of cellular proteins from heat-shocked and non-shocked C. sakazakii, while the results of image analysis with Multi-Gauge are shown in Table 2. Compared with the respective protein expression in the non-shocked C. sakazakii, which was set as 1.0, eleven proteins bands including B-L in the heat-shocked cells exhibited an increased expression level with a relative protein expression value of 1.05e2.70. This implied that heat shock enhanced the expression of these proteins. Among these proteins, the increased expression of protein L with a molecular mass of 20 KDa, was the highest in the heat-shocked cells followed by the 65 KDa protein (band C) which showed an increased expression level of 1.74. A relatively lower degree of increased expression with a level of 1.04e1.08 was noted with the other five proteins bands (D, F, G, I) in the heat-shocked cells of C. sakazakii. In contrast, the expression of protein A, having a molecular mass of 80 KDa in the heat-shocked C. sakazakii showed a reduced level of 0.77 compared with that in the control cells. The alternation of protein expression observed might relate to the changes in the susceptibility of C. sakazakii to some lethal stresses as previously reported by Chang et al. (2009, 2010). It was reported that proteins separated by isoelectric focusing in a linear pH gradient in first dimension followed by SDS-PAGE in the second dimension could provide more detailed protein patterns (Chiang
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Although heat shock at 47 C for 15 min enhanced the resistance of C. sakazakii to lethal stresses such as 58 C, pH 3.3, 15% ethanol 3 and 20 C, cells of the heat-shocked C. sakazakii were found to be injured (Chang et al., 2009, 2010). The heat-shocked cells of C. sakazakii showed greater leakage of nucleic acid and protein than did the control cells. These results concerning the effect of various carbon and nitrogen sources on the growth behavior of heat-shocked and non-shocked cells of C. sakazakii warranted further investigation. As shown in Fig. 2, the growth of C. sakazakii, regardless of heat shock, varied with the nitrogen source present in the basal medium. Generally, both control and heat-shocked C. sakazakii in the media started to grow after 2 h of incubation except in the medium supplemented with malt extract as the nitrogen source. The control cells in the medium supplanted with beef extract exhibited the highest maximum population after 7 h of cultivation (Fig. 2A). On the contrary, the highest maximum population of heat-shocked C. sakazakii was noted in the medium having either tryptone or yeast extract as the nitrogen source (Fig. 1B). Yeast extract is an aqueous extract of yeast cells. While tryptone is a pancreatic digest of casein. Both, containing plenty of amino acids and peptides, are rich sources of nitrogen. Additionally, they also contain vitamins, growth factors of various microorganisms (BD BionutrientsÔ Technical Manual, 2006). These may thus led to the highest maximum growth of test organism observed (Fig. 2). 0.7
Fig. 1. SDS-PAGE (10%) pattern of cellular proteins from C. sakazakii. Lane 1, marker; lane 2, non-heat-shocked cells; lane 3, heat-shocked cells.
3.3. The effect of heat shock on the growth of C. sakazakii in presence of various nitrogen and carbon sources
Absorbance (600nm)
and Chou, 2008). Therefore, analyses of protein patterns by twodimensional electrophoresis and LC/MS/MS were performed currently in our laboratory in an attempt to further identify, characterize and elucidate the function of the proteins induced by heat shock treatment in the cells of C. sakazakii.
0.5
0.4
0.3
0.2
Nutrient availability is an important factor which affects the repair and growth of injured cells (Moss and Speck, 1966; Morichi, 1969). Busch and Donnelly (1992) reported that supplementing glucose, lactose or sucrose in TSB facilitated the repair and growth of the heat-injured cells of L. monocytogenes. Chou and Cheng (2000) observed that the growth of low temperature-injured cells of E. coli O157:H7 varied with carbon and nitrogen sources in the medium.
0.1
Table 2 One-dimensional SDS-PAGE pattern of heat-shocked and non-heat-shocked cells of C. sakazakii.
0.5
A B C D E F G H I J K L a
% of total protein
Relative protein
Non-heat-shocked
Heat-shocked
Expressiona
1.92 2.85 2.47 0.70 4.45 1.96 1.57 1.80 1.77 2.96 2.81 1.04
1.48 3.39 4.31 0.74 5.87 2.03 1.66 1.94 1.85 3.74 3.23 2.81
0.77 1.19 1.74 1.05 1.32 1.04 1.06 1.08 1.04 1.26 1.15 2.70
Relative protein expression was obtained by dividing the protein expression of heat-shocked cells with that of non-heat-shocked cells which was assigned as 1.0.
0.0 0
2
4
6
8
Time (h) 0.7
(B)
N-Z-Amine Beef extract Casamino acid Malt extract Peptone Soytone Tryptone Yeast extract
0.6
Absorbance (600nm)
Band
(A)
N-Z-Amine Beef extract Casamion acid Malt extract Peptone Soytone Tryptone Yeast extract
0.6
0.4
0.3
0.2
0.1
0.0 0
2
4
6
8
Time (h) Fig. 2. Effect of heat shock on the growth of C. sakazakii in the presence of various nitrogen sources. Basal medium containing 0.25% glucose, 0.5% NaCl and 0.25% K2HPO4 (pH 7) was added with 2% (w/v) nitrogen sources. (A) Non-heat-shocked cell; (B) heatshocked cell.
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Fig. 3 shows the growth of C. sakazakii in media containing 2% tryptone and supplemented with 0.25% of various carbon sources. All the carbon sources examined were found to support the growth of the heat-shocked and non-shocked cells of C. sakazakii. Regardless of the carbon source added or the heat shock treatment employed, C. sakazakii in the medium exhibited a lag phase of ca. 2 h. Generally, the growth patterns of heat-shocked and nonshocked cells in medium supplemented with same carbon source was similar. Among the various carbon sources examined, the growth of the test organism, regardless of heat shock, was greatest in the medium having glucose as the carbon source. In contrast, using sorbitol as the carbon source in the supplementation medium yielded the lowest level of maximum growth. This may be due to the failure of test organism to utilize sorbitol (Nazarowec-White and Farber, 1997). However, the exact reason remained to be further explored. 3.4. The effect of heat shock on the viability of C. sakazakii exposed to disinfectants To prevent the contamination and the proliferation of microorganisms, chemical disinfectants are frequently applied to food contact and non-food contact surfaces in the food industry, food
service kitchens, day-care centers and formula preparation areas in hospitals. Susceptibility to disinfectant varies with microorganisms. Moreover, factors such as concentration, exposure time, temperature, pH, equipment cleanliness and water hardness affect the efficacy of a disinfectant (Marriott and Gravani, 2006). Sub-lethal stress has been observed to alter the susceptibility of L. monocytogenes and Salmonella Typhimurium to disinfectants (Lin et al., 2011). They indicated that the changes of microbial susceptibility varied with the kinds of sublethal stress, as well as the microorganisms and disinfectants examined. Therefore, the viability of heat-shocked and non-shocked C. sakazakii exposed to Clidox-S and Quatricide was further compared in the present study. Fig. 4 shows the survival of heat-shocked and non-shocked C. sakazakii during exposure to Clidox-S at 25 and 40 C. Clidox-S is a chlorine-containing disinfectant. It has been suggested that the mode actions of the chlorine-containing disinfectants include: the inhibition of glucose oxidation, oxidative decarboxylation of amino acids, disruption of protein synthesis, reactions with nucleic acids, purines, and pyrimidines, induction of deoxyribonucleic acid lesions, creation of chromosomal alterations and formation of toxic pyrimidines, leading to the injury and death of microbial cells (Marriott and Gravani, 2006). As shown in Fig. 4A, the viability of C. sakazakii, regardless of heat shock, reduced as the period of exposure to Clidox-S at 25 C was
0.7
0.5
0.4
(A) 25 oC
102 101 Survival (%)
Absorbance (600nm)
0.6
103
(A)
Fructose Galactose Glucose Lactose Maltose Mannitol Sorbitol Sucrose
0.3
0.2
100 10-1 10-2
0.1
10-3
0.0 0
2
4
6
Non-heat-shocked cells Heat-shocked cells
10-4
8
0
30
60
120
Time (h) 0.8
0.5
10
300
(B) 40 oC
2
101 Survival (%)
Absorbance (600nm)
0.6
103
(B)
Fructose Galactose Glucose Lactose Maltose Mannitol Sorbitol Sucrose
0.7
180 Time (sec)
0.4 0.3
100 10-1 10-2
0.2 0.1
10-3
0.0
10-4 0
2
4
6
8
Time (h) Fig. 3. Effect of heat shock on the growth of C. sakazakii in the presence of various carbon sources. Basal medium containing 2% tryptone, 0.5% NaCl and 0.25% K2HPO4 (pH 7) was added with 0.25% (w/v) carbon sources. (A) Non-heat -shocked cell; (B) heat-shocked cell.
Non-heat-shocked cells Heat-shocked cells 0
30
60
120
*
*
180
300
Time (sec) Fig. 4. Viability of heat-shocked and non-shocked C. sakazakii exposed to Clidox-S at 25 C (A) and 40 C (B). * None detectable. Five milliliters of cell suspension containing test organism at a concentration of ca. 7.0 log cfu/ml were mixed with 5 ml Clidox-S or 15 ml Quatricide and incubated at 25 or 40 C.
P.-T. Li et al. / Food Microbiology 36 (2013) 142e148
extended. However, the heat shocked C. sakazakii exhibited a higher viability than did the control cells during the entire exposure period. At the end of the exposure to Clidox-S at 25 C, the viable population of control cells reduced from the initial viable population of 6.62e 1.79 log cfu/ml, exhibiting a population reduction of 4.83 log cfu/ml. While at the end of a similar exposure period, the heat shocked C. sakazakii showed a population reduction of 2.09 log cfu/ml which is significantly less (p < 0.05) than that of the control cells. This demonstrated that heat shock enhanced the survival of C. sakazakii exposed to Clidox-S at room temperature. Similar to that noted at 25 C (Fig. 4A), viability of heat shocked and control C. sakazakii reduced during exposure to Clidox-S at 40 C (Fig. 4B). However, the rate of reduction in the viability of the heat shocked or control C. sakazakii is greater at 40 C than that of the respective cells noted at 25 C. This demonstrated that Clidox-S exhibited a greater disinfectant effect on the test organism at 40 C than at 25 C. Quatricide, the sanitizer examined, is QACs with n-alkyl dimethyl benzyl ammonium chlorides and n-alkyl dimethyl ethylbenzyl ammonium chlorides as the active ingredients (Pharmacal Research Labs. Inc., 2010). Being QACs, they are capable of interacting with the cytoplasmic membrane, causing cell leakage and membrane damage, primarily due to their absorption by the bacterial membrane (Ioannou et al., 2007). 103
(A) 25 oC
147
Similar to observations in the presence of Clidox-S, the viability of heat-shocked and non-shocked C. sakazakii exposed to Quatricide reduced steadily during the entire exposure period (Fig. 5). Having an initial population of ca. 106 cfu/ml, the viable population of the heat-shocked and control C. sakazakii reduced to 3.63 and 2.15 log cfu/ml after 300 s of exposure to Quatricide at room temperature, the control C. sakazakii showed a population reduction of 4.28 log cfu/ml compared to a significantly lower (p < 0.05) population reduction of 2.87 log cfu/ml observed in the heat-shocked cells (Fig. 5A). As the exposure temperature was raised to 40 C, a marked reduction in the viable population of C. sakazakii, regardless of heat shock, was observed throughout the entire exposure period (Fig. 5B). For example, control C. sakazakii showed a viable population of 2.15 log cfu/ml with a population reduction of 4.28 log cfu/ml at the end of exposure to Quatricide at 25 C (Fig. 5A). In comparison, the viable population of the control cells reduced to a non-detectable level with a population reduction of >6.00 log cfu/ml after a similar period of exposure to Quatricide at 40 C (Fig. 5B). Additionally, the viability of the heat-shocked C. sakazakii was also noted to exhibit a relatively lower reduction rate than the control cells. The viable population of heat-shocked C. sakazakii detected at all the specific exposure intervals, except at 0 time, was significantly higher (p < 0.05) than that of the control cells (Fig. 5B). These results, in addition to showing the greater disinfectant effect of Quatricide at 40 C than at 25 C, demonstrated that heat shock also reduced the susceptibility of C. sakazakii to Quatricide.
102
4. Conclusion
Survival (%)
101
Data obtained from the present study revealed that heat shock altered the protein and fatty acid profiles of C. sakazakii. This might relate to the altered response of the test organism when exposed to subsequent stress conditions as observed previously (Chang et al., 2009, 2010). Furthermore, heat shock enhanced the resistance of C. sakazakii to the disinfectants examined. These observations stress the importance of taking into account heat shock response in developing improved safety measures. With improved measures in place, disinfectants can be used more effectively against C. sakazakii and the food system can be made safer.
100 10-1 10-2 10-3 Non-heat-shocked cells Heat-shocked cells
10-4 10-5 0
30
60
120
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300
Acknowledgment
Time (sec)
Survival (%)
103 102
This research was financially supported by the National Science Council, ROC (Taiwan). (NSC 98-2313-B-002-037-MY3).
101
References
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(B) 40 oC
0
10-1 10-2 10-3 Non-heat-shocked cells Heat-shocked cells
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60
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*
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Time (sec) Fig. 5. Viability of heat-shocked and non-shocked C. sakazakii exposed to Quatricide at 25 C (A) and 40 C (B). * None detectable. Five milliliters of cell suspension containing test organism at a concentration of ca. 7.0 log cfu/ml were mixed with 15 ml Quatricide and incubated at 25 or 40 C.
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Effect of heat shock on the fatty acid and protein profiles of Cronobacter sakazakii BCRC 13988 as well as its growth and survival in the presence of various carbon, nitrogen sources and disinfectants Po-Ting Li, Wan-Ling Hsiao, Roch-Chui Yu, Cheng-Chun Chou* Graduate Institute of Food Science and Technology, National Taiwan University, 59, Lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2012 Received in revised form 14 June 2012 Accepted 28 April 2013 Available online 15 May 2013
In the present study, Cronobacter sakazakii, a foodborne pathogen, was first subjected to heat shock at 47 C for 15 min. Effect of heat shock on the fatty acid and protein profiles, carbon and nitrogen source requirements as well as the susceptibilities of C. sakazakii to Clidox-S, a chlorine-containing disinfectant and Quatricide, a quaternary ammonium compound were investigated. Results revealed that heat shock increased the proportion of myristic acid (14:0), palmitic acid (16:0) and the ratio of saturated fatty acid to unsaturated fatty acid, while reducing the proportion of palmitoleic acid (16:1) and cis-vacceric acid (18:1). In addition, eleven proteins showed enhanced expression, while one protein showed decreased expression in the heat-shocked compared to the non-heat-shocked cells. Non-heat-shocked cells in the medium supplemented with beef extract exhibited the highest maximum population. On the contrary, the highest maximum population of heat-shocked C. sakazakii was noted in the medium having either tryptone or yeast extract as the nitrogen source. Among the various carbon sources examined, the growth of the test organism, regardless of heat shock, was greatest in the medium having glucose as the carbon source. Furthermore, heat shock enhanced the resistance of C. sakazakii to Clidox-S or Quatricide. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Cronobacter sakazakii Heat shock Fatty acid and protein profiles Nitrogen and carbon sources Disinfectant
1. Introduction Cronobacter sakazakii, formerly Enterobacter sakazakii, is an emerging foodborne pathogen that has been implicated as a cause of meningitis and enterocolitis in premature infants and has resulted in high mortality rates in those affected (Van Acker et al., 2001; Iversen et al., 2008). This pathogen has also been reported to be the cause of death of the elderly people and immunocompromised adults (Lehner et al., 2005). It appears to be ubiquitous in nature and has been isolated from various food sources (Iversen and Forsythe, 2003; Beuchat et al., 2009). However, it is its presence in dried infant formula milk that has been linked to the severe systemic neonatal infections (Clark et al., 1990; Van Acker et al., 2001; Himelright et al., 2002). The International Commission of Microbiological Specifications for Foods has ranked this pathogen as a “severe hazard for restricted population, life threatening or substantial chronic sequelae or long duration” (ICMSF, 2002). Thus, this pathogen has garnered the increased interest and concern of
* Corresponding author. Tel.: þ886 2 3366 4111; fax: þ886 2 2362 0849. E-mail addresses: [email protected], [email protected] (C.-C. Chou). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.04.018
regulatory agencies, health care providers, the food industry and the scientific community. Various degrees of heat treatment are commonly employed in the food industry and in households when foods and food products are prepared or processed. These treatments may inactivate, reduce and prevent the proliferation of pathogenic and spoilage microorganisms that are present in the food. This treatment extends the shelf-life of prepared food and increases its safety. Nevertheless, sub-lethal heat treatment (heat shock) might induce heat shock response on microorganisms. Cells of microorganisms exposed to temperatures a few degrees centigrade above their optimal growth temperature may develop enhanced tolerance to more adverse heat stress or other stresses otherwise lethal to them (Bunning et al., 1990; Linton et al., 1992; Murano and Pierson, 1993). This phenomenon of heat shock response has been observed in various foodborne pathogens and has important implications in food safety (Bunning et al., 1990; Farber and Brown, 1990; Mackey and Derrick, 1990; Chang et al., 2004; Lin and Chou, 2004). These implications are more pressing because currently employed control measures were developed based on data collected with non-heat-shocked microorganisms, and therefore may be inadequate in controlling the proliferation of the more resilient heat-shocked microorganisms.
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In the past few years, a series of experiments concerning the heat shock response of C. sakazakii has been conducted in our laboratory to obtain the data necessary for developing adequate control measures and risk assessments for C. sakazakii. We found that heat-shocked C. sakazakii exhibited enhanced survival when exposed to 51 C and other lethal stress conditions such as ethanol (15%, v/v), acidity (pH 3.3) (Chang et al., 2009, 2010) and simulated gastric juice (Hsiao et al., 2010a). In addition, heat shock enhanced the survival of C. sakazakii during the lactic fermentation of milk, after freeze-drying or spray-drying of milk (Hsiao et al., 2010b). In this study, we further investigated the effect of heat shock on the fatty acid and protein profile of C. sakazakii to elucidate the heat response of this organism. The susceptibility of the heat-shocked and non-shocked cells of C. sakazakii to disinfectants was also examined. Furthermore, considering the possible effect of nutrient availability on the growth of heat shocked and non-heat shocked microorganisms as observed for Listeria monocytogenes and Vibrio parahaemolyticus (Busch and Donnelly, 1992; Chiang and Chou, 2008), growth pattern of heat-shocked and non-shocked C. sakazakii in presence of various nitrogen and carbon sources was examined and compared in the present study. 2. Materials and methods 2.1. C. sakazakii and preparation of the heat-shocked cells In the present study, C. sakazakii BCRC 13988 (ATCC 29544), obtained from the Bioresources Collection and Research Center in Hsinchu, Taiwan, was used as the test organism. It is a clinical strain originally isolated from throat of an infant patient (Farmer et al., 1980). Procedures as described previously (Chang et al., 2009) were followed to prepare the heat-shocked cells of C. sakazakii. Briefly, the activated cultures obtained by two successive transfers of the test organism in Tryptic soy broth (TSB) (Acumedia Manufactures, Lansing, MI, USA) at 37 C for 24 h, were grown in TSB (pH 7.2) at 37 C for 6 h to reach late exponential growth. The culture was centrifuged at 8000 g for 10 min. After re-suspending the pellet in fresh TSB which was pre-warmed at 47 C, the cultures were subjected to heating at 47 C for 15 min to obtain the heat-shocked cells of C. sakazakii. Cultures not exposed to the heat shock treatment were included as control. 2.2. Growth study with various carbon and nitrogen sources In this study, the prepared heat-shocked and control were first centrifuged at 4000 g for 15 min at 4 C. Cells were washed and re-suspended in phosphate buffer solution (PBS, pH 7.0) to give a population of ca. 107 cfu/ml. The nitrogen sources tested were N-Z amine (Sigma, St. Louis, MO, USA), beef extract (Difco), casamino acid (Difco), malt extract (Difco), peptone (Difco), soytone (Difco), tryptone (Difco), tryptone and yeast extract (Difco). On the other hand, fructose (Sigma), galactose (Sigma), glucose (Sigma), lactose (Sigma), maltose (Sigma), mannitol (Sigma), sorbitol (Sigma) and sucrose (Sigma) were tested as carbon sources. To study the effects of nitrogen sources, 2% (w/v) of each nitrogen source was added to a basal medium containing 0.25% glucose, 0.5% NaCl and 0.25% K2HPO4, pH 7.0. To examine the effects of carbon sources, a basal medium containing 2% tryptone, 0.5% NaCl and 0.25% K2HPO4, pH 7.0 was supplemented with 0.25% of each carbon source. After inoculation with the heat-shocked or non-shocked cells at 106 cfu/ ml, the media were incubated at 37 C for 7 h. Growth was determined from increases of the absorbance at 600 nm, measured using a spectrophotometer (Helios Alpha, Spectronic Unicam, Cambridge, UK).
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2.3. Study on the susceptibility of heat-shocked and control C. sakazakii to disinfectants In the present study, the susceptibility of the test organism to disinfectants including Clidox-S, a chlorine base disinfectant, and Quatricide, a quaternary ammonium compound (QACs) was investigated. Both disinfectants are products of Pharmacal Research Labs, Inc. (Naugatuck, CT, USA) Formulation of Clidox-S solution was performed by mixing Clidox-S, distilled water and Clidox-S activator at a ratio of 1:18:1 (v:v:v) while ca. 59.1 ml (two ounces) of Quatricide was mixed with 3785.4 ml (one gallon) of distilled water (w/v) according to the manufacture’s instructions. To examine the susceptibility of heat-shocked and control C. sakazakii to disinfectants, the procedures of Kim et al. (2007) were followed. Essentially, one ml of C. sakazakii cells was first mixed with 9.0 ml of reconstituted infant formula (Wyeth Nutritionals Inc., Singapore) to give a population of ca. 7.0 log cfu/ml. The reconstituted infant formula was prepared by dissolving 8.5 g S-26 powder infant formula in 60 ml distilled water and autoclaving at 121 for 15 min. Five milliliters of cell suspension were then mixed with 5 ml Clidox-S or 15 ml Quatricide and incubated at 25 or 40 C for 300 s. At specific time intervals during incubation, one ml of sample was withdrawn and combined with one ml of Dey-Engley (DE) neutralizing broth (Sigma) to neutralize those compounds that might be lethal to the test organism (Sutton et al., 1991). Then the viability of test organism was determined. 2.4. Enumeration of C. sakazakii To determine the viable population of C. sakazakii, samples were serially diluted in PBS containing 0.1% peptone. The viable counts were then made by spread plating 0.1 ml on Tryptic soy agar (Acumedia Manufactures). Colonies were counted after 18 h of incubation at 37 C. 2.5. The preparation of cell extract and protein determination The method described by Wouters et al. (1999) was followed to prepare the protein extract, cells of C. sakazakii were collected by centrifugation at 3000 g for 10 min and were suspended in 50 mM phosphate buffer (pH 7.0) containing 0.1 M NaCl, 0.1 mM EDTA. The cell suspension, maintained in an ice bath, was then sonicated with a sonicator (Model 3000, Misonix, Farmingdale, NY, USA) for sixteen 30-sec intervals. Cell debris was removed by centrifugation at 10,000 g for 20 min. The supernatant extracted from cells served as the cellular proteins. The content of protein in the cell lysate was measured by the colorimetric method of Bradford (1976), with bovine serum albumin (BSA) (Sigma) as the standard. SDS-PAGE was performed according to the method of Laemmli et al. (1970) using a separating gel of 12.5% acrylamide. The cell extract was mixed with an equal volume of sample buffer (250 mM Tris, 4 mM EDTA-2Na, 4% SDS, 10% b-mercaptoethanol) and then a small amount of bromophenol blue (5 ml) was added, after which it was heated in a water bath at 100 C for 5 min. After cooling to room temperature, the same quantity of extracted protein (10 mg) was loaded onto a gel and separated with a mini-vertical electrophoresis unit (Model SE260, Hoefer, San Francisco, CA, USA) at 150 V for 1.5 h. The standard marker proteins (11e98 KDa) (Bio-Rad Laboratories, Inc., Hercules, California, USA) were used to run concurrently as a size marker. The gels were stained with colloidal Coomassie blue (2 mM Coomassie Brilliant R-250, 45% methanol, 10% acetic acid for 1 h and destained with a solution containing 20% methanol and 10% acetic acid. The images of the gels were scanned and analyzed using Multi-Gauge (Fuji Photo Film Co., Tokyo, Japan).
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2.6. Analysis of fatty acids To determine the acid profile of C. sakazakii, cultures containing control or heat-shocked cells were first centrifuged (3000 g, 10 min) and twice washed with phosphate buffer solution (pH 7.0, PBS). Transesterification of fatty acids as described by Lepage and Roy (1986) was then followed to prepare the methylesters of cellular fatty acids. The prepared methylated fatty acids were dissolved in n-hexane and subjected to GC analysis. Chromatography was conducted on a 30-m fused silica column with an internal diameter of 0.32 mm. The column was wall-coated with 0.2 mm RTXÒ-2330 (Restekcorp Co., PA, USA). Analysis was performed on a Hewlett Packard Model 5890 SERIES II gas chromatograph equipped with a flame ionization detector (Hewlett Packard, Palo Alto, CA, USA). Hydrogen was used as a carrier gas and nitrogen as a make-up gas. The split ratio was 1:25 (column flow:split vent flow). The injection port temperature was 230 C and the detector temperature was 250 C. The column temperature was held at 130 C for 2 min, and then the temperature was increased to 180 C by 3 C per min and held for 1 min. After that, the temperature was increased again to 195 C by 2 C per min and applied for 0.2 min. A final increase in temperature to 205 C by 3 C per min was then held for 28 min. Commercial standards of fatty acid methylesters (Sigma) were run under identical conditions and the chromatograms were evaluated with reference to the retention time standards. 2.7. Statistical analysis Mean values and standard deviations were calculated from the data obtained from the three separate experiments. Data were analyzed using an unpaired two-tailed Student’s t-test. Statistical significance was set at p < 0.05 (SAS, 2001). 3. Results and discussion 3.1. The effect of heat shock on fatty acid composition Table 1 shows the fatty acid composition of the heat shocked and control cells of C. sakazakii. It was noted that palmitic acid (16:0) and cis-vaccenic acid (18:1) was, respectively, the major saturated and unsaturated fatty acid noted in the non heat-shocked cells of C. sakazakii. Heat shock resulted in an increase in the content of myristic acid (14:0) and palmitic acid (16:0) in the cells of C. sakazakii. On the other hand, palmitoleic acid (16:1) and cis-vaccenic acid (18:1) content reduced from 15.94% to 25.79%, respectively, to 10.01 and 13.15% (heat-shocked cells) after heat shock. Compared with non-shocked cells, the proportion of unsaturated fatty acid (USFA) Table 1 Fatty acid composition of heat-shocked and non- heat-shocked C. sakazakii cells. Fatty acida
Fatty acid proportion (%)b Non-heat-shocked
12:0 14:0 16:0 16:1 18:1 SFA USFA SFA/USFA
1.68 5.82 50.84 15.94 25.79 58.34 41.66 1.44
0.16 0.28 3.97 2.39 1.72 0.56 1.99 0.06
Ac B B A A B A B
Heat-shocked 1.54 12.52 62.78 10.01 13.15 76.84 23.16 3.31
0.14 0.98 2.21 1.22 1.25 0.67 1.23 0.07
A A A B B A B A
a 12:0, Lauric acid; 14:0 Myristic acid: 16:0, Palmitic acid; 16:1 Palmitoleic acid; 18:1 cis-vaccenic acid; SFA, saturated fatty acid; USFA, unsaturated fatty acid. b Values are proportion of total fatty acid (%). c Values in the same row with different upper case letters (A, B) are significantly different (p < 0.05).
decreased, while content of saturated fatty acids (SFA) and the proportion of SFA to USFA increased in the heat-shocked cells of C. sakazakii. A similar phenomenon has also been observed on Escherichia coli (Sinensky, 1974), V. parahaemolyticus (Chiang et al., 2005) and Pediococcus sp. (Annous et al., 1999). The relative proportions of SFA and USFA affected the fluidity and permeability of cell membranes (Sinensky, 1974; Mejia et al., 1995). Hansen (1971) and Chang et al. (2004), respectively, reported that there was a relationship between increased SFA/USFA ratios in lipids and the enhanced heat resistance of E. coli and V. parahaemolyticus 690 incubated at elevated temperatures. Previously, it was noted that the induced formation of the heat shock protein Ibp B, which both facilitates the fouling of protein and reduces the heat coagulation of protein, might lead to the increased thermal resistance (Pérez et al., 2007). The alteration of fatty acid profile observed in the present study might also contribute to the increased thermal resistance and the alteration of the cell membrane permeability of the heat-shocked cells of C. sakazakii as observed by Chang et al. (2009). 3.2. The effect of heat shock on protein profile Thermal tolerance has been identified as an important factor relevant to the pathogenicity of C sakazakii. Previously, Williams et al. (2005) reported that thermotolerant Cronobacter strains contained a protein, Mfla_1165, which is a homologue of a hypothetical protein from the thermotolerant bacterium Methylobacillus flagellatus KT.126. Besides, Gajdosova et al. (2011) showed that the gene encoding this homologue, orfI, is associated with an 18 kbp region containing 22 open reading frames which were upregulated under heat adaptation conditions. The major feature of the region is a cluster of conserved genes, most of them having significant homologies with known bacterial proteins involved in some type of stress response, including heat, oxidation and acid stress. Furthermore, heat shock response has been proposed as one of the adaptive responses with its induction of heat-shocked proteins (HpsP). Analysis with SDS-PAGE has shown an alteration in the expression of proteins due to sublethal heat shock and has been observed on various microorganisms (Sahu et al., 1994; Jeevanjyot and Ghosh, 1995; Chiang et al., 2008). The elevated levels of some proteins in microbial cells are believed to be connected with an increase in thermal as well as other forms of stress. Fig. 1 shows the SDS-PAGE pattern of cellular proteins from heat-shocked and non-shocked C. sakazakii, while the results of image analysis with Multi-Gauge are shown in Table 2. Compared with the respective protein expression in the non-shocked C. sakazakii, which was set as 1.0, eleven proteins bands including B-L in the heat-shocked cells exhibited an increased expression level with a relative protein expression value of 1.05e2.70. This implied that heat shock enhanced the expression of these proteins. Among these proteins, the increased expression of protein L with a molecular mass of 20 KDa, was the highest in the heat-shocked cells followed by the 65 KDa protein (band C) which showed an increased expression level of 1.74. A relatively lower degree of increased expression with a level of 1.04e1.08 was noted with the other five proteins bands (D, F, G, I) in the heat-shocked cells of C. sakazakii. In contrast, the expression of protein A, having a molecular mass of 80 KDa in the heat-shocked C. sakazakii showed a reduced level of 0.77 compared with that in the control cells. The alternation of protein expression observed might relate to the changes in the susceptibility of C. sakazakii to some lethal stresses as previously reported by Chang et al. (2009, 2010). It was reported that proteins separated by isoelectric focusing in a linear pH gradient in first dimension followed by SDS-PAGE in the second dimension could provide more detailed protein patterns (Chiang
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Although heat shock at 47 C for 15 min enhanced the resistance of C. sakazakii to lethal stresses such as 58 C, pH 3.3, 15% ethanol 3 and 20 C, cells of the heat-shocked C. sakazakii were found to be injured (Chang et al., 2009, 2010). The heat-shocked cells of C. sakazakii showed greater leakage of nucleic acid and protein than did the control cells. These results concerning the effect of various carbon and nitrogen sources on the growth behavior of heat-shocked and non-shocked cells of C. sakazakii warranted further investigation. As shown in Fig. 2, the growth of C. sakazakii, regardless of heat shock, varied with the nitrogen source present in the basal medium. Generally, both control and heat-shocked C. sakazakii in the media started to grow after 2 h of incubation except in the medium supplemented with malt extract as the nitrogen source. The control cells in the medium supplanted with beef extract exhibited the highest maximum population after 7 h of cultivation (Fig. 2A). On the contrary, the highest maximum population of heat-shocked C. sakazakii was noted in the medium having either tryptone or yeast extract as the nitrogen source (Fig. 1B). Yeast extract is an aqueous extract of yeast cells. While tryptone is a pancreatic digest of casein. Both, containing plenty of amino acids and peptides, are rich sources of nitrogen. Additionally, they also contain vitamins, growth factors of various microorganisms (BD BionutrientsÔ Technical Manual, 2006). These may thus led to the highest maximum growth of test organism observed (Fig. 2). 0.7
Fig. 1. SDS-PAGE (10%) pattern of cellular proteins from C. sakazakii. Lane 1, marker; lane 2, non-heat-shocked cells; lane 3, heat-shocked cells.
3.3. The effect of heat shock on the growth of C. sakazakii in presence of various nitrogen and carbon sources
Absorbance (600nm)
and Chou, 2008). Therefore, analyses of protein patterns by twodimensional electrophoresis and LC/MS/MS were performed currently in our laboratory in an attempt to further identify, characterize and elucidate the function of the proteins induced by heat shock treatment in the cells of C. sakazakii.
0.5
0.4
0.3
0.2
Nutrient availability is an important factor which affects the repair and growth of injured cells (Moss and Speck, 1966; Morichi, 1969). Busch and Donnelly (1992) reported that supplementing glucose, lactose or sucrose in TSB facilitated the repair and growth of the heat-injured cells of L. monocytogenes. Chou and Cheng (2000) observed that the growth of low temperature-injured cells of E. coli O157:H7 varied with carbon and nitrogen sources in the medium.
0.1
Table 2 One-dimensional SDS-PAGE pattern of heat-shocked and non-heat-shocked cells of C. sakazakii.
0.5
A B C D E F G H I J K L a
% of total protein
Relative protein
Non-heat-shocked
Heat-shocked
Expressiona
1.92 2.85 2.47 0.70 4.45 1.96 1.57 1.80 1.77 2.96 2.81 1.04
1.48 3.39 4.31 0.74 5.87 2.03 1.66 1.94 1.85 3.74 3.23 2.81
0.77 1.19 1.74 1.05 1.32 1.04 1.06 1.08 1.04 1.26 1.15 2.70
Relative protein expression was obtained by dividing the protein expression of heat-shocked cells with that of non-heat-shocked cells which was assigned as 1.0.
0.0 0
2
4
6
8
Time (h) 0.7
(B)
N-Z-Amine Beef extract Casamino acid Malt extract Peptone Soytone Tryptone Yeast extract
0.6
Absorbance (600nm)
Band
(A)
N-Z-Amine Beef extract Casamion acid Malt extract Peptone Soytone Tryptone Yeast extract
0.6
0.4
0.3
0.2
0.1
0.0 0
2
4
6
8
Time (h) Fig. 2. Effect of heat shock on the growth of C. sakazakii in the presence of various nitrogen sources. Basal medium containing 0.25% glucose, 0.5% NaCl and 0.25% K2HPO4 (pH 7) was added with 2% (w/v) nitrogen sources. (A) Non-heat-shocked cell; (B) heatshocked cell.
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Fig. 3 shows the growth of C. sakazakii in media containing 2% tryptone and supplemented with 0.25% of various carbon sources. All the carbon sources examined were found to support the growth of the heat-shocked and non-shocked cells of C. sakazakii. Regardless of the carbon source added or the heat shock treatment employed, C. sakazakii in the medium exhibited a lag phase of ca. 2 h. Generally, the growth patterns of heat-shocked and nonshocked cells in medium supplemented with same carbon source was similar. Among the various carbon sources examined, the growth of the test organism, regardless of heat shock, was greatest in the medium having glucose as the carbon source. In contrast, using sorbitol as the carbon source in the supplementation medium yielded the lowest level of maximum growth. This may be due to the failure of test organism to utilize sorbitol (Nazarowec-White and Farber, 1997). However, the exact reason remained to be further explored. 3.4. The effect of heat shock on the viability of C. sakazakii exposed to disinfectants To prevent the contamination and the proliferation of microorganisms, chemical disinfectants are frequently applied to food contact and non-food contact surfaces in the food industry, food
service kitchens, day-care centers and formula preparation areas in hospitals. Susceptibility to disinfectant varies with microorganisms. Moreover, factors such as concentration, exposure time, temperature, pH, equipment cleanliness and water hardness affect the efficacy of a disinfectant (Marriott and Gravani, 2006). Sub-lethal stress has been observed to alter the susceptibility of L. monocytogenes and Salmonella Typhimurium to disinfectants (Lin et al., 2011). They indicated that the changes of microbial susceptibility varied with the kinds of sublethal stress, as well as the microorganisms and disinfectants examined. Therefore, the viability of heat-shocked and non-shocked C. sakazakii exposed to Clidox-S and Quatricide was further compared in the present study. Fig. 4 shows the survival of heat-shocked and non-shocked C. sakazakii during exposure to Clidox-S at 25 and 40 C. Clidox-S is a chlorine-containing disinfectant. It has been suggested that the mode actions of the chlorine-containing disinfectants include: the inhibition of glucose oxidation, oxidative decarboxylation of amino acids, disruption of protein synthesis, reactions with nucleic acids, purines, and pyrimidines, induction of deoxyribonucleic acid lesions, creation of chromosomal alterations and formation of toxic pyrimidines, leading to the injury and death of microbial cells (Marriott and Gravani, 2006). As shown in Fig. 4A, the viability of C. sakazakii, regardless of heat shock, reduced as the period of exposure to Clidox-S at 25 C was
0.7
0.5
0.4
(A) 25 oC
102 101 Survival (%)
Absorbance (600nm)
0.6
103
(A)
Fructose Galactose Glucose Lactose Maltose Mannitol Sorbitol Sucrose
0.3
0.2
100 10-1 10-2
0.1
10-3
0.0 0
2
4
6
Non-heat-shocked cells Heat-shocked cells
10-4
8
0
30
60
120
Time (h) 0.8
0.5
10
300
(B) 40 oC
2
101 Survival (%)
Absorbance (600nm)
0.6
103
(B)
Fructose Galactose Glucose Lactose Maltose Mannitol Sorbitol Sucrose
0.7
180 Time (sec)
0.4 0.3
100 10-1 10-2
0.2 0.1
10-3
0.0
10-4 0
2
4
6
8
Time (h) Fig. 3. Effect of heat shock on the growth of C. sakazakii in the presence of various carbon sources. Basal medium containing 2% tryptone, 0.5% NaCl and 0.25% K2HPO4 (pH 7) was added with 0.25% (w/v) carbon sources. (A) Non-heat -shocked cell; (B) heat-shocked cell.
Non-heat-shocked cells Heat-shocked cells 0
30
60
120
*
*
180
300
Time (sec) Fig. 4. Viability of heat-shocked and non-shocked C. sakazakii exposed to Clidox-S at 25 C (A) and 40 C (B). * None detectable. Five milliliters of cell suspension containing test organism at a concentration of ca. 7.0 log cfu/ml were mixed with 5 ml Clidox-S or 15 ml Quatricide and incubated at 25 or 40 C.
P.-T. Li et al. / Food Microbiology 36 (2013) 142e148
extended. However, the heat shocked C. sakazakii exhibited a higher viability than did the control cells during the entire exposure period. At the end of the exposure to Clidox-S at 25 C, the viable population of control cells reduced from the initial viable population of 6.62e 1.79 log cfu/ml, exhibiting a population reduction of 4.83 log cfu/ml. While at the end of a similar exposure period, the heat shocked C. sakazakii showed a population reduction of 2.09 log cfu/ml which is significantly less (p < 0.05) than that of the control cells. This demonstrated that heat shock enhanced the survival of C. sakazakii exposed to Clidox-S at room temperature. Similar to that noted at 25 C (Fig. 4A), viability of heat shocked and control C. sakazakii reduced during exposure to Clidox-S at 40 C (Fig. 4B). However, the rate of reduction in the viability of the heat shocked or control C. sakazakii is greater at 40 C than that of the respective cells noted at 25 C. This demonstrated that Clidox-S exhibited a greater disinfectant effect on the test organism at 40 C than at 25 C. Quatricide, the sanitizer examined, is QACs with n-alkyl dimethyl benzyl ammonium chlorides and n-alkyl dimethyl ethylbenzyl ammonium chlorides as the active ingredients (Pharmacal Research Labs. Inc., 2010). Being QACs, they are capable of interacting with the cytoplasmic membrane, causing cell leakage and membrane damage, primarily due to their absorption by the bacterial membrane (Ioannou et al., 2007). 103
(A) 25 oC
147
Similar to observations in the presence of Clidox-S, the viability of heat-shocked and non-shocked C. sakazakii exposed to Quatricide reduced steadily during the entire exposure period (Fig. 5). Having an initial population of ca. 106 cfu/ml, the viable population of the heat-shocked and control C. sakazakii reduced to 3.63 and 2.15 log cfu/ml after 300 s of exposure to Quatricide at room temperature, the control C. sakazakii showed a population reduction of 4.28 log cfu/ml compared to a significantly lower (p < 0.05) population reduction of 2.87 log cfu/ml observed in the heat-shocked cells (Fig. 5A). As the exposure temperature was raised to 40 C, a marked reduction in the viable population of C. sakazakii, regardless of heat shock, was observed throughout the entire exposure period (Fig. 5B). For example, control C. sakazakii showed a viable population of 2.15 log cfu/ml with a population reduction of 4.28 log cfu/ml at the end of exposure to Quatricide at 25 C (Fig. 5A). In comparison, the viable population of the control cells reduced to a non-detectable level with a population reduction of >6.00 log cfu/ml after a similar period of exposure to Quatricide at 40 C (Fig. 5B). Additionally, the viability of the heat-shocked C. sakazakii was also noted to exhibit a relatively lower reduction rate than the control cells. The viable population of heat-shocked C. sakazakii detected at all the specific exposure intervals, except at 0 time, was significantly higher (p < 0.05) than that of the control cells (Fig. 5B). These results, in addition to showing the greater disinfectant effect of Quatricide at 40 C than at 25 C, demonstrated that heat shock also reduced the susceptibility of C. sakazakii to Quatricide.
102
4. Conclusion
Survival (%)
101
Data obtained from the present study revealed that heat shock altered the protein and fatty acid profiles of C. sakazakii. This might relate to the altered response of the test organism when exposed to subsequent stress conditions as observed previously (Chang et al., 2009, 2010). Furthermore, heat shock enhanced the resistance of C. sakazakii to the disinfectants examined. These observations stress the importance of taking into account heat shock response in developing improved safety measures. With improved measures in place, disinfectants can be used more effectively against C. sakazakii and the food system can be made safer.
100 10-1 10-2 10-3 Non-heat-shocked cells Heat-shocked cells
10-4 10-5 0
30
60
120
180
300
Acknowledgment
Time (sec)
Survival (%)
103 102
This research was financially supported by the National Science Council, ROC (Taiwan). (NSC 98-2313-B-002-037-MY3).
101
References
10
(B) 40 oC
0
10-1 10-2 10-3 Non-heat-shocked cells Heat-shocked cells
10-4 10-5 0
30
60
120
*
*
180
300
Time (sec) Fig. 5. Viability of heat-shocked and non-shocked C. sakazakii exposed to Quatricide at 25 C (A) and 40 C (B). * None detectable. Five milliliters of cell suspension containing test organism at a concentration of ca. 7.0 log cfu/ml were mixed with 15 ml Quatricide and incubated at 25 or 40 C.
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