Elucidation of antibacterial mechanism of 405±5 nm light-emitting diode against Salmonella spp. at

40 Pages·2016·1.16 MB·English

AEM Accepted Manuscript Posted Online 21 December 2016
Appl. Environ. Microbiol. doi:10.1128/AEM.02582-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1 Elucidation of antibacterial mechanism of 405±5 nm light-emitting diode
2 against Salmonella spp. at refrigeration temperature
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4 Running title: Antibacterial mechanism of LED illumination
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6 Min-Jeong Kim1, Hyun-Gyun Yuk1* o
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8 1Food Science and Technology Programme, Department of Chemistry, National University of e
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9 Singapore, Science Drive 4, Singapore 117543 ro
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22 *Corresponding author: Tel: [phone]; Fax: [phone]; E-mail:
23 [email protected] (H.G. Yuk)
24
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25 ABSTRACT
26 The aim of this study was to elucidate the antibacterial mechanism of 405±5 nm light-
27 emitting diode (LED) illumination against Salmonella at 4 °C in phosphate-buffered saline
28 (PBS) by determining endogenous coproporphyrin content, DNA oxidation, damage to
29 membrane function, and morphological change. Gene expression levels, including oxyR, recA,
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30 rpoS, sodA, and soxR, were also examined to understand the response of Salmonella to LED
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31 illumination. Results showed that Salmonella spp. responded differently to LED illumination, n
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32 revealing that S. Enteritidis (ATCC 13076) and S. Saintpaul (ATCC 9712) were more d
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33 susceptible and resistant, respectively, than 16 other test strains. There was no difference in f
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34 the amount of endogenous coproporphyrin in the two strains. Compared to non-illuminated
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35 cells, DNA oxidation levels in illuminated cells increased. A loss of efflux pump activity, p
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36 damage to the glucose uptake system, and changes in membrane potential and integrity were e
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37 observed in illuminated cells. Transmission electron microscopy revealed disorganization of a
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38 chromosomes and ribosomes due to LED illumination. All five genes in both non-illuminated .o
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39 and illuminated S. Saintpaul cells were upregulated in PBS at a set temperature of 4 °C, /
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40 indicating that increased gene expression levels might be due to temperature-shift and A
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41 nutrient deficiency rather than LED illumination. In contrast, only oxyR in S. Enteritidis cells il
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42 was upregulated. Thus, different sensitivities of the two strains to LED illumination are 20
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43 attributed to differences in gene regulation.
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44 g
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45 IMPORTANCE st
46 Bacterial inactivation using visible light has recently received attention as a safe and
47 environmentally friendly technology over the ultraviolet light which has detrimental effect on
48 human health and environment. The present study was designed to understand how 405±5 nm
49 LED illumination kills Salmonella spp. at refrigeration temperature. Data clearly demonstrate
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50 that the effectiveness of LED illumination on Salmonella spp. was highly dependent on
51 serotype and strain. Our finding also reveals that its antibacterial mechanism is mainly
52 attributed to DNA oxidation and loss of membrane functions rather than membrane lipid
53 peroxidation which has been proposed by other researchers who studied the antibacterial
54 effect of LED illumination by addition of exogenous photosensitizers such as chlorophyllin
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55 and hypericin. Therefore, the present study suggests that the detailed antibacterial
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56 mechanisms of 405 nm LED illumination without additional photosensitizers may be n
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57 different from that by exogenous photosensitizers. Furthermore, change in stress-related gene d
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58 regulation may make a difference in the susceptibility of Salmonella cells to LED f
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59 illumination at refrigeration temperature. Thus, our study provides new insights into the
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60 antibacterial mechanism of 405±5 nm LED illumination on Salmonella cells. p
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62 Keywords: 405 nm light-emitting diode, antibacterial mechanism, Salmonella, DNA a
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63 oxidation, membrane functions, gene expression .o
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64 INTRODUCTION
65 Salmonella is a major causative agent of foodborne illness worldwide, resulting in
66 approximately 80.3 million cases of infection and 155,000 deaths each year (1). According to
67 the Foodborne Disease Active Surveillance Network (FoodNet) in the United States (US),
68 among a total of 19,542 cases of infections, 7,452 infections, 2,141 hospitalizations, and 30
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69 deaths were caused by Salmonella in 2014 (2). In Singapore, Salmonella also has been
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70 identified as the top contributor to foodborne illness, accounting for more than half of the n
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71 reported cases in 2001–2010 (3). To minimize the risk of salmonellosis, storing perishable d
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72 and ready-to-eat foods in the refrigerator is a common practice. However, Salmonella cells, f
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73 having a cold shock protein (CspH), are able to survive at temperatures less than 5 °C (4).
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74 Moreover, refrigerators in retail shops are often held at temperatures above 5 °C (5), enabling p
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75 potential growth of Salmonella and thus increasing risk of salmonellosis. For these reasons, a e
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76 secondary hurdle in combination with low temperature control should be employed to more a
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77 effectively control Salmonella in foods during storage. .o
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78 A light-emitting diode (LED) with visible wavelengths has recently received increased /
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79 attention as an emerging food preservation technology due to its antibacterial effect described A
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80 as photodynamic inactivation (PDI). Previous studies have shown that blue LEDs have il
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81 antibacterial efficacy against pathogenic bacteria in buffered solutions and food matrices, 20
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82 with or without the addition of exogenous photosensitizers such as chlorophyll and porphyrin
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83 (6–8). Our previous studies have demonstrated the antibacterial effects of 405 and 460 nm g
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84 LEDs against Bacillus cereus, E. coli O157:H7, Listeria monocytogenes, S. Typhimurium, st
85 Shigella sonnei, and Staphylococcus aureus in buffered solution and bacterial growth media
86 under chilling conditions without the aid of a photosensitizer (9–12).
87 The proposed mechanism of PDI requires three components: visible light in the
88 wavelength range of 400–430 nm, photosensitizers, and oxygen (6). Once bacterial cells are
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89 exposed to light, the photosensitizer, such as intracellular porphyrin compounds, absorbs light
90 energy and then is excited, resulting in the production of reactive oxygen species (ROS) by
91 transferring energy to oxygen. ROS, such as hydrogen peroxide, superoxide, and singlet
92 oxygen, can result in cytotoxicity by reacting with DNA, RNA, proteins, and lipids,
93 eventually causing death of bacteria (6, 11). However, the detailed mechanism of
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94 antimicrobial activity of 405±5 nm LED illumination in the absence of an exogenous
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95 photosensitizer has not been described. We hypothesized that the mode of bacterial n
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96 inactivation of LED illumination without additional photosensitizers may be different from d
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97 that by exogenous photosensitizers since genomic DNA and membrane proteins of f
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98 Salmonella cells may be major targets of ROS generated by 405±5 nm LED illumination due
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99 to the cellular localization of an intracellular endogenous photosensitizer. p
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100 It is crucial to understand in the response of Salmonella to oxidative stress by ROS e
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101 generated from LED illumination in order to elucidate its antibacterial mechanism. It is a
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102 known that resistance of Salmonella to oxidative stress is enhanced by the induction of .o
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103 regulators such as OxyR, SodA, SoxRS, and RpoS (13). The oxyR gene of the OxyR /
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104 regulator is activated via the formation of disulfide bonds under hydrogen peroxide stress A
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105 (13). The oxidized OxyR also activates the expression of katG (encoding catalase il
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106 hydroperoxidase I), dps (encoding non-specific DNA-binding proteins), gorA (encoding 20
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107 glutathione reductase), and oxyS (encoding small regulatory RNA) involved in the bacterial
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108 resistance to oxidative stress (13–15). Bacteria possess at least two superoxide dismutases g
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109 (SODs), Mn–SOD encoded by sodA and Fe–SOD encoded by sodB, which catalyzes the st
110 conversion of superoxide anions into hydrogen peroxide and oxygen. SoxRS regulator,
111 encoded by two regulatory genes, soxR and soxS, regulates SODs under high levels of
112 superoxide stress (13–15). The rpoS-encoded σS subunit of RNA polymerase is another
113 regulator that survives under oxidative stress as well as general stress, including low
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114 temperature and starvation (16, 17). It is known that UV light and hydrogen peroxide (low
115 and high concentrations) are able to induce recA as a global regulator of SOS response (13);
116 recA expression is associated with DNA topological change, DNA gyrase, and single-
117 stranded DNA (13, 18).
118 Although the molecular response of Salmonella to general oxidative stress has been
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119 well documented (13), its response to LED illumination at a molecular level is unknown.
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120 Furthermore, studies to determine if differences in gene regulation influence bacterial n
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121 susceptibility to LED illumination have not been reported. Thus, the objective of this study d
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122 was to elucidate the detailed antibacterial mechanism of 405±5 nm LED illumination on f
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123 Salmonella by determining changes in endogenous coproporphyrin content, DNA oxidation,
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124 damage to membrane function, morphological change, and regulatory gene expression level p
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125 during LED illumination at refrigeration temperature. e
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126 a
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127 MATERIALS AND METHODS .o
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128 Bacterial culture conditions. Eighteen Salmonella enterica strains were used in this /
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129 study; S. Agona BAA-707 (alfalfa sprouts), S. Enteritidis ATCC 13076 (CDC), S. Gaminara A
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130 BAA-711 (orange juice), S. Heidelberg ATCC 8326, S. Montevideo BAA-710 (clinical isolate il
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131 from tomato outbreak), S. Newport ATCC 6962 (food poisoning fatality, England), S. Poona 20
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132 BAA-1673 (iguana), S. Saintpaul ATCC 9712 (cystitis, Panama), S. Tennessee ATCC 10722,
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133 S. Typhimurium ATCC 14028 (poultry), S. Typhimurium ATCC 13311 (human feces), S. g
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134 Typhimurium ATCC 25241 (methionine auxotroph derived from strain LT-2), S. st
135 Typhimurium ATCC 29269, and S. Typhimurium ATCC 51812 (human blood) were
136 purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA); S.
137 Enteritidis 109 (phage type Group D1; Peter Holt), S. Enteritidis 124 (phage type 8; Maryland
138 Department of Health and Mental Hygiene, MD, USA), S. Enteritidis 125 (phage type 13A;
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139 U.S. Department of Agriculture, WA, USA), and S. Enteritidis 130 (phage type 2; CDC, NY,
140 USA) were obtained from Dr. Kun-Ho Seo at Konkuk University, Republic of Korea. All
141 Salmonella strains were stored at –70 °C into cryoinstant vials with porous beads (DeltaLab,
142 Barcelona, Spain). Cells were activated in 10 mL of sterile tryptone soy broth (TSB; Oxoid,
143 Basingstoke, UK) for 18–24 h at 37 °C. After two consecutive transfers at 18–24h intervals at
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144 37 °C, 1 mL of stationary phase culture was centrifuged at 6,000 × g for 10 min at 4 °C and
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145 washed twice with 1 mL of sterilized phosphate-buffered saline (PBS; Vivantis Technologies n
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146 Sdn Bhd, Malaysia). Cells in the resultant pellet were resuspended in 1 mL of PBS and d
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147 serially diluted to initial populations of approximately 106, 108, or 109 CFU/mL for LED f
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148 illumination.
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149 Light-emitting diode (LED) illumination system. High intensity 405±5 nm LEDs (8 p
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150 × 8 mm) were purchased from Shenzhen Getian Opto-Electronics Co., Ltd. (Shenzhen, e
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151 Guangdong, China). The LED illumination system is described elsewhere (9, 10). Briefly, a
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152 LEDs were attached to a heat sink and a fan to minimize heat transfer to the cell suspension. .o
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153 To prevent penetration of external light during illumination, an acrylonitrile butadiene styrene /
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154 (ABS) housing was constructed for each LED system. The suspension (10 mL and 1 cm A
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155 depth) in a sterile Petri dish (60 mm diameter) or streaked on tryptone soy agar (1 cm depth, il
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156 TSA, Oxoid) plates (90 mm diameter) was placed directly below the LED bulbs at a distance 20
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157 of 4.5 cm to illuminate the entire surface of the inoculum (Fig. 1). The surface temperatures
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158 of TSA and PBS were monitored with a Fluke 5.4 thermocouple thermometer (Everett, WA, g
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159 USA) during exposure to LED illumination at doses up to 576 J/cm2. The irradiance (W/cm2) st
160 of a 405±5 nm LED unit was measured using a Compact Power and Energy Meter Console
161 (PM100D; Thorlabs GmbH, Dachau, Germany), with 20±2 mW/cm2 at the surface of the cell
162 suspension. The dosage applied to the sample was calculated using the following equation (8):
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164 E = Pt
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166 where E = dose in J/cm2, P = irradiance in W/cm2, and t = time in second.
167 Sensitivity of Salmonella to LED illumination. To screen for sensitivity and
168 resistance of Salmonella to LED illumination, a 10-µL bacterial suspension of each of the 18
169 Salmonella strains (ca. 106 CFU/mL) was streaked in individual lines ca. 3 cm length using D
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170 an inoculating loop onto the surface of TSA plates. The inoculated plates were placed in the n
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171 LED system as previously described and exposed to 288 J/cm2 at a set temperature of 4 °C in d
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172 a temperature controlled incubator (MIR-154, Panasonic Healthcare Co., Ltd., Osaka, Japan). f
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173 Non-illuminated control plates were placed in a temperature-controlled incubator in the dark.
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174 After illumination, plates were incubated at 37 °C for 24 h and then visualized using a G:Box p
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175 EF2 Imaging System (Syngene, Frederick, MD, USA). e
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176 LED illumination of the Salmonella suspension and enumeration. Based on results a
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177 from the above experiments, 10-mL suspensions of S. Enteritidis ATCC 13076 and S. .o
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178 Saintpaul at an initial population of ca. 106 CFU/mL were deposited in sterile Petri dishes and /
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179 placed in the LED illumination system, followed by exposure to LED illumination at doses A
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180 up to 576 J/cm2 at a set temperature of 4 °C. The same volume of cell suspension in a sterile il
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181 Petri dish placed in an incubator without LED illumination (dark condition) served as non- 20
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182 illuminated control. A 0.5-mL aliquot was taken at selected treatment times and analyzed for
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183 number of surviving Salmonella. Aliquots from non-illuminated control and illuminated cell g
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184 suspensions were serially diluted with 0.1% sterile peptone water (PW; Oxoid) and pour- st
185 plated in TSA. After incubation at 37 °C for 24–48 h, the number of viable cells was
186 enumerated using a colony counter (Rocker Scientific Co. Ltd., Taipei, Taiwan) and
187 expressed as log CFU/mL. Bacterial survival curves were constructed by plotting log number
188 of survivors against dose. The line of best fit for the survival curve was determined by linear
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189 regression with Microsoft Excel (Microsoft Corp. Redmond, WA, USA). The D-values (J/cm2)
190 for the first-order inactivation kinetics were calculated as the negative reciprocal of the slope.
191 Determination of intracellular coproporphyrin content. Cultures (100 mL) of S.
192 Enteritidis ATCC 13076 and S. Saintpaul in stationary growth phase in TSB were centrifuged
193 at 6,000 × g for 10 min at 4 °C, washed thrice with 20 mL of PBS, and dried at 45 °C for 2 h.
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194 Intracellular coproporphyrin was extracted from the dried pellets with a 0.1 M
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195 NH4OH:acetone solution (1:9, v/v). The extracts were quantified using a C-18 modified silica nlo
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196 column, a reversed phase system and a Waters 2695 HPLC system equipped with a Multi λ d
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197 fluorescent detector at 407 nm excitation and 612 nm emission. Elution was carried out with f
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198 a gradient separation consisting of water (Solvent A) and acetonitrile (Solvent B). A standard
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199 curve was prepared in a concentration range of 0–10 μg/mL of coproporphyrin I p
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200 dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA). The amount of coproporphyrin is e
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201 expressed in ng coproporphyrin/mg dried cells. a
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202 Determination of cellular DNA oxidation. The degree of cellular DNA oxidation .o
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203 caused by LED illumination was measured according to Kim, Bang, and Yuk (19). Briefly, /
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204 suspensions (10 mL) of S. Enteritidis ATCC 13076 and S. Saintpaul (ca. 108 CFU/mL) were A
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205 illuminated (576 J/cm2) at a set temperature of 4 °C. Genomic DNA of LED-illuminated cells il
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206 was extracted using a GeneJET Genomic DNA Purification Kit (Thermo Scientific, Waltham, 20
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207 MA, USA) according to manufacturer’s instructions. The concentration and purity of
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208 extracted genomic DNA dissolved in a 100 μL of elution buffer were measured with a g
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209 BioDrop Touch Duo Spectrophotometer (BioDrop, Cambridge, UK). Genomic DNAs of st
210 freshly cultured cells and non-illuminated cells held at 9.5 °C without LED illumination were
211 extracted by the same method.
212 For the DNA oxidation assay, an OxiSelect™ Oxidative DNA Damage ELISA kit (Cell
213 Biolabs, San Diego, CA, USA) was used. The DNA extract (1.3–3.1 μg) was converted to
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214 single-strand DNA at 95 °C for 5 min, immediately chilled in an ice bath, and incubated with
215 6 units of nuclease PI (Wako, Osaka, Japan) in 20 mM sodium acetate buffer (Sigma-Aldrich)
216 at 37 °C for 2 h. Two units of Escherichia coli alkaline phosphatase (Takara Bio Inc., Shiga,
217 Japan) were added to the mixture containing a 10% (v/v) 10 × alkaline phosphatase buffer
218 (Takara Bio Inc.) and incubated at 37 °C for 1 h. The mixtures were centrifuged at 6,000 × g
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219 for 5 min at 4 °C and 8-hydroxydeoxyguanosine (8-OHdG) in the supernatant was quantified
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220 according to manufacturer’s instructions. Measurement was made at 450 nm with a Synergy n
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221 HT multi-detection Microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The d
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222 amount of 8-OHdG was calculated with a standard curve (0–10 ng/mL) and is reported as ng f
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223 8-OHdG per mg of DNA.
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224 Determination of damage to cell membrane function. To investigate damage to cell p
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225 membrane function by LED illumination, five fluorescent probes were used: SYTO®9 and e
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226 propidium iodide (PI) from LIVE/DAED® BacLight Kit (Molecular Probes™, ThermoFisher a
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227 Scientific, Eugene, OR, USA), ethidium bromide (EtBr; Sigma-Aldrich), bis-(1,3- .o
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228 dibutylbarbituric acid) trimethine oxonol [DiBAC4(3); Molecular Probes™], and 2-[N-(7- o/
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229 nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG; Molecular Probes™). A
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230 Ten-mL suspensions (ca. 106 CFU/mL) of S. Enteritidis ATCC 13076 and S. Saintpaul were il
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231 exposed to 576 J/cm2 at a set temperature of 4 °C; 0.5-mL or 1-mL aliquots were withdrawn 20
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232 at 288 and 578 J/cm2 of illumination, respectively. Aliquots (200 μL) were immediately 9
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233 stained with two mixed probes (SYTO®9/PI or SYTO®9/EtBr) and three single probes g
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234 (SYTO®9, DiBAC4(3), and 2-NBDG). Before staining cells, 2.0 mM of 2,4-dinitrophenol st
235 (2,4-DNP; Sigma-Aldrich) was added to 2-NBDG. The working concentrations of SYTO®9,
236 PI, EtBr, DiBAC4(3), and 2-NBDG were 5, 30, 30, 10, and 5 μM, respectively. The mixtures
237 were incubated in the dark for 10 min at 37 °C for 2-NBDG and for 15 min at room
238 temperature for SYTO®9, PI, EtBr, and DiBAC4(3). Measurements were made using a BD
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