Elucidating Antibacterial Activity and Mechanism of Daphnetin against Pseudomonas fluorescens and Shewanella putrefaciens

In this research, the antibacterial activity and mechanism of daphnetin against Pseudomonas ﬂuorescens and Shewanella putrefaciens were evaluated. The minimum inhibitory concentration (MIC) of daphnetin on P. ﬂuorescens and S. putrefaciens was 0.16 and 0.08mg · mL − 1 , respectively. The growth curve test also showed that daphnetin had a good antibacterial eﬀect. The results of intracellular component leakage and cell viability analysis illustrated that daphnetin destroyed the morphology of the cell membrane. According to scanning electron microscope and transmission electron microscope observations, the treated bacterial cells displayed obvious morphological and ultrastructural changes in the cell membrane of the two tested strains, whichconﬁrmed daphnetin’s damage to the integrity of the cell membrane. The ﬁndings indicated that daphnetin mainly exerted its antibacterial eﬀect by destroying the membrane and suggested that it had good potential to be as a natural food preservative.


Introduction
Fish is a nutritious food source and is recommended because of its multiple nutritional values [1]. It is low in saturated fats and rich in poly-unsaturated fatty acids (PUFA) and protein.
It also contains many other healthy nutrients, such as selenium, iodine, and vitamin D [2]. e World Health Organization (WHO) suggests eating 1-2 servings of fish regularly each week to provide an equivalent of 200-500 mg of omega-3-PUFA [3].
However, the quality of fresh fish can easily deteriorate after being slaughtered. Most fish are degraded by digestive enzymes and lipases, microbial spoilage, and oxidation of surface bacteria [4]. P. fluorescens and S. putrefaciens are Gram-negative bacteria and considered to be specific spoilage organisms (SSOs) that are common during refrigeration of fish and fish products [5][6][7]. Furthermore, changes in the composition of fish during decay not only lead to lipid oxidation and protein degradation but also the loss of other valuable molecules. What's more, high-quality and less processed products are increasingly demanded by consumers. e soft or mushy texture of fish will affect the shelf life and thus hinder the sale of the product. erefore, fish are traditionally cooled and stored in flake ice, frozen seawater, or ice slurry, and some are even preserved by exposure to chemical agents [8,9].
In recent years, researchers have made considerable efforts to find natural preservatives in order to slow or inhibit bacteria and fungi growth in fish. Meanwhile, an increasing number of consumers are conscious of the potential negative health effects of synthetic preservatives and the benefits of natural additives, which prompt food industry to seek natural products as alternatives. Many preservatives derived from plants have been shown to have antioxidant or antibacterial properties. Daphnetin (7,8-dihydroxycoumarin) is a dihydroxylated derivative of coumarin derived from plants [10] (structure is shown in Figure 1). It is an active element of plant extracted from Daphne Korean Nakai [11], which has been reported to possess antimicrobial, antioxidant, antimalarial, anticoagulation, and immunomodulating activity [12][13][14][15]. However, the potential effects of daphnetin on food-borne spoilage bacteria were rarely reported. erefore, P. fluorescens and S. putrefaciens, as the SSOs in fish and fish products, were selected as targeted bacteria to evaluate the antibacterial activity and effect mechanism of daphnetin on vegetative cells to provide a basis for daphnetin to replace chemical preservatives in the food industry.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
Broth microdilution method was used to detect the antimicrobial effect of daphnetin on P. fluorescens and S. putrefaciens. e test bacterial suspension (10 5 CFU·mL −1 ) was mixed with daphnetin (0.01-1.28 mg·mL −1 ) and cultured with shaking at 30°C for 24 h. e minimum concentration of daphnetin added which inhibits the growth of visible bacteria is defined as MIC. In addition, the bacterial suspensions of the test groups without visible bacteria growth were subcultured on nutrient agar to determine that the minimum concentration of daphnetin was defined as MBC, which resulted in no colony growth at all [16].

Determination of Growth Curve.
e growth curves in medium at 30°C were determined as previously described [17]. Bacteria were grown to an OD 595 value of 0.1 in medium, and then, 150 L of the culture was transferred into each well of a 100-well microtiter plate (Bioscreen, Finland). e bacterial suspensions were mixed with 1 ×, 2 ×, and 4 × MIC daphnetin, respectively. 0 × MIC was used as a control. Bacteria were further cultured at 30°C, and cell growth was monitored at 600 nm using an automatic microbial growth curve analyzer (Bioscreen C MBR, Bioscreen, Finland). e growth curve was drawn with time as the abscissa and OD 600 as the ordinate.

Integrity of P. fluorescens and S. putrefaciens Cells and
Spores.
e integrity of cell membranes can be assessed by detecting the leakage of nucleic acids and proteins in the cells [18]. e steps were as follows: the bacterial suspensions were treated by daphnetin at the concentration of 0 ×, 1 ×, 2 ×, and 4 × MIC daphnetin at 30°C for 4 h, respectively. After treatment, the bacterial solution was centrifuged (3000 r/min, 4°C, 10 min) to obtain the supernatant and then filtered through a 0.22 μm filter. e amount of nucleic acids and proteins leaking from the cytoplasm was determined by measuring the absorbance readings of the filtrate at 260 nm and 595 nm with a UV spectrophotometer (UV-2100, UNICO Instrument Co., Ltd, Shanghai, China). e control group was a 0 × MIC sample.

Determination of Protein Concentration.
e concentration of intracellular protein was obtained by the bicinchoninic acid (BCA) protein assay kit (Jiancheng Bioengineering Institute, Jiangsu, China) absorbance at 562 nm with a UV spectrophotometer [19].

Leakage of Intracellular
Constituents. Different concentrations of daphnetin were added to the bacterial culture so that the final concentrations were 1 ×, 2 ×, and 4 × MIC, respectively. A control group (0×MIC) was established at the same time.
e ATPase activity was determined by the ATPase assay kit (Jiancheng Bioengineering Institute, Jiangsu, China) after 4 h, and the absorbance at 636 nm was recorded using Microplate Reader (iMark, BIO-RAD, US) [20].

Integrity of Cell Wall Assays.
e prepared bacteria were treated with different concentrations of daphnetin for 4 h at 30°C. en, the AKP assay kit (Jiancheng Bioengineering Institute, Jiangsu, China) was used to measure the activity of alkaline phosphatase (AKP). e absorption value of the supernatant was determined by Microplate Reader at 490 nm [21].

Scanning Electron Microscope (SEM) Analysis.
Daphnetin with different concentrations was added to the initial suspension. en, it was cultured with shaking at 30°C for 6 h, and then, the bacteria cells were obtained by centrifugation. e samples were fixed with 2.5% glutaraldehyde solution for 4 h and washed with 0.01 mol/L PBS solution. en, they were eluted once with 30-90% ethanol solution and eluted twice with 100% ethanol solution. e samples were air-dried on the coverslip, sprayed with gold, and observed by using scanning electron microscope (Tescan

Transmission Electron Microscopy (TEM) Analysis.
e control and treated samples were collected and fixed with 2.5% (m/v) glutaraldehyde, washed with 0.01 mol/L phosphate buffer, postfixed in 1% osmium tetroxide, washed with the buffer, continuously dehydrated with ethanol (30,50,70,80,90,95, and 100% (v/v)), and then treated with pure acetone for 20 min. e samples were embedded with epoxy resin (SPURR) for polymerization and then sliced into thin slices with an ultramicrotome (EMUC7, Leica Microsystems GMBH, Germany). Double staining was done with uranyl acetate and lead citrate. TEM images were obtained using a JEM-2100 TEM (JEOL Ltd., Japan) [23].

FTIR Spectroscopic Analysis.
e prepared bacterial suspension was added into the daphnetin solution with a concentration of 0 ×, 1 ×, 2 ×, and 4 × MIC daphnetin, respectively. Subsequently, the culture was shaken at 30°C for 4 h, and the bacteria were then collected by centrifugation at 3000 rpm for 10 min. e obtained bacterial samples were washed three times with PBS, freeze-dried for 48 h, and then tested with a Fourier infrared spectrometer (Nicolet is 5, ermo Fisher, USA) [24].

Statistical Analysis.
e experimental results were reported as means ± SD and statistically analyzed by SPSS 22.0 software. e one-way ANOVA procedure and Duncan's multiple range tests were used to determine significant differences (p < 0.05) between mean values of treatments.

Antibacterial Activity of Daphnetin.
e antibacterial activity of daphnetin was evaluated by MIC and MBC values. e MIC of daphnetin against P. fluorescens and S. putrefaciens cells was 0.16 and 0.08 mg·mL −1 , respectively. e MBC of daphnetin against the two tested strains was 0.16 mg·mL −1 . e results of Yang et al. [25] showed that the MIC and MBC of daphnetin anti-R. solanacearum were both 0.064 mg·mL −1 , which were slightly lower than those tested in the present study. e results indicated that daphnetin had a good antibacterial effect on P. fluorescens and S. putrefaciens. e growth of the two test bacteria in the presence of different concentrations of daphnetin was plotted to further analyse the antibacterial activity of daphnetin. As shown in Figure 2(a), the growth of bacteria in the control group was consistent with the S-shaped growth curve. P. fluorescens grew rapidly in the absence of daphnetin and began to enter the exponential phage after 6 h and then entered the stationary phase after 26 h. However, the groups treated with daphnetin at 1 ×, 2 ×, and 4 × MIC concentrations completely inhibited the growth of bacteria. S. putrefaciens (Figure 2(b)) without daphnetin addition began to be at an exponential phase after 14 h and then stationary phase after 28 h. e growth rate of S. putrefaciens with the addition of 1 × MIC daphnetin was slower than that of the control. e inhibitory effect gradually weakened after 30 h, and the bacterial growth began to enter the logarithmic phase. However, when 2 × MIC and 4 × MIC concentrations of daphnetin were added, the bacterial growth could be completely inhibited.

3.2.
Effect on the Integrity of Cell Wall. By separating intracellular enzymes and macromolecular substances, the cell wall plays an important role in maintaining normal growth [26]. AKP is an intracellular enzyme located between the cell wall and membrane, so its activity cannot be detected in the extracellular environment under normal conditions [27]. However, if the cell wall is damaged, it will cause AKP to leak into the extracellular environment, thereby increasing the enzyme activity in the environment. erefore, the integrity of bacterial cell wall can be evaluated by AKP activity in cell suspension [28]. As presented in Figure 3(a), compared with the control group, the release of AKP was significantly increased in the two bacterial samples treated with daphnetin for 4 h. e AKP activity in the supernatant increased with the increase in daphnetin concentration. After treating with daphnetin, the OD 520 value increased from 1.49 to 3.68 in P. fluorescens and from 1.47 to 3.02 in S. putrefaciens samples, which suggested that damage to the integrity of the cell wall is probably the primary cause of AKP release into the supernatant. Zheng [29] claimed chlorogenic acid could cause the Escherichia coli cell wall to be destroyed in a short time and had obvious destructive effects. Cao et al. [30] showed that the OD 520 value of AKP in Staphylococcus aureus cell suspension increased significantly treated with lactobionic acid for 5 h compared with the control. Similarly, the results of He [31] and Ma et al. [32] can also explain this phenomenon. ese proved that daphnetin may first act on the cell wall, causing AKP to leak from the cell and cell wall integrity loss by inducing cell wall damage [33].

Effect on the Integrity of Cell Membrane.
Cell membrane can not only maintain the cell environment stable metabolism but also regulate and select substances that enter and leave the cells [26]. Information about the release of cellular components reveals the cytomembrane integrity [34]. When the bacterial membrane is damaged, small ions flow out first, followed by large molecules such as nucleic acids and proteins [35]. erefore, the leakage of nucleic acids and proteins can reflect the integrity of the membrane. Nucleic acids are one of the most basic substances that allow bacteria to maintain normal life activities. As the concentration of daphnetin increased, the leakage of nucleic acids in bacteria also increased (Figure 3(b)). After 4 h of treatment with different concentrations of daphnetin (0 ×, 1 ×, 2 ×, and 4 × MIC, respectively), the values at 260 nm were 0.160, 0.223, 0.285, and 0.318 for P. fluorescens and 0.247, 0.254, 0.282, and 0.343 for S. putrefaciens, respectively. Cui et al. [36] proved that the structure of bacteria was destroyed with the action of clove oil, which resulted in the changes in cell membrane permeability and release of nucleic acids. Zhang et al. [37] demonstrated that the cell membrane integrity of bacteria had been impaired after exposure to cinnamon essential oil. Based on these results, daphnetin could damage P. fluorescens and S. putrefaciens cell membranes and causes nucleic acid leak. Another possible cause is that daphnetin could prevent the synthesis of nucleic acids, and thus, the nucleic acid contents of the treated samples were significantly lower than that of the control [38].
Protein is essential for the life activities of bacteria, it is the material basis and main carrier of life activity, and it is closely related to many life forms. Figure 3(c) shows the protein leakage in the cells or spores of P. fluorescens and S. putrefaciens treated with different concentrations of daphnetin (0 ×, 1 ×, 2 ×, and 4 × MIC, respectively) for 4 h. e addition of daphnetin significantly increased the leakage. Compared with the control, the OD 595 of P. fluorescens and S. putrefaciens treated with 4 × MIC daphnetin for 4 h was increased by 85.55% and 93.03%, respectively. Similarly, Meng et al. [39] proved that the essential oil from the leaves of Juniperus rigida destroyed the integrity of Klebsiella pneumoniae membrane, thereby releasing intracellular components such as nucleic acids and proteins. ese results indicated that daphnetin could damage the cells and spores of P. fluorescens and S. putrefaciens, resulting in leakage of proteins.

Intracellular Protein Leakage.
e bacterial membrane is damaged and treated with the antimicrobial agents, and the protein, small ions, and nucleic acids in the cell will be lost [40]. erefore, an important indicator of membrane damage is the release of proteins. e change in intracellular protein contents of P. fluorescens and S. putrefaciens is shown in Figure 3 [41] showed that the soluble protein content of the treated samples was lower than that of the control, indicating that these proteins were lost through the damaged cell membrane. Wang et al. [42] found that lactic acid may cause a large amount of protein leakage from Salmonella, E. coli, and Listeria cells. Fei et al. [43] noticed that the antibacterial effect of olive oil polyphenol extract on Cronobacter sakazakii was related to the release of protein. Moreover, He et al. [44] observed that when flavonoids were added to the E. coli strain, bacterial proteins leaked into extracellular suspensions during the detection period. ese proved that daphnetin can reduce the cellular protein content by destroying the bacterial membrane and inhibiting its synthesis [45].

Determination of ATPase Activity.
Enzyme, as a biocatalyst, participates in various chemical reactions of biological metabolism and is affected by various factors so that organisms can adapt to changes in external conditions to keep normal life activities [46]. ATP is a glycolysis product in bacterial cell walls and cell membranes. ATPase is one of the main enzymes that promotes the formation and metabolism of ATP [19]. As shown in Figure 3(e), the activity of ATPase decreased when compared with the control group. After 4 h of exposure to daphnetin, the ATPase content of P. fluorescens and S. putrefaciens was reduced to 2.107 U·mL −1 prot and 0.315 U·mL −1 prot, respectively, which may be due to the destruction of cell membranes and inhibition of ATPase activity by daphnetin leading to the loss of ATP balance inside and outside the cell. Previous studies considered that the decrease in ATPase level was one of the main factors leading to bacterial cell death [20]. Joung et al. [47] reported that luteolin could inhibit the effect of ATPase in. Moreover, Cai [48] speculated that Polygonum orientale extracts damaged Clavibacter michiganensis subsp. michiganensis cell membrane and caused the inhibition of ATPase activity in the plasma membrane. Hu et al. [16] revealed that the addition of Litsea cubeba essential oil to MRSA reduced four typical ATPases (Na + /K + -ATPase, Mg 2+ -ATPase, Ca 2+ -ATPase, and Ca 2+ /Mg 2+ -ATPase) activity. In addition, Cui et al. [49] showed that the activity of ATPase was related to the energy metabolism carried out by bacteria, and oregano essential oil could reduce the activity of Na + /K + -ATPase, Mg 2+ -ATPase, and Ca 2+ -ATPase in MRSA bacteria. ese results indicated that one of the main factors leading to bacterial cell death is the reduction in ATPase levels.

FTIR Spectroscopy.
e secondary structure of the biomacromolecule conjugate in the strain was detected by FTIR technology to verify the antibacterial activity of daphnetin against the tested strain. e characteristic absorption bands observed around 3279 cm −1 , 2924 cm −1 , 2358 cm −1 , 1640 cm −1 , 1538 cm −1 , 1231 cm −1 , and 1066 cm −1 correspond to the deformation of OH stretching vibration, CH stretching vibration, CN stretching vibration, CO stretching vibration, proteinamide II, SO stretching vibration, and nucleic acid, respectively [9,50,51]. As can be seen from Figure 4(a), the bands of P. fluorescens decreased at 1640 cm −1 and 1538 cm −1 , meaning that the proteins in the strain leaked into the extracellular environment. At the same time, the band of 1074 cm −1 also dropped, suggesting that the growth of P. fluorescens was inhibited due to nucleic acid leakage. As shown in Figure 4(b), the changes in absorption peaks at 3274 cm −1 and 1231 cm −1 indicated that daphnetin disrupted the phospholipid structure on the cell membrane. e decrease of 1633 cm −1 band indicated that the protein leaked into the extracellular environment. Meanwhile, the absorption peak at 1066 cm −1 reduced, indicating that the nucleic acids were leaking, so the growth of S. putrefaciens was inhibited. ese phenomena are also the same as the findings regarding cell membrane integrity. However, there was no significant difference in other absorption peaks.

Effect on the Morphology of P. fluorescens and S. putrefaciens Cells.
e morphological changes in cells or spores from P. fluorescens and S. putrefaciens treated with different amounts of daphnetin (0 ×, 1 ×, 2 ×, and 4 × MIC, respectively) for 6 h were observed by SEM. In the control ( Figure 5, A1 and B1), it showed a typical rod-like structure with a flat and complete surface, and the protoplasts in cells were filled and dense with high density. In contrast, the cell structure changed significantly after treatment with different concentrations of daphnetin. e two bacterial cell membrane surfaces at 1 × MIC showed dents and ruffles. When the daphnetin concentration achieved 2 × MIC, the structure of the two bacterial membranes changed noticeably. e cell surface of the two bacteria became twisted, dissolved, and even contracted together. At 4 × MIC, the surface of the two bacterial cells had many pits and wrinkles.
ese results were also consistent with a previous report [52] and showed SEM changes in S. putrefaciens cells treated with cinnamon oil were damaged; some cells were ruptured, and some were not smooth. Another article reported [53] that, after treatment with rLc-P5L, the SEM images of P. fluorescens had similar changes.
ese results were reminiscent of those reported by Ferreira et al. [54], who found that, after benzyldimethyldodecylammonium chloride treatment, the cell volume of Pseudomonas fluorescens seemed to be rougher, wrinkled, and deformed for the membrane. ese phenomena indicated that, after 6 h of incubation with daphnetin, the cell membranes and cell walls of the bacteria were destroyed to varying degrees. e loss of cell integrity leads to cell destruction and division.
TEM was used to observe the morphological changes in P. fluorescens and S. putrefaciens cells or spores after treatment for 4 h with different concentrations of daphnetin (0 × and 4 × MIC), respectively. e surface interactions were studied by TEM, and the leakage of cellular secretions (in the treated cells) in the surrounding medium was found [55]. e control P. fluorescens and S. putrefaciens maintained typical bacterial morphology with clear nuclei, cytoplasm, cell membranes, and cell walls ( Figure 6, A1 and B1). Treated P. fluorescens (Figure 6, A2) displayed significant variability. First, the bacterial cell walls and cell membranes were destroyed, lysed, and broken. Secondly, bacterial cytoplasm extravasated, forming a clear cavity. S. putrefaciens after treatment (Figure 6, B2) also showed obvious change in characteristics. e cells were highly deformed, the cell walls ruptured, and the cytoplasm leaked. Similarly, Lee et al. [56] confirmed that SKN has the activity of anti-MRSA, which leads to cell dissolution and leakage of cytoplasmic content by destroying bacterial cell walls and plasma membranes. Chen et al. [57] reported that the strain cells treated with sugar beet molasses induced the disorder of cell membrane function and showed obvious intracellular damage. Lee and Je [58] showed that the treatment of S. aureus and E. coli with gallic acid-g-chitosan (I) could promote the outflow of intracellular substances. Wang et al. [59] explained that the cell walls and membranes of S. aureus and E. coli treated with SE (the synergistic combination between alcohol extracts from the Chimonanthus salicifolius S. Yi et al. leaves and streptomycin) were broken and dissolved, and the cytoplasm of bacteria leaked out, forming a clear cavity. ese phenomena indicated that the addition of polyphenols may interfere with the division of bacterial cells, causing the cells to change from a typical long rod shape to a short rod shape or coccus shape [60]. Obviously, according to the results, the cells of the strain treated with daphnetin will suffer significant internal damage by inducing cell membrane dysfunction [57].

Conclusions
is research reported the antibacterial activity and mechanism of daphnetin on P. fluorescens and S. putrefaciens. e MIC of daphnetin acting on P. fluorescens was 0.16 mg·mL −1 and that of S. putrefaciens was 0.08 mg·mL −1 . Daphnetin can destroy the cell wall and membrane integrity of P. fluorescens and S. putrefaciens, which causes nucleic acid and protein leakage and affects intracellular AKP and ATPase activities. SEM and TEM results showed that daphnetin induced alterations in the morphology and caused the leakage of intracellular contents in bacterial cells. e FTIR spectroscopy of P. fluorescens and S. putrefaciens indicted daphnetin destroyed the phospholipid structure on the membrane and the nucleic acid and protein leakage resulting in the growth inhibition of P. fluorescens and S. putrefaciens. Overall, our results indicate that daphnetin is a promising natural preservative used as a potential substitute for synthetic preservatives in food industry providing a viable strategy for enhancing microbial inactivation.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.  Journal of Food Quality