Nanolayered Double Hydroxide Inhibits the Pathogenicity of Vibrio parahaemolyticus

Nanolayered double hydroxide (LDH) is a type of anion layered inorganic compound whose bacteriostatic properties have recently garnered much attention. Vibrio parahaemolyticus is a marine pathogen that can lead to aquaculture diseases and substantial economic losses. Therefore, our study assessed the mechanisms by which Mg/Al-LDH prevents V. parahaemolyticus infection. Our results demonstrated that Mg/Al-LDH not only inhibited V. parahaemolyticus growth but also biofilm formation. Moreover, coupling Mg/Al-LDH with hydrogen peroxide and UV irradiation further inhibited the growth and biofilm formation of V. parahaemolyticus. Additionally, Mg/Al-LDH was found to adversely affect DNA and the gelling ability of chitosan. Furthermore, exposing V. parahaemolyticus to Mg/Al-LDH led to a 54.73% and 4.3% inhibition in the expression of the toxic genes tlh and trh, respectively. Mg/Al-LDH also improved the symptoms of V. parahaemolyticus infection in Penaeus vannamei, making this a promising candidate to prevent pathogenic bacteria infection in aquaculture.


Introduction
Nanolayered double hydroxide (LDH), also known as anionic clay [1], is a type of anionic layered compound consisting of a positively charged metal hydroxide outlayer and a negative anion interlayer [2]. The molecular formula of LDH is [M II 1-x M III x (OH) 2 ] x+ (A n-) X/n ·yH 2 O (x = 0:2 -0:33, y = 0:5 -1), where M III represents trivalent metal ions, M II represents divalent metal ions, A nrepresents n-valent anions, and LDH represents the different structures that can be synthesized by changing the value of x [3,4]. LDH can be cost-effectively prepared in the laboratory with good biocompatibility, adjustable layer charge density and particle size, low storage requirements, and low toxicity [1,5]. LDH with various unique properties can be synthesized through different combinations of divalent metal ions and trivalent metal ions, as well as changes in proportion [1,6]. LDH has many unique characteristics, including the adjust-ability of laminate element composition, exchangeability of the negative anion interlayer, thermal stability, pH sensitivity, and memory, all of which make this material uniquely well suited for a variety of applications in several fields such as biomedicine [7][8][9], adsorption [10][11][12], catalysis [13][14][15], and environmental remediation [16][17][18].
Particularly, the antibacterial properties of LDH have garnered much attention in recent years. Ding et al. [19] assessed LDH toxicity in Chlorophyta and found that this compound was highly toxic to Streptococcus, with an EC 50 of 10 mg/L after 72 h of LDH exposure. Further, Streptococcus growth was completely inhibited when the LDH concentration reached 50 mg/L. An LDH prepared by Moaty et al. exhibited long-lasting antibacterial activity against Gramnegative bacteria (Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) and Grampositive bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Aspergillus, Streptococcus pyogenes, and Salmonella) [20]. Many studies have also confirmed that LDH has a good bacteriostatic effect when combined with other compounds or materials [21][22][23][24][25].
V. parahaemolyticus was first discovered by Tsunesaburo Fujino of Osaka University after a seafood poisoning outbreak in Japan in 1950 [26]. V. parahaemolyticus is a Gram-negative halophilic bacterium that is commonly found in estuaries and marine environments and various types of seafood [27], which can cause aquaculture diseases and considerable economic losses to the aquaculture industry. Moreover, people who consume raw or undercooked seafood are at high risk of becoming infected with V. parahaemolyticus. The symptoms of V. parahaemolyticus infection include diarrhea that lasts for 2 to 10 days, abdominal cramps, nausea, vomiting, and headaches [28]. Wound infection with V. parahaemolyticus can also lead to septicemia, which can be fatal [29][30][31]. Marine animals such as bony fish and grouper, mud crabs, and shrimp infected with V. parahaemolyticus often exhibit marked histopathological changes and increased mortality, thus resulting in huge economic losses to the aquaculture industry [32][33][34].
Several studies have evaluated the virulence factors and pathogenesis of V. parahaemolyticus. One such study found that the pathogenicity of V. parahaemolyticus was closely related to many virulence factors, including hemolysin, type III secretion system, type VI secretion system, adhesion factor, and iron uptake system [28,35]. It has also been reported that V. parahaemolyticus produces three kinds of hemolysin: thermally stable direct hemolysin (tdh), tdhassociated hemolysin (trh), and heat-intolerant hemolysin (tlh), which are encoded by the tdh, trh, and tlh genes, respectively [36,37]. Additionally, V. parahaemolyticus often forms biofilms and becomes embedded in its own extracellular polymeric matrix, making this pathogen uniquely resistant to cleaning and disinfection processes [38][39][40]. Therefore, biofilms protect microbial communities and facilitate quorum sensing.
Our study investigated the effects of exposed Mg/Al-LDH on V. parahaemolyticus when the Mg/Al-LDH and V. parahaemolyticus were exposed together. Furthermore, the mechanisms of effects were explored. The purpose is to reduce the chance of humans and aquaculture being infected with V. parahaemolyticus. Our findings indicated that Mg/ Al-LDH prevented V. parahaemolyticus in Penaeus vannamei, thus highlighting the promising potential of Mg/Al-LDH as an antibacterial agent to increase aquaculture yields.

2.2.
Effect of Mg/Al-LDH on V. parahaemolyticus Growth. V. parahaemolyticus was inoculated into TSB broth containing different Mg/Al-LDH concentrations (0-100 mg/L). The strain was incubated at 30°C and 180 r/min for 24 h, and the optical density at 600 nm was detected in a 96-well plate using a microplate reader (Infinite M1000 Pro, Tecan, Switzerland).

Effect of Mg/Al-LDH When Coupled with UV Irradiation
or Hydrogen Peroxide. The activated V. parahaemolyticus was inoculated into media containing Mg/Al-LDH (100 mg/L) and then irradiated with ultraviolet light for 0-300 s. The strain was also grown without Mg/Al-LDH as a control treatment. Similarly, different concentrations of hydrogen peroxide (0-500 mM) were added to the growth media containing Mg/Al-LDH (100 mg/L), after which OD 600 of the broth was measured.
2.4. Effect of Mg/Al-LDH on V. parahaemolyticus Biofilm Formation. V. parahaemolyticus biofilm formation was assessed as described by Yin et al. [40] with slight modifications. Specifically, this study explored the effect of Mg/Al-LDH on the biofilm formation of V. parahaemolyticus. The activated V. parahaemolyticus (50 μL), fresh medium, and Mg/Al-LDH were mixed in microplates to a 250 μL total volume. The final concentrations of Mg/Al-LDH were 0-100 mg/L. Then, the microplates were incubated at 30°C for 24 h. Afterward, the medium was removed and the wells were washed three times with 250 μL of phosphate-buffered saline (PBS). The microplates were then dried in a 60°C oven for 1 h. The biofilms were stained with 250 μL of 0.1% crystal violet solution at room temperature for 15 min. After gently washing the samples with PBS to remove excess crystal violet, the stained biofilm was dried again. Afterward, 250 μL of glacial acetic acid (30%) was added to dissolve the crystal violet stain and the optical density was measured at 590 nm.
Next, V. parahaemolyticus (50 μL), fresh medium, Mg/ Al-LDH (100 mg/L), and hydrogen peroxide were mixed in microplates to a 250 μL total volume. The final hydrogen peroxide concentration ranged between 0 and 500 mM. A blank control was prepared without Mg/Al-LDH. Biofilm formation was detected after incubation.   3 Journal of Nanomaterials Finally, V. parahaemolyticus (50 μL), fresh medium, Mg/ Al-LDH (100 mg/L), and hydrogen peroxide (50 mM) were mixed in microplates to a 250 μL total volume and then irradiated with ultraviolet light for 0-300 s. A blank control was prepared without Mg/Al-LDH. Biofilm formation was detected after incubation.
2.5. Effects of Mg/Al-LDH on Polysaccharides. Chitosan and 2.5% glutaraldehyde form a gel after being placed in a water bath at 70°C for 1 h. We then added 200 μL of 0.5% chitosan solution, 20 μL of 2.5% glutaraldehyde, and Mg/Al-LDH (100 mg/L) to glass bottles to a final 250 μL volume, after which they were treated with hydrogen peroxide (50 mM) and UV irradiation (2 min). The samples were taken out of the water bath (70°C) after 1 h. Upon gel formation, the weight of the gel was measured.
2.6. Effects of Mg/Al-LDH on Proteins. Bovine serum protein (BSA) is a typical functional protein. A 250 μL system was prepared by mixing Mg/Al-LDH (100 mg/L), bovine serum protein, followed by hydrogen peroxide (50 mM) or UV irradiation (2 min). SDS-PAGE gel electrophoresis and quantitative analysis were then performed after incubation for 2 h in a 37°C water bath.
2.7. Effects of Mg/Al-LDH on DNA Integrity. Salmon DNA was added to a 50 μL system with Mg/Al-LDH (100 mg/L), hydrogen peroxide (50 mM), or UV irradiation (2 min) and then incubated in a 37°C water bath for 2 h. Afterward, 1% agar electrophoresis was conducted for verification and quantitative analysis was performed using a microanalyzer.
2.9. P. vannamei Infection Experiments. Healthy white shrimp (P. vannamei; 6 cm long) were purchased from the Lianyungang Wholesale Market. Twelve P. vannamei were placed in each water tank outfitted with a circulating filter device to maintain water quality. The water temperature and salinity of the water tank were controlled to 25°Cand 20‰, respectively. Once the shrimp were adapted to the growth environment, 20 mL of V. parahaemolyticus (ATCC17802; 1:21 × 108 CFU/mL) was added to  2.10. Statistical Analysis. All experiments were conducted in triplicate, from which means and standard deviations were calculated. Significance was conducted using the S-N-K(S) test in SPSS.

Effect of Mg/Al-LDH on V. parahaemolyticus Growth.
Mg/Al-LDH significantly inhibited V. parahaemolyticus growth at a 100 mg/L concentration (Figure 1(a)), with OD values reaching only 90% compared to the control. The growth of V. parahaemolyticus treated with Mg/Al-LDH was more severely inhibited within a short time of UV irradiation (Figure 1(b)). However, once the irradiation time reached 2 min, the growth inhibition of V. parahaemolyticus was largely unaffected by Mg/Al-LDH. Combining hydrogen peroxide with Mg/Al-LDH had a stronger inhibitory effect on the growth of the strain. Particularly, hydrogen peroxide concentrations ranging from 0 to 50 mM effectively inhibited the growth of V. parahaemolyticus when coupled with Mg/Al-LDH (100 mg/L) (Figure 1(c)). However, when the concentration of hydrogen peroxide reached 100 mM, the turbidity of the bacterial liquid was lower than 0.2. At this point, bacterial growth was mainly inhibited by hydrogen peroxide. Therefore, Mg/Al-LDH (100 mg/L) inhibited V. parahaemolyticus growth most efficiently when combined with low concentrations of hydrogen peroxide.
3.1.2. Effect of Mg/Al-LDH on V. parahaemolyticus Biofilm Formation. We next assessed the effect of Mg/Al-LDH on the formation of V. parahaemolyticus biofilm. With increasing Mg/Al-LDH concentrations, V. parahaemolyticus biofilm formation was gradually inhibited (Figure 2(a)). The results showed that OD 590 decreased from 2.75 to 1.76 and the inhibition rate reached 36%.
V. parahaemolyticus biofilms were severely damaged during the formation process (Figure 2(b)). As the concentration of hydrogen peroxide increased, the biofilm of the experimental group was more severely damaged. However, when the concentration of hydrogen peroxide reached 100 mM, the growth of V. parahaemolyticus was severely inhibited and biofilm formation was reduced. At this point, Mg/Al-LDH had little effect on V. parahaemolyticus biofilm formation. However, when the concentration of hydrogen peroxide was 50 mM, the growth of V. parahaemolyticus was not severely inhibited. The results showed that OD 590 of the control group was 1.74 and OD 590 of the experimental group treated with Mg/Al-LDH was 0.89.
The effects of UV irradiation coupled with hydrogen peroxide and Mg/Al-LDH on V. parahaemolyticus biofilm formation in microplates were also assessed. V. parahaemolyticus biofilm formation was more severely inhibited when all three factors were combined. Our results demonstrated that OD 590 of the control group decreased from 1.99 to 0.92, indicating that the biofilm formation could be inhibited   Journal of Nanomaterials by UV irradiation and the inhibition increased with increasing irradiation time (Figure 2(c)). In the experimental group, OD 590 decreased from 1.01 to 0.66.

Effects of Mg/Al-LDH on Polysaccharides.
Chitosan and glutaraldehyde undergo a gelling reaction when heated to 70°C. However, upon adding Mg/Al-LDH (100 mg/L), the gelling reaction is greatly reduced. This indicated that Mg/Al-LDH can inhibit the gelation ability of polysaccharides. Moreover, polysaccharides were further weakened when treated with Mg/Al-LDH, hydrogen peroxide, and UV light (Figure 3(b)).

Effect of Mg/Al-LDH on Protein.
Based on the comparisons between 1 and 2, 3 and 4, 5 and 6, and 7 and 8 in Figure 3(d), the protein residue in the experimental group with Mg/Al-LDH was slightly higher than that without Mg/Al-LDH. However, as illustrated by the comparison between 2 and 8, the most significant protein degradation   3.1.5. Effect of Mg/Al-LDH on DNA. Our study also explored whether DNA is degraded under Mg/Al-LDH, hydrogen peroxide, and ultraviolet conditions. To achieve this, DNA, Mg/Al-LDH, and hydrogen peroxide were mixed, irradiated with UV rays, and incubated in a 37°C water bath for 2 h. After subsequent quantitative analysis using a microanalyzer, the values of 1, 3, 5, and 7 were all higher than those of 2, 4, 6, and 8 ( Figure 4(b)). This indicated that Mg/Al-LDH addition caused DNA degradation.
3.1.6. Effect of Mg/Al-LDH on the Expression of Toxicity-Associated Genes in V. parahaemolyticus. The extracted DNA was quantitatively analyzed via fluorescence quantitative PCR. As shown in Figure 4(c), Mg/Al-LDH exerted 4.3% and 54.73% inhibition rates for the trh and tlh genes of strain ATCC17802. In contrast, Mg/Al-LDH promoted the expressions of the tdh and tlh genes by 60.21% and 35.19% in the ATCC33846 strain.
3.1.7. Infection Experiment. The shrimps (P. vannamei) were placed in each water tank outfitted with a circulating filter (Figure 5(a)). After breeding for a period of time under different conditions, as illustrated in Figures 5(b) and 5(c), the symptoms of P. vannamei upon V. parahaemolyticus infection in experimental group 1 were more severe than those in experimental group 2. The shrimp in experimental group 2 did not appear sluggish but presented hepatopancreas ulcers and hyperemia. Ulcers were not detected in the parotid glands, tail fans, appendages, and swimming appendages. Additionally, as illustrated in Figure 5(d), shrimp mortality in experimental group 2 was much lower than that in experimental group 1 after 3-11 days of infection.
3.2. Discussion. Mg/Al-LDH significantly inhibited V. parahaemolyticus growth that was consistent with the excellent antibacterial properties of Zn/Fe-LDH reported by Moaty et al. on several common bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus [20]. Previous studies have shown that LDHs had a strong inhibitory effect on the growth of some pathogenic bacteria and might therefore be used to treat and prevent bacterial infection. Ultraviolet (UV) light, an important component of Earth's natural lighting, kills microorganisms by changing and damaging the structure of their DNA [41]. Particularly, UVC can effectively kill bacteria, viruses, protists, and other microorganisms [42] and is therefore often used as a disinfectant. Within a short time of UV irradiation, the growth of V. parahaemolyticus treated with Mg/Al-LDH was more severely inhibited. This may have been because short-term UVC irradiation destroyed the DNA of V. parahaemolyticus, thereby inhibiting its growth. The decomposition of hydrogen peroxide can produce free radicals, which can inactivate microorganisms by interfering with cell membranes, DNA, and proteins [43,44]. Therefore, combining hydrogen peroxide with Mg/Al-LDH had a stronger inhibitory effect on the growth of the strain.
Biofilms are composed of a variety of biological macromolecules such as proteins, polysaccharides, DNA, RNA,   11 Journal of Nanomaterials peptidoglycans, lipids, and phospholipids. These complex matrices render bacteria highly resistant to antibiotics, as well as cleaning and disinfection processes [40]. Adding Mg/Al-LDH could effectively inhibit the formation of bio-films that might be because Mg/Al-LDH destroys major macromolecules such as polysaccharides and DNA during biofilm formation. And hydrogen peroxide, Mg/Al-LDH, and UV irradiation could inhibit the formation of V. parahaemolyticus biofilm more effectively, in which hydrogen peroxide and ultraviolet radiation can destroy the biofilm to varying degrees. Chitosan is a natural polysaccharide derived from chitin. It is the second most ubiquitous naturally occurring polysaccharide after cellulose. Chitosan is obtained when chitin is deacetylated to approximately 50% of free amines. As a biopolymer, chitosan has attracted much attention due to its biomedical applications [45]. In our study, Mg/Al-LDH can inhibit the gelation ability of polysaccharides. This might be because Mg/Al-LDH was adsorbed on the polysaccharide, thus affecting its gelling ability.
Bovine serum albumin (BSA), a typical functional protein molecule, is cost-efficient, safe, degradable, rich in chemical groups, and relatively stable, all of which make this protein uniquely well suited for medical care and pharmaceutical applications [46]. Our research results showed that Mg/Al-LDH did not degrade bovine serum proteins. Therefore, we speculated that Mg/Al-LDH may not disrupt most proteins in cells. In many reports, LDH is used for drug delivery [47]. Mg/Al-LDH was likely adsorbed on the protein, thus preventing its degradation.
Deoxyribonucleic acid (DNA) is a kind of polynucleotide that carries essential genetic information and can be used to encode messenger RNA and proteins. DNA has been widely used in gene therapy, biosensing, and information storage. But Mg/Al-LDH could degrade salmon DNA. Therefore, Mg/Al-LDH may damage the DNA of V. parahaemolyticus, which provides an important basis for the further exploration of the molecular mechanisms of Mg/Al-LDH-mediated antibacterial effects. The results of subsequent toxic gene experiments were also consistent with these observations. It was confirmed that Mg/Al-LDH can inhibit the expression of virulence-associated genes in some V. parahaemolyticus strains. Therefore, our findings indicated that Mg/Al-LDH not only inhibits the growth of V. parahaemolyticus but also downregulates the expression of toxic genes.
Shrimp mortality in experimental group 2 was much lower than that in experimental group 1 after infection. These observations were consistent with our previous results, thus confirming that Mg/Al-LDH can inhibit the pathogenicity of V. parahaemolyticus. This was mainly due to the cytotoxicity of Mg/Al-LDH to V. parahaemolyticus, which disrupts its biofilm and affects the expression of toxic genes. Ahmed et al. [48] reported that the effect of nanoparticles (NPs) on bacteria mainly depends on certain mechanisms, including the interaction with cell barriers, penetration through diffusion and adsorption, inhibition of bacterial proteins and DNA synthesis, regulation of metabolic gene expression, and inhibition of biofilm formation.

Conclusions
LDH, a novel low-toxicity nanomaterial, plays an important role in various industries. In this study, this nanomaterial was used to prevent and control bacterial infection in Pacific white leg shrimp (i.e., a widely cultured aquaculture species).
The following are the key conclusions of this study: (1) Mg/Al-LDH had a certain inhibitory effect on the growth of V. parahaemolyticus, which could be enhanced when coupled with hydrogen peroxide and UV irradiation (2) Mg/Al-LDH also effectively inhibited V. parahaemolyticus biofilm formation. However, the addition of hydrogen peroxide and UV radiation did not significantly enhance the inhibitory effects of Mg/Al-LDH on V. parahaemolyticus biofilm formation These five conclusions confirm the important role of Mg/Al-LDH in the prevention and treatment of V. parahaemolyticus. Therefore, our study provides a robust basis for the development of novel strategies to protect human health and control bacterial diseases in the seafood industry.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
All authors have declared that (i) no support, financial or otherwise, has been received from any organization that may have an interest in the submitted work and (ii) there are no other relationships or activities that could appear to have influenced the submitted work.