Inhibition of Quorum Sensing and Biofilm Formation of Esculetin on Aeromonas Hydrophila

Quorum sensing (QS) and biofilm formation inhibition activity of esculetin on Aeromonas hydrophila SHAe 115 were evaluated. Exposure to esculetin at 25, 50, and 100μg/ml significantly inhibited the production of protease and hemolysin, the formation of biofilms and attenuated the swarming motility of A. hydrophila SHAe 115. Biofilm forming inhibition was also observed through confocal laser scanning microscopy and scanning electron microscope. Quantitative real-time PCR analysis indicated that genes positively related to QS and biofilm formation were downregulated to varying degrees, while gene (litR) negatively related to biofilm formation was significantly upregulated. The phenotypic results were in good agreement with gene expression levels. These results indicated that esculetin would be a potential QS inhibitor for A. hydrophila.

INTRODUCTION biosynthesis, degrading the synthesized AHL molecules and/ or inactivating the AHL receptor protein (Belapurkar et al., 2014).
Aeromonas hydrophila is a conditional pathogen that is common to humans, livestock, and aquatic animals (Zhou and Zhou, 2012). A. hydrophila can infect molluscs (Grizzle and Brunner, 2009), crustaceans (Ma et al., 2012), fish (Liang and Xie, 2013;Zhao and Wang, 2015), amphibians (Meng et al., 2009), reptiles (Chen et al., 2003), and poultry (Pan et al., 2003). Humans can suffer from diarrhea, food poisoning, and secondary infections due to pathogenic A. hydrophila infection (Yang and Wang, 2006). A. hydrophila not only poses a threat to human health, but also cause huge economic losses to the aquaculture industry, which has attracted great attention over the world.
Many natural compounds have been reported as QS inhibitors (Jakobsen et al., 2012;Figueroa et al., 2014;Venkadesaperumal et al., 2015), and some of the most effective QS inhibition molecules derived from plants are coumarins, a large structurally diverse family of plant phenolic compounds characterized by their pharmacological properties (Venugopala et al., 2013). Biological studies on coumarins demonstrated that these compounds have potential activities, such as antitumor (Vanamala et al., 2006;Tehsina et al., 2011;Jamier et al., 2014), antiinflammatory (Fylaktakidou et al., 2004;Bucolo et al., 2009), anticoagulant (Payá et al., 1992;Fereshteh et al., 2014), and antibacterial (Ojala et al., 2000;Souza et al., 2005). Among coumarins, hydroxylated coumarins, such as umbelliferone, daphnetin, and esculetin, showed stronger bioactivities. Wang et al. (2017) demonstrated that esculetin has obvious antibacterial effect on KPC-producing Klebsiella pneumoniae. Esculetin also has superior antibacterial activity against the phytopathogen Ralstonia solanacearum and can inhibit its biofilm formation (Yang et al., 2016). Thomas et al. (2017) showed that esculetin has a good inhibitory effect on the QS-regulated transcription factor SdiA of Salmonella typhi through molecular docking. Recent reports on coumarins inhibiting biofilm formation and reducing virulence factors of Escherichia coli and Pseudomonas aeruginosa (Lee et al., 2014) have drawn the attention of researchers to the potential of coumarins as QS inhibitors and anti-biofilm agents. According to Duncan et al. (1998), esculetin and umbelliferone could inhibit the growth of E. coli O157:H7. Dürig et al. (2010) reported that esculetin was able to prevent biofilm formation of Staphylococcus aureus without affecting its cell growth. D' Almeida et al. (2017) conducted a comparison of seven structurally related coumarins (coumarin and different hydroxylated derivatives) on the QS inhibitory and anti-biofilm activities against P. aeruginosa and Chromobacterium violaceum. The results showed that molecules with hydroxyl groups on the aromatic ring have higher activity on virulence factors inhibition and biofilm formation. Besides, research of Lee et al. (2014) showed that hydroxylation in position C-4 dramatically diminishes the anti-biofilm activity on E. coli O157:H7 while hydroxylation in position C-7 enhances it.
According to the above introduction, esculetin has good inhibitory activity against many bacteria, and many plants contain this compound. Our previous phytochemical work also isolated this compound. However, no study on the inhibitory effect of esculetin against A. hydrophila has been reported. Hereby, we investigated the influence of esculetin on QS-related virulence factors and biofilm formation of A. hydrophila SHAe115. We hope that esculetin can mitigate human disease caused by A. hydrophila and/or reduce the loss of aquaculture caused by A. hydrophila.

Bacterial Strain and Culture Conditions
Aeromonas hydrophila SHAe 115 used in this study was obtained from China General Microbiological Culture Collection Center. All experiments were conducted at 37°C in Luria-Bertani (LB) medium.
Esculetin was isolated from Onosma bracteatum Wall. in our previous study (Sun et al., 2021). It was dissolved in DMSO to prepare a stock solution of 50 mg/ml.

The Minimum Inhibitory Concentration and Growth Measurement
Esculetin was tested against A. hydrophila SHAe 115 to determine the minimum inhibitory concentration (MIC) according to Clinical and Laboratory Standards Institute (2015) (Zhou et al., 2017) and with an inoculum of 1-5 × 10 5 CFU ml −1 . The OD 620 value of bacterial cultures of A. hydrophila SHAe 115 at this concentration was approximately 0.1. Two-fold dilution method was used and a series of diluted esculetin solution (25,50,100,200,400, and 800 μg/ml) was performed in LB broth. The experiment was carried out in 96-well polystyrene microtiter plate, with 10 wells for each concentration. MIC is defined as the minimum concentration of esculetin which inhibited the visible growth of A. hydrophila SHAe 115 and sub-MICs were selected for the assessment of anti-virulence and anti-biofilm activity.
For growth measurement, overnight cultures of A. hydrophila SHAe 115 were inoculated into fresh LB medium and the optical density (OD) value was adjusted to 0.1 at 620 nm. The cultures were transferred to 96-well polystyrene microtiter plate and supplemented with esculetin of different final concentrations (25, 50, and 100 μg/ml), and then incubated continuously at 37°C for 24 h with shaking (180 rpm). DMSO was set as the negative control, and each concentration was set for 10 wells. The bacterial growth was monitored at 1 h intervals, and the OD 620 was recorded by a microplate reader (Biotek, United States).

Hemolysin Assay
The hemolysin assay was performed with a few changes based on the method of Zhou et al. (2019). 1% overnight culture of A. hydrophila SHAe 115 was added to fresh LB medium (OD 620 ≈ 0.1) and cultivated in 24-well polystyrene microtiter plate with or without esculetin at 37°C for 24 h with shaking (180 rpm). The final concentrations of esculetin were 25, 50, and 100 μg/ml. DMSO was set as the negative control, and three parallel groups were set for each concentration. After the cultivation, the cultures were centrifuged at 10000 rpm for 10 min (4°C), and the cell free supernatants were collected. 100 μl of cell free supernatants of esculetin treated and untreated cultures were mixed with 900 μl of 2% washed sheep blood (in PBS, pH 7.2). The mixture was incubated for 1 h at 37°C followed by 10 min of centrifugation at 3000 rpm. Absorbance of the supernatant was measured at 530 nm.

Azocasein Assay
Azocasein assay was conducted to evaluated the total proteolytic activity of A. hydrophila SHAe 115, and the activity was determined by the method of Ding et al. (2018). 75 μl of the abovementioned cell free supernatant obtained from each group was added to 125 μl of 0.3% azocasein solution (in 50 mM Tris-HC1 and 0.5 mM CaCl 2 ) and incubated at 30°C for 15 min. The reaction was ended by adding 600 μl of 10% trichloroacetic acid. After centrifugation for 10 min at 10000 rpm, 700 μl of NaOH (1 M) was mixed with the supernatant and the OD 440 was recorded by a microplate reader (Biotek, United States).

Swarming Motility Assay
The swarming agar was freshly prepared with 0.8% nutrient broth (NB) medium, 0.5% glucose, and 0.3% agar (pH 7.2). 2 μl overnight culture of A. hydrophila SHAe 115 was inoculated at the center of the agar plate containing a series of esculetin (25, 50, and 100 μg/ml). DMSO was set as the control, and three parallel groups were set for each concentration. The plates were cultured at 37°C for 24 h, and the swarming migration diameters were recorded (Zhou et al., 2019).

Biofilm Inhibition Assay
The effect of esculetin at sub-MICs on biofilm formation was measured according to Ding et al. (2018) with some modifications. Briefly, 1% overnight culture of A. hydrophila SHAe 115 was added to fresh LB medium (OD 620 ≈ 0.1) and transferred to a 96-well polystyrene microtiter plate then incubated in the presence and absence of esculetin for 24 h at 37°C without shaking. DMSO served as the negative control with 10 wells for each concentration. After the incubation, planktonic cells and spent media were discarded and the biofilms were washed with PBS (pH 7.2) for three times. After being fixed with methanol for 15 min, the biofilms were stained with 0.05% crystal violet (CV). Further, excess stain was removed and the biofilms were rinsed three times with PBS (pH 7.2) and bound CV was dissolved with 95% ethanol. Biofilm biomass was quantified by measuring the absorbance of crystal violet-ethanol solutions at 570 nm (Biotek, United States).

Microscopy Analysis
One percent overnight culture of A. hydrophila SHAe 115 was added to fresh LB medium (OD 620 ≈ 0.1), and cultivated in 24-well polystyrene microtiter plate containing glass slides (d = 14 mm) with and without esculetin. Culture was incubated without agitation at 37°C for 24 h. DMSO was set as the negative control, and three parallel groups were set for each concentration. After the incubation, planktonic cells and spent media were removed and the glass slides were gently rinsed three times with PBS (pH 7.2).
For scanning electron microscopy (SEM) observation, samples were prepared with the method described by Zhou et al. (2017).
Method used in confocal laser scanning microscopy (CLSM) observation was according to Zhou et al. (2018). Briefly, the dried samples were stained with acridine orange (0.1%) for 15 min and excess dye was discarded. After being washed with PBS (pH 7.2), the slides were then fixed with paraformaldehyde (4%) for 15 min in the dark and subsequently subjected to CLSM (Nikon, A1+ SIM-S). For each group, we randomly selected five areas for image analysis.

Quantitative Real-Time PCR Analysis
The quantitative real-time PCR (qRT-PCR) assay was carried out under the guidance of Zhou et al. (2018) with slight modification. A. hydrophila SHAe 115 was grown in LB medium supplemented with or without esculetin (100 μg/ml) at 37°C at 180 rpm for 24 h. After incubation, cells were washed with sterile PBS (pH 7.2) three times and collected after 10 min centrifugation at 4°C. Total RNA was extracted from the bacterial cells using an RNA extraction kit (Biofit Biotechnologies, Chengdu, China) following the manufacturer's instruction. Reverse transcript reaction was performed with a commercial reverse-transcription enzyme (Tsingke Biotechnology, Beijing, China) according to the manufacturer's instruction. Quantitative real-time PCR was carried out with an ABI 7300 Plus realtime PCR system. The amplification was carried out in a 20 μl reaction volume containing 2 × T5 Fast qPCR Mix (SYBR Green I; 10 μl, Tsingke Biotechnology, Beijing, China), primers (0.8 μl of each), diluted cDNA (1 μl), and ddH 2 O (7.4 μl). The thermocycling conditions were as follows: incubation for 10 min at 95°C, followed by denaturation for 15 s at 95°C, annealing and extension at 60°C for 60 s. PCR amplification consisting of 45 cycles was conducted. All samples were run in triplicate. The primers used in this study were listed in Table 1 (Kozlova et al., 2011). 16S rRNA served as an internal control (Patel et al., 2017). The relative expression of target genes was calculated by the conventional 2 −ΔΔCT method proposed by Pfaffl (2001).

Statistical Analysis
All of the experiments were performed in triplicate, and the data were presented as mean ± standard deviation (SD). Statistical analysis was carried out with GraphPad Prism 8 software (San Diego, CA, United States). One-way ANOVA plus post-hoc Tukey test or two-tail paired t-test was used to evaluate the statistical significance between groups. The following terminology is used to denote the statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Determination of MIC and Growth Inhibition Analysis
The MIC of esculetin evaluated by two-fold dilution was 200 μg/ml. All the following experiments were conducted at sub-MICs (25, 50, and 100 μg/ml). The bacterial cultures treated with esculetin at sub-MICs did not show any significant inhibitory effect on growth of A. hydrophila SHAe 115 compared with the control (Figure 1).

Inhibition of Hemolysin
As shown in Figure 2, esculetin has a significant inhibitory effect on the production of hemolysin of A. hydrophila SHAe 115 at sub-MICs. Compared to the DMSO control group, treatment with esculetin at 25, 50, and 100 μg/ml caused reduction in hemolysin production of approximately 77, 86, and 87%, respectively. Bleeding is a common phenomenon that A. hydrophila infects animals, and the hemolytic activity can be detected in vivo and in vitro. Hemolysin is considered to be the main virulence factor of A. hydrophila, so the role of hemolysin is highly valued (James and Trevor, 1988). The hemolysin is a single polypeptide molecule, it is one of the exotoxins (also known as aerolysin) produced by A. hydrophila. Allan and Stevenson (1981) injected the exotoxin of A. hydrophila on rainbow trout and speckled trout, which caused disease in these fish. Thune et al. (1986) isolated β-hemolysin from a protease-deficient strain of A. hydrophila that can kill catfish. Most studies proved that aerolysin genes are highly conserved in Aeromonas spp. (Chacón et al., 2003;Lu et al., 2004;Nam and Joh, 2007), indicating that they play an important role in pathogenicity. Our results showed that esculetin significantly inhibited the hemolysin production of A. hydrophila SHAe 115, which proved that esculetin can effectively attenuate the pathogenicity of this bacteria.

Inhibition of Protease Activity
This assay was carried out to analyze the potential of esculetin in inhibiting the production of protease in A. hydrophila SHAe 115. Azocasein was used as the substrate. The obtained results indicated that the production of protease was significantly reduced. And the inhibitory ability of esculetin on protease increased with concentration. Protease production was inhibited in the level of 31, 41, and 46%, respectively, in groups supplied with esculetin at concentrations of 25, 50, and 100 μg/ml. Extracellular proteases (Chu and Lu, 2000) are one of the virulence factors of A. hydrophila. At present, metalloproteases and serine proteases are widely studied. They are widely present in A. hydrophila. Some extracellular proteases have direct pathogenicity, and some can activate other pathogenic factors. For example, the exotoxins secreted by A. hydrophila are in the form of an inactive precursor and require extracellular proteases to activate them (Chacón et al., 2003). In our study, as concentration increased, the inhibitory ability of esculetin on proteases gradually increased, as shown in Figure 3. On one hand, the reduction of protease activity can reduce the pathogenicity of A. hydrophila SHAe 115; on the other hand, the lack of protease activation of exotoxins (such as hemolysin) will also reduce the pathogenicity of the bacteria.

Inhibition of Swarming Motility
The swarming motility of A. hydrophila SHAe 115 was clearly visible in DMSO control ( Figure 4A). However, on esculetin supplements at concentrations from 25 to 100 μg/ml, the swarming motility of A. hydrophila SHAe 115 was repressed (Figures 4B-D). And, the diameter of swarming zone decreased as the concentration increased. The bacteria exhibited a total swarming diameter of 24 mm. When treated with esculetin at sub-MICs, the swarming diameter decreased to 16, 12, and 8 mm, respectively, with an inhibition of 32, 48, and 66% ( Figure 4E). The swarming motility of bacteria has been characterized as flagellar-mediated motility which is regulated by QS system (Köhler et al., 2000;Déziel et al., 2003). And the motility driven by flagellum played an import part in the pathogenicity of the bacteria. Previous studies have suggested that swarming motility can contribute to biofilm formation (Shrout et al., 2010). The swarming motility of A. hydrophila SHAe 115 was significantly inhibited by esculetin at sub-MICs (Figure 4). As shown in the figure, the diameters of swarming zone of the bacteria treated with esculetin were much smaller than that of the control, suggesting that esculetin had some inhibitory effects on the swarming motility of the bacteria. And the reason might be the interference of QS system in the pathogen caused by esculetin.

Inhibition of Biofilm Formation
As shown in Figure 5A, esculetin significantly inhibited the biofilm formation. As the concentration increased, the inhibitory activity of esculetin acted in a concentration-dependent manner. The biofilm inhibition rate was approximately 38, 60, and 79%, respectively, as the bacteria treated with esculetin at 25, 50, and 100 μg/ml.
In addition to the quantitative analysis of the biofilm biomass with CV staining method, we also observed the development of biofilm structure through SEM and CLSM after incubation in the presence and absence of esculetin. SEM images showed that the biofilm was thick and dense in the DMSO control group (Figure 5Ba), while with esculetin at sub-MICs treatment, the biofilm was hindered and finally turned sparse as the concentration increased (Figure 5Bb-d). CLSM images also demonstrated similar results. After incubated in the presence of esculetin, the biofilm got sparser with the increased concentration and its thickness reduced from 16 to 11 μm (Figures 5Ca-d).
Biofilms are microbial communities, in which bacterial cells are embedded in a self-generated matrix of lipids, exopolysaccharides (EPS), proteins, and nucleic acids that can block the entry of antimicrobial agents into cells (Ramanathan et al., 2017;Zhou et al., 2017). Biofilms are closely related to the multi-drug resistance of many bacteria (Morohoshi et al., 2007). Therefore, destroying or inhibiting the formation of biofilm can be an effective way to attenuate the pathogenicity and drug resistance of bacteria. Our results suggested that esculetin significantly inhibited the biofilm formation of A. hydrophila SHAe 115, making it loose and sparse. The possible reason was that esculetin affected the synthesis of the matrix (such as EPS) composing the biofilm.

Effect of Esculetin on Gene Expression
The qRT-PCR assay was carried out to examine the effect of esculetin at 100 μg/ml on changes in the gene expression of motility and biofilm formation. The results showed that ahyI, ahyR, luxS, csgAB, and fleQ were significantly downregulated and their expression levels were reduced by 32, 29, 39, 12, and 65%, respectively. While litR was upregulated, the expression level increased by 40%. Esculetin had no obvious effect on fleN (Figure 6).
According to the reports of Kozlova et al. (2008 and and Khajanchi et al. (2009), the ahyI/R genes in A. hydrophila are homologs of lasI/R that are responsible for regulating the AHL-mediated AI-1 QS system. They demonstrated that the ahyI/R system was mainly responsible for the QS-related virulence production and biofilm formation in A. hydrophila. After treatment with esculetin, the expression levels of ahyI and ahyR decreased, which interfered the AHL-mediated AI-1 QS system. As a result, the production of virulence factors related to this system and the biofilm formation were inhibited. litR gene in A. hydrophila is a homolog of hapR gene. This gene in Vibrio cholerae encodes HapR protein that can negatively regulate bacterial biofilm formation and EPS biosynthesis (Hammer and Bassler, 2003) by lowering the intracellular level of c-di-GMP (Waters et al., 2008). Further, HapR can also negatively affect the transcription of vpsT, and the product of vpsT (VpsT) is a transcriptional activator required for the expression of EPS biosynthesis operon in V. cholerae (Yildiz et al., 2001;Casperlindley and Yildiz, 2004). While in A. hydrophila, the homologous gene of vpsT is csgAB (Kozlova et al., 2011). The increase of litR and decrease of csgAB gene may affect the biosynthesis of EPS together, thereby affecting the formation of biofilm. FleQ, encoded by fleQ, is a master regulator of flagellar gene expression in P. aeruginosa (Hickman and Harwood, 2010). FleN, encoded by fleN, is an anti-activator of FleQ which downregulates FleQ activity through direct interactions. Kozlova et al. (2011) reported that they found fleQ and fleN genes in A. hydrophila. Hence, the transcription of fleQ and fleN and interactions between FleQ and FleN play a crucial role in the motility of A. hydrophila. Another study of Kozlova et al. (2008) demonstrated that the luxS system also existed in the bacteria and it was mainly responsible for the motility. On one hand, the expression level of fleQ reduced, which affected the development of bacterial flagella. On the other hand, the expression level of luxS also reduced, and the inhibitory effect of esculetin on these two genes may jointly affect the motility of the bacteria.

CONCLUSION
The present study explored the inhibitory effect of esculetin on QS-related virulence factors and biofilm formation of A. hydrophila SHAe 115. The results showed that esculetin can significantly inhibit the production of hemolysin and protease, affect the swarming motility of A. hydrophila SHAe 115 at sub-MICs. They are all the main virulence factors of the bacteria. The formation of biofilm was also inhibited, the biofilm biomass decreased, and the biofilm structure turned thinner and sparser compared to the control group. qRT-PCR analysis indicated that genes positively related to motility and biofilm formation were downregulated to varying degrees, while gene (litR) negatively related to biofilm formation was significantly upregulated. The results of swarming motility and biofilm formation were in good agreement with gene expression analysis. Therefore, in combination with the activities against other pathogenic bacteria, esculetin has the potential to be a QS inhibitor.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
FIGURE 6 | Effect of esculetin at 100 μg/ml on the expression of QS-related genes in A. hydrophila SHAe 115. Data are presented as the expression fold changes of mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the DMSO control group by t-test.