Vibrio parahaemolyticus ATCC17802 (Guangdong Institute of Microbiology) was used as a test bacterial pathogen in this study. The cells were cultured in Tryptic Soy Broth (TSB) (3% NaCl) (Guangdong HuanKai Microbial, China) for 8 h at 37°C and then resuspended through centrifugation (2,506 × g for 10 min) in 10⁸ CFU/mL. Protocatechuic aldehyde (3,4-dihydroxybenzaldehyde, 98%) was purchased from Macklin (Shanghai, China) and the stock solutions were prepared using TSB or sterile water.

The minimum inhibitory concentrations and minimum bactericidal concentrations at different PCA concentrations were calculated using twofold dilutions. The PCA concentrations ranged from 7.8 to 1,000 μg/mL (Shili et al., 2016). The bacterial suspension was prepared using a nutrient broth containing 3% NaCl to give 10⁶ CFU/mL as a final concentration. The optical density was measured at 600 nm every 1 h for 24 h at 37°C using an automatic growth curve analyzer (Oy Growthcurves Ab Ltd.). The positive control was TSB (3% NaCl) with the bacterial suspension and without PCA. The negative control was an uninoculated TSB (3% NaCl).

Crystal violet staining was used to evaluate biofilm formation (Wang et al., 2022). Briefly, 100 μL of bacterial suspension (10⁶ CFU/mL) was seeded into 96-well plates with different concentrations of PCA (0, 9.375, 18.25, and 37.5 μg/mL) to form a biofilm. Inoculated or PCA-treated TSB (3% NaCl) was used as background. In addition, PCA (0, 9.375, 18.25, and 37.5 μg/mL) was added for 24 and 48 h. The scavenging effect of PCA on mature biofilms was detected as described by Qiao et al. (2021). The plate was incubated at 37°C for 24 h. Then, the plate was washed twice with sterile saline, dried for 30 min (25°C), stained with 200 μL of crystal violet (0.1% w/v), and incubated at 25°C for 20 min. Then, we washed the plate once with sterile saline. Subsequently, we added 200 μL of 33% (v/v) glacial acetic acid and measured the absorbance at 570 nm.

Vibrio parahaemolyticus biofilm metabolic activity was determined using MTT (Wang et al., 2010). After forming biofilms in 96-well plates for 72 h, a PBS wash was used to remove the loosely attached cells and planktonic cells (Liu et al., 2022). To each well, thiazole blue (MTT) [3,4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium bromide solution (5 mg/mL, prepared in sterile water) was added. The mixture was incubated at 37°C in the dark for 3 h. We then added 200 μL of dimethyl sulfoxide, incubated at 37°C for 30 min, and then measured the absorbance at 490 nm.

The extracellular polysaccharides in biofilms were determined using the phenol-sulfuric acid method (Cao et al., 2021). V. parahaemolyticus was treated with PCA at 37°C for 24 h. The cells were centrifuged (2,500 × g for 15 min), and the supernatant was separately mixed with a threefold volume of 95% (v/v) ethanol and precipitated at 4°C for 24 h. The mixture was then centrifuged to collect the precipitates (2,500 × g for 15 min). Then, 5% phenol and 90% H₂SO₄ were added to the precipitate, mixed, and placed in the dark for 1 h at 25°C. The final mixture was centrifuged at 9600 × g for 10 min, and the absorbance of the supernatant at 490 nm was measured.

The survival of V. parahaemolyticus under PCA inhibition was determined using a live/dead staining kit (Yu et al., 2022). Briefly, the PCA (0 and 75 μg/mL)-treated bacterial suspension was centrifuged (6,000 × g for 5 min), then 10 μL of NucGreen and EthD-III mix was added, and left in the dark for 15 min. Then the suspension was analyzed using a Zeiss fluorescence confocal microscopy (30×). Additionally, the bacterial suspension (200 μL, 10⁶ CFU/mL) and PCA solution (200 μL) were added to the 8-well chamber slides (Bhardwaj et al., 2021). The final concentrations of PCA were 75 and 300 μg/mL. The mixture was incubated at 37°C for 48 h. Then slides were washed with PBS; FITC conA (20 μg/mL) was added and stained at 25°C in the dark (30 min). Finally, the slides were washed with PBS and visualized using a Zeiss fluorescence confocal microscope (10×). The image acquisition was performed using the ZEN software. The micrographs were analyzed using the ImageJ software to evaluate the biomass and the surface volume ratio of the biofilms (Lu et al., 2021).

For the swimming exercise test (Faleye et al., 2021), PCA was added to warm (45°C) TSB medium (15 mL) containing 0.3% (w/v) agar to obtain final concentrations of 0, 7.81, 15.625, 31.25, 150, and 300 μg/mL. A swarming exercise was performed using 15 mL of TSB medium containing 0.5% (w/v) agar. The plate was then dried for 1 h; then 5 μL of bacterial suspension (1 × 10⁶ CFU/mL) was added to its center and incubated at 37°C for 12 h. The diameter of the bacterial movement zone was measured using a vernier caliper (mm), and pictures were taken with Gel DOCTM XR +. A medium without PCA was used as a control.

Vibrio parahaemolyticus was treated with 75 μg/mL of PCA (B sample) or without PCA (A sample) as a control. Two samples of PCA were added to the logarithmic growth phase bacteria and shaken at 37°C and 150 rpm for 8 h. We extracted RNA using an RNA extraction kit (TIANGEN BIOTECH CO., Ltd.). Gene library sequencing was performed using the Illumina Hiseq platform, and quality screening was performed using FastQC (Zhang Q. et al., 2020). Reference genome alignments were performed at https://www.ncbi.nlm.nih.gov/nucleotide/CP014046.2 using Bowtie2. Differential expression analysis between the two groups of samples was performed with the DESeq2 R software package (1.16.1) [Sangon Biotech (Shanghai) Co., Ltd.] with a corrected P-value of 0.05 and an absolute fold change ≥ 2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were further profiled using the cluster Profiler package.

mRNA was extracted and transcribed into cDNA using an RNA extraction kit (Vazyme, Nanjing, China) and a reverse transcription kit (HiScript II Q RT Super Mix) (Vazyme) according to the kit instructions. Primer sequences (5′–3′) were designed with Primer software (Table 1). In a RT-qPCR reaction, the total volume was 20μL, which contained 0.6 μL of 10 μM of each primer F/R, 10 μL of SYBR Mix (Vazyme Biotech, Nanjing, China), 1 μL of cDNA template, and 7.8 μL of nuclease-free water. The temperature was first maintained at 95°C for 1 min in the qPCR reaction. Then, 40 cycles of temperature change at 95°C for 10 s, annealing at 55°C for 34 s, and extension at 72°C for 15 s were performed. Changes in the expression levels of target mRNAs were calculated using the 2^–△△CT method (Zhang J. et al., 2020).

The mean and standard deviation (SD) were calculated for triplicates. Differences between variables were tested for significance using SPSS. Standard curves and other figures were completed using Origin 2021, Graph 7, and ImageJ.

The ability of PCA to inhibit the growth of V. parahaemolyticus was determined. With an increase in PCA concentration, the inhibitory ability of PCA against V. parahaemolyticus gradually increased, and the growth of V. parahaemolyticus almost completely stopped at 1,000 μg/mL (Figure 1). Using SPSS software for analysis, compared with the control group, PCA concentration (300 μg/mL) had a remarkable inhibitory effect on V. parahaemolyticus. These results indicate that PCA dose-dependently inhibited the growth of V. parahaemolyticus.

Vibrio parahaemolyticus is a typical bacterium that forms biofilms (Bhardwaj et al., 2021). The two main methods of processing biofilms are to prevent their formation or eliminate already formed biofilms (Faleye et al., 2021). Therefore, the ability of PCA to disrupt prefabricated biofilms and its ability to clear mature biofilms was investigated. At different PCA concentrations (9.375, 18.75, and 37.5 μg/mL), the biofilm formation rate decreased by 73, 80, and 83%, respectively, after 24 h and by 69.3, 73.3, and 87.4%, respectively after 48 h (Figure 2A). Under the action of PCA (37.5 μg/mL), the clearance rates of mature biofilms at 24 and 48 h reached 76.8 and 68% (Figure 2B), respectively. We speculate that PCA not only inhibits the formation of biofilms but also considerably inhibits the adhesion of bacterial.

We determined the metabolic capacity of the biofilm by measuring the metabolic activity of the cells in the biofilm (Liu et al., 2022). The intensity of the MTT releases positively correlated with cellular metabolic activity. After treatment with PCA, the cell metabolic activity increased by 23.8 and 63.5% at low PCA concentrations (9.37 and 18.75 μg/mL) (Figure 3). As shown in Figure 1, the number of bacteria did not increase at the concentration of 15.5 μg/mL compared with the control group. With an increase in PCA (75 μg/mL), the inhibitory effect on the metabolic activity of the biofilm of pathogenic bacteria increased continuously, and the metabolic activity decreased sharply by 88.59%. Then, the metabolic state of the bacteria remained stable.

EPSs are the major part of the biofilm and directly contribute to the properties of the biofilm, especially with their strong water-binding capacity. They usually account for more than 90% of the biofilm mass (Liu et al., 2022). We used the sulfate and phenol methods to measure the polysaccharide content for evaluating the effect of PCA on EPS production. As shown, the polysaccharide content in the V. parahaemolyticus biofilms decreased significantly in the presence of PCA. When PCA concentration increased from 37.5 to 75 μg/mL, the polysaccharide content decreased by 27.6, 44.3, and 48% (Figure 4). The amount of biofilm formed, the biofilm metabolic activity, and the amount of polysaccharide produced decreased significantly at 75 μg/mL.

During the initial phase of biofilm formation, movement of V. parahaemolyticus is critical for the attachment to host surfaces (Faleye et al., 2021). Loss of movement may affect bacterial adhesion, impairing biofilm formation (Zhu et al., 2020). As shown, PCA remarkably inhibited the swimming and swarming ability of V. parahaemolyticus at 75 μg/mL concentration (Figures 5C,D). Compared with the untreated cells, the swimming Bacterial colonies area (75 and 150 μg/mL) decreased by 81.4% (Figure 5B) and 93.1%, respectively. Meanwhile, the swarming Bacterial colonies area decreased by 56.9 and 73.7% (Figure 5A), respectively.

As shown in the control group, the bacteria were wrapped in large amounts of polysaccharides, forming a large biofilm structure with a thick membrane structure (Figures 6A,D). The number of dead bacteria increased after 48 h of treating the mature biofilms with a high concentration (300 μg/mL) of PCA. In addition, biofilm formation decreased by 83.5%, and the biofilms were dispersed with a substantial reduction in thickness. The fluorescence intensity in the three-dimensional optical microscopy images changed remarkably (Figures 6C,F; Lu et al., 2021).

The number of viable bacteria was remarkably lower than that of the dead bacteria accounting for 63.7% of the total bacteria (Figure 6C). Visual results obtained with a microscope indicate that PCA reduced the biofilm of V. parahaemolyticus accounting for 4% (Figure 6A; Shangguan et al., 2021). The polysaccharide matrix was considerably reduced in the biofilms formed at 75 μg/mL PCA concentration. The overall membrane structure was dispersed. The biofilm area decreased by 71%, and showed low thickness variation (Figures 6B,E). The number of viable bacteria decreased by 88% under PCA (75 μg/mL) inhibition (Figure 6B).

Global transcriptional analysis revealed a differential expression of 142 genes, approximately 8 h after exposure to 75 μg/mL PCA, with 63 genes upregulated and 79 genes downregulated. This mainly manifested in cell motility (Figure 7A), cell growth and death, signaling, and energy metabolism. Combined with the metabolic pathways provided by the KEGG database (Figure 7B). We found that the pathways significantly enriched in V. parahaemolyticus mainly included Carbohydrate metabolism, Amino acid metabolism, ABC transport process, Ribosome, Two-component system (i.e., not complete). Up-regulated or down-regulated genes are shown in Table 2.

We analyzed the data by combining the metabolic pathways and GO annotations provided by the KEGG database. We found up-regulation of Nitrate/nitrite response regulator (narL) (Figure 7C) and down-regulation of ATP-dependent 6-phosphofructokinase (pfkA). Regulation of these two genes inhibited the expression of frdA and frdB, thereby regulating the two-component system and pentose phosphate pathway. Previous studies have found that inhibition of the bacterial two-component system (Cho and Sung-il, 2021) and pentose phosphate pathway (Kong et al., 2022) may promote carbohydrate intake and dephosphorylation of the phosphotransferase system. Kong et al. (2022) found that the inhibition of the pentose phosphate pathway may reduce the intracellular accumulation of carbohydrates such as glucose, thereby inhibiting bacterial exopolysaccharide synthesis and biofilm formation; Meanwhile, it may also reduce the efficiency of glucose transport, thereby destroying the energy metabolism of bacteria and inhibiting its cell viability.

After PCA was applied to V. parahaemolyticus, glycolysis and TCA cycle were also involved in the internal regulation of the bacteria. The up-regulation of isopropyl malate synthase (IMS) and alanine dehydrogenase (ald) affect acetyl-CoA formation, thereby regulating glycolysis and TCA cycle. In addition, the up-regulation of transcription factor glucose-6-phosphate 1 dehydrogenase (G6PD) promotes the conversion of NADP to NADPH. Tan et al. (2022) found that after antibiotic inhibition, internal carbon and nitrogen metabolism in V. parahaemolyticus is involved in the activation of bacterial glycolysis and TCA cycle to promote ATP accumulation, and the increase of NADPH maintains a stable cellular state and also promotes amino acid metabolism to enhance antibiotic tolerance. At the same time, we found that enoyl-CoA hydratase (paaF) and 3-hydroxyalkyl-CoA dehydrogenase (fadB) were up-regulated in V. parahaemolyticus, which may lead to enhanced tryptophan or β-alanine metabolism. Dukes et al. (2015) found that by promoting tryptophan or β-alanine metabolism, producing pyruvate and acetyl-CoA to activate mTOR, it would promote glycolysis and TCA cycle. However, this promotion of glycolysis and the TCA cycle by pyruvate and acetyl-CoA reaches a threshold (Yang et al., 2020).

The metabolism of amino acids in V. parahaemolyticus also changes after PCA inhibition. The down-regulation of L-ectoine synthase (ectC), aspartate kinase (lysC), cysteine synthetase (cysK), and asparagine synthetase (asnB) related genes involved in amino acid synthesis (Figure 7C) may result in the decreased amino acid synthesis of aspartic acid, glutamic acid, threonine, and cysteine. Down-regulation of small subunit ribosomal protein S18 (rpsR) also directly affects amino acid substitutions. In ribosomes, the small subunit ribosomal protein S15 (IF) responsible for adhesion and invasion is downregulated. At the same time, the iron (III) transport system substrate-binding protein (fbpA) and putative amino acid ABC transporter substrate-binding protein (yxeM) were also significantly down-regulated. In biofilm proteomics of V. parahaemolyticus, Guo et al. (2020) found that increased glutamate and threonine promoted the synthesis of extracellular proteins in biofilms and down-regulation of cysK gene may regulate the secretion of toxins by bacteria to inhibit the growth of neighboring cells. Zhu et al. (2020) found in their proteomic studies on V. parahaemolyticus that the ABC transport system is closely related to bacterial adhesion ability. Thereby down-regulation of genes related to the ABC transport system may hinder material transport and reduce bacterial adhesion and biofilm formation.

In addition, under the inhibition of PCA, the chemotaxis receptor response regulator (cheY) in the internal genes of V. parahaemolyticus was down-regulated, and cheY could directly inhibit Flageller motor switch adaptation. A gene associated with bacterial quorum sensing, oligopeptide transport system substrate-binding protein (oppA), was down-regulated. Upregulation of 5’-deoxynucleotides (yfbR) associated with eDNA synthesis in biofilms is upregulated. This can lead to reduced motility, adhesion, and biofilm production (Kong et al., 2020). Chang et al. (2020) found that cheY could modulate Flageller motor in bacteria, thereby affecting bacterial motility. Sun et al. (2022) hypothesized that quorum sensing may be involved in regulating pilus production to control biofilm generation. Renfei et al. (2019) and Wu et al. (2022) found that quorum-sensing regulators affect gene transcription in the lateral flagella of V. parahaemolyticus to regulate swarming movement. In general, PCA inhibits the biofilm of V. parahaemolyticus by regulating a variety of genes.

We screened biofilm-related genes by high-throughput sequencing and verified the reliability of the data by RT-qPCR. Four significantly decreased genes and one significantly increased gene were screened. The gene narL related to bacterial metabolism was up-regulated. The relative expression of this gene with values of 217% of the control group (Figure 3). The ABC transport system genes fbpA and yxeM related to biofilm clearance and production were down-regulated. rpsR gene related to amino acid synthesis was down-regulated. The bacterial growth-related gene ectC was down-regulated. The relative expression of these genes was significantly reduced with values of 1.4%, 13.5, 3.7, and 7.5% of the control group, respectively. These results demonstrated that 75 μg/mL PCA could effectively inhibit the biofilm formation of V. parahaemolyticus.