Bacteriostatic effects of benzyl isothiocyanate on Vibrio parahaemolyticus: Transcriptomic analysis and morphological verification

Background Foodborne illness caused by Vibrio parahaemolyticus (V. parahaemolyticus) is generally associated with the consumption of seafood. Fish and other seafood can be contaminated with V. parahaemolyticus, natural inhabitants of the marine, estuarine, and freshwater environment. In this study, the antibacterial activities of benzyl isothiocyanate (BITC) against V. parahaemolyticus were investigated by both transcriptomic analysis and morphological verification. Results Treatment with 1/8 minimum inhibitory concentration (1/8 MIC) BITC resulted in 234 upregulated genes and 273 downregulated genes. The results validated by quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the relative expression levels of the six genes VP0820, VP0548, VP2233, VPA2362, fliA and fliG were only 31.0%, 31.1%, 55.8%, 57.0%, 75.3%, and 79.9% of the control group, respectively. Among them, genes VP2233, fliA and fliG are related to flagella and VP2362 can regulate a protein relevant to biofilm formation. Morphologically, we verified that the swimming diffusion diameter of V. parahaemolyticus was significantly reduced by 14.9% by bacterial swimming ability, and biofilm formation was significantly inhibited by treatment with 1/8 MIC BITC by crystal violet quantification assay. Conclusions These results indicated that 1/8 MIC BITC had antibacterial effect on V. parahaemolyticus by inhibiting virulence gene expression related to flagella and biofilm. Supplementary Information The online version contains supplementary material available at 10.1186/s12896-021-00716-4.


Background
Vibrio parahaemolyticus is a moderately halophilic gram-negative bacterium mainly in the form of rods, arcs, etc., without capsules and spores, and has become the main reason of aquatic product poisoning worldwide since it was identified in 1950 [1]. The colonies of V. parahaemolyticus CGMCC 1.1614, known as ATCC 33,845, are round, smooth, complete and cream colored. It can grow at temperature of 5 to 44 °C, and survive in the pH range from 4.8 to 11.0. It is common in coastal estuaries and marine environments and is usually associated with seawater and marine organisms such as zooplankton, plankton, mollusks and shellfish, fish and crabs [1]. Bacterial gastroenteritis related to seafood is mainly caused by direct or cross-contamination by V. parahaemolyticus [1]. Poisoning usually manifests as diarrhea, headache, vomiting, nausea, abdominal cramps and other symptoms. Therefore, it is necessary to take various measures to reduce the contamination of V. parahaemolyticus.
Antibiotics have long been applied to prevent and control marine pollution, but their long-term use has led to bacterial resistance to antibiotics and even the induction of aplastic anemia in human [2]. The hydrolysis of glucosinolate can produce isothiocyanates (ITCs), which are organic sulfides in cruciferous plants. ITCs have shown various beneficial effects, including antibacterial, antiviral, anti-inflammatory, anticancer, neuroprotective, chemical prevention and anti-parasitic properties. Benzyl isothiocyanate (BITC) is a type of ITC strong antibacterial capacity. Studies have found that BITC indicates antifungal activity and can effectively inhibit various molds [3] and bacteria, such as Campylobacter jejuni [4], Salmonella typhimurium [5], Staphylococcus aureus [6]. Studies have indicated the antibacterial mechanism of BITC. For example, BITC can affect the biofilm integrity, bacterial morphology, and membrane potential of Pseudomonas aeruginosa, E. coli and S. aureus [7]. However, knowledge about the antibacterial effects of BITC on V. parahaemolyticus is limited.
Scholars have conducted studies on the antibacterial mechanisms of natural products in V. parahaemolyticus. The major constituent of black seed volatile oil could affect quorum sensing, biofilm, and virulence-associated genes to reduce the virulence of V. parahaemolyticus [8]. Dihydromyricetin is the main bioactive component of Ampelopsis grossedentata, which can inhibit V. parahaemolyticus by decreasing the activity of proline dehydrogenase and inducing an increase in cell injury, proline content and cell surface hydrophobicity [9]. Banu et al. [10] studied the effect of essential oil from Cinnamomum tamala on V. parahaemolyticus and found that the virulence factors were regulated by polysaccharides, cytotoxins, flagella, lipopolysaccharides and biofilms. Song et al. [11] used the transcriptome to study the antibacterial effect of 1/4 MIC BITC on V. parahaemolyticus. However, no study has reported the bacteriostatic effect of BITC on V. parahaemolyticus by combining transcriptional level and morphological characteristics. Therefore, the antibacterial mechanism of BITC against V. parahaemolyticus can be studied from the genetic level and the destruction of the bacterial membrane.
In the current study, we determined the antibacterial effect of BITC at a 1/8 MIC subinhibitory concentration on V. parahaemolyticus. The differentially expressed genes (DEGs) were detected by RNA-seq, from which we screened out genes related to virulence and verified their expression by qRT-PCR. Morphologically, the influence of BITC on V. parahaemolyticus motility and biofilm formation was also verified.

Bacterial strain and culture
The V. parahaemolyticus CGMCC 1.1614 (tdh + , tlh + , trh -) was purchased from China General Microbiological Culture Collection Center. The strain was stored in physiological saline with 10% glycerol at − 80 °C. 100 µL of frozen bacteria solution was added into 10 mL tryptone soy broth medium with 3% sodium chloride (3% NaCl-TSB) liquid medium and cultured at 37 °C overnight. The cultured bacterial cells were streaked onto tryptone soy agar medium with 3% sodium chloride (3% NaCl-TSA) medium and incubated at 37 °C. Next, the activated single colony was inoculated into 3% NaCl-TSB liquid medium and cultured with shaking at 37 °C.

Antimicrobial tests
BITC was purchased from Sigma-Aldrich (CAS: 622-78-6), and its Flavor and Extract Manufacturing Association (FEMA) number is 4428. The MIC of BITC of V. parahaemolyticus was determined to be 9.54 µmol/L by the broth microdilution method. 1/8 MIC BITC was added to 100 mL 3% NaCl-TSB liquid culture medium with V. parahaemolyticus in log phase. Bacterial solution without BITC was incubated at 37 °C on a shaker (150 r/min) for 6 h. Bacterial solution without BITC was used as a control.

Enrichment and sequencing of RNA
V. parahaemolyticus was treated with 1/8 MIC BITC (E_BITC) or without BITC (C_BITC) for 6 h. Total RNA was extracted by the RNAprep Pure Cell/Bacterial Kit (Tiangen Biotech, Beijing, China) as recommended by the manufacturer. The preparation of sequencing library used the Illumina's NEBNext UltraTM Directed RNA Library Preparation Kit (NEB, Ipswich, MA, USA), and then added the index codes. All the samples were evaluated for product and library quality using an Agilent Bioanalyzer 2100 system (G2939B; Agilent Technologies, Palo Alto, CA, USA). The recount data obtained in the gene expression level analysis by DEGseq 2 software were used to analyze and screen the DEGs, wherein the screening standard was p < 0.05. DEGs is analyzed using volcanic plots, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The statistical enrichment of DEGs in the KEGG pathway was analyzed by KOBAS software.

qRT-PCR verification of the RNA-seq results
We used qRT-PCR to investigate the differential gene expression between different groups with 16S rRNA as the reference gene. The annealing temperature was set at 58 °C and the obtainment of melting curve was 60 to 95 °C. Additionally, the relative expression was calculated by 2 −∆∆Ct method. The specific primer sequences for qRT-PCR are listed in Additional file 1. The genomic DNA was removed from the total RNA as recommended by instructions. The cDNA templates were reverse transcribed using the PrimeScript ™ RT Reagent kit with gDNA Eraser (TaKaRa, Dalian, China). qRT-PCR was performed by using SYBR ® Premix Ex Taq ™ II (TliRNaseH Plus) (Takara, Dalian, China) as recommended by the manufacturer.

Mobility measurement
The mobility of V. parahaemolyticus was determined based on the method of Butler et al. [12]. One milliliter of the log phase of V. parahaemolyticus was added to 99 mL of fresh 3% NaCl-TSB liquid medium, followed by incubation for 6 h. Next, 15 mL of swimming medium was sterilized and cooled to approximately 45 °C, then added 500 μL of 1/8 MIC BITC stock solution or physiological salin as the experimental group or control group, respectively. Thereafter, 3 μL of bacterial solution was added dropwise to the plate, and then the diameter of the inhibition zone was counted after incubation at 37 °C for 12 h. The average diameters of inhibition zone of the control group and experimental group were obtained to show the antibacterial effect of BITC on V. parahaemolyticus mobility. The data comparison of each group was analyzed by student's t test. P < 0.05 indicated a statistically significant difference.

Biofilm measurement
The biofilm formation of V. parahaemolyticus affected by BITC was investigated according to the method of Pratt and Kolter [13]. Three-percent NaCl-TSB liquid medium, bacterial solution and 1/8 MIC BITC were added to a 96-well plate at ratio of 8:1:1, followed by incubating at 25 °C to form the biofilm. 3% NaCl-TSB liquid medium with and without bacterial culture was used as the controls. After treatment with phosphate buffer saline (PBS), anhydrous methanol, crystal violet and glacial acetic acid, the absorbance was measured at 590 nm using a microplate reader (Spectra Max M2, Molecular Devices, CA, USA). The experiment was repeated three times, and each group included three parallel determinations. The data comparison of two groups was analyzed by student's t test. P < 0.05 indicated a statistically significant difference.

Transcriptome results
In our previous studies, Song et al. [11] determined the sensitivity of V. parahaemolyticus CGMCC 1.1614 to BITC, and the MIC was 9.54 μmol/L. Transcriptome analysis can reveal the underlying molecular mechanisms by which food additives act on pathogens. We performed transcriptome sequencing by the Illumina Hi Seq TM2500 high-throughput sequencing platform, generating two transcriptome databases for the control group without BITC (C_BITC) and the experimental group with 1/8 MIC BITC treatment (E_BITC) to understand the bacteriostatic effect of BITC on V. parahaemolyticus. The RNA-seq results were uploaded to the Gene Expression Omnibus (GEO) database, numbered GSE152671. The total quantity of sequencing data we obtained was 9.52 Gb, the error rate of the single-base position of all samples was less than 1%, and Q20 was greater than 80%, which were up to the standards of sequencing quality control (see Additional file 2).
The volcano map shows the distribution of DEGs. According to Fig. 1, E_BITC obtained 507 DEGs compared with C_BITC, among which 234 were upregulated and 273 were downregulated.
The top 10% genes with the most significantly differential expression were shown in the Table 1, of which 19 were up-regulated and 31 were down-regulated. It was shown that BITC regulated 14 enzymerelated genes, including cryptic beta-D-galactosidase subunit alpha, membrane-bound lytic murein transglycosylase D, orotate phosphoribosyltransferase, longchain-fatty-acid-CoA ligase, manganese-dependent inorganic pyrophosphatase, oxaloacetate decarboxylase subunit gamma, carbamoyl phosphate synthase small subunit, GTP cyclohydrolase I, ribonuclease PH, quinolinate phosphoribosyltransferase, 5'-deoxynucleotidase, short chain dehydrogenase, glycerol kinase and cytidylate kinase. Oxaloacetate decarboxylase α/β/γ complex is a membrane-bound enzyme complex in Vibrio cholerae, and its assembly is affected the oxaloacetate decarboxylase subunit gamma regulated by VP2545 [14]. The influence of BITC on the activities of these enzymerelated genes should be discussed in the further study. BITC also showed effects on the other genes related to bacterial virulence. For examples, BITC upregulated molecular chaperone DnaK, which is related to protein folding [15]. BITC also affected the expression of transfer protein genes such as uhpT and VP2869, which regulate sugar phosphate antiporter and sodium/solute symporter, respectively (Table 1). Membrane associated protein genes such as VP1286 and VP1091, which regulate integral membrane protein and transmembrane protein, respectively, affecting cell membrane permeability, biofilm formation and septum formation (Table 1). BITC also regulated some functional proteins, such as sodium/solute symporter, carbon starvation protein A, integral membrane protein, heavy metal membrane efflux protein, RhlE protein, sugar phosphate antiporter, transmembrane protein affecting septum formation and cell membrane permeability, lipoprotein, heat shock protein 90, phosphate ABC transporter ATP-binding protein, integral membrane protein transporter, SpoOM-like protein, 30S ribosomal protein S21, thiamine biosynthesis protein ThiC, NadC family protein and nucleoid occlusion protein. Among them VP0821-regulated heat shock protein 90 is important for the virulence and spread of protozoan parasites [16].
Besides, rpsU-encoded ribosomal protein S21 can affect the motility and biofilm formation of Bacillus subtilis [17]. The down-regulated genes VP1892 and FliG are related to bacterial chemotaxis (see Additional file 3). FliG regulates flagellar motor switch protein G (Table 2) and VP1892 regulates methyl-accepting chemotaxis protein. VP2826 and VPA1006 were down-regulated, regulating transporter and LysR family transcriptional regulator, respectively. According to the objective of present study, we focused on the antibacterial mechanism of BITC by screening differentially expressed genes related to virulence, as shown in Table 2 (VP0820, VP0548, VP2233, VPA2362, fliA and fliG), in which VP0820, VP0548 can regulate ToxR, thereby affecting the virulence of V. parahaemolyticus; VP2233, fliA and fliG genes are related to flagella, thereby affecting motility, and VP2362-encoded outer membrane protein that is related to the biofilm formation.
GO analysis was performed on the DEGs in V. parahaemolyticus treated with 1/8 MIC BITC. We found that these genes were annotated into 1679 GO terms, and the enrichment was selected from each group. Thirty significant GO terms were identified, with three main domains, molecular functions (12 subclasses), biological processes (10 subclasses) and cellular components  , and gidB, which regulate methylated-DNA-protein -cysteine S-methyltransferase, ubiquinone/menaquinone biosynthesis methyltransferase, RNA methyltransferase, 16S rRNA (cytosine(1407)-C(5))-methyltransferase RsmF, 3-demethylubiquinone-9 3-methyltransferase, 16S rRNA methyltransferase and 16S rRNA methyltransferase GidB (see Additional file 4). Some studies have confirmed that methyltransferase can affect bacterial motility and biofilm formation [19]. These terms might be related to the motility and biofilm formation in V. parahaemolyticus. The analysis of KEGG enrichment was conducted following GO analysis. In total, 507 DEGs were enriched into 78 pathways compared with the control group. Bacterial chemotaxis pathways can affect the direction of bacterial movement by adjusting the rotation direction of flagella movement. Bacterial chemotaxis pathways included 7 differentially expressed genes, of which VP1628, VP1892, VP1904, VP2629, and VPA1000 regulate methyl-accepting chemotaxis protein, while VP2230 and fliG regulate chemotaxis protein CheZ and flagellar motor switch protein G, respectively (see Additional file 3). Flagellar assembly pathways included three DEGs flgF, fliI, and fliG, which are related to flagellar basal body rod protein FlgF, flagellar-specific ATP synthase and flagellar motor switch protein G, respectively (see Additional file 3). Figure 2b shows 20 pathways with the most enrichment, such as ABC transporters, biosynthesis of secondary metabolites, carbon metabolism, microbial metabolism in diverse environments and metabolic pathways.

qRT-PCR verification of the RNA-seq results
qRT-PCR is a common and important technical method to study the gene expression levels. The DEGs related to virulence were screened by RNA-seq, and the reliability of the data was verified by qRT-PCR. According to RNA-seq, six genes related to virulence with obviously decreased expression were screened ( Table 2). The expression of six virulence-related genes was investigated in V. parahaemolyticus treated with 1/8 MIC BITC by qRT-PCR. The virulence-related genes VP0820, VP0548, VP2233, VPA2362, fliA, and fliG were downregulated (Fig. 3). The relative expression of these genes was significantly reduced with values of 31.0%, 31.1%, 55.8%, 57.0%, 75.3%, and 79.9% of the control group, respectively. These results verified that BITC at 1/8 MIC could effectively inhibit virulence-related gene expression of V. parahaemolyticus.

Mobility measurement
The motility of V. parahaemolyticus is affected by the flagella of bacterium, which are connected with the virulence of bacteria. According to the transcriptome results, the expression of flagella-related genes VP2233, fliA, and fliG was downregulated. By detecting the movement of V. parahaemolyticus, we verified the antibacterial effect of BITC on V. parahaemolyticus morphologically. The motility of V.
parahaemolyticus is related to the flagella of bacterium. As shown in Fig. 4, BITC at 1/8 MIC significantly suppressed the mobility of V. parahaemolyticus. BITC significantly reduced the diameter of swimming diffusion by 14.9% (p < 0.05) compared with that of control group. This finding agreed with the downregulation of VP2233, fliA, and fliG gene expression in the transcriptome results (Table 2). Therefore, swimming is related to the bacteriostatic effect of BITC in V. parahaemolyticus.

Biofilm measurement
The formation of biofilm is related to the virulence of bacteria. In most cases, bacteria adhere to the surface of object during formation of biofilms to maintain survival, causing food safety problems. The mechanism of BITC in food-borne pathogenic bacteria can be investigated by exploring the biofilm changes of V. parahaemolyticus after BITC treatment. VP2362 encodes an outer membrane protein, which is related to the biofilm formation of V. parahaemolyticus. According to the transcriptome results, the relative expression of this gene was downregulated by treatment with BITC. We expected to verify the transcriptome results by measuring the biofilm formation of V. parahaemolyticus and understand the mechanism of BITC effects. The formation of biofilm of V. parahaemolyticus was significantly inhibited by 1/8 MIC BITC treatment (p < 0.05), and BITC functioned to scavenge biofilm (Fig. 5). These results were consistent with the downregulation of VP2362 gene expression (see Additional file 4).

Discussion
Transcriptome sequencing technology is an important technical approach to screen DEGs and determine gene functions. It has advantages in terms of time, data yield, cost, coverage, and accuracy of data [20]. It is also possible to obtain gene-related biological function data by analyzing multilevel gene regulation expression. Studies based on prokaryotic transcriptome sequencing have included E. coli [21], S. aureus [22], and Listeria monocytogenes [23]. Moreover, the difference in gene expression of V. parahaemolyticus in seawater has been studied under different oxygen environments [24]. Therefore, RNA sequencing was applied in this study to understand the corresponding changes of V. parahaemolyticus CGMCC 1.1614 under the action of 1/8 MIC BITC. According to the research purpose, we screened the DEGs related to virulence at the molecular level and then verified the gene expression using qRT-PCR. From a molecular biology perspective, the impact of BITC on both the virulence and morphology of V. parahaemolyticus was also investigated. Studies have shown that the motility of bacteria will be inhibited when sodium ion transport is disturbed [25]. Oxidoreductase and   transferase are very important in the formation of biofilms [26,27]. RNA sequencing results of Salmonella Enteritidis under acid stress have shown that the terms of macromolecular complexes, transferase activity, and transferring one-carbon groups are the most highly represented [28]. Combined with the GO analysis results, multiple pathways related to motility and biofilm were significantly enriched (sodium ion transport, oxidoreductase activity, transferase activity, transferring onecarbon groups and macromolecular complex). Therefore, it is necessary to investigate the influence of BITC on V. parahaemolyticus of motility and biofilm formation.
Recent findings have confirmed that flagella are adhesive and invasive as a potential class of virulence factors [29]. Bacterial motility is closely related to flagella, and V. parahaemolyticus has two different types of flagella to accommodate life in different situations [24]. Studies have shown that bacterial motility is also inhibited when flagella are affected. When histone-like nucleoid structuring protein (H-NS) acted on the flagellin lateral flagellar A (lafA), the swarming motilities of Vibrio hemolyticus [30] was inhibited. Quinazoline-2, 4-diamino analogs inhibit the aggregation of polar flagella and movement of V. cholerae [31], and phenethyl isothiocyanate (PEITC) can reduce the migration capacity of E. coli [32]. In this study, the transcriptome results indicated that the flagella-related gene expression of VP2233, VP2232, and VP2248 were significantly inhibited by 1/8 MIC BITC treatment. Additionally, the morphological verification results of Fig. 4 were similar to the transcriptome results, further proving that the flagella and bacterial motility are related to the bacteriostatic effects of BITC on V. parahaemolyticus.
The antimicrobial effects of ITCs are thought to be associated with damage to bacterial membrane integrity [33]. ITCs, including BITC, prevent the formation of biofilms in various bacteria such as P. aeruginosa, L. monocytogenes, S. aureus, and E. coli [32]. VP2362 is a gene encoding an outer membrane protein, which is connected with the biofilm formation of V. parahaemolyticus. By BITC treatment, gene expression level of VP2362 was significantly downregulated compared with that of control group. Previous findings have confirmed that the cell surface integrity, neutralizing host defense mechanisms, cell adhesion and invasion, and inhibition of the complement system are all affected by outer membrane proteins [34]. Therefore, the outer membrane protein is considered to be a virulence factor. The expression of membrane genes was significantly downregulated after BITC treatment at 1/8 MIC (p < 0.05). We found that the formation of V. parahaemolyticus of biofilm was inhibited by BITC treatment, which was consistent with the gene expression results.
In this paper, the bacteriostatic effect of BITC on V. parahaemolyticus was reported by RNA-seq technology from the gene level, showing multiple pathways of secondary metabolites. Various virulence genes were inhibited that were further verified morphologically. Further studies will focus on other important DEGs and the corresponding proteomics studies to obtain more comprehensive mechanism for bacteriostatic effects of BITC.

Conclusions
In this paper, the bacteriostatic effect of BITC on V. parahaemolyticus was reported by RNA-seq technology from the gene level, showing multiple pathways of secondary metabolites. Various virulence genes were inhibited that were further verified morphologically. Further studies will focus on other important DEGs and the corresponding proteomics studies to obtain more comprehensive mechanism for bacteriostatic effects of BITC.