Effect of sublethal dose of chloramphenicol on biofilm formation and virulence in Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus isolates are generally very sensitive to chloramphenicol. However, it is usually necessary to transfer a plasmid carrying a chloramphenicol resistance gene into V. parahaemolyticus to investigate the function of a specific gene, and the effects of chloramphenicol on bacterial physiology have not been investigated. In this work, the effects of sublethal dose of chloramphenicol on V. parahaemolyticus were investigated by combined utilization of various phenotypic assays and RNA sequencing (RNA-seq). The results showed that the growth rate, biofilm formation capcity, c-di-GMP synthesis, motility, cytoxicity and adherence activity of V. parahaemolyticus were remarkably downregulated by the sublethal dose of chloramphenicol. The RNA-seq data revealed that the expression levels of 650 genes were significantly differentially expressed in the response to chloramphenicol stress, including antibiotic resistance genes, major virulence genes, biofilm-associated genes and putative regulatory genes. Majority of genes involved in the synthesis of polar flagellum, exopolysaccharide (EPS), mannose-sensitive haemagglutinin type IV pilus (MSHA), type III secretion systems (T3SS1 and T3SS2) and type VI secretion system 2 (T6SS2) were downregulated by the sublethal dose of chloramphenicol. Five putative c-di-GMP metabolism genes were significantly differentially expressed, which may be the reason for the decrease in intracellular c-di-GMP levels in the response of chloramphenicol stress. In addition, 23 genes encoding putative regulators were also significantly differentially expressed, suggesting that these regulators may be involved in the resistance of V. parahaemolyticus to chloramphenicol stress. This work helps us to understand how chloramphenicol effect on the physiology of V. parahaemolyticus.

Introduction

Vibrio parahaemolyticus (V. parahaemolyticus), the leading cause of seafood-related gastroenteritis, produces multiple virulence factors playing different roles in its pathogenesis, including thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), type III secretion systems (T3SS1 and T3SS2), type VI secretion systems (T6SS1 and T6SS2), lipopolysaccharide and extracellular proteases (Cai and Zhang, 2018; Osei-Adjei et al., 2018; Li et al., 2019; Pazhani et al., 2021). Both TDH and TRH have hemolytic activity, but only TDH can cause β-type hemolysis on Wagatsuma agar called the Kanagawa phenomenon (Xu et al., 1994; Cai and Zhang, 2018). TDH also possesses the lethal toxicity, cytotoxicity, and enterotoxicity (Park et al., 2004; Hiyoshi et al., 2010; Raghunath, 2014; Cai and Zhang, 2018). T3SS, a needle-like injection system, can manipulate host cell functioning by injecting toxic proteins into host cells (Pais et al., 2023). Both T3SS1 and T3SS2 are required for the full virulence of V. parahaemolyticus (Hiyoshi et al., 2010; Matsuda et al., 2012; Zhou et al., 2013; Gu et al., 2020; Hu et al., 2021; Lin et al., 2021). However, T3SS1 plays significant roles in cytotoxic activity against various cell lines and lethal activity in mice, whereas T3SS2 is the major contributor to enterotoxicity of V. parahaemolyticus (Hiyoshi et al., 2010). Moreover, T6SS1 possesses antibacterial activity and thus is required for the environmental fitness of V. parahaemolyticus, whereas T6SS2 plays a role in cell adhesion (Yu et al., 2012; Salomon et al., 2013; Coulthurst, 2019).

Vibrio parahaemolyticus is often found in the form of biofilms, which are matrix-enclosed, surface associated communities (Yildiz and Visick, 2009; Ruhal and Kataria, 2021). Some specific structures such as flagella, exopolysaccharide (EPS) and type IV pili are required for the mature biofilm formation of V. parahaemolyticus (Enos-Berlage et al., 2005; Shime-Hattori et al., 2006; Yildiz and Visick, 2009; Jung et al., 2019; Li et al., 2020; Ruhal and Kataria, 2021; Liu et al., 2022). V. parahaemolyticus expresses two kinds of flagellar systems, a polar flagellum for swimming in liquid and lateral flagella for swarming on the surface (McCarter, 2004). Loss of polar flagellum prevents V. parahaemolyticus to form mature biofilms (Enos-Berlage et al., 2005). The cpsA-K and scvA-O gene clusters are responsible for the synthesis of EPS in V. parahaemolyticus (Liu et al., 2022). Strains without cps genes form smooth colonies on the agar plate, whereas those without scv genes form wrinkly colonies (Liu et al., 2022). The wrinkly phenotype spreader has stronger biofilm formation capacities than the smooth phenotype strain (Wu et al., 2022). V. parahaemolyticus possesses two kinds of type IV pili, mannose-sensitive haemagglutinin type IV pili (MSHA) and chitin-regulated pili (ChiRP) (Shime-Hattori et al., 2006). MSHA is required for bacterial attachment, whereas ChiRP plays a role in bacterial agglutination (Shime-Hattori et al., 2006). V. parahaemolyticus biofilm formation is also strongly correlated with the contents of proteins and DNA in the extracellular polymeric substance (Li et al., 2020). In addition, some regulators such as QsvR (Zhang et al., 2019, 2023b), H-NS (Sun et al., 2014; Zhang et al., 2018; Xue et al., 2022) and ToxR (Chen et al., 2018; Zhang et al., 2018) and some regulatory processes such as quorum sensing (QS) pathways (Wang et al., 2013; Zhang et al., 2019; Liu et al., 2021; Zhang et al., 2023b) and cyclic di-GMP (c-di-GMP) (Kim and McCarter, 2007; Tamayo et al., 2007; Kimbrough et al., 2020; Kimbrough and McCarter, 2021; Zhong et al., 2022) are involved in the regulation of virulence and biofilm formation of V. parahaemolyticus.

Almost all V. parahaemolyticus isolates exhibited multiple antibiotics resistances, especially to ampicillin and colistin (Amalina et al., 2019; Mok et al., 2021; Sun et al., 2022; Zaafrane et al., 2022). Previously, two class 1 integrons, dfrA14-bla_VEB-1-aadB and bla_VEB-1-aadB-arr2-cmlA-bla_OXA-10-aadA1, which are strongly associated with the resistance to multiple antibiotics such as ampicillin, ceftazidime and cefotaxime, were detected in V. parahaemolyticus (Lei et al., 2020). Additionally, a total of 12 resistance nodulation cell division (RND)-type efflux transporters playing important roles in intrinsic resistance to antibiotics were estimated in V. parahaemolyticus, among which VmeAB, VmeCD, VmeEF, VmeYZ have already been characterized (Matsuo et al., 2007, 2013). Majority of V. parahaemolyticus isolates are sensitivity to chloramphenicol (Amalina et al., 2019; Lei et al., 2020; Mok et al., 2021; Zaafrane et al., 2022). However, a novel chloramphenicol acetyltransferase gene, catC, is distributed among the V. parahaemolyticus strains, and heterologously expressing this gene was able to enhance chloramphenicol resistance of E. coli (Zhang et al., 2019).

The suicide plasmid pDS132 and the L-arabinose induced plasmid pBAD33 are widely used to construct specific mutants and complemented mutants, respectively (Guzman et al., 1995; Philippe et al., 2004). Both pDS132 and pBAD33 harbor a chloramphenicol resistance gene, but V. parahaemolyticus strains transformed with pDS132 or pBAD33 were still unable to grow in the conditions containing chloramphenicol higher than 5 μg/mL (Sun et al., 2014; Zhang et al., 2018, 2023b; Zhang Y. et al., 2023). Therefore, chloramphenicol probably has great effect on the growth and physiology of V. parahaemolyticus. In this work, we aimed to investigate the sublethal dose of chloramphenicol on the physiology of V. parahaemolyticus. Notably reductions in growth, motility, biofilm formation, c-di-GMP production, cytoxicity and adhesion activity were evident in the response to sublethal doses of chloramphenicol. Sublethal dose of chloramphenicol greatly affected the expression of 650 genes in V. parahaemolyticus, including the antibiotic resistance genes, major virulence genes, biofilm-associated genes and putative regulatory genes. This work helps us to understand how chloramphenicol effect on the physiology of V. parahaemolyticus.

Materials and methods

Growth conditions

Vibrio parahaemolyticus RIMD2210633, which was isolated from a diarrhea patient in 1996 (Makino et al., 2003) and kindly provided by Dr. Dongsheng Zhou from Beijing Institute of Microbiology and Epidemiology, was used throughout in this work. Approximately 20 μL glycerol stock of bacterial cells was inoculated into 5 mL 2.5% (w/v) Bacto heart infusion broth (HI broth; BD Bioscience, USA), followed by incubated at 37°C with shaking at 200 rpm for 12 h. The resultant cultures were diluted 50-fold into 5 mL of fresh HI broth for the second round of growth, which were grown to an OD₆₀₀ value of 1.5. The resultant cultures were then diluted 1000-fold into 5 mL of fresh HI broth for a third round of growth, and were harvested at an OD₆₀₀ value of approximately 1.5. When necessary, the medium was supplemented with different concentrations of chloramphenicol (0, 0.2, 0.3 or 0.4 μg/mL).

Growth inhibition assay

The second round of bacterial cell cultures was diluted 1000-fold into 10 mL HI broth containing 0, 0.2, 0.3 or 0.4 μg/mL chloramphenicol in a glass tube, and allowed to grow continuously at 37°C with shaking at 200 rpm. Three biological replicates were set for each concentration. The growth curves were created by monitoring the OD₆₀₀ values of each culture at 1 h intervals.

Biofilm formation assay

Biofilm formation capacities of V. parahaemolyticus under different concentrations of chloramphenicol were assessed by the crystal violet (CV) staining method, which was performed similarly as previously described (Fang et al., 2013; Zhang et al., 2021). Briefly, the second round of bacterial cell cultures was diluted 50-fold into 2 mL Difco marine (M) broth 2,216 (M broth; BD Bioscience, USA) containing 0, 0.2 or 0.4 μg/mL chloramphenicol in a 24-well cell culture plate, and allowed to grow at 30°C with shaking at 150 rpm for 48 h. The surface-attached cells were washed gently three times with deionized water after removing the planktonic cells, incubated at 80°C for 15 min to fix the biofilm cells, and then stained with 3 mL of 0.1% CV for 30 min. The CV stained biofilm cells were washed for another three times with deionized water. The bound CV was dissolved with 20% acetic acid, and the OD₅₇₀ values were measured as an index of CV staining.

Colony morphology

Colony morphology assay was performed similarly as previously described (Zhang et al., 2021). Briefly, the second round of bacterial cell cultures was diluted 50-fold into 2 mL M broth in a 24-well cell culture plate, and allowed to grow without shaking at 30°C for 48 h. The cell culture was mixed thoroughly, and then 2 μL of the culture was spotted onto a HI agar containing 0, 0.2 or 0.4 μg/mL chloramphenicol, followed by incubated at 30°C for 48 h.

Opaque (OP)-translucent (TR) colony phenotypes

The OP-TR colony phenotypes were detected as previously described (Zhang et al., 2023b). Briefly, a small amount of overnight cell culture in HI broth was streaked onto a HI plate containing 0, 0.2 or 0.4 μg/mL chloramphenicol, and then statically incubated at 37°C for 24 h.

Swimming motility

Swimming motility assay was performed as previously described (Wu et al., 2022). Briefly, 2 μL of the second round of bacterial cell culture were inoculated into a swimming plate [1% Oxoid tryptone, 2% NaCl (Merck, Germany), and 0.5% Difco Noble agar (BD Biosciences, USA)] containing 0, 0.2 or 0.4 μg/mL chloramphenicol. The swimming plate was statically incubated at 37°C. Starting from the 3rd hour after incubation, the diameter of swimming area was measured per hour until the 7th hour.

Swarming motility

Swarming motility assay was performed as previously described (Wu et al., 2022). Briefly, 2 μL of the second round of bacterial culture were spotted on a solid swarm plate [2.5% Bacto heart infusion, 1.5% NaCl (Merck), and 2.0% Difco noble agar (BD Bioscience)] containing 0, 0.2 or 0.4 μg/mL chloramphenicol. The diameters of swarming zones were measured at the 24th, 48th, and 72th hours after incubation statically at 37°C.

Intracellular c-di-GMP quantification

Intracellular c-di-GMP levels were measured as previously described (Wu et al., 2022). Briefly, the second round of bacterial culture was diluted 1000-fold into 5 mL of HI broth containing 0, 0.2 and 0.4 μg/mL chloramphenicol, respectively, and incubated at 37°C with shaking to an OD₆₀₀ value of 1.5. Bacterial cells were harvested and resuspended in 2 mL ice-cold phosphate buffered saline (PBS), and then incubated at 100°C for 5 min, followed by sonicated for 15 min (power 100%, frequency 37 kHz) in an ice-water bath. The supernatant containing c-di-GMP was collected, and the pellet was resuspended in 2 mL ice-cold PBS and re-extracted for another two times. The intracellular c-di-GMP levels were determined with a c-di-GMP Enzyme-linked Immunosorbent Assay (ELISA) Kit (Mskbio, Beijing, China). In addition, total protein in the supernatant was also determined by a Pierce BCA Protein Assay kit (ThermoFisher Scientific, USA). Intracellular c-di-GMP levels were expressed as pmol/g protein.

Cytotoxicity assay

Cytotoxicity assay was performed as previously described (Sun et al., 2014). Briefly, the second round of bacterial culture was diluted 1000-fold into 5 mL of HI broth containing 0, 0.2 and 0.4 μg/mL chloramphenicol, respectively, and incubated at 37°C with shaking to an OD₆₀₀ value of 1.5. Bacterial cells were harvested, washed and then serially diluted with the pre-warmed Dulbecco’s modified Eagle’s medium (DMEM) lacking phenol red for colony forming unit (CFU) measurement and infection. HeLa cells were infected with 10⁶ CFU of bacteria for 3 h at a multiplicity of infection (MOI) of 2.5. The release of lactate dehydrogenase (LDH) was quantified with a CytoTox 96^® Non-Radioactive Cytotoxicity Assay kit (Promega, USA) according to the manufacturer’s instructions.

Adhesion assay

Adhesion assay was performed similarly as previously described (Zhang et al., 2017). Briefly, HeLa cell monolayers were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Invitrogen) at 37°C with 5% CO₂. The second round of bacterial culture was diluted 1,000-fold into 5 mL of HI broth containing 0, 0.2 and 0.4 μg/mL chloramphenicol, respectively, and incubated at 37°C with shaking to an OD₆₀₀ value of 1.5. Bacterial cells were collected, washed, and then re-suspended in DMEM. The cell monolayers were infected at multiplicity of infection (MOI) of 1:10. Bacterial cells were also added to empty wells to determine the amount of input as the final total number of V. parahaemolyticus. After incubation for 90 min, the monolayers were washed thrice with PBS and lysed with 1% Triton X-100. The lysates and input bacteria were serially diluted 10-fold and counted on LB agar plates. Percent adherence was calculated as bacterial cells adhered/input bacterial cells.

RNA sequencing (RNA-seq)

The second round of bacterial culture was diluted 1000-fold into 5 mL of HI broth containing 0 and 0.2 μg/mL chloramphenicol, respectively, and incubated at 37°C with shaking to an OD₆₀₀ value of 1.5. Bacterial cells were harvested, and total RNA was extracted using TRIzol Reagent (Invitrogen, USA). RNA concentrations were measured using a Nanodrop 2000, and RNA integrity was evaluated using the agarose gel electrophoresis (Wu et al., 2022). rRNA removal and mRNA enrichment were performed using an Illumina/Ribo-Zero™ rRNA Removal Kit (bacteria) (Illumina, USA). cDNA library construction and sequencing were performed on an Illumina Hiseq platform at the GENEWIZ Biotechnology Co. Ltd. (Suzhou, China). The significantly differentially expressed genes (DEGs) were analyzed using the DESeq2 (V1.6.3) software with at least 2-fold changes in the ratio of mRNA levels (test/reference) and a p value of <0.05.

Quantitative PCR (qPCR)

qPCR assays were performed similarly as previously described (Gao et al., 2011). Briefly, the second round of bacterial culture was diluted 1000-fold into 5 mL of HI broth containing 0 and 0.2 μg/mL chloramphenicol, respectively, and incubated at 37°C with shaking to an OD₆₀₀ value of 1.5. Bacterial cells were harvested, and total RNA was extracted using TRIzol Reagent. cDNA was generated from 1 μg total RNA using a FastKing First Strand cDNA Synthesis Kit (Tiangen Biotech, China). The relative mRNA level of each target gene was determined using the classical 2^−ΔΔCt method with the 16S rRNA gene as the internal control. Primers used in this work were synthesized by GENEWIZ Biotechnology Co. Ltd. (Suzhou, China). Primers used in this work (Table 1) were synthesized by GENEWIZ Biotechnology Co., Ltd. (Suzhou, China) based on the solid phase subphosphorylation, and purified using the C18 column desalt method.

Experimental replicates and statistical methods

The growth curves, CV staining, c-di-GMP quantification, swarming, swimming, cytotoxicity and adhesion assays were performed at least three independent times with three biological replicates in each time, and the results were expressed as the mean ± standard deviation (SD). Paired Student’s t-tests were used to calculate statistical the significance with a p-value less than 0.05 considered significant. Colony morphology and OP-TR colony switching assays were performed at least three independent times.

Results

Chloramphenicol inhibited the growth of Vibrio parahaemolyticus

The growth curves of V. parahaemolyticus in HI broth supplemented with 0, 0.2, 0.3, and 0.4 μg/mL chloramphenicol were measured to investigate whether sublethal doses of chloramphenicol have some effects on the growth of the bacterium. As shown in Figure 1, the growth rates of V. parahaemolyticus in the presence of chloramphenicol were significantly slower than in the absence of chloramphenicol, and V. parahaemolyticus manifested slower and slower growth rates with the increasing of chloramphenicol. These results suggested that sublethal doses of chloramphenicol were able to inhibit the growth of V. parahaemolyticus.

Figure 1

Sublethal doses of chloramphenicol reduced the biofilm formation capacity of Vibrio parahaemolyticus

The effects of sublethal doses of chloramphenicol on the biofilm formation capacity of V. parahaemolyticus were assessed using the CV staining and colony morphology assays. As shown in Figure 2A, V. parahaemolyticus grown in the presence of sublethal doses of chloramphenicol exhibited significantly less normalized CV staining relative to that grown in the absence of chloramphenicol (p < 0.05), and the higher chloramphenicol concentration was added, the less normalized CV staining was detected. As further confirmed by the colony morphology (Figure 2B), the colonies of V. parahaemolyticus on the HI plate supplemented with 0.4 μg/mL of chloramphenicol were smooth, whereas those on other concentrations of chloramphenicol containing plates were wrinkly. However, the colonies of V. parahaemolyticus on the HI plate without chloramphenicol were much more wrinkled than those on the plate supplemented with 0.2 μg/mL of chloramphenicol (Figure 2B). These results suggested that chloramphenicol dose-dependently inhibited the biofilm formation capacity of V. parahaemolyticus.

Figure 2

Chloramphenicol did not affect the OP-TR colony variation of Vibrio parahaemolyticus

Vibrio parahaemolyticus naturally switches between OR and TP colony phenotypes based on whether capsular polysaccharide (CPS) production or not (Chen et al., 2010). In this study, the data showed that V. parahaemolyticus manifested the OP colony phenotype no matter whether the chloramphenicol presence or not (Figure 3), suggesting that sublethal doses of chloramphenicol did not affect the OP-TR colony variation of V. parahaemolyticus. However, the lawn of V. parahaemolyticus on the HI plate supplemented with 0.4 μg/mL of chloramphenicol seemed to be much drier than those under the other two conditions (Figure 3), which may be related to the growth inhibition of chloramphenicol (Figure 1).

Figure 3

Sublethal doses of chloramphenicol reduced the motor capacity of Vibrio parahaemolyticus

Effect of sublethal doses of chloramphenicol on the motor capacity of V. parahaemolyticus was investigated using the swimming and swarming assays. As shown in Figure 4, the swimming and swarming motility of V. parahaemolyticus were significantly decreased in the presence of sublethal doses of chloramphenicol relative to the conditions without chloramphenicol at all time points tested. Higher concentration of chloramphenicol was able to reduce more the motor ability of V. parahaemolyticus. These results suggested that chloramphenicol dose-dependently inhibited swimming and swarming motility of V. parahaemolyticus.

Figure 4

Sublethal doses of chloramphenicol inhibited the production of intracellular c-di-GMP

The intracellular c-di-GMP levels were investigated to assess whether sublethal doses of chloramphenicol affect the c-di-GMP pool of V. parahaemolyticus. As shown in Figure 5, the intracellular c-di-GMP levels of V. parahaemolyticus were remarkably reduced in the presence of sublethal doses of chloramphenicol, suggesting that sublethal doses of chloramphenicol was able to inhibit the synthesis of intracellular c-di-GMP in V. parahaemolyticus.

Figure 5

Sublethal doses of chloramphenicol reduced the adherence and cytotoxicity of Vibrio parahaemolyticus

Effect of sublethal doses of chloramphenicol on the adherence and cytotoxicity of V. parahaemolyticus was investigated using the following virulence-associated phenotypes. First, cytotoxicity against HeLa cells of V. parahaemolyticus grown in the presence or absence of chloramphenicol was investigated regarding to the release of LDH from cultured cells (Figure 6A). The cytotoxicity of V. parahaemolyticus grown in the conditions with chloramphenicol was significantly reduced relative to that grown in the condition without chloramphenicol, suggesting that chloramphenicol inhibited the cytotoxicity of V. parahaemolyticus against HeLa cells. Second, adhesion activity of V. parahaemolyticus to HeLa cells was investigated using the adhesion assay (Figure 6B). The results showed that sublethal doses of chloramphenicol were able to influence the adhesion of V. parahaemolyticus to HeLa cells in a dose-dependent manner, as that the adhesion rates of V. parahaemolyticus under the conditions of 0.2 and 0.4 μg/mL chloramphenicol were only approximately 30 and 20% of that under the condition without chloramphenicol, respectively. Taken together, these results suggested that sublethal doses of chloramphenicol inhibited the adherence and cytotoxicity of V. parahaemolyticus.

Figure 6

Sublethal dose of chloramphenicol affected global gene expression of Vibrio parahaemolyticus

The growth of V. parahaemolyticus was severely inhibited by 0.4 μg/mL chloramphenicol (Figure 1). Therefore, the gene expression profiles of V. parahaemolyticus grown in HI broth supplemented with 0.2 (test) chloramphenicol were compared with those grown in HI broth containing 0 μg/mL chloramphenicol (reference) by RNA-seq to investigate the cellular pathways involved in the response to the sublethal dose of chloramphenicol. As shown in Figure 7A, a total of 650 genes were significantly differentially expressed in the response to chloramphenicol stress, of these 354 were down-regulated and 296 were up-regulated. The enrichment of gene ontology (GO) term showed that the DEGs were involved in molecular function (13 GO terms, 62 DEGs), followed by biological process (11 GO terms, 70 DEGs) and cellular component (6 GO terms, 48 DEGs) (Figure 7B). The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 15, 163, 17, 56 and 62 DEGs were involved in organismal systems, metabolism, human diseases, environmental information processing and cellular processes, respectively (Figure 7C). The results of Cluster of Orthologous Groups of proteins (COG) enrichment showed that the DEGs were divided into 20 functional categories, and the 5 top pathways were function unknown, general function prediction only, energy production and conversion, cell motility and amino acid transport and metabolism (Figure 7D). The detailed information of DEGs was listed in Supplementary Table S1.

Figure 7

Selected DEGs of interest

A total of 6 genes associated with antibiotic resistance were significantly differentially expressed in the response of chloramphenicol (Table 2), of these 3 were downregulated (VP2167, VPA0520, and VPA1590) and 5 were upregulated (VP3019, VPA0168, VPA1190, VPA1191, and VPA1647). Five genes encoding GGDEF and/or EAL-domain proteins were significantly differentially expressed, including 2 downregulated genes (VP1768 and VPA0202) and 3 upregulated genes (VPA0360, VPA0556, and VPA0609). Additionally, 1 lateral flagellar gene (VPA1546), 43 polar flagellar genes, 1 CPS-related gene (VP0228), 6 scv genes (VP1458, VP1461, VP1463, VP1464, VP1466, and VP1467), and 5 MSHA genes (VP2699, VP2700, VP2701, VP2702, and VP2703), 28 T3SS1-associated genes including exsD, 5 T3SS2-associated genes (VPA1342, VPA1345, VPA1346, VPA1361, and VPA1370), 1 T6SS1-related gene (hcp1), and 13 T6SS2-related genes were remarkably downregulated in the presence of chloramphenicol (Table 2). In addition, at least 23 putative regulatory genes were significantly differentially expressed including 11 downregulated and 12 upregulated genes (Table 2). The DEGs also contained other of interest genes including 2 antioxidative genes (katE1 and ahpC1) and 12 outer membrane protein (OMP) encoding genes (Table 2).

Validation of RNA-seq data by qPCR

A total of 26 DEGs were selected as the target genes (Table 2) to subject to the qPCR assays to validate the RNA-seq data. As shown in Figure 8, the results of qPCR for all the tested genes manifested consistent trends with the RNA-seq data (Table 2), confirming the reliability of RNA-seq data. In addition, the qPCR results demonstrated that transcriptional levels of cpsA and cpsE were also remarkably downregulated in response to the chloramphenicol stress.

Figure 8

Discussion

Chloramphenicol has potent inhibition on bacterial protein biosynthesis via binding to the 50S subunit of ribosome (Bale et al., 2023), and thus has a significant impact on gene expression, bacterial physiology and behaviors. Addition of chloramphenicol to culture media significantly inhibited the growth and respiration of Escherichia coli (Smirnova et al., 2023). The biofilm formation abilities of Klebsiella sp., Pseudomonas aeruginosa, Achromobacter sp., Klebsiella pneumoniae, and Bacillus pumilis were significantly decreased in the presence of chloramphenicol (Liaqat et al., 2009). Chloramphenicol down-regulated flagellar gene expression and swimming and swarming motility of Salmonella enterica serovar Typhimurium, but up-regulated genes involved in invasion, attachment, and intracellular survival (Brunelle et al., 2014, 2017). Transcriptome analysis revealed that chloramphenicol affects global gene expression in multiple bacterial species including such as Streptococcus pneumoniae (Ng et al., 2003), Staphylococcus equorum (Heo et al., 2022), Bacillus subtilis (Lin et al., 2005), Yersinia pestis (Qiu et al., 2006), Thermotoga maritima (Montero et al., 2007), Pseudomonas putida (Fernandez et al., 2012), Myxococcus xanthus (Yang et al., 2019), Enterococcus faecalis (Tatta et al., 2023), and Escherichia coli (Bie et al., 2023). The genes that showed altered expression in response to chloramphenicol are involved in multiple cellular pathways including metabolism, stress response, regulation, and protein biosynthesis.

In this study, the data showed that sublethal doses of chloramphenicol remarkably inhibited the growth rate, biofilm formation, motility, c-di-GMP biosynthesis, cytoxicity and adherence activity of V. parahaemolyticus (Figures 1–6). The RNA-seq data disclosed that sublethal dose of chloramphenicol strikingly affected the expression of 650 genes in V. parahaemolyticus, of these 296 were upregulated and 354 were downregulated (Figure 7; Table 2). Majority of virulence genes (T3SS1, T6SS2, etc.), polar flagellar genes and biofilm-associated genes (msh, scv., cps, etc.) were down-regulated by sublethal dose of chloramphenicol (Figure 7; Table 2). Therefore, the reduction in functional phenotypes of V. parahaemolyticus is likely due to growth defect and downregulation of related gene expression. In addition, the results in this study also suggested that sublethal dose of chloramphenicol could be used as an anti-biofilm or anti-virulence regent to combat with V. parahaemolyticus contamination. There are currently many studies exploring the anti-virulence and anti-biofilm strategies toward bacteria (Imchen et al., 2022; Law and Tan, 2022). Findings show that some substances including cationic surfactants such as benzalkonium chloride (Park and Yoon, 2023), chemicals such as Rhein (Folliero et al., 2022) and 2,6-Di-tert-butyl-4-methylphenol (Santhakumari et al., 2018), peptides such as MSI-I (Ye et al., 2021) and PV-Q5 (Dai et al., 2023), and traditional chinese medicine such as gingerol (Shukla et al., 2021) and Ulva fasciata (Qiao et al., 2021) possess anti-virulence and/or anti-biofilm activity. Compared to antibiotics such as chloramphenicol, these substances have obvious advantages, such as less susceptibility to bacterial resistance. Further research is needed to discover compounds with strong applicability that can reduce the virulence and biofilm formation capacity of V. parahaemolyticus.

Drug efflux from bacterial cells is one of the key mechanisms of drug resistance (Stephen et al., 2022). There are five major groups of drug efflux pumps, that is, the RND family, the MF (major facilitator) family, the MATE (multidrug and toxic compound extrusion) family, the SMR (small multidrug resistance) family and the ABC (ATP binding cassette) superfamily (Stephen et al., 2022). V. parahaemolyticus possesses 12 RND-type efflux pumps (Matsuo et al., 2013), but only VmeYZ was induced by the sublethal dose of chloramphenicol (Table 2), suggesting that VmeYZ may play the key role in the resistance of V. parahaemolyticus to chloramphenicol. Moreover, VP3019, emrD, VPA0520 and VPA1647 encoding the MF-type pumps (Makino et al., 2003) were also upregulated by the sublethal dose of chloramphenicol (Table 2), but their roles in V. parahaemolyticus remain ill-defined.

Reducing antibiotic influx through OMP channels is another important mechanism of drug resistance in bacteria (Rodrigues et al., 2022). For example, in E. coli, OmpC is associated with β-lactams resistance, OmpF with β-lactams and fluoroquinolone resistance, and TolC, YddB, OmpX and TosA with the resistances of enrofloxacin, novobiocin, fluoroquinolones, β-lactams and globomycin (Rodrigues et al., 2022). Seventeen OMPs including OmpU, OmpN, OmpA, OmpV and OmpK are associated with the resistance of V. alginolyticus to erythromycin, tetracycline, kanamycin, streptomycin, nalidixic acid and chloromycetin (Xiong et al., 2010). OmpN is involved in the indole-dependent tetracycline resistance of V. splendidus (Zhang et al., 2020). Twelve OMP genes were regulated by the sublethal dose of chloramphenicol including the known porins such as OmpU, OmpK and OmpN (Table 2), indicating that OMPs were remodeled in the response to the chloramphenicol. However, whether these putative OMPs are associated with chloramphenicol resistance of V. parahaemolyticus remains unknown.

Vibrio parahaemolyticus has a strong ability to form biofilms, which are also closely linked to the antibiotic resistance (Mishra et al., 2023). V. parahaemolyticus undergoes the phase variation between the wrinkly and smooth colony phenotypes, which is based on whether the EPS production or not (Wu et al., 2022). EPS production is associated with the cpsA-K and scvA-O gene clusters that positively correlate with the biofilm formation by V. parahaemolyticus, but the wrinkly colony phenotype is only attributed to the expression of cpsA-K (Liu et al., 2022). Sublethal doses of chloramphenicol inhibited V. parahaemolyticus to form the wrinkly colony and biofilms (Figure 2), but no transcripts of cps genes were detected by RNA-seq. By contrast, six scv genes were remarkably low expressed in the response to chloramphenicol (Table 2). However, the results of qPCR showed that the transcriptional levels of cpsA and cpsE were notably decreased in the response to chloramphenicol (Figure 8), suggesting that the undetectable of cps genes probably be caused by the preparation of RNA-seq samples.

One CPS gene, VP0228, was remarkably decreased in the presence of sublethal dose of chloramphenicol (Table 2). There are 25 genes in the CPS locus (VP0214-0238) (Okura et al., 2008), and only one of them was regulated, which was likely to have no impact on the synthesis of CPS. Indeed, CPS production was not affected by sublethal dose of chloramphenicol (Figure 3). V. parahaemolyticus expresses dual flagellar systems: a single polar flagellum for swimming in liquid and peritrichous lateral flagella for swarming over surface (McCarter, 2004). Strains with defective polar flagellum failed to form mature biofilms, which can be restored by exogenous addition of recombinant polar flagellins (Enos-Berlage et al., 2005; Jung et al., 2019). Although lacks detailed mechanisms, lateral flagella are thought to be required for the mature biofilm formation (Yildiz and Visick, 2009). A total of 43 polar flagellar genes were remarkably downregulated in the presence of sublethal dose of chloramphenicol (Table 2). However, only 2 lateral flagellar genes were significantly differentially expressed, of these 1 was upregulated and 1 was downregulated (Table 2). Therefore, the inhibitory effect of sublethal dose of chloramphenicol on swimming probably be mainly attributed to the downregulation of polar flagellar genes, whereas on swarming may be more related to the growth inhibition of V. parahaemolyticus. In addition, V. parahaemolyticus produces two kinds of type IV pili: MSHA and ChiRP (Makino et al., 2003). MSHA and ChiRP are required for the mature biofilm formation of V. parahaemolyticus (Shime-Hattori et al., 2006). The RNA-seq data showed that five MSHA genes were downregulated but one ChiRP gene was upregulated in the response to sublethal dose of chloramphenicol (Table 2), indicating that chloramphenicol-dependent biofilm inhibition was closely related to the different expression of type IV pili.

c-di-GMP, a ubiquitous second messenger, is involved in controlling multiple physiological roles of bacteria including motility, adherence, biofilm formation, virulence and cell cycle progression (Jenal et al., 2017). c-di-GMP is synthesized by guanylate cyclase (DGC) carrying a GGDEF domain, whereas is degraded by phosphodiesterase (PDF) carrying either a EAL or HD-GYP domain (Jenal et al., 2017). More than 50 proteins may be involved in the metabolism of c-di-GMP in V. parahaemolyticus (Seshasayee et al., 2010). However, only a small portion of them were demonstrated to be required for the c-di-GMP metabolism, including the GGDEF-EAL-containing proteins, ScrG and ScrC, the EAL-containing protein LafV, and the GGDEF-containing proteins, ScrO, GefA, ScrJ and ScrL (Kim and McCarter, 2007; Ferreira et al., 2008; Kimbrough et al., 2020; Kimbrough and McCarter, 2021; Zhong et al., 2022). In this study, a total of 5 genes probably encoding DGCs or PDFs were significantly differentially expressed in the stimulation of sublethal dose of chloramphenicol (Table 2), of these 2 (VP1768 and VPA0202) were downregulated and 3 (VPA0360, VPA0556 and VPA0609) were upregulated. The clarifying functions of these genes will be beneficial for us to understand the regulatory mechanisms of sublethal dose of chloramphenicol on c-di-GMP metabolism in V. parahaemolyticus.

The expression levels of 28 T3SS1 genes including exsD were remarkably decreased in the presence of sublethal dose of chloramphenicol (Table 2). Transcription of T3SS1 genes was positively regulates by ExsA (Zhou et al., 2008). ExsD binds ExsA to block the regulatory activity of ExsA and thereby inhibiting the expression of T3SS1 genes (Zhou et al., 2010). In addition, five T3SS2 genes, one T6SS1 gene and 13 T6SS2 genes were remarkably downregulated in the response to sublethal dose of chloramphenicol (Table 2). T3SS1 is mainly involved in cytotoxicity, whereas T3SS2 predominantly contributes to enterotoxicity, and both are required for the full virulence of V. parahaemolyticus (Park et al., 2004; Hiyoshi et al., 2010). By contrast, T6SS1 is required for the antibacterial activity of V. parahaemolyticus, whereas T6SS2 mainly acts as an adherence factor (Yu et al., 2012; Salomon et al., 2013). In brief, downregulating the major virulence genes may be beneficial for V. parahaemolyticus to combat the killing effect of chloramphenicol.

RNA-seq data also revealed that at least 23 genes encoding putative regulators were significantly differentially expressed in the response to sublethal dose of chloramphenicol, of these 11 were downregulated and 12 were upregulated (Table 2). Some genes encode global regulators, including LysR family transcriptional regulators (VP0624, VPA0299, VPA0602, and VPA0717), TetR/AcrR family transcriptional regulators (VP1229 and VPA0053), MarR family transcriptional regulator (VP2450) and GntR family transcriptional regulator (VP1649). In addition, the sublethal dose of chloramphenicol remarkably induced the expression of CpsQ, a c-di-GMP binding regulatory protein that promotes the expression of cpsA-K and biofilm formation by V. parahaemolyticus (Ferreira et al., 2012), suggesting that this regulator may also be required for resistance to chloramphenicol stress. However, the functions of the other regulatory genes are currently unknown, and future experiments should be designed to elucidate the roles of these regulators.

In conclusion, the present data demonstrated that the growth rate, biofilm formation capacity, c-di-GMP synthesis, motility, cytoxicity and adherence activity of V. parahaemolyticus were remarkably downregulated by the sublethal of chloramphenicol. A total of 650 genes were significantly differentially expressed in the response to chloramphenicol, including antibiotic resistance genes, major virulence genes, biofilm-associated genes and putative regulatory genes. Majority of genes involved in polar flagellum, EPS, MSHA, T3SS1, T3SS2 and T6SS2 were downregulated. In addition, five putative c-di-GMP metabolism genes were significantly differentially expressed, which may be the reason for the decrease in intracellular c-di-GMP levels in the response to chloramphenicol. Moreover, 23 putative regulatory genes were also significantly differentially expressed due to the stimulation of sublethal of chloramphenicol, suggesting that these regulators may be involved in the resistance of V. parahaemolyticus to chloramphenicol stress. This work helps us to understand how chloramphenicol effect on the physiology of V. parahaemolyticus. However, transcriptome analysis is only a preliminary study on the adaptation mechanism of V. parahaemolyticus to the sublethal dose of chloramphenicol, and more research should be performed to discover the underlying molecular mechanisms especially the regulation mechanisms involved in the resistance to chloramphenicol.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the Research Projects of Nantong Health Commission (Grant No. QN2022044).

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

References

• 1

  AmalinaN. Z.SanthaS.ZulperiD.AmalM. N. A.YusofM. T.Zamri-SaadM.et al. (2019). Prevalence, antimicrobial susceptibility and plasmid profiling of vibrio spp. isolated from cultured groupers in peninsular Malaysia. BMC Microbiol.19:251. doi: 10.1186/s12866-019-1624-2

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 2

  BaleB. I.ElebesunuE. E.ManikavasagarP.AgwunaF. O.OgunkolaI. O.SowA. U.et al. (2023). Antibiotic resistance in ocular bacterial infections: an integrative review of ophthalmic chloramphenicol. Trop. Med. Health51:15. doi: 10.1186/s41182-023-00496-x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 3

  BieL.ZhangM.WangJ.FangM.LiL.XuH.et al. (2023). Comparative analysis of transcriptomic response of Escherichia coli K-12 MG1655 to nine representative classes of antibiotics. Microbiol. Spectr.11:e0031723. doi: 10.1128/spectrum.00317-23

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 4

  BrunelleB. W.BearsonB. L.BearsonS. M. (2014). Chloramphenicol and tetracycline decrease motility and increase invasion and attachment gene expression in specific isolates of multidrug-resistant Salmonella enterica serovar typhimurium. Front. Microbiol.5:801. doi: 10.3389/fmicb.2014.00801

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 5

  BrunelleB. W.BearsonB. L.BearsonS. M. D.CaseyT. A. (2017). Multidrug-resistant Salmonella enterica Serovar typhimurium isolates are resistant to antibiotics that influence their swimming and swarming motility. mSphere2:e00306-17. doi: 10.1128/mSphere.00306-17

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 6

  CaiQ.ZhangY. (2018). Structure, function and regulation of the thermostable direct hemolysin (TDH) in pandemic Vibrio parahaemolyticus. Microb. Pathog.123, 242–245. doi: 10.1016/j.micpath.2018.07.021

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 7

  ChenY.DaiJ.MorrisJ. G.Jr.JohnsonJ. A. (2010). Genetic analysis of the capsule polysaccharide (K antigen) and exopolysaccharide genes in pandemic Vibrio parahaemolyticus O3:K6. BMC Microbiol.10:274. doi: 10.1186/1471-2180-10-274

  • CrossRef
  • Google Scholar

• 8

  ChenL.QiuY.TangH.HuL. F.YangW. H.ZhuX. J.et al. (2018). ToxR is required for biofilm formation and motility of Vibrio Parahaemolyticus. Biomed. Environ. Sci.31, 848–850. doi: 10.3967/bes2018.112

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 9

  CoulthurstS. (2019). The type VI secretion system: a versatile bacterial weapon. Microbiology165, 503–515. doi: 10.1099/mic.0.000789

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 10

  DaiJ.JinR.GaoJ.AweyaJ. J.LinR.LiG.et al. (2023). Antibacterial activity and mechanism of peptide PV-Q5 against Vibrio parahaemolyticus and Escherichia coli, derived from salt-fermented Penaeus vannamei. Foods12:12. doi: 10.3390/foods12091804

  • CrossRef
  • Google Scholar

• 11

  Enos-BerlageJ. L.GuvenerZ. T.KeenanC. E.McCarterL. L. (2005). Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus. Mol. Microbiol.55, 1160–1182. doi: 10.1111/j.1365-2958.2004.04453.x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 12

  FangN.GaoH.WangL.QuS.ZhangY. Q.YangR. F.et al. (2013). Optimized methods for biofilm analysis in Yersinia pestis. Biomed. Environ. Sci.26, 408–411. doi: 10.3967/0895-3988.2013.05.012

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 13

  FernandezM.CondeS.de la TorreJ.Molina-SantiagoC.RamosJ. L.DuqueE. (2012). Mechanisms of resistance to chloramphenicol in Pseudomonas putida KT2440. Antimicrob. Agents Chemother.56, 1001–1009. doi: 10.1128/AAC.05398-11

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 14

  FerreiraR. B.AntunesL. C.GreenbergE. P.McCarterL. L. (2008). Vibrio parahaemolyticus ScrC modulates cyclic dimeric GMP regulation of gene expression relevant to growth on surfaces. J. Bacteriol.190, 851–860. doi: 10.1128/JB.01462-07

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 15

  FerreiraR. B.ChodurD. M.AntunesL. C.TrimbleM. J.McCarterL. L. (2012). Output targets and transcriptional regulation by a cyclic dimeric GMP-responsive circuit in the Vibrio parahaemolyticus Scr network. J. Bacteriol.194, 914–924. doi: 10.1128/JB.05807-11

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 16

  FollieroV.Dell’AnnunziataF.RoscettoE.AmatoA.GasparroR.ZannellaC.et al. (2022). Rhein: a novel antibacterial compound against Streptococcus mutans infection. Microbiol. Res.261:127062. doi: 10.1016/j.micres.2022.127062

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 17

  GaoH.ZhangY.YangL.LiuX.GuoZ.TanY.et al. (2011). Regulatory effects of cAMP receptor protein (CRP) on porin genes and its own gene in Yersinia pestis. BMC Microbiol.11:40. doi: 10.1186/1471-2180-11-40

  • CrossRef
  • Google Scholar

• 18

  GuD.ZhangY.WangQ.ZhouX. (2020). S-nitrosylation-mediated activation of a histidine kinase represses the type 3 secretion system and promotes virulence of an enteric pathogen. Nat. Commun.11:5777. doi: 10.1038/s41467-020-19506-1

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 19

  GuzmanL. M.BelinD.CarsonM. J.BeckwithJ. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol.177, 4121–4130. doi: 10.1128/jb.177.14.4121-4130.1995

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 20

  HeoS.KimT.NaH. E.LeeG.LeeJ. H.JeongD. W. (2022). Transcriptomic analysis of Staphylococcus equorum KM1031 from the high-salt fermented seafood jeotgal under chloramphenicol, erythromycin and lincomycin stresses. Sci. Rep.12:15541. doi: 10.1038/s41598-022-19897-9

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 21

  HiyoshiH.KodamaT.IidaT.HondaT. (2010). Contribution of Vibrio parahaemolyticus virulence factors to cytotoxicity, enterotoxicity, and lethality in mice. Infect. Immun.78, 1772–1780. doi: 10.1128/IAI.01051-09

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 22

  HuM.ZhangY.GuD.ChenX.WaldorM. K.ZhouX. (2021). Nucleolar c-Myc recruitment by a vibrio T3SS effector promotes host cell proliferation and bacterial virulence. EMBO J.40:e105699. doi: 10.15252/embj.2020105699

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 23

  ImchenM.AnjuV. T.BusiS.MohanM. S.SubhaswarajP.DyavaiahM.et al. (2022). Metagenomic insights into taxonomic, functional diversity and inhibitors of microbial biofilms. Microbiol. Res.265:127207. doi: 10.1016/j.micres.2022.127207

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 24

  JenalU.ReindersA.LoriC. (2017). Cyclic di-GMP: second messenger extraordinaire. Nat. Rev. Microbiol.15, 271–284. doi: 10.1038/nrmicro.2016.190

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 25

  JungY. C.LeeM. A.LeeK. H. (2019). Role of Flagellin-homologous proteins in biofilm formation by pathogenic vibrio species. MBio10:10. doi: 10.1128/mBio.01793-19

  • CrossRef
  • Google Scholar

• 26

  KimY. K.McCarterL. L. (2007). ScrG, a GGDEF-EAL protein, participates in regulating swarming and sticking in Vibrio parahaemolyticus. J. Bacteriol.189, 4094–4107. doi: 10.1128/JB.01510-06

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 27

  KimbroughJ. H.CribbsJ. T.McCarterL. L. (2020). Homologous c-di-GMP-binding Scr transcription factors orchestrate biofilm development in Vibrio parahaemolyticus. J. Bacteriol.202:e00723-19. doi: 10.1128/JB.00723-19

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 28

  KimbroughJ. H.McCarterL. L. (2021). Identification of three new GGDEF and EAL domain-containing proteins participating in the Scr surface colonization regulatory network in Vibrio parahaemolyticus. J. Bacteriol.203:e00409-20. doi: 10.1128/JB.00409-20

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 29

  LawS. K. K.TanH. S. (2022). The role of quorum sensing, biofilm formation, and iron acquisition as key virulence mechanisms in Acinetobacter baumannii and the corresponding anti-virulence strategies. Microbiol. Res.260:127032. doi: 10.1016/j.micres.2022.127032

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 30

  LeiT.ZhangJ.JiangF.HeM.ZengH.ChenM.et al. (2020). Characterization of class 1 integrons harboring Bla(VEB-1) in Vibrio parahaemolyticus isolated from ready-to-eat foods in China. Int. J. Food Microbiol.318:108473. doi: 10.1016/j.ijfoodmicro.2019.108473

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 31

  LiL.MengH.GuD.LiY.JiaM. (2019). Molecular mechanisms of Vibrio parahaemolyticus pathogenesis. Microbiol. Res.222, 43–51. doi: 10.1016/j.micres.2019.03.003

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 32

  LiW.WangJ. J.QianH.TanL.ZhangZ.LiuH.et al. (2020). Insights into the role of extracellular DNA and extracellular proteins in biofilm formation of Vibrio parahaemolyticus. Front. Microbiol.11:813. doi: 10.3389/fmicb.2020.00813

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 33

  LiaqatI.SumbalF.SabriA. N. (2009). Tetracycline and chloramphenicol efficiency against selected biofilm forming bacteria. Curr. Microbiol.59, 212–220. doi: 10.1007/s00284-009-9424-9

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 34

  LinJ. T.ConnellyM. B.AmoloC.OtaniS.YaverD. S. (2005). Global transcriptional response of Bacillus subtilis to treatment with subinhibitory concentrations of antibiotics that inhibit protein synthesis. Antimicrob. Agents Chemother.49, 1915–1926. doi: 10.1128/AAC.49.5.1915-1926.2005

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 35

  LinI. T.TulmanE. R.GearyS. J.ZhouX. (2021). A gatekeeper protein contributes to T3SS2 function via interaction with an ATPase in Vibrio parahaemolyticus. Microbiol. Res.252:126857. doi: 10.1016/j.micres.2021.126857

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 36

  LiuM.NieH.LuoX.YangS.ChenH.CaiP. (2022). A polysaccharide biosynthesis locus in Vibrio parahaemolyticus important for biofilm formation has homologs widely distributed in aquatic bacteria mainly from Gammaproteobacteria. mSystems7:e0122621. doi: 10.1128/msystems.01226-21

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 37

  LiuM.ZhuX.ZhangC.ZhaoZ. (2021). LuxQ-LuxU-LuxO pathway regulates biofilm formation by Vibrio parahaemolyticus. Microbiol. Res.250:126791. doi: 10.1016/j.micres.2021.126791

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 38

  LuR.SunJ.QiuY.ZhangM.XueX.LiX.et al. (2021). The quorum sensing regulator OpaR is a repressor of polar flagellum genes in Vibrio parahaemolyticus. J. Microbiol.59, 651–657. doi: 10.1007/s12275-021-0629-3

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 39

  MakinoK.OshimaK.KurokawaK.YokoyamaK.UdaT.TagomoriK.et al. (2003). Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae. Lancet361, 743–749. doi: 10.1016/S0140-6736(03)12659-1

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 40

  MatsudaS.OkadaN.KodamaT.HondaT.IidaT. (2012). A cytotoxic type III secretion effector of Vibrio parahaemolyticus targets vacuolar H+-ATPase subunit c and ruptures host cell lysosomes. PLoS Pathog.8:e1002803. doi: 10.1371/journal.ppat.1002803

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 41

  MatsuoT.HayashiK.MoritaY.KoterasawaM.OgawaW.MizushimaT.et al. (2007). VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus. Microbiology153, 4129–4137. doi: 10.1099/mic.0.2007/009597-0

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 42

  MatsuoT.NakamuraK.KodamaT.MikamiT.HiyoshiH.TsuchiyaT.et al. (2013). Characterization of all RND-type multidrug efflux transporters in Vibrio parahaemolyticus. Microbiology2, 725–742. doi: 10.1002/mbo3.100

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 43

  McCarterL. L. (2004). Dual flagellar systems enable motility under different circumstances. J. Mol. Microbiol. Biotechnol.7, 18–29. doi: 10.1159/000077866

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 44

  MishraS.GuptaA.UpadhyeV.SinghS. C.SinhaR. P.HäderD. P. (2023). Therapeutic strategies against biofilm infections. Life13:172. doi: 10.3390/life13010172

  • CrossRef
  • Google Scholar

• 45

  MokJ. S.ChoS. R.ParkY. J.JoM. R.HaK. S.KimP. H.et al. (2021). Distribution and antimicrobial resistance of Vibrio parahaemolyticus isolated from fish and shrimp aquaculture farms along the Korean coast. Mar. Pollut. Bull.171:112785. doi: 10.1016/j.marpolbul.2021.112785

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 46

  MonteroC. I.JohnsonM. R.ChouC. J.ConnersS. B.GeougeS. G.TachdjianS.et al. (2007). Responses of wild-type and resistant strains of the hyperthermophilic bacterium Thermotoga maritima to chloramphenicol challenge. Appl. Environ. Microbiol.73, 5058–5065. doi: 10.1128/AEM.00453-07

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 47

  NgW. L.KazmierczakK. M.RobertsonG. T.GilmourR.WinklerM. E. (2003). Transcriptional regulation and signature patterns revealed by microarray analyses of Streptococcus pneumoniae R6 challenged with sublethal concentrations of translation inhibitors. J. Bacteriol.185, 359–370. doi: 10.1128/JB.185.1.359-370.2003

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 48

  OkuraM.OsawaR.TokunagaA.MoritaM.ArakawaE.WatanabeH. (2008). Genetic analyses of the putative O and K antigen gene clusters of pandemic Vibrio parahaemolyticus. Microbiol. Immunol.52, 251–264. doi: 10.1111/j.1348-0421.2008.00027.x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 49

  Osei-AdjeiG.HuangX.ZhangY. (2018). The extracellular proteases produced by Vibrio parahaemolyticus. World J. Microbiol. Biotechnol.34:68. doi: 10.1007/s11274-018-2453-4

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 50

  PaisS. V.KimE.WagnerS. (2023). Virulence-associated type III secretion systems in gram-negative bacteria. Microbiology169:169. doi: 10.1099/mic.0.001328

  • CrossRef
  • Google Scholar

• 51

  ParkK. S.OnoT.RokudaM.JangM. H.IidaT.HondaT. (2004). Cytotoxicity and enterotoxicity of the thermostable direct hemolysin-deletion mutants of Vibrio parahaemolyticus. Microbiol. Immunol.48, 313–318. doi: 10.1111/j.1348-0421.2004.tb03512.x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 52

  ParkS.YoonH. (2023). Transcriptional insight into the effect of benzalkonium chloride on resistance and virulence potential in Salmonella Typhimurium. Microbiol. Res.266:127240. doi: 10.1016/j.micres.2022.127240

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 53

  PazhaniG. P.ChowdhuryG.RamamurthyT. (2021). Adaptations of Vibrio parahaemolyticus to stress during environmental survival, host colonization, and infection. Front. Microbiol.12:737299. doi: 10.3389/fmicb.2021.737299

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 54

  PhilippeN.AlcarazJ. P.CoursangeE.GeiselmannJ.SchneiderD. (2004). Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria. Plasmid51, 246–255. doi: 10.1016/j.plasmid.2004.02.003

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 55

  QiaoY.JiaR.LuoY.FengL. (2021). The inhibitory effect of Ulva fasciata on culturability, motility, and biofilm formation of Vibrio parahaemolyticus ATCC17802. Int. Microbiol.24, 301–310. doi: 10.1007/s10123-021-00165-1

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 56

  QiuY.HuL.YangW.YinZ.ZhouD.YangH.et al. (2020). The type VI secretion system 2 of Vibrio parahaemolyticus is regulated by QsvR. Microb. Pathog.149:104579. doi: 10.1016/j.micpath.2020.104579

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 57

  QiuJ.ZhouD.QinL.HanY.WangX.duZ.et al. (2006). Microarray expression profiling of Yersinia pestis in response to chloramphenicol. FEMS Microbiol. Lett.263, 26–31. doi: 10.1111/j.1574-6968.2006.00394.x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 58

  RaghunathP. (2014). Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus. Front. Microbiol.5:805. doi: 10.3389/fmicb.2014.00805

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 59

  RodriguesI. C.RodriguesS. C.DuarteF. V.CostaP. M. D.CostaP. M. D. (2022). The role of outer membrane proteins in UPEC antimicrobial resistance: a systematic review. Membranes12:981. doi: 10.3390/membranes12100981

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 60

  RuhalR.KatariaR. (2021). Biofilm patterns in gram-positive and gram-negative bacteria. Microbiol. Res.251:126829. doi: 10.1016/j.micres.2021.126829

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 61

  SalomonD.GonzalezH.UpdegraffB. L.OrthK. (2013). Vibrio parahaemolyticus type VI secretion system 1 is activated in marine conditions to target bacteria, and is differentially regulated from system 2. PLoS One8:e61086. doi: 10.1371/journal.pone.0061086

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 62

  SanthakumariS.JayakumarR.LogalakshmiR.PrabhuN. M.Abdul NazarA. K.Karutha PandianS.et al. (2018). In vitro and in vivo effect of 2,6-Di-tert-butyl-4-methylphenol as an antibiofilm agent against quorum sensing mediated biofilm formation of vibrio spp. Int. J. Food Microbiol.281, 60–71. doi: 10.1016/j.ijfoodmicro.2018.05.024

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 63

  SeshasayeeA. S.FraserG. M.LuscombeN. M. (2010). Comparative genomics of cyclic-di-GMP signalling in bacteria: post-translational regulation and catalytic activity. Nucleic Acids Res.38, 5970–5981. doi: 10.1093/nar/gkq382

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 64

  Shime-HattoriA.IidaT.AritaM.ParkK. S.KodamaT.HondaT. (2006). Two type IV pili of Vibrio parahaemolyticus play different roles in biofilm formation. FEMS Microbiol. Lett.264, 89–97. doi: 10.1111/j.1574-6968.2006.00438.x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 65

  ShuklaA.ParmarP.PatelB.GoswamiD.SarafM. (2021). Breaking bad: better call gingerol for improving antibiotic susceptibility of Pseudomonas aeruginosa by inhibiting multiple quorum sensing pathways. Microbiol. Res.252:126863. doi: 10.1016/j.micres.2021.126863

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 66

  SmirnovaG.TyulenevA.MuzykaN.UshakovV.SamoilovaZ.OktyabrskyO. (2023). Influence of growth medium composition on physiological responses of Escherichia coli to the action of chloramphenicol and ciprofloxacin. BioTech12:12. doi: 10.3390/biotech12020043

  • CrossRef
  • Google Scholar

• 67

  StephenJ.LekshmiM.AmminiP.KumarS. H.VarelaM. F. (2022). Membrane efflux pumps of pathogenic vibrio species: role in antimicrobial resistance and virulence. Microorganisms10:382. doi: 10.3390/microorganisms10020382

  • CrossRef
  • Google Scholar

• 68

  SunJ.LiX.HuZ.XueX.ZhangM.WuQ.et al. (2022). Characterization of Vibrio parahaemolyticus isolated from stool specimens of diarrhea patients in Nantong, Jiangsu, China during 2018-2020. PLoS One17:e0273700. doi: 10.1371/journal.pone.0273700

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 69

  SunF.ZhangY.QiuY.YangH.YangW.YinZ.et al. (2014). H-NS is a repressor of major virulence gene loci in Vibrio parahaemolyticus. Front. Microbiol.5:675. doi: 10.3389/fmicb.2014.00675

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 70

  TamayoR.PrattJ. T.CamilliA. (2007). Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annu. Rev. Microbiol.61, 131–148. doi: 10.1146/annurev.micro.61.080706.093426

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 71

  TattaE. R.PaulS.KumavathR. (2023). Transcriptome analysis revealed the synergism of novel rhodethrin inhibition on biofilm architecture, antibiotic resistance and quorum sensing Inenterococcus faecalis. Gene871:147436. doi: 10.1016/j.gene.2023.147436

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 72

  WangL.LingY.JiangH.QiuY.QiuJ.ChenH.et al. (2013). AphA is required for biofilm formation, motility, and virulence in pandemic Vibrio parahaemolyticus. Int. J. Food Microbiol.160, 245–251. doi: 10.1016/j.ijfoodmicro.2012.11.004

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 73

  WuQ.LiX.ZhangT.ZhangM.XueX.YangW.et al. (2022). Transcriptomic analysis of Vibrio parahaemolyticus underlying the wrinkly and smooth phenotypes. Microbiol. Spectr.10:e0218822. doi: 10.1128/spectrum.02188-22

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 74

  XiongX. P.WangC.YeM. Z.YangT. C.PengX. X.LiH. (2010). Differentially expressed outer membrane proteins of Vibrio alginolyticus in response to six types of antibiotics. Mar. Biotechnol. (N.Y.)12, 686–695. doi: 10.1007/s10126-009-9256-4

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 75

  XuM.YamamotoK.HondaT.MingX. (1994). Construction and characterization of an isogenic mutant of Vibrio parahaemolyticus having a deletion in the thermostable direct hemolysin-related hemolysin gene (trh). J. Bacteriol.176, 4757–4760. doi: 10.1128/jb.176.15.4757-4760.1994

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 76

  XueX. F.ZhnagM. M.SunJ. F.LiX.WuQ. M.YinZ.et al. (2022). H-NS represses biofilm formation and c-di-GMP synthesis in Vibrio parahaemolyticus. Biomed. Environ. Sci.35, 821–829. doi: 10.3967/bes2022.106

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 77

  YangY. J.SinghR. P.LanX.ZhangC. S.ShengD. H.LiY. Q. (2019). Whole transcriptome analysis and gene deletion to understand the chloramphenicol resistance mechanism and develop a screening method for homologous recombination in Myxococcus xanthus. Microb. Cell Factories18:123. doi: 10.1186/s12934-019-1172-3

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 78

  YeX.HuangY.ZhouC.LiuX.ZhaoW.ZhaoX.et al. (2021). MSI-1 combats drug-resistant S. aureus by affecting bacterial viability and inhibiting carotenoid pigment production. Microbiol. Res.255:126909. doi: 10.1016/j.micres.2021.126909

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 79

  YildizF. H.VisickK. L. (2009). Vibrio biofilms: so much the same yet so different. Trends Microbiol.17, 109–118. doi: 10.1016/j.tim.2008.12.004

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 80

  YuY.YangH.LiJ.ZhangP.WuB.ZhuB.et al. (2012). Putative type VI secretion systems of Vibrio parahaemolyticus contribute to adhesion to cultured cell monolayers. Arch. Microbiol.194, 827–835. doi: 10.1007/s00203-012-0816-z

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 81

  ZaafraneS.MaatoukK.AlibiS.BenM. H. (2022). Occurrence and antibiotic resistance of Vibrio parahaemolyticus isolated from the Tunisian coastal seawater. J. Water Health20, 369–384. doi: 10.2166/wh.2022.243

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 82

  ZhangY.GaoH.Osei-AdjeiG.ZhangY.YangW.YangH.et al. (2017). Transcriptional regulation of the type VI secretion system 1 genes by quorum sensing and ToxR in Vibrio parahaemolyticus. Front. Microbiol.8:2005. doi: 10.3389/fmicb.2017.02005

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 83

  ZhangY.HuL.Osei-AdjeiG.ZhangY.YangW.YinZ.et al. (2018). Autoregulation of ToxR and its regulatory actions on major virulence gene loci in Vibrio parahaemolyticus. Front. Cell. Infect. Microbiol.8:291. doi: 10.3389/fcimb.2018.00291

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 84

  ZhangY.HuL.QiuY.Osei-AdjeiG.TangH.ZhangY.et al. (2019). QsvR integrates into quorum sensing circuit to control Vibrio parahaemolyticus virulence. Environ. Microbiol.21, 1054–1067. doi: 10.1111/1462-2920.14524

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 85

  ZhangM.LuoX.LiX.ZhangT.WuF.LiM.et al. (2023a). L-arabinose affects the growth, biofilm formation, motility, c-di-GMP metabolism, and global gene expression of Vibrio parahaemolyticus. J. Bacteriol.e0010023. doi: 10.1128/jb.00100-23. [Epub ahead of print].

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 86

  ZhangY.Osei-AdjeiG.NiB.FangH.ZhangL.ZhaoX.et al. (2016). Transcription of exsD is repressed directly by H-NS in Vibrio parahaemolyticus. Microb. Pathog.97, 221–225. doi: 10.1016/j.micpath.2016.06.003

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 87

  ZhangL.Osei-AdjeiG.ZhangY.GaoH.YangW.ZhouD.et al. (2017). CalR is required for the expression of T6SS2 and the adhesion of Vibrio parahaemolyticus to HeLa cells. Arch. Microbiol.199, 931–938. doi: 10.1007/s00203-017-1361-6

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 88

  ZhangY.QiuY.GaoH.SunJ.LiX.ZhangM.et al. (2021). OpaR controls the metabolism of c-di-GMP in Vibrio parahaemolyticus. Front. Microbiol.12:676436. doi: 10.3389/fmicb.2021.676436

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 89

  ZhangY.QiuY.XueX.ZhangM.SunJ.LiX.et al. (2021). Transcriptional regulation of the virulence genes and the biofilm formation associated operons in Vibrio parahaemolyticus. Gut Pathog13:15. doi: 10.1186/s13099-021-00410-y

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 90

  ZhangS.ShaoY.ZhaoX.LiC.GuoM.LvZ.et al. (2020). Indole contributes to tetracycline resistance via the outer membrane protein OmpN in Vibrio splendidus. World J. Microbiol. Biotechnol.36:36. doi: 10.1007/s11274-020-02813-6

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 91

  ZhangG.SunK.AiG.LiJ.TangN.SongY.et al. (2019). A novel family of intrinsic chloramphenicol acetyltransferase CATC in Vibrio parahaemolyticus: naturally occurring variants reveal diverse resistance levels against chloramphenicol. Int. J. Antimicrob. Agents54, 75–79. doi: 10.1016/j.ijantimicag.2019.03.012

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 92

  ZhangL.WengY.WuY.WangX.YinZ.YangH.et al. (2018). H-NS is an activator of exopolysaccharide biosynthesis genes transcription in Vibrio parahaemolyticus. Microb. Pathog.116, 164–167. doi: 10.1016/j.micpath.2018.01.025

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 93

  ZhangM.XueX.LiX.WuQ.ZhangT.YangW.et al. (2023b). QsvR and OpaR coordinately repress biofilm formation by Vibrio parahaemolyticus. Front. Microbiol.14:1079653. doi: 10.3389/fmicb.2023.1079653

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 94

  ZhangY.XueX.SunF.LiX.ZhangM.WuQ.et al. (2023). Quorum sensing and QsvR tightly control the transcription of vpa0607 encoding an active RNase II-type protein in Vibrio parahaemolyticus. Front. Microbiol.14:1123524. doi: 10.3389/fmicb.2023.1123524

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 95

  ZhongX.LuZ.WangF.YaoN.ShiM.YangM. (2022). Characterization of GefA, a GGEEF domain-containing protein that modulates Vibrio parahaemolyticus motility, biofilm formation, and virulence. Appl. Environ. Microbiol.88:e0223921. doi: 10.1128/aem.02239-21

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 96

  ZhouX.GewurzB. E.RitchieJ. M.TakasakiK.GreenfeldH.KieffE.et al. (2013). A Vibrio parahaemolyticus T3SS effector mediates pathogenesis by independently enabling intestinal colonization and inhibiting TAK1 activation. Cell Rep.3, 1690–1702. doi: 10.1016/j.celrep.2013.03.039

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 97

  ZhouX.KonkelM. E.CallD. R. (2010). Regulation of type III secretion system 1 gene expression in Vibrio parahaemolyticus is dependent on interactions between ExsA, ExsC, and ExsD. Virulence1, 260–272. doi: 10.4161/viru.1.4.12318

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 98

  ZhouX.ShahD. H.KonkelM. E.CallD. R. (2008). Type III secretion system 1 genes in Vibrio parahaemolyticus are positively regulated by ExsA and negatively regulated by ExsD. Mol. Microbiol.69, 747–764. doi: 10.1111/j.1365-2958.2008.06326.x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar