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It is well known that most microbial populations develop their intrinsic antibiotics resistance at low concentrations in antibiotics environments, but the factors influencing spontaneous resistance are still largely unknown. In this study, Aeromonas hydrophila strains with different resistance levels to oxytetracycline (OXY) were induced by sublethal antibiotic selection pressure, and differential expression of proteins was compared among them using iTRAQ-based quantitative proteomics. Our following bioinformatic analysis showed that energy metabolism-related proteins were downregulated, while several iron-related proteins were upregulated in high OXY-resistant strains. To further investigate the role of spontaneous OXY resistance evolution, four TonB-dependent receptor-coded genes were deleted, and their OXY susceptibility capabilities and antibiotic evolutionary assays were performed, respectively. Our results showed that the deletion of these genes did not affect the susceptibility to OXY, but showed different evolution rates in the spontaneous OXY evolution compared with wild-type strain, especially for AHA_0971 and AHA_4251. Therefore, this study indicates the important role of TonB-dependent receptor proteins during the bacterial antibiotics resistance evolution and may provide a new prophylactic strategy against the development of antibiotic resistance.

Vibrio alginolyticus, a bacterial pathogen in fish and humans, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. In this study, the T3SS chaperone protein gene vscB was cloned from V. alginolyticus wild-type strain HY9901and the vscB gene has a length of 429 bp and encodes 142 amino acids. The amino acid sequence homology comparison results showed that VscB had the highest similarity with that of Vibrio parahaemolyticus VscB, reaching 91%. The mutant strain HY9901ΔvscB was constructed by the in-frame deletion method. The HY9901ΔvscB showed an attenuated swarming phenotype and a 23-fold decrease in virulence to pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu). However, the HY9901ΔvscB showed no difference in morphology, growth, biofilm formation and extracellular protease (ECPase) activity. The results of antibiotic susceptibility testing showed that the wild-type HY9901 is more sensitive to Doxycycline, Minocyline and Kanamycin than in the HY9901ΔvscB. A total of 95 differentially expressed genes were screened by transcriptome sequencing analysis of HY9901 and strain ΔvscB, revealing 57 genes up-regulated and 38 genes were down-regulated in the ΔvscB compared to HY9901. qRT-PCR was employed to analyze the transcription levels of T3SS-related genes showing that ΔvscB had decreased expression of vopN and vscO and increased expression of hop, vscN, vscK, vscL and vopS compared to HY9901. Finally, grouper vaccinated via intraperitoneal (IP) injection with HY9901ΔvscB had a relative percent survival (RPS) of 77.6% following challenge with HY9901. Real-time qPCR assays showed that vaccination with HY9901ΔvscB enhanced the expression of immune-related genes, including MHC-I, IgM, and CD8α both in the liver and spleen, indicating that HY9901ΔvscB is able to induce humoral and cell-mediated immune responses in pearl gentian grouper. These results demonstrate that the HY9901ΔvscB mutant could be used as an effective live attenuated vaccine to combat V. alginolyticus infection in pearl gentian grouper.

Antibiotic resistance among aquatic bacterial pathogens has become a serious concern in aquaculture environments, which has increased research interest to develop solutions to overcome this problem. Moreover, the activities of several Aeromonas hydrophila gene pathways have remained elusive concerning antibiotic resistance evolution. Therefore, in this study, we have performed a transcriptomic analysis to compare differentially expressed genes between oxytetracycline (OXY) susceptible and resistant strains of A. hydrophila. Compared to the A. hydrophila susceptible strain, a total of 22 and 185 genes were differentially expressed in the 4-fold minimal inhibitory concentration (MIC) and 8-fold MIC resistant strains, respectively. Furthermore, the bioinformatics analysis revealed that the sulfur metabolism-related genes were down-regulated. The genes responsible for mannitol metabolism and the efflux pump system were up-regulated in resistant strains, compared to the susceptible strain. Therefore, it suggests that these three pathways may be involved in the OXY resistance evolution in A. hydrophila. The outcome of the transcriptomic data was further validated through quantitative reverse transcription-PCR (qRT-PCR) and Western blot analysis. Overall, the obtained data provides a deeper insight into the intrinsic molecular mechanism of OXY resistance evolution in A. hydrophila.

The survival and immune responses of Litopenaeus vannamei were evaluated during white spot syndrome virus (WSSV) or Vibrio parahaemolyticus single and concurrent infections. The mortality, WSSV load, activities of 4 immune enzymes: acid phosphatase (ACP), alkaline phosphatase (AKP), peroxidase (POD) and superoxide dismutase (SOD), and the transcription of Evolutionarily Conserved Signaling Intermediate in Toll pathways of L.vannamei (LvECSIT) were quantified at 0, 3, 6, 12, 24, 48, 72 and 96 h post-infection (pi). The results showed: (i) the cumulative mortality of the co-infection group (WSSV and V. Parahaemolyticus 83%) was significantly lower than the WSSV infection group (97%) (P < 0.05) at 96 hpi; (ii) copies of WSSV in the co-infection group were significantly lower than that of the single infection group from 24 to 96 hpi (P < 0.05); (iii) ACP, AKP,POD and SOD activity in the gills of the co-infection group was higher than that of the WSSV group at12, 48 and 96 hpi (P < 0.05).The expression of LvECSIT mRNA in the co-infection group was significantly higher than in the WSSV infection group from 12 to 72 hpi (P < 0.05).The results indicate that proliferation of WSSV is inhibited by V.parahaemolyticus infection. In addition, infection with WSSV alone causes a significant reduction in some immune responses of shrimp than co-infection with WSSV and V.parahaemolyticus occurs at 26 °C. Third, LvECSIT, an essential member of TLR signaling pathway might play a crucial role in shrimp defense against WSSV – Vibrio co-infection.

AhyI is homologous to the protein LuxI and is conserved throughout bacterial species including Aeromonas hydrophila. A. hydrophila causes opportunistic infections in fish and other aquatic organisms. Furthermore, this pathogennot only poses a great risk for the aquaculture industry, but also for human public health. AhyI (expressing acylhomoserine lactone) is responsible for the biosynthesis of autoinducer-1 (AI-1), commonly referred to as a quorum sensing (QS) signaling molecule, which plays an essential role in bacterial communication. Studying protein structure is essential for understanding molecular mechanisms of pathogenicity in microbes. Here, we have deduced a predicted structure of AhyI protein and characterized its function using in silico methods to aid the development of new treatments for controlling A.hydrophila infections. In addition to modeling AhyI, an appropriate inhibitor molecule was identified via high throughput virtual screening (HTVS) using mcule drug-like databases.The AhyI-inhibitor N-cis-octadec-9Z-enoyl-l-Homoserine lactone was selected withthe best drug score. In order to understand the pocket sites (ligand binding sites) and their interaction with the selected inhibitor, docking (predicted protein binding complex) servers were used and the selected ligand was docked with the predicted AhyI protein model. Remarkably, N-cis-octadec-9Z-enoyl-l-Homoserine lactone established interfaces with the protein via16 residues (V24, R27, F28, R31, W34, V36, D45, M77, F82, T101, R102, L103, 104, V143, S145, and V168), which are involved with regulating mechanisms of inhibition. These proposed predictions suggest that this inhibitor molecule may be used as a novel drug candidate for the inhibition of auto-inducer-1 (AI-1) activity.The N-cis-octadec-9Z-enoyl-l-Homoserine lactone inhibitor molecule was studied on cultured bacteria to validate its potency against AI-1 production. At a concentration of 40 μM, optimal inhibition efficiency of AI-1 was observedin bacterial culture media.These results suggest that the inhibitor molecule N-cis-octadec-9Z-enoyl-l-Homoserine lactone is a competitive inhibitor of AI-1 biosynthesis.

A mutant strain of Vibiro alginolyticus with an in-frame deletion of the toxR gene was constructed to reveal the role of ToxR in the physiology and virulence of V. alginolyticus. The statistical analysis showed no significant difference in the growth ability, swarming motility, activity of extracellular protease and the virulence by injection (the value of LD50) between the wild-type and the toxR mutant. However, the deletion of toxR could decrease the level of biofilm formation. The comparative proteomic analysis demonstrated the deletion mutation of toxR could up-regulate the expression of glutamine synthetase and levansucrase, and down-regulate the expression of 10 proteins such as OmpU, DnaK, etc. These results suggest that ToxR may be involved in the early stages of infection by influencing colonization of the bacteria on the surface of the intestine through enhancing the biofilm information of V. alginolyticus via modulating the expression of glutamine synthetize, levansucrase and OmpU.

DNA vaccines had been widely used against microbial infection in animals. The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been increasingly studied in recent years. MyD88 is one of the adapter molecules to activate the signaling cascades and produces inflammatory mediators, and its immunological role and adjuvant potential which had been proved in mammals were rarely reported in fish species. In this study, plasmid pcMyD88 was constructed and the capacity of MyD88 as molecular adjuvant was explored by co-injecting with a DNA vaccine encoding AcfA against Vibrio alginolyticus infection in orange spotted grouper. The results suggested that it needed at least 7 days to transported DNA vaccine pcacfA or molecular adjuvant pcMyD88 from the injected muscle to kidney and spleens and stimulate host's immune system for later protection. The co-injection of pcMyD88 with DNA vaccine pcacfA could increase significantly specific antibody levels and the expression levels of the immune-related genes including MHCIα, MHCIIα, CD4, CD8α, IL-1β and TNFα. Furthermore, pcMyD88 enhanced the immunoprotection of pcacfA against V. alginolyticus infection, with the significantly higher RPS of 83.3% in pcMyD88 + pcacfA group compared with that of pcacfA alone (73.3%) at challenging test of 10 weeks post vaccination. Together, these results clearly demonstrate that MyD88 is an effective adjuvant for the DNA vaccine pcacfA in orange spotted grouper.

Vibrio alginolyticus, a Gram-negative bacterium, has been recognized as an opportunistic pathogen in marine animals as well as humans. Type III secretion system (T3SS) is critical for pathogen virulence and disease development. However, no more information is known about the C-ring component VscQ and its physiological role. In this study, gene vscQ was cloned from V. alginolyticus wild-type strain HY9901 and the mutant strain HY9901ΔvscQ was constructed by the in-frame deletion method. The HY9901ΔvscQ mutant showed an attenuated swarming phenotype and a closely 4.6-fold decrease in the virulence to Danio rerio. However, the HY9901ΔvscQ mutant showed no difference in growth, biofilm formation and ECPase activity. HY9901ΔvscQ reduces the release of LDH, NO and caspase-3 activity of infected FHM cell, which are involved in fish cell apoptosis. Deletion of gene vscQ downregulates the expression level of T3SS-related genes including vscL, vopB, hop, vscO, vscK, vopD, vcrV and vopS and flagellum-related genes (flaA and fliG). And Danio rerio vaccinated via i.m injection with HY9901ΔvscQ induced a relative percent survival (RPS) value of 71% after challenging with the wild-type HY9901. Real-time PCR assays showed that vaccination with HY9901ΔvscQ enhanced the expression of immune-related genes, including TNF-α, TLR5, IL-6R, IgM and c/ebpβ in liver and spleen after vaccination, indicating that it is able to induce humoral and cell-mediated immune response in zebrafish. These results demonstrate that the HY9901ΔvscQ mutant could be used as an effective live vaccine to combat V. alginolyticus infection.

Vibrio alginolyticus is an opportunistic and halophilic Gram-negative pathogen in limiting the development of aquatic industry and affecting human health. SODs are oxidative enzymes that play a critical role in oxidative defense. In this study, an in-frame deleted mutant strain (ΔsodB) was constructed by allelic exchange mutagenesis to investigate physiological role of sodB in pathogenicity of V. alginolyticus. The results exhibited that ΔsodB showed no differences in growth compared with wild-type strain HY9901 (WT), but led to increasing in biofilm formation, ECPase activity and sensitivity to hydrogen peroxide, decreasing in swarming motility, adherence to CIK cells, SOD activity and virulence. In addition, ΔsodB induced a high antibody titer and provided a valid protection with a relative percent survival value of 86.5% without inducing clinical symptoms after challenging with WT. These results suggest that sodB is important for normal physiological function, oxidation resistance and virulence in V. alginolyticus, and ΔsodB may be considered as an effective live attenuated vaccine against V. alginolyticus.

V. alginolyticus is an important opportunistic pathogen which causes vibriosis in aquatic animals. AcfA, as an accessory colonization factor, is hypothesized to be involved in the pathogenesis of infection. In this study, a mutant strain with an in-frame deletion removed nucleotides 86 to 561 of the acfA gene was constructed to reveal the role of AcfA in the physiology and virulence from V. alginolyticus. An acfA mutant showed a similar growth level, an obvious decrease in swarming motility and the activity of ECPase, a higher LD50 value by intraperitoneal injection of grouper fish compared to that of the wild-type. Furthermore, the deletion of acfA could enhance the level of biofilm formation and suppress the polar flagellum forming. The comparative proteomic analysis demonstrated the deletion mutation of acfA could up-regulate the expression of 4 proteins of p4alcd, deoD, phb and DctP, and down-regulate the expression of 8 proteins of Clp, hpV36980, ABCtp, pepD, arA, aggp, fla and ompA compared to that of the wild-type. The analysis of RT-qPCR showed the mRNA levels of DctP and deoD were significantly induced, and the mRNA levels of pepD, arA, fla and ompA were significantly reduced in acfA mutant compared with the wild-type. The results suggest that acfA may contribute to the overall success in the pathogenesis of V. alginolyticus by regulating the expression of some relevant genes.

Vibrio alginolyticus is one of the most serious diseases in cultured marine and freshwater fish and shellfish. The absence of suitable vaccine or virulent marker can be the bottleneck to control V. alginolyticus infection. In this study, immunoproteomic approaches were undertaken to study the immunogenicity of the whole-cell protein of V. alginolyticus HY9901. The whole-cell proteins were analysed by two-dimensional gel electrophoresis and subsequent immunoblotting using the rabbit anti-V. alginolyticus HY9901 serum. A total of 55 immunogenic proteins were identified by immunoproteomic analysis. Of the 55 proteins, 51 are specific immunoreactive proteins and four are nonspecific immunoreactive proteins. Furthermore, outer membrane protein N (spot 2) was used as immunogens to immunize Epinephelus coioides for investigation of protective abilities and activities. The E. coioides immunized with OmpN has abilities to fight against infections caused by V. alginolyticus. The other novel immunogenic proteins may be developed as alternative antigens for further study of V. alginolyticus vaccine and diagnostics. These data show that immunoproteomics methods can be successfully applied in identifying immunogenic proteins of V. alginolyticus, which helps to search for the protective antigens in future.

Vibrio spp. represent a serious threat to the culture of Epinephelus coioides (Orange-spotted Grouper) in Southeast Asia. In this study we used two-dimensional electrophoresis (2-DE) and Western blotting to identify common immunogenic proteins of Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus. Membranes were probed with orange-spotted grouper anti-V. alginolyticus sera and accordingly 60, 58 and 48 immunogenic protein spots were detected. By matching analysis for the three Western blotting membranes, 6 cross immunogenic spots for the three Vibrio species were identified. They were Outer membrane protein W (OmpW), dihydrolipoamide dehydrogenase (DLD), succinate dehydrogenase flavoprotein subunit(SDHA), elongation factor Ts(Ts), peptide ABC transporter periplasmic peptide-binding protein and phosphoenolpyruvate carboxykinase(PEPCK). One of the proteins, DLD, was used to evaluate the cross protective function for E. coioides with a bacterial immunization and challenge method. The relative percent survival rate of E. coioides against V. alginolyticus, V. harveyi and V. parahaemolyticus was 90%, 86% and 80%, respectively. This work may provide potential cross protective vaccine candidate antigens for three Vibrio species, and DLD may be considered as an effective cross-protective immunogen against three Vibrio species.

Type III secretion system (T3SS) in Vibrio alginolyticus is essential for its pathogenesis. VscO's homologous proteins FliJ, InvI and YscO have been suggested to be putative chaperone escorts although its function in V. alginolyticus is unclear. To investigate the physiological role of VscO, a mutant strain of V. alginolyticus with an in-frame deletion of the vscO gene was constructed in the present study. One finding was that the mRNA expression levels of SycD, VopB and VopD proteins decreased in the ΔvscO mutant. In addition, the ΔvscO mutant showed an attenuated swarming ability and a ten-fold decrease in the virulence to fish. However, the ΔvscO mutant showed no difference in the biofilm formation and ECPase activity. Complementation of the mutant strain with the vscO gene could restore the phenotypes of the wild-type strain. Finally, the recombinant VscO protein caused a high antibody titer and an effective protection against lethal challenge with the wild-type strain V. alginolyticus. These results indicated that VscO protein has a specific role in the pathogenesis of V. alginolyticus and it may be a candidate antigen for development of a subunit vaccine against vibriosis.

Vibrio harveyi is an important pathogen that causes vibriosis in various aquatic organisms. Here, we announce the draft genome sequence of V. harveyi strain ZJ0603, which was isolated from diseased Orange-spotted grouper ( Epinephelus coioides ) in Guangdong, China.