Roles of linker region flanked by transmembrane and peptidoglycan binding region of PomB in energy conversion of the Vibrio flagellar motor.

The flagellar components of Vibrio spp., PomA and PomB, form a complex that transduces sodium ion and contributes to rotate flagella. The transmembrane protein PomB is attached to the basal body T-ring by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. Previously we showed that PomB deleted from E41 to R120 (Δ41-120) was functionally comparable to the full-length PomB. In this study, three deletions after the plug region, PomB (Δ61-120), PomB (Δ61-140), and PomB (Δ71-150), were generated. PomB (Δ61-120) conferred motility, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed comparable for the wild-type cells. When the two PomB mutants were introduced into a wild-type strain, the swimming ability was not affected by the mutant PomBs. Then, we purified the mutant PomAB complexes to confirm the stator formation. When plug mutations were introduced into the PomB mutants, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletions prevent the stator activation and the linker and plug regions, from E41 to S150, are not essential for the motor function of PomB but are important for its regulation.

Vibrio alginolyticus PEPCK Mediates Florfenicol Resistance through Lysine Succinylation Modification.

Protein succinylation modification is a common post-translational modification (PTM) that plays an important role in bacterial metabolic regulation. In this study, quantitative analysis was conducted on the succinylated proteome of wild-type and florfenicol-resistant Vibrio alginolyticus to investigate the mechanism of succinylation regulating antibiotic resistance. Bioinformatic analysis showed that the differentially succinylated proteins were mainly enriched in energy metabolism, and it was found that the succinylation level of phosphoenolpyruvate carboxyl kinase (PEPCK) was highly expressed in the florfenicol-resistant strain. Site-directed mutagenesis was used to mutate the lysine (K) at the succinylation site of PEPCK to glutamic acid (E) and arginine (R), respectively, to investigate the function of lysine succinylation of PEPCK in the florfenicol resistance of V. alginolyticus. The detection of site-directed mutagenesis strain viability under florfenicol revealed that the survival rate of the E mutant was significantly higher than that of the R mutant and wild type, indicating that succinylation modification of PEPCK protein may affect the resistance of V. alginolyticus to florfenicol. This study indicates the important role of PEPCK during V. alginolyticus antibiotic-resistance evolution and provides a theoretical basis for the prevention and control of vibriosis and the development of new antibiotics.

DUF1127-containing protein and ProQ had opposite effects on biofilm formation in Vibrio alginolyticus.

The RNA binding protein is crucial for gene regulation at the post transcription level. In this study, functions of the DUF1127-containing protein and ProQ, which are RNA-binding proteins, were revealed in Vibrio alginolyticus. DUF1127 deletion increased the ability of biofilm formation, whereas ProQ deletion reduced the amount of biofilm. Moreover, extracellular proteinase secretion was significantly reduced in the DUF1127 deletion strain. ProQ, not DUF1127-containing protein, can help the cell to defense oxidative stress. Deletion of DUF1127 resulted in a higher ROS level in the cell, however, ProQ deletion showed no difference. RNA-seq unveiled the expression of genes involved in extracellular protease secretion were significantly downregulated and biofilm synthesis-related genes, such as rbsB and alsS, were differentially expressed in the DUF1127 deletion strain. ProQ affected the expression of genes involved in biofilm synthesis (flgC and flgE), virulence (betB and hutG), and oxidative stress. Moreover, the DUF1127-containing and ProQ affected the mRNA levels of various regulators, such as LysR and BetI. Overall, our study revealed that the DUF1127-containing protein and ProQ have crucial functions on biofilm formation in V. alginolyticus.

Isolation and Characterization of a Novel Vibrio Phage vB_ValA_R15Z.

Vibrio phages have emerged as a potential alternative to antibiotic therapy for treating Vibrio infections. In this study, a lytic Vibrio phage, vB_ValA_R15Z against Vibrio alginolyticus ATCC 17749T, was isolated from an aquatic water sample collected in Xiamen, China. The phage had an icosahedral head (diameter 69 ± 2 nm) and a short, non-contractile tail measuring 16 ± 2 nm. The genome of vB_ValA_R15Z was found to be a double-stranded DNA consisting of 43, 552 bp, containing 54 coding sequences (CDSs) associated with phage packaging, structure, DNA metabolism, lysis and additional functions. The BLASTN results indicated that vB_ValA_R15Z shared less than 90.18% similarity with known phages recorded in the NCBI GenBank database, suggesting that vB_ValA_R15Z was a novel Vibrio phage. Furthermore, phylogenetic analysis revealed that vB_ValA_R15Z belongs to the genus Kaohsiungvirus. In addition, a typical lytic mechanism (holin-endolysim) was found in the genome of vB_ValA_R15Z, while no antibiotic resistance- or virulence factor-related gene was detected. Overall, the study provides valuable insights into the isolation and characterization of vB_ValA_R15Z, highlighting its potential as an effective phage therapy option for combating Vibrio alginolyticus infections.

Deletion of ArmPT, a LamB-like protein, increases cell membrane permeability and antibiotic sensitivity in Vibrio alginolyticus.

Vibrio bacterial species are dominant pathogens in mariculture animals. However, the extensive use of antibiotics and other chemicals has increased drug resistance in Vibrio bacteria. Despite rigorous investigative studies, the mechanism of drug resistance in Vibrio remains a mystery. In this study, we found that a gene encoding LamB-like outer membrane protein, named ArmPT, was upregulated in Va under antibiotic stress by RT-qPCR. We speculated that ArmPT might play a role in Va's drug resistance. Subsequently, using ArmPT gene knockout and gene complementation experiments, we confirmed its role in resistance against a variety of antibiotics, particularly kanamycin (KA). Transcriptomic and proteomic analyses identified 188 and 83 differentially expressed genes in the mutant strain compared with the wild-type (WT) before and after KA stress, respectively. Bioinformatic analysis predicted that ArmPT might control cell membrane permeability by changing cadaverine biosynthesis, thereby influencing the cell entry of antibiotics in Va. The higher levels of intracellular reactive oxygen species and the infused content of KA showed that antibiotics are more likely to enter the Va mutant strain. These results uncover the drug resistance mechanism of Va that can also exist in other similar pathogenic bacteria.

Isolation and Characterization of a Novel Phage against Vibrio alginolyticus Belonging to a New Genus.

Vibrio alginolyticus causes substantial economic losses in the aquaculture industry. With the rise of multidrug-resistant Vibrio strains, phages present a promising solution. Here, a novel lytic Vibrio phage, vB_ValC_RH2G (RH2G), that efficiently infects the pathogenic strain V. alginolyticus ATCC 17749T, was isolated from mixed wastewater from an aquatic market in Xiamen, China. Transmission electron microscopy revealed that RH2G has the morphology of Siphoviruses, featuring an icosahedral head (73 ± 2 nm diameter) and long noncontractile tail (142 ± 4 nm). A one-step growth experiment showed that RH2G had a short latent period (10 min) and a burst size of 48 phage particles per infected cell. Additionally, RH2G was highly species-specific and was relatively stable at 4-55 °C and pH 4-10. A genomic analysis showed that RH2G has a 116,749 bp double-stranded DNA genome with 43.76% GC content. The intergenomic similarity between the genome sequence of RH2G and other phages recorded in the GenBank database was below 38.8%, suggesting that RH2G represents a new genus. RH2G did not exhibit any virulence or resistance genes. Its rapid lysis capacity, lytic activity, environmental resilience, and genetic safety suggested that RH2G may be a safe candidate for phage therapy in combatting vibriosis in aquaculture settings.

The Effect of the Lysine Acetylation Modification of ClpP on the Virulence of Vibrio alginolyticus.

Acetylation modification has become one of the most popular topics in protein post-translational modification (PTM) research and plays an important role in bacterial virulence. A previous study indicated that the virulence-associated caseinolytic protease proteolytic subunit (ClpP) is acetylated at the K165 site in Vibrio alginolyticus strain HY9901, but its regulation regarding the virulence of V. alginolyticus is still unknown. We further confirmed that ClpP undergoes lysine acetylation (Kace) modification by immunoprecipitation and Western blot analysis and constructed the complementation strain (C-clpP) and site-directed mutagenesis strains including K165Q and K165R. The K165R strain significantly increased biofilm formation at 36 h of incubation, and K165Q significantly decreased biofilm formation at 24 h of incubation. However, the acetylation modification of ClpP did not affect the extracellular protease (ECPase) activity. In addition, we found that the virulence of K165Q was significantly reduced in zebrafish by in vivo injection. To further study the effect of lysine acetylation on the pathogenicity of V. alginolyticus, GS cells were infected with four strains, namely HY9901, C-clpP, K165Q and K165R. This indicated that the effect of the K165Q strain on cytotoxicity was significantly reduced compared with the wild-type strain, while K165R showed similar levels to the wild-type strain. In summary, the results of this study indicate that the Kace of ClpP is involved in the regulation of the virulence of V. alginolyticus.

Formation of multiple flagella caused by a mutation of the flagellar rotor protein FliM in Vibrio alginolyticus.

Marine bacterium Vibrio alginolyticus forms a single flagellum at a cell pole. In Vibrio, two proteins (GTPase FlhF and ATPase FlhG) regulate the number of flagella. We previously isolated the NMB155 mutant that forms multiple flagella despite the absence of mutations in flhF and flhG. Whole-genome sequencing of NMB155 identified an E9K mutation in FliM that is a component of C-ring in the flagellar rotor. Mutations in FliM result in defects in flagellar formation (fla) and flagellar rotation (che or mot); however, there are a few reports indicating that FliM mutations increase the number of flagella. Here, we determined that the E9K mutation confers the multi-flagellar phenotype and also the che phenotype. The co-expression of wild-type FliM and FliM-E9K indicated that they were competitive in regard to determining the flagellar number. The ATPase activity of FlhG has been correlated with the number of flagella. We observed that the ATPase activity of FlhG was increased by the addition of FliM but not by the addition of FliM-E9K in vitro. This indicates that FliM interacts with FlhG to increase its ATPase activity, and the E9K mutation may inhibit this interaction. FliM may control the ATPase activity of FlhG to properly regulate the number of the polar flagellum at the cell pole.

Characterization of a Filamentous Phage, Vaf1, from Vibrio alginolyticus AP-1.

Filamentous phages are ubiquitously distributed in the global oceans. However, little is known about their biological contribution to their host's genetic and phenotypic diversity. In this study, a filamentous phage, Vaf1, was isolated and characterized from the emerging marine pathogen strain Vibrio alginolyticus AP-1. We explored the effects of the resident phage Vaf1 on the host physiology under diverse conditions by precisely deleting the entire phage Vaf1. Our results demonstrate that the presence of phage Vaf1 significantly increased biofilm formation, swarming motility, and contact-dependent competition. Furthermore, the gene expression profile suggests that several phage genes were upregulated in response to low-nutrient conditions. Unexpectedly, an in vivo study of zebrafish shows that fish infected with strain ΔVaf1 survived longer than those infected with wild-type strain AP-1, indicating that Vaf1 contributes to the virulence of V. alginolyticus. Together, our results provide direct evidence for the effect of Vaf1 phage-mediated phenotypic changes in marine bacteria V. alginolyticus. This further emphasizes the impressive complexity and diversity that filamentous phage-host interactions pose and the challenges associated with bacterial disease control in marine aquaculture. IMPORTANCE Non-lytic filamentous phages can replicate without killing their host, establishing long-term persistence within the bacterial host. In contrast to the well-studied CTXφ phage of the human-pathogenic Vibrio cholerae, little is known about the filamentous phage Vaf1 and its biological role in host fitness. In this study, we constructed a filamentous phage-deleted strain, ΔVaf1, and provided direct evidence on how an intact phage, φVaf1, belonging to the family Inoviridae, helps the bacterial host AP-1 to overcome adverse environmental conditions. Our results likely open new avenues for fundamental studies on how filamentous phage-host interactions regulate different aspects of Vibrio cell behaviors.

Occurrence of virulence determinants in vibrio cholerae, vibrio mimicus, vibrio alginolyticus, and vibrio parahaemolyticus isolates from important water resources of Eastern Cape, South Africa.

BACKGROUND: Virulence determinants are crucial to the risk assessment of pathogens in an environment. This study investigated the presence of eleven key virulence-associated genes in Vibrio cholerae (n = 111) and Vibrio mimicus (n = 22) and eight virulence determinants in Vibrio alginolyticus (n = 65) and Vibrio parahaemolyticus (n = 17) isolated from six important water resources in Eastern Cape, South Africa, using PCR techniques. The multiple virulence gene indexes (MVGI) for sampling sites and isolates as well as hotspots for potential vibriosis outbreaks among sampling sites were determined statistically based on the comparison of MVGI. RESULT: The PCR assay showed that all the V. cholerae isolates belong to non-O1/non-O139 serogroups. Of the isolates, Vibrio Cholera (84%), V. mimicus (73%), V. alginolyticus (91%) and V. parahaemolyticus (100%) isolates harboured at least one of the virulence-associated genes investigated. The virulence gene combinations detected in isolates varied at sampling site and across sites. Typical virulence-associated determinants of V. cholerae were detected in V. mimicus while that of V. parahaemolyticus were detected in V. alginolyticus. The isolates with the highest MVGI were recovered from three estuaries (Sunday river, Swartkopps river, buffalo river) and a freshwater resource (Lashinton river). The cumulative MVGI for V. cholerae, V. mimicus, V. alginolyticus and V. parahaemolyticus isolates were 0.34, 0.20, 0.45, and 0.40 respectively. The targeted Vibrio spp. in increasing order of the public health risk posed in our study areas based on the MVGI is V. alginolyticus > V. parahaemolyticus > V. cholerae > V. mimicus. Five (sites SR, PA5, PA6, EL4 and EL6) out of the seventeen sampling sites were detected as the hotspots for potential cholera-like infection and vibriosis outbreaks. CONCLUSIONS: Our findings suggest that humans having contact with water resources in our study areas are exposed to potential public health risks owing to the detection of virulent determinants in human pathogenic Vibrio spp. recovered from the water resources. The study affirms the relevancy of environmental Vibrio species to the epidemiology of vibriosis, cholera and cholera-like infections. Hence we suggest a monitoring program for human pathogenic Vibrio spp. in the environment most especially surface water that humans have contact with regularly.

Comprehensive insights into the metabolism characteristics of small RNA Qrr4 in Vibrio alginolyticus.

Vibrio alginolyticus is an important foodborne pathogen that can infect both humans and marine animals and cause massive economic losses in aquaculture. Small noncoding RNAs (sRNAs) are emerging posttranscriptional regulators that affect bacterial physiology and pathological processes. In the present work, a new cell density-dependent sRNA, Qrr4, was characterized in V. alginolyticus based on a previously reported RNA-seq analysis and bioinformatics approach. The effects of Qrr4 actions on the physiology, virulence, and metabolism of V. alginolyticus were comprehensively investigated based on molecular biology and metabolomics approaches. The results showed that qrr4 deletion markedly inhibited growth, motility and extracellular protease activities. Additionally, nontargeted metabolism and lipidomics analyses revealed that qrr4 deletion induced significant disturbance of multiple metabolic pathways. The key metabolic remodelling that occurred in response to qrr4 deletion was found to involve phospholipid, nucleotide, carbohydrate and amino acid metabolic pathways, providing novel clues about a potential mechanism via which mutation of qrr4 could interfere with cellular energy homeostasis, modulate membrane phospholipid composition and inhibit nucleic acid and protein syntheses to regulate the motility, growth and virulence characteristics of V. alginolyticus. Overall, this study provides a comprehensive understanding of the regulatory roles of the new cell density-dependent sRNA Qrr4 in V. alginolyticus. KEY POINTS: • A novel cell density-dependent sRNA, Qrr4, was cloned in V. alginolyticus. •Qrr4 regulated growth and virulence factors of V. alginolyticus. • Phospholipid, nucleotide and energy metabolisms were modulated obviously by Qrr4.

Binding site profiles and N-terminal minor groove interactions of the master quorum-sensing regulator LuxR enable flexible control of gene activation and repression.

LuxR is a TetR family master quorum sensing (QS) regulator activating or repressing expression of hundreds of genes that control collective behaviors in Vibrios with underlying mechanism unknown. To illuminate how this regulator controls expression of various target genes, we applied ChIP-seq and DNase I-seq technologies. Vibrio alginolyticus LuxR controls expression of ∼280 genes that contain either symmetric palindrome (repDNA) or asymmetric (actDNA) binding motifs with different binding profiles. The median number of LuxR binding sites for activated genes are nearly double for that of repressed genes. Crystal structures of LuxR in complex with the respective repDNA and actDNA motifs revealed a new mode of LuxR DNA binding that involves contacts of its N-terminal extension to the minor groove. The N-terminal contacts mediated by Arginine-9 and Arginine-11 differ when LuxR binds to repDNA vs actDNA, leading to higher binding affinity at repressed targets. Moreover, modification of LuxR binding sites, binding profiles, and N-terminal extension have important consequences on QS-regulated phenotypes. These results facilitate fundamental understanding of the high flexibility of mechanisms of LuxR control of gene activation and repression in Vibrio QS, which may facilitate to design QS inhibiting chemicals that interfere with LuxR regulation to effectively control pathogens.

A strain of Vibrio alginolyticus isolated from Azumapecten farreri and its pathogenic mechanism using CRISPR-Cas9 technology.

Scallops have become an important aquaculture species in China because they contain high-quality protein, and scallops are important health food that combines multiple effects and high economic benefits. However, scallop aquaculture is perennially threatened by various pathogenic Vibrio species, leading to great economic losses. We obtained a strain of pathogenic bacteria, identified as Vibrio alginolyticus, from the diseased Azumapecten farreri in the scallop farming area of Huangdao District in 2018, and V. alginolyticus is one of the major shellfish pathogens. We showed that V. alginolyticus was isolated and identified as a pathogen in A. farreri for the first time. In this study, we evaluated its morphology and performed a phylogenetic analysis based on 16S rRNA gene sequencing. In addition, we performed a preliminary analysis of its pathogenic mechanisms. The Hfq protein in V. alginolyticus is an important RNA-binding protein in the quorum-sensing system that not only affects the sensitivity of Vibrio to environmental stress but also regulates a variety of functions, such as cell membrane formation, motility, and virulence towards the host. However, its effect on the pathogenesis of V. alginolyticus to A. farreri is unclear. To further investigate the pathogenic mechanism of the Hfq protein in V. alginolyticus to A. farreri, we used the CRISPR-Cas9 system to target and deplete the hfq gene fragment in V. alginolyticus and obtained the mutant strain V. ΔHfq-. We found that the peripheral flagellum of the mutant strain was lost, which reduced the motility of V. alginolyticus. Therefore, the deletion of target genes by the CRISPR/Cas9 genome editing system confirmed that the Hfq protein played a key role in reducing the ability of V. alginolyticus to infect A. farreri. In conclusion, our current findings provided valuable insights into the healthy culture of scallops.

Genome Sequencing-Based Mining and Characterization of a Novel Alginate Lyase from Vibrio alginolyticus S10 for Specific Production of Disaccharides.

Alginate oligosaccharides prepared by alginate lyases attracted great attention because of their desirable biological activities. However, the hydrolysis products are always a mixture of oligosaccharides with different degrees of polymerization, which increases the production cost because of the following purification procedures. In this study, an alginate lyase, Alg4755, with high product specificity was identified, heterologously expressed, and characterized from Vibrio alginolyticus S10, which was isolated from the intestine of sea cucumber. Alg4755 belonged to the PL7 family with two catalytic domains, which was composed of 583 amino acids. Enzymatic characterization results show that the optimal reaction temperature and pH of Alg4755 were 35 °C and 8.0, respectively. Furthermore, Alg4755 was identified to have high thermal and pH stability. Moreover, the final hydrolysis products of sodium alginate catalyzed by Alg4755 were mainly alginate disaccharides with a small amount of alginate trisaccharides. The results demonstrate that alginate lyase Alg4755 could have a broad application prospect because of its high product specificity and desirable catalytic properties.

Cloning and expression of two anti-lipopolysaccharide factors in Eriocheir hepuensis under Vibrio alginolyticus-induced stress.

Anti-lipopolysaccharide factors (ALFs) are small basic proteins that exhibit broad-spectrum antiviral properties and antibacterial activity. In this research, we cloned and studied two Eriocheir hepuensis ALFs, EhALF2 and EhALF3. The results showed that the open reading frame lengths of EhALF2 and EhALF3 were 363 and 372 bp, encoding 120 and 123 amino acids, respectively. Their sequences both contained an Lipopolysaccharide-binding (LPS) domain and were highly similarity to other crab ALFs. qRT-PCR showed that EhALF2 and EhALF3 were detected in nine examined tissues and were expressed the highest in the haemocytes. After challenge with Vibrio alginolyticus, in the hepatopancreas, the expression levels of EhALF2 and EhALF3 reached the highest levels at 48 and 3 h, respectively. In the heart, the expression levels of the two genes were highest at 12 h. These results indicate that EhALF2 and EhALF3 could participate in the resistance of E. hepuensis to V. alginolyticus stress within a short time. They have potential applications in the study of environmental stress markers and disease-resistance factors in E. hepuensis.

Rapid visual nucleic acid detection of Vibrio alginolyticus by recombinase polymerase amplification combined with CRISPR/Cas13a.

Vibrio alginolyticus (V. alginolyticus) is a common pathogen in the ocean. In addition to causing serious economic losses in aquaculture, it can also infect humans. The rapid detection of nucleic acids of V. alginolyticus with high sensitivity and specificity in the field is very important for the diagnosis and treatment of infection caused by V. alginolyticus. Here, we established a simple, fast and effective molecular method for the identification of V. alginolyticus that does not rely on expensive instruments and professionals. The method integrates recombinase polymerase amplification (RPA) technology with CRISPR system in a single PCR tube. Using this method, the results can be visualized by lateral flow dipstick (LFD) in less than 50 min, we named this method RPA-CRISPR/Cas13a-LFD. The method was confirmed to achieve high specificity for the detection of V. alginolyticus with no cross-reactivity with similar Vibrio and common clinical pathogens. This diagnostic method shows high sensitivity; the detection limit of the RPA-CRISPR/Cas13a-LFD is 10 copies/µL. We successfully identified 35 V. alginolyticus strains from a total of 55 different bacterial isolates and confirmed their identity by (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS). We also applied this method on infected mice blood, and the results were both easily and rapidly obtained. In conclusion, RPA-CRISPR/Cas13a-LFD offers great potential as a useful tool for reliable and rapid diagnosis of V. alginolyticus infection, especially in limited conditions.

Transcriptome analysis of Pacific white shrimp (Litopenaeus vannamei) hepatopancreas challenged by Vibrio alginolyticus reveals lipid metabolic disturbance.

Vibrio alginolyticus is a devastating bacterial pathogen of Pacific white shrimp (Litopenaeus vannamei), which often causes acute hepatopancreatic necrosis syndrome (AHPNS) and early mortality syndrome (EMS). Elucidation of molecular mechanisms of L. vannamei in responding to infection is essential for controlling the epidemic. In the present study, transcriptomic profiles of L. vannamei hepatopancreas were explored by injecting with PBS or V. alginolyticus. Hepatopancreas morphology of L. vannamei was also assessed. The result reveals that compared with the hepatopancreas of PBS group, the storage cells (R-cell), secretory cells (B-cell) and star-shaped polygonal structures of the lumen were disappeared and necrotic after challenged by V. alginolyticus at 24 h. Transcriptome data showed that a total of 314 differential expression genes were induced by V. alginolyticus, with 133 and 181 genes up- and down-regulated, respectively. These genes were mainly associated with lysosome pathway, glycerophospholipid metabolism, drug metabolism-other enzymes, cysteine and methionine metabolism, aminoacyl-tRNA biosynthesis and PPAR signal pathway. Among these pathways, the lysosome pathway, glycerophospholipid metabolism and PPAR signal pathway were both related with lipid metabolism. Therefore, we detected the lipid accumulation in hepatopancreas by Oil Red O staining, TG and CHOL detection and the relative mRNA expression of several lipid metabolism related genes in the hepatopancreas of shrimp after challenge to V. alginolyticus. The present data reveals that lipids from the L. vannamei are nutrient sources for the V. alginolyticus and define the fate of the infection by modulating lipid homeostasis. These findings may have important implication for understanding the L. vannamei and V. alginolyticus interactions, and provide a substantial dataset for further research and may deliver the basis for preventing the bacterial diseases.

Vibrio pathogenicity island and phage CTX genes in Vibrio alginolyticus isolated from different aquatic environments.

In the present study, we investigated the presence of four Vibrio cholerae virulence genes (ctxA, VPI, Zot and ace) in 36 Vibrio alginolyticus isolates obtained from different seawater, sediments and aquatic organisms. We tested the virulence of 13 V. alginolyticus strains against juveniles of Sparus aurata and this virulence was correlated with the presence of V. cholerae virulence genes. A positive amplification for the virulence pathogenicity island was produced by five V. alginolyticus strains and four for cholerae toxin. Some of the V. alginolyticus strains are pathogenic to aquatic animals and might have derived their virulence genes from V. cholerae. V. alginolyticus strains can be considered as a possible reservoir of V. cholerae virulence genes.

Fast and sensitive graphene oxide-DNAzyme-based biosensor for Vibrio alginolyticus detection.

DNAzymes have been widely and effectively used for the detection of pathogenic bacteria, which pose a serious public health threat. However, the rapid and cost-effective detection of such bacteria remains a major challenge. In this study, we successfully selected Vibrio alginolyticus-specific DNAzymes. The activity of the candidates was assessed via fluorescence intensity and gel electrophoresis. The DNAzyme DT1 had a detection limit of 31 CFU/ml for V. alginolyticus and exhibited high specificity. Graphene oxide (GO) was used to develop a DNAzyme-based fluorescent sensor for the detection of V. alginolyticus, which significantly improved detection performance and shortened the reaction time as little as 10 s. The proposed method was then validated using crab, shrimp, fish, clam, and oyster samples. This study thus provides a new method for the rapid and sensitive detection of V. alginolyticus.

The role of dctP gene in regulating colonization, adhesion and pathogenicity of Vibrio alginolyticus strain HY9901.

Vibriosis caused by Vibrio alginolyticus has severely affected the development of mariculture industry in recent decades. DctP, a tripartite ATP-independent periplasmic transporter solute-binding subunit, is thought to be one of the virulence factors in Vibrio. In this study, the results displayed no difference in morphological characteristics and growth between ΔdctP (dctP mutant strain) and WT (wild-type strain). Nevertheless, the ability of swarming motility, biofilm formation, ECPase formation, cell adhesion and colonized ability of ΔdctP significantly decreased compared to those of WT. The LD50 of ΔdctP significantly increased by 40-fold compared to that of WT. The transcriptome analysis demonstrated the deletion mutation of dctP could regulate the expression levels of 22 genes related to colonization, adhesion and pathogenicity in V. alginolyticus. The analysis of qRT-PCR showed the transcriptome data were reliable. These results reveal the effect of attenuated function of DctP on colonization, adherence and pathogenicity by controlling the expression of related gene.