Mobile-CRISPRi protocol optimization for Vibrionaceae.

Mobile clustered regularly interspaced palindromic repeats interference (Mobile-CRISPRi) is an established method for bacterial gene expression knockdown. The deactivated Cas9 protein and guide RNA are isopropyl ß-D-1-thiogalactopyranoside inducible, and all components are integrated into the chromosome via Tn7 transposition. Here, we optimized methods specific for applying Mobile-CRISPRi in multiple Vibrio species.

Bimetallic nanoparticles with sulfated galactan eliminate Vibrio parahaemolyticus in shrimp Penaeus vannamei.

Bimetallic (Au/Ag) nanoparticles (BNPs) have shown enhanced antibacterial activity compared to their monometallic counterparts. Sulfated galactans (SG) are a naturally occurring polymer commonly found in red seaweed Gracilaria fisheri. They are biocompatible and biodegradable and environmentally friendly. In this study, we utilized SG in combination with BNPs to develop composite materials that potentially enhance antibacterial activity against shrimp pathogens Vibrio parahaemolyticus and Vibrio harveyi, compared to BNPs or SG alone. BNPs were coated with sulfated galactan (SGBNPs) and characterized using UV-vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, zeta potential, and transmission electron microscopy (TEM). UV-vis spectroscopy analysis revealed that the surface plasmon peaks of BNPs and SGBNPs appeared at 530 nm and 532 nm, respectively. Zeta potential measurements showed that SGBNPs had a negative charge of -32.4 mV, while the BNPs solution had a positive charge of 38.7 mV. TEM images demonstrated the spherical morphology of both BNPs and SGBNPs with narrow size distributions (3-10 nm). Analysis of the FTIR spectra indicated that SG maintained its backbone structure in SGBNPs, but some functional groups were altered. Notably, SGBNPs showed superior antimicrobial and antibiofilm activities against V. parahaemolyticus and V. harveyi compared to SG and BNPs. Furthermore, treatment with SGBNPs significantly down-regulated the expression of virulence-related genes (toxR, cpsQ, and mfpA) for V. parahaemolyticus 3HP compared to the respective control, bacteria treated with BNPs or SG. Diets supplemented with SGBNPs, BNPs, or SG showed no detrimental impact on the growth of shrimp Penaeus vannamei. Shrimp fed with SGBNPs-supplemented feed showed significantly higher survival rates than those fed with BNPs-supplemented feed when infected with 3HP after being on the supplemented feed for seven days and a subsequent number of fifteen days. These findings collectively demonstrate the benefit of using SG capped Au-Ag BNPs as an antibacterial agent for the prevention and control of Vibrio sp. infection in shrimp while reducing the risk of environmental contamination.

Characterization of Vibrio parahaemolyticus isolated from clinical specimens and oysters in Thailand.

INTRODUCTION: Vibrio parahaemolyticus is a common pathogen that can cause seafood-borne gastroenteritis in humans. We determined the prevalence and characteristics of V. parahaemolyticus isolated from clinical specimens and oysters in Thailand. METHODOLOGY: Isolates of V. parahaemolyticus from clinical specimens (n = 77) and oysters (n = 224) were identified by biochemical testing, polymerase chain reaction (PCR) assays, and serotyping. The toxin genes, antimicrobial resistance, and ß-lactamase production were determined. RESULTS: A total of 301 isolates were confirmed as V. parahaemolyticus by PCR using specific primers for the toxR gene. The majority of clinical isolates carried the tdh+/trh- genotype (82.1%), and one of each isolate was tdh-/trh+ and tdh+/trh+ genotypes. One isolate from oyster contained the tdh gene and another had the trh gene. Twenty-six serotypes were characterized among these isolates, and O3:K6 was the most common (37.7%), followed by OUT:KUT, and O4:K9. In 2010, most clinical and oyster isolates were susceptible to antibiotics, with the exception of ampicillin. In 2012, clinical isolates were not susceptible to cephalothin (52.4%), streptomycin (95.2%), amikacin (66.6%), kanamycin (61.9%), and erythromycin (95.2%), significantly more frequently than in 2010. More than 95% of isolates that were not susceptible to ampicillin produced ß-lactamase enzymes. CONCLUSIONS: We found toxin genes in two oyster isolates, and the clinical isolates that were initially determined to be resistant to several antibiotics. Toxin genes and antimicrobial susceptibility profiles of V. parahaemolyticus from seafood and environment should be continually monitored to determine the spread of toxin and antimicrobial resistance genes.

Establishment and application of a rapid visualization method for detecting Vibrio parahaemolyticus nucleic acid.

Background: Swift and accurate detection of Vibrio parahaemolyticus, which is a prominent causative pathogen associated with seafood contamination, is required to effectively combat foodborne disease and wound infections. The toxR gene is relatively conserved within V. parahaemolyticus and is primarily involved in the expression and regulation of virulence genes with a notable degree of specificity. The aim of this study was to develop a rapid, simple, and constant temperature detection method for V. parahaemolyticus in clinical and nonspecialized laboratory settings. Methods: In this study, specific primers and CRISPR RNA were used to target the toxR gene to construct a reaction system that combines recombinase polymerase amplification (RPA) with CRISPR‒Cas13a. The whole-genome DNA of the sample was extracted by self-prepared sodium dodecyl sulphate (SDS) nucleic acid rapid extraction reagent, and visual interpretation of the detection results was performed by lateral flow dipsticks (LFDs). Results: The specificity of the RPA-CRISPR/Cas13a-LFD method was validated using V. parahaemolyticus strain ATCC-17802 and six other non-parahaemolytic Vibrio species. The results demonstrated a specificity of 100%. Additionally, the genomic DNA of V. parahaemolyticus was serially diluted and analysed, with a minimum detectable limit of 1 copy/µL for this method, which was greater than that of the TaqMan-qPCR method (102 copies/µL). The established methods were successfully applied to detect wild-type V. parahaemolyticus, yielding results consistent with those of TaqMan-qPCR and MALDI-TOF MS mass spectrometry identification. Finally, the established RPA-CRISPR/Cas13a-LFD method was applied to whole blood specimens from mice infected with V. parahaemolyticus, and the detection rate of V. parahaemolyticus by this method was consistent with that of the conventional PCR method. Conclusions: In this study, we describe an RPA-CRISPR/Cas13a detection method that specifically targets the toxR gene and offers advantages such as simplicity, rapidity, high specificity, and visual interpretation. This method serves as a valuable tool for the prompt detection of V. parahaemolyticus in nonspecialized laboratory settings.

A pH ultra-sensitive hydrated iridium oxyhydroxide films electrochemical sensor for label-free detection of Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is a major foodborne pathogen, which can cause serious foodborne illnesses like diarrhoea. Rapid on-site detection of foodborne pathogens is an ideal way to respond to foodborne illnesses. Herein, we provide an electrochemical sensor for rapid on-site detection. This sensor utilized a pH-sensitive metal-oxide material for the concurrent isothermal amplification and label-free detection of nucleic acids. Based on a pH-sensitive hydrated iridium oxide oxyhydroxide film (HIROF), the electrode transforms the hydrogen ion compound generated during nucleic acid amplification into potential, so as to achieve a real-time detection. The results can be transmitted to a smartphone via Bluetooth. Moreover, HIROF was applied in nucleic acid device detection, with a super-Nernst sensitivity of 77.6 mV/pH in the pH range of 6.0-8.5, and the sensitivity showed the best results so far. Detection of V. parahaemolyticus by this novel method showed a detection limit of 1.0×103 CFU/mL, while the time consumption was only 30 min, outperforming real-time fluorescence loop-mediated isothermal amplification (LAMP). Therefore, the characteristics of compact, portable, and fast make the sensor more widely used in on-site detection.

Genomic and Transcriptomic Analyses Reveal Multiple Strategies for Vibrio parahaemolyticus to Tolerate Sub-Lethal Concentrations of Three Antibiotics.

Vibrio parahaemolyticus can cause acute gastroenteritis, wound infections, and septicemia in humans. The overuse of antibiotics in aquaculture may lead to a high incidence of the multidrug-resistant (MDR) pathogen. Nevertheless, the genome evolution of V. parahaemolyticus in aquatic animals and the mechanism of its antibiotic tolerance remain to be further deciphered. Here, we investigated the molecular basis of the antibiotic tolerance of V. parahaemolyticus isolates (n = 3) originated from shellfish and crustaceans using comparative genomic and transcriptomic analyses. The genome sequences of the V. parahaemolyticus isolates were determined (5.0-5.3 Mb), and they contained 4709-5610 predicted protein-encoding genes, of which 823-1099 genes were of unknown functions. Comparative genomic analyses revealed a number of mobile genetic elements (MGEs, n = 69), antibiotic resistance-related genes (n = 7-9), and heavy metal tolerance-related genes (n = 2-4). The V. parahaemolyticus isolates were resistant to sub-lethal concentrations (sub-LCs) of ampicillin (AMP, 512 µg/mL), kanamycin (KAN, 64 µg/mL), and streptomycin (STR, 16 µg/mL) (p < 0.05). Comparative transcriptomic analyses revealed that there were significantly altered metabolic pathways elicited by the sub-LCs of the antibiotics (p < 0.05), suggesting the existence of multiple strategies for antibiotic tolerance in V. parahaemolyticus. The results of this study enriched the V. parahaemolyticus genome database and should be useful for controlling the MDR pathogen worldwide.

Construction of PCR-SERS Method for Detection of Vibrio parahaemolyticus.

A paper-based surface enhancement of a Raman scattering substrate consisting of silver-nanowires stacked on glass-fiber filter paper was prepared. At the same time, the DNA-embedding molecule Eva Green was introduced as a signaling molecule for surface-enhanced Raman scattering (SERS) detection. Polymerase chain reaction (PCR) was used to amplify target genes and the method was developed into a rapid molecular diagnostic system. The total detection time of the developed detection method was 40 min, including 30 min of PCR amplification and 10 min of SERS measurement. After 30 PCR cycles, bacterial DNA with an initial concentration of 20 fg/µL and a bacterial suspension with an initial concentration of 7.2 × 101 CFUs/mL could be detected. When the enrichment culture time was 4 h, target bacteria with an initial contamination inoculation volume of 1.5 CFUs/mL could be detected in artificially contaminated samples. The method is fast and highly sensitive, and has not been applied to the detection of V. parahaemolyticus.

A 3D-Printed Integrated Handheld Biosensor for the Detection of Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the important seafood-borne pathogens that cause a serious gastrointestinal disorder in humans. Recently, biosensors have attracted serious attention for precisely detecting and tracking risk factors in foods. However, a major consideration when fabricating biosensors is to match the low cost of portable devices to broaden its application. In this study, a 3D-printed integrated handheld biosensor (IHB) that combines RPA-CRISPR/Cas12a, a lateral flow strip (LFS), and a handheld device was developed for the ultrasensitive detection of V. parahaemolyticus. Using the preamplification of RPA on tlh gene of V. parahaemolyticus, a specific duplex DNA product was obtained to activate the trans-cleavage activity of CRISPR/Cas12a, which was then utilized to cleave the ssDNA probe. The ssDNA probe was then detected by the LFS, which was negatively correlated with the content of amplified RPA products of the tlh gene. The IHB showed high selectivity and excellent sensitivity for V. parahaemolyticus detection, and the limit of detection was 4.9 CFU/mL. The IHB also demonstrated great promise for the screening of V. parahaemolyticus in samples and had the potential to be applied to the rapid screening of other pathogen risks for seafood and marine environmental safety.

Complete genome sequence of Vibrio parahaemolyticus ST36 strain MAVP-26, a clinical isolate from an oyster-borne human gastric infection.

A Pacific native lineage of Vibrio parahaemolyticus ST36 serotype O4:K12 was introduced into the Atlantic, which increased local source illnesses. To identify genetic determinants of virulence and ecological resiliency and track their transfer into endemic populations, we constructed a complete genome of a 2013 Atlantic-traced clinical isolate by hybrid assembly.

Unveiling the biofloc culture potential: Harnessing immune functions for resilience of shrimp and resistance against AHPND -causing Vibrio parahaemolyticus infection.

In shrimp aquaculture, disease mitigation may be accomplished by reducing the virulence of the pathogen or by boosting the shrimp's immunity. Biofloc technology is an innovative system that improves the health and resistance of shrimp to microbial infections while providing a viable option for maintaining the quality of culture water through efficient nutrient recycling. This review aimed at demonstrating the efficacy of the biofloc system in boosting the immune responses and protective processes of shrimp against Vibrio parahaemolyticus infection, which is known to cause Acute Hepatopancreatic Necrosis Disease (AHPND). Numerous studies have revealed that the biofloc system promotes the immunological capability of shrimp by raising multiple immune -related genes e.g. prophenoloxidase, serine proteinase gene, ras-related nuclear gene and penaeidinexpression and cellular and humoral responses such as hyperaemia, prophenoloxidase activity, superoxide dismutase activity, phagocytic activity; the protection and survival of shrimp when faced with a challenge from the V. parahaemolyticus strain have been enhanced. Furthermore, the use of the biofloc system improves water quality parameters and potentially bolstering their immune and overall health to effectively resist diseases; hence, promotes the growth of shrimp. The present review suggests that biofloc can serve as an effective therapy for both preventing and supporting the management of probable AHPND infection in shrimp culture. This approach exhibits potential for the progress of sustainable shrimp farming, higher productivity, and improved shrimp health.

Sustainable double-synergistic silver-hydroxyapatite composite catalyst derived from fish bones for efficient disinfection of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a food-borne pathogen that poses a serious threat to seafood safety and human health. An efficient, nontoxic, and sustainable disinfection material with a stable structure is urgently needed. Herein, silver (Ag)-hydroxyapatite (HAP) composite catalysts were prepared using HAP derived from waste fish bones. The Ag2.50%-HAP showed a 100% disinfection rate against V. parahaemolyticus, disinfecting nearly 7.0 lg CFU mL-1 within 15 min at a low concentration of 300 µg mL-1. This efficient disinfection activity could be attributed to the double-synergistic effect of Ag and superoxide radicals, which resulted in the destruction of bacterial cell structures and the leakage of intracellular proteins. Importantly, the composite also exhibited high activity in controlling the growth of pathogens during the storage process of Penaeus vannamei. These findings provided sustainable composite catalysts for disinfecting V. parahaemolyticus in seafood and a high-value utilization strategy for waste fish bones.

Effects of Taiwanese indigenous cinnamon (Cinnamomum osmophloeum) leaf hot-water extract on nonspecific immune responses, resistance against Vibrio parahaemolyticus, nonviable cells, and haemocyte subpopulations in white shrimp (Penaeusvannamei).

This study investigated the effects of Cinnamomum osmophloeum leaf hot-water extract (CLWE) on nonspecific immune responses and resistance to Vibrio parahaemolyticus in white shrimp (Penaeus vannamei). Firstly, a cell viability assay demonstrated that the CLWE is safe to white shrimp heamocytes in the concentration of 0-500 mg L-1. Haemocytes incubated in vitro with 10 and 50 mg L-1 of CLWE showed significantly higher response in superoxide anion production, PO activity, and phagocytic activity. In the in vivo trials, white shrimp were fed with 0, 0.5, 1, 5, and 10 g kg-1 CLWE supplemented feeds (designated as CLWE 0, CLWE 0.5, CLWE 1, CLWE 5, and CLWE 10, respectively) over a period of 28 days. In vivo experiments demonstrated that CLWE 0.5 feeding group resulted in the highest total haemocyte count, superoxide anion production, phenoloxidase activity, and phagocytic activity. Moreover, CLWE 0.5 supplemented feed significantly upregulated the clotting system, antimicrobial peptides, pattern recognition receptors, pattern recognition proteins, and antioxidant defences in white shrimp. Furthermore, the shrimp were infected with V. parahaemolyticus injections after 14 days of feeding as challenge test. Based on the challenge test result, both CLWE 0.5 and CLWE 5 demonstrated a strong resistance to V. parahaemolyticus. These two dosages effectively reduced the number of nonviable cells and activated different haemocyte subpopulations. These findings indicated that treatment with CLWE 0.5 could promote nonspecific immune responses, immune-related gene expression, and resistance to V. parahaemolyticus in white shrimp.

The mannose-binding lectin (MBL) gene cloned from Exopalaemon carinicauda plays a key role in resisting infection by Vibrio parahaemolyticus.

Mannose-binding lectin (MBL) is a vital member of the lectin family, crucial for mediating functions within the complement lectin pathway. In this study, following the cloning of the mannose-binding lectin (MBL) gene in the ridgetail white prawn, Exopalaemon carinicauda, we examined its expression patterns across various tissues and its role in combating challenges posed by Vibrio parahaemolyticus. The results revealed that the MBL gene spans 1342 bp, featuring an open reading frame of 972 bp. It encodes a protein comprising 323 amino acids, with a predicted relative molecular weight of 36 kDa and a theoretical isoelectric point of 6.18. The gene exhibited expression across various tissues including the eyestalk, heart, gill, hepatopancreas, stomach, intestine, ventral nerve cord, muscle, and hemolymph, with the highest expression detected in the hepatopancreas. Upon challenge with V. parahaemolyticus, RT-PCR analysis revealed a trend of MBL expression in hepatopancreatic tissues, characterized by an initial increase followed by a subsequent decrease, peaking at 24 h post-infection. Employing RNA interference to disrupt MBL gene expression resulted in a significant increase in mortality rates among individuals challenged with V. parahaemolyticus. Furthermore, we successfully generated the Pet32a-MBL recombinant protein through the construction of a prokaryotic expression vector for conducting in vitro bacterial inhibition assays, which demonstrated the inhibitory effect of the recombinant protein on V. parahaemolyticus, laying a foundation for further exploration into its immune mechanism in response to V. parahaemolyticus challenges.

IcmF2 of the type VI secretion system 2 plays a role in biofilm formation of Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses two distinct type VI secretion systems (T6SS), namely T6SS1 and T6SS2. T6SS1 is predominantly responsible for adhesion to Caco-2 and HeLa cells and for the antibacterial activity of V. parahaemolyticus, while T6SS2 mainly contributes to HeLa cell adhesion. However, it remains unclear whether the T6SS systems have other physiological roles in V. parahaemolyticus. In this study, we demonstrated that the deletion of icmF2, a structural gene of T6SS2, reduced the biofilm formation capacity of V. parahaemolyticus under low salt conditions, which was also influenced by the incubation time. Nonetheless, the deletion of icmF2 did not affect the biofilm formation capacity in marine-like growth conditions, nor did it impact the flagella-driven swimming and swarming motility of V. parahaemolyticus. IcmF2 was found to promote the production of the main components of the biofilm matrix, including extracellular DNA (eDNA) and extracellular proteins, and cyclic di-GMP (c-di-GMP) in V. parahaemolyticus. Additionally, IcmF2 positively influenced the transcription of cpsA, mfpA, and several genes involved in c-di-GMP metabolism, including scrJ, scrL, vopY, tpdA, gefA, and scrG. Conversely, the transcription of scrA was negatively impacted by IcmF2. Therefore, IcmF2-dependent biofilm formation was mediated through its effects on the production of eDNA, extracellular proteins, and c-di-GMP, as well as its impact on the transcription of cpsA, mfpA, and genes associated with c-di-GMP metabolism. This study confirmed new physiological roles for IcmF2 in promoting biofilm formation and c-di-GMP production in V. parahaemolyticus.

Antimicrobial susceptibility profile and molecular characterization of Vibrio parahaemolyticus strains isolated from imported shrimps.

Vibrio parahaemolyticus is a threat to human health and one of the leading bacterial causes of seafood-borne infection worldwide. This pathogen is autochtonous in the marine environment and is able to acquire antimicrobial resistance (AMR) mechanisms, which is a global concern. However, the emergence of AMR V. parahaemolyticus strains in seafood is still understudied, as interpretation criteria for this species for antimicrobial susceptibility tests are limited in the literature. In this study, we investigated the susceptibility profiles to clinically important antibiotics and the associated genetic determinants of V. parahaemolyticus isolates cultured from imported shrimps. Based on the analysis of the resistance phenotypes of 304 V. parahaemolyticus isolates, we have defined experimental epidemiological cutoff values (COWT) for 14/15 antibiotics tested. We observed that 19.1% of the bacterial isolates had acquired resistance to at least one antibiotic class. The highest number of resistance was associated with tetracycline (14.5% of the strains) and trimethoprim-sulfamethoxazole (3.6%). Moreover, seven strains were multidrug-resistant (MDR, resistant to at least three antibiotic classes). The most frequently identified genes in these strains were aph(3″)-Ib/aph(6)-Id (aminoglycoside resistance), sul2 (sulfonamide), tet(59) (tetracycline), and floR (chloramphenicol). The SXT/R391 family ICE and class 1 integron-integrase genes were detected by PCR in three and one MDR V. parahaemolyticus strains, respectively. Consequently, V. parahaemolyticus in seafood can act as a reservoir of AMR, constituting a health risk for the consumer.IMPORTANCEOur study on "Antimicrobial Resistance Profiles and Genetic Determinants of Vibrio parahaemolyticus Isolates from Imported Shrimps" addresses a critical gap in understanding the emergence of antimicrobial resistance (AMR) in this seafood-associated pathogen. Vibrio parahaemolyticus is a major cause of global seafood-borne infections, and our research reveals that 19.1% of isolates from imported shrimps display resistance to at least one antibiotic class, with multidrug resistance observed in seven strains. Importantly, we establish experimental epidemiological cutoff values for antibiotic susceptibility, providing valuable criteria specific to V. parahaemolyticus. Our findings underscore the potential risk to consumers, emphasizing the need for vigilant monitoring and intervention strategies. This study significantly contributes to the comprehension of AMR dynamics in V. parahaemolyticus, offering crucial insights for global public health. The dissemination of our research through Microbiology Spectrum ensures broad accessibility and impact within the scientific community and beyond.

Identification of an LysR family transcriptional regulator that activates motility and flagellar gene expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus utilizes a polar flagellum for swimming in liquids and employs multiple lateral flagella to swarm on surfaces and in viscous environments. The VPA0961 protein is an LysR family transcriptional regulator that can regulate the swimming and swarming motility of V. parahaemolyticus, but the detailed regulatory mechanisms are not yet fully understood. Herein, we designated the protein as AcsS, which stands for activator of swimming and swarming motility. Our data provided evidence that deleting the acsS gene significantly reduced both swimming and swarming motility of V. parahaemolyticus. Furthermore, AcsS was found to activate the expression of both polar (flgA, flgM, flgB, and flgK) and lateral (motY, fliM, lafA, and fliD) flagellar genes. Overexpression of AcsS in Escherichia coli induced the expression of flgA, motY, and lafA, but did not affect the expression of flgB, flgK, flgM, fliM, and fliD. Interestingly, His-tagged AcsS did not bind to the upstream DNA regions of all the tested genes, suggesting indirect regulation. In conclusion, AcsS positively regulated the swimming and swarming motility of V. parahaemolyticus by activating the transcription of polar and lateral flagellar genes. This work enriched our understanding of the gene expression regulation within the dual flagellar systems of V. parahaemolyticus.

Dietary vitamin C reduces mortality of pacific white shrimp (Penaeus vannamei) post-larvae by Vibrio parahaemolyticus challenge.

This study was conducted to investigate whether optimal vitamin C (VC) levels can enhance non-specific immune response and antioxidant capacity and reduce mortality of Pacific white shrimp (Penaeus vannamei) post-larvae when infected with Vibrio parahaemolyticus. Six experimental diets were formulated to contain six different VC levels of 0, 40, 80, 120, 160 and 320 mg/kg diet (designated as C0, C40, C80, C120, C160 and C320, respectively). Shrimp post-larvae (39.1 ± 0.47 mg) were randomly distributed to 24 tanks with 40 shrimp per tank. Four replicate groups of shrimp were fed one of the diets for 43 days. VC supplemented groups showed significantly higher growth performance than C0 group. Shrimp fed C120 diet had significantly improved feed utilization efficiency than shrimp fed C0 diet. VC concentrations in hepatopancreas and gills were significantly higher with the increase in dietary VC levels. Optimal dietary VC levels significantly upregulated the expressions of growth and digestive enzyme-related genes such as IGF-1, IGF-BP, amylase, trypsin and chymotrypsin, and also upregulated the expressions of innate immunity and antioxidant-related genes such as prophenoloxidase, crustin, penaiedin-3a, superoxide dismutase, glutathione peroxidase and catalase in hepatopancreas. Shrimp fed C80, C120 and C160 diets showed significantly increased resistance to V. parahaemolyticus than shrimp fed C0 diet. The optimum dietary VC level for the shrimp post-larvae was established to be 80.2 mg/kg diet by a broken-line regression analysis based on the growth. The findings from the challenge test indicated that VC levels over 83.0 mg/kg diet could enhance disease resistance of the shrimp against V. parahaemolyticus.

The effectiveness of gliding arc discharge plasma in sterilizing artificial seawater contaminated with Vibrio parahaemolyticus.

The rapid proliferation of the halophilic pathogen Vibrio parahaemolyticus poses a severe health hazard to halobios and significantly impedes intensive mariculture. This study aimed to evaluate the potential application of gliding arc discharge plasma (GADP) to control the infection of Vibrio parahaemolyticus in mariculture. This study investigated the inactivation ability of GADP against Vibrio parahaemolyticus in artificial seawater (ASW), changes in the water quality of GADP-treated ASW, and possible inactivation mechanisms of GADP against Vibrio parahaemolyticus in ASW. The results indicate that GADP effectively inactivated Vibrio parahaemolyticus in ASW. As the volume of ASW increased, the time required for GADP sterilization also increased. However, the complete sterilization of 5000 mL of ASW containing Vibrio parahaemolyticus of approximately 1.0 × 104 CFU/mL was achieved within 20 min. Water quality tests of the GADP-treated ASW demonstrated that there were no significant changes in salinity or temperature when Vibrio parahaemolyticus (1.0 ×104 CFU/mL) was completely inactivated. In contrast to the acidification observed in plasma-activated water (PAW) in most studies, the pH of ASW did not decrease after treatment with GADP. The H2O2 concentration in the GADP-treated ASW decreased after post-treatment. The NO2-concentration in the GADP-treated ASW remained unchanged after post-treatment. Further analysis revealed that GADP induced oxidative stress in Vibrio parahaemolyticus, which increased cell membrane permeability and intracellular ROS levels of Vibrio parahaemolyticus. This study provides a viable solution for infection with the halophilic pathogen Vibrio parahaemolyticus and demonstrates the potential of GADP in mariculture.

The effect of environmental calcium on gene expression, biofilm formation and virulence of Vibrio parahaemolyticus.

Calcium (Ca2+) can regulate the swarming motility and virulence of Vibrio parahaemolyticus BB22. However, the effects of Ca2+ on the physiology of V. parahaemolyticus RIMD2210633, whose genomic composition is quite different with that of BB22, have not been investigated. In this study, the results of phenotypic assays showed that the biofilm formation, c-di-GMP production, swimming motility, zebrafish survival rate, cytoxicity against HeLa cells, and adherence activity to HeLa cells of V. parahaemolyticus RIMD2210633 were significantly enhanced by Ca2+. However, Ca2+ had no effect on the growth, swarming motility, capsular polysaccharide (CPS) phase variation and hemolytic activity. The RNA sequencing (RNA-seq) assay disclosed 459 significantly differentially expressed genes (DEGs) in response to Ca2+, including biofilm formation-associated genes and those encode virulence factors and putative regulators. DEGs involved in polar flagellum and T3SS1 were upregulated, whereas majority of those involved in regulatory functions and c-di-GMP metabolism were downregulated. The work helps us understand how Ca2+ affects the behavior and gene expression of V. parahaemolyticus RIMD2210633.

Virulence phenotypes differ between toxigenic Vibrio parahaemolyticus isolated from western coasts of Europe.

Vibrio parahaemolyticus is the leading bacterial cause of gastroenteritis associated with seafood consumption worldwide. Not all members of the species are thought to be pathogenic, thus identification of virulent organisms is essential to protect public health and the seafood industry. Correlations of human disease and known genetic markers (e.g. thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH)) appear complex. Some isolates recovered from patients lack these factors, while their presence has become increasingly noted in isolates recovered from the environment. Here, we used whole-genome sequencing in combination with mammalian and insect models of infection to assess the pathogenic potential of V. parahaemolyticus isolated from European Atlantic shellfish production areas. We found environmental V. parahaemolyticus isolates harboured multiple virulence-associated genes, including TDH and/or TRH. However, carriage of these factors did not necessarily reflect virulence in the mammalian intestine, as an isolate containing TDH and the genes coding for a type 3 secretion system (T3SS) 2α virulence determinant, appeared avirulent. Moreover, environmental V. parahaemolyticus lacking TDH or TRH could be assigned to groups causing low and high levels of mortality in insect larvae, with experiments using defined bacterial mutants showing that a functional T3SS1 contributed to larval death. When taken together, our findings highlight the genetic diversity of V. parahaemolyticus isolates found in the environment, their potential to cause disease and the need for a more systematic evaluation of virulence in diverse V. parahaemolyticus to allow better genetic markers.