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.

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.

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.

Mobile-CRISPRi as a powerful tool for modulating Vibrio gene expression.

CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.

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.

Prophages carrying Zot toxins on different Vibrio genomes: A comprehensive assessment using multilayer networks.

Vibrios, a group of bacteria that are among the most abundant in marine environments, include several species such as Vibrio cholerae and Vibrio parahaemolyticus, which can be pathogenic to humans. Some species of Vibrio contain prophages within their genomes. These prophages can carry genes that code for toxins, such as the zonula occludens toxin (Zot), which contribute to bacterial virulence. Understanding the association between different Vibrio species, prophages and Zot genes can provide insights into their ecological interactions. In this study, we evaluated 4619 Vibrio genomes from 127 species to detect the presence of prophages carrying the Zot toxin. We found 2030 potential prophages with zot-like genes in 43 Vibrio species, showing a non-random association within a primarily modular interaction network. Some prophages, such as CTX or Vf33, were associated with specific species. In contrast, prophages phiVCY and VfO3K6 were found in 28 and 20 Vibrio species, respectively. We also identified six clusters of Zot-like sequences in prophages, with the ZOT2 cluster being the most frequent, present in 34 Vibrio species. This analysis helps to understand the distribution patterns of zot-containing prophages across Vibrio genomes and the potential routes of Zot-like toxin dissemination.

Antimicrobial susceptibility and genomic characterization of Vibrio parahaemolyticus isolated from aquatic foods in 15 provinces, China, 2020.

Prevalent in marine, estuarine and coastal environments, Vibrio parahaemolyticus is one of the major foodborne pathogens which can cause acute gastroenteritis through consumption of contaminated food. This study encompassed antimicrobial resistance, molecular characteristics and phylogenetic relationships of 163 V. parahaemolyticus isolated from aquatic foods across 15 provinces in China. The isolates showed high resistance rates against ampicillin (90.80 %, 148/163) and cefazolin (72.39 %, 118/163). Only 5 isolates demonstrated multi-drug resistance (MDR) phenotypes. A total of 37 different antibiotic resistance genes (ARGs) in correlation with seven antimicrobial categories were identified. tet(34) and tet(35) were present in all 163 isolates. Other most prevalent ARGs were those conferring resistance to ß-lactams, with prevalence rate around 18.40 % (30/163). The virulence genes tdh and trh were found in 17 (10.43 %) and 9 (5.52 %) isolates, respectively. Totally 121 sequence types (STs) were identified through whole genome analysis, among which 60 were novel. The most prevalent sequence type was ST3 (9.20 %, 15/163), which shared the same genotype profile of trh_, tdh+ and blaCARB-22+. Most of the tdh+V. parahaemolyticus isolates was clustered into a distinctive clade by the phylogenetic analysis. Our study showed that the antimicrobial resistance of V. parahaemolyticus in aquatic foods in China was moderate. However, the emerging of MDR isolates implicate strengthened monitoring is needed for the better treatment of human V. parahaemolyticus infections. High genetic diversity and virulence potential of the isolates analyzed in this study help better understanding and evaluating the risk of V. parahaemolyticus posed to public health.

The role of rcpA gene in regulating biofilm formation and virulence in Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common seafood-borne pathogen that can colonize the intestine of host and cause gastroenteritis. Biofilm formation by V. parahaemolyticus enhances its persistence in various environments, which poses a series of threats to food safety. This work aims to investigate the function of rcpA gene in biofilm formation and virulence of V. parahaemolyticus. Deletion of rcpA significantly reduced motility, biofilm biomass, and extracellular polymeric substances, and inhibited biofilm formation on a variety of food and food contact surfaces. In mice infection model, mice infected with ∆rcpA strain exhibited a decreased rate of pathogen colonization, a lower level of inflammatory cytokines, and less tissue damage when compared to mice infected with wild type strain. RNA-seq analysis revealed that 374 genes were differentially expressed in the rcpA deletion mutant, which include genes related to quorum sensing, flagellar system, ribosome, type VI secretion system, biotin metabolism and transcriptional regulation. In conclusion, rcpA plays a role in determining biofilm formation and virulence of V. parahaemolyticus and further research is necessitated to fully understand its function in V. parahaemolyticus.

Characterization of a secondary hydroxy-acyltransferase for lipid A in Vibrio parahaemolyticus.

Lipid A plays a crucial role in Vibrio parahaemolyticus. Previously we have reported the diversity of secondary acylation of lipid A in V. parahaemolyticus and four V. parahaemolyticus genes VP_RS08405, VP_RS01045, VP_RS12170, and VP_RS00880 exhibiting homology to the secondary acyltransferases in Escherichia coli. In this study, the gene VP_RS12170 was identified as a specific lipid A secondary hydroxy-acyltransferase responsible for transferring a 3-hydroxymyristate to the 2'-position of lipid A. Four E. coli mutant strains WHL00, WHM00, WH300, and WH001 were constructed, and they would synthesize lipid A with different structures due to the absence of genes encoding lipid A secondary acyltransferases or Kdo transferase. Then V. parahaemolyticus VP_RS12170 was overexpressed in W3110, WHL00, WHM00, WH300, and WH001, and lipid A was isolated from these strains and analyzed by using thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry. The detailed structural changes of lipid A in these mutant strains with and without VP_RS12170 overexpression were compared and conclude that VP_RS12170 can specifically transfer a 3-hydroxymyristate to the 2'-position of lipid A. This study also demonstrated that the function of VP_RS12170 is Kdo-dependent and its favorite substrate is Kdo-lipid IVA. These findings give us better understanding the biosynthetic pathway and the structural diversity of V. parahaemolyticus lipid A.

The sensor of the bacterial histidine kinase CpxA is a novel dimer of extracytoplasmic Per-ARNT-Sim domains.

Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the Escherichia coli CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of Vibrio parahaemolyticus CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function cpxA∗ alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologs revealed the sensor domain of Pseudomonas aeruginosa sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation.

Recent advances in understanding the fitness and survival mechanisms of Vibrio parahaemolyticus.

The presence of Vibrio parahaemolyticus (Vp) in different production stages of seafood has generated negative impacts on both public health and the sustainability of the industry. To further better investigate the fitness of Vp at the phenotypical level, a great number of studies have been conducted in recent years using plate counting methods. In the meantime, with the increasing accessibility of the next generation sequencing and the advances in analytical chemistry techniques, omics-oriented biotechnologies have further advanced our knowledge in the survival and virulence mechanisms of Vp at various molecular levels. These observations provide insights to guide the development of novel prevention and control strategies and benefit the monitoring and mitigation of food safety risks associated with Vp contamination. To timely capture these recent advances, this review firstly summarizes the most recent phenotypical level studies and provide insights about the survival of Vp under important in vitro stresses and on aquatic products. After that, molecular survival mechanisms of Vp at transcriptomic and proteomic levels are summarized and discussed. Looking forward, other newer omics-biotechnology such as metabolomics and secretomics show great potential to be used for confirming the cellular responses of Vp. Powerful data mining tools from the field of machine learning and artificial intelligence, that can better utilize the omics data and solve complex problems in the processing, analysis, and interpretation of omics data, will further improve our mechanistic understanding of Vp.

Genome-based characterization of a novel prophage of Vibrio parahaemolyticus, VPS05ph1, a novel member of Peduoviridae.

Vibrio parahaemolyticus is a globally important bacterium related to climate warming and health threat to human and marine animals. Yet, there is limited knowledge about its polylysogeny harboring multiple prophages and the genetic information. In this study, two prophages (VPS05ph1 and VPS05ph2) were identified in a V. parahaemolyticus isolate through genomic and transcriptional analyses. Both prophages were determined as HP1-like phages, located in a novel phylogenetic lineage of Peduoviridae. They shared a moderate genome-wide sequence similarity with each other and high synteny with the closest relatives, but showed low identities to the repressor counterparts of the representative phages within the family. In addition, no bacterial virulence genes, antibiotic resistance genes and known phage-encoded lytic proteins were identified on both prophage genomes. Moreover, the V. parahaemolyticus isolate was induced with mitomycin, which caused aberrant cellular morphology and nonviability of bacterial cells and excision of prophage VPS05ph1, accompanied by the respective inhibition and promotion of transcriptions of the cI-like and cox-like regulator genes for phage decision making. Results in this study provide the genetic context of polylysogeny in the V. parahaemolyticus isolate, support the diversity and prevalence of HP1-like phages in vibrios, and promote to explore interactions between the HP1-like prophage and its vibrio host.

Epidemiological and Genomic analysis of Vibrio parahaemolyticus isolated from imported travelers at the port of Shanghai, China (2017-2019).

BACKGROUND: Vibrio parahaemolyticus is the predominant etiological agent of seafood-associated foodborne illnesses on a global scale. It is essential to elucidate the mechanisms by which this pathogen disseminates. Given the existing research predominantly concentrates on localized outbreaks, there is a pressing necessity for a comprehensive investigation to capture strains of V. parahaemolyticus cross borders. RESULTS: This study examined the frequency and genetic attributes of imported V. parahaemolyticus strains among travelers entering Shanghai Port, China, between 2017 and 2019.Through the collection of 21 strains from diverse countries and regions, Southeast Asia was pinpointed as a significant source for the emergence of V. parahaemolyticus. Phylogenetic analysis revealed clear delineation between strains originating from human and environmental sources, emphasizing that underlying genome data of foodborne pathogens is essential for environmental monitoring, food safety and early diagnosis of diseases. Furthermore, our study identified the presence of virulence genes (tdh and tlh) and approximately 120 antibiotic resistance-related genes in the majority of isolates, highlighting their crucial involvement in the pathogenesis of V. parahaemolyticus. CONCLUSIONS: This research enhanced our comprehension of the worldwide transmission of V. parahaemolyticus and its antimicrobial resistance patterns. The findings have important implications for public health interventions and antimicrobial stewardship strategies, underscoring the necessity for epidemiological surveillance of pathogen at international travel hubs.

ZntA maintains zinc and cadmium homeostasis and promotes oxidative stress resistance and virulence in Vibrio parahaemolyticus.

Although metals are essential for life, they are toxic to bacteria in excessive amounts. Therefore, the maintenance of metal homeostasis is critical for bacterial physiology and pathogenesis. Vibrio parahaemolyticus is a significant food-borne pathogen that mainly causes acute gastroenteritis in humans and acute hepatopancreatic necrosis disease in shrimp. Herein, we report that ZntA functions as a zinc (Zn) and cadmium (Cd) homeostasis mechanism and contributes to oxidative stress resistance and virulence in V. parahaemolyticus. zntA is remarkably induced by Zn, copper, cobalt, nickel (Ni), and Cd, while ZntA promotes V. parahaemolyticus growth under excess Zn/Ni and Cd conditions via maintaining Zn and Cd homeostasis, respectively. The growth of ΔzntA was inhibited under iron (Fe)-restricted conditions, and the inhibition was associated with Zn homeostasis disturbance. Ferrous iron supplementation improved the growth of ΔzntA under excess Zn, Ni or Cd conditions. The resistance of ΔzntA to H2O2-induced oxidative stress also decreased, and its virulence was attenuated in zebrafish models. Quantitative real-time PCR, mutagenesis, and ß-galactosidase activity assays revealed that ZntR positively regulates zntA expression by binding to its promoter. Collectively, the ZntR-regulated ZntA is crucial for Zn and Cd homeostasis and contributes to oxidative stress resistance and virulence in V. parahaemolyticus.

Detection of non-pathogenic and pathogenic populations of Vibrio parahaemolyticus in various samples by the conventional, quantitative and droplet digital PCRs.

In this study, three generations of polymerase chain reaction (PCR) assays: (i) conventional PCR, (ii) qPCR and (iii) droplet digital PCR (ddPCR), were systematically tested for their abilities to detect non-pathogenic and pathogenic populations of Vibrio parahaemolyticus. The limit of detection (LOD) for the ddPCR was 1.1 pg/µL of purified DNA, followed by the qPCR (5.6 pg/µL) and the conventional PCR (8.8 pg/µL). Regarding the LOD for V. parahaemolyticus cells, the ddPCR assay was able to detect 29 cells, followed by the conventional PCR assay (58 cells) and the qPCR assay (115 cells). Regarding the sensitivities to detect this pathogen from PCR inhibition prone samples (naturally contaminated mussels), the ddPCR assay significantly outperformed the conventional PCR and qPCR. The ddPCR assay was able to consistently detect non-pathogenic and pathogenic populations of V. parahaemolyticus from naturally contaminated mussels, indicating its tolerance to various PCR inhibitors. This study also revealed the significant difference between conventional PCR and qPCR. The conventional PCR assay showed significantly greater sensitivity than that of the qPCR assay in detecting V. parahaemolyticus in crude samples, whereas the qPCR assay showed better sensitivity in detecting the presence of V. parahaemolyticus in purified DNA samples.

Assessing biofilm formation and resistance of vibrio parahaemolyticus on UV-aged microplastics in aquatic environments.

UV degradation of marine microplastics (MPs) could increase their vector potential for pathogenic bacteria and threaten human health. However, little is known about how the degree of UV aging affects interactions between MPs and pathogens and how various types of MPs differ in their impact on seafood safety. This study investigated five types of UV-aged MPs and their impact on Vibrio parahaemolyticus, a seafood pathogen. MPs exposed to UV for 60 days showed similar physicochemical changes such as surface cracking and hydrophobicity reduction. Regardless of the type, longer UV exposure of MPs resulted in more biofilm formation on the surface under the same conditions. V. parahaemolyticus types that formed biofilms on the MP surface showed 1.4- to 5.0-fold upregulation of virulence-related genes compared to those that did not form biofilms, independently of UV exposure. However, longer UV exposure increased resistance of V. parahaemolyticus on MPs to chlorine, heat, and human gastrointestinal environment. This study implies that the more UV degradation occurs on MPs, the more microbial biofilm formation is induced, which can significantly increase virulence and environmental resistance of bacteria regardless of the type of MP.

Genomic mining of Vibrio parahaemolyticus highlights prevalence of antimicrobial resistance genes and new genetic markers associated with AHPND and tdh + /trh + genotypes.

BACKGROUND: Acute Hepatopancreatic Necrosis Disease (AHPND) causes significant mortality in shrimp aquaculture. The infection is primarily instigated by Vibrio parahaemolyticus (Vp) strains carrying a plasmid encoding the binary toxin PirAB. Yet, comprehension of supplementary virulence factors associated with this relatively recent disease remains limited. Furthermore, the same holds for gastroenteritis in humans caused by other Vp genotypes. Additionally, given the prevalent use of antibiotics to combat bacterial infections, it becomes imperative to illuminate the presence of antimicrobial resistance genes within these bacteria. RESULTS: A subsampled number of 1,036 Vp genomes was screened for the presence of antimicrobial resistance genes, revealing an average prevalence of 5 ± 2 (SD) genes. Additional phenotypic antimicrobial susceptibility testing of three Vp strains (M0904, TW01, and PV1) sequenced in this study demonstrated resistance to ampicillin by all tested strains. Additionally, Vp M0904 showed multidrug resistance (against ampicillin, tetracycline, and trimethoprim-sulfamethoxazole). With a focus on AHPND, a screening of all Vibrio spp. for the presence of pirA and/or pirB indicates an estimated prevalence of 0.6%, including four V. campbellii, four V. owensii, and a Vibrio sp. next to Vp. Their pirAB-encoding plasmids exhibited a highly conserved backbone, with variations primarily in the region of the Tn3 family transposase. Furthermore, an assessment of the subsampled Vp genomes for the presence of known virulence factors showed a correlation between the presence of the Type 3 Secretion System 2 and tdh, while the presence of the Type 6 Secretion System 1 was clade dependent. Furthermore, a genome-wide association study (GWAS) unveiled (new) genes associated with pirA, pirB, tdh, and trh genotypes. Notable associations with the pirAB genotype included outer membrane proteins, immunoglobulin-like domain containing proteins, and toxin-antitoxin systems. For the tdh + /trh + genotypes (containing tdh, trh, or both genes), associations were found with T3SS2 genes, urease-related genes and nickel-transport system genes, and genes involved in a 'minimal' type I-F CRISPR mechanism. CONCLUSIONS: This study highlights the prevalence of antimicrobial resistance and virulence genes in Vp, identifying novel genetic markers associated with AHPND and tdh + /trh + genotypes. These findings contribute valuable insights into the genomic basis of these genotypes, with implications for shrimp aquaculture and food safety.

QsvR and OpaR coordinately regulate the transcription of cpsS and cpsR in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis, has a strong capacity to form biofilms on surfaces, which is strictly regulated by the CpsS-CpsR-CpsQ regulatory cascade. OpaR, a master regulator of quorum sensing, is a global regulator that controls multiple cellular pathways including biofilm formation and virulence. QsvR is an AraC-type regulator that works coordinately with OpaR to control biofilm formation and virulence gene expression of V. parahaemolyticus. QsvR and OpaR activate cpsQ transcription. OpaR also activates cpsR transcription, but lacks the detailed regulatory mechanisms. Furthermore, it is still unknown whether QsvR regulates cpsR transcription, as well as whether QsvR and OpaR regulate cpsS transcription. In this study, the results of quantitative real-time PCR and LacZ fusion assays demonstrated that deletion of qsvR and/or opaR significantly decreased the expression levels of cpsS and cpsR compared to the wild-type strain. However, the results of two-plasmid lacZ reporter and electrophoretic mobility-shift assays showed that both QsvR and OpaR were unable to bind the regulatory DNA regions of cpsS and cpsR. Therefore, transcription of cpsS and cpsR was coordinately and indirectly activated by QsvR and OpaR. This work enriched our knowledge on the regulatory network of biofilm formation in V. parahaemolyticus.

Genome-wide analysis of ATP-binding cassette (ABC) transporter in Penaeus vannamei and identification of two ABC genes involved in immune defense against Vibrio parahaemolyticus by affecting NF-κB signaling pathway.

The ATP-binding cassette (ABC) transporters have crucial roles in various biological processes such as growth, development and immune defense in eukaryotes. However, the roles of ABC transporters in the immune system of crustaceans remain elusive. In this study, 38 ABC genes were systematically identified and characterized in Penaeus vannamei. Bioinformation analysis revealed that PvABC genes were categorized into ABC A-H eight subfamilies with 17 full-transporters, 11 half transporters and 10 soluble proteins, and multiple immunity-related cis-elements were found in gene promoter regions. Expression analysis showed that most PvABC genes were widely and highly expressed in immune-related tissues and responded to the stimulation of Vibrio parahaemolyticus. To investigate whether PvABC genes mediated innate immunity, PvABCC5, PvABCF1 and PvABCB4 were selected for dsRNA interference experiment. Knockdown of PvABCF1 and PvABCC5 not PvABCB4 increased the cumulative mortality of P. vannamei and bacterial loads in hepatopancreas after infection with V. parahaemolyticus. Further analysis showed that the PvABCF1 and PvABCC5 knockdown decreased expression levels of NF-κB pathway genes and antimicrobial peptides (AMPs). Collectively, these findings indicated that PvABCF1 and PvABCC5 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway and then promoting the expression of AMPs, which would contribute to overall understand the function of ABC genes in innate immunity of invertebrates.