Structural basis of the activation of MARTX cysteine protease domain from Vibrio vulnificus.

The multifunctional autoprocessing repeat-in-toxin (MARTX) toxin is the primary virulence factor of Vibrio vulnificus displaying cytotoxic and hemolytic properties. The cysteine protease domain (CPD) is responsible for activating the MARTX toxin by cleaving the toxin precursor and releasing the mature toxin fragments. To investigate the structural determinants for inositol hexakisphosphate (InsP6)-mediated activation of the CPD, we determined the crystal structures of unprocessed and ß-flap truncated MARTX CPDs of Vibrio vulnificus strain MO6-24/O in complex with InsP6 at 1.3 and 2.2Å resolution, respectively. The CPD displays a conserved domain with a central seven-stranded ß-sheet flanked by three α-helices. The scissile bond Leu3587-Ala3588 is bound in the catalytic site of the InsP6-loaded form of the Cys3727Ala mutant. InsP6 interacts with the conserved basic cleft and the ß-flap inducing the active conformation of catalytic residues. The ß-flap of the post-CPD is flexible in the InsP6-unbound state. The structure of the CPD Δß-flap showed an inactive conformation of the catalytic residues due to the absence of interaction between the active site and the ß-flap. This study confirms the InsP6-mediated activation of the MARTX CPDs in which InsP6-binding induces conformational changes of the catalytic residues and the ß-flap that holds the N terminus of the CPD in the active site, facilitating hydrolysis of the scissile bond.

In vivo examination of pathogenicity and virulence in environmentally isolated Vibrio vulnificus.

Human exposure to Vibrio vulnificus, a gram-negative, halophilic environmental pathogen, is increasing. Despite this, the mechanisms of its pathogenicity and virulence remain largely unknown. Each year, hundreds of infections related to V. vulnificus occur, leading to hospitalization in 92% of cases and a mortality rate of 35%. The infection is severe, typically contracted through the consumption of contaminated food or exposure of an open wound to contaminated water. This can result in necrotizing fasciitis and the need for amputation of the infected tissue. Although several genes (rtxA1, vvpE, and vvhA) have been implicated in the pathogenicity of this organism, a defined mechanism has not been discovered. In this study, we examine environmentally isolated V. vulnificus strains using a zebrafish model (Danio rerio) to investigate their virulence capabilities. We found significant variation in virulence between individual strains. The commonly used marker gene of disease-causing strains, vcgC, did not accurately predict the more virulent strains. Notably, the least virulent strain in the study, V. vulnificus Sept WR1-BW6, which tested positive for vcgC, vvhA, and rtxA1, did not cause severe disease in the fish and was the only strain that did not result in any mortality. Our study demonstrates that virulence varies greatly among different environmental strains and cannot be accurately predicted based solely on genotype.

Regulatory role of VvsB protein on serine protease activity of VvsA in Vibrio vulnificus.

BACKGROUND: Vibrio vulnificus NCIMB2137, a Gram-negative, metalloprotease negative estuarine strain was isolated from a diseased eel. A 45 kDa chymotrypsin-like alkaline serine protease known as VvsA has been recently reported as one of the major virulence factor responsible for the pathogenesis of this strain. The vvsA gene along with a downstream gene vvsB, whose function is still unknown constitute an operon designated as vvsAB. OBJECTIVE: This study examines the contribution of VvsB to the functionality of VvsA. METHOD: In this study, VvsB was individually expressed using Rapid Translation System (RTS system), followed by an analysis of its role in regulating the serine protease activity of VvsA. RESULT: The proteolytic activity of VvsA increased upon the addition of purified VvsB to the culture supernatant of V. vulnificus. However, the attempts of protein expression using an E. coli system revealed a noteworthy observation that protein expression from the vvsA gene exhibited higher protease activity compared to that from the vvsAB gene within the cytoplasmic fraction. These findings suggest an intricate interplay between VvsB and VvsA, where VvsB potentially interacts with VvsA inside the bacterium and suppress the proteolytic activity. While outside the bacterial milieu, VvsB appears to stimulate the activation of inactive VvsA. CONCLUSION: The findings suggest that Vibrio vulnificus regulates VvsA activity through the action of VvsB, both intracellularly and extracellularly, to ensure its survival.

Virulent properties and genomic diversity of Vibrio vulnificus isolated from environment, human, diseased fish.

The incidence of Vibrio vulnificus infections, with high mortality rates in humans and aquatic animals, has escalated, highlighting a significant public health challenge. Currently, reliable markers to identify strains with high virulence potential are lacking, and the understanding of evolutionary drivers behind the emergence of pathogenic strains is limited. In this study, we analyzed the distribution of virulent genotypes and phenotypes to discern the infectious potential of V. vulnificus strains isolated from three distinct sources. Most isolates, traditionally classified as biotype 1, possessed the virulence-correlated gene-C type. Environmental isolates predominantly exhibited YJ-like alleles, while clinical and diseased fish isolates were significantly associated with the nanA gene and pathogenicity region XII. Hemolytic activity was primarily observed in the culture supernatants of clinical and diseased fish isolates. Genetic relationships, as determined by multiple-locus variable-number tandem repeat analysis, suggested that strains originating from the same source tended to cluster together. However, multilocus sequence typing revealed considerable genetic diversity across clusters and sources. A phylogenetic analysis using single nucleotide polymorphisms of diseased fish strains alongside publicly available genomes demonstrated a high degree of evolutionary relatedness within and across different isolation sources. Notably, our findings reveal no direct correlation between phylogenetic patterns, isolation sources, and virulence capabilities. This underscores the necessity for proactive risk management strategies to address pathogenic V. vulnificus strains emerging from environmental reservoirs.IMPORTANCEAs the global incidence of Vibrio vulnificus infections rises, impacting human health and marine aquacultures, understanding the pathogenicity of environmental strains remains critical yet underexplored. This study addresses this gap by evaluating the virulence potential and genetic relatedness of V. vulnificus strains, focusing on environmental origins. We conduct an extensive genotypic analysis and phenotypic assessment, including virulence testing in a wax moth model. Our findings aim to uncover genetic and evolutionary factors that drive pathogenic strain emergence in the environment. This research advances our ability to identify reliable virulence markers and understand the distribution of pathogenic strains, offering significant insights for public health and environmental risk management.

Wildfire Ashes from the Wildland-Urban Interface Alter Vibrio vulnificus Growth and Gene Expression.

Climate change-induced stressors are contributing to the emergence of infectious diseases, including those caused by marine bacterial pathogens such as Vibrio spp. These stressors alter Vibrio temporal and geographical distribution, resulting in increased spread, exposure, and infection rates, thus facilitating greater Vibrio-human interactions. Concurrently, wildfires are increasing in size, severity, frequency, and spread in the built environment due to climate change, resulting in the emission of contaminants of emerging concern. This study aimed to understand the potential effects of urban interface wildfire ashes on Vibrio vulnificus (V. vulnificus) growth and gene expression using transcriptomic approaches. V. vulnificus was exposed to structural and vegetation ashes and analyzed to identify differentially expressed genes using the HTSeq-DESeq2 strategy. Exposure to wildfire ash altered V. vulnificus growth and gene expression, depending on the trace metal composition of the ash. The high Fe content of the vegetation ash enhanced bacterial growth, while the high Cu, As, and Cr content of the structural ash suppressed growth. Additionally, the overall pattern of upregulated genes and pathways suggests increased virulence potential due to the selection of metal- and antibiotic-resistant strains. Therefore, mixed fire ashes transported and deposited into coastal zones may lead to the selection of environmental reservoirs of Vibrio strains with enhanced antibiotic resistance profiles, increasing public health risk.

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.

Characterization of the transcriptionally active form of dephosphorylated DctD complexed with dephospho-IIAGlc.

Bacterial enhancer-binding proteins (bEBPs) acquire a transcriptionally active state via phosphorylation. However, transcriptional activation by the dephosphorylated form of bEBP has been observed in DctD, which belongs to Group I bEBP. The formation of a complex between dephosphorylated DctD (d-DctD) and dephosphorylated IIAGlc (d-IIAGlc) is a prerequisite for the transcriptional activity of d-DctD. In the present study, characteristics of the transcriptionally active complex composed of d-IIAGlc and phosphorylation-deficient DctD (DctDD57Q) of Vibrio vulnificus were investigated in its multimeric conformation and DNA-binding ability. DctDD57Q formed a homodimer that could not bind to the DNA. In contrast, when DctDD57Q formed a complex with d-IIAGlc in a 1:1 molar ratio, it produced two conformations: dimer and dodecamer of the complex. Only the dodecameric complex exhibited ATP-hydrolyzing activity and DNA-binding affinity. For successful DNA-binding and transcriptional activation by the dodecameric d-IIAGlc/DctDD57Q complex, extended upstream activator sequences were required, which encompass the nucleotide sequences homologous to the known DctD-binding site and additional nucleotides downstream. This is the first report to demonstrate the molecular characteristics of a dephosphorylated bEBP complexed with another protein to form a transcriptionally active dodecameric complex, which has an affinity for a specific DNA-binding sequence.IMPORTANCEResponse regulators belonging to the bacterial two-component regulatory system activate the transcription initiation of their regulons when they are phosphorylated by cognate sensor kinases and oligomerized to the appropriate multimeric states. Recently, it has been shown that a dephosphorylated response regulator, DctD, could activate transcription in a phosphorylation-independent manner in Vibrio vulnificus. The dephosphorylated DctD activated transcription as efficiently as phosphorylated DctD when it formed a complex with dephosphorylated form of IIAGlc, a component of the glucose-phosphotransferase system. Functional mimicry of this complex with the typical form of transcriptionally active phosphorylated DctD led us to study the molecular characteristics of this heterodimeric complex. Through systematic analyses, it was surprisingly determined that a multimer constituted with 12 complexes gained the ability to hydrolyze ATP and recognize specific upstream activator sequences containing a typical inverted-repeat sequence flanked by distinct nucleotides.

A One-Step RPA-CRISPR Assay Using crRNA Based on Suboptimal Protospacer Adjacent Motif for Vibrio vulnificus Detection.

Vibrio vulnificus is a hazardous foodborne pathogen responsible for approximately 95% of seafood-related deaths. This highlights the urgent requirement for specialized detection tools to be developed and used by food enterprises and food safety authorities. The DETECTR (DNA endonuclease targeted CRISPR trans reporter) system that combines CRISPR/Cas and recombinase polymerase amplification (RPA) has been utilized to develop a molecular detection assay for V. vulnificus. However, because the incompatibility between RPA and Cas12a cleavage has not been addressed, it is a two-step assay that lacks convenience and presents contamination risk. Here, we developed a one-step RPA-CRISPR assay for V. vulnificus using a special crRNA targeting a sequence with a suboptimal protospacer adjacent motif (PAM). The entire assay, conducted at 37°C, takes only 40-60 min, yields results visualized under blue light, and exhibits exceptional specificity and sensitivity (detecting 4 pathogen genome copies per reaction). This study offers a valuable tool for detecting V. vulnificus, aiding in foodborne infection prevention, and exemplifies one-step RPA-CRISPR assays managing Cas-cleavage activity through PAM adjustments.

[Clinical characteristics of 11 patients with Vibrio vulnificus infection and the establishment of a rapid diagnosis procedure for this disease].

Objective: To analyze the clinical characteristics of patients with Vibrio vulnificus infection, share diagnosis and treatment experience, and establish a rapid diagnosis procedure for this disease. Methods: This study was a retrospective case series study. From January 2009 to November 2022, 11 patients with Vibrio vulnificus infection who met the inclusion criteria were admitted to the Department of Burns and Wound Repair of Guangdong Provincial People's Hospital Affiliated to Southern Medical University. The gender, age, time of onset of illness, time of admission, time of diagnosis, route of infection, underlying diseases, affected limbs, clinical manifestations and signs on admission, white blood cell count, hemoglobin, platelet count, C-reactive protein (CRP), alanine transaminase (ALT), aspartate transaminase (AST), creatinine, procalcitonin, albumin, N-terminal pro-B-type natriuretic peptide (NT-proBNP), and blood sodium levels on admission, culture results and metagenomic next-generation sequencing (mNGS) results of pathogenic bacteria and the Vibrio vulnificus drug susceptibility test results during hospitalization, treatment methods, length of hospital stay, and outcomes of all patients were recorded. Comparative analysis was conducted on the admission time and diagnosis time of patients with and without a history of exposure to seawater/marine products, as well as the fatality ratio and amputation of limbs/digits ratio of patients with and without early adequate antibiotic treatment. For the survived patients with hand involvement, the hand function was assessed using Brunnstrom staging at the last follow-up. Based on patients' clinical characteristics and treatment conditions, a rapid diagnosis procedure for Vibrio vulnificus infection was established. Results: There were 7 males and 4 females among the patients, aged (56±17) years. Most of the patients developed symptoms in summer and autumn. The admission time was 3.00 (1.00, 4.00) d after the onset of illness, and the diagnosis time was 4.00 (2.00, 8.00) d after the onset of illness. There were 7 and 4 patients with and without a history of contact with seawater/marine products, respectively, and the admission time of these two types of patients was similar (P>0.05). The diagnosis time of patients with a history of contact with seawater/marine products was 2.00 (2.00, 5.00) d after the onset of illness, which was significantly shorter than 9.00 (4.25, 13.00) d after the onset of illness for patients without a history of contact with seawater/marine products (Z=-2.01, P<0.05). Totally 10 patients had underlying diseases. The affected limbs were right-hand in 8 cases, left-hand in 1 case, and lower limb in 2 cases. On admission, a total of 9 patients had fever; 11 patients had pain at the infected site, and redness and swelling of the affected limb, and 9 patients each had ecchymosis/necrosis and blisters/blood blisters; 6 patients suffered from shock, and 2 patients developed multiple organ dysfunction syndrome. On admission, there were 8 patients with abnormal white blood cell count, hemoglobin, and albumin levels, 10 patients with abnormal CRP, procalcitonin, and NT-proBNP levels, 5 patients with abnormal creatinine and blood sodium levels, and fewer patients with abnormal platelet count, ALT, and AST levels. During hospitalization, 4 of the 11 wound tissue/exudation samples had positive pathogenic bacterial culture results, and the result reporting time was 5.00 (5.00, 5.00) d; 4 of the 9 blood specimens had positive pathogenic bacterial culture results, and the result reporting time was 3.50 (1.25, 5.00) d; the mNGS results of 7 wound tissue/exudation or blood samples were all positive, and the result reporting time was 1.00 (1.00, 2.00) d. The three strains of Vibrio vulnificus detected were sensitive to 10 commonly used clinical antibiotics, including ciprofloxacin, levofloxacin, and amikacin, etc. A total of 10 patients received surgical treatment, 4 of whom had amputation of limbs/digits; all patients received anti-infection treatment. The length of hospital stay of 11 patients was (26±11) d, of whom 9 patients were cured and 2 patients died. Compared with that of the 6 patients who did not receive early adequate antibiotic treatment, the 5 patients who received early adequate antibiotic treatment had no significant changes in the fatality ratio or amputation of limbs/digits ratio (P>0.05). In 3 months to 2 years after surgery, the hand function of 8 patients was assessed, with results showing 4 cases of disabled hands, 2 cases of incompletely disabled hands, and 2 cases of recovered hands. When a patient had clinical symptoms of limb redness and swelling and a history of contact with seawater/marine products or a pre-examination triage RiCH score of Vibrio vulnificus sepsis ≥1, the etiological testing should be initiated immediately to quickly diagnose Vibrio vulnificus infection. Conclusions: Vibrio vulnificus infection occurs most frequently in summer and autumn, with clinical manifestations and laboratory test results showing obvious infection characteristics, and may be accompanied by damage to multiple organ functions. Both the fatality and disability ratios are high and have a great impact on the function of the affected limbs. Early diagnosis is difficult and treatment is easily delayed, but mNGS could facilitate rapid detection. For patients with red and swollen limbs accompanied by a history of contact with seawater/marine products or with a pre-examination triage RiCH score of Vibrio vulnificus sepsis ≥1, the etiological testing should be initiated immediately to quickly diagnose Vibrio vulnificus infection.

Evaluation of a novel lysis-based sample processing method to optimize Vibrio vulnificus detecting by loop-mediated isothermal amplification assay.

BACKGROUND: Vibrio vulnificus exists as one of the most serious foodborne pathogens for humans, and rapid and sensitive detection methods are needed to control its infections. As an emerging method, The Loop-Mediated Isothermal Amplification (LAMP) assay has been applied to the early detection of various foodborne pathogens due to its high efficiency, but sample preprocessing still prolongs the complete detection. To optimize the detection process, our study established a novel sample preprocessing method that was more efficient compared to common methods. RESULT: Using V. vulnificus as the detecting pathogen, the water-lysis-based detecting LAMP method shortened the preprocessing time to ≤ 1 min with 100% LAMP specificity; the detection limits of the LAMP assay were decreased to 1.20 × 102 CFU/mL and 1.47 × 103 CFU/g in pure culture and in oyster, respectively. Furthermore, the 100% LAMP specificity and high sensitivity of the water-lysis method were also obtained on detecting V. parahaemolyticus, V. alginolyticus, and P. mirabilis, revealing its excellent LAMP adaption with improvement in sensitivity and efficiency. CONCLUSION: Our study provided a novel LAMP preprocessing method that was more efficient compared to common methods and possessed the practical potential for LAMP application in the future.

Optimizing Conditions for the Production of Bacterial Extracellular Vesicles of Vibrio vulnificus and Analysis of the Inner Small RNA Compositions.

Chemical and physical elements affecting the production of bacterial extracellular vesicles (BEVs) of the human pathogen Vibrio vulnificus were quantitatively assessed to optimize the conditions for the BEV production by using the western blot quantification for an outer membrane porin OmpU and by fluorescent dye FM4-64. When cells were cultured at 37°C in an enriched medium (2 × Luria Bertani; 2 × LB) in the presence of EDTA, they produced about 70% more BEVs. BEVs were purified from the cells cultured in the established optimal conditions by the density gradient ultracentrifugation. The dynamic light scattering measurement of the purified BEVs showed that the diameter of them ranged from approximately 25 nm to 161 nm. We hypothesized that there may be some features in nucleotide sequences specific to RNAs packaged in BEVs compared to those in cellular RNA molecules. We compared the nucleotide sequences and abundance of sRNAs between in the cellular fraction and in BEVs through next-generation sequencing (NGS). While no distinct feature was observed in the nucleotide sequences of sRNAs between two groups, the length of sRNA fragments from BEVs were significantly shorter than those in cytoplasm.

RNA-seq analysis revealed the pathogenicity of Vibrio vulnificus to American eel (Anguilla rostrata) and the strategy of host anti-V. vulnificus infection.

Vibrio vulnificus is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-V. vulnificus infection remains uncertain. In this study, American eels were infected with different dose of V. vulnificus to determine the LD50. Then, bacterial load in the liver and kidney histopathology were assessed post the LD50 of V. vulnificus infection. Additionally, gene expressions of 18 immune related genes in the liver, spleen and kidney were detected. Furthermore, transcriptome sequencing and enrichment of differentially expressed genes (DEGs) were analyzed in the eel spleens between pre-infection (Con_0), post-36 h (Vv_36), and post-60 h (Vv_60) infection. The results showed that LD50 of V. vulnificus to American eels was determined to be 5.0 × 105 cfu/g body weight, and the bacterial load peaked at 24 and 12 h post the infection (hpi) in the kidney and liver, respectively. The histopathology was highlighted by necrotic hepatocytes and splenic cells, congestion blood vessels in liver and spleen, atrophied glomeruli and vacuolization of renal tubular epithelial cells. The results of RT-PCR revealed that 18 host immune-related genes showed significantly up or downregulated expression post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 16 DEGs play essential role to the immunosuppression in American eels, and the protein-protein interactions shed light on the widespread upregulation GEGs related to metabolism and immune response maintained the host cell homeostasis post the V. vulnificus infection, shedding new light on our understanding of the V. vulnificus pathogenesis towards understudied American eel and the host anti-V. vulnificus infection strategies in gene transcript.

Functional conservation of specialized ribosomes bearing genome-encoded variant rRNAs in Vibrio species.

Heterogeneity of ribosomal RNA (rRNA) sequences has recently emerged as a mechanism that can lead to subpopulations of specialized ribosomes. Our previous study showed that ribosomes containing highly divergent rRNAs expressed from the rrnI operon (I-ribosomes) can preferentially translate a subset of mRNAs such as hspA and tpiA in the Vibrio vulnificus CMCP6 strain. Here, we explored the functional conservation of I-ribosomes across Vibrio species. Exogenous expression of the rrnI operon in another V. vulnificus strain, MO6-24/O, and in another Vibrio species, V. fischeri (strain MJ11), decreased heat shock susceptibility by upregulating HspA expression. In addition, we provide direct evidence for the preferential synthesis of HspA by I-ribosomes in the V. vulnificus MO6-24/O strain. Furthermore, exogenous expression of rrnI in V. vulnificus MO6-24/O cells led to higher mortality of infected mice when compared to the wild-type (WT) strain and a strain expressing exogenous rrnG, a redundant rRNA gene in the V. vulnificus CMCP6 strain. Our findings suggest that specialized ribosomes bearing heterogeneous rRNAs play a conserved role in translational regulation among Vibrio species. This study shows the functional importance of rRNA heterogeneity in gene expression control by preferential translation of specific mRNAs, providing another layer of specialized ribosome system.

Enhancing Vibrio vulnificus infection diagnosis for negative culture patients with metagenomic next-generation sequencing.

Objective: To evaluate the diagnostic value of metagenomic next-generation sequencing (mNGS) in Vibrio vulnificus (V. vulnificus) infection. Methods: A retrospective analysis of patients with V. vulnificus infection at the Fifth Affiliated Hospital of Sun Yat-Sen University from January 1, 2020 to April 23, 2023 was conducted. 14 enrolled patients were diagnosed by culture or mNGS. The corresponding medical records were reviewed, and the clinical data analyzed included demographics, epidemiology laboratory findings, physical examination, symptoms at presentation, antibiotic and surgical treatment, and outcome. Results: In this study, 78.6% (11/14) patients had a history of marine trauma (including fish stab, shrimp stab, crab splints and fish hook wounds), 7.1% (1/14) had eaten seafood, and the remaining 14.3% (2/14) had no definite cause. Isolation of V. vulnificus from clinical samples including blood, tissue, fester and secreta. 9 cases were positive for culture, 5 cases were detected synchronously by mNGS and got positive for V. vulnificus. 85.7% (12/14) cases accepted surgical treatment, with 1 patient suffering finger amputated. 14 enrolled patients received appropriate antibiotic therapy, and all of them had recovered and discharged. 9 strains V. vulnificus isolated in this study were sensitive to most beta-lactam antibiotics, aminoglycosides, quinolones, etc. Conclusion: Vibrio vulnificus infection is a common water-exposed disease in Zhuhai, which requires identification of a number of pathogens. Of severe infections with unknown pathogen, mNGS can be used simultaneously, and the potential to detect multiple pathogens is of great help in guiding treatment.

Rifampicin-resistant RpoB S522L Vibrio vulnificus exhibits disturbed stress response and hypervirulence traits.

Rifampicin resistance, which is genetically linked to mutations in the RNA polymerase ß-subunit gene rpoB, has a global impact on bacterial transcription and cell physiology. Previously, we identified a substitution of serine 522 in RpoB (i.e., RpoBS522L ) conferring rifampicin resistance to Vibrio vulnificus, a human food-borne and wound-infecting pathogen associated with a high mortality rate. Transcriptional and physiological analysis of V. vulnificus expressing RpoBS522L showed increased basal transcription of stress-related genes and global virulence regulators. Phenotypically these transcriptional changes manifest as disturbed osmo-stress responses and toxin-associated hypervirulence as shown by reduced hypoosmotic-stress resistance and enhanced cytotoxicity of the RpoBS522L strain. These results suggest that RpoB-linked rifampicin resistance has a significant impact on V. vulnificus survival in the environment and during infection.

Identification of potential pathogenic targets and survival strategies of Vibrio vulnificus through population genomics.

Vibrio vulnificus, a foodborne pathogen, has a high mortality rate. Despite its relevance to public health, the identification of virulence genes associated with the pathogenicity of currently known clinical isolates of V. vulnificus is incomplete and its synergistic pathogenesis remains unclear. Here, we integrate whole genome sequencing (WGS), genome-wide association studies (GWAS), and genome-wide epistasis studies (GWES), along with phenotype characterization to investigate the pathogenesis and survival strategies of V. vulnificus. GWAS and GWES identified a total of six genes (purH, gmr, yiaV, dsbD, ramA, and wbpA) associated with the pathogenicity of clinical isolates related to nucleotide/amino acid transport and metabolism, cell membrane biogenesis, signal transduction mechanisms, and protein turnover. Of these, five were newly discovered potential specific virulence genes of V. vulnificus in this study. Furthermore, GWES combined with phenotype experiments indicated that V. vulnificus isolates were clustered into two ecological groups (EGs) that shared distinct biotic and abiotic factors, and ecological strategies. Our study reveals pathogenic mechanisms and their evolution in V. vulnificus to provide a solid foundation for designing new vaccines and therapeutic targets.

Genome analysis of clinical genotype Vibrio vulnificus isolated from seafood in Mangaluru Coast, India provides insights into its pathogenicity.

Vibrio vulnificus an opportunistic human pathogen native to marine/estuarine environment, is one of the leading causes of death due to seafood consumption and exposure of wounds to seawater worldwide. The present study involves the whole genome sequence analysis of an environmental strain of V. vulnificus (clinical genotype) isolated from seafood along the Mangaluru coast of India. The sequenced genome data was subjected to in-silico analysis of phylogeny, virulence genes, antimicrobial resistance determinants, and secretary proteins using suitable bioinformatics tools. The sequenced isolate had an overall genome length of 4.8 Mb and GC content of 46% with 4400 coding DNA sequences. The sequenced strain belongs to a new sequence type (Multilocus sequence typing) and was also found to branch with a phylogenetic lineage that groups the most infectious strains of V. vulnificus. The seafood isolate had complete genes involved in conferring serum resistance yet showed limited serum resistance. The study identified several genes against the antibiotics that are commonly used in their treatment, highlighting the need for alternative treatments. Also, the secretory protein analysis revealed genes associated with major pathways like ABC transporters, two-component systems, quorum sensing, biofilm formation, cationic antimicrobial peptide (CAMP) resistance, and others that play a critical role in the pathogenesis of the V. vulnificus. To the best of our knowledge, this is the first report of a detailed analysis of the genomic information of a V. vulnificus isolated from the Indian subcontinent and provides evidence that raises public health concerns about the safety of seafood.

Integration of a CRISPR Cas12a-assisted multicolor biosensor and a micropipette tip enables visible point-of-care testing of foodborne Vibrio vulnificus.

Foodborne pathogens cause numerous food safety problems, and as a virulent bacterium falling under this category, Vibrio vulnificus (V. vulnificus) poses a huge threat to public health. The conventional methods used for the detection of V. vulnificus, including culture-based and molecular detection methods, have a variety of drawbacks, including being time-consuming and labor-intensive, the requirement of large-scale equipment, and the lack of professional operators. This paper establishes a visible detection platform for V. vulnificus based on CRISPR/Cas12a, which is integrated with nucleic acid isothermal amplification and ß-galactosidase-catalyzed visible color reaction. The specific vvhA gene and a conservative segment in the 16S rDNA gene of the Vibrio genus were selected as the detection targets. By using spectrum analysis, this CRISPR detection platform achieved sensitive detection of V. vulnificus (1 CFU per reaction) with high specificity. Through the color transformation system, as low as 1 CFU per reaction of V. vulnificus in both bacterial solution and artificially contaminated seafood could be visibly observed with the naked eye. Furthermore, the consistency between our assay and the qPCR assay in the detection of V. vulnificus spiked seafood was confirmed. In general, this visible detection platform is user-friendly, accurate, portable, and equipment-free, and is expected to provide a powerful supplement in point-of-care testing of V. vulnificus and also holds good promise for future application in foodborne pathogen detection.

CarRS Two-Component System Essential for Polymyxin B Resistance of Vibrio vulnificus Responds to Multiple Host Environmental Signals.

Enteropathogenic bacteria express two-component systems (TCSs) to sense and respond to host environments, developing resistance to host innate immune systems like cationic antimicrobial peptides (CAMPs). Although an opportunistic human pathogen Vibrio vulnificus shows intrinsic resistance to the CAMP-like polymyxin B (PMB), its TCSs responsible for resistance have barely been investigated. Here, a mutant exhibiting a reduced growth rate in the presence of PMB was screened from a random transposon mutant library of V. vulnificus, and response regulator CarR of the CarRS TCS was identified as essential for its PMB resistance. Transcriptome analysis revealed that CarR strongly activates the expression of the eptA, tolCV2, and carRS operons. In particular, the eptA operon plays a major role in developing the CarR-mediated PMB resistance. Phosphorylation of CarR by the sensor kinase CarS is required for the regulation of its downstream genes, leading to the PMB resistance. Nevertheless, CarR directly binds to specific sequences in the upstream regions of the eptA and carRS operons, regardless of its phosphorylation. Notably, the CarRS TCS alters its own activation state by responding to several environmental stresses, including PMB, divalent cations, bile salts, and pH change. Furthermore, CarR modulates the resistance of V. vulnificus to bile salts and acidic pH among the stresses, as well as PMB. Altogether, this study suggests that the CarRS TCS, in responding to multiple host environmental signals, could provide V. vulnificus with the benefit of surviving within the host by enhancing its optimal fitness during infection. IMPORTANCE Enteropathogenic bacteria have evolved multiple TCSs to recognize and appropriately respond to host environments. CAMP is one of the inherent host barriers that the pathogens encounter during the course of infection. In this study, the CarRS TCS of V. vulnificus was found to develop resistance to PMB, a CAMP-like antimicrobial peptide, by directly activating the expression of the eptA operon. Although CarR binds to the upstream regions of the eptA and carRS operons regardless of phosphorylation, phosphorylation of CarR is required for the regulation of the operons, resulting in the PMB resistance. Furthermore, the CarRS TCS determines the resistance of V. vulnificus to bile salts and acidic pH by differentially regulating its own activation state in response to these environmental stresses. Altogether, the CarRS TCS responds to multiple host-related signals, and thus could enhance the survival of V. vulnificus within the host, leading to successful infection.

Validation of the Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay for the Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in Seafood Matrixes: AOAC Performance Tested MethodsSM 022301.

BACKGROUND: The Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay method is a real-time PCR method for the multiplex detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in seafood. OBJECTIVE: The Thermo Scientific SureTect Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus Assay was evaluated for AOAC Performance Tested MethodsSM certification. METHOD: Inclusivity/exclusivity, matrix, product consistency/stability, and robustness studies were conducted to assess the method's performance. For the matrix study, the method was validated using the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR Food Safety Instrument and the Applied Biosystems™ 7500 Fast Real-Time PCR Food Safety Instrument against the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 9 (2004), Vibrio and ISO 21872-1:2017 Microbiology of the food chain-Horizontal method for the determination of Vibrio spp.-Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus reference methods. RESULTS: Matrix studies showed equivalent or superior performance of the candidate method compared to the reference method and, overall, no difference between presumptive and confirmed results, except for one matrix due to high background flora. The inclusivity/exclusivity study correctly identified/excluded all strains analyzed. Robustness testing showed no statistically significant differences in assay performance under varied test conditions. Product consistency and stability studies demonstrated no statistically significant differences between assay lots with different expiration dates. CONCLUSIONS: The data presented show that the assay constitutes a rapid and reliable workflow for the detection of V. cholerae, V. parahaemolyticus, and V. vulnificus in seafood matrixes. HIGHLIGHTS: The SureTect PCR Assay method allows for fast, reliable detection of stipulated strains in seafood matrixes with results obtained in as little as 80 min post-enrichment.