Investigation of potentially pathogenic Vibrionaceae in Saint-Louis city, Senegal.

Introduction: as cholera, due to toxigenic bacteria Vibrio cholera (serogroups O1 and O139), is a major public health threat in Africa, the aim of this work was to investigate potentially pathogenic Vibrionaceae bacteria firstly from human stool samples, and secondly from various environmental water points of Saint-Louis city in Senegal. Methods: a hospital-based study was conducted between 2013 and 2015. Stool samples were taken and cultured from daily incoming patients or hospitalized for acute diarrhea at Saint-Louis´ regional hospital. For environment, a monthly longitudinal sampling from January to October 2016 was carried out at 10 sites in the city. We used total DNA extracted from APW (alkaline peptone water) broth solutions and on suspect bacterial colonies to run PCR Multiplex targeting specific DNA fragments to detect Vibrio genus and specific species. In case of positivity, a simplex PCR was performed to test for cholera toxins Ctx, and V. parahaemolyticus TRH and TDH. Results: for 43 patients screened, bacterial culture was positive in 6% of cases but no strain of V. cholerae or other Vibrio sp. was isolated. PCR on 90 APW solutions were positive for Vibrio sp.(n = 43), V. cholera(n = 27), V. mimicus(n = 16), V. parahaemolyticus(8), V. alginolyticus(n = 4), and V. vulnificus(n = 2). Unlike for those on suspected colonies which were positive for a majority of V. parahaemolyticus (n = 40) and V. cholerae non-O1 / O139 (n = 35). Six strains of V. parahaemolyticus carried TRH gene, 3 of which expressed simultaneously virulence TRH and TDH genes. For physicochemical parameters, all temperatures varied similarly according to a unimodal seasonality, as well as salinity. Conclusion: despite the presence of natural populations of Vibrionaceae, even toxigenic ones, was noted in water environment, along with favorable habitat conditions that could play a role in transmission of Vibriosis in the Saint Louis population, we did not isolate any of them from patients screened at the hospital.

Epidemiological investigation on diseases of Larimichthys crocea in Ningbo culture area.

Introduction: Due to the high-density farming of Larimichthys crocea over the years, diseases caused by pathogens such as bacteria, viruses, and parasites frequently occur in Ningbo, posing a huge threat and challenge to the sustainable and healthy development of the L. crocea's bay farming industry. In order to understand the diseases occurrence in L. crocea farming in Ningbo area, an epidemiological investigation of L. crocea diseases was carried out through regular sampling in 2023. Methods: From April to October 2023, routine sampling of L. crocea was conducted monthly in various farming areas in Ningbo. Each time, live or dying L. crocea with obvious clinical symptoms were sampled, with a total number of 55 L. crocea collected. The samples were preserved in ice bags and transported to the laboratory for pathogen detection(including bacterial isolation and identification,virus identification, and parasites detection). Results: A total of fifty-five fish dying L. crocea with obvious clinical symptoms were collected in this study, of which 78.18% (43/55) were detected with symptoms caused by pathogenic infection, while 21.82% (12/55) did not have identified pathogens, which were presumed to be breeding abrasions, nutritional metabolic disorders, unconventional pathogens infection or other reasons. A total of twenty-five pathogenic bacteria strains were isolated, which mainly were Pseudomonas plecoglossicida and Vibrio harveyi, accounting for 52% (13/25) and 32% (8/25) of the pathogenic bacteria strains, respectively. Among them, both V. harveyi and Streptococcus. iniae co-infected one fish. Additionally, three other bacterial strains including Nocardia seriolae, Staphylococcus Saprophyticus, and Photobacterium damselae subsp.damselae were isolated. Microscopic examination mainly observed two parasites, Cryptocaryon irritans and Neobenedenia girellae. In virus detection, the red sea bream iridovirus (RSIV) was mainly detected in L. crocea. Statistical analysis showed that among the fish with detected pathogens, 55.81% (24/43) had bacterial infections, 37.21% (16/43) had parasitic infections, and 37.21% (16/43) had RSIV infections. Among them, five fish had mixed infections of bacteria and parasites, three had mixed infections of bacteria and viruses, three had mixed infections of parasites and viruses, and one L. crocea had mixed infections of viruses, bacteria, and parasites. Discussion: These findings indicate that these three major types of diseases are very common in the L. crocea farming area in Ningbo, implying the complexity of mixed infections of multiple diseases.

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors.

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

The Description of Pseudoalteromonas apostichopi sp. nov., Vibrio apostichopi sp. nov., and Marinobacter apostichopi sp. nov. from the Fertilized Eggs and Larvae of Apostichopus japonicus.

Three novel bacterial strains, FE4T, FE10T, and LA51T, which are phylogenetically affiliated to the genera Pseudoalteromonas, Vibrio, or Marinobacter, respectively, isolated from fertilized eggs and juveniles of sea cucumber Apostichopus japonicus were characterized by a genome-based taxonomical approach including multilocus sequence analysis (MLSA) combined with classical phenotypic and chemotaxonomic characterizations. A molecular network reconstructed on the basis of nucleotide sequences of four phylogenetic maker protein genes revealed that the strains FE4T, FE10T, and LA51T were closely related to Pseudoalteromonas shioyasakiensis, Vibrio lentus, and Marinobacter similis, respectively. Average nucleotide identity (ANI) comparisons against phylogenetically related species to FE4T, FE10T, and LA51T demonstrated that each newly described strain could not be identified as any previously described species within each genus showing < 95% ANI: 91.3% of FE4T against P. shioyasakiensis JCM 18891 T, 92.6% of FE10T against "V. bathopelagicus" Sal10, and 92.6% of LA51T against M. similis A3d10T, in maximum, respectively. Here, we show molecular phylogenetic, genomic, phenotypic, and chemotaxonomic features of the newly described species FE4T, FE10T, and LA51T. We also propose Pseudoalteromonas apostichopi sp. nov. with FE4T (JCM 36173 T = LMG 33143 T) as the type strain, Vibrio apostichopi sp. nov. with FE10T (JCM 36174 T = LMG 33144 T) as the type strain, and Marinobacter apostichopi sp. nov. with LA51T (JCM 36175 T = LMG 33145 T) as the type strain.

Core Transcriptome of Hydrogen Producing Marine Vibrios Reveals Contribution of Glycolysis in Their Efficient Hydrogen Production.

Pyruvate (Pyr) is the end product of the glycolysis pathway. Pyr is also renewable and is further metabolized to produce formate, which is the precursor of H2, via pyruvate formate lyase (PFL) under anaerobic conditions. The formate is excluded and re-imported via the formate channel and is then converted to H2 via the formate hydrogenlyase (FHL) complex. In H2 producing marine vibrios, such as Vibrio tritonius and Vibrio porteresiae in the Porteresiae clade of the family Vibrionaceae, apparent but inefficient H2 production from Pyr has been observed. To elucidate the molecular mechanism of why this inefficient H2 production is observed in Pry-metabolized marine vibrio cells and how glycolysis affects those H2 productions of marine vibrios, the "Core Transcriptome" approach to find common gene expressions of those two major H2 producing Vibrio species in Pyr metabolism was first applied. In the Pyr-metabolized vibrio cells, genes for the "Phosphoenolpyruvate (PEP)-Pyruvate-Oxalate (PPO)" node, due to energy saving, and PhoB-, RhaR-, and DeoR-regulons were regulated. Interestingly, a gene responsible for oxalate/formate family antiporter was up-regulated in Pyr-metabolized cells compared to those of Glc-metabolized cells, which provides new insights into the uses of alternative formate exclusion mechanics due to energy deficiencies in Pyr-metabolized marine vibrios cells. We further discuss the contribution of the Embden-Meyerhof-Parnas (EMP) pathway to efficient H2 production in marine vibrios.

A "Pro-Asp-Thr" Amino Acid Repeat from Vibrio sp. QY108 Alginate Lyase Exhibits Alginate-Binding Capacity and Enhanced Soluble Expression and Thermostability.

Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single catalytic domains, research on multi-module alginate lyases has been lfiguimited. This study identified VsAly7A, a multi-module alginate lyase present in Vibrio sp. QY108, comprising a "Pro-Asp-Thr(PDT)" fragment and two PL-7 catalytic domains (CD I and CD II). The "PDT" fragment enhances the soluble expression level and increases the thermostability and binding affinity to the substrate. Moreover, CD I exhibited greater catalytic efficiency than CD II. The incorporation of PDT-CD I resulted in an increase in the optimal temperature of VsAly7A, whereas CD II displayed a preference for polyG degradation. The multi-domain structure of VsAly7A provides a new idea for the rational design of alginate lyase, whilst the "PDT" fragment may serve as a fusion tag in the soluble expression of recombinant proteins.

The ArgR-Regulated ADI Pathway Facilitates the Survival of Vibrio fluvialis under Acidic Conditions.

Vibrio fluvialis is an emerging foodborne pathogenic bacterium that can cause severe cholera-like diarrhea and various extraintestinal infections, posing challenges to public health and food safety worldwide. The arginine deiminase (ADI) pathway plays an important role in bacterial environmental adaptation and pathogenicity. However, the biological functions and regulatory mechanisms of the pathway in V. fluvialis remain unclear. In this study, we demonstrate that L-arginine upregulates the expression of the ADI gene cluster and promotes the growth of V. fluvialis. The ADI gene cluster, which we proved to be comprised of two operons, arcD and arcACB, significantly enhances the survival of V. fluvialis in acidic environments both in vitro (in culture medium and in macrophage) and in vivo (in mice). The mRNA level and reporter gene fusion analyses revealed that ArgR, a transcriptional factor, is necessary for the activation of both arcD and arcACB transcriptions. Bioinformatic analysis predicted the existence of multiple potential ArgR binding sites at the arcD and arcACB promoter regions that were further confirmed by electrophoretic mobility shift assay, DNase I footprinting, or point mutation analyses. Together, our study provides insights into the important role of the ArgR-ADI pathway in the survival of V. fluvialis under acidic conditions and the detailed molecular mechanism. These findings will deepen our understanding of how environmental changes and gene expression interact to facilitate bacterial adaptations and virulence.

Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria.

Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery.

Shewanella phaeophyticola sp. nov. and Vibrio algarum sp. nov., isolated from marine brown algae.

Two Gram-stain-negative, rod-shaped bacteria, designated as strains KJ10-1T and KJ40-1T, were isolated from marine brown algae. Both strains were catalase-positive, oxidase-positive, and facultative aerobic. Strain KJ10-1T exhibited optimal growth at 25 °C, pH 7.0, and 3 % NaCl, whereas strain KJ40-1T showed optimal growth at 25 °C, pH 7.0, and 2 % NaCl. The respiratory quinones of strain KJ10-1T were ubiquinone-8, ubiquinone-7, menaquinone-7, and methylated menaquinone-7, while the respiratory quinone of strain KJ40-1T was only ubiquinone-8. As major fatty acids, strain KJ10-1T contained C16 : 0, C17 : 1 ω8c, iso-C15 : 0, and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and strain KJ40-1T contained C16 : 0 and summed features 3 and 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The major polar lipids in strain KJ10-1T were phosphatidylethanolamine, phosphatidylglycerol, and an unidentified aminolipid, whereas those in strain KJ40-1T were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G+C contents of strains KJ10-1T and KJ40-1T were 42.1 and 40.8 mol%, respectively. Based on 16S rRNA gene sequences, strains KJ10-1T and KJ40-1T exhibited the closest relatedness to Shewanella saliphila MMS16-UL250T (98.6 %) and Vibrio rumoiensis S-1T (95.4 %), respectively. Phylogenetic analyses, based on both 16S rRNA and 92 housekeeping genes, showed that the strains formed distinct phylogenic lineages within the genera Shewanella and Vibrio. Digital DNA-DNA hybridization and orthologous average nucleotide identity values between strain KJ10-1T and other Shewanella species, as well as between strain KJ40-1T and other Vibrio species, were below the thresholds commonly accepted for prokaryotic species delineation. Based on the phenotypic, chemotaxonomic, and phylogenetic data, strains KJ10-1T and KJ40-1T represent novel species of the genera Shewanella and Vibrio, respectively, for which the names Shewanella phaeophyticola sp. nov. and Vibrio algarum sp. nov. are proposed, respectively. The type strains of S. phaeophyticola and V. algarum are KJ10-1T (=KACC 22589T=JCM 35409T) and KJ40-1T (=KACC 22588T=JCM 35410T), respectively.

Deciphering the Nitrogen Fixation Gene Cluster in Vibrio natriegens: A Study on Optimized Expression and Application of Nitrogenase.

Microbial nitrogen fixation presents a viable alternative to chemical fertilizers, yet the limited colonization and specificity of naturally occurring nitrogen-fixing microorganisms present significant challenges to their widespread application. In this study, we identified a nitrogen fixation gene cluster (VNnif) in Vibrio natriegens (VN) and tested its nitrogenase activity through the acetylene reduction assay. We investigated the potential utilization of nitrogenase by incorporating the nitrogenase gene cluster from VN into plant growth-promoting rhizosphere bacteria Pseudomonas protegens CHA0 and enhancing its activity to 48.16 nmol C2H2/mg/h through promoter replacement and cluster rearrangement. The engineered strain CHA0-PVNnif was found to positively impact the growth of Arabidopsis thaliana col-0 and Triticum aestivum L. (wheat). This study expanded the role of plant growth-promoting rhizobacteria (PGPR) and provided a research foundation for enhancing nitrogenase activity.

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.

Rationally designed chromosome fusion does not prevent rapid growth of Vibrio natriegens.

DNA replication is essential for the proliferation of all cells. Bacterial chromosomes are replicated bidirectionally from a single origin of replication, with replication proceeding at about 1000 bp per second. For the model organism, Escherichia coli, this translates into a replication time of about 40 min for its 4.6 Mb chromosome. Nevertheless, E. coli can propagate by overlapping replication cycles with a maximum short doubling time of 20 min. The fastest growing bacterium known, Vibrio natriegens, is able to replicate with a generation time of less than 10 min. It has a bipartite genome with chromosome sizes of 3.2 and 1.9 Mb. Is simultaneous replication from two origins a prerequisite for its rapid growth? We fused the two chromosomes of V. natriegens to create a strain carrying one chromosome with a single origin of replication. Compared to the parental, this strain showed no significant deviation in growth rate. This suggests that the split genome is not a prerequisite for rapid growth.

Whole-genome sequencing of Pseudoalteromonas piscicida 2515 revealed its antibacterial potency against Vibrio anguillarum: a preliminary invitro study.

Pseudoalteromonas piscicida 2515, isolated from Litopenaeus vannamei culture water, is a potential marine probiotic with broad anti-Vibrio properties. However, genomic information on P. piscicida 2515 is scarce. In this study, the general genomic characteristics and probiotic properties of the P. piscicida 2515 strain were analysed. In addition, we determined the antibacterial mechanism of this bacterial strain by scanning electron microscopy (SEM). The results indicated that the whole-genome sequence of P. piscicida 2515 contained one chromosome and one plasmid, including a total length of 5,541,406 bp with a G + C content of 43.24%, and 4679 protein-coding genes were predicted. Various adhesion-related genes, amino acid and vitamin metabolism and biosynthesis genes, and stress-responsive genes were found with genome mining tools. The presence of genes encoding chitin, bromocyclic peptides, lantibiotics, and sactipeptides showed the strong antibacterial activity of the P. piscicida 2515 strain. Moreover, in coculture with Vibrio anguillarum, P. piscicida 2515 displayed vesicle/pilus-like structures located on its surface that possibly participated in its bactericidal activity, representing an antibacterial mechanism. Additionally, 16 haemolytic genes and 3 antibiotic resistance genes, including tetracycline, fluoroquinolone, and carbapenem were annotated, but virulence genes encoding enterotoxin FM (entFM), cereulide (ces), and cytotoxin K were not detected. Further tests should be conducted to confirm the safety characteristics of P. piscicida 2515, including long-term toxicology tests, ecotoxicological assessment, and antibiotic resistance transfer risk assessment. Our results here revealed a new understanding of the probiotic properties and antibacterial mechanism of P. piscicida 2515, in addition to theoretical information for its application in aquaculture.

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.

Prevalence and antimicrobial resistance of major foodborne pathogens isolated from pangas and tilapia fish sold in retail markets of Dhaka city, Bangladesh.

Fish sold at retail markets are often contaminated with harmful bacterial pathogens, posing significant health risks. Despite the growing aquaculture industry in Bangladesh to meet high demand, little attention has been paid to ensuring the safety of fish. The objective of this study was to evaluate the microbiological quality of tilapia and pangas fish sold in retail markets across Dhaka city, Bangladesh. Specifically, the study aimed to compare the quality of fish from traditional wet markets and modern supermarkets, as well as fish samples collected during morning and evening hours. A total of 500 raw cut-fish samples (250 tilapia and 250 pangas) were collected at the point of sale from 32 wet markets and 25 supermarkets. All samples were tested for Escherichia coli, extended-spectrum ß-lactamase-producing E. coli (ESBL-Ec), along with the foodborne pathogens Salmonella, Shigella, Vibrio, and Cryptosporidium spp. Bacterial isolates were characterized using antibiotic susceptibility tests (AST) and the presence of common virulence and antibiotic-resistant genes. Fish samples from retail markets had higher prevalence of tested bacteria including E. coli (92 %), V. cholerae (62 %), ESBL-Ec (48 %), and Salmonella spp. (24 %). There was a significant difference in the prevalence of E. coli (97 % vs. 71 %), ESBL-Ec (58 % vs. 8 %) and Salmonella spp. (28 % vs. 8 %) on the wet market samples compared to supermarket samples (p < 0.005). The mean concentration of E. coli on fish from the wet market was 3.0 ± 0.9 log10 CFU/g, while that from supermarkets was 1.6 ± 0.9 log10 CFU/g. The mean concentration of ESBL-Ec in fish from wet markets and supermarkets were 2.3 ± 0.8 log10 CFU/g and 1.6 ± 0.5 log10 CFU/g, respectively. AST revealed that 46 % of E. coli isolates were multi-drug resistant (MDR), while 4 %, 2 % and 5 % of E. coli, Salmonella spp. and Vibrio spp. isolates, respectively, were resistant to carbapenems. At least 3 % of total E. coli isolates were found to be diarrheagenic, while 40 % of Salmonella isolates harbored pathogenic genes (stn, bcfC, ssaQ, avrA and sodC1), and none of the V. cholerae isolates harbored ctxA and tcpA. Our research shows that raw-cut fish samples from retail markets are contaminated with pathogenic and antibiotic-resistant bacteria, which could be a significant food safety concern. Public health interventions should be implemented to improve food safety and hygiene practices in the retail fish markets.

Virulence, antibiotic resistance phenotypes and molecular characterisation of Vibrio furnissii isolates from patients with diarrhoea.

BACKGROUND: Vibrio furnissii is an emerging human pathogen closely related to V. fluvialis that causes acute gastroenteritis. V. furnissii infection has been reported to be rarer than V. fluvialis, but a multi-drug resistance plasmid has recently been discovered in V. furnissii. METHODS: During daily monitoring at a general hospital in Beijing, China, seven V. furnissii strains were collected from patients aged over 14 years who presented with acute diarrhoea between April and October 2018. Genome analysis and comparison were performed for virulence and antimicrobial resistance genes, plasmids and transposon islands, together with phylogenetic analysis. Antimicrobial resistance to 19 antibiotics was investigated using the microbroth dilution method. Virulence phenotypes were investigated based on type VI secretion system (T6SS) expression and using a bacterial killing assay and a haemolysin assay. RESULTS: Phylogenetic analysis based on single-nucleotide polymorphisms revealed a closer relationship between V. furnissii and V. fluvialis than between other Vibrio spp. The seven V. furnissii isolates were in different monophyletic clades in the phylogenetic tree, suggesting that the seven cases of gastroenteritis were independent. High resistance to cefazolin, tetracycline and streptomycin was found in the V. furnissii isolates at respective rates of 100.0%, 57.1% and 42.9%, and intermediate resistance to ampicillin/sulbactam and imipenem was observed at respective rates of 85.7% and 85.7%. Of the tested strains, VFBJ02 was resistant to both imipenem and meropenem, while VFBJ01, VFBJ02, VFBJ05 and VFBJ07 were multi-drug resistant. Transposon islands containing antibiotic resistance genes were found on the multi-drug resistance plasmid in VFBJ05. Such transposon islands also occurred in VFBJ07 but were located on the chromosome. The virulence-related genes T6SS, vfh, hupO, vfp and ilpA were widespread in V. furnissii. The results of the virulence phenotype assays demonstrated that our isolated V. furnissii strains encoded an activated T6SS and grew in large colonies with strong beta-haemolysis on blood agar. CONCLUSION: This study showed that diarrhoea associated with V. furnissii occurred sporadically and was more common than expected in the summer in Beijing, China. The antibiotic resistance of V. furnissii has unique characteristics compared with that of V. fluvialis. Fluoroquinolones and third-generation cephalosporins, such as ceftazidime and doxycycline, were effective at treating V. furnissii infection. Continua laboratory-based surveillance is needed for the prevention and control of V. furnissii infection, especially the dissemination of the antibiotic resistance genes in this pathogen.

Expanding the capabilities of MuGENT for large-scale genetic engineering of the fastest-replicating species, Vibrio natriegens.

The fastest replicating bacterium Vibrio natriegens is a rising workhorse for molecular and biotechnological research with established tools for efficient genetic manipulation. Here, we expand on the capabilities of multiplex genome editing by natural transformation (MuGENT) by identifying a neutral insertion site and showing how two selectable markers can be swapped at this site for sequential rounds of natural transformation. Second, we demonstrated that MuGENT can be used for complementation by gene insertion at an ectopic chromosomal locus. Additionally, we developed a robust method to cure the competence plasmid required to induce natural transformation. Finally, we demonstrated the ability of MuGENT to create massive deletions; the 280 kb deletion created in this study is one of the largest artificial deletions constructed in a single round of targeted mutagenesis of a bacterium. These methods each advance the genetic potential of V. natriegens and collectively expand upon its utility as an emerging model organism for synthetic biology. IMPORTANCE: Vibrio natriegens is an emerging model organism for molecular and biotechnological applications. Its fast growth, metabolic versatility, and ease of genetic manipulation provide an ideal platform for synthetic biology. Here, we develop and apply novel methods that expand the genetic capabilities of the V. natriegens model system. Prior studies developed a method to manipulate multiple regions of the chromosome in a single step. Here, we provide new resources that diversify the utility of this method. We also provide a technique to remove the required genetic tools from the cell once the manipulation is performed, thus establishing "clean" derivative cells. Finally, we show the full extent of this technique's capability by generating one of the largest chromosomal deletions reported in the literature. Collectively, these new tools will be beneficial broadly to the Vibrio community and specifically to the advancement of V. natriegens as a model system.

Molecular identification of Proteus mirabilis, Vibrio species leading to CRISPR-Cas9 modification of tcpA and UreC genes causing cholera and UTI.

Heavy metal accumulation increases rapidly in the environment due to anthropogenic activities and industrialization. The leather and surgical industry produces many contaminants containing heavy metals. Cadmium, a prominent contaminant, is linked to severe health risks, notably kidney and liver damage, especially among individuals exposed to contaminated wastewater. This study aims to leverage the natural cadmium resistance mechanisms in bacteria for bioaccumulation purposes. The industrial wastewater samples, characterized by an alarming cadmium concentration of 29.6 ppm, 52 ppm, and 76.4 ppm-far exceeding the recommended limit of 0.003 ppm-were subjected to screening for cadmium-resistant bacteria using cadmium-supplemented media with CdCl2. 16S rRNA characterization identified Vibrio cholerae and Proteus mirabilis as cadmium-resistant bacteria in the collected samples. Subsequently, the cadmium resistance-associated cadA gene was successfully amplified in Vibrio species and Proteus mirabilis, revealing a product size of 623 bp. Further analysis of the identified bacteria included the examination of virulent genes, specifically the tcpA gene (472 bp) associated with cholera and the UreC gene (317 bp) linked to urinary tract infections. To enhance the bioaccumulation of cadmium, the study proposes the potential suppression of virulent gene expression through in-silico gene-editing tools such as CRISPR-Cas9. A total of 27 gRNAs were generated for UreC, with five selected for expression. Similarly, 42 gRNA sequences were generated for tcpA, with eight chosen for expression analysis. The selected gRNAs were integrated into the lentiCRISPR v2 expression vector. This strategic approach aims to facilitate precise gene editing of disease-causing genes (tcpA and UreC) within the bacterial genome. In conclusion, this study underscores the potential utility of Vibrio species and Proteus mirabilis as effective candidates for the removal of cadmium from industrial wastewater, offering insights for future environmental remediation strategies.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Frequent nonhomologous replacement of replicative helicase loaders by viruses in Vibrionaceae.

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene.