Microbial and transcriptional response of Acropora valida and Turbinaria peltata to Vibrio coralliilyticus challenge: insights into corals disease resistance.

BACKGROUND: Coral diseases are significant drivers of global coral reef degradation, with pathogens dominated by Vibrio coralliilyticus playing a prominent role in the development of coral diseases. Coral phenotype, symbiotic microbial communities, and host transcriptional regulation have been well-established as factors involved in determining coral disease resistance, but the underlying mechanisms remain incompletely understood. METHODS: This study employs high-throughput sequencing to analyse the symbiotic microbial and transcriptional response of the hosts in order to evaluate the disease resistance of Acropora valida and Turbinaria peltata exposed to Vibrio coralliilyticus. RESULTS: A. valida exhibited pronounced bleaching and tissue loss within 7 h of pathogen infection, whereas T. peltata showed no signs of disease throughout the experiment. Microbial diversity analyses revealed that T. peltata had a more flexible microbial community and a higher relative abundance of potential beneficial bacteria compared to A. valida. Although Vibrio inoculation resulted in a more significant decrease in the Symbiodiniaceae density of A. valida compared to that of T. peltata, it did not lead to recombination of the coral host and Symbiodiniaceae in either coral species. RNA-seq analysis revealed that the interspecific differences in the transcriptional regulation of hosts after Vibrio inoculation. Differentially expressed genes in A. valida were mainly enriched in the pathways associated with energy supply and immune response, such as G protein-coupled receptor signaling, toll-like receptor signaling, regulation of TOR signaling, while these genes in T. peltata were mainly involved in the pathway related to immune homeostasis and ion transport, such as JAK-STAT signaling pathway and regulation of ion transport. CONCLUSIONS: Pathogenic challenges elicit different microbial and transcriptional shifts across coral species. This study offers novel insights into molecular mechanisms of coral resistance to disease.

Producing recombinant proteins in Vibrio natriegens.

The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli.

Pan-Genome Provides Insights into Vibrio Evolution and Adaptation to Deep-Sea Hydrothermal Vents.

This study delves into the genomic features of 10 Vibrio strains collected from deep-sea hydrothermal vents in the Pacific Ocean, providing insights into their evolutionary history and ecological adaptations. Through sequencing and pan-genome analysis involving 141 Vibrio species, we found that deep-sea strains exhibit larger genomes with unique gene distributions, suggesting adaptation to the vent environment. The phylogenomic reconstruction of the investigated isolates revealed the presence of 2 main clades: The first is monophyletic, consisting exclusively of Vibrio alginolyticus, while the second forms a monophyletic clade comprising both Vibrio antiquarius and Vibrio diabolicus species, which were previously isolated from deep-sea vents. All strains carry virulence and antibiotic resistance genes related to those found in human pathogenic Vibrio species which may play a wider ecological role other than host infection in these environments. In addition, functional genomic analysis identified genes potentially related to deep-sea survival and stress response, alongside candidate genes encoding for novel antimicrobial agents. Ultimately, the pan-genome we generated represents a valuable resource for future studies investigating the taxonomy, evolution, and ecology of Vibrio species.

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.

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.

Direct Itaconate Production from Brown Macroalgae Using Engineered Vibrio sp. dhg.

Itaconate is a promising platform chemical with broad applicability, including the synthesis of poly(methyl methacrylate). Most studies on microbial itaconate production entail the use of crop-based feedstock, which imposes constraints due to its limited supply. Brown macroalgae have recently gained attention as next-generation biomass owing to their high biomass productivity and carbohydrate content and amenability to mass production. Therefore, the use of macroalgae for itaconate production warrants exploration. In this study, the direct production of itaconate from brown macroalgae was demonstrated using engineered Vibrio sp. dhg, which has emerged as an efficient platform host for brown macroalgal biorefineries. Specifically, to enhance production, cis-aconitate decarboxylase (Cad) from Aspergillus terreus was heterologously expressed and isocitrate dehydrogenase (icd) was deleted. Notably, the resulting strain, VIC, achieved itaconate titers of 2.5 and 1.5 g/L from a mixture of alginate and mannitol (10 g/L of each) and 40 g/L of raw Saccharina japonica (S. japonica), respectively. Overall, this study highlights the utility of brown macroalgae as feedstock, as well as that of Vibrio sp. dhg as a platform strain for improving itaconate bioproduction.

Isolation and Characterization of a Novel Vibrio Phage vB_ValA_R15Z.

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

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.

Graded-CRISPRi, a Tool for Tuning the Strengths of CRISPRi-Mediated Knockdowns in Vibrio natriegens Using gRNA Libraries.

In recent years, the fast-growing bacterium Vibrio natriegens has gained increasing attention as it has the potential to become a next-generation chassis for synthetic biology. A wide range of genetic parts and genome engineering methods have already been developed. However, there is still a need for a well-characterized tool to effectively and gradually reduce the expression levels of native genes. To bridge this gap, we created graded-CRISPRi, a system utilizing gRNA variants that lead to varying levels of repression strength. By incorporating multiple gRNA sequences into our design, we successfully extended this concept to simultaneously repress four distinct reporter genes. Furthermore, we demonstrated the capability of using graded-CRISPRi to target native genes, thereby examining the effect of various knockdown levels on growth.

Natural transformation of Vibrio natriegens with large genetic cluster enables alginate assimilation for isopentenol production.

Alginate is a major component of brown macroalgae, and its efficient utilization is critical for developing sustainable technologies. Vibrio natriegens is a fast-growing marine bacterium that has gained massive attention due to its potential as an alternative industrial chassis. However, V. natriegens cannot naturally metabolize alginate, limiting its usage in marine biomass conversion. In this study, V. natriegens was engineered to utilize marine biomass, kelp, as a carbon source. A total of 33.8 kb of the genetic cluster for alginate assimilation from Vibrio sp. dhg was integrated into V. natriegens by natural transformation. Engineered V. natriegens was further modified to produce 1.8 mg/L of isopentenol from 16 g/L of crude kelp powder. This study not only presents the very first case in which V. natriegens can be naturally transformed with large DNA fragments but also highlights the potential of this strain for converting marine biomass into valuable products.

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.

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.

Deciphering nutritional stress responses via knowledge-enriched transcriptomics for microbial engineering.

Understanding diverse bacterial nutritional requirements and responses is foundational in microbial research and biotechnology. In this study, we employed knowledge-enriched transcriptomic analytics to decipher complex stress responses of Vibrio natriegens to supplied nutrients, aiming to enhance microbial engineering efforts. We computed 64 independently modulated gene sets that comprise a quantitative basis for transcriptome dynamics across a comprehensive transcriptomics dataset containing a broad array of nutrient conditions. Our approach led to the i) identification of novel transporter systems for diverse substrates, ii) a detailed understanding of how trace elements affect metabolism and growth, and iii) extensive characterization of nutrient-induced stress responses, including osmotic stress, low glycolytic flux, proteostasis, and altered protein expression. By clarifying the relationship between the acetate-associated regulon and glycolytic flux status of various nutrients, we have showcased its vital role in directing optimal carbon source selection. Our findings offer deep insights into the transcriptional landscape of bacterial nutrition and underscore its significance in tailoring strain engineering strategies, thereby facilitating the development of more efficient and robust microbial systems for biotechnological applications.

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.

Osmotic stress response of the coral and oyster pathogen Vibrio coralliilyticus: acquisition of catabolism gene clusters for the compatible solute and signaling molecule myo-inositol.

Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known as osmolytes. The osmotic stress response and compatible solutes used by the coral and oyster pathogen Vibrio coralliilyticus were unknown. In this study, we showed that to alleviate osmotic stress V. coralliilyticus biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not myo-inositol. Myo-inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified myo-inositol (iol) catabolism clusters in V. coralliilyticus and other Vibrio, Photobacterium, Grimontia, and Enterovibrio species. Growth pattern analysis demonstrated that V. coralliilyticus utilized myo-inositol as a sole carbon source, with a short lag time of 3 h. An iolG deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on myo-inositol. Within the iol clusters were an MFS-type (iolT1) and an ABC-type (iolXYZ) transporter and analyses showed that both transported myo-inositol. IolG and IolA phylogeny among Vibrionaceae species showed different evolutionary histories indicating multiple acquisition events. Outside of Vibrionaceae, IolG was most closely related to IolG from a small group of Aeromonas fish and human pathogens and Providencia species. However, IolG from hypervirulent A. hydrophila strains clustered with IolG from Enterobacter, and divergently from Pectobacterium, Brenneria, and Dickeya plant pathogens. The iol cluster was also present within Aliiroseovarius, Burkholderia, Endozoicomonas, Halomonas, Labrenzia, Marinomonas, Marinobacterium, Cobetia, Pantoea, and Pseudomonas, of which many species were associated with marine flora and fauna.IMPORTANCEHost associated bacteria such as Vibrio coralliilyticus encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that myo-inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of myo-inositol could contribute to a breakdown of the coral-algae symbiosis during thermal stress or disrupt the coral microbiome. Phylogenetic analyses showed that the evolutionary history of myo-inositol metabolism is complex, acquired multiple times in Vibrio, but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved iol cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.

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.

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.

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.