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 isscarce. 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 participated in its bactericidal activity, representing a novel antibacterial mechanism. Nevertheless, 16 haemolytic genes and 3 antibiotic resistance genes (tetracycline, �uoroquinolone, and carbapenem) were annotated, but virulence genes encoding enterotoxin FM (entFM), cereulide (ces), and cytotoxin K were not detected. Further toxicity tests should be conducted to con�rm the safety of P. piscicida 2515. The results here provide a new understanding of the probiotic properties and antibacterial mechanism of P. piscicida 2515 and theoretical information for its application in aquaculture.


Introduction
Vibrio are gram-negative bacteria and critical pathogens in the marine aquaculture industry that cause the common aquaculture disease vibriosis and signi cant economic losses (Manchanayake et al., 2023;Sanches-Fernandes et al., 2022).Antibiotics, chemical drugs, and probiotics are commonly used to control Vibrio infection in the aquaculture industry (Oh et al., 2006;Won and Park, 2008;Etyemez Büyükdeveci et al., 2023).However, the emergence of resistant strains caused by antibiotic abuse and the pollution caused by drug abuse are not compatible with aquaculture sustainability (Dawood et al., 2015;Yi et al., 2019).
Expanding the use of nonantibiotic agents is essential for the management of aquaculture biosafety.Probiotics, which are live microorganisms, modulate the gut micro ora and reduce pathogenic bacterial abundance (Farzanfar, 2006;Wanna et al., 2021).Pseudoalteromonas is a marine bacterial genus belonging to the Pseudoalteromonadaceae family, Alteromonadales order, and Gammaproteobacteria class (Gauthier et al., 1995).Pseudoalteromonas can produce a series of secondary metabolites, including antimicrobial, antifouling, and algicidal molecules (Bowman, 2007;Sánchez-Porro et al., 2003).A previous study con rmed the protective effect of Pseudoalteromonas sp.NC201 against Vibrio.nigripulchritudo infection (Sorieul et al., 2018).In addition, Pseudoalteromonas.avipulchra CDM8 was presented as a candidate probiotic that can improve the survival rate of shrimp infected by Vibrio.parahaemolyticus (Wang et al., 2018).In this study, Pseudoalteromonas.piscicida strain 2515 was introduced as a potential probiotic for shrimp and olive ounder aquaculture since it shows a broad anti-Vibrio effect (Wang et al., 2021(Wang et al., , 2022)).However, it is important to investigate its probiotic characteristics prior to aquaculture application (Farzanfar, 2006;Wanna et al., 2021).Genomic information is an essential part of probiotic evaluations (Farzanfar, 2006).A few studies have investigated functional genes using bioinformatics analysis of the bacterial genome (Wang et al., 2021;Yu et al., 2013).Pseudoalteromonas genetic information is scarce.The P. piscicida 36Y_RITHPW genome contains many genes related to the production of bacteriocins and ribosomally synthesized antibacterial peptides (Sánchez-Díaz et al., 2019).P. piscicida DE1-A and DE2-A genome annotation revealed that these strains contain 6 and 7 serine proteases, respectively, that contribute to their antibacterial properties (Richards et al., 2019).P. avipulchra JG1 has some genes that are involved in the synthesis of various antimicrobial substances.These compounds enriched the antagonistic mechanisms (Yu et al., 2013).As a potential probiotic bacterium, genome information on P. piscicida 2515 is needed for strain evaluation prior to aquaculture application.
We hypothesized that genomic analysis of P. piscicida 2515 may reveal its potential and application safety in aquaculture through investigation of the adhesion-related genes, amino acid and vitamin metabolism and biosynthesis genes, virulence factor and antibiotic resistance genes, and stressresponsive genes, including thermal, acid, bile salt, and oxidative stressors, which are essential parameters for probiotic evaluation.This study aimed to analyse the antibacterial gene cluster in the P. piscicida 2515 genome and observe P. piscicida 2515 in coculture with Vibrio.anguillarum by scanning electron microscopy (SEM) to clarify its antimicrobial mechanism.To the best of our knowledge, this is the rst study to investigate the probiotic properties of P. piscicida 2515 using whole genome information, which will provide theoretical data for its application in aquaculture.

Materials and methods
Bacterial sample preparation P. piscicida 2515 was isolated from the water body of an aquaculture shrimp pond (Weifang, China) and preserved in the China Center for Type Culture Collection in Wuhan with number CCTCC NO.M2020731.P. piscicida 2515 was cultured in 2216E liquid medium at 28°C for 24 h, and then the fermented broth was centrifuged at 6000×g and resuspended three times in PBS solution.Finally, P. piscicida 2515 cells were harvested in a 1.5 mL Eppendorf tube and stored at -80°C for genomic DNA extraction.
DNA extraction, library construction, sequencing, and genome assembly Genomic DNA was extracted from the sample with the TGuide S96 Magnetic Universal Genomic DNA kit (QIAGEN) following the manufacturer's instructions.The harvested DNA was assessed by agarose gel electrophoresis and quanti ed by a Nanodrop 2000 (Thermo) and QubitTM 3 uorometer assays (Invitrogen).The sequencing library was constructed from high-quality genomic DNA (10 )  database, Swiss-Prot, TrEMBL and Pfam.In addition, carbohydrate-active enzymes (CAZy) were annotated using the CAZy database (Cantarel et al., 2009).After the structural and functional annotations were performed, the graphic circular map of the genome was constructed by using Circos version 0.66 (Krzywinski et al., 2009).
Antibacterial assays by SEM P. piscicida 2515 and V. anguillarum MN were cultured in marine 2216E broth medium overnight.Then, 100 µL of overnight culture of V. anguillarum MN (concentration approximately 10 8 CFU/mL) was plated on 2216E agar plates at 28°C for 24 h, followed by adding 5 µL of the overnight culture of P. piscicide 2515 (concentration approximately 10 9 CFU/mL).The agar including P. piscicide 2515 colonies surrounded by zones of clearing on the lawns of V. anguillarum MN was cut with a sterile scalpel and used for SEM observations according to a method described previously (Kanehisa, 2004).The cut agar was placed in a single well of a 6-well cell culture plate and xed for 30 min with 2.5% glutaraldehyde (Solarbio, Beijing, China).Afterwards, the agar pieces were washed twice for 30 min each time with 0.1 M imidazole buffer (pH 7.0), dehydrated in ethanol ( rst time with 50% and 70%, second time with 100%, 30 min each), and rinsed with ethanol-isoamyl acetate (1:1, v/v) one time for 10 min and in isoamyl acetate two times for 10 min each.The agar pieces isolated by lter paper were collected in a metal basket and placed in a critical point drying device.Finally, the samples were treated by critical point drying and coated with gold.The samples were observed and photographed by scanning electron microscopy (TESCAN, Czech Republic).

Results and discussion
Genomic analysis of P. piscicida 2515 showed that it had some probiotic properties and strong antibacterial activity due to the presence of chitin, bromocyclic peptide, lantibiotic, and sactipeptide genes.In addition, it was detected that P. piscicida 2515 can kill V. anguillarum by a novel antibacterial mechanism of vesicle/pilus-like structure transfer.

General genomic characteristics of P. piscicida 2515
The general features of the P. piscicida 2515 genome are summarized in Table 1.De novo assembly generated a circular chromosome (Fig. 1a) and a circular plasmid (Fig. 1b), with a total length of 5,541,406 bp and a G+C content of 43 there have been no reports regarding GIs in P. piscicida.Thus, the speci c functions of the GIs in the P. piscicida 2515 genome need further analysis.The genome sequence of P. piscicida 2515 was submitted to NCBI with the accession numbers CP074587 and CP074588.
The 3,125 genes were annotated in the GO database, including cellular components (1,693 genes), molecular function (2,481 genes), and biological process (2,289 genes) (Fig. 2b).Membrane, cell, and cell part were at the second level with the largest numbers of genes annotated to "cellular component".
Catalytic activity and binding were at the second level with the largest number of genes annotated to "molecular function".Metabolic process, cellular process, and single-organism process were at the second level with the largest numbers of genes annotated to "biological process".
Among these, 36 genes were annotated to "two-component system", 120 genes were annotated to "biosynthesis of amino acids", 86 genes were annotated to "carbon metabolism", 62 genes were annotated to "purine metabolism", and 47 genes were annotated to "pyrimidine metabolism" at level 2.
These results indicated that P. piscicida 2515 may be able to synthesize a variety of amino acids, polypeptides, and macromolecular proteins to adapt to the complex marine environment, but whether it has an antibacterial function needs further study.
Carbohydrate enzyme-related genes were analysed using Carbohydrate-Active Enzymes (CAZy) databases.The whole genome of P. piscicida 2515 contained 187 CAZymes, while carbohydrate-binding modules (CBMs) accounted for 31.55%,glycoside hydrolases (GHs) accounted for 21.92%, carbohydrate esterases (CEs) accounted for 20.85%, and polysaccharide lyases (PLs) accounted for 3.2% (Fig. 2d).GHs play a signi cant role in the breakdown of polysaccharides, including cellulose, xylan, and chitin (wang et al., 2023).Notably, the P. piscicida 2515 genome harboured 12 genes coding for chitinase.Such genes included GH18, GH19, and GH23, three enzymatic families.Previous studies have also reported that genes encoding chitin degradation machinery are common in Pseudoalteromonas strains (Paulsen et  ).In addition, the chitinase enzyme has shown hydrolysis activity against the cell wall of fungi and lysozyme activity against bacteria (Chang et al., 2003).Therefore, the chitinase annotated in P. piscicida 2515 can be regarded as an antibacterial protein with an antagonizing mechanism.

Assessment of probiotic properties
Stress-responsive genes in the whole genome P. piscicida is a potential probiotic that must overcome the harsh conditions (extreme temperature, pH, osmotic, bile, and oxidative stresses) of the gastrointestinal tract and successfully colonize the host's intestine.
Genome annotation and analysis showed that there are several genes in the P. piscicida 2515 genome encoding proteins that are related to stress tolerance (Table S1).A number of adaptive stress response genes have been identi ed, including some proteases and chaperones, such as energydependent intracellular proteases and chaperones, chaperone protein clpB, and hslUV proteases (van de Guchte et al., 2002).Among them, clpB, clpA, and clpX can engage in the heat stress response.Moreover, the presence of molecular chaperones (groES-groEL and dnaJ-dnaK-grpE-hrcA-htpG), heat shock protein genes (hslR, hslO, and htpX), and a small heat shock protein gene (ibpA) also showed a potential heat shock response (Liang et al., 2020;Liu et al., 2022).In addition, three cold shock protein genes, cspA, cspD, and cspE, were extracted from the P. piscicida 2515 genome, which could help this strain recover gradually during prolonged cold exposure (Derzelle et al., 2000).
Stomach acid resistance and bile salt tolerance are two essential properties of probiotics, as they dictate the capacity to survive in the host's intestine (Naissinger da Silva et al., 2021).We detected a bile acid sodium symporter family protein gene, a glucosamine-6-phosphate deaminase, and a CTP synthase gene in the P. piscicida 2515 genome (Table S1).F0F1-ATPase is considered to be the main regulator of intracellular pH (Wu et al., 2022).Eight genes encoding the F0F1-ATPase subunit system were determined in the P. piscicida 2515 genome, including atpC, atpD, atpG, atpA, atpH, atpE, atpF, and atpB.Furthermore, seven (Na + /H + ) antiporter genes, an (H + /Cl − ) antiporter gene clcA, and a (K + /H + ) antiporter gene were identi ed (Table 2), which have been demonstrated to be essential genes in pH regulation and ion homeostasis (Papadimitriou et al., 2016).These results potentially contributed to the adaptation of this strain to bile salt and stomach acid stressors in addition to its competitiveness with other bacteria in the intestinal environment.
Probiotics can be used as antioxidants to reduce ROS levels in cells and maintain the oxidationreduction balance in the intestine (Liu et al., 2022;Sundararaman et al., 2021).The genome of P. piscicida 2515 showed at least 38 genes related to the oxidative stress response, including one catalase gene, three catalase-peroxidase genes, three alkyl hydroperoxide reductase genes, one thiol peroxidase gene, four glutathione peroxidase genes, two thioredoxin genes, one thioredoxin reductase gene, one DyP-type peroxidase gene, and three glutaredoxin genes (Table S1).The presence of thiol peroxidase contributes to reversing oxidative damage (Schell et al., 2002).Catalase and glutathione peroxidase are engaged directly in hydrogen peroxide and ROS detoxi cation (Liang et al., 2020;Wang et al., 2023).These genes indicated the high antioxidant ability of P. piscicida 2515, which could thereby increase host immunity.

Adhesion and aggregation abilities
The adhesion of probiotics to the host intestinal epithelium is vital to increase their dwell time in the intestine for colonization and the inhibition of pathogens (Granato et al., 2004;Kuebutornye et al., 2022).Adhesion-related genes were found in the P. piscicida 2515 genome, including seven agellar hook-associated protein genes, one chitin-binding protein gene, two segregation and condensation protein genes, and one capsular polysaccharide biosynthesis protein gene (Table S1).It is suggested that P. piscicida 2515 has the potential to adhere to the host gut or other matrices.

Amino acid and vitamin metabolism and biosynthesis
The secretion of amino acids and vitamins is a crucial property of probiotics.Previous studies have reported that amino acids can enhance the growth, antioxidant, immune, disease resistance, digestion and absorption abilities of aquatic animals (Hardy, 2010;Hoseini et al., 2020).Vitamins are used as immunostimulants and for growth promotion in aquaculture (Wang et al., 2017).The amino acid metabolic pathway in P. piscicida 2515 involves genes that are responsible for threonine, tryptophan, methionine, leucine, lysine, cysteine, histidine, and arginine metabolic biosynthesis.Furthermore, thiamine, ribo avin, pyridoxine, biotin, and folate biosynthesis metabolic pathways were also determined in the genome of P. piscicida 2515 (Table S1).Therefore, P. piscicida 2515 has nutritional value for the host.

SEM observations of bactericidal activity
SEM observations revealed that P. piscicida 2515 had a multitude of vesicle/pilus-like structures on its surface (Fig. 3a), while V. anguillarum showed a smooth surface (Fig. 3b).V. anguillarum cells were observed to be lysed when they were cocultured with P. piscicida 2515 (Fig. 3c, 3d), indicating the antibacterial activity of P. piscicida 2515.Moreover, numerous vesicle/pilus-like structures were observed on the surface of V. anguillarum and their lysed cells (Fig. 3c, 3d).These results demonstrated that P. piscicida 2515 probably needed to transfer vesicles/pilus-like structures to kill V. anguillarum, which is in agreement with previous studies (Richards et al., 2017b;Wang et al., 2021).Unlike typical outer membrane vesicles (OMVs), the vesicles/pilus-like structures are tethered to the surface of P. piscicida 2515, not secreted or extracellular.This transfer mechanism was previously unknown because the vesicle/pilus-like structures could not be quickly observed by SEM during the transfer process.Therefore, the full function of this structure and underlying antibacterial mechanism need further study.

Synthesis of secondary metabolites
Antimicrobial activity is regarded as an essential approach by which probiotics ght against pathogens in the gut.Previous studies have shown that P. piscicida 2515 has broad-spectrum antibacterial activity, and dietary supplementation with P. piscicida 2515 has a positive effect on shrimp and olive ounder intestinal microbiota (Wang et al., 2021(Wang et al., , 2022)).The availability of whole-genome sequence information and the variety of genome mining tools make it possible to predict the antimicrobial compounds of this bacterium and their underlying mechanism.
The results of the secondary metabolite gene cluster analysis by antiSMASH 6.0 showed that the P. piscicida 2515 genome had 6 cluster types in 11 gene clusters, including two RiPP-like, one ladderane-NRPS, four NRPS-T1pks, one thiopeptide, two NRPSs, and one lantipeptide (Table 2).
Cluster 3 and Cluster 7 showed only 30% and 6% similarity to crochelin A and N-tetradecanoyl tyrosine, respectively.Nevertheless, eight gene clusters were unknown in the P. piscicida 2515 genome, which indicated that P. piscicida 2515 could potentially synthesize new secondary metabolites and antibacterial compounds.
Three gene clusters with the potential for lanthipeptide, bottromycin, and sactipeptide synthesis were observed in the P. piscicida 2515 genome by the BAGEL4 database (Fig. 4).Lantibiotics are an essential class of antimicrobial compounds that often damage the integrity of the cell walls of gram-positive bacteria (van der Donk and Nair, 2014), and sactipeptides also show antibacterial activity (Chen et al., 2021).Therefore, these metabolites could be helpful for the antibacterial activities of probiotics.Further studies are needed to purify and identify these metabolites and determine their mode of action.
Safety evaluation of P. piscicida 2515 Safety evaluations of probiotics are required for animal and human health protection (Feito et al., 2022).
Based on the CARD database prediction, three antibiotic resistance genes were determined in the P. piscicida 2515 genome, including tetracycline, uoroquinolone, and carbapenem resistance using resistance mechanisms including antibiotic e ux and antibiotic inactivation (Table .3).However, the P. piscicida 2515 strain did not show full resistance to these antibiotics in the in vitro study (data not shown).Therefore, it is necessary to verify the expression of these genes in vitro.In addition, the assessment of virulence factor genes is essential in probiotic safety evaluations.A total of 1002 virulence genes were identi ed in the P. piscicida 2515 genome based on the VFDB database (Fig. 5), such as nutritional/metabolic factors (23.85%), motility (14.77%), immune modulation (11.98%), effector delivery systems (11.68%), regulation (10.08%), adherence (8.08%), and exotoxins (5.99%).Among the exotoxins, 16 genes were annotated as related to haemolysin, including cylA, cylF, cylG, cylI, cyaB, cyaD, hlyB, and hlyD (Table S2).These results were in agreement with previous reports regarding other probiotics that contain haemolytic genes (Jiménez et al., 2013;Trapecar et al., 2011).In our previous study, P. piscicida 2515 showed haemolytic activity on sh blood cells (Zhang et al., 2021).Moreover, heat-killed P. piscicida 2515 could enhance nonspeci c immunity and resistance against V. anguillarum in olive ounder aquaculture (Wang et al., 2022).In addition, virulence genes, including enterotoxin FM (entFM), cereulide (ces), and cytotoxin K, were not detected in the P. piscicida 2515 genome.After thermal exposure, the haemolytic effect of P. piscicida 2515 was eliminated (Zhang et al., 2021), and the administration of this bacterium has potent and safe application prospects in sh and shrimp aquaculture (Wang et al., 2021(Wang et al., , 2022)).Further comprehensive investigations, including food biosecurity and human health aspects, must be considered.

Conclusion
Genome analysis revealed the potential of the P. piscicida 2515 strain as a probiotic agent due to its antibacterial activity, amino acid and vitamin synthesis abilities, and adhesion ability, in addition to the adaptive genes against environmental stresses.Moreover, P. piscicida 2515 killed V. anguillarum by transferring vesicle/pilus-like structures, which is a novel antibacterial mechanism.Therefore, the P. piscicida 2515 possesses many probiotic attributes, and further studies should be conducted to con rm its antibacterial substances and to provide theoretical support for its use in aquaculture.

Figure 2 Distribution
Figure 2

Table 2 .
Summary of predicted gene clusters of secondary metabolite synthesis strain P. piscicida 2515