Controlling edwardsiellosis caused by Edwardsiella piscicida and mitigating drug-resistant gene dissemination: Bacteriophage EPP-1, a promising antibiotic alternative

Controlling edwardsiellosis caused by Edwardsiella piscicida and mitigating drug-resistant gene dissemination: Bacteriophage EPP-1, a promising antibiotic alternative | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Controlling edwardsiellosis caused by Edwardsiella piscicida and mitigating drug-resistant gene dissemination: Bacteriophage EPP-1, a promising antibiotic alternative Ganghua Han, Ting Huang, Xinchun Liu, Ruyin Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3844797/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Edwardsiella piscicida causes significant economic losses to the aquaculture industry worldwide. Phage-based biocontrol methods are experiencing a renaissance because of the spread of drug-resistant genes and bacteria resulting from the heavy use of antibiotics. Here, we showed that the novel Edwardsiella phage EPP-1 could achieve comparable efficacy to florfenicol using a zebrafish model of Edwardsiella piscicida infection and could reduce the content of the floR resistance gene in zebrafish excreta. Specifically, phage EPP-1 inhibited bacterial growth in vitro and significantly improved the zebrafish survival rate in vivo ( P = 0.0035), achieving an efficacy comparable to that of florfenicol ( P = 0.2304). Notably, integrating the results of 16S rRNA sequencing, metagenomic sequencing, and qPCR, although the effects of phage EPP-1 converged with those of florfenicol in terms of the community composition and potential function of the zebrafish gut microbiota, it reduced the floR gene content in zebrafish excreta and aquaculture water. Overall, our study highlights the feasibility and safety of phage therapy for edwardsiellosis control, which has profound implications for the development of antibiotic alternatives to address the antibiotic crisis. Biological sciences/Biotechnology Biological sciences/Microbiology Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences Phage therapy Drug-resistant gene Edwardsiella piscicida Edwardsiellosis Antibiotic alternative Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Edwardsiella piscicida is a notorious fish pathogen that infects its host mainly through the gut (Shao et al., 2022 ), leading to severe edwardsiellosis and causing substantial economic losses to the aquaculture industry worldwide (Abayneh et al., 2013 ; Buján et al., 2018 ). The Food and Drug Administration (FDA) recommends a dose of 10‒15 mg florfenicol/kg fish for no more than 10 consecutive days to combat edwardsiellosis in aquaculture ( https://www.fda.gov/media/84229/download ). Recent studies, however, have unveiled that exposure to standard therapeutic doses of florfenicol alters the structure of the gut microbiota and increase the abundance of florfenicol-resistance genes in aquaculture systems (Sáenz et al., 2019 ; Zeng et al., 2019 ). The use of other antibiotics like oxytetracycline, sulfamethoxazole, or enrofloxacin similarly induced rise of antibiotic resistance gene (ARG) levels or emergence of novel antibiotic resistant bacteria (ARB) across a wide range of habitats (Jin et al., 2023 ; Kayani et al., 2021 ). These ARGs or ARB possess the potential to disseminate into the environment (He et al., 2023 ; Su et al., 2023 ; Xu et al., 2023 ), thereby heightening their risks in global public health sector (Gao et al., 2023 ; Luo et al., 2022 ). Accordingly, the excessive use of antibiotics overshadows the sustainable development of the aquaculture industry, and particularly in the global context of “One Health,” novel antibacterial agents are urgently needed. Bacteriophages (phages), viruses that specifically infect bacteria, are ubiquitous and abundant worldwide, with an estimated total number exceeding 10 31 particles (Mokili et al., 2012 ). Phage-based biocontrol methods, or phage therapy, have been reinvigorated with unprecedented momentum and are emerging as important strategies in the post-antibiotic era due to the fact that it can reduce the use of antibiotics at the source and alleviate a series of issues caused by the use of antibiotics (Altamirano and Barr 2019 ; Domingo-Calap and Delgado-Martínez 2018 ). Phage therapy in the field of aquaculture is now being researched against Vibrio , Flavobacterium , Aeromonas , Pseudomonas , and Lactococcus and has yielded favorable outcomes (Culot et al., 2019 ; Liu et al., 2022 ). Available evidence suggests that administration of E. tarda phage vB_EtaM_ET-ABTNL-9 by feeding, injection, or immersion significantly reduced mortality as well as nonspecific immune-related enzyme activities of turbot Scophthalmus maximus (Cui et al., 2022a ). Furthermore, a phage cocktail composed of E. tarda phage PETp9 and V. harveyi phage PVHp5 significantly reduced host levels of pathogens and maintained the normal gut microbiota profile for ascites prevention in turbot (Cui et al., 2022b ). As a novel antibiotic alternative, the limitation of these studies on phage therapy in aquaculture is that they do not consider the production of ARGs, which is the most severe problem resulting from antibiotic use. Furthermore, there is a considerable dearth of research on phage therapy for the control of edwardsiellosis caused by E. piscicida , its potential to substitute for antibiotics and its impacts on ARGs production in the control of edwardsiellosis is similarly unknown. Elucidation of these issues is a prerequisite for implementing phage therapy in aquaculture and addressing the associated antibiotic resistance issues. Zebrafish ( Danio rerio ) is the most commonly used model species for environmental monitoring and toxicological evaluations (Li et al., 2023b ; Ricarte et al., 2023 ). Therefore, it was selected as the experimental subject in this study. Here, we isolated a novel E. piscicida phage and characterized its physiological and genomic properties. We also investigated its bactericidal effect on E. piscicida in vitro and its protective effect on zebrafish in vivo . In addition, we evaluated the safety of phage therapy for edwardsiellosis control in zebrafish in terms of the antioxidant capacity and inflammatory cytokines in the gut and liver of the model species, the diversity and function of the gut microbial community, and the content of the floR gene in fish excreta and environmental media. To the best of our knowledge, this is the first attempt to use a novel phage to control edwardsiellosis caused by E. piscicida and to comprehensively evaluate its safety for aquaculture. This study thus provides novel insights into the biocontrol of edwardsiellosis in aquaculture and has profound implications for the development of antibiotic alternatives to address the antibiotic crisis. 2. Materials and Methods 2.1 Bacterial strains and zebrafish culture Bacteria for phage isolation and host spectrum determination (Table S1 ) were obtained from the Marine Culture Collection of China (MCCC) and cultivated in Trypticase Soy Broth medium (Solarbio, Beijing, China) at 30℃ with 120 rpm of shaking. Wild-type zebrafish ( D. rerio , AB line, 3‒4 cm, 0.3‒0.4 g) were purchased from Zhongke Water Quality Environmental Technology Co., Ltd. (Wuxi, Jiangsu, China), raised in a standard aquaculture system with a photoperiod of 14 h:10 h light/dark, and fed Artemia salina twice daily. To avoid water quality deterioration and potential impacts on subsequent experiments, one-third of the aquaculture water was replaced daily with tap water that was aerated for 24 h, water quality (Table S2) was monitored every 2 days, and feces produced by zebrafish was collected thoroughly on days 2, 4, and 7 during the whole experiment. All zebrafish used in this study were acclimatized for at least seven days under the standard aquaculture system prior to the next step in the experiment, and the conditions were consistent throughout the experiment. 2.2 Phage isolation, characterization, and genome analysis The double-layer agar method, as described by Thung (Thung et al., 2017 ), was used to isolate Edwardsiella phages from aquaculture wastewater and natural surface water (Supplementary Information). After purification and proliferation, several characteristics of the isolated phages were characterized, including morphology via transmission electron microscopy (TEM), host spectrum, and temperature and pH tolerance (see Supplementary Information for details). Phage nucleic acids were extracted and purified, as described by Kim et al. (Supplementary Information) (Kim et al., 2019 ). Purified phage DNA was randomly broken into 350 bp fragments and then linked to a specific adapter for library preparation using the standard NEBNext® Ultra™ II DNA Library Preparation Kit for Illumina®. After checking the library, paired-end 2 × 150 sequencing was performed on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA) at Fixgene Technology Co., Ltd. (Beijing, China). The raw sequencing data were subjected to quality control using fastp (Chen et al., 2018 ) and then assembled using SPAdes v.3.12.0 (Nurk et al., 2013 ). The resulting sequences were corrected using PhageTerm (Garneau et al., 2017 ). Subsequent bioinformatic analysis is presented in the Supplementary Information. 2.3 Phage therapy versus antibiotic therapy for edwardsiellosis To compare the efficacy of phage therapy with that of conventional antibiotic therapy for edwardsiellosis control in the zebrafish model, we set up four different treatment scenarios based on intraperitoneal injection, namely the PBS + SM group (negative control group, PBS buffer (137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na 2 HPO 4 , 1.76 mM of KH 2 PO 4 , pH = 7.4) + SM buffer (200 mM of NaCl, 10 mM of MgSO 4 , 50 mM of Tris-HCl, pH = 7.5)), E. p. + SM group (positive control group, 10 5 CFU E. piscicida (MCCC 1K00246)/fish, determined in the study), E. p. + FLO group (florfenicol therapy group, 10 5 CFU E. piscicida (MCCC 1K00246)/fish + 10 mg florfenicol/kg fish weight), and E. p. + EPP-1 group (phage therapy group, 10 5 CFU E. piscicida (MCCC 1K00246)/fish + MOI of 1 for the phage EPP-1, determined in the study), with 30 zebrafish per tank. In the described above, the E. piscicida was diluted with PBS, phage EPP-1 and florfenicol were diluted with SM buffer. The number of dead zebrafish was recorded daily for seven consecutive days to plot survival curves. The effects of the different treatments on the antioxidant capacity and levels of inflammatory cytokines in the gut and liver of zebrafish were evaluated. Finally, zebrafish feces and farmed water were collected on days 2, 4, and 7 for microbial-related analyses. 2.3.1 Oxidative stress and inflammatory cytokines in the gut and liver On days 1, 3, and 7 after intraperitoneal injection, 6 zebrafish were randomly selected and divided into two groups with three biological replicates each, one group for the antioxidant capacity assay and the other group for the inflammatory cytokine assay. Briefly, the zebrafish were sacrificed to harvest the gut and liver samples. These samples were then weighed and prepared as 10% homogenates with saline (for antioxidant capacity determination) and PBS (for inflammatory cytokine determination) and centrifuged at 6000 × g for 10 min, and the supernatant was collected. Superoxide dismutase (SOD) and catalase (CAT) activity and reduced glutathione (GSH) content were determined using SOD, CAT, and GSH assay kits (Nanjing Jiancheng Bioeng Inst., Nanjing, China). Inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) were quantified using fish tissue-specific enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioeng Inst., Nanjing, China). Enzyme activity and inflammatory cytokine levels were determined according to the manufacturer’s instructions using a microplate reader (Synergy™ H1; BioTek Inc., Winooski, VT, USA) at specific wavelengths. Each measurement was conducted in triple biological replication. T-test analysis was performed to examine the difference among different treatments. 2.3.2 Nucleic acid extraction from water and zebrafish feces Feed water and zebrafish fecal samples were collected from different treatment groups for DNA extraction on days 2, 4, and 7 after intraperitoneal injection, that is, 3 water samples and 3 fecal samples for each treatment. Approximately 600 mL of water samples were filtered through 0.22 µm-pore-size membranes (Merck Millipore, Bedford, MA, USA) within 24 h, and the membranes were then cut with sterile scissors for DNA extraction. Fecal samples were aspirated from the bottom of the tank, centrifuged at 6000 × g for 10 min, washed three times with PBS, and weighed, after which they were ready for DNA extraction. Genomic DNA was extracted from water and fecal samples using the FastDNA™ Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA), following the manufacturer’s instructions. Finally, the extracted DNA was fractionated and stored at − 20°C for further analysis. 2.3.3 Quantification of E. piscicida and the floR gene The host pathogen E. piscicida and the florfenicol-resistance gene floR were measured in feeding water and zebrafish feces via quantitative polymerase chain reaction (qPCR) using previously reported available primers ( gyrB and floR genes for E. piscicida and florfenicol-resistance gene quantification, respectively) and programs (Li et al., 2013 ), respectively. A standard curve was first plotted based on constructed plasmids with known content of target genes, and then the content of target genes in the samples was calculated based on this standard curve. The primer sequences, product sizes, and detailed qPCR programs are shown in Supplementary Information Table S3. Three replicates for each are used for quantification and the content of each gene is presented as the mean ± standard deviation. 2.3.4 16S rRNA gene sequencing and analysis The V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified via PCR using the barcoded primer pair 338F and 806R (Table S3). Amplicon sequencing was performed on an Illumina MiSeq platform (Illumina, Woburn, MA, USA) by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China), and sequencing data were analyzed using the online Majorbio Cloud Platform ( https://cloud.majorbio.com/ ) (Ren et al., 2022 ). Briefly, the raw paired sequences were subjected to quality control and merged using fastp v0.19.6 (Chen et al., 2018 ) and FLASH v1.2.11 (Magoc and Salzberg 2011 ). The resulting tags were analyzed using QIIME 1.9.1 (Caporaso et al., 2010 ), following the steps reported by Guo et al . (Guo et al., 2019 ). The quality-filtered sequences were clustered into operational taxonomic units (OTUs) with a 97% identity cut-off using UPARSE v11 (Edgar 2013 ) and annotated based on taxonomy with the Silva (SSU138) 16S rRNA database as a reference, with a 70% classification confidence threshold (Quast et al., 2013 ), using the naïve Bayesian-based RDP Classifier v2.13 (Wang et al., 2007 ). Alpha diversity indices and Bray-Curtis distance-based beta diversity indices were calculated using QIIME 1.9.1 (Caporaso et al., 2010 ). Principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) analysis at the OTU level were performed using RStudio according to the Bray-Curtis distance matrix. 2.3.5 Metagenomic analysis of the gut microbial communities Metagenomic sequencing of 12 fecal samples was conducted on an Illumina HiSeq 4000 platform (Illumina, Woburn, MA, USA) with a 2 × 150 bp paired-end sequencing strategy by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Adapters, sequences with a length < 50 bp or an average quality score < 20, or reads containing the “N” base were removed from raw reads using fastp v0.20.0 (Chen et al., 2018 ). The resulting clean reads were assembled using the succinct de Bruijn graph method in Megahit v1.1.2 (Li et al., 2015 ). Open reading frames (ORFs) were predicted from assembled contigs > 100 bp using Prodigal v2.6.3 (Hyatt et al., 2010 ). The predicted ORFs were clustered using CD-HIT v4.6.1 (Fu et al., 2012 ) with 90% identity and 90% coverage, to construct a non-redundant gene set. The high-quality reads of each sample were then matched to the gene set using SOAPaligner v2.21 (Li et al., 2009 ), based on the Reads Per Kilobase per Million (RPKM) algorithm (Lawson et al., 2017 ), with 95% identity to determine the abundance of target genes. Gene function was annotated by aligning the sequences of the non-redundant gene set against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using DIAMOND v0.8.35 (Buchfink et al., 2021 ) with an E-value ≤ 10 − 5 . Carbohydrate-active enzymes and antibiotic resistance genes were annotated, and their abundances were calculated based on the Carbohydrate-Active enZYmes Database (CAZy) (Drula et al., 2022 ) and the Comprehensive Antibiotic Resistance Database (CARD) (Alcock et al., 2023 ), respectively. In addition, PCoA, NMDS, and Kruskal-Wallis H tests of gene function at multiple taxonomic levels were performed based on RPKM abundance using the Bray-Curtis distance matrix. 2.4 Statistical analysis Student’s t -test, One-way ANOVA with Sidak’s post-hoc test, and two-way ANOVA were performed in IBM SPSS v25.0. Survival analyses with Mantel-Cox test was performed in Graphpad Prism v9.0. P < 0.05 was considered statistically significant. Stress value < 0.05 in NMDS indicates their good conformity. 2.5 Data availability All sequencing data have been deposited in the NCBI Sequence Read Archive under the BioProject ID PRJNA964478. 3. Results 3.1 Isolation and characterization of phage EPP-1 In this study, an Edwardsiella phage, named EPP-1, was isolated from aquaculture wastewater in Henan, China, and preserved at the China General Microbiological Culture Collection Center (CGMCC; preservation number, CGMCC No. 45078). The complete genome of the phage was uploaded to NCBI with GenBank accession number OQ910326. Phage EPP-1 lysed host E. piscicida MCCC 1K00246 and formed clear plaques on a double-layer plate (Fig. 1 a). TEM-based morphology showed that phage EPP-1 has a head of 37 nm in diameter and a tail that is 103 nm in length (Fig. 1 b) and belongs to the Myoviridae family in Caudovirales, according to the classification criteria of the International Committee on Taxonomy of Viruses. The host spectrum assay showed that this phage also lysed E. piscicida MCCC 1K03230, E. tarda MCCC 1K00241, Edwardsiella sp. MCCC 1K00239, Edwardsiella sp. MCCC 1K00240, and Edwardsiella sp. MCCC 1K00242, indicating their broad-spectrum properties (Table S1 ). Phage EPP-1 was found to have strong tolerance to low and moderate temperatures; its titer began to decrease at temperatures above 50℃ and completely vanished at 80℃ (Fig. 1 c). The phage was highly stable between pH 5 and 11, but its numbers decreased from 8.51 log 10 PFU/mL at pH 5 to 3.48 log 10 PFU/mL at pH 3, and it was completely inactivated under more acidic or alkaline conditions (Fig. 1 d). According to whole-genome sequencing, the genome of phage EPP-1 was structured as linear dsDNA, with a size of 38,641 bp and a GC content of 52.29%. PHASTER predicted 66 CDSs from the EPP-1 genome, of which 28 appeared to encode functional proteins, such as phage structural proteins, DNA replication-related enzymes, and lysis-related enzymes. Notably, lysogeny-related genes, such as the integrase and recombination loci attL and attR , were also predicted in the phage EPP-1 genome (Fig. 1 e), suggesting that the virus might enter the lysogenic cycle in phage–host interactions. No virulence factors, ARGs, or tRNA were identified in the genome of EPP-1. According to TerL -based phylogenic analysis, phage EPP-1 was distantly related to other Edwardsiella phages but closely related to the Yersinia phages YeP1, YeP2, and YeP3 and Salmonella typhimurium phage ST64B (Fig. 1 f). The average nucleotide identity (ANI) values for whole-genome comparisons were calculated using the same genome collections. The highest ANI value was 98.11% for phage EPP-1 with Edwardsiella phage GF-2, which has a genome size of 43,129 bp, whereas the remaining ANI values were lower than 95% (Fig. 1 g), indicating the distinctiveness of phage EPP-1 isolated in this study. 3.2 Phage EPP-1 treatment is comparable to florfenicol in efficacy The in vitro antibacterial effect of phage EPP-1 and its protective effect in vivo in a zebrafish model were investigated to evaluate its feasibility for use in edwardsiellosis control in aquaculture. Phage EPP-1 showed strong antibacterial activity in vitro , effectively inhibiting the growth of its host, making their OD 600 values lower than 0.2 even at a low MOI of 0.01, as a comparison, the group without phage addition had an OD 600 value of more than 0.6 (Fig. 2 a). For in vivo assays, 10 5 CFU/fish of E. piscicida 1K00246 was used (Fig. S1 a). Given the complex components of the zebrafish gut, which contribute to a harsher condition than that used in the in vitro assay, different doses (MOIs of 0, 0.1, 1, 5, and 10) of phage EPP-1 were administered to determine the optimal therapeutic dose. An MOI of 1 significantly improved the zebrafish survival compared to that in the challenge group (MOI = 1 vs challenge group, P = 0.0008) and was comparable to that in the higher dose groups (MOI = 1 vs MOI = 5, P = 0.6491; MOI = 1 vs MOI = 10, P = 0.1572) (Fig. S1 b), and thus was therefore selected for further trials. To compare the therapeutic efficacy of phage EPP-1 with that of the antibiotic florfenicol for edwardsiellosis control, four different treatment scenarios were established as follows: PBS + SM negative control group, E. p. + SM positive control group, E. p. + FLO treatment group, and E. p. + EPP-1 treatment group (Fig. 2 b). Phage EPP-1 alleviated the signs of ascites in zebrafish caused by E. piscicida infection (Fig. 2 c). Both treatment groups showed significantly decreased zebrafish mortality ( E. p. + SM vs E. p. + EPP-1, P = 0.0035; E. p. + SM vs E. p. + FLO, P < 0.0001), and the efficacy of phage EPP-1 was comparable to that of conventional florfenicol ( E. p. + FLO vs E. p. + EPP-1, P = 0.2304) (Fig. 2 d). Pathogenic E. piscicida , introduced into the guts of zebrafish, can enter the environment via excrement. Edwardsiella -specific qPCR showed that both EPP-1 and florfenicol therapy groups harbored substantial Edwardsiella in their feeding water and zebrafish feces on day 2 (7.08 and 5.65 log 10 GC/L of feeding water in FLO and EPP-1 groups, and 8.65 and 7.38 log 10 GC/g wet feces in the FLO and EPP-1 groups, respectively), but decreased rapidly with feeding time, reaching levels comparable to those in the PBS + SM group (5.14 log 10 GC/L of feeding water and 4.26 log 10 GC/g wet feces in the PBS + SM group) (Fig. 2 e). Edwardsiella contents in zebrafish feces of the two therapy groups were comparable (4.41 and 4.47 log 10 GC/g wet feces in FLO and EPP-1 groups, respectively), which were both lower than that in the E. p. + SM challenge group on day 7 (6.29 log 10 GC/g wet feces) (Fig. 2 e). 3.3 Phage EPP-1 treatment alleviates oxidative stress and immune responses in the zebrafish gut Enzyme activities and inflammatory cytokines in the gut and liver after different treatments were measured to further compare the effects of phage EPP-1 and florfenicol on the antioxidant capacity and immune response in zebrafish. The results showed that, compared to those with florfenicol treatment, phage EPP-1 could efficiently alleviate changes in the GSH content and CAT activity in the guts of fish infected with E. piscicida , especially the changes in GSH on day 1 and CAT activity on day 7; however, the changes in SOD activity were not significantly altered within 7 days (Fig. 3 a-c). In the liver, there were no significant changes in SOD and CAT activities among the different treatment groups within 7 days; however, the GSH content in the EPP-1 and florfenicol treatment groups decreased significantly on days 1 and 3 compared with that in the negative control group (Fig. 3 d). In addition, we found that phage EPP-1 alleviated immune responses in different organs of zebrafish for a longer time than that in the florfenicol treatment. Specifically, the treatment effect of florfenicol was faster than that of EPP-1 in alleviating the gut immune response caused by E. piscicida infection, as reflected by the changes in IL-1β, IL-6, TNF-α, and IFN-γ levels (Fig. 3 e- 3 h). In contrast, phage therapy performed better in the middle and later phases of treatment, especially in moderating the response of IL-1β, TNF-α, and IFN-γ on day 3 (Fig. 3 e- 3 h). The differences in IL-1β and IL-6 contents in the phage and florfenicol therapy groups became insignificant on day 7 over a prolonged post-injection period (Fig. 3 e, f). It was also evident that phage EPP-1 moderated the upregulation of IL-6 levels in the liver compared to the effects of florfenicol (Fig. 3 i). 3.4 Composition of bacterial community in the zebrafish gut In total, 673,007 optimized sequences were generated through 16S rRNA gene sequencing. The rarefaction curve for each sample tended to become saturated, indicating a sufficient sequencing depth for community composition analysis (Fig. S2a). We identified 464 OTUs, of which 101 were shared by different experimental groups. The number of OTUs in the negative control group was approximately two-fold lower than that in the other groups injected with pathogenic E. piscicida (Fig. 4 a). Furthermore, the richness (Sobs, Chao 1, and ACE estimators) of the gut bacterial community was significantly lower in the negative control group than in the other experimental groups (Fig. 4 b). Proteobacteria and Bacteroidetes were consistently dominant in gut communities, whereas the relative abundance of Fusobacteria in the phage therapy group decreased from 20.89% on day 2 to 0.57% on day 7. Except for the negative control group, all experimental groups had a higher abundance (> 5%) of Actinobacteria on day 7 (Fig. S2b). At the genus level, Rheinheimera and Paucibacter , the two dominant genera (average relative abundances of 11.08 and 7.25%, respectively) in the negative control communities, almost disappeared in the other groups (Fig. 4 c). Compared to that in other gut communities, the relative abundance of Pseudomonas declined remarkably in the treatment group communities on day 7, whereas that of Haliscomenobacter increased significantly (Fig. 4 c). An analysis of similarity (ANOSIM) showed that different treatments significantly altered the gut bacterial community of zebrafish ( P = 0.023). According to hierarchical clustering at the genus level, the negative control group without the injection of E. piscicida maintained a similar gut bacterial community composition during the entire experimental period. The gut bacterial communities of the phage EPP-1 and florfenicol treatment groups clustered well with those of the positive control group ( E. p. + SM) at the early stages (days 2 and 4), whereas their gut bacterial communities clustered individually on the day 7, distantly from other communities (Fig. 4 c). PCoA and NMDS analyses at the OTU level also revealed a pattern of gut bacterial community composition similar to that of the hierarchical clustering analysis (Fig. 4 d and Fig. S2c). Overall, the gut microbiota of the negative control group remained nearly stable and was distinct from that of the other groups. In contrast, the bacterial community in the groups in which pathogens were injected changed dramatically at the end of the experiment (day 7), and interestingly, the phage and antibiotic treatment groups showed a convergent bacterial community shift. 3.5 Functional profiles of the microbial communities of the zebrafish gut Metagenomic sequencing was used to explore the gut microbial function profiles in different experimental groups, with 6.9 GB of clean data per fecal sample. In total, 1,513,523 non-redundant genes were predicted from 2,779,370 contigs assembled using MEGAHIT. The obtained genes were assigned to 12,142 KEGG orthologies and mainly classified into the following KEGG categories: Global and overview maps, Carbohydrate metabolism, and Amino acid metabolism (Fig. S3a). According to the CAZy classification, the affiliated genes ( n = 496) were mainly distributed among Glycosyl Transferases (GTs), Glycoside Hydrolases (GHs), Carbohydrate Esterases (CEs), and Auxiliary Activities (AAs) families (Fig. S3b). Among them, the RPKM abundances of GH- and AA-related genes were significantly different ( P = 0.012 and 0.048 for GHs and AAs, respectively) in the four different treatment groups, as revealed through ANOSIM. Specifically, for oxidase genes in the AA family, the abundances of AA7, AA1_1, AA1_3, AA5_2, and AA3_4 in the groups injected with E. piscicida were significantly higher than those in the control group, but the abundances of AA1, AA3_2, and AA1_2 were significantly lower (Fig. 5 a). Regarding the phosphorylase genes in the GH family, the abundance of GH94 was decreased by 96.73%, 95.90%, and 93.84% in the E. p. + SM, E. p. + FLO, and E. p. +EPP groups, respectively, compared to that in the PBS + SM group (Fig. 5 b). In addition, glycosidase abundance in the GH family also changed, with GH5_45 and GH43_29 increasing, whereas the others decreased, in the negative control group (Fig. 5 c). Lysozyme GH24 and hydrolase GH153 also increased in the PBS + SM group, but hydrolase GH88 decreased (Fig. S3c). Similar to the pattern of bacterial community dynamics, CAZy-based NMDS analysis revealed that the gut microbial function in the negative control group was different from that in the other groups; whereas the former showed little change, the latter (especially the phage and antibiotic therapy groups) showed large but similar changes on the day 7 (Fig. 5 d). 3.6 Phage EPP-1 treatment reduces floR gene content The most important prospect of phage therapy, as an alternative to antibiotics, is its ability to reduce antibiotic consumption and decrease the risk of antibiotic resistance. In total, 784 ARGs were identified from 12 gut microbial metagenomes, which were dominated by multidrug-, MLS-, glycopeptide-, tetracycline-, peptide-, and beta-lactam-related genes (Fig. S4). The average RPKM abundance of 181.37, 237.40, 225.41, and 198.15 for phenicol-like resistance genes were recognized from the metagenome sequences of the PBS + SM, E. p. + SM, E. p. + FLO, and E. p. + EPP-1 groups, respectively (Fig. 6 a). The RPKM abundance of phenicol-class ARGs in the E. p. + EPP-1 group was lower than that in the E. p. + SM and E. p. + FLO groups. The floR resistance gene belongs to the phenicol class and confers bacteria-specific resistance to florfenicol. Bioinformatics analysis identified an average RPKM values of 5.34, 30.22, 57.01, and 21.77 for floR -related gene in the metagenomic sequences of the PBS + SM, E. p. + SM, E. p. + FLO, and E. p. + EPP-1 groups, respectively (Fig. 6 b). Furthermore, qPCR was used to quantify floR gene contents in zebrafish excreta and aquaculture water. Results showed that the floR gene content in aquaculture water of the E. p. + FLO group increased from 1.04 × 10 7 GC/µg DNA on day 1 to 1.48 × 10 8 GC/µg DNA on day 7. In contrast, the gene content in the water of the E. p. + EPP-1 group was significantly lower than that of the E. p. + FLO group ( P = 0.0003, two-way ANOVA) and reached a level comparable to that in the PBS + SM group on day 7 (1.19 × 10 7 GC/µg DNA). In addition, the floR gene content in zebrafish feces from the E. p. +EPP group was significantly lower than that in the E. p. + FLO group ( P = 0.0127, two-way ANOVA; Fig. 6 c). In summary, our results indicate that phage EPP-1 reduces the production of phenicol-class and floR ARGs relative to that with florfenicol treatment. 4. Discussion The extensive use of antibiotics induces the residual of antibiotics, the generation of ARGs, and the emergence of ARB, which demonstrate significant health risks. Multiple ARGs and ARB have been detected in aquaculture wastewater and the relevant environments (Li et al., 2023a ; Zhu et al., 2023 ). Phages, as novel antibiotic alternatives, can reduce the antibiotic dosage at source and further alleviate a series of issues caused by antibiotic use. In this study, we have isolated an Edwardsiella phage named EPP-1, confirmed its feasibility for edwardsiellosis control in aquaculture, and assessed its impacts on the production of ARGs in relevant habitats. Relatively few Edwardsiella phages have been isolated, the whole genomes of only 14 phages have currently been released at the NCBI. To our knowledge, phage EPP-1 isolated in this study is the first Edwardsiella phage carrying the integrase gene and recombinant loci attR - attL sites required for a lysogenic lifecycle. However, EPP-1 is not lysogenic. Regardless, it still managed to lyse the host robustly, which could be related to the presence of genes encoding endolysin and Lambda Rz-like phage lysis proteins in the phage genome. Phage containing similar lysogenic genes but with lytic properties has also been isolated from the environment recently, however, studies of their practical application have not been conducted (Li et al., 2021 ). The isolation of this novel phage enriches the Edwardsiella phage bank and provides an alternative biomaterial for the biocontrol of edwardsiellosis. In this study, the phage EPP-1 treatment group showed an improved zebrafish survival rate, by 16.7%, relative to that in the challenge group, and this efficacy was comparable to that in the florfenicol therapy group ( P = 0.2304). Additionally, some studies have suggested that phage therapy can achieve results comparable to those with antibiotics (Chen et al., 2019 ) or other novel antibiotic alternatives, such as polymyxin B (Xu et al., 2021 ), for fish disease control. Xu and colleagues (Xu et al., 2022 ) obtained an enhanced outcome; a phage cocktail composed of two Edwardsiella phages increased the survival rates of zebrafish and turbot by 35%. Interestingly, phage EPP-1 was potent in suppressing host growth even at a low dose (MOI = 0.01) in vitro , but failed to reduce zebrafish mortality in vivo at the same dose. This could be related to complex biological and chemical substances in the zebrafish gut interfering with the phage titer and infection activity. Furthermore, as opposed to florfenicol, phage EPP-1 alleviated the dysregulation of antioxidant capacity and immune system dysfunction in the gut caused by E. piscicida infection. Therefore, we conclude that phage EPP-1 is feasible in the biocontrol of edwardsiellosis. The guts of normal animals inherently harbor numerous bacteria and phages in a dynamic balance, and theoretically, the introduction of exogenous phages does not affect the community structure of gut microbes beyond the host due to their strong specificity. Some studies support this hypothesis. The Aeromonas phage PZL-Ah152, used as a biocontrol agent, can reduce the colony numbers of pathogenic hosts, but does not affect the alpha and beta diversity of the crucian carp gut microbiota (Feng et al., 2022 ). An Acinetobacter -specific phage cocktail containing nine different phage lysates showed similar results in green turtles (Ahasan et al., 2019 ). In our study, survivors in the pathogen injection group (regardless of the presence of antibiotics or phage treatment) exhibited similar gut microbial community composition and function profiles. Treatment with neither phage EPP-1 nor the antibiotic florfenicol seemed to restore the intestinal microflora of the survivors to their original state within a short time. The changes in the intestinal microflora might be mainly attributed to pathogenic bacterial perturbations. The unique composition of the gut microbial community imparts distinctive functions to the microorganisms. The introduction of exogenous substances ( Edwardsiella in this study) can disrupt originally established homeostasis and drive multiple responses in bacteria, which in turn alter the functions of the bacteria that reside in the gut. Corresponding to the uniqueness of the gut microbial community composition, the CAZyme functions of the gut microbes in the groups injected with pathogens exhibited differences from those in the control groups. The precise regulation of gut microbial function is a promising direction for the future, and intervention through phage is potentially one of the important approaches. However, the precise regulation of gut microbial functions in the context of a complex gut is considerably challenging because of the following: first, the lytic spectrum of most isolated phages is relatively unclear and the gut could contain other potential hosts; second, the introduction of exogenous substances inevitably causes an immune response in the organism, affecting other bacterial populations; and finally, the cascading effects caused by the change in the abundance of one bacterium seem to be unavoidable, at least in several closely linked ecological niches. The dissemination of ARGs arising from the misuse of antibiotics is a major concern to the sustainable development of the aquaculture industry. Our metagenomic and qPCR-based results demonstrated that phage EPP-1 could significantly reduce the levels of floR genes in zebrafish excreta and associated environmental waters compared to those with florfenicol. This implies that phage for bacterial disease control in aquaculture is environmentally friendly. The major weakness of this study is that the efficacy was not as high as expected. The combination of phage with antibiotics or probiotics and the construction of phage cocktails to enhance therapeutic efficacy and reduce the use of antibiotics in a synergistic manner may be a promising path for phage therapy in aquaculture in the future. 5. Conclusions In this study, we isolated a novel Edwardsiella phage EPP-1 and employed it for the control of Edwardsiella piscicida infection in the zebrafish model. Our findings demonstrate that phage EPP-1 achieved therapeutic efficacy comparable to florfenicol, alleviating the dysregulation of antioxidant capacity and immune dysfunction in the zebrafish gut. Importantly, treatment with phage EPP-1 effectively reduced the content of the floR resistance gene in zebrafish excreta and aquaculture water. These results suggest that phage therapy holds promise as an effective antibiotic alternative for controlling E. piscicida infections in fish, with the potential to mitigate the dissemination of antibiotic resistance genes in aquaculture environments. Declarations Ethics in publishing All zebrafish experiments in this work were in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. This work has received approval for research ethics from the Institutional Animal Care at University of Chinese Academy of Sciences, where the experiment was conducted. All experiments were performed in accordance with ARRIVE guidelines ( https://arriveguidelines.org ) Author Contribution G.H.H. carried out the experiment, analyzed data and wrote the manuscript; T.H. administrated the project and carried out the experiment; X.C.L. supervised the project; R.Y.L. designed the experiment and reviewed of the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 42377120). References Abayneh, T., Colquhoun, D.J., Sorum, H., 2013. Edwardsiella piscicida sp. nov., a novel species pathogenic to fish. J. Appl. Microbiol. 114 (3), 644–654. Ahasan, M.S., Kinobe, R., Elliott, L., Owens, L., Scott, J., Picard, J., Huerlimann, R., Ariel, E., 2019. Bacteriophage versus antibiotic therapy on gut bacterial communities of juvenile green turtle, Chelonia mydas . Environ. Microbiol. 21 (8), 2871–2885. Alcock, B.P., Huynh, W., Chalil, R., Smith, K.W., Raphenya, A.R., Wlodarski, M.A., Edalatmand, A., Petkau, A., Syed, S.A., Tsang, K.K., Baker, S.J.C., Dave, M., McCarthy, M.C., Mukiri, K.M., Nasir, J.A., Golbon, B., Imtiaz, H., Jiang, X.J., Kaur, K., Kwong, M., Liang, Z.C., Niu, K.C., Shan, P., Yang, J.Y.J., Gray, K.L., Hoad, G.R., Jia, B.F., Bhando, T., Carfrae, L.A., Farha, M.A., French, S., Gordzevich, R., Rachwalski, K., Tu, M.M., Bordeleau, E., Dooley, D., Griffiths, E., Zubyk, H.L., Brown, E.D., Maguire, F., Beiko, R.G., Hsiao, W.W.L., Brinkman, F.S.L., Van Domselaar, G., McArthur, A.G., 2023. CARD 2023: Expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res. 51 (D1), D690-D699. Altamirano, F.L.G., Barr, J.J., 2019. Phage therapy in the postantibiotic era. Clin. Microbiol. Rev. 32 (2), e00066-18. Buchfink, B., Reuter, K., Drost, H.G., 2021. Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat. Methods. 18 (4), 366–368. Buján, N., Toranzo, A.E., Magariños, B., 2018. Edwardsiella piscicida : A significant bacterial pathogen of cultured fish. Dis. Aquat. Org. 131 (1), 59–71. Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Peña, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T., Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D., Muegge, B.D., Pirrung, M., Reeder, J., Sevinsky, J.R., Tumbaugh, P.J., Walters, W.A., Widmann, J., Yatsunenko, T., Zaneveld, J., Knight, R., 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods. 7 (5), 335–336. Chen, L., Fan, J.Q., Yan, T.W., Liu, Q., Yuan, S.J., Zhang, H.R., Yang, J.F., Deng, D., Huang, S.Q., Ma, Y.F., 2019. Isolation and characterization of specific phages to prepare a cocktail preventing Vibrio sp. Va-F3 Infections in Shrimp ( Litopenaeus vannamei ). Front. Microbiol. 10, 2337. Chen, S.F., Zhou, Y.Q., Chen, Y.R., Gu, J., 2018. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 34 (17), 884–890. Cui, H.J., Cong, C., Wang, L.L., Li, X.Y., Li, J.B., Yang, H., Li, S.Y., Xu, Y.P., 2022a. Control of Edwardsiella tarda infection in turbot Scophthalmus maximus (L.) using phage vB_EtaM_ET-ABTNL-9. Aquacult. Res. 53 (8), 3010–3024. Cui, H.J., Xu, Y.P., Cong, C., Li, C.X., Li, X.Y., Li, S.Y., Li, J.B., Wang, L.L., 2022b. Evaluation of the preventive effect of phage cocktails on turbot ascites and its influence on main physiological indicators. Aquaculture. 547, 737539. Culot, A., Grosset, N., Gautier, M., 2019. Overcoming the challenges of phage therapy for industrial aquaculture: A review. Aquaculture. 513, 734423. Domingo-Calap, P., Delgado-Martínez, J., 2018. Bacteriophages: Protagonists of a post-antibiotic era. Antibiotics-Basel. 7 (3), 66. Drula, E., Garron, M.L., Dogan, S., Lombard, V., Henrissat, B., Terrapon, N., 2022. The carbohydrate-active enzyme database: Functions and literature. Nucleic Acids Res. 50 (D1), D571-D577. Edgar, R.C., 2013. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods. 10 (10), 996–998. Feng, C., Jia, K.X., Chi, T., Chen, S.M., Yu, H.B., Zhang, L., Raza, S.H.A., Alshammari, A.M., Liang, S., Zhu, Z.S., Li, T.X., Qi, Y.L., Shan, X.F., Qian, A.D., Zhang, D.X., Zhang, L., Sun, W.W., 2022. Lytic bacteriophage PZL-Ah152 as biocontrol measures against lethal Aeromonas hydrophila without distorting gut microbiota. Front. Microbiol. 13, 898961. Fu, L.M., Niu, B.F., Zhu, Z.W., Wu, S.T., Li, W.Z., 2012. CD-HIT: Accelerated for clustering the next-generation sequencing data. Bioinformatics. 28 (23), 3150–3152. Gao, F.-Z., He, L.-Y., Bai, H., He, L.-X., Zhang, M., Chen, Z.-Y., Liu, Y.-S., Ying, G.-G., 2023. Airborne bacterial community and antibiotic resistome in the swine farming environment: Metagenomic insights into livestock relevance, pathogen hosts and public risks. Environ. Int. 172, 107751. Garneau, J.R., Depardieu, F., Fortier, L.C., Bikard, D., Monot, M., 2017. PhageTerm: A tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Sci. Rep. 7, 8292 Guo, L., Wang, G.C., Sheng, Y.Z., Shi, Z.M., Sun, X.Y., 2019. Groundwater microbial communities and their connection to hydrochemical environment in Golmud, Northwest China. Sci. Total Environ. 695, 133848. He, L.X., He, L.Y., Gao, F.Z., Zhang, M., Chen, J., Jia, W.L., Ye, P., Jia, Y.W., Hong, B., Liu, S.S., Liu, Y.S., Zhao, J.L., Ying, G.G., 2023. Mariculture affects antibiotic resistome and microbiome in the coastal environment. J. Hazard. Mater. 452, 131208. Hyatt, D., Chen, G.L., LoCascio, P.F., Land, M.L., Larimer, F.W., Hauser, L.J., 2010. Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 11, 119. Jin, X.X., Liu, S.Z., Zhang, Z.T., Liu, T., Li, N., Liang, Y.X., Zheng, J.S., Peng, N., 2023. Enrofloxacin-induced transfer of multiple-antibiotic resistance genes and emergence of novel resistant bacteria in red swamp crayfish guts and pond sediments. J. Hazard. Mater. 443 (B), 130261. Kayani, M.U.R., Yu, K., Qiu, Y.S., Shen, Y., Gao, C.X., Feng, R., Zeng, X.X., Wang, W.Y., Chen, L., Su, H.L., 2021. Environmental concentrations of antibiotics alter the zebrafish gut microbiome structure and potential functions. Environ. Pollut. 278, 116760. Kim, S.G., Jun, J.W., Giri, S.S., Yun, S., Kim, H.J., Kim, S.W., Kang, J.W., Han, S.J., Jeong, D., Park, S.C., 2019. Isolation and characterisation of pVa-21, a giant bacteriophage with anti-biofilm potential against Vibrio alginolyticus . Sci. Rep. 9, 6284. Lawson, C.E., Wu, S., Bhattacharjee, A.S., Hamilton, J.J., McMahon, K.D., Goel, R., Noguera, D.R., 2017. Metabolic network analysis reveals microbial community interactions in anammox granules. Nat. Commun. 8, 15416. Li, C.C., Wang, Z.M., Zhao, J.L., Wang, L., Xie, G.S., Huang, J., Zhang, Y.Y., 2021. A novel Vibriophage vB_VcaS_HC containing lysogeny-related gene has strong lytic ability against pathogenic bacteria. Virol. Sin. 36 (2), 281–290. Li, D.H., Liu, C.M., Luo, R.B., Sadakane, K., Lam, T.W., 2015. MEGAHIT: An ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 31 (10), 1674–1676. Li, J., Shao, B., Shen, J.Z., Wang, S.C., Wu, Y.N., 2013. Occurrence of chloramphenicol-resistance genes as environmental pollutants from swine feedlots. Environ. Sci. Technol. 47 (6), 2892–2897. Li, R.Q., Yu, C., Li, Y.R., Lam, T.W., Yiu, S.M., Kristiansen, K., Wang, J., 2009. SOAP2: An improved ultrafast tool for short read alignment. Bioinformatics. 25 (15), 1966–1967. Li, R.X., Wu, X.D., Han, Z.F., Xu, L.J., Gan, L., Zhang, Y.Q., Lu, F.R., Lin, H., Yang, X., Yan, M.T., Chu, W., Gong, H., 2023a. Removal of antibiotic-resistant bacteria and genes by Solar-activated Ferrate/ Peroxymonosulfate: Efficiency in aquaculture wastewater and mechanism. Chem. Eng. J. 474, 145547. Li, Z., Ru, S.G., Li, J.L., Yang, Y.J., Wang, W.W., 2023b. Continuous exposure to bisphenol S increases the accumulation of endogenous metabolic toxicants by obstructing the glucuronic acid pathway. Environ. Pollut. 336, 122433. Liu, R.Y., Han, G.H., Li, Z., Cun, S.J., Hao, B., Zhang, J.P., Liu, X.C., 2022. Bacteriophage therapy in aquaculture: Current status and future challenges. Folia Microbiol. 67 (4), 573–590. Luo, J.Y., Du, W., Chu, S.Q., Xu, Y.L., Zhang, Q., Zhang, L., Cheng, X.S., Huang, W.X., Cao, J.S., Su, Y.L., 2022. Effects of persulfate treatment on the fates of antibiotic resistance genes in waste activated sludge fermentation process and the underlying mechanism. Bioresour. Technol. 345, 126474. Magoc, T., Salzberg, S.L., 2011. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics. 27 (21), 2957–2963. Mokili, J.L., Rohwer, F., Dutilh, B.E., 2012. Metagenomics and future perspectives in virus discovery. Curr. Opin. Virol. 2 (1), 63–77. Nurk, S., Bankevich, A., Antipov, D., Gurevich, A.A., Korobeynikov, A., Lapidus, A., Prjibelski, A.D., Pyshkin, A., Sirotkin, A., Sirotkin, Y., Stepanauskas, R., Clingenpeel, S.R., Woyke, T., McLean, J.S., Lasken, R., Tesler, G., Alekseyev, M.A., Pevzner, P.A., 2013. Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J. Comput. Biol. 20 (10), 714–737. Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., Glöckner, F.O., 2013. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 41 (D1), D590-D596. Ren, Y., Yu, G., Shi, C., Liu, L., Guo, Q., Han, C., Zhang, D., Zhang, L., Liu, B., Gao, H.J.I., 2022. Majorbio Cloud: A one-stop, comprehensive bioinformatic platform for multiomics analyses. iMeta. 1 (2), e12. Ricarte, M., Prats, E., Montemurro, N., Bedrossiantz, J., Bellot, M., Gómez-Canela, C., Raldúa, D., 2023. Environmental concentrations of tire rubber-derived 6PPD-quinone alter CNS function in zebrafish larvae. Sci. Total Environ. 896, 165240. Sáenz, J.S., Marques, T.V., Barone, R.S.C., Cyrino, J.E.P., Kublik, S., Nesme, J., Schloter, M., Rath, S., Vestergaard, G., 2019. Oral administration of antibiotics increased the potential mobility of bacterial resistance genes in the gut of the fish Piaractus mesopotamicus . Microbiome. 7, 24. Shao, S., Zhang, Y., Yin, K.Y., Zhang, Y.X., Wei, L.F., Wang, Q.Y., 2022. FabR senses long-chain unsaturated fatty acids to control virulence in pathogen Edwardsiella piscicida . Mol. Microbiol. 117 (4), 737–753. Su, Z., Wen, D., Gu, A.Z., Zheng, Y., Tang, Y., Chen, L., 2023. Industrial effluents boosted antibiotic resistome risk in coastal environments. Environ. Int. 171, 107714. Thung, T.Y., Norshafawatie, B., Premarathne, J., Chang, W.S., Loo, Y.Y., Kuan, C.H., New, C.Y., Ubong, A., Ramzi, O.S.B., Mahyudin, N.A., Dayang, F.B., Jasimah, W., Son, R., 2017. Isolation of food-borne pathogen bacteriophages from retail food and environmental sewage. Int. Food Res. J. 24 (1), 450–454. Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73 (16), 5261–5267. Xu, Y., Zhu, L., Chen, S., Wu, H., Li, R., Li, J., Yuan, J., Wen, T., Xue, C., Shen, Q., 2023. Risk assessment and dissemination mechanism of antibiotic resistance genes in compost. Environ. Int. 178, 108126. Xu, Z.H., Jin, P., Zhou, X.S., Zhang, Y.X., Wang, Q.Y., Liu, X.H., Shao, S., Liu, Q., 2021. Isolation of a virulent Aeromonas salmonicida subsp. masoucida bacteriophage and its application in phage therapy in Turbot ( Scophthalmus maximus ). Appl. Environ. Microbiol. 87 (21), e01468-21. Xu, Z.H., Shao, S., Ding, Z.H., Zhang, Y.X., Wang, Q.Y., Liu, X.H., Liu, Q., 2022. Therapeutic efficacies of two newly isolated Edwardsiella phages against Edwardsiella piscicida infection. Microbiol. Res. 263, 127043. Zeng, Q.F., Liao, C., Terhune, J., Wang, L.X., 2019. Impacts of florfenicol on the microbiota landscape and resistome as revealed by metagenomic analysis. Microbiome 7 (1), 155. Zhu, M.J., Li, Y., Wang, L.Q., Zhang, W.L., Niu, L.H., Hu, T., 2023. Unraveling antibiotic resistomes associated with bacterial and viral communities in intertidal mudflat aquaculture area. J. Hazard. Mater. 459, 132087. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3844797","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":267644681,"identity":"c802400f-20be-4488-ab8e-a4ba4897bfb9","order_by":0,"name":"Ganghua Han","email":"","orcid":"","institution":"University of Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ganghua","middleName":"","lastName":"Han","suffix":""},{"id":267644682,"identity":"c9577f71-aa3a-4ed5-ba0a-f939d99fd21f","order_by":1,"name":"Ting Huang","email":"","orcid":"","institution":"University of Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Huang","suffix":""},{"id":267644683,"identity":"c4733088-068a-47b6-893e-8243f5c1d634","order_by":2,"name":"Xinchun Liu","email":"","orcid":"","institution":"University of Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xinchun","middleName":"","lastName":"Liu","suffix":""},{"id":267644684,"identity":"0871223c-20d1-44b2-ab86-d83c810171e0","order_by":3,"name":"Ruyin Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACAyA+8KFCQo4kLYwHZ5yxMCZJC/Nh3raKxAbitUifMTjMc0YifcPxw48/MNTYMfDPJqDbgC/H4OCcConcDWfSzCQYjiUzSNw5QEALD4/BgTdngFpuMJgxMLAdYDCQSCBCC2+bRLrBDfbPHxj+EanlIFBLgsENHgMJxjaitLAVAANZwnDmmZwyicS+ZB6JGwS02Pcwb/7woaJOnu/4cSDjm50c/wwCWhgYOAwQbKBiHkLqgYD9ARGKRsEoGAWjYEQDAIaMQk111gqVAAAAAElFTkSuQmCC","orcid":"","institution":"University of Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Ruyin","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-01-08 07:30:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3844797/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3844797/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49837411,"identity":"73d20bfe-afcc-4a9e-962a-f7e27d51f5e7","added_by":"auto","created_at":"2024-01-18 19:33:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7440207,"visible":true,"origin":"","legend":"\u003cp\u003eThe morphology, physiology, phylogenetic and genomic characteristics of phage EPP-1. (a) Clear plaques formed by phage EPP-1 on the double layer plate, and (b) the morphology of phage EPP-1 under TEM. (c) Temperature and (d) pH tolerance of phage EPP-1, shown as mean ± SD (n=3). (e) The circle genetic map of phage EPP-1. GC skew and GC content are shown in the inner circle, and the CDSs predicted are shown in the outer circle. The green pattern represents CDSs of known function and the brown pattern represents hypothetical proteins. (f) Phylogenetic tree of phage EPP-1 based on the \u003cem\u003eTerL\u003c/em\u003egene. (g) Heatmap of the ANI values calculated using 19 related phage genomes.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3844797/v1/4bfb6ff9b2c5b8d41a67b00b.png"},{"id":49837412,"identity":"b82b664f-a9a7-4486-8307-97db4678d2ad","added_by":"auto","created_at":"2024-01-18 19:33:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4183335,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the efficacy of phage EPP-1 and florfenicol treatments. (a) The bactericidal effects of phage EPP-1 under different MOIs \u003cem\u003ein vitro\u003c/em\u003e. (b) Experimental setup for\u003cem\u003e in vivo\u003c/em\u003ecomparison of phage EPP-1 and florfenicol treatments. (c) Clinical symptoms of zebrafish under different treatments on day 3. (d) Survival curves of zebrafish under different treatments within 7 days (n=30, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001, Mantel-Cox test). (e) The qPCR results of\u003cem\u003e Edwardsiella\u003c/em\u003e content in water and zebrafish feces.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3844797/v1/9c1eaa072c80d74043d4dc5d.png"},{"id":49837406,"identity":"0a467934-69f0-465c-a22a-ffadbf00c669","added_by":"auto","created_at":"2024-01-18 19:33:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1337773,"visible":true,"origin":"","legend":"\u003cp\u003eImpacts of different treatments on antioxidant capacity and inflammatory cytokines in the gut and liver of zebrafish. Bar plots of (a) SOD activity, (b) CAT activity, and (c) GSH content in the gut of zebrafish. (d) Heat map of SOD activity, CAT activity, and GSH content log\u003csub\u003e10\u003c/sub\u003e fold changes in the liver of zebrafish. Bar plot of (e) IL-1β, (f) IL-6, (g) TNF-α, and (h) IFN-γ content in the gut of zebrafish. (i) Heat map of IL-1β, IL-6, TNF-α, and IFN-γ content (log\u003csub\u003e10\u003c/sub\u003e−transformed) changes in the liver of zebrafish. One-way ANOVA with Sidak’s post-hoc test (a-c, e-h) was performed for significant difference test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. For the heat maps (d) and (i), the PBS+SM group served as the baseline for standardization, red representing an up-regulation of enzyme activities or inflammatory cytokines in the gut and liver, and blue representing a down-regulation.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3844797/v1/ee0e44190fc6eeba4f739e6a.png"},{"id":49837876,"identity":"3cd45687-69c6-4359-9425-3a8e6f95374e","added_by":"auto","created_at":"2024-01-18 19:49:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1678888,"visible":true,"origin":"","legend":"\u003cp\u003eProfiles of the zebrafish gut bacterial community based on 16S rRNA gene under different treatments. (a) Venn diagram of OTU numbers in the PBS + SM, \u003cem\u003eE. p. \u003c/em\u003e+ SM, \u003cem\u003eE. p. \u003c/em\u003e+ FLO, and \u003cem\u003eE. p. \u003c/em\u003e+ EPP treatment groups. The bar plot at the bottom shows the total number of OTUs in different groups. (b) Comparison of alpha diversity of gut bacterial communities in different treatment groups, including Sobs, ACE, and Chao 1 estimators. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, Student’s \u003cem\u003et\u003c/em\u003e-test. (c) Hierarchical clustering tree based on Bray-Curtis distances at the genus level. The genera with a relative abundance less than 5% are merged into others. (d) Principal co-ordinates analysis(PCoA) of the bacterial community of the PBS + SM, \u003cem\u003eE. p. \u003c/em\u003e+ SM, \u003cem\u003eE. p. \u003c/em\u003e+ FLO, and \u003cem\u003eE. p. \u003c/em\u003e+EPP treatment groups based on Bray-Curtis distances at the OTU level. The analysis of similarities (ANOSIM) was used for statistical testing. The CK, E_p_, FLO, and EPP in the legend refer to the PBS + SM, \u003cem\u003eE. p. \u003c/em\u003e+ SM, \u003cem\u003eE. p. \u003c/em\u003e+ FLO, and \u003cem\u003eE. p. \u003c/em\u003e+EPP groups, respectively.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3844797/v1/3fd5782dbcefdcb2dd6ddc63.png"},{"id":49837704,"identity":"d1e22a28-af03-4dae-8db1-7f9301eb0521","added_by":"auto","created_at":"2024-01-18 19:41:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":763752,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of (a) oxidase genes, (b) phosphorylase genes, and (c) glycosidase genes in four different treatment groups. (d) NMDS analysis based on the RPKM values of genes affiliated to CAZyme. The CK, E_p_, FLO, and EPP in the legend refer to the PBS + SM, \u003cem\u003eE. p. \u003c/em\u003e+ SM, \u003cem\u003eE. p. \u003c/em\u003e+ FLO, and \u003cem\u003eE. p. \u003c/em\u003e+EPP groups, respectively.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3844797/v1/6e310754f980fa7c13a62e35.png"},{"id":49837408,"identity":"248c4ac2-eccd-4fb7-8547-8148626c8e83","added_by":"auto","created_at":"2024-01-18 19:33:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":744524,"visible":true,"origin":"","legend":"\u003cp\u003eAntibiotic resistance gene (ARG) profiles of different treatment groups based on metagenomic sequencing and qPCR. (a) Average RPKM abundance of phenicol-class related ARGs in zebrafish fecal metagenomes from different treatment groups. (b) Average RPKM abundance of the \u003cem\u003efloR\u003c/em\u003e gene in zebrafish fecal metagenomes from different treatment groups. (c) qPCR results of \u003cem\u003efloR\u003c/em\u003e gene in zebrafish feces and feeding water on days 2, 4, and 7. The black line corresponds to \u003cem\u003efloR\u003c/em\u003egene content in water and the red one corresponds to \u003cem\u003efloR\u003c/em\u003e gene content in zebrafish feces.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3844797/v1/10e7114b2f827a8278092467.png"},{"id":49838360,"identity":"2d1ae8a3-46ef-4549-beec-2447993e6d00","added_by":"auto","created_at":"2024-01-18 19:57:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2462364,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3844797/v1/3a9ea825-a2f1-41ae-8d93-785a91c9cdbd.pdf"},{"id":49837410,"identity":"e4281aa0-957a-40ad-8d56-aa6e5e9a39a5","added_by":"auto","created_at":"2024-01-18 19:33:47","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":470080,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3844797/v1/38bb596c61397693fc92019f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Controlling edwardsiellosis caused by Edwardsiella piscicida and mitigating drug-resistant gene dissemination: Bacteriophage EPP-1, a promising antibiotic alternative","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eEdwardsiella piscicida\u003c/em\u003e is a notorious fish pathogen that infects its host mainly through the gut (Shao et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), leading to severe edwardsiellosis and causing substantial economic losses to the aquaculture industry worldwide (Abayneh et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Buj\u0026aacute;n et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The Food and Drug Administration (FDA) recommends a dose of 10‒15 mg florfenicol/kg fish for no more than 10 consecutive days to combat edwardsiellosis in aquaculture (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fda.gov/media/84229/download\u003c/span\u003e\u003cspan address=\"https://www.fda.gov/media/84229/download\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Recent studies, however, have unveiled that exposure to standard therapeutic doses of florfenicol alters the structure of the gut microbiota and increase the abundance of florfenicol-resistance genes in aquaculture systems (S\u0026aacute;enz et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zeng et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The use of other antibiotics like oxytetracycline, sulfamethoxazole, or enrofloxacin similarly induced rise of antibiotic resistance gene (ARG) levels or emergence of novel antibiotic resistant bacteria (ARB) across a wide range of habitats (Jin et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kayani et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These ARGs or ARB possess the potential to disseminate into the environment (He et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Su et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), thereby heightening their risks in global public health sector (Gao et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Luo et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Accordingly, the excessive use of antibiotics overshadows the sustainable development of the aquaculture industry, and particularly in the global context of \u0026ldquo;One Health,\u0026rdquo; novel antibacterial agents are urgently needed.\u003c/p\u003e \u003cp\u003eBacteriophages (phages), viruses that specifically infect bacteria, are ubiquitous and abundant worldwide, with an estimated total number exceeding 10\u003csup\u003e31\u003c/sup\u003e particles (Mokili et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Phage-based biocontrol methods, or phage therapy, have been reinvigorated with unprecedented momentum and are emerging as important strategies in the post-antibiotic era due to the fact that it can reduce the use of antibiotics at the source and alleviate a series of issues caused by the use of antibiotics (Altamirano and Barr \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Domingo-Calap and Delgado-Mart\u0026iacute;nez \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Phage therapy in the field of aquaculture is now being researched against \u003cem\u003eVibrio\u003c/em\u003e, \u003cem\u003eFlavobacterium\u003c/em\u003e, \u003cem\u003eAeromonas\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eLactococcus\u003c/em\u003e and has yielded favorable outcomes (Culot et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Available evidence suggests that administration of \u003cem\u003eE. tarda\u003c/em\u003e phage vB_EtaM_ET-ABTNL-9 by feeding, injection, or immersion significantly reduced mortality as well as nonspecific immune-related enzyme activities of turbot \u003cem\u003eScophthalmus maximus\u003c/em\u003e (Cui et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Furthermore, a phage cocktail composed of \u003cem\u003eE. tarda\u003c/em\u003e phage PETp9 and \u003cem\u003eV. harveyi\u003c/em\u003e phage PVHp5 significantly reduced host levels of pathogens and maintained the normal gut microbiota profile for ascites prevention in turbot (Cui et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). As a novel antibiotic alternative, the limitation of these studies on phage therapy in aquaculture is that they do not consider the production of ARGs, which is the most severe problem resulting from antibiotic use. Furthermore, there is a considerable dearth of research on phage therapy for the control of edwardsiellosis caused by \u003cem\u003eE. piscicida\u003c/em\u003e, its potential to substitute for antibiotics and its impacts on ARGs production in the control of edwardsiellosis is similarly unknown. Elucidation of these issues is a prerequisite for implementing phage therapy in aquaculture and addressing the associated antibiotic resistance issues.\u003c/p\u003e \u003cp\u003eZebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) is the most commonly used model species for environmental monitoring and toxicological evaluations (Li et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Ricarte et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, it was selected as the experimental subject in this study. Here, we isolated a novel \u003cem\u003eE. piscicida\u003c/em\u003e phage and characterized its physiological and genomic properties. We also investigated its bactericidal effect on \u003cem\u003eE. piscicida in vitro\u003c/em\u003e and its protective effect on zebrafish \u003cem\u003ein vivo\u003c/em\u003e. In addition, we evaluated the safety of phage therapy for edwardsiellosis control in zebrafish in terms of the antioxidant capacity and inflammatory cytokines in the gut and liver of the model species, the diversity and function of the gut microbial community, and the content of the \u003cem\u003efloR\u003c/em\u003e gene in fish excreta and environmental media. To the best of our knowledge, this is the first attempt to use a novel phage to control edwardsiellosis caused by \u003cem\u003eE. piscicida\u003c/em\u003e and to comprehensively evaluate its safety for aquaculture. This study thus provides novel insights into the biocontrol of edwardsiellosis in aquaculture and has profound implications for the development of antibiotic alternatives to address the antibiotic crisis.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bacterial strains and zebrafish culture\u003c/h2\u003e \u003cp\u003eBacteria for phage isolation and host spectrum determination (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were obtained from the Marine Culture Collection of China (MCCC) and cultivated in Trypticase Soy Broth medium (Solarbio, Beijing, China) at 30℃ with 120 rpm of shaking. Wild-type zebrafish (\u003cem\u003eD. rerio\u003c/em\u003e, AB line, 3‒4 cm, 0.3‒0.4 g) were purchased from Zhongke Water Quality Environmental Technology Co., Ltd. (Wuxi, Jiangsu, China), raised in a standard aquaculture system with a photoperiod of 14 h:10 h light/dark, and fed \u003cem\u003eArtemia salina\u003c/em\u003e twice daily. To avoid water quality deterioration and potential impacts on subsequent experiments, one-third of the aquaculture water was replaced daily with tap water that was aerated for 24 h, water quality (Table S2) was monitored every 2 days, and feces produced by zebrafish was collected thoroughly on days 2, 4, and 7 during the whole experiment. All zebrafish used in this study were acclimatized for at least seven days under the standard aquaculture system prior to the next step in the experiment, and the conditions were consistent throughout the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Phage isolation, characterization, and genome analysis\u003c/h2\u003e \u003cp\u003eThe double-layer agar method, as described by Thung (Thung et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), was used to isolate \u003cem\u003eEdwardsiella\u003c/em\u003e phages from aquaculture wastewater and natural surface water (Supplementary Information). After purification and proliferation, several characteristics of the isolated phages were characterized, including morphology via transmission electron microscopy (TEM), host spectrum, and temperature and pH tolerance (see Supplementary Information for details). Phage nucleic acids were extracted and purified, as described by Kim et al. (Supplementary Information) (Kim et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Purified phage DNA was randomly broken into 350 bp fragments and then linked to a specific adapter for library preparation using the standard NEBNext\u0026reg; Ultra\u0026trade; II DNA Library Preparation Kit for Illumina\u0026reg;. After checking the library, paired-end 2 \u0026times; 150 sequencing was performed on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA) at Fixgene Technology Co., Ltd. (Beijing, China). The raw sequencing data were subjected to quality control using fastp (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and then assembled using SPAdes v.3.12.0 (Nurk et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The resulting sequences were corrected using PhageTerm (Garneau et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Subsequent bioinformatic analysis is presented in the Supplementary Information.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Phage therapy versus antibiotic therapy for edwardsiellosis\u003c/h2\u003e \u003cp\u003eTo compare the efficacy of phage therapy with that of conventional antibiotic therapy for edwardsiellosis control in the zebrafish model, we set up four different treatment scenarios based on intraperitoneal injection, namely the PBS\u0026thinsp;+\u0026thinsp;SM group (negative control group, PBS buffer (137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.76 mM of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, pH\u0026thinsp;=\u0026thinsp;7.4)\u0026thinsp;+\u0026thinsp;SM buffer (200 mM of NaCl, 10 mM of MgSO\u003csub\u003e4\u003c/sub\u003e, 50 mM of Tris-HCl, pH\u0026thinsp;=\u0026thinsp;7.5)), \u003cem\u003eE. p.\u003c/em\u003e + SM group (positive control group, 10\u003csup\u003e5\u003c/sup\u003e CFU \u003cem\u003eE. piscicida\u003c/em\u003e (MCCC 1K00246)/fish, determined in the study), \u003cem\u003eE. p.\u003c/em\u003e + FLO group (florfenicol therapy group, 10\u003csup\u003e5\u003c/sup\u003e CFU \u003cem\u003eE. piscicida\u003c/em\u003e (MCCC 1K00246)/fish\u0026thinsp;+\u0026thinsp;10 mg florfenicol/kg fish weight), and \u003cem\u003eE. p.\u003c/em\u003e + EPP-1 group (phage therapy group, 10\u003csup\u003e5\u003c/sup\u003e CFU \u003cem\u003eE. piscicida\u003c/em\u003e (MCCC 1K00246)/fish\u0026thinsp;+\u0026thinsp;MOI of 1 for the phage EPP-1, determined in the study), with 30 zebrafish per tank. In the described above, the \u003cem\u003eE. piscicida\u003c/em\u003e was diluted with PBS, phage EPP-1 and florfenicol were diluted with SM buffer. The number of dead zebrafish was recorded daily for seven consecutive days to plot survival curves. The effects of the different treatments on the antioxidant capacity and levels of inflammatory cytokines in the gut and liver of zebrafish were evaluated. Finally, zebrafish feces and farmed water were collected on days 2, 4, and 7 for microbial-related analyses.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Oxidative stress and inflammatory cytokines in the gut and liver\u003c/h2\u003e \u003cp\u003eOn days 1, 3, and 7 after intraperitoneal injection, 6 zebrafish were randomly selected and divided into two groups with three biological replicates each, one group for the antioxidant capacity assay and the other group for the inflammatory cytokine assay. Briefly, the zebrafish were sacrificed to harvest the gut and liver samples. These samples were then weighed and prepared as 10% homogenates with saline (for antioxidant capacity determination) and PBS (for inflammatory cytokine determination) and centrifuged at 6000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min, and the supernatant was collected. Superoxide dismutase (SOD) and catalase (CAT) activity and reduced glutathione (GSH) content were determined using SOD, CAT, and GSH assay kits (Nanjing Jiancheng Bioeng Inst., Nanjing, China). Inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) were quantified using fish tissue-specific enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioeng Inst., Nanjing, China). Enzyme activity and inflammatory cytokine levels were determined according to the manufacturer\u0026rsquo;s instructions using a microplate reader (Synergy\u0026trade; H1; BioTek Inc., Winooski, VT, USA) at specific wavelengths. Each measurement was conducted in triple biological replication. T-test analysis was performed to examine the difference among different treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Nucleic acid extraction from water and zebrafish feces\u003c/h2\u003e \u003cp\u003eFeed water and zebrafish fecal samples were collected from different treatment groups for DNA extraction on days 2, 4, and 7 after intraperitoneal injection, that is, 3 water samples and 3 fecal samples for each treatment. Approximately 600 mL of water samples were filtered through 0.22 \u0026micro;m-pore-size membranes (Merck Millipore, Bedford, MA, USA) within 24 h, and the membranes were then cut with sterile scissors for DNA extraction. Fecal samples were aspirated from the bottom of the tank, centrifuged at 6000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min, washed three times with PBS, and weighed, after which they were ready for DNA extraction. Genomic DNA was extracted from water and fecal samples using the FastDNA\u0026trade; Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA), following the manufacturer\u0026rsquo;s instructions. Finally, the extracted DNA was fractionated and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Quantification of E. piscicida and the floR gene\u003c/h2\u003e \u003cp\u003eThe host pathogen \u003cem\u003eE. piscicida\u003c/em\u003e and the florfenicol-resistance gene \u003cem\u003efloR\u003c/em\u003e were measured in feeding water and zebrafish feces via quantitative polymerase chain reaction (qPCR) using previously reported available primers (\u003cem\u003egyrB\u003c/em\u003e and \u003cem\u003efloR\u003c/em\u003e genes for \u003cem\u003eE. piscicida\u003c/em\u003e and florfenicol-resistance gene quantification, respectively) and programs (Li et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), respectively. A standard curve was first plotted based on constructed plasmids with known content of target genes, and then the content of target genes in the samples was calculated based on this standard curve. The primer sequences, product sizes, and detailed qPCR programs are shown in Supplementary Information Table S3. Three replicates for each are used for quantification and the content of each gene is presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4 16S rRNA gene sequencing and analysis\u003c/h2\u003e \u003cp\u003eThe V3\u0026ndash;V4 hypervariable regions of the bacterial 16S rRNA gene were amplified via PCR using the barcoded primer pair 338F and 806R (Table S3). Amplicon sequencing was performed on an Illumina MiSeq platform (Illumina, Woburn, MA, USA) by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China), and sequencing data were analyzed using the online Majorbio Cloud Platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com/\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Ren et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, the raw paired sequences were subjected to quality control and merged using fastp v0.19.6 (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and FLASH v1.2.11 (Magoc and Salzberg \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The resulting tags were analyzed using QIIME 1.9.1 (Caporaso et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), following the steps reported by Guo \u003cem\u003eet al\u003c/em\u003e. (Guo et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The quality-filtered sequences were clustered into operational taxonomic units (OTUs) with a 97% identity cut-off using UPARSE v11 (Edgar \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and annotated based on taxonomy with the Silva (SSU138) 16S rRNA database as a reference, with a 70% classification confidence threshold (Quast et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), using the na\u0026iuml;ve Bayesian-based RDP Classifier v2.13 (Wang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Alpha diversity indices and Bray-Curtis distance-based beta diversity indices were calculated using QIIME 1.9.1 (Caporaso et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) analysis at the OTU level were performed using RStudio according to the Bray-Curtis distance matrix.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5 Metagenomic analysis of the gut microbial communities\u003c/h2\u003e \u003cp\u003eMetagenomic sequencing of 12 fecal samples was conducted on an Illumina HiSeq 4000 platform (Illumina, Woburn, MA, USA) with a 2 \u0026times; 150 bp paired-end sequencing strategy by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Adapters, sequences with a length\u0026thinsp;\u0026lt;\u0026thinsp;50 bp or an average quality score\u0026thinsp;\u0026lt;\u0026thinsp;20, or reads containing the \u0026ldquo;N\u0026rdquo; base were removed from raw reads using fastp v0.20.0 (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The resulting clean reads were assembled using the succinct de Bruijn graph method in Megahit v1.1.2 (Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Open reading frames (ORFs) were predicted from assembled contigs\u0026thinsp;\u0026gt;\u0026thinsp;100 bp using Prodigal v2.6.3 (Hyatt et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The predicted ORFs were clustered using CD-HIT v4.6.1 (Fu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) with 90% identity and 90% coverage, to construct a non-redundant gene set. The high-quality reads of each sample were then matched to the gene set using SOAPaligner v2.21 (Li et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), based on the Reads Per Kilobase per Million (RPKM) algorithm (Lawson et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), with 95% identity to determine the abundance of target genes. Gene function was annotated by aligning the sequences of the non-redundant gene set against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using DIAMOND v0.8.35 (Buchfink et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) with an E-value\u0026thinsp;\u0026le;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. Carbohydrate-active enzymes and antibiotic resistance genes were annotated, and their abundances were calculated based on the Carbohydrate-Active enZYmes Database (CAZy) (Drula et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and the Comprehensive Antibiotic Resistance Database (CARD) (Alcock et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), respectively. In addition, PCoA, NMDS, and Kruskal-Wallis H tests of gene function at multiple taxonomic levels were performed based on RPKM abundance using the Bray-Curtis distance matrix.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Statistical analysis\u003c/h2\u003e \u003cp\u003eStudent\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, One-way ANOVA with Sidak\u0026rsquo;s post-hoc test, and two-way ANOVA were performed in IBM SPSS v25.0. Survival analyses with Mantel-Cox test was performed in Graphpad Prism v9.0. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Stress value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 in NMDS indicates their good conformity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Data availability\u003c/h2\u003e \u003cp\u003eAll sequencing data have been deposited in the NCBI Sequence Read Archive under the BioProject ID PRJNA964478.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Isolation and characterization of phage EPP-1\u003c/h2\u003e \u003cp\u003eIn this study, an \u003cem\u003eEdwardsiella\u003c/em\u003e phage, named EPP-1, was isolated from aquaculture wastewater in Henan, China, and preserved at the China General Microbiological Culture Collection Center (CGMCC; preservation number, CGMCC No. 45078). The complete genome of the phage was uploaded to NCBI with GenBank accession number OQ910326. Phage EPP-1 lysed host \u003cem\u003eE. piscicida\u003c/em\u003e MCCC 1K00246 and formed clear plaques on a double-layer plate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). TEM-based morphology showed that phage EPP-1 has a head of 37 nm in diameter and a tail that is 103 nm in length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and belongs to the Myoviridae family in Caudovirales, according to the classification criteria of the International Committee on Taxonomy of Viruses. The host spectrum assay showed that this phage also lysed \u003cem\u003eE. piscicida\u003c/em\u003e MCCC 1K03230, \u003cem\u003eE. tarda\u003c/em\u003e MCCC 1K00241, \u003cem\u003eEdwardsiella\u003c/em\u003e sp. MCCC 1K00239, \u003cem\u003eEdwardsiella\u003c/em\u003e sp. MCCC 1K00240, and \u003cem\u003eEdwardsiella\u003c/em\u003e sp. MCCC 1K00242, indicating their broad-spectrum properties (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Phage EPP-1 was found to have strong tolerance to low and moderate temperatures; its titer began to decrease at temperatures above 50℃ and completely vanished at 80℃ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The phage was highly stable between pH 5 and 11, but its numbers decreased from 8.51 log\u003csub\u003e10\u003c/sub\u003e PFU/mL at pH 5 to 3.48 log\u003csub\u003e10\u003c/sub\u003e PFU/mL at pH 3, and it was completely inactivated under more acidic or alkaline conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to whole-genome sequencing, the genome of phage EPP-1 was structured as linear dsDNA, with a size of 38,641 bp and a GC content of 52.29%. PHASTER predicted 66 CDSs from the EPP-1 genome, of which 28 appeared to encode functional proteins, such as phage structural proteins, DNA replication-related enzymes, and lysis-related enzymes. Notably, lysogeny-related genes, such as the integrase and recombination loci \u003cem\u003eattL\u003c/em\u003e and \u003cem\u003eattR\u003c/em\u003e, were also predicted in the phage EPP-1 genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), suggesting that the virus might enter the lysogenic cycle in phage\u0026ndash;host interactions. No virulence factors, ARGs, or tRNA were identified in the genome of EPP-1. According to \u003cem\u003eTerL\u003c/em\u003e-based phylogenic analysis, phage EPP-1 was distantly related to other \u003cem\u003eEdwardsiella\u003c/em\u003e phages but closely related to the \u003cem\u003eYersinia\u003c/em\u003e phages YeP1, YeP2, and YeP3 and \u003cem\u003eSalmonella typhimurium\u003c/em\u003e phage ST64B (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). The average nucleotide identity (ANI) values for whole-genome comparisons were calculated using the same genome collections. The highest ANI value was 98.11% for phage EPP-1 with \u003cem\u003eEdwardsiella\u003c/em\u003e phage GF-2, which has a genome size of 43,129 bp, whereas the remaining ANI values were lower than 95% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), indicating the distinctiveness of phage EPP-1 isolated in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Phage EPP-1 treatment is comparable to florfenicol in efficacy\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e antibacterial effect of phage EPP-1 and its protective effect \u003cem\u003ein vivo\u003c/em\u003e in a zebrafish model were investigated to evaluate its feasibility for use in edwardsiellosis control in aquaculture. Phage EPP-1 showed strong antibacterial activity \u003cem\u003ein vitro\u003c/em\u003e, effectively inhibiting the growth of its host, making their OD\u003csub\u003e600\u003c/sub\u003e values lower than 0.2 even at a low MOI of 0.01, as a comparison, the group without phage addition had an OD\u003csub\u003e600\u003c/sub\u003e value of more than 0.6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). For \u003cem\u003ein vivo\u003c/em\u003e assays, 10\u003csup\u003e5\u003c/sup\u003e CFU/fish of \u003cem\u003eE. piscicida\u003c/em\u003e 1K00246 was used (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Given the complex components of the zebrafish gut, which contribute to a harsher condition than that used in the \u003cem\u003ein vitro\u003c/em\u003e assay, different doses (MOIs of 0, 0.1, 1, 5, and 10) of phage EPP-1 were administered to determine the optimal therapeutic dose. An MOI of 1 significantly improved the zebrafish survival compared to that in the challenge group (MOI\u0026thinsp;=\u0026thinsp;1 \u003cem\u003evs\u003c/em\u003e challenge group, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0008) and was comparable to that in the higher dose groups (MOI\u0026thinsp;=\u0026thinsp;1 \u003cem\u003evs\u003c/em\u003e MOI\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.6491; MOI\u0026thinsp;=\u0026thinsp;1 \u003cem\u003evs\u003c/em\u003e MOI\u0026thinsp;=\u0026thinsp;10, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1572) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb), and thus was therefore selected for further trials.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo compare the therapeutic efficacy of phage EPP-1 with that of the antibiotic florfenicol for edwardsiellosis control, four different treatment scenarios were established as follows: PBS\u0026thinsp;+\u0026thinsp;SM negative control group, \u003cem\u003eE. p.\u003c/em\u003e + SM positive control group, \u003cem\u003eE. p.\u003c/em\u003e + FLO treatment group, and \u003cem\u003eE. p.\u003c/em\u003e + EPP-1 treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Phage EPP-1 alleviated the signs of ascites in zebrafish caused by \u003cem\u003eE. piscicida\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Both treatment groups showed significantly decreased zebrafish mortality (\u003cem\u003eE. p.\u003c/em\u003e + SM \u003cem\u003evs E. p.\u003c/em\u003e + EPP-1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0035; \u003cem\u003eE. p.\u003c/em\u003e + SM \u003cem\u003evs E. p.\u003c/em\u003e + FLO, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and the efficacy of phage EPP-1 was comparable to that of conventional florfenicol (\u003cem\u003eE. p.\u003c/em\u003e + FLO \u003cem\u003evs E. p.\u003c/em\u003e + EPP-1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2304) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003ePathogenic \u003cem\u003eE. piscicida\u003c/em\u003e, introduced into the guts of zebrafish, can enter the environment via excrement. \u003cem\u003eEdwardsiella\u003c/em\u003e-specific qPCR showed that both EPP-1 and florfenicol therapy groups harbored substantial \u003cem\u003eEdwardsiella\u003c/em\u003e in their feeding water and zebrafish feces on day 2 (7.08 and 5.65 log\u003csub\u003e10\u003c/sub\u003e GC/L of feeding water in FLO and EPP-1 groups, and 8.65 and 7.38 log\u003csub\u003e10\u003c/sub\u003e GC/g wet feces in the FLO and EPP-1 groups, respectively), but decreased rapidly with feeding time, reaching levels comparable to those in the PBS\u0026thinsp;+\u0026thinsp;SM group (5.14 log\u003csub\u003e10\u003c/sub\u003e GC/L of feeding water and 4.26 log\u003csub\u003e10\u003c/sub\u003e GC/g wet feces in the PBS\u0026thinsp;+\u0026thinsp;SM group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). \u003cem\u003eEdwardsiella\u003c/em\u003e contents in zebrafish feces of the two therapy groups were comparable (4.41 and 4.47 log\u003csub\u003e10\u003c/sub\u003e GC/g wet feces in FLO and EPP-1 groups, respectively), which were both lower than that in the \u003cem\u003eE. p.\u003c/em\u003e + SM challenge group on day 7 (6.29 log\u003csub\u003e10\u003c/sub\u003e GC/g wet feces) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Phage EPP-1 treatment alleviates oxidative stress and immune responses in the zebrafish gut\u003c/h2\u003e \u003cp\u003eEnzyme activities and inflammatory cytokines in the gut and liver after different treatments were measured to further compare the effects of phage EPP-1 and florfenicol on the antioxidant capacity and immune response in zebrafish. The results showed that, compared to those with florfenicol treatment, phage EPP-1 could efficiently alleviate changes in the GSH content and CAT activity in the guts of fish infected with \u003cem\u003eE. piscicida\u003c/em\u003e, especially the changes in GSH on day 1 and CAT activity on day 7; however, the changes in SOD activity were not significantly altered within 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). In the liver, there were no significant changes in SOD and CAT activities among the different treatment groups within 7 days; however, the GSH content in the EPP-1 and florfenicol treatment groups decreased significantly on days 1 and 3 compared with that in the negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, we found that phage EPP-1 alleviated immune responses in different organs of zebrafish for a longer time than that in the florfenicol treatment. Specifically, the treatment effect of florfenicol was faster than that of EPP-1 in alleviating the gut immune response caused by \u003cem\u003eE. piscicida\u003c/em\u003e infection, as reflected by the changes in IL-1β, IL-6, TNF-α, and IFN-γ levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). In contrast, phage therapy performed better in the middle and later phases of treatment, especially in moderating the response of IL-1β, TNF-α, and IFN-γ on day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). The differences in IL-1β and IL-6 contents in the phage and florfenicol therapy groups became insignificant on day 7 over a prolonged post-injection period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f). It was also evident that phage EPP-1 moderated the upregulation of IL-6 levels in the liver compared to the effects of florfenicol (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Composition of bacterial community in the zebrafish gut\u003c/h2\u003e \u003cp\u003eIn total, 673,007 optimized sequences were generated through 16S rRNA gene sequencing. The rarefaction curve for each sample tended to become saturated, indicating a sufficient sequencing depth for community composition analysis (Fig. S2a). We identified 464 OTUs, of which 101 were shared by different experimental groups. The number of OTUs in the negative control group was approximately two-fold lower than that in the other groups injected with pathogenic \u003cem\u003eE. piscicida\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Furthermore, the richness (Sobs, Chao 1, and ACE estimators) of the gut bacterial community was significantly lower in the negative control group than in the other experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Proteobacteria and Bacteroidetes were consistently dominant in gut communities, whereas the relative abundance of Fusobacteria in the phage therapy group decreased from 20.89% on day 2 to 0.57% on day 7. Except for the negative control group, all experimental groups had a higher abundance (\u0026gt;\u0026thinsp;5%) of Actinobacteria on day 7 (Fig. S2b). At the genus level, \u003cem\u003eRheinheimera\u003c/em\u003e and \u003cem\u003ePaucibacter\u003c/em\u003e, the two dominant genera (average relative abundances of 11.08 and 7.25%, respectively) in the negative control communities, almost disappeared in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Compared to that in other gut communities, the relative abundance of \u003cem\u003ePseudomonas\u003c/em\u003e declined remarkably in the treatment group communities on day 7, whereas that of \u003cem\u003eHaliscomenobacter\u003c/em\u003e increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn analysis of similarity (ANOSIM) showed that different treatments significantly altered the gut bacterial community of zebrafish (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023). According to hierarchical clustering at the genus level, the negative control group without the injection of \u003cem\u003eE. piscicida\u003c/em\u003e maintained a similar gut bacterial community composition during the entire experimental period. The gut bacterial communities of the phage EPP-1 and florfenicol treatment groups clustered well with those of the positive control group (\u003cem\u003eE. p.\u003c/em\u003e + SM) at the early stages (days 2 and 4), whereas their gut bacterial communities clustered individually on the day 7, distantly from other communities (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). PCoA and NMDS analyses at the OTU level also revealed a pattern of gut bacterial community composition similar to that of the hierarchical clustering analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and Fig. S2c). Overall, the gut microbiota of the negative control group remained nearly stable and was distinct from that of the other groups. In contrast, the bacterial community in the groups in which pathogens were injected changed dramatically at the end of the experiment (day 7), and interestingly, the phage and antibiotic treatment groups showed a convergent bacterial community shift.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Functional profiles of the microbial communities of the zebrafish gut\u003c/h2\u003e \u003cp\u003eMetagenomic sequencing was used to explore the gut microbial function profiles in different experimental groups, with 6.9 GB of clean data per fecal sample. In total, 1,513,523 non-redundant genes were predicted from 2,779,370 contigs assembled using MEGAHIT. The obtained genes were assigned to 12,142 KEGG orthologies and mainly classified into the following KEGG categories: Global and overview maps, Carbohydrate metabolism, and Amino acid metabolism (Fig. S3a).\u003c/p\u003e \u003cp\u003eAccording to the CAZy classification, the affiliated genes (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;496) were mainly distributed among Glycosyl Transferases (GTs), Glycoside Hydrolases (GHs), Carbohydrate Esterases (CEs), and Auxiliary Activities (AAs) families (Fig. S3b). Among them, the RPKM abundances of GH- and AA-related genes were significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012 and 0.048 for GHs and AAs, respectively) in the four different treatment groups, as revealed through ANOSIM. Specifically, for oxidase genes in the AA family, the abundances of AA7, AA1_1, AA1_3, AA5_2, and AA3_4 in the groups injected with \u003cem\u003eE. piscicida\u003c/em\u003e were significantly higher than those in the control group, but the abundances of AA1, AA3_2, and AA1_2 were significantly lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Regarding the phosphorylase genes in the GH family, the abundance of GH94 was decreased by 96.73%, 95.90%, and 93.84% in the \u003cem\u003eE. p.\u003c/em\u003e + SM, \u003cem\u003eE. p.\u003c/em\u003e + FLO, and \u003cem\u003eE. p.\u003c/em\u003e +EPP groups, respectively, compared to that in the PBS\u0026thinsp;+\u0026thinsp;SM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In addition, glycosidase abundance in the GH family also changed, with GH5_45 and GH43_29 increasing, whereas the others decreased, in the negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Lysozyme GH24 and hydrolase GH153 also increased in the PBS\u0026thinsp;+\u0026thinsp;SM group, but hydrolase GH88 decreased (Fig. S3c). Similar to the pattern of bacterial community dynamics, CAZy-based NMDS analysis revealed that the gut microbial function in the negative control group was different from that in the other groups; whereas the former showed little change, the latter (especially the phage and antibiotic therapy groups) showed large but similar changes on the day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Phage EPP-1 treatment reduces \u003cem\u003efloR\u003c/em\u003e gene content\u003c/h2\u003e \u003cp\u003eThe most important prospect of phage therapy, as an alternative to antibiotics, is its ability to reduce antibiotic consumption and decrease the risk of antibiotic resistance. In total, 784 ARGs were identified from 12 gut microbial metagenomes, which were dominated by multidrug-, MLS-, glycopeptide-, tetracycline-, peptide-, and beta-lactam-related genes (Fig. S4). The average RPKM abundance of 181.37, 237.40, 225.41, and 198.15 for phenicol-like resistance genes were recognized from the metagenome sequences of the PBS\u0026thinsp;+\u0026thinsp;SM, \u003cem\u003eE. p.\u003c/em\u003e + SM, \u003cem\u003eE. p.\u003c/em\u003e + FLO, and \u003cem\u003eE. p.\u003c/em\u003e + EPP-1 groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The RPKM abundance of phenicol-class ARGs in the \u003cem\u003eE. p.\u003c/em\u003e + EPP-1 group was lower than that in the \u003cem\u003eE. p.\u003c/em\u003e + SM and \u003cem\u003eE. p.\u003c/em\u003e + FLO groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe \u003cem\u003efloR\u003c/em\u003e resistance gene belongs to the phenicol class and confers bacteria-specific resistance to florfenicol. Bioinformatics analysis identified an average RPKM values of 5.34, 30.22, 57.01, and 21.77 for \u003cem\u003efloR\u003c/em\u003e-related gene in the metagenomic sequences of the PBS\u0026thinsp;+\u0026thinsp;SM, \u003cem\u003eE. p.\u003c/em\u003e + SM, \u003cem\u003eE. p.\u003c/em\u003e + FLO, and \u003cem\u003eE. p.\u003c/em\u003e + EPP-1 groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Furthermore, qPCR was used to quantify \u003cem\u003efloR\u003c/em\u003e gene contents in zebrafish excreta and aquaculture water. Results showed that the \u003cem\u003efloR\u003c/em\u003e gene content in aquaculture water of the \u003cem\u003eE. p.\u003c/em\u003e + FLO group increased from 1.04 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e GC/\u0026micro;g DNA on day 1 to 1.48 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e GC/\u0026micro;g DNA on day 7. In contrast, the gene content in the water of the \u003cem\u003eE. p.\u003c/em\u003e + EPP-1 group was significantly lower than that of the \u003cem\u003eE. p.\u003c/em\u003e + FLO group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003, two-way ANOVA) and reached a level comparable to that in the PBS\u0026thinsp;+\u0026thinsp;SM group on day 7 (1.19 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e GC/\u0026micro;g DNA). In addition, the \u003cem\u003efloR\u003c/em\u003e gene content in zebrafish feces from the \u003cem\u003eE. p.\u003c/em\u003e +EPP group was significantly lower than that in the \u003cem\u003eE. p.\u003c/em\u003e + FLO group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0127, two-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). In summary, our results indicate that phage EPP-1 reduces the production of phenicol-class and \u003cem\u003efloR\u003c/em\u003e ARGs relative to that with florfenicol treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe extensive use of antibiotics induces the residual of antibiotics, the generation of ARGs, and the emergence of ARB, which demonstrate significant health risks. Multiple ARGs and ARB have been detected in aquaculture wastewater and the relevant environments (Li et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Phages, as novel antibiotic alternatives, can reduce the antibiotic dosage at source and further alleviate a series of issues caused by antibiotic use. In this study, we have isolated an \u003cem\u003eEdwardsiella\u003c/em\u003e phage named EPP-1, confirmed its feasibility for edwardsiellosis control in aquaculture, and assessed its impacts on the production of ARGs in relevant habitats.\u003c/p\u003e \u003cp\u003eRelatively few \u003cem\u003eEdwardsiella\u003c/em\u003e phages have been isolated, the whole genomes of only 14 phages have currently been released at the NCBI. To our knowledge, phage EPP-1 isolated in this study is the first \u003cem\u003eEdwardsiella\u003c/em\u003e phage carrying the integrase gene and recombinant loci \u003cem\u003eattR\u003c/em\u003e-\u003cem\u003eattL\u003c/em\u003e sites required for a lysogenic lifecycle. However, EPP-1 is not lysogenic. Regardless, it still managed to lyse the host robustly, which could be related to the presence of genes encoding endolysin and Lambda Rz-like phage lysis proteins in the phage genome. Phage containing similar lysogenic genes but with lytic properties has also been isolated from the environment recently, however, studies of their practical application have not been conducted (Li et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The isolation of this novel phage enriches the \u003cem\u003eEdwardsiella\u003c/em\u003e phage bank and provides an alternative biomaterial for the biocontrol of edwardsiellosis.\u003c/p\u003e \u003cp\u003eIn this study, the phage EPP-1 treatment group showed an improved zebrafish survival rate, by 16.7%, relative to that in the challenge group, and this efficacy was comparable to that in the florfenicol therapy group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2304). Additionally, some studies have suggested that phage therapy can achieve results comparable to those with antibiotics (Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or other novel antibiotic alternatives, such as polymyxin B (Xu et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), for fish disease control. Xu and colleagues (Xu et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) obtained an enhanced outcome; a phage cocktail composed of two \u003cem\u003eEdwardsiella\u003c/em\u003e phages increased the survival rates of zebrafish and turbot by 35%. Interestingly, phage EPP-1 was potent in suppressing host growth even at a low dose (MOI\u0026thinsp;=\u0026thinsp;0.01) \u003cem\u003ein vitro\u003c/em\u003e, but failed to reduce zebrafish mortality \u003cem\u003ein vivo\u003c/em\u003e at the same dose. This could be related to complex biological and chemical substances in the zebrafish gut interfering with the phage titer and infection activity. Furthermore, as opposed to florfenicol, phage EPP-1 alleviated the dysregulation of antioxidant capacity and immune system dysfunction in the gut caused by \u003cem\u003eE. piscicida\u003c/em\u003e infection. Therefore, we conclude that phage EPP-1 is feasible in the biocontrol of edwardsiellosis.\u003c/p\u003e \u003cp\u003eThe guts of normal animals inherently harbor numerous bacteria and phages in a dynamic balance, and theoretically, the introduction of exogenous phages does not affect the community structure of gut microbes beyond the host due to their strong specificity. Some studies support this hypothesis. The \u003cem\u003eAeromonas\u003c/em\u003e phage PZL-Ah152, used as a biocontrol agent, can reduce the colony numbers of pathogenic hosts, but does not affect the alpha and beta diversity of the crucian carp gut microbiota (Feng et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). An \u003cem\u003eAcinetobacter\u003c/em\u003e-specific phage cocktail containing nine different phage lysates showed similar results in green turtles (Ahasan et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In our study, survivors in the pathogen injection group (regardless of the presence of antibiotics or phage treatment) exhibited similar gut microbial community composition and function profiles. Treatment with neither phage EPP-1 nor the antibiotic florfenicol seemed to restore the intestinal microflora of the survivors to their original state within a short time. The changes in the intestinal microflora might be mainly attributed to pathogenic bacterial perturbations.\u003c/p\u003e \u003cp\u003eThe unique composition of the gut microbial community imparts distinctive functions to the microorganisms. The introduction of exogenous substances (\u003cem\u003eEdwardsiella\u003c/em\u003e in this study) can disrupt originally established homeostasis and drive multiple responses in bacteria, which in turn alter the functions of the bacteria that reside in the gut. Corresponding to the uniqueness of the gut microbial community composition, the CAZyme functions of the gut microbes in the groups injected with pathogens exhibited differences from those in the control groups. The precise regulation of gut microbial function is a promising direction for the future, and intervention through phage is potentially one of the important approaches. However, the precise regulation of gut microbial functions in the context of a complex gut is considerably challenging because of the following: first, the lytic spectrum of most isolated phages is relatively unclear and the gut could contain other potential hosts; second, the introduction of exogenous substances inevitably causes an immune response in the organism, affecting other bacterial populations; and finally, the cascading effects caused by the change in the abundance of one bacterium seem to be unavoidable, at least in several closely linked ecological niches.\u003c/p\u003e \u003cp\u003eThe dissemination of ARGs arising from the misuse of antibiotics is a major concern to the sustainable development of the aquaculture industry. Our metagenomic and qPCR-based results demonstrated that phage EPP-1 could significantly reduce the levels of \u003cem\u003efloR\u003c/em\u003e genes in zebrafish excreta and associated environmental waters compared to those with florfenicol. This implies that phage for bacterial disease control in aquaculture is environmentally friendly. The major weakness of this study is that the efficacy was not as high as expected. The combination of phage with antibiotics or probiotics and the construction of phage cocktails to enhance therapeutic efficacy and reduce the use of antibiotics in a synergistic manner may be a promising path for phage therapy in aquaculture in the future.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, we isolated a novel Edwardsiella phage EPP-1 and employed it for the control of \u003cem\u003eEdwardsiella piscicida\u003c/em\u003e infection in the zebrafish model. Our findings demonstrate that phage EPP-1 achieved therapeutic efficacy comparable to florfenicol, alleviating the dysregulation of antioxidant capacity and immune dysfunction in the zebrafish gut. Importantly, treatment with phage EPP-1 effectively reduced the content of the \u003cem\u003efloR\u003c/em\u003e resistance gene in zebrafish excreta and aquaculture water. These results suggest that phage therapy holds promise as an effective antibiotic alternative for controlling \u003cem\u003eE. piscicida\u003c/em\u003e infections in fish, with the potential to mitigate the dissemination of antibiotic resistance genes in aquaculture environments.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003cb\u003eEthics in publishing\u003c/b\u003e \u003c/p\u003e \u003cp\u003e All zebrafish experiments in this work were in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. This work has received approval for research ethics from the Institutional Animal Care at University of Chinese Academy of Sciences, where the experiment was conducted. All experiments were performed in accordance with ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eG.H.H. carried out the experiment, analyzed data and wrote the manuscript; T.H. administrated the project and carried out the experiment; X.C.L. supervised the project; R.Y.L. designed the experiment and reviewed of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 42377120).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbayneh, T., Colquhoun, D.J., Sorum, H., 2013. \u003cem\u003eEdwardsiella piscicida\u003c/em\u003e sp. nov., a novel species pathogenic to fish. J. Appl. Microbiol. 114 (3), 644\u0026ndash;654.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhasan, M.S., Kinobe, R., Elliott, L., Owens, L., Scott, J., Picard, J., Huerlimann, R., Ariel, E., 2019. Bacteriophage versus antibiotic therapy on gut bacterial communities of juvenile green turtle, \u003cem\u003eChelonia mydas\u003c/em\u003e. Environ. Microbiol. 21 (8), 2871\u0026ndash;2885.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlcock, B.P., Huynh, W., Chalil, R., Smith, K.W., Raphenya, A.R., Wlodarski, M.A., Edalatmand, A., Petkau, A., Syed, S.A., Tsang, K.K., Baker, S.J.C., Dave, M., McCarthy, M.C., Mukiri, K.M., Nasir, J.A., Golbon, B., Imtiaz, H., Jiang, X.J., Kaur, K., Kwong, M., Liang, Z.C., Niu, K.C., Shan, P., Yang, J.Y.J., Gray, K.L., Hoad, G.R., Jia, B.F., Bhando, T., Carfrae, L.A., Farha, M.A., French, S., Gordzevich, R., Rachwalski, K., Tu, M.M., Bordeleau, E., Dooley, D., Griffiths, E., Zubyk, H.L., Brown, E.D., Maguire, F., Beiko, R.G., Hsiao, W.W.L., Brinkman, F.S.L., Van Domselaar, G., McArthur, A.G., 2023. CARD 2023: Expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res. 51 (D1), D690-D699.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltamirano, F.L.G., Barr, J.J., 2019. Phage therapy in the postantibiotic era. Clin. Microbiol. Rev. 32 (2), e00066-18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuchfink, B., Reuter, K., Drost, H.G., 2021. Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat. Methods. 18 (4), 366\u0026ndash;368.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuj\u0026aacute;n, N., Toranzo, A.E., Magari\u0026ntilde;os, B., 2018. \u003cem\u003eEdwardsiella piscicida\u003c/em\u003e: A significant bacterial pathogen of cultured fish. Dis. Aquat. Org. 131 (1), 59\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Pe\u0026ntilde;a, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T., Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D., Muegge, B.D., Pirrung, M., Reeder, J., Sevinsky, J.R., Tumbaugh, P.J., Walters, W.A., Widmann, J., Yatsunenko, T., Zaneveld, J., Knight, R., 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods. 7 (5), 335\u0026ndash;336.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, L., Fan, J.Q., Yan, T.W., Liu, Q., Yuan, S.J., Zhang, H.R., Yang, J.F., Deng, D., Huang, S.Q., Ma, Y.F., 2019. Isolation and characterization of specific phages to prepare a cocktail preventing \u003cem\u003eVibrio\u003c/em\u003e sp. \u003cem\u003eVa-F3\u003c/em\u003e Infections in Shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e). Front. Microbiol. 10, 2337.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, S.F., Zhou, Y.Q., Chen, Y.R., Gu, J., 2018. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 34 (17), 884\u0026ndash;890.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui, H.J., Cong, C., Wang, L.L., Li, X.Y., Li, J.B., Yang, H., Li, S.Y., Xu, Y.P., 2022a. Control of \u003cem\u003eEdwardsiella tarda\u003c/em\u003e infection in turbot \u003cem\u003eScophthalmus maximus\u003c/em\u003e (L.) using phage vB_EtaM_ET-ABTNL-9. Aquacult. Res. 53 (8), 3010\u0026ndash;3024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui, H.J., Xu, Y.P., Cong, C., Li, C.X., Li, X.Y., Li, S.Y., Li, J.B., Wang, L.L., 2022b. Evaluation of the preventive effect of phage cocktails on turbot ascites and its influence on main physiological indicators. Aquaculture. 547, 737539.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCulot, A., Grosset, N., Gautier, M., 2019. Overcoming the challenges of phage therapy for industrial aquaculture: A review. Aquaculture. 513, 734423.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDomingo-Calap, P., Delgado-Mart\u0026iacute;nez, J., 2018. Bacteriophages: Protagonists of a post-antibiotic era. Antibiotics-Basel. 7 (3), 66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrula, E., Garron, M.L., Dogan, S., Lombard, V., Henrissat, B., Terrapon, N., 2022. The carbohydrate-active enzyme database: Functions and literature. Nucleic Acids Res. 50 (D1), D571-D577.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdgar, R.C., 2013. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods. 10 (10), 996\u0026ndash;998.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng, C., Jia, K.X., Chi, T., Chen, S.M., Yu, H.B., Zhang, L., Raza, S.H.A., Alshammari, A.M., Liang, S., Zhu, Z.S., Li, T.X., Qi, Y.L., Shan, X.F., Qian, A.D., Zhang, D.X., Zhang, L., Sun, W.W., 2022. Lytic bacteriophage PZL-Ah152 as biocontrol measures against lethal \u003cem\u003eAeromonas hydrophila\u003c/em\u003e without distorting gut microbiota. Front. Microbiol. 13, 898961.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu, L.M., Niu, B.F., Zhu, Z.W., Wu, S.T., Li, W.Z., 2012. CD-HIT: Accelerated for clustering the next-generation sequencing data. Bioinformatics. 28 (23), 3150\u0026ndash;3152.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao, F.-Z., He, L.-Y., Bai, H., He, L.-X., Zhang, M., Chen, Z.-Y., Liu, Y.-S., Ying, G.-G., 2023. Airborne bacterial community and antibiotic resistome in the swine farming environment: Metagenomic insights into livestock relevance, pathogen hosts and public risks. Environ. Int. 172, 107751.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarneau, J.R., Depardieu, F., Fortier, L.C., Bikard, D., Monot, M., 2017. PhageTerm: A tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Sci. Rep. 7, 8292\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo, L., Wang, G.C., Sheng, Y.Z., Shi, Z.M., Sun, X.Y., 2019. Groundwater microbial communities and their connection to hydrochemical environment in Golmud, Northwest China. Sci. Total Environ. 695, 133848.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe, L.X., He, L.Y., Gao, F.Z., Zhang, M., Chen, J., Jia, W.L., Ye, P., Jia, Y.W., Hong, B., Liu, S.S., Liu, Y.S., Zhao, J.L., Ying, G.G., 2023. Mariculture affects antibiotic resistome and microbiome in the coastal environment. J. Hazard. Mater. 452, 131208.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHyatt, D., Chen, G.L., LoCascio, P.F., Land, M.L., Larimer, F.W., Hauser, L.J., 2010. Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 11, 119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin, X.X., Liu, S.Z., Zhang, Z.T., Liu, T., Li, N., Liang, Y.X., Zheng, J.S., Peng, N., 2023. Enrofloxacin-induced transfer of multiple-antibiotic resistance genes and emergence of novel resistant bacteria in red swamp crayfish guts and pond sediments. J. Hazard. Mater. 443 (B), 130261.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKayani, M.U.R., Yu, K., Qiu, Y.S., Shen, Y., Gao, C.X., Feng, R., Zeng, X.X., Wang, W.Y., Chen, L., Su, H.L., 2021. Environmental concentrations of antibiotics alter the zebrafish gut microbiome structure and potential functions. Environ. Pollut. 278, 116760.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, S.G., Jun, J.W., Giri, S.S., Yun, S., Kim, H.J., Kim, S.W., Kang, J.W., Han, S.J., Jeong, D., Park, S.C., 2019. Isolation and characterisation of pVa-21, a giant bacteriophage with anti-biofilm potential against \u003cem\u003eVibrio alginolyticus\u003c/em\u003e. Sci. Rep. 9, 6284.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawson, C.E., Wu, S., Bhattacharjee, A.S., Hamilton, J.J., McMahon, K.D., Goel, R., Noguera, D.R., 2017. Metabolic network analysis reveals microbial community interactions in anammox granules. Nat. Commun. 8, 15416.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, C.C., Wang, Z.M., Zhao, J.L., Wang, L., Xie, G.S., Huang, J., Zhang, Y.Y., 2021. A novel Vibriophage vB_VcaS_HC containing lysogeny-related gene has strong lytic ability against pathogenic bacteria. Virol. Sin. 36 (2), 281\u0026ndash;290.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, D.H., Liu, C.M., Luo, R.B., Sadakane, K., Lam, T.W., 2015. MEGAHIT: An ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 31 (10), 1674\u0026ndash;1676.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, J., Shao, B., Shen, J.Z., Wang, S.C., Wu, Y.N., 2013. Occurrence of chloramphenicol-resistance genes as environmental pollutants from swine feedlots. Environ. Sci. Technol. 47 (6), 2892\u0026ndash;2897.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, R.Q., Yu, C., Li, Y.R., Lam, T.W., Yiu, S.M., Kristiansen, K., Wang, J., 2009. SOAP2: An improved ultrafast tool for short read alignment. Bioinformatics. 25 (15), 1966\u0026ndash;1967.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, R.X., Wu, X.D., Han, Z.F., Xu, L.J., Gan, L., Zhang, Y.Q., Lu, F.R., Lin, H., Yang, X., Yan, M.T., Chu, W., Gong, H., 2023a. Removal of antibiotic-resistant bacteria and genes by Solar-activated Ferrate/ Peroxymonosulfate: Efficiency in aquaculture wastewater and mechanism. Chem. Eng. J. 474, 145547.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Z., Ru, S.G., Li, J.L., Yang, Y.J., Wang, W.W., 2023b. Continuous exposure to bisphenol S increases the accumulation of endogenous metabolic toxicants by obstructing the glucuronic acid pathway. Environ. Pollut. 336, 122433.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, R.Y., Han, G.H., Li, Z., Cun, S.J., Hao, B., Zhang, J.P., Liu, X.C., 2022. Bacteriophage therapy in aquaculture: Current status and future challenges. Folia Microbiol. 67 (4), 573\u0026ndash;590.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo, J.Y., Du, W., Chu, S.Q., Xu, Y.L., Zhang, Q., Zhang, L., Cheng, X.S., Huang, W.X., Cao, J.S., Su, Y.L., 2022. Effects of persulfate treatment on the fates of antibiotic resistance genes in waste activated sludge fermentation process and the underlying mechanism. Bioresour. Technol. 345, 126474.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagoc, T., Salzberg, S.L., 2011. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics. 27 (21), 2957\u0026ndash;2963.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMokili, J.L., Rohwer, F., Dutilh, B.E., 2012. Metagenomics and future perspectives in virus discovery. Curr. Opin. Virol. 2 (1), 63\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNurk, S., Bankevich, A., Antipov, D., Gurevich, A.A., Korobeynikov, A., Lapidus, A., Prjibelski, A.D., Pyshkin, A., Sirotkin, A., Sirotkin, Y., Stepanauskas, R., Clingenpeel, S.R., Woyke, T., McLean, J.S., Lasken, R., Tesler, G., Alekseyev, M.A., Pevzner, P.A., 2013. Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J. Comput. Biol. 20 (10), 714\u0026ndash;737.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., Gl\u0026ouml;ckner, F.O., 2013. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 41 (D1), D590-D596.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen, Y., Yu, G., Shi, C., Liu, L., Guo, Q., Han, C., Zhang, D., Zhang, L., Liu, B., Gao, H.J.I., 2022. Majorbio Cloud: A one-stop, comprehensive bioinformatic platform for multiomics analyses. iMeta. 1 (2), e12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicarte, M., Prats, E., Montemurro, N., Bedrossiantz, J., Bellot, M., G\u0026oacute;mez-Canela, C., Rald\u0026uacute;a, D., 2023. Environmental concentrations of tire rubber-derived 6PPD-quinone alter CNS function in zebrafish larvae. Sci. Total Environ. 896, 165240.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026aacute;enz, J.S., Marques, T.V., Barone, R.S.C., Cyrino, J.E.P., Kublik, S., Nesme, J., Schloter, M., Rath, S., Vestergaard, G., 2019. Oral administration of antibiotics increased the potential mobility of bacterial resistance genes in the gut of the fish \u003cem\u003ePiaractus mesopotamicus\u003c/em\u003e. Microbiome. 7, 24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao, S., Zhang, Y., Yin, K.Y., Zhang, Y.X., Wei, L.F., Wang, Q.Y., 2022. FabR senses long-chain unsaturated fatty acids to control virulence in pathogen \u003cem\u003eEdwardsiella piscicida\u003c/em\u003e. Mol. Microbiol. 117 (4), 737\u0026ndash;753.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu, Z., Wen, D., Gu, A.Z., Zheng, Y., Tang, Y., Chen, L., 2023. Industrial effluents boosted antibiotic resistome risk in coastal environments. Environ. Int. 171, 107714.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThung, T.Y., Norshafawatie, B., Premarathne, J., Chang, W.S., Loo, Y.Y., Kuan, C.H., New, C.Y., Ubong, A., Ramzi, O.S.B., Mahyudin, N.A., Dayang, F.B., Jasimah, W., Son, R., 2017. Isolation of food-borne pathogen bacteriophages from retail food and environmental sewage. Int. Food Res. J. 24 (1), 450\u0026ndash;454.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73 (16), 5261\u0026ndash;5267.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, Y., Zhu, L., Chen, S., Wu, H., Li, R., Li, J., Yuan, J., Wen, T., Xue, C., Shen, Q., 2023. Risk assessment and dissemination mechanism of antibiotic resistance genes in compost. Environ. Int. 178, 108126.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, Z.H., Jin, P., Zhou, X.S., Zhang, Y.X., Wang, Q.Y., Liu, X.H., Shao, S., Liu, Q., 2021. Isolation of a virulent \u003cem\u003eAeromonas salmonicida\u003c/em\u003e subsp. \u003cem\u003emasoucida\u003c/em\u003e bacteriophage and its application in phage therapy in Turbot (\u003cem\u003eScophthalmus maximus\u003c/em\u003e). Appl. Environ. Microbiol. 87 (21), e01468-21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, Z.H., Shao, S., Ding, Z.H., Zhang, Y.X., Wang, Q.Y., Liu, X.H., Liu, Q., 2022. Therapeutic efficacies of two newly isolated \u003cem\u003eEdwardsiella\u003c/em\u003e phages against \u003cem\u003eEdwardsiella piscicida\u003c/em\u003e infection. Microbiol. Res. 263, 127043.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng, Q.F., Liao, C., Terhune, J., Wang, L.X., 2019. Impacts of florfenicol on the microbiota landscape and resistome as revealed by metagenomic analysis. Microbiome 7 (1), 155.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, M.J., Li, Y., Wang, L.Q., Zhang, W.L., Niu, L.H., Hu, T., 2023. Unraveling antibiotic resistomes associated with bacterial and viral communities in intertidal mudflat aquaculture area. J. Hazard. Mater. 459, 132087.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Phage therapy, Drug-resistant gene, Edwardsiella piscicida, Edwardsiellosis, Antibiotic alternative","lastPublishedDoi":"10.21203/rs.3.rs-3844797/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3844797/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eEdwardsiella piscicida\u003c/em\u003e causes significant economic losses to the aquaculture industry worldwide. Phage-based biocontrol methods are experiencing a renaissance because of the spread of drug-resistant genes and bacteria resulting from the heavy use of antibiotics. Here, we showed that the novel \u003cem\u003eEdwardsiella\u003c/em\u003e phage EPP-1 could achieve comparable efficacy to florfenicol using a zebrafish model of \u003cem\u003eEdwardsiella piscicida\u003c/em\u003e infection and could reduce the content of the \u003cem\u003efloR\u003c/em\u003e resistance gene in zebrafish excreta. Specifically, phage EPP-1 inhibited bacterial growth \u003cem\u003ein vitro\u003c/em\u003e and significantly improved the zebrafish survival rate \u003cem\u003ein vivo\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0035), achieving an efficacy comparable to that of florfenicol (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2304). Notably, integrating the results of 16S rRNA sequencing, metagenomic sequencing, and qPCR, although the effects of phage EPP-1 converged with those of florfenicol in terms of the community composition and potential function of the zebrafish gut microbiota, it reduced the \u003cem\u003efloR\u003c/em\u003e gene content in zebrafish excreta and aquaculture water. Overall, our study highlights the feasibility and safety of phage therapy for edwardsiellosis control, which has profound implications for the development of antibiotic alternatives to address the antibiotic crisis.\u003c/p\u003e","manuscriptTitle":"Controlling edwardsiellosis caused by Edwardsiella piscicida and mitigating drug-resistant gene dissemination: Bacteriophage EPP-1, a promising antibiotic alternative","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 19:33:42","doi":"10.21203/rs.3.rs-3844797/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-13T07:16:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-26T00:27:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65a27ecf-2646-4b2a-bcf3-f61b3dc78b7e","date":"2024-01-17T04:35:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-17T02:41:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-17T00:20:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-16T11:00:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-16T10:49:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-08T07:29:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"35d52b42-e7da-4f10-814b-4e5273e1b008","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":28200551,"name":"Biological sciences/Biotechnology"},{"id":28200552,"name":"Biological sciences/Microbiology"},{"id":28200553,"name":"Earth and environmental sciences/Ecology"},{"id":28200554,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2024-04-19T11:03:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-18 19:33:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3844797","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3844797","identity":"rs-3844797","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}