Crocodile-derived lactobacillus salivarius and application thereof

Abstract

The application belongs to the technical field of microorganisms, and provides crocodile-derived lactobacillus salivarius and application thereof. The crocodile-derived lactobacillus salivarius is named lactobacillus salivarius H7 Ligilactobacillus salivarius H7, which was preserved in China Center for Type Culture Collection (CCTCC) on January 30, 2026, and the address of the preservation center is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2026301. The lactobacillus salivarius H7 has in-vivo and in-vitro antibacterial activity of preventing infection of zebrafish model organisms of aeromonas dhakini, and has a good application prospect in aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to Lactobacillus salivarius of crocodile origin and its applications. Background Technology

[0002] In aquatic animal farming, diseases caused by Gram-negative bacteria have the highest incidence. Among them, Aeromonas dacarbazina (… Aeromonas dhakensis ) and Aeromonas hydrophila ( ) and its genus Aeromonas hydrophila ( A. hydrophila Compared to other strains, this bacterium exhibits significantly higher virulence. It can cause severe septicemia in infected species, with a mortality rate as high as 90%, and typical clinical symptoms include edema, hemorrhage, and ulceration syndrome. Currently, the standard practice in aquaculture to combat this type of bacterial infection is to spray antibiotics onto the feed surface before administration. For example, in the United States, the main antibiotic approved by the FDA for use against this highly virulent strain in aquatic animals is florfenicol. However, research indicates that six genes are known to mediate bacterial resistance to florfenicol, and at least three of these resistance genes have been detected in Aeromonas. This situation underscores the urgent need to develop novel antibiotic alternatives. Summary of the Invention

[0003] Based on the above, the purpose of this invention is to provide crocodile-derived Lactobacillus salivarius and its application, which is a type of Lactobacillus salivarius that can antagonize Aeromonas dacarinae, an important pathogenic bacterium in water, isolated from crocodile feces, and can be used in aquatic animal farming.

[0004] The technical solution adopted in this invention is as follows:

[0005] This invention provides a crocodile-derived Lactobacillus salivarius, named Lactobacillus salivarius H7. Ligilactobacillus salivarius H7 was deposited on January 30, 2026 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026301.

[0006] Furthermore, the total genome length of the Lactobacillus salivarius H7 is 1,886,039 bp, and the average GC content is 32.64%.

[0007] Furthermore, the genome of the Lactobacillus salivarius H7 contains ribosome synthesis and post-translational modified peptide analog (RiPP-like) gene clusters, as well as type III polyketide synthase (T3 PKS) and terpene-precursor related gene clusters.

[0008] Furthermore, the whole genome of *Lactobacillus salivarius* H7 contains six families of carbohydrate-active enzymes (CAZymes): helper enzymes (AA), carbohydrate binding modules (CBM), carbohydrate esterases (CE), glycoside hydrolases (GH), glycosyltransferases (GT), and polysaccharide lyases (PL).

[0009] Furthermore, the *Lactobacillus salivarius* H7 is sensitive to the following antibiotics, including norfloxacin, polymyxin B, minocycline, trimethoprim-sulfamethoxazole, ciprofloxacin, chloramphenicol, and tobramycin.

[0010] The present invention also provides a probiotic agent for aquatic animals, including the above-mentioned crocodile-derived Lactobacillus salivarius.

[0011] The present invention also provides the application of the above-mentioned crocodile-derived Lactobacillus salivarius in aquatic animal farming.

[0012] Furthermore, the application includes the use of Lactobacillus salivarius H7 as an antagonistic bacterium against pathogenic bacteria in aquatic animals.

[0013] Furthermore, the aquatic animal pathogens include Aeromonas dacarba.

[0014] Furthermore, the pathogenic bacterium of the aquatic animals is Aeromonas dacarbazina C160501.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention isolates *Lactobacillus salivarius* H7, an antagonistic bacterium against *Aeromonas dacarina*, an important pathogenic bacterium in aquatic animals, from crocodile feces at a crocodile farm in Dongfang City, Hainan Province. The isolate demonstrates good efficacy, and further verification confirms that *Lactobacillus salivarius* H7 possesses in vivo and in vitro antibacterial activity against *Aeromonas dacarina* infection, reducing mortality in zebrafish, a model organism infected with *Aeromonas dacarina*. This invention shows promising application prospects in aquatic animal husbandry. Attached Figure Description

[0017] Figure 1 The growth curve of Lactobacillus salivarius H7 is shown.

[0018] Figure 2 The results are for the hemolytic activity assay of Lactobacillus salivarius H7.

[0019] Figure 3 Phylogenetic tree of 16S rRNA of Lactobacillus salivarius H7.

[0020] Figure 4 This is the complete genome map of Lactobacillus salivarius H7.

[0021] Figure 5The results are predicted for potential antagonistic-related secondary metabolite biosynthesis gene clusters in the genome of Lactobacillus salivarius H7.

[0022] Figure 6 The results of annotation analysis of carbohydrate-active enzyme-related genes in the genome of Lactobacillus salivarius H7.

[0023] Figure 7 The results show the effect of antagonizing the growth of Aeromonas dacarba by co-culturing Lactobacillus salivarius H7 in vitro.

[0024] Figure 8 This study investigated the inhibitory effect of Lactobacillus salivarius H7 on the adhesion of Aeromonas daca to Caco-2 cells.

[0025] Figure 9 To illustrate the inhibitory effect of Lactobacillus salivarius H7 on Aeromonas daca biofilm formation, Figure a shows the crystal violet staining results; Figure b shows the AO fluorescence microscopy observation results; and Figure c shows the SBF quantitative analysis results.

[0026] Figure 10 Design and survival rate analysis of an in vivo protective assay against Aeromonas dacarba in zebrafish infected with Lactobacillus salivarius H7.

[0027] Figure 11 In vivo fluorescence imaging (Figure a) and quantitative analysis (Figure b) of zebrafish infected with Aeromonas daca after gavage pretreatment with Lactobacillus salivarius H7.

[0028] Figure 12 Effect of Lactobacillus salivarius H7 pretreatment via gavage on the relative abundance of gut microbiota genera in zebrafish infected with Aeromonas daca. Detailed Implementation

[0029] To more clearly illustrate the present invention, the invention will be further described in detail below with reference to embodiments and accompanying drawings. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0030] Lactobacillus salivarius H7 Ligilactobacillus salivarius H7 was deposited on January 30, 2026 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCCNO: M 2026301.

[0031] Because it originates from crocodile feces, it is also known as crocodile-derived Lactobacillus salivarius.

[0032] Example

[0033] I. Isolation and Identification of Lactobacillus salivarius H7

[0034] A 50 g sample of crocodile feces was collected from a crocodile farm in Dongfang Crocodile Town, Dongfang City, Hainan Province, China. This sample was added to an Erlenmeyer flask containing 200 mL of MRS liquid culture medium and shaken at 180 r / min for 2 hours on a constant-temperature shaker to thoroughly mix and prepare a bacterial suspension. The MRS liquid culture medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., product model HB0384, and was prepared according to the instructions and then autoclaved at 121℃ for 15 min before use. Subsequently, 2 mL of the bacterial suspension was inoculated into an Erlenmeyer flask containing 100 mL of MRS liquid culture medium and incubated at 37℃ at 180 r / min for 2 days.

[0035] Transfer 1 mL of the enrichment culture medium to a sterile test tube and perform 10-fold serial dilutions using sterile physiological saline. Select a dilution of 10... -3 and 10 -4 100 µL of each bacterial suspension was evenly spread onto MRS solid agar plates. The plates were incubated upside down in a 37°C incubator for 24–48 h. After incubation, single colonies with distinct morphological characteristics were observed and selected. These colonies were then purified by streaking on fresh medium 2–3 times to obtain single colonies of uniform purity for identification.

[0036] The growth curve of Lactobacillus salivarius is shown in the figure. Figure 1 It can be clearly observed that this strain is in the adaptation phase (lag phase) for the first 2-4 hours after inoculation, with relatively slow proliferation; subsequently, it rapidly enters the logarithmic growth phase, with a significant increase in absorbance (OD). 600nm The OD value rises sharply, indicating rapid and massive proliferation of the bacteria; after approximately 8 hours of continuous culture, the strain enters a plateau phase of growth. 600nm The value no longer increased significantly and remained at a stable high level. The results indicate that *Lactobacillus salivarius* H7 has strong growth vitality and a short generation time, and can reach maximum biomass in a relatively short period of time.

[0037] II. Hemolytic activity

[0038] Two equal aliquots (5 μL) of purified *Lactobacillus salivarius* strains were incubated on Columbia agar plates containing 5% sterile defibrinated sheep blood. The diameter and color of the halo indicating hemolytic activity were then recorded. A clear transparent zone around the colony (β-hemolysis) indicated positive hemolytic activity, while a green area (α-hemolysis) or no clear area (γ-hemolysis) indicated negative hemolytic activity. Each potential probiotic strain was tested twice using a triplet assay.

[0039] Hemolytic activity test results as follows Figure 2As shown in the figure, compared to the pathogen (Aeromonas dacarina) within the white box that produced a clear transparent hemolytic zone, Lactobacillus salivarius H7 within the black box grew normally on blood agar plates, and its colonies did not show a clear transparent hemolytic zone or a green area, exhibiting typical non-hemolytic characteristics (i.e., γ-hemolysis). This result clearly indicates that Lactobacillus salivarius H7 has negative hemolytic activity, confirming its potential as a safe strain for safe application as a microecological preparation in aquatic animal husbandry.

[0040] III. Drug Sensitivity Testing

[0041] The Kirby–Bauer disk diffusion method was used to assess the susceptibility of *Lactobacillus salivarius* H7 to seven antibiotics. The tested antibiotics were norfloxacin, polymyxin B, minocycline, trimethoprim-sulfamethoxazole, ciprofloxacin, chloramphenicol, and tobramycin. In short, 200 μL of bacterial suspension was evenly spread on the surface of MRS solid medium. After the medium surface had slightly dried, antibiotic susceptibility testing disks were attached, and the mixture was incubated at 37°C for 24 h. After incubation, the diameter of the inhibition zone was measured, and the susceptibility of the strain to each antibiotic was evaluated according to the judgment criteria. All experiments were performed in triplicate, with each replicate independently twice. The results are shown in Table 1.

[0042] Table 1. Analysis of Drug Susceptibility Test Results

[0043]

[0044] Note: R indicates drug resistance; I indicates moderate sensitivity; S indicates sensitivity.

[0045] Lactobacillus salivarius H7 is sensitive to protein synthesis inhibitors (minocycline, chloramphenicol, and tobramycin), fluoroquinolones (norfloxacin and ciprofloxacin), antibiotics that affect folic acid metabolism (trimethoprim-sulfamethoxazole), and cell membrane-acting antibiotics (polymyxin B), which ensures its good safety during later feed preparation.

[0046] IV. Bacterial framework diagram and resequencing

[0047] Genomic DNA was isolated from fresh bacterial cultures using a DNA extraction kit (FastPure® Bacteria DNA Isolation Mini Kit, Novizan, catalog number DC103). Quantification was performed using a Nanodrop spectrophotometer (Thermo Fisher Scientific).

[0048] Bacterial framework diagram: After the DNA sample is received, it is tested, and then a library is constructed using the qualified samples: First, the DNA sample is randomly fragmented to produce DNA fragments of the required length. The sticky ends formed by fragmentation are repaired into blunt ends, and then an "A" base is added to the 3' end so that the DNA fragment can be ligated to a special adapter with a "T" base at the 3' end. Finally, PCR technology is used to amplify the DNA fragments with adapters at both ends, thus completing the entire library construction. The constructed qualified libraries are then subjected to cluster preparation and sequencing.

[0049] Bacterial resequencing: (1) After the DNA sample is received, the sample is tested. If the DNA length, total amount and purity meet the requirements for library construction, subsequent experiments are carried out; (2) DNA samples that pass the electrophoresis test are randomly broken into fragments of about 350 bp in length using a Covaris ultrasonic disruptor. After the DNA fragments are processed, the library is prepared by end repair, A-tailing, sequencing adapter addition, purification, PCR amplification and other steps; (3) After the library is constructed, the library is first initially quantified using Qubit2.0 and diluted to 2 ng / ffL. Then, the insert size of the library is detected using Agilent 2100. After the fragments meet the expectations, the effective concentration of the library is accurately quantified using Q-PCR to ensure the quality of the library; (4) After the library is qualified, different libraries are mixed in proportion according to the effective concentration and the target amount of data to be sequenced and then sequenced using Illumina Novaseq 6000.

[0050] Figure 3 Based on the 16S phylogenetic tree of the genomic data, it is clear that the sequenced strain is more closely related to *Lactobacillus salivarius*. Ligilactobacillus salivarius ).

[0051] Mean nucleic acid similarity (ANI) is one of the most powerful measures for determining the closeness of genomic relationships in bacteria. It reflects the evolutionary distance of the genome based on the average of comparisons of all orthologous protein sequences in the genome quality check. When ANI > 95%, it indicates that the two genomes belong to the same species. The ANI analysis results of the genome and the reference genome are shown in Table 2. The results indicate that the detected strain is *Lactobacillus salivarius*.

[0052] Table 2. ANI analysis results of the genome and the reference genome

[0053]

[0054] Figure 4This is the complete genome map of Lactobacillus salivarius H7. Whole-genome sequencing results show that the total genome length of Lactobacillus salivarius H7 is 1,886,039 bp, with an average GC content of 32.64%; further prediction indicates that the genome encodes a total of 1,858 protein-coding genes.

[0055] like Figure 5 As shown, antiSMASH analysis of the Lactobacillus salivarius H7 genome detected two potential secondary metabolism-related regions. One region on H7_ctg011 was predicted to be a ribosome synthesis and post-translational modification peptide analog gene cluster (RIPP-like cluster), suggesting that this strain may have the potential to synthesize RIPP-like bioactive peptides or bacteriocin-like molecules. The other region on H7_ctg001 was annotated as a type III polyketide synthase (T3PKS) and terpene-precursor-related gene cluster, suggesting that H7 may possess the metabolic potential related to polyketides or isoprene precursors, but the types of its metabolites and their specific contributions to antagonistic effects remain unclear.

[0056] like Figure 6 As shown, a total of 79 carbohydrate-active enzymes (CAZymes) were identified in the whole genome of *Lactobacillus salivarius* H7, covering 6 functional families, including helper enzymes (AA, 2), carbohydrate binding modules (CBM, 13), carbohydrate esterases (CE, 8), glycoside hydrolases (GH, 15), glycosyltransferases (GT, 40), and polysaccharide lyases (PL, 1). Among them, GT is the dominant family, followed by GH and CBM, indicating that this strain has strong potential for glycosyltransfer and carbohydrate metabolism.

[0057] V. Preparation of cell-free supernatant (CFS) and bacterial culture of Lactobacillus salivarius H7

[0058] Preparation of sterile fermentation supernatant (CFS): *Lactobacillus salivarius* H7 was cultured in MRS medium at 37°C and 180 r / min for 18 h. The cells were collected by centrifugation at 4°C and 6000 r / min for 15 min. The cells were washed three times with sterile 0.1 mol / L phosphate buffer, resuspended in PBS, and the bacterial concentration was adjusted to 1 × 10⁻⁶. 9 CFU / mL. The supernatant was filtered through a 0.22 μm aqueous microfiltration membrane to obtain CFS and stored at -80 °C.

[0059] Preparation of bacterial culture: Lactobacillus salivarius H7 was cultured in MRS medium at 37°C and 180 r / min for 18 h. The cells were collected by centrifugation at 4°C and 6000 r / min for 15 min. The cells were washed three times with sterile 0.1 mol / L phosphate buffer, resuspended in PBS, and the bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL.

[0060] VI. In vitro co-culture of Lactobacillus salivarius H7 antagonizes the growth of Aeromonas dacarba.

[0061] The Aeromonas dacarba used in this application is the wild-type Aeromonas dacarba C160501 strain, isolated from farmed Siamese crocodiles in Hainan Province, and described in the literature (Pu,W., G.Guo, N.Yang, Q.Li, F.Yin, P.Wang, J.Zheng, and J.Zeng. "Three Species of Aeromonas (A. Dhakensis, A. Hydrophila and A. Jandaei) Isolated from Fresh water Crocodiles (Crocodylus Siamensis) with Pneumonia and Septicemia." Lett Appl Microbiol 68, no.3 (2019):212 18.), which was isolated and preserved by our laboratory.

[0062] To evaluate the inhibitory effect of *Lactobacillus salivarius* H7 on the growth of *Aeromonas daca*, two groups were established: a *Aeromonas daca* culture-only group (single culture group) and a *Lactobacillus salivarius* and *Aeromonas daca* co-culture group (co-culture group). In the single culture group, a freshly cultured *Aeromonas daca* C160501 suspension (20 μL, 1×10⁻⁶ cells / mL) was cultured. 9 CFU / mL) was inoculated into 200 mL of MRS / LB (MRS and LB mixed volume ratio of 1:1) broth and cultured at 37℃ for 48 h; in the co-culture group, 20 μL of Lactobacillus salivarius H7 suspension (3×10) was inoculated into the broth. 5 CFU / mL) and Aeromonas dacca C160501 bacterial suspension (20 μL, 1×10⁻⁶ CFU / mL) 9 (CFU / mL) were co-inoculated into 200 mL of MRS / LB (MRS and LB volume ratio 1:1) mixed broth and incubated at 37℃ for 48 h. Samples were taken at 0, 8, 16, 24, 32, 40, and 48 h to determine the viable count of Aeromonas dacarbazin, and expressed as log... 10 (CFU / mL) indicates the quantity.

[0063] The results are as follows Figure 7 As shown, *Lactobacillus salivarius* H7 significantly inhibited the growth of *Aeromonas dacarba* C160501. In the single-culture group, the pathogenic bacterial load rapidly peaked after 4 h and remained at approximately 8.7-9.1 log₂O₅. 10 (CFU / mL); while in the co-culture group, the bacterial count continued to decrease after 4 h, and dropped to approximately 2.4 log at 48 h. 10 (CFU / mL), indicating that H7 has a significant and time-dependent antagonistic effect on Aeromonas dacca.

[0064] VII. Inhibitory effect of Lactobacillus salivarius H7 on Aeromonas dacarbamate adhesion to Caco-2 cells

[0065] To evaluate the effect of *Lactobacillus salivarius* H7 on the adhesion of *Aeromonas dacarina* to host intestinal epithelial cells, an adhesion inhibition experiment was conducted using a Caco-2 cell monolayer model. Caco-2 cells were seeded in 12-well plates and cultured at 37°C and 5% CO2 until a dense monolayer formed. The experiment included a control group, a competition group, an exclusion group, and a displacement group. Before the experiment, *Aeromonas dacarina* C160501 and *Lactobacillus salivarius* H7 were cultured to the logarithmic growth phase, centrifuged, washed, and resuspended in antibiotic-free DMEM medium. The bacterial concentration of *Aeromonas dacarina* C160501 was adjusted to 1 × 10⁻⁶. 7 CFU / mL, Lactobacillus salivarius H7 bacterial concentration is 1 × 10⁻⁶. 7 CFU / mL. The total volume of bacterial suspension added to each well was 1 mL. In the control group, 1 mL of Aeromonas dacca C160501 suspension was added to each well and incubated at 37 ℃ for 2 h. In the competition group, 500 μL of Lactobacillus salivarius H7 suspension and 500 μL of Aeromonas dacca C160501 suspension were added to each well and incubated together for 2 h. In the rejection group, 1 mL of Lactobacillus salivarius H7 suspension was added to each well and incubated for 2 h. After gentle washing with PBS, 1 mL of Aeromonas dacca C160501 suspension was added to each well and incubated for another 2 h. In the replacement group, 1 mL of Aeromonas dacca C160501 suspension was added to each well and incubated for 2 h. After gentle washing with PBS, 1 mL of Lactobacillus salivarius H7 suspension was added to each well and incubated for another 2 h. After incubation, the cells were gently washed three times with PBS to remove unadhered cells. 0.1% Triton X-100 was added to lyse the cells, and the adherent bacterial solution was collected. After serial dilution, the solution was plated on LB plates for counting. The adhesion inhibition rate was calculated with the control group as the baseline.

[0066] The results showed that *Lactobacillus salivarius* H7 significantly inhibited the adhesion of *Aeromonas daca* to Caco-2 cells. In the control group, *Aeromonas daca* exhibited strong adhesion to the surface of Caco-2 cells; however, after the addition of H7, as shown in the previous results... Figure 8 As shown, the number of pathogens adhering to the experimental groups (competition group, rejection group, and replacement group) was significantly reduced. Specifically, the adhesion inhibition rates in the competition group, rejection group, and replacement group were 61.6% ± 2.7%, 45.5% ± 6.0%, and 34.3% ± 2.5%, respectively, corresponding to (1.97 ± 0.09) × 10⁻⁶ pathogens. 6 (1.46 ± 0.19) × 10 6 and (1.10 ± 0.08) × 10 6 CFU / mL. The adhesion inhibition rate was calculated using the formula: (Adhesion rate of *Aeromonas daca* to Caco-2 cells - Adhesion rate of experimental group cells) / Adhesion rate of *Aeromonas daca* to Caco-2 cells. The results showed that *Lactobacillus salivarius* H7 significantly inhibited the adhesion of *Aeromonas daca*, with displacement being the strongest, followed by repulsion, and competition being relatively weak. This suggests that H7 can reduce the colonization of *Aeromonas daca* on the host cell surface through multiple mechanisms, including competing for binding sites, preferentially colonizing to form a biological barrier, and interfering with already adhered pathogens.

[0067] 8. Inhibitory effect of Lactobacillus salivarius H7 on biofilm formation of Aeromonas dacca

[0068] To evaluate the inhibitory effect of *Lactobacillus salivarius* H7 on biofilm formation in *Aeromonas daca*, *Aeromonas daca* was treated with different concentrations of H7 cell-free supernatant (CFS), and the results were observed using crystal violet staining, acridine orange (AO) fluorescence staining, and quantitative analysis of specific biofilm formation (SBF). *Aeromonas daca* C160501 bacterial suspension was inoculated into culture plates, with control, 25% CFS treatment, 50% CFS treatment, and 75% CFS treatment groups. The plates were incubated at 37 ℃ for 24 h to induce biofilm formation. After incubation, some samples were stained with crystal violet to observe the overall biofilm formation; other samples were gently washed with PBS to remove airborne bacteria, stained with AO, and the distribution and density of the biofilm were observed under a fluorescence microscope. The biofilm formation capacity was then quantitatively evaluated using SBF values.

[0069] The results showed that cell-free supernatant of *Lactobacillus salivarius* H7 significantly inhibited biofilm formation of *Aeromonas dacarba*. Figure 9 The crystal violet staining results in Figure a show that the control group appeared deep blue-purple, indicating that *Aeromonas dacarina* had formed a relatively dense biofilm. However, after treatment with 25% CFS, 50% CFS, and 75% CFS, the staining gradually lightened. Figure 9 The AO fluorescence microscopy observation results in Figure b show that the control group exhibited a large area of ​​continuous and strong green fluorescence signal, indicating the formation of a structurally complete and densely distributed biofilm on the well plate surface. However, after treatment with 25% CFS, 50% CFS, and 75% CFS, the green fluorescence signal significantly weakened, the biofilm coverage area gradually decreased, and the structure became looser and more fragmented. The 75% CFS group showed the weakest fluorescence, with only a few scattered fluorescent spots. Figure 9 The quantitative analysis of SBF in Figure c showed that, consistent with microscopic observations, the SBF values ​​of all treatment groups were significantly lower than those of the control group, and gradually decreased with increasing CFS concentration, indicating that H7-CFS has a significant concentration-dependent inhibitory effect on Aeromonas dacarbamate biofilm formation. These results demonstrate that the metabolites secreted by Lactobacillus salivarius H7 can effectively interfere with the surface attachment, cell aggregation, and biofilm establishment processes of Aeromonas dacarbamate, thereby weakening its colonization ability and potential pathogenicity.

[0070] 9. Antagonism of Aeromonas dacarba by Lactobacillus salivarius H7 in zebrafish

[0071] In the in vivo challenge protection experiment, healthy AB strain zebrafish were randomly divided into 5 groups: a PBS group, an injection-only group, and groups receiving Lactobacillus salivarius H7 via gavage for 1, 2, and 3 weeks before challenge (H7-1 week, H7-2 week, and H7-3 week groups). Twenty zebrafish were used in each group for survival rate statistics, in vivo imaging analysis, and intestinal sample collection. All zebrafish were fasted for 12 hours before the experiment. From day 0, the PBS group and the injection-only group received an equal volume of sterile buffer via gavage daily, while the H7-1 week, H7-2 week, and H7-3 week groups received a live Lactobacillus salivarius H7 suspension via gavage daily at a dose of 1×10⁻⁶. 8 CFU / tail, administered via gavage for 7, 14, and 21 days, respectively. After completing the corresponding pretreatment time, only the injection group and each H7 pretreatment group received intraperitoneal injection of Aeromonas dacarbazina C160501 bacterial suspension, with a challenge dose of 1×10⁻⁶. 8 CFU / tail; the PBS group was injected with an equal volume of sterile PBS. After challenge, the survival of zebrafish in each group was continuously recorded, and in vivo fluorescence imaging and intestinal sample collection were performed at set time points. The collected intestinal contents or tissue were used for total DNA extraction and 16S rRNA high-throughput sequencing analysis to compare the changes in the composition and relative abundance of the gut microbiota in zebrafish from different treatment groups.

[0072] like Figure 10As shown, after intraperitoneal injection of Aeromonas dacarbazin C160501, the survival rate of zebrafish in all groups gradually decreased over time, but the decrease in the H7 pretreatment group was significantly lower than that in the injection-only group. The injection-only group experienced rapid death earlier after infection, resulting in the lowest final survival rate. In contrast, the H7-1 week, H7-2 week, and H7-3 week groups all improved the survival rate of zebrafish after challenge to varying degrees, with the H7-2 week and H7-3 week groups showing more significant protective effects, which were generally superior to the H7-1 group. The results indicate that Lactobacillus salivarius H7 pretreatment can effectively enhance the resistance of zebrafish to Aeromonas dacarbazin C160501 infection, and its protective effect generally increases with the extension of gavage time.

[0073] In vivo fluorescence imaging results further validated the aforementioned protective effect. For example... Figure 11 As shown in Figure a, the PBS group showed almost no obvious fluorescence signal, while the injection-only group injected with mCherry-labeled Aeromonas dacarpa C160501 showed the strongest red fluorescence aggregation, indicating that the pathogen colonized or amplified extensively in zebrafish. The mCherry-labeled Aeromonas dacarpa C160501 used in the experiment was constructed and preserved in our laboratory. Compared with the injection-only group, the fluorescence signals in the H7-1, H7-2, and H7-3 week groups were significantly weakened, and generally showed a decreasing trend with prolonged gavage time, with the H7-3 week group exhibiting the lowest fluorescence range and intensity. Quantitative analysis results ( Figure 11 Figure b shows that the total radiation efficiency of the injection group was significantly higher than that of each H7 pretreatment group. Meanwhile, the fluorescence load of the H7-2 and H7-3 week groups was further lower than that of the H7-1 week group, while there was no significant difference between the H7-2 and H7-3 week groups. These results indicate that H7 pretreatment with *Lactobacillus salivarius* can effectively reduce the load of *Aeromonas dacarba* labeled with *mCherry* in zebrafish, and longer-term gavage intervention has better antibacterial and protective effects.

[0074] like Figure 12 As shown, compared with the injection-only group, the intestinal microbiota structure of the H7 pretreatment groups treated with Lactobacillus salivarius H7 for 1, 2, and 3 weeks by gavage all underwent varying degrees of change. With prolonged gavage, the relative abundance of Aeromonas generally decreased, while the relative abundance of other genera gradually increased, indicating a more diversified and balanced intestinal microbiota composition. The improvement in microbiota structure was most significant in the H7-3 week group, suggesting that longer-term Lactobacillus salivarius H7 intervention is more beneficial for maintaining the gut microecological homeostasis of zebrafish, thereby enhancing the host's resistance to pathogen infection.

[0075] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. Alligator-derived Lactobacillus salivarius, characterized in that, The name is Lactobacillus salivarius H7 Ligilactobacillus salivarius H7 was deposited on January 30, 2026 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026301.

2. The crocodile-derived Lactobacillus salivarius according to claim 1, characterized in that, The total genome length of the Lactobacillus salivarius H7 is 1,886,039 bp, and the average GC content is 32.64%.

3. The alligator-derived Lactobacillus salivarius according to claim 1, characterized in that, The genome of *Lactobacillus salivarius* H7 contains a gene cluster for ribosome synthesis and post-translational modified peptide analogs, as well as a gene cluster related to type III polyketide synthase and terpene precursors.

4. The alligator-derived Lactobacillus salivarius according to claim 1, characterized in that, The genome of *Lactobacillus salivarius* H7 contains six families of carbohydrate-active enzymes: helper enzymes, carbohydrate-binding modules, carbohydrate esterases, glycoside hydrolases, glycosyltransferases, and polysaccharide lyases.

5. The alligator-derived Lactobacillus salivarius according to claim 1, characterized in that, The *Lactobacillus salivarius* H7 is sensitive to the following antibiotics, including norfloxacin, polymyxin B, minocycline, trimethoprim-sulfamethoxazole, ciprofloxacin, chloramphenicol, and tobramycin.

6. A probiotic agent for aquatic animals, characterized in that, Includes the crocodile-derived Lactobacillus salivarius as described in any one of claims 1 to 5.

7. The application of the crocodile-derived Lactobacillus salivarius according to any one of claims 1 to 5 in aquatic animal farming, wherein the application is the use of Lactobacillus salivarius H7 as an antagonistic bacterium against Aeromonas dacarba.

8. The application according to claim 7, characterized in that, The Aeromonas daca strain is Aeromonas daca C160501.

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