Bacillus subtilis strain and application thereof for resisting aeromonas hydrophila

Abstract

The bacillus subtilis strain against aeromonas and its application, the bacillus subtilis strain of the present application is named bacillus subtilis (wgb0621), and the preservation number is CCTCC NO: M 2026135. Bacillus subtilis The bacillus subtilis strain has good contact inhibition activity on aeromonas veronii and aeromonas hydrophila, can improve the survival rate of grass carp after being infected with aeromonas, has good safety, and is not dependent on diffusible bacteriostatic substances. The bacillus subtilis strain is suitable for biological prevention and control of aeromonas disease in aquaculture, and has good application prospect.

Description

Technical Field

[0001] This invention relates to a technology for the prevention and control of aquatic pathogens, specifically to a Bacillus subtilis strain with contact antibacterial activity and its application. Background Technology

[0002] Among the many diseases affecting aquatic animals, bacterial diseases are a major cause of death in farmed animals.

[0003] Aeromonas spp. is a group of opportunistic pathogens that are widely distributed in water bodies, sediments and various aquatic animals. They can cause acute or chronic diseases such as hemorrhagic septicemia and enteritis in fish and are one of the most prevalent and harmful pathogens in freshwater fish farming in my country.

[0004] Currently, the control of bacterial diseases in aquatic organisms still mainly relies on antibiotics and chemical disinfectants. Although these methods have a certain control effect in the short term, long-term or improper use can easily lead to increased drug resistance in pathogens, imbalance of the microecology in the aquaculture environment, and problems such as drug residues and environmental pollution. Utilizing beneficial microorganisms for the biological control of aquatic diseases is gradually becoming an important development direction for green and healthy aquaculture.

[0005] Microorganisms interact with other microorganisms in specific ecological niches through various mechanisms, among which antagonism is an important mechanism for maintaining microecological stability. Traditional research suggests that beneficial microorganisms mainly inhibit the growth of pathogens through non-contact methods such as secreting diffusible antimicrobial substances, competing for nutrients and space, and interfering with biofilm formation. Based on this, various aquatic microecological preparations have been developed. For example, CN111733117B discloses a Bacillus subtilis strain whose secreted antimicrobial peptide has a significant inhibitory effect on Aeromonas hydrophila.

[0006] However, with the deepening research into microbial interaction mechanisms, it has been discovered that in addition to non-contact inhibition, there are also antagonistic mechanisms between microorganisms that rely on direct cell-to-cell contact. Compared to the mode of action that relies on diffusible antimicrobial products, contact-dependent inhibition, through direct contact between bacterial cells, precisely targets toxic effectors to neighboring competing strains. This can disrupt the cell wall or cell membrane structure of target bacteria, interfere with their energy metabolism and key physiological processes, thereby achieving highly efficient antimicrobial inhibition. This type of inhibition is characterized by toxic agents that are not easily diluted, strong targeting, and low likelihood of inducing drug resistance, giving it a significant ecological advantage in high-density microbial communities.

[0007] In aquaculture systems, especially in nutrient-rich environments with high bacterial density and intense microbial competition, such as the fish gut, contact-dependent antagonistic mechanisms are considered more advantageous for microorganisms in competing for niches and maintaining community stability. Previous studies have shown that under such conditions, microorganisms tend to compete with other microbial community members through cell-to-cell contact, which is more efficient than inhibition mechanisms relying on diffusible antimicrobial products. Currently, there are no reports on the contact-dependent antagonistic mechanisms of Gram-positive bacteria, especially resilient Bacillus spp. and other commonly used aquatic probiotics, and their application in the control of aquatic pathogens.

[0008] Existing aquatic Bacillus preparations still suffer from problems in practical applications, such as unclear strain sources, limited spectrum of antagonistic pathogens, and insufficient stability of antagonistic effects. In particular, there is a lack of endogenous Bacillus strains derived from the intestines of aquatic animals that can stably colonize the intestinal microenvironment and efficiently antagonize important pathogens such as Aeromonas through contact-dependent mechanisms.

[0009] Therefore, screening new strains of Bacillus that have good adaptability to the fish intestinal environment and can stably antagonize Aeromonas through a contact-dependent mechanism, and applying them to the prevention and control of aquatic diseases, is of great significance for enriching the resources of biological control strains for aquatic diseases, reducing antibiotic use, and promoting the green and sustainable development of aquaculture. Summary of the Invention

[0010] The first technical problem to be solved by the present invention is to provide a Bacillus subtilis strain that has contact inhibitory activity against Aeromonas hydrophila.

[0011] The second technical problem to be solved by the present invention is to provide an application of the aforementioned Bacillus subtilis in the preparation of aquatic pathogen inhibitors.

[0012] The third technical problem to be solved by the present invention is to provide a fish feed additive.

[0013] The fourth technical problem to be solved by the present invention is to provide an application of the fish feed additive described above in grass carp farming.

[0014] The technical solution adopted by the present invention to solve its first technical problem is that a strain of Bacillus subtilis, named Bacillus subtilis WGB0621, was deposited at the China Center for Type Culture Collection on January 16, 2026, with accession number CCTCC NO: M 2026135.

[0015] The technical solution adopted by the present invention to solve its second technical problem is the application of Bacillus subtilis in the preparation of aquatic pathogen inhibitors, wherein the aquatic pathogen is Aeromonas hydrophila.

[0016] Furthermore, the Bacillus subtilis exerts an inhibitory effect through direct contact between the bacterial cells and the aquatic pathogens, and the fermentation supernatant of the Bacillus subtilis does not have significant antibacterial activity.

[0017] Furthermore, the aquatic pathogen inhibitor is a microbial preparation containing live Bacillus subtilis cells.

[0018] The technical solution adopted by the present invention to solve its third technical problem is a fish feed additive containing live Bacillus subtilis cells.

[0019] Furthermore, the concentration of the live bacteria is ≥10. 8 CFU / g.

[0020] The technical solution adopted by the present invention to solve its fourth technical problem is the application of the feed additive in grass carp farming.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention isolates a strain of Bacillus subtilis WGB0621 from the intestinal contents of disease-resistant grass carp. This strain is derived from the host intestine and has good host adaptability and application safety, and is suitable for aquaculture systems.

[0023] (2) The Bacillus subtilis WGB0621 of the present invention has a significant antagonistic effect on Aeromonas hydrophila, and its fermentation supernatant does not show obvious antibacterial activity, which is different from the traditional probiotic action mode that depends on secretory antibacterial substances.

[0024] (3) Through comprehensive verification using a variety of methods such as Transwell co-culture system, CFU counting and quantitative analysis of fluorescence signals, it was confirmed that the inhibitory effect of Bacillus subtilis WGB0621 on pathogens mainly depends on the contact inhibition effect produced by direct contact between the bacteria, and this effect has obvious contact dependence, providing a new technical path for the antagonistic mechanism of aquatic probiotics.

[0025] (4) Feeding and challenge experiments showed that feed containing Bacillus subtilis WGB0621 could significantly improve the survival rate of grass carp infected with Aeromonas hydrophila and reduce the risk of death caused by pathogen infection, demonstrating its potential as a probiotic and inhibitor of aquatic pathogens.

[0026] (5) The Bacillus subtilis WGB0621 of the present invention did not show hemolytic activity in the safety evaluation, the antibiotic resistance spectrum is controllable, and it is safe and reliable to use. It helps to reduce the use of antibiotics and chemical drugs in aquaculture, which meets the development needs of green and healthy aquaculture.

[0027] Information on the preservation of biological materials

[0028] Bacillus subtilis WGB0621 was deposited on January 16, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China; accession number: CCTCC NO: M 2026135. Attached Figure Description

[0029] Figure 1 Phase contrast microscopy images of Bacillus subtilis strain WGB0621 cultured in LB liquid medium for different times (12 h, 24 h, 36 h, 48 h, 60 h, 72 h).

[0030] Figure 2 Scanning electron microscopy images of vegetative cells (A) and spores (B) of Bacillus subtilis WGB0621.

[0031] Figure 3 Photographs show the plate inhibition zones of Bacillus subtilis WGB0621 cells and supernatant against Aeromonas hydrophila BCY X-1 after 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h of culture in LB broth. The supernatant (200 μl) of the control strain, Bacillus belyss ZFB19, contained a diffusible inhibitory product and exhibited antibacterial activity against Aeromonas hydrophila BCY X-1.

[0032] Figure 4 This is a phylogenetic tree of Bacillus subtilis WGB0621 constructed based on the gyrB gene.

[0033] Figure 5 Fluorescence microscopy observation of the antagonistic effect between green fluorescently labeled Bacillus subtilis WGB0621 and red fluorescently labeled Aeromonas hydrophila BCY X-1 under different co-culture conditions in a Transwell (24-well plate) system.

[0034] Figure 6 Figure (A) shows the quantitative analysis results of fluorescence signal intensity of Aeromonas hydrophila under different co-culture conditions, and Figure (B) shows the CFU statistics of survival status.

[0035] Figure 7 To analyze the contact inhibition phenotype of Bacillus subtilis WGB0621 against fluorescently labeled Aeromonas hydrophila using plate co-culture technology.

[0036] Figure 8 The results of safety testing for Bacillus subtilis WGB0621;

[0037] Among them, A shows the hemolysis test results of strain WGB0621 on 5% goat blood plates, and compares them with the hemolytic phenotype of Aeromonas hydrophila; B shows the drug susceptibility test results of strain WGB0621 to 16 commonly used antibiotics.

[0038] Figure 9 Survival curves of grass carp challenged with Aeromonas hydrophila after 30 days of feeding with feed containing Bacillus subtilis strain WGB0621. The control group consisted of grass carp challenged directly with Aeromonas hydrophila without being fed with strain WGB0621.

[0039] Figure 10 Histopathological observation results of the liver, spleen, intestines and gills of grass carp;

[0040] Among them, A represents the histopathological observation results of the liver, spleen, intestines and gills of healthy grass carp; B represents the histopathological observation results of the liver, spleen, intestines and gills of healthy grass carp after being challenged with Aeromonas hydrophila; C represents the histopathological observation results of the liver, spleen, intestines and gills of grass carp after being fed with feed containing Bacillus subtilis WGB0621 for 30 days and then challenged with Aeromonas hydrophila. Detailed Implementation

[0041] Many specific details of the invention are set forth in the following description to enable those skilled in the art to implement and fully understand the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make various modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the reagents and other materials used in this embodiment are all commercially available products. Sequencing was commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0044] Example 1: Screening and Identification of Strains

[0045] Healthy, disease-resistant grass carp were collected from aquaculture ponds and dissected under aseptic conditions in the laboratory. Their intestinal contents were treated at 100 °C for 8 min to enrich heat-resistant Bacillus subtilis. The heat-treated intestinal samples were plated on LB agar plates (formula: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 20 g agarose, 1000 mL H2O, pH 6.8-7.2) and incubated at 30 °C for 12-16 h. After incubation, single colonies were picked and transferred to GYS agar plates (formula: 1 g glucose, 2 g ammonium sulfate, 2 g yeast extract, 0.5 g K2HPO4, 1 mL 8% CaCl2, 1 mL 20% MgSO4, 1 mL 5% MnSO4·H2O, 1000 mL H2O, pH 6.8-7.2) for streaking purification to obtain single bacterial strains.

[0046] After confirming the ability of the isolated and purified strains to form spores by phase-contrast microscopy, their antagonistic activity against Aeromonas hydrophila was analyzed. Aeromonas hydrophila BCY X-1 was used as an indicator bacterium, spread on the surface of LB solid medium. Candidate strains were then cultured in LB liquid medium. The collected bacterial pellets were resuspended in PBS buffer and inoculated onto designated locations on plates. The plates were incubated at 30 ℃ for 12–16 h to observe whether the candidate strains exhibited antibacterial activity against the indicator bacterium.

[0047] Based on the antibacterial ability of the candidate strains against the indicator bacteria, a dominant strain was selected and named WGB0621. Phase contrast microscopy and scanning electron microscopy observations showed that this strain was a rod-shaped bacterium during the vegetative stage, and produced spores and released them from the mother cell in the later stage of culture. Figure 1 and Figure 2 Bacillus spp. WGB0621 was cultured in LB liquid medium with shaking at 180 r / min and 30 ℃. Samples were taken at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, and the culture medium was centrifuged. The bacterial precipitate and supernatant were collected separately, and their antibacterial activity against Aeromonas hydrophila BCY X-1 was analyzed. The results are as follows: Figure 3 As shown, the cells of Bacillus strain WGB0621 exhibited significant antibacterial activity against BCY X-1 strain, but its supernatant (200 μl) did not show any inhibitory effect on BCY X-1 strain. This suggests that the antagonistic effect of Bacillus strain WGB0621 may not depend on diffusible extracellular metabolites, but rather on direct contact between the cells of the antagonist and the target bacteria.

[0048] Genomic DNA was extracted from Bacillus subtilis strain WGB0621, and the gyrB gene was amplified by PCR. The obtained gyrB sequence (see sequence listing) was then subjected to BLAST sequence alignment analysis in GenBank. Homologous Bacillus subtilis gyrB sequences were selected and aligned using Clustal X v3.0. A phylogenetic tree was constructed using the Neighbor-Joining method with MEGA 7.0 software. The results showed that this strain was closely related to Bacillus subtilis strain 168. Figure 4 The results indicate that the Bacillus strain obtained from the intestines of disease-resistant grass carp is Bacillus subtilis.

[0049] Example 2: Identification of the antibacterial pattern of Bacillus subtilis WGB0621 using a co-culture system

[0050] This embodiment utilizes the Transwell co-culture system and the plate co-culture system to identify the antibacterial mode of Bacillus subtilis WGB0621 against Aeromonas hydrophila, and further determines that this inhibitory effect depends on direct contact between bacterial cells.

[0051] (I) Construction of the Transwell co-culture system

[0052] A contactless and contact co-culture model was constructed using Transwell chambers with a pore size of 0.4 μm. Bacillus subtilis WGB0621 (pGFP4412) labeled with GFP fluorescently was used as the attack bacterium, and Aeromonas hydrophila BCYX-1 (pBBR1MCS2-Tac-mCherry-Gen) labeled with mCherry fluorescently was used as the target bacterium.

[0053] The experiment was set up with the following three groups:

[0054] (1) Control group: Fluorescently labeled Bacillus subtilis WGB0621 and Aeromonas hydrophila BCY X-1 were cultured separately in Transwell chambers;

[0055] (2) Non-contact co-culture group: mCherry fluorescently labeled Aeromonas hydrophila BCY X-1 was cultured in the lower chamber of the Transwell, and GFP fluorescently labeled Bacillus subtilis WGB0621 was cultured in the upper chamber. The two were separated by a semi-permeable membrane to avoid direct contact between the bacteria.

[0056] (3) Co-culture group: GFP fluorescently labeled Bacillus subtilis WGB0621 and mCherry fluorescently labeled Aeromonas hydrophila BCY X-1 were mixed at a ratio of 1:1 and co-inoculated in the same culture system so that the two could come into direct contact.

[0057] After being cultured under the same conditions for 16 h, each treatment group was used for subsequent observation under an inverted fluorescence microscope and quantitative analysis of CFU.

[0058] (II) Fluorescence microscopic observation of contact inhibition phenomenon

[0059] After cultivation, the fluorescence signals of the strains in each treatment group were observed using an inverted fluorescence microscope. The results are as follows: Figure 5 As shown, green fluorescence signals emitted by Bacillus subtilis WGB0621 (pGFP4412) were observed in both single-culture and co-culture groups. Aeromonas hydrophila BCY X-1 (pBBR1MCS2-Tac-mCherry-Gen) maintained strong fluorescence signals in both the single-culture and non-contact co-culture groups, while the fluorescence signal of Aeromonas hydrophila was significantly weakened in the mixed co-culture group. The fluorescence intensity of each well was read using a multi-mode microplate reader. Using the fluorescence intensity of single-culture as a baseline, the fluorescence signals of the non-contact co-culture group and the mixed co-culture group were normalized, and the relative fluorescence intensity of each treatment group was calculated. Figure 6 A) The results showed that the fluorescence intensity of Bacillus subtilis remained basically consistent across groups. The relative fluorescence intensity of Aeromonas hydrophila in the non-contact co-culture group did not differ significantly from the control group, while the relative fluorescence intensity in the mixed co-culture group was significantly reduced. These results indicate that Bacillus subtilis has a significant inhibitory effect on Aeromonas hydrophila under bacterial contact conditions.

[0060] (III) Quantitative analysis of CFU of viable strains

[0061] To further quantitatively analyze the survival of Aeromonas hydrophila under different culture conditions, culture media from each treatment group were collected, serially diluted, and spread onto selective media. Colony forming units (CFU) were counted after incubation. Results are shown below. Figure 6 As shown in B.

[0062] Compared with the control group, the number of CFUs in *Aeromonas hydrophila* BCY X-1 (pBBR1MCS2-Tac-mCherry-Gen) decreased slightly under contactless co-culture conditions, while the number of CFUs decreased significantly under mixed co-culture conditions. In contrast, the number of CFUs in *Bacillus subtilis* WGB0621 (pGFP4412) did not decrease significantly in either the contactless or mixed co-culture groups. These results further indicate that the inhibitory effect of *Bacillus subtilis* WGB0621 on *Aeromonas hydrophila* BCY X-1 mainly occurs under direct bacterial contact conditions, exhibiting a clear contact-dependent characteristic.

[0063] (iv) Determining contact-dependent antibacterial mechanisms using plate co-culture systems

[0064] The contact-dependent inhibitory effect of Bacillus subtilis WGB0621 on Aeromonas hydrophila BCY X-1 was further verified by plate co-culture method.

[0065] OD was adjusted by overnight culture of Bacillus subtilis WGB0621 and Aeromonas hydrophila BCY X-1 (pBBR1MCS2-Tac-mCherry-Gen). 600 After washing the bacterial cells with PBS buffer, a bacterial enrichment solution was prepared. Then, 2 μL of each strain, individually and in combination, was spotted onto LB agar plates. Furthermore, the two strains were separated using a 0.22 μm filter membrane, with 2 μL of bacterial solution spotted onto each side of the membrane onto LB agar plates. The plates were incubated at 30°C for 24 h, and the fluorescence changes of *Aeromonas hydrophila* were observed using a fluorescence stereomicroscope. The results showed that ( Figure 7 Aeromonas hydrophila BCY X-1 (pBBR1MCS2-Tac-mCherry-Gen) exhibited strong fluorescence. When Bacillus subtilis and Aeromonas hydrophila were mixed, the fluorescence intensity of Aeromonas hydrophila significantly decreased. After separating the two bacteria with a filter membrane, the fluorescence intensity of Aeromonas hydrophila on the left side of the filter membrane was not significantly different from that of Aeromonas hydrophila cultured alone. This further indicates that the inhibitory effect of Bacillus subtilis WGB0621 on Aeromonas hydrophila BCY X-1 does not depend on diffusible inhibitory products, but requires direct contact between Bacillus subtilis and Aeromonas hydrophila, exhibiting a contact-dependent characteristic.

[0066] Example 3 Safety assessment of Bacillus subtilis WGB0621

[0067] This embodiment evaluates the biosafety of Bacillus subtilis WGB0621 as a probiotic for aquaculture by testing its hemolytic activity and antibiotic resistance.

[0068] (a) Hemolytic activity assay

[0069] The hemolytic characteristics of Bacillus subtilis WGB0621 were detected using a blood agar plate test. Strain WGB0621 was inoculated onto blood agar plates containing 5% goat blood and incubated at 37 °C for 16 h. The formation of a clear hemolytic zone around the colonies was observed. Results showed that strain WGB0621 grew well on blood agar plates, and no obvious clear hemolytic zone or incomplete hemolysis was observed around the colonies (see [link to test results]). Figure 8 A) indicates that the strain does not have hemolytic activity.

[0070] (II) Analysis of Antibiotic Resistance

[0071] The antibiotic susceptibility of Bacillus subtilis WGB0621 was determined using the disk diffusion method. Logarithmic growth phase bacterial suspension was evenly spread on the surface of LB solid medium, followed by placing antimicrobial susceptibility test strips containing different antibiotics on top. The medium was incubated at 37°C for 16 h, and the diameter of the inhibition zone was measured to determine resistance. Sixteen commonly used antibiotics were selected for testing in this example, including: penicillin, kanamycin, streptomycin, neomycin, tetracycline, doxycycline, norfloxacin, ofloxacin, erythromycin, polymyxin B, sulfamethoxazole, trimethoprim-sulfamethoxazole, rifampin, chloramphenicol, trimethoprim, and enrofloxacin.

[0072] The results showed that Bacillus subtilis WGB0621 was sensitive or moderately sensitive to most antibiotics, and showed resistance to only some antibiotics (streptomycin, polymyxin B). No broad-spectrum resistance was detected. Figure 8 B. Table 1). The above results indicate that this strain does not carry a significant high-risk drug resistance phenotype and has good application safety.

[0073]

[0074] Example 4: Evaluation of the function of Bacillus subtilis strain WGB0621 in grass carp's resistance to Aeromonas infection.

[0075] (a) Feeding grass carp with antagonistic bacteria

[0076] Bacillus subtilis WGB0621 was administered at a concentration of 1×10⁻⁶. 8 CFU·g -1 The probiotic feed was prepared by uniformly mixing the probiotics into the basal feed and feeding it to grass carp (12±1 cm, 20±1.6 g). The experiment was conducted in 3 replicates, with 16 fish per replicate. The fish were fed continuously for 30 days, once a day, with the amount of feed calculated as 2% of the grass carp's body weight. The basal feed without the added strain served as a control.

[0077] (II) Protection rate of grass carp against Aeromonas hydrophila challenge and antagonistic bacteria

[0078] After feeding with antagonistic bacteria, grass carp were challenged with Aeromonas hydrophila BCY X-1 via intraperitoneal injection at a dose of 3 × 10⁻⁶. 7 CFU·Tail -1 After the initial challenge, the grass carp were kept in captivity, and their survival rate was recorded daily to generate a survival curve (see [link]). Figure 9 The results showed that grass carp fed with strain WGB0621 in advance had a higher survival rate after challenge than grass carp not fed with strain WGB0621 in advance, indicating that this strain can enhance the grass carp's ability to resist Aeromonas hydrophila infection. Based on the challenge test results, the relative percentage survival (RPS) was further calculated using the following formula:

[0079]

[0080] The calculation results show that feeding grass carp with Bacillus subtilis WGB0621 provides a relative protection rate of 40.79%.

[0081] (III) Histopathological observation

[0082] Grass carp from different treatment groups were dissected, and liver, spleen, intestines, and gill tissues were collected. After fixation with 4% paraformaldehyde, the tissues were dehydrated, embedded, sectioned, and stained with hematoxylin and eosin (HE). Histopathological changes were observed under a light microscope (see [link to article]). Figure 10 The results showed that healthy grass carp had intact tissue structure without abnormalities. After 3 days of challenge with Aeromonas hydrophila BCY X-1 alone, significant vacuolation and loose structure of hepatocytes were observed; inflammatory changes were found in the spleen and intestines; and the integrity of the gill structure decreased. When challenged with BCY X-1 again after 30 days of feeding with strain WGB0621, the degree of tissue damage was significantly reduced, hepatocytes were more intact, and vacuolation was less severe. This indicates that Bacillus subtilis WGB0621 can effectively reduce tissue damage caused by BCY X-1 infection and has a certain tissue protective effect.

[0083] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis It was named Bacillus subtilis WGB0621, and its accession number is CCTCC NO: M 2026135.

2. The application of Bacillus subtilis according to claim 1 in the preparation of aquatic pathogen inhibitors, characterized in that, The aquatic pathogen is Aeromonas hydrophila.

3. The application according to claim 2, characterized in that, The Bacillus subtilis exerts an inhibitory effect through direct contact between the bacterial cells and the aquatic pathogens, and the fermentation supernatant of the Bacillus subtilis does not have significant antibacterial activity.

4. The application according to claim 2 or 3, characterized in that, The aquatic pathogen inhibitor is a microbial preparation containing live Bacillus subtilis cells.

5. A fish feed additive comprising live Bacillus subtilis cells as described in claim 1.

6. The fish feed additive according to claim 5, characterized in that, The concentration of the live bacteria is ≥10 8 CFU / g.

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