Enterobacter cloacae against aeromonas hydrophila and application thereof

Abstract

The present application discloses a strain of Enterobacter mori against Aeromonas veronii and its application, which is classified and named as Enterobacter mori (SCH0241). Enterobacter mori SCH0241, preserved in China Center for Type Culture Collection on October 17, 2024, with a preservation number of CCTCC NO: M 20242236. The Enterobacter mori SCH0241 of the present application is derived from the intestine of a highly stress-resistant healthy hybrid crucian carp “Hefang crucian carp No. 2”, has good contact-dependent bacteriostatic activity against Aeromonas veronii, and can improve the ability of zebrafish to resist Aeromonas veronii infection. Through the construction of an insertion mutation library and phenotype screening, it is found that the gene cluster of T6SS-2 of SCH0241 plays a contact inhibition toxicity. The Enterobacter mori SCH0241 of the present application provides a new powerful tool for the biological control of fish diseases caused by Aeromonas veronii.

Description

Technical Field

[0001] This invention relates to a microbial strain, Enterobacter mori, and more particularly to a strain of Enterobacter mori SCH0241 and its applications. Background Technology

[0002] Aeromonas veronii is a common fish pathogen that seriously threatens the sustainable development of aquaculture in my country. Existing aquatic disease control strategies mainly include immunization, ecological control, and drug control. Long-term use of chemical drugs leads to drug resistance in pathogens and disrupts the microecological balance of the aquaculture environment, increasing the difficulty of disease control in aquatic animals. Furthermore, chemical drugs easily remain in fish and the environment, polluting the environment and threatening human health. Ecological control of fish pathogens using antagonistic strains is receiving increasing attention due to its safety and environmental friendliness.

[0003] For Aeromonas bacteria infected via the oral-gut route, colonization of the host's gut by antagonistic bacteria is a crucial prerequisite for their antagonistic effect. Therefore, antagonistic bacteria derived from the fish gut are far superior to those screened from other environments in terms of both safety and efficacy. Furthermore, based on the different spatial modes of interaction between antagonistic bacteria and target bacteria, they can be broadly classified into two categories: non-contact inhibition and contact-dependent inhibition. The first category is typically represented by diffusible antibiotics and other small molecule compounds; the second category acts on adjacent bacteria through direct contact, including the CDI (Contact-dependent inhibition) system, the type IV secretion system (T4SS), and the type VI secretion system (T6SS). In aquaculture, currently reported antagonistic strains mainly achieve non-contact inhibition of pathogens by producing diffusible antimicrobial products. The mechanism of action of this antimicrobial method has been studied extensively, making the discovery of novel antimicrobial products unlikely. Meanwhile, research on the screening and application of beneficial antagonistic aquatic bacteria resources with contact inhibitory activity is rarely reported. Currently, there is still much to be explored regarding fish gut microbiota resources that have antagonistic activity against Aeromonas, especially strains with contact-dependent antagonistic activity are severely lacking.

[0004] To date, there have been no reports of using *Enterobacter molluscioides* for the prevention and control of animal pathogens, especially fish pathogens (including *Aeromonas verrucosa*). Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a probiotic strain of Enterobacter mulberry that has contact inhibitory activity against Aeromonas verrucosa, a pathogenic bacterium of fish. This Enterobacter mulberry strain has good safety activity, intestinal colonization and protective effect on zebrafish juveniles and adult fish.

[0006] The technical solution adopted by the present invention to solve its technical problem is that a strain of Enterobacter mori, classified and named Enterobacter mori SCH0241, was deposited at the China Center for Type Culture Collection on October 17, 2024, with accession number: CCTCC NO: M 20242236.

[0007] The *Corydalis molluscioides* strain SCH0241 of this invention was screened from the intestine of a highly resistant and healthy hybrid crucian carp named "Hefangji No. 2".

[0008] The gyrB gene sequence of *Enterobacter mulberry* SCH0241 of this invention is shown in Table SEQ ID No. 1.

[0009] The *Enterobacter mollusc SCH0241* of this invention exhibits contact-dependent toxicity against *Aeromonas verrucosa* HS1906.

[0010] The *Enterobacter mulberry* SCH0241 of this invention is relatively safe. It can colonize the intestines of healthy zebrafish juveniles and protect zebrafish from infection by *Aeromonas verrucosa* HS1906, thereby improving the survival rate of zebrafish.

[0011] The contact inhibition virulence gene cluster of *Enterobacter mollusc SCH0241* against *Aeromonas verrucosa* HS1906 in this invention is T6SS-2.

[0012] The *Enterobacter molluscioides* SCH0241 of this invention can be used as an antagonist to fish pathogens, especially *Aeromonas vesiculosus*, for the prevention and control of fish pathogens, particularly *Aeromonas vesiculosus*; including for the prevention and treatment of fish diseases, especially those caused by *Aeromonas vesiculosus*.

[0013] Using the *Enterocera molluscica* SCH0241 of this invention, drugs for the prevention and treatment of fish diseases, especially those caused by *Aeromonas vesiculosus*, can be prepared.

[0014] The *Enterobacter mulberryii* strain SCH0241 of this invention exhibits good contact inhibition activity against the fish pathogen *Aeromonas vesiculosus*, and the strain itself demonstrates good safety, intestinal colonization ability, and protection rate in zebrafish. Insertion mutant library analysis revealed that the T6SS-2 gene cluster of SCH0241 plays a crucial role in the contact inhibition activity against HS1906. Based on this, further research into the contact inhibition mechanism of *Enterobacter mulberryii* is conducted to provide a novel strategy for the biological control of fish diseases and the prevention and control of fish pathogens.

[0015] Description of Microbial Preservation

[0016] The Enterobacter mori of this invention, classified and named as Enterobacter mori (Em) SCH0241, was deposited on October 17, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with the accession number CCTCC NO: M 20242236. Attached Figure Description

[0017] Figure 1 This is a red fluorescence signal intensity diagram (partial strains) for the initial screening of antagonistic bacteria using a 96-well plate according to the present invention.

[0018] Among them, (A) is the red fluorescence signal intensity diagram of the first batch of screened strains, (B) is the red fluorescence signal intensity diagram of the second batch of screened strains, (C) is the red fluorescence signal intensity diagram of the third batch of screened strains, and (D) is the red fluorescence signal intensity diagram of the fourth batch of screened strains.

[0019] Figure 2 This is a view of the antibacterial activity analysis of the supernatant of the antagonistic bacterium Enterobacter mollusc SCH0241 of the present invention (no inhibition zone appeared in the supernatant).

[0020] Figure 3 The image shows the intensity of red fluorescence signals for identifying the cell contact inhibition phenotype of the antagonistic bacterium *Enterobacter molluscica* SCH0241, as presented in this invention.

[0021] Figure 4 The image shows the hemolysis test results of the antagonistic bacterium Enterobacter mollusc SCH0241 of this invention.

[0022] Figure 5 Morphological images of zebrafish ZF4 cells under different treatments (scale bar: 20 μm).

[0023] Figure 6 Phylogenetic tree of *Enterobacter mulberry* strain SCH0241 constructed based on the housekeeping gene gyrB.

[0024] Figure 7 The complete genome map of *Enterobacter mollusciculatus* SCH0241;

[0025] Among them, GC content refers to the amount of GC, GC Skew+ refers to positive GC bias, and GC Skew- refers to negative GC bias.

[0026] Figure 8 The image shows the safety test results of Cercobacterium mulberryii SCH0241 on adult zebrafish.

[0027] Figure 9 Bar graph showing the functional analysis of *Enterobacter molluscica* SCH0241 in zebrafish juveniles against *Aeromonas verrucosa* infection;

[0028] Among them, (A) the survival rate of juvenile fish in each treatment group; (B) the survival rate of HS1906 in different treatment groups. mCherry The count; ** indicates p < 0.01, * indicates p < 0.05.

[0029] Figure 10 SCH0241 EGFP Figure showing the colonization observation results in juvenile zebrafish (scale bar: 1 mm).

[0030] Figure 11 To counteract the weakened antagonistic activity of the SCH0241 mutant strain against HS1906 mCherry The antagonistic effect diagram.

[0031] Figure 12 The hcp2 mutant strain and complement strain of SCH0241 against HS1906 mCherry The antagonistic effect diagram.

[0032] Figure 13 The graph shows the effect of SCH0241 on protecting germ-free zebrafish juveniles from Aeromonas verrucosa infection through contact inhibition activity. * indicates p < 0.05. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0034] The material "Hefangji No. 2" used in the following embodiments was provided by the team of Academician Liu Shaojun of Hunan Normal University (see the invention patent application number CN201811288974.9, entitled: Method for Establishing a Hybrid Strain of Japanese White Carp and Red Carp and Method for Cultivating Hefangji No. 2); healthy adult zebrafish used for in vivo safety tests were purchased from zebrafish breeding bases in Guangzhou; sexually mature zebrafish (Tu strain) used to prepare sterile zebrafish juveniles were provided by the Animal Nutrition and Human Health Laboratory of Hunan Normal University; zebrafish ZF4 cell line (embryonic fibroblasts) was purchased from the National Zebrafish Resource Center. Unless otherwise specified, all other reagents and equipment used were obtained through conventional commercial channels.

[0035] 1. Initial screening of Aeromonas versicolor antagonistic bacteria

[0036] The intestines of healthy "Hefangji No. 2" crucian carp were aseptically dissected. The intestinal contents were mixed thoroughly with 0.85% sterile saline, serially diluted, and then spread onto CFC, BHIA, M17, and TSA media for culture. Intestinal isolates and Aeromonas villus HS1906 carrying a red fluorescent protein expression plasmid were then cultured. mCherryMix the samples at a 1:1 ratio and add them evenly to a 96-well plate. After incubating at 30°C for 24–48 h, observe the changes in red fluorescence signal intensity using a small animal imaging system. The results showed that 66 strains, including strain 2-41, caused a decrease in red fluorescence signal (see [link to article]). Figure 1 This indicates that these strains have antibacterial activity against Aeromonas versicolor.

[0037] 2. Identification of contact inhibition activity

[0038] To analyze the antibacterial activity of the bacterial supernatant, different antagonistic strains were cultured overnight. The supernatant was collected by centrifugation and filtered through a 0.22 µm filter membrane. 50 μL of the supernatant filtrate was added to wells of LB agar plates containing Aeromonas vermicularis HS1906, with gentamicin as a control. The plates were incubated overnight at 30°C. To further analyze the antibacterial activity of the bacteria, the overnight cultured antagonistic bacteria and HS1906 were... mCherry Adjusting the OD600 to 5, a bacterial cell enrichment solution was prepared, followed by the addition of antagonistic bacteria and HS1906. mCherry Two μL samples of each bacterial mixture were spotted onto LB agar plates. Additionally, the antagonistic bacteria and AvHS1906 were filtered through a 0.22 μm filter membrane. mCherry Physical separation (2 μL of sample was spotted onto each side of the filter membrane onto an LB solid plate), incubated at 30℃ for 24–48 h, and observed using a fluorescence stereomicroscope.

[0039] The results showed that the supernatants of strains 2-41, 2-54, 5-80, 5-82, 5-94, 6-2, 6-14, and 6-38 had no antibacterial activity (see [link to study]). Figure 2 When separated from HS1906 by a filter membrane, the antagonistic activity against HS1906 disappears (see [link]). Figure 3 This indicates that these eight antagonistic bacteria have contact inhibitory toxicity against HS1906.

[0040] 3. Hemolytic and Cytotoxic Analysis

[0041] Hemolytic activity analysis showed that among the eight strains with contact inhibitory virulence, only strains 2-41 lacked hemolytic activity (see [link to relevant documentation]). Figure 4 Cytotoxicity analysis showed that after treatment of zebrafish ZF4 cell lines with strain 2-41 at 100 MOI for 48 h, the cells were uniform in size and maintained a good fibroblast state, almost identical to the cytotoxic negative control E. coli DH5α strain. In contrast, treatment with strain HS1906 at 100 MOI resulted in severe cell damage (see [link to relevant documentation]). Figure 5 ).

[0042] 4. Identification and genome sequencing of strain SCH0241

[0043] (1) gyrB gene sequence analysis

[0044] Genomic DNA was extracted from strain 2-41 and used as a PCR template. Amplification was performed using gyrB primers. The PCR products were analyzed by agarose gel electrophoresis and then sequenced. A phylogenetic tree was constructed using the gyrB gene sequence, revealing that strain 2-41 is a *Ceratophyllum demersum* strain (see [link to PCR]). Figure 6 The strain was named *Enterobacter mulberry* SCH0241.

[0045] (2) Whole genome sequencing

[0046] SCH0241 bacterial culture in the logarithmic growth phase was collected by centrifugation for whole-genome sequencing. The genome size was found to be 5,010,480 bp, consisting of one chromosome and one circular plasmid. The chromosome size was 5,005,438 bp, the plasmid size was 5,042 bp, and the GC content was 55.42% (see [link to original text]). Figure 7 ).

[0047] 5. In vivo safety, colonization, and protective efficacy studies.

[0048] (1) In vivo safety test of adult zebrafish

[0049] To further evaluate the safety of strain SCH0241, 8.5 × 10⁻⁶ μL of the strain was used. 6 CFU SCH0241, administered intraperitoneally to adult zebrafish, showed a 96.7% survival rate after 7 days (see [link to relevant documentation]). Figure 8 Furthermore, the fish showed no obvious lesions or abnormalities on their body surface. However, after being injected with 1.0×10... 6 The 7-day survival rate of zebrafish using CFU HS1906 was only 20.0%, and dead zebrafish showed abdominal congestion and swelling, as well as anal redness and swelling, indicating that SCH0241 has high in vivo safety for adult zebrafish.

[0050] (2) Colonization and protection rate analysis using sterile zebrafish juveniles

[0051] First, the constitutive expression vector pBBR1MCS2-Tac-EGFP of green fluorescent protein was introduced into strain SCH0241 via conjugation transfer to prepare fluorescently labeled strain SCH0241. EGFP The bacterial suspension was prepared at a concentration of 1.7 × 10⁻⁶. 8 SCH0241 CFU / mL EGFP Zebrafish juveniles were soaked in the infected solution for 24 hours, and their growth was observed. The colonization ability of strain SCH0241 was assessed by fluorescence stereomicroscopy and CFU counting on plates. The results showed that SCH0241… EGFP No juvenile fish died after soaking for 24 hours (see...) Figure 9 (A) The number of fluorescently labeled strains in juvenile fish reached 2.3 × 10⁻⁶. 5 CFU / species, and bright green fluorescence was observed in the intestines of juvenile fish (see [link]). Figure 10 Using SCH0241 EGFP Pre-treat the juvenile fish for 24 hours, then use 2.2×10 8 CFU / mL HS1906 mCherry Immersion infection for 24 hours was performed to assess the ability of *Enterobacter molluscica* SCH0241 to protect the host from *Aeromonas vesiculosus* infection. See [link to relevant documentation]. Figure 9 (A) It was found that the relative protection rate of zebrafish after pretreatment with *Enterobacter mulberryis* was 37.5%. (See [reference]) Figure 9 (B) reduced the colonization of Aeromonas verrucosa in juvenile fish by about 3 times. This indicates that SCH0241 has high safety in juvenile zebrafish, can colonize the intestines of juvenile fish, and can protect them from Aeromonas verrucosa infection to a certain extent after colonization.

[0052] 6. Using a random mutant library, the T6SS-2 gene cluster of *Ceratophyllum demersum* was found to mediate contact inhibition toxicity.

[0053] To identify the functional gene clusters in *Enterobacter mollusc* SCH0241 that exert contact inhibitory toxicity against *Aeromonas vesiculosus* HS1906, a random mutant library of *SCH0241* strain was constructed using the pUT-mini-Tn5-Km2-Gen plasmid containing the Tn5 transposon, and mutants with weakened inhibitory activity were screened. A transposon mutant library containing 1362 mutant strains was constructed using the pUT-mini-Tn5-Km2-Gen plasmid. To verify the randomness of Tn5 transposition, 20 mutant strains were selected, and the flanking sequences of the transposons were amplified using hiTAIL-PCR and sequenced. The sequencing results were aligned to the *SCH0241* genome sequence using BioEdit. The results showed that the mutation sites in the 20 mutant strains occurred at different locations in the genome, indicating that the Tn5 transposon can undergo random mutation in this strain.

[0054] The SCH0241 mutant strain and HS1906 mCherry Co-culture was performed to identify the antagonistic activity of mutant strains against HS1906. The results showed that the antagonistic activity of five mutant strains (M0176, M0353, M0409, M0331, and M0532) was weakened (see [link to original text]). Figure 11Using hiTAIL-PCR to identify the mutation sites, it was found that the mutation sites M0176, M0353, M0409, and M0331 were all located within the T6SS-2 gene cluster of strain SCH0241, while the mutation site M0532 was located in the promoter region of the T6SS-2 gene cluster. This indicates that the T6SS-2 gene cluster of strain SCH0241 may play a key role in the contact inhibition activity against HS1906.

[0055] To verify the results of the above random mutations, site-directed mutagenesis and complementation were performed on the hcp2 mutation site of M0176 from mutant strains with weakened antagonistic activity. The hcp2 gene in strain SCH0241 was knocked out without scarring using the suicide vector pRE112 based on homologous recombination. Then, the Hcp2 expression vector pBBR1-Rha-Hcp2 was constructed using the wide-host shuttle vector pBBR1-Rha-eGFP. It was found that after knocking out the hcp2 gene, strain SCH0241 lost its antagonistic activity against HS1906. mCherry The SCH0241 strain exhibited contact inhibitory toxicity, but after the gene was reintroduced, it regained its contact inhibitory toxicity (see [link to gene].) Figure 12 ).

[0056] 7. Strain SCH0241 protects the host from Aeromonas versicolor infection through contact inhibition activity.

[0057] Knocking out the key functional gene tssK2 in the T6SS-2 gene cluster of strain SCH0241 revealed that the knockout strain SCH0241 ΔtssK2 lost its contact inhibitory virulence against strain HS1906. Using approximately 1.7 × 10⁻⁶... 8 CFU / mL SCH0241△tssK2 EGFP After immersion in infected, sterile zebrafish juveniles for 24 hours, the colonization rate in the juveniles was 2.6 × 10⁻⁶. 5 CFU / fish, while the colonization rate of wild-type SCH0241 in juvenile fish was 2.3 × 10⁻⁶. 5 CFU / each, with no significant difference between the two. SCH0241△tssK2 was used respectively. EGFP After pretreating sterile zebrafish with SCH0241 for 24 hours, they were then treated with 2.2 × 10⁻⁶ mol / L mol / L. 8 CFU / mL HS1906 mCherry After 24 hours of immersion infection, the wild-type strain SCH0241 was found to provide better protection against Aeromonas versicolor infection than tssK2 (see [link to original text]). Figure 13 The above results indicate that strain SCH0241 can protect the host from Aeromonas versicolor infection through contact inhibition activity.

[0058] In summary, based on culturamics methods, the inventors isolated 1312 bacterial strains from the intestines of hybrid crucian carp 'Hefangji No. 2', and used fluorescent labeling and high-throughput screening technology to screen out 8 antagonistic bacteria with contact inhibitory toxicity against Aeromonas verrucosa. Among them, *Enterobacter mulberryii* SCH0241 showed good safety, could colonize the intestines of zebrafish juveniles, and effectively protect the host from *Aeromonas verrucosa* infection after colonization. Further research showed that strain SCH0241 mainly mediated contact inhibitory toxicity against *Aeromonas verrucosa* through the T6SS-2 gene cluster, and protected the host from *Aeromonas verrucosa* infection through contact inhibition.

Claims

1. A strain of Enterococcus saccharolyticus against Aeromonas sp. of fish, characterized in that, Named *Ceratophyllum demersum* ( Enterobacter mori SCH0241 was deposited at the China Center for Type Culture Collection on October 17, 2024, with accession number CCTCCNO: M 20242236.

2. The use of the A. sanchengii against A. wautersii in the preparation of a drug for preventing and treating fish diseases caused by A. wautersii. Aeromonas veronii )

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