Marine bacterium with antibacterial function

Abstract

The invention relates to the field of marine bacteria, and discloses a marine bacterium with a bacteriostatic function, the bacterial strain is separated from seawater, is named as Bacillus velezensis NDB, is preserved in China General Microbiological Culture Collection Center (CGMCC), has a preservation number of CGMCC NO.32286, and is preserved on October 21, 2024, the bacterial strain is extracted from seawater, and the bacterial strain is named as Bacillus velezensis NDB and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.32286, and the preservation date is October 21, 2024. The strain has good eco-friendliness, and the bacterial strain increases antibacterial varieties, so that the marine bacteria can be efficiently inhibited, and the influence on marine ecology is small.

Description

Technical Field

[0001] This invention relates to the field of marine bacteria, and more particularly to a marine bacterium with antibacterial function. Background Technology

[0002] In aquaculture, pathogens present in the marine environment often cause large-scale disease and mortality in aquatic animals, resulting in severe economic losses for the industry. Traditional treatments for pathogens rely on antibiotics, which can alleviate disease occurrence to some extent. However, the overuse of antibiotics has led to drug resistance in many pathogens, and antibiotics can also suppress the immune system function of aquatic animals. Furthermore, antibiotic residues in aquatic products may pose a potential threat to consumer health. Therefore, there is an urgent need to develop a green and effective antimicrobial strategy to replace antibiotics.

[0003] With the rise of antibiotic-free aquaculture and the gradual verification of the effectiveness of microbial control, probiotics have become the most popular means of pathogen control. Recent studies have shown that Bacillus, as a non-pathogenic bacterium with broad-spectrum antibacterial activity, has been considered a strong candidate for antibiotic alternatives, significantly reducing the dependence of aquaculture on antibiotics while inhibiting bacteria.

[0004] Chinese Patent Application No. 201910770446.5 discloses a Bacillus belyssus YFI-4 and its application in the preparation of aquatic bacterial antibacterial agents. The Bacillus belyssus YFI-4 has the accession number CCTCC NO: M2019653 and has an inhibitory effect on Aeromonas vera, Aeromonas hydrophila, Pseudomonas fluorescens, Yersinia rumenella, Elizabethany meningitidis, or Pseudomonas putida.

[0005] The aforementioned strains were isolated from water samples from aquaculture ponds, and their ability to inhibit a limited number of pathogens is limited. However, marine pathogens are diverse, therefore, it is necessary to continue searching for Bacillus beleilliformis strains that can inhibit a wider range of pathogens. Furthermore, since this bacterium is ultimately intended as an additive in aquaculture feed, its impact on the marine ecosystem must be minimized. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies where strains are isolated from aquaculture pond water samples and can only inhibit a limited number of pathogenic bacteria. It provides a marine bacterium with antibacterial properties that increases the number of antibacterial species and has a minimal impact on the marine ecosystem when added to seawater.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A marine bacterium with antibacterial function, named Bacillus velezensis NDB, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 32286 and deposit date of October 21, 2024.

[0008] Using the above method, this bacterium was isolated from seawater obtained at a depth of 0.5 m in the central waters of Xiangshan Port, Ningbo, Zhejiang Province (121°43'06''E, 29°34'44''N). 16S rRNA gene sequence analysis revealed that the bacterium belongs to the genus *Bacillus* and clusters with strain *B. velezensis* SRCM102752. Therefore, we named it *B. velezensis* NDB, i.e., *Bacillus. velezensis* NDB. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 32286, and the deposit date is October 21, 2024. Because it was isolated from seawater, its addition to seawater will have a relatively small impact on the marine ecosystem.

[0009] As a preferred option, Bacillus velezensis NDB can inhibit the following pathogens: Vibrio splendidus, Staphylococcus aureus, Vibrio mimicus, Escherichia coli, Aeromonas hydrophila, Listeria esculenta, Vibrio harveyi, Enterobacter sakazakii, Acinetobacter baumannii, Bacillus cereus, Pseudomonas putida, and Shewanella putrefactive.

[0010] Using the above scheme, in subsequent experiments, purified *Bacillus velezensis* NDB was first cultured, and its growth was observed. *Bacillus velezensis* NDB showed excellent growth rate. Based on the genomic data of *Bacillus velezensis* NDB, gene mining was performed on potential antibacterial active gene clusters in *Bacillus velezensis* NDB to comprehensively reveal the antibacterial secondary metabolic potential of the strain. Extracellular polypeptide products of *Bacillus velezensis* NDB were detected, including surfactant nonribosomal peptide synthase srfAB and difficidin synthase pksR. Based on these results, related pathogens were screened for detection, and it was found that *Bacillus velezensis* NDB could inhibit twelve pathogens, increasing the number of inhibited bacteria.

[0011] The present invention has significant technical effects due to the adoption of the above technical solutions: the B. velezensis NDB can inhibit twelve kinds of pathogens, increasing the types of bacteria inhibited; at the same time, the bacteria is isolated from seawater and can coexist with marine organisms, which is eco-friendly. Attached Figure Description

[0012] Figure 1 This is a schematic diagram illustrating the isolation of B. velezensis NDB on the culture medium in the example; Figure 2 This is the phylogenetic tree of B. velezensis NDB in the example; Figure 3 This is the phylogenetic tree of B. velezensis NDB in the embodiment; Figure 4 This is a growth curve of B. velezensis NDB in the embodiment; Figure 5 This is a spectrum of extracellular product detection of B. velezensis NDB in the example; Figure 6 This is a diagram showing the antibacterial test results of B. velezensis NDB in the examples. Detailed Implementation

[0013] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0014] Example A marine bacterium with antibacterial properties.

[0015] Isolation of bacteria Seawater samples were collected from the central waters of Xiangshan Port in Ningbo City, Zhejiang Province (121°43'06''E, 29°34'44''N) at a depth of 0.5 m.

[0016] 50 μL of seawater sample was diluted 8-12 times and inoculated into liquid culture medium. The culture was then incubated at 36-38 °C and a shaking speed of 100-150 r / min for 12-16 h to form a bacterial culture. In this example, the seawater sample was diluted 10 times, incubated at 37 °C and a shaking speed of 120 r / min for 14 h. The liquid culture medium was a seawater beef extract peptone medium containing 2% NaCl, comprising: 0.3% beef extract, 1% peptone, 2% sodium chloride, and the remainder water.

[0017] Take 100 μL of bacterial culture and spread it on a plate medium. After incubation for 10–14 h, pick well-grown mixed bacterial colonies with different morphologies and streak them to obtain a mixed bacterial culture. In this example, the incubation time was 12 h, and the plate medium was solid beef extract peptone medium. Solid beef extract peptone medium contains the following components: 0.3% beef extract, 1% peptone, 2% sodium chloride, 2% agar, and the remainder is water.

[0018] Glycerol magnetic beads were used to adsorb mixed bacterial cultures to form a bacterial glycerol preservation solution, which was then stored at -80°C. The glycerol magnetic beads and the adsorption method for the mixed bacterial cultures are existing technologies and will not be described in detail here. Furthermore, the glycerol magnetic beads only extend the preservation time of the mixed bacterial cultures and do not affect subsequent bacterial purification, culturing, or experiments.

[0019] Take 10 μL of bacterial glycerol preservation solution and dilute it 10-20 times with sterile water to form a bacterial solution. In this example, the solution is diluted 12 times with sterile water.

[0020] Spread 100 μL of bacterial solution evenly onto the liquid culture medium and incubate for 10–14 h at 36–38 °C and a shaking speed of 180–230 r / min. Then, refer to… Figure 1 The bacteria formed white colonies, and an inhibition zone was formed around the white colonies. Therefore, it is speculated that the white bacterial strain has an antibacterial effect.

[0021] White colonies were isolated and purified by streak plating on agar plates. In this example, the temperature was 37 °C, the shaker speed was 200 r / min, and the incubation time was 12 h.

[0022] The purified bacteria were stored in a refrigerator at -80 °C.

[0023] Bacterial culture The purified bacteria were inoculated into a liquid culture medium with an initial pH of 7-9 at a rate of 0.5-1.5%, and cultured for 8-16 hours at a temperature of 36-38 °C and a shaking speed of 180-230 r / min. In this example, the initial pH of the liquid culture medium was 7, the inoculation amount was 1%, the culture temperature was 37 °C, the shaking speed was 200 r / min, and the culture time was 12 hours.

[0024] Identification and nomenclature of bacteria The phylogenetic position of this bacterium was determined by 16S rRNA gene sequence analysis. DNA was isolated from this strain, and the 16S rRNA gene was amplified by PCR using universal bacterial primers 27F and 1492R. The 16S rRNA sequence of this bacterium was compared with the GeneBank online database using the BLASTN program, and a phylogenetic tree was constructed using Mega 7.0 software, as detailed in [reference needed]. Figure 2 , Figure 3 It is a phylogenetic tree of strains based on 16S rDNA, used to supplement the evolutionary process of strains.

[0025] 16S rRNA sequence:

[0026] Based on morphological characteristics, 16S rDNA, and whole-genome sequencing analysis, this strain was identified as belonging to the genus Bacillus and clustered with strain B. velezensis SRCM102752. Therefore, we named it B. velezensis NDB, i.e., Bacillus velezensis NDB.

[0027] Determination of bacterial growth curve Prepare liquid culture medium and sterilize at 121 °C for 20 min. Dispense the liquid medium into 50 mL cell culture flasks, 25 mL per flask. Pick a single colony and incubate at 37 °C with shaking for 12 h at a shaking speed of 200 r / min. Measure the OD. 600 The OD value (i.e., primary bacterial culture) was then measured. 10 μL of the primary bacterial culture was then transferred to a culture flask and cultured under the same conditions (i.e., secondary bacterial culture), with OD measured every 2 hours. 600 Value, result reference Figure 4 It can be seen that the NDB concentration of B. velezensis began to increase significantly after 4 h of culture; during the period of 4 to 16 h, the bacteria grew rapidly and proliferated in large quantities, entering the logarithmic growth phase; after 16 h of culture, the concentration of the bacterial solution did not change much, indicating that cell growth had entered the plateau phase.

[0028] Discovery of antibacterial gene clusters in bacteria Gene clusters synthesizing secondary metabolites with antibacterial potential were analyzed in the whole genome of *B. velezensis* NDB using the online software antiSMASH. Specific results are shown in Table 1.

[0029] Table 1. Prediction of secondary metabolites from the NDB genome of strain NDB Gene clusters Synthetic enzyme types Homology analysis Starting position End position Region1 NRPS Surfactin (91%) 315,286 377,575 Region2 Ladderance 675,203 716,402 Region3 PKS-like butirosin A / butirosin B (7%) 94,5717 98,6961 Region4 Terpene 1,074,774 1,091,945 Region5 Trans AT-PKS macrolactin H (100%) 1,441,766 1,529,598 Region6 Trans AT-PKS bacillaene (100%) 1,749,137 1,849,698 Region7 NRPS fengycin(100%) 1,441,766 1,529,598 Region8 Terpene 1,749,137 1,849,698 Region9 T3pks 2,314,515 2,355,615 Region10 Trans AT-PKS difficidin (100%) 2,555,409 2,649,200 Region11 NRPS bacillibactin (100%) 3,264,220 3,316,008 Region12 Other bacilysin(100%) 3,857,773 3,899,191 The results showed that B. velezensis NDB contains a gene cluster that synthesizes three nonribosomal peptide synthases (NRPS) and three free AT-type polyketide synthases (TransAT-PKS).

[0030] Further analysis revealed that the gene clusters synthesizing NRPS contained gene clusters highly homologous to those synthesizing surfactant, fengycin, and bacillibactin synthases, with homology rates of 91%, 100%, and 100%, respectively. Surfactin and fengycin have been widely proven to have potent activity in inhibiting Gram-positive bacteria and fungi, while bacillibactin plays a crucial role in iron ion competition, further enhancing the strain's survival advantage in a competitive microbial environment.

[0031] Secondly, the gene cluster synthesizing TransAT-PKS shows 100% similarity to the gene clusters of known macrolide H synthase, bacillaene synthase, and difficidin synthase. Macrolide H, bacillaene, and difficidin are all broad-spectrum antimicrobial compounds with complex structures, exhibiting significant inhibitory effects against various pathogenic bacteria (such as Escherichia coli, Salmonella, and Staphylococcus aureus).

[0032] Comprehensive analysis shows that B. velezensis NDB integrates multiple known highly active antimicrobial metabolite synthesis pathways in its genome, possessing significant potential as a novel natural antimicrobial agent resource, and providing a theoretical basis and genetic foundation for the development of novel antimicrobial agents to replace antibiotics.

[0033] Detection of extracellular products of bacteria B. velezensis NDB was cultured in a protein-free medium for 24 h and then centrifuged at high speed. The protein-free medium consisted of the following components: potassium dihydrogen phosphate 0.01%, trace vitamins, (complex) amino acids 1%, ammonium acetate 0.2%, glucose 0.2%, sodium chloride 0.5%, magnesium sulfate 0.01%, and the remainder being sterile water. The centrifugation speed was 10000 r / min, and the centrifugation time was 3 min. Take the supernatant, transfer it to a 10 KD ultrafiltration tube and centrifuge it again at a high speed of 10000 r / min for 3 min. The liquid in the ultrafiltration tube was freeze-dried and dried, then redissolved in deionized water, desalted by a C18 column, and eluted with 50% acetonitrile-0.1% formic acid solution. Take 1 μL of eluent and spot it onto a target plate. After drying, add 1 μL of CHCA matrix to the spotted area. After drying again, send the sample to a RaffixX® MALDI (Bruker) mass spectrometer for analysis. The mass spectrometry range is 200–5000 m / z. The MASCOT software was used to search and compare the results in the database. Figure 5 .

[0034] The test results showed that the extracellular metabolites of strain NDB included surfactant nonribosomal peptide synthase and difficidin synthase. Surfactin is a typical lipopeptide surfactant, whose structure consists of a cyclic lipopeptide composed of β-hydroxy fatty acid and 7 amino acids. It exerts its antibacterial effect by attacking the cell membrane of pathogens, causing cell membrane disintegration or osmotic pressure imbalance; inhibiting the protein synthesis of pathogens and preventing cell proliferation; and inhibiting the enzyme activity of pathogens, affecting the normal metabolism of cells. Difficidin is an unsaturated 22-membered macrocyclic polyene lactone phosphate ester with broad-spectrum antibacterial activity against aerobic and anaerobic bacteria. Studies have found that it can effectively treat fatal bacteremia caused by Klebsiella pneumoniae in mice.

[0035] Antibacterial effect test of bacteria The antibacterial activity was determined using the paper disc method. B. velezensis NDB strain was inoculated into seawater beef extract and peptone liquid medium, and cultured at 37°C in a constant-temperature shaker at 200 rpm until a concentration of 1×10⁻⁶ was achieved. 7 CFU / mL bacterial suspension. Pipette 20 μL of bacterial suspension onto a 6 mm diameter sterile antibiotic susceptibility test strip, and then place the strip onto a prepared pathogen culture plate. Incubate at 37°C for 24 h. Measure the diameter of the inhibition zone to determine the antibacterial effect. Results are referenced from [reference needed]. Figure 6 . Figure 6 In Chinese: Sp stands for Shewanella; Lm for Listeria europaea; Sa for Staphylococcus aureus; Esc for Escherichia coli; Bc for Bacillus cereus; Cs for Enterobacter sakazakii; Vs for Vibrio splendidus; Pp for Pseudomonas putida; Ab for Acinetobacter baumannii; Ah for Aeromonas hydrophila; Vm for Vibrio mimicus; Vh for Vibrio harveyi.

[0036] The experimental results show that strain B. velezensis NDB has a certain inhibitory effect on Escherichia coli, Vibrio mimicus, Cronobacter sakazakii, Vibrio splenti, Acinetobacter baumannii, Shewanella putrefactive bacteria, Bacillus cereus, Vibrio harzianum, Pseudomonas putida, and Listeria ergella. Among them, the inhibitory effect on Aeromonas hydrophila is the greatest, followed by Staphylococcus aureus.

[0037] In summary, *B. velezensis* NDB is extracted from seawater, making it an eco-friendly additive for marine feed when added to marine feed. Furthermore, *B. velezensis* NDB can be cultured on beef extract peptone medium without the need for additional carbon or nitrogen sources, resulting in inexpensive and readily available raw materials. Additionally, *B. velezensis* NDB exhibits excellent growth rates, reaching the logarithmic growth phase within hours, making its cultivation simple, rapid, and easy to manage. Most importantly, *B. velezensis* NDB demonstrates strong antibacterial effects against many foodborne pathogens and aquaculture pathogens.

[0038] Therefore, R. marisflavi NDB is a marine bacterium that is highly effective in inhibiting bacteria, easy to mass-produce, and has a relatively small impact on the marine ecosystem.

[0039] The above description is merely a preferred embodiment of the present invention. 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 marine bacterium with antibacterial function, characterized by: The strain was named Bacillus velezensis NDB and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 32286 and deposit date of October 21, 2024.

2. The marine bacterium with antibacterial function according to claim 1, characterized in that: Bacillus velezensis NDB can inhibit the following pathogens: Vibrio splendidus, Staphylococcus aureus, Vibrio mimicus, Escherichia coli, Aeromonas hydrophila, Listeria eluti, Vibrio harveyi, Enterobacter sakazakii, Acinetobacter baumannii, Bacillus cereus, Pseudomonas putida, and Shewanella putrefactive.

Images