Salt-tolerant bacillus with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria and application of salt-tolerant bacillus

Abstract

The invention discloses a salt-tolerant bacillus strain with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria and application thereof, and belongs to the field of microbial animal cell lines, the salt-tolerant bacillus strain is separated from intestinal tracts of patients in the recovery period of chronic diarrhea, is named as SH1, has an obvious bacteriostatic effect on clinically common gram-positive drug-resistant pathogenic bacteria, and can be used for preparing drugs for treating chronic diarrhea. A certain bacteriostatic effect is achieved on gram-negative drug-resistant bacteria. The size of a genome of the salt-tolerant bacillus strain SH1 is 4152283 bp, the salt-tolerant bacillus strain SH1 comprises 12 plasmids, the salt-tolerant bacillus strain SH1 is preserved in China General Microbiological Culture Collection Center (CGMCC) on November 23, 2023, and the preservation number of the salt-tolerant bacillus strain SH1 is CGMCC No. 29118. The invention also relates to an application of the salt-tolerant bacillus SH1 in a medicine for resisting gram-positive drug-resistant bacteria. According to the present invention, the bacterial strain having broad-spectrum antibacterial activity on the clinical common drug-resistant pathogenic bacteria is separated from the intestinal flora of the patient in the recovery phase of the chronic diarrhea, such that the foundation is laid for the exploration of the novel antibacterial molecules having characteristics of low toxicity, no harm, drug resistance generation resistance, and strong pertinence, wherein the novel antibacterial molecules have characteristics of low toxicity, no harm, and strong pertinence.

Description

Technical Field

[0001] The present invention belongs to the field of microbial animal cell lines, and specifically relates to a halodurable Bacillus having broad-spectrum antibacterial activity against Gram-positive drug-resistant bacteria and its application, especially to a new antibacterial molecule against Gram-positive drug-resistant bacteria and its application in the field of antibacterial liquid for common pathogenic bacteria Staphylococcus aureus and Escherichia coli. Background Art

[0002] Drug-resistant strains have become common pathogens of clinical infections, leading to more and more difficult-to-treat infections and posing a threat to global health. In 2017, the World Health Organization announced the priority pathogens for new antimicrobial drugs, collectively known as ESKAPE, including Enterococcus faecium (E), Staphylococcus aureus (S), Klebsiella pneumoniae (K), Acinetobacter baumannii (A), Pseudomonas aeruginosa (P), and Enterobacter (E), which are the main components of highly drug-resistant bacteria in hospital infections. Among them, methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Enterobacteriaceae (CRE) have become the most difficult pathogens for clinical anti-infection treatment due to multidrug resistance. As the resistance of pathogens to antibiotics continues to increase, new anti-infective drugs need to be developed clinically to compete with them. Most existing antibiotics focus on mining antimicrobial molecules from environmental microorganisms. With the development of human microbiome in recent years, researchers have begun to focus on the human intestinal symbiotic flora / colonization flora that compete and restrict human diseases.

[0003] Millions of microorganisms inhabit the human intestinal microbiome. The intestinal microbiome encodes highly diverse genes. As a result of long-term competition and co-evolution, they check and balance each other to maintain the microecological balance of human health. Many common opportunistic pathogens, such as Staphylococcus aureus and Escherichia coli, are symbiotic flora of the human body. There are microorganisms and their antibacterial substances in the human intestine that check and balance these drug-resistant pathogens. The development of sequencing technology has enabled people to gain a deeper understanding of the microbiome, especially in the human intestine. The contribution of intestinal microorganisms to host metabolism and immune health is increasingly recognized.

[0004] The intestinal microbiome encodes highly diverse genes and is one of the largest reservoirs of antibiotic resistance genes. At the same time, due to long-term competition and co-evolution, it is expected that antimicrobial drugs against multidrug-resistant (MDR) bacteria will be found. Spore-forming bacteria in the intestinal flora have a high tolerance to oxygen and can grow both inside and outside the intestine. The presence of spore bacteria is of great significance for the transmission of intestinal flora between mothers and infants and between people.

[0005] At present, there is no literature reporting the isolation of halotolerant Bacillus from the human intestine and its association with Gram-positive resistant bacteria. Summary of the invention

[0006] The purpose of the present invention is to isolate a strain with broad-spectrum antibacterial activity against common clinical drug-resistant pathogens from the human intestinal microbial flora, specifically to a halotolerant Bacillus isolated from the intestinal symbiotic / colonizing flora of patients with chronic diarrhea in the recovery period. The antibacterial molecules produced by the strain have broad-spectrum antibacterial activity against common clinical Gram-positive drug-resistant bacteria.

[0007] The technical solution of the present invention is as follows:

[0008] The first object of the present invention is to provide a halodurable Bacillus having broad-spectrum antibacterial activity against Gram-positive resistant bacteria, wherein the halodurable Bacillus has a size of 4152283 bp, a G+C content of 44.51%, and contains 4 prophages, 7 genomic islands and 12 plasmids.

[0009] The morphological and physiological and biochemical characteristics of the strain with broad-spectrum antibacterial activity against common clinical drug-resistant pathogens are as follows: the bacterium is a facultative anaerobic Gram-positive bacterium that grows better under aerobic conditions. It is salt-resistant and heat-resistant, can oxidize and ferment a variety of sugars to produce acid, and can utilize citrate and other characteristics. It forms colorless, transparent, pinpoint-like small colonies on eosin-methylene blue agar medium; the colonies cultured on nutrient agar plates are white, large, flat, moist, with irregular edges, and a milky halo is produced on the edge of the colony; the colonies cultured on Columbia blood agar plates are white, large, flat, moist, with irregular edges, and have a transparent hemolytic ring. During the growth process, a milky halo is produced on the edge of the colony, and a depression is formed in the center of the colony.

[0010] Furthermore, the halotolerant Bacillus is named SH1, its classification name is Halotolerant Bacillus (Bacillushalodurans, CGMCC No.29118), its preservation number is CGMCC No.29118, the preservation date is November 23, 2023, the preservation unit is the General Microbiology Center (CMCC) of the China Culture Collection Administration, and the preservation unit address is No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0011] Furthermore, the Gram-positive drug-resistant bacteria include oxacillin-resistant Staphylococcus aureus, β-lactam-resistant Staphylococcus aureus, macrolide-resistant and clindamycin-resistant Staphylococcus aureus. Gram-negative bacteria include Escherichia coli and its drug-resistant strains (extended-spectrum β-lactamase (ESBL)-producing Escherichia coli).

[0012] The second object of the present invention is to provide a use of the above-mentioned salt-tolerant Bacillus in the preparation of drugs against Gram-positive drug-resistant bacteria.

[0013] Furthermore, the anti-Gram-positive drug-resistant bacteria drugs include oxacillin-resistant Staphylococcus aureus drugs, clindamycin, and erythromycin-resistant Staphylococcus aureus drugs.

[0014] The present invention isolates a strain with broad-spectrum antibacterial activity against common clinical drug-resistant pathogens from the symbiotic / colonizing flora in the human intestine, specifically a halodurous Bacillus SH1 isolated from anal swab specimens of patients with chronic diarrhea. The antibacterial molecules produced by the strain have broad-spectrum antibacterial activity against common clinical Gram-positive drug-resistant bacteria, laying the foundation for discovering new antibacterial molecules that are low in toxicity and harmless to the human body, not prone to drug resistance, and more targeted. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The halotolerant Bacillus halotolerans with broad-spectrum antibacterial activity against Gram-positive resistant bacteria described in the present invention is named SH1, its classification name is (Bacillus halotolerans), its preservation number is CGMCC No.29118, the preservation date is November 23, 2023, the preservation unit is the General Microbiology Center (CMCC) of the China Culture Collection Administration, and the preservation unit address is No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0016] Figure 1 A schematic diagram showing that the halodurable Bacillus has a broad-spectrum antibacterial effect on pathogenic Gram-positive resistant bacteria from different sources;

[0017] Figure 2 Schematic diagram showing the results of whole genome sequencing analysis of the halodurable Bacillus SH1 strain: Using the second-generation platform sequencing, the genome size of the halodurable Bacillus SH1 strain is 4152283bp, the G+C content is 44.51%, and it contains 4 prophages, 7 genomic islands and 12 plasmids.

[0018] Figure 3 A diagram showing the antibacterial test of SH1 sterile supernatant on different clinical Staphylococcus aureus strains. DETAILED DESCRIPTION

[0019] The present invention discloses a halogen-tolerant Bacillus having broad-spectrum antibacterial activity against Gram-positive drug-resistant bacteria, which is isolated from a halogen-tolerant Bacillus in the intestinal flora of a chronic diarrhea patient, named SH1, which has been deposited on November 23, 2023, and its deposit number is CGMCC No. 29118. The present invention also relates to a method for screening the halogen-tolerant Bacillus.

[0020] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0021] 1. Culture plate for inhibiting Staphylococcus aureus

[0022] Example 1

[0023] This embodiment is a screening step for halotolerant Bacillus having antibacterial activity against Gram-positive bacteria and drug-resistant bacteria thereof, which comprises:

[0024] Step 1) Aseptically collect anal swabs from patients with a history of chronic diarrhea during the recovery period of diarrhea, and spread the swabs on aerobic Bacillus plates.

[0025] Step 2) The inhibitory effect of the salt-tolerant Bacillus on the growth of common clinical Staphylococcus aureus and its drug-resistant bacteria, Escherichia coli and its drug-resistant bacteria was analyzed by plate antibacterial experiment. (Specific types of common clinical drug-resistant pathogens tested are shown in Table 1)

[0026] Step 3) The salt-tolerant Bacillus spores that coexist / colonize in the intestine can produce antibacterial molecules with different effects, effectively resisting the colonization and invasion of clinically resistant pathogens. Among them, the SH1 strain has a significant inhibitory effect on all tested Staphylococcus aureus, while the Bacillus cereus isolated from the anal swabs of diarrhea patients cannot inhibit all tested Staphylococcus aureus. (The inhibitory results are shown in Figure 1 )

[0027] The present invention subjects the human intestinal symbiotic / colonized halobacterium resistant Bacillus SH1 to liquid culture fermentation for 15 hours, collects the supernatant, and vacuum-dries the supernatant after organic extraction. The antibacterial effect of the supernatant still exists, indicating that the antibacterial molecules produced by halobacterium resistant Bacillus SH1 can be secreted into its culture supernatant.

[0028] Example 2

[0029] This example performs gene sequencing on the halotolerant Bacillus spp. that has broad-spectrum antibacterial activity against common clinical Staphylococcus aureus and Escherichia coli verified in the above Example 1. The results are as follows:

[0030] In this example, the whole genome sequencing analysis of the halodurous Bacillus strain SH1, which produces antibacterial active molecules against Staphylococcus aureus and Escherichia coli, was performed, and it was found that its size was 4152283bp, the G+C content was 44.51%, and it contained 4 prophages, 7 GI-like regions, and 12 plasmids. Figure 2 )

[0031] The halodurable Bacillus SH1 described in this embodiment was deposited on November 23, 2023, and its classification name is halodurable Bacillus, its deposit number is CGMCC No.29118, and the depository is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), and the depository address is No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0032] Example 3

[0033] This example is a bacteriostatic verification experiment of halodurable Bacillus SH1, which takes Staphylococcus aureus as an example, and the verification steps are as follows:

[0034] A single clone of the halodurous Bacillus SH1 strain isolated from a patient with a history of diarrhea was selected and inoculated into 10 ml of fresh TSB liquid culture medium, and the culture was incubated at 37°C, 220 rpm, and shaken overnight.

[0035] Transfer to 100 ml of fresh TSB liquid culture medium at a ratio of 1:100 and continue culturing at 37°C and 220 rpm for 15 h.

[0036] The supernatant was collected by centrifugation at 8000 g, filtered through a 0.22 μm filter membrane, and organically extracted with ethyl acetate 1:1 three times. The supernatant component was obtained by vacuum rotary evaporation, and its properties were yellow solid dry powder.

[0037] The above supernatant components were dissolved in sterile ddH2O to prepare a 50ug / ml suspension. After filtering through 0.22μm, a blank drug-sensitive paper was placed in the filtered solution and allowed to stand at 4°C for 4h.

[0038] Pick up the monoclonal Staphylococcus aureus cultured overnight on the blood plate and put it into 0.45% sterile saline. Use a turbidimeter to adjust the OD value of the bacterial suspension to 0.5-0.6. Use a sterile cotton swab to dip the suspension and evenly spread it on the MH plate.

[0039] Pick a single clone of the halotolerant Bacillus SH1 cultured overnight on a blood plate and put it into 0.45% sterile saline. Use a turbidimeter to adjust the OD value of the bacterial suspension to 0.5-0.6. Suspend a sterile blank drug-sensitive paper in the bacterial suspension of the halotolerant Bacillus SH1.

[0040] The paper pieces of the sterile supernatant component in (4) and the paper pieces of the halodurogenic Bacillus SH1 in (6) were aseptically inoculated into MH plates evenly coated with Staphylococcus aureus, and after drying, they were cultured in a 37°C incubator overnight, and the inhibition zone was observed for 18 to 24 hours.

[0041] It can be seen from the antibacterial verification experiment that the halotolerant Bacillus SH1 of the present invention has a significant antibacterial effect on Staphylococcus aureus.

[0042] It can be seen from the above embodiments that the present invention provides a halotolerant Bacillus strain SH1 isolated from the intestinal symbiosis / colonization of patients with a history of chronic diarrhea. The antibacterial molecules produced by this strain have broad-spectrum antibacterial activity against common clinical Gram-positive resistant bacteria, which lays the foundation for discovering new antibacterial molecules that are low-toxic, harmless to the human body, not easy to develop drug resistance, and more targeted.

[0043] The specific embodiments of the present invention are described in detail above, but are not limited to the specific embodiments described above. Any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Equivalent modifications and substitutions under the spirit and scope of the present invention should all be included in the scope of the present invention.

Claims

1. A halodurogenic Bacillus having broad-spectrum antibacterial activity against Gram-positive drug-resistant bacteria, characterized in that: The size of the halodurable Bacillus is 4152283 bp, the G+C content is 44.51%, and it contains 4 prophages, 7 genomic islands and 12 plasmids.

2. A halodurable Bacillus according to claim 1, characterized in that: The salt-tolerant Bacillus strain is named SH1, and its preservation number is CGMCC No.29118, and the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration.

3. The salt-tolerant Bacillus according to claim 1 is a facultative anaerobic spore-forming Bacillus that grows better under aerobic conditions. It is salt-resistant and heat-resistant, can oxidize and ferment a variety of sugars to produce acid, and can utilize citrate and other characteristics. It forms colorless, transparent, pinpoint-like small colonies on eosin-methylene blue agar medium; the colonies cultured on nutrient agar plates are white, large, flat, moist, with irregular edges, and a milky halo is produced on the edges of the colonies; the colonies cultured on Columbia blood agar plates are white, large, flat, moist, with irregular edges and a transparent hemolytic ring. During the growth process, a milky halo is produced on the edges of the colonies, and a depression is formed in the center of the colonies.

4. Use of the halodurable Bacillus according to any one of claims 1 to 3 in the preparation of drugs against Gram-positive drug-resistant bacteria.

5. Use of the halodurable Bacillus according to any one of claims 1 to 3 in the preparation of an antibacterial catheter against Gram-positive drug-resistant bacteria.

6. The halotolerant Bacillus according to any one of claims 1 to 3, characterized in that Application of antimicrobial molecules on the surface of catheters for clinical catheterization.

7. The halotolerant Bacillus according to any one of claims 1 to 6, wherein The Gram-positive drug-resistant bacteria include: oxacillin-resistant Staphylococcus aureus, β-lactam-resistant Staphylococcus aureus, macrolide-resistant and clindamycin-resistant Staphylococcus aureus. Gram-negative bacteria include Escherichia coli and its drug-resistant strains (extended-spectrum β-lactamase (ESBL)-producing Escherichia coli).

8. The halotolerant Bacillus according to any one of claims 1 to 3, characterized in that Application in the preparation of antibacterial liquid against common bacteria Staphylococcus aureus and Escherichia coli.

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