Lactobacillus plantarum against multiple drug-resistant bacteria and application thereof

Abstract

The application discloses a lactobacillus plantarum resisting multiple drug-resistant bacteria and application thereof, and a lactobacillus plantarum 6-14 is separated from collected wild bird excrement, the bacterium has obvious inhibiting effect on multiple drug-resistant bacteria and non-drug-resistant bacteria, including inhibiting methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococcus faecalis, penicillin-resistant streptococcus pneumoniae, staphylococcus aureus, escherichia coli, salmonella enteritidis and salmonella typhimurium, and the application lays a foundation for developing good antibacterial probiotic preparations.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a plant lactobacillus resistant to multiple drug-resistant bacteria and its applications. Background Technology

[0002] It was discovered long ago that some microorganisms inhibit the growth and reproduction of others. With the advancement of science, the nature of this inhibition was finally revealed, and substances that inhibit the growth and reproduction of other microorganisms were found within some microorganisms. These substances were named antibiotics, such as penicillin produced by *Penicillium* and streptomycin produced by *Streptomyces griseus*. Since the 1990s, scientists have broadened the scope of antibiotics, collectively referring to them as biopharmaceuticals. They are mainly used to treat various bacterial infections or diseases caused by pathogenic microorganisms, and generally do not produce serious side effects on their hosts. However, with the widespread use of antibiotics, more and more bacteria have developed resistance, seriously threatening the safety of animals and humans. Antibiotic alternatives have emerged as a result. To date, many antibiotic alternatives have been discovered, mainly including probiotics, oligosaccharides, antimicrobial peptides, enzyme preparations, traditional Chinese medicine preparations, and acidifiers. Currently, probiotics are considered the most promising.

[0003] Probiotic preparations are live bacterial preparations for animals made from known beneficial microorganisms through special processes such as cultivation, extraction, and drying. Probiotic preparations can participate in regulating the balance of the gastrointestinal tract microecology, stimulating specific or non-specific immune function, helping to promote animal growth, enhance animal immune function, and improve the internal and external ecological environment. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0005] The Lactiplantibacillus plantarum 6-14 of this invention is deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number: GDMCC No: 64771, deposit date: June 20, 2024, address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] As one aspect of the present invention, the present invention provides a *Lactiplantibacillus plantarum* 6-14 resistant to multiple drug-resistant bacteria, characterized in that: the *Lactiplantibacillus plantarum* 6-14 has the accession number GDMCC No: 64771.

[0007] The present invention also provides the application of the aforementioned Lactobacillus plantarum 6-14, which is resistant to multiple drug-resistant bacteria, in the preparation of probiotic formulations.

[0008] The present invention also provides the application of the aforementioned Lactobacillus plantarum 6-14, which is resistant to multiple drug-resistant bacteria, in the preparation of microbial preparations with antibacterial effects, characterized in that: the antibacterial effect includes inhibition of drug-resistant bacteria and / or non-drug-resistant bacteria.

[0009] The antibacterial effect includes inhibiting one or more of the following: methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, and Salmonella typhimurium.

[0010] The microbial preparations include those for use on animals.

[0011] The animals mentioned include poultry, livestock, or pets.

[0012] Wherein, the microbial preparations include oral preparations and / or topical preparations.

[0013] Among them, Lactiplantibacillus plantarum 6-14 is acid-resistant.

[0014] Among them, Lactiplantibacillus plantarum 6-14 is bile salt tolerant.

[0015] The beneficial effects of this invention are as follows: This invention isolates a strain of *Lactobacillus plantarum* 6-14 from collected wild bird droppings. This bacterium exhibits significant inhibitory effects against various drug-resistant and non-drug-resistant bacteria. The *Lactobacillus plantarum* 6-14 involved in this invention possesses broad-spectrum antibacterial activity. It is effective against both drug-resistant and non-drug-resistant bacteria. Drug-resistant bacteria include: methicillin-resistant *Staphylococcus aureus* (ATCC43300), vancomycin-resistant *Enterococcus faecalis* (ATCC51299), and penicillin-resistant *Streptococcus pneumoniae* (ATCC51915); non-drug-resistant bacteria include: *Staphylococcus aureus* (ATCC25923), *Escherichia coli* (ATCC25922), *Salmonella enteritidis* (ATCC19585), and *Salmonella typhimurium* (CVCC542). This invention lays the foundation for the development of effective antibacterial probiotic preparations. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0017] Figure 1 This is a colony morphology diagram of Lactobacillus plantarum 6-14.

[0018] Figure 2 The image shows the cell morphology of Lactobacillus plantarum after Gram staining (6-14).

[0019] Figure 3 This is a colony morphology diagram of Lactobacillus plantarum 6-14 on a blood agar plate.

[0020] Figure 4 Electrophoretic identification diagram of 16S rRNA from Lactobacillus plantarum 6-14.

[0021] Figure 5 This is a comparison diagram of the inhibition zones of *Lactobacillus plantarum* 6-14 with two other *Lactobacillus plantarum* strains against drug-resistant bacteria.

[0022] Figure 6 A comparison of the inhibition zones of *Lactobacillus plantarum* 6-14 with two other *Lactobacillus plantarum* strains against non-drug-resistant bacteria.

[0023] Figure 7 The image shows the results of the drug susceptibility test for Lactobacillus plantarum 6-14. Detailed Implementation

[0024] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0025] Example 1:

[0026] Isolation and purification of strains

[0027] 1. Sample Source

[0028] The strain used in this invention was isolated from collected wild bird droppings. More than 60 samples were collected in total for subsequent isolation, purification, and identification.

[0029] 2. Isolation and purification of strains

[0030] A suitable amount of fecal matter was collected using a sterile cotton swab and placed in an EP tube. The tube was immediately sent to the laboratory for bacterial isolation. MRS liquid medium was added to the EP tube containing the fecal matter, and the mixture was vortexed. The mixture was then streaked onto MRS solid medium in a clean bench and incubated at 37°C for 18 hours. After incubation, typical colonies from the agar medium were selected and repeatedly streaked onto MRA solid medium for purification until all colonies on the plate exhibited uniform morphology. Single colonies were then picked and cultured in MRS liquid medium. The resulting strain was stored in MRS liquid medium containing 25% glycerol at -80°C.

[0031] Example 2:

[0032] Oxford Cup Antibacterial Experiment

[0033] The Oxford cup method was used to conduct antibacterial experiments on the purified strains. Pathogenic bacteria included methicillin-resistant Staphylococcus aureus (ATCC43300), vancomycin-resistant Enterococcus faecalis (ATCC51299), penicillin-resistant Streptococcus pneumoniae (ATCC51915), Staphylococcus aureus (ATCC25923), Escherichia coli (ATCC25922), Salmonella enteritidis (ATCC19585), and Salmonella typhimurium (CVCC542). The probiotic bacterial concentration was adjusted to 5 × 10⁻⁶. 8 The concentration of pathogenic bacteria was adjusted to 10 CFU / ml by centrifugation. 5 -10 6 CFU / ml, 100 μl of pathogenic bacterial suspension was evenly spread onto LB solid medium. Oxford cups were placed on the medium, and 200 μl of probiotic supernatant was transferred into the Oxford cups. The mixture was incubated at 37℃ for 18 h, and the diameter of the inhibition zone was measured (Table 1).

[0034] Table 1. Diameter of inhibition zone (mm) in Oxford cup antibacterial test

[0035]

[0036]

[0037] Strains 6-14, which exhibited the best antibacterial effect against all the above pathogens, were selected. Strains 6-14 were sequenced using universal bacterial primers (16S rRNA). They were identified as *Lactobacillus plantarum*, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No. 64771, deposit date: June 20, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences).

[0038] Example 3:

[0039] Analysis of stress resistance of Lactobacillus plantarum 6-14

[0040] 1. Acid resistance of Lactobacillus plantarum 6-14

[0041] Artificial gastric juice was prepared by adjusting the pH of autoclaved PBS to 3.0 with hydrochloric acid. 3 mg of pepsin was added per milliliter of the artificial gastric juice. The mixture was filtered through a 0.22 μm filter, and *Lactobacillus plantarum* 6-14 (1 × 10⁻⁶) was added at a ratio of 10%. 8CFU / ml). The cultures were incubated at 37℃, and counts were performed at 0, 0.5, 1, 2, and 3 h to calculate the survival rate. The results are shown in Table 2. The survival rate of *Lactobacillus plantarum* 6-14 after treatment in simulated gastric fluid at pH 3.0 for 3 h was 76.3%, significantly higher than the other two *Lactobacillus plantarum* strains (65.6% and 43.2%), indicating that *Lactobacillus plantarum* 6-14 has better acid resistance.

[0042] Table 2. Stress resistance of Lactobacillus plantarum 6-14 under acidic conditions.

[0043]

[0044]

[0045] 2. Bile salt tolerance of Lactobacillus plantarum 6-14

[0046] MRS liquid culture medium containing 0.3% bile salts was prepared by adding 3 mg of porcine bile salts per milliliter of autoclaved medium, filtering through a 0.22 μm filter, and adding Lactobacillus plantarum 6-14 (1×10⁻⁶) at a ratio of 10%. 8 CFU / ml). The cultures were incubated at 37℃, and counts were performed at 0, 0.5, 1, 2, and 3 h to calculate the survival rate. The results are shown in Table 3. The survival rate of *Lactobacillus plantarum* 6-14 after 3 h in MRS liquid culture containing 0.3% bile salts was 60.2%, significantly higher than the other two *Lactobacillus plantarum* strains (50.6% and 40.0%), indicating that *Lactobacillus plantarum* 6-14 has a certain tolerance to bile salts.

[0047] Table 3. Stress resistance of Lactobacillus plantarum 6-14 under bile salt conditions.

[0048] strain name 0.5h survival rate 1-hour survival rate 2-hour survival rate 3-hour survival rate Lp 6-14 84.2％ 79.3％ 65.1％ 60.2％ Lp 8-3 75.8％ 60.0％ 55.2％ 50.6％ Lp 7-8 74.7％ 61.6％ 50.4％ 40.0％

[0049] Example 4:

[0050] Safety analysis of Lactobacillus plantarum 6-14

[0051] Weigh 3.9g of Columbia agar medium, dissolve it in 100ml of distilled water by heating, autoclave at 121℃ for 15 minutes, and after the medium cools to about 50℃, add 5% sterile defibrinated sheep blood, mix well, and pour into sterile Petri dishes. Inoculate the bacterial suspension onto the plates using the three-zone streak method, and incubate at 37℃ for 18 hours, observing for hemolysis. It can be observed that the colonies are intact, with no hemolysis around them.

[0052] Example 5:

[0053] Drug susceptibility test of Lactobacillus plantarum 6-14

[0054] The concentration of Lactobacillus plantarum 6-14 was adjusted to 1.5 × 10⁻⁶. 8 CFU / ml, adjust antibiotic concentrations: erythromycin (1.5 mg / ml), vancomycin (3 mg / ml), ampicillin (1 mg / ml), tetracycline (3 mg / ml). Take 100 μl of the adjusted bacterial suspension and spread it evenly on MRS solid medium. Place a circular filter paper disc firmly onto the solid medium, add 10 μl of antibiotic to the filter paper disc, and incubate at 37°C for 18 h. Measure the diameter of the inhibition zone (Table 3).

[0055] Table 3. Diameter of inhibition zone (mm) in drug susceptibility test

[0056] antibiotic name Erythromycin Vancomycin Ampicillin tetracycline Lp 6-14 28±6.5 20±7.5 21±7 27±7

[0057] The above experiments show that *Lactobacillus plantarum* 6-14 is sensitive to erythromycin, vancomycin, ampicillin, and tetracycline.

[0058] Example 6:

[0059] Application of Lactobacillus plantarum 6-14

[0060] 1. Application in pet food: Lactobacillus plantarum 6-14 is processed into freeze-dried bacterial powder, and 2 x 10 g of the powder is added per gram of regular dog food. 6 Mix CFU of active Lactobacillus plantarum at a ratio of 6-14 and feed.

[0061] 2. Adding to pet shampoo: Adjust the concentration of Lactobacillus plantarum 6-14 to 1×10⁻⁶. 9 CFU / ml, centrifuge and collect the supernatant, add the bacterial supernatant to regular pet shampoo at a 1:1 ratio.

[0062] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A *Lactiplantibacillus plantarum* 6-14 resistant to multiple drug-resistant bacteria, characterized in that: The Lactobacillus plantarum 6-14 has the accession number GDMCC No: 64771.

2. The application of Lactobacillus plantarum 6-14, which is resistant to multiple drug-resistant bacteria according to claim 1, in the preparation of microbial preparations with antibacterial activity, characterized in that: The Lactiplantibacillus plantarum 6-14 strain can inhibit methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, and Salmonella typhimurium.

3. The application according to claim 2, characterized in that: The microbial preparations include those for use in animals.

4. The application according to claim 3, characterized in that: The animals mentioned include poultry, livestock, or pets.

5. The application according to claim 3, characterized in that: The microbial preparations include oral preparations and / or topical preparations.

6. The application according to claim 2, characterized in that: The Lactobacillus plantarum 6-14 is acid-resistant.

7. The application according to claim 2, characterized in that: The Lactobacillus plantarum 6-14 strain is tolerant to bile salts.

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