Lactobacillus rhamnosus and application thereof

Abstract

The invention belongs to the field of microorganisms, and relates to lactobacillus rhamnosus and application thereof. The lactobacillus rhamnosus KC3 is separated from Xinjiang healthy bactrian camel milk, and the lactobacillus rhamnosus KC3 has the activity of inhibiting various pathogenic bacteria such as escherichia coli, klebsiella pneumoniae, salmonella, proteus mirabilis, staphylococcus aureus, staphylococcus epidermidis, listeria monocytogenes and streptococcus agalactiae. The metabiotics of the strain can enhance the sensitivity of pathogenic bacteria such as escherichia coli and staphylococcus aureus to antibiotics such as beta-lactam antibiotics, and can synergistically enhance the antibacterial effect of the beta-lactam antibiotics to the pathogenic bacteria such as escherichia coli, staphylococcus aureus, CRE or MRSA. The lactobacillus rhamnosus KC3 metagen can also destroy cell walls of pathogenic bacteria such as escherichia coli, staphylococcus epidermidis, CRE or MRSA and the like, and the formation of bacterial biofilms is inhibited.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a Lactobacillus rhamnosus strain and application thereof. Background Art

[0002] Antibiotic microbial resistance (AMR) has become a major global public health issue. In 2019, approximately 4.95 million deaths worldwide were related to AMR, of which approximately 1.27 million deaths were directly related to AMR. If no measures are taken, by 2050, AMR will cause 10 million deaths each year and economic losses will reach 100 trillion US dollars. In May 2024, the World Health Organization released the "2024 List of Priority Bacterial Pathogens". Among the 15 drug-resistant bacteria involved, carbapenem-resistant Enterobacteriaceae (CRE) and methicillin-resistant Staphylococcus aureus (MRSA) are key priority bacteria and high priority bacteria, respectively. Data from the China Antimicrobial Resistance Monitoring Network show that CRE isolated in Chinese hospitals is on the rise. For example, the resistance rate of Klebsiella pneumoniae to meropenem has increased nearly tenfold from 2005 (2.9%) to 2021 (24.4%); the national average MRSA detection rate in 2023 is 29.1%, which is also on the rise and remains at a high level. At present, antibiotics are still the main means of treating bacterial infectious diseases, but data from the China Antimicrobial Resistance Monitoring Network show that common pathogens have shown serious resistance to conventional antibiotics such as ampicillin, cefazolin, ciprofloxacin, penicillin G, etc., and are impacting the "last line of defense of antibiotics" - carbapenem antibiotics. Therefore, it is particularly important to take necessary measures to address the problem of bacterial antibiotic resistance.

[0003] Drug-resistance genes are the main reason for the resistance of pathogens. For example, CRE often carries genes such as blaSHV, blaCTX-M, blaOXA and blaKPC, which can produce extended-spectrum β-lactamases and mediate resistance to β-lactams, aminoglycosides and fluoroquinolones. MRSA often carries genes such as mecA and mecC. The penicillin-binding protein (PBP2a) encoded by mecA is a new penicillin-binding protein with low affinity for almost all β-lactam antibiotics. In addition, biofilm is another important cause of drug resistance in pathogens. Biofilm refers to a large number of bacterial aggregates formed by bacteria adhering to the contact surface, secreting polysaccharide matrix, fibrin, lipids and extracellular DNA, etc., and wrapping themselves in it. Compared with planktonic bacteria, the drug resistance of bacteria in biofilms is increased by more than a thousand times, and it can promote the horizontal transmission of drug-resistance genes and virulence genes. In the host, biofilms can mediate the immune escape of pathogens to the host, allowing pathogens to exist in the host for a long time and recur repeatedly. The strong biofilm-forming ability of CRE and MRSA is also an important reason why it is difficult to effectively contain them clinically. It may also cause the persistent residue of pathogens and the horizontal transmission of drug resistance, posing a serious threat to public health, food health and animal husbandry.

[0004] At present, scientists have invested a lot of time and energy in the hope of discovering new active substances that can curb the problem of bacterial resistance from the perspectives of plants, microorganisms (lactic acid bacteria) and bacteriophages. Among them, lactic acid bacteria is an ideal biological antibacterial agent that can inhibit pathogens by producing bacteriocins, competing for nutrients, and regulating host immune responses. Bacteriocins produced by lactic acid bacteria are a class of proteins or polypeptides with antibacterial activity. They are safe, non-toxic, have a relatively wide antimicrobial spectrum, and have no cross-resistance with antibiotics. They have great application potential. And when bacteriocins produced by lactic acid bacteria are used in combination with other antimicrobial agents, they can increase the sensitivity of pathogens to antibiotics. For example, the antibacterial activity of nisin in the food system increases after combining with sodium lactate. When Nisin is used in combination with conventional antibiotics (amoxicillin and ceftriaxone, etc.), it can synergistically increase the inhibitory effect of antibiotics on Streptococcus suis. In addition, the combined use of bacteriocins and antibiotics can also synergistically inhibit the biofilm. For example, when Nisin and its derivatives are used in combination with antibiotics, they can synergistically inhibit the biofilm of Pseudomonas and Staphylococcus.

[0005] In summary, lactic acid bacteria and their metabolites have great application potential in the prevention and treatment of drug-resistant bacteria. They can be used as new natural antibacterial agents to reduce the use of antibiotics and provide new solutions to address the threat of super bacteria. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the problem of existing antibiotic resistance, and provide a Lactobacillus rhamnosus strain and application thereof.

[0007] The present invention isolates and screens a strain of Lactobacillus rhamnosus KC3 that can inhibit multiple pathogens from the milk of healthy Bactrian camels in Changji Hui Autonomous Prefecture, Xinjiang. The postbiotics of this strain (Lactobacillus rhamnosus KC3 postbiotics) can synergistically increase the antibacterial effect of antibiotics such as β-lactam antibiotics on Escherichia coli and Staphylococcus aureus; it can enhance the sensitivity of CRE to imipenem and enhance the sensitivity of MRSA to cefoxitin. Lactobacillus rhamnosus KC3 postbiotics can also destroy the cell walls of Escherichia coli and Staphylococcus epidermidis, inhibit the formation of bacterial biofilms; it also has a good inhibitory effect on the formation of CRE and MRSA biofilms. The strain has a complete gene cluster for synthesizing new bacteriocins, which can provide strain resources and gene information for the development and application of new bacteriocins.

[0008] The purpose of the present invention is to provide a strain of Lactobacillus rhamnosus.

[0009] The object of the present invention is to provide the use of the Lactobacillus rhamnosus in preparing products that inhibit pathogens.

[0010] The purpose of the present invention is to provide a Lactobacillus rhamnosus postbiotic.

[0011] The object of the present invention is to provide the application of the Lactobacillus rhamnosus postbiotic in the preparation of a product for inhibiting bacteria.

[0012] The present invention also aims to provide the use of the Lactobacillus rhamnosus postbiotic in the preparation of a product for inhibiting bacterial biofilm formation.

[0013] The present invention also aims to provide the use of the Lactobacillus rhamnosus postbiotic in the preparation of products for enhancing the sensitivity of bacteria to antibiotics.

[0014] The present invention also aims to provide the use of the Lactobacillus rhamnosus postbiotic in combination with an antibiotic in the preparation of a product that inhibits bacteria.

[0015] The above-mentioned purpose of the present invention is achieved by the following technical means:

[0016] The present invention provides a strain of Lactobacillus rhamnosus, named Lactobacillus rhamnosus KC3, which has been deposited in China Center for Type Culture Collection on October 15, 2024, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC NO: M 20242212. The nucleotide sequence of 16S rDNA of the strain is shown in SEQ ID NO: 1.

[0017] The Lactobacillus rhamnosus is used in preparing a product for inhibiting pathogens, wherein the pathogens include Escherichia coli, Klebsiella pneumoniae, Salmonella, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes or Streptococcus agalactiae.

[0018] A Lactobacillus rhamnosus postbiotic is a postbiotic of the Lactobacillus rhamnosus.

[0019] The preparation method of the Lactobacillus rhamnosus postbiotic comprises the following steps: culturing Lactobacillus rhamnosus KC3 in a liquid culture medium, centrifuging to obtain a supernatant, filtering and sterilizing to obtain the Lactobacillus rhamnosus postbiotic.

[0020] The application of the Lactobacillus rhamnosus postbiotic in the preparation of products that inhibit bacteria.

[0021] Preferably, the bacteria include Escherichia coli, Klebsiella pneumoniae, Salmonella, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus (MRSA) or carbapenem-resistant Enterobacteriaceae (CRE).

[0022] The application of the Lactobacillus rhamnosus postbiotic in the preparation of a product that inhibits bacterial biofilm formation.

[0023] Preferably, the bacteria include Escherichia coli, Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus (MRSA) or carbapenem-resistant Enterobacteriaceae (CRE).

[0024] The application of the Lactobacillus rhamnosus postbiotic in the preparation of products that enhance the sensitivity of bacteria to antibiotics.

[0025] Preferably, the bacteria include Escherichia coli or Staphylococcus aureus; the antibiotics include ampicillin (AMP), amoxicillin (AM), meropenem (MEM), ceftiofur (FUR), cefotaxime (CTX), cefazolin (KZ), penicillin (P), tetracycline (TE) or rifampicin (RD).

[0026] The application of the Lactobacillus rhamnosus postbiotic in combination with an antibiotic in the preparation of a product that inhibits bacteria.

[0027] Preferably, the bacteria include Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) or carbapenem-resistant Enterobacteriaceae (CRE). The antibiotics include ampicillin sodium, amoxicillin (AM), cefotaxime (CTX), cefazolin (KZ), imipenem or cefoxitin (FOX).

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The Lactobacillus rhamnosus KC3 of the present invention has the activity of inhibiting multiple pathogens such as Escherichia coli, Klebsiella pneumoniae, Salmonella, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Streptococcus agalactiae, etc. The postbiotics of the strain can enhance the sensitivity of pathogens such as Escherichia coli and Staphylococcus aureus to antibiotics such as β-lactam antibiotics, and can synergistically increase the antibacterial effect of β-lactam antibiotics on pathogens such as Escherichia coli, Staphylococcus aureus, CRE or MRSA. The postbiotics of the Lactobacillus rhamnosus KC3 of the present invention can also destroy the cell walls of pathogens such as Escherichia coli, Staphylococcus epidermidis, CRE or MRSA, and inhibit the formation of bacterial biofilms. The strain has a complete gene cluster for synthesizing new bacteriocins, which can provide strain resources and gene information for the development and application of new bacteriocins, and has a far-reaching practical application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the phylogenetic tree of the KC3 strain in Example 1 of the present invention.

[0031] Figure 2 This is the colony morphology of the KC3 strain in Example 1 of the present invention.

[0032] Figure 3 This is the effect of the Lactobacillus rhamnosus KC3 postbiotics on Escherichia coli and drug-resistant Escherichia coli in Example 3 of the present invention.

[0033] Figure 4 This is the effect of the Lactobacillus rhamnosus KC3 postbiotics in Example 3 of the present invention on Staphylococcus aureus and drug-resistant Staphylococcus aureus.

[0034] Figure 5 This is the effect of the combined use of Lactobacillus rhamnosus KC3 postbiotics and antibiotics on ATCC25922 and ATCC29213 in Example 4 of the present invention, A represents ATCC 25922; B represents ATCC 29213.

[0035] Figure 6 This is the result of the inhibitory effect of the Lactobacillus rhamnosus KC3 postbiotic on DE17 in Example 5 of the present invention.

[0036] Figure 7 This is the result of the inhibitory effect of the Lactobacillus rhamnosus KC3 postbiotic on ATCC 35984 in Example 5 of the present invention.

[0037] Figure 8The scanning electron microscopy results of the inhibitory effect of the postbiotics of Lactobacillus rhamnosus KC3 on pathogenic bacteria in Example 5 of the present invention are shown. In the figure, A1-D1 represent DE17; A2-D2 represent ATCC 29213.

[0038] Fig. 9 This is the result of the inhibitory effect of the Lactobacillus rhamnosus KC3 postbiotic on CRE-1 in Example 5 of the present invention.

[0039] Fig.10 The results of the inhibitory effect of the Lactobacillus rhamnosus KC3 postbiotic on MRSA-2 to MRSA-3 in Example 5 of the present invention are shown. In the figure, A represents MRSA-1, B represents MRSA-2, and C represents MRSA-3.

[0040] Fig.11 This is the change result of the antibacterial activity of Lactobacillus rhamnosus KC3 after postbiotic enzyme treatment in Example 6 of the present invention.

[0041] Fig.12 This is the synthesis pathway of bacteriocin in the genome of Lactobacillus rhamnosus KC3 predicted by AntiSMASH in Example 7 of the present invention.

[0042] Fig.13 It is the gene cluster encoding bacteriocin in the genome of Lactobacillus rhamnosus KC3 in Example 7 of the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0044] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0045] Example 1

[0046] 1. Isolation and identification of Lactobacillus rhamnosus

[0047] Healthy camel milk samples were collected from Changji Hui Autonomous Prefecture, Xinjiang, and stored and transported at low temperature. Under the sterile environment of the laboratory, 2 mL of camel milk sample was centrifuged at 8000r / min for 2 minutes and resuspended with sterile saline. After gradient dilution, the milk sample of appropriate concentration was spread on MRS agar medium and cultured at 37℃ for 24h~48h. Single colonies with round, medium-sized, slightly white protrusions and neat edges were picked and inoculated on MRS solid medium containing 1% CaCO3 and cultured at 37℃ for 24h~48h. Single colonies with calcium-dissolving rings were selected for pure culture, and then Gram staining was performed for microscopic examination. Gram-positive bacteria with spherical or rod-shaped shapes were stored in glycerol tubes at -80℃ for later use. The 16S rRNA gene of the preserved strains was amplified and sequenced using the universal primers 27F (5′-AGA GTT GAT CTG GTC AG-3′) and 1492R (5′-TAC GGT ACC TTG TTA CGA CTT-3′) of the bacterial 16S rRNA gene. The sequencing work was completed by Shanghai Shenggong Biotechnology Co., Ltd. After obtaining the 16S rRNA gene sequence, the sequence was compared on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) to determine the genus and species of the strains. Among them, the strain numbered KC3 (KC3 strain) has the highest homology with Lactobacillus rhamnosus and is named Lactobacillus rhamnosus KC3. The strain has been deposited in the China Center for Type Culture Collection (Wuhan University) on October 15, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20242212. The nucleotide sequence of 16S rDNA of KC3 strain is shown in SEQ ID NO: 1. The phylogenetic tree of KC3 strain is shown in Figure 1 shown.

[0048] The KC3 strain appears as milky white colonies on MRS solid medium, which are round, medium in size, and have neat edges. Figure 2 shown.

[0049] 2. Biochemical identification

[0050] The biochemical identification tube set of lactic acid bacteria (Qingdao Haibo Biotechnology Co., Ltd.) was used for biochemical identification of Lactobacillus rhamnosus KC3.

[0051] The fermentation ability of Lactobacillus rhamnosus KC3 on 11 substances is shown in Table 1 below.

[0052] Table 1

[0053] Strain number Esculin Cellobiose maltose Mannitol Salicin Sorbitol sucrose Raffinose Inulin lactose 1% Sodium Hippurate KC3 + + + + + + + - + + +

[0054] Note: + indicates that it can be fermented, - indicates that it cannot be fermented.

[0055] The results in Table 1 show that Lactobacillus rhamnosus KC3 can ferment conventional sugars such as esculin, cellobiose, maltose, and sodium hippurate, but does not have the ability to ferment raffinose.

[0056] 3. Determination of acid resistance, alkali resistance and bile salt resistance

[0057] Lactobacillus rhamnosus KC3 was inoculated into MRS solid culture medium and cultured at 37°C overnight, and then a single colony was picked and transferred to fresh MRS liquid culture medium and cultured until the bacterial liquid became cloudy, thereby obtaining activated Lactobacillus rhamnosus KC3.

[0058] The activated Lactobacillus rhamnosus KC3 was inoculated at a ratio of 1:100 into MRS liquid culture medium with pH values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and cultured at 37°C for 24 h to determine the acid and alkali resistance of the KC3 strain.

[0059] The activated Lactobacillus rhamnosus KC3 was inoculated into MRS liquid culture medium with ox bile salt contents of 0, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, and 0.30%, and cultured at 37°C for 24 hours to determine the bile salt tolerance of the KC3 strain.

[0060] The results showed that the KC3 strain could grow in the pH range of 4 to 8, but not in the pH range of 1 to 3 and 9 to 10. The KC3 strain could grow when the ox bile salt content was 0.05 to 0.20%, but not when the ox bile salt content was 0.25% and 0.30%.

[0061] In summary, Lactobacillus rhamnosus KC3 has potential prebiotic function, and its growth ability shows a wide range of pH tolerance and bile salt tolerance. It has the potential to withstand the killing of gastrointestinal fluid and bile in animals and colonize in animals for a long time.

[0062] 4. Determination of the antibacterial spectrum of Lactobacillus rhamnosus KC3

[0063] Target strains: ATCC 25922 (Escherichia coli), ATCC 13883 (Klebsiella pneumoniae), ATCC 14028 (Salmonella), ATCC 43071 (Proteus mirabilis), ATCC 29213 (Staphylococcus aureus), ATCC 25923 (Staphylococcus aureus), ATCC 35984 (Staphylococcus epidermidis), ATCC 19115 (Listeria monocytogenes), ATCC 13813 (Streptococcus agalactiae).

[0064] The Oxford cup method was used to determine the antibacterial spectrum of Lactobacillus rhamnosus KC3. The target strain cultured to the logarithmic phase was adjusted to 1×10 8 CFU / mL and set aside; first spread 1.5% agar on the bottom of the plate, and evenly place the sterilized Oxford cup on it after solidification, and pour in the test strain (10 8 CFU / mL) of TSA semi-solid medium, take out the Oxford cup after it solidifies, add 100μL CFS into the well, and use sterile MRS as a control. After culturing at 37℃ for 24h, take out and observe the presence of inhibition zone to judge the presence of antibacterial activity.

[0065] The results showed that Lactobacillus rhamnosus KC3 had antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Salmonella, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Streptococcus agalactiae.

[0066] In summary, the KC3 strain can inhibit Escherichia coli, Klebsiella pneumoniae, Salmonella, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Streptococcus agalactiae.

[0067] Example 2

[0068] The preparation of Lactobacillus rhamnosus KC3 postbiotic powder is as follows:

[0069] Lactobacillus rhamnosus KC3 was inoculated into 5 mL of MRS liquid culture medium, cultured at 37°C for 24 hours, and then transferred to 195 mL of MRS liquid culture medium for further culture for 48 hours, centrifuged (4°C, 6000 r / min, 10 min), the supernatant was filtered and sterilized, and then placed in a -80°C refrigerator for overnight freezing, and then vacuum freeze-dried to obtain Lactobacillus rhamnosus KC3 postbiotic powder. Lactobacillus rhamnosus KC3 postbiotic powder was dissolved in sterile water to a final dry matter concentration of 0.25 g / mL, filtered and sterilized, and stored in a 4°C refrigerator for later use.

[0070] Example 3

[0071] 1. Determination of the minimum inhibitory concentration (MIC) and sub-inhibitory concentration (SIC) of Lactobacillus rhamnosus KC3 postbiotics against pathogens

[0072] Lactobacillus rhamnosus KC3 postbiotic powder was diluted in a 96-well cell culture plate using the serial dilution method, and the final concentrations were 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.906, 1.953, 0.977 and 0.489 mg / mL, respectively. The activated bacterial suspensions of ATCC 25922 (Escherichia coli), ATCC 13883 (Klebsiella pneumoniae), ATCC 14028 (Salmonella), ATCC 43071 (Proteus mirabilis), ATCC 29213 (Staphylococcus aureus), ATCC 25923 (Staphylococcus aureus), ATCC35984 (Staphylococcus epidermidis), ATCC 19115 (Listeria monocytogenes), and ATCC 13813 (Streptococcus agalactiae) were inoculated into culture plates at a ratio of 1:100 after 0.5 McFarland turbidimetry, cultured at 37°C overnight, and the MIC and SIC of Lactobacillus rhamnosus KC3 were determined.

[0073] The results of MIC and SIC determination of Lactobacillus rhamnosus KC3 against 9 pathogens are shown in Table 2.

[0074] Table 2

[0075] Pathogens MIC (mg / mL) SIC (mg / mL) ATCC25922 (Escherichia coli) 31.25 3.906 ATCC13883 (Klebsiella pneumoniae) 31.25 1.953 ATCC14028 (Salmonella) 31.25 7.8125 ATCC43071 (Proteus mirabilis) 31.25 1.953 ATCC29213 (Staphylococcus aureus) 31.25 3.906 ATCC25923 (Staphylococcus aureus) 31.25 3.906 ATCC35984 (Staphylococcus epidermidis) 31.25 7.8125 ATCC19115 (Listeria monocytogenes) 31.25 7.8125 ATCC13813 (Streptococcus agalactiae) 31.25 3.906

[0076] The results in Table 2 show that the postbiotics of Lactobacillus rhamnosus KC3 have antibacterial activity against 9 test strains including different genera and species of Gram-negative and Gram-positive bacteria.

[0077] 2. Determination of the antibacterial activity of Lactobacillus rhamnosus KC3 postbiotics against pathogenic bacteria

[0078] The Oxford cup double-layer agar diffusion method was used to detect the antibacterial activity of Lactobacillus rhamnosus KC3 postbiotics against pathogens. The target strains included: ATCC 25922 (Escherichia coli), ATCC 29213 (Staphylococcus aureus), 30 randomly selected clinically isolated Staphylococcus aureus (including MRSA and wild Staphylococcus aureus), and 30 randomly selected carbapenem-resistant Enterobacteriaceae.

[0079] All 30 clinically isolated Staphylococcus aureus and 30 avian pathogenic Escherichia coli were isolated from diseased samples sent for inspection after clinical death. After culture with selective culture medium, 16S rRNA sequencing and homology comparison were performed to determine the genus and species of the strains. All the above strains were preserved by the Tarim Animal Disease Diagnosis and Prevention Engineering Laboratory of the Xinjiang Production and Construction Corps. The 30 strains of avian pathogenic Escherichia coli were tested for drug sensitivity using the KB paper disc method in accordance with the CLSI (2021) experimental standards to determine the strains' drug resistance and multidrug resistance characteristics (the multidrug resistance judgment standard is: strains that are resistant to 3 or more types of antimicrobial drugs at the same time are regarded as multidrug-resistant "multiple drug resistance, MDR" strains). The steps and judgment criteria for the confirmation experiments of methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Enterobacteriaceae (CRE) were referred to CLSI (2021).

[0080] Take the target strain cultured to the logarithmic phase and perform 0.5 McFarland turbidimetry for later use; first spread 1.5% agar on the bottom of the plate, and after solidification, evenly place a sterilized Oxford cup on it, and pour in the test strain (10 8 CFU / mL) of TSA semi-solid medium, take out the Oxford cup after it solidifies, add 100μL of Lactobacillus rhamnosus KC3 postbiotic powder dilution (250mg / mL) into the well (3 parallels), and use sterile MRS as a control. After culturing at 37℃ for 24h, take out and observe whether there is an inhibition zone (if there is an inhibition zone, there is antibacterial activity), and measure the diameter of the inhibition zone.

[0081] Effects of Lactobacillus rhamnosus KC3 postbiotics on Escherichia coli and drug-resistant Escherichia coli Figure 3 As shown in Figure 2, the effects of Lactobacillus rhamnosus KC3 postbiotics on Staphylococcus aureus and drug-resistant Staphylococcus aureus are as follows Figure 4 As shown,

[0082] The results showed that Lactobacillus rhamnosus KC3 postbiotics had antibacterial activity against 30 strains of avian pathogenic Escherichia coli and 30 strains of clinically isolated Staphylococcus aureus.

[0083] In summary, the postbiotics of Lactobacillus rhamnosus KC3 have antibacterial activity against 9 pathogens including different genera and species of Gram-negative and Gram-positive bacteria, including ATCC 25922 (Escherichia coli), ATCC 13883 (Klebsiella pneumoniae), ATCC 14028 (Salmonella), ATCC 43071 (Proteus mirabilis), ATCC 29213 (Staphylococcus aureus), ATCC 25923 (Staphylococcus aureus), ATCC 35984 (Staphylococcus epidermidis), ATCC 19115 (Listeria monocytogenes), and ATCC 13813 (Streptococcus agalactiae). Using Escherichia coli and Staphylococcus aureus as model organisms for Gram-negative and Gram-positive pathogens, Lactobacillus rhamnosus KC3 postbiotics also showed antibacterial activity against 30 randomly selected avian pathogenic Escherichia coli and 30 clinical isolates of Staphylococcus aureus. Lactobacillus rhamnosus KC3 postbiotics can be prepared into inhibitors of pathogenic bacteria (including Escherichia coli, Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae and methicillin-resistant Staphylococcus aureus, etc.).

[0084] Example 4

[0085] 1. Effect of Lactobacillus rhamnosus KC3 postbiotics on antibiotic sensitivity

[0086] ATCC 29213 (Staphylococcus aureus) and ATCC 25922 (Escherichia coli) were used as target strains. After the MH agar medium was sterilized by high pressure, the medium was cooled to 40-50°C, and Lactobacillus rhamnosus KC3 postbiotic powder was added to make the final concentrations of SIC for each pathogen, respectively. The MH agar medium without Lactobacillus rhamnosus KC3 postbiotic powder was used as the control group. After culturing ATCC29213 and ATCC 25922 strains overnight, the KB paper disk method was used to detect whether the postbiotics of Lactobacillus rhamnosus KC3 had an effect on enhancing the sensitivity of pathogens to antibiotics. The antibiotics selected were ampicillin (AMP), amoxicillin (AM), meropenem (MEM), cefuroxime (CPT), ceftiofur (FUR), ceftriaxone (CTX), cefazolin (KZ), penicillin (P), cefoxitin (FOX), tetracycline (TE), rifampicin (RD), ciprofloxacin (CIP), tilmicosin (TIL); amikacin (AK); and clindamycin (DA).

[0087] Effects of combined use of Lactobacillus rhamnosus KC3 postbiotics and antibiotics on ATCC 25922 and ATCC29213 Figure 5As shown, A represents ATCC 25922; B represents ATCC 29213; MH is the blank control, the amount of postbiotic powder added to Lactobacillus rhamnosus KC3 is 0 mg / mL; SIC: the amount of postbiotic powder added to Lactobacillus rhamnosus KC3 is 3.906 mg / mL. The results showed that under the treatment of Lactobacillus rhamnosus KC3 postbiotics, the pathogen showed an enhanced sensitivity to some or all of the nine selected β-lactam antibiotics. For the target strain ATCC 25922, after treatment with Lactobacillus rhamnosus KC3 postbiotics, its sensitivity to AMP, AM, MEM, KZ, and TE was significantly increased, and there was no difference in sensitivity to CPT, FUR, CTX, P, FOX, RD, CIP, TIL, AK, and DA; for the target strain ATCC 29213, after treatment with Lactobacillus rhamnosus KC3 postbiotics, its sensitivity to AMP, AM, MEM, FUR, CTX, KZ, P, TE, and RD was significantly increased, there was no difference in sensitivity to CPT, FOX, and CIP, and the sensitivity to TIL, AK, and DA was significantly decreased. This shows that Lactobacillus rhamnosus KC3 postbiotics can enhance the sensitivity of pathogens (including bacteria such as Staphylococcus aureus and Escherichia coli) to β-lactam antibiotics.

[0088] 2. Test on the effect of combined use of Lactobacillus rhamnosus KC3 and antibiotics on Escherichia coli and Staphylococcus aureus

[0089] Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213) were used as target strains. The checkerboard method was used to detect the combined effect of Lactobacillus rhamnosus KC3 postbiotics and antibiotics on a 96-well cell culture plate. The antibiotic detection concentrations were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml; the detection concentrations of Lactobacillus rhamnosus KC3 postbiotics were MIC, 1 / 2MIC, 1 / 4MIC, SIC, 1 / 2SIC, and 1 / 4SIC for each target strain. 50 μL of fresh MH medium was added to well A12 and row H, and 50 μL of bacterial solution (0.5 McFarland turbidimetric) was added to each of the remaining wells. Among them, H1 is the growth control well (containing 100 μL diluted bacterial solution + 100 μL MH blank medium), and A12 is the blank control well (100 μL antibiotics + 50 μL Lactobacillus rhamnosus KC3 postbiotics + 50 μL MH blank medium). After the 96-well plate was placed at 37°C for 24 hours, the OD 600nm To determine the MIC and judge the changes of the MIC.

[0090] The FIC index was introduced as an evaluation standard for the effect of the combined use of Lactobacillus rhamnosus KC3 postbiotics and antibiotics. The formula of the FIC index is as follows:

[0091] FIC index = FIC(A) + FIC(B) = [A] / MIC A +[B] / MIC B ,

[0092] FIC(A) is the FIC of drug A against the microorganism, where [A] is the concentration of drug A and MIC A is the MIC of drug A against the indicator strain; FIC(B), MIC B and [B] is defined the same as for drug A.

[0093] The FIC index judgment criteria are: <0.5, judged as synergy; 0.5-0.75, judged as partial synergy; 0.76-1, judged as cumulative effect; 1.0-4.0, judged as no difference; >4.0, judged as antagonism.

[0094] The effects of Lactobacillus rhamnosus KC3 postbiotics combined with antibiotics on ATCC 25922 and ATCC 29213 are shown in Table 3.

[0095] Table 3

[0096]

[0097] The results in Table 3 show that the postbiotics of Lactobacillus rhamnosus KC3 can synergistically increase the antibacterial effect of β-lactam antibiotics on ATCC25922 and ATCC 29213, and the postbiotics of Lactobacillus rhamnosus KC3 have a synergistic effect with ampicillin sodium, amoxicillin, cefotaxime, and cefazolin. This indicates that the postbiotics of Lactobacillus rhamnosus KC3 can synergistically increase the antibacterial effect of β-lactam antibiotics on Escherichia coli and Staphylococcus aureus. The postbiotics of Lactobacillus rhamnosus KC3 can be combined with β-lactam antibiotics to prepare an antibacterial agent that inhibits bacteria such as Escherichia coli and Staphylococcus aureus.

[0098] 3. Test on the effect of combined use of Lactobacillus rhamnosus KC3 and antibiotics on carbapenem-resistant Enterobacteriaceae and methicillin-resistant Staphylococcus aureus

[0099] The carbapenem-resistant Enterobacteriaceae (CRE-2 to CRE-9) and methicillin-resistant Staphylococcus aureus (MRSA-1 to MRSA-3) isolated in Example 3 were used as target strains, and the chessboard method was used to detect the combined use effect of Lactobacillus rhamnosus KC3 postbiotics and antibiotics on a 96-well cell culture plate. For the target strains CRE-2 to CRE-9, imipenem was selected as the antibiotic; for the target strains MRSA-1 to MRSA-3, cefoxitin was selected as the antibiotic. The antibiotic detection concentrations were 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / ml, and the specific steps were the same as the experiment on the combined use of Lactobacillus rhamnosus KC3 and antibiotics on Escherichia coli and Staphylococcus aureus. The above FIC index was used as the effect evaluation standard.

[0100] The effects of Lactobacillus rhamnosus KC3 postbiotics combined with antibiotics on CRE and MRSA are shown in Table 4.

[0101] Table 4

[0102]

[0103] The results in Table 4 show that there is no difference in the antibacterial effect of Lactobacillus rhamnosus KC3 postbiotics combined with imipenem on CRE-1; on the antibacterial effect of CRE-2 to CRE-9, Lactobacillus rhamnosus KC3 postbiotics can synergistically increase the antibacterial effect of imipenem on CRE; on the antibacterial effect of MRSA-1 to MRSA-3, Lactobacillus rhamnosus KC3 postbiotics can synergistically increase the antibacterial effect of cefoxitin on MRSA. Lactobacillus rhamnosus KC3 postbiotics can be combined with β-lactam antibiotics to prepare antibacterial agents that inhibit MRSA and other bacteria.

[0104] Example 5

[0105] 1. Semi-quantitative detection of the effect of Lactobacillus rhamnosus KC3 postbiotics on DE17 and ATCC 35984 biofilms

[0106] DE17 (avian pathogenic Escherichia coli, from Hu J, Lv X, Niu X, Yu F, Zuo J, Bao Y, et al. Effect of nutritional and environmental conditions on biofilm formation of avian pathogenic Escherichia coli. J Appl Microbiol 2022; 132 (6): 4236-4251.) was used as the standard strain of negative bacteria with film-forming ability, and ATCC 35984 (Staphylococcus epidermidis) was used as the standard strain of positive bacteria with film-forming ability. The crystal violet staining method was used to semi-quantitatively detect the effect of postbiotics on bacterial biofilm. After strain culture (target strain ATCC 35984, target strain DE17 was cultured at 25°C), 0.5 McFarland turbidimetry was performed first, and then 1:50 dilution was performed. The detection concentrations of postbiotics of Lactobacillus rhamnosus KC3 were 15.625, 7.8125, 3.906, and 1.953 mg / mL. Take 100 μL of diluted bacterial solution and add it to a 96-well plate. The target strain without postbiotic treatment of Lactobacillus rhamnosus KC3 was used as the positive control, and the sterile TSB liquid medium was used as the blank control. After overnight culture, fixation, staining, and dissolution were performed, and the OD was measured. 570 nm value.

[0107] The results of the inhibitory effect of Lactobacillus rhamnosus KC3 postbiotics on DE17 are shown in Figure 6 As shown in Figure 2, the inhibitory effect of Lactobacillus rhamnosus KC3 postbiotics on ATCC 35984 was shown in Figure 2. Figure 7 As shown, ns: P ≥ 0.05; *: 0.01 ≤ P ≤ 0.05; **: P ≤ 0.01. The results showed that Lactobacillus rhamnosus KC3 postbiotics showed good biofilm inhibition on DE17 and ATCC 35984. When the addition amount of Lactobacillus rhamnosus KC3 postbiotics reached 1.953 mg / mL, it began to show significant biofilm inhibition, and the biofilm inhibition effect continued to increase with the increase of the content of Lactobacillus rhamnosus KC3 postbiotics. This shows that Lactobacillus rhamnosus KC3 postbiotics can inhibit the formation of bacterial biofilms such as Escherichia coli and Staphylococcus epidermidis.

[0108] 2. Scanning electron microscopy observation of the effect of Lactobacillus rhamnosus KC3 postbiotics on DE17 and ATCC 35984 biofilms

[0109] DE17 (avian pathogenic Escherichia coli): DE17 without treatment with Lactobacillus rhamnosus KC3 postbiotics was used as a blank control. The treatment group added Lactobacillus rhamnosus KC3 postbiotics to LB medium to make its content 1.953 (1 / 2SIC), 3.906 (SIC), and 7.8125 mg / mL (2SIC). The final total volume of the solution was 1 mL. The activated DE17 bacterial solution (0.5 turbidity) was added at a 1% inoculum amount and cultured at 25°C for 12 hours. The scanning electron microscopy sample processing operation was then performed and observed on the microscope.

[0110] ATCC 35984 (Staphylococcus epidermidis): ATCC 35984 without Lactobacillus rhamnosus KC3 postbiotic treatment was used as a blank control. In the treatment group, Lactobacillus rhamnosus KC3 postbiotics were added to LB medium to make its content 3.906 (1 / 2SIC), 7.8125 (SIC) mg / mL, and 15.625 (2SIC) mg / mL. The total volume of the final solution was 1 mL. The activated ATCC 35984 bacterial solution was added at a 1% inoculum amount and cultured at 37°C for 24 hours. The scanning electron microscope sample processing operation was then performed and observed on the microscope.

[0111] Scanning electron microscopy results of the inhibitory effect of Lactobacillus rhamnosus KC3 postbiotics on pathogenic bacteria Figure 8 As shown, in the figure, A1-D1 represents DE17; A2-D2 represents ATCC 29213; A1: Lactobacillus rhamnosus KC3 postbiotic addition amount 0 mg / mL; B1: Lactobacillus rhamnosus KC3 postbiotic addition: 1.953 mg / mL; C1: Lactobacillus rhamnosus KC3 postbiotic addition: 3.906 mg / mL; D1: Lactobacillus rhamnosus KC3 postbiotic addition amount 7.8125 mg / mL; A2: Lactobacillus rhamnosus KC3 postbiotic addition amount 0 mg / mL; B2: Lactobacillus rhamnosus KC3 postbiotic addition amount: 3.906 mg / mL; C2: Lactobacillus rhamnosus KC3 postbiotic addition amount: 7.8125 mg / mL; D2: Lactobacillus rhamnosus KC3 postbiotic addition amount 15.625 mg / mL.

[0112] The results showed that after treatment with Lactobacillus rhamnosus KC3 postbiotics, the biofilm formation of target strains DE17 and ATCC 35984 was significantly inhibited, and the bacteria also underwent morphological changes. With the increase in the amount of Lactobacillus rhamnosus KC3 postbiotics added, the changes in the biofilm and bacterial morphology of target strains DE17 and ATCC 35984 became more significant. This shows that Lactobacillus rhamnosus KC3 postbiotics can inhibit the formation of bacterial biofilms such as Escherichia coli and Staphylococcus epidermidis.

[0113] 3. Semi-quantitative detection of the effect of Lactobacillus rhamnosus KC3 postbiotics on CRE and MRSA biofilms

[0114] The carbapenem-resistant Enterobacteriaceae (CRE-1, note: CRE-2 to CRE-9 have no biofilm forming ability) and methicillin-resistant Staphylococcus aureus (MRSA-1 to MRSA-3) isolated in Example 3 were used as target strains. The crystal violet staining method was used to semi-quantitatively detect the biofilm inhibition effect of postbiotics. The detection concentrations of Lactobacillus rhamnosus KC3 postbiotics were 15.625, 7.8125, 3.906, and 1.953 mg / mL, and the detection method was the same as DE17 and ATCC 35984.

[0115] The results of the inhibitory effect of Lactobacillus rhamnosus KC3 postbiotics on CRE-1 are as follows Fig. 9 As shown in Figure 2, the results of the inhibitory effect of Lactobacillus rhamnosus KC3 postbiotics on MRSA-1 to MRSA-3 are as follows Fig.10 As shown, A represents MRSA-1, B represents MRSA-2, and C represents MRSA-3; ns: P ≥ 0.05; *: 0.01 ≤ P ≤ 0.05; **: P ≤ 0.01. The results showed that Lactobacillus rhamnosus KC3 postbiotics had a good inhibitory effect on CRE-1 (carbapenem-resistant Enterobacteriaceae). When the addition amount of Lactobacillus rhamnosus KC3 postbiotics reached 1.953 mg / mL, it began to show a significant inhibitory effect, and the inhibitory effect continued to increase with the increase of the content of Lactobacillus rhamnosus KC3 postbiotics. Lactobacillus rhamnosus KC3 postbiotics did not show a significant inhibitory effect on MRSA-1 (methicillin-resistant Staphylococcus aureus), but showed a good inhibitory effect on MRSA-2 to MRSA-3. This indicates that Lactobacillus rhamnosus KC3 postbiotics can inhibit the formation of biofilms of drug-resistant bacteria such as carbapenem-resistant Enterobacteriaceae (CRE) and some methicillin-resistant Staphylococcus aureus (MRSA).

[0116] In summary, Lactobacillus rhamnosus KC3 postbiotics have the ability to inhibit the formation of pathogenic biofilm and synergize with β-lactam antibiotics.

[0117] Example 6

[0118] Using the Oxford cup method, Lactobacillus rhamnosus KC3 postbiotics that were only subjected to pH adjustment (ultimately restoring the original pH) in a water bath but not subjected to enzyme treatment and MRS broth culture medium with only enzyme addition were used as the treatment group, and untreated Lactobacillus rhamnosus KC3 postbiotics and blank MRS broth culture medium were used as the control group to preliminarily explore the antibacterial active substances of Lactobacillus rhamnosus KC3 postbiotics.

[0119] The experimental groups containing enzyme treatment were added with proteinase K, pepsin, trypsin, papain, peroxidase, catalase, and peptidase (final concentration was 1.0 mg / mL), respectively. The pH was first adjusted to the optimal pH of each enzyme and then bathed in 37°C water for 2 h, and then adjusted to the initial pH for antibacterial experiments.

[0120] The results of changes in the antibacterial activity of Lactobacillus rhamnosus KC3 after postbiotic enzyme treatment are as follows Fig.11 As shown, ns: P ≥ 0.05; *: 0.01 ≤ P ≤ 0.05; **: P ≤ 0.01.

[0121] The results showed that the blank MRS broth medium and the MRS broth medium control group with only enzyme addition had no antibacterial activity. After different enzyme treatments, the antibacterial effect of Lactobacillus rhamnosus KC3 postbiotics changed to varying degrees. After catalase treatment, the antibacterial activity of Lactobacillus rhamnosus KC3 postbiotics did not change significantly. After peroxidase treatment, the antibacterial effect of Lactobacillus rhamnosus KC3 postbiotics decreased significantly, which may be related to its participation in the decomposition of certain fatty acids in postbiotics. Under the treatment of five enzymes such as trypsin and proteinase K, the diameter of the antibacterial zone of Lactobacillus rhamnosus KC3 postbiotics showed a very significant decrease, among which the decrease of peptidase was the largest. This shows that Lactobacillus rhamnosus KC3 postbiotics contain protein or peptide antibacterial substances.

[0122] Example 7

[0123] The inventors sequenced the whole genome of Lactobacillus rhamnosus KC3 using the second-generation Illumina NovaSeq NGS platform and the third-generation PacBio Sequel single-molecule sequencing technology. The sequencing work was entrusted to Shanghai Lingen Biotechnology Co., Ltd. The sequencing data were spliced ​​using Unicycler software (mixed splicing: using second-generation short reads and third-generation long reads sequencing data at the same time), and the assembly results were evaluated, and finally the whole genome sequence was obtained. The entire genome sequence was then analyzed using GeneMarkS software for gene prediction. AntiSMASH (https: / / antismash.secondarymetabolites.org) was used to analyze the secondary metabolite gene clusters produced by Lactobacillus rhamnosus KC3. BAGEL4 (http: / / bagel4.molgenrug.nl.) was then used to find the types of bacteriocins produced by Lactobacillus rhamnosus KC3 and the number of genes encoding bacteriocins.

[0124] A bacteriocin synthesis pathway was predicted from the whole genome of Lactobacillus rhamnosus KC3 using AntiSMASH and BAGEL4. The bacteriocin synthesis pathway in the genome of Lactobacillus rhamnosus KC3 predicted by AntiSMASH is as follows: Fig.12 shown.

[0125] Using AntiSMASH and BAGEL4, a gene cluster encoding an unnamed new class II bacteriocin was predicted from the whole genome of Lactobacillus rhamnosus KC3. The gene cluster encoding bacteriocin in the genome of Lactobacillus rhamnosus KC3 is as follows: Fig.13 As shown, the gene cluster contains 32 genes, and the core peptides are Enterocin-X-chain-beta, orf00028 and Camocin-CP52. Enterocin-X-chain-beta and orf00028 are mainly described as Bacteriocin class II with double-glycine leader peptide, and Camocin-CP52 is mainly described as canomycin. This gene cluster has 26 coding genes, including two core biosynthetic genes: ABC-type bacterial transporter-related gene (TIGR01193) and bacterial signal sequence (TIGR01847), encoding ABC-type bacteriocin transporter and Protein with bacterial signal sequence, respectively; one additional biosynthetic gene: (PF02517.19), encoding membrane-bound protease C AAX family immunity protein PlnI; and one transport-related gene: Major Facilitator Superfamily (PF07690.19), encoding major facilitator superfamily MFS_1transporter.

[0126] In summary, Lactobacillus rhamnosus KC3 has a complete gene cluster for synthesizing new bacteriocins.

[0127] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus, characterized in that It was named Lactobacillus rhamnosus KC3, and was deposited in the China Center for Type Culture Collection on October 15, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20242212.

2. The use of Lactobacillus rhamnosus according to claim 1 in preparing a product that inhibits pathogens, characterized in that: The pathogenic bacteria include Escherichia coli, Klebsiella pneumoniae, Salmonella, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes or Streptococcus agalactiae.

3. A Lactobacillus rhamnosus postbiotic, characterized in that It is a postbiotic of the Lactobacillus rhamnosus according to claim 1.

4. Use of the Lactobacillus rhamnosus postbiotic according to claim 3 in the preparation of a product that inhibits bacteria.

5. The use according to claim 4, characterized in that: The bacteria include Escherichia coli, Klebsiella pneumoniae, Salmonella, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus or carbapenem-resistant Enterobacteriaceae.

6. Use of the Lactobacillus rhamnosus postbiotic according to claim 3 in the preparation of a product that inhibits bacterial biofilm formation.

7. The use according to claim 6, characterized in that: The bacteria include Escherichia coli, Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus or carbapenem-resistant Enterobacteriaceae.

8. Use of the Lactobacillus rhamnosus postbiotic according to claim 3 in the preparation of a product that enhances the sensitivity of bacteria to antibiotics.

9. The use according to claim 8, characterized in that: The bacteria include Escherichia coli or Staphylococcus aureus; And / or, the antibiotics include ampicillin, amoxicillin, meropenem, ceftiofur, ceftriaxone, cefazolin, penicillin, tetracycline or rifampicin.

10. Use of the Lactobacillus rhamnosus postbiotics in combination with antibiotics in the preparation of a product that inhibits bacteria according to claim 3; preferably, the bacteria include Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus or carbapenem-resistant Enterobacteriaceae; the antibiotics include ampicillin sodium, amoxicillin, cefotaxime, cefazolin, imipenem or cefoxitin.

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