Lactobacillus paracasei W1 and application thereof in inhibiting clostridium difficile infection
The application of Lactobacillus paracasei W1 has solved the problem of the lack of probiotics in the existing technology to inhibit Clostridium difficile infection, and has achieved effective inhibition of Clostridium difficile and treatment of colitis, showing good safety and therapeutic potential.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHONGQING MEDICAL UNIVERSITY
- Filing Date
- 2024-09-05
- Publication Date
- 2026-05-12
AI Technical Summary
Currently, there is a lack of effective probiotic drugs or drug combinations to inhibit Clostridium difficile infection. Existing treatments have the risk of drug resistance and recurrence, and there is an urgent clinical need for treatments with high safety.
A strain of Lactobacillus paracasei W1 was provided, which is tolerant to low pH and high concentration of bile salts in the gastrointestinal tract. Its sterile supernatant can inhibit the growth and biofilm formation of Clostridium difficile in vitro and effectively reduce the mortality rate of infected mice in animal experiments.
Lactobacillus paracasei W1 showed good antibacterial effects both in vitro and in vivo, and can be used to prevent or treat colitis caused by Clostridium difficile infection, reduce the degree of infection and improve colitis symptoms.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microbial engineering technology, and in particular to a Lacticaseibacillus paracasei W1 and its application in inhibiting Clostridioides difficile infection. BACKGROUND
[0002] Clostridioides difficile is a gram-positive anaerobic spore-forming bacterium that is a common pathogen of diarrhea in hospitalized patients. Clostridioides difficile can form spores, and has strong tolerance to physical and chemical factors such as heat, dryness and disinfectants, so that it can survive for a long time in the environment. Therefore, spores are the main form of its transmission in the population. Toxin A and toxin B are the main pathogenic factors of Clostridioides difficile, both of which can act on colon epithelial cells and immune cells, induce innate immunity and cause inflammatory response. It is worth noting that toxin B may be a trigger factor for multiple organ dysfunction syndrome. At the same time, Clostridioides difficile can obtain antibiotic resistance through forming biofilm, plasmid transfer and other ways, thereby affecting the therapeutic effect of antibiotics. Clostridioides difficile infection (CDI) accounts for 30% of antibiotic-associated diarrhea (AAD). Clinical manifestations include mild to moderate diarrhea, pseudomembranous colitis, toxic megacolon and even death. At present, the treatment of Clostridioides difficile infection (CDI) depends on antibiotics such as metronidazole, vancomycin and fidaxomicin, but with the increase of bacterial drug resistance and the risk of CDI recurrence, fecal microorganism transplantation (FMT) has gradually become a more advantageous alternative therapy for the treatment of recurrent CDI. FMT aims to treat CDI by restoring the diversity of intestinal microbiota, rebuilding the intestinal mucosal barrier and restoring the metabolism of microbiota. Some studies have shown that FMT can not only treat colitis-related diseases, but also has potential applications in the treatment of irritable bowel syndrome, depression and even cancer. However, we should still consider the potential risks of FMT. FMT treatment may have some uncertain associations with certain diseases, including peripheral neuropathy, idiopathic thrombocytopenic purpura, Sjogren's syndrome and rheumatoid arthritis. Therefore, there is an urgent need in clinic for new treatment methods with high safety.
[0003] As probiotics play an increasingly important role in the treatment of colitis, the antibacterial activity of specific probiotics against Clostridium difficile remains poorly understood. Previous studies have shown that probiotic combinations containing Lactobacillus acidophilus CL1285 and Lactobacillus casei LBC80R are well-tolerated and effectively reduce the risk of antibiotic-associated diarrhea. Furthermore, a meta-analysis showed that Lactobacillus casei is more effective and safer in preventing antibiotic-associated diarrhea compared to other lactobacilli. However, there are currently no in vitro or in vivo studies on the inhibitory effect of Lactobacillus paracasei on Clostridium difficile.
[0004] Based on the above analysis, the existing technology has the following problems and shortcomings: Currently, there are no probiotic drugs or drug compositions specifically designed to inhibit Clostridium difficile on the domestic market. Therefore, the development of such lactic acid bacteria to reduce the severity of infection has great application potential. Summary of the Invention
[0005] The purpose of this invention is to solve the above-mentioned problems and provide a strain of Lactobacillus paracasei W1 and its application in inhibiting Clostridium difficile infection. This strain of Lactobacillus paracasei has the ability to tolerate low pH and high concentration of bile salts in the gastrointestinal tract. Its sterile supernatant can inhibit the growth and biofilm formation of Clostridium difficile in vitro. In animal experiments, this bacterium can effectively reduce the mortality rate of mice infected with Clostridium difficile. It has good antibacterial effect against Clostridium difficile both in vitro and in vivo.
[0006] To achieve the above objectives, the technical solution of the present invention is as follows:
[0007] A strain of Lactobacillus paracasei W1, characterized in that: the Lactobacillus paracasei has the preservation number CCTCC No: M 2024619.
[0008] Furthermore, the nucleotide sequence of the 16S rRNA of *Lactobacillus paracasei* W1 is shown in SEQ ID No. 1.
[0009] Application of Lactobacillus paracasei W1 in inhibiting Clostridium difficile infection.
[0010] Furthermore, the sterile supernatant of Lactobacillus paracasei W1 inhibits the growth of Clostridium difficile and the formation of biofilms.
[0011] The use of a strain of Lactobacillus paracasei W1 in the preparation of a medicament or pharmaceutical composition for the prevention and treatment of colitis caused by Clostridium difficile infection.
[0012] Furthermore, the content of *Lactobacillus paracasei* in the drug or drug composition is not less than 1.0 × 10⁻⁶.9 cfu / mL or 1.0×10 9 cfu / g.
[0013] Compared with existing technologies, the beneficial effects of this solution are as follows: *Lactobacillus paracasei* W1 of this invention exhibits good tolerance to gastrointestinal fluids, and its sterile supernatant can effectively inhibit the growth of *Clostridium difficile* in vitro, and has good therapeutic and preventive effects against infections caused by *Clostridium difficile*. It can be used to prepare drugs or pharmaceutical compositions for the prevention or treatment of colitis caused by *Clostridium difficile* infection. Attached Figure Description
[0014] Figure 1 This is a colony morphology diagram of Lactobacillus paracasei W1 in an embodiment of the present invention;
[0015] Figure 2 This is a Gram staining image of Lactobacillus paracasei W1 in an embodiment of the present invention;
[0016] Figure 3 This is a schematic diagram illustrating the effect of sterile supernatant of Lactobacillus paracasei W1 on the growth of Clostridium difficile in an embodiment of the present invention.
[0017] Figure 4 This is a schematic diagram illustrating the effect of sterile supernatant of Lactobacillus paracasei W1 on the formation of Clostridium difficile biofilm in an embodiment of the present invention.
[0018] Figure 5 This is a schematic diagram illustrating the effect of Lactobacillus paracasei W1 on colon length in CDI mice in an embodiment of the present invention;
[0019] Figure 6 This is a schematic diagram illustrating the effect of Lactobacillus paracasei W1 on the survival rate of CDI mice in an embodiment of the present invention;
[0020] Figure 7 This is a schematic diagram illustrating the effect of Lactobacillus paracasei W1 on the CSS score of CDI mice in an embodiment of the present invention;
[0021] Figure 8 This is a diagram illustrating the effect of Lactobacillus paracasei W1 on intestinal tissue damage in CDI mice in an embodiment of the present invention. Detailed Implementation
[0022] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.
[0023] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.
[0024] The Lactobacillus paracasei W1 provided by this invention was isolated from human oral saliva and taxonomically named Lacticaseibacillus paracasei. It was deposited on April 3, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No: M 2024619, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.
[0025] The *Lactobacillus paracasei* provided by this invention is present in a content of not less than 1.0 × 10⁻⁶ in the drug or drug composition. 9 CFU / mL or 1.0×10 9 CFU / g.
[0026] The technical solution of the present invention will be further described below with reference to specific embodiments.
[0027] Isolation and purification of Lactobacillus paracasei W1:
[0028] Take 1 mL of saliva sample from a normal person's mouth and add it to 9 mL of sterile water. After thorough shaking and dilution, take 100 μL of the sample suspension diluted 10-7 times and spread it on MRS solid medium. Incubate in an anaerobic incubator at 37 ℃ with the medium upside down for 48 h. After a single colony grows on the plate, pick the single colony for purification. Repeat this process three or more times until pure colonies are cultured.
[0029] Example 1: Lactobacillus paracasei W1 has the following microbiological characteristics:
[0030] (1) Colony characteristics: This bacterium forms milky white colonies on MRS solid medium. The colony surface is smooth, moist, and raised, with neat edges. It is opaque and does not produce pigments. See Appendix. Figure 1 .
[0031] (2) Staining characteristics: Gram positive, blue, and appears as rods or bars under a light microscope, see Figure 2.
[0032] (3) Growth characteristics: Under constant temperature aerobic or anaerobic conditions at 37 ℃, the growth reaches the logarithmic phase after about 16 hours of culture in MRS medium.
[0033] (4) Carbohydrate fermentation test of Lactobacillus paracasei W1. Lactic acid bacteria biochemical identification tubes (purchased from Hangzhou Microbial Reagent Co., Ltd.) were used to identify the reaction of Lactobacillus paracasei W1 to 11 carbohydrates.
[0034] The results are shown in Table 1. As can be seen from Table 1, Lactobacillus paracasei W1 was able to utilize, decompose and metabolize 11 carbohydrates in the fermentation experiment of 11 carbohydrates, including aescin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, inulin, lactose and 1% sodium hippurate.
[0035] Table 1. Biochemical identification results of Lactobacillus paracasei W1
[0036]
[0037] Example 2: Lactobacillus paracasei W1 exhibits good tolerance to simulated gastrointestinal fluid.
[0038] Frozen *Lactobacillus paracasei* W1 was inoculated into MRS solid medium and anaerobically cultured at 37°C for 48 h. After three subcultures on MRS solid medium, single colonies were inoculated into liquid MRS medium and cultured for 24 h. Then, 1 mL of *Lactobacillus paracasei* W1 culture was inoculated into 9.0 mL of MRS liquid medium at pH 2 and pH 3, respectively, with a control group included. After anaerobic culture at 37°C for 3 h, the culture was removed, serially diluted, and plated at 100 μL onto MRS solid medium. After anaerobic culture for 48 h, the plates were removed. Finally, the number of viable bacteria on the plates was counted, and the survival rate was calculated (see Table 2).
[0039] Survival rate = viable count of MRS plate after pH=2 or pH=3 treatment / viable count of MRS plate × 100%.
[0040] Table 2. Determination of acid resistance of Lactobacillus paracasei W1
[0041]
[0042] One mL of Lactobacillus paracasei W1 culture was inoculated into 9.0 mL of MRS liquid medium containing 0.3% ox bile salt and MRS liquid medium without ox bile salt, respectively. After anaerobic culture at 37°C for 3 h, the culture was removed, serially diluted, and spread into 100 μL plate on MRS solid medium. After anaerobic culture for 48 h, the plate was removed, colony count was performed, viable bacteria count was determined, and survival rate was calculated (see Table 3).
[0043] Survival rate = (MRS plate viable count after 0.3% bovine bile salt treatment) / (MRS plate viable count) × 100%.
[0044] Table 3. Determination of bile salt resistance in Lactobacillus paracasei W1
[0045]
[0046] The experimental results are shown in Tables 2 and 3. The results indicate that *Lactobacillus paracasei* W1 has good acid and bile salt resistance.
[0047] Example 3: Effect of sterile supernatant of Lactobacillus paracasei W1 on the growth of Clostridium difficile
[0048] After resuscitating and subculturing *Lactobacillus paracasei* W1 2-3 times, it was inoculated into MRS liquid medium and anaerobically cultured at 37°C for 48 h. The fermentation supernatant of *Lactobacillus paracasei* W1 was collected, centrifuged at 12000g for 10 min, and filtered through a 0.22μm filter membrane to obtain a sterile supernatant (CFS). Then, sterile supernatants were prepared using fresh BHI broth at different dilution ratios (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32) and stored at 4°C for later use. The experimental strain *Clostridium difficile* ATCC 43255 was thawed from a -80°C freezer, and 100μL was spread onto CCFA plates. After activation and subculturing 3 times, a single colony was picked and inoculated into BHI broth and cultured until the OD600 reached approximately 0.5. 400 μL of the exponential growth phase bacterial culture was inoculated into test tubes containing 4 mL of sterile supernatant at different dilution ratios (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32). A blank control group containing sterile supernatant and a positive control group containing only *C. difficile* (CD) were set up, and this point was recorded as time 0. The culture was then anaerobic at 37°C, and the turbidity (OD 600 nm) was used as the growth index of CD. At different time points, 200 μL samples were taken and their absorbance was measured at 600 nm to plot growth curves (see Appendix). Figure 3 The results showed that CFS at dilutions of 1 / 2, 1 / 4, and 1 / 8 significantly inhibited the growth of Clostridium difficile.
[0049] Example 4: Effect of sterile supernatant of Lactobacillus paracasei W1 on Clostridium difficile biofilm formation
[0050] The lowest dilution with no turbidity change during Example 3 was used as the inhibitory concentration, and dilutions with turbidity changes were used for the experiment. 400 μL of the bacterial culture in the exponential growth phase was taken and inoculated into test tubes containing 4 mL of sterile supernatant at different dilutions (1 / 16, 1 / 32), with three replicates for each dilution. 1 mL was inoculated into each well of a 24-well polystyrene plate and incubated anaerobically at 37 °C for 72 h. After incubation, the 24-well plate was opened, the supernatant was aspirated, and the biofilm was gently washed twice with sterile distilled water to remove residual bacterial culture. The plate was then dried at 37 °C for 30 min. The biofilm was again gently washed twice with sterile distilled water and dried at 37 °C for 30 min. Then, 0.2% crystal violet staining solution was added for 30 min, excess staining solution was discarded, and the plate was gently washed 6 times with distilled water to remove residual staining solution. The plate was then dried at 37 °C. Finally, add 200 μL of 95% ethanol to dissolve the staining solution bound to the biofilm (30 min), and add 200 μL to a 96-well plate. Measure the absorbance of each well at 570 nm (see appendix). Figure 4 The results showed that CFS at a 1 / 16 dilution significantly inhibited Clostridium difficile biofilm formation.
[0051] Example 5: Effect of Lactobacillus paracasei W1 on colonic shortening in CDI mice
[0052] Thirty-one female C57 mice (6 weeks old, 14-16g) were randomly divided into four groups: a control group (n=7) and the remaining groups (n=8 each). The groups were: Control Group (CDI), Model Group (CDI), *Lactobacillus paracasei* W1 Prevention Group (LP W1 pre), and *Lactobacillus paracasei* W1 Treatment Group (LP W1 post). For the three experimental groups, an antibiotic mixture was prepared as drinking water for the mice. The antibiotic mixture consisted of kanamycin (0.4 mg / mL), gentamicin (0.035 mg / mL), colistin (850 U / mL), metronidazole (0.215 mg / mL), and vancomycin (0.045 mg / mL). This treatment was administered for 5 consecutive days, followed by one day of sterile drinking water. The *Lactobacillus paracasei* W1 Prevention Group (W1 pre) received 200 μL of *Lactobacillus paracasei* W1 bacterial suspension (1×10⁻⁶) via gavage for 6 consecutive days in addition to the antibiotic treatment. 9 (CFU / mL), while the control group mice drank sterile water continuously.
[0053] One day prior to Clostridium difficile infection, all experimental group mice were intraperitoneally injected with clindamycin (10 mg / kg). From 17 hours prior to infection until 2 hours after infection, mice were fasted and deprived of water. On day 1 post-infection, the Lactobacillus paracasei W1 treatment group (LPW1 post) was treated with a bacterial suspension (1×10⁻⁶). 9Mice were administered CFU / mL via gavage once daily for 6 consecutive days. Observation was conducted to detect diarrhea and death, and the mice were weighed and their condition recorded.
[0054] In addition, the mice's fecal characteristics, behavioral status, and weight changes were observed daily to calculate the Clinical Sickness Score (CSS). The scores for each item are shown in Table 4. On day 6, the mice were dissected, their colons were harvested, and their length was measured. Figure 5 ), and calculate the survival rate of the mice ( Figure 6 ) and CSS score (Figure 7).
[0055] Table 4. Clinical Sickness Score (CSS)
[0056]
[0057] The experimental results are shown in Figure 5. Compared with the control group, the colon length of mice in the CDI group was significantly shortened, while the colon length of mice in the LPW1 post and LP W1 pre groups was significantly restored. This indicates that *Lactobacillus paracasei* W1 has an ameliorative effect on colon shortening in *Clostridium difficile* colitis. The survival rate of mice in the CDI group was significantly reduced, while the survival rates of mice in the LP W1 post and LP W1 pre groups were improved, especially the LP W1 pre group. Furthermore, the CSS score of the CDI group was significantly increased, while the scores of the LP W1 post and LP W1 pre groups were lower than those of the CDI group, with the LP W1 pre group showing the most significant decrease. This indicates that *Lactobacillus paracasei* W1 has a remedial and restorative effect on *Clostridium difficile* colitis. Figure 6 , Figure 7 .
[0058] Example 6: Effects of Lactobacillus paracasei W1 on intestinal tissue damage in CDI mice
[0059] Mouse cecum and colon tissues were taken, and about 1 cm of cecum and colon tissue was cut and soaked in 4% paraformaldehyde fixative for 24 hours. After sectioning, HE staining was performed to observe tissue changes.
[0060] HE staining of colon and cecum tissue can reveal (…) Figure 8In the CDI group, the colonic and cecal mucosal epithelial structures were severely damaged, with goblet cells and crypts missing, and significant inflammation was observed in the colon and cecum, with a large number of inflammatory cells infiltrating the submucosa. In contrast, the Control, LP W1 treatment, and LP W1 prevention groups showed intact colonic mucosal epithelial cells, normal morphology of the lamina propria glands, normal crypts, and only a small number of inflammatory cells infiltrating the submucosa. This indicates that *Lactobacillus paracasei* W1 can prevent and treat colitis symptoms caused by *Clostridium difficile*.
[0061] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.
Claims
1. A strain of Lactobacillus paracasei W1, characterized by: The Lactobacillus paracasei W1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No: M 2024619.
2. The *Lactobacillus paracasei* W1 strain as described in claim 1, characterized in that: The nucleotide sequence of the 16S rRNA of Lactobacillus paracasei W1 is shown in SEQ ID No.
1.
3. The use of the Lactobacillus paracasei W1 strain according to claim 1 in the preparation of a medicament for inhibiting Clostridium difficile.
4. The use of the Lactobacillus paracasei W1 strain according to claim 1 in a medicament for the prevention and / or treatment of colitis caused by Clostridium difficile infection.
5. The use of a strain of *Lactobacillus paracasei* W1 as described in claim 4 in a medicament for the prevention and / or treatment of colitis caused by *Clostridium difficile* infection, characterized in that: The content of Lactobacillus paracasei in the drug is not less than 1.0 × 10⁻⁶. 9 CFU / mL or 1.0×10 9 CFU / g.