Escherichia coli YC49 and application of Escherichia coli YC49 in inhibiting clostridium difficile in cooperation with bacteroides

Abstract

The invention provides Escherichia coli YC49 and application of the Escherichia coli YC49 in inhibiting clostridium difficile in cooperation with bacteroides, and belongs to the technical field of microorganisms. The invention provides Escherichia coli YC49, and the preservation number of the Escherichia coli YC49 is CGMCC (China General Microbiological Culture Collection Center) No. 34488. The invention also provides an effect of the Escherichia coli YC49 and bacteroides on synergistically inhibiting clostridium difficile, and further provides a compound probiotic composition, an in-vitro antibacterial product and a coprophilous fungus transplantation product. The Escherichia coli YC49 can accelerate the whole process from growth to decline of clostridium difficile, and a culture solution obtained after the Escherichia coli YC49 is combined with common bacteroides or the Escherichia coli YC49 and the common bacteroides are co-cultured can remarkably inhibit growth of early-stage clostridium difficile, so that the Escherichia coli YC49 has application significance in treatment of clostridium difficile infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to Escherichia coli YC49 and its synergistic Bacteroides in inhibiting Clostridium difficile. BACKGROUND

[0002] Clostridium difficile infection (CDI) is usually treated with antibiotics, but antibiotic treatment can disrupt the intestinal microbiota and increase susceptibility to recurrent CDI (rCDI). Fecal microbiota transplantation (FMT) and other donor-derived formulations have been shown to be effective for rCDI4-6, however, due to the differences between donors and the natural temporal fluctuations in the composition of the microbiome within donors, the composition of FMT products is not fixed, resulting in unstable quality, which also affects the therapeutic effect of FMT. In addition, to prevent pathogen transfer, extensive screening of FMT donor feces is required, which also increases the cost of use and additional financial burden on patients. In contrast, by fermenting purified bacteria to produce a fixed combination of microbiota for improved FMT products, there are standardized composition and dosage, repeatable quality attributes, and lower risk of pathogen transfer. Therefore, it is of great significance to find bacteria or bacterial combinations with specific functions for patients in urgent need of treatment by FMT. However, the research in this aspect is still not perfect, therefore, screening and providing probiotics with significant inhibitory effect on Clostridium difficile and developing related preparations are still the key to research in the field. SUMMARY

[0003] To solve the problems in the prior art, the first purpose of the present application is to provide an Escherichia coli (E. coli) YC49 with a preservation number of CGMCC No. 34488. Escherichia coli )YC49, with a preservation number of CGMCC No. 34488.

[0004] The second purpose of the present application is to provide the use of the above-mentioned Escherichia coli YC49 in the preparation of a drug for promoting the decline of Clostridium difficile.

[0005] The third purpose of the present application is to provide the use of the above-mentioned Escherichia coli YC49 in combination with Bacteroides in the preparation of a drug for inhibiting the growth of Clostridium difficile and treating Clostridium difficile infection.

[0006] The fourth purpose of the present application is to provide a composite probiotic composition, a product for inhibiting Clostridium difficile in vitro, and a fecal bacteria transplantation product, which can significantly inhibit the growth of early Clostridium difficile.

[0007] The fifth purpose of the present application is to provide the use of the above-mentioned composite probiotic composition or fecal bacteria transplantation product in the preparation of a drug for treating Clostridium difficile infection.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a strain of Escherichia coli (Escherichia coli) Escherichia coli The Escherichia coli YC49 has the accession number CGMCC No. 34488.

[0009] The present invention also provides the use of the above-mentioned Escherichia coli YC49 in the preparation of a drug that promotes the death of Clostridium difficile.

[0010] The present invention also provides the use of the above-mentioned Escherichia coli YC49 combined with Bacteroides in the preparation of a drug for inhibiting the growth of Clostridium difficile or treating Clostridium difficile infection.

[0011] Preferably, the Bacteroides includes Bacteroides vulgaris.

[0012] The present invention also provides a compound probiotic composition comprising the above-mentioned Escherichia coli YC49 and Bacteroides vulgaris.

[0013] Preferably, the ratio of viable Escherichia coli YC49 to Bacteroides vulgaris is 1:1.

[0014] The present invention also provides a product for in vitro inhibition of Clostridium difficile, comprising a co-culture medium of the aforementioned Escherichia coli YC49 and Bacteroides vulgaris.

[0015] Preferably, glycocholic acid is added during the co-culture of Escherichia coli YC49 and Bacteroides vulgaris.

[0016] The present invention also provides a fecal microbiota transplantation product, comprising a suspension of Escherichia coli YC49 and a suspension of Bacteroides var. mongolica; or comprising powder of Escherichia coli YC49 and powder of Bacteroides var. mongolica.

[0017] The present invention also provides the use of the above-mentioned compound probiotic composition or the above-mentioned fecal microbiota transplantation product in the preparation of a medicament for treating Clostridium difficile infection.

[0018] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention provides the first yield of a strain of Escherichia coli (Escherichia coli). Escherichia coliYC49, and found that it can speed up the whole process of C. difficile from growth to decline, while E. coli YC49 and Bacteroides combination can synergistically inhibit the early growth of C. difficile, avoid the recurrence of C. difficile, quickly clean up and inhibit the existence of C. difficile in the intestine, and has good application prospect for the treatment of C. difficile infection (CDI). Based on the synergistic bacteriostatic effect of E. coli YC49 and Bacteroides vulgaris, a probiotic composition, a product for inhibiting C. difficile in vitro or a fecal bacteria transplantation product is provided, which has application significance for treating C. difficile infection.

[0019] Biological preservation instructions E. coli YC49, classified as E. coli (Escherichia coli) Escherichia coli ), the preservation unit: China General Microbiological Culture Collection Center (CGMCC for short), the preservation unit address: No. 3, Beichen West Road, Chaoyang District, Beijing, the preservation number: CGMCC No. 34488, the preservation date: May 9, 2025. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 : Macroscopic morphology of E. coli YC49 colony in a petri dish; Figure 2 : Microscopic morphology of E. coli YC49 strain; Figure 3 : C. difficile growth curves of different treatment groups. DETAILED DESCRIPTION

[0021] The present application provides an E. coli (Escherichia coli) Escherichia coli ) YC49, the preservation number of the E. coli YC49 is CGMCC No. 34488. The E. coli YC49 described in the present application is isolated and purified from the intestine (feces) of a 13-year-old healthy boy, and is identified as E. coli (Escherichia coli) Escherichia coli ) on May 9, 2025, and is preserved in China General Microbiological Culture Collection Center (CGMCC for short), the preservation unit address: No. 3, Beichen West Road, Chaoyang District, Beijing.

[0022] The present application provides the application of the above-mentioned E. coli YC49 in the preparation of a drug for promoting the decline of C. difficile, and it is found that it can speed up the whole process of C. difficile from growth to decline, and has good application prospect for the treatment of C. difficile infection (CDI), effectively replacing the use of antibiotics.

[0023] The application also provides application of the above-mentioned Escherichia coli YC49 and Bacteroides in preparation of a medicine for inhibiting growth of Clostridium difficile and treating Clostridium difficile infection. Phocaeicola vulgatus As an optional embodiment, the Bacteroides of the application is Bacteroides vulgaris ATCC8482. The earlier the combination of the Escherichia coli YC49 and Bacteroides in the application inhibits the growth of Clostridium difficile, the stronger the synergistic inhibition effect is, and preferably, the combination inhibits germination of Clostridium difficile, which is more meaningful for treating Clostridium difficile infection (CDI) and recurrent CDI (rCDI).

[0024] The application also provides a composite probiotic composition comprising the above-mentioned Escherichia coli YC49 and Bacteroides Phocaeicola vulgatus As an optional embodiment, the Bacteroides of the application is Bacteroides vulgaris ATCC8482. Preferably, the ratio of viable cell count of the Escherichia coli YC49 and Bacteroides is 1:1.

[0025] The application also provides a product for inhibiting Clostridium difficile in vitro, comprising a co-culture solution of the above-mentioned Escherichia coli YC49 and Bacteroides. Preferably, glycoglycholic acid is supplemented during the co-culture of the Escherichia coli YC49 and Bacteroides. As an optional embodiment, the Escherichia coli YC49 and Bacteroides are inoculated into a culture medium containing glycoglycholic acid, and the fermentation liquor or centrifugal supernatant obtained by fermentation has a significant inhibitory effect on growth of Clostridium difficile. Preferably, the fermentation is static culture at 37℃, and the fermentation liquor is obtained after 12-36h of culture. Preferably, the centrifugal supernatant of the fermentation liquor is obtained by centrifuging the fermentation liquor at 4000g-6000g for 10-30min to collect the supernatant. Preferably, the final concentration of glycoglycholic acid in the culture medium is 50-200μg / ml, and more preferably, 100μg / ml. The glycoglycholic acid is metabolized into free bile acid by the Bacteroides, and then the free bile acid is metabolized into secondary bile acid by the Escherichia coli YC49, so as to synergistically inhibit growth of Clostridium difficile. As an optional embodiment, the Bacteroides of the application is Bacteroides vulgaris ATCC8482. Preferably, the ratio of viable cell count of the Escherichia coli YC49 and Bacteroides is 1:1.

[0026] The present application also provides a fecal bacteria transplantation product, which comprises the bacterial body suspension of the above-mentioned Escherichia coli YC49 and the bacterial body suspension of Bacteroides vulgatus; or comprises the bacterial powder of the above-mentioned Escherichia coli YC49 and the bacterial powder of Bacteroides vulgatus. As an optional embodiment, the present application obtains a culture solution after culturing the Escherichia coli YC49 and Bacteroides vulgatus respectively, centrifuges the culture solution to obtain bacterial bodies, and resuspends the bacterial bodies in physiological saline (concentration of 0.85%) to obtain a bacterial body suspension, then mixes the bacterial body suspensions of the two in a proportion of 1:1 according to the number of viable bacteria to obtain the fecal bacteria transplantation product; the centrifugation condition is preferably 4000g-6000g, 10-30min. As another optional embodiment, the present application centrifuges the bacterial bodies after culturing the Escherichia coli YC49 and Bacteroides vulgatus according to the requirements of General Principles of Microecological Living Bacteria Products in the Chinese Pharmacopoeia (Volume III), and mixes the bacterial bodies with suitable dispersing agents and stabilizers. The bacterial bodies are dried by vacuum freeze-drying method, then are crushed and sieved into powder to obtain a powder, and then the powder is mixed with excipients to prepare an oral product in the form of powder, granules or capsules; the centrifugation condition is preferably 4000g-6000g, 10-30min; the preparation method of the bacterial powder is preferably vacuum freeze-drying method, and the freeze-drying condition is preferably: fast pre-freezing to-50℃; pressure <10 Pa, vacuum freeze-drying at-50℃; sublimation temperature is-35℃--45℃. As an optional embodiment, the Bacteroides vulgatus is Bacteroides vulgatus ATCC8482.

[0027] The present application also provides the use of the above-mentioned composite probiotic composition or fecal bacteria transplantation product in the preparation of a medicine for treating Clostridium difficile infection, and the earlier the growth of Clostridium difficile, the stronger the synergistic inhibition.

[0028] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0029] In the embodiments of the present application, the Bacteroides vulgatus strain is purchased from Beinaelieli Biotechnology Co., Ltd. (platform number BNCC340630, other collection center number ATCC8482). Clostridium difficile (platform strain number TS344347, ATCC 43593) is purchased from Ningbo Tastuk Biological Technology Co., Ltd.

[0030] In the following examples, all are conventional methods unless otherwise specified.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Example 1 Isolation and identification of Escherichia coli YC49: Escherichia coli was isolated from the intestines (feces) of a healthy 13-year-old boy. The fecal samples were serially diluted with 0.85% physiological saline, and 1 mL was spread onto Columbia blood agar plates and incubated at 37°C for 24–48 h. After incubation, all single colonies were identified by 16S rRNA testing, revealing four strains of Escherichia coli. These Escherichia coli were then tested according to the requirements of "Food Microbiology Examination: Examination of Diarrheal-Causing Escherichia coli" (GB4789.6—2016). A single strain was selected, namely Escherichia coli YC49.

[0033] Escherichia coli YC49 was inoculated onto LB solid medium and incubated overnight at 37°C. Colony morphology was observed through petri dishes. The results are as follows. Figure 1 As shown in the image. The results showed that *Escherichia coli* YC49 was round with neat edges; milky white in color; the colonies were translucent or opaque; and the surface was smooth, moist, and glossy, typical of *Escherichia coli* morphology. The staining and microscopic examination results are as follows. Figure 2 As shown, the results indicate that Escherichia coli YC49 cells are red or pink, rod-shaped, and Gram-negative, consistent with the results for Escherichia coli.

[0034] Whole-genome sequencing of Escherichia coli YC49 was performed, and its 16S rDNA sequence is shown in SEQ ID No. 1, confirming that it belongs to Escherichia coli. Escherichia coli ).

[0035] Escherichia coli ( Escherichia coli YC49 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34488 and deposit date May 9, 2025.

[0036] Example 2 1. Co-culture of Bacteroides ATCC8482 and Escherichia coli YC49 with glycocholic acid: Common Bacteroides culture: Open the inoculum tube, use a pipette or pipette to draw 300 μl of thawed inoculum, and inoculate it into a test tube containing 3 ml of modified enhanced Clostridium broth medium (purchased from Ningbo Mingzhou Biotechnology Co., Ltd., catalog number KDM145) (the inoculation is complete and it is the first generation inoculum). Place the common Bacteroides in an anaerobic incubator and incubate at 37°C overnight.

[0037] E. coli YC49 culture: Open the strain tube, use pipette or pipet to take 300 μl of thawed strain, inoculate in 1 test tube containing 3 ml of LB nutrient broth medium (purchased from Ningbo Mingzhou Biotechnology Co., Ltd., item number KDM159) (inoculation is completed as a generation of strain), E. coli is placed in a shaker, 37℃, 200 rpm for overnight culture.

[0038] Test 1: B. vulgatus subculture (supplemented with glycocholic acid) B. vulgatus was inoculated at a ratio of 1:10 (v / v), 1 ml of bacterial solution was added to a large test tube containing 10 ml of modified Clostridium fortis broth medium (1 mg / ml of glycocholic acid was added to the medium to a final concentration of 100 μg / ml), and it was cultured at 37℃. After 24 hours of culture, the OD value reached 2.68, and the supernatant was collected by centrifugation (5000g, 20 min).

[0039] Test 2: E. coli YC49 subculture (supplemented with glycocholic acid) E. coli YC49 was taken 1 ml of bacterial solution was added to a large test tube containing 10 ml of LB nutrient broth medium (1 mg / ml of glycocholic acid was added to the medium to a final concentration of 100 μg / ml), and it was cultured in an anaerobic incubator. After overnight culture at 37℃, the OD value reached 2.37, and the supernatant was collected by centrifugation (5000g, 20 min).

[0040] Test 3: E. coli YC49 and B. vulgatus mixed culture (supplemented with glycocholic acid) 1 ml of E. coli YC49 bacterial solution and 1 ml of B. vulgatus bacterial solution were added to a large test tube containing 10 ml of modified Clostridium fortis broth medium (1 mg / ml of glycocholic acid was added to the medium to a final concentration of 100 μg / ml), and it was cultured at 37℃. After 24 hours of culture, the supernatant was collected by centrifugation (5000g, 20 min).

[0041] 2, the supernatant obtained by test 1, test 2, test 3 was used to inhibit the growth of C. difficile respectively: C. difficile recovery: Open the strain tube, use pipette or pipet to take 300 μl of thawed strain, inoculate in 1 test tube containing 3 ml of modified Clostridium fortis medium liquid medium (inoculation is completed as a generation of strain), and place it in an anaerobic incubator, 37℃ static culture for 24h.

[0042] C. difficile subculture: the cultured C. difficile was inoculated at a ratio of 1:10 (v / v). 2 ml of the bacterial solution was added to a large test tube containing 20 ml of fresh modified and enhanced C. difficile culture medium at 37°C and incubated; 2 ml of the bacterial solution was added to a large test tube containing 15 ml of fresh modified and enhanced C. difficile culture medium, and 5 ml of B. vulgatus culture supernatant was added, 2 ml of the bacterial solution was added to a large test tube containing 15 ml of fresh modified and enhanced C. difficile culture medium, and 5 ml of E. coli YC49 culture supernatant was added, 2 ml of the bacterial solution was added to a large test tube containing 15 ml of fresh modified and enhanced C. difficile culture medium, and 5 ml of mixed culture supernatant was added; and incubated at 37°C for 24 hours. The OD value was detected during the culture process. The results are shown in Table 1. The growth curve of C. difficile is shown in Figure 1. 600 Figure 3

[0043] Table 1 OD value of the bacterial solution during the culture process

[0044] Tube 3: The supernatant of the culture of E. coli YC49 and glycocholic acid. The growth curve shows that the C. difficile in tube 3 grows rapidly in the early stage, has no plateau period, and then dies rapidly, indicating that E. coli YC49 accelerates the entire process of C. difficile from growth to death, which is a discovery for the first time. This effect of E. coli YC49 has a good application prospect for the treatment of C. difficile infection (CDI). At present, the treatment of C. difficile mainly relies on antibiotics, but C. difficile has spores. When the antibiotic treatment is over, the C. difficile spores germinate again, which is the main reason why C. difficile is difficult to treat. The effect of E. coli YC49 shows that the treatment of antibiotics can be avoided because after the colonization of E. coli YC49, it will first promote the rapid growth of C. difficile in the intestine until it dies, and after the death of the living C. difficile, E. coli YC49 can also inhibit the early growth of C. difficile by taking advantage of the synergistic effect with B. vulgatus, so as to quickly clean up and inhibit the existence of C. difficile in the intestine.

[0045] Tube 4: The test group supplemented with mixed culture supernatant of E. coli YC49 and B. vulgatus. The C. difficile grows most slowly, indicating that the mixed culture supernatant of E. coli YC49 and B. vulgatus has the strongest inhibitory effect on the growth of C. difficile.

[0046] Calculation of FIC index: ​​The growth inhibition of C. difficile by the supernatant of the bacterial culture was expressed by the growth rate of C. difficile (growth rate = ΔOD / culture time). The stronger the inhibition, the slower the growth rate. The control group of C. difficile entered the plateau phase after 8 hours of culture. The synergistic effect of the supernatant of the three groups of culture on the growth inhibition of C. difficile was calculated at the latent period, the logarithmic growth phase and the plateau phase, respectively, and the calculation results are shown in Table 2.

[0047] Table 2 Growth rate of C. difficile during culture

[0048] The FIC index was calculated by taking the growth rate of tube 4 as EoBS, the growth rate of tube 2 as EA, and the growth rate of tube 3 as EB. The results show that the supernatant of mixed culture has a synergistic effect on the growth inhibition of C. difficile at different stages of C. difficile growth, and the earlier the culture, the more obvious the synergistic effect, indicating that the combination of B. vulgatus and E. coli YC49 has the best effect on the synergistic inhibition of C. difficile at the germination stage of C. difficile.

[0049] The possible mechanism of the synergistic inhibition of C. difficile by the combination of E. coli YC49 and B. vulgatus is that B. vulgatus has cholylglycine hydrolase, which can degrade conjugated bile acids such as glycocholic acid to release free bile acids, and E. coli YC49 amplifies the inhibitory effect of secondary bile acids on C. difficile.

[0050] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An Escherichia coli (E. coli) YC49, characterized in that, Escherichia coli The preservation number of the Escherichia coli YC49 is CGMCC No. 34488. ​ 2. Use of the Escherichia coli YC49 of claim 1 in the preparation of a drug for promoting the decline of Clostridium difficile.

3. Use of the Escherichia coli YC49 of claim 1 in combination with Bacteroides in the preparation of a drug for inhibiting the growth of Clostridium difficile or treating Clostridium difficile infection.

4. Use according to claim 3, characterized in that, The Bacteroides includes Bacteroides vulgatus.

5. A probiotic composition comprising, The Bacteroides includes Bacteroides vulgatus.

6. The combination probiotic composition according to claim 5, wherein, The ratio of viable cell number of the Escherichia coli YC49 to that of the Bacteroides is 1:

1.

7. A product for inhibiting Clostridium difficile in vitro, characterized in that, The co-culture solution of the Escherichia coli YC49 and the Bacteroides of claim 1.

8. The product of claim 7, wherein, Glycocholic acid is supplemented during the co-culturing of the Escherichia coli YC49 and the Bacteroides.

9. A fecal microbiota transplant product, characterized by, The bacterial body suspension of the Escherichia coli YC49 of claim 1 and the bacterial body suspension of the Bacteroides; or the bacterial powder of the Escherichia coli YC49 of claim 1 and the bacterial powder of the Bacteroides.

10. Use of the complex probiotic composition of any one of claims 5-6 or the fecal bacteria transplantation product of claim 9 in the preparation of a drug for treating Clostridium difficile infection.

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