Synthetic flora capable of antagonizing methicillin-resistant staphylococcus aureus and application thereof

Abstract

The application discloses a synthetic flora capable of antagonizing methicillin-resistant Staphylococcus aureus and application thereof, and belongs to the technical field of microorganisms. The synthetic flora comprises Faecalibacterium prausnitzii, Bacteroides uniformis, Ditiola putrida, Bacteroides massiliosus and Bacteroides thetaiotaomicron, and each strain is preferably compounded in an equal viable bacterial number ratio. The synthetic flora can be prepared into a viable bacterial preparation, a freeze-dried bacterial powder, a bacterial suspension, a capsule, a granule, an oral liquid or a microcapsule preparation. In an in-vitro experiment, the synthetic flora can effectively antagonize methicillin-resistant Staphylococcus aureus; in an animal experiment, the synthetic flora can significantly inhibit the colonization of MRSA in a host intestinal tract, and reduce the load of MRSA which has established colonization, and has the effects of preventing colonization and treating colonization, thereby providing a new technical means for the microecological prevention and control of methicillin-resistant Staphylococcus aureus and having a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and specifically to a synthetic bacterial community capable of antagonizing methicillin-resistant Staphylococcus aureus and its applications. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus, S. aureus Staphylococcus aureus (S. aureus) is a common Gram-positive opportunistic pathogen that can cause various diseases such as skin and soft tissue infections, pneumonia, and sepsis, making it one of the important clinical pathogens. With the long-term irrational use of antibiotics, the problem of drug resistance in Staphylococcus aureus has become increasingly serious. Since the discovery of penicillin-resistant strains in the 1940s, methicillin-resistant Staphylococcus aureus (MRSA) has subsequently emerged. Staphylococcus aureus MRSA (Mec-like Antibiotic Syndrome) carries drug resistance genes such as mecA, which encode the penicillin-binding protein PBP2a, leading to widespread resistance to β-lactam antibiotics and a significant decline in the effectiveness of traditional anti-infective treatments. In recent years, MRSA has gradually spread from hospital environments to community and livestock farming environments, posing a serious threat to public health and safety.

[0003] Currently, clinical treatment of MRSA infections mainly relies on antibiotics such as vancomycin and linezolid. However, with the widespread use of antibiotics, MRSA resistance to various "last line of defense" antibiotics continues to increase, and even vancomycin-intermediate or drug-resistant strains have emerged, posing a significant challenge to clinical treatment. At the same time, MRSA also possesses a strong biofilm-forming ability and environmental adaptability. The biofilms it forms can effectively hinder antibiotic penetration and enhance its immune evasion ability, which is one of the important reasons for persistent infection and increased drug resistance.

[0004] Recent studies have shown that normal flora can develop colonization resistance against pathogens through nutrient competition, space occupation, secretion of antimicrobial metabolites, and regulation of host immunity, thereby inhibiting pathogen colonization and infection. Compared with traditional antibiotics, using functional strains to construct synthetic microbial consortia (SynCom) to intervene in drug-resistant bacterial infections has advantages such as being less likely to induce drug resistance and maintaining microecological balance, showing promising application prospects in the field of drug-resistant bacterial control. However, research on functional synthetic microbial consortia targeting MRSA and their applications is still relatively lacking.

[0005] Therefore, developing a synthetic microbial community that can effectively antagonize methicillin-resistant Staphylococcus aureus (MRSA) and its application is of great significance for reducing the risk of MRSA colonization and infection, reducing antibiotic use, and maintaining the health of the host's microecology. Summary of the Invention

[0006] To address the problems of methicillin-resistant Staphylococcus aureus (MRSA) easily colonizing the intestines, persistently carrying it, and being difficult to eliminate in existing technologies, this invention provides a synthetic bacterial community that can effectively antagonize MRSA and its application.

[0007] First, this invention provides a synthetic bacterial community capable of antagonizing methicillin-resistant Staphylococcus aureus (MRSA), the synthetic bacterial community comprising the following species: Clostridium plasmidonum (… Faecalibacterium prausnitzii ), Bacteroides monomorpha ( Bacteroides uniformis ), putrefactive bacteria ( Alistipes putredinis Bacteroides masei ( Phocaeicola massiliensis ) and Bacteroides multiforme ( Bacteroides thetaiotaomicron ).

[0008] Furthermore, the synthetic microbial community includes Clostridium plasmidon CCFM1205, Bacteroides monomorpha FSDTAHCK_B1, Alternaria rotica CCUG 45780T, Bacteroides masei FHNXY-54-K-2, and Bacteroides polymorpha FTJS6K5.

[0009] Furthermore, the strains contained in the synthetic microbial community can be compounded in a ratio of equal live bacteria count; in some embodiments, when the synthetic microbial community contains the above 5 strains, the strains can be compounded in a live bacteria count ratio of 1:1:1:1:1.

[0010] Furthermore, the synthetic microbial community can be a live bacterial preparation, a freeze-dried bacterial powder, a bacterial suspension, a capsule preparation, a granule, an oral liquid, or a microcapsule preparation.

[0011] Furthermore, the synthetic microbiota can be administered to the host via gavage, oral administration, feeding, or other methods suitable for intestinal delivery.

[0012] Furthermore, the number of cells in the synthesized bacterial community is not less than 1 × 10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0013] The present invention also provides products containing the aforementioned synthetic microbial community.

[0014] Furthermore, the products include pharmaceuticals, probiotics, food, health products, or feed additives.

[0015] Furthermore, the drug also contains a pharmaceutical carrier and / or pharmaceutical excipients.

[0016] The present invention also provides the use of the synthetic microbial community in the preparation of medicaments for the prevention and / or treatment of methicillin-resistant Staphylococcus aureus infections.

[0017] Furthermore, the application includes inhibiting the growth, colonization, amplification, and persistent presence of methicillin-resistant Staphylococcus aureus in the host, particularly in the gut.

[0018] Furthermore, the application includes administering the synthetic microbial community prior to MRSA exposure to enhance the host's colonization resistance to MRSA.

[0019] Furthermore, the application also includes administering the synthetic microbiota after MRSA has colonized the gut to reduce pathogen load and promote decolonization.

[0020] Furthermore, the synthetic microbial community can be administered continuously for 3 days or more.

[0021] Furthermore, the dosage of the synthetic bacterial flora is 1×10⁻⁶. 8 ~1×10 10 CFU / session, preferably 1×10 9 CFU / time.

[0022] This invention verified the antagonistic effect of the aforementioned synthetic bacterial flora in in vivo experiments on germ-free mice. In a preventative intervention experiment, germ-free mice were first given the synthetic bacterial flora to establish a stable bacterial background, followed by gavage administration of methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. The results showed that, compared with the blank control group, the synthetic bacterial flora of this invention significantly reduced the colonization level of MRSA in the mouse intestine and maintained a sustained and stable colonization resistance during the observation period.

[0023] In a therapeutic intervention experiment, germ-free mice were first orally administered MRSA ATCC 43300 to establish a stable colonization model, followed by intervention with the synthetic microbiota described herein. The results showed that the synthetic microbiota of this invention significantly reduced the intestinal burden of colonized MRSA, exhibiting a good decolonization effect.

[0024] Furthermore, compared with the control probiotic preparation VSL#3, the synthetic microbial community described in this invention has a more lasting and stable effect in inhibiting MRSA colonization and reducing persistent MRSA carriage.

[0025] Therefore, this invention provides a functional synthetic microbial community with clearly defined components, stable functions, and feasible applications. It can be used for preventive antagonism and therapeutic decolonization of methicillin-resistant Staphylococcus aureus, and has good application prospects.

[0026] The present invention also provides the application of the synthetic bacterial community in in vitro antagonism against methicillin-resistant Staphylococcus aureus.

[0027] Beneficial effects: 1. This invention provides a functional synthetic bacterial flora capable of effectively antagonizing MRSA. The synthetic bacterial flora described in this invention can significantly reduce the colonization level of methicillin-resistant Staphylococcus aureus (MRSA) in the host intestine, exhibiting a clear in vivo antagonistic effect against MRSA. The synthetic bacterial flora possesses both preventative colonization resistance and therapeutic decolonization capabilities. Pre-infection intervention with Neg_M can continuously inhibit MRSA colonization for up to 10 days post-infection, maintaining its viral load at approximately 10^6. 7 CFU / g; Intervention after MRSA colonization can reduce MRSA load to approximately 4.93 × 10⁻⁶. 6 CFU / g showed a good decolonization effect.

[0028] 2. The synthetic bacterial flora described in this invention can antagonize MRSA proliferation in vitro, and the MRSA load is reduced by 97.2% compared with the control group.

[0029] Therefore, this invention provides a novel microecological intervention scheme for the prevention and control of methicillin-resistant Staphylococcus aureus, which is different from traditional antibiotics and has advantages such as high application safety, good sustainability and low likelihood of inducing drug resistance. Attached Figure Description

[0030] Figure 1 A schematic diagram of the experimental design for a germ-free mouse prophylactic intervention model.

[0031] Figure 2 Dynamic changes in the absolute abundance of MRSA in the feces of mice in different treatment groups under preventive intervention conditions.

[0032] Figure 3 A schematic diagram of the experimental design for a therapeutic intervention model in germ-free mice.

[0033] Figure 4 Dynamic changes in the absolute abundance of MRSA in the feces of mice in different treatment groups under therapeutic intervention conditions.

[0034] Figure 5 Absolute abundance of MRSA in feces of mice in different treatment groups at the endpoint of the therapeutic intervention model.

[0035] Figure 6 Antagonistic effect of synthetic bacteria against MRSA outside the community. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. In the embodiments, all original reagent materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0037] The culture media involved in the following examples are as follows: The culture media, including modified peptone-yeast extract-glucose medium (mPYG) and Chopped MeatCarbohydrate Broth (CMC), were purchased from Qingdao Haibo Biotechnology.

[0038] M2GSC medium is a self-prepared medium with the following formula (1L): 10g casein, 5g yeast extract, 5g glucose, 2g cellobiose, 2g fructose, 1g cysteine, 4g sodium bicarbonate, 0.45g dipotassium hydrogen phosphate, 0.45g potassium dihydrogen phosphate, 0.9g ammonium sulfate, 0.9g sodium chloride, 0.09g calcium chloride, 0.09g magnesium sulfate heptahydrate, 1mg resazurin, 10mL clarified rumen fluid, and pH adjusted to 6.5-6.8.

[0039] The strains involved in the following examples are from the following sources: Methicillin-resistant Staphylococcus aureus (ATCC) 43300 and *Alternaria rotundifolia* CCUG 45780T were purchased from Mingzhou Biotechnology Co., Ltd.; *Clostridium plasmidonense* FWXPL1Y45 (CCFM1205, published in patent CN115093999B, accession number GDMCC No: 62240), *Bacteroides masei* FHNXY-54-K-2 (published in a dissertation from Jiangnan University, "The Influence of the Structural Composition of Intestinal Bacteroides on the Development of Ulcerative Colitis," *Bacteroides masei* FHNXY-54-K-2 is the *Bacteroides masei* isolated in the literature, sourced from Xiayi County, Henan Province), and *Bacteroides monomorpha* FSDTAHCK_B1 (published in *Structure–function relationship of Konjac glucomannan with varying acetylation degrees in modulating gut microbiota and alleviating prediabetes*, *CarbohydratePolymers*, 2026: 125103) and Bacteroides FTJS6K5 (published in A CAZymes atlas of the human gut microbiota: From 40,000+ genomes to bench validation, Food Research International 2026: 118573) from the Food Biotechnology Center of Jiangnan University; VSL#3 probiotic preparation was purchased from VSLExperience overseas company.

[0040] Example 1: Preparation of the synthetic bacterial community Neg_M 1. Experimental strains The synthetic bacterial community Neg_M used in this embodiment includes the following 5 strains: Clostridium plasminoides ( Faecalibacterium prausnitzii CCFM1205, Bacteroides monomorpha ( Bacteroides uniformis FSDTAHCK_B1, Rotten Fungi ( Alistipes putredinis CCUG 45780T, Bacteroides masei ( Phocaeicola massiliensis FHNXY-54-K-2 and Bacteroides multiforme ( Bacteroides thetaiotaomicron FTJS6K5.

[0041] The control probiotic preparation was VSL#3, and the pathogen was methicillin-resistant Staphylococcus aureus ATCC 43300.

[0042] 2. Strains culture Bacteroides monomorphosus, Bacteroides masei, and Bacteroides polymorphosus were inoculated at a 4% inoculum in mPYG medium, while Cronobacter spp. were inoculated in CMC medium, and Clostridium plasmidonum inoculated in M2GSC medium. All strains were cultured under strictly anaerobic conditions at 37°C. The cultures were subcultured twice to ensure viability.

[0043] 3. Preparation of synthetic microbial communities Each bacterial strain was cultured to the logarithmic growth phase, and the OD of each bacterial culture was measured under anaerobic conditions. 600 The values ​​were adjusted according to the bacterial concentration. Then, the bacterial strains were mixed at a ratio of 1:1:1:1:1 to obtain the synthetic bacterial community Neg_M. The mixed bacterial solution was centrifuged at 3000×g for 10 min at 4℃ to collect the bacterial cells. The supernatant was discarded, and the bacterial cells were resuspended in 15% glycerol, aliquoted, and stored at -80℃ for later use.

[0044] 4. Preparation of the bacterial culture for administration Before animal experiments, the frozen synthetic bacterial culture Neg_M was revived under anaerobic conditions, and the bacterial concentration was adjusted. During gavage, each mouse was given 200 μL of the bacterial suspension, ensuring a total dose of 1 × 10⁻⁶ synthetic bacterial culture per mouse. 9 CFU.

[0045] Example 2: The preventive antagonistic effect of the synthetic microbial community Neg_M on MRSA intestinal colonization 1. Laboratory animals and grouping Fifteen male germ-free mice were randomly divided into three groups of five each: the Neg_M group, the VSL#3 group, and the control group.

[0046] 2. Dosing and Infection Control Protocol according to Figure 1 The experimental procedure shown was as follows: from day -7 to day -5, mice in the Neg_M group were administered the synthetic microbial community Neg_M once daily by gavage, mice in the VSL#3 group were administered VSL#3 once daily by gavage, and mice in the control group were administered an equal volume of sterile saline by gavage daily. The gavage volume for each group was 200 μL, with each mouse in the Neg_M and VSL#3 groups receiving a dose of 1 × 10⁻⁶ μL. 9 CFU. After pretreatment, on day 0, all mice were administered 200 μL of methicillin-resistant Staphylococcus aureus (ATCC) 43300 bacterial suspension by gavage, with a dose of 1 × 10⁻⁶ CFU per mouse. 9 CFU was used to establish an intestinal colonization model.

[0047] 3. Sample collection and testing Fecal samples were collected from mice on days 0, 1, 3, 5, 7, and 10 post-infection to determine the absolute viral load of methicillin-resistant Staphylococcus aureus (MRSA). Quantitative analysis of MRSA was performed using qPCR, and the results were converted to absolute viral load using a standard curve. Results are expressed as CFU / g feces. Some fecal samples were used for subsequent non-targeted metabolomics analysis.

[0048] 4. Experimental Results The results are as follows Figure 2 As shown, both the Neg_M group and the VSL#3 group reduced the colonization level of methicillin-resistant Staphylococcus aureus (MRSA) in the mouse intestine, but the sustained effects of the two groups differed significantly. The Neg_M group maintained a relatively stable inhibitory effect throughout the entire 10-day infection observation period, with the MRSA viral load remaining at approximately 10. 7 CFU / g fecal level, compared to 10 in the control group. 9 The CFU / g fecal level decreased by approximately 100-fold; while the protective effect of the VSL#3 group was present in the early stage of infection, but gradually weakened after day 5. These results indicate that the synthetic bacterial flora Neg_M described in this invention can provide sustained and stable colonization resistance in vivo, exhibiting a more durable antagonistic effect against methicillin-resistant Staphylococcus aureus.

[0049] Example 3: Therapeutic decolonization effect of synthetic microbial community Neg_M on established MRSA colonies 1. Laboratory animals and grouping Eighteen germ-free mice, half male and half female, were randomly divided into three groups of six mice each: the Neg_M group, the VSL#3 group, and the control group.

[0050] 2. Infection and Treatment Plan according to Figure 3 The experimental procedure shown was as follows: on day 0, all mice were administered 200 μL of methicillin-resistant Staphylococcus aureus (ATCC) 43300 bacterial suspension by gavage, with each mouse receiving a dose of 1 × 10⁻⁶. 9 CFU was used to establish a stable colonization model. After pathogen colonization was established, therapeutic intervention was administered for three consecutive days from day 7 to day 9. The Neg_M group received Neg_M via gavage once daily, the VSL#3 group received VSL#3 via gavage once daily, and the control group received 200 μL of sterile saline via gavage daily. The dosage of bacterial culture in each group was 1×10⁻⁶. 9 CFU / each.

[0051] 3. Sample collection and testing Fecal samples were collected from mice on days 3, 6, 10, 12, and 16, and fecal bacterial DNA was extracted. The absolute MRSA load was detected by qPCR. Results are expressed as CFU / g feces. Animal weight was monitored regularly during the experiment.

[0052] 4. Experimental Results The results are as follows Figure 4 As shown, before the microbial intervention (days 3 and 6), the MRSA load in the feces of mice in all groups remained at a high level, approximately 10. 9 CFU / g. After intervention began on day 7, the MRSA load in the Neg_M group continued to decrease; while the VSL#3 group showed only a limited decrease, subsequently plateauing. Endpoint results are as follows: Figure 5 As shown, compared with the control group, Neg_M treatment significantly reduced MRSA viral load by nearly three orders of magnitude; simultaneously, the MRSA viral load in the Neg_M group was significantly lower than that in the VSL#3 group. The endpoint MRSA viral load in the Neg_M group was approximately 4.93 × 10⁻⁶. 6 CFU / g, while group VSL#3 remained at 10. 8 CFU / g or higher.

[0053] The above results demonstrate that the synthetic microbial community Neg_M described in this invention can not only prevent MRSA colonization, but also has a significant decolonization effect on established MRSA intestinal colonies.

[0054] Example 4: Antagonistic effect of synthetic bacterial community Neg_M on MRSA in vitro 1. Strains activation and co-culture protocol Methicillin-resistant Staphylococcus aureus ATCC 43300 seed culture was inoculated into mPYG medium at an inoculation rate of 2% (v / v) and continuously passaged for two generations to activate it.

[0055] Following the method described in Example 1, bacterial suspensions of *Bacteroides monomorpha*, *Bacteroides masei*, *Bacteroides polymorpha*, *Alternaria rotundifolia*, and *Clostridium plasmidon* were prepared. The strains were mixed at a viable count ratio of 1:1:1:1:1 to obtain the synthetic bacterial community Neg_M. Neg_M was inoculated into mPYG medium at a 2% (v / v) inoculum (initial OD 0.03) and continuously passaged for two generations. VSL#3 was inoculated into mPYG medium (initial OD 0.03) and anaerobic cultured at 37°C for 12 h, continuously activating for two generations. Using mPYG medium, the OD values ​​of Neg_M, VSL#3, and methicillin-resistant Staphylococcus aureus (ATCC) 43300 were adjusted to the same level. 600 Value 0.2.

[0056] Grouping and culturing were carried out in the following manner: Control group: Activated MRSA was inoculated into fresh mPYG medium at an inoculation rate of 2% (v / v), without inoculating other bacterial groups, and physiological saline was added at an inoculation rate of 2% (v / v) as a control. Neg_M group: Activated MRSA and synthetic bacteria Neg_M were co-inoculated into fresh mPYG medium at an inoculation rate of 2% (v / v); VSL#3 group: Activated MRSA and VSL#3 were co-inoculated into fresh mPYG medium at an inoculum of 2% (v / v). Each group was divided into 3 replicates and cultured anaerobically at 37°C for 12 h.

[0057] 3. Sample collection and testing After the culture was completed, the culture medium of each group was collected, and the MRSA in the culture medium was quantitatively detected. The results were expressed as CFU / mL. Unpaired two-tailed t-tests were used to compare the differences between the two groups, and p < 0.05 was considered statistically significant.

[0058] 4. Experimental Results The results are as follows Figure 6 As shown, after 12 h of anaerobic culture, the MRSA load in the control group cultured alone was approximately 5.94 × 10⁻⁶. 8 CFU / mL; after co-culturing with the synthetic bacterial community Neg_M, the MRSA load decreased to approximately 1.69 × 10⁻⁶ CFU / mL. 7 The CFU / mL level was reduced by approximately 97.2% compared to the control group, equivalent to a reduction of approximately 1.55 orders of magnitude. The MRSA load in group VSL#3 was approximately 9.53 × 10⁻⁶. 7 The CFU / mL was also lower than that of the control group, but its load was still higher than that of the Neg_M group.

[0059] Further comparison revealed that the MRSA load in the Neg_M group was approximately 17.8% of that in the VSL#3 group, which is a decrease of about 82.2% compared to the VSL#3 group. This indicates that under the in vitro co-culture conditions of this embodiment, the synthetic bacterial community Neg_M exhibits a strong inhibitory effect on MRSA growth, and its inhibitory effect is greater than that in the VSL#3 group.

[0060] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A synthetic bacterial community antagonistic to methicillin-resistant Staphylococcus aureus, characterized in that, The synthetic microbial community includes any two or more of the following species: Clostridium perfringens (… Faecalibacterium prausnitzii ), Bacteroides monomorpha ( Bacteroides uniformis ), putrefactive bacteria ( Alistipes putredinis Bacteroides masei ( Phocaeicola massiliensis ) and Bacteroides multiforme ( Bacteroides thetaiotaomicron ).

2. The synthetic microbial community according to claim 1, characterized in that, The synthetic microbial community includes Clostridium praosporum CCFM1205, Bacteroides monomorpha FSDTAHCK_B1, Alternaria rotica CCUG 45780T, Bacteroides masei FHNXY-54-K-2, and Bacteroides polymorpha FTJS6K5.

3. The synthetic microbial community according to claim 2, characterized in that, In the synthesized bacterial community, the viable count ratio of Clostridium pralis CCFM1205, Bacteroides monomorphosum FSDTAHCK_B1, Alternaria rotica CCUG 45780T, Bacteroides masei FHNXY-54-K-2, and Bacteroides polymorphosum FTJS6K5 was 1:1:1:1:

1.

4. The synthetic microbial community according to any one of claims 1 to 3, characterized in that, The synthetic microbial community is a live bacterial preparation, freeze-dried bacterial powder, bacterial suspension, capsule preparation, granule, oral liquid or microcapsule preparation.

5. The synthetic microbial community according to claim 4, characterized in that, The number of cells in the synthetic bacterial community is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

6. A product containing any of the synthetic microbial communities described in claims 1 to 5.

7. The product according to claim 6, characterized in that, The products include pharmaceuticals, probiotics, food, health products, or feed additives.

8. The product according to claim 7, characterized in that, The drug also contains a pharmaceutical carrier and / or pharmaceutical excipients.

9. The use of the synthetic microbial community according to any one of claims 1 to 5 in the preparation of a medicine for the prevention and / or treatment of methicillin-resistant Staphylococcus aureus infection.

10. The use of the synthetic bacterial group according to any one of claims 1 to 5 in in vitro antagonism against methicillin-resistant Staphylococcus aureus.

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