CN117085050A

CN117085050A - Application of candida tropicalis in control of clostridium difficile

Info

Publication number: CN117085050A
Application number: CN202310767936.6A
Authority: CN
Inventors: 曹阳春; 姚军虎; 王腊梅; 王菁; 赵聪聪
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2023-06-27
Filing date: 2023-06-27
Publication date: 2023-11-21

Abstract

The application belongs to the technical field of microorganisms and biological medicines, and particularly relates to application of candida tropicalis in clostridium difficile prevention and treatment. Specifically, the application provides a novel thinking and theoretical basis for preventing and relieving clostridium diarrhea by changing the traditional method for treating clostridium diarrhea by using antibiotics, and creatively providing fungi which can inhibit clostridium difficile growth or relieve toxin generation from the perspective of symbiotic fungi. The research result is expected to provide a product which is safe, efficient, cost-saving and easy to operate and is used for preventing and treating clostridium diarrhea, which is used for replacing antibiotic therapy, has important significance for treating and relieving clostridium diarrhea and other related diseases, and has good practical application value.

Description

Application of candida tropicalis in control of clostridium difficile

Technical Field

The application belongs to the technical field of microorganisms and biological medicines, and particularly relates to application of candida tropicalis in clostridium difficile prevention and treatment.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the application and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Diarrhea is a common disease of an intensive dairy cow farm, is frequently generated in calves, can cause massive dehydration of organisms and unbalanced water and salt metabolism, finally causes metabolic syndrome, and is an important factor affecting the health of calves, reducing the production efficiency and increasing the feeding cost (Ma et al 2020). Research shows that the detection rate of clostridium difficile in the intestinal tracts of diarrhea calves is between 10 and 40 percent, and some clostridium difficile can reach 60 percent, and the diarrhea calves are frequently born to 2 months of age. Clostridium difficile infection (Clostridiume difficile infection, CDI) is a multi-step, multi-stage process, affected by a number of factors, and the infected person may develop symptoms of severe diarrhea and even die. The use of broad-spectrum antibacterial agents inhibits or kills normal flora such as lactobacillus and bifidobacteria in the intestinal tract, causing dysbacteriosis in the intestinal tract, thereby weakening or losing antagonism to clostridium difficile, overgrowing clostridium difficile and releasing toxins, causing antibiotic-associated diarrhea (Antibiotic associated diarrhea, AAD) in animals, and causing death in severe cases.

Previous studies have shown that clostridium difficile infection has a correlation with microorganisms and metabolites in the gut, and at present, studies of other microorganisms interacting with clostridium difficile and the effect on clostridium difficile pathogenicity have been focused mainly on bacterial aspects, while less and less comprehensive studies are conducted from a fungal point of view.

Disclosure of Invention

Aiming at the defects existing in the prior art, the application provides the application of candida tropicalis in preventing and treating clostridium difficile, successfully screens out the fungus candida tropicalis which can inhibit the growth of clostridium difficile, reveals the influence of the fungus candida tropicalis on the pathogenicity of clostridium difficile, provides a theoretical basis for preventing and treating clostridium difficile diarrhea of calves, and has important significance in maintaining the health of milk animals and improving the production benefit of livestock industry. Based on the above results, the present application has been completed.

Specifically, the application relates to the following technical scheme:

in a first aspect of the application there is provided the use of candida tropicalis for in vitro inhibition of clostridium difficile.

According to the application, researches show that the candida tropicalis and clostridium difficile are subjected to in vitro contact co-culture, so that the clostridium difficile growth can be inhibited, but the expression of clostridium difficile toxin genes can be promoted; and the clostridium difficile is subjected to in-vitro non-contact co-culture, the inhibition effect of the candida tropicalis on the clostridium difficile growth is eliminated, but the expression of clostridium difficile toxin genes can be reduced, and the exercise capacity of the clostridium difficile is slowed down, so that the pathogenicity of the clostridium difficile is relieved. Thus, in particular, the application comprises:

(a1) The candida tropicalis and clostridium difficile inhibit the growth of clostridium difficile when being subjected to in-vitro contact co-culture;

(a2) The candida tropicalis and clostridium difficile are subjected to in-vitro non-contact co-culture, so that the expression of clostridium difficile toxin genes is reduced, the exercise capacity of clostridium difficile is slowed down, and the pathogenicity of clostridium difficile is relieved.

Wherein, in the step (a 2), the in vitro non-contact co-culture can be performed by any known prior art, and in the present application, the non-contact culture is performed using a Transwell chamber, which is actually used for exploring the effect of candida tropicalis metabolites on clostridium difficile. Wherein the candida tropicalis is cultured by adopting YM culture medium.

The clostridium difficile toxin genes include, but are not limited to TcdA, tcdB, tcdC, tcdR.

In a second aspect of the application there is provided the use of candida tropicalis in the manufacture of a product for the prevention and/or treatment of clostridium difficile infection.

According to the application, the concept of "prevention and/or treatment" means any suitable measure for the treatment of clostridium difficile associated diseases, such as diarrhea, or for the prophylactic treatment of such an expressed disease or of an expressed symptom, or for the avoidance of recurrence of such a disease, for example after the end of a treatment period or for the treatment of symptoms of an already-developed disease, or for the pre-interventional prevention or inhibition or reduction of the occurrence of such a disease or symptom.

In a third aspect of the application there is provided the use of candida tropicalis in the manufacture of a product against clostridium difficile.

In a fourth aspect of the application there is provided the use of candida tropicalis in the manufacture of a product for inhibiting clostridium difficile growth.

In the second to fourth aspects, the product may be a drug or an experimental reagent for use in basic research, for use in relevant model construction, for research of clostridium difficile pathogenic mechanism, etc.

In a fifth aspect of the application there is provided a pharmaceutical composition for combating clostridium difficile infection, said pharmaceutical composition consisting of candida tropicalis and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

In particular, other non-pharmaceutically active ingredients include pharmaceutically acceptable excipients and/or carriers.

The pharmaceutical composition of the present application comprising the above candida tropicalis may be administered in unit dosage form. The administration dosage form may be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.

The pharmaceutical compositions or formulations of the present application may also contain conventional carriers, which include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum proteins, buffer substances such as phosphates, glycerol, sorbitol, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of 1% to 98% by weight, typically about 80% by weight. For convenience, local anesthetics, preservatives, buffers, and the like may be directly dissolved in the carrier.

In a sixth aspect of the present application, there is provided a method of preventing and/or treating a disease associated with clostridium difficile infection, the method comprising: the candida tropicalis or the pharmaceutical composition is administered to the subject.

Wherein the clostridium difficile infection-associated disease comprises diarrhea;

the subject may be a human or non-human mammal including, but not limited to, pigs, cows, dogs, horses, rabbits, cats, and sheep.

It must be recognized that the optimal dosage and spacing of the active ingredients of the present application is determined by its nature and external conditions such as the form, route and site of administration and the particular mammal being treated, and that such optimal dosage may be determined by conventional techniques. It must also be appreciated that the optimal course of treatment, i.e. the daily dosage of the simultaneous compounds over the nominal time period, can be determined by methods well known in the art.

The beneficial technical effects of the technical scheme are that:

according to the technical scheme, the traditional method for treating clostridium diarrhea by using antibiotics is changed, the novel thinking and theoretical basis for preventing and relieving clostridium diarrhea are provided for creatively screening fungi which can inhibit clostridium difficile growth or relieve toxin production from the perspective of symbiotic fungi. The research result is expected to provide a product which is safe, efficient, cost-saving and easy to operate and is used for preventing and treating clostridium diarrhea, which is used for replacing antibiotic therapy, has important significance for treating and relieving clostridium diarrhea and other related diseases, and has good practical application value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application.

FIG. 1 is a graph showing the growth of strains in example 1 of the present application; a is C.difficile; b is P.fermentions (1); c is C.dublininensis; d is C.tropicalis (2) ₆₀₀ A curve of the value over time.

FIG. 2 shows the OD600 measurement of in vitro co-culture of Clostridium difficile with each fungus in example 1 of the present application; a1 and A2 are P.fermantans (1) and P.fermantans (2) OD, respectively ₆₀₀ A value measurement result; B-D are C.dublininensis, A.niger and A.prograndosOD, respectively ₆₀₀ A value measurement result; E1-E4 are C.tropicalis (1) - (4) OD, respectively ₆₀₀ The measurement results were different, indicating that the difference was significant (P<0.05 Representing that the difference is extremely significant (P<0.01 Representing that the difference is extremely significant (P<0.001)。

FIG. 3 shows the effect of clostridium difficile co-cultivation in vitro with various fungi on clostridium difficile growth in example 1 of the present application; a1 and A2 are the measurement results of clostridium difficile bacteria of P.fermentum (1) and P.fermentum (2) respectively; B-D are C.dublininensis, A.niger and A.prolifferans clostridium difficile assay results, respectively; figures E1-E4 are c.tropicalis (1) - (4) clostridium difficile measurements, respectively, different values indicating differences in significance, differences in significance (P < 0.05), differences in significance (P < 0.01), differences in significance (P < 0.001).

FIG. 4 shows the effect of co-culture of various fungi with Clostridium difficile in vitro on genes related to Clostridium difficile in example 1 of the present application; A-D are the relative expression of TcdA, tcdB, tcdC, tcdR genes, respectively. By performing single-factor analysis of variance, letters (AB) and (AB) respectively represent that the relative expression conditions of genes in the clostridium difficile single culture group and the co-culture group have obvious differences among 5 time points, and the different letters represent that the differences are obvious; by performing independent sample T-test analysis, each gene was expressed differently between the two groups at each time point, expressed as P <0.05, P <0.01, and P < 0.001.

FIG. 5 shows the results of the in vitro non-contact co-culture OD600 measurement of Clostridium difficile and C.tropicalis (3) in example 2 of the present application. A is a graph of non-contact co-cultivation of clostridium difficile and C.tropicalis (3) using a Transwell chamber, B is the result of OD600 value at each time point of the lower chamber of the clostridium difficile single culture group in non-contact co-cultivation, and C is the result of OD600 value at each time point of two treatment groups.

FIG. 6 shows the effect of clostridium difficile on clostridium difficile growth by non-contact co-cultivation with C.tropicalis (3) in example 2 of the present application.

FIG. 7 shows the effect of clostridium difficile co-cultivation with C.tropicalis (3) on clostridium difficile toxin-associated gene expression in example 2 of the present application; A-D are the relative expression of TcdA, tcdB, tcdC, tcdR genes, respectively. By performing one-way analysis of variance, letters (AB) and (AB) respectively indicate that there are significant differences between the relative expression of the genes in the clostridium difficile individual culture group and the co-culture group at 5 time points; by performing independent sample T-test analysis, each gene was expressed differently between the two groups at each time point, expressed as P <0.05, P <0.01, and P < 0.001.

FIG. 8 is the effect of clostridium difficile on clostridium difficile motility by non-contact co-cultivation with C.tropicalis (3) in example 2 of the present application; A-E are the results of the motility test of Clostridium difficile in the group of Clostridium difficile, the non-contact co-culture of Clostridium difficile with C.tropicalis (3), respectively, for 0-48 h.

FIG. 9 shows the effect of clostridium difficile on the biofilm formation capacity of clostridium difficile by non-contact co-cultivation with C.tropicalis (3) in example 2 of the present application; a is the expression level of Cwp84 gene, and B is the result of staining the biofilm by crystal violet staining method.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The application will be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not to be construed as limiting the application. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the sales company; materials, reagents and the like used in the examples were commercially available unless otherwise specified.

Example 1Screening fungi inhibiting clostridium difficile growth in vitro

In the test, the in vitro contact co-culture test is carried out on each fungus and clostridium difficile respectively, the effect of each fungus on the in vitro growth of clostridium difficile is explored, the fungus with the most obvious inhibition on the clostridium difficile growth is screened, and the influence of the fungus on the clostridium difficile toxin gene expression is explored.

1 materials and methods

1.1 test strains

Clostridium difficile ATCC 43255 (Clostridium difficile, C.diffiiile) used in the test was purchased from ATCC company in the United states, pichia fermentans (Pichia fermantis) 2 strains, designated as F3YB-3-2 and F10YB-3-3, respectively (presented below as P.fermantans (1) and P.fermantans (2) for convenience of description), candida dubliensis (Candida dubliensis) 1 strain, designated as PC35 (presented below as C.dubliensis) for convenience of description, candida tropicalis (Candida trompilis) 4 strain, the numbers are 150-1 to 10-3, 149-1 to 11-4, 6-9 to 1 and 31 to 10-8 (which are respectively represented by C.tropicalis (1), C.tropicalis (2), C.tropicalis (3) and C.tropicalis (4) for convenience of description), the Aspergillus niger strain 1 is 13JKQ84, and the Aspergillus oryzae strain 1 (which is respectively represented by A. Niger, A. Prograns for convenience of description) is presented by the institute of China academy of sciences microbiological culture, wherein C.tropicalis (3) is CICC 1681 and C.tropicalis 4) is CICC 1316.

1.2 test reagents

During the cultivation, the respective fungi and clostridium difficile correspond to the culture medium used as shown in table 1 below.

TABLE 1 Strain and Medium correspondence table

1.3 design of experiments

The test is divided into a negative control group (pure BHI liquid culture medium), a clostridium difficile independent culture group and a clostridium difficile co-culture group and fungus co-culture treatment groups respectively, 15 replicates of each group are respectively obtained, 3 replicates are respectively reserved for 0, 12, 24, 36 and 48 hours for preservation to be detected.

1.4 Co-cultivation of bacteria and fungi in contact

According to the measured results of the growth curves of clostridium difficile and fungi, selecting strains in a logarithmic growth phase (the strain state is good at the moment), namely selecting clostridium difficile which grows for about 8 hours, fermenting pichia pastoris, candida dublinii, candida tropicalis and aspergillus niger and aspergillus breeder which grow for about 36 hours, and performing a co-culture experiment. The specific operation steps are as follows:

(1) in an ultra-clean workbench, subpackaging BHI liquid culture medium into 12mL shaking tubes with a 5mL pipette, wherein each tube is 10mL, and the BHI liquid culture medium is divided into a negative control group, a clostridium difficile and fungus co-culture group, and 3 repeats of each group are prepared for 0, 12, 24, 36 and 48 hours;

(2) taking Clostridium difficile and fungus liquid in logarithmic growth phase, and obtaining OD of the (2) bacteria in 2.1.3.1 ₆₀₀ Value determination method for detecting OD ₆₀₀ The values are respectively marked as OD1 and OD2, and the bacterial liquid concentrations of clostridium difficile and fungus are respectively calculated to be C1 and C2 according to the corresponding relation between the bacterial liquid OD value and the concentration established in the earlier stage;

(3) to achieve a1 x 10 initial clostridium difficile and fungus broth concentration in the 10mLBHI broth ⁶ cfu/mL, recording that the volume of clostridium difficile bacteria liquid to be inoculated is V1, the volume of fungus to be inoculated is V2, and according to the molar law formula N=C, V (N, the amount of substances; C, the concentration of the substances; V, the volume of solution), the amounts of the substances are equal, then:

C1*V1＝1*10 ⁶ (cfu/mL)*10(mL)，V1＝1*10 ⁷ /C1(mL)；

C2*V2＝1*10 ⁶ (cfu/mL)*10(mL)，V2＝1*10 ⁷ /C2(mL)。

(4) after bacterial liquid is sequentially added into the corresponding fungus shaking tube according to the time point sequence from 0h to 48h, the mouth of the fungus shaking tube and the tube cover are burnt by the outer flame of the alcohol lamp, the tube cover is quickly covered, the tube cover is put on a test tube rack, the tube cover is put into an anaerobic culture bag, an anaerobic gas producing bag is quickly put into the anaerobic culture bag, the sealed bag mouth is put into a bacteria incubator at 37 ℃ for 0, 12, 24, 36 and 48h culture.

1.5 collection of bacterial liquid

After reaching the culture time point, fully and uniformly mixing each tube of bacterial liquid, blowing by a pipetting gun, and then sucking 2 1mL of bacterial liquid into a 1.5mL sterile centrifuge tube respectively, wherein 1 part of bacterial liquid is used for immediately detecting OD ₆₀₀ The value, 1 part frozen at-80 ℃ for subsequent extraction of bacterial DNA, and another 5mL frozen at-80 ℃ in a 10mL centrifuge tube for bacterial RNA extraction.

1.6 determination of growth curves

(1) Bacterial growth curve detection

Taking purified clostridium difficile bacterial liquid and measuring OD thereof ₆₀₀ Value according to OD ₆₀₀ And calculating the proportional relation between the value and the bacterial liquid concentration, and recording the bacterial liquid concentration as C1. To each shake tube, 5mLBHI broth was added to give an initial Clostridium difficile broth concentration of 1X 10 inoculated in 5mLBHI broth ⁶ cfu/mL (i.e. OD ₆₀₀ =0.01), note that the volume of clostridium difficile fluid to be inoculated is V1, V (N, amount of substance according to moore's law formula n=c; c, the mass concentration of the substance; v, solution volume), the amount of material is equal, then:

C1*V1＝1*10 ⁶ (cfu/mL)*5(mL)，V1＝0.5*10 ⁷ c1 (mL); immediately covering the tube cover after the inoculation is finished, filling the tube cover into an anaerobic culture bag, rapidly placing an anaerobic gas producing bag, sealing the mouth of the bag, placing the bag into a bacterial incubator at 37 ℃ for culture, and respectively measuring the OD (optical density) of bacterial solutions at 0, 4, 8, 12, 14, 16, 25 and 31 hours ₆₀₀ Values.

(2) Fungal growth curve detection

The method of "detection of bacterial growth curve" was the same as that of the above-mentioned "(1).

1.7OD ₆₀₀ Determination of the relation between the value and the concentration of the bacterial liquid

Early team experiments showed that when clostridium difficile bacteria liquid OD ₆₀₀ When=0.5, the bacterial liquid concentration is about 1×10 ⁸ cfu/mL。

To establish the quantitative relationship between the OD value of each fungus and the corresponding concentration of the fungal liquid, we selected three strains of fungi P.fermentans (1), C.dublininensis and C.tropicalis (2) (from P.fermentans (1) and P.fermentans (2) OD ₆₀₀ Value, C.tropicalis (1) -C.tropicalis (4) OD ₆₀₀ The values were almost uniform with time, so that P.fermentions (1) and C.tropicalis (2) were randomly selected for dilution plating, and OD was measured separately ₆₀₀ The bacterial liquid is respectively subjected to gradient dilution and then is plated for 48 hours, on the premise of no pollution of a negative control group, single colonies on each plate are counted, the numerical value with larger difference is removed, and the initial concentration of the bacterial liquid is calculated, thereby establishing OD ₆₀₀ Relationship between the value and the concentration of the bacterial liquid.

The method is not suitable for obtaining colony concentration because no turbidity phenomenon of bacterial liquid is observed in the growth process of the A. Niger and the A. Prograners. In the actual culture process, the growth state of the strain can be judged according to the increase of the number of the A. Niger and A. Proliferrins meristematic bacteria balls.

1.8 treatment group OD ₆₀₀ Value detection

200 mu L of bacterial liquid is sucked into a 96-hole ELISA plate (when sucking, the mixture is blown and evenly mixed) from a 1.5mL sterile centrifuge tube by a pipetting gun, 3 repeated holes are added, the ELISA plate is placed at a wavelength of 600nm for detection, the OD value is the average value of 3 holes, and the OD values of the other two tubes can be obtained. Taking bacterial liquid of a negative control group with the same culture time, sucking 200 mu L of liquid from a tube into a 96-hole ELISA plate by a pipetting gun, detecting the ELISA plate at a wavelength of 600nm for 3 repeated holes, obtaining an OD value of the negative control group which is a 3-hole average value, obtaining the OD values of the other two negative control groups, calculating the OD average value of 3 tubes, marking the OD value as OD1, and subtracting the OD value of the negative control group from the OD value of each tube of each treatment group.

1.9RT-qPCR detection of Clostridium difficile

And (3) according to the established standard curve, carrying the Ct value of the sample into an equation, and obtaining the corresponding clostridium difficile concentration of each sample.

1.10RT-qPCR detection of clostridium difficile toxin-related Gene expression

Based on the Gene database in NCBI, searching the complete sequence of each Gene, carrying out primer design for SYBR Green dye method fluorescence quantitative PCR by a 'free primer design' plate of a webpage of Shanghai Biotechnology Co., ltd, and carrying out primer synthesis by Shanghai Biotechnology Co., ltd, wherein the used primers and sequences are shown in annex F. By AG CoqPCR operation is carried out according to the specification by the Green Pro Taq HS premixed qPCR kit, and the amplification reaction system and conditions are shown in annex G. After the program is finished, parameters such as Ct value and the like are recorded, the bacterial rpoc gene is taken as an internal reference gene, and the method is according to 2 ^-△△Ct And calculating the gene expression quantity. 3 duplicate wells were made for each DNA template and the average of 3 detection results was taken for analysis to reduce experimental error.

1.11 data sorting and analysis

Statistical analysis of the data using SPSS software, multiple comparisons using One-way ANOVA (One-way ANOVA), duncan method; a difference comparison between the two groups was performed using independent sample T-test.

2. Test results

2.1 results of measurement of growth curves of strains

The change in OD values of the bacterial liquids C.difficile, P.fermentans (1), C.dublininensis and C.tropicalis (2) with time are shown in FIG. 1.

2.2 contact Co-culture OD ₆₀₀ Value determinationResults

Clostridium difficile and each fungus were subjected to in vitro co-culture experiments at OD at time points 0, 12, 24, 36, 48h, respectively ₆₀₀ The results of the value measurement are shown in FIG. 2. From each OD ₆₀₀ From the values, clostridium difficile and fungi are well conditioned at each time point, conforming to the change in strain growth from log-plateau curves.

2.3 Effect of fungi on Clostridium difficile growth in vitro

Clostridium difficile was co-cultivated in vitro with fungus by liquid contact and absolute quantitative detection of clostridium difficile in different treatment groups was performed, the results are shown in figure 3. (1) Clostridium difficile was co-cultivated with p.fermentum (1), with clostridium difficile amounts lower than in the 12, 36h co-cultivated group (P < 0.05) alone, with p.fermentum (2), and with clostridium difficile amounts lower than in the 36h co-cultivated group (P < 0.05) alone. (2) Clostridium difficile was co-cultivated with c.dublinensis, with no significant difference in clostridium difficile amounts (P > 0.05) in both treatment groups at each time point. (3) Clostridium difficile was co-cultivated with a. Niger, with a lower clostridium difficile amount in the 24h co-cultivated group than in the alone cultivated group (P < 0.01). (4) Clostridium difficile was co-cultured with a.prograns, with no significant difference in clostridium difficile amounts (P > 0.05) in the co-and individual culture groups at each time point. (5) Clostridium difficile was co-cultivated with c.tropicalis (1), with a lower clostridium difficile content in the 12h co-cultivated group than in the alone cultivated group (P < 0.05); co-cultivation with C.tropicalis (2) the amount of Clostridium difficile in the 12, 24h co-cultivated group was significantly lower than in the alone cultivated group (P < 0.01); co-cultivation with c.tropicalis (3), the clostridium difficile amount in the 12h co-cultivated group was significantly lower than in the alone cultivated group (P < 0.001); co-culture with C.tropicalis (4) there was no significant difference in Clostridium difficile amounts (P > 0.05) in the co-culture group versus the individual culture groups at each time point.

2.4 effects of Clostridium difficile Co-culture with C.tropicalis (3) on Clostridium difficile toxin-related Gene expression

The results of measuring the expression level of TcdA, tcdB, tcdC, tcdR gene at 5 time points in the two treatment groups are shown in FIG. 4. The relative expression quantity of TcdA in the two treatment groups is highest for 12h, and then gradually decreases; the relative expression of TcdA in 24-48h co-culture group was significantly higher than in Clostridium difficile group (P < 0.001). The relative expression of TcdB in both groups was also highest at 12h, then gradually decreased, and the relative expression of TcdB in the co-culture group at the time point of 12-48h was significantly higher than that in the Clostridium difficile group (P < 0.001). The relative expression quantity of TcdC in the two groups is high in 12h, and then gradually decreases, and the groups have no difference. The relative expression of TcdR in the two groups is highest in 12h, and then gradually decreases, and the relative expression of TcdR in the co-culture group is obviously higher than that of clostridium difficile group in 12, 36 and 48h (12 h, P <0.01;36, 48h, P < 0.05).

In this study, according to the growth curve of clostridium difficile measured in the early stage, clostridium difficile is in the growth plateau phase in 8-12h, the amount of the new bacteria is equal to the amount of dead bacteria, and the growth environment of clostridium difficile is limited, so that TcdA and TcdB genes are highly expressed from 12h in a clostridium difficile single culture group, and the mRNA expression amounts of TcdA and TcdB genes in a 12-48h co-culture group have a tendency to be higher than that of clostridium difficile single culture group or are extremely higher than that of clostridium difficile single culture group, which is probably related to competition of nutrient substances caused by Ct (3) and clostridium difficile. the tcdC gene is highly expressed in the early exponential phase and decreases with increasing growth phase. the tcdR gene is used as a positive regulatory gene of a toxin gene, has a trend or significance higher than that of clostridium difficile in a 12-48h co-culture group, and is consistent with the fact that the co-culture groups of TcdA and TcdB at corresponding time points are higher than that of the clostridium difficile.

The inhibition of clostridium difficile growth by tropicalis (3) was most pronounced. Co-cultivation of Clostridium difficile with C.tropilis (3) promotes expression of the Clostridium difficile toxin gene.

Example 2Influence of in vitro non-contact co-culture on clostridium difficile growth and pathogenicity

The test is based on a fungus C.tropicalis (3) which is screened by in vitro contact co-culture and has the most obvious effect of inhibiting clostridium difficile growth, and the fungus C.tropicalis and clostridium difficile are subjected to in vitro non-contact co-culture test, so that the effect of the fungus C.tropicalis on clostridium difficile growth is explored, and the influence of the fungus C.difficile on clostridium difficile pathogenicity is illustrated by detecting the clostridium difficile toxin gene expression condition, motility, adhesiveness and biofilm formation capability.

1 materials and methods

1.1 test strains

Clostridium difficile ATCC 43255 (Clostridium difficile, C.difficicle), candida tropicalis (Candida tropicalis 6-9-1, C.tropicalis 3).

1.2 design of experiments

The experiments were divided into a negative control group (pure BHI broth), clostridium difficile alone, clostridium difficile co-culture with c.tropicalis (3) two treatment groups, 3 replicates each, and the co-culture time was divided into 0, 12, 24, 36, 48h 5 time points.

1.3 non-contact Co-cultivation of bacteria and fungi

(1) Taking clostridium difficile growing for 8 hours and 36hC.tropicalis bacterial liquid, and detecting OD ₆₀₀ The values are respectively marked as OD1 and OD2, and the bacterial liquid concentrations of clostridium difficile and C.tropicalis (3) are respectively calculated to be C1 and C2 according to the corresponding relation between the bacterial liquid OD value and the bacterial liquid concentration established in the earlier stage;

(2) to achieve an initial bacterial concentration of clostridium difficile or c.tropicalis (3) in 5mL BHI broth of 1 x 10 ⁶ cfu/mL, recording that the volume of clostridium difficile bacteria liquid to be inoculated is V1, the volume of fungus to be inoculated is V2, and according to the molar law formula N=C, V (N, the amount of substances; C, the concentration of the substances; V, the volume of solution), the amounts of the substances are equal, then:

C1*V1＝1*10 ⁶ (cfu/mL)*5(mL)，V1＝0.5*10 ⁷ /C1(mL)；

C2*V2＝1*10 ⁶ (cfu/mL)*5(mL)，V2＝0.5*10 ⁷ /C2(mL)。

(3) 5mL of BHI liquid culture medium was placed in a 6-well culture plate, 2mL of C.tropicalis (3) stock solution V was inoculated into each of the A1-C1 wells, a Transwell chamber (shown in Table 2) was placed in each well, 5mL of BHI liquid culture medium was added to the chamber, and 1mL of Clostridium difficile stock solution V was inoculated into each of the 6 chambers (plate layout shown in Table 2). Covering the plate cover, rapidly placing into anaerobic culture bag, rapidly placing into an anaerobic gas producing bag, and placing into a bacteria incubator at 37deg.C for 0, 12, 24, 36, 48 hr.

TABLE 2 Clostridium difficile C.tropicalis (3) non-contact Co-culture plates

1.4 cultivation of bacterial biofilms

(1) Taking clostridium difficile bacterial liquid cultured for 8 hours, fully and uniformly mixing, and measuring OD thereof ₆₀₀ Values, noted OD1;

(2) mixing the cultured C.tropicalis (3) bacterial liquid for 36 hr, and measuring OD ₆₀₀ Values, noted OD2;

(3) taking 10mL of C.tropicalis (3) bacterial liquid cultured for 48h, centrifuging at 12000rpm for 10min, transferring the supernatant into a sterilized 10mL centrifuge tube, and re-suspending bacterial sediment with 5mLPBS solution for later use;

(4) plates were plated on sterile 24-well plates with fresh pure BHI broth as negative control as follows:

TABLE 3 Clostridium difficile C.tropicalis (3) non-contact Co-culture biofilm plates

Each treatment was performed in 3 replicates, with c.diffiile wells, each with 1mL of fresh pure BHI broth medium, μl of clostridium difficile stock solution (OD 1/0.01) (such that the initial clostridium difficile concentration was 1 x 10) ⁶ cfu/mL), and uniformly mixing by blowing;

difficile+C.tropicalis (3) bacterial liquid holes, 1mL of fresh pure BHI broth culture medium is added into each hole, mu L of clostridium difficile original bacterial liquid (OD 1/0.01) and mu L of C.tropicalis (3) original bacterial liquid (OD 2/0.01) are blown and evenly mixed;

PBS solution is added into the rest holes, and the 12-hole plates with the plates are respectively subjected to anaerobic culture for 0h, 12h, 24h, 36h and 48h at 37 ℃.

1.5 detection of Clostridium difficile movement Capacity

Adding 5mL of 0.5% BHI semisolid culture into each shaking tube, carrying out ultraviolet irradiation in an ultra-clean workbench, taking and mixing the grown clostridium difficile bacterial liquid and co-culture bacterial liquid fully and uniformly after solidification (about 30 min), dipping the bacterial liquid by a disposable sterile inoculating needle, vertically penetrating into the right center of the semisolid culture medium, quickly covering a tube cover, filling into an anaerobic culture bag, quickly putting into an anaerobic production bag, and culturing in a bacterial incubator at 37 ℃ for 0, 12, 24, 36 and 48h.

1.6 detection of Clostridium difficile biofilm

After reaching the incubation time point:

(1) the medium in each well was aspirated and removed with ddH ₂ O washing for 2-3 times to remove bacteria floating in the holes, ventilating in an ultra-clean workbench for 15min for air drying, adding 500 mu L of 1% crystal violet solution into each hole, and dyeing for 20min;

(2) the crystal violet solution was removed by pipetting and blotting with ddH ₂ O is washed until the solution is no longer purple (about 5 times), at the moment, a round biological film dyed into purple is seen at the bottom of the pore plate, and the pore plate is air-dried for 10min in an ultra-clean workbench;

(3) 1mL of 95% ethanol was added to each well to blow and dissolve the purple pellet, and after complete dissolution, absorbance was measured at 570nm using an microplate reader and recorded.

1.7 data arrangement and analysis

2. Test results

2.1 non-contact Co-culture OD ₆₀₀ Value measurement results

Non-contact culture experiments using Transwell chambers, it was first observed that BHI solution was clear and transparent in the chambers of clostridium difficile individual culture groups at each time point (as shown in fig. 5A), indicating that clostridium difficile solution in the upper chamber did not permeate the lower chamber; OD is performed ₆₀₀ Value determination (as shown in FIG. 5B), it was found that at each time point the BHI solution OD in the lower chamber of Clostridium difficile individual culture group ₆₀₀ The values are about 0.1, and are the same as the OD of the negative control group ₆₀₀ The values are almost identical; by culturing Clostridium difficile alone in group B in the lower chamberHI solution coated CCFA plates showed little clostridium difficile growth, and therefore, it is feasible to use a Transwell chamber for non-contact culture experiments.

Clostridium difficile was subjected to in vitro non-contact co-culture with C.tropicalis (3) at each time point of 0, 12, 24, 36, 48h in two treatment groups OD ₆₀₀ The result of the value measurement is shown in FIG. 5C. From OD ₆₀₀ From the values, clostridium difficile and c.tropilis (3) performed well at each time point, conforming to the change in growth curve.

2.2 Effect of non-contact Co-cultivation on Clostridium difficile growth

Absolute quantitative detection of clostridium difficile in both treatment groups showed that clostridium difficile growth was not affected as seen from the results of figure 6.

2.3 Effect of non-contact Co-culture on clostridium difficile toxin-associated Gene expression

The results of measurement of the expression of TcdA, tcdB, tcdC, tcdR gene at 5 time points in the two treatment groups are shown in FIG. 7. The relative expression quantity of TcdA in the two treatment groups is highest for 12h, and then gradually decreases; the relative expression amount of TcdA in the 24-48h co-culture group is obviously lower than that of clostridium difficile group. The relative expression quantity of TcdB in the two groups is the highest in 12h, and gradually decreases after 12h, and the relative expression quantity of TcdB in the co-culture group at the time point of 12-48h is obviously lower than that of the clostridium difficile group. The relative expression quantity of the TcdC in the two groups is high in 12h and gradually decreases after 12h, and the relative expression quantity of the TcdC in the 24-48h co-culture group is obviously lower than that of the clostridium difficile group. The relative expression quantity of TcdR in the two groups is highest in 12h, and gradually decreases after 12-48h, and the relative expression quantity of TcdR in the co-culture group is obviously lower than that of clostridium difficile group.

2.4 Effect of non-contact Co-cultivation on the ability of Clostridium difficile to move

The semi-solid puncture assay was performed on clostridium difficile motility in clostridium difficile groups, clostridium difficile and c.tropicalis (3) non-contact co-culture groups, and the results are shown in fig. 8. A decrease in the ability of Clostridium difficile to exercise was observed in the 24h co-culture group, followed by a gradual recovery.

2.5 Effect of non-contact Co-cultivation on the biofilm Forming Capacity of Clostridium difficile

For clostridium difficile group and clostridium difficileThe expression condition of a regulatory gene Cwp84 gene formed by a biological film in a non-contact co-culture group of bacteria and C.tropicalis (3) is detected, the expression capability of the gene in the co-culture group of 24h and 48h is reduced, a clostridium difficile biological film is dyed by a crystal violet dyeing method, and the OD (optical density) of the clostridium difficile biological film is measured ₅₇₀ The biofilm forming ability of clostridium difficile was observed to be significantly higher in the 48h co-culture group than in the culture group alone. The results are shown in FIG. 9.

The influence of C.tropicalis (3) metabolites on clostridium difficile is mainly observed by non-contact co-culture, the inhibition effect of C.tropicalis (3) on clostridium difficile growth is disappeared after non-contact co-culture, analysis is performed from the aspect that fungi influence clostridium difficile, and the effect is possible because certain metabolites of fungi are available for clostridium difficile growth, so clostridium difficile growth is not inhibited, and the increase of clostridium difficile toxin gene expression caused by nutrition competition can be relieved; it is also possible that the expression of clostridium difficile toxin genes may be regulated by certain metabolites of the fungus, such that the co-cultivated group toxin gene expression is significantly lower than that of clostridium difficile alone. Analysis was performed from the standpoint of clostridium difficile affecting fungi, probably because metabolites produced by clostridium difficile have an inhibitory effect on fungal growth, fungi are growth-restricted, clostridium difficile can utilize more nutrients, and therefore no growth inhibition occurs. Clostridium difficile requires movement to the site of colonization by flagella and adhesion to host cells by secretion of adhesins, so that a strong motility is an important part of the pathogenic process of clostridium difficile, and our results have been observed to reduce the motility of clostridium difficile at 24h by non-contact co-cultivation with c.tropicalis (3), which may be a result of affecting flagella-related genes. The biofilm as a "reservoir" of clostridium difficile plays an important role in the persistent, recurrent process of clostridium difficile infection, and we detected that co-culture can reduce the expression of the regulatory gene Cwp84 of the biofilm, but no consistent result was observed by a crystal violet staining method, indicating that the analysis of co-culture from the Cwp84 gene level alone is not comprehensive in the effect on the biofilm.

The results of this study show that candida tropicalis affects clostridium difficile growth and virulence in vitro, indicating that the candida tropicalis may have potential effects of preventing and treating CDI, but specific effects and interaction mechanisms need to be further explored through in vivo experiments.

Clostridium difficile and c.tropicalis (3) are subjected to in vitro non-contact co-culture, so that the inhibition effect of c.tropicalis (3) on clostridium difficile in vitro growth is eliminated, and the expression of clostridium difficile toxin protein genes tcdA and tcdB can be reduced, and the motility of clostridium difficile can be slowed down, so that c.tropicalis (3) has the potential effect of reducing the pathogenicity of clostridium difficile.

Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present application, and the present application is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present application has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. The application of candida tropicalis in inhibiting clostridium difficile in vitro; specifically, the application includes:

2. The use of claim 1, wherein in (a 2) the in vitro noncontact co-culture is performed using a Transwell chamber; the candida tropicalis is cultured by adopting YM culture medium.

3. The use of claim 1 wherein the clostridium difficile toxin genes comprise TcdA, tcdB, tcdC and TcdR.

4. Use of candida tropicalis for the preparation of a product for the prevention and/or treatment of clostridium difficile infection.

5. Use of candida tropicalis in the preparation of a clostridium difficile resistant product.

6. Use of candida tropicalis in the preparation of a product for inhibiting clostridium difficile growth.

7. The use according to any one of claims 4 to 6, wherein the product is a pharmaceutical or an experimental agent for use in basic research.

8. A pharmaceutical composition against clostridium difficile infection, comprising candida tropicalis and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

9. The pharmaceutical composition of claim 8, wherein the additional non-pharmaceutically active ingredient comprises a pharmaceutically acceptable adjuvant and/or carrier.

10. A method of preventing and/or treating a disease associated with clostridium difficile infection, the method comprising: administering candida tropicalis or the pharmaceutical composition of claim 8 or 9 to a subject.