TW202110464A - Probiotic and probiotic combination for inhibition of vancomycin-resistant enterococci and use thereof - Google Patents

Abstract

A probiotic combination for inhibition of vancomycin-resistant enterococci includes Bacillus coagulans, Lactobacillus rhamnosus GG, Lactobacillus reuteri, and Lactobacillus acidophilus. The probiotic combination is used for preparing medical and health products for inhibiting growth, adherence, or virulence of vancomycin-resistant enterococci, and is used for inhibiting virulence gene expression of vancomycin-resistant enterococci.

Description

Probiotics for inhibiting vancomycin-resistant enterococci and their combinations and uses thereof

This case is about a combination of probiotics, especially a combination of probiotics that inhibits vancomycin-resistant enterococci.

Enterococci is a kind of intestinal bacteria resident in the human intestines. It has now become one of the most important pathogens of opportunistic infections in the world. When the patient's immune status is low, such as serious underlying diseases, low white blood cell count, and urethra Or when the vascular catheter is placed and the hospital stay is too long, it may cause urinary tract infection, myocarditis, meningitis, or sepsis, and cause serious medical problems that cannot be effectively treated with late-line antibiotics. The proportion of enterococcus clinical isolates around the world found to be vancomycin-resistant enterococci (vancomycin-resistant enterococci, referred to as VRE) is 4.0% in Europe, 6.0% in Canada, and 11.9% in Asia. It is 12.9% in South America, and even as high as 35.5% in the United States. In addition, the incidence of VRE among patients in intensive care units has been increasing in recent years and has reached 4.4-12.3%. Taiwan discovered the first VRE in 1996, and in the 2013 annual report of Taiwan's Opportunistic Infection Surveillance System, as many as 28.7% of the enterococcus isolates from patients in the intensive care unit were vancomycin resistant, and they were found in National Taiwan University. The rate of hospitalization in the intensive care unit of hospitals is as high as 21.9 people received new VRE per 1,000 person-days. It can be seen that VRE has become a very serious threat to human health and medical treatment in the world.

In recent years, there have been a few studies on the use of probiotics to remove the colonization of VRE in the human intestines, but the probiotic strains used are not the same, and whether it is effective has not yet been determined. For example, Lactobacillus rhamnosus GG ( L GG for short) has seen a decrease in VRE colonization in human intestines in two clinical trials (Manley et al. (2007) Med J Aust 186: 454-457; Szachta et al. (2011) J Clin Gastroenterol 45: 872-877), but no effective removal effect was seen in another small experiment (Doron et al. (2015) Antimicrob Agents Chemother. 59(8): 4593- 9). In addition, another strain of Lactobacillus rhamnosus Lcr35 (Lactobacillus rhamnosus Lcr35) has not seen specific effects in a small-scale test in adults (Vidal et al. (2010) J Clin Microbiol 48: 2595-2598). Two other studies have shown that multiple combinations of probiotic strains have no preventive effect on VRE colonization in the human intestine, and there are even doubts about the transmission of antibiotics to intestinal bacteria (de Regt et al. (2010) Antimicrob Agents Chemother 54: 2801-2805 ; Topcuoglu et al. (2015) J Matern Fetal Neonatal Med 28: 1491-1494).

Due to the different strains, dosages, time, and research methods used in these few clinical studies, there is still an urgent need to find a combination of probiotics that can effectively inhibit or remove the VRE colonization effect.

The purpose of this case is to provide a combination of probiotics, which can inhibit the growth of vancomycin-resistant enterococci, attach to host cells or virulence, and thereby reduce the harm of vancomycin-resistant enterococci to the human body.

In order to achieve the above objective, this case provides a probiotic combination for inhibiting vancomycin-resistant enterococci. The probiotic combination is composed of Bacillus coagulans , Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG), Roy Lactobacillus reuteri and Lactobacillus acidophilus .

In order to achieve the above-mentioned purpose, this case further provides the use of a combination of probiotics, which is used to prepare medicines and health products that inhibit the growth of vancomycin-resistant enterococci, adhere to host cells or have virulence, wherein the combination of probiotics is composed of It is composed of Bacillus coagulans , Lactobacillus rhamnosus GG, Lactobacillus reuteri , and Lactobacillus acidophilus.

In order to achieve the above purpose, this case also provides a combination of probiotics, which is used to inhibit the expression of virulence genes of vancomycin-resistant enterococci, and the virulence genes include asa1 , acm , ebpA , ebpB , and ebpC at least one efaA, sagA, esp, sgrA and scm, wherein the probiotic composition of Bacillus coagulans system (Bacillus coagulans), Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG), Lactobacillus reuteri (Lactobacillus reuteri ), and Lactobacillus acidophilus (Lactobacillus acidophilus).

In order to achieve the above-mentioned purpose, this case further provides a probiotic that inhibits vancomycin-resistant enterococci. The probiotics include Bacillus coagulans , Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG), and Roy's milk. At least one of Lactobacillus reuteri and Lactobacillus acidophilus.

In order to achieve the above-mentioned purpose, the present case further provides a use of probiotics, which is used to prepare medicines and health products that inhibit the growth of vancomycin-resistant enterococci, adhere to host cells or have virulence, wherein the probiotics include Bacillus coagulans At least one of Bacillus coagulans , Lactobacillus rhamnosus GG, Lactobacillus reuteri , and Lactobacillus acidophilus.

In order to achieve the above purpose, this case also provides a use of probiotics, which is used to inhibit the expression of virulence genes of vancomycin-resistant enterococci, and the virulence genes include asa1 , acm , ebpA , ebpB , ebpC , At least one of efaA , sagA , esp , sgrA, and scm , wherein the probiotics include Bacillus coagulans , Lactobacillus rhamnosus GG, Lactobacillus reuteri , And at least one of Lactobacillus acidophilus.

In one embodiment, the probiotic or combination of probiotics inhibits the attachment of vancomycin-resistant enterococci to the intestinal epithelial cells of the host.

In one embodiment, the vancomycin-resistant enterococci include Enterococcus faecium and Enterococcus faecalis .

In one embodiment, the medical and health care product is a capsule, and the capsule includes an excipient.

In one embodiment, the excipient is corn starch.

Some embodiments embodying the features and advantages of this case will be described in detail in the following description. It should be understood that this case can have various changes in different aspects, which do not depart from the scope of this case, and the description and drawings therein are essentially for illustrative purposes, rather than limiting the case.

In this case, a directional microbial interaction network combined with biological experiments was selected to effectively inhibit the growth, attachment to host cells or virulence of vancomycin-resistant enterococci (VRE). And a combination of probiotics that inhibits the expression of its virulence genes. This probiotic combination is made up of Bacillus coagulans , Lactobacillus rhamnosus GG ( L GG), Lactobacillus reuteri , and Lactobacillus acidophilus The composition can effectively inhibit the growth of VRE in in vitro experiments, at the same time significantly inhibit the expression of VRE virulence genes, and reduce the attachment of VRE to human intestinal epithelial cell lines. The combination of probiotics provided in this case will help in the clinical application of treatment and decolonization of VRE from the host's intestines, thereby reducing the harm of VRE to the human body.

The following examples will further illustrate the method of selecting the probiotic combination in this case and related experimental verification. Figure 1 shows the flow chart of the method for selecting a combination of probiotics in this case. As shown in Figure 1, the method for selecting a combination of probiotics in this case includes selection of strains (step S1), co-cultivation (step S2), microbial analysis (step S3), selection of probiotic combinations (step S4), and experimental verification (Step S5) and other steps are described as follows.

First of all, the vancomycin-resistant enterococcus (VRE) used in this case was obtained from the infection control room of the hospital attached to Taipei Medical University, and it was confirmed by the disc diffusion assay that the vancomycin (VRE) vancomycin), and confirm the existence of vanA , vanB , vanC , vanD , vanE , vanG and other drug resistance genes by PCR. Among them, VRE contains at least two bacterial species, namely Enterococcus faecium and Enterococcus faecalis .

In step S1, in order to achieve the diversity of microbial species, this case uses classification from the edible probiotic species listed in the announcement of the Food and Drug Administration of the Ministry of Health and Welfare of Taiwan, and considers the availability of species and co-cultivation For the feasibility of selecting two of different genera, 10 probiotics listed in Table 1 were initially screened. Table 1 Bacteria scientific name Bacillus coagulans Bacillus coagulans Bifidobacterium bifidum Bifidobacterium bifidum Bifidobacterium longum subsp. infantis Bifidobacterium longum subsp. infantis Lactobacillus rhamnosus GG Lactobacillus rhamnosus GG Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis Lactobacillus plantarum Lactobacillus plantarum subsp. plantarum Lactobacillus reuteri Lactobacillus reuteri Spore-forming lactic acid bacteria Sporolactobacillus inulinus Streptococcus salivarius subsp.thermophilus Streptococcus salivarius subsp. thermophilus Lactobacillus acidophilus Lactobacillus acidophilus

Next, in step S2, the 10 kinds of probiotics that have been preliminarily screened are cultured in a shaking manner ^{, and the number of 1×10 8} CFU of each is mixed in equal amounts, and cultured together with VRE for a certain period of time. During the co-cultivation of probiotics and VRE, the amount of VRE was quantified with VRE chromogenic medium (CHROM VRE agar) to test whether these 10 probiotics can effectively inhibit VRE. Figure 2 shows the results of co-cultivation of VRE and 10 kinds of probiotics. As shown in Figure 2, compared to the independent culture of VRE, the amount of VRE decreased significantly on the second day after the co-cultivation of VRE and 10 kinds of probiotics, and there was almost no viable VRE on the third day, indicating that these 10 kinds of probiotics The combination of bacteria can inhibit the growth of VRE.

Subsequently, the bacterial liquids at 11 time points (two replicates) during the three-day co-cultivation process were selected to extract bacterial genomic DNA, and the next generation sequencing (NGS) technology was used to identify and quantify the 16s rDNA of all bacteria.

Then in step S3, using the microbiome analysis pipeline (MAP) technology, the analysis software QIIME (Caporaso et al. (2010) Nat Methods. 7(5): 335-336) is used to further obtain the results of each culture time point. The relative abundance analysis results of each bacterial race group. Figure 3 shows the relative expression of microorganisms when VRE is co-cultured with 10 kinds of probiotics, and the relative proportions of various bacterial species to the whole can be observed at each time point. Among them, Enterococcus_s represents VRE, Lactobacillus_s represents L. plantarum or L. acidophilus , and Lactobacillus_rhamnosus represents L GG. In addition, Streptococcus salivarius ( Sporolactobacillus inulinus ) was added in the original experiment, but it was not classified by QIIME in the next generation sequencing (NGS) analysis, so there is no such bacterium in Figure 3.

After obtaining the relative abundance analysis results of each strain population at each cultivation time point, use the rule-based microbial network algorithm (RMN) (Tsai et al., (2015) BMC Syst Biol 9: 54) Numerical analysis of the relative performance of any three microorganisms can obtain the cooperative and competitive relationships between microorganisms, and then generate a microbial interaction network. Figure 4 shows the predicted microbial interaction relationship diagram, in which the solid arrow is the inhibitory effect, and the hollow arrow is the assisting effect. In this case, the rule-based microbial network algorithm (RMN) was used to infer the cooperative and competitive relationship between VRE and probiotics, as well as the cooperative and competitive relationship between probiotics, and then remove the probiotics that have a cooperative relationship with VRE. , And remove the probiotics that are competing between the probiotics, and finally select the probiotic combination with the effect of inhibiting VRE as shown in Figure 4 (step S4), including Bacillus coagulans and Lactobacillus rhamnosus GG ( Lactobacillus rhamnosus GG, L GG for short), Lactobacillus reuteri , and Lactobacillus_s.

Next, in step S5, an in vitro experiment is used to verify the effect of the found combination of probiotics in inhibiting VRE.

First, since Lactobacillus_s contains L. plantarum and L. acidophilus , in order to confirm which of L. plantarum and L. acidophilus is more effective, this case compares L. plantarum (ATCC14917 strain) with B. coagulans (ATCC7050 strain), L GG (Hansen strain), L. reuteri (BioGaia strain), and L. acidophilus (Infloran strain) with B. coagulans (ATCC7050 strain), L GG (Hansen strain), L. reuteri (BioGaia strain) ) These two different combinations (all four bacteria are combined in equal proportions), the effect of inhibiting the growth of VRE during the co-cultivation process. Also in the process of co-cultivation, the amount of VRE was quantified with VRE chromogenic medium (CHROM VRE agar). Figure 5 shows the results of co-cultivation with VRE containing L. acidophilus or L. plantarum in a combination of probiotics. The results can be seen from FIG. 5, L. acidophilus with B. coagulans, the combination L GG, L. reuteri compared to the L. plantarum with B. coagulans, the combination of L. reuteri and L GG, it has better VRE The growth inhibition effect also proves that the probiotic combination inferred by the rule-based microbial network algorithm (RMN) can indeed successfully inhibit the growth of VRE.

Since L. acidophilus has a better VRE growth inhibitory effect than L. plantarum , four probiotics, B. coagulans , L GG, L. reuteri and L. acidophilus, were selected as the probiotic combination in this case.

In order to use these four probiotics in clinical trials and possible product development in the future, this case also purchases strains from different sources, including the BC1031 strain of B. coagulans, the DSMZ32250 strain of L GG, the BR101 strain of L. reuteri , and the L. acidophilus The LA1063 strain was tested, and the 16s rDNA sequence was compared with the original four edible strains. According to the sequence comparison results, the 16s rDNA of the ATCC7050 strain of B. coagulans and the BC1031 strain have 99% sequence similarity in the V3-V4 region, and the 16s rDNA of the Hansen strain of L GG and the DSMZ32250 strain have 100% in the V3-V4 region. % Sequence similarity, the 16s rDNA of the BioGaia strain of L. reuteri and the BR101 strain has 100% sequence similarity in the V3-V4 region, and the 16s rDNA of the Infloran strain of L. acidophilus and the LA1063 strain has 100% in the V3-V4 region. % Sequence similarity. In other words, the 16s rDNA of strains from different sources has almost the same sequence of the V3-V4 region, so it can be predicted that strains from different sources should also have comparable inhibitory effects.

Figure 6 shows the co-cultivation results of VRE and a combination of probiotics from different strains, in which all four types of bacteria are combined in equal proportions. According to the results in Figure 6, it can be seen that the combination of B. coagulans , L GG, L. reuteri and L. acidophilus composed of strains from different sources has a considerable inhibitory effect, which also proves the effectiveness of this combination of probiotics. The effect is indeed not limited to strains from specific sources.

In addition, a combination of the four probiotics in equal proportions and a single strain with the same amount of bacteria (4×10 ⁸ CFU) can compare the effect of inhibiting the growth of VRE. Figures 7 to 11 show the results of co-cultivation of VRE with four combinations of probiotics and individual single strains. According to the results shown in Figures 7 to 11, it can be seen that whether it is a probiotic combination composed of B. coagulans _ATCC7050, L GG_Hansen, L. reuteri _BioGaia, and L. acidophilus _Infloran, or a combination of B. coagulans _BC1031, L. The VRE inhibitory effect of the probiotic combination composed of GG_DSMZ32250, L. reuteri _BR101, and L. acidophilus _LA1063 is better than that of a single strain. B. coagulans _ATCC7050 alone does not have the effect of inhibiting the growth of VRE (as shown in Figure 7). Although L GG_Hansen, L. reuteri _BioGaia or L. acidophilus _Infloran also has the effect of inhibiting the growth of VRE, the inhibition rate is slower (such as (Shown in Figure 8 and Figure 9). B. coagulans _BC1031 alone also did not have the effect of inhibiting the growth of VRE, and the effect of L GG_DSMZ32250 alone was also inferior to the combination of four bacteria. It took 48 hours to significantly reduce the number of VRE (as shown in Figure 10). The effect of L. reuteri _BR101 alone in inhibiting the growth of VRE was slightly inferior to the combination of four bacteria at the 41-hour time point, while L. acidophilus _LA1063 alone had no effect on inhibiting the growth of VRE (as shown in Figure 11). Therefore, the probiotic combination of the four probiotics B. coagulans , L GG, L. reuteri and L. acidophilus in this case is more effective in inhibiting the growth of VRE than a single strain, which confirms that the probiotic combination provided in this case is indeed effective In vitro experiments significantly inhibited the growth of VRE.

In order to find a better combination ratio of the four probiotics for further application in clinical human trials, this case combines the four bacteria in different ratios and conducts co-cultivation experiments to compare the VRE inhibitory effects of different combination ratios. Since the predicted microbial interaction relationship diagram (as shown in Figure 4) shows that L. reuteri is the most important strain inhibiting VRE, the adjusted ratios are all based on L. reuteri as the main strain, and the other three bacteria are the secondary strains. . Figures 12 to 15 show the co-cultivation results of VRE and four probiotics in different combination ratios. In some embodiments, the four probiotics B. coagulans , L GG, L. reuteri and L. acidophilus are tested in four combination ratios, which are 1:1:1:1 (that is, the contents of the four bacteria are respectively 25%, 25%, 25% and 25%), 1.2: 0.5: 1.8: 0.5 (that is, the contents of the four bacteria are 30%, 12.5%, 45% and 12.5%), 0.5: 0.5: 1.8: 1.2 ( That is, the contents of the four bacteria are 12.5%, 12.5%, 45%, and 30%), and 0.5:1.2:1.8:0.5 (that is, the contents of the four bacteria are 12.5%, 30%, 45%, and 12.5%) . In other words, the contents of the four bacteria B. coagulans , L GG, L. reuteri and L. acidophilus are 12.5-30 %, 12.5-30%, 25-45% and 12.5-30%, respectively.

As shown in Figures 12 and 13, the probiotic combination (represented by the first source) composed of B. coagulans _ATCC7050, L GG_Hansen, L. reuteri _BioGaia and L. acidophilus _Infloran (represented by the first source) is 1:1:1 :1, 1.2: 0.5: 1.8: 0.5, 0.5: 0.5: 1.8: 1.2, or 0.5: 1.2: 1.8: 0.5, the effects of inhibiting the growth of VRE are similar. As shown in Figures 14 and 15, a combination of probiotics (represented by the second source) composed of B. coagulans _BC1031, L GG_DSMZ32250, L. reuteri _BR101, and L. acidophilus _LA1063 from another source At the combination ratio of 1:1:1:1 or 0.5:1.2:1.8:0.5, the effect of inhibiting the growth of VRE is equivalent, and the inhibition effect of the combination ratio of 0.5:0.5:1.8:1.2 is slightly inferior, while the inhibitory effect of the combination ratio of 1.2:0.5:1.8 : The inhibitory effect of the combination ratio of 0.5 is also slightly inferior to that of the equal ratio combination at the 41st hour.

According to the above experimental results, the four bacteria B. coagulans , L GG, L. reuteri and L. acidophilus are probiotics composed of 12.5-30%, 12.5-30%, 25-45% and 12.5-30% respectively. The combination of bacteria has an obvious inhibitory effect on the growth of VRE, and the combination of roughly equal proportions has a better inhibitory effect on the growth of VRE. Since the aforementioned combination ratio has a significant inhibitory effect on the growth of VRE, and the different combination ratio does not significantly affect the effect of this probiotic combination to restrict the growth of VRE, it can be reasonably inferred that the combination of equal or unequal proportions can be effective The growth of VRE is restricted, and the combination ratio used in the above embodiments is only used to demonstrate the scope of possible implementation, not to limit the case. Therefore, other combination ratios of the same four bacteria should not deviate from the scope of protection of this case.

On the other hand, this case also further analyzed the probiotic combination composed of B. coagulans , L GG, L. reuteri and L. acidophilus . During the co-cultivation process, the virulence genes acm and acm related to the adhesion and survival of the VRE host were further analyzed. The performance of asa1 is affected. Among them, the acm gene is mainly related to the adhesion and colonization of Enterococcus faecium (E. faecium ), while the asa1 gene is mainly related to the adhesion and colonization of Enterococcus faecalis (E. faecalis). In this experiment, samples were taken at 0 hours and 16 hours during the co-cultivation process, and bacterial RNA was extracted to perform quantitative real-time polymerase chain reaction (qRT-PCR), and then calculate the acm gene or The expression level of the asa1 gene after 16 hours of co-cultivation was compared with the expression level at the beginning of the co-cultivation to analyze the changes in the expression level of the two virulence genes after the co-cultivation of VRE and the probiotic combination of this case.

Figure 16 shows the effect of the probiotic combination from the first source on the acm gene expression of Enterococcus faecium. Figure 17 shows the effect of the probiotic combination from the first source on the expression of the asa1 gene of Enterococcus faecalis. Figure 18 shows the effect of the probiotic combination from the first source on the acm gene expression of Enterococcus faecalis. The effect of the combination of two sources of probiotics on the expression of the acm gene of Enterococcus faecium and the asa1 gene of Enterococcus faecalis. It can be seen from the schematic result that the probiotic combination (the first source) composed of B. coagulans _ATCC7050, L GG_Hansen, L. reuteri _BioGaia and L. acidophilus _Infloran is at 1:1:1:1 and 0.5:1.2: The combination ratio of 1.8:0.5 can effectively inhibit the expression of acm gene and asa1 gene, and the effect is similar. In addition, the probiotic combination (second source) composed of B. coagulans _BC1031, L GG_DSMZ32250, L. reuteri _BR101, and L. acidophilus _LA1063, which is in the range of 1:1:1:1 and 0.5:0.5:1.8:1.2 The combination ratio can significantly inhibit the expression of acm gene, while the expression of asa1 gene is in the combination ratio of 1:1:1:1, 0.5:1.2:1.8:0.5, and 0.5:0.5:1.8:1.2 Down, can effectively suppress its performance. Therefore, the probiotic combination provided in this case can directly and effectively inhibit the expression of the virulence genes acm and asa1 of VRE. Since the acm gene and asa1 gene are related to the adhesion and colonization of VRE in the host intestine, the acm gene and asa1 gene are inhibited. The performance of VRE will help to colonize VRE from the host's intestines, thereby reducing the harm of VRE to the human body.

In addition, this case also further observed the interaction of the probiotic combination composed of B. coagulans , L GG, L. reuteri and L. acidophilus with VRE and human intestinal epithelial cell lines, which was performed by a competition assay. In comparison, in the presence of the human intestinal epithelial cell line Caco-2, after adding the probiotic combination and VRE for co-cultivation, the effect of the probiotic combination on reducing the attachment of VRE to Caco-2 cells was observed. In a demonstration experiment, in order to simplify the experimental design, only Enterococcus faecium, which is clinically more resistant to vancomycin, was used as the representative of VRE for the experiment. The probiotic combination in this case was co-cultured with Enterococcus faecium and the human intestinal epithelial cell line Caco-2. Figure 19 shows the effect of different ratios of probiotic combinations on the attachment of Enterococcus faecium to epithelial cells. From the graphical results, it can be seen that the probiotic combination (second source) composed of B. coagulans _BC1031, L GG_DSMZ32250, L. reuteri _BR101, and L. acidophilus _LA1063 is 1:1:1:1, 1.2:0.5: The combination ratios of 1.8:0.5, 0.5:1.2:1.8:0.5, and 0.5:0.5:1.8:1.2 can significantly reduce the number of Enterococcus faecium attached to Caco-2 cells in a competitive manner. That is, the different ratios of probiotic combinations provided in this case can significantly reduce VRE attachment to human intestinal epithelial cells.

On the other hand, this case also tried to compare the combination of four bacteria and the difference in the effect of a single strain in reducing the adhesion of VRE to epithelial cells. This experiment can be divided into the aforementioned competition assay and displacement assay two different analytical methods. In the competitive analysis method, probiotics and VRE are added to co-culture with the intestinal epithelial cell line. After a period of time, compared with the control group without any probiotics, whether the number of VRE attached is reduced. The substitution analysis rule is to first co-culture VRE with the intestinal epithelial cell line for a period of time, then add probiotics for co-cultivation, and finally compare with the control group without any probiotics to observe whether the subsequent probiotics added can reduce the first added probiotics The effect of VRE attached to the intestinal epithelial cell line. The intestinal epithelial cell line used in this experiment is the human intestinal epithelial cell line Caco-2, and the VRE is Enterococcus faecium.

Figure 20 shows the comparison of the effects of different probiotics on the attachment of Enterococcus faecium to epithelial cells by the competitive analysis method, Figure 21 shows the comparison of the effects of different probiotics on the attachment of Enterococcus faecium to the epithelial cells by the substitution analysis method . From the results in Figure 20, it can be seen that a combination of probiotics (second source) composed of B. coagulans _BC1031, L GG_DSMZ32250, L. reuteri _BR101 and L. acidophilus _LA1063 in equal proportions, or a single B. coagulans _BC1031 Both L GG_DSMZ32250 and L. reuteri _BR101 alone can reduce the adhesion of Enterococcus faecium to Caco-2 cells to 30% to 40% of the control group without probiotics, and there is no significant difference between these groups. The group using L. acidophilus _LA1063 did not significantly reduce the adhesion of Enterococcus faecium to Caco-2 cells. In addition, from the results in Figure 21, it can be seen that the combination of the four bacteria or B. coagulans _BC1031 alone, L GG_DSMZ32250 alone, and L. reuteri _BR101 alone can reduce the number of Enterococcus faecium attached to Caco-2 cells first. Up to 65% to 77% of the control group without probiotics, and there was no significant difference between these groups, and the group using L. acidophilus _LA1063 alone could not significantly reduce the adhesion of Enterococcus faecium. According to the aforementioned experiment, in addition to the combination of the four bacteria can significantly reduce the attachment of VRE to host cells, B. coagulans , L GG and L. reuteri alone can also significantly reduce the attachment of VRE to host cells.

In order to understand why the combination of probiotics in this case affects the attachment of VRE to host cells, in this case, after VRE is co-cultured with the human intestinal epithelial cell line Caco-2, we observe the virulence genes related to VRE attachment to host cells and biofilms. Expression level, the analyzed virulence genes include acm , ebpA , ebpB , ebpC , efaA , sagA , esp , sgrA and scm, etc., and observe whether the addition of probiotics combination will affect the expression level of these VRE virulence genes. In a demonstration experiment, the probiotic combination in this case was combined in equal proportions with B. coagulans _BC1031, L GG_DSMZ32250, L. reuteri _BR101 and L. acidophilus _LA1063, and was combined with Enterococcus faecium and the human intestinal epithelial cell line Caco-2. Co-cultivation for 2.5 hours, after which sampling and analysis of the expression of each virulence gene.

Figures 22 to 30 show the effects of the combination of probiotics from the second source on the virulence genes of Enterococcus faecium in the presence of host cells. The main analysis is after the co-cultivation of VRE and Caco-2 (VRE+Caco-2) , The expression level of each virulence gene relative to the control group (VRE) where VRE is not in contact with Caco-2, and when the probiotic combination also exists in the culture environment (VRE+Caco-2+probiotic combination) . The expression level is based on the control group (VRE), showing the relative value of the experimental group, and the value is taken as Log ² . From Fig. 23 to Fig. 25, it can be observed that when VRE is co-cultured with Caco-2 (VRE+Caco-2), the expression levels of ebpA , ebpB and ebpC genes have approximately a 2-fold increase. On the other hand, it is obvious from Figure 22 to Figure 30 that if the probiotic combination of this case is added at the same time during the co-cultivation process, acm , ebpA , ebpB , ebpC , efaA , sagA , esp , sgrA and scm The expression levels of the nine genes have decreased significantly. It is speculated that this may be the reason why the combination of probiotics in the competitive analysis method can effectively reduce the VRE attached to the intestinal epithelial cell line Caco-2. In other words, the probiotic combination in this case has the effect of down-regulating (inhibiting) the expression of VRE virulence genes, which can effectively reduce the attachment of VRE to host cells, help to colonize VRE from the host intestine, and reduce VRE The virulence (virulence), thereby reducing the harm of VRE to the human body.

Therefore, the probiotic combination provided in this case can indeed significantly inhibit the growth of VRE, attach to host cells or virulence, so it can be further developed and prepared as medical and health products to effectively prevent or treat VRE infection. For example, the probiotic combination provided in this case can be further prepared as a probiotic capsule, which contains B. coagulans , L GG, L. reuteri and L. acidophilus , as well as excipients. In one embodiment, the four strains are combined in equal proportions, and the excipient may be corn starch, but it is not limited thereto. In some other embodiments, the four strains are respectively composed of 12.5% to 30%, 12.5% to 30%, 25% to 45%, and 12.5% to 30%, and the excipient may be corn starch, but it is not limited to this. . In addition to taking a probiotic combination to treat VRE infection after being infected with VRE, you can also take a probiotic combination to prevent VRE infection when the patient is hospitalized or when the immune status is low, or even take it daily to promote intestinal health.

It is worth noting that the most important spirit of this case is to use a directional microbial interaction network to select a combination of probiotics that can effectively inhibit the growth of VRE, and combine biological experiments to further verify its effectiveness in inhibiting VRE. The combination ratios of the four bacteria used in the above embodiments are only used to demonstrate the scope of possible implementation, and are not used to limit the case, and the other combination ratios of the four bacteria should not deviate from the scope of protection of the case.

In addition, in addition to the probiotic combination of the four bacteria provided in this case, which can effectively inhibit the growth of VRE, attach to host cells, or virulence, B. coagulans , L GG, L. reuteri or L. acidophilus alone can also inhibit the growth of VRE, The effect of attachment to host cells or virulence. For example, Figures 8 to 11 show the effect of L GG, L. reuteri or L. acidophilus alone in inhibiting the growth of VRE, as shown in Figures 20 and 21. It can be seen that B. coagulans , L GG or L. reuteri alone inhibit the effect of VRE on host cells. Therefore, the efficacy of a single strain to inhibit VRE should also be covered by the scope of protection of this case.

In summary, this case uses a directional microbial interaction network combined with biological experiments to select a combination of probiotics that can effectively inhibit the growth of vancomycin-resistant Enterococcus (VRE), including Bacillus coagulans , Four types of bacteria are Lactobacillus rhamnosus GG ( L GG for short), Lactobacillus reuteri , and Lactobacillus acidophilus. This combination of probiotics can effectively inhibit the growth of VRE in in vitro experiments, and the four probiotics are not limited to strains from specific sources, and the combination ratio of the four bacteria is not limited. In addition, this probiotic combination can reduce the attachment of VRE to human intestinal epithelial cells and significantly inhibit VRE virulence genes, including asa1 , acm , ebpA , ebpB , ebpC , efaA , sagA , esp , sgrA, and scm . which performed. Based on the above, the probiotic combination provided in this case can effectively inhibit the growth of VRE, attach to host cells or virulence, and is expected to be further developed and prepared as medicines and health products, which will help in clinical treatment or prevention applications, and reduce VRE from Decolonization in the host intestine reduces the virulence of VRE, thereby reducing the harm of VRE to the human body.

Even though the present invention has been described in detail by the above-mentioned embodiments, it can be modified in many ways by those skilled in the art, but it does not deviate from the scope of the attached patent application.

S1: Step 1 S2: Step 2 S3: Step 3 S4: Step 4 S5: Step 5

Figure 1 shows the flow chart of the method for selecting a combination of probiotics in this case. Figure 2 shows the results of co-cultivation of VRE and 10 kinds of probiotics. Figure 3 shows the relative expression of microorganisms when VRE is co-cultured with 10 kinds of probiotics. Figure 4 shows the predicted microbial interaction relationship. Figure 5 shows the results of co-cultivation of Lactobacillus acidophilus or Lactobacillus plantarum in a probiotic combination with VRE. Figure 6 shows the co-cultivation results of VRE and a combination of probiotics from different strains. Figures 7 to 11 show the results of co-cultivation of VRE with four combinations of probiotics and individual single strains. Figures 12 to 15 show the co-cultivation results of VRE and four probiotics in different combination ratios. Figure 16 shows the effect of the probiotic combination from the first source on the acm gene expression of Enterococcus faecium. Figure 17 shows the effect of the probiotic combination from the first source on the expression of the asa1 gene of Enterococcus faecalis. Figure 18 shows the effect of the probiotic combination from the second source on the expression of the acm gene of Enterococcus faecium and the asa1 gene of Enterococcus faecalis. Figure 19 shows the comparison of the effects of different ratios of probiotic combinations on the attachment of Enterococcus faecium to epithelial cells using a competitive analysis method. Figure 20 shows the comparison of the effects of different probiotics on the attachment of Enterococcus faecium to epithelial cells using a competitive analysis method. Figure 21 shows the comparison of the effects of different probiotics on the attachment of Enterococcus faecium to epithelial cells by the substitution analysis method. Figures 22 to 30 show the effects of a combination of probiotics from the second source on the virulence genes of Enterococcus faecium in the presence of host cells.

Claims (13)

A use of a combination of probiotics, which is used to prepare medicines and health products that inhibit the growth of vancomycin-resistant enterococci, adhere to host cells or have virulence, wherein the combination of probiotics is composed of Bacillus coagulans , Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG), Lactobacillus reuteri , and Lactobacillus acidophilus . The use of the probiotic combination according to claim 1, wherein the probiotic combination inhibits the attachment of vancomycin-resistant enterococci to the intestinal epithelial cells of the host. The use of the probiotic combination according to claim 1, wherein the vancomycin-resistant enterococci include Enterococcus faecium and Enterococcus faecalis . The use of the probiotic combination according to claim 1, wherein the medicine and health product is a capsule, and the capsule contains an excipient. The use of the probiotic combination according to claim 4, wherein the excipient is corn starch. A combination of probiotics, which is used to inhibit the expression of virulence genes of vancomycin-resistant enterococci, and the virulence genes include asa1 , acm , ebpA , ebpB , ebpC , efaA , sagA , esp , sgrA And at least one of scm , wherein the probiotic combination is composed of Bacillus coagulans , Lactobacillus rhamnosus GG, Lactobacillus reuteri , and Lactobacillus acidophilus ( Lactobacillus acidophilus ). The use of the probiotic combination according to claim 6, wherein the vancomycin-resistant enterococci include Enterococcus faecium and Enterococcus faecalis . A probiotic combination for inhibiting vancomycin-resistant enterococci, the probiotic combination is composed of Bacillus coagulans , Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG), and Lactobacillus reuteri , And Lactobacillus acidophilus (Lactobacillus acidophilus). The probiotic combination according to claim 8, wherein the probiotic combination inhibits the attachment of vancomycin-resistant enterococci to the intestinal epithelial cells of the host. The probiotic combination according to claim 8, wherein the vancomycin-resistant enterococcus comprises Enterococcus faecium and Enterococcus faecalis . A use of probiotics, which is used to prepare medicines and health products that inhibit the growth of vancomycin-resistant enterococci, adhere to host cells or have virulence, wherein the probiotics include Bacillus coagulans and rhamnose At least one of Lactobacillus GG ( Lactobacillus rhamnosus GG), Lactobacillus reuteri , and Lactobacillus acidophilus. A use of probiotics, which is used to inhibit the expression of virulence genes of vancomycin-resistant enterococci, and the virulence genes include asa1 , acm , ebpA , ebpB , ebpC , efaA , sagA , esp , sgrA, and at least one of scm , wherein the probiotics include Bacillus coagulans , Lactobacillus rhamnosus GG, Lactobacillus reuteri , and Lactobacillus acidophilus ) At least one of them. A probiotic that inhibits vancomycin-resistant enterococci, the probiotics include Bacillus coagulans , Lactobacillus rhamnosus GG, Lactobacillus reuteri , and Lactobacillus reuteri At least one of Lactobacillus acidophilus.

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