TW202329997A - Lactic acid bacterial composition and its use in preparation of oral composition of inhibiting drug-resistant enterobacteriaceae - Google Patents

Abstract

The invention provides a lactic acid bacterial composition and its use in preparation of an oral composition of inhibiting drug-resistant Enterobacteriaceae. The aforementioned lactic acid bacterial composition includes Lacticaseibacillus rhamnosusJJ101 as an active ingredient. The lactic acid bacterial composition can inhibit the growth of the drug-resistant Enterobacteriaceaeafter oral administration in a subject, and thus can potentially be used to prevent, improve and/or treat the infection of the drug-resistant Enterobacteriaceae.

Description

Lactic acid bacteria composition and its use for preparing oral composition for inhibiting drug-resistant Enterobacteriaceae

The present invention relates to a composition of lactic acid bacteria, in particular to a composition of lactic acid bacteria and its use for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae.

Enterobacteriaceae ( Enterobacteriaceae ) are Gram-negative bacteria, belonging to the γ-proteobacteria Enterobacteriaceae. Enterobacter is ubiquitous in the environment (such as: soil and water) and organisms (such as: animals and plants), and is one of the intestinal bacteria of the human body. Enterobacteriaceae contain beneficial commensal bacteria as well as pathogenic bacteria that are opportunistic. These pathogens can cause dysentery, enteric fever, urinary tract infection, wound infection, liver abscess, sepsis, meningitis, pneumonia and other diseases, and are one of the main pathogens of nosocomial and community infections.

Antibiotics are the main drugs for the treatment of Enterobacteriaceae infection, among which carbapenem antibiotics have a wide range of antibacterial activity, and there are few drug-resistant strains. They are currently the last line of defense against multidrug-resistant Enterobacteriaceae. However, in recent years, enterobacteria such as Klebsiella pneumoniae ( Klebsiella pneumoniae ) have evolved methods to reduce their susceptibility to carbapenem antibiotics, such as: carbapenemase-producing Enterobacteriaceae (CPE) show Carbapenemase, which can decompose carbapenem antibiotics, thereby increasing the morbidity and mortality of infected patients, is one of the major threats to global public health.

In view of the limitations of drugs such as antibiotics for the control of bacterial infections, it is urgent to provide a non-drug composition for inhibiting drug-resistant Enterobacteriaceae and solve the above problems.

Therefore, one aspect of the present invention is to provide a lactic acid bacteria composition. The lactic acid bacteria composition contains Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101 as an active ingredient to inhibit the growth of drug-resistant Enterobacteriaceae.

Another aspect of the present invention is to provide a use of Lactobacillus rhamnosus for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the oral composition contains Lactobacillus rhamnosus JJ101 as an active ingredient.

According to the above aspects of the present invention, a lactic acid bacteria composition is proposed. This lactic acid bacteria composition contains Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101 as an active ingredient to inhibit the growth of drug-resistant Enterobacteriaceae. The Lactobacillus rhamnosus JJ101 was deposited in the Food Industry Foundation on December 22, 2021 The Bioresource Collection and Research Center (BCRC) of the Development Institute, with the registration number BCRC 911088.

In one embodiment of the present invention, the lactic acid bacteria composition may optionally contain 1% to 5% by weight of prebiotics. In one embodiment of the present invention, the prebiotic includes lactulose and/or isomaltooligosaccharide. In an embodiment of the present invention, the drug-resistant Enterobacteriaceae has carbapenemase ( Klebsiella pneumoniae carbapenemase, KPC)-2 of Klebsiella pneumoniae. In one embodiment of the present invention, the subject is administered with an effective dose of the lactic acid bacteria composition for at least 14 days. In an embodiment of the present invention, when the subject is a mouse, the effective dosage may be, for example, 5.0×10 ¹⁰ CFU/kg body weight/day to 1.5×10 ¹¹ CFU/kg body weight/day.

According to another aspect of the present invention, a use of Lactobacillus rhamnosus for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae is proposed, wherein the oral composition contains Lactobacillus rhamnosus JJ101 as an active ingredient, wherein rhamnosus The registration number of Lactobacillus sugarus JJ101 is BCRC 911088, and the lactic acid bacteria composition is administered to the subjects for at least 14 days. In an embodiment of the present invention, the oral composition may optionally include 1% to 5% by weight of prebiotics, and the prebiotics may include but not limited to lactulose and/or isomaltooligosaccharide. In one embodiment of the present invention, the drug-resistant Enterobacter has KPC-2.

The use of the lactic acid bacteria composition of the present invention and its use for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae can inhibit the growth of drug-resistant Enterobacteriaceae with KPC-2 in vitro and/or in vivo, so the lactic acid bacteria of the present invention The composition has the potential to be applied to prevent, improve and/or treat drug-resistant Enterobacteriaceae infection.

Based on the above, the present invention provides a lactic acid bacteria composition and its use for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the lactic acid bacteria composition may include but not limited to Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) as an effective Components to inhibit the growth of drug-resistant Enterobacteriaceae.

The "lactic acid bacteria" mentioned herein refers to bacteria that can decompose sugars (such as lactose, glucose, sucrose, fructose, etc.) to produce acidic substances (such as organic acids such as lactic acid and/or acetic acid). Some lactic acid bacteria can be used as probiotics. Probiotics refer to microorganisms that can promote the health of the host when administered to the host in an appropriate dose, such as: Lactobacillus, Pediococcus, Bacillus, Bifidobacterium and Rhamna Lactobacillus sugar.

In one embodiment, the above-mentioned Lactobacillus rhamnosus can be, for example, stored in the Bioresource Collection and Research Center (BCRC, BCRC, Address: 30062 Food Industry, Hsinchu City, Taiwan) on December 22, 2021. Road 331), and Lactobacillus rhamnosus JJ101 with deposit number BCRC 911088. Animal experiments have confirmed that compared with other strains of the same genus, after oral administration of Lactobacillus rhamnosus JJ101 for 3 days, the number of viable bacteria remaining in the animal's body is more, indicating that the animal's body (that is, the intestinal tract) persists better ability. It should be added that the term "probiotics have a good retention capacity in the animal body (i.e. the intestinal tract)" mentioned in this article refers to the probiotics remaining in the animal body (i.e. the intestinal tract) for a long time and/or the number of viable bacteria remaining more. The viable count of probiotics retained in the intestinal tract can be assessed, for example, by calculating the viable count per unit weight of feces. In one embodiment, the probiotics are resistant to acid and bile salts, so they have better intestinal persistence.

Animal experiments and in vitro co-culture experiments have confirmed that Lactobacillus rhamnosus JJ101 has the effect of drug-resistant Enterobacteriaceae. The "drug-resistant Enterobacteriaceae" mentioned herein refers to strains of Enterobacteriaceae that are resistant to antibiotics. The "inhibition of drug-resistant Enterobacteriaceae" mentioned herein refers to the co-cultivation of Lactobacillus rhamnosus JJ101 and drug-resistant Enterobacteriaceae in vitro, which can effectively inhibit the growth of drug-resistant Enterobacteriaceae (for example, reduce by at least 2 orders of magnitude, which is equivalent to inhibiting the growth of drug-resistant Enterobacteriaceae). The rate is at least 99%), or after oral administration, the level of drug-resistant Enterobacteriaceae in animals is reduced (for example, after administration of Lactobacillus rhamnosus JJ101 for at least 14 consecutive days, the level of drug-resistant Enterobacteriaceae in the feces of infected animals is reduced by at least 2 orders of magnitude, corresponding to an inhibition rate of at least 99%). It is supplemented that the inhibition rate is the percentage of the difference between the initial bacterial count and the treated bacterial count to the initial bacterial count, where the initial bacterial count is the number of viable bacteria of drug-resistant Enterobacteriaceae that have not been co-cultured with Lactobacillus rhamnosus JJ101 , and the amount of bacteria after treatment is the number of live bacteria of drug-resistant Enterobacteriaceae after co-cultivation with Lactobacillus rhamnosus JJ101, or the initial amount of bacteria is the number of infected animals that have not been orally administered with Lactobacillus rhamnosus JJ101 The content of drug-resistant Enterobacteriaceae, and the amount of bacteria after treatment is the content of drug-resistant Enterobacteriaceae in the feces of infected animals after oral administration of Lactobacillus rhamnosus JJ101.

In one embodiment, the above-mentioned antibiotics may be, for example, β-lactam antibiotics, which can inhibit the growth of bacteria by interfering with the synthesis of cell walls. β-lactam antibiotics may include, but are not limited to, penicillins, cephalosporins, carbapenems, and monoamide rings. In one embodiment, the drug-resistant Enterobacteriaceae can be, for example, β-lactam-resistant Enterobacteriaceae. In one embodiment, the drug-resistant Enterobacteriaceae may be, for example, carbapenem-resistant Enterobacteriaceae (CRE). In some specific examples, the drug-resistant Enterobacteriaceae can be, for example, carbapenemase-producing Enterobacteriaceae (CPE).

It is supplemented that carbapenemase is a kind of β-lactamases (β-lactamases), which can hydrolyze β-lactam antibiotics (such as carbapenems), thereby reducing the effect of CPE on β-lactams. susceptibility to antibiotics. Klebsiella pneumoniae carbapenemase ( Klebsiella pneumoniae carbapenemase, KPC) is a kind of carbapenemase, which was first discovered in Klebsiella pneumoniae in 1996, hence its name. The KPC gene is located on the plastid, so it can be transmitted across strains. At present, other enterobacteriaceae (such as: Citrobacter freundii, E. Lebsiella, Proteus mirabilis, Salmonella enterica, Serratia marcescens) and other non-enterobacterial Gram-negative bacteria (such as: Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter ) have been found to produce KPC strains. According to the different gene sequences, KPC can be classified into KPC-1, KPC-2, KPC-3 and so on. Among them, drug-resistant Enterobacteriaceae with KPC-2 are relatively common in clinical practice, such as Klebsiella pneumoniae of sequence type (sequence type, ST) 11.

In one embodiment, the lactic acid bacteria composition may optionally contain prebiotics to form "synbiotics". "Synbiotics" as used herein refers to a mixture of probiotics and prebiotics. The "prebiotics" mentioned herein refer to substances that cannot be digested by the host, but are beneficial to the growth and/or metabolic activity of specific strains in the digestive tract of the host, thereby improving the health of the host. Common prebiotics include disaccharides, oligosaccharide carbohydrates (OSCs), resistant starch and other non-sugar substances, including but not limited to fructooligosaccharides (lactulose), inulin, milk Fructo-oligosaccharides and/or isomalto-oligosaccharides. It has been confirmed by in vitro co-culture experiments that fructooligosaccharides, inulin, lactulose and/or isomaltooligosaccharides can promote the production of acidic substances (such as: organic acids) by Lactobacillus rhamnosus JJ101, making the pH of the co-culture solution less than 5. To inhibit the growth of drug-resistant enterobacteria, among which lactulose and/or isomaltooligosaccharides are better.

In one embodiment, the content of prebiotics is not limited, as long as it does not exceed a safe dose. The daily safe dose of prebiotics for adults may be less than 10 g, so as not to cause discomfort symptoms such as abdominal distension and diarrhea. In one embodiment, the content of prebiotics can be, for example, 1% to 5% by weight, 1.5% to 2.5% by weight, or 2% by weight, so as to fully stimulate the growth and/or metabolism of Lactobacillus rhamnosus JJ101 active, but does not exceed the above safe daily dose.

When using the above-mentioned Lactobacillus rhamnosus JJ101, the route of administration is not particularly limited, such as oral administration, which can be adjusted according to actual needs. The dosage and times of administration of Lactobacillus rhamnosus JJ101 above can also be flexibly adjusted according to demand. In one embodiment, the effective dose of Lactobacillus rhamnosus JJ101 in the in vitro culture medium is 10 ⁵ CFU/mL to 10 ⁷ CFU/mL. In one embodiment, when the subject is a mouse, the effective dose of Lactobacillus rhamnosus JJ101 can be, for example, 5.0×10 ¹⁰ CFU/kg body weight/day to 1.5×10 ¹¹ CFU/kg body weight/day. For example, in the above animal experiments, the effective dose of Lactobacillus rhamnosus JJ101 to mice is 1.0×10 ¹¹ CFU/kg body weight/day, that is, 2.0×10 ⁹ CFU/mouse (20 g body weight)/day .

It should be added that in animal experiments, mice were directly orally administered drug-resistant Enterobacteriaceae, so the content of drug-resistant Enterobacteriaceae in the intestines of mice was much higher than that of clinical patients. Second, mice have the habit of eating feces and will repeatedly ingest drug-resistant Enterobacteriaceae in feces. Therefore, mice in animal experiments need to be orally administered a higher dose of Lactobacillus rhamnosus JJ101 in order to effectively reduce drug-resistant Enterobacteriaceae. In other words, the effective dose of Lactobacillus rhamnosus JJ101 in clinical application to adults can be lower than the effective dose to mice in animal experiments, and it can also effectively inhibit drug-resistant Enterobacteriaceae. In a specific example, the effective dose of Lactobacillus rhamnosus JJ101 for an adult can be, for example, 1.0×10 ⁸ CFU/60 kg body weight/day to 1.0×10 ¹⁰ CFU/60 kg body weight/day. In one embodiment, the subject is administered the effective dose of Lactobacillus rhamnosus JJ101 for several consecutive days. In one embodiment, the subject is administered Lactobacillus rhamnosus JJ101 for at least several consecutive days, such as 7 days to 12 months or 14 days to 6 months.

Lactobacillus rhamnosus JJ101 has the effect of inhibiting the growth of drug-resistant enterobacteria, so it can be used as an active ingredient of lactic acid bacteria composition. In one embodiment, the lactic acid bacteria composition can be an oral composition, for example. In one embodiment, the lactic acid bacteria composition may be, for example, a food composition or a medical composition. In one embodiment, the lactic acid bacteria composition may optionally include food or pharmaceutically acceptable carriers, excipients, diluents, adjuvants and/or additives, such as solvents, emulsifiers, suspending agents, and disintegrating agents , adhesives, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants and/or absorption delaying agents, etc.

The dosage form of the lactic acid bacteria composition is not particularly limited. In one embodiment, the dosage form of the lactic acid bacteria composition can be, for example, an aqueous solution, a suspension, a dispersion, an emulsion (single-phase or multi-phase dispersion system, single-chamber or multi-chamber liposomes), hydrogel, gel, solid lipid gel, etc. Rice grains, lozenges, granules, powder or capsules, etc.

Several examples are used below to illustrate the application of the present invention, but it is not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various modifications and changes without departing from the spirit and scope of the present invention. retouch. Embodiment one, lactic acid bacteria isolation, cultivation and microbiological properties

Lactic acid bacteria (LAB) strain LYC1504 and strain JJ101 were isolated from fruit fermentation liquid. LAB were inoculated on de Man, Rogosa and Sharpe (MRS) agar medium in a four-section method and incubated at 37°C for 16 to 18 hours to obtain single colonies. Next, a single colony was inoculated into the MRS culture solution, and cultured at 37° C. for 16 to 24 hours to obtain a LAB culture solution. The LAB culture solution was centrifuged to obtain a bacterial pellet.

Perform RNA purification and reverse transcription on the bacterial precipitate of LAB, and then use the nucleic acid sequence such as sequence identification numbers (SEQ ID NOs.): 1 and 2 to perform polymerase chain reaction (polymerase chain reaction, PCR) to obtain 16S rDNA nucleic acid fragments, and perform nucleic acid sequencing to obtain the 16S rDNA nucleic acid sequence of LAB. The above LAB was identified as Lactobacillus rhamnosus (strain LYC1504 and strain JJ101) by using Basic Local Alignment Search Tool (BLAST) for comparison. The strain JJ101 was deposited in BCRC on December 22, 2021, and the deposit number is BCRC 911088.

It is supplemented that the colonies of the strain JJ101 are milky white, opaque, round, with smooth and protruding surfaces and neat edges. Present, no flagella, no motility, no sporulation, and Gram stain positive. Example 2: Evaluating the Persistence Ability of Lactic Acid Bacteria and Drug-resistant Enterobacteriaceae in Animals 1. Persistence of lactic acid bacteria in animals

BALB/c mice (hereinafter referred to as mice) were used as experimental animals. The 5-week-old female mice were kept in independent ventilated cages in the animal room to allow the mice to adapt to the environment. During acclimatization, mice had free access to standard pelleted chow and sterile distilled water. The temperature of the animal room was 23±3°C, the relative humidity was 60±10%, and there were 12 hours of light and 12 hours of darkness per day. Follow-up evaluations were performed after the mice had grown to 6 weeks of age.

First, antibiotics were given to mice daily, and the bacterial content of mouse feces was tested to confirm whether the feces were sterile. The detection method is described as follows: after weighing the fresh feces of the mice, add 1 mL of physiological experiment water (normal saline, NS) to grind into a test solution, and then spread the test solution on Enterobacteriaceae culture medium, Miller Hunton ( Mueller Hinton broth, MHB) agar and LAB medium, and count the number of colonies after culturing at 37°C for 24 hours. The above Enterobacteriaceae medium was eosin methylene blue containing 16 μg/mL of vancomycin, 64 μg/mL of ampicillin and 16 μg/mL of cefotaxime. , EMB) agar can be used to detect Enterobacteriaceae. LAB medium is MRS agar containing 32 μg/mL vancomycin, and the pH value is 5.0, which can be used to detect LAB.

After the feces of the mice were confirmed to be sterile, the mice were fed with different LAB solutions, in which the LAB solution was obtained by dissolving the precipitates of the strain LYC1504 and the strain JJ101 in phosphate buffered saline (PBS). The LAB content was obtained and adjusted, and mice were orally administered 2.0×10 ⁹ CFU/day of LAB for 3 consecutive days. Then, stop the tube feeding, and after stopping the tube feeding for 1 day, 3 days and 7 days, use the above-mentioned LAB medium to detect the LAB content of the mouse feces, wherein the LAB content is the ratio of the number of viable LAB bacteria to the weight of the mouse feces (unit : CFU/g).

Please refer to Fig. 1, wherein Fig. 1 shows the content of Lactobacillus rhamnosus in the feces of mice according to one embodiment of the present invention after oral administration of different Lactobacillus rhamnosus for 3 consecutive days and stop tube feeding In the line graph, the horizontal axis represents time (unit: day), and the vertical axis represents the content of Lactobacillus rhamnosus (unit: CFU/g). Line 101 and line 103 are bacterial strain LYC1504 and bacterial strain JJ101, respectively. As shown in Figure 1, after stopping tube feeding for 1 day and 3 days, the content of bacterial strain JJ101 (broken line 103) in mouse feces was higher than that of strain LYC1504 (broken line 101), and after stopping tube feeding for 3 days, the content of bacterial strain JJ101 was higher At 10 ⁷ CFU/g, it was confirmed that the intestinal persistence of the strain JJ101 was better. 2. Persistence of drug-resistant Enterobacteriaceae in animals

The strains KPC001, KPC011, KPC021 and KPC035 are drug-resistant Enterobacteriaceae expressing KPC-2 (hereinafter referred to as CPE) isolated from the clinical laboratory of the Medical Research Center of Chi Mei Hospital. CPE was inoculated on Enterobacteriaceae culture medium by four-section streak method, and cultured at 37°C for 16 hours to 18 hours to obtain a single colony. Next, a single colony was inoculated into MHB and cultured at 37° C. for 16 hours to 24 hours to obtain a CPE culture solution. The CPE culture solution was centrifuged to obtain a pellet of CPE cells.

Antibiotics were administered daily to the mice until the feces were sterile. Then, the mice were fed with CPE solution, wherein the CPE solution was to redissolve the CPE bacterial sediment in an aqueous solution containing 20% by weight of skimmed milk powder, and adjust the CPE content of the CPE solution so that the mice were orally administered with 3.0×10 Infected mice were obtained by administering ⁸ CFU/day of CPE for 3 consecutive days. Then, tube feeding was stopped, and infected mice were collected again after tube feeding was stopped for 1 day, 2 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, 31 days and 35 days Feces, and the CPE content of the feces was detected with MHB agar, where the CPE content was the ratio of the number of viable CPE bacteria to the weight of the feces (unit: CFU/g).

Please refer to Fig. 2, wherein Fig. 2 is a line graph showing the CPE content of feces of infected mice according to one embodiment of the present invention, wherein the horizontal axis represents time (unit: day), and the vertical axis represents CPE content (unit: CFU /g), broken line 201, broken line 203, broken line 205 and broken line 207 represent bacterial strain KPC001, bacterial strain KPC011, bacterial strain KPC021 and bacterial strain KPC035 respectively. As shown in Figure 2, after stopping tube feeding for 1 day, the CPE content of different strains of mouse feces was about 10 ¹⁰ CFU/g, and after stopping tube feeding for 4 days to 35 days, the CPE content of different strains of mouse feces Still maintained at 10 ⁴ CFU/g to 10 ⁶ CFU/g. The above results showed that there was no difference in the intestinal persistence ability of different strains of CPE in mice. Subsequent evaluations were performed with strain KPC001. Example 3. Evaluation of the efficacy of bacterial strain JJ101 in inhibiting drug-resistant Enterobacteriaceae

Antibiotics were administered daily to the mice until the feces were sterile. Then, 3.0×10 ⁸ CFU/day of CPE was orally administered to the mice for 3 consecutive days to obtain infected mice. Then, the CPE content in feces of infected mice was detected, and it was used as the CPE content of infected mice that were not orally administered with LAB. Then, the infected mice were divided into blank group and experimental group. The infected mice in the blank group were orally administered with PBS for 21 consecutive days, and the infected mice in the experimental group were orally administered with 2.0×10 ⁹ CFU/day of strain JJ101 for 21 consecutive days. After the mice were orally administered with PBS or strain JJ101 for 4 days, 7 days, 11 days, 14 days, 18 days and 21 days, the CPE content in the feces of the infected mice was detected.

Please refer to Fig. 3, wherein Fig. 3 is a line graph showing the CPE content of feces of different groups of infected mice according to an embodiment of the present invention, wherein the horizontal axis represents the concentration of infected mice orally administered with PBS or bacterial strain JJ101 The number of consecutive days (unit: day), the vertical axis represents the CPE content of infected mouse feces (unit: CFU/g), the broken line 301 and the broken line 303 represent the blank group and the experimental group respectively, and "*" and "**" represent the Statistically significant difference ( p value range is p <0.05 and p <0.01, respectively).

As shown in Figure 3, after the infected mice were orally administered bacterial strain JJ101 for 7 days to 21 consecutive days, the CPE content (broken line 303) of the feces of the infected mice in the experimental group was significantly lower than that of the blank group (broken line 301), reducing at least 2 orders of magnitude, equivalent to an inhibition rate of at least 99%, confirming that the strain JJ101 has the effect of inhibiting the growth of CPE in vivo. Example 4. Evaluating the efficacy of synbiotics in inhibiting drug-resistant Enterobacteriaceae 1. Co-cultivation test of strain JJ101 and CPE with different prebiotics

LAB can decompose sugars to produce acidic substances (such as lactic acid and/or acetic acid), thereby reducing the pH value of the environment (such as: intestinal tract), thereby inhibiting CPE. Therefore, if the LAB can utilize prebiotics more effectively, the effect of this prebiotic and the synbiotics composed of LAB on CPE growth inhibition will be better.

The strain JJ101 and CPE (strain KPC001) were added to the co-culture solution at pH 6.5 to carry out the co-cultivation experiment, wherein the initial LAB (strain JJ101) content of the co-culture solution was 10 ⁷ CFU/mL, and the initial CPE content was 10 ⁶ CFU/mL. Then, LAB content detection, CPE content detection and pH value detection were performed on the co-culture solution to obtain the initial LAB content, CPE content and pH value (equivalent to 0 hours of companionship). The LAB content detection system spread the co-culture solution on the MRS agar medium with pH 5.5, and cultured at 37°C to obtain a single colony of LAB. The LAB content (unit: CFU/mL) of the co-culture solution can be estimated by the number of single LAB colonies. The co-culture solution for the detection of the number of CPE bacteria was spread on the EMB agar medium containing 16 μg/mL ampicillin, and cultured at 37°C to obtain a single colony of CPE. The CPE content (unit: CFU/mL) of the co-culture solution can be estimated by the single colony count of CPE.

The co-culture solution was prepared from glucose-free MRS culture solution and MHB at a volume ratio of 1:1, and sugar was added or not added according to the group. The co-culture solution of the NON group did not contain sugar, and the co-culture solution of the SUC group The co-culture solution of the group contains 2% by weight of sucrose, the co-culture solution of the FOS group contains 2% by weight of fructo-oligosaccharides, the co-culture solution of the IN group contains 2% by weight of inulin, and the co-culture solution of the XOS group contains 2% by weight The co-culture solution of the LU group contained 2% by weight of lactulose, and the co-culture solution of the IMO group contained 2% by weight of isomalto-oligosaccharide.

The co-culture solution was cultured at 37°C. After culturing for 3 hours, 6 hours, 24 hours and 48 hours, LAB content detection, CPE content detection and pH value detection were carried out. The results are as follows: After culturing for 48 hours, SUC The content of strain JJ101 in the co-culture solution of group, FOS group, IN group, XOS group, LU group and IMO group was higher than that of NON group, which was greater than 1.0×10 ⁸ CFU/mL and less than 1.0×10 ⁹ CFU/mL (not shown shown in the figure), confirming that prebiotics are beneficial to the growth of strain JJ101 in vitro.

Please refer to Figure 4A and Figure 4B, which respectively depict the CPE content (Figure 4A) and pH value of the co-culture solution after the co-cultivation of the bacterial strain JJ101 and CPE according to an embodiment of the present invention in the co-culture solution containing different prebiotics (Figure 4B) the line chart. The horizontal axis of Fig. 4A represents time (unit: hour), and the vertical axis represents CPE content (unit: CFU/mL). The horizontal axis in FIG. 4B represents time (unit: hour), and the vertical axis represents pH. The folded lines 401, 403, 405, 407, 409, 411, and 413 in FIGS. 4A and 4B represent the NON group, the SUC group, the FOS group, the IN group, the XOS group, the LU group, and the IMO group, respectively.

As shown in Figure 4A, after culturing for 24 hours, the CPE content of the co-culture solution of the SUC group (broken line 403), FOS group (broken line 405), IN group (broken line 407) and LU group (broken line 411) was lower than detection limit. After 48 hours of culture, the CPE content of the co-culture solution of the IMO group (broken line 413) was 2 orders of magnitude less than the initial CPE content (0 hour) (ie, the inhibition rate was 99%). However, the CPE content of the co-culture solution of the NON group (line 401 ) and the XOS group (line 409 ) was higher than the initial CPE content (0 hour) after culturing for 48 hours. As shown in Figure 6B, after culturing for 24 hours to 48 hours, the SUC group (broken line 403), the FOS group (broken line 405), the IN group (broken line 407), the LU group (broken line 411) and the IMO group (broken line 413) The pH value of the co-culture solution is less than 5, but the pH value of the co-culture solution of the XOS group (line 409) and the NON group (line 401) is greater than 5. The above results confirmed that sucrose, fructooligosaccharides, inulin, lactulose and isomaltooligosaccharides can promote the production of acidic substances by the strain JJ101, making the pH value of the co-culture solution less than 5, thereby inhibiting the growth of CPE. It should be noted that sucrose can be digested by animals and cannot be used as a prebiotic. Therefore, the preferred prebiotics of the strain JJ101 can be, for example, fructooligosaccharides, inulin, lactulose and isomaltooligosaccharides. 2. Cultivation test of strain JJ101 with different prebiotics

As mentioned above, in the co-cultivation test of strain JJ101 and CPE, if fructo-oligosaccharide, inulin, lactulose and isomalto-oligosaccharide are preferred prebiotics, the pH of the co-culture solution should be less than 5. However, the CPE content in clinical patients is much lower than that in co-culture experiments. Therefore, the choice of prebiotics also needs to consider the pH value after the culture test of strain JJ101 with different prebiotics.

Strain JJ101 was inoculated into different formulations of glucose-free MRS culture solution, and cultured at 37°C for 24 hours to obtain cultures. Then, measure the pH value of culture, and the result (average ± standard deviation of 3 repetitions) is recorded in table 1, wherein NON group represents that MRS culture solution does not add carbohydrate, SUC group represents that MRS culture solution adds 2% by weight of Sucrose, FOS group means MRS culture medium with 2% by weight fructooligosaccharide added, IN group means MRS culture medium with 2% by weight inulin added, IMO group means MRS culture medium with 2% by weight isomaltooligosaccharide added, LU group Indicates that 2% by weight of lactulose was added to the MRS culture solution, and the XOS group indicated that 2% by weight of xylooligosaccharide was added to the MRS culture solution.

Table 1 group pH (Lactobacillus rhamnosus) JJ101 LYC1504 NON 6.08±0.02 6.13±0.02 SUC 5.31±0.01 5.29±0.01 FOS 5.89±0.07 5.85±0.03 IN 5.40±0.01 5.41±0.03 XOS 5.21±0.01 5.18±0.02 LU 3.78±0.02 3.83±0.01 IMO 4.29±0.01 4.28±0.01

As shown in Table 1, the pH values of the cultures of the strain JJ101 in the LU group and the IMO group were lower than 5.0, confirming that lactulose and isomaltooligosaccharides have the effect of promoting the production of acidic substances on the strain JJ101, so the probiotics of the strain JJ101 Lactulose and isomaltooligosaccharide should be used as the element.

In summary, Lactobacillus rhamnosus JJ101 can inhibit the growth of drug-resistant Enterobacteriaceae, which means that Lactobacillus rhamnosus JJ101 has the potential to be used in the prevention, improvement and/or treatment of drug-resistant Enterobacteriaceae infection. Secondly, the addition of prebiotics can enhance the activity of Lactobacillus rhamnosus JJ101 to produce acidic substances and inhibit the growth of drug-resistant Enterobacteriaceae.

In summary, although the present invention uses specific strains of Lactobacillus rhamnosus, specific manufacturing processes, specific effective doses, specific administration methods, specific experimental models, and specific evaluation methods as examples to illustrate the composition of lactic acid bacteria of the present invention substances and their use in the preparation of oral compositions for inhibiting drug-resistant Enterobacteriaceae, but those with ordinary knowledge in the technical field of the present invention should understand that the present invention is not limited thereto, and without departing from the spirit and scope of the present invention, The present invention can also be carried out with other strains of Lactobacillus rhamnosus, other production processes, other effective doses, other administration methods, other experimental models and other evaluation methods.

Although the present invention has been disclosed above with several specific embodiments, various modifications, changes and substitutions can be made to the foregoing disclosure, and it should be understood that certain situations will be changed without departing from the spirit and scope of the present invention. Some features of the embodiments of the present invention are used but not others. Therefore, the spirit of the present invention and the scope of claims should not be limited to those described in the above exemplary embodiments.

101,103,201,203,205,207,301,303,401,403,405,407,409,411,413: polyline

In order to make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, the detailed description of the accompanying drawings is as follows: [ Fig. 1 ] is a line graph showing the content of Lactobacillus rhamnosus in feces of mice according to an embodiment of the present invention after oral administration of different Lactobacillus rhamnosus for 3 consecutive days and stop tube feeding. [ FIG. 2 ] is a line graph showing the CPE content in feces of infected mice according to an embodiment of the present invention. [ FIG. 3 ] is a line graph showing the CPE content in feces of different groups of infected mice according to an embodiment of the present invention. [FIG. 4A] to [FIG. 4B] are line graphs showing the CPE content and pH value of the co-culture solution after the co-cultivation of the strain JJ101 and CPE according to an embodiment of the present invention in the co-culture solution containing different prebiotics .

Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101 was deposited on December 22, 2021 in the Bioresource Collection and Research Center (BCRC, Address: No. 331, Food Road, Hsinchu City, Taiwan, 30062) , the deposit number is BCRC 911088.

301,303: polyline

Claims (10)

A lactic acid bacteria composition comprising Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101 as an active ingredient to inhibit the growth of drug-resistant Enterobacteriaceae, wherein the Lactobacillus rhamnosus JJ101 was deposited in the consortium on December 22, 2021 Legal person Bioresource Collection and Research Center (BCRC), Food Industry Development Institute, deposit number is BCRC 911088. The lactic acid bacteria composition according to claim 1 further comprises a prebiotic in an amount of 1% to 5% by weight. The lactic acid bacteria composition according to claim 2, wherein the prebiotics include lactulose and/or isomaltooligosaccharide. The lactic acid bacteria composition according to claim 1, wherein the drug-resistant enterobacteriaceae have Klebsiella pneumoniae carbapenemase ( Klebsiella pneumoniae carbapenemase, KPC)-2. The lactic acid bacteria composition as described in Claim 1, wherein the lactic acid bacteria composition is an oral composition. The lactic acid bacteria composition as described in Claim 1, wherein a test subject is administered with an effective dose of the lactic acid bacteria composition for at least 14 days. The lactic acid bacteria composition according to claim 1, wherein when the subject is a mouse, the effective dose is 5.0×10 ¹⁰ CFU/kg body weight/day to 1.5×10 ¹¹ CFU/kg body weight/day. A use of Lactobacillus rhamnosus for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the oral composition contains Lactobacillus rhamnosus JJ101 as an active ingredient, and the registration number of Lactobacillus rhamnosus JJ101 is BCRC 911088, and the oral composition is administered to a subject for at least 14 days. The use of Lactobacillus rhamnosus as described in Claim 8 in the preparation of an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the oral composition further comprises 1% to 5% by weight of a prebiotic, and the prebiotic Contains lactulose and/or isomaltooligosaccharides. Use of Lactobacillus rhamnosus as described in Claim 8 for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the drug-resistant Enterobacteriaceae has KPC-2.

Images