TWI819483B - 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 lactic acid bacteria composition, in particular to a lactic acid bacteria composition and its use for preparing an oral composition for inhibiting drug-resistant enterobacteriaceae.

Enterobacteriaceae are Gram-negative bacteria belonging to the order Enterobacteriaceae of the class Gammaproteobacteria. Enterobacteriaceae are commonly found in the environment (such as soil and water) and organisms (such as animals and plants), and are one of the intestinal bacteria in the human body. Enterobacteriaceae include beneficial commensal bacteria as well as pathogenic bacteria that cause opportunistic infections. These pathogenic bacteria can cause dysentery, enteric fever, urinary tract infection, wound infection, liver abscess, sepsis, meningitis, pneumonia and other diseases, and are one of the main pathogenic bacteria of nosocomial infections and community infections.

Antibiotics are the main drugs used to treat Enterobacteriaceae infections. Among them, carbapenem antibiotics have a wide antibacterial range and have few drug-resistant bacterial species. They are currently the last line of defense against multi-drug-resistant Enterobacteriaceae. However, in recent years, Enterobacteriaceae such as Klebsiella pneumoniae have evolved methods to reduce their susceptibility to carbapenem antibiotics, such as the performance of carbapenemase-producing Enterobacteriaceae (CPE). Carbapenemase can decompose carbapenem antibiotics, thereby increasing the morbidity and mortality of infected patients. It is currently one of the major threats to global public health.

In view of the limitations of antibiotics and other drugs in controlling bacterial infections, there is an urgent need to provide a non-drug composition that can be used to inhibit drug-resistant enterobacteriaceae and solve the above problems.

Therefore, one aspect of the present invention is to provide a lactic acid bacteria composition. This lactic acid bacteria composition contains Lacticaseibacillus rhamnosus JJ101 as an active ingredient, thereby inhibiting 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 aspect of the present invention, a lactic acid bacteria composition is proposed. This lactic acid bacteria composition contains Lacticaseibacillus rhamnosus JJ101 as an active ingredient to inhibit the growth of drug-resistant enterobacteriaceae. Lacticaseibacillus rhamnosus JJ101 was deposited in the Food Industry on December 22, 2021 Bioresource Collection and Research Center (BCRC) of the Institute of Development Studies, registration number is BCRC 911088.

In one embodiment of the present invention, the lactic acid bacteria composition may optionally include 1 to 5% by weight of prebiotics. In one embodiment of the invention, the prebiotic includes lactulose and/or isomaltooligosaccharide. In one embodiment of the present invention, the drug-resistant Enterobacteriaceae possess Klebsiella pneumoniae carbapenemase (KPC)-2. In one embodiment of the present invention, the subject is administered an effective dose of the lactic acid bacteria composition for at least 14 days. In one embodiment of the present invention, when the subject is a mouse, the effective dose 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, the 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 R. rhamnosus The registration number of Lactobacillus saccharus JJ101 is BCRC 911088, and the lactic acid bacteria composition was administered to the subject for at least 14 days. In one embodiment of the present invention, the oral composition may optionally include 1 to 5% by weight of prebiotics, and the prebiotics may include but are not limited to lactulose and/or isomaltooligosaccharide. In one embodiment of the invention, the drug-resistant Enterobacteriaceae has KPC-2.

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. Therefore, the lactic acid bacteria of the present invention The composition has the potential to be used to prevent, improve and/or treat drug-resistant Enterobacteriaceae infections.

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. The lactic acid bacteria composition may include but is not limited to Lacticaseibacillus rhamnosus as an effective ingredients to inhibit the growth of drug-resistant Enterobacteriaceae.

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

In one embodiment, the above-mentioned Lactobacillus rhamnosus can be deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Research Institute (BCRC) on December 22, 2021, address: 30062 Hsinchu City, Taiwan Food Road No. 331), and the deposit number is Lactobacillus rhamnosus JJ101 with the number BCRC 911088. Animal experiments have confirmed that compared with other strains of the same genus, after animals were orally administered Lactobacillus rhamnosus JJ101 for 3 days, more viable bacteria remained in the animal's body (i.e., in the intestine). Better ability. It should be supplemented that the "good retention ability of probiotics in the animal body (i.e. intestinal tract)" mentioned in this article refers to the longer time that probiotics remain in the animal body (i.e. intestinal tract) and/or the number of viable bacteria that remain. More. The number of viable bacteria remaining in the intestinal tract of probiotics can be assessed, for example, by calculating the number of viable bacteria per unit weight of feces. In one embodiment, the probiotics are acid-resistant and bile-salt-resistant, so their intestinal retention ability is better.

It has been confirmed by animal experiments and in vitro co-culture experiments that Lactobacillus rhamnosus JJ101 has the efficacy of drug-resistant Enterobacteriaceae. "Antimicrobial-resistant Enterobacteriaceae" as used herein refers to Enterobacteriaceae strains that are resistant to antibiotics. The term "inhibition of drug-resistant Enterobacteriaceae" as used herein refers to the fact that Lactobacillus rhamnosus JJ101 and drug-resistant Enterobacteriaceae are co-cultured in vitro and can effectively inhibit the growth of drug-resistant Enterobacteriaceae (for example, by at least 2 orders of magnitude, which is equivalent to inhibiting The rate is at least 99%), or after oral administration, the content of drug-resistant Enterobacteriaceae in the animal body is reduced (for example: Lactobacillus rhamnosus JJ101 is administered for at least 14 consecutive days, and the content of drug-resistant Enterobacteriaceae in the feces of infected animals is reduced by at least 2 orders of magnitude, equivalent to an inhibition rate of at least 99%). It should be added that the inhibition rate is the percentage of the difference between the initial bacterial load and the treated bacterial load to the initial bacterial load, where the initial bacterial load is the number of viable bacteria that are not co-cultured with Lactobacillus rhamnosus JJ101. , and the bacterial load after treatment is the number of viable bacteria of drug-resistant Enterobacteriaceae after co-culture with Lactobacillus rhamnosus JJ101, or the initial bacterial load is the feces of infected animals without oral administration of 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 antibiotics can be, for example, β-lactam antibiotics, which can inhibit the growth of bacteria by interfering with cell wall synthesis. Beta-lactam antibiotics may include, but are not limited to, penicillins, cephalosporins, carbapenems and monoamide rings. In one embodiment, the drug-resistant Enterobacteriaceae may 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 may be, for example, carbapenemase-producing Enterobacteriaceae (CPE).

It should be added that carbapenemase is a type of β-lactamases (β-lactamases), which can hydrolyze β-lactam antibiotics (such as carbapenems), thereby reducing the impact of CPE on β-lactamases. Antibiotic susceptibility. Klebsiella pneumoniae carbapenemase (KPC) is a type of carbapenemase. It 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 bacterial species. Currently, other Enterobacteriaceae (such as: Citrobacter flexneri, Escherichia coli, Enterobacter vitiligo, Enterobacter aerogenes, Enterobacter cloacae, Gram acidophilus Lebsiella pneumoniae, Proteus mirabilis, Salmonella enterica, Serratia marcescens) and other non-enterobacteria Gram-negative bacteria (such as: Pseudomonas aeruginosa, Pseudomonas odorifera, Acinetobacter spp. ) have all found KPC-producing strains. According to different gene sequences, KPC can be classified into KPC-1, KPC-2, KPC-3, etc. Among them, drug-resistant Enterobacteriaceae with KPC-2 are relatively common in clinical practice, such as Klebsiella pneumoniae of sequence type (ST) 11.

In one embodiment, the lactic acid bacteria composition may optionally include prebiotics, thereby forming "synbiotics". "Synbiotics" as used herein refers to a mixture of probiotics and prebiotics. "Prebiotics" as used 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-saccharide substances. Specifically, they may include but are not limited to fructooligosaccharides (lactulose), inulin (inulin), lactose Fructose (fructo-oligosaccharide) and/or isomaltooligosaccharide (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 value of the co-culture solution less than 5, thereby inhibiting the growth of drug-resistant Enterobacteriaceae, among which lactulose and/or isomaltooligosaccharide have the better effect.

In one embodiment, the content of prebiotics is not limited, as long as it does not exceed a safe dose. The safe daily dose of prebiotics for adults can be, for example, less than 10 g to avoid causing uncomfortable symptoms such as bloating and diarrhea. In one embodiment, the content of the prebiotic can be, for example, 1 to 5% by weight, 1.5 to 2.5% by weight, or 2% by weight to fully stimulate the growth and/or metabolism of Lactobacillus rhamnosus JJ101. activity, but not exceeding the above safe daily dosage.

When applying the above-mentioned Lactobacillus rhamnosus JJ101, its administration route is not particularly limited. It can be administered orally, for example, and it can be adjusted according to actual needs. The dosage and number of administrations of the above-mentioned Lactobacillus rhamnosus JJ101 can also be adjusted according to the elasticity of 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 may 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 for mice was 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 noted 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. Secondly, mice have the habit of coprophagy and will repeatedly ingest drug-resistant Enterobacteriaceae in their feces. Therefore, mice in animal experiments need to be orally administered higher doses of Lactobacillus rhamnosus JJ101 to effectively reduce drug-resistant Enterobacteriaceae. In other words, the effective dose of Lactobacillus rhamnosus JJ101 in clinical applications for adults can be lower than the effective dose for 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 adults 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 above effective dose of Lactobacillus rhamnosus JJ101 for several consecutive days. In one embodiment, the subject is administered Lactobacillus rhamnosus JJ101 continuously for at least several days, such as from 7 days to 12 months or from 14 days to 6 months.

Lactobacillus rhamnosus JJ101 has the effect of inhibiting the growth of drug-resistant Enterobacteriaceae, and therefore can be used as an active ingredient in lactic acid bacteria compositions. In one embodiment, the lactic acid bacteria composition can be, for example, an oral composition. In one embodiment, the lactic acid bacteria composition can be, for example, a food composition or a pharmaceutical composition. In one embodiment, the lactic acid bacteria composition may optionally include food or pharmaceutically acceptable carriers, excipients, diluents, auxiliaries and/or additives, which may be, for example, solvents, emulsifiers, suspending agents, and disintegrants. , 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 liposome), hydrocolloid, gel, solid lipid nanoparticles Rice grains, tablets, granules, powders or capsules, etc.

Several examples are used below to illustrate the application of the present invention, but they are not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention. polish. Example 1. Isolation, culture and microbiological properties of lactic acid bacteria

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

The bacterial pellet of LAB was subjected to RNA purification and reverse transcription, and then the upstream primer and downstream primer as shown in the nucleic acid sequences (SEQ ID NOs.): 1 and 2 were used 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. Basic Local Alignment Search Tool (BLAST) was used for comparison, and the above-mentioned LAB was identified as Lactobacillus rhamnosus (strain LYC1504 and strain JJ101). Strain JJ101 was deposited with BCRC on December 22, 2021, and the deposit number is BCRC 911088.

It should be added that the colonies of strain JJ101 are milky white, opaque, round, with smooth and protruding surfaces and neat edges. The bacterial cells are short rod-shaped, with blunt ends at both ends, in the form of single, paired, short chain or chain. There are no flagella, no motility, no sporulation, and the Gram stain is positive. Example 2: Evaluating the persistence ability of lactic acid bacteria and drug-resistant Enterobacteriaceae in animals 1. The ability of lactic acid bacteria to survive in animals

BALB/c mice (hereinafter referred to as mice) were used as experimental animals. Five-week-old female mice were raised in independent ventilation cages in the animal room to allow the mice to adapt to the environment. During the acclimation period, mice had ad libitum access to standard pelleted chow and sterile distilled water. The temperature of the animal room is 23±3°C, the relative humidity is 60±10%, and there is a 12-hour light period and a 12-hour dark period every day. Follow-up evaluations will be conducted after the mice reach 6 weeks of age.

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

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

Please refer to Figure 1, which illustrates the Lactobacillus rhamnosus content in the feces of mice after oral administration of different Lactobacillus rhamnosus to mice for 3 consecutive days and discontinuation of tube feeding according to one embodiment of the present invention. A line chart, in which the horizontal axis represents time (unit: day), the vertical axis represents the content of Lactobacillus rhamnosus (unit: CFU/g), and the broken lines 101 and 103 are strain LYC1504 and strain JJ101 respectively. As shown in Figure 1, after stopping tube feeding for 1 and 3 days, the content of strain JJ101 (broken line 103) in mouse feces was higher than that of strain LYC1504 (broken line 101). Among them, after stopping tube feeding for 3 days, the content of strain JJ101 was higher. At 10 ⁷ CFU/g, it was confirmed that strain JJ101 has better intestinal retention ability. 2. The ability of drug-resistant Enterobacteriaceae to persist in animals

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

The mice were administered antibiotics daily until their feces became sterile. Then, the mice were fed CPE solution by tube, in which the CPE bacterial precipitate was back-dissolved in an aqueous solution containing 20% by weight of skim milk powder, and the CPE content of the CPE solution was adjusted, so that the mice were orally administered 3.0×10 CPE at ⁸ CFU/day for 3 consecutive days to obtain infected mice. Then, tube feeding was stopped, and infected mice were collected again after 1 day, 2 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, 31 days and 35 days after stopping tube feeding. feces, and use MHB agar to detect the CPE content of the feces, where the CPE content is the ratio of the number of viable CPE bacteria to the weight of the feces (unit: CFU/g).

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

The mice were administered antibiotics daily until their feces became sterile. Then, the mice were orally administered with 3.0×10 ⁸ CFU/day of CPE for 3 consecutive days to obtain infected mice. Then, the CPE content in the feces of the infected mice was detected as the CPE content of the infected mice without oral administration of 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 PBS or strain JJ101 for 4 consecutive days, 7 days, 11 days, 14 days, 18 days and 21 days, the CPE content of the feces of the infected mice was detected.

Please refer to Figure 3, which is a line graph illustrating the CPE content in the feces of infected mice of different groups according to one embodiment of the present invention. The horizontal axis represents the CPE content of infected mice orally administered with PBS or strain JJ101. The number of consecutive days (unit: day), the vertical axis represents the CPE content of the feces of infected mice (unit: CFU/g), the polyline 301 and the polyline 303 represent the blank group and the experimental group respectively, and "*" and "**" represent those with Statistically significant differences ( p value ranges are p <0.05 and p <0.01 respectively).

As shown in Figure 3, after the infected mice were orally administered strain JJ101 for 7 to 21 days, the CPE content of the feces of the infected mice in the experimental group (broken line 303) was significantly lower than that of the blank group (broken line 301), which was reduced by at least 2 orders of magnitude, equivalent to an inhibition rate of at least 99%, confirming that strain JJ101 has the effect of inhibiting CPE growth in vivo. Example 4. Evaluation of the efficacy of Synylin in inhibiting drug-resistant Enterobacteriaceae 1. Co-culture 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 the intestine), thereby inhibiting CPE. Therefore, if LAB can utilize prebiotics more effectively, the synergistic factor composed of prebiotics and LAB will be better at inhibiting the growth of CPE.

Strain JJ101 and CPE (strain KPC001) were added to the co-culture solution at pH 6.5 to conduct a co-culture test. 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, the co-culture fluid was tested for LAB content, CPE content and pH value to obtain the initial LAB content, CPE content and pH value (equivalent to 0 hours of co-culture). To detect LAB content, the co-culture solution is spread on 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 colonies of LAB. The CPE bacterial count detection system co-culture solution was spread on 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 number of single colonies of CPE.

The co-culture medium is prepared from glucose-free MRS culture medium and MHB in a volume ratio of 1:1, with or without adding sugar according to the group. The co-culture medium of the NON group does not contain sugar, and the co-culture medium of the SUC group does not contain sugar. The co-culture medium of the FOS group contains 2 wt% sucrose, the co-culture medium of the FOS group contains 2 wt% fructooligosaccharide, the co-culture medium of the IN group contains 2 wt% inulin, and the co-culture medium of the XOS group contains 2 wt% Of xylo-oligosaccharides, the co-culture fluid of the LU group contained 2 wt% lactulose, and the co-culture fluid of the IMO group contained 2 wt% isomaltooligosaccharide.

The co-culture solution was cultured at 37°C. After 3 hours, 6 hours, 24 hours and 48 hours of culture, LAB content detection, CPE content detection and pH value detection were performed. The results are as follows: After 48 hours of culture, SUC The strain JJ101 content in the co-culture fluid of the group, FOS group, IN group, XOS group, LU group and IMO group was higher than that of the 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), it was confirmed that prebiotics were beneficial to the growth of strain JJ101 in vitro.

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

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

As mentioned above, in the co-culture test of strain JJ101 and CPE, if fructooligosaccharides, inulin, lactulose and isomaltooligosaccharides are used as the better prebiotics, the pH of the co-culture solution should be less than 5. However, the CPE content in clinical patients is much lower than the CPE content in co-culture experiments. Therefore, the selection of prebiotics also needs to consider the pH value of strain JJ101 after culture tests with different prebiotics.

Strain JJ101 was inoculated into different formulas of glucose-free MRS culture medium and cultured at 37°C for 24 hours to obtain cultures. Then, measure the pH value of the culture, and record the results (mean ± standard deviation of 3 replicates) in Table 1, where the NON group indicates that the MRS culture solution does not add sugar, and the SUC group indicates that the MRS culture solution adds 2% by weight of sugar. The sucrose and FOS groups indicate that the MRS culture medium is supplemented with 2 wt% of fructooligosaccharides, the IN group indicates that the MRS culture medium is supplemented with 2 wt% of inulin, the IMO group indicates that the MRS culture medium is supplemented with 2 wt% of isomaltooligosaccharide, and the LU group It means that the MRS culture medium is supplemented with 2 wt% of lactulose, and the XOS group means that the MRS culture medium is supplemented with 2 wt% of xylo-oligosaccharide.

Table 1 Group pH value (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 value of the culture of strain JJ101 in the LU group and IMO group was lower than 5.0. It was confirmed that lactulose and isomaltooligosaccharide have the effect of promoting the production of acidic substances in strain JJ101. Therefore, the probiotics of strain JJ101 Lactulose and isomaltooligosaccharide should be used.

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 to prevent, improve and/or treat drug-resistant Enterobacteriaceae infections. Secondly, adding appropriate prebiotics can enhance the activity of Lactobacillus rhamnosus JJ101 in producing acidic substances and inhibiting the growth of drug-resistant Enterobacteriaceae.

In summary, although the present invention uses a specific strain of Lactobacillus rhamnosus, a specific manufacturing process, a specific effective dose, a specific administration method, a specific experimental model and a specific evaluation method as examples to illustrate the composition of the lactic acid bacteria of the present invention Materials and their use for preparing oral compositions for inhibiting drug-resistant Enterobacteriaceae. However, those with ordinary knowledge in the technical field to which the present invention belongs will understand that the present invention is not limited thereto. Without departing from the spirit and scope of the present invention, The present invention can also be used with other strains of Lactobacillus rhamnosus, other processes, other effective dosages, other administration methods, other experimental models and other evaluation methods.

Although the present invention has been disclosed above in terms of several specific embodiments, various modifications, changes and substitutions may be made to the foregoing disclosure, and it should be understood that, in some cases, without departing from the spirit and scope of the present invention, Certain features of embodiments of the invention may be employed without corresponding use of other features. Therefore, the spirit and scope of the claims of the present invention should not be limited to the above illustrated 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 apparent and understandable, the detailed description of the accompanying drawings is as follows: [Fig. 1] is a line graph illustrating the Lactobacillus rhamnosus content in the feces of mice after oral administration of different Lactobacillus rhamnosus to mice for 3 consecutive days and discontinuation of tube feeding according to one embodiment of the present invention. [Fig. 2] is a line graph illustrating the CPE content of feces of infected mice according to one embodiment of the present invention. [Fig. 3] is a line graph illustrating the CPE content of feces of infected mice in different groups according to one embodiment of the present invention. [Figure 4A] to [Figure 4B] are line graphs respectively illustrating the CPE content and pH value of the co-culture solution after strain JJ101 and CPE are co-cultured in a co-culture solution containing different prebiotics according to one embodiment of the present invention. .

Lacticaseibacillus rhamnosus JJ101 strain was deposited at the Bioresource Collection and Research Center (BCRC, address: No. 331, Shishi Road, Hsinchu City, Taiwan 30062) on December 22, 2021 at the Food Industry Development Research Institute Biological Resource Center (BCRC) , registration number is BCRC 911088.

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Claims (9)

A lactic acid bacteria composition, including Lacticaseibacillus rhamnosus JJ101 as an active ingredient and a prebiotic, to inhibit the growth of drug-resistant Enterobacteriaceae, wherein the Lacticaseibacillus rhamnosus JJ101 was produced in December 2021 It was deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute on the 22nd. The deposit number is BCRC 911088, and the prebiotic contains lactulose and/or isomaltooligosaccharide. The lactobacillus composition as claimed in claim 1, wherein the content of the prebiotic is 1% to 5% by weight. The lactic acid bacteria composition according to claim 1, wherein the drug-resistant Enterobacter has Klebsiella pneumoniae carbapenemase (KPC)-2. The lactic acid bacteria composition according to claim 1, wherein the lactic acid bacteria composition is an oral composition. The lactic acid bacteria composition as described in claim 1, wherein a subject is administered an effective dose of the lactic acid bacteria composition for at least 14 days. The lactic acid bacteria composition as described in 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 a prebiotic, wherein the lactobacillus rhamnosus The registration number of Bacillus JJ101 is BCRC 911088, the prebiotic includes lactulose and/or isomaltooligosaccharide, and the oral composition is administered to a subject for at least 14 days. The use of Lactobacillus rhamnosus as described in claim 7 for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the oral composition further contains 1 to 5% by weight of a prebiotic. The use of Lactobacillus rhamnosus as described in claim 7 for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the drug-resistant Enterobacteriaceae has KPC-2.