TWI810805B - Lactic acid bacteria complex composition and its use in preparation of oral composition of inhibiting drug-resistant enterobacteriaceae - Google Patents

Abstract

The invention provides a lactic acid bacteria complex composition and its use in preparation of an oral composition of inhibiting drug-resistant Enterobacteriaceae. The aforementioned lactic acid bacteria composition includes a lactic acid bacteria complex as an active ingredient, in which the lactic acid bacteria complex is consisted of Lacticaseibacillus rhamnosusJJ101, Lacticaseibacillus paracaseiJJ102 and Lactiplantibacillus plantarumJJ103. The lactic acid bacteria complex 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

Composite 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 composite lactic acid bacteria composition 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 complex lactic acid bacteria composition, wherein the complex lactic acid bacteria composition contains complex lactic acid bacteria as an active ingredient. Compound lactic acid bacteria are composed of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101, Lactobacillus paracasei ( Lacticaseibacillus paracasei ) JJ102 and Lactobacillus plantarum ( Lactiplantibacillus plantarum , also known as Lactobacillus plantarum) JJ103 to inhibit the growth of drug-resistant Enterobacteriaceae.

Another aspect of the present invention is to provide a compound lactic acid bacteria composition for the preparation of an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the oral composition contains an effective dose of compound lactic acid bacteria as an active ingredient to inhibit drug-resistant Enterobacteriaceae of growth.

According to the above aspects of the present invention, a composite lactic acid bacteria composition is proposed, wherein the composite lactic acid bacteria composition contains composite lactic acid bacteria as an active ingredient to inhibit the growth of drug-resistant Enterobacteriaceae. The compound lactic acid bacteria is composed of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103. The above-mentioned Lactobacillus rhamnosus JJ101 was deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute (BCRC) on December 22, 2021, with the deposit number BCRC 911088, and the Lactobacillus paracasei JJ102 was deposited in It was deposited in BCRC on December 22, 2021, with the deposit number BCRC 911089, and the Lactobacillus germinus JJ103 was deposited in BCRC on December 22, 2021, with the deposit number BCRC 911090.

In an embodiment of the present invention, the ratio of the number of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103 may be, for example, 1-5:1-5:1-10. In an embodiment of the present invention, the composite lactic acid bacteria composition may be, for example, an oral composition. 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 complex lactic acid bacteria for at least 14 days. In one embodiment of the present invention, when the subject is a mouse, the effective dose can be, for example, 5.0×10 ¹⁰ colony-forming unit (colony-forming unit, 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 a compound lactic acid bacteria composition for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae is proposed, wherein the oral composition includes an effective dose of compound lactic acid bacteria as an active ingredient. This complex lactic acid bacteria is composed of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus plantarum JJ103. The registration number of Lactobacillus rhamnosus JJ101 is BCRC 911088, and the registration number of Lactobacillus paracasei JJ102 is BCRC 911089. And the deposit number of lactic acid bacteria JJ103 is BCRC 911090. The oral composition is administered to the subject for at least 14 days. In one embodiment of the present invention, the drug-resistant Enterobacter has KPC-2. In an embodiment of the present invention, the ratio of the number of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103 may be, for example, 1-5:1-5:1-10.

Application of the compound 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 present invention The composite lactic acid bacteria composition has the potential to be used in the prevention, improvement and/or treatment of drug-resistant Enterobacteriaceae infection.

Based on the above, the present invention provides a compound lactic acid bacteria composition and its use for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the compound lactic acid bacteria composition may include but not limited to compound lactic acid bacteria as an active ingredient. Animal experiments have proved that compound lactic acid bacteria are better than single lactic acid bacteria in inhibiting drug-resistant Enterobacteriaceae.

The "lactic acid bacteria" mentioned in this article refers to bacteria that can decompose sugars (such as: lactose, glucose, sucrose, fructose, etc.) and then secrete acidic substances (such as: organic acids such as lactic acid and/or acetic acid), such as: Bacillus, Bacillus and Bifidobacterium. It is worth noting that different strains of lactic acid bacteria may interfere with each other and affect the efficacy, but a combination of specific strains may also produce synergistic effects, thereby improving the persistence of the strains in the animal body (ie, the intestinal tract). Therefore, when using lactic acid bacteria, it is necessary to select a single strain of lactic acid bacteria or multiple strains of lactic acid bacteria (called compound lactic acid bacteria) according to the strain, the subject and/or the desired effect (such as: inhibiting drug-resistant Enterobacteriaceae). It should be added that the good retention of lactic acid bacteria in the animal body (i.e., the intestinal tract) means that the lactic acid bacteria remain in the animal body (i.e., the intestinal tract) for a long time and/or have a large number of viable bacteria. Among them, the lactic acid bacteria in the animal body (i.e. The number of viable bacteria remaining in the intestinal tract) can be assessed, for example, by calculating the number of viable bacteria per unit weight of feces. In one embodiment, the lactic acid bacteria are acid-resistant and bile-salt resistant, so their ability to persist in the intestinal tract is better.

In one embodiment, compound lactic acid bacteria are selected as the active ingredients of the compound lactic acid bacteria composition. The compound lactic acid bacteria can be composed of, for example, Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), Lactobacillus paracasei ( Lacticaseibacillus paracasei ) and lactic acid bacteria ( Lactiplantibacillus plantarum, also known as Lactobacillus plantarum ). In one embodiment, Lactobacillus rhamnosus can be, for example, Lactobacillus rhamnosus JJ101 (also known as bacterial strain JJ101) of the accession numbering system BCRC 911088, and Lactobacillus paracasei can be, for example, paracheese milk of the accession numbering system BCRC 911089 Bacillus JJ102 (also known as strain JJ102), and the lactic acid bacteria plantarum can be, for example, Lactobacillus plantarum JJ103 (also known as strain JJ103) of the accession number system BCRC 911090. It should be added that Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus plantarum JJ103 were all deposited in the Bioresource Collection and Research Center (BCRC) on December 22, 2021. ; Address: No. 331, Food Road, Hsinchu City, Taiwan, 30062), and the survival test was completed on January 7, 2022. In one embodiment, the bacterial count ratio of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103 can be, for example, 1~5:1~5:1~10, so that Lactobacillus rhamnosus JJ101, paracasei JJ101, paracasei Lactobacillus casei JJ102 and Lactobacillus plantarum JJ103 can maintain a synergistic effect in animals, thereby more effectively inhibiting the growth of drug-resistant Enterobacteriaceae. In a specific example, the bacterial count ratio of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102, and Lactobacillus malulosus JJ103 can be, for example, 1:1:1.

It has been confirmed by animal experiments that compared with other strains of the same genus, after oral administration of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102, and Lactobacillus malus JJ103 for 3 consecutive days, the number of viable bacteria remaining in the intestinal tract is lower than that of other strains of the same genus. More, it means that Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103 have better intestinal retention ability. Secondly, compared with the infected animals administered with any one of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 or Lactobacillus plantarum JJ103, the simultaneous administration of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus plantarum JJ103 The amount of drug-resistant Enterobacteriaceae in the intestinal tract of infected animals was less, indicating that the effect of compound lactic acid bacteria on inhibiting the growth of drug-resistant Enterobacteriaceae was better than that of single strains of lactic acid bacteria.

The "drug-resistant Enterobacteriaceae" mentioned herein refers to strains of Enterobacteriaceae that are resistant to antibiotics. The above-mentioned "compound lactic acid bacteria suppress drug-resistant Enterobacteriaceae" means that the compound lactic acid bacteria can reduce the content of drug-resistant Enterobacteriaceae in the body (for example, after oral administration of compound lactic acid bacteria for at least 14 consecutive days, the content of drug-resistant Enterobacteriaceae in the feces of infected animals is reduced by at least 5 orders of magnitude, equivalent to an inhibition rate of at least 99.999%). It is added 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 drug-resistant Enterobacteriaceae content in the feces of infected animals that have not been orally administered complex lactic acid bacteria , and the amount of bacteria after treatment is the content of drug-resistant Enterobacteriaceae in the feces of infected animals after compounding lactic acid bacteria.

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 include 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.

When using the above-mentioned lactic acid bacteria, the route of administration is not particularly limited, such as oral administration, which can be adjusted according to actual needs. The dosage and frequency of the above-mentioned lactic acid bacteria can also be flexibly adjusted according to demand. In one embodiment, when the subject is a mouse, the effective dose of the compound lactic acid bacteria can be, for example, 5.0×10 ¹⁰ colony-forming unit (colony-forming unit, 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 compound lactic acid bacteria for mice is 1.0×10 ¹¹ CFU/kg body weight/day, that is, 2.0×10 ⁹ CFU/mouse (20 g body weight)/day of compound lactic acid bacteria.

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 need to be orally administered a higher dose of compound lactic acid bacteria to effectively reduce drug-resistant Enterobacteriaceae. In other words, when the effective dose of compound lactic acid bacteria in clinical application to adults is lower than the effective dose to mice in animal experiments, it can effectively inhibit drug-resistant Enterobacteriaceae. In a specific example, the effective dose of compound lactic acid bacteria 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-mentioned effective dose of compound lactic acid bacteria for several consecutive days. In one embodiment, the subject is administered the complex lactic acid bacteria for at least 14 consecutive days, such as 14 days to 1 year, or 30 days to 6 months.

The compound lactic acid bacteria has the effect of inhibiting the growth of drug-resistant enterobacteria, so it can be used as an active ingredient of the compound lactic acid bacteria composition. In one embodiment, the composite lactic acid bacteria composition may be, for example, an oral composition. In one embodiment, the composite lactic acid bacteria composition may be, for example, a food composition or a medical composition. In one embodiment, the composite lactic acid bacteria composition may optionally include food or pharmaceutically acceptable carriers, excipients, diluents, adjuvants and/or additives, such as solvents, emulsifiers, suspending agents, disintegrating agent, binder, stabilizer, chelating agent, diluent, gelling agent, preservative, lubricant and/or absorption delaying agent, etc. The dosage form of the compound lactic acid bacteria composition is not particularly limited. In one embodiment, the dosage form of the compound 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 Nanoparticles, lozenges, granules, powders 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

Eleven strains of lactic acid bacteria including strain LYC1504, strain JJ101, strain LYC1119, strain JJ102, strain LYC1129, strain LYC1031, strain LYC1112, strain LYC1117, strain LYC1146, strain LYC1159 and strain JJ103 , LAB) is isolated from fruit fermented liquor . 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. Using the basic local alignment search tool (Basic Local Alignment Search Tool, BLAST) to compare, identify 2 strains of Lactobacillus rhamnosus (bacterial strain LYC1504 and bacterial strain JJ101), 3 strains of Lactobacillus paracasei (bacterial strain LYC1119) among the 11 strains of LAB. , bacterial strain JJ102 and bacterial strain LYC1129) and 6 strains of lactic acid bacteria (strain LYC1031, bacterial strain LYC1112, bacterial strain LYC1117, bacterial strain LYC1146, bacterial strain LYC1159 and bacterial strain JJ103).

The 16S rDNA nucleic acid sequence of the above strain JJ101 is shown in SEQ ID NOs:3. The 16S rDNA nucleic acid sequence of strain JJ102 is shown in SEQ ID NOs:4. The 16S rDNA nucleic acid sequence of strain JJ103 is shown in SEQ ID NOs:5. The strains JJ101, JJ102 and JJ103 were deposited in BCRC on December 22, 2021, and the survival test was completed on January 7, 2022. The registration number of strain JJ101 is BCRC 911088, and the registration number of strain JJ102 is BCRC 911089, and the deposit number of strain JJ103 is BCRC 911090.

It is supplemented that the colony of the strain JJ101 (Lactobacillus rhamnosus) is milky white, opaque, round, smooth and protruding, with neat edges, and its cells are short rods with blunt round ends. Short chains or chain-like forms exist, with no flagella, no motility, no sporulation, and Gram-stain positive. The colony of strain JJ102 (Lactobacillus paracasei) is milky white, opaque, round or almost round, with smooth and protruding surfaces and neat edges. There is a flagellum-like form, no motility, no sporulation, and the Gram stain is positive. The colonies of strain JJ103 (lactic acid bacteria with germ) are milky white, opaque, round to slightly irregular in shape, with smooth and protruding surfaces and neat edges. Short chain forms exist, are motile without flagella, do not sporulate, and are Gram-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 respectively, wherein the LAB solution was obtained by dissolving the precipitates of the above 11 strains of LAB respectively in phosphate buffered saline (PBS) , and adjust the LAB content, so that the 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.

Please refer to Figure 2, wherein Figure 2 is a broken line showing the content of Lactobacillus paracasei in the feces of mice according to an embodiment of the present invention after oral administration of different Lactobacillus paracasei for 3 consecutive days and stopping tube feeding Figure, wherein horizontal axis represents time (unit: day), and vertical axis represents Lactobacillus paracasei content (unit: CFU/g), broken line 201, broken line 203 and broken line 205 are bacterial strain LYC1119, bacterial strain JJ102 and bacterial strain LYC1229 respectively. As shown in Figure 2, after stopping tube feeding for 1 day, the content of strain JJ102 in mouse feces (line 203) was higher than that of strain LYC1119 and strain LYC1229 (line 201 and line 205), and the content of strain JJ102 was higher than 10 ⁷ CFU /g, confirming that the intestinal persistence ability of strain JJ102 is better.

Please refer to Fig. 3, wherein Fig. 3 is a line graph showing the content of lactic acid bacteria in the feces of mice according to an embodiment of the present invention after oral administration of different lactic acid bacteria to mice for 3 consecutive days and stopping tube feeding. The axis represents time (unit: day), and the vertical axis represents the content of germ lactic acid bacteria (unit: CFU/g). The broken line 301, broken line 303, broken line 305, broken line 307, broken line 309 and broken line 311 are strain LYC1031, strain LYC1112 and strain LYC1117 respectively , strain LYC1146, strain LYC1159 and strain JJ103.

As shown in Figure 3, after stopping tube feeding for 1 day, 3 days and 7 days, the content of bacterial strain JJ103 in mouse feces (broken line 311) was higher than that of other bacterial strains (broken line 301 to broken line 309 and broken line 313). After 3 days, the content of strain JJ103 was 10 ⁶ CFU/g to 10 ⁷ CFU/g, and 7 days after stop tube feeding, the content of strain JJ103 was still more than 10 ⁵ CFU/g, confirming that compared with other lactic acid bacteria, the content of strain JJ103 Intestinal retention is 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 bacteria.

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. 4, wherein Fig. 4 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 401, broken line 403, broken line 405 and broken line 407 represent bacterial strain KPC001, bacterial strain KPC011, bacterial strain KPC021 and bacterial strain KPC035, respectively. As shown in Figure 4, 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 of CPE among different strains. Subsequent evaluations were performed with strain KPC001. Example 3. Evaluation of the efficacy of compound lactic acid bacteria 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 4 groups (blank group, control group 1, control group 2, control group 3 and experimental group 1). The infected mice in the blank group were orally administered with PBS for 21 consecutive days, the infected mice in the control group 1 were orally administered with 2.0×10 ⁹ CFU/day of strain JJ101 for 21 consecutive days, and the infected mice in the control group 2 were Orally administered 2.0×10 ⁹ CFU/day of strain JJ102 for 21 consecutive days, the infected mice in control group 3 were orally administered 2.0×10 ⁹ CFU/day of strain JJ103 for 21 consecutive days, and the infected mice in experimental group 1 Mice were orally administered 2.0×10 ⁹ CFU/day of compound LAB for 21 consecutive days, wherein the compound LAB was composed of strain JJ101, strain JJ102 and strain JJ103 at a ratio of 1:1:1. After mice were orally administered LAB for 4 days, 7 days, 11 days, 14 days, 18 days and 21 days, the CPE content in feces of infected mice was detected.

Please refer to FIG. 5, wherein FIG. 5 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 consecutive days of oral administration of LAB to infected mice ( Unit: day), the vertical axis represents the CPE content (unit: CFU/g) of infected mouse feces, broken line 501, broken line 503, broken line 505, broken line 507 and broken line 509 represent blank group, control group 1, control group 2, Control group 3 and experimental group 1, and different letters A, B, C and D indicate statistically significant differences (p<0.05).

As shown in Figure 5, the CPE content of the infected mouse feces of experimental group 1, matched group 1, matched group 2 and matched group 3 (broken line 509, broken line 503 to broken line 507) is lower than that of blank group (broken line 501), wherein The CPE content in feces of infected mice in experimental group 1 was significantly lower than that in control group 1, control group 2, and control group 3, confirming that the effect of compound LAB on inhibiting the growth of CPE was better than that of single LAB. In detail, compared with the CPE content of infected mice without oral administration of LAB, the CPE content of feces of infected mice in experimental group 1 was reduced by at least 5 orders of magnitude after oral administration of compound LAB to infected mice for 21 consecutive days , which is equivalent to an inhibition rate of at least 99.999%, but the CPE content of the feces of infected mice in the control group 1, control group 2, and control group 3 (line 503 to line 507) was significantly higher than that of the infected mice orally administered with different LAB strains for 21 consecutive days. Tianhou is only reduced by 2 to 3 orders of magnitude, which is equivalent to an inhibition rate of only 99% to 99.9%.

In summary, the specific compound lactic acid bacteria can inhibit the growth of drug-resistant Enterobacteriaceae in vivo, indicating that compound lactic acid bacteria have the potential to be used in the prevention, improvement and/or treatment of drug-resistant Enterobacteriaceae infection.

To sum up, although the present invention uses the compound lactic acid bacteria with specific strain combination, specific process, specific effective dose, specific administration method, specific experimental model and specific evaluation method as an example to illustrate the compound lactic acid bacteria composition of the present invention and its 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 the present invention can be included within the spirit and scope of the present invention. The invention can also be carried out by compound lactic acid bacteria with other bacterial strain combinations, other manufacturing processes, other effective doses, other delivery 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.

101,103,201,203,205,301,303,305,307,309,311,401,403,405,407,501,503,505,507,509: 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 and stopping tube feeding. [ Fig. 2 ] is a line graph showing the content of Lactobacillus paracasei in the feces of mice according to an embodiment of the present invention after oral administration of different Lactobacillus paracasei for 3 consecutive days and stop tube feeding. [ Fig. 3 ] is a line graph showing the content of lactic acid bacteria in the feces of mice according to an embodiment of the present invention after orally administering different lactic acid bacteria to mice for 3 consecutive days and stopping tube feeding. [ FIG. 4 ] is a line graph showing the CPE content in feces of infected mice according to an embodiment of the present invention. [ FIG. 5 ] is a line graph showing the CPE content in feces of different groups of infected mice according to an embodiment of the present invention.

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. The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) JJ102 line was deposited in BCRC on December 22, 2021, and the deposit number is BCRC 911089. Lactiplantibacillus plantarum JJ103 was deposited in BCRC on December 22, 2021, with the deposit number BCRC 911090.

501,503,505,507,509: polyline

Claims (10)

A compound lactic acid bacteria composition, comprising a compound lactic acid bacterium as an active ingredient, so as to inhibit the growth of a drug-resistant enterobacteriaceae, wherein the compound lactic acid bacteria is composed of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101, Lactobacillus paracasei ( Lacticaseibacillus paracasei ) JJ102 and Lactobacillus plantarum ( Lactiplantibacillus plantarum ) JJ103. The Lactobacillus rhamnosus JJ101 was deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute on December 22, 2021. The deposit number is BCRC 911088, the Lactobacillus paracasei JJ102 line was deposited in BCRC on December 22, 2021, and the deposit number is BCRC 911089, and the Lactobacillus germinus JJ103 line was deposited in BCRC on December 22, 2021, and the deposit number is BCRC 911090, and the drug-resistant Enterobacteriaceae has Klebsiella pneumoniae carbapenemase ( Klebsiella pneumoniae carbapenemase, KPC)-2. The composite lactic acid bacteria composition as described in Claim 1, wherein the ratio of the number of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102, and Lactobacillus germ JJ103 is 1~5:1~5:1~10. The compound lactic acid bacteria composition as described in Claim 1, wherein the compound lactic acid bacteria composition is an oral composition. The composite lactic acid bacteria composition as described in Claim 1 further includes food or pharmaceutically acceptable carriers, excipients, diluents, adjuvants and/or additives. The compound lactic acid bacteria composition as described in Claim 1, wherein a test subject is administered with an effective dose of the compound lactic acid bacteria for at least 14 days. The compound lactic acid bacteria composition as described in claim 5, wherein when the subject is a mouse, the effective dose is 5.0×10 ¹⁰ colony-forming units (colony-forming unit, CFU)/kg body weight/day to 1.5 ×10 ¹¹ CFU/kg body weight/day. A compound lactic acid bacteria composition is used to prepare an oral composition for inhibiting drug-resistant enterobacteria, wherein the oral composition contains an effective dose of a compound lactic acid bacterium as an active ingredient, and the compound lactic acid bacteria is produced by Lactobacillus rhamnosus JJ101 , Lactobacillus paracasei JJ102 and Lactobacillus plantarum JJ103, the registration number of Lactobacillus rhamnosus JJ101 is BCRC 911088, the registration number of Lactobacillus paracasei JJ102 is BCRC 911089, and the registration number of Lactobacillus paracasei JJ103 is BCRC 911090 , the oral composition is administered to a subject for at least 14 days, and the drug-resistant enterobacteriaceae have KPC-2. The compound lactic acid bacteria composition as described in claim item 7 is used for making Preparation of an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the composite lactic acid bacteria composition further includes food or pharmaceutically acceptable carriers, excipients, diluents, adjuvants and/or additives. The compound lactic acid bacteria composition as described in claim 7 is used to prepare the purposes of the oral composition of inhibiting drug-resistant Enterobacteriaceae, wherein the number of bacteria of the Lactobacillus rhamnosus JJ101, the Lactobacillus paracasei JJ102 and the Lactobacillus lactobacillus JJ103 The ratio is 1~5:1~5:1~10. Use of the composite lactic acid bacteria composition as described in Claim 7 for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, 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.