Application of lactobacillus plantarum in inhibition of carbapenem-resistant klebsiella pneumoniae intestinal colonization
By using Lactobacillus plantarum LP-Y01 to regulate the bacterial community structure, the infection problem of carbapenem-resistant Klebsiella pneumoniae was solved, achieving effective prevention and treatment of drug-resistant bacteria and reducing the use of antibiotics.
Patent Information
- Application Number
- CN202511354815.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-22
- Publication Date
- 2025-12-23
AI Technical Summary
The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has exacerbated the difficulty of treating clinical bacterial infections. Current technologies lack effective methods to prevent infections in livestock and poultry, and antibiotic use has led to drug resistance.
Lactobacillus plantarum LP-Y01 and its inoculum can be used to prevent and treat carbapenem-resistant bacterial infections by regulating the bacterial community structure, thereby reducing the use of antibiotics.
Lactobacillus plantarum LP-Y01 has shown significant inhibitory effects on carbapenem-resistant Klebsiella pneumoniae in vitro and in vivo, and regulates the bacterial community structure, providing a precise strategy for the prevention and control of drug-resistant bacteria, and has good application prospects.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of microorganisms, and relates to a lactobacillus plantarum and application of the lactobacillus plantarum in inhibiting carbapenem-resistant klebsiella pneumoniae. BACKGROUND
[0002] In recent years, the wide use of carbapenem antibiotics has led to serious drug resistance, and the emergence of carbapenem-resistant klebsiella pneumoniae (CRKP) has aggravated the difficulty of clinical bacterial infection treatment. The applicant's early research found that the high carrying rate (55.5%, 233 / 420) of bla NDM in the migratory bird samples collected from Qinghai Lake, among which klebsiella pneumoniae is the dominant epidemic strain, and the isolation rate is as high as 66.5% %. The high prevalence of CRKP in migratory birds, as well as the wide area and diversity of transmission modes of birds, greatly increase the difficulty of prevention and control of CRKP.
[0003] At present, the research on klebsiella pneumoniae in veterinary clinics mainly focuses on the high isolation rate and high drug resistance of strains in breeding environments, and there is no clear method for preventing infection of livestock and poultry animals at this stage. SUMMARY
[0004] The application establishes a mouse colonization model to explore the intestinal colonization rules of bla NDM-5 positive CRKP from migratory birds, and explores the transfer of bla NDM-5 in mice and the colonization resistance of probiotics to CRKP, uses the colonization resistance of probiotics to prevent and treat CRKP infection, reduces the use of antibiotics, and slows down the generation of drug resistance. The results can provide a new idea for the occurrence and diagnosis and treatment of intestinal colonization CRKP, and have important application value for early prevention and treatment of infection.
[0005] To achieve the above purpose, the application provides the following technical scheme:
[0006] The application of lactobacillus plantarum LP-Y01 and its bacterial agent in the preparation of a medicine for preventing and / or treating infection of carbapenem-resistant bacteria.
[0007] Preferably, the strain of carbapenem-resistant bacteria in the medicine for carbapenem-resistant bacteria is an animal source strain.
[0008] Preferably, the strain of carbapenem-resistant bacteria carries a bla NDM-5 gene.
[0009] Preferably, the carbapenem-resistant bacteria is escherichia coli and / or klebsiella pneumoniae.
[0010] Compared with the prior art, the application has the following beneficial effects:
[0011] The application verifies that the above-mentioned Lactobacillus plantarum can produce direct inhibitory effect on carbapenem-resistant Klebsiella pneumoniae in vitro, and has certain effective activity against colonization of carbapenem-resistant Klebsiella pneumoniae in vivo through regulation of flora. The probiotic pretreatment shows significant clinical potential by regulating the structure of flora, provides precise strategy for prevention and control of drug-resistant bacteria, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0012] Figure 1 The colonization of CRKP under different conditions; note: blank control group (Con), high concentration infection group (H-KP), low concentration infection group (L-KP), and control group without antibiotic intestinal flora disturbance (Un-pre).
[0013] Figure 2 The schematic diagram of the inhibition zone of Lactobacillus plantarum cell-free supernatant.
[0014] Figure 3 The growth of Lactobacillus plantarum and Klebsiella pneumoniae in different culture environments. A and B are the growth of two strains in LB and BHI medium, respectively; C-F: growth in LB medium containing different proportions of MRS medium.
[0015] Figure 4 The content of QH179-1 in the co-culture system.
[0016] Figure 5 The anti-colonization effect of Lactobacillus plantarum on Klebsiella pneumoniae in mice. "*" indicates significant difference between groups (p<0.05), and no mark indicates no significant difference between groups (p>0.05). DETAILED DESCRIPTION
[0017] The technical solutions of the present application will be described in detail below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. The test methods used in the embodiments of the present application are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.
[0018] Lactobacillus plantarum LP-Y01 is a strain with strong activity isolated from food leavening provided by the Food College of South China Agricultural University, and Lactobacillus plantarum LP-Y02 is another strain isolated from the same food leavening. Standard strains: Escherichia coli ATCC 25922 and streptomycin-resistant Escherichia coli C600.
[0019] The strain used in the experiment is a clinically isolated strain: a verified bla NDM-5 carrying carbapenem-resistant Klebsiella pneumoniae isolated from bird samples and their surrounding environment collected from Qinghai Lake Bird Island in China, stored in the laboratory.
[0020] Experimental animals: SPF C57BL / 6J mice, 6-8 weeks old, purchased from Guangdong Zhuhai Baitishun Biotechnology Co., Ltd. All experimental animals were raised in a pre-screening system, and free access to food and water (SPF). The animal experiment plan was approved by the institutional ethics committee (approval number: 2024c043), and the whole process followed the experimental animal welfare and 3R principle operation specification.
[0021] I. Probiotics and Klebsiella pneumoniae co-culture experiment:
[0022] Culture medium co-culture system screening: BHI, LB, LB+MRS (1:1), 10% MRS+LB, 20% MRS+LB, 30% MRS+LB, a total of 6 systems, respectively inoculated with LP (Lactobacillus plantarum), KP (carbapenem-resistant Klebsiella pneumoniae carrying blaNDM-5), and the growth curve was measured. Take 5mL broth in a 15ml centrifuge tube, and set up a blank control group. The test strain was cultured overnight, and the OD600 was measured after 18h (the blank group was used for zero adjustment). Dilute 1 / 10 or dilute to OD600 0.1. Add 1 mL of culture medium to each well and add 10 μL of diluted bacterial solution. After adding the system, put it into the monitor for 24h, and draw the growth curve.
[0023] II. Growth monitoring of Lactobacillus plantarum and Klebsiella pneumoniae co-culture
[0024] The concentration of Lactobacillus plantarum LP-Y02, LP-Y01 bacterial suspension was 1.0×10 8 CFU / mL. Experimental group: add equal volume (100 μL) of LP and KP to 25 mL co-culture system; control group: equal volume of PBS and KP. Incubate at 37°C, and count KP in the co-culture system at 0, 2, 4, 6, 8, 10, 12h.
[0025] III. Bacteriostatic test of Lactobacillus plantarum cell-free supernatant
[0026] 1) Preparation of cell-free supernatant
[0027] Lactobacillus plantarum LP-Y02, LP-Y01 was cultured in MRS liquid medium at 37°C for 36h, centrifuged at 8000g for 15min, and the supernatant was filtered with a 0.22 micron sterile filter membrane to obtain cell-free supernatant, which was stored at 4°C for use. Measure pH.
[0028] 2) Bacteriostatic test by punching method on plate agar
[0029] LP-Y02 and LP-Y01 were subcultured at a ratio of 1% for 36 h, centrifuged at 8000 g for 15 min, and the supernatant was retained. After 18 h of culture, the bacterial solution was diluted to 1.0 x 10 8 CFU / mL with LB liquid broth, 200 μL was taken and spread on LB plates, and after the surface of the plate was dry, a sterile puncher with a diameter of 6 mm was used to punch the plate with the bacterial solution, 100 μL of each probiotic fermentation supernatant was added to each hole, and MRS and LB medium were used as negative controls. After 18 hours of culture in a 37°C incubator, the presence or absence of the bacteriostatic ring and its size were recorded, and the diameter of each bacteriostatic ring was accurately measured with a vernier caliper. The above test was repeated 3 times, and the results were averaged.
[0030] 3) Determination of bacteriostatic curve
[0031] First, the test strain was cultured overnight in LB broth medium, and the OD 600 value was measured, and the OD 600 value of the bacterial solution was diluted to 0.1. After adding 900 μL of LB broth medium to each well of the microbial growth plate, 10 μL of the diluted bacterial solution was added, and then 100 μL of cell-free supernatant of Lactobacillus plantarum LP-Y02 and LP-Y01 or MRS medium (control group) was added. Each group was set up in triplicate, and the OD value of each group was measured every 2 hours for 24 hours.
[0032] Four, bla NDM-5 Positive K.P infection mouse colonization model experiment
[0033] 1) Strain culture
[0034] Klebsiella pneumoniae QH179-1 (from blaNDM-5 carrying carbapenem-resistant Klebsiella pneumoniae culture) was cultured overnight at 37°C, 200 rpm in LB liquid medium, centrifuged at 5000 rpm for 3 min, the bacterial pellet was discarded, and the supernatant was discarded, the bacterial pellet was washed twice with PBS. The bacterial concentration was adjusted to 1 x 10 5 CFU / mL and 1 x 10 8 CFU / mL with PBS.
[0035] 2) Determine the best colonization model experiment group
[0036] 40 C57BL / 6J mice were randomly divided into 4 groups, 10 in each group, namely blank control group (Con), high concentration infection group (H-KP), low concentration infection group (L-KP) and antibiotic-free intestinal flora disturbance control group (Un-pre).
[0037] After 5 days of adaptive feeding, the mice were pre-treated with antibiotics. Ampicillin (1 g / L), metronidazole (1 g / L), neomycin (1 g / L) and vancomycin (1 g / L) were added to the drinking water of the mice, and the treatment lasted for 72 hours. The Un-pre group added the same volume of PBS in the drinking water. 12 hours before the gavage treatment, the mice were subjected to water and food deprivation. The mice were inoculated with 200 µL of bacterial cell suspension by gavage, and the blank control group was inoculated with 200 µL of PBS.
[0038] Five, the establishment of mouse anti-colonization model: experimental strain: QH179-1 (resuscitated blaNDM-5 carrying carbapenem-resistant Klebsiella pneumoniae), LP-Y01. Experimental grouping: divided into 5 groups, 10 mice in each group. Respectively, blank control group (Con), colonization infection group (KP), antibiotic 50 mg / kg.d exposure group (K-Mem), probiotic treatment group (K-LP) and probiotic prevention treatment group (K-pLP).
[0039] The antibiotic exposure group was given 200 µL of 1 × 10 5 CFU / mL CRKP by gavage after 3 days (72 h) of mixed antibiotic pretreatment, and 200 µl of 50 mg / kg meropenem was injected subcutaneously at the same time for 3 consecutive days; the probiotic treatment group was treated with 200 µL of 1 × 10 8 CFU / mL of Lactobacillus plantarum orally and gavaged for 3 consecutive days; the probiotic prevention treatment group was given 200 µL of 1 × 10 8 CFU / mL of Lactobacillus plantarum orally and gavaged for 3 consecutive days; the probiotic prevention treatment group was given 200 µL of 1 × 10 5 CFU / mL CRKP by gavage after 3 days (72 h) of mixed antibiotic pretreatment, and 200 µl of 50 mg / kg meropenem was injected subcutaneously at the same time for 3 consecutive days; the probiotic treatment group was treated with 200 µL of 1 × 10
[0040] Six, determination of the absolute expression of bla NDM-5 gene in feces
[0041] Establishment of standard curve: the purity and concentration of recombinant plasmid were determined by ultramicro UV spectrophotometer, and the absolute abundance (copy number) of plasmid DNA was calculated by the following formula:
[0042] In the formula, c DNA is the plasmid concentration, 2692 is the size of the vector, and X is the size of the inserted target gene.
[0043] After calculating the copy number of the plasmid, 10-fold gradient dilution was performed. After 7 gradients of sequential dilution, the concentration of bla NDM-5 was (8.27 × 103 8.27 × 10 10 copies / μl, with sterile water as negative control for Real-time fluorescence quantitative PCR (qPCR) determination. The reaction system of qPCR was as follows: qPCR Mix 10.0 μl, each 0.4 μl of upstream and downstream primers, 1 μl of DNA template, 8.2 μl of sterile water. The amplification reaction program was pre-denaturation: 95℃, 30 s; cycle reaction: 95℃, 10 s, 60℃, 30 s, 40 cycles; melting curve: 95℃, 15 s, 60℃, 60 s, 95℃, 15 s. qPCR was set up 3 repeats for each sample. The primer sequences were shown in Table 1.
[0044] Table 1 Gene primer sequences
[0045] The standard plasmid was subjected to qPCR analysis in a fluorescence quantitative PCR instrument (Bio-rad CFX96) and a standard curve was constructed. If R 2 > 0.99, the amplification efficiency E was between 90% and 110%, and the melting peak was single, it was indicated that the standard plasmid of the target gene could be used for qPCR analysis of the subsequent sample DNA.
[0046] Experimental results:
[0047] 1. Strain drug sensitivity experiment results
[0048] 180 strains of Klebsiella pneumoniae (KP) isolated from Qinghai migratory birds were recovered and identified in the laboratory, and 7 strains of bla NDM-5 positive KP were successfully recovered, including 5 strains of type A (QH179-1, QH241, QH281-4, QH283-2, QH288-1) and 2 strains of type B (QH194, QH230-4).
[0049] The sensitivity of 7 strains of CRKP to antibiotics was determined by double dilution agar method. Different strains showed certain similarity in drug resistance to different categories of antibiotics. All 7 strains of CRKP showed multi-drug resistance (resistant to three or more than three categories of drugs), among which cephalosporins, beta-lactams and sulfonamides showed high-level resistance, and all strains were resistant to gentamicin and tetracycline, but all strains were sensitive to amikacin and polymyxin B. Strains QH241, QH281-4 and QH194 were resistant to gentamicin, tetracycline, tigecycline, cefotaxime, ceftazidime, ciprofloxacin, ampicillin and trimethoprim. All strains were resistant to carbapenems, and the MIC value of QH179-1 was the highest.
[0050] 2. Verification of conjugative transfer ability
[0051] Seven strains of Klebsiella pneumoniae carrying bla NDM-5 were used as donor bacteria, and Escherichia coli C600 was used as recipient bacteria for conjugative transfer experiment. The results showed that after conjugative transfer, 6 strains of bacteria could grow on double-antibiotic plates containing meropenem and streptomycin. PCR verification of bla NDM-5 gene and 16s RNA sequencing identification of the 6 strains of bacteria as Escherichia coli showed that the 6 strains of bacteria were conjugants, and the 6 strains of Klebsiella pneumoniae carrying carbapenem-resistant genes could horizontally transfer between different genera in vitro. The strain QH179-1 with high MIC value and producing conjugants was selected for mouse intestinal colonization experiment.
[0052] 3. Effect of infection dose on the abundance of CRKP in feces
[0053] In order to establish the optimal infection dose, high and low concentration animal models were established, and fecal samples were collected on days 1, 4, 6 and 8 for plate counting of carbapenem-resistant Klebsiella pneumoniae (CRKP) numbers. As shown in the figure, Figure 1 , the colonization of Klebsiella pneumoniae in mice did not show an upward trend with the increase of infection dose, and there was no significant difference between high and low concentration infection (p<0.05). It was observed that the colonization of CRKP in the mouse intestine in the colonization group without intestinal disturbance before infection was not stable, and the number of bacteria detected on days 1, 4, 6 and 8 was significantly lower than that in the two groups with intestinal flora disturbance before infection. As can be seen from the figure, the abundance of CRKP detected in feces showed a continuous downward trend, especially in H-KP and L-KP, which showed an intensified downward trend on day 6, and the abundance of UN-pre group showed a temporary upward trend on day 6, but showed a downward trend on day 8.
[0054] After 1x105 CFU / ml or 1 x 10 8 The number of CRKP in feces in the colonization model at the infection dose of 1 x 10 5 CFU / ml bacteria concentration was performed.
[0055] Therefore, in the process of building the CRKP colonization model, it was found that the detection abundance in feces decreased with the longer colonization time. The detection amount of CRKP on the eighth day of colonization was extremely low without disturbing the intestinal flora, indicating that CRKP was not easy to colonize in the intestinal tract. After the low-concentration antibiotic treatment disturbed the structure of the intestinal flora, it could promote the colonization of CRKP in the intestinal tract. The intestinal microbial community is a protective barrier in the body, which plays a role in the invasion of some potential pathogenic organisms. Once the intestinal microbial community is stable, the external pathogenic microorganisms are more likely to colonize the body and cause disease infection.
[0056] 4. Inhibition of Klebsiella pneumoniae by Lactobacillus plantarum:
[0057] 1) Agar punching inhibition zone experiment was performed by preparing two strains of Lactobacillus plantarum fermentation to generate cell-free supernatant (CFS), and the CFS was tested with three different Klebsiella pneumoniae.
[0058] As shown in Figure 2 , the diameters of the inhibition zones produced by the CFS of Lactobacillus plantarum LP-Y02 on Klebsiella pneumoniae QH179-1, QH281-4, and QH283-2 were 16.50 ± 0.20 mm, 15.81 ± 0.11 mm, and 14.68 ± 0.21 mm, respectively. The diameters of the inhibition zones produced by the CFS of LP-Y01 on QH179-1, QH281-4, and QH283-2 were 19.03 ± 0.12 mm, 18.35 ± 0.17 mm, and 12.86 ± 0.15 mm, respectively. According to the resistance and sensitivity values (≥ 18 mm) and intermediate values (15-17 mm) of the β-lactam complex preparation (meropenem-velpatin) in CLSI for measuring the inhibition zone in enterobacteriaceae, it can be seen that the cell-free supernatant of Lactobacillus plantarum LP-Y01 has a significant inhibitory effect on Klebsiella pneumoniae. The inhibition zones formed by LP-Y01 on QH179-1 and QH281-4 were larger than those formed by LP-Y02, and the inhibition effect of LP-Y01 was stronger.
[0059] 2) Inhibition of Klebsiella pneumoniae growth by Lactobacillus plantarum
[0060] In the foregoing, the supernatant of the culture has certain bacteriostatic effect on Klebsiella pneumoniae. Next, in order to further evaluate the in vitro inhibitory effect of LP-Y02 and LP-Y01 on Klebsiella pneumoniae, the two strains of Lactobacillus plantarum were co-cultured with Klebsiella pneumoniae to study their bacteriostatic effect.
[0061] Firstly, the suitable culture environment of the two groups of bacteria was explored. According to the growth of Lactobacillus plantarum LP-Y01 and Klebsiella pneumoniae QH179-1 in different culture systems (Fig. 1), Figure 3 it can be seen from the figure that the ordinary LB and BHI culture medium cannot provide the growth environment for LP-Y01, but the growth trend of the two strains in the LB broth containing 30% MRS is relatively close, so it is used as the co-culture system.
[0062] Subsequently, equal amounts of LP-Y02 and LP-Y01 were inoculated into equal amounts of QH179-1 in LB broth containing 30% MRS medium, and Klebsiella pneumoniae QH179-1 was counted at different time points. It can be seen from Fig. 2 that under the co-culture system, both strains of Lactobacillus plantarum have inhibitory effect on the growth of QH179-1. LP-Y01 can completely inhibit the growth of Klebsiella pneumoniae after 8 hours of co-culture, and the inhibitory effect of Lactobacillus plantarum LP-Y01 is better than that of LP-Y02. Figure 4
[0063] 3) Anti-colonization effect of Lactobacillus plantarum on Klebsiella pneumoniae in mice
[0064] In vitro experiments show that Lactobacillus plantarum has inhibitory effect on the growth of Klebsiella pneumoniae. Next, animal experiments were conducted to verify whether Lactobacillus plantarum can inhibit the colonization of Klebsiella pneumoniae in vivo. Klebsiella pneumoniae was isolated from mouse feces collected at different times to obtain the bacterial load in feces, and the results are shown in Fig. 3. Figure 5 Compared with the blank group Con, the fecal bacterial load of the model group KP, the Lactobacillus plantarum LP-Y01 treatment group K-Lp and the LP-Y01 pretreatment group was higher in the first 3 days after colonization and infection. There was no significant difference in the bacterial load between the KP group and K-Lp in the first 5 days, but the bacterial load in K-Lp was significantly reduced on the 7th day. Most significantly, the fecal bacterial load in the pretreatment group K-pLp was significantly lower than that in the KP and K-Lp groups, and Klebsiella pneumoniae was not detected in the 5th to 7th days after colonization and infection. The results show that Lactobacillus plantarum can interfere with the colonization of Klebsiella pneumoniae in mice, and pretreatment with Lactobacillus plantarum can completely inhibit the colonization.
[0065] In summary, protecting the body from pathogenic bacteria is one of the important functions of probiotics, and many studies have shown that lactic acid bacteria strains have antibacterial activity against a variety of pathogens, including Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae The present application selects specific Lactobacillus plantarum as a prevention and treatment factor for the colonization of Klebsiella pneumoniae in the intestinal tract. In vitro experiments were conducted to study the inhibitory effect of Lactobacillus plantarum on Klebsiella pneumoniae. The cell-free supernatant (CFS) of Lactobacillus plantarum LP-Y01 and LP-Y02 had an inhibition zone diameter of 19.03 ± 0.12 mm and 16.50 ± 0.20 mm, respectively, for CRKP strain (QH179-1), significantly inhibiting its growth. Co-culture experiments further showed that LP-Y01 completely inhibited the proliferation of QH179-1 within 8 hours, and the antibacterial effect was better than that of LP-Y02, which may be due to the strong antibacterial activity of its metabolic products (such as organic acids and bacteriocins). The antibacterial mechanism of lactic acid bacteria is related to the competition for nutrients and binding sites, immune stimulation, the ability to lower the pH of the intestinal tract by producing organic acids (such as acetic acid and lactic acid), and the production of antibacterial compounds (such as fatty acids and bacteriocins) After the Lactobacillus genus entered the mouse, the intestinal colonization of Klebsiella pneumoniae in mice treated with tigecycline was significantly reduced In vitro studies showed that in the mouse model, the CRKP load in the pre-treatment group with Lactobacillus plantarum (K-pLp group) dropped below the detection limit on the 5th day after infection, which was significantly lower than that in the untreated group (KP group) and the post-treatment group (K-Lp group). This result suggests that Lactobacillus plantarum can effectively block the colonization of CRKP by occupying the intestinal niche in advance, enhancing the intestinal barrier function, or secreting antibacterial substances.
[0066] Obviously, the above-described specific embodiments are only a further detailed description of the purpose, technical solutions and beneficial effects of the present application, and it should be understood that the above-described is only a specific example of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims
1. Application of Lactobacillus plantarum LP-Y01 and its inoculum in the preparation of drugs for the prevention and / or treatment of carbapenem-resistant bacterial infections.
2. The application according to claim 1, characterized in that, The carbapenem-resistant bacteria strains in the drug are animal-derived strains.
3. The application according to claim 1, characterized in that, The carbapenem-resistant bacteria strains carry bla NDM-5 Gene.
4. The application according to claim 1, characterized in that, The carbapenem-resistant bacteria are Escherichia coli and / or Klebsiella pneumoniae.