Use of ethanolamine in the preparation of a drug for inhibiting polymyxin heteroresistance in klebsiella pneumoniae
By combining ethanolamine and colistin, the heterogeneous drug-resistant subgroups of Klebsiella pneumoniae were specifically inhibited, solving the problem of colistin treatment failure and improving the therapeutic efficacy and safety of colistin.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-04-16
- Publication Date
- 2026-06-16
AI Technical Summary
In existing technologies, the heterogeneous resistance of colistin to Klebsiella pneumoniae leads to treatment failure and recurrent infections, which are difficult to effectively address through traditional drug susceptibility testing.
The combined use of ethanolamine and colistin enhances the antibacterial activity of colistin against heterogeneous drug-resistant strains by exogenously adding ethanolamine, and specifically inhibits the growth of heterogeneous drug-resistant subgroups.
It significantly reduces the minimum inhibitory concentration (MIC) of colistin against heterogeneous drug-resistant strains, improves the therapeutic effect of colistin, reduces the risk of treatment failure and recurrent infection, and has broad applicability and phenotypic selectivity.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of antibacterial agent technology, specifically relating to the application of ethanolamine in the preparation of drugs that inhibit heterogeneous resistance to colistin in Klebsiella pneumoniae. Background Technology
[0002] Klebsiella pneumoniae is an important Gram-negative opportunistic pathogen that can cause various infections, including pneumonia, bacteremia, and urinary tract infections, and is particularly common in hospital-acquired infections and in immunocompromised populations. In recent years, with the continuous increase in multidrug-resistant strains, especially carbapenem-resistant Klebsiella pneumoniae, its clinical treatment has become significantly more difficult, making it one of the key pathogens in the prevention and control of drug-resistant infections.
[0003] Colistin is an important drug for treating infections caused by multidrug-resistant Gram-negative bacteria, and is often considered a last-line treatment against some drug-resistant Klebsiella pneumoniae infections. It primarily works by electrostatically interacting with lipid A in the lipopolysaccharide of the outer membrane of Gram-negative bacteria, displacing the divalent cations that stabilize the outer membrane structure, thereby disrupting the integrity of the bacterial outer membrane and leading to bacterial death.
[0004] Antibiotic susceptibility testing is considered a primary basis for designing clinical bacterial disease treatment regimens. However, studies have indicated that even bacteria showing susceptibility in susceptibility testing can still experience treatment failure. Research suggests that heterogeneous resistance may be associated with an increased risk of recurrent infections and antibiotic treatment failure. Bacterial heterogeneous resistance phenotype typically refers to a bacterial population not exhibiting uniform sensitivity to a particular antimicrobial agent, but rather possessing subpopulations with varying levels of resistance. While most cells remain sensitive or show low-level tolerance to the drug, a smaller proportion of cells may survive or grow at higher drug concentrations, exhibiting higher minimum inhibitory concentrations (MICs) or stronger survivability. Several studies have found that Klebsiella pneumoniae exhibits heterogeneous resistance to multiple antibiotics. One study found that among 288 Klebsiella pneumoniae strains isolated from intensive care patients in different hospitals in European countries, 108 strains showed heterogeneous resistance to colistin. In a Korean hospital, 3 strains of colistin-sensitive Klebsiella pneumoniae were found to exhibit heterogeneous colistin resistance. Of the 60 colistin-sensitive Klebsiella pneumoniae isolates collected from Chilean hospitals, 8 were heterogeneously resistant. In six Chinese hospitals, the colistin heterogeneity rate of carbapenem-resistant Klebsiella pneumoniae was 6.2% (28 / 45 strains), while in US hospitals it was 8.4% (24 / 286 strains), showing similar probabilities. Of the 96 carbapenem-resistant Klebsiella pneumoniae isolates collected from Beijing hospitals, 69 (71.9%) exhibited a heterogeneous colistin resistance phenotype. Summary of the Invention
[0005] To overcome the aforementioned problems in the prior art, this invention provides the application of ethanolamine in the preparation of drugs that inhibit heterogeneous resistance to colistin in Klebsiella pneumoniae. Addressing the key issue of "how to effectively inhibit heterogeneous resistance to colistin in Klebsiella pneumoniae, thereby improving the therapeutic effect of colistin," this invention provides the relevant application of ethanolamine (CAS No.: 141-43-5). This invention provides a new strategy for inhibiting heterogeneous resistance to colistin in Klebsiella pneumoniae and a combined drug regimen of ethanolamine and colistin. Furthermore, the inhibitory effect has been verified in various heterogeneous resistant strains from different sources (KP_336, KP_416, KP_428, KP_604, D23).
[0006] To achieve the above objectives, the present invention employs the following technical solution:
[0007] This invention provides the application of ethanolamine (EA) in the preparation of drugs that inhibit heterogeneous resistance to colistin in Klebsiella pneumoniae. Ethanolamine is a small molecule compound widely present in biomembrane-related metabolic processes and can be produced by the degradation of membrane phospholipids.
[0008] Preferably, the drug is used in combination with colistin to enhance the antibacterial activity of colistin against heterogeneous drug-resistant strains.
[0009] Preferably, the effective concentration of the ethanolamine is 1.56 mM to 12.5 mM.
[0010] Preferably, the Klebsiella pneumoniae is one or more of the colistin-resistant strains KP_336, KP_416, KP_428, KP_604, or D23.
[0011] Preferably, the dosage form of the drug is an oral preparation, an injectable preparation, or a topical preparation.
[0012] Compared with the prior art, the advantages and beneficial effects of the present invention are:
[0013] This invention specifically inhibits the growth of heterogeneous colistin-resistant subgroups in Klebsiella pneumoniae by exogenously adding ethanolamine, without significantly affecting the overall MIC of sensitive strains or the original bacteria. This indicates that its action has phenotypic selectivity, which helps to precisely intervene in heterogeneous drug resistance without exacerbating overall drug resistance pressure.
[0014] In this invention, ethanolamine, at a low concentration range of 1.56 mM to 6.25 mM, can significantly reduce the MIC value of heterogeneous drug-resistant subclones (such as 428HR) against colistin (from 64 μg / mL to 4 μg / mL), and almost completely eliminates the resistant subgroup in in vitro bactericidal experiments. Combined drug susceptibility testing showed a FICI value of 0.2812, indicating a good synergistic effect between ethanolamine and colistin.
[0015] The present invention has also verified the inhibitory effect of ethanolamine in various heterogeneous colistin-resistant strains of Klebsiella pneumoniae (KP_336, KP_416, KP_428, KP_604, D23) from different sources, indicating that its effect is not limited to a specific strain and has broad applicability and universality.
[0016] This invention can be prepared as an adjuvant drug to reduce the risk of heterogeneous resistance to colistin. When used in combination with colistin, it is expected to improve the therapeutic effect of colistin on heterogeneous drug-resistant strains, reduce the risk of treatment failure and recurrent infection, and provide a new strategy and tool for clinical management of multidrug-resistant Klebsiella pneumoniae infection. Attached Figure Description
[0017] Figure 1 This is a line graph for bacterial community typology analysis. Note: HR: Heteroresistant, R: Resistant, S: Susceptible.
[0018] Figure 2 This is a heatmap of the checkerboard method synergistic experiment between EA and colistin.
[0019] Figure 3 The bacterial survival rate after adding EA at the sub-inhibitory concentration of colistin.
[0020] Figure 4 The graph shows the inhibitory effect of EA on the colistin PAP of KP_428.
[0021] Figure 5 This is a bar chart showing the differences in bacterial concentration during the PAP test.
[0022] Figure 6 Figure 1 shows the inhibitory effect of colistin PAP on each strain under 3 mM EA treatment. Note: Figure A is the PAP test of KP_336, Figure B is the PAP test of KP_416, Figure C is the PAP test of KP_604, and Figure D is the PAP test of D23. Detailed Implementation
[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0024] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.
[0025] Special notes on materials: Colistin-resistant strains KP_336, KP_428, KP_604, and D23.
[0026] The heterogeneous drug-resistant subclone 428HR is a single colony of KP_428 grown on an 8-fold colistin MIC plate.
[0027] The stock concentration of colistin solution is 5120 μg / mL.
[0028] The purity of ethanolamine is ACS, ≥99%.
[0029] Example 1 Drug sensitivity test
[0030] The MIC of colistin in the test sample strains was determined using the microbroth dilution method:
[0031] (1) Using an inoculation loop, pick a single colony from an LB plate and inoculate it into 4 mL of MH broth medium. Incubate at 37 ℃ and 180 rpm for 4 h. After reaching the logarithmic growth phase, adjust the bacterial culture to approximately 1×10⁻⁶. 6 CFU / mL.
[0032] (2) In a 96-well plate, add 180 μL of MH broth to the first column and 100 μL of MH broth to the remaining columns. Add 20 μL of 5120 μg / mL colistin solution to the first column, mix well by pipetting, and then transfer 100 μL of the mixture to the second column. Continuous serial dilutions are performed until the last column, and the 100 μL mixture in the last column is discarded. Add 100 μL of diluted bacterial culture to each well. Perform three biological replicates.
[0033] (3) At the same time, the positive control well was set with 100 μL of MH broth and 100 μL of bacterial solution, and the negative control well was set with 200 μL of MH broth.
[0034] (4) The final inoculum size is approximately 5 × 10⁻⁶. 5CFU / mL, with final colistin concentration gradients of 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL. The 96-well plates were incubated at 37 ℃ for 16 h.
[0035] (5) After the culture is completed, the bacterial growth status is judged by observing whether the broth in each well becomes cloudy or whether cell deposits form at the bottom. The bacterial MIC value is read.
[0036] Example 2: Microbial Spectrum Analysis (PAP) Experiment
[0037] The bacterial community profile analysis (PAP) experiment was performed on the strains to assess their survival distribution at different antimicrobial drug concentrations and to detect heterogeneous drug-resistant subgroups.
[0038] (1) Use a sterile inoculation loop to pick a single colony on an LB agar plate, inoculate it into MH broth, and culture it to the logarithmic growth phase. The bacterial suspension is serially diluted 10 times to prepare a series of bacterial suspensions with different inoculation amounts.
[0039] (2) Prepare colistin MH agar plates with different concentration gradients using the double agar dilution method. First, add colistin solutions of different concentrations to the petri dishes. After the autoclaved MH agar medium is cooled to below 50 °C at room temperature, use an electric pipette to add the medium to the petri dishes. Shake the petri dishes to mix the drug with the medium thoroughly and let them stand to solidify.
[0040] (3) Multiple concentration points were set to cover the range from complete sensitivity to high-level resistance. In this experiment, MH agar plates with two-fold gradient concentrations of colistin were prepared with the following specific concentrations: 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL.
[0041] (4) Take the bacterial suspensions of each dilution and spread them on the surface of plates of different drug concentrations using a spreader. Set up 3 parallel replicates for each concentration and dilution. Incubate the plates upside down at 37 ℃ for 18-24 h until visible single colonies are formed.
[0042] (5) Count the visible colony-forming units (CFU) on each plate and convert them to the number of surviving bacteria at the corresponding initial inoculum amount and drug concentration, expressed as log. 10 Expressed as CFU / mL. Plot the drug concentration on the x-axis and logarithmically... 10A microbial community profile analysis curve was plotted with CFU / mL as the ordinate to compare the survival characteristics of different strains or the same strain under different conditions.
[0043] (6) Criteria for identifying heterogeneous drug-resistant strains include assessing the ratio of antibiotic concentrations exhibiting higher inhibitory activity to the highest non-inhibitory concentrations, i.e., the frequency of resistant subpopulations, representing the percentage of resistant cells in the total cell count. If a resistant subpopulation exists, its MIC is at least 8 times that of the main population, and the number of colonies growing at 2×MIC or 4×MIC is at least 0.0001% (1 / 10) of the number of colonies growing on antibiotic-free plates. 6 If the strain is classified as heterogeneous resistance, then the strain is classified as heterogeneous resistance.
[0044] Example 3 Combined drug susceptibility test
[0045] The synergistic effect of EA and colistin was evaluated using a 96-well plate with a checkerboard method. The specific steps are as follows:
[0046] A single colony was inoculated into 4 mL of MH broth and incubated at 37 °C and 180 rpm for 4 h. The bacterial culture was then diluted with MH broth to a concentration of 1 × 10⁻⁶. 6 CFU / mL available for use.
[0047] In 96-well plates, drug or compound concentration gradients were set according to the MIC. Colistin was serially diluted 2-fold longitudinally, and EA was serially diluted 2-fold transversely. The maximum starting concentration was set at 2×MIC or 4×MIC. Drug-only groups, drug-free growth control groups, and sterile culture blank control groups were set up. Incubation was performed at 37 °C for 16 h.
[0048] After the culture was completed, the OD was measured using a microplate reader. 600 Value. The lowest combination of drug concentrations that completely inhibits bacterial growth is used as the combined MIC. The fractional inhibitory concentration index (FICI) is calculated.
[0049] Example 4 In vitro sterilization experiment
[0050] (1) Inoculate a single colony into 4 mL of LB broth and incubate at 37 °C and 180 rpm for 4 h. Centrifuge at 5000 rpm for 5 min, collect the bacterial cells, wash twice with M9 minimum medium, and finally adjust the bacterial concentration to approximately 1 × 10⁻⁶ with M9 medium. 6 CFU / mL available for use.
[0051] (2) Add the test substance to the bacterial suspension, and use a bacterial solution with an equal volume of solvent as a control group. Place each mixture at 37 ℃ for 6 h.
[0052] (3) After incubation, samples from each group were serially diluted. The diluted solution was spread onto LB plates. The plates were placed in a 37 ℃ constant temperature incubator for 12 h, and the CFU of each group was counted.
[0053] (4) Bacterial survival rate was calculated based on the CFU ratio of the treatment group to the control group.
[0054] Example 5: Determination of the inhibitory effect of EA on heterogeneous drug resistance phenotypes in bacterial strains
[0055] The effect of adding a fixed concentration of EA to the PAP test was verified.
[0056] (1) Use a sterile inoculation loop to pick a single colony on an LB agar plate, inoculate it into MH broth, and culture it to the logarithmic growth phase. The bacterial suspension is serially diluted 10 times to prepare a series of bacterial suspensions with different inoculation amounts.
[0057] (2) Prepare MH agar plates with two-fold gradient concentrations of colistin, specifically 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL. Simultaneously prepare MH agar plates containing two-fold gradient concentrations of colistin to a final concentration of 3 mM EA. Set up a drug-free MH agar plate as a blank control.
[0058] (3) Spread 100 µL of each diluted bacterial suspension onto the surface of plates with different drug concentrations, and set up 3 parallel replicates for each concentration and dilution. Incubate the plates upside down at 37°C for 18-24 h.
[0059] (4) Count the visible colony-forming units (CFU) on each plate and convert them to the number of surviving bacteria at the corresponding initial inoculum amount and drug concentration, expressed as log. 10 Expressed as CFU / mL. Plot the drug concentration on the x-axis and logarithmically... 10 A microbial community profile analysis curve was plotted with the number of surviving bacteria as the ordinate.
[0060] (5) Calculate the effect of adding EA on the frequency of colistin heterogeneous resistance phenotypes based on the number of colonies.
[0061] Experimental results:
[0062] 1. Results of drug sensitivity test
[0063] The MIC values for KP_336, KP_428, KP_604, and D23 were determined to be 1 μg / mL using the microdilution broth method, while the MIC value for the heterogeneous drug-resistant subclone 428HR of KP_428 was 64 μg / mL.
[0064] Table 1. MIC values of colistin
[0065] 2 PAP Results
[0066] According to the PAP line graph, susceptible, resistant, and heterogeneous resistant strains exhibited different survival distribution characteristics. Susceptible strains could grow normally in antibiotic environments below the MIC concentration, but the colony count decreased exponentially with increasing drug concentration, and no visible growth was observed at 2×MIC (2 μg / mL), exhibiting a typical susceptibility curve. Resistant strains maintained high colony counts at drug concentrations ≤16 μg / mL, showing a persistent resistance phenotype, but no colonies were detected at 32 μg / mL. In contrast, heterogeneous resistant strains generally behaved similarly to susceptible strains below the MIC concentration, while the colony count decreased significantly with increasing concentration. However, a small number of surviving colonies could still be observed on high-concentration antibiotic plates, suggesting the existence of a small subgroup within the bacterial community capable of tolerating high concentrations of antibiotics.
[0067] 3. Results of combined drug susceptibility testing
[0068] Combined antimicrobial susceptibility testing showed that strain 428HR exhibited high resistance to colistin (MIC = 64 μg / mL) in the absence of exogenous EA. However, with increasing exogenous EA concentration, the sensitivity of 428HR to colistin gradually increased. EA concentrations of 1.56 mM and 3.12 mM reduced the colistin MIC to 16 μg / mL, and at an EA concentration of 6.25 mM, the colistin MIC decreased to 4 μg / mL. Figure 2 B). This indicates that low concentrations of EA can inhibit drug resistance in heterogeneous drug-resistant subclones. However, in the original strain KP_428, EA at concentration gradients of 1.56 mM to 12.5 mM did not show significant inhibitory effects on its colistin MIC, only showing a slight decrease in OD600 value, but without reducing the colistin MIC value. Figure 2 A). Based on the FICI calculation of EA and colistin in three biological replicates, the FICI was 0.2812 in 428HR and 0.75 in KP_428, indicating that EA may be coordinated with colistin.
[0069] 4. Results of in vitro sterilization
[0070] According to the checkerboard test, low concentrations of EA effectively inhibited the drug resistance of 428HR, but had no significant effect on the original strain KP_428. To further verify the bacterial survival results under drug stress, EA was added at sub-inhibitory concentrations of colistin, with final concentrations of 0 mM, 1.56 mM, 3.12 mM, and 6.25 mM. After 6 h of incubation, bacterial survival rates were calculated by plating. The results showed that the survival rate of 428HR decreased significantly at an EA concentration of 1.56 mM, and was almost completely killed at EA concentrations of 3.12 mM and 6.25 mM. Figure 3 A). The survival rate of KP_428 after adding EA was close to that before adding it, with no significant change. Figure 3 B). This indicates that exogenous EA restored the sensitivity of 428HR to colistin in a dose-dependent manner, while no significant effect was observed in KP_428.
[0071] Results of 5 EA assay for inhibiting heterogeneous drug resistance phenotypes in strains
[0072] The combined drug susceptibility test results showed that 1.5–3 mM EA had an inhibitory effect on the drug-resistant subclone 428HR, but had no effect on the MIC results of the original KP_428 strain, indicating that EA may have an inhibitory effect on heterogeneous drug resistance phenotypes. Therefore, a combined action was conducted using low concentrations of EA in the colistin PAP test of KP_428 to explore the inhibitory effect of EA on heterogeneous drug resistance phenotypes in the original strain. The results showed that compared with the PAP results without EA, the addition of 1.5 mM EA reduced the frequency of drug-resistant subgroups at a colistin concentration of 16 μg / mL, and completely inhibited them at a colistin concentration of 32 μg / mL. The addition of 3 mM EA reduced the frequency of drug-resistant subgroups at colistin concentrations of 4 μg / mL and 8 μg / mL, and completely inhibited them from 16 μg / mL. Figure 4 ).
[0073] The differences in bacterial counts at PAP test concentrations of colistin at 4×MIC, 8×MIC, and 16×MIC were compared. The results showed that 1.5 mM EA had an inhibitory effect on the resistant subgroup at 16×MIC, reducing bacterial concentration by 80%. However, 3 mMEA significantly reduced the survival frequency of the resistant subgroup at 4×MIC and 8×MIC, with bacterial concentrations decreasing by 78% and 88%, respectively. Figure 5 ).
[0074] The colistin PAP test of heterogeneous drug-resistant strains KP_336, KP_428, KP_604, and D23 was also tested by adding exogenous EA to examine the generalizability of the inhibitory effect of EA on the PAP test of heterogeneous drug-resistant strains. The results showed that with the addition of 3 mM EA, the peak survival concentration of colistin for the drug-resistant subgroups of KP_336 and KP_604 decreased from 64 μg / mL to 16 μg / mL, the peak survival concentration of colistin for the drug-resistant subgroup of KP_416 decreased from 32 μg / mL to 16 μg / mL, and the peak survival concentration of colistin for the drug-resistant subgroup of D23 decreased from 32 μg / mL to 8 μg / mL. Figure 6 Low concentrations of EA have a general inhibitory effect on heterogeneous colistin-resistant subgroups of Klebsiella pneumoniae.
[0075] Obviously, the specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. Application of ethanolamine in the preparation of drugs that inhibit heterogeneous resistance to colistin in Klebsiella pneumoniae.
2. The application according to claim 1, characterized in that, The drug is used in combination with colistin to enhance the antibacterial activity of colistin against heterogeneous drug-resistant strains.
3. The application according to claim 1, characterized in that, The effective concentration of the ethanolamine is 1.56 mM to 12.5 mM.
4. The application according to claim 1, characterized in that, The Klebsiella pneumoniae strain is one or more of the colistin-resistant strains KP_336, KP_416, KP_428, KP_604, or D23.
5. The application according to claim 1, characterized in that, The dosage form of the drug is an oral preparation, an injectable preparation, or a topical preparation.