Amikacin composition capable of effectively inhibiting pseudomonas aeruginosa biofilm bacteria

Abstract

The invention discloses a composition of N-(3-cyclobutyrolactone)-4-nitrobenzene butyramide and amikacin, and an application of the composition in treatment of chronic infection of drug-resistant bacteria. After the N-(3-cyclobutyrolactone)-4-nitrobenzenebutyramide and amikacin are combined for use, not only can the sensitivity of the pseudomonas aeruginosa to amikacin be remarkably improved, but also the formation amount of a pseudomonas aeruginosa biological membrane is greatly reduced, and the N-(3-cyclobutyrolactone)-4-nitrobenzenebutyramide can be applied to prevention and treatment of chronic infection of drug-resistant pseudomonas aeruginosa.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a composition of N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide and amikacin. Background Art

[0002] Pseudomonas aeruginosa (PA) is a common clinical conditional pathogen. When the human immune function is low, the tissue barrier is damaged, or invasive medical operations are performed, it can cause local suppurative inflammation in human tissues and systemic infections, such as respiratory tract infections, pneumonia, sepsis, etc. In addition, due to the strong adaptability and drug resistance of PA, as well as the role of biofilm, common antimicrobial drugs usually cannot effectively act on it.

[0003] Biofilm is a structured film-like bacterial group formed by bacteria and their secreted extracellular polysaccharides and other substances. The biofilm structure plays a protective role and prevents antibiotics from entering bacterial cells. Moreover, the formation of biofilm will prompt PA to convert into a state with low metabolic activity. In this state, their sensitivity to antibiotics will be significantly reduced, and even the phenomenon of insensitivity to antibiotics will occur. What is more serious is that the presence of biofilm can also lead to chronic infection and repeated infection, making it difficult to completely cure the infection. Clinical data show that in pulmonary cystic fibrosis, the biofilm formed by PA hinders the efficacy of antibiotics and leads to repeated infection. In addition, chronic wound infection, chronic sinusitis, chronic otitis media, etc. are also related to the formation of PA biofilm. We envision the use of biofilm inhibitors in combination with antibiotics to solve the bacterial resistance caused by biofilm. The present invention unexpectedly found that the compound N-(3-cyclobutyrolactone)-4-nitrophenylbutyric acid amide can achieve the effect of synergistically reducing the amount of biofilm formed and increasing the sensitivity of biofilm bacteria to amikacin after being used in combination with amikacin. Compared with the combination with other antibiotics, the inhibitory effect is the most significant. The composition has the effect of treating diseases caused by Pseudomonas aeruginosa biofilm bacterial infection, and the result provides a new method for the clinical treatment of PA infection. Summary of the invention

[0004] The purpose of the present invention is to provide a composition for enhancing the sensitivity of biofilm bacteria to antibiotics and inhibiting the formation of bacterial biofilm, which specifically comprises the following contents:

[0005] In a first aspect, the present invention provides a composition for enhancing the sensitivity of biofilm bacteria to antibiotics and inhibiting the formation of bacterial biofilms, wherein the active ingredients of the composition include amikacin and N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide (Formula II), and the structural formula is:

[0006]

[0007] Preferably, the bacterium is Pseudomonas aeruginosa.

[0008] Preferably, the mass ratio of amikacin to N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide is 1:0.1 to 1:8.70.

[0009] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0010] In a second aspect, the present invention provides a use of N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide in the preparation of a drug for enhancing the sensitivity of biofilm bacteria to antibiotics, wherein the antibiotic is amikacin.

[0011] Preferably, the biofilm bacteria is Pseudomonas aeruginosa.

[0012] Preferably, the Pseudomonas aeruginosa is drug-resistant Pseudomonas aeruginosa.

[0013] Preferably, any composition described in the first aspect is used in the preparation of a drug for treating chronic infectious diseases caused by Pseudomonas aeruginosa.

[0014] Preferably, the chronic infectious diseases caused by Pseudomonas aeruginosa include chronic wound infection, chronic sinusitis, and chronic otitis media.

[0015] Beneficial effects of the present invention:

[0016] The combined use of N-(3-cyclobutyrolactone)-4-nitrophenylbutyric acid amide and amikacin can not only significantly increase the sensitivity of Pseudomonas aeruginosa to amikacin, but also significantly reduce the amount of biofilm formed by Pseudomonas aeruginosa, and can be used to prevent and treat chronic infections of drug-resistant Pseudomonas aeruginosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of the combination of antibiotics and compound L2 on the growth of PAO1 biofilm bacteria. (A) Carbenicillin combined with compound L2; (B) Amikacin combined with compound L2; (C) Trimethoprim combined with compound L2; (D) Gentamycin combined with compound L2; (E) Ceftazidime combined with compound L2;

[0018] Figure 2 The effect of the combination of antibiotics and compound L2 on the amount of biofilm formed by PAO1. (A) Carbenicillin combined with compound L2; (B) Amikacin combined with compound L2; (C) Trimethoprim combined with compound L2; (D) Gentamycin combined with compound L2; (E) Ceftazidime combined with compound L2;

[0019] Figure 3 CLSM was used to observe the changes in PA biofilm after the combination of amikacin and L2. DETAILED DESCRIPTION

[0020] The following specific embodiments are provided to implement the technical solutions described in the present invention, but are not limited to these embodiments.

[0021] Example 1 Determination of MIC of different antibiotics against PAO1 planktonic bacteria and biofilm bacteria

[0022] Pick a single PAO1 colony in LB liquid culture medium and culture it at 200 rpm and 37°C for 15 h. After the culture is completed, dilute the bacterial solution to 5×10 5 CFU / mL. Amikacin was diluted to the corresponding concentration by 2-fold serial dilution. Next, the serially diluted amikacin was added to a 5×10 5 CFU / mL of bacterial solution, the final concentration of amikacin was 0.16μg / mL~5μg / mL. Finally, the bacterial solution containing different concentrations of amikacin was inoculated in a 96-well plate at 150μL / well, with 6 replicates for each concentration and the group without antibiotics and the blank culture medium group as controls. The 96-well plate was cultured in a 37℃ constant temperature incubator for 18 to 20 hours. The experiment is valid if the bacteria in the group without antibiotics grow normally. The MIC value refers to the minimum drug concentration that can inhibit the growth of PA planktonic bacteria in the culture medium after 18 to 20 hours of culture. The detection method of MIC of antibiotics such as gentamicin, ceftazidime, ciprofloxacin, carbenicillin and trimethoprim is consistent with that of amikacin.

[0023] When determining the MIC of biofilm bacteria, PA cultured in LB medium containing 1% glucose needs to be added to a 96-well plate in advance. After culturing for 24 hours to form a biofilm, the planktonic bacteria are removed, and the plate is washed twice with PBS to wash away the planktonic bacteria before the determination is performed according to the determination method of the planktonic bacteria MIC.

[0024] The results are shown in Table 1. The MICs of amikacin against PAO1 planktonic bacteria and biofilm bacteria were 2.5 μg / mL and 5 μg / mL, respectively, and the MIC of biofilm bacteria was twice that of planktonic bacteria. The MICs of ceftazidime against PAO1 planktonic bacteria and biofilm bacteria were 1.25 μg / mL and 40 μg / mL, respectively, and the MIC of biofilm bacteria was 32 times that of planktonic bacteria. The MICs of gentamicin, carbenicillin, and trimethoprim against PAO1 biofilm bacteria and planktonic bacteria were also significantly different, and the MICs of biofilm bacteria were 2, 2, and 8 times that of planktonic bacteria, respectively. Ciprofloxacin has a good ability to penetrate bacterial biofilms because it does not carry a positive charge. Therefore, there is no significant difference in the MICs of ciprofloxacin against PAO1 biofilm bacteria and planktonic bacteria. The above data show that after PA forms a biofilm, the MICs of most antibiotics against biofilm bacteria increase significantly, and the sensitivity of biofilm bacteria to antibiotics decreases. It can be seen that the formation of bacterial biofilms is one of the causes of bacterial resistance.

[0025] Table 1 Determination of MIC of different antibiotics against PAO1 planktonic bacteria and biofilm bacteria

[0026]

[0027] Example 2 Inhibitory effect of antibiotics combined with compound L2 on PAO1 biofilm bacteria

[0028] First, allow PAO1 to form a mature biofilm, and gently rinse twice with 1×PBS to remove floating bacteria. Then, different concentrations of L2 were mixed with amikacin in LB medium, so that the final concentrations of L2 were 1.56μM, 3.125μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM, and 200μM, and the final concentration of amikacin was 4μg / mL, and 150μL / well was transferred to a 96-well plate, with 6 replicates in each group, and the treatment groups containing only L2 and only amikacin were used as controls. The 96-well plate was statically cultured in a 37°C constant temperature incubator for 18 to 20 hours. After the culture, the OD of the bacterial solution was detected using an enzyme marker. 600 The value was used to determine the effect of L2 and antibiotics combined on the growth of biofilm bacteria.

[0029] The effect of gentamicin, ceftazidime, carbenicillin and trimethoprim combined with compound L2 on the growth of biofilm bacteria was determined in the same manner as amikacin. The final concentrations were set at 5 μg / mL, 10 μg / mL, 2.5 μg / mL and 4 μg / mL, respectively.

[0030] Next, discard the above bacterial solution and gently rinse twice with 1×PBS to remove floating bacteria. After the last wash, completely discard the remaining liquid in the well and place the plate in a 37°C constant temperature incubator until the plate is completely dry. Then add 150μL of 0.5% crystal violet solution to each well and stain at room temperature for 15 minutes. After staining, aspirate the crystal violet staining solution in the well, add 280μL of sterile water and gently wash 3 times. After it is dry, add 150μL of 33% acetic acid solution to each well to dissolve the bacterial biofilm for 30 minutes, and use an enzyme reader to detect the OD of the dissolved solution. 570 The inhibitory activity of the drug combination on PA biofilm formation was determined.

[0031] In this experiment, antibiotics at a certain concentration were used in combination with compound L2 at different concentrations. Figure 1 , Figure 2 It can be seen that when the concentration of antibiotics is fixed, after adding different concentrations of compound L2, the antibiotics have different degrees of inhibition on the growth of PA biofilm bacteria. When the antibiotic concentration is constant, the enhancement effect of compound L2 on antibiotics in the concentration range of 25-200μM and 1.56-12.5μM is concentration-dependent, and the enhancement effect of compound L2 on the inhibition of biofilm bacterial growth is most obvious when the concentration of compound L2 is 25μM. Figure 1 It can be seen that when the concentration of compound L2 is 25 μM, the growth inhibition rates of ceftazidime, gentamicin, amikacin, carbenicillin, and trimethoprim on biofilm bacteria are 26%, 63%, 72%, 30%, and 24%, respectively. Figure 2 It can be seen that when the concentration of compound L2 is 25 μM, the inhibition rates of ceftazidime, gentamicin, amikacin, carbenicillin, and trimethoprim on biofilm formation of biofilm bacteria are 48%, 68%, 60%, 63%, and 66%, respectively. The above results show that compound L2 can enhance the inhibitory effect of antibiotics on the growth and biofilm formation of PA biofilm bacteria. In summary, compound L2 can significantly enhance the inhibitory effect of antibiotics on PA biofilm bacteria and can significantly improve the sensitivity of PA biofilm bacteria to antibiotics.

[0032] Implementation case 3: CLSM observation of the changes in PA biofilm after the combination of amikacin and L2.

[0033] Compounds L2 and No.10 were added to the diluted PAO1 bacterial solution to make the final concentration of 25μM, and the final concentration of amikacin was 4μg / mL, where No.10 was used as a positive control, and the same volume of DMSO treatment group was added as a negative control. Take 1mL of the above-treated bacterial solution and add it to the culture wells respectively, place 1 glass slide in each experimental well, and 3 replicates per group. Place the 6-well culture plate in a constant temperature incubator, culture at 37℃ for 24h, and then gently rinse three times with PBS to remove free bacteria. Fix the slide with 2.5% glutaraldehyde solution at 4℃ for 4h, then gently rinse three times with PBS buffer for 10min each time, and use absorbent paper to absorb the remaining liquid on the slide. Evenly add 20μL of 1mg / mL FITC-ConA solution on the slide and stain at room temperature for 20min. After staining, discard the excess dye, rinse gently with PBS buffer 3 times to remove the excess dye, and use absorbent paper to absorb the remaining liquid on the slide. Then, add 20μL of 50μg / mL PI solution evenly on the slide again, and leave the slide to stain at room temperature for 15min. After staining, discard the excess dye, rinse gently with PBS buffer 3 times to remove the excess dye, and use absorbent paper to absorb the remaining liquid on the slide. Finally, place the slide under a laser confocal microscope to observe the changes in the biofilm structure.

[0034] like Figure 3 As shown in the figure, the biofilm thickness of the control group was 14 μm. After treatment with amikacin, the biofilm thickness decreased to 10 μm, and only a weak red fluorescence was shown in the figure. Amikacin can reduce the formation of biofilm to a certain extent, but there is almost no killing effect on biofilm bacteria. After L2 treatment, the biofilm thickness decreased to 8 μm, and there was no strong red fluorescence, indicating that L2 can reduce the formation of biofilm to a certain extent, but there is no killing effect on biofilm bacteria. However, when L2 and amikacin were used in combination, the thickness of the biofilm was reduced to 8 μm, and the red fluorescence was significantly enhanced, indicating that the killing effect of the combination on bacteria was enhanced. The above results further illustrate that L2 increases the sensitivity of PAO1 biofilm bacteria to amikacin, and the combination of L2 and amikacin achieves a more significant inhibitory activity of biofilm formation.

Claims

1. A pharmaceutical composition for treating drug-resistant bacterial infection, wherein the active ingredients of the composition include amikacin and N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide (Formula II), and the structural formula is:

2. The pharmaceutical composition according to claim 1, characterized in that The bacteria is Pseudomonas aeruginosa.

3. The composition according to claim 1, characterized in that The mass ratio of amikacin to N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide is 1:0.1-8.

70.

4. The composition according to claim 1, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

5. Use of N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide in the preparation of a drug for enhancing the sensitivity of biofilm bacteria to antibiotics, wherein the antibiotic is amikacin.

6. The use according to claim 5, characterized in that The biofilm bacteria is Pseudomonas aeruginosa.

7. The use according to claim 6, characterized in that The Pseudomonas aeruginosa is drug-resistant Pseudomonas aeruginosa.

8. Use of the composition according to any one of claims 1 to 4 in the preparation of a drug for treating chronic infectious diseases caused by Pseudomonas aeruginosa.

9. The use according to claim 8, characterized in that The chronic infectious diseases caused by Pseudomonas aeruginosa include chronic wound infection, chronic sinusitis, and chronic otitis media.

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