An amikacin composition effective to inhibit pseudomonas aeruginosa biofilm bacteria

Abstract

The application discloses an N-(3-cyclobutyrolactone)-4-nitrophenyl butyryl amide and amikacin composition and application of the composition in treating chronic infection of drug-resistant bacteria. After the N-(3-cyclobutyrolactone)-4-nitrophenyl butyryl amide and the amikacin are combined, not only the sensitivity of pseudomonas aeruginosa to the amikacin can be obviously increased, but also the formation amount of the pseudomonas aeruginosa biofilm can be greatly reduced, so that the composition can be applied to preventing and treating chronic infection of drug-resistant pseudomonas aeruginosa.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a composition of N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide and amikacin. Background Technology

[0002] Pseudomonas aeruginosa (PA) is a common opportunistic pathogen in clinical practice. When the body's immune function is weakened, tissue barriers are compromised, or invasive medical procedures are performed, it can cause localized purulent inflammation of tissues and systemic infections, such as respiratory infections, pneumonia, and sepsis. Furthermore, due to PA's adaptability and strong drug resistance, as well as the effect of biofilms, common antibacterial drugs are usually ineffective against it.

[0003] Biofilms are structured, membrane-like bacterial communities formed by bacteria and their secreted extracellular polysaccharides and other substances. The biofilm structure provides protection, preventing antibiotics from entering bacterial cells. Furthermore, biofilm formation causes basal ganglia (PAs) to transition to a metabolically less active state, significantly reducing their sensitivity to antibiotics, and even leading to antibiotic insensitivity. More seriously, the presence of biofilms can lead to chronic and recurrent infections, making complete cure difficult. Clinical data shows that in cystic fibrosis, PA biofilms hinder the efficacy of antibiotics and cause recurrent infections. Additionally, chronic wound infections, chronic sinusitis, and chronic otitis media are also associated with PA biofilm formation. We envisioned combining biofilm inhibitors with antibiotics to address bacterial resistance caused by biofilms. This invention unexpectedly discovered that the compound N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide, when used in combination with amikacin, synergistically reduces biofilm formation and increases the sensitivity of biofilm-bearing bacteria to amikacin, exhibiting the most significant inhibitory effect compared to combinations with other antibiotics. This composition is effective in treating diseases caused by Pseudomonas aeruginosa biofilm infection, providing a new approach for the clinical treatment of PA infection. Summary of the Invention

[0004] The present invention aims to provide a composition that enhances the antibiotic sensitivity of biofilm bacteria and inhibits bacterial biofilm formation, specifically comprising the following:

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

[0006]

[0007] Formula II.

[0008] Preferably, the bacteria is Pseudomonas aeruginosa.

[0009] Preferably, the mass ratio of amikacin to N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide is 1:0.1 to 1:8.70.

[0010] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

[0011] Secondly, the present invention provides the application of N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide in the preparation of a drug that enhances the antibiotic sensitivity of biofilm bacteria, wherein the antibiotic is amikacin.

[0012] Preferably, the biofilm bacteria are Pseudomonas aeruginosa.

[0013] Preferably, the Pseudomonas aeruginosa is a drug-resistant Pseudomonas aeruginosa.

[0014] Preferably, the use of any of the compositions described in the first aspect in the preparation of a medicament for treating chronic infectious diseases caused by Pseudomonas aeruginosa.

[0015] Preferably, the chronic infectious diseases caused by Pseudomonas aeruginosa include chronic wound infection, chronic sinusitis, and chronic otitis media.

[0016] The beneficial effects of this invention are:

[0017] When N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide is used in combination with amikacin, it can not only significantly increase the sensitivity of Pseudomonas aeruginosa to amikacin, but also greatly reduce the amount of Pseudomonas aeruginosa biofilm formation. It can be used to prevent and treat chronic infections of drug-resistant Pseudomonas aeruginosa. Attached Figure Description

[0018] Figure 1 The effects of combined use 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) Gentamicin combined with compound L2; (E) Ceftazidime combined with compound L2;

[0019] Figure 2The effect of combined use of antibiotics and compound L2 on the amount of biofilm formed in PAO1. (A) Carbenicillin combined with compound L2; (B) Amikacin combined with compound L2; (C) Trimethoprim combined with compound L2; (D) Gentamicin combined with compound L2; (E) Ceftazidime combined with compound L2;

[0020] Figure 3 To observe the changes in PA biofilm after the combined use of amikacin and L2 in CLSM. Detailed Implementation

[0021] The following specific embodiments are provided to implement the technical solutions described in this invention, but are not limited to these embodiments.

[0022] Example 1: Determination of MIC of different antibiotics against PAO1 planktonic bacteria and biofilm bacteria

[0023] Single colonies of PAO1 were picked and incubated in LB broth at 37°C for 15 hours at 200 rpm. After incubation, the bacterial culture was diluted to 5 × 10⁻⁶ with fresh, sterile LB broth. 5 CFU / mL. Amikacin was then serially diluted 2-fold to the corresponding concentration. Next, the serially diluted amikacin was added to a 5×10⁻⁶ solution. 5 Amikacin was prepared at CFU / mL bacterial suspensions to achieve final concentrations ranging from 0.16 μg / mL to 5 μg / mL. Finally, bacterial suspensions containing different concentrations of amikacin were inoculated at 150 μL / well into 96-well plates, with six replicates per concentration. Controls included a no-antibiotic group and a blank culture group. The 96-well plates were incubated at 37°C for 18–20 h. The experiment was considered valid if bacteria in the no-antibiotic group grew normally. The MIC value refers to the lowest drug concentration that inhibits the growth of PA planktonic bacteria in the culture medium after 18–20 h of incubation. The detection methods for the MICs of antibiotics such as gentamicin, ceftazidime, ciprofloxacin, carbenicillin, and trimethoprim are the same as for amikacin.

[0024] For the determination of MIC of biofilm bacteria, PA cultured in LB medium containing 1% glucose should be added to a 96-well plate beforehand. After culturing for 24 hours to form a biofilm, the planktonic bacteria should be removed, and the plate should be washed twice with PBS to remove the planktonic bacteria before the determination of MIC of planktonic bacteria is performed.

[0025] 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, with the MIC for biofilm bacteria being 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, with the MIC for biofilm bacteria being 32 times that of planktonic bacteria. Gentamicin, carbenicillin, and trimethoprim also showed significant differences in their MICs against PAO1 biofilm bacteria and planktonic bacteria, with the MICs for biofilm bacteria being 2, 2, and 8 times that of planktonic bacteria, respectively. Ciprofloxacin, due to its lack of a positive charge, had a good ability to penetrate bacterial biofilms. Therefore, there was no significant difference in the MICs of ciprofloxacin against PAO1 biofilm bacteria and planktonic bacteria. These data indicate that after PA biofilm formation, the MICs of most antibiotics against biofilm bacteria significantly increase, and the sensitivity of biofilm bacteria to antibiotics decreases. This suggests that bacterial biofilm formation is one of the causes of bacterial resistance.

[0026] Table 1. Determination of MIC of different antibiotics against PAO1 planktonic bacteria and biofilm bacteria.

[0027] antibiotic Pseudomonas aeruginosa PAO1 airborne bacteria (μg / mL) Pseudomonas aeruginosa PAO1 biofilm bacteria (μg / mL) Amika Star 2.5 5 Ceftazidime 1.25 40 Gentamicin 1.25 2.5 Ciprofloxacin 0.16 0.16 Carbenicillin 2.5 5 Trimethoprim 1.25 10

[0028] Case Study 2: Inhibitory Effect of Combined Antibiotic and Compound L2 on PAO1 Biofilm Bacteria

[0029] First, PAO1 was allowed to form a mature biofilm, and then gently rinsed twice with 1×PBS to remove floating bacteria. Next, different concentrations of L2 were mixed with amikacin in LB medium to achieve final L2 concentrations of 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, and a final amikacin concentration of 4 μg / mL. 150 μL / well was transferred to 96-well plates, with 6 replicates per group. Treatments containing only L2 and only amikacin served as controls. The 96-well plates were incubated at 37℃ for 18–20 h. After incubation, the OD of the bacterial culture was measured using a microplate reader. 600 The value was used to determine the effect of L2 and the combined use of antibiotics on the growth of biofilm bacteria.

[0030] The effects of combined use of antibiotics such as gentamicin, ceftazidime, carbenicillin, and trimethoprim with compound L2 on the growth of biofilm bacteria were determined using the same method as with amikacin. The final concentrations were set at 5 μg / mL, 10 μg / mL, 2.5 μg / mL, and 4 μg / mL, respectively.

[0031] Next, discard the bacterial culture and gently rinse twice with 1×PBS to remove any floating bacteria. After the final wash, completely discard any remaining liquid in the wells and incubate the plates at 37 °C until completely dry. Add 150 μL of 0.5% crystal violet solution to each well and stain for 15 min at room temperature. After staining, aspirate the crystal violet staining solution from the wells, then gently wash three times with 280 μL of sterile water. After drying, add 150 μL of 33% acetic acid solution to each well to dissolve the bacterial biofilm for 30 min. Detect the OD of the lysate using a microplate reader. 570 The value was used to determine the inhibitory activity of the combined drug combination on PA biofilm formation.

[0032] In this experiment, antibiotics were used in combination with different concentrations of compound L2 at a certain concentration. Figure 1 , Figure 2 It can be seen that, at a fixed antibiotic concentration, the addition of different concentrations of compound L2 enhanced the inhibitory effect of the antibiotic on the growth of PA biofilm bacteria to varying degrees. Under a constant antibiotic concentration, the enhancing effect of compound L2 on the antibiotic showed a concentration-dependent effect in the concentration ranges of 25-200 μM and 1.56-12.5 μM, with the most significant enhancement of the antibiotic's inhibitory effect on biofilm bacterial growth occurring at a concentration of 25 μM. Figure 1 It can be seen that at a compound L2 concentration of 25 μM, the growth inhibition rates of ceftazidime, gentamicin, amikacin, carbenicillin, and trimethoprim against biofilm bacteria were 26%, 63%, 72%, 30%, and 24%, respectively. Figure 2 The results showed that at a compound L2 concentration of 25 μM, the inhibition rates of ceftazidime, gentamicin, amikacin, carbenicillin, and trimethoprim against biofilm formation in PA bacteria were 48%, 68%, 60%, 63%, and 66%, respectively. These results indicate that compound L2 can enhance the inhibitory effect of antibiotics on the growth and biofilm formation of PA biofilm bacteria. In conclusion, 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.

[0033] Implementation Case 3: CLSM observation of changes in PA biofilm after combined use of amikacin and L2.

[0034] Compounds L2 and No. 10 were added to the diluted PAO1 bacterial suspension to a final concentration of 25 μM, and amikacin was added to a final concentration of 4 μg / mL. No. 10 served as a positive control, and the same volume of DMSO was added as a negative control. 1 mL of the treated bacterial suspension was added to each well, with one slide placed in each well, and three replicates per group. The 6-well plate was placed in a constant temperature incubator and incubated at 37°C for 24 h. Afterward, the plate was gently rinsed three times with PBS to remove free bacteria. The slides were fixed with 2.5% glutaraldehyde solution at 4°C for 4 h, then gently rinsed three times with PBS buffer for 10 min each time, and the remaining liquid was blotted dry with absorbent paper. 20 μL of 1 mg / mL FITC-ConA solution was evenly added to the slide, and the plate was stained at room temperature for 20 min. After staining, discard any excess dye, gently rinse three times with PBS buffer to remove any remaining dye, and blot the slide dry with absorbent paper. Then, add 20 μL of 50 μg / mL PI solution evenly to the slide again, and incubate at room temperature for 15 min. After staining, discard any excess dye, gently rinse three times with PBS buffer to remove any remaining dye, and blot the slide dry with absorbent paper. Finally, observe the changes in biomembrane structure under a laser confocal microscope.

[0035] like Figure 3 As shown, the biofilm thickness in the control group was 14 μm. After amikacin treatment, the biofilm thickness decreased to 10 μm, and only weak red fluorescence was observed in the figure. Amikacin can reduce biofilm formation to some extent, but it has almost no killing effect on biofilm bacteria. After L2 treatment, the biofilm thickness decreased to 8 μm, and no strong red fluorescence appeared, indicating that L2 can reduce biofilm formation to some extent, but it has no killing effect on biofilm bacteria. However, when L2 and amikacin were used in combination, the biofilm thickness decreased to 8 μm, and the red fluorescence was also significantly enhanced, indicating that the combined use enhanced the killing effect on bacteria. These 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 on biofilm formation.

Claims

1. A pharmaceutical composition for treating drug-resistant bacterial infections, the active ingredients of the pharmaceutical composition comprising amikacin and N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide, wherein the bacteria are Pseudomonas aeruginosa (…). Pseudomonas aeruginosa The concentration of amikacin is 4 μg / mL, the concentration of N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide is 25 μM, and the structural formula of N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide is shown in Formula II. Formula II.

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

3. The use of the pharmaceutical composition according to any one of claims 1-2 in the preparation of a medicament for treating chronic infectious diseases caused by Pseudomonas aeruginosa.

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