Homoserine lactone compound for effectively inhibiting formation of multi-drug-resistant pseudomonas aeruginosa biofilm

Abstract

The invention discloses a homoserine lactone compound N-(3-cyclobutyrolactone)-4-nitrobenzene butyramide which can effectively inhibit the formation of a multi-drug-resistant pseudomonas aeruginosa biofilm, and an application of the N-(3-cyclobutyrolactone)-4-nitrobenzene butyramide in the treatment of chronic infection of drug-resistant bacteria. The N-(3-cyclobutyrolactone)-4-nitrobenzene butyramide not only can inhibit QS systems of pseudomonas aeruginosa standard strains and clinical strains and enhance the inhibitory activity of antibiotics to pseudomonas aeruginosa, but also is good in safety and can be used for treating chronic infection caused by pseudomonas aeruginosa.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of a homoserine lactone compound in the preparation of a drug that inhibits biofilm formation in Pseudomonas aeruginosa. Background Technology

[0002] *Pseudomonas aeruginosa* is a common and highly pathogenic bacterium. As an important nosocomial pathogen, *P. aeruginosa* can threaten patients with prolonged intubation and immunodeficiency, as well as those with cystic fibrosis, corneal trauma, burns, Gustilo open fractures, and can lead to fatal infections. The pathogenicity of *P. aeruginosa* is closely related to its biofilm formation on surfaces and the secretion of virulence factors. Recent studies have found that the quorum sensing system of *P. aeruginosa* plays an important role in its biofilm formation and virulence factor secretion. Biofilm formation is associated with 80% of bacterial infections in humans. Biofilms are surface-associated microbial communities. These sessile microbial communities are constructed by planktonic bacteria attaching to surfaces, subsequently interacting with the extracellular matrix to develop into ever-growing colonies with complex three-dimensional structures, thereby promoting chronic infections. Antibiotic resistance is partly mediated by surface-attached biofilms, which act as a physical barrier to antibiotic penetration. Therefore, inhibiting the development of quorum sensing is one of the effective methods for controlling Pseudomonas aeruginosa infection and antibiotic resistance. In this invention, a series of compounds were designed by structurally modifying existing quorum sensing inhibitors (QSIs). Among the modified compounds, compound L2 not only exhibits excellent inhibitory activity against multiple virulence factors, but also significantly enhances the inhibitory activity of antibiotics against Pseudomonas aeruginosa in vitro. Therefore, compound L2 can be used as an effective QSI for further development against bacterial infections. Summary of the Invention

[0003] The purpose of this invention is to provide the application of homoserine lactone compounds in the preparation of drugs that inhibit the formation of biofilms by Pseudomonas aeruginosa, specifically including the following:

[0004] This invention provides the application of homoserine lactone compounds in the preparation of drugs that inhibit biofilm formation in Pseudomonas aeruginosa.

[0005] Preferably, the general formula of the homoserine lactone compound (Formula I) is:

[0006]

[0007] Preferably, the homoserine lactone compound is N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide (L2), and the structural formula of N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide (L2) is:

[0008]

[0009] Furthermore, the application of the homoserine lactone compound (L2) in the preparation of drugs for treating chronic infectious diseases caused by Pseudomonas aeruginosa.

[0010] Preferably, the dosage form of the drug for chronic infectious diseases is selected from oral liquid, tablets, capsules, granules, ear drops, nasal drops, suppositories, pills, liniments, ointments, creams, patches, pastes, sprays, aerosols, or powder sprays.

[0011] Preferably, the chronic infectious diseases caused by Pseudomonas aeruginosa include chronic wound infection, chronic sinusitis, and chronic otitis media.

[0012] The beneficial effects of this invention are:

[0013] The N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide (L2) of the present invention can not only significantly inhibit the QS system of standard and clinical strains of Pseudomonas aeruginosa and significantly enhance the inhibitory activity of antibiotics against Pseudomonas aeruginosa, but also has good safety and can be used to treat chronic infections caused by Pseudomonas aeruginosa. Attached Figure Description

[0014] Figure 1 The inhibitory effects of different concentrations of compound L2 on the growth of Pseudomonas aeruginosa were investigated.

[0015] Figure 2 To evaluate the inhibitory effect of different concentrations of compound L2 on biofilm formation in Pseudomonas aeruginosa;

[0016] Figure 3 To observe the inhibitory effect of 25 μM compound L2 on biofilm formation in Pseudomonas aeruginosa using CLSM;

[0017] Figure 4 To observe the inhibitory effect of 25 μM compound L2 on biofilm formation of Pseudomonas aeruginosa using SEM, A, B, and C were observed at 20,000x field of view, while D, E, and F were observed at 2,000x field of view.

[0018] Figure 5 The inhibitory effect of L2 on the motility of Pseudomonas aeruginosa is shown. (A)(E) represent the swimming motility of P. aeruginosa PAO1, (B)(F) represent the swarming motility of P. aeruginosa PAO1, and (C)(G) represent the twitching motility of P. aeruginosa PAO1.

[0019] Figure 6 The effect of 25 μM compound L2 on the secretion of psoriatic toxin by Pseudomonas aeruginosa PAO1;

[0020] Figure 7 The effect of 25 μM compound L2 on the elastase activity of Pseudomonas aeruginosa PAO1;

[0021] Figure 8 To evaluate the effect of 25 μM compound L2 on the expression level of PAO1 QS-regulated genes in Pseudomonas aeruginosa;

[0022] Figure 9 The effect of 25 μM compound L2 on Pseudomonas aeruginosa PAO1 fluorescent reporter strains in different quorum sensing systems;

[0023] Figure 10 Evaluation of the toxicity of 25 μM compound L2 to nematodes;

[0024] Figure 11 The effect of different concentrations of compound L2 on hemolysis of mouse erythrocytes. Detailed Implementation

[0025] The following specific embodiments are provided to implement the technical solutions described in this invention, but are not limited to these embodiments.

[0026] Example 1: Synthesis and Characterization of Homoserine Lactone Analogs

[0027]

[0028] The raw materials (Formula III and Formula IV) were mixed in a 1:1 mass ratio and dissolved in dichloromethane (DCM). 2.5 eq N,N-diisopropylethylamine (DIEPA) and 1.0 eq 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were added to the system as condensing agents. The mixture was stirred at room temperature for 18-24 h. After the reaction, the reaction solution was washed three times in a separatory funnel with equal volumes of 10% (v / v) HCl solution and saturated NaCl solution, respectively. The organic phase solution was then collected, and anhydrous MgSO4 was added to remove residual water. The crude product was obtained by drying in a rotary evaporator at 50 °C. The crude product was subjected to SiO2 column chromatography and TLC monitoring. The product was eluted with dichloromethane:ethyl acetate = 10:1. The eluent was then subjected to rotary evaporation at 50°C and vacuum filtration with an oil pump to obtain the target products L1, L2, L6, L9, and L10. Their structural spectral characterization is shown in Formula VI above.

[0029] Example 2 Evaluation of the activity of the compound in inhibiting the formation of Pseudomonas aeruginosa biofilm

[0030] Single colonies of *Pseudomonas aeruginosa* PAO1 and clinical strains C1, C2, and C3 were picked and inoculated into LB medium and cultured at 180 rpm and 37°C until the logarithmic growth phase. Next, the bacterial suspension was diluted to 5 × 10⁻⁶ with fresh, sterile LB medium. 7 CFU / mL, then compounds L1, L2, L6, L9, and L10 dissolved in DMSO were added to the diluted PAO1 bacterial culture to create final concentration gradients of 6.25, 12.5, 25, 50, and 100 μM, serving as the experimental groups. An equal volume of DMSO was added to the negative control group. The positive control group consisted of N-(3-cyclobutyrolactone)-4-bromophenylbutyramide (NO. 10) at final concentrations of 6.25, 12.5, 25, 50, and 100 μM. Each group had 6 replicates, with 150 μL of bacterial culture per well. The 96-well plate was placed in a 37°C incubator and incubated statically for 24 h. After incubation, the bacterial culture was aspirated from the 96-well plate, and each well was gently washed twice with 280 μL of sterile water. After drying, 150 μL of 0.5% crystal violet solution was added to each well, and staining was performed at room temperature for 15 min. After staining, the crystal violet staining solution was aspirated from the wells, and each well was gently washed three times with 280 μL of sterile water. After drying, 150 μL of 33% acetic acid solution was added to each well to dissolve the biofilm for 30 min. The OD values ​​were then measured using a microplate reader. 570 Value. The evaluation test procedures for clinical strains C1, C2, and C3 were the same as above, and the experimental results are shown in the table below:

[0031] Table 1. Inhibitory activity of compounds against biofilm formation in standard and clinical strains of Pseudomonas aeruginosa.

[0032]

[0033] As shown in Table 1, all compounds L1, L2, L6, L9, and L10 exhibited inhibitory activity against biofilm formation in both standard and clinical strains of *Pseudomonas aeruginosa*. Among all analogues, compound L2 showed more significant inhibitory activity against both the standard strain and the three clinical strains of *Pseudomonas aeruginosa* compared to L1, L6, L9, L10, and NO.10, and was identified as a candidate compound for further experimental evaluation.

[0034] Example 3: Evaluation of the inhibitory effect of compound L2 on the growth of Pseudomonas aeruginosa

[0035] Pseudomonas aeruginosa PAO1 was cultured overnight and then diluted in LB medium. The L2 concentration in the diluted LB culture was adjusted to range from 10 to 400 μM, and the culture was incubated at 37°C. Then, 1 mL of sample was collected every 2 hours, and the OD was measured using a spectrophotometer. 600 Then, growth curves were generated. NO.10 was used as a control.

[0036] like Figure 1 As shown, similar to NO.10, compound L2 did not inhibit the growth of Pseudomonas aeruginosa PAO1 at concentrations ranging from 10 to 400 μM.

[0037] Example 4: Evaluation of the inhibitory effect of compound L2 on Pseudomonas aeruginosa biofilm formation

[0038] The inhibitory effect of compound L2 on biofilm formation was further identified at concentration gradients from 6.25 to 400 μM, with N-(3-cyclobutyrolactone)-4-bromophenylbutyramide (NO.10) serving as a positive control. Single colonies of *Pseudomonas aeruginosa* PAO1 and clinical strains C1, C2, and C3 were picked and inoculated into LB medium and cultured at 37°C and 180 rpm until the logarithmic growth phase. The bacterial culture was then diluted to 5 × 10⁻⁶ with fresh, sterile LB medium. 7 CFU / mL, and then compound L2 dissolved in DMSO was added to the diluted PAO1 bacterial culture to create final concentration gradients of 6.25, 12.5, 25, 50, and 100 μM as experimental groups. An equal volume of DMSO was added to the control group. Each group had 6 replicates, with 150 μL of bacterial culture per well. The 96-well plate was placed in a 37°C incubator for 24 h. After incubation, the bacterial culture was aspirated from the 96-well plate, and each well was gently washed twice with 280 μL of sterile water. After drying, 150 μL of 0.5% crystal violet solution was added to each well, and staining was performed at room temperature for 15 min. After staining, the crystal violet staining solution was aspirated from the wells, and the plates were gently washed three times with 280 μL of sterile water. After drying, 150 μL of 33% acetic acid solution was added to each well to dissolve the bacterial biofilm for 30 min. The OD values ​​were then measured using a microplate reader. 570 value.

[0039] like Figure 2 As shown, compound L2 exhibited a more significant inhibitory effect on biofilm formation in the concentration range of 6.25 to 25 μM, and this effect was dose-dependent. In contrast, NO.10 showed a more significant inhibitory effect on biofilm formation in the concentration range of 25 to 200 μM, with the highest inhibition rate of 50% at 200 μM. Notably, compound L2 achieved an inhibition rate of 56% on PA biofilm formation at a low concentration of 25 μM, demonstrating a more significant inhibitory effect.

[0040] Example 5: Observation of the inhibitory effect of compound L2 on biofilm formation of Pseudomonas aeruginosa using CLSM.

[0041] Compounds L2 and NO.10 were added to the diluted PAO1 bacterial suspension to a final concentration of 25 μM, with NO.10 serving as a positive control and an equal volume of DMSO as a negative control. 1 mL of the treated bacterial suspension was added to each well of a 6-well plate, with one slide placed in each well and three replicates per group. The 6-well plates were incubated at 37°C for 24 h, followed by gentle rinsing 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 staining was performed at room temperature for 20 min. After staining, excess dye was discarded, and the slides were gently rinsed three times with PBS buffer to remove any remaining dye, and the remaining liquid was blotted dry with absorbent paper. Next, add 20 μL of 50 μg / mL PI solution evenly to the slide again, and incubate the slide 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 the biomembrane structure under a laser confocal microscope.

[0042] Fluorescence microscopy observation results as follows Figure 3 As shown, the bacterial cell count in the L2 treatment group was significantly reduced compared to the control group, and the reduction was even more pronounced in the L2 treatment group compared to the NO.10 treatment group. The green fluorescence thickness in the control group was approximately 18 μm. After treatment with NO.10 and L2, the thickness of the green fluorescence in the biofilm decreased to 8 μm and 5 μm, respectively. This result further demonstrates that the candidate compound L2 has more significant biofilm inhibitory activity.

[0043] Example 6: SEM observation of the inhibitory effect of compound L2 on biofilm formation of Pseudomonas aeruginosa.

[0044] After the bacterial culture was treated and fixed according to the experimental method described above for observing changes in biofilm structure using CLSM, it was subjected to a gradient dehydration treatment with 30%, 50%, 70%, 85%, 95%, and 100% ethanol solutions, each for 10 minutes, with the 100% ethanol dehydration treatment performed twice. The biofilm samples were then air-dried in a desiccator for 24 hours, followed by gold sputtering. Images were then captured using a scanning electron microscope to observe the formation of the bacterial biofilm.

[0045] Depend on Figure 4It is evident that the control group bacteria formed a multi-layered, overlapping biofilm, resulting in a thicker biofilm with dense bacterial distribution. In contrast, the treated groups produced thinner, less uniform biofilms with a looser structure and less dense bacterial distribution. Furthermore, the biofilm structure in the compound L2 treatment group was even looser than that in the NO.10 treatment group. These results indicate that compound L2 can reduce bacterial biofilm formation and alter its structure.

[0046] Example 7: Evaluation of the inhibitory effect of L2 on the motility of Pseudomonas aeruginosa

[0047] 1. Swimming

[0048] Using sterile toothpicks, swimming agar plates containing 25 μM compound L2 (10 g / L peptone, 5 g / L NaCl, and 0.3% agarose) and blank control plates were punctured at the center with Pseudomonas aeruginosa PAO1. Compound NO.10 was used as a positive control. After incubation at 30°C for 12–14 h, the swimming motility zone was determined by measuring the diameter of the blue area at the center of the plate.

[0049] 2. Swarming

[0050] The swarming plates consisted of nutrient agar (8 g / L) and glucose (5.0 g / L). 25 μM of compounds L2 and NO.10 were added to the swarming plates, with a swarming plate without added compounds serving as a blank control and NO.10 as a positive control. Then, 5 μL of overnight cultured *Pseudomonas aeruginosa* was added to the plate. The swarming plates were then incubated at 30°C for 18–24 h, and the swarming diameter at the center of the plate was measured.

[0051] 3. Twitching

[0052] The culture medium used in this experiment was LB medium. 25 μM of compounds L2 and NO.10 were added to the LB medium. LB plates without the compounds were used as a blank control, and NO.10 was used as a positive control. Overnight cultured Pseudomonas aeruginosa PAO1 was added to the bottom of the stab plate and incubated at 37°C for 48 h. The agar was then removed, the plate was air-dried, and stained with crystal violet. The stain was washed off with sterile water, and the twitching area was measured.

[0053] like Figure 5As shown, the swimming, swarming, and twitching ranges of P. aeruginosa PAO1 in the blank control group were 2.70±0.06 cm, 3.50±0.15 cm, and 8.00±0.12 cm, respectively. In the presence of NO.10, the swimming, swarming, and twitching ranges of P. aeruginosa PAO1 were 1.40±0.04 cm, 2.80±0.12 cm, and 6.40±0.18 cm, respectively. In the presence of compound L2, it significantly reduced the motility zone by 51.85±0.05%, 60.00±0.11%, and 31.25±0.06%, respectively. In conclusion, both NO.10 and compound L2 have significant inhibitory effects on the motility of PAO1, with compound L2 exhibiting stronger inhibitory activity.

[0054] Example 8: Evaluation of the inhibitory effect of L2 on the production of virulence factors in Pseudomonas aeruginosa

[0055] 1. Analysis of Pseudomonas aeruginosa

[0056] After selecting a single colony of PAO1 and allowing it to grow aerobically in a shaker at 37°C for 24 hours, the overnight cultured *Pseudomonas aeruginosa* PAO1 was subcultured into 25 μM LB medium (L2) and cultured for 24 hours. The color of the culture was then observed. Compound NO.10 was used as a positive control. Finally, the OD was measured. 520 To determine the content of pyocyanin.

[0057] Experimental results are as follows Figure 6 As shown, at the same concentration, both NO.10 and compound L2 can inhibit the production of PAO1 aureomycin. Compound NO.10 reduced the amount of aureomycin produced by 36.98±0.23%, while L2 reduced the amount of PA aureomycin produced by 46.69±0.21%. Compared with compound NO.10, L2 has a more significant inhibitory effect on the production of Pseudomonas aeruginosa aureus aureomycin.

[0058] 2. Analysis of elastase

[0059] After overnight culture of *Pseudomonas aeruginosa*, the overnight cultured PAO1 was passaged into fresh LB medium containing 25 μM L2. A group without the compound was used as a blank control, and a group containing compound NO.10 was used as a positive control. The cultures were incubated at 37°C and 180 rpm for 24 h. After 24 h, the cultures were centrifuged at 10,000 rpm for 15 min, and the supernatant was collected. 100 μL of the supernatant was then added to the wells of skim milk agar plates, which were then incubated at 37°C for 24 h. Elastase activity was determined by measuring the diameter of the clear zone on the plates.

[0060] Experimental results are as follows Figure 7 As shown, compared with the control group, both compound 10 and compound L2 inhibited elastase activity. After treatment with compound 10, the diameter of the transparent area on the skim milk plate decreased by 26 ± 1.02%, and after treatment with L2, the diameter decreased by 41 ± 0.87%. In conclusion, both compound NO.10 and compound L2 can inhibit elastase activity, with compound L2 showing a more significant inhibitory effect.

[0061] Example 9: Evaluation of the effect of L2 on the expression level of QS-regulated genes in Pseudomonas aeruginosa

[0062] The relative expression levels of key QS regulatory genes (lasI, lasR, rhlI, rhlR, mvfR) and antibiotic resistance gene ampC in Pseudomonas aeruginosa PAO1 were detected in the presence of compound L2, with compound NO.10 as a positive control and the 16S rRNA housekeeping gene as an internal reference gene. Primers for the genes detected are shown in Table 2.

[0063] Primers for the genes detected in Table 2

[0064]

[0065] Total RNA was isolated using an ultrapure RNA kit (CWBIO, Beijing, China) and purified according to the manufacturer's instructions. III. First-strand cDNA Synthesis for qPCR: First-strand cDNA was synthesized using SuperMix (Yeasen, Beijing, China). The reaction was performed using Hieff according to the manufacturer's instructions. The assay was performed using Universal Blue qPCR SYBR Green premixed buffer (Yeasen, Beijing, China). Two [units / components] were used. -ΔΔCt The method calculates relative gene expression.

[0066] Experimental results are as follows Figure 8 As shown, compared with the untreated group, NO.10 inhibited the expression of lasI, lasR, rhlI, mvfR, and ampC, but upregulated the expression of rhlR. After NO.10 treatment, the relative expression levels of lasI, lasR, rhlI, mvfR, and ampC decreased by 16±0.76%, 47±2.01%, 25±0.39%, 38±0.83%, and 38±1.21%, respectively. Compared with NO.10, L2 downregulated the expression of all six genes, with the relative expression levels of lasI, lasR, rhlI, rhlR, mvfR, and ampC decreasing by 48±0.34%, 68±0.62%, 16±0.28%, 17±1.01%, 79±0.36%, and 50±0.48%, respectively.

[0067] Example 10: Effect of L2 on fluorescent reporter strains in different quorum sensing systems

[0068] Single colonies of PAO1-lasB-gfp, PAO1-rhlA-gfp, and PAO1-pqsA-gfp were picked and cultured for 12-14 h. They were then subcultured at a 1:100 ratio into fresh LB medium and cultured to the logarithmic growth phase. The logarithmic growth phase bacteria were then added to 96-well plates, with the concentrations of compounds L2 and NO.10 reaching 25 μM. A control group without added compounds was used as the blank control, and NO.10 as the positive control. The inoculated bacterial plates were placed in a microplate reader at 37℃, with excitation light at 485 nm and emission light at 528 nm. Fluorescence was measured every 15 min, and the optical density of the reporter strain was simultaneously monitored at 600 nm (OD). 600 To ensure that the compound would not have any effect on bacterial growth, measurements were taken continuously for 16 hours to detect the effect of the compound on three pathways.

[0069] Experimental results are as follows Figure 9 As shown, compound L2 slightly inhibited the expression of fluorescent protein in PAO1-rhlA-gfp and PAO1-pqsA-gfp green fluorescent protein reporter strains of the rhl and pqs systems, while it significantly inhibited the expression of fluorescent protein in PAO1-lasB-gfp green fluorescent protein reporter strains of the las system. However, compound NO.10 did not show any significant inhibitory effect on the three pathways. This result indicates that compound L2 has a significantly stronger inhibitory effect on LasR than compound NO.10.

[0070] Implementation Case 11: Rapid Killing Experiment of Nematodes

[0071] The inhibitory effect of 25 μM compound L2 on the production of virulence factors in *Pseudomonas aeruginosa* (PA) was evaluated using a rapid kill assay in *C. elegans*. Compound NO.10 was used as a positive control. *C. elegans* were grown to the L4 stage at 20°C and then synchronized before being inoculated into fresh NGM medium and cultured at 20°C for 48 h to reach the L4 stage. During nematode growth, compound L2 was added to *Pseudomonas aeruginosa* PAO1 to a final concentration of 25 μM and cultured at 37°C for 24 h. DMSO was used as a blank control, and NO.10 as a positive control. The overnight cultured PAO1 was then inoculated onto solid medium containing PGS agar (1% peptone, 1% NaCl, 1% glucose, 150 mM sorbitol, and 1.7% agar) and cultured at 37°C for 24 h. Subsequently, synchronized L4-stage nematodes were inoculated onto the PAO1-inoculated plates, with 25-30 nematodes per plate. Then, the number of surviving nematodes was counted every 3 hours at 25°C.

[0072] Experimental results are as follows Figure 10 As shown, within 18 hours, compound L2 significantly improved the survival rate of *C. elegans* (a type of *Pseudomonas aeruginosa*) compared to compound NO.10. Compared to the untreated group, the survival rate of *C. elegans* in the compound L2 group was close to 80% within 6 hours of incubation and still reached 60% within 18 hours. The experimental results demonstrate that compound L2 has a more significant inhibitory effect on the QS system of *Pseudomonas aeruginosa* than NO.10.

[0073] Implementation Case 12 Hemolysis Test

[0074] Freshly isolated mouse erythrocytes were washed three times with PBS buffer to prepare a 5% erythrocyte solution. Compound L2 was serially diluted with PBS buffer to obtain concentrations of 12.5 μM, 25 μM, 50 μM, and 100 μM in a 200 μL system. The mixture of compound L2 and 5% erythrocytes was then incubated at 37°C for 1 h. The incubation solution was centrifuged at 1500 rpm for 10 min. The supernatant (30 μL) was transferred to 96-well plates, with each well containing 70 μL of PBS buffer. The absorbance of all wells was recorded at 540 nm using a microplate reader. PBS buffer was used as a negative control, and 2% Triton X-100 was used as a positive control.

[0075] Experimental results are as follows Figure 11 As shown, the hemolysis rates of compound L2 at concentrations of 12.5, 25, 50, and 100 μM were 0.05%, 1.5%, 2.1%, and 2.3%, respectively. Even at a high concentration of 100 μM, the hemolysis rate of compound L2 was less than 2.5%, indicating that compound L2 has a very small hemolytic effect on mouse erythrocytes.

Claims

1. The application of a homoserine lactone compound, characterized in that, The application of the homoserine lactone compound in the preparation of a drug to inhibit the formation of biofilms in Pseudomonas aeruginosa, wherein the homoserine lactone compound is N-(3-cyclobutyrolactone)-4-nitrophenylbutyramide (L2), and its structure has the following molecular structural formula:

2. The use of the homoserine lactone compound according to claim 1 in the preparation of a drug for treating chronic infectious diseases caused by Pseudomonas aeruginosa.

3. The application according to claim 2, characterized in that, The dosage form of the drug for chronic infectious diseases is selected from oral liquid, tablets, capsules, granules, ear drops, nasal drops, suppositories, pills, liniments, ointments, creams, patches, pastes, sprays, aerosols, or powders.

4. The application according to claim 3, characterized in that, Chronic infectious diseases caused by Pseudomonas aeruginosa include chronic wound infections, chronic sinusitis, and chronic otitis media.

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