A composition effective to inhibit pseudomonas aeruginosa biofilm bacteria

Abstract

The application discloses a N-(3-cyclobutyrolactone)-4-bromobenzamide and meropenem composition and application of the composition in treatment of chronic infection of drug-resistant bacteria. After the N-(3-cyclobutyrolactone)-4-bromobenzamide and the meropenem are combined, not only can the sensitivity of a biofilm bacterium to the meropenem be significantly increased, the killing effect of the meropenem on the biofilm bacterium be improved, but also the formation amount of a Pseudomonas aeruginosa biofilm can be greatly reduced; the composition can be applied to preventing and treating chronic infection of drug-resistant Pseudomonas aeruginosa.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a combination of N-(3-cyclobutyrolactone)-4-bromobenzamide and meropenem. BACKGROUND

[0002] Pseudomonas aeruginosa (PA) is a common opportunistic pathogen in clinic, which can cause local pyogenic inflammation and systemic infection of human body, such as respiratory tract infection, pneumonia, septicemia, etc., when the human body is in a state of low immunity, tissue barrier is damaged or invasive medical operation is performed. Moreover, due to the strong adaptability and drug resistance of PA and the effect of biofilm, common antibacterial drugs usually cannot play an effective role on PA. In addition, the existence of biofilm also leads to chronic infection and repeated infection, which makes the infection difficult to be completely cured.

[0003] Biofilm is a structured film-like bacterial population formed by bacteria and their secreted extracellular polysaccharides and other substances. The structure of biofilm plays a protective role, preventing antibiotics from entering bacterial cells. Moreover, the formation of biofilm can promote PA to transform into a state with lower metabolic activity, in which state, the sensitivity of PA to antibiotics will be significantly reduced, and even PA will become insensitive to antibiotics. What is more serious is that the existence of biofilm also leads to chronic infection and repeated infection, which makes the infection difficult to be completely cured. Clinical data shows that the biofilm formed by PA in pulmonary cystic fibrosis 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 biofilm formation of PA. In view of the above problems, some studies have proposed the idea of combining biofilm inhibitors with antibiotics to solve the bacterial resistance caused by biofilm. However, our previous research results show that the combination of biofilm inhibitors with Gentamicin (GM), Tobramycin (TOB) and Amikacin (AK) cannot achieve the effects of reducing the amount of PA biofilm formation and increasing the sensitivity of PA to antibiotics at the same time, and the drug composition cannot kill the bacteria in biofilm more effectively.

[0004] The present application unexpectedly found that the combination of compound N-(3-cyclobutyrolactone)-4-bromobenzamide and meropenem (MEPM) can achieve the effects of synergistically reducing the amount of biofilm formation and increasing the sensitivity of bacteria in biofilm to MEPM, which provides a new method for the clinical treatment of PA infection. SUMMARY

[0005] The purpose of the present application is to provide a composition for enhancing the sensitivity of bacteria in biofilm to antibiotics and inhibiting the formation of bacterial biofilm, which specifically comprises the following contents:

[0006] In a first aspect, the present application provides a composition for enhancing the sensitivity of biofilm bacteria to antibiotics and the formation of bacterial biofilm, wherein the active ingredients of the composition comprise meropenem and N-(3-cyclobutyrolactone)-4-bromophenylbutyramide.

[0007] Preferably, the bacteria is Pseudomonas aeruginosa.

[0008] Preferably, the mass ratio of the meropenem and N-(3-cyclobutyrolactone)-4-bromophenylbutyramide is 1:4.4-1:35.

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

[0010] In a second aspect, the present application provides the use of N-(3-cyclobutyrolactone)-4-bromophenylbutyramide in the first aspect in the preparation of a drug for enhancing the sensitivity of biofilm bacteria to antibiotics.

[0011] Preferably, the antibiotic is meropenem.

[0012] Preferably, the bacterial biofilm is Pseudomonas aeruginosa biofilm.

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

[0014] In a third aspect, the present application provides the use of the composition in the first aspect in the preparation of a drug for treating chronic infection diseases caused by Pseudomonas aeruginosa.

[0015] In a fourth aspect, the present application provides the use of the composition in the first aspect in the preparation of a drug for treating chronic infection diseases caused by Pseudomonas aeruginosa biofilm.

[0016] Preferably, the chronic infection diseases caused by Pseudomonas aeruginosa include chronic wound infection, chronic sinusitis, chronic otitis media.

[0017] Advantages of the present application:

[0018] (1) N-(3-cyclobutyrolactone)-4-bromobenzamide can be used as a synergist for increasing the sensitivity of PA to MEPM, and after N-(3-cyclobutyrolactone)-4-bromobenzamide is used in combination with MEPM, the sensitivity of the biofilm PA bacteria to MEPM can be significantly increased, and the killing effect of MEPM on the biofilm bacteria is improved, (2) after the compound N-(3-cyclobutyrolactone)-4-bromobenzamide is used in combination with MEPM, the formation amount of PA biofilm can be significantly reduced. (3) The composition of N-(3-cyclobutyrolactone)-4-bromobenzamide and MEPM as active ingredients can be applied to prevent and treat chronic infection diseases caused by Pseudomonas aeruginosa biofilm, such as chronic wound infection, chronic sinusitis, chronic otitis media and the like. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Effect of the composition of the present application on the growth of Pseudomonas aeruginosa biofilm bacteria that have been formed

[0020] Figure 2 Effect of the composition of the present application on the formation of Pseudomonas aeruginosa biofilm that has been formed

[0021] Figure 3 Effect of the composition of the present application on the growth of Pseudomonas aeruginosa biofilm bacteria that have been formed under observation by a laser confocal microscope

[0022] Figure 4 Effect of N-(3-cyclobutyrolactone)-4-bromobenzamide and TOB on the growth of Pseudomonas aeruginosa biofilm bacteria that have been formed

[0023] Figure 5 Effect of N-(3-cyclobutyrolactone)-4-bromobenzamide and TOB on the formation of Pseudomonas aeruginosa biofilm that has been formed

[0024] Figure 6 Effect of N-(3-cyclobutyrolactone)-4-bromobenzamide and TOB used in combination on the growth of Pseudomonas aeruginosa biofilm bacteria that have been formed under observation by a laser confocal microscope

[0025] Figure 7 Effect of N-(3-cyclobutyrolactone)-4-bromobenzamide and AK on the growth of Pseudomonas aeruginosa biofilm bacteria that have been formed

[0026] Figure 8 Effect of N-(3-cyclobutyrolactone)-4-bromobenzamide and AK on the formation of Pseudomonas aeruginosa biofilm that has been formed

[0027] Figure 9 Effect of N-(3-cyclobutyrolactone)-4-bromobenzamide and MEPM on the growth of Pseudomonas aeruginosa biofilm bacteria that have been formed

[0028] Figure 10Effect of N-(3-cylobutyrolactone)-4-bromophenylacetamide and MEPM on the growth of preformed PA biofilm bacteria

[0029] Figure 11 Effect of N-(3-cylobutyrolactone)-4-bromophenylacetamide and MEPM on the growth of preformed PA biofilm bacteria

[0030] Figure 12 Effect of N-(3-cylobutyrolactone)-4-bromophenylacetamide and MEPM on the growth of preformed PA biofilm bacteria DETAILED DESCRIPTION

[0031] The following specific examples are provided to achieve the technical solutions described in the present application, but are not limited to these examples.

[0032] Example 1. MIC detection of PAOl free bacteria and biofilm bacteria to different antibiotics

[0033] The experimental method is referred to the experimental method of (Liu et al., 2019), and is briefly adjusted on this basis. The specific experimental method is as follows: pick a single colony of PAOl in LB liquid medium, 200 rpm, 37°C constant temperature culture for 15h. After 15h, the bacterial solution was diluted to 5x10 5 CFU / mL with fresh sterile LB medium. And dilute the antibiotics (MEPM, GM, TOB, AK and CIP) in a 2-fold serial dilution manner. Next, add the serially diluted antibiotics to the diluted bacterial solution, so that the concentration of the bacterial solution is maintained at 5x10 5 CFU / mL. Finally, inoculate the bacterial solution containing different concentrations of antibiotics into 96-well plates at 150μL / well, and take the group without adding antibiotics and the medium group as controls. The 96-well plates were incubated at 37°C for 24h. When the bacteria in the group without adding antibiotics grow normally, the experiment is valid, and the MIC value is the lowest concentration showing complete inhibition of visible growth. The experimental method for biofilm bacterial MIC detection is referred to (Zhou et al., 2018), and is briefly adjusted on this basis. The specific experimental method is as follows: pick a single colony of PAOl in LB liquid medium, 200 rpm, 37°C constant temperature culture for 15h. After 15h, the bacterial solution was diluted to 5x10 5CFU / mL. The diluted bacteria were inoculated into 96-well plates at 150 μL / well, and the plates were incubated at 37 °C for 24 h to form mature biofilms. After 24 h, the bacteria in the wells were discarded, and the wells were gently washed twice with 1 x PBS to remove the planktonic bacteria. After the last wash, the residual liquid in the wells was completely discarded. Next, LB medium containing different antibiotics was inoculated into 96-well plates at 150 μL / well, and the groups without antibiotics and the medium group were used as controls, respectively. The 96-well plates were incubated at 37 °C for 24 h. The experiment was valid when the bacteria in the group without antibiotics grew normally, and the MIC value of the biofilm bacteria was the lowest concentration at which visible growth was completely inhibited.

[0034] As shown in Table 1, the MIC of the remaining three antibiotics for PAOl biofilm bacteria was greater than that for planktonic bacteria, except for CIP. Among them, the biofilm bacterial MIC of MEPM was 32 times that of planktonic bacteria, the biofilm bacterial MIC of AK was 4 times that of planktonic bacteria, and the biofilm bacterial MIC of TOB was 4 times that of planktonic bacteria. Thus, the formation of bacterial biofilm is one of the reasons for bacterial drug resistance.

[0035] Table 1 MIC of different antibiotics for PAOl planktonic bacteria and biofilm bacteria

[0036]

[0037]

[0038] Example 2 Effect of combined use of biofilm inhibitors and antibiotics on the growth of PA biofilm bacteria

[0039] After the recovered PA was inoculated in LB liquid medium and cultured for 15 h, the bacterial concentration was adjusted to 5.0 x 10 5 CFU / mL, and was added to a 96-well plate at 150 μL / well and incubated at 37 °C in a constant temperature incubator for 24 h to form mature biofilms. After 24 h, the bacteria in the wells were discarded, and the wells were gently washed twice with sterile PBS to remove the planktonic bacteria and retain the bacterial biofilms formed by the bacteria. Subsequently, N-(3-cyclobutyrolactone)-4-bromophenylbutyramide at a final concentration of 25, 50, 100, and 200 μM and MEPM at a final concentration of 2.5 μg / mL were added to the LB medium at 150 μL / well, respectively, and the blank medium and the medium containing only MEPM were used as controls, respectively. The 96-well plates were incubated at 37 °C in a constant temperature incubator for 18-20 h. The OD value at 600 nm was detected by a microplate reader, and 6 parallel wells were prepared for each group. The data are expressed as mean ± SD, and the comparison between groups was performed by one-way ANOVA, with P < 0.05 being considered statistically significant.

[0040] The experimental method of AK and TOB combined with N-(3-oxetanoyl)-4- bromophenylbutyramide is consistent with MEPM, which uses concentrations of 4 μg / mL and 1.25 μg / mL, respectively. In addition, the method of MEPM combined with N-(3-oxetanoyl)-4-bromophenylacetamide and N-(3-oxetanoyl)-4-nitrophenylacetamide is also consistent with the above.

[0041] As shown in Figure 1 , compared with the DMSO group, the compound can increase the inhibition of MEPM on the growth of biofilm bacteria in a dose-dependent manner in the use concentration range of 25-200 μM. The inhibition rate of MEPM on the growth of biofilm bacteria is 26.1% when used alone, and the inhibition rate increases to 39.72% after combined with 200 μM of N-(3-oxetanoyl)-4-bromophenylbutyramide, which significantly increases the inhibition of meropenem on the growth of biofilm bacteria. As shown in Figure 4 , the inhibition rate of TOB on the growth of biofilm bacteria is 38.67% when used alone, and the inhibition rate increases to 54.93% after combined with 200 μM of N-(3-oxetanoyl)-4-bromophenylbutyramide. As shown in Figure 7 , the inhibition rate of AK on the growth of biofilm bacteria is 13.95% when used alone, and the inhibition rate increases to 26.92% after combined with 200 μM of N-(3-oxetanoyl)-4-bromophenylbutyramide. The above results show that the compound N-(3-oxetanoyl)-4-bromophenylbutyramide can synergistically increase the inhibition of MEPM, TOB and AK on the growth of PAO1 biofilm bacteria.

[0042] And as shown in Figure 9 and Figure 11 , after MEPM is combined with N-(3-oxetanoyl)-4-bromophenylacetamide and N-(3-oxetanoyl)-4-nitrophenylacetamide, respectively, these two combinations cannot increase the killing ability of MPEM on biofilm bacteria. Therefore, N-(3-oxetanoyl)-4-bromophenylacetamide and N-(3-oxetanoyl)-4-nitrophenylacetamide cannot increase the sensitivity of biofilm bacteria to antibiotics.

[0043] Studies have shown that in addition to the activity of bacteria, the amount of biofilm formation is also one of the key points affecting the treatment of chronic infection caused by PA, so we continue to evaluate the inhibitory effect of the above drug combination on biofilm formation.

[0044] Example 3 Influence of combination of biofilm inhibitors and antibiotics on PA biofilm

[0045] After the incubation of P. aeruginosa PA as described in Example 2, the bacterial solution in the 96-well plate was aspirated, 200 μL of sterile PBS was added to each well for washing twice to remove free bacteria, then the residual liquid in the wells was absorbed with a clean filter paper, 150 μL of 0.1% crystal violet solution was added to each well, and the staining was performed for 15 min. After the staining, the crystal violet solution in the wells was aspirated, 200 μL of sterile PBS was added to each well for washing three times, and the residual liquid in the wells was absorbed with a clean filter paper. 150 μL of 33% acetic acid solution was added to each well, and after the dissolution of the biofilm-crystal violet complex, the OD value at 570 nm was detected by an enzyme-labeled instrument, and six parallel wells were prepared for each group. The data were expressed as mean ± SD, and the comparison between groups was performed by one-way ANOVA, with P < 0.05 being considered statistically significant.

[0046] As shown in Figure 2 , when MEPM and 200 μM N-(3-cyclobutyrolactone)-4-bromophenylbutyramide were used alone, their inhibition rates on biofilm formation were only 0% and 19.93%, respectively, while the compound N-(3-cyclobutyrolactone)-4-bromophenylbutyramide could inhibit biofilm formation in a dose-dependent manner (15.76%-47.49%) in the concentration range of 25-200 μM in combination with meropenem. The results showed that MEPM and N-(3-cyclobutyrolactone)-4-bromophenylbutyramide could synergistically inhibit the formation of PA bacterial biofilm. As shown in Figure 5 and Figure 8 , the use of TOB and AK alone promoted the formation of bacterial biofilm, which was consistent with the phenomenon reported by (Tahrioui et al., 2019) and others. However, after the combination, the phenomenon of promoting biofilm formation was alleviated to a certain extent, but there was no synergistic effect.

[0047] As shown in Figure 10 and Figure 12 , N-(3-cyclobutyrolactone)-4-bromophenylacetamide and N-(3-cyclobutyrolactone)-4-nitrophenylacetamide could reduce the amount of biofilm formation in a dose-dependent manner in the concentration range of 25 μM-200 μM, and the degree of reduction of biofilm formation was consistent with that of single use when the above two compounds were combined with MEPM. Therefore, N-(3-cyclobutyrolactone)-4-bromophenylacetamide and N-(3-cyclobutyrolactone)-4-nitrophenylacetamide cannot synergistically reduce the amount of biofilm formation.

[0048] In order to further describe the above results, we next observed the effect of the combination of biofilm inhibitors and antibiotics on PA biofilm by fluorescence staining (FITC-ConA labeled extracellular polysaccharide, PI labeled dead bacteria) and laser confocal observation.

[0049] Example 4: Effects of combined use of biofilm inhibitors and antibiotics on PA biofilm bacteria observed by laser confocal microscopy

[0050] Following the experimental procedure in Example 3, a sterile glass slide (14 mm in diameter) was placed in each well of a 24-well culture plate. 1 mL of the prepared bacterial solution was added to each well, with three replicates. The 24-well culture plate was placed in an incubator and incubated for 24 hours. The slides were then removed and gently rinsed twice with 1×PBS to remove any floating bacteria. The slides were fixed with 2.5% glutaraldehyde solution at 4°C for 1.5 hours, followed by gentle rinsing three times with 1×PBS for 10 minutes each time. Any remaining PBS on the slides was then blotted dry with absorbent paper. 20 μL of 1 mg / mL FITC-ConA solution was evenly added to the slide, and the slides were stained at room temperature for 30 minutes. After 30 minutes, excess dye was discarded, and the slides were gently rinsed once with 1×PBS to remove any remaining dye. Any remaining PBS on the slides was then blotted dry with absorbent paper. Next, add 20 μL of 50 μg / mL PI solution evenly to the slide again, and stain the slide at room temperature for 15 min. After 15 min, discard the excess dye, gently rinse once with 1×PBS to remove any remaining dye, and blot the slide dry with absorbent paper. Finally, observe the changes in the biofilm and biofilm bacteria under a laser confocal microscope. All staining processes must be conducted in the dark to prevent fluorescence quenching.

[0051] Depend on Figure 3 As shown, compared to the DMSO group (40 μm), N-(3-cyclobutyrolactone)-4-bromophenylbutyramide alone can reduce biofilm formation to some extent, reducing the biofilm thickness to 25 μm, and no red fluorescence is observed, indicating that this compound has no antibacterial effect when used alone. After using MEPM alone, the biofilm thickness is 35 μm, and only weak red fluorescence is observed in the figure, indicating that MEPM at this concentration can reduce biofilm formation to some extent and has only a weak bactericidal ability. However, when N-(3-cyclobutyrolactone)-4-bromophenylbutyramide and MEPM are used in combination, the biofilm thickness is reduced to 9 μm, and the red fluorescence is significantly enhanced, indicating that the combined use of N-(3-cyclobutyrolactone)-4-bromophenylbutyramide and MEPM can both increase the sensitivity of biofilm bacteria to MEPM and synergistically reduce the amount of biofilm formation.

[0052] like Figure 6As shown, N-(3-cyclobutyrolactone)-4-bromobenzamide alone can reduce the amount of biofilm formation to a certain extent, reduce the biofilm thickness to 25 μm, and no red fluorescence appears, indicating that the compound alone has no bacteriostatic effect. After using TOB alone, the biofilm thickness increases to 80 μm, and only weak red fluorescence appears in the figure, indicating that TOB at this concentration can promote the formation of bacterial biofilm and only has weak bactericidal ability. When N-(3-cyclobutyrolactone)-4-bromobenzamide and TOB are used in combination, the thickness of the biofilm is reduced to 30 μm, and the red fluorescence is also significantly enhanced, indicating that N-(3-cyclobutyrolactone)-4-bromobenzamide and TOB in combination can increase the sensitivity of biofilm bacteria to TOB to a certain extent, but cannot synergistically reduce the amount of biofilm formation.

[0053] In summary, the combination of MEPM and N-(3-cyclobutyrolactone)-4-bromobenzamide not only increases the bactericidal ability of MEPM to biofilm bacteria, but also synergistically reduces the amount of biofilm formation. The combination of AK and TOB with N-(3-cyclobutyrolactone)-4-bromobenzamide can only increase the bactericidal ability of antibiotics to biofilm bacteria, but cannot significantly reduce the amount of biofilm formation. Studies have shown that the presence of biofilm increases the risk of repeated infection in patients, making it difficult to cure infections caused by P. aeruginosa. Therefore, the combination of AK and TOB with N-(3-cyclobutyrolactone)-4-bromobenzamide cannot be used to treat chronic infectious diseases. In addition, the combination of N-(3-cyclobutyrolactone)-4-bromobenzamide analogs N-(3-cyclobutyrolactone)-4-bromobenzamide and N-(3-cyclobutyrolactone)-4-nitrobenzamide with MEPM cannot increase the sensitivity of biofilm bacteria to MEPM, nor can it synergistically reduce the amount of bacterial biofilm formation, so the above combinations cannot be used to treat chronic infectious diseases. The composition of the present application can significantly reduce the formation of PA biofilm and increase the sensitivity of biofilm PA to antibiotics, and can be used to treat PA chronic infection, and has broad clinical application prospects.

Claims

1. A composition for enhancing the sensitivity of biofilm bacteria to antibiotics and inhibiting the formation of bacterial biofilm, wherein the active ingredients of the composition comprise meropenem and N-(3-cyclobutyrolactone)-4-bromophenylbutyramide.

2. The composition of claim 1, wherein The bacteria are Pseudomonas aeruginosa.

3. The composition of claim 1, wherein The mass ratio of the meropenem and N-(3-cyclobutyrolactone)-4-bromophenylbutyramide is 1:4.4-1:

35.

4. The composition of claim 1, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

5. Use of N-(3-cyclobutyrolactone)-4-bromophenylbutyramide in the preparation of a drug for enhancing the sensitivity of Pseudomonas aeruginosa to meropenem.

6. Use according to claim 5, wherein The Pseudomonas aeruginosa is drug-resistant Pseudomonas aeruginosa.

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

8. Use of the composition according to claim 7 in the preparation of a drug for treating a chronic infectious disease caused by Pseudomonas aeruginosa biofilm.

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