Composition and application thereof in resisting multi-drug-resistant acinetobacter baumannii

Abstract

The invention discloses a composition and application thereof in resisting multi-drug resistant acinetobacter baumannii, and belongs to the technical field of bacterial infection resistance. The invention provides a composition for resisting multi-drug resistance acinetobacter baumannii. The composition is prepared from emodin, aloe-emodin and rhein, the mass ratio of the emodin to the aloe-emodin to the rhein is 1: 1: 1. The invention also provides an application of the composition in preparation of an antibacterial preparation. According to the composition disclosed by the invention, the sensitivity of the multi-drug-resistant acinetobacter baumannii to polymyxin and carbapenem antibiotics can be recovered again, and the killing time of the polymyxin and carbapenem antibiotics to the multi-drug-resistant acinetobacter baumannii can be shortened; the healing of a mouse skin wound infected by the multi-drug-resistant acinetobacter baumannii can be accelerated. In addition, the compositions of the present invention do not exhibit cytotoxicity at lower concentrations. Therefore, the composition disclosed by the invention can be used for preparing a medicine for resisting multi-drug-resistant acinetobacter baumannii infection.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-bacterial infection, and in particular relates to a composition and application thereof in resisting multi-drug resistant Acinetobacter baumannii. Background Art

[0002] Although Acinetobacter baumannii was once considered a low-toxic bacterium, it is now recognized by major medical institutions around the world as one of the most dangerous conditional pathogens. Acinetobacter baumannii has been involved in a variety of hospital-acquired infections associated with high morbidity or mortality, such as ventilator-associated pneumonia, wound infection, blood infection, secondary meningitis, skin and soft tissue infection, and urinary tract infection. With the extensive use of antibiotics in clinical practice, the multidrug resistance of Acinetobacter baumannii has gradually increased. Carbapenem antibiotics (such as imipenem and meropenem) are considered to be the basic drugs for the treatment of Acinetobacter baumannii infections. However, in recent years, the global prevalence of carbapenem-resistant Acinetobacter baumannii has increased year by year. Polymyxin antibiotics, such as polymyxin B and polymyxin E (also known as colistin), have a good effect on multidrug-resistant Acinetobacter baumannii and are the "last line of defense" for multidrug-resistant Acinetobacter baumannii infections. However, with the long-term and large-scale use of polymyxins, polymyxin-resistant Acinetobacter baumannii began to appear clinically, making first-line antibiotics almost ineffective against such resistant bacteria.

[0003] At present, the discovery of new antibiotics is extremely challenging. The global antibiotic market is still dominated by old antibiotics discovered more than half a century ago. In addition, the development cycle of new antibiotics is long and the cost is very high. Only by investing a lot of money can the drugs be successfully brought to the market. Therefore, people are in urgent need of actively seeking new solutions for infections caused by multidrug-resistant Acinetobacter baumannii, especially those resistant to polymyxins and carbapenems. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a composition and its application in fighting multidrug-resistant Acinetobacter baumannii. The composition provided by the present invention can be used to fight infection by multidrug-resistant Acinetobacter baumannii, especially infection by Acinetobacter baumannii resistant to polymyxins and carbapenems.

[0005] The present invention provides the following technical solutions:

[0006] Technical solution 1: A composition for resisting multidrug-resistant Acinetobacter baumannii, consisting of rhein, aloe-rhein and rhein.

[0007] Furthermore, the mass ratio of the emodin, aloe-emodin and rhein is 1:1:1.

[0008] Technical solution 2: Use of the composition in the preparation of antibacterial preparations.

[0009] Technical solution three: an antibacterial preparation, the active ingredients of which include the composition and EDTA.

[0010] Furthermore, the concentration of EDTA is 256 mg / L.

[0011] Furthermore, the antibacterial preparation is a pharmaceutical preparation for treating diseases caused by Acinetobacter baumannii.

[0012] Furthermore, the Acinetobacter baumannii is a multidrug-resistant Acinetobacter baumannii.

[0013] Technical solution 4: Use of the composition in the preparation of drugs for sensitizing multidrug-resistant Acinetobacter baumannii.

[0014] Technical Solution 5: Use of the composition in the preparation of a drug for shortening the killing time of polymyxins and carbapenems antibiotics on multidrug-resistant Acinetobacter baumannii.

[0015] Technical Solution 6: Use of the composition in the preparation of a drug for accelerating the healing of skin wounds infected with multidrug-resistant Acinetobacter baumannii.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] In view of the deficiencies of the prior art, the present invention provides a composition that can be used to resist infection by multidrug-resistant Acinetobacter baumannii, especially infection by polymyxin- and carbapenem-resistant Acinetobacter baumannii. The composition contains rhein, aloe-rhein and rhein. The mass ratio of rhein, aloe-rhein and rhein is 1:1:1. The final concentration of rhein is preferably 10.667 mg / L; the final concentration of aloe-rhein is preferably 10.667 mg / L; the final concentration of rhein is preferably 10.667 mg / L; the final concentration of EDTA is preferably 256 mg / L.

[0018] The composition provided by the present invention and polymyxin B, polymyxin E, imipenem and meropenem all show synergy in inhibiting multidrug-resistant Acinetobacter baumannii. The sensitivity of multiple multidrug-resistant Acinetobacter baumannii clinical isolates to polymyxins and carbapenems is significantly enhanced, and the resistant type is converted into a sensitive type. Not only that, the composition of the present invention can also significantly improve the killing activity of polymyxins and carbapenems against multidrug-resistant Acinetobacter baumannii. With the gradual increase of the concentration of the mass ratio composition (EAR) of emodin, aloe-emodin and rhein, the killing efficiency of polymyxin B and meropenem is gradually improved. Especially when the concentration of EAR is 32mg / L, 2mg / L of polymyxin B and meropenem can completely kill Acinetobacter baumannii at 6h and 24h respectively.

[0019] In addition, the composition of the present invention showed no toxicity to human liver cancer cells (Hep3B) and human kidney cancer cells (Hep293) at a lower concentration (no more than 32 mg / L). When the composition of the present invention was used in combination with polymyxin B, it had a good therapeutic effect on the skin wounds of mice infected with multidrug-resistant Acinetobacter baumannii 1415E. Compared with the control group, the skin wounds of mice in the drug group showed a significant accelerated healing trend on the 8th day of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 is the chemical structural formula of rhein;

[0022] Figure 2 is the chemical structural formula of aloe-emodin;

[0023] Figure 3 is the chemical structural formula of rhein;

[0024] Figure 4 is the killing curve of the combination and polymyxin B against Acinetobacter baumannii 1415E;

[0025] Figure 5 is the killing curve of the combination and meropenem against Acinetobacter baumannii 1415E;

[0026] Figure 6 The inhibitory analysis of different concentrations of EAR on the viability of Hep3B and Hep293 cells;

[0027] Figure 7 The present invention shows the therapeutic effect of the combination of the composition and polymyxin B on mice infected with Acinetobacter baumannii 1415E. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The Acinetobacter baumannii 1415E, 10213, 07AC366, 189B, 6588E, 038E, 245B, AC36, 89E, P68, P95 and P171 used in this article were all isolated from culture samples such as sputum, urine, blood, secretions, puncture fluid, pus, ascites, pleural effusion, and exudate received by the Laboratory Department of Qilu Hospital of Shandong University.

[0034] The chemical structure of rhein is shown in Figure 1 The chemical structure of aloe-emodin is shown in Figure 2The chemical structure of rhein is shown in Figure 3 .

[0035] Example 1 Isolation and drug sensitivity analysis of clinical strains of Acinetobacter baumannii

[0036] Samples such as sputum, urine, blood, secretions, feces, puncture fluid, pus, ascites, pleural effusion, and exudate were streaked onto blood plates and incubated at 37°C for 24 hours before picking the dominant colonies for purification culture. The VITEK 2COMPACT fully automatic microbial identification and drug sensitivity analyzer was used to identify the types of pathogens in each culture sample, and the drug sensitivity test was automatically completed using the corresponding drug sensitivity card that came with the instrument. The drug sensitivity results were interpreted according to the standards set by the Clinical and Laboratory Standards Institute (CLSI).

[0037] Table 1 shows the drug sensitivity interpretation results of clinical isolates of Acinetobacter baumannii. Among them, S means sensitive; I means intermediate; R means resistant. The results showed that Acinetobacter baumannii 1415E, 10213, 07AC366, 189B, 6588E, 038E, 245B, AC36, 89E, P68, P95 and P171 were resistant to more than two antibiotics (R), showing the characteristics of multidrug resistance.

[0038] Table 1

[0039]

[0040] Example 2 Screening of EDTA concentration

[0041] The minimum inhibitory concentration of the combination (components include rhein, aloe-rhein and rhein) under the combined action of drugs was determined by the square chessboard dilution method. In a sterile 96-well plate, the concentration of the bacterial solution of Acinetobacter baumannii 1415E was adjusted to 5×10 5 CFU / mL. Rhein (abbreviated as E), aloe-rhein (abbreviated as A), and rhein (abbreviated as R) were mixed at a mass ratio of 1:1:1 (abbreviated as EAR). EAR solution, EDTA solution, polymyxin B solution, and polymyxin E solution of different final concentrations were added to each well (the amount added is detailed in Tables 2 and 3), pipetted evenly, placed in a 37°C constant temperature incubator, and cultured for 18-24 hours. The absence of visible bacterial growth was used as the basis for determining the minimum inhibitory concentration (MIC).

[0042] The fractional inhibitory concentration index FICI (Fractional inhibitory concentration index) was calculated as follows: FICI = MIC (group A combined) / MIC (group A alone) + MIC (group B combined) / MIC (group B alone). After calculation, the minimum FICI value was selected as the optimal FICI for a group of combined drugs. When FICI is less than 0.5, the two drugs have a synergistic effect; when FICI is 0.5-1, the two drugs have an additive effect; when FICI is greater than 1 and less than 2, the two drugs have an irrelevant effect; when FICI is greater than 2, the two drugs have an antagonistic effect. Tables 2 and 3 show the antibacterial activity analysis of EAR combined with polymyxin B or E in the presence of different concentrations of EDTA. As shown in Tables 2 and 3, when EDTA is present, the antibacterial activity of EAR and polymyxin B or E against Acinetobacter baumannii 1415E showed synergy (FICI < 0.5). When the EDTA concentration was 256 mg / L, EAR made Acinetobacter baumannii 1415E extremely sensitive to polymyxin B or E.

[0043] Table 2

[0044]

[0045] Table 3

[0046]

[0047] Example 3 Screening of EAR mass ratio

[0048] Through Example 1, it was found that the EDTA concentration when EAR and polymyxin B or E had a synergistic effect was 256 mg / L. Then, at this EDTA concentration, the mass ratio scheme of EAR was continued to be optimized. EAR solutions of different mass ratios were prepared according to the scheme shown in Table 4. The bacterial suspension of Acinetobacter baumannii 1415E was diluted to 5×10 5 cfu / mL. 100 μL of 5×10 5 cfu / mL bacterial suspension, add different ratios of EAR solution to 200μL in the first column. Then perform two-fold gradient dilution, and the drug concentration in each subsequent column is half of the previous column. The last column is used as a blank control. The MIC of EAR is determined after culturing in a 37℃ incubator for 24 hours.

[0049] Table 4 shows the antibacterial activity analysis of EAR at different mass ratios in the presence of EDTA (256 mg / L). The results show that when EDTA is present and the mass ratio of E, A, and R is 1:1:1, the antibacterial activity of the combination EAR against Acinetobacter baumannii 1415E is most obvious, and its MIC is 4 mg / L.

[0050] Table 4

[0051]

[0052]

[0053] Example 4 Analysis of the antibacterial activity of the composition of the present invention in other clinical isolates of Acinetobacter baumannii A

[0054] The present invention tests the synergy of the composition with polymyxins and carbapenems in more clinical isolates of Acinetobacter baumannii (see Table 5-Table 8 for details). Tables 5 and 6 are the antibacterial activity analysis of EAR in combination with polymyxin B or E under the preferred scheme. The results show that when the composition of the present invention (EAR mass ratio is 1:1:1) is used in combination with polymyxin B or E, the antibacterial activity of multiple clinical isolates of Acinetobacter baumannii is synergistic, and the sensitivity of these clinical isolates of Acinetobacter baumannii to polymyxin B or E is significantly improved, so that the polymyxin-resistant bacteria are converted into polymyxin-sensitive bacteria. Moreover, Tables 7 and 8 show the antibacterial activity analysis of EAR in combination with meropenem or imipenem under the preferred scheme. The results show that the composition of the present invention also exhibits synergy when used in combination with carbapenem antibiotics, which significantly enhances the sensitivity of clinical isolates of Acinetobacter baumannii to meropenem or imipenem, and transforms the carbapenem-resistant type into the sensitive type.

[0055] Table 5

[0056] Table 6

[0057]

[0058] Table 7

[0059]

[0060] Table 8

[0061]

[0062]

[0063] Example 5 Analysis of bactericidal activity of the composition of the present invention in combination with polymyxins or carbapenems

[0064] A single colony of multidrug-resistant Acinetobacter baumannii 1415E was picked and inoculated into TSB liquid culture medium and cultured overnight at 37°C and 200 rpm. The next day, the bacterial solution was diluted with fresh sterile TSB medium to OD 600nmThe concentration of EDTA was 256 mg / L, and the concentration of EAR was 8, 16 or 32 mg / L. 20 μL of bacterial solution was taken out at 0h, 2h, 4h, 6h, 12h, and 24h, respectively, and diluted 10 times in a 96-well plate with 0.85% saline. 20 μL of the diluted bacterial solution was taken onto a TSB solid plate, spread with a sterile coating rod, and inverted in a 37°C incubator overnight. Three biological replicates were set up.

[0065] The killing curve results of the combination (EAR mass ratio of 1:1:1) and polymyxin B against Acinetobacter baumannii 1415E are as follows Figure 4 As shown, the killing curve of the combination (EAR mass ratio is 1:1:1) and meropenem against Acinetobacter baumannii 1415E is as shown Figure 5 As shown. Since Acinetobacter baumannii 1415E is resistant to polymyxins and carbapenems, 2 mg / L polymyxin B or meropenem cannot effectively kill 1415E. When 2 mg / L polymyxin B or meropenem is used in combination with 256 mg / L EDTA and EAR, the bactericidal activity of polymyxin B or meropenem against Acinetobacter baumannii 1415E is significantly improved. As the concentration of EAR gradually increases, the bactericidal time of polymyxin B or meropenem is gradually shortened. Especially when the concentration of EAR is 32 mg / L, 2 mg / L polymyxin B can completely kill 1415E in 6 hours.

[0066] Example 6 Cytotoxicity Analysis of the Composition of the Present Invention

[0067] Human hepatoma cell (Hep3B) and human renal cell (Hep293) cells were used to determine the in vitro cytotoxicity of the drug combination (EAR mass ratio of 1:1:1). First, the cells were uniformly seeded in a 48-well plate (final density of 2×10 4 Cells / well) were added and cultured overnight in an incubator at 37°C with 5% CO2. The final concentration of EDTA in each well was adjusted to 265 mg / L, and the final concentration of EAR was adjusted to 0, 2, 4, 8, 16, 32, 64 and 128 mg / L. The 48-well plate was treated with 5% CO2 at 37°C for 24 hours, and then 25 μL of CCK-8 reagent was added to each well and incubated for 1 hour. The absorbance of each well at 450 nm was measured using a microplate reader. The cell suspension plus CCK-8 reagent was used as a positive control, and the culture medium plus CCK-8 reagent was used as a negative control. The experiment was repeated twice.

[0068] The results showed that when the EAR concentration did not exceed 32 mg / L, the composition had no significant effect on the viability of Hep3B and Hep293 cells; however, when the EAR concentration was 64 and 128 mg / L, the viability of Hep3B and Hep293 cells was significantly inhibited ( Figure 6 ). The above results showed that EAR showed no toxicity to Hep3B and Hep293 cells at lower concentrations.

[0069] Example 7 Analysis of the therapeutic effect of the composition of the present invention combined with polymyxin B on infected mice

[0070] BALB / c mice were randomly divided into groups, with 3 mice in each group. After the back skin of the mice was burned with high temperature, OD 600nm The wound was smeared with 20μL of 1415E bacterial solution of Acinetobacter baumannii, and the infection lasted for 3 consecutive days. After that, the wound was gently wiped with a cotton swab every day at a dosage of 32mg / kg EDTA+4mg / kg EAR+2mg / kg PMB, as the drug group, with an EAR mass ratio of 1:1:1. An equal volume of 1×PBS solution was used as the control group to observe and record the changes in the mouse wounds, and the diameter of the wound area was measured in two directions every two days. The results showed that when the treatment reached the 8th day, the skin wound healing speed of the mice in the drug group was significantly faster than that of the control group ( Figure 7 ). On the 12th day of treatment, the skin wound area of ​​mice in the drug group had increased from the initial 94.98 mm 2 Down to 13.95±6.60mm 2 ; At this time, the skin wound area of ​​mice in the control group was 34.25±3.89mm 2 , the wound area was significantly larger than that in the treatment group.

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composition for resisting multidrug-resistant Acinetobacter baumannii, characterized in that: It is composed of rhein, aloe-emodin and rhein.

2. The composition according to claim 1, characterized in that The mass ratio of the rhein, aloe-rhein and rhein is 1:1:

1.

3. Use of the composition according to claim 1 or 2 in the preparation of an antibacterial preparation.

4. An antibacterial preparation, characterized in that The active ingredients include the composition according to claim 1 or 2 and EDTA.

5. The antibacterial preparation according to claim 4, characterized in that The concentration of the EDTA is 256 mg / L.

6. The antibacterial preparation according to claim 4, characterized in that The antibacterial preparation is a pharmaceutical preparation used for treating diseases caused by Acinetobacter baumannii.

7. The antibacterial preparation according to claim 6, characterized in that The Acinetobacter baumannii is a multidrug-resistant Acinetobacter baumannii.

8. Use of the composition according to claim 1 or 2 in the preparation of a drug for sensitizing multidrug-resistant Acinetobacter baumannii.

9. Use of the composition according to claim 1 or 2 in the preparation of a drug for shortening the killing time of polymyxins and carbapenems antibiotics against multidrug-resistant Acinetobacter baumannii.

10. Use of the composition according to claim 1 or 2 in the preparation of a medicament for accelerating the healing of skin wounds infected by multidrug-resistant Acinetobacter baumannii.

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