A pharmaceutical preparation containing tryptophan for use against biofilm formation of acinetobacter baumannii and uses thereof

Abstract

The application relates to a medicine preparation containing tryptanthrin for resisting biofilm formation of Acinetobacter baumannii and application thereof, and belongs to the technical field of biological medicine. The medicine preparation containing tryptanthrin for resisting biofilm formation of Acinetobacter baumannii contains tryptanthrin and dimethyl sulfoxide, and the Acinetobacter baumannii is selected from one of standard strains, drug-resistant strains and sensitive strains. The tryptanthrin in the medicine preparation can affect the development process of the biofilm, thereby inhibiting the biofilm of the Acinetobacter baumannii, and the tryptanthrin does not affect the growth of the bacteria, so that the drug resistance is not easily generated. When the concentration of the tryptanthrin is 20 mu g / mL, the effect of more than 50% of the biofilm being inhibited can be achieved without affecting the growth of the bacteria, so that the tryptanthrin as the biofilm inhibitor can be used as an anti-biofilm medicine in the clinic, and has a good application prospect as an anti-biofilm infection coating material of medical implant equipment and artificial organs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of tryptophan in inhibiting biofilms in Acinetobacter baumannii infection. Background Technology

[0002] Acinetobacter baumannii (Ab) is an aerobic, non-fermenting, Gram-negative opportunistic pathogen listed by the Infectious Diseases Society of America (IDSA) as one of the pathogens of "ESKAPE" bacteria. It can cause nosocomial infections such as respiratory tract infections, urinary tract infections, bacteremia, or secondary meningitis. Furthermore, Acinetobacter baumannii exhibits high resistance to β-lactam antibiotics, carbapenems, fluoroquinolones, and aminoglycosides, significantly limiting treatment efficacy. Multiple studies have shown a positive correlation between drug resistance and biofilm formation in Acinetobacter baumannii.

[0003] Generally, antimicrobial agents or bactericides are used to control biofilms to inhibit bacterial growth, but a major problem with this approach is that continued use can lead to increased bacterial resistance. Recent research has found that natural plant extracts can act as biofilm inhibitors; these inhibitors can interfere with biofilm development mechanisms without affecting bacterial growth, thereby reducing the development of resistance.

[0004] Tryptanthrin is an indolequinazoline, an alkaloid derived from the indigo plant. It is a naturally occurring compound obtained from various plants and cell cultures, including yeast. Studies have shown that tryptanthrin possesses multiple activities related to immune inflammation, regulating the expression levels of IL-2, IL-10, and TNF-α. Furthermore, tryptanthrin is an orally effective inhibitor of leukotriene (LT) biosynthesis. Additionally, tryptanthrin has been found to have antiparasitic and antibacterial activities, exhibiting activity against Gram-positive bacteria, Gram-negative bacteria, fungi, and parasites. However, its inhibitory effect on Acinetobacter baumannii biofilms remains unstudied. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide tryptamine ketone that can inhibit the formation of Acinetobacter baumannii biofilm without affecting bacterial growth.

[0006] Studies on the effects of tryptophan on the surface motility and extracellular eDNA content of Acinetobacter baumannii revealed that tryptophan can reduce the aggregation of Acinetobacter baumannii, thereby controlling the biofilm development process and ultimately inhibiting biofilm formation. Tryptophan significantly reduced the surface motility and extracellular eDNA of clinically observed Acinetobacter baumannii. This demonstrates that tryptophan can effectively inhibit the formation of Acinetobacter baumannii biofilms. At a concentration of 20 μg / ml, tryptophan can achieve a biofilm inhibition rate of over 50% without affecting bacterial growth, proving that tryptophan, as a biofilm inhibitor, has promising applications as a clinically used anti-biofilm drug and as an anti-biofilm coating material for medical implants and artificial organs.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] On one hand, the present invention provides a pharmaceutical formulation for inhibiting biofilm formation by Acinetobacter baumannii, the pharmaceutical formulation comprising tryptophan and dimethyl sulfoxide (DMSO).

[0009] The aforementioned pharmaceutical preparation for inhibiting biofilm formation by Acinetobacter baumannii, wherein Acinetobacter baumannii is selected from one of the following: standard strains, drug-resistant strains, and susceptible strains.

[0010] The aforementioned pharmaceutical preparation for inhibiting biofilm formation by Acinetobacter baumannii, wherein the mass concentration of tryptophan in the pharmaceutical preparation is 10-40 μg / mL, preferably 10-20 μg / mL.

[0011] The pharmaceutical preparation for inhibiting biofilm formation by Acinetobacter baumannii is characterized in that the dosage form of the pharmaceutical preparation is selected from one of injection, ointment or tablet.

[0012] Secondly, the present invention provides the use of any of the pharmaceutical preparations described herein in the treatment of Acinetobacter baumannii infection for purposes other than disease diagnosis and treatment.

[0013] In the aforementioned application, the anti-Acinetobacter baumannii drug preparation effectively inhibits the formation of Acinetobacter baumannii biofilm without affecting bacterial growth.

[0014] Thirdly, the present invention provides the use of tryptophan in inhibiting biofilm formation in Acinetobacter baumannii for non-disease diagnosis and treatment purposes.

[0015] Fourthly, the present invention provides a medical implant material for preventing biofilm infection, comprising a medical implant material and a tryptamine solution coated thereon, wherein the solvent of the tryptamine solution is dimethyl sulfoxide.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention provides the application of tryptophan in the preparation of formulations for treating Acinetobacter baumannii infection. Tryptophan can reduce the aggregation of Acinetobacter baumannii, thereby controlling the biofilm development process and ultimately inhibiting biofilm formation. When tryptophan is at 20 μg / mL, bacterial growth is not affected, thus reducing the likelihood of drug resistance. Furthermore, over 50% biofilm inhibition was observed in all three bacterial strains, demonstrating that tryptophan can effectively inhibit Acinetobacter baumannii biofilm formation without affecting bacterial growth. Attached Figure Description

[0018] Figure 1 The effects of tryptophan on the biofilm of Acinetobacter baumannii standard strain ATCC17978, drug-resistant strain AB108, and susceptible strain AB102 were investigated. In this study, A represents the changes in biofilm of strain ATCC17978 after treatment with different concentrations of the drug; B represents the changes in biofilm of strain AB108 after treatment with different concentrations of the drug; and C represents the changes in biofilm of strain AB102 after treatment with different concentrations of the drug.

[0019] Figure 2 The effect of different concentrations of tryptophan on the growth of Acinetobacter baumannii;

[0020] Figure 3 The effect of tryptophan treatment on the surface motility of Acinetobacter baumannii; where *P<0.05, **P<0.01;

[0021] Figure 4 The effect of tryptophan treatment on the release of Acinetobacter baumannii eDNA; where *P<0.05, **P<0.01;

[0022] Figure 5 Different concentrations of tryptophan significantly inhibited biofilm formation in Acinetobacter baumannii. Detailed Implementation

[0023] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0024] Example 1: Effect of tryptophan on biofilm formation ability of Acinetobacter baumannii using crystal violet staining method under different materials

[0025] The strains used in the testing process of this invention were the standard strain ATCC17978, the drug-resistant strain AB108, and the sensitive strain AB102, all of which were deposited in the Pathogenic Microbiology Laboratory of Yangzhou University. Tryptamine was used as the solvent, DMSO, and after preparing a stock solution, it was diluted to the required concentration with MH broth medium.

[0026] The ability of Acinetobacter baumannii to form static biofilms, including clinical isolates and standard strains, was analyzed using sterile polystyrene plates and glass tubes. OD was collected. 600nm A bacterial suspension with a concentration of 0.5 was diluted 1:100 with MH broth. 200 μL of the diluted bacterial suspension was inoculated into sterile 6-well plates or glass tubes, with culture medium used as a negative control. The plates were then incubated at 37°C for 24 h. The bacterial suspension was discarded. The plates were washed with phosphate-buffered saline (PBS) to remove airborne bacteria and then air-dried. Next, 200 μL of 0.4% (w / v) crystal violet was used for staining in the dark for approximately 30 min. After staining, the plates were rinsed with phosphate-buffered saline (PBS) until no obvious crystal violet residue remained in the negative control wells, and then air-dried again.

[0027] The results are as follows Figure 1 As shown, *Acinetobacter baumannii* formed a thin, ring-shaped biofilm on the glass surface, but a robust ring-shaped biofilm on the polyethylene surface. Furthermore, tryptophan exhibited more significant anti-ring biofilm activity on the glass surface than on the polystyrene surface. These results indicate that all three *Acinetobacter baumannii* strains can form biofilms on different materials, and tryptophan can effectively inhibit biofilm formation on all materials.

[0028] Example 2: Effect of tryptophan on the growth of Acinetobacter baumannii

[0029] In the absence of tryptophan and in the presence of different concentrations of tryptophan, the test strain was inoculated into MH broth at a concentration of 1% and cultured at 37°C and 200 rpm for 24 hours. Starting from the start of inoculation, absorbance of the bacterial suspension was measured at OD 600 nm at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 16h, 20h, and 24h, and bacterial counts were performed. A growth curve was plotted with time on the x-axis. Figure 2 The figure shows the growth curves of different concentrations of tryptophan on the growth of Acinetobacter baumannii.

[0030] Example 3: Effect of tryptophan on the motion of Baumann-stationary surfaces

[0031] First, a culture medium containing 0.25% agarose, 1% tryptone, and 0.5% yeast (Oxoid product from the UK) was prepared for surface motility testing. Tryptophan was added to the medium to a final concentration of 5 μg / mL, with DMSO (0.1%) used as a negative control. 0.2 μL of Acinetobacter baumannii cultured overnight was placed on a culture plate using a sterile pipette tip. After incubation at 37°C for approximately 12 hours, the size of the halos produced by the bacteria on the agar plates was measured. Results are shown below. Figure 3 It can be seen that adding 5 μg / mL tryptophan can inhibit its surface movement, indicating that tryptophan has a certain inhibitory effect on the initial formation and movement of biofilm.

[0032] Example 4: eDNA content detection of the effect of tryptophan on inhibiting biofilm formation

[0033] To further demonstrate that tryptophan can effectively inhibit Acinetobacter baumannii biofilm, the cDNA content of the three strains of Acinetobacter baumannii that were previously shown to be effective was detected. In 6-well polystyrene plates, the test strains were inoculated into MH broth at a concentration of 1%. Different concentrations of tryptophan were added to the wells, and the plates were incubated at 37°C for 24 h to form a biofilm. The bacterial culture was discarded, the biofilm was scraped off, resuspended in TE buffer, and vortexed vigorously for 1 h. The supernatant was then collected by centrifugation at 8000 rpm for 10 min, and loading buffer was added for color development. The amount of cDNA present in the supernatant was separated by 1.5% (w / v) agarose gel electrophoresis. Results are shown below. Figure 4 It can be seen that the eDNA release content of Acinetobacter baumannii was significantly reduced after treatment with tryptophan, proving that tryptophan has an inhibitory effect on biofilm formation.

[0034] Example 5: Different concentrations of tryptophan can significantly inhibit the formation of Acinetobacter baumannii biofilm.

[0035] The effect of tryptophan on biofilm formation in Acinetobacter baumannii was analyzed using sterile polystyrene plates with crystal violet staining. OD was collected. 600nm A bacterial suspension with a concentration of 0.5 was diluted 1:100 with MH broth. 200 μL of the diluted bacterial suspension was inoculated into a sterile 96-well plate, with culture medium used as a negative control. The plate was then incubated at 37°C for 24 h. The bacterial suspension was discarded. The plate was washed with phosphate-buffered saline (PBS) to remove airborne bacteria and then air-dried. Next, it was stained with 200 μL of 0.4% (w / v) crystal violet in the dark for approximately 30 min. After staining, the plate was rinsed with PBS until no obvious crystal violet residue remained in the negative control wells and then air-dried again. Finally, 200 μL of 95% ethanol was added to each well to dissolve the bacteria, and the absorbance was measured at 590 nm using a multi-plate reader.

[0036] See results Figure 5It was found that tryptophan showed a concentration-dependent biofilm inhibition effect on all three strains. At a concentration of 20 μg / mL, tryptophan showed strong anti-biofilm activity against Acinetobacter baumannii, with inhibition rates of 59% (ATCC17978), 85% (AB102), and 63% (AB108), respectively.

[0037] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a pharmaceutical formulation containing tryptophan and dimethyl sulfoxide in the preparation of a drug for inhibiting Acinetobacter baumannii biofilm formation, characterized in that, The Acinetobacter baumannii is selected from one of the standard strain, drug-resistant strain, and sensitive strain; the mass concentration of tryptophan in the drug preparation is 10-40 μg / mL.

2. The application as described in claim 1, characterized in that, The mass concentration of tryptophan in the pharmaceutical preparation is 10–20 μg / mL.

3. The application as described in claim 1, characterized in that, The dosage form of the pharmaceutical preparation is selected from one of injections, ointments, or tablets.

4. Application of tryptophan in inhibiting biofilm formation of Acinetobacter baumannii for in vitro non-disease diagnosis and treatment purposes.

5. The application of medical implant materials and the tryptophan solution coated thereon in the preparation of implant materials for preventing biofilm infection, characterized in that, The solvent for the tryptophan solution is dimethyl sulfoxide, and the biofilm is an Acinetobacter baumannii biofilm.

6. Application of tryptophan in the preparation of formulations for treating Acinetobacter baumannii infection.

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