CN114539378B - An antimicrobial peptide and its application in preventing and treating multi-drug resistant bacteria - Google Patents

Abstract

The invention provides an antibacterial peptide and application thereof in preventing and treating multi-drug-resistant bacteria, the polypeptide has good antibacterial effect, and meanwhile, the antibacterial peptide and antibiotics are combined to kill the multi-drug-resistant bacteria. The amino acid sequence of the antibacterial peptide provided by the invention is SEQ ID NO. 1. The antibacterial peptide provided by the invention is used for preparing antibacterial products. The antibacterial peptide provided by the invention has obvious antibacterial effect on common pathogenic bacteria, and can effectively kill multi-drug resistant bacteria when being used in combination with antibiotics.

Description

An antimicrobial peptide and its application in preventing and treating multi-drug resistant bacteria

technical field

The invention belongs to the technical field of antimicrobial peptide applications, and in particular relates to an antimicrobial peptide and its application in preventing and treating multidrug-resistant bacteria.

Background technique

The overuse and abuse of antibiotics has led to the emergence of multi-drug resistant (MDR) bacteria. It is estimated that infections caused by antibiotic-resistant bacteria currently kill about 70,000 people worldwide each year, and without new treatments, infections caused by multi-drug-resistant bacteria could kill an additional 10 million people worldwide each year by 2050, more than cancer . Therefore, antibiotic resistance has become a growing threat to global human public health, and there is an urgent need to explore and develop new antibacterial strategies.

The two most promising strategies to address this problem are the use of antimicrobial peptides (AMPs) and combination therapy. AMPs are short peptides of 10 to 50 amino acids in length, naturally produced by various organisms including fungi, plants and animals, and were first isolated from human leukocyte extracts by Zeya and Spitznagel. AMPs, as components of the host's first line of defense against pathogen invasion, exhibit broad antibacterial activity against different Gram-positive and Gram-negative bacteria, including MDR bacteria. Despite highly diverse amino acid sequences, most AMPs are amphipathic, with cationic (positively charged) and hydrophobic properties, which allow AMPs to efficiently interact with anionic microbial surfaces and enter cell membranes, altering membrane permeability and thereby impair internal homeostasis.

Combination therapy refers to the combination of traditional antibiotics with other antibiotics or AMPs to improve the therapeutic effect compared with single drug therapy. Combination therapy with multiple conventional antibiotics has become a common treatment for multidrug-resistant infections, but the combination of AMPs and antibiotics has largely lacked extensive research.

Contents of the invention

The invention firstly provides an antimicrobial peptide and its application in preventing and treating multidrug-resistant bacteria. The polypeptide has good antibacterial effect, and at the same time, the antimicrobial peptide is used in combination with antibiotics to kill multidrug-resistant bacteria.

The antimicrobial peptide provided by the present invention has an amino acid sequence as follows:

VFILSKYRWVMRFVPWCKALLKRFGKYKKHAK (SEQ ID NO: 1)

The antibacterial peptide provided by the present invention is used to prepare antibacterial products;

Another aspect of the present invention also provides an antibacterial product, which contains the above-mentioned antimicrobial peptide and antibiotic;

The antibiotics include Oxacillin, Vancomycin, Streptomycin, Azithromycin and the like.

The antimicrobial peptide provided by the invention has obvious antibacterial effect on common pathogenic bacteria, and can effectively kill multi-drug resistant bacteria when used in combination with antibiotics.

Detailed ways

The present invention will be described in detail below through specific examples.

Embodiment 1: Screening and detection of antimicrobial peptides

The antimicrobial peptide with amino acid sequence VFILSKYRWVMRFVPWCKALLKRFGKYKKHAK (SEQ ID NO: 1) was screened from chicken bursa and named AUDap. After the Oxford cup experiment, it was found that the antimicrobial peptide AUDAP has obvious antibacterial effect on non-resistant Gram-negative bacteria Escherichia coli and Gram-positive bacteria Bacillus subtilis; However, the antibacterial effect on Escherichia coli 577 (ATCC 577, E.coli 577) and Klebsiella pneumoniae 2182 (ATCC 2182, K.pneumonia 2182) with drug-resistant bacteria was not significant.

The minimum inhibitory concentration (MIC) results of the AUDAP antimicrobial peptides against the above four strains are shown in Table 1.

Table 1: Minimum inhibitory concentration MIC (μg/mL) of AUDAP antimicrobial peptides against four strains

Therefore, the AUDAP antimicrobial peptide screened by the present invention has antibacterial and antibacterial effects on both Gram-negative bacteria and Gram-positive bacteria.

Embodiment 2: Cytotoxicity detection of AUDAP antimicrobial peptide

The survival rate of mouse macrophage cells under the action of AUDAP antimicrobial peptide was determined by MTT colorimetry, and the results showed that AUDAP antimicrobial peptide had no obvious toxicity to RAW264.7 mouse macrophages at a concentration of 15 μg/ml Injury effect, indicating that AUDAP antimicrobial peptide does not have significant toxicity to mammalian normal tissue cells.

Determination of erythrocyte hemolysis of the antimicrobial peptide AUDAP, the specific steps are as follows:

1) Anticoagulate fresh mouse whole blood with heparin, centrifuge at 3000 r/min at 4°C for 10 min, wash the precipitate with normal saline, and resuspend according to 8% (v/v);

2) Mix 100 μL of erythrocyte suspension with 100 μL of antimicrobial peptide solutions of different concentrations. The mixed system was placed in a 37°C incubator and incubated for 1h, taken out and centrifuged at 3000r/min at 4°C for 10min, the supernatant was taken, and the absorbance at 570nm was measured with a microplate reader.

The results show that the AUDAP antimicrobial peptide of the present invention has hemolysis at a concentration of 75 μg/mL, indicating that the antimicrobial peptide of the present invention has almost no cytotoxicity to cells and can be developed into an antibacterial drug

Embodiment 3: AUDAP antimicrobial peptide is used in combination with antibiotics

The detected strains were Escherichia coli 577 (ATCC 577, E.coli 577), Klebsiella pneumoniae 2182 (ATCC 2182, K.pneumonia 2182) and methicillin-resistant Staphylococcus aureus USA500 ( S. aureus USA500).

The AUDAP antimicrobial peptide was synthesized by a biological company, and the carboxyl terminal was amidated during synthesis, and the purity of the synthetic product was greater than 98%.

The synthesized AUDAP antimicrobial peptide was dissolved in 1% (v/v) trifluoroethanol solution, configured as a mother solution with a final concentration of 5 mg/ml, and stored at -80°C after aliquoting.

Inoculate the strains to be tested in LB liquid medium to expand the culture until the bacteria are cultured to the logarithmic growth phase. After the bacteria are collected by centrifugation, the bacterial sediment is repeatedly washed with PBS solution, and the obtained bacteria are finally resuspended with PBS. Use a hemocytometer to count under a microscope, and dilute the concentration to about 2×10 ⁶ CFU/ml for use.

To examine the synergistic effect between AUDAP antimicrobial peptides and antibiotics (oxacillin, vancomycin, streptomycin and azithromycin), checkerboard microdilution was performed using the method described by Sopirala et al. Dilute the antimicrobial peptides and antibiotics with LB, respectively, and combine the antimicrobial peptides and antibiotics with different concentrations in 96 plate wells: keep the concentration of antimicrobial peptides in each well in each row of the 96-well plate unchanged, add 10 μl of antibiotics to make The concentrations are 2×, 1×, 0.5×, 0.25×, 0.125×, 0.0625× and 0.03125×MIC in turn; the concentration of antibiotics in each column of the 96-well plate is constant, and 10 μl of antimicrobial peptide solution is added to make the concentrations 2 ×, 1×, 0.5×, 0.25×, 0.125×, 0.0625× and 0.03125×MIC, add 10ul and 170ul of fresh LB with a concentration of 3× ¹⁰⁶ CFU/ml bacterial solution to each well, set up 3 parallel samples, and shake Evenly, incubate at 37°C for 10h. A PBS negative control group without antibiotic antimicrobial peptides and a blank control group containing only LB medium were set up. The lowest concentration without bacterial growth is the MIC when combined, and the absorbance at 600nm is measured under the Multiskan MK3 microplate reader to determine the MIC. The synergistic effect between antimicrobial peptides and antibiotics was assessed using the Fractional Inhibitory Concentration Index FIC Index (FICI) method.

FICA = MIC when drug A is used in combination/MI when drug A is used alone,

FICB = MIC when drug B is used in combination/MIC when used alone,

FICI=FICA+FICB,

Synergistic effect: FICI≤0.5; additive effect: 0.5<FICI≤1; no effect: 1<FICI≤4; antagonism FICI>4.

The results showed that AUDAP antimicrobial peptide and oxacillin treated S.aureus USA500, and AUDAP antimicrobial peptide and azithromycin treated K.pneumoniae 2182 had FICI≤0.4, all of which showed synergistic antibacterial effect. AUDAP antimicrobial peptide and vancomycin against S.aureus USA500 and AUDAP antimicrobial peptide and streptomycin against E.coli 577 were 0.5<FICI≤0.6, showing a synergistic antibacterial effect. When AUDAP antimicrobial peptides were combined with oxacillin to treat S.aureus USA500, the concentration of AUDAP antimicrobial peptides decreased to 0.04×MIC. When AUDAP antimicrobial peptide was combined with vancomycin, the concentration of AUDAP antimicrobial peptide decreased to 0.6×MIC.

The above results indicated that the combination of AUDAP antimicrobial peptides and conventional antibiotics (oxacillin, vancomycin, streptomycin and azithromycin) showed a synergistic antibacterial effect on drug-resistant strains.

sequence listing

<110> Qingdao Agricultural University

<120> An antimicrobial peptide and its application in the prevention and treatment of multi-drug resistant bacteria

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 32

<212> PRT

<213> Artificial Sequence

<400> 1

Val Phe Ile Leu Ser Lys Tyr Arg Trp Val Met Arg Phe Val Pro Trp

1 5 10 15

Cys Lys Ala Leu Leu Lys Arg Phe Gly Lys Tyr Lys Lys His Ala Lys

20 25 30

Claims (5)

1. The antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is SEQ ID NO. 1.

2. Use of the antibacterial peptide of claim 1 for the preparation of antibacterial products.

3. An antimicrobial preparation comprising a pharmacologically effective concentration of the antimicrobial peptide of claim 1.

4. The antimicrobial article of claim 3, further comprising an antibiotic.

5. The antimicrobial article of claim 4, wherein the antibiotic is any one or more of oxacillin, vancomycin, streptomycin, and azithromycin.