Application of L-lysine in enhancing the susceptibility of Vibrio alginolyticus to clindamycin

Abstract

This invention discloses the application of L-lysine in improving the sensitivity of Vibrio alginolyticus to clindamycin. This invention discovers that the combined use of L-lysine and clindamycin can significantly increase reactive oxygen species within Vibrio alginolyticus, thereby increasing the amount of clindamycin entering the bacteria and ultimately leading to bacterial death. Therefore, L-lysine can improve bacterial sensitivity to clindamycin, thus overcoming the problem of bacterial resistance. The small molecule metabolite provided by this invention, when used in combination with clindamycin, can significantly enhance the bactericidal effect of clindamycin, exhibiting better efficacy, higher safety, and greater operability compared to existing methods that use clindamycin alone as an antibacterial drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of L-lysine in improving the sensitivity of Vibrio alginolyticus to clindamycin. Background Technology

[0002] Vibrio alginolyticus is one of the most abundant pathogens in the marine environment, found in seawater worldwide. Antibiotics are the primary means of controlling Vibrio diseases. Initially, antibiotics offered advantages such as rapid effectiveness and low cost; however, overuse can lead to bacterial resistance and even the emergence of superbugs. Developing new, safe, and efficient methods for disease control has become an urgent problem to solve.

[0003] Currently, treatment for drug-resistant bacteria mainly relies on the development of novel antibiotics, the combined use of antibiotics, and increasing the dosage of antibiotics. In recent years, small molecule metabolites, as end products of gene and protein expression, can synergistically enhance the bactericidal effect of antibiotics, which is of great significance for controlling bacterial infections, especially those caused by drug-resistant bacteria.

[0004] L-Lysine is an essential amino acid required for protein biosynthesis and plays an important regulatory role in bacterial growth, physiological characteristics, and cell morphology. Studies have found that L-lysine can induce oxidative stress and cell membrane damage in Pseudomonas aeruginosa, indicating that L-lysine has an important regulatory role in bacterial physiological metabolism. However, there are currently no reports on whether L-lysine can improve the clearance of pathogens by antibiotics. Summary of the Invention

[0005] This invention discovers that L-lysine can enhance the sensitivity of Vibrio alginolyticus to clindamycin, thereby overcoming the problem of bacterial resistance.

[0006] To achieve the above objectives, the present invention is implemented through the following solution:

[0007] Application of L-lysine in improving the sensitivity of Vibrio alginolyticus to clindamycin, wherein the Vibrio alginolyticus is a highly virulent wild-type strain of Vibrio alginolyticus, specifically strain EPGS02040 Vibrio alginolyticus EPGS020401, strain preservation number CCTCC No. AB209306.

[0008] The molar ratio of L-lysine to clindamycin is 1:0.18-60.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] This invention is the first to discover that L-lysine can enhance the sensitivity of Vibrio alginolyticus to clindamycin, thereby overcoming the problem of bacterial resistance. Further research revealed that L-lysine induces an increase in the concentration of reactive oxygen species (ROS) within the bacteria, leading to bacterial death or cessation of growth. Compared to existing methods that use clindamycin alone as an antibacterial drug, this approach offers better efficacy, higher safety, and greater operability, demonstrating promising application prospects. Attached Figure Description

[0011] Figure 1 As an example, the growth of Vibrio alginolyticus after adding L-lysine to clindamycin.

[0012] Figure 2 As an example, the growth of Vibrio alginolyticus was observed after adding different concentrations of L-lysine to clindamycin.

[0013] Figure 3 The following is an example of how the ROS levels in bacteria changed after the addition of L-lysine. Detailed Implementation

[0014] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention includes, but is not limited to, the scope represented by the following embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials.

[0015] This invention discovered that the addition of L-lysine significantly enhanced the bactericidal effect of clindamycin against Vibrio alginolyticus resistant bacteria, exhibiting an L-lysine concentration-dependent effect. The results indicate that L-lysine can enhance the sensitivity of Vibrio alginolyticus to clindamycin.

[0016] This invention discovers that L-lysine increases the concentration of reactive oxygen species (ROS) in Vibrio alginolyticus, thereby causing oxidative damage to the cells and ultimately leading to bacterial death.

[0017] The Vibrio alginolyticus used in this invention is a highly virulent wild-type strain, specifically strain EPGS020401, classified as *Vibrio alginolyticus* EPGS020401, with accession number CCTCC No. AB209306, accession date October 24, 2009, and deposited at the China Center for Type Culture Collection in Wuhan, China. The L-lysine used in this invention is produced by Shanghai Lanji Biotechnology Co., Ltd., with CAS number 56-87-1. Example

[0018] 1. Determination of drug resistance of Vibrio alginolyticus strain EPGS02040

[0019] First, the minimum inhibitory concentration (MIC) of Vibrio alginolyticus EPGS02040 against clindamycin was determined. The results showed that the MIC of Vibrio alginolyticus strain EPGS02040 against clindamycin was 76.6 μg / mL, indicating that Vibrio alginolyticus EPGS02040 was resistant to clindamycin.

[0020] 2. Preparation of test samples

[0021] Vibrio alginolyticus EPGS02040 was inoculated at a rate of 1% into 5 mL of LBS (3% (w / v) NaCl, 1% (w / v) Tryptone, 0.5% (w / v) Yeast Extract) medium and cultured with shaking at 30°C and 200 rpm until OD was reached. 600 =1.

[0022] 3. L-Lysine enhances the susceptibility of Vibrio alginolyticus EPGS02040 to clindamycin.

[0023] To investigate whether the addition of L-lysine improved the sensitivity of Vibrio alginolyticus EPGS02040 to clindamycin, the prepared bacterial samples were divided into two groups: a control group (control group 1: clindamycin only at 38.3 μg / mL (1 / 2 MIC)) and an experimental group (clindamycin and 10 mL L-lysine were added). After 24 h of treatment, 200 μL of bacterial culture was taken to determine its OD. 600 .

[0024] The results are as follows Figure 1 As shown, the sensitivity of Vibrio alginolyticus to clindamycin increased by 2.69 times after the addition of L-lysine.

[0025] This result indicates that the addition of L-lysine can significantly improve the sensitivity of Vibrio alginolyticus to clindamycin.

[0026] 4. L-Lysine enhances the sensitivity of Vibrio alginolyticus to clindamycin in a concentration-dependent manner.

[0027] To investigate whether the enhancement of clindamycin sensitivity in Vibrio alginolyticus EPGS02040 by L-lysine is concentration-dependent, experiments were conducted with different concentrations of L-lysine added before the addition of clindamycin. In prepared bacterial samples, 38.3 μg / mL (1 / 2 MIC) of clindamycin was added first, followed by the addition of 0, 0.5, 1, 1.5, 2, 2.5, 3, 6, and 8 mM of L-lysine, respectively. After incubation at 30℃ and 200 rpm for 12 h on a shaker, 200 μL of the bacterial solution was taken to determine its OD value. 600 .

[0028] The results are as follows Figure 2As shown, in the presence of clindamycin, within a certain range, clindamycin inhibits bacterial growth as the concentration of added L-lysine increases; however, when the concentration of L-lysine continues to increase, the bactericidal efficiency of kanamycin against bacteria decreases.

[0029] 5. L-Lysine increases ROS in Vibrio alginolyticus.

[0030] As shown in the examples, the addition of L-lysine significantly increased the sensitivity of Vibrio alginolyticus to clindamycin, but the specific mechanism of action remains unclear. To investigate the mechanism of action of L-lysine entering the bacterial interior, the experiment (sample preparation was performed according to step 2 of the examples) was divided into two groups: one experimental group (added with 38.3 μg / mL (1 / 2 MIC) clindamycin and 10 mL L-lysine) and one control group (added with only 38.3 μg / mL (1 / 2 MIC) clindamycin). Both groups were incubated on a shaker at 30°C and 200 rpm for 9 h. One mL of bacterial culture from each group was centrifuged at 4°C and 5000 rpm for 5 min, the supernatant was discarded, and then 1 mL of 10 μmol / L 2,7-dichlorodihydrofluorescein diacetate solution was added. After incubation at 37°C for 20 min, the supernatant was removed by centrifugation. The bacterial cells were washed three times with phosphate buffer and then resuspended in phosphate buffer. The fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. This process was repeated three times.

[0031] The results are as follows Figure 3 As shown, in the presence of clindamycin, the addition of L-lysine increased the ROS in bacteria by 4.91 times. This result indicates that the addition of L-lysine can induce oxidative damage in bacteria, ultimately leading to bacterial death.

[0032] In summary, the addition of L-lysine to clindamycin significantly enhances the sensitivity of Vibrio alginolyticus to clindamycin, thereby achieving the purpose of bacteriostasis or bactericidal action. Therefore, this invention seeks protection for the application of L-lysine in improving the sensitivity of Vibrio alginolyticus to clindamycin.

[0033] The above content provides a further detailed description of the present invention. It should not be construed that the specific embodiments of the present invention are limited to this. For those skilled in the art, various experiments can be conducted without departing from the concept of the present invention, and all such experiments should be considered to fall within the scope of protection of the present invention as determined by the submitted claims.

Claims

1. The application of L-lysine in the preparation of drugs that enhance the sensitivity of Vibrio alginolyticus to clindamycin, characterized in that, The Vibrio alginolyticus strain mentioned is a wild-type virulent strain of Vibrio alginolyticus, specifically EPGS020401 Vibrio alginolyticus EPGS020401, strain preservation number CCTCC No. AB209306.

2. The application according to claim 1, characterized in that, The molar ratio of L-lysine to clindamycin is 1:0.18-60.

Images