阅读说明：本技术 血根碱在多重耐药雷氏普罗威登斯菌生物被膜抑制和清除中的应用 (Application of sanguinarine in inhibition and removal of multiple-drug-resistant providencia rettgeri biofilm ) 是由 钱卫东 刘淼 付玉婷 孙照欢 何维维 李康帆 高悦 李靖原 于 2019-12-10 设计创作，主要内容包括：本发明公开了血根碱在多重耐药雷氏普罗威登斯菌生物被膜抑制和清除中的应用,用于解决耐药雷氏普罗威登斯菌生物被膜耐药性以及一旦形成难以清除的技术问题。根据血根碱对雷氏普罗威登斯菌具有较好的生物被膜抑制和清除作用,能够抑制雷氏普罗威登斯菌生物被膜的形成,生物被膜最低抑制浓度为7.81μg/mL。血根碱能有效清除成熟的雷氏普罗威登斯菌生物被膜,且生物被膜最低清除浓度为125μg/mL。其采用天然产物血根碱对多重耐药雷氏普罗威登斯菌生物被膜抑制和清除作用,可有效缓解或解决雷氏普罗威登斯菌生物被膜引起的感染治疗困难,提高治愈率,为抑制雷氏普罗威登斯菌生物被膜的形成和清除成熟的生物被膜提供新的先导化合物,具有重要的实践意义。(The invention discloses application of sanguinarine in inhibition and removal of multiple drug-resistant providencia rettgeri biofilm, which is used for solving the technical problems of drug resistance of drug-resistant providencia rettgeri biofilm and difficulty in removal once the drug-resistant providencia rettgeri biofilm is formed. According to the characteristic that sanguinarine has better biofilm inhibition and removal effects on providencia rettgeri, the formation of the biofilm of the providencia rettgeri can be inhibited, and the minimum inhibition concentration of the biofilm is 7.81 mu g/mL. The sanguinarine can effectively remove mature providencia rettgeri biofilm, and the minimum removal concentration of the biofilm is 125 mug/mL. The natural product sanguinarine is adopted to inhibit and remove the multiple drug-resistant providencia rettgeri biofilm, so that the infection treatment difficulty caused by the providencia rettgeri biofilm can be effectively relieved or solved, the cure rate is improved, a new lead compound is provided for inhibiting the formation of the providencia rettgeri biofilm and removing the mature biofilm, and the method has important practical significance.)

1. Application of sanguinarine in inhibition and removal of multiple drug-resistant providencia rettgeri biofilm is provided.

2. The use of sanguinarine for multiple drug resistant providencia rettgeri biofilm inhibition and removal according to claim 1, wherein the multiple drug resistant providencia rettgeri is human providencia rettgeri.

3. Use of sanguinarine for multiple drug resistant providencia retzii biofilm inhibition and removal according to claim 1, wherein the multiple drug resistant providencia retzii resistant antibiotic is selected from a plurality of ampicillin, ampicillin/sulbactam, ceftizole, cefotetan, ceftriaxone, ceftazidime, tobramycin, piperacillin tazobactam, ciprofloxacin, levofloxacin, amikacin, co-trimoxazole, cefepime, ertapenem, imipenem, furadantin, aztreonam, gentamicin.

4. Use of sanguinarine for multiple drug resistant providencia retzii biofilm inhibition and removal according to claim 1, wherein said multiple drug resistant providencia retzii is selected from human providencia retzii of ampicillin resistance, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, tobramycin, piperacillin tazobactam, ciprofloxacin, levofloxacin, amikacin, zinomine, cefepime, ertapenem, imipenem, aztreonam, gentamicin.

5. The use of sanguinarine for the inhibition and removal of multiple drug resistant providencia rettgeri biofilm according to claim 1, wherein the concentration that minimally inhibits providencia rettgeri biofilm formation is determined by crystal violet staining; the concentration of minimally cleared mature providencia rettgeri biofilm was determined by crystal violet staining.

6. The use of sanguinarine for the inhibition and clearance of multiple drug resistant providencia rettgeri biofilm according to claim 1, wherein the sanguinarine has a minimum inhibitory concentration of 7.81 μ g/mL and a minimum clearance concentration of 125 μ g/mL against providencia rettgeri biofilm.

7. The use of sanguinarine for the inhibition and removal of multiple drug resistant providencia rettgeri biofilm according to claim 1, wherein sanguinarine has inhibitory and removal effects on multiple drug resistant providencia rettgeri biofilm.

8. The application of sanguinarine in the preparation of medicine for resisting multiple drug-resistant providencia rettgeri biofilm is provided.

9. The use of sanguinarine in the manufacture of a medicament against multiple drug resistant providencia rettgeri biofilm according to claim 8, wherein the multiple drug resistant providencia rettgeri is providencia rettgeri.

10. Use of sanguinarine in a medicament against multiple drug resistant providencia retzii biofilm according to claim 8, wherein the multiple drug resistant providencia retzii is selected from multiple antibiotic resistant providencia retzii of ampicillin, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, tobramycin, piperacillin tazobactam, ciprofloxacin, levofloxacin, amikacin, ziram, cefepime, ertapenem, imipenem, aztreonam, gentamicin.

Technical Field

The invention belongs to the technical field of medicine and food safety, and relates to application of sanguinarine in inhibition and removal of multiple-drug-resistant providencia rettgeri biofilm.

Background

Providencia is a facultative anaerobic gram-negative bacillus, can cause food poisoning to cause vomiting and diarrhea, cause urinary tract infection and other parenteral infections, can cause fulminant epidemics in hospitals, and is increasingly regarded as a pathogen with extensive existence conditions to threaten human health. In recent years, reports of clinical sporadic cases caused by providencia in China have been increasing. At the same time, this strain was also detected in food. In a food processing environment, the risk of cross-contamination is increased due to contaminated food, with consequent economic losses. Bacterial biofilms are a serious global health problem due to their ability to withstand antibiotics, host defense systems and other external stresses, thus leading to chronic infections for a long period of time. Biofilms are a non-living microbial community on the surface of medical implants such as sutures, catheters and dental implants where self-generated extracellular polymeric substances colonize and grow, causing infections that can only be treated by removing them. The biofilm consists of an extracellular matrix-packed bacterial population, which has exopolysaccharides, exoproteins, eDNA, and the like. Biofilms not only protect microorganisms from pH changes, osmotic pressure, nutrient deprivation, mechanical forces and shear forces. In addition, bacterial biofilm populations are prevented from entering from antibiotics and host immune cells. Thus, the biofilm matrix confers additional resistance to the bacteria, allowing them to not only withstand harsh environments, but also to antibiotics, leading to the emergence of undesirable bacterial infections such as multidrug, extensively and fully resistant.

The antibacterial agent is a biological film agent which selectively eliminates a persistent biological film so as to promote the diffusion of antibiotics into a complex biological film environment. Sanguinarine is an extract from macleaya cordata and has been shown to have antioxidant, antitumor and anti-inflammatory properties (fanhuining, zhangjing, zhugui. sanguinarine. research on antitumor mechanisms advances in modern oncology, 2019,27(18): 3323-. Sanguinarine has been reported to significantly inhibit the growth of ovarian cancer cells, induce apoptosis, increase the production of active oxygen, and inhibit the growth of ovarian cancer tumors (Zhang bud, Liyi, Wutao, et al. sanguinarine inhibits the growth of ovarian cancer tumors by inducing apoptosis [ J ]. J.Utility J.Oncology, 2019,33(04): 305-. In addition, sanguinarine has a potent antifungal effect and is found to have a good inhibitory effect on biofilm, and the concentration at which biofilm-resistant activity is achieved is below its minimum inhibitory concentration (Zhonghua. sanguinarine's antifungal effect and mechanism research [ D ]. university of naval military, people's liberation force, China, 2019.). Sanguinarine is reported in the literature (Zhong H, Hu D, Hu G H, et al, Activity of Sanguinarine against Candida albicans Biofilms [ J ]. Antichronomicrobiological Agents and Chrohotherapy, 2017,61 (5)) to have a strong ability against Candida albicans Biofilms. More importantly, it not only inhibits biofilm formation, but also disrupts the maintenance of mature biofilms. In addition, the inhibition and removal of multiple drug-resistant providencia biofilm by sanguinarine have been reported in the literature (Dinghao, Dingjing, Song, Suzhijun, Yulinxue, Aixiahui 36 kinds of traditional Chinese medicines for the in vitro bacteriostatic and biofilm-eliminating effects of tilapia-derived Streptococcus agalactiae [ J ]. freshwater fishery, 2019,49(03):71-77 ]).

Disclosure of Invention

The invention aims to provide the application of sanguinarine in inhibition and removal of multiple drug-resistant providencia rettgeri biofilm, and explores a potential biofilm remover from the existing medicinal plant resource library, so as to provide a practical basis for relieving or solving the infection problem and reducing the fatality rate caused by the multiple drug-resistant providencia rettgeri biofilm and solving the difficult problem that the drug-resistant providencia rettgeri biofilm is difficult to remove.

The invention is realized by the following technical scheme:

application of sanguinarine in inhibition and removal of multiple drug-resistant providencia rettgeri biofilm is provided.

Further, the multidrug-resistant providencia rettgeri is human providencia rettgeri.

Further, the antibiotic resistant to multiple drug resistant providencia rettgeri is selected from a plurality of ampicillin, ampicillin/sulbactam, ceftizolin, cefotetan, ceftriaxone, ceftazidime, tobramycin, piperacillin tazobactam, ciprofloxacin, levofloxacin, amikacin, sulfamethoxazole, cefepime, ertapenem, imipenem, nitrofurantoin, aztreonam, gentamicin.

Further, the multidrug-resistant providencia rettgeri is selected from human providencia rettgeri having ampicillin resistance, ampicillin/sulbactam resistance, cefazolin resistance, cefotetan resistance, ceftriaxone resistance, ceftazidime resistance, tobramycin resistance, piperacillin tazobactam resistance, ciprofloxacin resistance, levofloxacin resistance, amikacin resistance, compound sulfamethoxine resistance, cefepime resistance, ertapenem resistance, imipenem resistance, aztreonam resistance and gentamicin resistance.

Further, the concentration that minimally inhibits biofilm formation by providencia rettgeri was determined by crystal violet staining; the concentration of minimally cleared mature providencia rettgeri biofilm was determined by crystal violet staining.

Furthermore, the minimum inhibitory concentration of sanguinarine on the providencia rettgeri biofilm is 7.81 mu g/mL, and the minimum clearing concentration is 125 mu g/mL.

Furthermore, sanguinarine has the effects of inhibiting and removing multiple drug-resistant providencia rettgeri biofilm.

The application of sanguinarine in the preparation of medicine for resisting multiple drug-resistant providencia rettgeri biofilm is provided.

Further, the multidrug-resistant providencia rettgeri is providencia rettgeri.

Further, the multiple drug resistant providencia rettgeri is selected from the group consisting of multiple antibiotic resistant providencia rettgeri of ampicillin, ampicillin/sulbactam, ceftizolid, cefotetan, ceftriaxone, ceftazidime, tobramycin, piperacillin tazobactam, ciprofloxacin, levofloxacin, amikacin, sulfamethoxazole, cefepime, ertapenem, imipenem, aztreonam, gentamicin.

Compared with the prior art, the invention has the following beneficial technical effects:

based on the research of the effect of sanguinarine on multiple drug-resistant providencia rettgeri, the sanguinarine can be used for inhibiting and removing multiple drug-resistant providencia rettgeri biofilm, a new thought is provided for the research, development and application of multiple drug-resistant providencia rettgeri scavenger, and the sanguinarine has wide application value in the field of medicine.

The invention further defines the inhibition effect of sanguinarine on drug resistance of Lei's providencia bacteria of antibiotics ampicillin, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, tobramycin, piperacillin tazobactam, ciprofloxacin, levofloxacin, amikacin, compound sulfamethoxazole, cefepime, ertapenem, imipenem, aztreonam and gentamicin, effectively relieves or solves the infection problem caused by multiple drug resistance providencia bacteria biofilms, and reduces the fatality rate.

Detailed Description

The present invention will be described in further detail with reference to examples. The examples are only for explaining the contents of the present invention and do not limit the present invention.

Sanguinarine is named as Sanguinarine in English name and has a molecular formula of C₂₀H₁₄ClNO₄The molecular weight is 367.78, the melting point is 281-285 ℃ and the CAS number is 5578-73-4. The molecular structure is:

1. drug susceptibility test of providencia rettgeri

The invention takes 5 strains of providencia rettgeri as starting strains, and selects 18 common antibiotics such as ampicillin, ampicillin/sulbactam, ceftizoxime, cefotetan, ceftriaxone, ceftazidime, tobramycin, piperacillin tazobactam, ciprofloxacin, levofloxacin, amikacin, compound sulfamethoxine, cefepime, ertapenem, imipenem, nitrofurantoin, aztreonam, gentamicin and the like for testing.

The pure colonies cultured for 24h were picked up and dissolved in 5mL of TSB uniformly, and the turbidity was adjusted to be equal to that of 0.5 McLeod turbiditube. Adding antibiotics, bacterial liquid and TSB liquid culture medium into a 96-hole culture plate by using a two-fold dilution method for overnight culture, and measuring the minimum inhibitory concentration MIC of the antibiotics to providencia rettgeri by using a microplate reader. The three liquid medicine groups with different concentrations are parallel, so that the reliability of experimental data is ensured. The bacteriostatic result is judged according to the national standard administration committee of the united states clinical laboratory (CLSl2017), and the judgment standard is shown in table 1. The results are shown in Table 2 and 1^#The providencia rettgeri strain is resistant to the most antibiotics, and is resistant to 17 antibiotics and sensitive to 1 antibiotic, so that the providencia rettgeri strain is used as a research object for the next experiment.

TABLE 1 results of the national Committee for standardization management of the clinical laboratory (CLSl2017) standards

TABLE 2 MIC results for antibiotics against providencia rettgeri

As can be seen from tables 1 and 2, sanguinarine has a good inhibitory effect on drug-resistant providencia retta bacteria resistant to ampicillin, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, tobramycin, piperacillin tazobactam, ciprofloxacin, levofloxacin, amikacin, sulfamethoxazole, cefepime, ertapenem, imipenem, aztreonam, gentamicin.

2. Inhibition of multiple drug-resistant bacterial strain biofilm by sanguinarine

The single active ingredient sanguinarine is taken as a research object, and a standard strain ATCC31052 of providencia rettgeri is taken as a control to carry out inhibition research on the biofilm. The pure colonies cultured for 24h are picked up and uniformly dissolved in 5ml LTSB liquid culture medium, and the OD600 value is measured to be 0.5 by an enzyme labeling instrument. The sanguinarine with the concentration of 500g/mL is prepared by dimethyl sulfoxide to be used as a liquid medicine, a sterile slide is added into a 24-hole plate, the liquid medicine, a bacterium liquid and a TSB liquid culture medium are added into the 24-hole culture plate by a double dilution method for overnight culture, and the final concentration of the liquid medicine is 125g/mL, 62.5g/mL, 31.25g/mL, 15.63g/mL, 7.811g/mL and 3.90 g/mL. Thereafter, the plate was washed three times with 0.1M PBS, stained with 200. mu.L of crystal violet dye for 20min, and excess dye was washed off and observed with a microscope. The three parallel liquid medicines with different concentrations ensure the reliability of experimental data.

3. Removing effect of sanguinarine on mature biofilm of multiple drug-resistant strains

The invention takes single active ingredient sanguinarine as a research object and takes a standard strain ATCC31052 of providencia rettgeri as a control to carry out the elimination research of mature biofilm. The pure colonies cultured for 24h are picked up and uniformly dissolved in a 5ml LTSB liquid culture medium, the turbidity is adjusted to be 0.5 McLeod, and the OD600 value is measured by an enzyme-labeling instrument. The method comprises preparing sanguinarine solution with concentration of 1000g/mL with dimethyl sulfoxide, adding sterile slide and 1mL bacterial solution into 24-well plate, culturing for 24h to form mature coating, adding the solution into each well, and culturing for 4h to obtain final concentrations of 62.5g/mL, 125g/mL, 500g/mL, and 1000 g/mL. Thereafter, the plate was washed three times with 0.1M PBS, stained with 200. mu.L of crystal violet dye for 20min, and excess dye was washed off and observed with a microscope. The three parallel liquid medicines with different concentrations ensure the reliability of experimental data. The results are shown in Table 3.

TABLE 3 inhibition of multiple drug resistant providencia rettgeri by sanguinarine

As can be seen from Table 3, the minimum inhibitory concentration of sanguinarine against providencia rettgeri biofilm was 7.81. mu.g/mL, and the minimum clearance concentration was 125. mu.g/mL.

According to the experimental results, by combining the characteristic that the traditional Chinese herbal medicines are difficult to remove the biofilm of the drug-resistant pathogenic bacteria, the characteristic that the biofilm of the drug-resistant pathogenic bacteria is difficult to remove can be directly taken as a clearing effect for clinically main multiple drug-resistant providencia rettgeri, so that the infection problem caused by the multiple drug-resistant providencia rettgeri biofilm can be effectively relieved or solved, the fatality rate is reduced, and the method has important practical significance.