阅读说明：本技术 白屈菜红碱在抑制多重耐药粘质沙雷菌生长中的应用 (Application of chelerythrine in inhibiting growth of multiple drug-resistant serratia marcescens ) 是由 李永东 倪红霞 许国章 焦素黎 刘昱慧 于 2019-12-20 设计创作，主要内容包括：本发明公开了白屈菜红碱在抑制多重耐药粘质沙雷菌生长中的应用,根据白屈菜红碱对多重耐药粘质沙雷菌具有较好的体外杀灭作用,能够抑制多重耐药粘质沙雷菌的生长,且最低杀菌浓度为30～250μg/mL,最小抑菌浓度为15～125μg/mL,本发明提出了白屈菜红碱对多重耐药粘质沙雷菌的抑制作用,在医药等领域具有广泛的应用价值。(The invention discloses an application of chelerythrine in inhibiting the growth of multiple drug-resistant serratia marcescens, and provides an inhibition effect of chelerythrine on multiple drug-resistant serratia marcescens according to the fact that the chelerythrine has a good in-vitro killing effect on the multiple drug-resistant serratia marcescens and can inhibit the growth of the multiple drug-resistant serratia marcescens, the minimum sterilization concentration is 30-250 mu g/mL, and the minimum inhibition concentration is 15-125 mu g/mL.)

1. Application of chelerythrine in inhibiting growth of multiple drug-resistant serratia marcescens is provided.

2. Use according to claim 1, characterized in that: the multiple drug-resistant serratia marcescens is humanized serratia marcescens.

3. Use according to claim 1, characterized in that: the drug resistance of the serratia marcescens is determined by a micro-double dilution method.

4. Use according to claim 1, characterized in that: the antibiotic which is tolerated by the multi-drug resistant serratia marcescens is selected from a plurality of ampicillin, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, piperacillin/tazobactam, compound neodimine, cefepime, aztreonam, gentamicin, ertapenem, imipenem, tobramycin, amikacin, ciprofloxacin, nitrofurantoin and levofloxacin.

5. Use according to claim 1, characterized in that: the multiple drug-resistant Serratia marcescens is selected from Serratia marcescens which is resistant to ampicillin, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, cefepime, ertapenem, aztreonam, tobramycin, amikacin, ciprofloxacin, levofloxacin, compound sulfamethoxazole, piperacillin/tazobactam and nitrofurantoin.

6. Use according to claim 5, characterized in that: the minimum bactericidal concentration of the chelerythrine to the multidrug-resistant serratia marcescens is 30-250 mu g/mL, and the minimum bacteriostatic concentration is 15-125 mu g/mL.

7. Use according to claim 1, characterized in that: the chelerythrine has in vitro killing effect on multiple drug-resistant serratia marcescens and can inhibit in vitro growth of multiple drug-resistant serratia marcescens.

8. Application of chelerythrine in preparing medicine for resisting multiple drug-resistant serratia marcescens is provided.

9. Use according to claim 8, characterized in that: the multiple drug-resistant serratia marcescens is humanized serratia marcescens.

10. Use according to claim 8, characterized in that: the multiple drug-resistant serratia marcescens is selected from serratia marcescens resistant to various antibiotics in ampicillin, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, piperacillin/tazobactam, compound neodimine, cefepime, aztreonam, gentamicin, ertapenem, imipenem, tobramycin, amikacin, ciprofloxacin, nitrofurantoin and levofloxacin.

Technical Field

The invention relates to the field of medicines, and in particular relates to application of chelerythrine in inhibition of growth of multiple drug-resistant serratia marcescens.

Background

Serratia marcescens (Serratia marcescens), a gram-negative bacterium of Serratia of Enterobacteriaceae, can cause various infections. Serratia marcescens is currently known to cause infections of the eye, lungs, urinary tract and bloodstream. Recent reports have shown that this pathogen is present in 2.5% -7.7% of catheter-related infections. In recent years, the drug resistance rate and the clinical separation rate of carbapenem-resistant serratia marcescens gradually rise, which brings great challenges to hospital infection prevention and control and clinical anti-infection treatment work.

Chelerythrine is a natural benzophenanthridine alkaloid extracted from plants such as semen Strychni, radix rehmanniae, and Canadian red Cephalotaxus fortunei. Chelerythrine has wide biological activity and plays an important role in resisting tumors, inflammation and diabetes. Studies have shown that chelerythrine has antibacterial activity. The document reports that chelerythrine has better inhibition effect on the growth of hyphae of gentian blight bacteria, corn small spot bacteria, walnut leaf blight bacteria, tomato blight bacteria, pumpkin blight bacteria and curvularia lunata. It is also reported in the literature that chelerythrine inhibits the growth of streptococcus mutans by inhibiting its cell division, and has a significant effect on the structure of capsule, cell wall, cell membrane, etc. Chinese patent reports the application of chelerythrine in treating poultry-resistant colibacillosis (CN106138054A), wherein the colibacillosis is Escherichia coli in Enterobacteriaceae. At present, no document reports that chelerythrine has an antibacterial effect on multiple drug-resistant serratia marcescens.

Disclosure of Invention

The invention aims to solve the problem of drug resistance in current clinical medicine and provides application of chelerythrine in inhibition of growth of multiple drug-resistant serratia marcescens.

In order to achieve the purpose, the invention adopts the technical scheme that:

the drug resistance of the serratia marcescens to the antibiotics is determined by a trace double dilution method, and then the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of chelerythrine to the serratia marcescens are determined. The results show that: the chelerythrine has good bacteriostatic action on the multiple drug-resistant serratia marcescens, and can be used for inhibiting the growth of the multiple drug-resistant serratia marcescens.

Preferably, the multidrug-resistant Serratia marcescens is human Serratia marcescens which is resistant to a plurality of antibiotics in ampicillin, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, piperacillin/tazobactam, compound neodimine, cefepime, aztreonam, gentamicin, ertapenem, imipenem, tobramycin, amikacin, ciprofloxacin, nitrofurantoin and levofloxacin.

Preferably, the multidrug-resistant Serratia marcescens is selected from the group consisting of Serratia marcescens of human origin resistant to ampicillin, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, cefepime, ertapenem, aztreonam, tobramycin, amikacin, ciprofloxacin, levofloxacin, sulfamethoxazole, piperacillin/tazobactam and nitrofurantoin.

Preferably, the minimum bactericidal concentration of the chelerythrine is 30-250 mug/mL, and the minimum bacteriostatic concentration of the chelerythrine is 15-125 mug/mL.

The invention has the beneficial effects that:

the invention starts from the existing medicinal plant resource library, develops a potential drug-resistant bacteria inhibitor, finds that the non-antibiotic compound chelerythrine can effectively inhibit the growth of the multiple drug-resistant serratia marcescens based on the research on the effect of the chelerythrine on the multiple drug-resistant serratia marcescens, provides a new thought and source for the research, development and application of the multiple drug-resistant serratia marcescens inhibitor, and has wide application value in the fields of medicine and the like.

Furthermore, the invention defines the inhibition effect of chelerythrine on the multiple drug-resistant serratia marcescens, and the chelerythrine can be used as an antibiotic substitute to effectively relieve or solve the drug-resistant and infection problems of the multiple drug-resistant serratia marcescens and reduce 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 present invention and do not limit the scope of protection of the present invention.

1. Drug sensitivity test of Serratia marcescens

The invention takes a plurality of strains of humanized Serratia marcescens (strain samples are taken from Ningbo women hospitals) as starting strains, and selects 18 common antibiotics of ampicillin, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, piperacillin/tazobactam, compound sulfamethoxazole, cefepime, aztreonam, gentamicin, ertapenem, imipenem, tobramycin, amikacin, ciprofloxacin, nitrofurantoin and levofloxacin for testing.

And (3) selecting and culturing the pure colonies for 18-24 hours, uniformly dissolving the pure colonies in 2-5 mL of sterile physiological saline, and adjusting the turbidity of the pure colonies to be equal to that of a 0.5 McLeod turbiditube. Adding antibiotics, bacterial liquid and TSB liquid culture medium into a 96-well culture plate by using a test tube double dilution method for overnight culture, and measuring the Minimum Inhibitory Concentration (MIC) of the antibiotics to Serratia marcescens 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 show that Serratia marcescens 1 is resistant to 16 antibiotics (2 mediators), and thus is the subject of the next experiment.

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

TABLE 2 MIC results of drug sensitivity test of human Serratia marcescens

2. Inhibition effect of chelerythrine on multiple drug-resistant strains

In order to fully consider the medication safety, the invention takes chelerythrine which is a single active component as a research object, and takes a standard strain (ATCC-14041, sensitive to antibiotics such as cephalexin, kanamycin, cephalosporin, gentamicin, neomycin, tetracycline and the like) as a reference to research the drug resistance inhibition effect. Selecting and culturing the pure bacterial colonies for 18-24 h, uniformly dissolving the pure bacterial colonies in 2-5 mL of TSB liquid culture medium, adjusting the turbidity of the TSB liquid culture medium to be equal to that of a 0.5 McLeod turbiditube, and measuring the OD of the TSB liquid culture medium by using an enzyme-labeling instrument₆₀₀The value is obtained. Preparing chelerythrine with concentration of 1000 μ g/mL with dimethyl sulfoxide as medicinal liquid, adding the medicinal liquid, bacterial liquid and TSB liquid culture medium into 96-well by test tube double dilution method, and culturingThe plates are cultured overnight, and three liquid medicine groups with different concentrations are parallel to ensure the reliability of experimental data.

And (3) determining the Minimum Inhibitory Concentration (MIC) of chelerythrine to the multiple drug-resistant Serratia marcescens by using a microplate reader. And transferring the culture solution with the concentration of the MIC diluted by the former drug to a sterile TSA solid culture medium for continuous culture, observing whether a single colony is generated, and if not, determining the concentration as the Minimum Bactericidal Concentration (MBC) of chelerythrine to the multidrug-resistant serratia marcescens, wherein the experimental results are shown in Table 3.

TABLE 3 results of chelerythrine inhibition of multidrug-resistant Serratia marcescens

As shown in Table 3, chelerythrine has a good inhibitory effect on drug-resistant Serratia marcescens with multiple drug resistance, wherein MIC is 125 mu g/mL, and MBC is 250 mu g/mL, against ampicillin, ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, ceftazidime, cefepime, ertapenem, aztreonam, tobramycin, amikacin, ciprofloxacin, levofloxacin, sulfamethoxazole, piperacillin/tazobactam, and nitrofurantoin. The results show that chelerythrine not only has the bacteriostatic action on the Serratia marcescens reference strain ATCC-14041, but also has the bacteriostatic action on the multiple drug-resistant Serratia marcescens (such as the No. 1 strain) in the invention.

According to the experimental results, by combining the characteristics of wide sources of Chinese herbal medicines, less adverse reaction, difficult generation of drug resistance and the like, the active single-product component chelerythrine can be obtained, the inhibition effect is directly exerted on clinically main multiple drug-resistant serratia marcescens, the infection problem of the multiple drug-resistant serratia marcescens can be effectively relieved or solved, the mortality rate of diseases is reduced, scientific basis is provided for researching clinically separated bacteriostatic agents of the serratia marcescens, and new ideas and sources are provided for developing drugs and antibiotic substitutes for inhibiting the multiple drug-resistant serratia marcescens.