CN110237058B

CN110237058B - Application of citral in inhibition of growth of multidrug-resistant enterobacter heumakii

Info

Publication number: CN110237058B
Application number: CN201910614507.9A
Authority: CN
Inventors: 钱卫东; 沈兰芳; 王婷; 杨敏; 刘淼; 李鑫辰; 王文静; 付玉婷
Original assignee: Shaanxi University of Science and Technology
Current assignee: Shaanxi University of Science and Technology
Priority date: 2019-07-09
Filing date: 2019-07-09
Publication date: 2022-04-29
Anticipated expiration: 2039-07-09
Also published as: CN110237058A

Abstract

The invention discloses an application of citral in inhibiting the growth of multidrug-resistant enterobacter hopcalis, which has a good in-vitro killing effect on the multidrug-resistant enterobacter hopcalis with piperacillin resistance, bromgertin resistance, imipenem resistance, ofloxacin resistance, nitrofurantoin resistance, cefotaxime resistance and nalidixic acid resistance, can inhibit the growth of the multidrug-resistant enterobacter hopcalis, has the minimum bactericidal concentration of 2.8mg/mL and the minimum bacteriostatic concentration of 2.4mg/mL, and has a wide application value in the field of medicine.

Description

Application of citral in inhibition of growth of multidrug-resistant enterobacter heumakii

Technical Field

The invention relates to research, development and application of a traditional Chinese medicine active ingredient citral as a bacterial inhibitor, in particular to application of citral in inhibition of growth of multiple drug-resistant enterobacter hophallii.

Background

Enterobacter hollandii (Enterobacter hormaechei) is a gram-negative bacterium, facultative anaerobic, readily grown on common media, and found in the human and animal intestines. Enterobacter hollisae can cause animal and human infection under specific conditions, is a conditional pathogenic bacterium, can be separated from infected animals or animal wounds, and the cases caused by the infection comprise cerebral apoplexy (Mingdong, Zhu Yan, Schering gold. the research on drug resistance and drug resistance mechanism of Enterobacter hollandii in sputum of patients with cerebral apoplexy [ J ]. the J.Hospital's journal of infectivity, 2011,21(05):1025 and 1026.), bacteremia of premature and weak infants and the like. At present, few domestic researches on the bacterium are reported (Liluyao, Liumengjia, Tengming, Wangle, Yanghuang, Liu jin. Enterobacter huoshi biology characteristics research [ J ]. animal husbandry veterinary magazine, 2017,36(04):1-2+6.), and the reports on the inhibition of the Enterobacter huoshi multiple drug resistance and traditional Chinese medicine extracts are less.

In recent years, with the variety increase and wide use of antibacterial drugs, particularly antibiotics, the problem of bacterial drug resistance is becoming more serious, and the treatment effect of infectious diseases and the recovery of patients are seriously affected. The traditional Chinese medicine has a long history of treating bacterial infection, and many researchers of the traditional Chinese medicine carry out related researches on the antibacterial treatment of the traditional Chinese medicine, and provide the advantages of the traditional Chinese medicine, such as low drug resistance, broad-spectrum antibacterial effect with multiple action targets, wide medicine source, low price and the like in the anti-infection aspect (Lupengye, Chuaihei. the research status of the in-vitro drug sensitivity test of the traditional Chinese medicine [ J ] test medicine and clinic, 2018,15(15): 2339-.

The litsea cubeba oil extracted from litsea cubeba is a natural spice with complex components, wherein citral is the main component, the content is generally between 60 and 80 percent, and researches show that the citral is effective in the aspects of treating cardiovascular diseases, relieving asthma and resisting allergy, inhibiting bacteria, treating colon diarrhea and the like (Zhang Qiang, Zhang Qi, Wang Suling, He Lin. citral beta-cyclodextrin inclusion compound research [ J ] of Chinese university of pharmacy, 1999(06):37 to 39.).

The influence of 10 compounds such as citral and derivatives thereof on the growth of the camellia oleosa anthracnose pathogen is researched by adopting a hypha growth rate method by Guoyang et al (Guoyang, Zhouyuyuihui, Wangpo, Chenshang \3804040, Van Guori, Wang Zong. citral and derivatives thereof on the bacteriostatic activity of the camellia oleosa anthracnose pathogen [ J ]. Jiangsu agricultural science 2016,44(12): 167-. Limbing et al (Limbing, Wang Ximan, Wang Peng, Chenshang \38040, Van Guo Rong, Wang Zong De. inhibitory activity of natural citral derivative on food spoilage bacteria [ J ]. Zhejiang agricultural science 2016,28(11): 1928) 1933) tested the inhibitory activity of 12 natural citral derivative compounds on Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa by a bacteriostatic circle method and an agar double dilution method, and found that 12 natural citral derivative compounds have a certain inhibitory effect on 4 bacteria. The inhibition effect of five plant source extracts on air source bacteria in museum exhibition halls [ J ] cultural relic protection and archaeological science, 2019,31(01):50-57.) of the tanghua et al (tanghuan, Wangchun, Zhouyun, Van Wenqui) utilizes 5 plant source extracts as purifying agents to carry out gas phase fumigation on the surface of a flat plate on air microorganisms collected in the museum exhibition halls, and further discovers that the plant source extract with the best inhibition effect is citral and the weakest is geranium oil through a viable count method and a polymerase chain reaction-denaturing gradient gel electrophoresis technology based on 16Sr RNA genes of bacteria. However, no research report about the inhibition of the enterobacter heuchei by the citral is found.

Disclosure of Invention

The invention aims to provide application of citral in inhibiting growth of multidrug-resistant enterobacter heumaei and provides a practical foundation for developing novel antibiotic substitutes.

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

the paper diffusion method is used for determining the drug resistance of the enterobacter hopcalis to antibiotics, and the double dilution method is used for determining the minimum bactericidal concentration and the minimum bacteriostatic concentration of citral to the enterobacter hopcalis. The results show that: the non-antibiotic compound citral has good bacteriostatic action on multiple drug-resistant enterobacter hopcalis and can be used for inhibiting the growth of multiple drug-resistant enterobacter hopcalis.

Preferably, the multidrug-resistant enterobacter hopcalis is enterobacter hopcalis which is of human or animal origin and is resistant to multiple antibiotics in piperacillin, bromgmelin, imipenem, ofloxacin, nitrofurantoin, cefotaxime and nalidixic acid.

Preferably, the minimum bactericidal concentration of the citral is 2.8mg/mL, and the minimum bacteriostatic concentration is 2.4 mg/mL.

Preferably, the citral has an inhibitory effect on enteric bacteria of the species Enterobacter huoshanense resistant to piperacillin, bromoglutenin, imipenem, ofloxacin, nitrofurantoin, cefotaxime and nalidixic acid.

The invention has the beneficial effects that:

the invention starts from the existing medicinal plant resource library, develops a potential drug-resistant bacteria inhibitor, utilizes citral to inhibit the growth of the multidrug-resistant enterobacter hophallus based on the research on the action of citral on the multidrug-resistant enterobacter hophallus, provides a new idea for the research, development and application of the multidrug-resistant enterobacter hophallus inhibitor, provides beneficial help for solving the drug resistance of antibiotics, and has wide application value in the field of medicine.

Furthermore, aiming at the difficult problem of clinical drug resistance, the invention utilizes the inhibiting effect of citral on the multiple drug-resistant enterobacter heumakii as a substitute of antibiotics, can relieve or solve the problem of drug-resistant infection of the multiple drug-resistant enterobacter heumakii, 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 present invention and do not limit the scope of protection of the present invention.

1. Drug sensitivity experiment of human enterobacter hopcalis

The invention takes 2 strains of human enterobacter hopcalis as starting strains (strain samples are taken from general hospitals of people's liberation force in China), and drug susceptibility test paper sheets of 8 antibiotics such as piperacillin, gentamicin, bromgemastine, imipenem, ofloxacin, nitrofurantoin, cefotaxime and nalidixic acid are selected for drug susceptibility test.

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. And (4) aseptically sucking 50-200 mu L of bacterial liquid, uniformly coating the bacterial liquid on a flat plate, and standing at room temperature for 3-5 min. The drug sensitive test paper pieces (diameter is 6mm) are tightly attached to the surface of the flat plate by using sterile forceps as required, and three drug sensitive test paper pieces are parallel. And incubating the inoculated plate at 36-37 ℃ for 16-24 h. The diameter of the inhibition zone is measured by a vernier caliper with the precision of 0.02mm, the sensitivity of the human enterobacter heumakii to the drugs is judged according to the diameter of the inhibition zone, the inhibition result is judged according to the standard of the national standard administration committee of the clinical laboratory (CLSl2017), and the judgment standard is shown in Table 1. The experimental results are shown in table 2, and show that one strain of human enterobacter heumai (strain # 1) can tolerate 7 clinically used antibiotics.

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

TABLE 2 diameter results of inhibition zone in drug sensitivity test of human Enterobacter Huoshanense

Note: the unit of the diameter of the inhibition zone is mm, and the numerical values are the average values of three parallels

2. Inhibition of multi-drug resistant strains by citral

In order to fully consider the medication safety, the invention takes the single active ingredient of the citral as a research object and takes the gentamicin as a positive control 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 LB liquid medium, adjusting the turbidity of the pure bacterial colonies to be equal to that of a 0.5 McLeod turbiditube, and measuring the OD of the pure bacterial colonies by using an enzyme-labeling instrument₆₀₀The value is obtained. The citral with the concentration of 50mg/mL is prepared by using dimethyl sulfoxide as a liquid medicine, the liquid medicine, a bacterial liquid and an LB liquid culture medium are added into a 96-hole culture plate by using a test tube double dilution method for overnight culture, three liquid medicine groups with different concentrations are parallel, and the reliability of experimental data is ensured.

The Minimum Inhibitory Concentration (MIC) of citral on multiple drug-resistant Enterobacter holtzeri (strain # 1 and strain # 2) was determined by a microplate reader. And the culture solution under the MIC concentration is transferred into a sterile LB liquid culture medium for continuous culture, the Minimum Bactericidal Concentration (MBC) of citral to the multidrug-resistant enterobacter hophallii is determined by using a microplate reader, and the experimental result is shown in Table 3.

TABLE 3 inhibition results of citral on human-derived multidrug-resistant Enterobacter hollisae

As shown in Table 3, citral has a good inhibitory effect on multidrug-resistant Enterobacter holtzii resistant to piperacillin, bromocriptine, imipenem, ofloxacin, nitrofurantoin, cefotaxime and nalidixic acid, and taking the strain # 1 as an example, the MIC is 2.4mg/mL and the MBC is 3.2 mg/mL.

According to the experimental results, the characteristics that the Chinese herbal medicines are not easy to generate drug resistance and the like are integrated, the citral active single-product component can be obtained, the inhibition effect can be directly exerted on the multiple drug-resistant enterobacter hopcalis, the problems of drug resistance, infection, pathogenicity and the like of the multiple drug-resistant enterobacter hopcalis can be effectively relieved or solved, the inhibition effect can be effectively exerted, the pathogenicity rate and the fatality rate can be reduced, a new thought is provided for inhibiting the multiple drug-resistant enterobacter hopcalis, and a solid foundation is laid for developing the inhibitor of the multiple drug-resistant enterobacter hopcalis.

Claims

1. The application of citral in preparing a medicament for resisting multiple drug-resistant Enterobacter holtzeri is characterized in that: the multiple drug-resistant enterobacter hopcalis is selected from enterobacter hopcalis which is resistant to various antibiotics in piperacillin, bromgertin, imipenem, ofloxacin, nitrofurantoin, cefotaxime and nalidixic acid.

2. Use according to claim 1, characterized in that: the multiple drug-resistant enterobacter heumakii is human or animal enterobacter heumai.

3. Use according to claim 1, characterized in that: the resistance of said enterobacter hopcalis to antibiotics was determined by the paper diffusion method.

4. Use according to claim 1, characterized in that: the minimum bactericidal concentration of the citral to the enterobacter heumakii is 3.2mg/mL, and the minimum bacteriostatic concentration is 2.4 mg/mL.

5. Use according to claim 1, characterized in that: the multiple drug-resistant enterobacter hopcalis is selected from enterobacter hopcalis which is resistant to piperacillin, bromgertin, imipenem, ofloxacin, nitrofurantoin, cefotaxime and nalidixic acid.

6. Use according to claim 1, characterized in that: the citral has in vitro killing effect on multiple drug-resistant Enterobacter hollisae, and can inhibit in vitro growth of multiple drug-resistant Enterobacter hollisae.