CN105168667B - Application of Chinese herbal medicine in delaying bacterial drug resistance - Google Patents
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Abstract
The invention relates to application of Chinese herbal medicines in delaying bacterial drug resistance, wherein the Chinese herbal medicines comprise isatis root, blackberrylily rhizome, lightyellow sophora root, indigowoad leaf, plantain herb, weeping forsythia capsule, baical skullcap root or wormwood. Its advantages are: the screened traditional Chinese medicine has the effect of delaying the drug resistance of aeromonas.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of Chinese herbal medicines in delaying bacterial drug resistance.
Background
It is well known that some substances kill microorganisms, but some microorganisms survive at normally lethal doses. In 1907, Ehrlich studied the development of p-rosaniline resistance by Trypanosoma brucei; in 1911, Morgenroth and Kaufmann reported the resistance of Streptococcus pneumoniae to ethyl hydrogen norquinine. Each of these antibacterial agents kills or inhibits microorganisms, but at the same time, resistance to the agents is also present.
In recent years, due to the widespread use of chemical antibacterial agents such as antibiotics, drug-resistant bacteria have rapidly grown, making effective treatment of infectious diseases a problem that seriously afflicts human and animal health. Therefore, the research on how to delay the drug resistance of bacteria becomes a focus of attention.
In 2001, Rayleigh et al reported that 146 pathogenic E.coli strains have different degrees of drug resistance, and the strains are resistant to 3 drugs at minimum and 15 drugs at maximum. In 2004, 180 strains of chicken-derived escherichia coli isolated by Yanghuochun et al have drug resistance to nalidixic acid, tetracycline, ampicillin, and amoxicillin.
Chinese patent document CN102600276A discloses a Chinese herbal compound capable of eliminating drug resistance of escherichia coli, which comprises the following components in parts by weight: 8-10 parts of honeysuckle, 8-10 parts of isatis root, 8-10 parts of scutellaria baicalensis, 8-10 parts of cape jasmine, 8-10 parts of chrysanthemum, 8-10 parts of buffalo horn, 8-10 parts of bovine and sheep cholic acid and 8-10 parts of mulberry leaf. Chinese patent document CN102247493A discloses a traditional Chinese medicine oral liquid capable of eliminating drug-resistant plasmids of chicken bacteria, which comprises the following raw material medicines: 45-55 g of astragalus membranaceus, 45-55 g of folium artemisiae argyi, 45-55 g of salvia miltiorrhiza, 45-55 g of dandelion, 25-35 g of rheum officinale, 45-55 g of garden burnet, 45-55 g of Chinese violet, 45-55 g of sweet wormwood, 25-35 g of red peony root and 15-25 g of liquorice. Chinese patent document CN101940698A discloses a pharmaceutical composition for eliminating neisseria gonorrhoeae drug-resistant plasmids, which consists of phellodendron, glabrous greenbrier rhizome and phellodendron. However, the application of the Chinese herbal medicine in delaying the drug resistance of bacteria is not reported at present.
Disclosure of Invention
The invention aims to provide application of Chinese herbal medicines in delaying bacterial drug resistance aiming at the defects in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that: the application of radix Isatidis, rhizoma Belamcandae, radix Sophorae Flavescentis, folium Isatidis, herba plantaginis, fructus forsythiae, Scutellariae radix or folium Artemisiae Argyi in delaying drug resistance of Aeromonas can be provided.
The application of radix Isatidis, fructus forsythiae, Scutellariae radix or radix Sophorae Flavescentis in delaying drug resistance of Aeromonas can be provided.
Application of fructus forsythiae in delaying drug resistance of aeromonas is provided.
Application of decoction extract of radix Isatidis, rhizoma Belamcandae, radix Sophorae Flavescentis, folium Isatidis, herba plantaginis, fructus forsythiae, Scutellariae radix or folium Artemisiae Argyi in delaying drug resistance of Aeromonas campestris
The preparation method of the extracting solution of the Chinese herbal medicine by the decoction method comprises the following steps: weighing traditional Chinese medicine decoction pieces, adding clear water, soaking for 30min, gradually decreasing the water addition amount of first, second and third decoctions, boiling with strong fire, then maintaining the slightly boiling state with slow fire, decocting for 20min after the first decoction is boiled, decocting for 15min after the second decoction is boiled, decocting for 10min after the third decoction is boiled, mixing all filtrates, concentrating to make the final concentration of the medicine be 1g/ml, adjusting pH to 7.2-7.4, and sterilizing for 20min with flowing steam.
The aeromonas is aeromonas hydrophila.
The aeromonas is aeromonas hydrophila standard strain ATCC 7966.
The drug resistance is caused by using enrofloxacin, florfenicol, doxycycline hydrochloride, compound m-methoxypyrimidine sodium or neomycin sulfate.
The invention has the advantages that:
1. the invention screens out the traditional Chinese medicine with the function of delaying drug resistance to aeromonas for the first time.
2. The invention also analyzes the drug resistance mechanism of the aeromonas hydrophila.
Drawings
FIG. 1 is a distribution of unigenes annotated to the GO database.
FIG. 2 is a distribution of the annotated unigrams of unigenes.
FIG. 3 is the amount of unigene significantly differentially expressed.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1 screening of Chinese herbal medicine having drug resistance delaying action
The main work includes: exploring the drug resistance delaying effect of Chinese herbal medicines on aeromonas or vibrio, and screening out the Chinese herbal medicines with the drug resistance delaying effect
1 screening of drug-resistant Strain
The method comprises the steps of selecting an aeromonas hydrophila standard strain ATCC7966(SS for short) as a target strain, screening drug resistance of the aeromonas hydrophila standard strain ATCC7966, preliminarily selecting five antibiotics (enrofloxacin, florfenicol, doxycycline hydrochloride, compound sodium metamethicillin and neomycin sulfate), and screening the strains with strong drug resistance to the five antibiotics as further experimental objects.
1.1 determination of Minimum Inhibitory Concentration (MIC) of five antibiotics on SS
The MIC of five antibiotics to SS is determined by two-fold dilution method, namely 0.2mL of liquid medicine with the concentration of 1280 mug/mL is respectively added into a liquid medicine containing 1.8mL of 5.0 × 105cfu/mL bacteria solution test tube, to drug final concentration of 0.031, 0.063, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 μ g/mL, and setting 1 negative control group, shaking and culturing at 30 deg.C 200r/min for 24h, observing the result, diluting the sterile growthMaximum drug solution concentration was used as MIC for each experimental strain
Table 1 minimum inhibitory concentration of each antibiotic against SS strain (n ═ 5)
The experimental results are shown in Table 1, and the MICs of enrofloxacin, florfenicol, doxycycline hydrochloride, compound m-methoxypyrimidine sodium and neomycin sulfate to SS are respectively 0.25, 2, 8, 1 and 1 mu g/mL.
1.2 Induction of drug-resistant strains
Respectively inoculating SS into MH culture medium with initial concentration of 1/2MIC drug for subculture, inoculating bacteria once every 2d, inoculating bacteria into MH culture medium with 2 times of drug concentration in the previous time, measuring MIC until the MIC of the cultured strain rises to a drug-resistant judgment concentration range, then inoculating into a drug-free plate for culturing, inoculating for 1 time every day, continuously subculturing for 12d, measuring the MIC of the drug to each strain every 3 days to judge the stability of drug-resistant bacteria, and finally, naming the strain with stable drug resistance as ATCC7966-RS (hereinafter referred to as RS) and reserving for later use.
A drug-resistant bacterium is induced, the drug resistance degree reaches 128-time MIC, namely, the drug resistance is generated to enrofloxacin with the concentration of 64mg/mL, and the gene is stably inherited.
2 Chinese herbal medicine induction of SS and RS
Culturing SS and selected RS with strong drug resistance on a culture medium added with Chinese herbal medicine components for several generations, stably culturing on a common culture medium, and performing drug resistance experiment on the strains subjected to Chinese herbal medicine induction.
Primarily selecting Chinese herbal medicines with antibacterial effect: jasmine flower, mint leaf, weeping forsythia, common anemarrhena rhizome, sweet wormwood herb, Chinese pulsatilla root, baical skullcap root, heartleaf houttuynia herb, wormwood, dark plum fruit, swordlike atractylodes rhizome, plantain herb, lightyellow sophora root, dandelion, indigowoad leaf, indigowoad root, amur corktree bark, blackberry lily, lithospermum, Mongolian snakegourd seed, golden thread, honeysuckle flower, Chinese gall, pomegranate bark, dahurian patrinia herb, common myrobalan fruit, common selfheal fruit-spike, arisaema tuber, rhubarb.
2.1 extraction of effective ingredients of Chinese herbs
The traditional Chinese medicine is extracted by adopting a decoction method. Weighing 50g of Chinese medicinal decoction pieces accurately, adding clear water with height of about 2-3cm, respectively, adding less water to heavy medicine and less water to light medicine, soaking for 30min, decocting for one time, decocting for two times, and gradually decreasing the water addition amount for three times. Boiling with strong fire, and then regulating with slow fire to keep slightly boiling state. Decocting the first decoction for 20min, the second decoction for 15min, and the third decoction for 10 min. All filtrates were mixed and concentrated to 50ml to a final concentration of 1g/ml drug, pH adjusted to 7.2-7.4 and flow through steam sterilization for 20 min. Cooling, and placing in a refrigerator at 4 deg.C for short-term use.
The selected Chinese herbal medicines are prepared into Chinese herbal medicine extracting solution, and the Chinese herbal medicine extracting solution is sterilized and then is put into a refrigerator with the temperature of 4 ℃ for standby.
2.2 Induction of the Chinese herbal medicine against RS resistant strains
RS (group 1) and SS (group 2) were inoculated into MH medium with an initial concentration of 30mg/mL of drug, respectively, for subculture, RS (group 3) and SS (group 4) were also inoculated into MH medium without drug, subcultured 1 time per day, MIC of drug to each strain was measured every 5 generations, and the presence or absence of change in MIC was observed.
TABLE 2MIC Change before and after action of Chinese herbal medicine of RS strain (n ═ 5)
Unit: mu g/mL
The initial screening of 26 herbs was performed and the MIC and RS (group 1) results were determined at passage 10 as shown in Table 2 above, while the MIC of the other three groups of SS (group 2) and RS (group 3) and SS (group 4) cultured in media without drug was unchanged.
The preliminary screening result shows that 8 Chinese herbal medicines have the effect of delaying the drug resistance of aeromonas hydrophila, and are respectively isatis root, blackberrylily rhizome, lightyellow sophora root, indigowoad leaf, plantain herb, weeping forsythia, baical skullcap root and wormwood, wherein the weeping forsythia and the indigowoad root have better effects.
3 delayed drug resistance verification experiment
In the 8 Chinese herbal medicines with the effect of restoring mutation screened in the experiment, the forsythia and the isatis root with better effect and the 2 commonly used Chinese herbal medicines which are allowed to be used in the breeding industry in the other 6 Chinese herbal medicines are respectively scutellaria baicalensis and sophora flavescens, and the four Chinese herbal medicines are subjected to a drug resistance verification test.
3.1 setting of the experimental groups
The experiment was divided into 5 groups, which were control group (containing no herbal medicine), group 1 (forsythia suspensa), group 2 (scutellaria baicalensis), group 3 (sophora flavescens ait), and group 4 (isatis root), respectively; chinese herbal medicines with the concentration of 30mg/mL are respectively added into 50mL MH liquid culture media of the groups 1 to 4, while the control group is not added with any Chinese herbal medicines; then, enrofloxacin (MIC) was added to the medium of 5 groups at a concentration of 0.25. mu.g/mL to complete the preparation of the medium for 5 experimental groups.
3.2 verification of the Experimental procedure
SS was inoculated into the drug-containing medium of each of the 5 experimental groups for subculture, and was transferred once a day, and whether there was a change in MIC of Aeromonas hydrophila was determined in each of the 5 experimental groups at passage 10 and 20.
The experimental results show that the lowest inhibitory concentration of the SS strains in the control group is 16 times of the initial inhibitory concentration after 20 generations of inoculation without the action of any Chinese herbal medicine, the lowest inhibitory concentration of the groups 2, 3 and 4 is 4 times of the initial inhibitory concentration, and the lowest inhibitory concentration of the group 1 is only increased by 2 times after 20 generations of inoculation with the action of fructus forsythiae. The four Chinese herbal medicines are proved to have the effect of delaying the drug resistance, and the fructus forsythiae has the most obvious effect of delaying the drug resistance.
TABLE 3 MIC Change of SS Strain after Simultaneous action of Chinese herbal medicine and enrofloxacin (n ═ 5)
Example 2 analysis of the mechanism of resistance to Aeromonas hydrophila
Aiming at the mechanism of the resistance of aeromonas hydrophila to enrofloxacin, the analysis is carried out through a transcriptome
1 Experimental strains
The experimental transcriptome sequencing strains had two strains, respectively, Aeromonas hydrophila ATCC7966(SS) with an MIC of 0.25. mu.g/mL and Aeromonas hydrophila ATCC7966-RS (RS) which had been previously induced to have an MIC of 64. mu.g/mL.
2 analysis of the partial data of transcriptome sequencing
After sample RNA was extracted, sequencing analysis was performed. After assembling and splicing the sequenced original data, 2353, 1821 and 4151 unigenes (non-repetitive sequence genes) belonging to RS, SS and RS & SS are obtained respectively. Of the 4151 unigenes in RS & SS, 3237 were annotated by Swiss-Prot database and 3944 were annotated by NCBI protein database. By BLAST and BLAST2GO software analysis, 2548, 3178 unigenes were annotated by the GO database (database defining and describing gene and protein functions) and the COG database (orthologous gene database), respectively, and 128 unigenes could be annotated by the KEGG database (database analyzing metabolic pathways of gene products in cells).
As a result of transcriptome analysis, 678 unigenes have significant difference in expression in two strains under the action of enrofloxacin, wherein 338 unigenes have up-regulated expression, and 340 unigenes have down-regulated expression. The up-regulated genes associated with the resistance mechanism are uvrA, gyrB (table 4), rmuC, SPO3184, pdxY, while the down-regulated genes are gyrA, umuC, lamb. Currently, the subject group is performing verification analysis of the above genes from the mRNA level by the method of fluorescent quantitative PCR.
TABLE 4 differential analysis of the gyrB genes
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (1)
1. The application of the Chinese herbal medicine in preparing the medicine for delaying the drug resistance of the aeromonas is characterized in that the Chinese herbal medicine is fructus forsythiae, the aeromonas is an aeromonas hydrophila standard strain ATCC7966, the drug resistance is caused by using enrofloxacin, and the preparation method of the Chinese herbal medicine comprises the following steps: weighing traditional Chinese medicine decoction pieces, adding clear water, soaking for 30min, gradually decreasing the water addition amount of first, second and third decoctions, boiling with strong fire, then maintaining the slightly boiling state with slow fire, decocting for 20min after the first decoction is boiled, decocting for 15min after the second decoction is boiled, decocting for 10min after the third decoction is boiled, mixing all filtrates, concentrating to make the final concentration of the medicine be 1g/ml, adjusting pH to 7.2-7.4, and sterilizing for 20min with flowing steam.
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| CN101757219A (en) * | 2009-09-18 | 2010-06-30 | 天津生机集团股份有限公司 | Compound pharmaceutical preparation for treating bacterial disease of eel and preparation method thereof |
| CN102240323A (en) * | 2011-05-11 | 2011-11-16 | 浙江海洋学院 | Traditional Chinese medicine for controlling aeromonas hydrophila disease of aquatic animals and utilizing method thereof |
| CN103239544A (en) * | 2013-05-29 | 2013-08-14 | 北京大北农动物保健科技有限责任公司 | Fishery Chinese and western compound medicine composition as well as premix compound, batch mixture and application thereof |
| CN103393778A (en) * | 2013-07-31 | 2013-11-20 | 西南大学 | Compound Chinese herbal preparation for preventing and treating fish bacteria and viral diseases |
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| CN101757219A (en) * | 2009-09-18 | 2010-06-30 | 天津生机集团股份有限公司 | Compound pharmaceutical preparation for treating bacterial disease of eel and preparation method thereof |
| CN102240323A (en) * | 2011-05-11 | 2011-11-16 | 浙江海洋学院 | Traditional Chinese medicine for controlling aeromonas hydrophila disease of aquatic animals and utilizing method thereof |
| CN103239544A (en) * | 2013-05-29 | 2013-08-14 | 北京大北农动物保健科技有限责任公司 | Fishery Chinese and western compound medicine composition as well as premix compound, batch mixture and application thereof |
| CN103393778A (en) * | 2013-07-31 | 2013-11-20 | 西南大学 | Compound Chinese herbal preparation for preventing and treating fish bacteria and viral diseases |
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