CN116396880A - Method for reducing bacterial drug resistance by using phage and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for reducing bacterial drug resistance by using phage and application thereof. A method for reducing bacterial drug resistance by using phage adopts phage to continuously co-culture drug resistant bacteria and its culture for more than three times. The invention adopts bacteriophage to treat drug-resistant bacteria, can directly inject bacteriophage into animal bodies, realizes multiple treatment effects on drug-resistant bacteria in animal bodies, reduces drug resistance of pathogenic bacteria in living bodies, reduces occurrence of drug resistance, and has lasting and stable effect. And compared with the traditional method, the method is safer and more efficient.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for reducing bacterial drug resistance by using phage and application thereof.
Background
Bacterial resistance is divided into intrinsic resistance (intrinsic bacterial resistance) and acquired resistance (acquired resistance). The inherent drug resistance is also called natural drug resistance, is determined by bacterial chromosome genes and is transmitted in a substitution phase, and can not be changed, such as the natural drug resistance of streptococcus to aminoglycoside antibiotics; the intestinal tract G-bacillus is naturally resistant to penicillin; pseudomonas aeruginosa is insensitive to most antibiotics. Acquired resistance is due to the fact that bacteria are mediated by plasmids after contact with antibiotics, and by changing their metabolic pathways, they are not killed by antibiotics. Acquired drug resistance of bacteria can disappear due to no longer contacting with antibiotics, or drug resistance genes can be transferred from plasmid to chromosome in a substitution way, so that the bacteria become inherent drug resistance.
At present, the methods for reducing or eliminating bacterial drug resistance are mainly as follows: traditional Chinese medicine elimination method, chemical elimination method and physical elimination method.
(1) The method for eliminating the traditional Chinese medicine comprises the following steps: the reports on the elimination of drug-resistant genes and drug-resistant plasmids of traditional Chinese medicines are gradually increased from the 80 s of the 20 th century, and researches show that most of the traditional Chinese medicines belong to heat-clearing and detoxicating and heat-clearing and dampness-drying medicines, and the effective components are diversified and are mainly volatile oil, flavone, organic acid, polysaccharide, alkaloid and the like (Marz 2011; xuan Hong Yuan and the like 2019; zhang Yingbing and the like 2017). The traditional Chinese medicine belongs to a pure natural medicine, has various advantages of low price, no drug residue and drug resistance, and has wide application prospect in animal husbandry in China.
(2) The chemical elimination method comprises the following steps: sodium dodecyl sulfate (Sodium dodecyl sulfate, SDS) is an anionic surfactant. The research shows that SDS can be used as an eliminator of drug-resistant plasmids, and the main action mechanism has two aspects: on the one hand, SDS can dissolve the inner membrane protein of the strain and destroy the binding site of the plasmid on the cell membrane; on the other hand, SDS enters bacterial cytoplasm to inactivate some proteins related to plasmid replication, so that the plasmid can not be normally replicated and distributed in the filial generation, thereby achieving the aim of eliminating drug-resistant plasmid. SDS was used to eliminate the drug-resistant plasmid of E.coli as early as 1968 by Tomoeda M et al, and the elimination effect was found to be closely related to both the test temperature and the concentration of SDS. Xin Du et al (2015) recovered sensitivity to some antibiotic drugs by alternately treating duck-origin resistant E.coli to passage 7 with high temperature-SDS.
(3) Physical elimination method: under various physical conditions, such as high temperature, high heat, ultraviolet irradiation, etc., the plasmid DNA molecules of the strain are decomposed and denatured, and the original functions are lost, so that the principle can be utilized to eliminate drug-resistant plasmids. The Yu's study (2001) found that the effect of eliminating drug-resistant plasmids was best when irradiated vertically with an ultraviolet lamp distance of 43 cm of 30W. There were researchers who eliminated 80% of drug-resistant E.coli drug-resistant plasmids by electric shock (Li Dong 2015). In practice, the conditions for physical elimination of the drug-resistant plasmid are not suitable for clinical use, and thus researchers often choose a combination of physical and chemical methods (e.g., high temperature-SDS) to eliminate the drug-resistant plasmid.
Although the three methods can eliminate the drug resistance of bacteria to a certain extent, the three methods are limited to in-vitro antibacterial tests and drug resistance elimination tests, the drug resistance of certain generation of drug resistant bacteria is eliminated, and the drug resistance of bacteria can be recovered along with the passage propagation of bacteria.
Disclosure of Invention
In view of the shortcomings of the conventional methods for eliminating bacterial resistance described above, an object of the present invention is to provide a method for reducing bacterial resistance using phage and application thereof.
The technical scheme adopted by the invention is as follows: a method for reducing bacterial drug resistance by using phage adopts phage to continuously co-culture drug resistant bacteria and its culture for more than three times.
Further, the phage is E.coli phage RDP-EC-16029.
Further, the processing titer of the phageIs 10 -2 MIC。
Further, the drug-resistant bacteria are drug-resistant escherichia coli, salmonella or klebsiella.
The invention also provides an application of the method for reducing the drug resistance of bacteria by using phage, and the method is used for treating drug-resistant bacteria to obtain bacteria with reduced or eliminated drug resistance.
A bacterium with reduced drug resistance, the drug resistant plasmid of which produces deletion mutation after the drug resistant bacterium is treated by the method.
Further, the phage was E.coli phage RDP-EC-16029 with a treatment titer of 10 -2 MIC
Compared with the prior art, the invention has the beneficial effects that: the invention adopts bacteriophage to treat drug-resistant bacteria, can directly inject bacteriophage into animal bodies, realizes multiple treatment effects on drug-resistant bacteria in animal bodies, reduces drug resistance of pathogenic bacteria in living bodies, reduces occurrence of drug resistance, and has lasting and stable effect. And compared with the traditional method, the method is safer and more efficient.
Drawings
FIG. 1 is an electrophoresis chart of R plasmid before treatment of drug-resistant bacteria in the embodiment of the invention, M: a Marker;1: EC-1;2: SA-2,3: KP-3;
FIG. 2 is an electrophoretogram of the eliminator R plasmid obtained after the treatment of E.coli resistant strain in the example of the present invention, M: a Marker; 1. 2,3, 4, 5 are respectively electrophoretogram of R plasmid of different eliminator colonies;
FIG. 3 shows an electrophoretogram of the eliminator R plasmid obtained after the salmonella resistant strain treatment in the example of the present invention, M: a Marker; 1. 2,3, 4, 5 are respectively electrophoretogram of R plasmid of different eliminator colonies;
FIG. 4 is an electrophoretogram of the eliminator R plasmid obtained after treatment of the Klebsiella resistant strain in the example of the present invention, M: a Marker; 1. 2,3, 4, 5 are electrophoretogram of R plasmid of different eliminator colonies, respectively.
Detailed Description
In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
EXAMPLE Elimination experiments with phages on E.coli, salmonella, klebsiella resistant strains
1. Isolation and identification of drug-resistant strains
Sampling from different disease farms in Shandong, taking diseased poultry livers through aseptic operation, streaking on a selective medium (MAIKAI agar), culturing for 18-24 hours at 37 ℃, forming round, flat, neat-edged red colonies with smooth and moist surfaces on the medium, continuously streaking and purifying typical colonies for 3 times, then picking single colonies, inoculating the single colonies into 5mL of LB broth, and culturing for 8 hours at 200rpm under shaking at 37 ℃ to obtain uniform and turbid bacterial suspension. And then the pathogenic escherichia coli is determined by 16sRNA molecular identification and serotype identification, which is named as EC-1, and the escherichia coli is stored in a refrigerator at the temperature of minus 80 ℃.
Streaking on a salmonella chromogenic medium of Ma Jia, culturing for 18-24 hours at 37 ℃, forming a circular, flat, neat-edged and smooth-surface purple colony on the medium, picking a typical colony, continuing streaking and purifying for 3 times, then picking a single colony, inoculating into 5mL of LB broth, and culturing for 8 hours at 200rpm at 37 ℃ to obtain a uniform and turbid bacterial suspension. And then the pathogenic salmonella is determined by 16sRNA molecular identification and serotype identification, and the salmonella is named SA-2 and is stored in a refrigerator at the temperature of minus 80 ℃.
Streaking on a positioning chromogenic medium of a urethra flora of Ma Jia of the family, culturing for 18-24 hours at 37 ℃, forming circular, flat, clean-edged and metallic-glossy colonies on the medium, picking typical colonies, continuing streaking and purifying for 3 times, then picking single colonies, inoculating the single colonies into 5mL LB broth, and culturing for 8 hours at 200rpm at 37 ℃ to obtain uniform and turbid bacterial suspension. Then the pathogenic klebsiella is determined by 16sRNA molecular identification and serotype identification, and is named as KP-3 and stored in a refrigerator at the temperature of minus 80 ℃.
2. Pathogenic drug-resistant bacteria drug-resistant gene detection and R plasmid extraction
Coli EC-1 was inoculated and streaked onto a MAIKAI plate and incubated at 37 ℃. Individual colonies characterized by this were selected and inoculated into the broth and cultured in a thermostatic water bath shaker at 37 ℃. Collecting bacterial liquid, centrifuging at room temperature to collect bacterial precipitate, and discarding supernatant. The mixed solution is added into each tube, bacterial sediment is resuspended, the sediment is ensured to be completely dispersed, and no bacterial agglomerate is visible. Adding into the resuspension mixture, gently reversing and mixing to completely lyse the bacteria, and the lysis reaction can not exceed 2min. And adding 350ul Solution III, immediately reversing the centrifuge tube, uniformly mixing for 4-6 times, and centrifuging for 10min at the room temperature of 13000r/min until white floccules are generated. Transfer supernatant to HiBind DNA binding column covered with 2mL collecting tube, centrifuge for 1min at 10000r/min at room temperature, and discard filtrate in collecting tube. The column was refilled into the collection tube, 500uL HB Buffer was added, centrifuged at 10000r/min for 1min at room temperature, and the filtrate was discarded. The column was refilled into the collection tube, 500uL Wash Buffer was added, centrifuged at 10000r/min for 1min as above, and the filtrate was discarded. The above procedure was repeated once. The filtrate was discarded, the column was refilled into the collection tube, and the empty column was centrifuged to remove residual liquid and to completely volatilize traces of ethanol. The column is put on a clean centrifuge tube, added into the column matrix, and is kept stand, and the plasmid is eluted by centrifugation, and the obtained liquid is the high-purity plasmid.
SA-2 and KP-3 were carried out in the same manner, and the corresponding plasmids were extracted.
3. Phage preparation
Firstly, preparing a proliferation culture medium, which mainly comprises the following components: LB liquid medium with 30% glycerol added. Inoculating host bacterium EC-1 into a proliferation culture medium according to an inoculum size of 5%; culturing the host bacteria at 37 ℃ to logarithmic phase; 100. Mu.l of phage RDP-EC-16029 stock was mixed with 300. Mu.l of host bacteria and allowed to stand for 15 minutes to infect. The mixture was inoculated into a test tube containing 10 ml fresh liquid medium and cultured with shaking at 37℃for about 8 hours. Centrifuging to remove impurities: centrifuging the mixed solution obtained in the last step at 3000r/min for 15min, removing impurities, and collecting the supernatant.
4. MIC determination of phage on bacteria
Experiments were performed using sterile 96-well plates, with 100ul of LB broth added to each row. 10 mu L phage RDP-EC-16029 is added into the first hole, and the mixture is blown and evenly mixed by a pipette. And then 10 mu L of mixed solution is taken from the first hole, added into the second hole, diluted by 10 series of times in sequence, sucked out to be lost when diluted to the tenth hole, and reserved for standby in 11 and 12 holes. 100 mu L of diluted escherichia coli bacterial liquid (1 multiplied by 10) is respectively added into 1-10 holes 5 ～2×10 5 cfu/m L); well 11 is a negative control, add 100 μl MH broth; and taking the 12 th hole as a positive control, and adding 100 mu L diluted bacterial liquid. 24. 24h was incubated at 37 ℃. Observing the growth condition of bacteria acting with phage, wherein the minimum concentration without bacterial growth is the minimum inhibitory concentration, and the minimum inhibitory concentration of phage on Escherichia coli is 10 -5 cfu/m L。
5. Treatment of drug-resistant strains with phages to obtain cancellers
Mixing phage RDP-EC-16029 with separated drug-resistant escherichia coli, salmonella and klebsiella, and culturing: diluting phage RDP-EC-16029 10 times, taking phage 7 times to test, respectively taking 100ul of escherichia coli EC-1, salmonella SA-2 and klebsiella KP-3, synchronously inoculating into LB broth without antibiotics, and culturing in a 37 ℃ incubator for 24 hours; 100ul of the mixture after 24 hours of culture was inoculated with phage RDP-EC-16029 in LB broth without antibiotics, respectively, and incubated in an incubator at 37℃for 24 hours; 100ul of the mixture after the secondary culture for 24 hours was inoculated with phage RDP-EC-16029 in LB broth without antibiotics, respectively, and incubated in an incubator at 37℃for 24 hours. Cultures were kept 3 rd after the action. The resulting bacteria were the bacteria after treatment (the eliminator).
The original drug-resistant strains EC-1, SA-2 and KP-3 before treatment and the treated strains EC-1, SA-2 and KP-3 are respectively subjected to drug sensitivity test comparison, and the results show that the drug resistance of bacteria after phage treatment is reduced, as shown in Table 1.
TABLE 1 sensitivity change to antibiotic drugs before and after treatment with drug-resistant strains
6. The effect of phage treatment on drug-resistant strains is compared with the prior method
Mixing phage RDP-EC-16029 with separated drug-resistant escherichia coli, salmonella and klebsiella, and culturing: serial dilution of phage RDP-EC-16029 by 10 times, testing phage by-7 times, respectively inoculating 100ul phage and 100ul Escherichia coli EC-1, salmonella SA-2, and Klebsiella KP-3 into LB broth without antibiotics, and culturing in 37 deg.C incubator for 24 hr; 100ul of the mixture after 24 hours of culture was inoculated with phage RDP-EC-16029 in LB broth without antibiotics, respectively, and incubated in an incubator at 37℃for 24 hours; 100ul of the mixture after the secondary culture for 24 hours was inoculated with phage RDP-EC-16029 in LB broth without antibiotics, respectively, and incubated in an incubator at 37℃for 24 hours. 3 cultures were taken after the action and stored.
The three resistant strains were treated with 0.02% SDS (treatment methods refer to reported literature) as an elimination control. The cultured bacterial liquid is fully diluted and then is coated on an NA agar plate, the bacterial liquid is cultured at 37 ℃ for 12-18 h, a large number of single colonies are obtained, 100 single colonies are respectively picked up and respectively spotted on the NA agar plate and the NA agar plate containing the antibiotics KA+, the bacterial liquid is inverted, the bacterial liquid is cultured at the constant temperature of 37 ℃ for 12 h, the bacterial liquid which does not grow on the medicine-containing plate and does not grow on the medicine-containing plate is selected, and the bacterial liquid is inoculated on a medicine-containing culture medium again for verification, and if the bacterial liquid does not grow, the bacterial liquid is an eliminator (bacterial liquid with eliminated medicine resistance). The results are shown in Table 2 below.
TABLE 2 results of drug resistance elimination test
7. Extraction and identification of the annihilator R plasmid
The delectants were subjected to plasmid extraction according to the method described above, and 5 parallel experimental groups were selected for each strain. And detecting whether plasmids of the original strain and the eliminator change or not through agarose gel electrophoresis, and whether the phenomenon of loss of the drug-resistant plasmids exists or not. As shown in the results of FIGS. 1-4, the eliminators obtained after the three pathogenic bacteria are treated by the phage RDP-EC-16029 all show loss of 2-3 plasmid bands, which indicates that the phage RDP-EC-16029 has good elimination effect on the drug-resistant bacteria R plasmid.
Claims (7)
1. A method for reducing bacterial resistance using phage, comprising: and adopting phage to continuously perform co-culture treatment on the drug-resistant bacteria and the culture thereof for more than three times.
2. The method for reducing bacterial resistance using phage of claim 1, wherein: the phage is coliphage RDP-EC-16029.
3. The method for reducing bacterial resistance using phage of claim 1, wherein: the phage had a processing titer of 10 -2 MIC。
4. The method for reducing bacterial resistance using phage of claim 1, wherein: the drug-resistant bacteria are drug-resistant escherichia coli, salmonella or klebsiella.
5. Use of a method for reducing bacterial resistance using phage according to any one of claims 1-4, wherein: by using the method, the bacteria with reduced or eliminated drug resistance are obtained by treating the drug-resistant bacteria.
6. A bacterium having reduced resistance, characterized in that: the method of any one of claims 1-4, wherein the drug resistant bacteria are subjected to deletion mutation on drug resistant plasmids.
7. The reduced resistance bacteria of claim 6, wherein: the phage was E.coli phage RDP-EC-16029 with a treatment titer of 10 -2 MIC。
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