CN113186169B - Serratia marcescens bacteriophage and medical application thereof - Google Patents
Serratia marcescens bacteriophage and medical application thereof Download PDFInfo
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Abstract
The invention discloses a serratia marcescens phage and medical application, which are named as: the serratia marcescens phage vB _ SamS-WG03 is preserved in China Center for Type Culture Collection (CCTCC) in 11 months and 9 days of 2020, and the preservation numbers are as follows: CCTCC NO: m2020712; the phage can not only crack serratia marcescens, but also crack part of yersinia pseudotuberculosis, can crack host bacteria SM2, can crack yersinia pseudotuberculosis separated from the environment or pathogenic animal materials, can be used for killing the environment independently or by being compounded with other phage, and is a safe and nontoxic product.
Description
Technical Field
The invention discloses a serratia marcescens phage (vB _ SamS-WG 03), simultaneously provides a separation and purification method and general biological characteristics thereof, can be used for preventing or treating infectious diseases caused by serratia marcescens and yersinia pseudoconjugate, and belongs to the technical field of biological engineering.
Background
Serratia marcescens is a conditioned pathogen existing in soil, water and plant surfaces, and also in intestinal tracts of animals and humans, and is one of important conditioned pathogens causing nosocomial infections, and can cause respiratory tract, urinary tract and wound infections and the like. In recent years, due to unreasonable use of antibiotics, bacteria have stronger and stronger tolerance to the antibiotics, and the serratia marcescens also has drug resistance to various antibiotics, so that the treatment of infection caused by the serratia marcescens becomes more and more difficult, an annual report from Mohnarin 2009 shows that the drug resistance rate of the serratia marcescens to cefuroxime is up to 87.5%, and the drug resistance rates to cefazolin and ampicillin respectively reach 87.1% and 76.2%. Therefore, the development of new drugs for controlling infection caused by drug-resistant serratia marcescens is urgent.
Bacteriophages (bacteriophages) are a class of viruses that can attack bacteria and may also be referred to as bacterial viruses. It can invade the interior of bacteria, then utilizes self-provided template and bacteria material and tool to synthesize its own genome and structural protein, and when the phage completes these steps and reaches the end of infection, it can utilize the hydrolysis action of enzyme to break the cell wall of bacteria so as to crack and kill bacteria. Bacteriophages are natural bacteria, "gram" that proliferate in large quantities upon infection of a host and release viral particles to the outside upon death of the host bacteria, allowingThe adjacent susceptible cells are infected. Since the beginning of phage discovery, they have been used to treat infections caused by various bacteria, and many countries have suspended the research on phage therapy as antibiotics are discovered and put into the treatment of bacterial infections in large quantities and widely. However, phage therapy has been applied in Grugia and other countries. The emergence of drug-resistant bacteria and the severity of the associated problems has made phage therapy a significant avenue of research. According to analysis, about 10 6 Species phages, the number of which is about 10, are present on the earth we live on 31 It is abundant as a gene bank, and thus it is also excellent as a subject of genetics. They are novel biological antibacterial substances which are harmless to human beings and animals, can specifically infect bacteria. Many studies from both home and abroad indicate that when used for treating bacterial infection, the bacteriophage can play a good role in treatment, namely, the application of bacteriophage has great potential in the aspects of preventing and controlling bacterial infection. Currently, there are few studies on Serratia marcescens phages.
Disclosure of Invention
The invention discloses a serratia marcescens phage, namely a serratia marcescens phage vB _ SamS-WG03;Serratia marcescensthe phage vB _ SamS-WG03 is deposited in China center for type culture Collection on 11/9/2020, and the deposit number is as follows: CCTCC NO: m2020712.
The invention relates to a novel bacteriophage, which has a regular icosahedron head and a non-contractive tail under an electron microscope; the phage can form clear bright plaques with clear edges and no halo around on a common LB semisolid culture medium, and the diameter of the plaques is about 0.5-2mm; agarose gel electrophoresis of the digested phage genome showed that the phage was a double stranded DNA (dsDNA) phage.
The invention also provides application of the serratia marcescens phage in preparing a medicament for preventing or treating infectious diseases caused by serratia marcescens and yersinia pseudoconjugate.
The invention discloses application of a serratia marcescens phage vB _ SamS-WG03 in killing serratia marcescens and yersinia pseudoconjugate in a space environment.
The serratia marcescens phage vB _ SamS-WG03 disclosed by the invention is applied to killing serratia marcescens and yersinia pseudoconjugate on the body surface and body of livestock.
A composition for preventing or treating infectious diseases caused by Serratia marcescens and Yersinia pseudobindans, which comprises the bacteriophage of the present invention as an effective ingredient.
The composition is characterized by being a liquid preparation, a freeze-dried preparation or an oral solid preparation.
The application of the phage or the composition thereof in preparing feed additives, feeds, drinking water additives, drinking water, disinfectants or detergents for killing serratia marcescens in bodies of livestock and poultry, body surfaces of livestock and poultry, livestock and poultry feeds, culture devices or culture space environments is disclosed.
The invention has the positive effects that:
a novel serratia marcescens phage vB _ SamS-WG03 is provided, which is a phage of the family of long-tailed phages separated from the nature and capable of specifically killing serratia marcescens and yersinia pseudoconjugate, can crack not only serratia marcescens but also part of yersinia pseudotuberculosis, can crack host bacteria SM2, can crack yersinia pseudotuberculosis separated from the environment or pathogenic materials of pathogenic animals, can be used for killing the environment alone or compounded with other phages, and is a safe and nontoxic product.
Drawings
FIG. 1 is a photograph of a bacteriophage vB _ SamS-WG03 plaque of the present invention;
FIG. 2 is a transmission electron micrograph of the bacteriophage vB _ SamS-WG03 of the present invention;
FIG. 3 is an optimal MOI map of bacteriophage vB _ SamS-WG03 of the present invention;
FIG. 4 is a graph showing the one-step growth of the bacteriophage vB _ SamS-WG03 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1
Phage isolation and preparation
The process of phage isolation is described in detail below. The sewage sample in the invention is taken from the roasted plum in Changchun city, and the host bacterium serratia marcescens SM2 is separated from the first college of Jilin university. Preparing an LB culture medium (100 mL) by using collected sewage instead of double distilled water; adding 1mL of host bacterium SM2 cultured overnight into the culture medium, and culturing at 37 ℃ for 10-12h; the mixed culture was centrifuged at 1mL and 12000 Xg for 5min, the supernatant was filtered through a 0.22 μm sterile filter to obtain a phage stock solution and stored, and the obtained filtrate was subjected to a plaque test to examine whether or not the filtrate contained a phage capable of cleaving Serratia marcescens SM 2.
And (3) plaque testing: taking serratia marcescens SM2 which is subjected to shaking culture at 37 ℃ overnight, and uniformly coating the bacterial liquid on an LB solid plate by using a sterile cotton swab; after drying, 10ul of the phage stocks were dropped onto the center of the plate; after the liquid drops are dried, the liquid drops are placed in an incubator at 37 ℃ upside down for 10 hours, and whether the specific area forms the plaque or not is observed.
If a plaque is formed in a particular area, it indicates that Serratia marcescens phage may be present in the filtrate. The phage stock solution obtained above was diluted by a series of 10-fold ratios. 0.1mL of each diluent with proper dilution times is uniformly mixed with 0.1mL of host bacteria cultured overnight, the mixture is quickly poured into the upper layer of an LB agar medium plate, after solidification, the mixture is placed in an incubator at 37 ℃ for 8 hours, and whether single plaques are formed on a double-layer plate or not is observed.
Example 2
Phage amplification and purification
Picking a single large, round and transparent plaque on a double-layer flat plate with the plaque, inoculating the single plaque into 5ml LB liquid culture medium containing 200 mu L of host bacteria liquid, uniformly mixing, carrying out shake culture at 37 ℃ for 3-5 h, then centrifuging at 12000 Xg for 10min at 4 ℃, and taking the supernatant; the double-layer plate experiment is repeated, and the operation is repeated for 4-5 times until plaques with uniform size can be formed on the double-layer plate.
5mL of freshly cultured host bacteria were mixed with 1mL of phage lysate (at optimal MOI), added to 800mL of LB liquid medium, and added to 1.25mM CaCl to final concentration 2 Shake culturing at 37 ℃ for 6-8h, centrifuging at 12000 Xg at 4 ℃ for 10min, and taking the supernatant to obtain the phage lysate.
And (3) PEG purification: adding RNase A and DNase I into the phage lysate until the final concentration is 1 mu g/mL, and acting for 0.5h at room temperature; adding NaCl to the final concentration of 1mol/L, and carrying out ice bath for 1-2h after completely mixing; centrifuging at 8000 Xg and 4 deg.C for 15-20min after ice bath, and keeping supernatant; adding PEG-8000 at a dose of 10g/100mL, stirring gently for dissolving, performing overnight ice bath, and precipitating bacteriophage under the action of PEG-8000; centrifuging the reacted product at 12000 Xg for 10-20min at 4 ℃, reserving the precipitate, adding 2mL of SM solution, and washing and resuspending the precipitate; adding chloroform with the same volume for extraction, and carrying out mild oscillation for 30s; centrifuging at 5000 Xg and 4 deg.C for 10min, collecting the upper hydrophilic phase, and obtaining the purified product of bacteriophage.
CsCl isopycnic gradient centrifugation purification, namely preparing CsCl gradient liquid according to the table 1, and sequentially adding 1mL of each gradient liquid into a 5mL translucent polyacrylamide high-speed centrifuge tube from high to low in density; slowly adding 700 μ L phage concentrate to CsCl gradient liquid upper layer, horizontally centrifuging at 35000r/min at 4 deg.C for 3 h; after the centrifugation is finished, the vacuum is required to be reduced to 0, then the bin gate is opened, the centrifuged centrifuge tube is taken out, and the machine is shut down; a layer of blue-color tape is arranged at the lower end of the mixed liquid in the centrifuge tube, and a thinner needle is inserted into the tape from the side surface to carefully absorb the blue-color tape; the resulting sample was placed in a dialysis bag and dialyzed against 10mM Tris-HCl,100mM MgCl 2 buffer at pH 7.4 for 2L (10-14 kd) at a time; finally, the sample was aspirated and the phage titer was determined.
TABLE 1 CsCl gradient liquid preparation
The phage titer is detected by a double-layer plate method: the phage liquid purified by the steps is serially diluted by 10 times, 100 mu L of phage diluent under each gradient is completely mixed with 100 mu L of host bacteria liquid, double-layer agar plates are paved and cultured at 37 ℃, and then the plaques appearing on each plate are counted. Selecting a plate containing about 30-300 plaques for effective counting, and calculating to obtain the titer of the phage. After purification, the phage can form a clear and regular bright plaque with a diameter of 0.5-2mm without a halo around in a common LB agar culture medium as shown in figure 1.
The purified phage is named vB _ SamS-WG03 by self, and is preserved in China center for type culture Collection (CGMCC), the preservation unit address: china, wuhan university, zip code 430072, deposit number: CCTCC NO: m2020712, category name: serratia marcescens phage vB _ SamS-WG03;Serratia marcescensthe phase vB _ SamS-WG03 has the preservation date of 2020, 11 months and 9 days.
Example 3
Phage vB _ SamS-WG03 transmission electron microscope observation
The phage purified by PEG-8000 in the embodiment 2 is taken for electron microscope observation, and the specific operation steps are as follows: dropping 10 μ L sample on copper mesh, precipitating for 15min, removing excessive liquid with filter paper, staining with 2% phosphotungstic acid (PTA) for 1-2min, drying, and observing with transmission electron microscope (Hitachi H-7650); as shown in FIG. 2, the head was a regular icosahedron, the diameter of the head was about 50. + -.3 nm, and the length of the tail was about 92. + -.3 nm. vB _ SamS-WG03 belongs to the family of the Long-tailed Virus (Siphoniviridae) according to the eighth report of the Committee for Classification of viruses, international Committee for Classification of viruses (ICTV), 2005.
Example 4
Optimal MOI assay for bacteriophage vB _ SamS-WG03
Optimal MOI determination: after culturing the bacterial liquid to logarithmic phase, the concentration of the bacterial liquid is adjusted to 10 7 cfu/mL, then 1X 10 in phage/bacteria ratio -6 、1×10 -5 、1×10 -4 、1×10 -3 、1×10 -2 、1×10 -1 And 1, mixing the phage with the bacterial liquid and transferring the mixture into an LB liquid culture medium for shaking culture at 37 ℃ for 8 hours. Then centrifuging the culture solution at 4 deg.C under 10000 Xg for 15min, filtering the supernatant with sterile disposable filter with pore size of 0.22 μm to obtain bacteriophage value-added solution, and determining its titer by double-layer plate method, wherein the ratio of bacteriophage/bacteria when reaching the highest titer is the optimal MOI, and the result is shown in FIG. 3.
Example 5
One-step growth Curve assay for bacteriophage vB _ SamS-WG03
One-step growth curve determination: mixing host bacteria and phage cultured to logarithmic phase according to the proportion of the optimal MOI, standing at 37 ℃ for 5min, suspending the precipitate with LB liquid culture medium, placing the suspension in a constant-temperature shaking table at 37 ℃ for shaking culture, sampling every 5min for the first 30min, sampling every 10min for the second 30min, respectively sampling in one hour to determine the titer of the phage at the point, and drawing a one-step growth curve of the phage infected bacteria, wherein the result is shown in figure 4.
Example 6
Analysis of phage vB _ SamS-WG03 host Spectroscopy
The phage vB _ SamS-WG03 titer obtained in example 2 was adjusted to 10 8 pfu/mL for use. In the test, a plurality of strains of serratia marcescens, escherichia coli, klebsiella pneumoniae and yersinia pseudotuberculosis are selected as objects, and the host spectrum of the phage vB _ SamS-WG03 is analyzed, and the specific operation is as follows:
determination of plaque test: taking bacterial liquid of each strain to be detected cultured for 12-16h, respectively dripping 100 mu L (part of the strains with special nutrition needs adopt specific culture medium) into the centers of different LB culture medium flat plates, and coating sterile cotton swabs to prepare uniform lawn. And (3) dripping 10 mu L of phage on the surface of the lawn, inverting the plate after the phage is sufficiently absorbed by the liquid drop, culturing at the constant temperature of 37 ℃ for 10-16 hours, taking out the plate, and observing the test result, wherein the plate is marked as "+" if plaque is generated, and the plate is marked as "-" if the plaque is not generated, and the result is shown in table 2.
Plaque assay determination: mu.L of phage stock was serially diluted 10-fold. Diluting respectively with 100 μ L of 10 4 、10 5 And 10 6 The multiplied liquid was uniformly mixed with 100. Mu.L of each culture solution of each strain to be tested which was cultured for 12 to 16 hours, and after sufficiently adsorbing at room temperature for 15 minutes, the mixture was added to about 7mL of LB semisolid medium at 45 ℃, the mixture was quickly poured onto the upper layer of a LB agar medium plate, shaken and laid flat, after solidification, the mixture was cultured at 37 ℃ for 6 to 8 hours, and after taking out the solidified mixture, the result was observed, and the result was marked as "+" when there was any plaque, and as "-" when there was no plaque, and the results are shown in Table 2.
TABLE 2 analysis of the phage vB _ SamS-WG03 host Spectroscopy
| Numbering | Name of bacteria to be tested | Plaque test | Double plate test |
| 1 | SM1 | - | - |
| 2 | SM3 | - | - |
| 3 | SM4 | - | - |
| 4 | SM6 | - | - |
| 5 | SM18 | - | - |
| 6 | SM20 | - | - |
| 7 | SM24 | - | - |
| 8 | SM26 | - | - |
| 9 | SM29 | + | + |
| 10 | SM36 | + | + |
| 11 | SM38 | - | - |
| 12 | pstⅠ | + | + |
| 13 | pstⅡ | - | - |
| 14 | pstⅢ | - | - |
| 15 | pstⅣ | - | - |
| 16 | pstⅥ | - | - |
| 17 | K7R | - | - |
| 18 | Salmonella typhimurium | - | - |
| 19 | AL | - | - |
| 20 | IEW | - | - |
| 21 | AE16-1 | - | - |
| 22 | PA15 | - | - |
| 23 | PA17 | - | - |
| 24 | N10 | - | - |
| 25 | 26 | - | - |
| 26 | 76 | - | - |
Of the 26 strains tested in the above table, there were 5 Yersinia pseudotuberculosis, 11 Serratia marcescens and several others.
As can be seen from Table 2, in the plaque assay, the phage vB _ SamS-WG03 was able to produce plaques with 3 test strains, while in the plaque assay, the phage vB _ SamS-WG03 was able to produce plaques with 2 test strains; the plaque is generated by 2 strains of serratia marcescens and 1 strain of yersinia pseudotuberculosis, wherein the plaque is generated by 1 strain of serratia marcescens without the plaque, which indicates that the bacteriophage has the characteristic of having the capability of cracking bacteria of different species.
Claims (6)
1. Serratia marcescens bacteriophage (A)Serratia marcescensphase) vB _ SamS-WG03, wherein the phage has been deposited in China Center for Type Culture Collection (CCTCC) at 11/9/2020 with the deposit number as follows: CCTCC NO: m2020712.
2. Use of a serratia marcescens bacteriophage of claim 1 in the manufacture of a medicament for preventing or treating infectious diseases caused by serratia marcescens and yersinia pseudobinderi.
3. The use of the serratia marcescens bacteriophage vB SamS-WG03 of claim 1 for killing serratia marcescens and yersinia pseudoconjugate in a spatial environment.
4. A composition for preventing or treating infectious diseases caused by Serratia marcescens and Yersinia pseudobindans, which comprises the bacteriophage of claim 1 as an effective ingredient.
5. The composition of claim 4, wherein the composition is a liquid formulation, a lyophilized formulation, or an oral solid formulation.
6. Use of the bacteriophage of claim 1 or the composition of claim 4 for the preparation of a feed additive, feed, drinking water additive, drinking water, disinfectant or detergent for killing serratia marcescens in vivo of livestock and poultry, on body surface of livestock and poultry, livestock and poultry feed, farming implement or farming space environment.
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