CN118308310A

CN118308310A - Salmonella typhimurium phage ZK22 and application thereof

Info

Publication number: CN118308310A
Application number: CN202410510499.4A
Authority: CN
Inventors: 王玮; 孙义虹; 周烁; 曲庆; 梁雅妍
Original assignee: Zhongkai University of Agriculture and Engineering
Current assignee: Zhongkai University of Agriculture and Engineering
Priority date: 2024-04-26
Filing date: 2024-04-26
Publication date: 2024-07-09

Abstract

The invention discloses a salmonella typhimurium phage (Salmonella bacteriophage) ZK22, which relates to the technical field of phage viruses, and has a preservation number of GDMCC No:64387-B1, the preservation unit name is Guangdong province microorganism strain preservation center, the preservation address is No. 59 building 5 of Dai 100 in Guangzhou martyr, and the preservation date is 2024, 3 months and 4 days. The invention also discloses application of the salmonella typhimurium phage ZK22 in inhibiting salmonella typhimurium proliferation and preparing a salmonella typhimurium inhibiting medicament. The phage ZK22 has high specificity to salmonella typhimurium, good stability, high cracking efficiency, strong safety and great application value.

Description

Salmonella typhimurium phage ZK22 and application thereof

Technical Field

The invention relates to the technical field of phage viruses, in particular to a salmonella typhimurium phage ZK22 and application thereof.

Background

Salmonella (Salmonella) belongs to gram-negative Brevibacterium, is one of the most common zoonotics, and is listed as a second type of infectious disease in China. Salmonella hosts are wide in range and are prone to symptoms such as septicemia, enteritis and abortion, and can be divided into 2600 serotypes according to different antigen carrying types, wherein Salmonella typhimurium is one of the most common serotypes in clinic. In recent years, the large-scale livestock and poultry cultivation in China is vigorously developed, and salmonellosis serving as one of bacterial diseases of livestock and poultry cultivation can be horizontally transmitted and vertically transmitted, so that huge losses are brought to the cultivation industry, and meanwhile, the human health is endangered. In the background of widely used antibiotics, the salmonella drug resistance is continuously enhanced, so that the livestock and poultry breeding industry in China is urgent to develop effective replacement resistant products.

Phage is a bacterial virus that is simple in structure, consists of only nucleic acid and protein shells, and must be propagated in dependence on the host bacteria. Phage studies have been carried out early in the beginning of the last century, but they have been put aside since antibiotics were developed, until now phage therapy has moved to the front of efficient alternative product development due to the production of superbacteria caused by antibiotic abuse. Compared with antibiotics, phage therapy has the advantages that the phage therapy is widely existing in nature, is huge in quantity and is easy to separate; secondly, the bacteriophage has strong specificity on the host bacteria, and usually only one or more strains of the host bacteria are infected, so that the bacteriophage cannot negatively influence other microbial groups in the same environment; thirdly, unlike chemicals, a phage will produce a large number of generations after its own life cycle is completed and repeat this process indefinitely until the host bacteria all die, but will also be metabolized by the host bacteria to be discharged outside the body after its own death, and will not remain in the body; finally, phages act as bacterial viruses that co-evolve with the host bacteria in their fight against them, thereby greatly reducing the bacterial resistance problem.

Although phage has great development value as a potential antibiotic substitute, some limiting factors still exist in the application process, such as low cleavage efficiency, poor physical and chemical resistance, and transfer of lysogenic genes and drug-resistant genes. Therefore, it is necessary to screen a phage which can efficiently, stably, specifically and safely lyse Salmonella typhimurium.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide the salmonella typhimurium phage ZK22 and the application thereof, wherein the phage ZK22 has high specificity on salmonella typhimurium, and has the advantages of good stability, high cracking efficiency, strong safety and great application value.

The technical scheme for solving the technical problems is as follows: a salmonella typhimurium phage (Salmonella bacteriophage) ZK22 is provided with a deposit number of GDMCC No:64387-B1, the preservation unit name is Guangdong province microorganism strain preservation center, the preservation address is No. 59 building 5 of Dai 100 in Guangzhou martyr, and the preservation date is 2024, 3 months and 4 days.

The phage ZK22 is separated from fresh market sewage, and can generate uniform transparent, clear-edged and surrounding halod plaques with a diameter of 1mm on a double-layer plate. Through observation of a transmission electron microscope, the phage ZK22 is in a polyhedral symmetrical head and long tail structure, the head is 62.86 +/-1.25 nm long, the tail is 164.54 +/-2.62 nm long, and the phage ZK22 belongs to the order of the end phages and belongs to the family of the end phages according to the eighth report of the International Commission on viral Classification, the International Commission on viral Classification (ICTV), and the taxonomic name of the phage ZK22 is Salmonella enterica subsp.

The invention also provides application of the salmonella typhimurium phage ZK22 in inhibiting salmonella typhimurium proliferation.

The invention also provides application of the salmonella typhimurium phage ZK22 in preparing a salmonella typhimurium inhibiting medicament.

The invention has the following beneficial effects:

1. The phage ZK22 is separated from the living sewage, which shows that the culture condition is simple and easy to enrich, the titer of the phage ZK22 after liquid amplification is higher, and the highest titer can reach 1.3 multiplied by 10 ¹⁵ PFU/mL.

2. The phage ZK22 of the invention has higher activity under different MOI conditions and higher titer, wherein the optimal multiplicity of infection is 10 ^-2, and the titer reaches 1.29 multiplied by 10 ¹⁵ PFU/mL.

3. The phage ZK22 has strong temperature stability and acid-base stability, and can keep high activity at the temperature of 4-70 ℃ and the pH value of 3-12.

4. The phage ZK22 has high specificity, only cracks salmonella typhimurium, has no cracking capability on salmonella pullorum, escherichia coli, lactobacillus and the like, has short latency, quick burst, strong cracking capability, high cracking capacity up to 393PFU/CFU, high efficiency on host salmonella typhimurium, and good clinical targeted application prospect.

5. After the phage ZK22 is subjected to whole genome sequencing, open reading frame annotation and protein function analysis, genes encoding currently known virulence genes, lysogenic genes and antibiotic resistance genes are not found, and the phage ZK22 has the advantages of small toxic and side effects and high safety.

Drawings

FIG. 1 is a graph of phage ZK22 double-layered plate plaques;

FIG. 2 is a morphological view of phage ZK22 transmission electron microscope;

FIG. 3 is an agarose gel electrophoresis diagram of phage ZK22 nucleic acid extraction and identification;

FIG. 4 is a graph showing the result of whole genome analysis of phage ZK 22;

FIG. 5 is a graph of the results of a determination of the optimal multiplicity of infection of phage ZK 22;

FIG. 6 is a graph of one-step growth of phage ZK 22;

FIG. 7 is a graph of phage ZK22 temperature stability;

FIG. 8 is a graph of acid-base stability of phage ZK 22;

FIG. 9 is a graph showing the inhibitory effect of phage ZK22 on the proliferation of Salmonella typhimurium.

Detailed Description

The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

EXAMPLE 1 isolation, purification, titre determination and preservation of phage ZK22

(1) Activating and culturing host bacteria: the host bacteria are salmonella typhimurium SS10 isolated and stored in a laboratory. Thawing salmonella typhimurium SS10 glycerol bacteria stored at-80 ℃ according to the following weight ratio of 1: inoculating 1000 proportion in LB liquid culture medium, culturing at 37 deg.C and 200rpm/min for 12 hr. The next day is inoculated to an SS solid culture medium, then the culture is inverted for 12 hours, single colony is selected and inoculated to an LB liquid culture medium, and the culture is carried out for 12 hours at 37 ℃ and 200rpm/min, thus obtaining the salmonella typhimurium SS10 pure culture bacterial liquid.

(2) Sample treatment: samples were taken from fresh market sewage in the sea ball region of Guangzhou, guangdong. The sample was filtered through gauze, centrifuged at 12000rpm/min for 15min, and the supernatant was filtered through a microporous membrane with a pore size of 0.22 μm and placed at 4℃for further use.

(3) Separating: mixing 5mL of filtrate, 5mL of LB liquid medium and 100 mu L of pure culture bacterial liquid of salmonella typhimurium SS10 in logarithmic phase, and carrying out shaking culture at 37 ℃ and 200rpm/min for 12 hours to amplify the salmonella typhimurium SS10 specific phage. Centrifuging the culture at 12000rpm/min for 15min, collecting supernatant, filtering with microporous membrane with 0.22 μm pore diameter to obtain stock solution, and standing at 4deg.C for use. Mixing 200 μl stock solution, 200 μl host bacteria solution and 5ml LB solid culture medium at 50deg.C, pouring onto the coagulated LB solid culture medium, making into double-layer plate, standing the upper-layer plate for coagulation, culturing in a constant temperature incubator at 37deg.C for 6 hr, and observing whether plaque appears.

(4) Purifying: selecting large transparent plaque, dispersing in 1mL sterile SM buffer solution for 30min at 37 ℃ under 200rpm/min, filtering with microporous filter membrane with 0.22 μm aperture, gradient diluting with sterile SM buffer solution, preparing double-layer plate culture by referring to the method of step (3), repeatedly purifying the large transparent plaque for 5 times until the shape and size of the plaque are consistent, and obtaining purified salmonella typhimurium phage ZK22. As a result, as shown in FIG. 1, phage ZK22 plaques were round, uniformly transparent, sharp-edged and halo-surrounding plaques, 1mm in diameter.

(5) Potency determination: after phage ZK22 filtrate is subjected to gradient dilution by sterile SM buffer solution, selecting proper dilutions to prepare a double-layer plate by referring to the method of the step (3), wherein each dilution is repeated three times, and after inversion culture, selecting plate count with plaque number of 30-300 and taking average value to calculate titer, wherein a titer calculation formula is as follows: phage titer (PFU/mL) =average plaque number x dilution x 5. The titer of phage ZK22 can reach 1.3X10 ¹⁵ PFU/mL.

(6) Preserving: storing the phage purified liquid obtained by filtration in a refrigerator at 4 ℃ for a short period of time; adding sterile glycerol with a final concentration of 30% into the phage purified liquid obtained by filtering, and storing in a refrigerator at-80 ℃ for long-term storage; and phage ZK22 is preserved in the microorganism strain collection center of Guangdong province, the preservation address is building 5 No. 59 of the university 100 in Guangzhou City martyr, the preservation time is 2024, 3 and 4 days, and the preservation number is GDMCC No:64387-B1.

Example 2 morphological observations of phage ZK22

Sucking 20 μl of phage purification liquid drop onto copper mesh by a pipette, naturally adsorbing for 5-10min, then sucking excessive liquid drop by a filter paper strip, slightly airing, sucking 20 μl of 2% phosphotungstic acid solution, dripping on copper mesh, standing for 3-5min, sucking excessive liquid drop by a filter paper strip, airing under an incandescent lamp, observing and photographing under a transmission electron microscope, and the result is shown in figure 2.

As can be seen from FIG. 2, under the observation of a transmission electron microscope, the phage ZK22 has a polyhedral symmetrical head and long tail structure, the head is 62.86 + -1.25 nm long, the tail is 164.54 + -2.62 nm long, and according to the eighth report of the International Commission on viral Classification (ICTV) of the International Commission on viral Classification, the phage ZK22 belongs to the order of the end-of-the-family of end-of-the-family phages.

Example 3 phage ZK22 host profiling

The host range of phage ZK22 was determined using the two-layer plate spotting method. In this example, 20 strains of Salmonella typhimurium, 1 strain of Salmonella pullorum, 12 strains of Escherichia coli, and 9 strains of lactic acid bacteria were selected as host bacteria for measurement. Respectively taking 200 mu L of log-phase host bacterial liquid and 5mL of LB solid culture medium at 50 ℃ to mix, pouring the mixture onto the solidified LB solid culture medium to prepare a double-layer flat plate, naturally solidifying the mixture in a sterile environment, taking 10 mu L of phage ZK22 purified liquid and 10 mu L of physiological salt to drop the purified liquid on different positions of the flat plate, naturally drying the mixture in the sterile environment, then inverting the mixture to a constant-temperature incubator at 37 ℃ for culturing for 6 hours, observing whether plaque appears, repeating the test three times, and the phage ZK22 host spectrum is shown in table 1.

TABLE 1 phage ZK22 host profile

Note that: "+" indicates that phage ZK22 has lytic ability to the strain, and "-" indicates that phage ZK22 has no lytic ability to the strain.

As shown in Table 1, phage ZK22 shows a lytic ability against 20 strains of Salmonella typhimurium, but has no lytic ability against Salmonella pullorum, E.coli and lactic acid bacteria, indicating that phage ZK22 is a phage that lyses Salmonella typhimurium with high specificity.

Example 4 phage ZK22 nucleic acid extraction identification and Whole genome sequencing

(1) Phage ZK22 nucleic acid extraction and identification: filtering phage ZK22 purified solution through a microporous filter membrane with a pore size of 0.22 μm, treating filtrate with 200U Benzo DNase I and 0.1mg/mL RNase A, performing heat inactivation on DNase I at 65 ℃ for 10min, and extracting phage genome DNA by using Qiagen MinElute virus spin kit; quantification of DNA concentration on a Qubit 3.0 fluorometer using Equalbit x-dsDNA HS assay kit; genomic DNA was identified by DNase I, RNase A and Mung Bean Nuclease and examined by 0.8% agarose gel electrophoresis, and the results are shown in FIG. 3, wherein M is Marker D15000, blank is blank, lane 1 is DNase I cleavage results, lane 2 is RNase A cleavage results, and lane 3 is Mung Bean Nuclease cleavage results.

As can be seen from FIG. 3, both DNase and mung bean nuclease were able to digest the nucleic acid of phage ZK22 completely, and RNase was unable to digest the nucleic acid of phage ZK22, indicating that phage ZK22 is of the single-stranded DNA (ssDNA) genome type.

(2) Phage ZK22 whole genome sequencing and characterization: genomic samples were submitted to sequencing by Chengdu New phagol Biotechnology Inc. Using a tool for IlluminaUniversal Plus DNA library preparation kit construction of Shotgun library, quality inspection of the library by an Agilent 4200 bioanalyzer, and double-ended sequencing on Illumina Nova Seq 6000 platform; filtering and quality controlling the obtained original sequencing data by fastp, cutting the adapter, and removing reads with high ratio of low quality reads to N to obtain CLEAN READS; CLEAN READS was de novo assembled using METASPADES software; carrying out annotation on coding genes and tRNA (transfer-RNA) of the genome by using Prokka, and then comparing a protein sequence with an NR library by using blastp to obtain sequence information with high similarity of each gene in the NR library; homologous comparison is carried out on the gene sequence and VFDB database by using blast, and virulence genes are predicted; the drug resistance gene was predicted by analysis using resfinder, and the results are shown in FIG. 4.

As can be seen from FIG. 4, the genome of phage ZK22 consisted of 47066bp single-stranded DNA with GC content of 45.71%, and tRNA encoding gene, virulence gene and drug resistance gene were not predicted.

Example 5 determination of the optimal multiplicity of infection of phage ZK22

The logarithmic phase host bacteria SS10 and phage ZK22 purified solution were mixed according to MOI＝10³、MOI＝10²、MOI＝10¹、MOI＝10⁰、MOI＝10^-1、MOI＝10^-2、MOI＝10^-3 and moi=10 ^-4 respectively, and after shaking culture at 37 ℃ and 200rpm/min for 6 hours, the mixture was filtered by a microporous filter membrane with a pore diameter of 0.22 μm, and after dilution with sterile SM buffer solution, a double-layered plate was prepared, after inversion culture, the plate count with plaque number between 30 and 300 was selected and the average value was calculated, the infection complex with the highest titer was the optimal infection complex, and the test was repeated three times, and the result is shown in fig. 5.

As can be seen from FIG. 5, phage ZK22 had an optimal multiplicity of infection of 10 ^-2, at which time the progeny phage titer was highest, reaching 1.29X 10 ¹⁵ PFU/mL.

Example 6 one-step growth Curve determination of phage ZK22

Mixing phage ZK22 purified solution with logarithmic phase salmonella typhimurium SS10 according to optimal infection complex number, adsorbing at 37deg.C for 10min, centrifuging at 4deg.C for 5min at 12000rpm/min, discarding supernatant, and suspending the precipitate in 10mL of 37 deg.C pre-heated LB liquid medium, and shake culturing at 37deg.C at 200rpm/min for 120min. 500 mu L of the mixed culture is taken every 10min from 0min, filtered by a microporous filter membrane with the pore diameter of 0.22 mu m, and the sterile SM buffer solution is subjected to gradient dilution to prepare a double-layer flat plate for measuring the titer of phage ZK 22. The test was repeated three times. The log of phage ZK22 titer is plotted on the abscissa with the sampling time as the abscissa, a one-step growth curve of phage ZK22 is drawn, the latency and lysis period are determined and the amount of lysis is calculated, and the amount of lysis = phage titer at the end of lysis/host bacterial liquid concentration at the initial stage of infection, and the results are shown in fig. 6.

As shown in FIG. 6, phage ZK22 had a incubation period of 0-10min, a lysis period of 10-80min, and a lysis amount of 393PFU/CFU.

Example 7 phage ZK22 temperature stability assay

Phage ZK22 purified solution was diluted to 1.5X10 ¹⁰ PFU/mL with sterile SM buffer solution, and then 200. Mu.L was packed in 1mL sterile centrifuge tubes, and placed in 4℃refrigerator and 25℃37℃50℃60℃70℃80℃thermostat water bath respectively, sampled at 0min 20min 40min 60min and diluted with sterile SM buffer solution in gradient to prepare double-layer plate to determine phage ZK22 titer, and the test was repeated three times, and the results are shown in FIG. 7.

As shown in FIG. 7, the titer of phage ZK22 is basically unchanged after the phage ZK22 is placed at 4 ℃,25 ℃ and 37 ℃ for 60min, the titer is 5.3X10 ⁷ PFU/mL after the phage ZK22 is placed at 50 ℃ for 60min, the titer is 1.5X10 ⁷ PFU/mL after the phage ZK22 is placed at 60 ℃ for 60min, the titer is 1.1X10 ⁷ PFU/mL after the phage ZK22 is placed at 70 ℃ for 60min, and the phage ZK22 is completely inactivated after the phage ZK22 is treated at 80 ℃, so that the phage ZK22 has good thermal stability and can maintain higher activity after the phage ZK22 is placed at 4-70 ℃ for 60 min.

Example 8 determination of acid-base stability of phage ZK22

The pH of the sterile SM buffer solution is adjusted to 2-13 by using HCl and NaOH solutions, 100 mu L of 1X 10 ¹¹ PFU/mL phage ZK22 purified solution is taken and mixed with 900 mu L of sterile SM buffer solutions with different pH values respectively, the initial titer is adjusted to 1X 10 ¹⁰ PFU/mL, the phage ZK22 titer is measured by using a double-layer flat plate after being subjected to gradient dilution in a constant temperature water bath at 37 ℃ for 1h, and the test is repeated three times, and the result is shown in FIG. 8.

As can be seen from fig. 8, phage ZK22 shows higher activity at pH 3-12, the titer is about 10 ⁷ PFU/mL in the ph=3-5 range, and the titer is about 10 ⁸ PFU/mL in the ph=6-12 range, wherein the maximum at ph=8 can reach 3×10 ⁹ PFU/mL, indicating that phage ZK22 has good acid-base stability.

Example 9 inhibition of the proliferation of the host Salmonella typhimurium by phage ZK22

100. Mu.L of phage ZK22 purified solution and 10 ⁸ CFU/mL of logarithmic phase Salmonella typhimurium SS10 were mixed in 96-well plates, respectively, in accordance with MOI=10 ¹、MOI＝10⁰、MOI＝10^-1 and MOI=10 ^-2, while 150. Mu.L of 10 ⁸ CFU/mL of logarithmic phase Salmonella typhimurium SS10 was mixed with 50. Mu.L of LB liquid medium as positive control, 200. Mu.L of LB liquid medium as negative control, 200. Mu.L of sterile water as blank control, and three replicates per group; the culture was performed in a microplate reader at 37℃under shaking at 200rpm/min for 12 hours, and OD ₆₀₀ _nm was measured every 0.5 hour, and the results are shown in FIG. 9.

As can be seen from fig. 9, the OD ₆₀₀ _nm of salmonella typhimurium SS10 in the positive control group started to increase at 0h and the increase rate was faster, the average OD ₆₀₀ _nm = 1.1326 by the end of the test, Indicating a faster proliferation. Whereas co-culture OD ₆₀₀ _nm mixed at moi=10 ¹、MOI＝10⁰、MOI＝10^-1、MOI＝10^-2 was lower all the way than the positive control, indicating that phage ZK22 had an inhibitory effect on the proliferation of salmonella typhimurium SS10, with co-culture OD ₆₀₀ _nm mixed at optimum multiplicity of infection moi=10 ^-2 beginning to slightly rise at 3.5h and to decline at 10.5h, Average OD ₆₀₀ _nm = 0.4772 by the end of the test, which is the lowest for all groups tested, shows that the best bacteriostatic effect can be obtained when phage ZK22 and salmonella typhimurium SS10 are mixed and cultured at the optimal multiplicity of infection. Furthermore, when moi=10 ¹, the co-culture started to rise in OD ₆₀₀ _nm at 5h, to an average OD ₆₀₀ _nm = 0.8820 at the end of the test, indicating that the inhibition effect of phage ZK22 on the proliferation of Salmonella typhimurium SS10 is in potency dependence. In conclusion, phage ZK22 shows good inhibition effect on the proliferation of salmonella typhimurium SS10, and the effect shows potency dependence, and has wide application prospect in the aspect of preventing and treating salmonella typhimurium.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. A salmonella typhimurium bacteriophage (Salmonella bacteriophage) ZK22 having a deposit number GDMCC No:64387-B1, the preservation unit name is Guangdong province microorganism strain preservation center, the preservation address is No. 59 building 5 of Dai 100 in Guangzhou martyr, and the preservation date is 2024, 3 months and 4 days.

2. Use of salmonella typhimurium bacteriophage ZK22 of claim 1, to inhibit salmonella typhimurium proliferation.

3. Use of salmonella typhimurium phage ZK22 of claim 1, in the preparation of a salmonella typhimurium inhibitory medicament.