Enterobacter hormaechei lytic bacteriophage Ehp-C109 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses an enterobacter hormaecheiphage Ehp-C109, the name of the enterobacter hormaecheiphage Ehp-C109 is Ehp-C109, the classification name of the enterobacter hormaecheiphage is Enterobacter hormaecheiphage, the preservation number of the enterobacter hormaecheiphage Ehp-C109 is CGMCC No.46361, the preservation date of the enterobacter hormaecheiphage Ehp-C109 is December 30, 2024, and the preservation unit of the enterobacter hormaecheiphage Ehp-C109 is China General Microbiological Culture Collection Center (CGMCC), No. 3, Yard 1, Beichen West Road, the bacteriophage Ehp-C109 disclosed by the invention is subjected to biological characteristic analysis and whole genome sequencing, results show that the range of a host spectrum of the bacteriophage Ehp-C109 is very wide, the bacteriophage Ehp-C109 still has relatively high titer and relatively high tolerance to an alkaline environment when the pH value is 12, and related virulence genes and drug-resistant genes are not found through predictive analysis; and a theoretical basis is provided for clinical prevention and control of enterobacter hormaechei and phage preparations thereof.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, in particular to a strain of Enterobacter hallii lytic bacteriophage Ehp-C109 and application thereof. Background Art

[0002] Enterobacter hormaechei is a zoonotic conditional pathogen. Enterobacter hormaechei is a common drug-resistant bacterium that carries multiple resistance genes, such as resistance genes to antibiotics such as quinolones, β-lactams, and peptides. Global distribution of multi-drug resistant Enterobacter hormaechei strains has been reported to be isolated from a variety of diseased animals, such as pigs, cattle, sheep, chickens, foxes, and shrimps. The main symptoms include bacteremia, endometritis, pneumonia, abortion, diarrhea, etc. In the case of nosocomial infections and animal infections in animal husbandry, the presence of multi-drug resistant bacteria may make traditional antibiotic treatments ineffective in controlling infections, resulting in prolonged infection duration and worsening of the disease, endangering the health of humans and animals.

[0003] Bacteriophages can kill bacteria specifically without destroying normal intestinal flora, and are not affected by bacterial resistance to antibiotics. Compared with antibiotics, phage preparations have the advantages of strong host specificity, self-reproduction, simple preparation, and no toxic side effects on animals. Therefore, the research on phages has received more and more attention in recent years. In addition, due to the simple structure of phages, they are easy to modify, and specific DNA fragments can be inserted into phages, so that phages can be used to target the host specificity of Escherichia coli.

[0004] At present, most of the existing technologies collect sewage samples, use the double-layer plate method to separate and purify the E. hallii phage; observe the morphology through transmission electron microscopy after phosphotungstic acid staining; determine its host range, temperature and pH stability, one-step growth curve and other biological characteristics. Perform whole genome sequencing analysis on the phage. Phages have high host specificity. A phage can usually only infect one or several closely related bacterial strains. Phages are difficult to eliminate multiple pathogens at the same time due to their narrow host spectrum. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an Enterobacter hallii lytic bacteriophage Ehp-C109 and its application.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] A strain of Enterobacter hormaecheiphage Ehp-C109, whose name is Ehp-C109, classification name is: Enterobacter hormaecheiphage, preservation number is: CGMCC No.46361, preservation date: December 30, 2024, preservation unit: General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, transparent plaques of uniform size were observed on the double-layer plate; transmission electron microscopy showed that the phage Ehp-C109 was an icosahedral phage with a tail that could not be retracted; the diameter of the head was 70.46 nm and the length of the tail was 140.41 nm.

[0009] Furthermore, the titer of the bacteriophage Ehp-C109 remained stable at pH 4-pH 11, and at pH 12, it still had a titer of 1.767×10 5 PFU / mL higher titer.

[0010] Furthermore, among the 82 bacterial strains tested in the host spectrum experiment, bacteriophage Ehp-C109 was able to lyse 20 of them, including 7 strains of Escherichia, 5 strains of Klebsiella, 6 strains of Salmonella and 2 strains of Streptococcus, indicating that the lysis range of bacteriophage Ehp-C109 was extremely wide.

[0011] The use of the Enterobacter hallii lytic phage Ehp-C109 as described above in the preparation of drugs for treating diseases caused by Enterobacter hallii.

[0012] Furthermore, among the 82 bacterial strains tested in the host spectrum experiment, bacteriophage Ehp-C109 was able to lyse 20 of them, including 7 strains of Escherichia, 5 strains of Klebsiella, 6 strains of Salmonella and 2 strains of Streptococcus, indicating that the lysis range of bacteriophage Ehp-C109 was extremely wide.

[0013] The advantages and positive effects achieved by the present invention are:

[0014] 1. The present invention uses Enterobacter hallii as the host bacteria to isolate bacteriophage Ehp-C109, analyzes its biological characteristics and performs whole genome sequencing. The results show that the host spectrum of bacteriophage Ehp-C109 is very wide, and it still has relatively high titers at a pH of 12, has high tolerance to alkaline environments, and no related virulence genes and drug-resistant genes were found through predictive analysis, which provides a theoretical basis for the clinical prevention and control of Enterobacter hallii and its phage preparations.

[0015] 2. Among the 82 strains of bacteria tested in the host spectrum test, bacteriophage Ehp-C109 was able to lyse 20 of them, including 7 strains of Escherichia coli, 5 strains of Klebsiella, 6 strains of Salmonella and 2 strains of Streptococcus, indicating that the lysis range of bacteriophage Ehp-C109 is extremely wide. Studies have shown that the isolated Enterobacter husnioides phage has good lysis ability against Enterobacter husnioides, but has no obvious lysis effect on other pathogens, and the lysis spectrum is narrow. The lysis spectrum of bacteriophage Ehp-C109 is wider, which solves the problem of the narrow lysis spectrum of phage, and can be better applied to the treatment and prevention of diseases related to Enterobacter husnioides. The titer of bacteriophage Ehp-C109 remains stable at pH 4-pH 11, and at pH 12 it still has 1.767×10 5 The phage Ehp-C109 showed strong tolerance to alkaline environment and had great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a plaque picture of the bacteriophage Ehp-C109 of the present invention;

[0017] Figure 2 is a transmission electron micrograph of the bacteriophage Ehp-C109 of the present invention;

[0018] Figure 3 This is the MOI diagram of the bacteriophage Ehp-C109 in the present invention;

[0019] Figure 4 is a one-step growth curve diagram of bacteriophage Ehp-C109 in the present invention;

[0020] Figure 5 It is a schematic diagram of the effect of temperature on the activity of bacteriophage Ehp-C109 in the present invention;

[0021] Figure 6 It is a schematic diagram of the effect of pH on the activity of bacteriophage Ehp-C109 in the present invention;

[0022] Figure 7 The CGView tool is used in the present invention to analyze the whole genome map of bacteriophage Ehp-C109;

[0023] Figure 8 The phylogenetic tree diagram of bacteriophage Ehp-C109 analyzed using Mega-X tool in the present invention;

[0024] Fig. 9 The virulence gene map of bacteriophage Ehp-C109 was predicted using the VFDB tool in the present invention;

[0025] Fig.10The CARD tool was used in the present invention to predict the drug resistance gene map of bacteriophage Ehp-C109.

[0026] A strain of Enterobacter hormaecheiphage Ehp-C109, whose name is Ehp-C109, classification name is: Enterobacter hormaecheiphage, preservation number is: CGMCC No.46361, preservation date: December 30, 2024, preservation unit: General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0028] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0029] A strain of Enterobacter hormaecheiphage Ehp-C109, whose name is Ehp-C109, classification name is: Enterobacter hormaecheiphage, preservation number is: CGMCC No.46361, preservation date: December 30, 2024, preservation unit: General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0030] Preferably, transparent plaques of uniform size were observed on the double-layer plate; transmission electron microscopy showed that the phage Ehp-C109 was an icosahedral phage with a tail, and the tail was not retractable; the diameter of the head was 70.46 nm, and the length of the tail was 140.41 nm.

[0031] Preferably, the bacteriophage Ehp-C109 maintains a stable titer at pH 4-pH 11, and still has a titer of 1.767×10 5 PFU / mL higher titer.

[0032] Preferably, among the 82 bacterial strains tested in the host spectrum test, bacteriophage Ehp-C109 has lytic activity against 20 strains, including 7 strains of Escherichia, 5 strains of Klebsiella, 6 strains of Salmonella and 2 strains of Streptococcus, indicating that bacteriophage Ehp-C109 has a very wide range of lytic activity.

[0033] The use of the Enterobacter hallii lytic phage Ehp-C109 as described above in the preparation of drugs for treating diseases caused by Enterobacter hallii.

[0034] Preferably, among the 82 bacterial strains tested in the host spectrum test, bacteriophage Ehp-C109 has lytic activity against 20 strains, including 7 strains of Escherichia, 5 strains of Klebsiella, 6 strains of Salmonella and 2 strains of Streptococcus, indicating that bacteriophage Ehp-C109 has a very wide range of lytic activity.

[0035] Specifically, the relevant preparation and detection are as follows:

[0036] The strains, reagents and culture media involved in the embodiment are:

[0037] MH liquid medium: weigh 21.0 g MH and dissolve it in 1000 mL ddHO 2 O, and sterilize at 121℃ for 20min.

[0038] MH semi-solid medium: weigh MH 21.0g, agar powder 7.5g, add ddH 2 0 to 1000 mL, autoclave at 121°C for 20 min.

[0039] MH solid medium: weigh 21.0 g of MH medium and 15.0 g of agar powder, add 1000 mL of ddH2O, sterilize at 121°C for 20 min, cool to 50°C, pour into a plate, cool and solidify, and then invert for later use.

[0040] Example 1

[0041] Isolation and purification of bacteriophages:

[0042] Wastewater from Tianjin Zoo, Tianjin, China, was centrifuged at 10,000 r / min for 10 min and filtered through a 0.22 μm filter. The filtered sample and 100 μL of the solution with a concentration of approximately 1×10 7 The same volume of Enterobacter hallii was mixed and added to the MH liquid medium and placed in a shaker for co-culture for 10 hours. The above substances were centrifuged at 8000r / min for 10 minutes and then filtered with a 0.22μm filter to obtain the enriched phage liquid. 100μL of the concentration was about 1×10 7 Add 5 mL of Enterobacter hallii to 5 mL of MH semi-solid medium using the double-layer plate method. Spot an appropriate amount of phage enrichment solution on the double-layer plate and place it in a 37°C incubator for 12 hours. Add the isolated plaques to the MH medium and place it in a shaker for 12 hours, centrifuge at 10,000 r / min for 5 minutes, and filter with a 0.22 μm filter to obtain a phage suspension. Dilute it to 10 -4 Take 100 μL and add 100 μL of the solution with a concentration of about 1×107 The plaques were purified by double-layer plate method and placed in a 37°C constant temperature incubator for 6 hours. The above steps were repeated 5 times to obtain purified phages.

[0043] Example 2

[0044] Phage morphology observation:

[0045] Add 1M NaCL and 10% final concentration of polyethylene glycol PEG8000 to the phage suspension, place on a shaker, and slowly mix until all solid particles are dissolved, then transfer to 4°C and let stand for 16 hours or more. Centrifuge at 10000r / min for 10min, discard the supernatant, absorb an appropriate amount of SM buffer to resuspend the precipitate, drop the concentrated phage liquid on a copper mesh, and then absorb the excess liquid with filter paper. Add 20μL of 2% phosphotungstic acid solution, wait for it to dry, and then observe its morphology under an electron microscope at 80kV low voltage. The results are as follows Figure 1 Figure 2 As shown, from Figure 1 Figure 2 As can be seen in the figure, transparent plaques of uniform size can be seen on the double-layer plate. Transmission electron microscopy shows that phage Ehp-C109 is an icosahedral tailed phage, and the tail cannot be retracted. The diameter of the head is 70.46nm, and the length of the tail is 140.41nm.

[0046] Example 3

[0047] Phage host spectrum:

[0048] The concentration of 100 μL is about 1×10 7 Add 5-6mL of Enterobacter hallii to 5-6mL of MH semi-solid medium, pour into solid medium, wait for the semi-solid medium to solidify, then drop 5μL of phage onto the double-layer plate, wait for the phage liquid to dry, put it into a 37℃ constant temperature incubator for 6 hours and observe the results. The test was repeated 3 times. The results are shown in Table 1. It can be seen from Table 1 that among the 82 strains tested, phage Ehp-C109 has lytic activity against 7 strains of Escherichia, 5 strains of Klebsiella, 6 strains of Salmonella and 2 strains of Streptococcus.

[0049] Example 4

[0050] Optimal infection multiplicity of phage:

[0051] Add the phage solution to MH liquid culture medium at a multiplicity of infection (MOI) of 0.001, 0.01, 0.1, 1, 10, and 100, and place it in a 37°C shaker for 6 hours. Take it out, centrifuge it at 10,000 r / min for 10 minutes, and filter it with a 0.22 μm filter. Dilute the phage solution obtained at different ratios and add 100 μL of the solution to a concentration of about 1×107 The same volume of E. coli was mixed and incubated into a double-layer plate, placed in a 37°C constant temperature incubator for 6 hours, and the phage titer was determined. The experiment was repeated 3 times. Figure 3 As shown, from Figure 3 It can be seen that the titer of bacteriophage Ehp-C109 was the highest when MOI was 0.1, reaching 1.069×10 9 PFU / mL.

[0052] Example 5

[0053] One-step growth curve of bacteriophage:

[0054] The phage solution with an MOI of 0.1 was mixed with the E. coli in the logarithmic phase at a volume ratio of 1:1, incubated in a 37°C constant temperature incubator for 5 minutes, centrifuged at 10,000 r / min for 2 minutes, the supernatant was aspirated, the precipitate was resuspended with MH liquid culture medium, and placed in a 37°C shaker. From 0 min to 120 min, samples were taken every 10 min, diluted to a suitable gradient, and the phage titer was determined by the double-layer plate method. The experiment was repeated 3 times. The results are as follows Figure 4 As shown, from Figure 4 It can be seen that the incubation period of bacteriophage Ehp-C109 is 60 minutes, the outbreak period is 70 minutes, and the titer remains stable and enters the plateau period after 70 minutes.

[0055] Example 6

[0056] Phage temperature stability assay:

[0057] The phage was placed at 25℃, 37℃, 50℃, 60℃, and 70℃, and samples were taken and diluted to a suitable gradient at 30min, 60min, and 90min. The phage titer was determined by the double-layer plate method, and the test was repeated 3 times. Figure 5 As shown, from Figure 5 It can be seen that the phage Ehp-C109 maintains a stable titer at 25-50°C, the titer begins to decrease significantly when the temperature is higher than 60°C, and the phage titer drops to 0 when the temperature reaches 70°C.

[0058] Example 7

[0059] Phage pH stability assay:

[0060] The pH of the MH medium was adjusted with HCL and NaCl, and the phage was placed in the MH medium with a pH of 2-12, placed in a 37°C shaker for 1 hour, and then taken out and diluted to a suitable gradient. The phage titer was determined by the double-layer plate method, and the experiment was repeated 3 times. Figure 6 As shown, from Figure 6It can be seen that the pH tolerance of bacteriophage Ehp-C109 remains stable between 4 and 11. When the pH is less than 4, the phage titer will be rapidly reduced, and it will drop to 0 at pH 2. At pH 12, there is still 1.767×10 5 PFU / mL higher titer.

[0061] Example 8

[0062] Phage whole genome sequencing and functional analysis:

[0063] The whole genome sequencing results were compared using BLASTn in NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.), and the highest homology with Klebsiella phage vB_Kpn2-P2 was 87.80%.

[0064] Use CGView (https: / / proksee.ca / ) to draw a genome circle map. The result is as follows Figure 7 As shown, from Figure 7 As can be seen from the data, the genome of bacteriophage Ehp-C109 is 60579bp in length and has a GC content of 50.82%. Bacteriophage Ehp-C109 is a linear double-stranded DNA. A total of 79 coding sequences (CDS) were predicted for the genome of bacteriophage Ehp-C109, of which 13 encode proteins of known function and the rest are hypothetical proteins. Among these CDS, 50 genes are forward transcribed, while the others are reverse transcribed.

[0065] The genome sequences of the other 14 phages obtained were selected for comparison with the genome sequence of Ehp-C109. The phylogenetic tree was constructed using the Neighbor-Joining algorithm using Mega-X software. The results are shown in Figure 8 As shown, from Figure 8 It can be seen that the 15 phages are scattered into two branches, and Ehp-C109 is most closely related to Klebsiellaphage vB_Kpn2-P2 and Klebsiella phage vB_KppS-Raw.

[0066] VFDB (https: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi) was used to predict whether the virulence gene was present. Fig. 9 As shown, from Fig. 9 It can be seen that no relevant virulence genes were found in the prediction analysis of bacteriophage Ehp-C109.

[0067] CARD (https: / / card.mcmaster.ca / ) was used to predict whether the patient had drug resistance genes. Fig.10 As shown, from Fig.10 It can be seen that no relevant drug-resistant genes were found in the prediction analysis of bacteriophage Ehp-C109.

[0068] Table 1 Host spectrum of bacteriophage Ehp-C109 in the present invention

[0069]

[0070]

[0071] Among the 82 strains of bacteria tested in the host spectrum test, phage Ehp-C109 was able to lyse 20 of them, including 7 strains of Escherichia coli, 5 strains of Klebsiella, 6 strains of Salmonella and 2 strains of Streptococcus, indicating that the lysis range of phage Ehp-C109 is extremely wide. Studies have shown that the isolated E. coli phage has good lysis against E. coli, but has no obvious lysis effect on other pathogens, and the lysis spectrum is narrow. The lysis spectrum of phage Ehp-C109 is wider, which solves the problem of the narrow lysis spectrum of phage, and can be better applied to the treatment and prevention of diseases related to E. coli. The titer of phage Ehp-C109 remains stable at pH 4-pH 11, and at pH 12, it still has 1.767×10 5 The phage Ehp-C109 showed strong tolerance to alkaline environment and had great application potential.

[0072] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A strain of Enterobacter hallii lytic bacteriophage Ehp-C109, characterized in that: Its name is Ehp-C109, the classification name is Enterobacter hormaecheiphage, the preservation number is CGMCC No.46361, the preservation date is December 30, 2024, and the preservation unit is General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The Enterobacter hallii lytic phage Ehp-C109 according to claim 1, characterized in that: Transparent plaques of uniform size were observed on the double-layer plate; transmission electron microscopy showed that the phage Ehp-C109 was an icosahedral phage with a tail that could not be retracted; the diameter of the head was 70.46 nm and the length of the tail was 140.41 nm.

3. The Enterobacter hallii lytic phage Ehp-C109 according to claim 1, characterized in that: The bacteriophage Ehp-C109 maintained a stable titer at pH 4-pH 11, and still had a titer of 1.767×10 5 PFU / mL higher titer.

4. The Enterobacter hallii lytic phage Ehp-C109 according to any one of claims 1 to 3, characterized in that: Among the 82 bacterial strains tested in the host spectrum experiment, bacteriophage Ehp-C109 had lytic effects on 20 strains, including 7 strains of Escherichia, 5 strains of Klebsiella, 6 strains of Salmonella and 2 strains of Streptococcus, indicating that the lytic range of bacteriophage Ehp-C109 is extremely wide.

5. Use of the Enterobacter hallii lytic phage Ehp-C109 according to any one of claims 1 to 4 in the preparation of a medicament for treating diseases caused by Enterobacter hallii.

6. The use according to claim 5, characterized in that: Among the 82 bacterial strains tested in the host spectrum experiment, bacteriophage Ehp-C109 had lytic effects on 20 strains, including 7 strains of Escherichia, 5 strains of Klebsiella, 6 strains of Salmonella and 2 strains of Streptococcus, indicating that the lytic range of bacteriophage Ehp-C109 is extremely wide.

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