A bacteriophage A155 and its application in antagonizing vancomycin-resistant Enterococcus faecalis

Abstract

The present invention discloses a bacteriophage A155 and its application in antagonizing vancomycin-resistant Enterococcus faecalis, and relates to the technical field of phage screening for antagonizing vancomycin-resistant Enterococcus faecalis. The bacteriophage (Enterococcus phage) A155 has a deposit number of CCTCC NO: M20241805, and was deposited in the China Center for Type Culture and General Microorganisms on August 16, 2024, and the deposit address is Wuhan University, Wuhan, China. The present invention screens out a virulent phage against vancomycin-resistant Enterococcus faecalis, performs biological characteristics and whole genome sequencing analysis on it, and evaluates its antibacterial ability in vitro and in vivo, laying a foundation for the development of phage therapy for vancomycin-resistant Enterococcus faecalis infection.

Description

Technical Field

[0001] The invention relates to the technical field of phage screening for antagonizing vancomycin-resistant Enterococcus faecalis, and more specifically to a phage A155 and application thereof in antagonizing vancomycin-resistant Enterococcus faecalis. Background Art

[0002] Enterococcus faecalis ( Enterococcus faecalis Efs (Efs) is a Gram-positive facultative anaerobic bacterium that is widely colonized in the intestines of humans and various animals and is considered a common intestinal commensal bacterium. However, Efs can also act as an opportunistic pathogen, causing diseases such as bacteremia, endocarditis, urinary tract and wound infections. With the widespread use of antibiotics, the drug resistance of Efs has become increasingly serious, especially the emergence of vancomycin-resistant enterococcus (VRE), which has brought difficulties to the treatment of clinical VRE infections. Studies have shown that VRE colonization in the intestine may not cause symptoms, but can persist for a long time, becoming a reservoir of VRE, with a huge risk of causing infection in patients and others. Therefore, it is urgent to develop alternative antibiotics for VRE infections.

[0003] Bacteriophages are viruses that can infect and lyse bacteria. They can target and kill specific bacteria without harming other microbial species, and have unique advantages in treating drug-resistant bacterial infections. In addition, bacteriophages are easy to obtain, have a fast killing speed, are harmless and safe to eukaryotic cells, and have a synergistic antibacterial effect with other antibacterial drugs such as antibiotics and antimicrobial peptides, making them a very promising alternative to antibiotics.

[0004] How to provide a virulent phage capable of lysing vancomycin-resistant Enterococcus faecalis is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention screened out a virulent phage against vancomycin-resistant Efs V583, analyzed its biological characteristics and whole genome sequencing, and evaluated its antibacterial ability in vitro and in vivo, in order to lay a foundation for the development of phage therapy for Efs infection.

[0006] In order to achieve the above object, the present invention adopts the following technical solution.

[0007] In a first aspect, an embodiment of the present invention provides a bacteriophage A155 that antagonizes vancomycin-resistant Enterococcus faecalis. (Enterococcus phage )The A155 deposit number is CCTCC NO: M20241805. It was deposited in the China Center for Type Culture Collection on August 16, 2024. The deposit address is Wuhan University, Wuhan, China.

[0008] The second aspect of the embodiments of the present invention provides the use of the bacteriophage A155 in preparing a preparation for treating and / or preventing vancomycin-resistant Enterococcus faecalis infection.

[0009] The third aspect of the embodiments of the present invention provides a preparation for treating and / or preventing vancomycin-resistant Enterococcus faecalis infection, comprising the bacteriophage A155.

[0010] Through the above technical solutions, it can be known that compared with the prior art, the present invention uses vancomycin-resistant Enterococcus faecalis V583 as the host bacteria, screens a phage that can specifically lyse Enterococcus faecalis, and performs lysis spectrum determination of multiple bacteria on phage A155. The results show that it has a broad spectrum of lysis activity against Enterococcus faecalis, with a coverage rate of 81.4% for the tested Enterococcus faecalis, and has no lysis activity against the tested Enterococcus faecium, Staphylococcus aureus, Listeria monocytogenes and Escherichia coli; and the mouse intestinal experiment shows that after treatment with phage A155, there are significant differences between the treatment group and the control group on both days after treatment (Day 3, 4), and the V583 load can be reduced by 1.13 orders of magnitude on the second day. This result confirms that A155 can effectively reduce the colonization of V583 in the mouse intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0012] Figure 1 The attached drawings show the plaque and electron microscopic morphology of bacteriophage A155; A is the plaque morphology of bacteriophage A155; B is the transmission electron microscopic morphology of bacteriophage A155.

[0013] Figure 2 The attached figure shows the results of whole genome sequencing analysis of bacteriophage A155.

[0014] Figure 3 The attached figure is a phylogenetic tree based on the amino acid sequence of the large subunit of the phage terminase; the serial numbers in brackets are the sequence numbers of the phage genome; the numbers on the branches represent the credibility, and the closer the value is to 100, the higher the credibility; the ruler represents the genetic distance.

[0015] Figure 4The attached figure is a one-step growth curve of bacteriophage A155.

[0016] Figure 5 The attached figure is a temperature and pH stability diagram of bacteriophage A155; A is the temperature stability of bacteriophage A155; B is the pH stability of bacteriophage A155.

[0017] Figure 6 The attached figure shows the in vitro inhibition curve of bacteriophage A155 at different MOIs.

[0018] Figure 7 The attached figure shows the growth of bacteria in two test tubes with and without the addition of bacteriophage A149.

[0019] Figure 8 The attached figures are the evaluation of the therapeutic effect of bacteriophage A155 on VRE colonization in the mouse intestine; A, the treatment flow chart of bacteriophage A155 on VRE colonization in the mouse intestine; B, the count of enterococci in feces after treatment with bacteriophage A155. *: p<0.05; **: p<0.01; ns: no significance. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Strains used in the examples and their sources: The 59 strains of Enterococcus faecalis, 5 strains of Enterococcus faecium, 2 strains of Staphylococcus aureus, 2 strains of Listeria monocytogenes, and 2 strains of Escherichia coli used in this study (see Table 2 for details) were all deposited by the Institute of Microbiology and Host Health of Linyi University.

[0022] Culture medium: brain-heart infusion media (BHI; g / L): 17.5 g of beef heart infusion powder, 10.0 g of tryptone, 5.0 g of sodium chloride, 2.5 g of Na2HPO4·12H2O, and 2.0 g of glucose.

[0023] BHI semisolid agar: BHI broth, 5 g / L agarose.

[0024] BHI solid agar: BHI broth, 15 g / L agar powder.

[0025] Enterococcus selective culture agar (g / L): peptone 20.0g, yeast extract powder 5.0g, ox bile 10.0g, sodium chloride 5.0g, esculin 1.0g, ammonium ferric citrate 0.5g, sodium azide 0.25g, sodium citrate 1.0g, agar 15.0g.

[0026] Main reagents and instruments: sterile solution (SM) buffer, Sangon Biotech (Shanghai) Co., Ltd.; PEG-8000, CsCl, chloroform and phosphotungstic acid, Aladdin Biochemical Technology Co., Ltd.; viral nucleic acid extraction kit, Tiangen Biochemical Technology (Beijing) Co., Ltd. Transmission electron microscope, Philips-FEI; ultracentrifuge, Beckman; 0.22 μm filter, MilliPore; bacterial growth curve instrument, Xinzhi Biotechnology Co., Ltd.

[0027] Example 1 Phage isolation and purification.

[0028] The collected farm wastewater was heated at 4 °C and 5000 r∙min. -1 After centrifugation for 10 min under the same conditions, the supernatant was filtered with a 0.22 μm filter and the filtrate was stored in a 4°C refrigerator for later use. The filtrate and 1.0 mL of Enterococcus faecalis V583 suspension were added to 100.0 mL of BHI liquid medium and cultured overnight at 37°C and 200 r / min. After 1.0 mL of the mixed culture was centrifuged at 12000 r / min for 5 min, the phage initial solution was obtained by filtering with a 0.22 μm filter. The phage initial solution was mixed with the host bacteria V583 suspension, and after standing for 10 min, the mixture was added to the BHI semi-solid medium and mixed evenly, and spread on the lower layer of BHI solid medium. After the upper layer of BHI semi-solid medium solidified, it was inverted and cultured in a 37°C incubator overnight. The next day, whether plaques appeared was observed. Pick a single plaque that is clear, bright, has clear edges, and is uniform in size, add it to SM buffer, and mix it with 100 μL of host bacteria in 10 mL of BHI medium, and culture it at 37°C and 200 r / min to obtain a clear and transparent culture solution. Repeat the operation 3-5 times to obtain purified phage.

[0029] The purified phage was amplified, PEG-8000 was added, and after overnight treatment at 4°C, the supernatant was discarded by centrifugation at 12000 r / min for 10 minutes, and the precipitate was resuspended with 2 mL SM buffer, and then an equal volume of chloroform was added for extraction to obtain a phage concentrate. The phage concentrate was slowly added to the CsCl gradient solution and centrifuged, and then the phage concentrate was collected with a syringe, and then transferred to phosphate buffered saline (PBS) for dialyzation overnight to obtain purified phage particles. The obtained sample was stained using the phosphotungstic acid negative staining method, and after 15 minutes, it was observed and recorded under a transmission electron microscope at 80 kV. It was named Enterococcus phage A155, such as Figure 1 As shown, Figure 1 The A in the figure represents the clear and transparent circular plaques formed by bacteriophage A155. Figure 1 B. Phage A155 was observed under a transmission electron microscope in Figure B. It has a typical head structure and a retractable tail structure, with a head diameter of about 100.1 nm and a tail length of about 156.9 nm. Based on the analysis of morphological characteristics, phage A155 belongs to the family Myoviridae of the order Caudovirales.

[0030] Example 2 Phage genome sequencing analysis.

[0031] The phage was amplified and concentrated, and the phage nucleic acid was extracted according to the instructions of the virus nucleic acid extraction kit and sent to Shanghai Paisono Company for whole genome sequencing and analysis. BRIG software was used to compare and analyze similar genomes. The results are shown in Figure 2 The genome of bacteriophage A155 is a circular double-stranded DNA molecule with a genome size of 143202 bp and a GC content of 35.76%. Through BLAST analysis, bacteriophage A155 contains 216 putative open reading frames (ORFs), of which 63 ORFs have been annotated, with an annotation rate of about 29% (63 / 216), and 153 ORFs have been annotated as hypothetical proteins. The length of the CDS nucleic acid sequence of this phage is between 135-5478 bp, and all ORFs contain a total of 127803 bp of bases, and the gene density in the genome can reach about 89.25%.

[0032] Genome sequencing analysis showed that bacteriophage A155 belongs to Viruses ; Duplodnaviria ; Heunggongvirae ; Uroviricota ; Caudoviricetes ; Herelleviridae ; Brockvirinae ; Kochikohdavirus Comparative genomic analysis showed that the whole genome sequence of this phage had a high similarity with the genomes of enterococcal phages EFLK1 (Identity: 98.01%), EF17H (97.88%), phiM1EF22 (98.56%), and Ef2.3 (98.66%), with the differences within 5%. Phage A155 and these four phages belong to the same enterococcal phage.

[0033] Through BLASTp protein sequence analysis, 216 ORFs of phage A155 contain genes related to phage structure and structural assembly, DNA replication and regulation, perforin and lytic enzymes, etc. It does not contain antibiotic resistance genes and virulence genes, will not cause cross-species gene transfer, and has potential value for phage therapy of Enterococcus faecalis.

[0034] At the same time, based on the amino acid sequence of the phage terminase large subunit, a phylogenetic tree analysis was performed on the phage and several other enterococcal phages. MAFFT version 7 was used to analyze the sequences and construct a phylogenetic tree.

[0035] Phylogenetic tree Figure 3 As shown, phage A155 interacts with EFLK1, EF17H, phiM1EF22, and Ef2.3 of the Myoviridae family ( Podoviridae ) phages, and are more closely related to the Brachyviridae ( Myoviridae ) and the family of Longicaudaviridae ( Spihoviridae )Bacteriophages are clearly distinguished in the phylogenetic tree.

[0036] Through the above experimental process, it was determined that bacteriophage A155 was Enterococcus phage A155, deposited in China Center for Type Culture Collection on August 16, 2024, with the deposit number CCTCC NO: M20241805, and the address is Wuhan University, Wuhan, China.

[0037] Example 3 Fragmentation spectrum determination.

[0038] The lysis spectra of phages against a variety of bacteria (including 59 strains of Enterococcus faecalis, 5 strains of Enterococcus faecium, 2 strains of Staphylococcus aureus, 2 strains of Listeria monocytogenes, and 2 strains of Escherichia coli) were determined by the spot method. The strains with spot spots were further verified by the double-layer plate method, and the strains with plaques were finally identified as lysis-positive. Each group of experiments was repeated 3 times in parallel.

[0039] The lysis spectrum of bacteriophage A155 was determined for various bacteria, and the results are shown in Table 1. A155 has a broad spectrum of lysis activity against Enterococcus faecalis, with a coverage rate of 81.4% (48 / 59) for the tested Enterococcus faecalis; it has no lysis activity against the tested Enterococcus faecium (0 / 5), Staphylococcus aureus (0 / 2), Listeria monocytogenes (0 / 2), and Escherichia coli (0 / 2). This result shows that A155 has a broad spectrum of lysis effect specific to Enterococcus faecalis.

[0040] Table 1 Determination of lysis spectrum of bacteriophage A155

[0041] Example 4 Determination of optimal multiplicity of infection.

[0042] The optimal infection multiplicity of phage refers to the ratio of the highest phage titer measured by mixing phage and host bacteria in different ratios for phage amplification. 600 The bacterial concentration was determined and the total amount was adjusted to 1×10 8 CFU. The phages were graded diluted at a multiplicity of infection (MOI) of 10, 1, 0.1, 0.01, and 0.001, and then mixed with Enterococcus faecalis V583 and added to BHI liquid culture medium, and cultured at 37°C and 200 r / min for 6 h. The titer of phages in mixed solutions of different ratios was determined by the double-layer plate method. The mixing ratio with the highest titer was the optimal MOI of the phages. The results are shown in Table 2.

[0043] Table 2 Determination of the optimal infection multiplicity of bacteriophage A155

[0044] As shown in Table 2, after culturing bacteriophage A155 and host bacteria at a ratio of 10, 1, 0.1, 0.01 and 0.001 for 6 h, the titer of bacteriophage A155 was determined by the double-layer plate method. When MOI = 0.001, the titer of bacteriophage A155 was the highest, reaching 9.2 × 10 10 PFU∙mL -1 , that is, the optimal infection multiplicity of bacteriophage A155 is 0.001.

[0045] Example 5 One-step growth curve determination.

[0046] Take 1.0 mL and culture until the mid-logarithmic growth period (OD 600The supernatant was discarded after centrifugation at 4℃ and 5000 r / min for 10 min, and the precipitate was resuspended in 1.0 mL sterile BHI culture medium. After adding phage according to the optimal MOI=0.001, it was placed in a 37℃ incubator for 10 min, then centrifuged at 12000 r / min for 10 min, the supernatant was discarded, the precipitate was resuspended in 10 mL sterile BHI culture medium, and cultured at 37℃ and 200 r / min with shaking. Within the first 30 min, 50 μL of culture medium was taken out every 5 min to measure the phage titer, and within the next 90 min, 50 μL of culture medium was taken out every 10 min to measure the phage titer. The total measurement was 120 minutes, and 3 parallel tests were performed at each time point to take the average value. The one-step growth curve of the Enterococcus faecalis phage was drawn with the sampling time and the logarithm of the phage titer as the horizontal and vertical coordinates, respectively. The results showed that the phage titer increased significantly within 15-50 minutes, and after 50 minutes, the phage titer stabilized and entered a plateau period. This indicates that the incubation period of phage A155 is 15 minutes, the burst volume is about 458 PFU / cell, and one lysis cycle is about 50 minutes ( Figure 4 ).

[0047] Example 6 Temperature and pH stability determination.

[0048] The concentration is 10 9 After incubating the phage A155 with a concentration of 10 PFU / mL in a constant temperature water bath at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 30 min and 60 min, 100 μL of the phage solution was taken out for 10-fold dilution, and the count was performed using the double-layer plate method. The results were repeated three times to obtain the average value and observe the changes in the phage titer at each temperature. 9 After incubating phage A155 at pH 2.0-14.0 (interval 1.0) at 37°C for 60 min, 100 μL of phage solution was diluted 10-fold and counted using the double-layer plate method. The results were repeated three times and the average value was taken to observe the changes in phage titer under different pH conditions.

[0049] The results are as follows Figure 5 As shown, Figure 5 A in the figure shows that A155 survives stably in a wide temperature range (20-50°C), the titer decreases slightly at 60°C, and is completely inactivated at 70°C and above; Figure 5 B in the figure shows that the stability of A155 at different pH values ​​was determined. A155 survived stably in the pH range of 4.0-9.0, with a slight decrease in titer at pH 10.0 and was completely inactivated at a pH lower than 3.0 or higher than 11.0.

[0050] Example 7 Inhibition curve determination.

[0051] In a 12-well plate, 12.5 μL of overnight cultured Enterococcus faecalis V583 was inoculated into an appropriate amount of BHI broth to make the total amount approximately 2.5 × 10 7 CFU, add the corresponding titer of phage A155 at a ratio of 0.01, 0.1, 1, 10, and 100, and adjust the total volume to 2.5 mL. Use a group without phage as the control group, and repeat 3 times for each MOI. Place the 12-well plate in a bacterial growth curve instrument and measure the absorbance value (OD) every 30 min. 600 ), a total of 16 hours were monitored. Figure 6 As shown in the figure, without the addition of bacteriophage, V583 reached the stable phase in about 8 hours; however, with the addition of bacteriophage A155, the growth of V583 was significantly inhibited and it did not start to grow until 11 hours later. There was no significant difference in the inhibition time of V583 with different MOI of bacteriophage addition. Figure 7 As shown, the control group showed a higher degree of turbidity (OD 600 =2.31, while the phage A155 treatment group (MOI=0.01) was still relatively clear (OD 600 =0.04). This result indicates that phage A155 has a strong in vitro inhibitory ability on the growth of V583.

[0052] Example 8 Evaluation of the therapeutic effect of bacteriophage on the mouse intestinal model infected with Enterococcus faecalis.

[0053] Establishment of a mouse intestinal model of Enterococcus faecalis infection.

[0054] The mouse intestinal model of Enterococcus faecalis infection was established by the following method. Figure 8 A in the figure: 16 6-8 week old SPF BALB / c mice were randomly divided into two groups. After one week of adaptation, antibiotics were taken orally to eliminate the intestinal flora in the mice, simulating the dysbiosis caused by hospitalized patients taking large amounts of antibiotics. First, mixed antibiotics (Abx: vancomycin 10 mg / mouse, neomycin 10 mg / mouse, ampicillin 10 mg / mouse, metronidazole 10 mg / mouse) were gavaged for 3 days, followed by mixed antibiotic drinking water (Abx: vancomycin 500 mg / L, neomycin 500 mg / L, ampicillin 500 mg / L, metronidazole 500 mg / L) for 7 days. During this period, the number of bacteria, especially enterococci, in the feces of mice was monitored by smear counting. After an interval of 2 days, 100 μL of Enterococcus faecalis V583 suspension (1×10 9CFU / mL) were gavaged to simulate the process of massive intestinal proliferation and infection of enterococci after dysbiosis of hospitalized patients. During the process, the number of enterococci in the feces of mice was monitored using enterococcus selective medium.

[0055] Evaluation of bacteriophage therapy in a mouse intestinal model of Enterococcus faecalis infection.

[0056] After stable enterococcal colonization (2 days after V583 gavage), the experimental group of mice were given 2.4×10 9 PFU / mice were treated with a single oral gavage of phage A155, and the control group was treated with the same dose of BHI broth as a control. After that, the feces of the two groups of mice were collected every day for enterococcal selective culture medium count to evaluate the clearance effect of phage on Enterococcus faecalis.

[0057] The experimental process and results are as follows Figure 8 As shown, according to Figure 8 The process of A in the above was used to treat VRE colonization in the intestinal tract of mice with bacteriophage A155. Enterococci in the feces of mice after treatment were counted to evaluate the therapeutic effect of A155. The results are shown in Figure 8 As shown in B, after mixed antibiotic treatment and V583 infection, V583 can achieve short-term stable colonization in the mouse intestine (Day 1, 2), and the enterococcal load in feces can reach 10 9 CFU / g. After treatment with bacteriophage A155, there were significant differences between the treatment group and the control group on Day 3 and Day 4. On the second day, the V583 load was reduced by 1.13 orders of magnitude. This result confirmed that A155 can effectively reduce the colonization of V583 in the mouse intestine.

[0058] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0059] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A bacteriophage A155 antagonizing vancomycin-resistant Enterococcus faecalis, characterized in that: The bacteriophage (Enterococcus phage) The A155 deposit number is CCTCC NO: M 20241805. It was deposited in the China Center for Type Culture Collection on August 16, 2024. The deposit address is Wuhan University, Wuhan, China.

2. Use of the bacteriophage A155 according to claim 1 in the preparation of a preparation for treating and / or preventing vancomycin-resistant Enterococcus faecalis infection.

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