CN118792265B - Phage A149 and application thereof in antagonizing vancomycin-resistant enterococcus faecalis - Google Patents
Phage A149 and application thereof in antagonizing vancomycin-resistant enterococcus faecalis Download PDFInfo
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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Abstract
本发明公开了一种噬菌体A149及其在拮抗耐万古霉素粪肠球菌中的应用，涉及拮抗耐万古霉素粪肠球菌的噬菌体筛选技术领域，噬菌体(Enterococcus phage)A149保藏编号为CCTCC NO:M 20241804，于2024年8月16日保藏于中国典型培养物保藏中心，保藏地址为中国.武汉.武汉大学。本发明筛选出一株针对耐万古霉素的粪肠球菌的烈性噬菌体，对其进行生物学特性及全基因组测序分析，并评价其在体外和体内的抗菌能力，为开发耐万古霉素的粪肠球菌感染的噬菌体疗法奠定了基础。
The present invention discloses a bacteriophage A149 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) A149 has a deposit number of CCTCC NO: M 20241804, and was deposited in the China Center for Type Culture Collection 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
The invention relates to the technical field of phage screening of antagonistic vancomycin-resistant enterococci, in particular to phage A149 and application thereof in antagonizing the vancomycin-resistant enterococci.
Background
Enterococcus faecalis (Enterococcus faecalis, efs) is a gram-positive facultative anaerobic bacterium that colonizes the intestines of humans and various animals and is considered a common gut symbiotic. Efs, however, may also be an opportunistic pathogen causing bacteremia, endocarditis, urinary tract and wound infections. With the widespread use of antibiotics, the resistance of Efs is becoming more severe, and particularly the emergence of Vancomycin-resistant enterococci (VRE) has rendered the treatment of clinical VRE infection difficult. Studies have shown that VRE colonization in the gut may not cause symptoms, but may persist for a longer period of time, becoming a repository of VRE, with a significant risk of causing infection in patients and others. Thus, there is an urgent need to develop antibiotic alternatives for VRE infection.
Phage is a virus that can infect and lyse bacteria, can target specific bacteria without damaging other microbial species, and has unique advantages in treating drug-resistant bacterial infections. In addition, the phage is easy to obtain, has high sterilization speed, is harmless and safe to eukaryotic cells, has synergistic antibacterial effect with other antibacterial drugs such as antibiotics, antibacterial peptides and the like, and is a very promising antibiotic substitution therapy.
How to provide a virulent phage capable of lysing vancomycin-resistant enterococcus faecalis is a technical problem to be solved by the person skilled in the art.
Disclosure of Invention
In view of this, the invention screens out a strain of virulent phage Efs V583 against vancomycin, performs biological characteristics and whole genome sequencing analysis on the phage, and evaluates the antibacterial ability of the phage in vitro and in vivo, so as to lay a foundation for developing phage therapy for Efs infection.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
an embodiment of the invention provides a phage A149 antagonizing vancomycin-resistant enterococcus faecalis, wherein the phage (Enterococcus phage) A149 is preserved in China center for type culture Collection (China center for type culture Collection) at a preservation address of China, university of Wuhan, and 8 th month for 2024 with a preservation number of CCTCC NO: M20241804.
In a second aspect, the embodiment of the invention provides an application of phage A149 in preparing a preparation for treating and/or preventing vancomycin-resistant enterococcus faecalis infection.
In a third aspect, embodiments of the present invention provide a formulation for the treatment and/or prophylaxis of vancomycin-resistant enterococcus faecalis infection comprising said phage A149.
Compared with the prior art, the invention takes vancomycin-resistant enterococcus faecalis V583 as host bacteria, screens out a phage which can specifically lyse the enterococcus faecalis, carries out the analysis of the lysis spectrum of a plurality of bacteria on phage A149, and shows that the invention has broad-spectrum lysis activity on the enterococcus faecalis, has 86.4 percent coverage rate on the tested enterococcus faecalis and has no lysis activity on the tested enterococcus faecalis, staphylococcus aureus, listeria monocytogenes and escherichia coli; moreover, the intestinal experiments in mice showed that after phage A149 treatment, the treatment group and the control group were significantly different on both days after treatment (Day 3, 4), and the V583 load could be reduced by 1.3 orders of magnitude on the next Day. This result demonstrates that a149 is effective in reducing V583 colonization in the mouse gut.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a drawing showing a plaque and electron microscope morphology of phage A149; a is the plaque morphology of phage A149; b is a transmission electron microscope morphology of phage A149.
FIG. 2 is a graph showing the results of a phage A149 whole genome sequencing analysis.
FIG. 3 is a drawing of a phylogenetic tree based on the amino acid sequence of the large subunit of the phage terminal enzyme; the serial number in brackets is the serial number of the phage genome; the number on the branch represents the confidence, the closer the number is to 100, the higher the confidence; ruler: representing the genetic distance.
FIG. 4 is a graph showing the one-step growth of phage A149.
FIG. 5 is a graph showing the temperature and pH stability of phage A149; a is the temperature stability of phage a 149; b is the pH stability of phage A149.
FIG. 6 is a graph showing in vitro bacteriostasis curves for phage A149 at different MOI.
FIG. 7 is a graph showing the growth of bacteria in both tubes with and without phage A149.
FIG. 8 is a graph showing the therapeutic evaluation of phage A149 on the colonization of the mouse intestinal tract by VRE; A. a therapeutic flow diagram of phage a149 to mice intestinal VRE colonization; B. enterococcus counts in feces after phage A149 treatment. * P < 0.05, p < 0.01, ns, no signalicant.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The examples use strains and sources: 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 (see table 2 for details) used in the study are all preserved by the university of near-Yi microorganisms and host health institute.
Culture medium: brain heart infusion broth (brain-heart infusion media, BHI; g/L) comprising bovine heart infusion 17.5g, tryptone 10.0g, sodium chloride 5.0g, na 2HPO4·12H2 O2.5 g, glucose 2.0g.
BHI semi-solid agar: BHI broth, 5g/L agarose.
BHI solid agar: BHI broth, 15g/L agar powder.
Enterococcus selection culture agar (g/L): 20.0g of peptone, 5.0g of yeast extract powder, 10.0g of oxgall, 5.0g of sodium chloride, 1.0g of esculin, 0.5g of ferric ammonium citrate, 0.25g of sodium azide, 1.0g of sodium citrate and 15.0g of agar.
Major reagents and instrumentation: sterile Solution (SM) buffer, division of bioengineering (Shanghai); PEG-8000, csCl, chloroform and phosphotungstic acid, alatin Biochemical technologies Co., ltd; viral nucleic acid extraction kit, tiangen Biochemical technology (Beijing) Co., ltd. Transmission electron microscope, philips-FEI company; ultracentrifuge, beckman Corp; 0.22 μm filter, millipore company. Bacterial growth profilometer, new Zhi Biotech Co.
Example 1 phage isolation and purification.
Centrifuging collected pasture sewage at 4deg.C for -1 min at 5000 r ∙ min for 10min, collecting supernatant, filtering with 0.22 μm filter, and storing filtrate in 4deg.C refrigerator. The filtrate was added to 100.0 mL BHI liquid medium with 1.0 mL enterococcus faecalis V583 suspension and cultured overnight at 37℃with 200 r/min shaking. After 1.0 mL of the mixed culture was centrifuged at 12000 r/min at 5.5 min, the phage primary liquid was obtained by filtration through a 0.22 μm filter. Mixing the phage primary liquid with host bacteria V583 suspension, standing for 10min, adding the mixed liquid into BHI semisolid culture medium, mixing, spreading on lower layer BHI solid culture medium, standing after the upper layer BHI semisolid culture medium is solidified, culturing in 37 deg.C incubator for overnight, and observing whether plaque appears the next day. Single plaques which are clear, bright, clear in edge and uniform in size are selected, added into SM buffer, mixed with 100 mu L of host bacteria in 10 mL BHI culture medium, and cultured at 37 ℃ for 200 r/min to obtain clear and transparent culture solution. Repeating the operation 3-5 times to obtain purified phage.
Amplifying the purified phage, adding PEG-8000, processing overnight at 4deg.C, centrifuging at 12000 r/min for 10: 10min, discarding supernatant, re-suspending the precipitate with 2mL SM buffer solution, and extracting with equal volume of chloroform to obtain phage concentrate. Slowly adding the phage concentrate into CsCl gradient liquid, centrifuging, collecting phage concentrate by using a syringe, and then transferring into phosphate buffer saline (phosphate buffered saline, PBS) for dialysis overnight to obtain purified phage particles. The obtained sample was stained with phosphotungstic acid by the negative staining method, and after 15 min, observed and recorded in a transmission electron microscope under the condition of 80: 80 kV. This is designated Enterococcus phageA as 149, and as shown in FIG. 1, in fact, FIG. 1A shows phage A149 forming a clear transparent circular plaque, and FIG. 1B shows phage A149 observed under a transmission electron microscope, having a typical head structure and a shorter tail structure, with a head diameter of about 46.2 nm and a tail length of about 28.8 nm. Based on morphological feature analysis, phage A149 belongs to the family of the Securoviridae of the order Rhinoceroviridae.
Example 2 phage genome sequencing analysis.
Phage nucleic acid was extracted according to the viral nucleic acid extraction kit instructions, and sent to the Shanghai Pair Nuo company for whole genome sequencing and analysis. Similar genomes were analyzed by alignment using BRIG software.
As a result, see FIG. 2, the genome of phage A149 is a circular double-stranded DNA molecule of 18561 bp in genome size and 33.13% GC content. Phage A149 contained 26 putative open reading frames (open READING FRAME, ORFs) by BLAST analysis, with 13 ORFs functioning annotated at approximately 50% (13/26) and 13 ORFs functioning annotated as putative proteins (hypothetical protein). The length of CDS nucleic acid sequence of the phage is between 138 and 2349 bp, all ORFs contain 17778 bp bases, and the gene density in genome can reach about 95.78%.
Genomic sequencing analysis showed that phage A149 belongs to Viruses; Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricetes; Rountreeviridae; Sarlesvirinae; Copernicusvirus. in classification by comparison of genomic analysis, and the whole genome sequence of this phage has higher similarity to the genomes of enterococcus phages AE4-17 (Identity: 91.01%), ef7.3 (95.94%) and Efmus4 (92.34%), the difference between the A149 genome and enterococcus phage Ef7.3 being within 5%, both belonging to the same enterococcus phage.
Through BLASTP protein sequence analysis, 26 ORFs of phage A149 have genes related to phage structure and structure assembly, DNA replication and regulation, perforin, lyase and the like, do not contain antibiotic resistance genes and virulence genes, do not cause trans-species gene transfer, and have potential value for enterococcus faecalis phage treatment.
Meanwhile, phylogenetic tree analysis was performed on phages and a number of other enterococcus phages based on the relatively conserved phage terminal enzyme large subunit sequences, and MAFFT version was used to analyze the sequences and construct phylogenetic trees.
Phylogenetic tree As shown in FIG. 3, phage A149 has closer relatedness to phages of the family of brachyoviridae (Podoviridae), such as AE4-17, ef7.3 and Efmus4, and has a more obvious distinction from phages of the family of Myoviridae (Myoviridae) and the family of longuroviridae (Spihoviridae) on phylogenetic trees.
As determined by the experimental process, phage A149 is classified and named Enterococcus phage and is preserved in China center for type culture Collection (CCTCC NO: M20241804) at the 8 th month and 16 th year of 2024, and the address is China university of Wuhan.
Example 3 cleavage spectrum determination.
The phage were subjected to lysis spectrometry by a spot method for a plurality of bacteria (including 59 enterococcus faecalis, 5 enterococcus faecium, 2 staphylococcus aureus, 2 listeria monocytogenes, 2 escherichia coli), and the strains with spot were further verified by a double-layer plate method, and the strains with spot were finally identified as positive. Each set of experiments was repeated 3 times in parallel.
The results of the lytic profiling of the various bacteria on phage A149 are shown in Table 1. A149 has broad-spectrum lytic activity against enterococcus faecalis and 86.4% coverage (51/59) against enterococcus faecalis tested; no lytic activity was observed against enterococcus faecium (0/5), staphylococcus aureus (0/2), listeria monocytogenes (0/2) and Escherichia coli (0/2). The results indicate that A149 has a broad-spectrum lysis effect specific to enterococcus faecalis.
TABLE 1 determination of the cleavage spectrum of phage A149
Example 4 optimal multiplicity of infection assay.
The optimal multiplicity of infection of phage refers to the ratio of the highest phage titers determined by mixing phage with host bacteria in different ratios to perform phage amplification. Enterococcus faecalis V583 was cultured to mid-log growth (OD 600 about 0.7), and the bacterial concentration was measured and the total amount was adjusted to 1X 10 8 CFU. Phage were diluted in a gradient at a ratio of multiplicity of infection (multiplicity of infection, MOI) of 10, 1, 0.1, 0.01 and 0.001, and mixed with enterococcus faecalis V583, respectively, and added to BHI liquid medium, and shake-cultured at 37℃and 200 r/min for 6 h. The titers of phages in the mixed solution with different ratios were determined by a double-layer plate method, and the mixed ratio with the highest titer was obtained as the optimal infection complex number of phages, and the results are shown in Table 2.
TABLE 2 determination of optimal multiplicity of infection for phage A149
As is clear from Table 2, after culturing phage A149 and host bacteria in a ratio of 10, 1, 0.1, 0.01 and 0.001 for 6h, the titer of phage A149 was measured by the double-layer plate method, and when MOI=0.001, the titer of phage A149 was the highest and could reach 1.0X10 10 PFU∙mL-1, i.e., the optimal multiplicity of infection of phage A149 was 0.001.
Example 5 one-step growth curve determination.
Enterococcus faecalis V583 cultured to mid-log growth (OD 600 about 0.7) at 1.0 mL was centrifuged at 5000 r/min at 4℃for 10 min, the supernatant was discarded, and the pellet was resuspended in 1.0 mL sterile BHI medium. After phage addition at optimal moi=0.001, the pellet was resuspended in 5mL sterile BHI medium and shake-cultured at 37 ℃, 200 r/min after 10 min at 37 ℃ incubator, followed by centrifugation at 12000 r/min for 10 min, the supernatant was discarded. 50 mu L of culture solution is taken out every 5min in the first 30min min for phage titer measurement, 50 mu L of culture solution is taken out every 10 min in the second 90min for phage titer measurement, total measurement is carried out for 120 min, 3 parallel experiments are carried out at each time point, and average value is obtained. And drawing a one-step growth curve of enterococcus faecalis by taking the logarithm of the sampling time and the phage titer as the horizontal and vertical coordinates respectively. The results showed a significantly rapid increase in phage titer within 5-20 min, with phage titers tending to plateau after 30: 30 min. This indicates that phage A149 had a latency of 5min or less, a burst size of about 912 PFU/cell and a lysis cycle of about 30min (FIG. 4).
Example 6 temperature and pH stability determination.
Phage A149 with the concentration of 10 9 PFU/mL is respectively placed in a constant-temperature water bath kettle with the temperature of 20 ℃,30 ℃,40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for incubation for 30min and 60min, 100 mu L of phage liquid is respectively taken out for 10-fold ratio dilution, counting is carried out by a double-layer plate method, the average value is taken three times repeatedly, and the titer change of phage at each temperature is observed. Meanwhile, phage A149 with the concentration of 10 9 PFU/mL is respectively placed under the condition of pH 2.0-14.0 (interval 1.0) and incubated for 60min at 37 ℃, 100 mu L of phage liquid is respectively taken for 10 times dilution, then counting is carried out by a double-layer flat plate method, the average value is obtained by repeating three times, and the titer change of phage under different pH conditions is observed.
As a result, as shown in FIG. 5, FIG. 5A shows that A149 survived stably over a wide temperature range (20-50 ℃) with a slight drop in titer at 60℃and complete inactivation at 70℃and above; in FIG. 5, B shows that A149 survived stably at pH 4.0-10.0, with a slight drop in titer at pH 11.0 and complete inactivation at pH below 3.0 or above 12.0, as measured by the stability of A149 at various pH values.
Example 7 bacteriostasis curve determination.
12.5. Mu.L of overnight cultured enterococcus faecalis V583 was inoculated in a 12-well plate to an appropriate amount of BHI broth to a total amount of about 2.5X10 7 CFU, phage A149 was added at a corresponding titer in a ratio of MOI of 0.01, 0.1, 1, 10, 100, respectively, and the total volume was adjusted to 2.5 mL, and each MOI was repeated 3 times in parallel using a group without phage as a control group. The 12-well plate was placed in a bacterial growth curve, and absorbance values (OD 600) were measured every 30 min, together with 16 h. As a result, as shown in FIG. 6, V583 reached the stationary phase at about 8h without phage addition; while growth of V583 was significantly inhibited with phage 149 added, growth did not begin until 11 h, and there was no significant difference in V583 inhibition time with phage addition at different MOI. 11 Bacterial turbidity at h is shown in fig. 7, with the control group having exhibited a higher degree of turbidity (OD 600 =2.31) while the phage a149 treated group (moi=0.01) was still clear (OD 600 =0.06). The results indicate that phage A149 has a strong in vitro inhibition of V583 growth.
Example 8 evaluation of phage treatment on intestinal models in enterococcus faecalis infected mice.
And (6) establishing an intestinal model of the enterococcus faecalis infected mice.
The establishment of intestinal models of enterococcus faecalis infected mice was performed according to the method of fig. 8 a: the 16 BALB/c mice with the SPF grade of 6-8 weeks are randomly divided into two groups, and after the mice are adapted to the environment for one week, antibiotics are orally taken to clear intestinal flora in the mice so as to simulate dysbacteriosis of hospitalized patients caused by taking a large amount of antibiotics. The number of bacteria, particularly enterococci, in the mouse feces was monitored by coating counts during which 3 days of mixed antibiotic (Abx: vancomycin 10 mg/neomycin 10 mg/ampicillin 10 mg/metronidazole 10mg /) and then 7 days of mixed antibiotic drinking (Abx: vancomycin 500 mg/L, neomycin 500 mg/L, ampicillin 500 mg/L, metronidazole 500 mg/L) were performed. After an interval of 2 days, both groups of mice were gastrinated with 100. Mu.L of enterococcus faecalis V583 suspension (1X 10 9 CFU/mL) to simulate the process of mass-proliferating enterococcus infection of the intestinal tract after dysbacteriosis in hospitalized patients, during which the number of enterococcus in the faeces of the mice was monitored using enterococcus selection medium.
Evaluation of phage treatment on intestinal models of enterococcus faecalis infected mice.
After stable colonisation with enterococci (day 2 after V583 intragastric administration), the experimental mice were treated with 5 x 10 9 PFU per single intragastric administration of phage a149, the control group was treated with the same dose of BHI broth as a control, and then faeces from both groups of mice were counted on enterococci selective medium daily to evaluate the effect of phage clearance from enterococci.
Experimental procedures and results are shown in fig. 8, phage a149 is treated according to the procedure of fig. 8 a for the colonization of the intestinal tract VRE of mice, enterococci in the feces of the mice after treatment are counted to evaluate the therapeutic effect of a149, and as shown in fig. 8B, V583 can achieve short-term stable colonization (Day 1, 2) in the intestinal tract of mice after mixed antibiotic treatment and V583 infection, and the enterococcus load in the feces can reach about 10 9 CFU/g. After phage a149 treatment, the treatment group and the control group were significantly different on both days after treatment (Day 3, 4), and V583 loading could be reduced by up to 1.3 orders of magnitude on the next Day. This result demonstrates that a149 is effective in reducing V583 colonization in the mouse gut.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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