CN118792264B - A bacteriophage A155 and its application in antagonizing vancomycin-resistant Enterococcus faecalis - Google Patents

Abstract

The invention discloses a phage A155 and application thereof in antagonizing vancomycin-resistant enterococcus faecalis, and relates to the technical field of phage screening antagonizing vancomycin-resistant enterococcus faecalis, wherein the phage (Enterococcus phage) A155 is preserved in China center for type culture (China center for type culture) with the preservation number of CCTCC NO: M20241805 and the preservation address of China university of Wuhan in 8 th month 16 of 2024. The invention screens out a virulent phage aiming at the vancomycin-resistant enterococcus faecalis, carries out biological characteristics and whole genome sequencing analysis on the virulent phage, evaluates the antibacterial capability of the virulent phage in vitro and in vivo, and lays a foundation for developing phage therapy of the vancomycin-resistant enterococcus faecalis infection.

Description

Phage A155 and application thereof in antagonizing vancomycin-resistant enterococcus faecalis

Technical Field

The invention relates to the technical field of phage screening of antagonistic vancomycin-resistant enterococci, in particular to phage A155 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 A155 antagonizing vancomycin-resistant enterococcus faecalis, wherein the phage (Enterococcus phage) A155 is preserved in China center for type culture Collection (China center for type culture Collection) at a preservation number of CCTCC NO: M20241805 and at a preservation address of university of Wuhan, china at 8-16 of 2024.

In a second aspect, the embodiment of the invention provides an application of phage A155 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 A155.

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 A155, shows that the invention has broad-spectrum lysis activity on the enterococcus faecalis, has 81.4 percent coverage rate on the tested enterococcus faecalis, has no lysis activity on the tested enterococcus faecalis, staphylococcus aureus, listeria monocytogenes and escherichia coli, and has obvious difference between a treatment group and a control group in two days (Day 3, 4) after the treatment of phage A155, and can reduce the V583 load by 1.13 orders of magnitude in the next Day. This result demonstrates that a155 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 the plaque and electron microscope morphology of phage A155, A showing the plaque morphology of phage A155, and B showing the transmission electron microscope morphology of phage A155.

FIG. 2 is a graph showing the results of a whole genome sequencing analysis of phage A155.

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 sequence number in brackets is the sequence number of the phage genome, the numbers on the branches indicate the confidence level, the closer the number is to 100, the higher the confidence level, and the scale represents the genetic distance.

FIG. 4 is a graph of a one-step growth curve of phage A155.

FIG. 5 is a graph showing the temperature and pH stability of phage A155, A is the temperature stability of phage A155, and B is the pH stability of phage A155.

FIG. 6 is a graph showing in vitro bacteriostasis curves for phage A155 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 evaluation of phage A155 on treatment of VRE colonization in the mouse intestinal tract, a flowchart of phage A155 on treatment of VRE colonization in the mouse intestinal tract, and B, enterococcus count in feces after phage A155 treatment. * P <0.05, p <0.01, ns, no signalisation.

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.

Examples the strains and sources used in this study were 59 enterococcus faecalis, 5 enterococcus faecium, 2 staphylococcus aureus, 2 listeria monocytogenes, 2 escherichia coli (see table 2 for details) all deposited by the university of near-net microorganisms and host health institute.

Culture medium, brain heart infusion broth (brain-heart infusion media, BHI; g/L) bovine heart infusion powder 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) comprising peptone 20.0g, yeast extract 5.0g, oxgall 10.0g, sodium chloride 5.0g, esculin 1.0g, ferric ammonium citrate 0.5g, sodium azide 0.25g, sodium citrate 1.0g, and agar 15.0g.

The main reagents and instruments are sterile Solution (SM) buffer, the biological engineering (Shanghai) Co., ltd., PEG-8000, csCl, chloroform and phosphotungstic acid, the Abin Biotechnology Co., ltd., and virus nucleic acid extraction kit, the Tiangen Biotechnology (Beijing) Co., ltd. Transmission electron microscope, philips-FEI company, ultracentrifuge, beckman company, 0.22 μm filter, millipore company. Bacterial growth profilometer, new Zhi Biotech Co.

Example 1 phage isolation and purification.

Centrifuging collected pasture sewage at 4 ℃ for 5000 r ∙ min ^-1 for 10min, collecting supernatant, filtering with 0.22 μm filter, and storing filtrate in a 4 ℃ 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. 5min, 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 semisolid 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 picked and added to SM buffer and mixed with 100. Mu.L of host bacteria in 10 mL BHI medium, and cultured at 37 ℃ and 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, centrifuging at 12000 r/min overnight at 4deg.C for 10: 10min, discarding supernatant, re-suspending the precipitate with 2 mL SM buffer, 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 was designated Enterococcus phageA as Enterococcus phageA and as shown in FIG. 1, A in FIG. 1 is phage A155 to form a clear transparent circular plaque. Phage A155 was observed under the B-transmission electron microscope in FIG. 1, with a typical head configuration and a telescoping tail configuration, with a head diameter of about 100.1 nm and a tail length of about 156.9 nm. Based on morphological feature analysis, phage A155 belongs to the family Myoviridae of the order Rheumatoid.

Example 2 phage genome sequencing analysis.

Phage nucleic acid was extracted according to the viral nucleic acid extraction kit instructions, sent to the Shanghai Pakino company for whole genome sequencing and analysis, and used BRIG software for comparison and analysis of similar genomes. As a result, see FIG. 2, the genome of phage A155 is a circular double-stranded DNA molecule of which the genome size is 143202 bp and the GC content is 35.76%. Phage A155 contains 216 putative open reading frames (open READING FRAME, ORFs) by BLAST analysis, with 63 ORFs functioning annotated at approximately 29% (63/216), and 153 ORFs functioning annotated as putative proteins (hypothetical protein). The length of CDS nucleic acid sequence of the phage is 135-5478bp, all ORFs contain 127803 bp bases, and the gene density in genome can reach about 89.25%.

Genomic sequencing analysis showed that phage A155, which belongs to Viruses; Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricetes; Herelleviridae; Brockvirinae;Kochikohdavirus. in classification by comparison of genomic analysis, had a higher similarity in whole genome sequence to the genomes of enterococcus phage EFLK1 (Identity: 98.01%), EF17H (97.88%), phiM EF22 (98.56%) and Ef2.3 (98.66%), within 5% of each, phage A155 and the four phages belonging to the same enterococcus phage.

By BLASTP protein sequence analysis, 216 ORFs of phage A155 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, based on the amino acid sequence of the large subunit of the phage terminal enzyme, phylogenetic tree analysis is carried out on phage and a plurality of other enterococcus phages, and MAFFT version is used for analyzing the sequence and constructing phylogenetic tree.

The phylogenetic tree is shown in FIG. 3, and phages A155 have closer relatedness with phages of the myoviridae (Podoviridae) such as EFLK1, EF17H, phiM1EF22 and Ef2.3, and are obviously distinguished from phages of the brachyoviridae (Myoviridae) and the longuroviridae (Spihoviridae) on the phylogenetic tree.

Through the experimental process, the phage A155 is Enterococcus phage A and is preserved in China Center for Type Culture Collection (CCTCC) No. M20241805 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 phage A155 was subjected to a lysis spectrum assay of various bacteria, and the results are shown in Table 1. A155 has broad-spectrum lytic activity against enterococcus faecalis, 81.4% coverage (48/59) against enterococcus faecalis tested, and no lytic activity against enterococcus faecium (0/5), staphylococcus aureus (0/2), listeria monocytogenes (0/2) and Escherichia coli (0/2) tested. The results indicate that A155 has a broad-spectrum lytic effect specific for enterococcus faecalis species.

TABLE 1 determination of the cleavage spectrum of phage A155

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 A155

As is clear from Table 2, after culturing phage A155 with host bacteria in a ratio of 10, 1, 0.1, 0.01 and 0.001 for 6 h, the titer of phage A155 was measured by the double-layer plate method, and when MOI=0.001, the titer of phage A155 was the highest and reached 9.2X10 ¹⁰ PFU∙mL^-1, i.e., the optimal multiplicity of infection of phage A155 was 0.001.

Example 5 one-step growth curve determination.

Enterococcus faecalis V583 cultured to mid-log growth (OD ₆₀₀ about 0.7) at 4℃under 5000 r/min was centrifuged for 10min, 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 sterile BHI medium 10 mL at 37 ℃ and shake-cultured 200 r/min by standing 10min in a 37 ℃ incubator, then centrifuging 10min at 12000 r/min, discarding the supernatant. 50 mu L of culture solution is taken out every 5: 5min in the first 30: 30min for phage titer measurement, 50 mu L of culture solution is taken out every 10min in the last 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 15-50 min, with phage titers tending to plateau after 50: 50 min. The incubation period for phage A155 was 15 min, the outbreak was about 458 PFU/cell, and one lysis cycle was about 50 min (FIG. 4).

Example 6 temperature and pH stability determination.

Phage A155 at a concentration of 10 ⁹ PFU/mL was incubated in a thermostatic water bath at 20℃30℃40℃50℃60℃70℃80℃60 min and 50℃ 30 min, respectively, 100. Mu.L of phage solution was taken out and diluted 10-fold, counted by the double-plate method and the average was taken three times to observe the titer change of phage at each temperature. Meanwhile, phage A155 with the concentration of 10 ⁹ PFU/mL is respectively placed under the conditions of pH 2.0-14.0 (interval 1.0) and incubated for 60 min 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, FIG. 5 shows that A155 survives stably in a wide temperature range (20-50 ℃) with a slight drop in 60 ℃ titer and complete inactivation at 70 ℃ and above, and FIG. 5 shows that A155 survives stably in a range of pH 4.0-9.0 with a slight drop in titer at pH10.0 and complete inactivation at pH below 3.0 or above 11.0, as measured by the stability of A155 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 A155 of the corresponding titer was added at 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 8 h without phage addition, while V583 growth was significantly inhibited with phage A155 addition, and growth did not begin until after 11: 11 h, with no significant difference in the inhibition time of V583 by 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 a155 treated group (moi=0.01) was still clear (OD ₆₀₀ =0.04). The results indicate that phage A155 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 carried out by randomly dividing 16 BALB/c mice of SPF grade of 6-8 weeks of age into two groups, and after one week of adaptation to the environment, taking antibiotics orally to clear intestinal flora in the mice, so as to simulate dysbacteriosis of hospitalized patients caused by taking a large amount of antibiotics, as shown in A of FIG. 8. 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 10 mg /) 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 2.4x ⁹ PFU per single intragastric administration of phage a155 alone, 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 an enterococci selective medium daily to evaluate the effect of phage clearance from enterococci.

Experimental procedures and results are shown in FIG. 8, phage A155 is used for treating VRE colonization of the intestinal tract of a mouse according to the flow of A in FIG. 8, enterococci in the feces of the mouse after treatment are counted to evaluate the treatment effect of A155, and as shown in B in FIG. 8, V583 can realize short-term stable colonization (Day 1, 2) of the intestinal tract of the mouse after mixed antibiotic treatment and V583 infection, and the enterococcus load in the feces can reach about 10 ⁹ CFU/g. After phage a155 treatment, the treatment group and the control group were significantly different for two days after treatment (Day 3, 4) and the V583 loading could be reduced by 1.13 orders of magnitude on the next Day, which results confirm that a155 could effectively reduce 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.

Claims (2)

1. A bacteriophage A155 that antagonizes vancomycin-resistant Enterococcus faecalis, characterized in that the bacteriophage (Enterococcus phage) A155 has a deposit number of CCTCC NO: M 20241805, and was deposited in the China Center for Type Culture Collection on August 16, 2024, and 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.