Phage PHS1 and application thereof
By isolating and identifying bacteriophage PHS1, the problem of preventing and controlling bacterial fibrosis in silkworms was solved. Stability in acidic and alkaline environments was achieved, improving the survival rate of silkworms and reducing the intestinal bacterial load.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-15
- Publication Date
- 2026-04-03
AI Technical Summary
The lack of effective methods for isolating Enterococcus montelukast from bacteriophages in the current technology makes it difficult to prevent and control bacterial fibrosis in silkworms, and limits the use of antibiotics.
Bacteriophage PHS1 was isolated and identified, which remains stable in both acidic and alkaline environments and can be used to prepare products for the prevention or treatment of bacterial enteropathy in silkworms, including silkworm feed and antibacterial agents.
Phage PHS1 significantly improves the survival rate of silkworms, significantly reduces the bacterial load in the intestine, and effectively prevents and treats bacterial fibrosis in silkworms.
Smart Images
Figure CN121780452A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of silkworm rearing technology, specifically to a bacteriophage PHS1 and its applications. Background Technology
[0002] Enterococci are a genus in the order Lactobacilliles and family Enterococciaceae. They are characterized by being Gram-positive, aerobic or facultative anaerobic, non-spore-forming, essentially non-motile, lacking a distinct capsule, and being cocci. Common enterococci in the human intestine are *Enterococcus faecalis* and *Enterococcus faecium*, while *Enterococcus montelukast* is commonly found in silkworms raised on artificial feed. Enterococcus mundtii ), is one of the pathogens that cause bacterial softening disease in silkworms.
[0003] Bacterial flacherie, also known as bacterial enteropathy, is caused by the uncontrolled proliferation of bacteria in the silkworm's intestines, leading to physiological changes that result in decreased appetite, stunted weight gain, diarrhea, and death. The characteristic feature of death is the softening of the silkworm's body, hence the name flacherie. Once it occurs, the disease is highly contagious, and no silkworm in the same rearing area can escape its infection.
[0004] The use of antibiotics in animal feed is currently restricted, but bacteriophages can serve as an alternative. They prevent the excessive proliferation of pathogenic bacteria by specifically lysing them, and are considered a novel type of antibacterial agent. Currently, no bacteriophages of Enterococcus montelukast have been reported, therefore, there is an urgent need to establish methods for isolating Enterococcus montelukast bacteriophages and to enrich the bacteriophage library for wider application. Summary of the Invention
[0005] In view of the shortcomings of the prior art, the present invention aims to provide a bacteriophage PHS1 and its application.
[0006] To address the problems of the prior art, the technical solution adopted by this invention is as follows: In one aspect, this application provides a bacteriophage PHS1, which lyses Enterococcus mongolicus and was deposited on October 11, 2025, at the Guangzhou Provincial Microbial Culture Collection Center with accession number GDMCC No:67071-B1, and taxonomically named... Enterococcus mundtii Phage, Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangzhou Academy of Sciences, Postcode 510070.
[0007] This application provides a bacteriophage isolated from nature that can lyse multiple strains of Enterococcus montelukastii, which can effectively treat bacterial enteritis in silkworms caused by Enterococcus montelukastii.
[0008] Preferably, the bacteriophage PHS1 is a myotail bacteriophage with a retractable tail, the tail sheath being 80.93±0.6 nm long when contracted and 170.54±1.13 nm long when extended.
[0009] Preferably, the phage PHS1 exhibits good 24-hour tolerance at pH 4-9 and good 4-hour tolerance at pH 10-10.8.
[0010] Preferably, the amount of bacteriophage added when lysing Enterococcus montelukast in feeding applications is 10^7 PFU / g.
[0011] Secondly, this application provides the application of the above-mentioned bacteriophage PHS1 in the preparation of related products for the prevention or treatment of bacterial enteropathy in silkworms caused by Enterococcus montelukast.
[0012] Thirdly, this application provides a silkworm feed comprising the aforementioned bacteriophage PHS1, which can be used to prevent or treat bacterial fibrosis in silkworms.
[0013] Fourthly, this application provides an antibacterial agent for bacterial enteropathy in silkworms, the active ingredient of which is bacteriophage PHS1.
[0014] Beneficial effects:
[0015] The *Enterococcus montelukas* phage PHS1 of this invention was isolated from activated sludge at the Hangzhou West Wastewater Treatment Plant. It is a newly discovered phage primarily targeting *Enterococcus montelukas*, belonging to the class Caudata, family Heller's Phage, and genus *Hickophage*. This phage can tolerate the acidic environment of the feed for extended periods and the alkaline environment of the silkworm midgut for short periods. Specifically, it exhibits good 24-hour tolerance at pH 4-9 and good 4-hour tolerance at pH 10-10.8, adapting to both the acidic feed environment and the alkaline intestinal environment of silkworms in artificial feed rearing settings. This phage also exhibits some lytic activity against *Enterococcus faecalis* and other bacteria besides *Enterococcus montelukas*, has a broad host spectrum, and lacks highly matched virulence genes, making it highly safe for use in silkworm rearing systems. This bacteriophage significantly improved the survival rate of silkworms and significantly reduced the bacterial load in their intestines. When used as an additive in finished feed, it showed a significant preventive and therapeutic effect against bacterial enteritis in silkworms caused by Enterococcus montelukastii. The Enterococcus montelukastii bacteriophage PHS1 of this invention has promising application prospects. Attached Figure Description
[0016] Figure 1 This is a transmission electron microscope image of bacteriophage PHS1.
[0017] Figure 2 This is a complete genome loop of bacteriophage PHS1.
[0018] Figure 3 pH stability of bacteriophage PHS1 over 24 hours.
[0019] Figure 4 The stability of bacteriophage PHS1 in an alkaline environment for 4 hours.
[0020] Figure 5 The survival curve for silkworms (n=36).
[0021] Figure 6 The intestinal bacterial load of silkworms on the fifth day of the fifth instar (n=4). Detailed Implementation
[0022] The reagents and culture media involved in the following examples are as follows: LB solid medium (500 mL): Weigh 2.5 g yeast extract, 5 g tryptone, 5 g sodium chloride, and 7.5 g agar powder, add 500 mL of purified water, stir well, autoclave at 121℃ for 15 min, cool to 60℃, pour into plates, and air dry for later use.
[0023] BHI semi-solid culture medium (100 mL): Weigh 3.85 g of culture medium powder and 0.5 g of agarose, add 100 mL of deionized water, melt in a microwave oven, dispense 4 mL / tube, and autoclave at 121℃ for 15 min before use.
[0024] BHI broth (500 mL): Weigh 19.25 g of culture medium powder, add 500 mL of deionized water, and autoclave at 121℃ for 15 min before use.
[0025] 2×Enterococcosel Broth (100 mL): Weigh 8.56 g of culture medium powder, dissolve it in 100 mL of deionized water, and autoclave at 121 °C for 15 min before use.
[0026] 2×PEG (100 mL): Weigh 20 g PEG8000 and 11.7 g sodium chloride, add to 100 mL of water and stir well. Sterilize by autoclaving at 121 °C for 15 min and set aside.
[0027] SM buffer (500 mL): Measure 25 mL of Tris-HCl (pH 7.4) buffer, weigh 2.9 g of sodium chloride and 2 g of magnesium chloride, dissolve them completely in 475 mL of deionized water, and autoclave at 121 °C for 15 min before use.
[0028] PBS buffer (500 mL): Measure 400 mL of deionized water, weigh 4 g of sodium chloride, 0.1 g of potassium chloride, 0.72 g of disodium hydrogen phosphate, and 0.12 g of potassium dihydrogen phosphate, add them to the water and stir well. Adjust the pH to 7.2-7.4, and finally add deionized water to make up to 500 mL. Autoclave at 121 °C for 15 min and set aside for use.
[0029] Example 1: Isolation of bacteriophage PHS1 Enrichment: Activated sludge from the Hangzhou West Wastewater Treatment Plant in China was used to isolate bacteriophages of Enterococcus montroxensis. Enterococcus montroxensis ZJU11 (GDMCC 1.4258) was used as the host bacterium and cultured in BHI broth at 37°C. The obtained activated sludge was allowed to stand at low temperature, and 5 mL of the lower layer was added to an equal volume of 2×Enterococcosel Broth (EB). After incubation overnight at 37°C on a shaker, the mixture was centrifuged (10000 g, 10 min), then filtered through a 0.22 μm filter for sterilization. The resulting filtrate was mixed with an equal volume of 2×PEG solution and allowed to settle overnight at 4°C. It was then centrifuged (10000 g, 30 min, 4°C), the supernatant was discarded, and the precipitate was resuspended in 2 mL of SM buffer to obtain the bacteriophage pool. Add 100 μL of fresh host bacterial culture (incubated overnight) to 3 mL of melted BHI semi-solid medium incubated at 60°C. Quickly and briefly vortex to mix, then rapidly pour onto an LB agar plate. After solidification, a top layer of agar containing the host bacteria will form, resulting in a double-layer agar plate. Place 5 μL of phage cells onto the double-layer agar plate containing the host bacteria, allow to dry, and incubate overnight at 37°C. The formation of a clear lysis zone indicates the presence of the target phage.
[0030] Isolation and Purification: Add 1 mL of host bacterial culture in the logarithmic growth phase to 10 μL of phage pooling solution and incubate at 37°C with shaking until clear. Centrifuge (5000 rpm, 5 min) and collect the supernatant for serial dilution. Add 100 μL of the supernatant dilution and 100 μL of host bacterial culture (incubated overnight) to 3 mL of melted BHI semi-solid medium incubated at 60°C to prepare double-layer agar plates. Incubate at 37°C for at least 12 h. Select the largest and clearest phage plaque on the plate, vertically puncture the soft agar containing the plaque with a 10 μL pipette tip, and then blow it into 500 μL of PBS. Shake well and incubate overnight at 4°C. Centrifuge (5000 rpm, 5 min) and collect the supernatant. Continue incubation with bacterial culture in the logarithmic growth phase and repeat the above procedure. Repeat 3 times to obtain purified phage. The final isolated bacteriophage PHS1 can form well-defined plaques with a diameter of 0.6±0.1 mm.
[0031] Propagation: The host bacteria were cultured in BHI broth to the logarithmic growth phase. The host bacteria culture and phage were mixed at a volume ratio of 3:1 and cultured in a shaker at 37°C until clear. After centrifugation (5000 rpm, 5 min), the supernatant was passed through a 0.22 μm filter membrane to remove the host bacteria, yielding the phage propagation solution with a titer of up to 3 × 10^9 PFU / mL.
[0032] Example 2: Genome sequencing of bacteriophage PHS1 The proliferation medium of phage PHS1 was sent to Chengdu Life Baseline Technology Co., Ltd., where the genome of the phage was sequenced using the DNBSEQ-T7 platform. The whole genome sequence was assembled, annotated, and analyzed.
[0033] The results are as follows Figure 2 As shown, the genome size of phage PHS1 is 150419 bp, belonging to the class Tailed Phages, family Heller Phages, and genus Hick Phage, with a GC content of 37%. 209 open reading frames (ORFs) were predicted. No highly matched virulence genes or any drug resistance genes were predicted.
[0034] Example 3 Host profile determination of bacteriophage PHS1 The host range of bacteriophages was determined using a simple protocol with seven Enterococcus strains. Briefly, overnight cultures of the seven Enterococcus strains (5 μL per spot) were spotted onto square LB agar plates (10 cm × 10 cm). After drying, 3 μL of phage dilution (10^7 PFU / mL) was spotted at the edge of each strain spot. The plates were air-dried, inverted, and incubated at 37°C for 24 hours. The clarity of the lysis zone indicated the ability of the phage to infect and multiply within each strain. The results are shown in Table 1, where "+" indicates lysis and "-" indicates non-lysis.
[0035] Table 1 Host spectrum of bacteriophage PHS1
[0036] Table 1 shows that phage PHS1 can lyse different strains of Enterococcus montelukastii, and also has lytic activity against Enterococcus faecalis and Enterococcus faecium, while Enterococcus pyogenes and Enterococcus avium are not within the lytic spectrum of PHS1.
[0037] Example 4: Transmission electron microscopy of bacteriophage PHS1 5 μL of host bacterium ZJU11 culture was spotted onto an LB agar plate. After drying, 3 μL of phage dilution (10^7 PFU / mL) was spotted at the edge of the host bacterium spot. The plate was air-dried, inverted, and incubated at 37°C for 24 hours to obtain a crescent-shaped colony lawn. Bacteria were carefully sampled from the boundary between the colony lawn and the phage lysis zone using a sterile toothpick and suspended in 50 μL of deionized water. 40 μL of the suspension was dropped onto a clean sealing film, and 40 μL of 1% phosphotungstic acid (PTA) was dropped onto another location for negative staining. A glow discharge copper grid was placed face down on the suspension drop and incubated for 5 minutes to allow for full phage adsorption. Excess liquid was then blotted away with filter paper, leaving a thin film on the copper grid surface. The copper grid was then placed face down on a PTA staining drop for 40 seconds, and then blotted away with filter paper. The copper grid was air-dried for at least 15 minutes. Observation and image acquisition were performed using a transmission electron microscope.
[0038] Image results as follows Figure 1 As shown, phage PHS1 is a myotail phage with a retractable tail. The tail sheath is 80.93±0.6 nm long when contracted and 170.54±1.13 nm long when extended.
[0039] Example 5: pH stability of bacteriophage PHS1 and confirmation of its application regimen In the process of raising silkworms with artificial feed, the use of bacteriophages faces two challenges: the acidity of the artificial feed and the alkalinity of the silkworm midgut.
[0040] Purchase silkworm compound feed from Chongqing Zhengjia Feed Co., Ltd., whose main components are soybean meal powder, corn starch, mulberry leaf powder, sorbic acid, compound vitamins, and inorganic salts. Prepare the artificial feed according to the manufacturer's instructions: weigh a certain amount of dry feed powder, add 1.8 times its weight of purified water, stir evenly, pack into a food bag, roll out into a uniformly thick cake, seal, and steam at 100℃ for 60 minutes. Remove and cool before use. Take 0.1g of the prepared artificial feed and add 10 times its weight of deionized water (pH=7.0) for homogenization. Use a Sartorius PB-10 benchtop pH meter equipped with a standard glass electrode to measure the pH value of the feed homogenate; the measured pH value is 4.88±0.04 (sample size n=10). Simultaneously, dissect and collect the midgut contents of fifth-instar silkworms raised on the fifth day of artificial feed and immediately homogenize using a handheld homogenizer; the measured pH value is 10.25±0.4 (sample size n=10).
[0041] The 24-hour stability of bacteriophages (10^9 PFU / mL) at different pH values (4-11) was tested based on the measured pH of the feed and the midgut pH of the silkworm. Specifically, at room temperature (25℃), the pH of PBS was adjusted using 3 M sodium hydroxide solution and 10% dilute hydrochloric acid to prepare PBS at different pH values (4, 5, 6, 7, 8, 9, 10, and 11), which were then sterilized by filtration through a 0.22 μm filter membrane. 99 μL of PBS corresponding to the pH value was added to a 200 μL PCR tube, followed by 1 μL of bacteriophage solution of known titer. The mixture was vortexed and incubated at 25℃ for 24 hours. The number of surviving bacteriophages was then quantified using double-layer agar plates. Each pH condition was set up in triplicate (sample size n=3).
[0042] The results are as follows Figure 3 As shown, phage PHS1 exhibits good stability within the pH range of 4-9, with a phage titer decrease of no more than one order of magnitude. However, it decreases by approximately two orders of magnitude at pH 10 and by approximately four orders of magnitude at pH 11. This demonstrates that phage PHS1 can maintain a stable phage titer in feed for an extended period (at least 24 hours), but it is unlikely to remain stable for such a long time in the alkaline environment of the midgut.
[0043] Since the food stays in the silkworm's intestines for no more than 3 hours, the stability of the phage titer was tested for 4 hours within the pH range of 10-11. The results are as follows... Figure 4 As shown, the titer of bacteriophage PHS1 decreases by no more than 0.5 orders of magnitude in the pH range of 10-10.6, and by nearly 1 order of magnitude at pH 10.8. This indicates that the bacteriophage can maintain a stable titer for a short period in the intestines of most silkworms.
[0044] In summary, when applying bacteriophage PHS1 in practice, the specific method is as follows: apply bacteriophage PHS1 to artificial feed and feed it continuously, and change the feed every 24 hours to maintain the presence of active bacteriophage in the silkworm's intestines for a long time.
[0045] Example 6: The effect of bacteriophage PHS1 in preventing and controlling Enterococcus montelukast infection in silkworms. Using formulated feed for large silkworms (same as in Example 5) purchased from Chongqing Zhengjia Feed Co., Ltd., 120 fifth-instar silkworms (Jingsong × Haoyue) were taken from the Shengzhou Artificial Feed Silkworm Farm and divided into 3 groups of 40 silkworms each. One group served as a blank control group, one group as an infection control group, and the remaining group as the PHS1 treatment group. The experimental treatments are shown in Table 2. The survival rate of silkworms was recorded over the 6-day experiment. The intestinal bacterial load was investigated on the fifth day of the fifth instar (n=4).
[0046] Table 2 Experimental arrangements for different treatment groups
[0047] like Figure 5 As shown, the mortality rate of silkworms in the infected control group was approximately 80%, while the mortality rate in the PHS1 treatment group was approximately 20%. This indicates that oral administration of 10^7 PFU / g of phage PHS1 can significantly increase the survival rate of silkworms.
[0048] like Figure 6 As shown, the midgut bacterial load in the infected control group was as high as 10^8 CFU / g, while the midgut bacterial load in the PHS1 treatment group was significantly reduced by about two orders of magnitude. This indicates that oral administration of 10^7 PFU / g of phage PHS1 indeed inhibited bacterial proliferation in the silkworm gut.
[0049] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.
Claims
1. A bacteriophage PHS1, characterized in that, The bacteriophage lysed Enterococcus mongolicus and was deposited on October 11, 2025, at the Guangzhou Provincial Microbial Culture Collection Center with accession number GDMCC No:67071-B1, and its taxonomic name is... Enterococcus mundtii Phage, Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangzhou Academy of Sciences, Postcode 510070.
2. The phage PHS1 according to claim 1, characterized in that, The phage PHS1 is a myotail phage with a retractable tail. The tail sheath is 80.93±0.6 nm long when contracted and 170.54±1.13 nm long when extended.
3. The phage PHS1 according to claim 1, characterized in that, The phage PHS1 exhibits good 24-hour tolerance at pH 4-9 and good 4-hour tolerance at pH 10-10.
8.
4. The phage PHS1 according to claim 1, characterized in that, The amount of the bacteriophage added to lyse Enterococcus montelukast in the feeding application is 10^7 PFU / g.
5. The use of a bacteriophage PHS1 according to any one of claims 1-4 in the preparation of related products for the prevention or treatment of bacterial enteropathy in silkworms caused by Enterococcus montelukast.
6. A type of feed for silkworms, characterized in that, The bacteriophage PHS1 comprises any one of claims 1-4.
7. An antibacterial agent for bacterial enteritis in silkworms, characterized in that, Its active ingredient is the bacteriophage PHS1 as described in any one of claims 1-4.