Phage132B bacteriophage strain with strong splitting action on MRSA strain and application of Phage132B bacteriophage strain

Abstract

The invention relates to the technical field of microorganisms, and discloses a Phage132B bacteriophage strain with a strong splitting action on an MRSA strain, and the Phage132B bacteriophage strain has the characteristics of wide bacteriophage spectrum, large outbreak amount and strong environmental tolerance. MRSA growth and biofilm formation can be remarkably inhibited in vitro, the survival rate of A549 cells can be improved by inhibiting MRSA infection in a cell experiment, and macrophage inflammatory response caused by MRSA is reduced; the phage gel prepared from the phage Phag132B can promote the recovery of skin abscess induced by MRSA through a local administration mode.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically to a Phage132B phage with strong lysis effect on MRSA and its application. Background Art

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant pathogen that can cause severe infections, causing various diseases worldwide, inducing high morbidity and mortality, and imposing a heavy burden on the global healthcare system. As an opportunistic pathogen, Staphylococcus aureus can colonize the nasal cavities of 20% to 30% of the population, and is a risk factor for skin and soft tissue infections (SSTIs). Severe SSTIs can trigger systemic symptoms, leading to serious complications or death. In recent years, the proportion of MRSA among the pathogens isolated from SSTIs has been increasing, complicating clinical treatment. Phages have the characteristic of specifically infecting and lysing bacteria, and are attracting attention as a potential antibacterial agent against bacterial infections. The control effect of phage therapy against antibiotic-resistant pathogen infections has been confirmed in many studies. Currently, there is a lack of phages against methicillin-resistant Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus poses a huge threat in livestock farming and clinical practice. It is difficult to achieve good prevention and control treatment effects with the limited existing phages. Summary of the Invention

[0003] The purpose of the present invention is to provide a Phage132B phage strain with strong lysis effect on MRSA strains and its application, so as to solve the problems raised in the above background art.

[0004] To achieve the above purpose, the present invention provides the following technical solutions: A Phage132B phage strain with strong lysis effect on MRSA strains, and the preservation number of the phage Phage132B is GDMCC No: 62838-B1.

[0005] A composition containing the above-mentioned Phage132B phage with strong lysis effect on MRSA strains.

[0006] An application of the above-mentioned phage Phage132B in the preparation of drugs against methicillin-resistant Staphylococcus aureus.

[0007] Compared with the prior art, the beneficial effects of the present invention are:

[0008] The phage strain of the present invention has the characteristics of a broad phage spectrum, a large burst amount, and strong environmental tolerance; it can significantly inhibit the growth and biofilm formation of MRSA in vitro, and can improve the survival rate of A549 cells and reduce the macrophage inflammatory response caused by MRSA in cell experiments; the phage gel prepared from Phag132B phage can promote the recovery of skin abscesses induced by MRSA through local administration. Brief Description of the Drawings

[0009] Figure 1 It is a double-layer plate plaque morphology diagram of Phag132B phage of the present invention;

[0010] Figure 2 It is a transmission electron microscope image of Phag132B phage of the present invention. Detailed Embodiments

[0011] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0012] Embodiment

[0013] Step 1: Isolate and identify MRSA strains from clinical samples, enrich phages from environmental samples using MRSA strains as host bacteria and isolate and purify them. Screen out phages with strong lytic ability and broad antibacterial spectrum according to the plaque morphology size and phage spectrum; measure the phage titer by the double-layer plate method, and detect the optimal multiplicity of infection (MOI) of the phage, one-step growth curve, and tolerance to temperature and acid-base environment, such as Figure 1 ; Determine the phage morphology by electron microscopy observation, such as Figure 2 ; Detect and analyze the genetic information of the phage by whole-genome sequencing.

[0014] Step 2: Determine the ability of the phage to inhibit the growth of MRSA in liquid medium by plotting an antibacterial curve; detect the ability of MRSA to form biofilms under the action of the phage using the crystal violet staining method; verify the protective effect of the phage on MRSA-infected cells according to the A549 cell survival experiment; detect the expression levels of THP-1 macrophage polarization-related cytokines by qPCR, and explore the anti-MRSA infection effect of the phage according to the inflammatory response.

[0015] Step 3: Establish a mouse skin abscess model by subcutaneous injection of MRSA culture. The treatment group is given 1×10 10The phage gel at PFU / mL was externally applied, the bacteria-infected group was given the empty gel externally, and the blank control group was injected with PBS. The status of mice in each group was observed daily, and the wound morphology and size were recorded. The mice in each group were sacrificed and sampled on the 3rd, 7th, and 12th days of treatment respectively. The bacterial load in the skin abscess of each group of mice was measured by the dilution plating method. The abscess site and the surrounding healthy skin were fixed with paraformaldehyde, and tissue sections were prepared by HE staining to observe the pathological characteristics of the skin tissues of each group of mice.

[0016] Results

[0017] 1. According to the identification of the drug resistance phenotype and mecA genotype of Staphylococcus aureus, 24 strains of MRSA strains were isolated and identified from clinical samples as host bacteria. A total of 93 water samples, fecal samples, and soil samples were collected from hospitals, wastewater treatment plants, farms, artificial lakes, etc. The treated environmental samples were enriched and separated using 24 strains of MRSA as host bacteria, and 33 strains of phages were isolated. The morphology of individual plaques was observed, and 5 phages with clear edges, transparent colors, and larger diameters of individual plaques were selected, namely Phage109B, Phage132B, Phage101E, Phage101M, and Phage109I. The phage spectra were detected and found that Phage101M could lyse 14 strains of MRSA, while Phage132B could lyse 11 strains of MRSA. The two phages had strong lytic ability against MRSA and a broad phage spectrum. Therefore, Phage101M and Phage132B were selected for further research.

[0018] 2. Observation under the electron microscope found that both Phage101M and Phage132B had polyhedral head structures and long tail filaments, belonging to the family Siphoviridae. The two phages could remain stable in the environment with a pH of 5 - 7.5 and 30 - 40 °C. In tryptic soy broth, with 30% glycerol as a protective agent, they could remain stable within 30 days at 4 °C and could adapt to general short-term and medium-term storage conditions. The optimal MOIs of Phage101M and Phage132B were 0.01 and 0.1 respectively, the latent periods were both 70 min, and the burst sizes were 2737 PFU / infected cell and 190 PFU / infected cell respectively, both having the characteristic of a large burst size. The results of the whole-genome analysis did not find known pathogenic genes and virulence genes.

[0019] 3. Through in vitro antibacterial curve and anti-biofilm activity analysis, it was found that the antibacterial and anti-biofilm effects of Phage132B in liquid medium were stronger than those of Phage101M. Therefore, Phage132B was selected to participate in further research. The results of the A549 cell survival experiment showed that when the multiplicity of infection of Phage132B was 0.01, 0.05, 0.1, 0.5, and 1.0, the cell survival rates were 61.1%, 64.5%, 69.1%, 78.9%, and 91.5% respectively, all higher than 42.0% of the control group (P < 0.05; P < 0.001). Analysis of the expression levels of THP-1 macrophage polarization-related factors found that under the intervention of Phage132B, the expression levels of M1-type polarization cytokines IL-1β, TNF-alpha, and TLR4 decreased, and the expression levels of M2-type polarization marker factors TGF-β and IL-10 increased, suggesting that Phage132B intervention reduced the inflammatory response caused by MRSA infection and decreased the pro-inflammatory M1-type macrophage polarization (P < 0.05; P < 0.01).

[0020] 4. Observing the skin conditions of mice in each group, it was found that abscesses appeared in the treatment group and the bacteria-infected group 24 hours after infection, while the skin of the control group mice was flat and normal. Comparing the abscess morphology and tissue sections of the treatment group and the bacteria-infected group mice, it was found that on the 3rd day of intervention, the abscess sites of the treatment group and the bacteria-infected group mice showed ulceration and scabbing, and different degrees of inflammatory infiltration were visible under the microscope. On the 5th day of intervention, the wounds of the bacteria-infected group mice showed a wound collapse state caused by tissue necrosis and loss. Correspondingly, on the 7th day of intervention, large abscess cavities appeared in the tissue sections of the bacteria-infected group mice. The treatment group mice were never observed to have wound collapse and showed signs of healing such as collagen fibers, new blood vessels, and new hair follicles earlier than the bacteria-infected group. Observing the skin abscess areas of the treatment group and the bacteria-infected group mice, it was found that on the 1st day of intervention, the abscess areas of the two groups of mice were balanced (P > 0.05), while on the 3rd day and the 12th day of intervention, the abscess areas of the treatment group mice were significantly smaller than those of the bacteria-infected group (P < 0.05; P < 0.01). Observing the bacterial load in the skin tissues of mice, it was found that on the 3rd day and the 7th day of intervention, the bacterial load in the abscess sites of the treatment group mice was significantly lower than that of the bacteria-infected group (P < 0.001). On the 12th day of intervention, the bacterial load of the treatment group mice was 0 CFU / mL, but there was no significant difference from the bacteria-infected group (P > 0.05). The healing speed of the treatment group mice was better than that of the bacteria-infected group. On the 12th day of intervention, the cure rate of the treatment group mice was 100% (6 / 6), while the cure rate of the bacteria-infected group mice was 16.7% (1 / 6) (P < 0.01).

[0021] The present invention systematically screens out Phage132B with strong lysis effect against common clinical MRSA strains in environmental samples, which has the characteristics of broad phage spectrum, large burst size and strong environmental tolerance; it can significantly inhibit the growth and biofilm formation of MRSA in vitro, and can improve the survival rate of A549 cells and reduce the macrophage inflammatory response caused by MRSA in cell experiments; the phage gel prepared from Phage132B can promote the recovery of MRSA-induced skin abscesses in mice by local administration.

[0022] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0023] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Phage132B phage strain with strong lytic effect on MRSA strains, characterized in that: The preservation number of the phage Phage132B is GDMCC No: 62838-B1.

2. A composition, characterized in that: The composition contains the Phage132B phage with strong lytic effect on MRSA strains described in claim 1.

3. Use of the phage Phage132B described in claim 1 in the preparation of drugs against methicillin-resistant Staphylococcus aureus.

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