Enrichment method and application of NDM-1 positive coliphage

Abstract

The invention discloses an enrichment method and application of NDM-1 positive coliphage, and belongs to the technical field of biological control. The method comprises the following steps: collecting a sewage mixed sample, carrying out vortex oscillation, filtration and ultrafiltration to obtain a bacteriophage concentrated solution, taking NDM-1 positive escherichia coli in the wastewater as host bacteria, and carrying out separation and purification by using a double-layer plate method to obtain the lytic bacteriophage PA2-3. The bacteriophage can efficiently and stably split host bacteria, has good acid and alkali resistance and thermal stability, and provides a technical basis for prevention, control, development and application of high-risk drug-resistant bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control in A / O process of pig farm sewage treatment, and specifically relates to an enrichment method of NDM-1 positive Escherichia coli phage and application thereof. Background Art

[0002] The long-term and unscientific use of antibiotics has led to the increasingly serious problem of bacterial resistance, which has posed a great threat to global public health security and the healthy development of animal husbandry. Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are considered to be new environmental pollutants, which have attracted widespread attention at home and abroad. Pig farm sewage and sludge are rich in ammonia nitrogen, nitrate nitrogen, phosphorus and organic matter. Existing studies have shown that pig farm sewage is a reservoir of ARGs, and the absolute abundance of ARGs is as high as 10 9 copies / mL, relative abundance is 10 -3 ~10 -1 The main component of pig farm sludge is pig manure sediment, which is also an important risk unit for resistance transmission. High-risk ARGs are discharged into the environment along with resource utilization, affecting environmental safety and even threatening human health through the food chain.

[0003] The anaerobic aerobic (A / O) wastewater treatment process in pig farms is one of the most widely used processes in pig farm wastewater treatment. It can effectively remove pollutants such as chemical oxygen demand (COD), ammonia nitrogen and phosphorus in wastewater, but it fails to effectively treat or reduce high-risk ARBs and ARGs. The pig farm wastewater A / O treatment system usually includes a sludge tank connected to the treatment system to treat and manage the generated sewage sludge. High-risk ARGs can enter the agricultural environment through the leakage or resource utilization of sewage and sludge, causing the enrichment of ARGs in the soil and water environment. The high-abundance ARGs present in pig farm wastewater, such as tetracyclines, sulfonamides and multidrug-resistant ARGs, account for more than 90% of the total ARGs, among which bla NDM-1 Escherichia coli has become a high-risk pathogen in pig farm wastewater due to its wide spectrum of drug resistance, multiple variants, and high risk of transmission. Intestinal damage, peritonitis, sepsis, and systemic infection caused by NDM-1 positive Escherichia coli have high morbidity and mortality.

[0004] Bacteriophage is a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes or spirochetes. Its sources are abundant and diverse. Among them, virulent phages specifically adsorb host bacteria and inject their DNA into the host, thereby reproducing and releasing progeny phages by lysing the host bacteria, and achieving the purpose of reduction with the death of the host bacteria. In pig farm sewage treatment, the good specificity of phages can achieve precise sterilization, while removing "super bacteria" without destroying the community structure of other functional microorganisms in the sewage treatment system. At present, phage-based biological control technologies are more in food and animal infections, and there are few studies on the prevention and control of high-risk ARBs and ARGs in sewage A / O treatment processes. Therefore, a method for enriching NDM-1-positive Escherichia coli phages and its application are needed. Summary of the invention

[0005] The purpose of the present invention is to provide an enrichment method and application of NDM-1 positive Escherichia coli phage in view of the current lack of effective prevention and control of environmental pollution and related diseases caused by high-risk pathogenic NDM-1 positive Escherichia coli in sewage A / O treatment processes. The phage has good titer, a wide range of acid and alkali resistance and high thermal stability.

[0006] The technical solutions specifically provided by the present invention are as follows:

[0007] The invention discloses an application of bacteriophage PA2-3 in lysing NDM-1 positive Escherichia coli, wherein the bacteriophage PA2-3 and NDM-1 positive Escherichia coli are mixed for lysing reaction.

[0008] Furthermore, the reaction conditions of the bacteriophage PA2-3 are a temperature of 30-50° C. and a pH of 3-12. The infection multiplicity of the bacteriophage PA2-3 and NDM-1 positive Escherichia coli is 0.1, and the lysis amount is 62.09 PFU / cell when mixed and lysed for 100-120 min.

[0009] A method for enriching NDM-1 positive Escherichia coli phage PA2-3 comprises the following steps:

[0010] (1) vortexing the sewage sample to disperse the bacteriophage in the sewage sample into a liquid phase;

[0011] (2) centrifuging the sewage sample, taking the supernatant and filtering it through a 0.22 μm sterile filter membrane to obtain a filtrate;

[0012] (3) using a 100KDa ultrafiltration tube to ultrafilter and concentrate the filtrate to obtain a phage concentrate;

[0013] (4) Using NDM-1 positive Escherichia coli as the host bacteria, the phage is separated and purified from the phage concentrate using a double-layer plate method to obtain the phage.

[0014] The main advantages of the present invention are:

[0015] The bacteriophage PA2-3 selected according to the present invention showed rapid and efficient lysis effect in the experiment of lysing NDM-1 positive Escherichia coli, and can be quickly and efficiently used for the prevention and treatment of the transmission risk and pathogenicity of high-risk NDM-1 positive Escherichia coli in the A / O process of pig farm sewage treatment wastewater and sludge; because the bacteriophage PA2-3 involved in the present invention has high lysis activity and good infection multiplicity, it can be used for the efficient prevention and control of NDM-1 positive Escherichia coli; because the bacteriophage PA2-3 involved in the present invention has a wide acid-base tolerance range and temperature stability, it can provide a new governance strategy for the further industrial production of bacteriophages and their use in blocking the spread of high-risk ARBs and ARGs in complex sewage environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A flow chart of the method for extracting and enriching bacteriophages from the A / O process of sewage treatment;

[0017] Figure 2 This is a schematic diagram of the separation and purification of NDM-1 positive Escherichia coli phage;

[0018] Figure 3 This is a schematic diagram of the electron microscopic morphology of bacteriophage PA2-3;

[0019] Figure 4 Schematic diagram of the optimal infection multiplicity of bacteriophage PA2-3;

[0020] Figure 5 Schematic diagram of the one-step growth curve of bacteriophage PA2-3;

[0021] Figure 6 Schematic diagram of the temperature stability of bacteriophage PA2-3;

[0022] Figure 7 Schematic diagram of the acid-base stability of bacteriophage PA2-3; DETAILED DESCRIPTION

[0023] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, further description is given in conjunction with embodiments. The specific implementation is only used to illustrate the present invention, and some non-essential improvements and adjustments made to the content of the present invention according to its modification also belong to the scope of protection of the present invention.

[0024] The phage host bacteria NDM-1 positive Escherichia coli used in the experiment of the present invention is separated, purified and preserved from pig farm sewage in this laboratory. The phage used is also a multivalent phage separated from pig farm sewage that can efficiently lyse the host bacteria.

[0025] Example 1 Extraction and enrichment of bacteriophages from sewage treatment A / O process

[0026] The samples used in the experiment of the present invention were collected from the A / O sewage treatment system of large-scale pig farms in Guangxi. The sewage and sludge samples in the anoxic tank (A), aerobic tank (O) and sludge tank (S) were collected in sterile plastic bottles, transported to the laboratory by cold chain, and stored in a 4°C refrigerator for isolating bacteriophages.

[0027] Add an appropriate amount of sewage and 5-6 mL of LB liquid culture medium to a 10 mL centrifuge tube, vortex and shake, let stand for 10 minutes, put it in a 37 ° C shaker for 4 hours, take it out, centrifuge it at 10000 r / min for 4 minutes, filter the supernatant with a 0.22 μm filter, and add the supernatant to a 100Kda ultrafiltration tube for ultrafiltration and concentration to obtain the initial phage liquid. The method for extracting and enriching phages from the sewage treatment A / O process is shown in the flow chart. Figure 1 .

[0028] Example 2 Isolation and purification of bacteriophage

[0029] The phage was isolated using the double-layer agar plate method. Take a 10mL centrifuge tube, add 100μL of NDM-1 positive Escherichia coli isolated from sewage and 5mL of LB liquid culture medium, mix well and prepare a double-layer plate. The plate is divided into six areas, 5μL of the initial phage solution is dripped into each area, cultured overnight, and the plate is observed for plaques. Use a blue gun tip to pick up the plaques and inject them into an EP tube containing 500μL SM buffer. Let it stand for a while, take the extract and the host bacteria to prepare a double-layer agar plate again, and purify 5-7 times until plaques of uniform size and transparency are obtained. The results show that the phage on the plate presents round plaques with neat edges and transparency, indicating that the phage is a lytic phage ( Figure 2 A) and named it PA2-3. Unpurified phages show plaques of inconsistent size and shape on the plate ( Figure 2 B); After multiple purifications, the plaques are clear, with neat and clear edges and no halo ( Figure 2 C).

[0030] Example 3 Morphological observation of bacteriophage PA2-3

[0031] After dropping 10 μL of PA2-3 concentrate on the copper mesh and waiting for 20-30 seconds, the remaining liquid was absorbed from the side with dry filter paper, and then a small amount of 2% phosphotungstic acid was added for staining. After drying for 5 minutes, it was observed under an electron microscope that PA2-3 had a long tail and a regular polyhedral head. The head plane profile was a regular hexagon with a diameter of 120 nm, a long tail of about 95 nm, and a distinct neck. Based on the morphology and structure and the classification of bacteriophages, PA2-3 can be determined to be a member of the order Caudata, family Longiphaga ( Figure 3 ).

[0032] Example 4 Titer determination of bacteriophage PA2-3

[0033] Adjust the concentration of host bacteria NDM-1 positive Escherichia coli to 10 8 CFU / mL, dilute the PA2-3 of known concentration in multiples so that the ratio of PA2-3 to NDM-1 positive Escherichia coli is 10-0.0001. Set the MOI to 10, 1, 0.1, 0.01, 0.001, and 0.0001 respectively. After incubating the mixed solutions of various proportions in a constant temperature oscillator at 37°C and 180r / min for 4h, centrifuge at 10000r / min for 5-6min, collect the supernatant and filter it with a 0.22μm filter membrane for sterilization. Take three parallel filtrates for gradient dilution, and use the double-layer plate method to measure the PA2-3 titer. The ratio with the highest PA2-3 titer is the optimal multiplicity of infection (MOI). The results showed that the PA2-3 titer was higher when the multiplicity of infection was 0.1, with a titer of 4.36×10 8 pfu / ml Figure 4 )

[0034] Example 5 One-step growth curve determination of bacteriophage PA2-3

[0035] NDM-1 positive Escherichia coli culture (10 8 CFU / mL) and PA2-3 lysate (10 6 PFU / mL) were mixed according to the optimal infection multiplicity ratio, and samples were taken every 10 minutes. After the phages were infected and adsorbed bacterial cells, they were centrifuged at 11000r / min for 6 minutes. The precipitate was resuspended with LB liquid, and the resuspension operation was repeated 2-3 times. The cells were cultured at 37°C, and samples were taken every 2 minutes for the first 10 minutes. After that, samples were taken every 10 minutes until 2 hours. The samples were centrifuged at 11000r / min for 6 minutes, filtered with a 0.22μm filter, and the PA2-3 titer was determined by the double-layer plate method. The PA2-3 characteristic curve was drawn with the infection time as the horizontal axis and the PA2-3 titer as the vertical axis. The results showed that the PA2-3 incubation period was 8 minutes, and then the titer increased steadily. The lysis period lasted for 92 minutes from 8 to 100 minutes, but the lysis effect was not obvious for 30 to 60 minutes in the middle. The phage concentration was at one level, and the plateau period lasted for 100 to 120 minutes. The lysis amount was about 62.09PFU / cell ( Figure 5 )

[0036] Example 6 Determination of temperature stability of bacteriophage PA2-3

[0037] The phage PA2-3 lysate (500μL / tube) was placed in a water bath at 30℃, 40℃, 50℃, and 60℃, and 100μL of PA2-3 solution was taken at 20min, 40min, and 60min, and the PA2-3 titer was measured by the double-layer plate method, and repeated three times. The results showed that PA2-3 had a certain temperature stability and could still maintain a high activity after being exposed to a 50℃ water bath for 1h, but the titer began to decrease after being exposed to 60℃ for 20min. As time went on, the PA2-3 titer dropped sharply. The results showed that PA2-3 had the best stability when the temperature was 30-50℃ ( Figure 6 ).

[0038] Example 7 Determination of acid-base stability of bacteriophage PA2-3

[0039] The pH value of LB liquid culture medium was adjusted to 1-13 using 10% HCl (1mol / L) and NaOH (1mol / L) solutions, and 100μL of PA2-3 was added to 900μL of LB culture medium with different pH values, mixed evenly, and cultured at 37℃ for 1h. The titer of PA2-3 under different pH was determined by double-layer plate method, and the experiment was repeated three times. The results showed that the titer of PA2-3 was 0 at pH 1, 2, and 13, and at pH 3-12, PA2-3 maintained a high titer, good stability, and a wide range of acid and alkali resistance ( Figure 7 ).

[0040] The above is a description of the relevant contents of the present invention, and those skilled in the art will be able to implement the present invention based on these descriptions. Based on the above contents of the present invention, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

Claims

1. An application of bacteriophage PA2-3 in lysing NDM-1 positive Escherichia coli, characterized in that: The bacteriophage PA2-3 and NDM-1 positive Escherichia coli were mixed and lysed.

2. The use of bacteriophage PA2-3 in lysing NDM-1 positive Escherichia coli according to claim 1, characterized in that: The reaction conditions of the bacteriophage PA2-3 are as follows: temperature of 30-50° C., pH 3-12, the infection multiplicity of the bacteriophage PA2-3 and NDM-1 positive Escherichia coli is 0.1, and the lysis amount is 62.09 PFU / cell when mixed and lysed for 100-120 min.

3. A method for enriching NDM-1 positive Escherichia coli phage PA2-3, characterized in that: The following steps are involved: (1) vortexing the sewage sample to disperse the bacteriophage in the sewage sample into a liquid phase; (2) centrifuging the sewage sample, taking the supernatant and filtering it through a 0.22 μm sterile filter membrane to obtain a filtrate; (3) using a 100KDa ultrafiltration tube to ultrafilter and concentrate the filtrate to obtain a phage concentrate; (4) Using NDM-1 positive Escherichia coli as the host bacteria, the phage is separated and purified from the phage concentrate using a double-layer plate method to obtain the phage.

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