Application of nano carbon material in reduction of antibiotic resistance gene transmission in phage-host system and method of nano carbon material in reduction of antibiotic resistance gene transmission in phage-host system

Abstract

The invention provides an application and a method of a nano carbon material in reduction of antibiotic resistance gene transmission in a bacteriophage-host system. The particle size of the nano carbon material is less than 1 mu m; the nanocarbon material can regulate and control the lifestyle of the bacteriophage in a host to be converted from a lyoprototype to a lysis type. The invention discloses a method for reducing antibiotic resistance gene transmission in a phage-earthworm intestinal system. The method comprises the following steps: providing a nano carbon material with the particle size of less than 1 mu m; and adding the nano carbon material and the earthworms into air-dried livestock and poultry manure for composting, so that the lifestyle of the bacteriophage in the intestinal tract of the earthworms is changed from a lyotype to a splitting type. The polypeptide can act on the lifestyle of the bacteriophage, has higher targeting property and more efficient ARGs blocking effect, and can realize resistance risk blocking on the micro-ecological level.

Description

Technical Field

[0001] This application relates to the field of organic solid waste resource utilization and environmental risk control technology, and in particular to the application and method of a nano-carbon material in reducing the spread of antibiotic resistance genes in a bacteriophage-host system. Background Technology

[0002] In recent years, antibiotic resistance genes (ARGs) and their carriers, resistant bacteria (ARBs), have proliferated widely in the environment, posing a significant challenge to global public health and ecological security. Bacteriophages have become important "invisible carriers" of resistance transmission, creating new pathways for resistance transmission and contamination in the environment.

[0003] Reducing the spread of antibiotic resistance genes is of great significance. Summary of the Invention

[0004] In view of this, the purpose of this application is to propose an application and method of nanocarbon materials in reducing the spread of antibiotic resistance genes in phage-host systems.

[0005] Based on the above objectives, this application provides the application of nanocarbon materials in reducing the spread of antibiotic resistance genes in phage-host systems; the nanocarbon materials have a particle size of less than 1 μm; the nanocarbon materials can regulate the lifestyle of phages in the host from lysate to lytic.

[0006] In some embodiments, the nanocarbon material is selected from at least one of nanobiocarbon, nanoactivated carbon, and graphene oxide.

[0007] In some embodiments, the nanocarbon material is selected from nanobiocarbon.

[0008] In some embodiments, the nano-biocarbon is selected from plant-derived nano-biocarbon.

[0009] In some embodiments, the plant-derived nano-biocarbon is prepared by pyrolysis of agricultural waste.

[0010] In some embodiments, the plant-derived nano-biochar has a particle size of 60-300 nm.

[0011] In some embodiments, the phage-host system is selected from a phage-earthworm gut system or a phage-anaerobic system in a wastewater treatment system.

[0012] This application also provides a method for reducing the spread of antibiotic resistance genes in the bacteriophage-earthworm gut system, including:

[0013] Provides nano-carbon materials with a particle size of less than 1 μm;

[0014] The nano-carbon material and earthworms were added to air-dried livestock and poultry manure for composting, so that the lifestyle of bacteriophages in the earthworm intestine changed from lysate to lysis.

[0015] In some embodiments, the nanocarbon material is plant-derived bio-carbon nanomaterial; the nanocarbon material providing a particle size of less than 1 μm includes:

[0016] Provide agricultural waste;

[0017] The agricultural waste is subjected to pyrolysis, pulverization and first sieving at 445-455℃ to obtain millimeter-sized biochar.

[0018] The millimeter-sized biochar is subjected to multiple grinding processes to obtain nano-carbon materials with a particle size of less than 1 μm; wherein the duration of each grinding process is the first duration, and there is an interval of the first duration after each grinding process.

[0019] In some embodiments, the earthworm is Eisenia fetida, Cyclocarya William, or Taiping No. 1 earthworm.

[0020] This application provides a novel method for reducing the spread of antibiotic resistance genes. By using nano-biocarbon materials to act on the lifestyle of bacteriophages, it inhibits the transduction potential of lysed ARGs while enhancing the ability of lysed bacteriophages to clear resistant bacteria, effectively reducing the risk of persistent ARG spread in the environment. It exhibits higher targeting and more efficient ARG blocking effects, achieving resistance risk blocking at the microecological level. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in this application or related technologies, the drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Morphological characteristics of biochar samples with different particle sizes;

[0023] Figure 2 A schematic diagram showing the changes in ARGs in each treatment;

[0024] Figure 3a This is a schematic diagram showing the changes in the relative abundance of HBP in each treatment;

[0025] Figure 3bA schematic diagram showing the relative abundance changes of phage lifestyles in each treatment;

[0026] Figure 4a This is a schematic diagram illustrating the interaction between bacteriophages and the host in a compost substrate sample.

[0027] Figure 4b This is a schematic diagram illustrating the interaction between bacteriophages and the host in an earthworm gut sample.

[0028] Figure 5 A schematic diagram illustrating the effect of adding nano-biochar on the relative abundance of bacteriophage hosts in the gut of earthworms.

[0029] Figure 6 This is a schematic diagram illustrating the effect of lysogenic and lytic phages on the relative abundance of ARGs.

[0030] Figure 7 This is a schematic diagram illustrating the effect of nano-biochar on bacteriophage titer and bactericidal rate. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings.

[0032] It should be noted that, unless otherwise defined, the technical or scientific terms used in the embodiments of this application shall have the ordinary meaning as understood by a person skilled in the art to which this application pertains.

[0033] Antimicrobial resistance genes (ARGs) are genetic materials that confer antibiotic resistance to microorganisms. These genes encode specific proteins that help microorganisms resist external chemicals, antibiotics, or other antimicrobial agents. These genes can be located on chromosomes, but are more commonly found on mobile genetic elements such as plasmids, transposons, and integrons, enabling resistance to spread rapidly horizontally between different strains and even species. The spread of ARGs not only affects public health but also has a significant impact on the microbial community in the environment, increasing the risk of antibiotic resistance and multidrug resistance. ARGs spread among microorganisms through multiple pathways, with horizontal gene transfer (HGT) being the core mechanism leading to their rapid spread.

[0034] Bacteriophages are a class of viruses that can infect and parasitize bacteria. They bind to specific receptors on the surface of bacteria, inject their genetic material, and then utilize the host cell's metabolic mechanisms to replicate and multiply. Bacteriophages are classified according to their lifestyle into lytic phages, which rapidly replicate and lyse host cells after infecting bacteria, causing cell death and releasing new phages; and lysogenic phages, which integrate their own DNA into the host genome, forming prophages that replicate alongside the host and can initiate a lysis cycle under appropriate conditions. As the most abundant biological entity on Earth, bacteriophages play a central role in regulating bacterial populations, driving community succession, and promoting horizontal gene transfer between bacteria (such as the spread of virulence factors and resistance genes). Their infection activity is regulated by host abundance and environmental factors, and they are also important tools for combating drug-resistant bacterial infections (phage therapy) and for molecular biology research. Phage-mediated transduction mechanisms, as an important process in hematologic tract development (HGT), have received increasing attention in recent years.

[0035] With the development of molecular biology and high-throughput sequencing technologies, environmental virology research has advanced rapidly. Related studies have found that bacteriophages are widely present in various environments, including wastewater treatment systems, kitchen waste, livestock manure, and airborne particulate matter, and carry multiple types of resistance genes (such as β-lactams, macrolides, and tetracyclines). This makes bacteriophages an important "hidden carrier" of resistance transmission, creating new pathways for resistance transmission and contamination in the environment.

[0036] After infecting bacteria, bacteriophages can choose between a lytic or lysosomal lifestyle, and these different infection strategies significantly influence the transmission pathways of ARGs (antibiotic resistant bacteria). When bacteriophages adopt a lytic lifestyle, they help control ARG numbers by lysing drug-resistant bacterial cells, thus alleviating the pressure of resistance transmission to some extent. Conversely, when bacteriophages adopt a lysosomal lifestyle, they can integrate ARGs into the host chromosome, enabling stable transmission and cross-population diffusion. Bacteriophages exhibit specific lifestyle preferences in different environmental niches; for example, they are predominantly lytic in dryland soils, while they are predominantly lysosomal in rice paddies and infant intestines. Therefore, the living environment plays a crucial regulatory role in their ecological behavior.

[0037] Vermicomposting is a biotechnology that utilizes specific earthworm species (such as Eisenia fetida) and their symbiotic microbial communities (combining native microorganisms and earthworms as key exogenous microbial processors) to co-process organic waste (kitchen waste, straw, livestock manure, etc.) under controlled conditions. Earthworms ingest and break down materials; enzymes and mucus secreted by their intestines, along with digestive microorganisms, initiate decomposition. The microbial community enriched in their excreted vermicomposts continues to undergo deep humification, ultimately efficiently transforming the waste into a high-quality organic fertilizer / soil conditioner (i.e., vermicompost) with a loose physical structure, rich in stable humus, balanced nutrients, and containing beneficial microorganisms and plant hormones. This process enables the resource utilization of organic waste and reduces environmental pollution. Its efficiency relies on the significant improvement of the microbial physicochemical environment (aeration, humidity, specific surface area) by earthworm activity.

[0038] Vermicomposting, as an important method for the resource utilization of livestock and poultry manure, is widely used in agriculture and ecological engineering. However, livestock and poultry manure is rich in antibiotics and their resistance genes, making vermicomposting a potential platform for resistance transmission. As an important organic waste treatment and resource utilization technology, research on ARGs transmission in vermicomposting systems mainly focuses on the interbacterial conjugation and transfer mechanisms. Related studies have found that in the vermicomposting gut or vermicomposting, due to abundant nutrients, dense microorganisms, and active conjugation transfer elements (such as plasmids), rapid transfer of ARGs between bacteria can be promoted. Control strategies mainly include: restricting conjugation conditions, adding metal ions, or blocking vector DNA activity. However, such studies have neglected the possible transduction role of bacteriophages in vermicomposting systems and have not paid attention to the microecological effects caused by bacteriophage-host interactions.

[0039] Based on this, this application provides a method for regulating phage-host interactions using nano-biochar. By controlling the conversion of phage lysis / lysate lifespan, it influences the phage's ability to transduce ARGs, thereby achieving resistance risk blocking at the microecological level. A phage-host network in a dual-niche environment (earthworm gut and vermicompost) is constructed to accurately identify the main ARG propagation pathway. High-confidence phage-host matching is obtained through the integration of multiple prediction methods, and key ARG propagation nodes in the composting system are identified by combining community structure differences. The method also verifies the material's ability to regulate phage lysis through an in vitro phage isolation simulation verification system combined with qPCR and plaque observation, establishing a complete experimental chain from microscopic infection mechanisms to community-level resistance load verification, forming a multi-scale closed-loop verification system. This method can actively intervene in the environmental propagation pathways of resistance genes, thereby reducing the propagation of antibiotic resistance genes (ARGs) in earthworm composting systems, and has a more efficient ARG blocking effect.

[0040] This application provides an application of nano-carbon materials in reducing the spread of antibiotic resistance genes (ARGs) in phage-host systems. The nano-carbon materials have a particle size of less than 1 μm. These materials possess a large specific surface area and high surface activity. The principle behind using nano-carbon materials to reduce the spread of antibiotic resistance genes in phage-host systems is to regulate the lifestyle of bacteriophages in the host, shifting them from a lysate to a lytic state. This application utilizes nano-biochar materials to influence the lifestyle of bacteriophages, inhibiting the transduction potential of lysate ARGs while simultaneously enhancing the ability of lytic bacteriophages to clear resistant bacteria, effectively reducing the risk of persistent ARG spread in the environment.

[0041] In some embodiments, the nanocarbon material may be selected from at least one of nano-biochar, nano-activated carbon, and graphene oxide. Nano-biochar materials possess mechanisms such as electrostatic adsorption, hydrogen bonding, and π-π interactions. The hydroxyl radicals generated on its surface also have a certain oxidative destructive ability. It can efficiently adsorb free ARGs DNA molecules in the environmental medium through mechanisms such as electrostatic adsorption, hydrogen bonding, and π-π interactions, thereby limiting their migration and diffusion; simultaneously, it can cause ARGs DNA strand breaks and structural damage through the hydroxyl radicals generated on the surface of nano-biochar, achieving the degradation and inactivation of ARGs. It can disperse in environments containing a large amount of free DNA or extracellular resistance factors, such as sewage and soil leachate, binding to resistance gene fragments or destroying their molecular structure, reducing their environmental mobility and functional activity. In the phage-host network, nano-biochar intervenes in the phage lifestyle, precisely intervening in the transition of the phage lifestyle from lysate to lytic, thereby intervening in the replication and transduction of ARGs within the microbial host. Both nano-activated carbon and graphene oxide have adsorption properties and biocompatibility, and can also play a role in intervening in the lifestyle of bacteriophages and the microecological effects caused by bacteriophage-host interactions.

[0042] In some embodiments, the nanocarbon material can be selected from nano-biochar. Conventional-sized biochar indirectly inhibits the spread of resistance genes by intervening in the microecological structure and weakening the ARGs propagation pathway. Its inhibition of elements related to horizontal gene transfer (such as integrons and transposases) only indirectly inhibits the environmental diffusion ability of ARGs. However, this application intervenes in the microecological effects caused by phage-host interactions through nano-biochar, precisely intervening in the (lysis / lysogen) transition of phage lifestyle, regulating the phage's lifestyle in the host from lysogenic to lytic, intervening in the transduction process of ARGs within the microbial host, and actively intervening in the environmental propagation pathway of resistance genes, thereby controlling the risk of phage transduction of ARGs at the microecological level. This results in stronger targeting and a more efficient ARGs blocking effect.

[0043] In some embodiments, the nano-biochar can be plant-derived nano-biochar. The plant-derived nano-biochar can be derived from agricultural waste. Agricultural waste sources can include sugarcane bagasse, corn stalks, rice husks, or coconut shells, etc. The plant-derived nano-biochar can be prepared by pyrolysis of agricultural waste. Specifically, the plant-derived nano-biochar can be prepared by pyrolysis of sugarcane bagasse, and its particle size can be 60-300 nm.

[0044] In some embodiments, the phage-host system may originate from phage-host systems in urban sewage sludge treatment systems, livestock and poultry manure high-temperature composting systems, and kitchen waste composting systems. For example, it may include phage-anaerobic bacteria systems in sewage treatment systems and phage-earthworm gut systems in livestock and poultry manure high-temperature composting systems.

[0045] Based on the same inventive concept, this application also provides a method for reducing the spread of antibiotic resistance genes (ARGs) in a bacteriophage-earthworm gut system. This method, by introducing nano-carbon materials into the earthworm composting system, can precisely regulate the lifestyle of bacteriophages in the earthworm gut, causing the bacteriophages to transform from a lysate form to a lytic form, thereby reducing the spread of antibiotic resistance genes (ARGs) in the earthworm composting system.

[0046] In some embodiments, methods for reducing the spread of antibiotic resistance genes in the bacteriophage-earthworm gut system may include:

[0047] We provide nano-carbon materials with a particle size of less than 1 μm.

[0048] The nano-carbon material and earthworms were added to air-dried livestock and poultry manure for composting, so that the lifestyle of bacteriophages in the earthworm intestine changed from lysate to lysis.

[0049] In some embodiments, the nanocarbon material may be selected from at least one of nanobiochar, nanoactivated carbon, and graphene oxide.

[0050] In some embodiments, the nanocarbon material is selected from nanobiochar.

[0051] In some embodiments, the nanocarbon material is plant-derived nanobiochar.

[0052] In some embodiments, the plant-derived biochar nanoparticles are prepared by pyrolysis of agricultural waste. The nanocarbon material providing a particle size of less than 1 μm may include:

[0053] Provide agricultural waste; sources of agricultural waste may include sugarcane bagasse, corn stalks, rice husks or coconut shells, etc.

[0054] The agricultural waste is subjected to pyrolysis, pulverization, and a first sieve treatment at 445-455℃ to obtain millimeter-sized biochar (i.e., biochar with conventional particle size). Typically, the particle size of millimeter-sized biochar can be 0.15-4 mm.

[0055] The millimeter-sized biochar was subjected to multiple grinding processes to obtain nano-carbon materials with a particle size of less than 1 μm. Each grinding process lasted for a first duration, followed by a first-duration interval. This grinding-rest interval mechanism prevents the biochar from agglomerating due to temperature rise, resulting in nano-carbon materials with a particle size of less than 1 μm. This improves the biological efficacy of the biochar material and is a crucial prerequisite for ensuring the reproducibility of the regulatory effects of plant-derived nano-biochar in controlling the lifestyle of bacteriophages within the host.

[0056] In some embodiments, the particle size of the plant-derived nanobiochar can be 60-300 nm.

[0057] In some embodiments, the mass ratio of the nano-carbon material to air-dried livestock manure can be (1-10):100, for example, 1:100, 1:20, or 1:10. The moisture content of the pile can be around 75%. The temperature of the pile can be around 30°C. In the pile, the mass concentration of the mixture of nano-carbon material and air-dried livestock manure can be 245 g / L; the concentration of earthworms can be 50-150 worms / kg, for example, 50 worms / kg, 100 worms / kg, or 150 worms / kg.

[0058] In some embodiments, the earthworm is Eisenia fetida, Cyclocarya William, or Taiping No. 1 earthworm.

[0059] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0060] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0061] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0062] Example 1: Method for reducing the spread of antibiotic resistance genes (ARGs) in vermicomposting systems

[0063] Step 1. Preparation of biochar with different particle sizes

[0064] Preparation method: (1) Weigh 1.5 kg of dried sugarcane bagasse as raw material and pyrolyze it in a tube furnace under anaerobic conditions at 450 °C for 2 hours. After cooling, the pyrolyzed product is crushed and passed through a 10-mesh sieve to obtain about 1.2 kg of biochar (B) with conventional particle size. Subsequently, the biochar is washed with deionized water to remove impurities and dried at 80 °C for later use.

[0065] (2) Conventional biochar was washed with deionized water and dried at 80°C. Part of it was mechanically ground and passed through a 100-mesh sieve to obtain micron-sized biochar (MC).

[0066] (3) In addition, 0.4 kg of conventional biochar was frozen at -80℃ for 24 hours, and then ball-milled for 18 minutes at 1800 r / min using a vibratory ball mill. After each ball milling for 3 minutes, the mixture was rested for 3 minutes to prevent agglomeration caused by temperature rise. Finally, nanoscale biochar (NC) with a particle size of <1 μm was obtained.

[0067] Detection methods: The prepared biochar samples with different particle sizes were characterized and analyzed by SEM (Zeiss Sigma 300, Germany) and TEM (JEOL JEM-F200, Japan) to ensure clear particle size classification.

[0068] Step 2. Building an Earthworm Composting System

[0069] (1) Mix 1.5 kg of air-dried cow dung with 45 g of biochar of different particle sizes and adjust the moisture content to about 75%.

[0070] (2) Pack 2.45 kg / barrel into 10L plastic buckets to construct the following 12 earthworm composting treatment groups, and set up 4 earthworm gut treatment groups. Each group has four replicates.

[0071] The earthworm composting treatment group includes: C (cow manure), CB (cow manure + ordinary biochar), CMB (cow manure + micronized biochar), CNB (cow manure + nano-biochar), CV (cow manure + earthworm), CBV (cow manure + earthworm + ordinary biochar), CMBV (cow manure + earthworm + micronized biochar), and CNBV (cow manure + earthworm + nano-biochar); the earthworm gut treatment group includes: CG (earthworms from CV treatment), CBG (earthworms from CBV treatment), CMBG (earthworms from CMBV treatment), and CNBG (earthworms from CNBV treatment).

[0072] (3) Add 150 mature Eisenia fetida to the earthworm treatment. The composting process lasts for 60 days, with the compost temperature controlled at about 30°C and the moisture content maintained at 75%.

[0073] Step 3. Multi-omics analysis of the phage-host system

[0074] (1) After composting, vermicompost and earthworm intestinal contents were collected. Total DNA was extracted and subjected to 16S amplicon sequencing, metagenomic and virological sequencing. The primers for 16S amplicon bacteria are shown in Table 1, including sequences with sequence numbers SEQ ID NO.1 and SEQ ID NO.2.

[0075] (2) After metagenomic assembly, quality control and species annotation, high-quality MAGs were constructed using MetaWRAP and their abundance (RPKM) was calculated.

[0076] (3) Predict phage hosts by combining three methods: CRISPR matching, tRNA homology, and whole genome alignment.

[0077] (4) Use DeepPhage to determine the phage lifestyle (lysate / lytic).

[0078] (5) Analyze the phage community structure and its regulatory effect on the ARGs host transmission pathway in earthworm gut and vermicompost samples, respectively.

[0079] After collecting vermicompost and earthworm intestinal contents, the samples were handed over to Meiji Biotechnology Co., Ltd. for subsequent experiments.

[0080] Table 1 Primer information for 16S amplicon bacteria

[0081]

[0082] Step 4. Verification experiment on the ability of nanocarbon to regulate bacteriophage infection.

[0083] (1) Isolation and concentration of bacteriophages: First, bacteriophages were extracted from the intestinal contents of earthworms. 1 g of intestinal contents was added to 100 mL of M9 medium, shaken at 180 rpm for 1 h, and then centrifuged at 8000 rpm for 5 min. The supernatant was filtered through a 2 μm filter membrane to remove large particulate impurities. Subsequently, a tangential flow filtration system (TFF; Sartorius Vivaflow 200, Germany) was used for secondary filtration and concentration, sequentially using filter membranes with a pore size of 0.22 μm and a molecular weight cutoff of 100 kDa. The fraction <100 kDa was extracellular phage suspension, and the fraction >0.22 μm particles was a suspension containing bacteria. The latter was incubated in the dark for 24 h with Mitomycin C (final concentration 1 μg / mL) to induce lysogenic phage activation, and then filtered through a 0.22 μm sterile filter membrane to obtain the lysogenic phage extract. The entire extraction process was completed in a clean bench, and all instruments were sterilized by autoclaving or disinfected with 70% ethanol before use.

[0084] (2) Co-culture experimental setup and qPCR detection: 0.75g of earthworm compost was added to 75mL of sterile M9 medium (150mL sterile Erlenmeyer flask), mixed well, and incubated at 180rpm for 2h as the microbial inoculum. Subsequently, 2mL of phage extract was mixed with 1mL of the microbial inoculum and placed in 10mL sterile centrifuge tubes, and incubated at 25℃ in the dark for 3 days. A total of 5 treatment groups were set up, with 5 replicates in each group. The treatment group setup is as follows:

[0085] (i) Group C: Only M9 medium and microbial inoculum were added (no bacteriophages were added).

[0086] (ii) Group P: Add extracellular phage (i.e., lysed phage) extract and microbial inoculum.

[0087] (iii) PI group: Add extracellular phage (i.e. lysed phage) extract and microbial inoculum after being inactivated by a 121°C water bath for 30 min.

[0088] (iv) Group L: Added lysed phage extract and microbial inoculum.

[0089] (v) LI group: Added inactivated lysate phage extract and microbial inoculum.

[0090] After culture, total DNA was extracted using the Tiangen Bacterial Genome Extraction Kit (DP302, Tiangen Biotech, Beijing). The abundance of ARGs was detected by quantitative PCR (qPCR). The reaction volume was 20 μL, containing 10 μL SYBR Premix ExTaq (Takara), 0.4 μL forward / reverse primers (10 μM), 1 μL template DNA, and 8.2 μL sterile water. Amplification was performed using an ABI 7500 Real-Time PCR System (Applied Biosystems). The amplification conditions were: 95℃ pre-denaturation for 30 s, followed by 40 cycles (95℃ for 5 s, 60℃ for 34 s). All samples were tested in triplicate. Primer information is shown in Table 2.

[0091] (3) Effect of Nano-Biochar on the Infectivity of Lytic Phages: A double-layer agar plate method was further constructed to verify the effect of nano-biochar on the activity of lystic phages. 0.1 g of nano-biochar (NB) inactivated at 121℃ was added to a 12 mL sterile test tube (NB treatment group), along with 2 mL of laboratory-purified lystic phage solution and 2 mL of Escherichia coli DH5α bacterial solution in the logarithmic growth phase (concentration approximately 10). 8Incubate at 180 rpm with shaking for 20-24 h (CFU / mL). Then centrifuge at 6000 rpm for 10 min, and collect the supernatant after filtering twice through a 0.22 μm filter membrane to collect any lysed phages that may be retained.

[0092] The control group (CK) received no nano-biochar, but all other conditions were the same. The resulting phage fluids were subjected to 10... 3 Up to 10 6 Serial dilutions were performed. For each group, 100 μL of the diluted solution was mixed with 100 μL of logarithmic growth phase DH5α, and then 4.5 mL of semi-solid LB medium (containing 0.7% agar) at 50–55 °C was added. The mixture was quickly shaken and poured onto LB solid plates to form the upper agar layer. The plates were incubated at 37 °C for 8–12 h, and lysis characteristics were observed. The appearance of plaques indicated phage lysis activity.

[0093] Table 2 PCR primer information

[0094]

[0095]

[0096]

[0097]

[0098] Test results: The morphological characteristics of biochar samples with different particle sizes obtained are as follows: Figure 1 As shown. Among them, Figure 1 Part (A) is a scanning electron microscope (SEM) image of ordinary biochar (B). Figure 1 Part (B) is a scanning electron microscope (SEM) image of micron-sized biochar (MC). Figure 1 Part (C) shows a scanning electron microscope (SEM) image of nano-biochar (NC). Red size markers indicate representative pore sizes. Figure 1 Parts (D), (E), (F), and (G) are transmission electron microscopy (TEM) images of the nano-biochar. The yellow size markers indicate particle size measurements.

[0099] The results of a multi-omics analysis of the phage-host system of vermicompost and earthworm gut contents are as follows: Figures 2 to 5 As shown in the figure. The changes in ARGs in each treatment are as follows: Figure 2 As shown. Figure 2 Part (A) in the figure represents the relative abundance of ARGs categories in each treatment. Figure 2Part (B) in the table shows the 40 most important ARGs in each treatment, which are the results after Z-score normalization of the relative abundance data. Figure 2 Part (C) in the table represents the total relative abundance of high-risk ARGs in each treatment. The changes in the relative abundance of HBP in each treatment group are shown below. Figure 3a As shown in the figure. The relative abundance changes of phage lifestyles in each treatment group are as follows. Figure 3b As shown. Analysis of phage-host interactions in compost substrate samples is as follows. Figure 4a As shown in the figure. From left to right, the four color groups represent the percentage of phages with or without a host, treatment, the phylum-level classification of host MAGs, the family-level classification of phages, and the lifestyle of phages, respectively. Analysis of phage-host interactions in earthworm gut samples is shown below. Figure 4b As shown in the figure. From left to right, the four groups of colors represent the percentage of phages with or without a host, treatment, the phylum-level classification of host MAGs, the family-level classification of phages, and the lifestyle of phages, respectively. The effect of adding nano-biochar on the relative abundance of phage hosts in the earthworm gut is shown in the figure. Figure 5 As shown.

[0100] The results of the verification experiment on the ability of nano-carbon to regulate bacteriophage infection are as follows: Figure 6 and Figure 7 As shown. Among them, Figure 6 The effects of lysogenic and lytic phages on the relative abundance of ARGs were investigated. Figure 6 C in the figure represents the effect of adding only the microbial inoculum; Figure 6 P in the figure represents the effect of adding lysozyme phage extract and microbial inoculum; Figure 6 The PI in the figure represents the effect of adding inactivated lysozyme phage extract and microbial inoculum; Figure 6 The L in the text indicates the addition of lysed phage extract and microbial inoculum; Figure 6 In the figure, LI represents the effect of adding inactivated lysogenic phage extract and microbial inoculum. Figure 7 The effect of nano-biochar on bacteriophage titer and bactericidal rate. Figure 7 The control group (CK) in the figure represents the effect of adding lysozyme and Escherichia coli DH5α, but without nano-biochar. Figure 7 NB in ​​the figure represents the effect of adding lysing phages and Escherichia coli DH5α to the treatment group containing nano-biochar. Infectivity was assessed by plaque formation on LB agar plates.

[0101] Results Analysis: Figure 1 It can be seen that compared with ordinary biochar and micron-sized biochar, nano-biochar has a smaller particle size, with a particle size of 60-300nm, and a more compact morphology.

[0102] Depend on Figure 2 It can be seen that, compared with the treatment without biochar (i.e., C, CV, and CG), the total abundance of high-risk ARGs (such as bacicaracin_bacA, sulfonamide_sul2, multidrug_msbA, and florfenicol_floR) decreased by 25.3%, 15.1%, and 27.8%, respectively, after treatment with nano-biochar (i.e., CNB, CNBV, and CNBG). Figure 3a It was found that earthworm intestines accumulated more human pathogenic bacteria (HBPs). Compared with CG treatment, the relative abundance of pathogenic Proteobacteria was significantly reduced (P<0.05), while the relative abundance of Firmicutes and Actinobacteria increased (P<0.05). Compared with CNBG treatment, the decrease in the relative abundance of pathogenic Proteobacteria was more significant (P<0.05), while the increase in the relative abundance of Firmicutes and Actinobacteria was more significant (P<0.05). Figure 3b It was found that in earthworm castings treatment (C, CB, CMB, CNB, CV, CBV, CMBV, and CNBV), the relative abundance of lysogenic phages accounted for 82.3%-89.1% of the total phage community. Conversely, the proportion of lysogenic phages (51.8%-62.9%) in the earthworm gut (CG, CBG, CMBG, and CNBG) was higher than that of lysogenic phages. Furthermore, the addition of nano-biochar significantly increased the proportion of lysogenic phages in the earthworm gut (P<0.05). Figure 4a and Figure 4b It can be seen that, compared to the treatment with earthworm castings (248 phage-host pairs), there are more phage-host interactions in the earthworm gut (2578 phage-host pairs). Among them, Figure 4a This indicates that *Bacteroidota*, *Proteobacteria*, and *Chloroflexota* are the three major hosts of lysing bacteriophages in earthworm castings. Figure 4b The results showed that, compared to earthworm castings, the main bacteriophage hosts in the earthworm gut included Actinobacteria, Proteobacteria, and Firmicutes, with lysogenic phages accounting for a higher proportion than lytic phages. Figure 5It was found that, compared with CG treatment, the abundance of host-associated lysative phages increased in CNBG treatment (p<0.05), especially Proteobacteriaphage. Conversely, compared with CG treatment, the abundance of lysogenic phages parasitized by actinomycetes (i.e., actinomycetophages) decreased significantly (p<0.05) in CNBG treatment, specifically by 76.3%. Figure 6 and Figure 7 Further laboratory validation experiments confirmed the effect of nano-biochar on the regulation of bacteriophages' influence on ARGs propagation. Among these, [the following is unclear due to incomplete sentence fragments]. Figure 6 It was found that, compared with the control group, the treatment with added lysogenic phage (L treatment) significantly increased the levels of multidrug, transposase, tetracycline, and plasmid resistance genes (P<0.05). Furthermore, the total abundance of ARGs in the L treatment was 2.1 times that in the treatment with added lysogenic phage (P treatment). Figure 7 The results showed that nano-biochar treatment (NB) enhanced lysis and infectivity, with the highest observed phage titer reaching 3.53E+07 pfu / mL, representing a 57.5% increase in bactericidal rate compared to the control group. In conclusion, nano-biochar treatment significantly reduced the abundance and transduction capacity of ARGs in the earthworm gut, demonstrating promising potential for widespread application. The laboratory simulation system can verify the phage regulatory mechanism and the effects of nanomaterials, providing a quantifiable pathway for subsequent engineering applications.

[0103] The above experimental results show that the addition of nano-biochar can significantly increase the proportion of lysing phages in the earthworm gut and significantly reduce the abundance and transduction capacity of ARGs in the earthworm gut. Therefore, nano-carbon materials can regulate the lifestyle of bacteriophages in the host from lysate to lytic.

[0104] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of this disclosure (including the claims) is limited to these examples; within the framework of this disclosure, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of the embodiments of this disclosure as described above, which are not provided in detail for the sake of brevity.

[0105] Although this disclosure has been described in conjunction with specific embodiments thereof, many substitutions, modifications and variations of these embodiments will be apparent to those skilled in the art from the foregoing description.

[0106] This disclosure is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure.

Claims

1. Application of nanocarbon materials in reducing the spread of antibiotic resistance genes in phage-host systems; wherein the particle size of the nanocarbon materials is less than 1 μm; wherein the nanocarbon materials can regulate the lifestyle of phages in the host from lysate to lytic.

2. The application according to claim 1, characterized in that, The nano-carbon material is selected from at least one of nano-biochar, nano-activated carbon, and graphene oxide.

3. The application according to claim 1, characterized in that, The nanocarbon material is selected from nano-biochar.

4. The application according to claim 3, characterized in that, The nanobiochar is selected from plant-derived nanobiochar.

5. The application according to claim 4, characterized in that, The plant-derived nano-biochar is prepared by pyrolysis of agricultural waste.

6. The application according to claim 5, characterized in that, The plant-derived nano-biochar has a particle size of 60-300 nm.

7. The application according to claim 1, characterized in that, The phage-host system is selected from the phage-earthworm gut microbial system or the phage-anaerobic system in the sewage treatment system.

8. A method for reducing the spread of antibiotic resistance genes in a bacteriophage-earthworm gut system, characterized in that, include: Provides nano-carbon materials with a particle size of less than 1 μm; The nano-carbon material and earthworms were added to air-dried livestock and poultry manure for composting, so that the lifestyle of bacteriophages in the earthworm intestine changed from lysate to lysis.

9. The method for reducing the spread of antibiotic resistance genes in the bacteriophage-earthworm intestinal system according to claim 8, characterized in that, The nano-carbon material is plant-derived nano-biochar; the nano-carbon material with a particle size of less than 1 μm includes: Provide agricultural waste; The agricultural waste is subjected to pyrolysis, pulverization and first sieving at 445-455℃ to obtain millimeter-sized biochar. The millimeter-sized biochar is subjected to multiple grinding processes to obtain nano-carbon materials with a particle size of less than 1 μm; wherein the duration of each grinding process is the first duration, and there is an interval of the first duration after each grinding process.

10. The method for reducing the spread of antibiotic resistance genes in the bacteriophage-earthworm intestinal system according to claim 9, characterized in that, The earthworms mentioned are Eisenia fetida, Cyclocarya William, or Taiping No. 1 earthworm.

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