Method of controlling antibiotic resistant biofilms with bacteriophage in combination with deinococcus radiodurans
By combining bacteriophages with radiation-resistant cocci, antibiotic-resistant bacteria in antibiotic-resistant biofilms are targeted to lyse and ARGs are degraded, solving the problem of removing antibiotic-resistant biofilms in pipeline networks and achieving efficient and environmentally friendly biofilm control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-05-12
- Publication Date
- 2026-07-14
Smart Images
Figure CN122187267B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental biotechnology and water treatment, specifically relating to a method for synergistic control of antibiotic-resistant biofilms by combining bacteriophages and radiation-resistant cocci. Background Technology
[0002] In the field of reclaimed water reuse, antibiotic-resistant bacteria (ARBs) and the biofilms they form in the distribution network pose a significant safety hazard. Currently, the most common method for controlling antibiotic-resistant biofilms in reclaimed water systems is to maintain the residual chlorine level in the network, but this measure also has significant limitations. The bacteria inside the biofilm are protected by extracellular polymeric substances (EPS), exhibiting strong resistance to traditional methods such as chlorine disinfection. Furthermore, chlorine disinfection generates harmful disinfection byproducts such as trihalomethanes (THMs) and haloacetic acids (HAAs), and disinfectants may also stimulate bacteria to secrete more EPS, making the biofilm even more difficult to remove.
[0003] Bacteriophages, as viruses capable of specifically lysing bacteria, are considered potential controllers for targeting and eliminating antibiotic-resistant biofilm organisms. However, using bacteriophages alone has significant drawbacks. On the one hand, it can easily accelerate the development of bacteriophage resistance; on the other hand, during bacterial lysis, intracellular antibiotic resistance genes (ARGs) may be released into the environment, exacerbating the risk of ARG environmental transmission. Meanwhile, bacteria of the genus *Deinococcus* can effectively scavenge reactive oxygen species (ROS) by producing reducing substances such as carotenoids, thereby reducing bacterial oxidative stress levels. Furthermore, they can secrete extracellular nucleases to degrade eDNA, a key component of the biofilm skeleton, thereby disrupting biofilm structure and reducing the risk of ARG transfer. However, the stable internal structure of mature biofilms can hinder the colonization of exogenous bacteria, limiting the effectiveness of their single application.
[0004] Therefore, there is an urgent need in this field for an innovative solution that can efficiently target and remove ARBs while sustainably inhibiting excessive biofilm growth and blocking the risk of ARGs transmission. Summary of the Invention
[0005] The purpose of this invention is to solve the problems of difficulty in efficiently targeting and eliminating antibiotic-resistant bacteria in the pipeline network and blocking the risk of ARGs transmission in the prior art, and to provide a method for controlling antibiotic-resistant biofilms by using bacteriophages in combination with radiation-resistant cocci.
[0006] The specific technical solution adopted in this invention is as follows:
[0007] In a first aspect, the present invention provides a method for controlling antibiotic-resistant biofilms by combining bacteriophage with Deinococcus radiodurans. The method involves adding a lysing bacteriophage and Deinococcus radiodurans as a compound bacterial agent to the environmental water body where the antibiotic-resistant biofilm is located, targeting and lysing the antibiotic-resistant bacteria in the antibiotic-resistant biofilm and degrading the accumulated intracellular antibiotic resistance genes.
[0008] The lytic phage is a phage capable of infecting and lysing antibiotic-resistant bacteria in antibiotic-resistant biofilms;
[0009] The radiodurans radioresistant strain was deposited on February 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37738.
[0010] As a preferred embodiment of the first aspect above, the antibiotic-resistant bacteria are Escherichia coli K12 and / or Pseudomonas aeruginosa PAO1.
[0011] As a preferred embodiment of the first aspect above, the lytic phage is obtained by continuously enriching and culturing a mixed phage source in a culture medium containing the antibiotic-resistant bacteria; the mixed phage source is phage-rich sewage, sludge, soil, or a phage library.
[0012] As a preferred embodiment of the first aspect above, the antibiotic-resistant biofilm is a biofilm in a pipeline network.
[0013] As a preferred embodiment of the first aspect above, the environmental water in which the antibiotic-resistant biofilm is located is a fluid transported in a distribution network and contains residual chlorine.
[0014] As a preferred embodiment of the first aspect described above, the lytic phage is introduced into the environmental water at an optimal multiple of infection (MOI) relative to antibiotic-resistant bacteria in the antibiotic-resistant biofilm.
[0015] As a preferred embodiment of the first aspect above, the concentration of the lytic bacteriophage in the environmental water body is controlled to be 0.5~1.5 × 10⁻⁶. 10 PFU / mL.
[0016] As a preferred embodiment of the first aspect above, the concentration of the radiation-resistant Deinococcus radiodurans in the environmental water body is controlled at 0.5~1.5 × 10⁻⁶. 8 CFU / mL.
[0017] Secondly, the present invention provides a compound bacterial agent comprising a lytic bacteriophage and *Deinococcus radiodurans*; the lytic bacteriophage is a bacteriophage capable of infecting and lysing antibiotic-resistant bacteria in antibiotic-resistant biofilms; the *Deinococcus radiodurans* was deposited on February 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNO.37738.
[0018] Thirdly, the present invention provides the application of the compound microbial agent as described in the second aspect above in the biological control of antibiotic-resistant biofilms.
[0019] Compared with the prior art, the present invention has the following advantages:
[0020] This invention combines a lytic bacteriophage with *Deinococcus radiodurans* to synergistically control antibiotic-resistant biofilms, achieving efficient and coordinated treatment by removing biofilm biomass and eliminating ARBs. The lytic bacteriophage targets and disrupts the biofilm structure, lysing antibiotic-resistant bacteria. Simultaneously, the extracellular nucleases of *Deinococcus radiodurans* effectively degrade ARGs released by phage lysis, solving the core problem of increased genetic contamination risk associated with using bacteriophages alone. Furthermore, this invention intervenes in the biofilm's structure, metabolism, and population through eDNA degradation, ROS removal, and ARB lysis, providing a comprehensive mechanism of action and minimizing the risk of resistance development. Therefore, this invention can be used for the biological control of antibiotic-resistant biofilms in water distribution networks, avoiding the byproduct problems of excessive chlorine disinfection, and represents a green and sustainable technology. Attached Figure Description
[0021] Figure 1 The result is the optimal multiple of infection (MOI) test result for phage ZJUN2;
[0022] Figure 2 The characterization results are shown during the screening of three radiation-resistant cocci.
[0023] Figure 3 The results characterize the effects of compound microbial agents on biofilm biomass and the number of antibiotic-resistant bacteria.
[0024] Figure 4 The results represent the characterization of the effects of compound bacterial agents on extracellular polysaccharides, eDNA, and oxidative stress indicators.
[0025] Figure 5 The results represent the characterization of the effect of compound bacterial agents on biofilm thickness.
[0026] Figure 6 The results represent the influence of the compound bacterial agent on the surface morphology and roughness of the biofilm.
[0027] Biological Preservation
[0028] Deinococcus radiodurans was deposited on February 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC), No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC NO. 37738. Detailed Implementation
[0029] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the present invention can be combined accordingly without mutual conflict.
[0030] This invention combines a lytic bacteriophage and *Deinococcus radiodurans* to form a composite bacterial agent. The lytic bacteriophage in the composite agent is a bacteriophage capable of infecting and lysing antibiotic-resistant bacteria in antibiotic-resistant biofilms. The *Deinococcus radiodurans* in the composite agent was deposited on February 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 37738.
[0031] Based on lytic bacteriophages and Deinococcus radiodurans, the two can be used in combination to control antibiotic-resistant biofilms. The specific method of using bacteriophages and Deinococcus radiodurans in combination to control antibiotic-resistant biofilms is as follows: The above-mentioned lytic bacteriophages and Deinococcus radiodurans are added as a compound bacterial agent to the environmental water body where the antibiotic-resistant biofilm is located, targeting and lysing the antibiotic-resistant bacteria in the antibiotic-resistant biofilm and degrading the accumulated and released intracellular antibiotic resistance genes.
[0032] It should be noted that the aforementioned antibiotic-resistant bacteria are not limited and can be any antibiotic-resistant bacteria that needs to be controlled. Typical antibiotic-resistant bacteria are *Escherichia coli* K12 and *Pseudomonas aeruginosa* PAO1. These two strains are not only typical model organisms for biofilm research, but also common species in reclaimed water distribution systems. Controlling these two strains is of great significance for ensuring the water quality safety of the distribution network.
[0033] However, it should be noted that if the antibiotic-resistant bacteria contained in the antibiotic-resistant biofilm to be controlled are different, the corresponding lytic phages should also be different, because the lytic phages must be able to target and lyse the antibiotic-resistant bacteria in the antibiotic-resistant biofilm. However, lytic phages are not limited to a specific type of phage; in fact, their function is to target and lyse antibiotic-resistant bacteria. Therefore, screening from nature or a known phage library based on this criterion is sufficient. There are many types of phages; generally, a series of lytic phages can be screened for any antibiotic-resistant bacteria, differing only in their lytic characteristics and environmental adaptability. Therefore, in the embodiments of this invention, lytic phages can be obtained by adding a mixed phage source to a culture medium containing the antibiotic-resistant bacteria to be controlled, and then isolating them using a continuous enrichment culture method. The aforementioned mixed phage source refers to a culture source rich in a large number of different phages, such as phage-rich sewage, sludge, soil, or an already constructed phage library.
[0034] Furthermore, if the antibiotic-resistant biofilm to be controlled is a biofilm in a pipeline network, then the environmental water body where such antibiotic-resistant biofilms are located is the fluid transported in the pipeline network, which often contains residual chlorine. Therefore, when screening for lytic bacteriophages, it is also necessary to ensure that they can stably survive in a water environment containing residual chlorine.
[0035] Theoretically, adding the aforementioned lytic phage and *Deinococcus radiodurans* as a compound bacterial agent to the environmental water containing antibiotic-resistant biofilms, regardless of the addition ratio, can achieve some effect. However, in practical applications, considering the maximization of control effect, it is recommended that the lytic phage be added to the environmental water at the optimal multiple of infection (MOI) relative to the antibiotic-resistant bacteria in the antibiotic-resistant biofilm. The specific MOI value can be determined and optimized based on actual experiments. The concentration of the lytic phage in the environmental water can be further calculated based on the determined optimal MOI value, thereby estimating the actual addition amount.
[0036] In addition, the amount of radiation-resistant Deinococcus radiodurans added to environmental water bodies should be as comparable as possible to that of Escherichia coli and Pseudomonas aeruginosa contained in antibiotic-resistant biofilms.
[0037] In an embodiment of the present invention, as a preferred control scheme, the concentration of lytic bacteriophages in environmental water is controlled to be 0.5~1.5 × 10⁻⁶. 10 The concentration of PFU / mL was controlled at 0.5–1.5 × 10⁻⁶ PFU / mL, while the concentration of *Deinococcus radiodurans* in environmental water was controlled at 0.5–1.5 × 10⁻⁶ PFU / mL. 8 CFU / mL.
[0038] The following examples illustrate the specific methods and technical effects of using bacteriophages in combination with radiation-resistant cocci to control antibiotic-resistant biofilms in this invention.
[0039] Example
[0040] In this embodiment, the specific method for controlling antibiotic-resistant biofilms by combining bacteriophages with radiation-resistant cocci is as follows:
[0041] Step 1: Selection and culture conditions of antibiotic-resistant bacteria
[0042] In this embodiment, bacterial biofilms were constructed using Escherichia coli K12 and Pseudomonas aeruginosa PAO1, common bacterial species in reclaimed water distribution systems. These two strains are not only typical model organisms for biofilm research but also common species in reclaimed water distribution systems. The Escherichia coli K12 strain carries the RP4 plasmid, which encodes ampicillin (bladderfungin). TEM-2 Resistance to tetracycline (tetA and tetR) and kanamycin (aphA).
[0043] To simulate the temperature and nutrient conditions in an actual pipeline network, the culture medium and conditions for bacteria were set as follows: Bacteria were cultured at 25°C using a modified M63 medium (formulation: 2.4 g / L KH₂PO₄, 5.6 g / L K₂HPO₄, 0.1 g / L MgSO₄, 1 g / L (NH₄)₂SO₄, 0.3 mg / L FeSO₄, 5.0 g / L acid-hydrolyzed casein, and 2.0 g / L glucose) until a relatively stable biofilm formed. Additionally, to simulate the residual chlorine environment in the pipeline network, a 5% NaClO solution was added to the culture medium to achieve a final effective chlorine concentration of 0.5 mg / L.
[0044] In addition, for the bacterial strains *Escherichia coli* K12 and *Pseudomonas aeruginosa* PAO1, a lytic phage capable of infecting these two bacteria was isolated from hospital wastewater using a continuous enrichment culture method, named ZJUN2. In this embodiment, phage ZJUN2 was multiplied overnight in logarithmic *E. coli* culture medium. The purified phage was stored in SM buffer at 4°C and then its performance was characterized. In this embodiment, the morphology of the phage was observed using transmission electron microscopy, the phage titer was determined using the double-layer agar plate method, the unit was plaque-forming units (PFU), and the optimal multiple of infection (MOI) test was performed. The specific procedure is as follows: First, the purified 10 10 PFU / mL phage solution was added to a carbon-coated copper grid surface, allowed to stand for 15 min, and excess liquid was blotted off with filter paper. The phages were then stained with a 2% (v / v) phosphotungstic acid (PTA) aqueous solution at pH 7.0, allowed to stand for 10 min, excess liquid was blotted off with filter paper, and the phages were air-dried for 10 min and observed under a microscope. Phages were screened based on plaque size and titer to determine the optimal MOI. The optimal MOI for phage ZJUN2, targeting *Escherichia coli* K12, was as follows: Figure 1 As shown in the figure, this indicates that selecting an MOI of 0.01 to 1 can produce a higher viral titer, with the highest viral titer achieved at an MOI of 0.01. In subsequent experiments, bacteriophage ZJUN2 will be added with an MOI of 0.01.
[0045] Furthermore, this embodiment reveals the lysis characteristics of phage ZJUN2 through further growth curve analysis, and demonstrates its good environmental adaptability and stable survival in water distribution networks through stability analysis. Simultaneously, host spectrum determination proves its infectivity against various Escherichia coli strains, and genomic analysis confirms that it does not contain gene clusters related to antibiotic resistance or virulence factors, thereby ensuring its biosafety in environmental applications.
[0046] Step 2: Screening for radiation-resistant cocci
[0047] Three *Radiatae* strains were used in this embodiment: wild-type (WT), a carotenoid synthesis-deficient mutant (Δ0862), and an extracellular nuclease synthesis-deficient mutant (Δ0067). The Δ0862 mutant lacks the homologous gene (crtB) encoding phytoene synthase, resulting in a significant reduction in the antioxidant activity of its secreted carotenoids. The Δ0067 mutant was constructed using homologous recombination technology, lacking the drb0067 gene encoding extracellular nuclease activity. The specific process for constructing the Δ0067 mutant through homologous recombination is as follows:
[0048] Using the genome of wild-type (WT) radiation-resistant *Streptococcus faecium* strain as a template, upstream and downstream homologous arm fragments were amplified using primers b0067-up-F / R (the sequence of b0067-up-F is shown in SEQ ID No. 1, and the sequence of b0067-up-R is shown in SEQ ID No. 2) and b0067-down-F / R (the sequence of b0067-down-F is shown in SEQ ID No. 5, and the sequence of b0067-down-R is shown in SEQ ID No. 6). The kanamycin resistance fragment carrying the DR groES gene promoter was amplified using primers pGroES-kana-F (sequence shown in SEQ ID No. 3) and kana-R (sequence shown in SEQ ID No. 4). The recovered upstream and downstream homologous arm fragments of drb0067 and the kanamycin resistance fragment carrying the DR groES gene promoter were ligated into a vector. The seamless cloning ligation product was transformed into E. coli DH5α competent cells (Shanghai Tulugang Biotechnology Co., Ltd.). The cells were incubated on ice for 30 min, heat-shocked at 42℃ for 45 sec, incubated on ice for 5 min, and then recovered at 37℃ for 1.5 h. 200 μL of the recovered cells were plated onto LB agar plates containing 50 μg / ml kanamycin antibiotic and incubated at 37℃ until single colonies were clearly visible. Single colonies from the antibiotic-resistant plates were picked for plasmid extraction and verification. Using the correctly sequenced plasmid as a template, the knockout fragment was amplified using b0067-p1 (sequence shown in SEQ ID No. 7) and b0067-p4 (sequence shown in SEQ ID No. 8) primers. Fresh wild-type (WT) radiation-resistant cocci strains were inoculated into TGY liquid medium and incubated at 30℃ until the exponential growth phase. 1 mL of the bacterial culture was transferred to a 1.5 mL centrifuge tube and centrifuged at 3000 g for 5 min to collect the bacterial cells; the supernatant was discarded. Bacterial cells were suspended in 250 μL of 4×TGY medium and 250 μL of 60 mM CaCl2 solution, rinsed, and centrifuged at 3000 g for 5 min to collect the cells. The supernatant was discarded, and the above operation was repeated once. Finally, the centrifuged cells were suspended in a mixture of 250 μL of 4×TGY medium and 250 μL of 60 mM CaCl2 solution and cultured in a shaker at 30℃ for 1.5 h to obtain competent cells. The drb0067 knockout homologous arm fusion fragment was added to 500 μL of competent cells and incubated on ice for 30 min. After the ice bath, the entire bacterial culture was inoculated into 5 mL of fresh TGY liquid medium and cultured in a shaker at 30℃ for at least 20 h. 400 μL of the above bacterial culture was plated onto TGY agar plates containing 35 µg / mL kanamycin and incubated at 30℃ for 3-4 days until colonies were visible to the naked eye. PCR was used to verify the results to ensure the successful construction of homozygous mutants.
[0049] To ensure the successful construction of the two core biological elements used in the synergistic control strategy, the extracellular nuclease functions of wild-type and Δ0067 mutant strains were verified through eDNA degradation and plasmid digestion experiments. Wild-type and Δ0067 mutant strains were co-incubated with eDNA, and changes in eDNA concentration were measured to analyze degradation efficiency. Simultaneously, plasmid digestion patterns were observed by agarose gel electrophoresis for further verification. Results are as follows: Figure 2 As shown, co-incubation with the wild-type strain resulted in a 65% decrease in eDNA concentration, with a degradation efficiency approaching 2.85-fold. In contrast, the Δ0067 mutant group showed only a slight decrease in eDNA concentration, demonstrating that the wild-type strain possesses potent extracellular nuclease activity, while the mutant strain lacking the drb0067 gene almost completely lost this function. The total antioxidant capacity (T-AOC) of the Δ0862 mutant and wild-type strains was measured using the rapid ABTS method to assess the effect of the crtB gene on carotenoid synthesis and its antioxidant function. Results are as follows... Figure 2 As shown, TEAC (Trolox equivalent antioxidant capacity) is the standard result of the ABTS method, indicating that the T-AOC of carotenoids secreted by the wild-type strain is 2.534±0.147 times that of the Δ0862 mutant strain. This suggests that knocking out the crtB gene impairs the synthesis of carotenoids in the strain, resulting in a weakened antioxidant capacity and increased sensitivity to oxidative stress.
[0050] Therefore, in this embodiment, a wild-type (WT) radiation-resistant coccus strain will be used as the strain to form a compound bacterial agent in combination with the lytic bacteriophage. This wild-type (WT) radiation-resistant coccus strain is named *Deinococcus radiodurans*, and was deposited on February 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.37738.
[0051] Step 3: Efficacy evaluation of treating biofilm with compound microbial agents
[0052] In this embodiment, a dual-channel flat-plate fluidized bed reactor (FC 274, BioSurface Technologies Corporation) is used to simulate the hydrodynamic conditions in a water supply network. This allows for controllable laminar flow and repeatable shear forces, thus accurately simulating biofilm growth under conditions similar to actual pipelines. The designed dual-channel flat-plate fluidized bed reactor is connected to two glass bottles via 2.4 mm silicone tubing to form a closed-loop simulated pipeline system. The silicone tubing passes through a bidirectional peristaltic pump, which, under the control of the pump, introduces the internal solution from one of the two glass bottles (denoted as A and B) into the internal pipeline of the dual-channel flat-plate fluidized bed reactor. Glass bottle A contains M63 medium containing *Escherichia coli* K12 in the logarithmic phase and *Pseudomonas aeruginosa* PAO1 in the logarithmic phase, with both bacteria at a concentration of 1 × 10⁻⁶. 8 CFU / mL. Another glass bottle B contains M63 medium containing the above-mentioned preserved wild-type (WT) radiation-resistant cocci strain and lytic phage.
[0053] The entire closed-loop simulated pipeline system was maintained at an ambient temperature of 25°C. The M63 culture medium formulation consisted of 2.4 g / L KH₂PO₄, 5.6 g / L K₂HPO₄, 0.1 g / L MgSO₄, 1 g / L (NH₄)₂SO₄, 0.3 mg / L FeSO₄, 5.0 g / L acid-hydrolyzed casein, and 2.0 g / L glucose. To simulate the slow and continuous flow of water in the distribution system, a peristaltic pump was controlled at 40 rpm, ensuring continuous circulation of the culture medium within the dual-channel flat-plate fluidized bed reactor. The biofilm experienced shear stresses similar to those in actual pipelines, and nutrient availability was also similar.
[0054] Based on the above closed-loop simulated pipeline system, the culture medium in glass bottle A is first introduced into the reactor for 24 hours, and a biofilm dominated by Escherichia coli K12 and Pseudomonas aeruginosa PAO1 is formed in the pipeline of the reactor.
[0055] Then, the wild-type *Radiata Streptococcus* strain with accession number CGMCC NO.37738 (denoted as Dr WT) and two other mutant strains (Δ0067 and Δ0862) were cultured in medium at 30°C in a shaker until the late logarithmic growth phase (OD600≈ 1.2). The cells were collected by centrifugation and resuspended in sterile PBS for the preparation of culture media. Four different culture media were prepared in glass bottle B based on four different closed-loop simulated pipeline systems for the experiments.
[0056] (i) Control group (labeled CK group) with only M63 medium added;
[0057] (ii) Simultaneously adding bacteriophage and wild-type *Radiataegilops* (labeled as ZJUN2 & Dr WT groups), the specific culture medium preparation is as follows: Based on M63 medium, Dr WT and bacteriophage ZJUN2 in the late logarithmic growth phase were added, with bacteriophage ZJUN2 added at the optimal multiple of infection (MOI=0.01). The final concentration of bacteriophage ZJUN2 in the culture medium solution was 1.09 × 10⁻⁶. 10 PFU / mL, while the Dr WT concentration is 1×10 8 CFU / mL.
[0058] (iii) Simultaneously adding bacteriophage and the radioresistant cocci Δ0067 mutant strain (labeled as ZJUN2 & Dr Δ0067 group), the specific culture medium preparation is as follows: based on M63 medium, add the extracellular nuclease synthesis defective mutant strain (Δ0067) in the late logarithmic growth stage and bacteriophage ZJUN2, wherein bacteriophage ZJUN2 is added at the optimal multiplicity of infection (MOI=0.01), and the final concentration of bacteriophage ZJUN2 in the culture medium solution is 1.09 × 10⁻⁶. 10 PFU / mL, while the concentration of the mutant strain Δ0067 was 1×10⁻⁶. 8 CFU / mL.
[0059] (iv) Simultaneously, bacteriophage and the *Radiataegilops* Δ0862 mutant strain (labeled as ZJUN2 & Dr Δ0862 group) were added. The specific culture medium preparation was as follows: Based on M63 medium, the carotenoid synthesis-deficient mutant strain (Δ0862) in late logarithmic growth and bacteriophage ZJUN2 were added. Bacteriophage ZJUN2 was added at the optimal multiplicity of infection (MOI = 0.01), resulting in a final concentration of 1.09 × 10⁻⁶ bacteriophage ZJUN2 in the culture medium solution. 10 PFU / mL, while the concentration of the mutant strain Δ0862 was 1×10⁻⁶. 8 CFU / mL.
[0060] To ensure that the simulated environment is identical to the chlorine-containing environment of the actual pipeline network, 5% NaClO solution was added to all culture media in glass bottle B to achieve a final effective chlorine concentration of 0.5 mg / L. A peristaltic pump was used to pump the culture media from glass bottle B into the simulated pipeline of the reactor where a biofilm had already formed. The mixture was incubated at 25°C for 12 hours, and then samples were taken for analysis.
[0061] In this embodiment, representative biofilm-related indicators such as biomass, bacterial count, EPS content, and resistance gene abundance were measured after different treatments.
[0062] Escherichia coli and Pseudomonas aeruginosa were counted on selective agar plates using the plate drop method, and the biomass of the biofilm and the number of antibiotic-resistant bacteria were assessed. The results are as follows: Figure 3 As shown in the figure, compared with the control group (CK group), the ZJUN2 & Dr WT group, ZJUN2 & Dr Δ0067 group, and ZJUN2 & Dr Δ0862 group reduced biofilm growth by 42.67%, 23.87%, and 35.69%, respectively, and the number of Escherichia coli in the biofilm also decreased by 18.750 times, 10.000 times, and 13.636 times, respectively.
[0063] In addition, extracellular polysaccharides (PS), proteins (PN), and eDNA were quantified using the phenol-sulfuric acid method, the BCA method, and the PicoGreen fluorescence method, respectively. The results are as follows: Figure 4 As shown in the figure, the results are similar to those observed during the screening of *Radiatae*, indicating that the combined treatment of bacteriophage and wild-type cDNA effectively removed eDNA. Simultaneously, the tetR gene, confers tetracycline resistance, was selected as a representative ARG to monitor changes in eARG abundance. In the control group (CK), the tetR gene copy number increased by 1.455-fold within 12 hours, while the copy number in the ZJUN2 & Dr Δ0067 group increased by 4.707-fold. However, in the ZJUN2 & Dr WT group, thanks to the degradation effect of extracellular nucleases, the increase in ARGs was limited to 2.386-fold. This result indicates that while killing ARBs, it may indeed lead to the accumulation of resistance genes in the environment. The introduction of *Radiatae*, through the secretion of extracellular nucleases, precisely compensated for this deficiency, thereby achieving synergistic control of the risk of ARB and ARG transmission.
[0064] Furthermore, biofilm thickness is a core indicator for measuring its role as a physical and chemical barrier. In this embodiment, the thickness of biofilm samples from different treatment groups was determined using Z-stack images obtained from a confocal laser scanning microscope. The results are as follows: Figure 5 As shown, the results indicated that, compared to the control group, the combined treatment with bacteriophage and wild-type radioresistant cocci (ZJUN2 & Dr WT group) reduced the biofilm thickness by 29.8%. Furthermore, after staining the biofilm with SYTO-9 and propidium iodide (PI), visualization analysis of dead / live cells and three-dimensional structures was performed using confocal laser scanning microscopy. The results are as follows... Figure 6 As shown in row A, the results indicate that the dead / live cell ratio (D / L) decreased in each treatment group compared to the control group. After staining with CalcofluorWhite, the distribution of extracellular polysaccharides was assessed using CLSM, and the results are shown below. Figure 6As shown in row B of the figure. Additionally, the surface morphology and roughness (Ra) of the biofilm were characterized using atomic force microscopy (AFM) and NanoScope Analysis software (version 1.80) with default parameters, as shown in the figure. Figure 6 As shown in row C. The results showed that, compared with the control group CK, the combined treatment of bacteriophage and wild-type radioresistant cocci (ZJUN2 & Dr WT group) reduced fluorescence intensity by 35.9% and had the lowest biofilm roughness. An uneven biofilm is more conducive to bacterial adsorption and reduces the problem of detachment caused by water flow shear force. This invention, however, avoids bacterial adsorption through a smoother biofilm surface, indicating that the combined treatment of bacteriophage and wild-type radioresistant cocci avoids passive enhancement of the biofilm under oxidative stress.
[0065] The embodiments described above are merely some preferred implementations of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.
Claims
1. A method for controlling antibiotic-resistant biofilms by combining bacteriophages with radiation-resistant cocci, characterized in that, Lysogenic bacteriophages and radiation-resistant cocci ( Deinococcus radiodurans As a compound bacterial agent, it is added to the environmental water body where antibiotic-resistant biofilms are located, targeting and lysing antibiotic-resistant bacteria in antibiotic-resistant biofilms and degrading the accumulated and released intracellular antibiotic resistance genes. The lytic phage is a phage capable of infecting and lysing antibiotic-resistant bacteria in antibiotic-resistant biofilms; The radiation-resistant Cocci ( Deinococcus radiodurans It was deposited on February 6, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37738.
2. The method for controlling antibiotic-resistant biofilms by combining bacteriophages with radiation-resistant cocci as described in claim 1, characterized in that, The antibiotic-resistant bacteria are Escherichia coli K12 and / or Pseudomonas aeruginosa PAO1.
3. The method for controlling antibiotic-resistant biofilms by combining bacteriophages with radiation-resistant cocci as described in claim 1, characterized in that, The lytic phage is obtained by continuously enriching and culturing a mixed phage source in a culture medium containing the antibiotic-resistant bacteria; the mixed phage source is phage-rich sewage, sludge, soil, or a phage library.
4. The method for controlling antibiotic-resistant biofilms by combining bacteriophages with radiation-resistant cocci as described in claim 1, characterized in that, The antibiotic-resistant biofilm is a biofilm in the pipeline network.
5. The method for controlling antibiotic-resistant biofilms by combining bacteriophages with radiation-resistant cocci as described in claim 4, characterized in that, The environmental water in which the antibiotic-resistant biofilm is located is a fluid transported in a distribution network and contains residual chlorine.
6. The method for controlling antibiotic-resistant biofilms by combining bacteriophages with radiation-resistant cocci as described in claim 1, characterized in that, The lytic phage is introduced into the environmental water body at the optimal infection multiple relative to antibiotic-resistant bacteria in the antibiotic-resistant biofilm.
7. The method for controlling antibiotic-resistant biofilms by combining bacteriophages with radiation-resistant cocci as described in claim 6, characterized in that, The concentration of the lytic bacteriophage in environmental water was controlled at 0.5–1.5 × 10⁻⁶. 10 PFU / mL.
8. The method for controlling antibiotic-resistant biofilms by combining bacteriophages with radiation-resistant cocci as described in claim 1, characterized in that, The radiation-resistant Cocci ( Deinococcus radiodurans The concentration of [specific substance] in environmental water bodies should be controlled at 0.5~1.5 × 10⁻⁶. 8 CFU / mL.
9. A compound microbial agent, characterized in that, Including lytic bacteriophages and radiation-resistant cocci ( Deinococcus radiodurans The lysing phage is a phage capable of infecting and lysing antibiotic-resistant bacteria in antibiotic-resistant biofilms; the radiation-resistant cocci ( Deinococcus radiodurans It was deposited on February 6, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37738.
10. The application of the compound microbial agent as described in claim 9 in the biological control of antibiotic-resistant biofilms.