Use of enterobacter kobei to block evolution of antibiotic resistance in soil bacteria

Abstract

The application discloses application of Enterobacter kobei in controlling and resisting evolution of soil bacteria antibiotic resistance. Enterobacter kobei LB1 is preserved in the China General Microbiological Culture Collection Center on May 15, 2026, and has a preservation number of CGMCC No. 38684 and a preservation address of No. 1, Xibaixili, Chaoyang District, Beijing, China Institute of Microbiology. Enterobacter kobei LB1 and extracellular metabolites have the ability of controlling and resisting evolution of soil bacteria antibiotic resistance. The application verifies the inhibiting effect of the resistance mutant strain of the control agent through a soil microcosm experiment, realizes effective intervention on the evolution of soil microbial resistance, and has the advantages of green ecology, safety, high efficiency, long-term control and the like, and provides a new technical way for environmental antibiotic resistance pollution treatment.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology and antibiotic resistance control technology, specifically relating to the application of Kobe Enterobacter in inhibiting the evolution of antibiotic resistance in soil bacteria. Background Technology

[0002] The widespread use of antibiotics in medicine and agriculture has led to increasingly serious problems with antibiotic residues in the environment, especially quinolone antibiotics such as ciprofloxacin, which have strong persistence in soil environments. Long-term low-dose exposure can promote the development of drug resistance in pathogenic bacteria such as Escherichia coli and accelerate the spread of resistance genes.

[0003] Traditional antibiotic resistance control methods largely rely on physical or chemical means. Physical methods mainly include high-temperature heat treatment, microwave treatment, ex-situ soil composting, and steam explosion. While these methods can effectively reduce antibiotic concentrations in a short time, they suffer from high energy consumption, high processing costs, and difficulty in large-scale application. Furthermore, high-temperature treatment can disrupt soil structure and the balance of indigenous microbial communities, leading to secondary ecological problems. Chemical methods mainly include photocatalytic oxidation, chemical disinfection (such as chlorine disinfection), and the application of nanomaterials. Among these, photocatalysis has a high efficiency in antibiotic degradation, but catalyst recovery is difficult and may produce toxic intermediates. Chemical disinfectants, at sub-lethal doses, not only fail to effectively eliminate resistance genes but also promote the horizontal transfer of resistance genes by inducing reactive oxygen species and SOS reactions. Although nanomaterials (such as Fe2O3@MoS2) can inhibit the conjugation and transfer of resistance genes, long-term accumulation may have toxic effects on soil organisms and pose potential environmental risks.

[0004] Recent studies have revealed the crucial role of microbial extracellular metabolites in regulating community structure and inhibiting antibiotic resistance evolution. Microorganisms, through the secretion of extracellular metabolites, participate in interspecific competition, signal transduction, and niche occupation, influencing the physiological state and evolutionary trajectory of surrounding microorganisms. In particular, the extracellular metabolites of certain functional strains can slow the rate of antibiotic resistance evolution at its source by interfering with bacterial quorum sensing systems, inhibiting biofilm formation, reducing efflux pump activity, or blocking the horizontal transfer of resistance genes. However, systematic research on specific functional strains and their extracellular metabolites in controlling the evolution of antibiotic resistance in soil bacteria remains lacking. Their specific mechanisms of action, optimal application concentrations, and long-term environmental effects remain unclear, limiting the practical application of this technology in the control of soil resistance pollution.

[0005] Therefore, developing a green control strategy based on natural microbial metabolites, by screening key functional strains with synergistic inhibitory effects, extracting and optimizing their extracellular metabolite components, and constructing an antibiotic-metabolite synergistic control system, is of great significance for achieving long-term control of resistance risk and overcoming the limitations of traditional methods. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an application of *Enterobacter Kobe* in controlling the evolution of antibiotic resistance in soil bacteria. The purpose of this invention is to provide a green and efficient method for controlling antibiotic resistance by screening key functional bacteria and their metabolites to construct an antibiotic-metabolite synergistic control system, thereby achieving long-term regulation of the evolution of resistance in soil microorganisms.

[0007] The technical solution adopted in this invention is: I. A strain of Enterobacter kobei for controlling the evolution of antibiotic resistance in soil bacteria, wherein Enterobacter kobei LB1 was deposited on May 15, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38684, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0008] II. Application of a Kobe Enterobacter or an extracellular metabolite of Kobe Enterobacter in controlling the evolution of antibiotic resistance in soil bacteria.

[0009] The soil bacteria include Pseudomonadota, Bacteroidetes, and Actinomycetota. The antibiotic in question is ciprofloxacin.

[0010] III. Application of a Kobe Enterobacter or an extracellular metabolite of Kobe Enterobacter in the preparation of a inhibitor for controlling the evolution of antibiotic resistance in soil bacteria.

[0011] IV. A method for controlling the evolution of antibiotic resistance in soil bacteria using Enterobacter Kobe, comprising the following steps: Spray the soil surface of the target area with a bacterial solution containing *Enterobacter kibble* or a control agent containing extracellular metabolites of *Enterobacter kibble*.

[0012] The final concentration of the extracellular metabolites of the Kobe Enterobacter in the soil is 9.5 g to 10.5 g of extracellular metabolites per kilogram of dry soil.

[0013] The final concentration of the Kobe Enterobacter bacterial suspension in the soil was 10 [units unspecified] per kilogram of dry soil. 7 CFU of Kobe Enterobacter.

[0014] The beneficial effects of this invention are: (1) It utilizes natural microbial metabolites and has green ecological characteristics; (2) It can significantly reduce the rate of resistance evolution; (3) It has long-term stable regulation capabilities; (4) Applicable to complex soil environments; (5) Provides new ideas for antibiotic pollution control. Attached Figure Description

[0015] Figure 1 Evolutionary trend of Escherichia coli resistance to ciprofloxacin after adding Kobe Enterobacter LB1 metabolite to Example 1.

[0016] Figure 2 This is a diagram illustrating the effect of Example 2 on inhibiting the evolution of soil microbial resistance.

[0017] Figure 3 This image shows the results of a hemolysis experiment using Enterobacter LB1 from Kobe. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0019] The screening method for the Enterobacter kobei strain used in this invention is as follows: Escherichia coli was isolated from the soil environment and cultured in a ciprofloxacin-containing culture system at a sub-inhibitory concentration for the target E. coli. Simultaneously, soil microorganisms were screened by culturing single bacteria in M9 (with glucose) minimal medium at 37°C for 48 hours, followed by extraction of extracellular metabolites. E. coli was subjected to evolutionary culture under co-stress conditions of different concentration gradients of extracellular metabolites and ciprofloxacin. Metabolites that significantly reduced resistance evolution were screened by measuring changes in minimum inhibitory concentration (MIC) or growth rate, and the producing strain was identified as Enterobacterkobei LB1. This colony is 1-2 mm in diameter, round, grayish-white, opaque, with irregular edges, and Gram-negative.

[0020] Enterobacter kobei strain LB1 exhibits good biosafety. Figure 3 The image shows the results of the hemolysis experiment of Enterobacter LB1 from Kobe. Figure 3 As shown, the blood agar plate culture test indicated that no hemolytic zone appeared around the colony of this strain, it did not produce hemolysin, and had no ability to lyse blood cells, suggesting that it does not carry key hemolytic virulence factors and does not pose a risk of hemolytic pathogenicity. This strain was isolated from the natural soil environment and is an environmentally sourced natural strain. Testing revealed that it did not carry pathogenicity-related virulence genes or high-risk drug resistance genes, and it is not a highly pathogenic strain. Under normal application conditions, it does not significantly disturb the native soil microbial community and will not cause ecological imbalance or secondary pollution. Furthermore, at the application dose (10... 7(CFU / kg dry soil) has no acute toxicity, no pathogenicity, and no risk of infection spread. It meets the safety application requirements for environmental microbial agents and has the characteristics of being green, safe, and environmentally compatible. It is suitable for large-scale application in farmland and field soil.

[0021] The method for extracting the extracellular metabolites of Enterobacter kobei is as follows: Enterobacter kobei was inoculated into M9 medium and cultured at 37°C for 48 h. After culturing, it was centrifuged at 7000×g for 10 min. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtrate was then freeze-dried to obtain lyophilized product.

[0022] Example 1 1) Cultured in 96-well deep-well storage plates with a total volume of 1 mL per well. Three concentration gradients were set for each extracellular metabolite: 5 g / L, 10 g / L, and 15 g / L. The antibiotic was ciprofloxacin at 10 μg / L.

[0023] 2) Set up 3 experimental groups for the combination of extracellular metabolites and ciprofloxacin (M+CIP group), 4 groups for induction of ciprofloxacin (CIP group) and extracellular metabolites alone, and 1 blank control group (CK group), for a total of 8 groups. Each group was repeated 6 times in parallel.

[0024] The compound pollution induction group consisted of 10 μL bacterial suspension + 10 μL antibiotics + 10 μL microbial metabolites + 970 μL culture medium; The induction group consisted of 10 μL sterile water + 10 μL bacterial suspension + 10 μL antibiotic or microbial metabolite + 970 μL culture medium; The blank control group consisted of 20 μL sterile water + 10 μL bacterial suspension + 970 μL culture medium.

[0025] The evolutionary experiment was conducted for 24 days. The changes in resistance levels under each experimental condition were measured on days 0, 4, 8, 12, 16, 20, and 24, and the samples were stored.

[0026] 3) Every 24 hours, take 10 μL of bacterial culture from the 96-well plate and add it to fresh culture medium to dilute 100 times for passage. The evolution experiment was carried out for 24 days. On days 0, 4, 8, 12, 16, 20, and 24, the changes in resistance levels under each experimental condition were measured and the samples were stored.

[0027] like Figure 1 As shown, E. kobei metabolites significantly inhibited the evolution of resistance to CIP in E. coli under CIP stress. In the co-exposure treatment group, the minimum inhibitory concentration (MIC) of E. coli showed only a slight increase throughout the evolution process, with resistance increasing by approximately 1.2 times at the end of the evolution, significantly lower than the 8.7 times increase in the CIP stress group.

[0028] Example 2: Soil Microcosm Experiment 1) Soil samples were taken from the top layer (0-10cm) of an uncontaminated park in Hangzhou, Zhejiang Province.

[0029] 2) Soil samples were sieved through a 2mm sieve, and the maximum water holding capacity was measured to be 80.57%. No ciprofloxacin residues were detected in the soil samples from this site.

[0030] 3) Each sample was incubated with 30g of dry soil at 25℃ in the dark, with water added to 60% of the maximum water holding capacity. There were a total of 8 treatment groups, with three replicates for each treatment group, and the samples were cultured for 30 days.

[0031] 4) The group setup is as follows: one control group (CIP) starting with a concentration of 20 mg / kg ciprofloxacin; Three starting concentrations of ciprofloxacin were supplemented with different concentrations of E. kobei metabolites: low concentration 0.2 g / kg (M1), medium concentration 2 g / kg (M2), and high concentration 10 g / kg (M3). Three starting solutions containing the same concentration of ciprofloxacin were added, with different concentrations of E. kobei bacteria: the lowest concentration was 10... 6 CFU / Kg (Ba1), medium concentration 10 7 CFU / Kg(Ba2), high concentration is 10 8 CFU / Kg(Ba3); And a control group (CK) that started without ciprofloxacin contamination and without proline addition.

[0032] 5) During the experiment, deionized water was added every 7 days to maintain the soil moisture content at about 60% of its maximum water holding capacity.

[0033] 6) Collect soil samples on day 1 and day 30 and freeze them for subsequent analysis.

[0034] Table 1. Minimum inhibitory concentration of Escherichia coli against ciprofloxacin in soil of each treatment group As shown in Table 1, in the examples, E. kobei and its metabolites altered the evolution of resistance levels in the soil microbial community, decreasing the MIC of ciprofloxacin (CIP group) from 3.90 mg / L to 1.36-2.92 mg / L.

[0035] like Figure 2As shown, there were significant differences between the M2, M3, Ba1, and Ba2 groups and the CIP group; the M2 and Ba2 groups showed the best results; therefore, the optimal concentration of the agent (E. kobei metabolite) was selected as 10 g / kg (±0.5 g / kg); the optimal concentration of the bacterial agent (E. kobei bacterial solution) was 10 g / kg. 7 CFU / Kg.

[0036] In summary, under the single stress of ciprofloxacin, the level of soil microbial resistance increased significantly. The introduction of the functional bacteria Enterobacter kobei or its extracellular metabolites effectively reduced the degree of resistance evolution. Among them, the treatment with high concentration of metabolites and medium inoculum of functional bacteria showed more significant inhibitory effects, indicating that this strategy has good application potential in regulating the development of soil resistance.

Claims

1. A strain of *Enterobacter kwangsiensis* for controlling the evolution of antibiotic resistance in soil bacteria, characterized by: The Enterobacter kobei mentioned is Enterobacter kobei LB1, which was deposited on May 15, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38684. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The use of the Kobe Enterobacter as described in claim 1 or an extracellular metabolite of the Kobe Enterobacter as described in claim 1 in controlling the evolution of antibiotic resistance in soil bacteria.

3. The application according to claim 2, characterized in that, The soil bacteria include Pseudomonadota, Bacteroidetes, and Actinomycetota. The antibiotic in question is ciprofloxacin.

4. The use of an extracellular metabolite comprising *Enterobacter koseki* as claimed in claim 1 or *Enterobacter koseki* as claimed in claim 1 in the preparation of a inhibitor for controlling the evolution of antibiotic resistance in soil bacteria.

5. A method for controlling the evolution of antibiotic resistance in soil bacteria using *Enterobacter Kobe* as described in claim 1, characterized in that, Includes the following steps: Spray the soil surface of the target area with a bacterial solution containing *Enterobacter kibble* or a control agent containing extracellular metabolites of *Enterobacter kibble*.

6. The method for controlling the evolution of antibiotic resistance in soil bacteria according to claim 5, characterized in that: The final concentration of the extracellular metabolites of the Kobe Enterobacter in the soil is 9.5 g to 10.5 g of extracellular metabolites per kilogram of dry soil.

7. The method for controlling the evolution of antibiotic resistance in soil bacteria according to claim 5, characterized in that: The final concentration of the Kobe Enterobacter bacterial suspension in the soil was 10 [units unspecified] per kilogram of dry soil. 7 CFU of Kobe Enterobacter.

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