A strain of *Microbacterium indicum* with antibiotic degradation function and its application

Abstract

This invention relates to the field of microbial technology, specifically disclosing a strain of *Exiguobacterium indicum* capable of degrading antibiotics and its applications. The *Exiguobacterium indicum* strain is named *Exiguobacterium indicum* WC54, and was deposited at the Guangdong Provincial Microbial Culture Collection Center on October 30, 2024, with accession number GDMCC NO.65371. This strain exhibits strong removal capabilities against five different types of antibiotics—tetracyclines, sulfacetamide, doxycycline, norfloxacin, and roxithromycin—in both farmland soil and livestock waste. Compared to traditional antibiotic treatment methods, this strain has fewer limitations in terms of application environment, is more convenient to use, lower in cost, and more environmentally friendly.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Microbacterium indicum* with antibiotic degradation function and its application. Background Technology

[0002] The rampant use of antibiotics globally has increasingly drawn attention to their threats to healthcare, food safety, and the environment. As researchers delve deeper into the side effects of antibiotics, they have revealed that the harmful effects of various antibiotics, such as tetracyclines, β-lactams, aminoglycosides, and macrolides, on soil, water sources, and the ecological environment are beyond imagination. Beyond the medical field, antibiotics are widely used throughout the entire process of livestock and poultry farming, becoming one of the main factors contributing to livestock and poultry pollution.

[0003] Current methods for controlling antibiotic pollution mainly focus on adsorption, precipitation, flotation, and membrane separation. These methods utilize physical and chemical means to separate antibiotics from the media in nature, thereby reducing their concentration in the environment. However, the characteristics of some antibiotics limit the effectiveness of these methods or result in high treatment costs. Currently, some microorganisms capable of degrading antibiotics are being discovered. Their high efficiency, safety, and low cost can compensate for the shortcomings of the aforementioned methods. However, the number of reported microorganisms is relatively small, resulting in a relatively narrow application scope for biological methods of antibiotic degradation. Summary of the Invention

[0004] One objective of this invention is to provide a strain of *Exiguobacterium indicum* capable of degrading antibiotics; a second objective is to provide an antibiotic-degrading bacterial agent containing *Exiguobacterium indicum* WC54; a third objective is to provide the application of *Exiguobacterium indicum* WC54 or the antibiotic-degrading bacterial agent in degrading antibiotic preparations, specifically, *Exiguobacterium indicum* WC54 degrading tetracycline, sulfacetamide, doxycycline, norfloxacin, and roxithromycin in two environmental media: farmland and livestock waste; a fifth objective is to provide a method for preparing the antibiotic-degrading bacterial agent.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] The present invention describes a strain of *Exiguobacterium indicum* with antibiotic degradation function. This strain is named *Exiguobacterium indicum* WC54 and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 30, 2024, with accession number GDMCC NO.65371.

[0007] The present invention discloses an antibiotic-degrading bacterial agent containing Exiguobacterium indicum WC54.

[0008] The application of Exiguobacterium indicum WC54 described in this invention in the degradation of antibiotic preparations.

[0009] The application of the antibiotic-degrading bacterial agent described in this invention in the degradation of antibiotic preparations.

[0010] The application described in this invention involves inoculating the *Exiguobacterium indicum* WC54 into the sample to be treated, followed by fermentation to degrade antibiotics.

[0011] The application described in this invention can also involve inoculating the antibiotic-degrading bacterial agent into a sample to be treated, followed by fermentation to degrade the antibiotic.

[0012] The antibiotic-degrading agent described in this invention is *Exiguobacterium indicum* WC54 or an antibiotic-degrading agent containing *Exiguobacterium indicum* WC54, which degrades five antibiotics—tetracycline, sulfacetamide, doxycycline, norfloxacin, and roxithromycin—in farmland soil environment.

[0013] The antibiotic degradation method described in this invention is: using *Exiguobacterium indicum* WC54 or an antibiotic degradation agent containing *Exiguobacterium indicum* WC54 to degrade four antibiotics—tetracycline, sulfacetamide, doxycycline, and norfloxacin—in the environmental medium of livestock and poultry waste.

[0014] The preparation method of the antibiotic-degrading bacterial agent of the present invention includes the following steps:

[0015] S1. In LB liquid medium, inoculate with Indian microbacterium WC54 and culture with shaking at 30°C and 180 rpm for 2 days to obtain the culture solution.

[0016] S2: Centrifuge the culture medium at room temperature and 3000-5000 rpm, discard the supernatant, resuspend in sterile water containing 15% skim milk powder, and then freeze dry to obtain solid bacterial agent.

[0017] The inoculation amount of *Microbacterium indicum* described in this invention is 0.5%-2%.

[0018] Furthermore, the inoculation amount of *Microbacterium indicum* described in this invention is 1%.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. This invention provides a novel microorganism capable of degrading multiple antibiotics in various media—Exiguobacterium indicum WC54.

[0021] 2. The *Indiana microbe* strain WC54 of this invention exhibits strong removal capabilities against five different antibiotics—tetracyclines, sulfacetamide, doxycycline, norfloxacin, and roxithromycin—in farmland soil. Degradation experiments of different antibiotics in farmland soil using *Indiana microbe* strain WC54 showed that the strain achieved the highest degradation efficiency (92%) for sulfacetamide, followed by tetracycline (88.23%), roxithromycin (84.13%), and norfloxacin (83.16%), with doxycycline showing the lowest efficiency (68.59%). This indicates that the strain can effectively degrade antibiotics in the tested complex farmland soil environment.

[0022] 3. The *Indiana microorganism* strain WC54 of this invention exhibits strong removal capabilities for four different antibiotics—tetracyclines, sulfacetamide, doxycycline, and norfloxacin—in livestock and poultry waste media. Through degradation experiments of antibiotics in livestock and poultry waste using *Indiana microorganism* strain WC54, the results showed that the strain achieved the highest degradation efficiency of tetracycline (91.81%), followed by doxycycline, sulfacetamide, and norfloxacin, at 90.86%, 71.39%, and 70.45%, respectively. This indicates that this strain can effectively degrade tetracyclines, sulfacetamide, doxycycline, and norfloxacin in the tested complex livestock and poultry waste media.

[0023] 4. Compared with traditional antibiotic treatment methods, the Exiguobacterium indicum WC54 strain isolated and screened by this invention has fewer limitations on the application environment. It can simultaneously remove multiple different types of antibiotics in both farmland soil and livestock and poultry waste media, making it more convenient to use and more environmentally friendly.

[0024] Preservation Information:

[0025] The isolated strain is named: Exiguobacterium indicum WC54

[0026] Preservation Institution: Guangdong Provincial Center for Microbial Culture Collection

[0027] Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou

[0028] Deposit date: October 30, 2024

[0029] Accession number: GDMCC NO.65371 Attached Figure Description

[0030] Figure 1 Morphological diagram of Exiguobacterium indicum WC54

[0031] Figure 2 Phylogenetic tree of Exiguobacterium indicum WC54

[0032] Figure 3 The degradation effect of Exiguobacterium indicum WC54 on various antibiotics in farmland soil

[0033] Figure 4 The degradation effect of Exiguobacterium indicum WC54 on various antibiotics in livestock and poultry waste. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by those skilled in the art.

[0035] Example 1

[0036] The Indian microbacterium, named *Exiguobacterium indicum* WC54, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 30, 2024, with accession number GDMCC NO.65371.

[0037] Example 2: Isolation and purification of strains

[0038] 1) Use the five-point sampling method to collect soil from the root zone of trees in the forest. The collection method is as follows: remove the dead branches and fallen leaves on the ground, loosen the soil with a shovel, collect the fine soil on the root surface, and put it into a sampling bag.

[0039] 2) Prepare LB liquid medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride). Add 10 g of soil sample to 100 mL of LB liquid medium and incubate at 30°C and 180 rpm with shaking for 2 days. Take 1 mL of the culture medium and dilute it 10 g / L. 4 10 5 10 6 Take 100 μL of each sample and spread it on an LB plate. Incubate at 30°C for 2 days.

[0040] 3) Select individual colonies from the plate and purify them on a new LB plate. Repeat the purification process until the colonies on the plate have a uniform morphology.

[0041] 4) Scrape the strain from the purified plate, resuspend it in sterile water containing 30% glycerol, and store the bacterial solution at -80°C.

[0042] Example 3: Identification of the strain

[0043] 1. Morphological identification of the strain

[0044] After inoculating *Microbacterium indicum* strains onto LB agar and culturing for 48 hours, the colonies on the LB agar were observed to be pale yellow (or orange-yellow), nearly round, semi-dry, with a smooth center and irregular edges. See [link / reference needed]. Figure 1 .

[0045] 2. Molecular biological identification of strains

[0046] After culturing the *Microbacterium indicum* strain in LB medium for 48 hours, the strain was sent to Guangzhou Aiji Biotechnology Co., Ltd. for 16S sequencing. The sequencing primer DNA sequence is as follows:

[0047] 27F:5-AGAGTTTGATCCTGGCTCAG-3

[0048] 1492R:5-GGTTACCTTGTTACGACTT-3

[0049] In the NCBI GenBank database, *Microsporum indicum* was identified using a BLAST search.

[0050] The sequencing results are as follows:

[0051] SEQ ID NO.1:

[0052]

[0053] Conclusion: Morphological and molecular biological identification revealed that strain WC54 consisted of pale yellow (orange-yellow) nearly circular colonies, semi-dry, with a smooth center and irregular edges. Comparison with the NCBI database showed that its 16S sequence had the highest similarity to *Indiana microbacterium*, indicating that strain WC54 is indeed *Indiana microbacterium*. See [link to citation]. Figure 2 .

[0054] Example 4: Preparation of solid inoculum of Microbacterium indicum

[0055] S1, In LB liquid medium, 1% of Inonotus indicus WC54 was inoculated and cultured with shaking at 30°C and 180 rpm for 2 days to obtain the culture solution;

[0056] S2: Centrifuge the culture medium at room temperature and 4000 rpm, discard the supernatant, resuspend in sterile water containing 15% skim milk powder, and then freeze dry to obtain solid bacterial agent.

[0057] Example 5: Preparation of solid inoculum of Microbacterium indicum

[0058] S1, In LB liquid medium, 0.5% of Inonotus indicus WC54 was inoculated and cultured with shaking at 30°C and 180 rpm for 2 days to obtain the culture solution;

[0059] S2: Centrifuge the culture medium at room temperature and 3000 rpm, discard the supernatant, resuspend in sterile water containing 15% skim milk powder, and then freeze dry to obtain solid bacterial agent.

[0060] Example 6: Preparation of *Microbacterium indicum* solid inoculum

[0061] S1, In LB liquid medium, 2% of Inonotus indicus WC54 was inoculated and cultured with shaking at 30°C and 180 rpm for 2 days to obtain the culture solution;

[0062] S2: Centrifuge the culture medium at room temperature and 5000 rpm, discard the supernatant, resuspend in sterile water containing 15% skim milk powder, and then freeze dry to obtain solid bacterial agent.

[0063] Example 7 Application

[0064] (1) Collect two environmental media, farmland soil and livestock wastewater, and collect them in the following ways: For farmland soil, take non-clumping soil with fine texture and put it into a sampling bag; for livestock waste, use sterilized black sealed bags to collect samples. The collection scope includes livestock and livestock wastewater, and store them at room temperature away from light.

[0065] (2) The collected samples were divided into two parts. One part was used to test the concentration of antibiotics such as tetracycline, sulfacetamide, doxycycline, norfloxacin, and roxithromycin. The test method was in accordance with the standard NY / T 3787-2020. The other part was used to complete the degradation test in the laboratory.

[0066] (3) Prepare solid bacterial agent of Microbacterium indicum according to Example 4, dissolve it with 5 times its weight of 10% glucose aqueous solution, and let it stand at room temperature for more than 4 hours to wait for the strain to be activated.

[0067] (4) The experimental soil was placed in flower pots, and the livestock and poultry waste samples were placed in sterilized pots. Inoculate the soil and water with 2% of Indo-Pacific mycobacterium.

[0068] (5) The experimental soil was placed at room temperature for 15 days, and the experimental water was tested in an open shaker at 50 rpm for 15 days. After the experiment, samples were collected again according to the method in (1), and the contents of tetracycline, sulfacetamide, doxycycline, norfloxacin and roxithromycin were detected using the same detection method.

[0069] (6) Calculation of antibiotic degradation rate: Degradation rate = [(initial antibiotic content - antibiotic content after the test) / initial antibiotic content] * 100%.

[0070] Experimental results: Antibiotic content detection is shown in Tables 1 and 2. Figures 3 to 4 The calculated results of antibiotic degradation rate are shown in Table 3.

[0071] Table 1. Detection of antibiotic content in farmland soil

[0072]

[0073] Table 2. Detection of Antibiotic Content in Livestock and Poultry Waste

[0074]

[0075] Table 3. Degradation rate (%) of antibiotics in different media by *Indiana microbes*

[0076]

[0077] 1) In complex soil media in farmland, *Microbacterium indicum* WC54 showed the highest degradation efficiency for sulfacetamide (92%), followed by tetracycline (88.23%), roxithromycin (84.13%), and norfloxacin (83.16%), with the lowest efficiency for doxycycline (68.59%). This indicates that this strain can effectively degrade antibiotics in the tested complex soil media.

[0078] 2) In the complex environmental medium of livestock and poultry waste, *Inonotus indicus* WC54 showed the highest degradation efficiency for tetracycline (91.81%), followed by doxycycline (90.86%), sulfacetamide (71.39%), and norfloxacin (70.45%). However, it did not degrade roxithromycin in the livestock and poultry waste environmental medium. This indicates that this strain can effectively degrade four different antibiotics—tetracycline, sulfacetamide, doxycycline, and norfloxacin—in the tested complex livestock and poultry waste medium.

[0079] In summary, the *Indiana microbacterium* WC54 strain of this invention exhibits significant degradation capabilities for antibiotics in farmland soil and livestock waste, with the highest degradation efficiency for sulfacetamide. Except for low anxiety regarding doxycycline degradation in farmland, this strain can effectively perform antibiotic degradation in both of the tested complex media.

[0080] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A strain of *Microbacterium indusia* with antibiotic-degrading capabilities, characterized in that, The strain was named Exiguobacterium indicum WC54 and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 30, 2024, with accession number GDMCC NO.65371.

2. An antibiotic-degrading bacterial agent, characterized in that, The antibiotic-degrading bacterial agent comprises *Exiguobacterium indicum* WC54 as described in claim 1.

3. The application of Exiguobacterium indicum WC54 as described in claim 1 or the antibiotic-degrading bacterial agent as described in claim 2 in the degradation of antibiotic preparations.

4. The application according to claim 3, characterized in that, The application involves inoculating the sample to be treated with *Exiguobacterium indicum* WC54 as described in claim 1 or the antibiotic-degrading agent as described in claim 2, and then fermenting and cultivating it to degrade the antibiotics.

5. The application according to claim 4, characterized in that, The antibiotic degrading agent is either *Exiguobacterium indicum* WC54 or the antibiotic degrading agent described in claim 2, which degrades tetracycline, sulfacetamide, doxycycline, norfloxacin, and roxithromycin in farmland soil environment.

6. The application according to claim 4, characterized in that, The antibiotic degrading agent is either *Exiguobacterium indicum* WC54 or the antibiotic degrading agent described in claim 2, which degrades tetracycline, sulfacetamide, doxycycline, and norfloxacin in the environmental medium of livestock and poultry waste.

7. A method for preparing the antibiotic-degrading bacterial agent as described in claim 2, characterized in that, Includes the following steps: S1, In LB liquid medium, inoculate with Indian microbacterium and culture with shaking at 30°C and 180 rpm for 2 days to obtain the culture solution; S2: Centrifuge the culture medium at room temperature and 3000-5000 rpm, discard the supernatant, resuspend in sterile water containing 15% skim milk powder, and then freeze dry to obtain solid bacterial agent.

8. The method for preparing the antibiotic-degrading bacterial agent according to claim 7, characterized in that, The inoculation amount of *Inonotus indicus* is 0.5%-2%.

9. The method for preparing the antibiotic-degrading bacterial agent according to claim 8, characterized in that, The inoculation amount of *Inonotus indicus* is 1%.

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