Fragrance-like fungus C8-3 and application thereof in degradation of tetracycline antibiotics

Abstract

The invention discloses aroma-like fungi C8-3 and an application of the aroma-like fungi C8-3 in degradation of tetracycline antibiotics. The strain is preserved in Guangdong Microbial Culture Collection Center on May 12, 2025, and the preservation number is GDMCC 66301. The C8-3 strain is a novel aroma-like fungus strain, has unique genome and physiological and biochemical characteristics, can significantly degrade tetracycline antibiotics, such as tetracycline (P = 0.0015), doxycycline (P < 0.0001) and tigecycline (P < 0.0001), especially can degrade (67.8 + / -2.4)% of tetracycline within 24 h, and has a good application prospect in the field of tetracycline drug microbial degradation.

Description

Technical Field

[0001] The present invention relates to microbial degradation of antibiotics, and in particular to a kind of aroma-like fungus ( Myroides sp.) C8-3 and its application in the degradation of tetracycline antibiotics. Background Art

[0002] Tetracycline antibiotics, as broad-spectrum antimicrobial agents, play an important role in human medicine and livestock farming. They are available from natural extracts (e.g., tetracycline), semi-synthetic derivatives (e.g., doxycycline), and chemically synthesized antibiotics (e.g., tigecycline). However, the low absorption rate of these drugs in vivo (approximately 75% is excreted in its active form) results in the parent compound and its active metabolites being excreted into the environment, where they accumulate in agricultural and aquatic ecosystems. These antibiotic residues not only directly disrupt microbial community structure but also contribute to the dynamic selection pressure for drug-resistant genes. Studies have shown that the slow degradation rate of tetracycline metabolites in soil promotes the spread of drug-resistant genes among pathogens, exacerbating the spread of multidrug-resistant bacteria and posing a serious threat to public health.

[0003] Microbial-mediated biotransformation, which degrades tetracycline antibiotic molecules into other products through enzymatic reactions, has become a research hotspot in environmental bioremediation due to its low cost and ease of operation. Currently, multiple bacterial species have been found to be capable of tetracycline degradation, including Pseudomonas (CN104403965A), Bacillus (CN117551570A), Acinetobacter (CN118126863A), Serratia marcescens (CN114134079A), Escherichia coli (CN110055192A), and Chryseobacterium (CN111088192A). However, given the widespread and urgent nature of antibiotic residues, the search for high-quality and efficient bacterial strains is intensified. Due to the broad adaptability of Aromatids, 17 officially named species have been identified. However, further exploration of new strain resources, particularly their applications, is needed. Summary of the Invention

[0004] The present invention provides a kind of fragrant fungus ( Myroides sp.) C8-3 and its application in the degradation of tetracycline antibiotics.

[0005] In order to achieve the above object, the present invention provides a kind of fragrant fungus Myroides sp. C8-3, the strain was deposited in Guangdong Provincial Microbiological Culture Collection on May 12, 2025, with the deposit number GDMCC 66301.

[0006] The second aspect of the present invention provides the use of the above-mentioned aroma-like bacteria C8-3 in the degradation of tetracycline antibiotics.

[0007] Preferably, the tetracycline antibiotic is tetracycline, doxycycline or tigecycline.

[0008] Through the above technical solution, the present invention achieves the following beneficial effects: The C8-3 strain of the present invention is a new type of aroma-like fungus with unique genome and physiological and biochemical characteristics, and can significantly degrade tetracycline antibiotics, such as tetracycline ( P =0.0015), doxycycline ( P＜ 0.0001) and tigecycline ( P＜ 0.0001), especially degrading (67.8±2.4)% of tetracycline within 24 h, which has good application prospects in the field of microbial degradation of tetracycline drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is the phylogenetic tree of the model species of the aroma-like fungus; Figure 2 These are the results of agar diffusion assays for the degradation of tetracycline, doxycycline, and tigecycline; Figure 3 This is the LC-MS / MS determination result of tetracycline. DETAILED DESCRIPTION

[0010] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0011] Example 1. Screening and preservation of strains On September 26, 2021, 0.2 g of chicken manure was collected from Zaozhuang City, Shandong Province. After vortexing and mixing with 1 mL of 0.9% saline, 100 μL of the sample was evenly spread on a LB agar plate containing 2 mg / L of tigecycline and incubated at 35°C for 20 hours. A single colony was picked and further purified using the same agar plate and culture conditions to obtain strain C8-3. Subsequently, a half-ring of the bacterial moss was scraped into 1 mL of 30% LB glycerol broth, pipetted, mixed, and stored in a -80°C freezer.

[0012] Example 2. Bacterial species identification based on 16S rRNA, dDDH, and ANI comparison The genomic DNA of strain C8-3 was extracted using the Tiangen Bacteria Genome Kit. The whole genome of C8-3 was sequenced by Annoroad Gene Technology (Beijing) Co., Ltd. The genome size was 3.6 Mb and the GC content was 37.6% (see Figure 1 ). With reference to the 16S rRNA sequence of the type strain of the myroides (SEQ ID No.1, https: / / lpsn.dsmz.de / genus / myroides), the 16S rRNA sequence of the C8-3 strain was extracted by blastn online comparison and compared with the Han Hanjiang myroides CJ210 ( Myroides fluvii , accession number MK129421) had a maximum similarity of 97.9%, but was below the intraspecific threshold of 99%. Further ddDH analysis showed that the strain had a similarity to the aroma fungus DSM 2801 ( Myroides odoratus , accession number GCA_000243275.1), showed the highest genome similarity, but only 24.0%, below the conventional threshold for de novo species classification (>70%). ANI analysis showed that C8-3 shared an average nucleotide similarity of 79.8% with the genome of the genus FDAARGOS_1131 (accession number GCA_016726985.1). While this is the closest species in the current database, it is still far below the intraspecies classification threshold (>95%). In summary, strain C8-3 was identified as a new species of the genus FDAARGOS.

[0013] SEQ ID No. 1:

[0014] Example 3. Physiological and biochemical characteristics of the aroma-like fungus C8-3 Based on the model strain pseudo-fragrant fungus ( Myroides odoratimimus) ATCC BAA-634, and systematically studied the physiological and biochemical characteristics of the aroma-like fungus C8-3 (see Table 1 for details): (1) The three-zone streak method was used to inoculate onto Huankai LB agar plates, and the plates were cultured at a temperature gradient of 20-43°C for 24 hours. The results showed that strain C8-3 formed typical regular round single colonies with smooth and plump surfaces, and the upper limit of growth temperature was 41°C. (2) Using Huankai LB semi-solid medium for puncture inoculation, no diffusive growth characteristics were observed after culturing at 35°C for 24 hours, indicating that strain C8-3 does not have the ability to move autonomously; (3) No colony formation was detected after three-zone streak culture at 35°C under anaerobic conditions for 24 hours, confirming that strain C8-3 was an aerobic microorganism; (4) By establishing a pH gradient (4-11) and a salt (sodium chloride) concentration gradient (0-5%), 5 μL of fresh bacterial solution was quantitatively inoculated into 4 mL of Huankai LB broth and cultured at 35°C and 200 rpm. The results showed that the survival pH range of the C8-3 strain was 5-9, and the salt tolerance was as high as 4%. (5) Other physiological and biochemical characteristics were determined using a non-fermentative bacterial identification tube. The results showed that the C8-3 strain was positive for oxidase, arginine dihydrolase, DNAase, and acetamide metabolism, but negative for glucose, maltose, xylose, mannitol, citrate utilization, and nitrate reduction.

[0015] Table 1 Physiological and biochemical identification results of the aroma-like fungus C8-3

[0016] Example 4. Determination of Minimum Inhibitory Concentration The minimum inhibitory concentrations (MICs) of the aroma-like bacteria C8-3 against three tetracyclines (first-generation tetracycline, second-generation doxycycline, and third-generation tigecycline) were determined using the two-fold broth microdilution method, according to the 2025 Clinical and Laboratory Standards Institute (CLSI) technical standard (M100-Ed35). Escherichia coli ATCC 25922 was used as a quality control strain. The results showed that the prepared solutions of tetracycline, doxycycline, and tigecycline met the quality control standards, with MICs of 32 mg / L, 8 mg / L, and 4 mg / L against the aroma-like bacteria C8-3, respectively.

[0017] Example 5. Agar Diffusion Assay for Degradation of Tetracycline, Doxycycline, and Tigecycline The strain C8-3 was co-cultured with Huankai MH broth containing tetracycline (64 mg / L), doxycycline (80 mg / L), or tigecycline (20 mg / L) at 35°C for 24 hours. Huankai MH broth containing only the corresponding concentration of antibiotics and blank MH broth without other ingredients were set as controls, with three replicates for each group. Subsequently, 100 μL of Escherichia coli ATCC25922 (OD 600 =0.4, tetracycline-susceptible strain) was evenly spread on a 90 mm diameter MH agar plate. Three 4 mm diameter agar wells were then randomly punctured and sealed with hot MH agar. After centrifugation, 15 μL of the supernatant was injected into the agar wells. After inverted incubation at 40°C for 16 h, the diameter of the inhibition zone was measured using a vernier caliper. Experimental data showed that the inhibition zone in the C8-3 strain treatment group was significantly smaller than that in the control groups for each antibiotic (tetracycline, P =0.0015; doxycycline, P <0.0001; tigecycline, P <0.0001), confirming that it has significant degradation efficiency against three tetracycline antibiotics (see Figure 2 ).

[0018] Example 6. Quantitative Determination of Tetracycline Degradation by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS) Take 40 μL of fresh C8-3 bacterial solution in the logarithmic growth phase (OD 600=0.6), inoculated in M9 complete medium (composed of 1×M9 minimal salt, 2 mM magnesium sulfate, 100 μM calcium chloride, 9 g / L glucose, 100 mg / L thiamine and 100 mg / L leucine, etc.) containing 10 mg / L tetracycline, with a final volume of 4 mL, and cultured in the dark in a constant temperature shaker at 35°C and 220 rpm for 24 hours. After high-speed centrifugation at 10,000×g for 5 minutes, sterilization with a 0.22 μm filter membrane, and ten-fold gradient dilution, the residual tetracycline was quantitatively detected by LC-MS / MS technology. At the same time, a culture medium with only tetracycline added was set up as a control group, and each group contained 6 biological replicates. Figure 3 As shown in Figure 2, the degradation efficiency of tetracycline by strain C8-3 reached (67.8±2.4)% within 24 hours, which was significantly higher than that of the control group ( P <0.0001).

[0019] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0020] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0021] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A novel aroma-like fungus C8-3, characterized in that: The deposit number is GDMCC 66301.

2. Use of the aroma-like fungus C8-3 according to claim 1 in the degradation of tetracycline antibiotics.

3. The use according to claim 2, characterized in that The tetracycline antibiotic is tetracycline, doxycycline or tigecycline.

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