Staphylococcus pseudovulgare AT8 for degrading antibiotics and application thereof

Abstract

This application provides a *Staphylococcus pseudoci* AT8 strain for degrading antibiotics and its application. The *Staphylococcus pseudoci* is a species of Staphylococcus (…). Staphylococcus The *Staphylococcus pseudocarpa* species, having a 16S rDNA sequence as shown in SEQ ID NO:1, is named... Staphylococcus pseudoxylosus AT8, deposited on November 20, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, accession number: GDMCC No: 67323. This application pertains to *Staphylococcus pseudocarpa*. Staphylococcus pseudoxylosus AT8 can achieve a degradation rate of over 90% for trimethoprim and tilmicosin, and over 78% for enrofloxacin. Furthermore, this application enriches the antibiotic-degrading bacterial strain library, promotes the control of new antibiotic pollutants in livestock and poultry manure and farmland soil, and facilitates the resource-based treatment and safe utilization of solid waste, demonstrating broad application prospects.

Description

Technical Field

[0001] This application relates to the field of microbiology, specifically to Staphylococcus pseudothomsonii AT8 for the degradation of antibiotics and its applications. Background Technology

[0002] Antibiotic residues in livestock and poultry manure are a significant source of antibiotic pollution in environmental media such as soil, water bodies, and groundwater. Statistics show that approximately 70% of antibiotics used globally each year are produced in livestock and poultry farming. After being fed to livestock and poultry, antibiotics are typically only partially metabolized; 30%-90% are excreted in feces and urine as the original drug or metabolites. Even after treatment methods such as aerobic composting, fermentation beds, stabilization ponds, and anaerobic fermentation, antibiotic residues remain and enter the ecological environment through the return of organic fertilizers and biogas slurry to the fields. In recent years, antibiotics have been detected in various environmental media in my country, including surface water, groundwater, soil, fertilizers, and plants.

[0003] Numerous studies have shown that aerobic composting can effectively remove residual antibiotics from livestock manure, but its effectiveness is also influenced by factors such as the type and concentration of antibiotics, compost temperature, and microbial community structure. Adding exogenous microbial agents or screening for functional strains can significantly improve the degradation rate of antibiotic contaminants. For example, patent CN202310172529.0 describes a Klebsiella pneumoniae LX strain capable of degrading fluoroquinolone antibiotics, and patent CN202511329394 describes a Bacillus bellis strain capable of degrading kanamycin antibiotics. However, these strains are typically only effective against one type of antibiotic.

[0004] Therefore, there is an urgent need to develop microbial strains that can simultaneously degrade multiple types of antibiotics in order to promote the resource-based treatment and safe utilization of solid waste, and protect the ecological environment and human health. Summary of the Invention

[0005] Therefore, embodiments of this application provide *Staphylococcus pseudotruncatula* strains for degrading antibiotics.

[0006] The first aspect of this application provides a *Staphylococcus pseudoci* species for degrading antibiotics, wherein the *Staphylococcus pseudoci* is a member of the genus *Staphylococcus*. Staphylococcus The *Staphylococcus pseudocarpa*, having the 16S rDNA sequence shown in SEQ ID NO: 1, is named... Staphylococcus pseudoxylosus AT8 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 20, 2025, accession number: GDMCC No: 67323.

[0007] In some embodiments, the antibiotic is at least one of the following antibiotics: trimethoprim antibiotics, tilmicosin antibiotics, and enrofloxacin antibiotics.

[0008] In some embodiments, the trimethoprim antibiotics include one or more of the following: trimethoprim, ormetoprim, baquiloprim; the tilmicosin antibiotics include one or more of the following: tilmicosin, tylosin, tilidipirosin, gamithromycin, tulathromycin; and the enrofloxacin antibiotics include one or more of the following: enrofloxacin, ciprofloxacin, sarafloxacin, danofloxacin, difloxacin, orbifloxacin, marbofloxacin, benifloxacin, and pradofloxacin.

[0009] A second aspect of this application provides a formulation comprising Staphylococcus pseudostaphylococcus as described in any embodiment of the first aspect of this application.

[0010] In some embodiments, the formulation further includes excipients.

[0011] In some embodiments, the formulation is a liquid formulation and / or a lyophilized formulation, and the dosage form of the formulation is selected from: wettable powder, water-dispersible granules, suspension concentrate, water emulsion, granules, seed coating agent, or a combination thereof.

[0012] The third aspect of this application proposes the use of Staphylococcus pseudotyphi as described in any embodiment of the first aspect of this application or an formulation as described in any embodiment of the second aspect of this application for the degradation of antibiotics in livestock and poultry excrement.

[0013] In some embodiments, the livestock and poultry include any one or more of the following: pigs, cattle, sheep, horses, chickens, ducks, geese; the excrement includes any one or more of the following: feces, urine, intestinal secretions.

[0014] The fourth aspect of this application provides a method for degrading antibiotics in livestock and poultry excrement, the method comprising preparing the livestock and poultry excrement into a mixture; and adding Staphylococcus pseudotuberculosis as described in any embodiment of the first aspect of this application or a preparation as described in any embodiment of the second aspect of this application to the mixture.

[0015] In some embodiments, Staphylococcus pseudostaphylococcus as described in any embodiment of the first aspect of this application or the preparation as described in any embodiment of the second aspect of this application is added at a mass ratio of 0.5% to 1% of the mixture.

[0016] The embodiments of this application achieve the following beneficial effects:

[0017] This application is the first to propose a strain of Staphylococcus pseudomalaecarpa AT8 capable of simultaneously degrading multiple antibiotics. Staphylococcus pseudoxylosus This strain (AT8) exhibits excellent degradation capabilities for three different types of antibiotics commonly found in pig manure: trimethoprim and enrofloxacin, and tilmicosin. At concentrations of approximately 1–2 mg / kg, the degradation rates for trimethoprim and tilmicosin can reach over 90%, and for enrofloxacin, over 78%. Furthermore, this application enriches the antibiotic-degrading bacterial strain library, promotes the control of new antibiotic pollutants in livestock and poultry manure and farmland soil, and facilitates the resource-based treatment and safe utilization of solid waste, demonstrating broad application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a clustering diagram of AT8 strains according to embodiments of this application; Figure 2 This is a diagram illustrating the growth morphology of the AT8 strain according to an embodiment of this application. Figure 3 For the verification of antibiotic resistance of the AT8 strain according to the embodiments of this application; wherein, TMP represents trimethoprim, TIL represents tilmicosin, ENR represents enrofloxacin, and AZM represents azithromycin; Figure 4 This is a Gram staining result diagram of the AT8 strain according to an embodiment of this application; Figure 5 This is a diagram of an antibiotic degradation test in composting according to an embodiment of this application.

[0020] Instructions for the preservation of microbial strains: The name of the provided biomaterial is Staphylococcus pseudoxylosus AT8, taxonomic name is Staphylococcus pseudoxylosus The deposit registration number is GDMCC No: 67323; the depository is Guangdong Provincial Center for Microbial Culture Collection; the address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the deposit date is November 20, 2025. Detailed Implementation

[0021] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit the scope of the present application. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0022] This application is based on the inventor's following understanding: Antibiotic residues in livestock and poultry manure are a significant source of antibiotic pollution in environmental media such as soil, water bodies, and groundwater. As the risks and harms of antibiotics to the ecological environment and human health become increasingly recognized, antibiotic residues in the environment have become a major social concern in my country, and antibiotics have been included in the "List of Key Controlled New Pollutants (2023 Edition)". New pollutants possess characteristics such as biotoxicity, environmental persistence, and bioaccumulation; even at low concentrations in the environment, they may pose significant environmental and health risks, and their hazards are potential and insidious. Livestock and poultry manure treatment, as the last barrier against antibiotics entering the environment from livestock manure, plays a crucial role in the control of new antibiotic pollutants and is an important component of the new pollutant control system proposed in the "Action Plan for the Control of New Pollutants" and the "Beijing Municipal Work Plan for the Control of New Pollutants".

[0023] Adding exogenous bacterial agents or screening for functional strains are important ways to improve the degradation rate of antibiotic pollutants. Previous studies have only involved obtaining strains that degrade single types of antibiotics, while the livestock and poultry farming industry currently uses dozens of types of antibiotics, such as tetracyclines, quinolones, and aminoglycosides. Therefore, screening for microbial strains that can simultaneously degrade multiple types of antibiotics is of great significance for effectively controlling antibiotic pollution, promoting the resource-based treatment and safe utilization of solid waste, and protecting the ecological environment and human health.

[0024] In this embodiment, a mixture of pig manure sample and sterilized sawdust was prepared and placed in a composting reactor for co-fermentation. The mixture at the high-temperature fermentation stage was then vortexed to prepare a bacterial suspension. A series of dilutions were prepared using a tenfold serial dilution method and plated onto TSB-based solid plates for cultivation. Vigorous single colonies were picked from the plates and repeatedly streaked for purification. By comparing growth patterns, a strain exhibiting good growth ability was named AT8. Preliminary identification of strain AT8 using 16S rRNA sequence alignment confirmed its genus. Staphylococcus pseudoxylosus Species. Furthermore, a phylogenetic tree was constructed using MAGA with neighbor-to-neighbor merging for closely related strains of AT8, revealing that AT8 is related to... Staphylococcus pseudoxylosusThe AT8 gene is closely related to the 16S ribosomal RNA gene, partial sequence (Genebank sequence number: MH643903.1), confirming that the isolated AT8 belongs to [the ribosomal RNA gene family]. Staphylococcus pseudoxylosus This is a new species of Staphylococcus. Furthermore, the embodiments of this application verified the multi-antibiotic degradation function of this strain through a series of experiments, including antimicrobial susceptibility testing and composting treatment experiments.

[0025] The first aspect of this application provides a *Staphylococcus pseudoci* species for degrading antibiotics, wherein the *Staphylococcus pseudoci* is a member of the genus *Staphylococcus*. Staphylococcus The *Staphylococcus pseudocarpa*, having the 16S rDNA sequence shown in SEQ ID NO: 1, is named... Staphylococcus pseudoxylosus AT8 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 20, 2025, accession number: GDMCC No: 67323.

[0026] In some embodiments, the antibiotic is at least one of the following antibiotics: trimethoprim antibiotics, tilmicosin antibiotics, and enrofloxacin antibiotics.

[0027] In some embodiments, the trimethoprim antibiotics include one or more of the following: trimethoprim, omeprine, baquipril; the tilmicosin antibiotics include one or more of the following: tilmicosin, tylosin, tylammycin, gamimycin, torammycin; and the enrofloxacin antibiotics include one or more of the following: enrofloxacin, ciprofloxacin, sarafloxacin, daflufloxacin, diflufloxacin, obibixacin, mebufloxacin, benifloxacin, and pralidoxacin.

[0028] Staphylococcus is a Gram-positive, catalase-positive, spherical bacterium arranged in grape-like clusters. The strain AT8 protected in this application forms round, smooth, moist, milky-white colonies after culturing on TSB solid medium. Phylogenetic analysis shows that AT8, as described in this application, is a novel species within the genus Staphylococcus. AT8 is a pseudostaphylococcus, belonging to the genus Staphylococcus, and is named accordingly. Staphylococcus pseudoxylosus AT8; deposit registration number: GDMCC No: 67323; depositary institution: Guangdong Provincial Center for Microbial Culture Collection; depositary address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; deposit date: November 20, 2025.

[0029] The second aspect of this application also provides a formulation comprising Staphylococcus pseudostaphylococcus as described in any embodiment of the first aspect of this application.

[0030] In the embodiments of this application, the formulation further includes excipients selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, and release inhibitors.

[0031] In some embodiments, the formulation is a liquid formulation and / or a lyophilized formulation. In specific embodiments, due to different needs in actual production applications, and combined with conventional technical means in the field of formulation preparation processes (e.g., "Microbial Inoculant Production Process Flowchart," "Encyclopedia of Pharmaceutical Technology," "Pharmaceutical Formulation Technology," etc.), those skilled in the art can select and formulate excipients, and use *Staphylococcus pseudomallei* with accession number GDMCC No: 67323. Staphylococcus pseudoxylosus AT8 can be formulated into various dosage forms, such as wettable powders, water-dispersible granules, suspensions, emulsions, granules, seed coatings, or combinations thereof. It is understood that the dosage forms used in the embodiments of this application are only required to ensure application in a specific form, and this application does not impose any limitations on this.

[0032] The formulation proposed in this application can effectively degrade various antibiotics in livestock and poultry excrement, such as the three common types of antibiotics: trimethoprim, enrofloxacin, and tilmicosin. The *Staphylococcus pseudocarpa* strain proposed in this application is of great significance for promoting the control of new antibiotic pollutants in livestock and poultry manure and farmland soil. Furthermore, it can promote the resource utilization and safe treatment of solid waste, showing broad application prospects.

[0033] The third aspect of this application also proposes the use of Staphylococcus pseudotyphi as described in any embodiment of the first aspect of this application or an formulation as described in any embodiment of the second aspect of this application in the degradation of antibiotics in livestock and poultry excrement.

[0034] In some embodiments, the livestock and poultry include any one or more of the following: pigs, cattle, sheep, horses, chickens, ducks, geese; the excrement includes any one or more of the following: feces, urine, intestinal secretions.

[0035] In some embodiments, the livestock or poultry is a pig.

[0036] In some embodiments, the excrement is feces.

[0037] In some embodiments, the livestock and poultry excrement is pig manure.

[0038] The fourth aspect of this application also provides a method for degrading antibiotics in livestock and poultry excrement, comprising preparing the livestock and poultry excrement into a mixture; and adding Staphylococcus pseudothomolecularis as described in any embodiment of the first aspect of this application or a preparation as described in any embodiment of the second aspect of this application to the mixture.

[0039] In some embodiments, the livestock and poultry include any one or more of the following: pigs, cattle, sheep, horses, chickens, ducks, geese; the excrement includes any one or more of the following: feces, urine, intestinal secretions.

[0040] In some embodiments, the livestock or poultry is a pig.

[0041] In some embodiments, the excrement is feces.

[0042] In some embodiments, the livestock and poultry excrement is pig manure.

[0043] In some embodiments, the mixture is required to have a moisture content of 55% to 65% and a pH value of 6 to 8.

[0044] In some embodiments, Staphylococcus pseudostaphylococcus as described in any embodiment of the first aspect of this application or the preparation as described in any embodiment of the second aspect of this application is added at a mass ratio of 0.5% to 1% of the mixture.

[0045] In some embodiments, the method further includes the steps of stirring and oxygenation, wherein the stirring frequency is 5-10 min / 6 h and the stirring rate is 5-15 r / min; further, the temperature is controlled not to exceed 65°C, and the temperature is maintained above 55°C for a duration of not less than 6-12 days.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0048] Example 1: Isolation and Screening of Strains 1.1 Sampling, composting and aerobic fermentation of pig manure A 10 kg sample of fresh pig manure was collected from a large-scale pig farm. Previous testing revealed that the manure sample contained residues of various antibiotics. The sample was thoroughly mixed with 2 kg of sterilized sawdust, and the moisture content of the mixture was adjusted to 60%-65%. The mixture was placed in a composting reactor and fermented in a laboratory setting, with stirring twice daily to maintain aerobic conditions. After three days of incubation, the temperature of the mixture reached 50°C.

[0049] 1.2 Isolation and screening of strains Take 1 g of the mixture during the high-temperature fermentation period, add 30 mL of sterile phosphate buffer, and vortex to prepare a bacterial suspension. Prepare a series of dilutions using a tenfold serial dilution method. Take 10 μL of each of the following solutions... -5 10 -6 10 -7 100 µL of isochronous dilutions were spread onto TSB-based solid plates. The plates were inverted and incubated at 37°C for 48–72 h. Vigorous single colonies were picked and repeatedly streaked onto solid plates for purification until the morphological characteristics of each colony were consistent and no other contaminating colonies appeared. By comparing growth performance, the strain exhibiting the best growth ability was named AT8. Figure 2 ).

[0050] 1.3 Validation of antibiotic resistance of the strain To verify the resistance of strain AT8 to multiple antibiotics, the Kirby-Bauer method was used for drug susceptibility testing. The purified strain was inoculated into liquid MH (Mueller-Hinton) medium and cultured at 37°C with shaking until the logarithmic growth phase. The bacterial suspension concentration was adjusted to 0.5 McFarland turbidity standard with sterile physiological saline. A sterile cotton swab was used to spread the bacterial suspension evenly onto the surface of an MH agar plate. After the plate surface had slightly dried, standard drug susceptibility discs (containing trimethoprim, enrofloxacin, tilmicosin, and azithromycin, respectively, with drug concentrations conforming to CLSI or relevant standards) were evenly spaced on the plate surface using sterile forceps. The plates were then incubated at 37°C for 16-18 hours. The results showed that strain AT8 exhibited significant resistance to trimethoprim (TMP), enrofloxacin (ENR), and tilmicosin (TIL) (no obvious inhibition zone), while it was sensitive to azithromycin (AZM) (forming a clear inhibition zone). Figure 3 ).

[0051] Example 2: Identification of the isolated strain The highly efficient antibiotic-degrading strain AT8, obtained from screening in Example 1, was identified. Genomic DNA was extracted from strain AT8, and PCR amplification and sequencing were performed using universal primers 27F and 1492R for bacterial 16S rDNA. The 16S rDNA gene sequence of strain AT8 was obtained, as shown in SEQ ID NO: 1 below:

[0052] The nearly full-length 16S rDNA sequence of AT8 (approximately 1.5 kb) obtained from sequencing (SEQ ID NO: 1) was subjected to BLAST homology comparison on the NCBI website. The comparison results showed that the strain with the highest homology to the AT8 gene in the database was... Staphylococcus pseudoxylosus (Sequence number: MH643903.1), with a similarity of 99.40%. Based on the 16S rRNA information, the AT8 strain can be preliminarily identified as belonging to... Staphylococcus A new species within the genus.

[0053] Furthermore, a phylogenetic tree was constructed using MAGA with neighbor-to-neighbor merging for closely related strains of AT8, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that AT8 does not cluster with other bacterial species, and... Staphylococcus pseudoxylosus The AT8 strain isolated in Example 1 is closely related to the 16S ribosomal RNA gene, partial sequence (Genebank sequence number: MH643903.1), which also proves that the AT8 strain is... Staphylococcus A new species within the genus.

[0054] Meanwhile, AT8 underwent whole-genome sequencing, and its genome sequence with the highest similarity was compared to it using FastANI software. Staphylococcus pseudoxylosus Genome-wide average nucleotide identity (ANI) analysis was performed on the genome of strain MH643903.1 (sequence number: MH643903.1). ANI is an indicator of nucleotide-level phylogenetic relationship between two genomes. ANI is defined as the average base similarity between homologous segments of two microbial genomes, characterized by high discriminative power between closely related species. The analysis results showed that the AT8 genome... Staphylococcus pseudoxylosus The genome ANI of (sequence number: MH643903.1) is only 91.27%, far lower than the 95% ANI value for the same species (see Jain C, Rodriguez-R LM, Phillippy AM, et al. Highthroughput ANI analysis of 90K prokaryotic genomes reveals clear speciesboundaries[J]. Nature Communications, 2018, 9(1): 5114, 95% is considered the threshold for determining whether they are the same species), therefore, AT8 is further determined to be... Staphylococcus A new strain of fungus belonging to the family.

[0055] Morphological and staining characteristics of strain AT8 were observed. After incubation on TSB solid medium at 37°C for 24 hours, strain AT8 formed round, smooth, moist, milky-white colonies. Gram staining was positive, and microscopic examination showed that the bacteria were spherical, mostly arranged in irregular clusters. Figure 4 Based on the bacterial growth morphology and referring to Bergey's Manual of Systematic Bacteriology and the Manual of Systematic Identification of Common Bacteria, and conforming to the physiological and biochemical characteristics of cocci, this strain was identified as *Staphylococcus pseudocarpa*. Staphylococcus pseudoxylosus ), and named Staphylococcus pseudoxylosus AT8, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number: GDMCC No: 67323, deposit date: November 20, 2025.

[0056] Example 3: Small-scale experiment on the treatment of pig manure compost containing antibiotic residues by Staphylococcus pseudotyphimurium AT8 3.1 Strain preparation: This example uses *Staphylococcus pseudocarpa* isolated and identified in Example 1. Staphylococcus pseudoxylosus AT8. This strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number: GDMCC No: 67323, deposit date: November 20, 2025.

[0057] 3.2 Preparation of Microbial Agent: The AT8 strain was inoculated into liquid seed culture medium for expansion culture to prepare a highly active microbial agent. The specific method is as follows: Prepare a liquid culture medium (components by weight percentage: peptone 1.0%, yeast extract 0.5%, sodium chloride 0.5%), adjust the pH to 7.0-7.2, and autoclave at 121℃ for 20 minutes. In a clean bench, the activated AT8 strain was inoculated into the sterilized culture medium and incubated in a constant temperature shaking incubator at 37℃ and 180 rpm for 24-36 hours, until the culture medium showed uniform turbidity (OD). 600 (Value stable). Plate count method was used to confirm that the viable bacteria concentration in the final inoculum reached 1×10⁻⁶. 9 CFU / mL or higher, for later use.

[0058] 3.3 Material Mixing and Inoculation: Take 10 kg of fresh pig manure containing antibiotic residues (preliminary measurements showed background concentrations of approximately 1.97 mg / kg for trimethoprim, 1.88 mg / kg for tilmicosin, and 1.91 mg / kg for enrofloxacin). Thoroughly mix the pig manure with 2 kg of sterilized sawdust. Adjust the initial moisture content of the mixture to 60%-65%. Then, uniformly spray the inoculum agent prepared in step 3.2 onto the mixture at a ratio of 0.5% of the total mass of the mixture, and stir thoroughly for 5-10 minutes to ensure uniform distribution of the inoculum agent, obtaining the final inoculated mixture. A parallel treatment without inoculation with inoculum agent AT8 was also set up as a blank control.

[0059] 3.4 Biodegradation Process and Monitoring: The final mixture and the blank control material were transferred to two identical 50 L self-made stainless steel aerobic composting reactors. The reactors were operated at room temperature (approximately 25°C). The automatic control system was activated, with intermittent stirring (frequency: 5 minutes every 6 hours, rotation speed 10 r / min) and intermittent forced ventilation (frequency: 10 minutes per hour, airflow 0.2 L air / (L material·min)). By adjusting the stirring and ventilation strategies, the core temperature of the material in the reactor was controlled to not exceed 65°C. The treatment cycle was set to 30 days. Representative samples were collected from each reactor periodically (on days 0, 3, 7, 14, 21, and 30) to monitor changes in the core temperature, moisture content, pH value, total nitrogen (TN), total phosphorus (TP), total potassium (TK), total organic matter content, seed germination index (GI), and concentrations of trimethoprim, enrofloxacin, and tilmicosin.

[0060] 3.5 Products and Emissions: After 30 days of aerobic composting, the reaction was completed. The treated solid material was discharged from the reactor outlet. Testing showed that in the treatment group inoculated with strain AT8, the residual concentrations of trimethoprim and tilmicosin in the products decreased to approximately 0.17 mg / kg and 0.13 mg / kg, respectively, with degradation rates exceeding 90% (compared to 75.57% and 74.23% in the control group, respectively). The residual concentration of enrofloxacin decreased to approximately 0.41 mg / kg, with a degradation rate exceeding 78% (compared to 66.43% in the control group). Figure 5 Meanwhile, the seed germination rate of the product was significantly improved, and the decomposition was good (Table 1). This product can be used directly or after further processing as a safe organic fertilizer or cultivation substrate for agricultural and forestry planting. The antibiotic residues in the blank control group were significantly higher than those in the inoculated group, and the decomposition was also poorer. The results of this experiment indicate that *Staphylococcus pseudocarpa* AT8 can effectively promote the harmless and resource-based treatment of pig manure containing multiple antibiotic residues.

[0061] Table 1. Operational effects after AT8 inoculation and pig manure composting.

[0062] Example 4: Pilot-scale experiment on the treatment of pig manure compost containing antibiotic residues by Staphylococcus pseudotyphimurium AT8 4.1 Experimental Overview This pilot-scale experiment was conducted at a large-scale pig farm in Beijing to verify the ability of Staphylococcus aureus AT8 enhanced composting technology to treat pig manure containing typical antibiotic residues. The system was designed to handle 3000 kg of wet pig manure, with a single-feed operation and aerobic fermentation. The total experimental period was 30 days.

[0063] 4.2 Preparation of inoculum and propagation of inoculum The strain used for enhancement in this experiment was *Staphylococcus pseudocarpa*. Staphylococcus pseudoxylosus AT8, its preservation information is the same as in Example 3.

[0064] Preparation of inoculum: Prepare a liquid expansion culture medium (components by weight percentage: peptone 1.0%, yeast extract 0.5%, sodium chloride 0.5%, glucose 0.2%, pH 7.0-7.2), and autoclave at 121℃ for 20 minutes. Inoculate with the activated inoculum under aseptic conditions and incubate at 37℃ and 180 rpm with constant temperature shaking for 36-48 hours, until the OD of the culture medium is reached. 600 The value is stable and the viable bacteria concentration is ≥1×10 9 CFU / mL was used to prepare a high-concentration bacterial agent for later use.

[0065] 4.3 Material mixing and inoculation Approximately 3000 kg of fresh pig manure containing antibiotic residues was collected daily (the background concentrations of trimethoprim, enrofloxacin, and tilmicosin were measured to be 1.5-2.0 mg / kg, 1.8-2.3 mg / kg, and 1.6-2.1 mg / kg, respectively). The pig manure was premixed with 600 kg of sterilized mushroom residue (used as a conditioner to adjust the C / N ratio and improve aeration). The initial moisture content of the mixture was adjusted to 60-65% by adding an appropriate amount of refluxed compost or water. Subsequently, the inoculum prepared that day was evenly sprayed onto the material at a ratio of 0.5% (approximately 18 L) of the total mass of the mixture, and then thoroughly mixed for 5 minutes using a twin-shaft mixer to form the inoculated fermented material.

[0066] 4.4 Biodegradation process and process control The inoculated mixture was fed into a trough-type aerobic fermentation reactor with an effective volume of 10 m³. The reactor was lined with stainless steel and equipped with an automatic turning, forced ventilation, and exhaust gas treatment system. Automatic turning was performed four times daily, with a 6-hour interval between each turn. The turning speed was adjustable to ensure loose material and uniform oxygen distribution. A forced ventilation mode with combined temperature feedback and time interval control was employed. When the core temperature of the pile exceeded 65°C, high-volume ventilation was activated to cool it down; when the temperature was between 55-65°C, intermittent ventilation (15 minutes per hour, air volume 0.3 m³ air / (m³ material·min)) was used to maintain the optimal aerobic environment. By adjusting the turning and ventilation frequency, the high-temperature period (>55°C) was ensured to last for at least 7 days to kill pathogens and weed seeds, while controlling the peak temperature to not exceed 65°C to protect the activity of functional microorganisms. Samples of the feed, reactor contents, and discharge were collected for key indicator analysis. The main data are shown in Table 2 below. Table 2. Composting operation effect under a treatment load of 3000 kg

[0067] Table 2 shows that the aerobic composting process enhanced with *Staphylococcus pseudocarpa* AT8 inoculant can stably treat pig manure containing multiple antibiotic residues on a pilot-scale of 3000 kg. The system can start up quickly and maintain high temperatures, achieving significant volume reduction while maintaining a stable degradation rate of over 90% for trimethoprim and tilmicosin, and nearly 80% for the more difficult-to-degrade enrofloxacin. The final product meets the requirements for organic fertilizer maturity and ecological safety. This pilot-scale experiment verifies the feasibility and reliability of this technical route from laboratory to engineering application.

[0068] 4.5 Products and Emissions After 30 days of intensive aerobic fermentation, approximately 1052 kg (dry basis) of mature compost was produced. The product is a dark brown, loose-textured, odorless solid material. The residues of the three target antibiotics were significantly lower than the relevant risk screening values, and the seed germination index (GI) was greater than 85%, indicating complete decomposition and low phytotoxicity. The product can be used directly for on-site fruit and vegetable cultivation or sold as raw material for commercial organic fertilizer. Water vapor and degradation exhaust gases generated during fermentation are treated by a biological filtration and deodorization system before being discharged in compliance with standards.

[0069] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0070] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A *Staphylococcus pseudocarpa* strain for degrading antibiotics, characterized in that, The *Staphylococcus pseudocarpa* is a member of the genus *Staphylococcus*. Staphylococcus The *Staphylococcus pseudocarpa*, having the 16S rDNA sequence shown in SEQ ID NO: 1, is named... Staphylococcus pseudoxylosus AT8 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 20, 2025, accession number: GDMCC No: 67323.

2. The *Staphylococcus pseudocarpa* according to claim 1, characterized in that, The antibiotic is one or more of the following: trimethoprim, tilmicosin, and enrofloxacin.

3. A formulation, characterized in that, The formulation contains *Staphylococcus pseudotruncatula* as described in claim 1 or 2.

4. The formulation according to claim 3, characterized in that, The formulation also includes excipients.

5. The formulation according to claim 3, characterized in that, The formulation is a liquid formulation and / or a lyophilized formulation, and the dosage form of the formulation is selected from: wettable powder, water-dispersible granules, suspension concentrate, water emulsion, granules, seed coating agent, or a combination thereof.

6. The use of Staphylococcus pseudotruncatula as described in claim 1 or 2, or the preparation as described in any one of claims 3-5, in the degradation of antibiotics in livestock and poultry excrement.

7. The application according to claim 6, characterized in that, The livestock and poultry mentioned are pigs; the excrement mentioned is feces.

8. A method for degrading antibiotics in livestock and poultry excrement, characterized in that, include: The livestock and poultry excrement is prepared into a mixture; Add Staphylococcus pseudothomolecularis according to claim 1 or 2, or the preparation according to any one of claims 3-5, to the mixture.

9. The method according to claim 8, characterized in that, The *Staphylococcus pseudotruncatula* or the preparation is added at a mass ratio of 0.5% to 1% of the mixture.

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