A strain of Stenotrophomonas maltophilia and its application in pollution control

Abstract

The present invention discloses a strain of Stenotrophomonas maltophilia and its application in the treatment of polluted environments. The present invention is the first to isolate and identify a strain of Stenotrophomonas maltophilia capable of degrading antibiotics from seawater samples. Studies have shown that the DA02 strain can degrade the antibiotics penicillin potassium, amoxicillin and ceftriaxone sodium, among which it has a highly efficient degradation effect on ceftriaxone sodium antibiotics, and also has a good degradation effect in high-concentration antibiotic drugs. Its degradation rate increases with the increase of antibiotic concentration, and the degradation effect remains stable over time. It shows that the DA02 strain, as a highly efficient ceftriaxone sodium degrading bacterium, is not affected by the concentration of the drug, and can be better applied to the degradation of high-concentration antibiotics in antibiotic-contaminated environments, providing more efficient treatment methods and approaches for antibiotic-contaminated environments.

Description

Technical Field

[0001] The present application relates to the technical field of microorganisms and environmental pollution control, and in particular to a strain of Stenotrophomonas maltophilia and its application in the control of polluted environments. Background Art

[0002] Approximately 100,000 tons of antibiotics are released into the environment annually worldwide through discharges from medical wastewater, aquaculture (feed additives), the pharmaceutical industry, and agricultural runoff. Antibiotic contamination can induce the spread of antimicrobial resistance genes (ARGs), threatening ecosystems and human health. Residues of tetracyclines, sulfonamides, cephalosporins, and β-lactam antibiotics are commonly found in water bodies (such as rivers and groundwater) and soil. Ceftriaxone sodium, a third-generation cephalosporin antibiotic with broad-spectrum anti-infective properties, is a serious problem of overuse. Ceftriaxone sodium can induce the production of extended-spectrum β-lactamases (ESBLs) in bacteria such as Escherichia coli and Klebsiella pneumoniae, leading to clinical treatment failure. It can also inhibit aquatic microbial communities, disrupt nitrogen / phosphorus cycles, and pose human health risks, such as allergic reactions and intestinal flora disturbances. Long-term low-dose exposure can increase the risk of antibiotic resistance genes establishing themselves. Ceftriaxone sodium has been detected in wastewater at concentrations ranging from 1.2 to 5.6 μg / L (Zhang et al., 2021).

[0003] The degradation pathways of ceftriaxone sodium in the environment are: (1) Traditional treatment methods, such as chlorine disinfection, activated sludge removal or photocatalysis (TiO2), but their degradation rate is low, toxic byproducts (such as halogenated disinfection byproducts) are easily generated, the risk of ARGs enrichment is high, the cost is high, and it is difficult to scale up. (2) Biodegradation technology, which can control antibiotic pollution through microbial strains, such as the use of Ochrobacterium ( Ochrobactrum sp.) can degrade ceftriaxone sodium by up to 85% within 72 hours (Feng et al., 2020). However, high concentrations of antibiotics inhibit microbial activity, limiting their ability to degrade antibiotics and their application in environmental remediation. Currently, there is a lack of microbial strains capable of efficiently degrading ceftriaxone sodium.

[0004] Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ) is a multidrug-resistant (MDR) Gram-negative bacterium that produces β-lactamases (L1 and L2), making it resistant to multiple antibiotics and making infection treatment extremely difficult. Existing studies have reported that Stenotrophomonas maltophilia is used as a drug-resistant bacterium or to promote plant growth, and few studies have revealed its ability to degrade antibiotics. In order to provide more diverse sources of Stenotrophomonas maltophilia, it is necessary to develop more microbial strains that can efficiently degrade antibiotics and tolerate higher antibiotic concentrations, which is of great significance for the environmental remediation of antibiotic pollution. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortage of existing microbial strain resources that are highly efficient in degrading antibiotics and can tolerate higher antibiotic concentrations, and to provide a strain of Stenotrophomonas maltophilia and its application in the treatment of polluted environments.

[0006] The purpose of the present invention is to provide a Stenotrophomonas maltophilia DA02 strain.

[0007] Another object of the present invention is to provide an application of the Stenotrophomonas maltophilia DA02 strain.

[0008] Another object of the present invention is to provide a biological preparation.

[0009] Another object of the present invention is to provide an antibiotic degradation product.

[0010] Another object of the present invention is to provide a method for degrading antibiotics.

[0011] Another object of the present invention is to provide a method for environmental remediation of antibiotic pollution.

[0012] The last object of the present invention is to provide an application of the Stenotrophomonas maltophilia DA02 strain.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] The present invention provides an antibiotic biodegrading bacterium isolated from a marine sample, Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ) strain DA02 was obtained and deposited with the Guangdong Provincial Center for Microbial Culture Collection on April 25, 2025, with accession number GDMCC No: 66215. The address of the deposit is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. This invention is the first to isolate and identify a strain of Stenotrophomonas maltophilia capable of degrading antibiotics from seawater samples, breaking the limitation of existing reports that Stenotrophomonas maltophilia is antibiotic-resistant. Studies have shown that the DA02 strain can effectively degrade penicillin potassium, amoxicillin, and ceftriaxone sodium antibiotics. It has a highly efficient degradation effect on ceftriaxone sodium, with a degradation rate of up to 100%. It also has a good degradation effect at high concentrations (300 mg / L) of ceftriaxone sodium. The degradation rate increases with increasing antibiotic concentration, and the degradation effect remains stable over time, still being able to degrade 99.12% of ceftriaxone sodium after 120 hours. The results showed that the DA02 strain is an efficient ceftriaxone sodium degrading bacterium and is not affected by the drug concentration. It can be better applied to the degradation of high-concentration antibiotics in antibiotic-contaminated environments, providing more efficient methods and approaches for the treatment of antibiotic-contaminated environments.

[0015] The present invention provides application of the DA02 strain in biodegrading antibiotics.

[0016] The present invention provides use of the DA02 strain in preparing an antibiotic degradation product.

[0017] Preferably, the antibiotic is one or more of ceftriaxone sodium, penicillin potassium, and amoxicillin.

[0018] More preferably, the antibiotic is ceftriaxone sodium.

[0019] The present invention provides a biological preparation containing a DA02 strain or a bacterial liquid thereof.

[0020] Preferably, the bacterial solution is prepared by placing DA02 in LB culture medium and shaking the culture at 30° C. and 180 rpm overnight to obtain the bacterial solution.

[0021] More preferably, the OD value of the bacterial solution is not less than 1.0.

[0022] The present invention provides an antibiotic degradation product containing the above biological preparation.

[0023] The present invention provides a method for degrading antibiotics, which uses DA02 strains or biological preparations to treat the antibiotics.

[0024] Preferably, the antibiotic is one or more of ceftriaxone sodium, penicillin potassium, and amoxicillin.

[0025] More preferably, the antibiotic is ceftriaxone sodium.

[0026] The present invention also provides an environmental treatment method for antibiotic pollution, which uses the DA02 strain or biological agents to treat polluted environmental samples.

[0027] Preferably, the polluted environment is an environment contaminated with ceftriaxone sodium at a concentration greater than 100 mg / L.

[0028] The present invention has the following beneficial effects:

[0029] This study, published in Nature Communications, isolated and identified a strain of Stenotrophomonas maltophilia capable of degrading antibiotics from seawater samples for the first time, overcoming the limitation of existing reports that Stenotrophomonas maltophilia harbors antibiotic resistance. Studies have shown that strain DA02 is highly effective at degrading penicillin potassium, amoxicillin, and ceftriaxone sodium, with a degradation rate reaching 100%. This degradation rate is also highly effective at high concentrations of ceftriaxone sodium (300 mg / L). The degradation rate increases with increasing antibiotic concentration, and the degradation rate remains stable over time, still capable of degrading 99.12% of ceftriaxone sodium after 120 hours. This demonstrates that strain DA02 is a highly efficient ceftriaxone degrader, unaffected by drug concentration, and is therefore well-suited for the degradation of high-concentration antibiotics in antibiotic-contaminated environments, providing additional, more efficient methods and approaches for the remediation of antibiotic-contaminated environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the colony morphology of the DA02 strain.

[0031] Figure 2 This is the phylogenetic tree of the DA02 strain.

[0032] Figure 3 This is the genome circle map of the DA02 strain.

[0033] Figure 4 This is the degradation result of ceftriaxone sodium by DA02 strain.

[0034] Figure 5 The results show the effects of different temperatures (A), initial pH (B) and initial drug concentrations (C) on the growth of DA02 strain.

[0035] Figure 6 This is a graph showing the effect of different drug concentrations on CTX degradation. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0038] Example 1 Isolation and identification of bacteria

[0039] 1. Isolation of bacteria

[0040] The samples were collected from the coastal seawater of Zhanjiang. After sampling, they were pretreated. In order to remove non-resistant bacteria and select bacteria that degrade β-lactam antibiotics, 1 mL of seawater was placed in 50 mL of LB broth (purchased from Beijing Luqiao Technology Co., Ltd.) containing 100 mg / L amoxicillin (purchased from Shanghai Macklin Company) and incubated at 30°C and 180 rpm overnight. 1 mL of the bacterial solution was centrifuged at 4°C and 12,000 rpm for 30 minutes, the supernatant was discarded, and the bacterial solution was resuspended in MSM medium (purchased from Beijing Coolabor Company) containing 100 mg / L amoxicillin and the bacterial solution concentration was adjusted until the OD value reached 0. 600 With nm = 1.0, inoculate 50 mL of the aforementioned MSM medium at a 1% (v / v) inoculum. After culturing at 30°C and 180 rpm for 5 days, spread 100 μL of the bacterial solution onto MSM agar solid medium containing 100 mg / L amoxicillin and incubate at 30°C for 48 hours. Pick a single colony and culture it in LB broth. After shaking overnight, add 20% (v / v) glycerol, mix thoroughly, and store at -20°C.

[0041] 2. Identification of bacteria

[0042] (1) Morphological identification

[0043] The colony morphology was observed by plate streak method. Figure 1 As shown, pinpoint-sized transparent colonies appeared on LB plate medium.

[0044] (2) Molecular identification

[0045] Single colonies were selected, and DNA was extracted using the boiling method. The DNA was then sent to Shanghai Sangon Biotechnology Service Co., Ltd. for 16S rRNA sequencing. The entire genome was sequenced by Sangon (Shanghai, China). After sequencing, BLAST comparisons were performed, and a phylogenetic tree was constructed.

[0046] The NCBI Prokaryotic Genome Annotation Pipeline (NCBI-PGAP) was used to predict the assembly of genetic elements. The genome of strain DA02 was mapped using CGView software. The predicted CDSs were compared and functionally annotated with the COG (Clusters of Orthologous Groups) and GO (Gene Ontology) databases using the Blastp (BLAST 2.3.0) sequence comparison tool. Furthermore, antibiotic resistance genes (ARGs) were predicted using the Comprehensive Antibiotic Research Database (CARD).

[0047] The phylogenetic tree results are as follows Figure 2 As shown, this strain is different from PQ451722.1 Stenotrophomonas maltophiliastrain MEPP0222 had a high homology, and combined with morphological identification, the taxonomic status of this strain was identified as Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ), and named it DA02 strain, and deposited it in Guangdong Provincial Microbiological Culture Collection Center on April 25, 2025, with the deposit number GDMCC No: 66215, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0048] The genome map of the sequenced DA02 strain is as follows Figure 3 The complete genome of this strain is shown to be 4,786,356 bp with a GC content of 66.58%. It contains 4,253 predicted genes, with a total predicted coding gene length of 4,199,394 bp. A total of 80 non-coding non-cDNAs, including 73 tRNAs and 7 rRNAs, are predicted in the genome. Its NCBI genome accession number is JBOBQJ000000000.

[0049] Example 2 Degradation of antibiotics by DA02 strain

[0050] The DA02 strain was placed in LB medium and shaken at 30°C and 180 rpm overnight. 3 mL of the bacterial solution was centrifuged at 4°C and 8000 rpm for 30 min and resuspended in MSM medium containing 100 mg / L antibiotics. The bacterial solution concentration was adjusted to OD 600 With nm = 1.0, a 1% inoculum was added to 3 mL of MSM culture medium containing 100 mg / L antibiotics. A control group without bacteria was established, and three replicates were performed for both the experimental and control groups. The culture medium was shaken at 30°C, 180 rpm, and degraded for 12 hours. The antibiotic concentration in the MSM culture medium was then measured. The above method was used to test the degradation of penicillin potassium (purchased from Shanghai Macklin), amoxicillin, and ceftriaxone sodium (purchased from Shanghai Macklin). The degradation ability of the DA02 strain against these antibiotics was tested.

[0051] Subsequently, quantitative detection of antibiotics was performed using high-performance liquid chromatography (HPLC, SHIMADZU LC-16P, Japan). The column used was a ShimNex CS C18 (4.6 × 250 mm, 5 μm, SHIMADZU, Japan). Mobile phases A and B consisted of ultrapure water + 0.1% formic acid and acetonitrile + 0.1% formic acid, respectively. All samples were filtered through a 0.22 μm filter membrane before injection. The analytical conditions are shown in Table 1.

[0052] Table 1 HPLC analysis conditions

[0053]

[0054] The detection conditions of penicillin potassium are as follows: mobile phase A:B=40%:60%, flow rate of 1 mL / min, detection wavelength of 210 nm, injection volume of 20 μL each time, column temperature the same as ambient temperature, and detection time of 13 min.

[0055] The detection conditions for amoxicillin are as follows: mobile phase A:B=80%:20%, flow rate of 1 mL / min, detection wavelength of 210 nm, injection volume of 20 μL each time, column temperature the same as ambient temperature, and detection time of 10 min.

[0056] The detection conditions of ceftriaxone sodium are as follows: mobile phase A:B=70%:30%, flow rate of 1 mL / min, detection wavelength of 254 nm, injection volume of 10 μL each time, column temperature the same as ambient temperature, and detection time of 6 min.

[0057] The antibiotic concentrations of the experimental group and the blank control group at 0 h and 12 h were measured by liquid chromatography, and the degradation capacity of the strain was calculated according to the following formula:

[0058] Biodegradation rate (%) = (C CK -C t ) / C0×100%

[0059] Where: C CK is the negative control antibiotic concentration at 12 h (mg / L);

[0060] C t is the antibiotic concentration after biodegradation (mg / L);

[0061] C0 is the antibiotic concentration in the culture medium at 0 h.

[0062] The test results showed that the DA02 strain had a high efficiency in degrading ceftriaxone sodium, and could degrade 100% of ceftriaxone sodium within 12 hours. However, the DA02 strain had a poor degradation effect on penicillin potassium and amoxicillin, only degrading 3.13% of penicillin potassium and 11.35% of amoxicillin.

[0063] Example 3: Efficient degradation of ceftriaxone sodium by DA02 strain

[0064] The DA02 strain was placed in LB medium and shaken at 30°C and 180 rpm overnight. 50 mL of the bacterial solution was centrifuged at 4°C and 8000 rpm for 30 min and resuspended in MSM medium containing 100 mg / L ceftriaxone sodium. The bacterial solution concentration was adjusted to OD 600nm = 1.0, and a 1% inoculum was added to 50 mL of MSM culture medium containing 100 mg / L ceftriaxone sodium. A control group without bacterial inoculation was set up, and three replicates were performed for both the experimental and control groups. The cells were placed in a shaker and degraded at 30°C, 180 rpm for 120 hours. The ceftriaxone sodium concentration was measured every 12 hours by high-performance liquid chromatography using the same method as in Example 2, and a degradation curve was plotted.

[0065] The degradation curve drawn is as follows Figure 4 As shown in the figure, the results showed that the DA02 strain had a good degradation effect on ceftriaxone sodium at different times. The degradation rate was 94.19% at 72 h, which would decrease slightly. At 120 h, the degradation rate could reach 99.12%, which was an efficient degradation effect on ceftriaxone sodium.

[0066] Example 4 Effects of different environmental factors on the growth and degradation of DA02 strain

[0067] 1. Effects of different environmental factors on the growth of DA02

[0068] The DA02 strain was placed in LB medium and shaken at 30°C and 180 rpm for 12 h. After taking it out, the OD 600 To 1.0. Inoculate the bacterial solution into LB medium at a ratio of 1% (v / v) and measure the OD every 2 hours. 600 The growth curves of the DA02 strain were plotted at different culture temperatures (20, 25, 30, 35, and 40°C), initial pH values ​​(5, 6, 7, 8, and 9), and initial antibiotic ceftriaxone sodium concentrations (0, 50, 100, 200, and 300 mg / L).

[0069] The effects of different environmental factors on the growth of DA02 strain were tested. Figure 5 As shown, the DA02 strain grew best at 30°C, followed by 35°C, and had difficulty growing rapidly at 20°C and 40°C ( Figure 5 A in the figure); it grows best at an initial pH of 7, but hardly grows at pH 5 ( Figure 5 B); DA02 strain has excellent tolerance to ceftriaxone sodium and can grow well even in a medium containing 300 mg / L ceftriaxone sodium ( Figure 5 C in the figure). Among the experimental groups with added antibiotics, the group containing 100 mg / L ceftriaxone sodium grew most vigorously.

[0070] 2. Effects of different ceftriaxone sodium concentrations on DA02 degradation

[0071] Bacteria were cultured according to the optimal growth conditions obtained by the above screening, and the degradation effect of the DA02 strain on a higher concentration of ceftriaxone sodium was detected according to the method of Example 3. At the same time, a negative control group with poor degradation effect on a higher concentration of ceftriaxone sodium was set up, and a degradation curve was drawn.

[0072] The results of the test are as follows Figure 6 As shown, the DA02 strain has a certain tolerance to ceftriaxone sodium at concentrations of 100, 200, and 300 mg / L, and has a good degradation effect; among them, it still has good resistance and degradation effect to ceftriaxone sodium up to 300 mg / L, and its degradation rate also increases with the increase of antibiotic concentration. It can be used for the rapid degradation of high-concentration ceftriaxone sodium, providing more efficient degradation bacteria for the pollution of antibiotic ceftriaxone sodium in the environment.

[0073] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ) DA02 strain, characterized in that This strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 25, 2025, with the collection number GDMCC No: 66215.

2. The use of the DA02 strain according to claim 1 in biodegradation of antibiotics, characterized in that: The antibiotics are one or more of ceftriaxone sodium and amoxicillin.

3. The use of the DA02 strain according to claim 1 in the preparation of antibiotic degradation products, characterized in that: The antibiotics are one or more of ceftriaxone sodium and amoxicillin.

4. A biological agent, characterized in that Containing the DA02 strain according to claim 1 or its bacterial liquid.

5. An antibiotic degradation product, characterized in that: Containing the preparation according to claim 4; the antibiotics degraded by the product are one or more of ceftriaxone sodium and amoxicillin.

6. A method for degrading antibiotics, characterized in that: The antibiotic is treated with the DA02 strain according to claim 1 or the preparation according to claim 4; the antibiotic is one or more of ceftriaxone sodium and amoxicillin.

7. A method for environmental remediation of antibiotic pollution, characterized in that: The DA02 strain according to claim 1 or the preparation according to claim 4 is used to treat the polluted environment; the antibiotic is one or more of ceftriaxone sodium and amoxicillin.

8. The method according to claim 7, characterized in that: The polluted environment is the pollution of ceftriaxone sodium with a concentration greater than 100 mg / L in the environment.

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