CN113308413B - Fluoroquinolone antibiotic degrading bacterium and application thereof in compost - Google Patents
Fluoroquinolone antibiotic degrading bacterium and application thereof in compost Download PDFInfo
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- CN113308413B CN113308413B CN202110782153.6A CN202110782153A CN113308413B CN 113308413 B CN113308413 B CN 113308413B CN 202110782153 A CN202110782153 A CN 202110782153A CN 113308413 B CN113308413 B CN 113308413B
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention relates to the technical field of microorganisms, in particular to a Brucella WXX-3 strain and application thereof in degrading fluoroquinolone antibiotics. The preservation number of the strain provided by the invention is CGMCC NO.22259. The WXX-3 strain provided by the invention can efficiently degrade fluoroquinolone antibiotics, and the embodiment shows that the strain is added into an LB liquid culture medium containing 20mg/L ciprofloxacin, and the highest degradation rate of the ciprofloxacin after light-resistant culture reaches 44.7%; in addition, the strain is inoculated into livestock and poultry manure for propagation for 8-10 days, and then conditioners such as sawdust, straws and the like are added for conventional composting for 30 days, so that the degradation rate of ciprofloxacin can reach 85.7%; meanwhile, the compost prepared by the strain can degrade antibiotic resistance genes, so that the safety of compost products is improved, and the strain is suitable for large-scale popularization and application.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a fluoroquinolone antibiotic degrading bacterium and application thereof in compost.
Background
In recent years, the risk of contamination with antibiotics and resistance genes has received increasing attention. By 2030, the consumption of antibiotics in livestock and poultry farming worldwide will reach 100 million tons. However, most antibiotics are difficult to be absorbed by the intestinal tract of animals, and about 30% to 90% of the antibiotics are excreted out of the body along with the feces and urine of the animals. The investigation shows that the antibiotic is detected in animal excrement, wastewater, manure and soil, and the concentration can reach the level of mg/kg. Antibiotic residues can also lead to the enrichment and spread of resistant bacteria and resistant genes in the environment, exacerbating the ecological stress and risks to human health.
The compost is a main means for realizing harmless and recycling treatment of the livestock and poultry manure, and can effectively remove part of antibiotics. Researches show that the compost can basically remove tetracycline antibiotics, sulfonamide antibiotics and macrolide antibiotics, but is difficult to remove fluoroquinolone antibiotics such as ciprofloxacin, oxyfloxacin and the like. Microbial degradation is an effective way for removing antibiotics, for example, chinese patent CN106929442A discloses a strain of Ochrobactum sp JOB which can remove up to 30% of ciprofloxacin in liquid, and the strain is prepared into an embedding agent which can be used for removing fluoroquinolone antibiotics in water and soil.
However, few reports about the microbial agents degrading antibiotics in the compost exist, and the high temperature generated by the compost can inhibit the activity of the antibiotic degrading bacteria, so that the effect of the microbial agents in the practical application is poor. In addition, the continuous existence of antibiotics can also cause the pollution of resistance genes, and meanwhile, the antibiotic degradation strain carries the resistance genes, and the strain can also have the risk of the pollution of the resistance genes in the application. Therefore, a strain capable of efficiently degrading the fluoroquinolone antibiotics needs to be screened, and a method for efficiently and safely utilizing the fluoroquinolone antibiotics in compost is provided.
Disclosure of Invention
In order to solve the problems, the invention provides a fluoroquinolone antibiotic degrading bacterium and application thereof in compost. The WXX-3 strain provided by the invention can efficiently degrade fluoroquinolone antibiotics, and the compost prepared by the strain has higher safety.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a Brucella (Brucella sp.) WXX-3 strain, wherein the preservation number of the strain is CGMCCNO.22259.
Preferably, the nucleotide sequence of 16S rDNA of said strain is as shown in SEQ ID NO:1 is shown.
The invention also provides a fermentation inoculant which comprises the strain.
Preferably, the viable count of the strain in each g or each mL of the zymophyte agent is (1.5-2.6) multiplied by 10 10 CFU。
The invention also provides a preparation method of the fermentation inoculant, which comprises the following steps:
and (3) inoculating the strain into an LB culture medium, and culturing for 24-40 h to obtain the fermentation inoculum.
Preferably, the temperature of the culture is 20 to 40 ℃.
The invention provides the application of the bacterial strain or the fermentation inoculant prepared by the preparation method in degrading antibiotics and/or antibiotic resistance genes.
Preferably, the antibiotic comprises a fluoroquinolone antibiotic;
the fluoroquinolone antibiotics comprise: one or more of ofloxacin, norfloxacin, ciprofloxacin and enrofloxacin.
The invention also provides a composting method for degrading the fluoroquinolone antibiotics and/or the fluoroquinolone antibiotic resistance genes, which comprises the following steps:
mixing the fermentation inoculum with the fecal raw material, carrying out propagation at 20-40 ℃ for 8-10 days, adjusting the carbon-nitrogen ratio, and fermenting for 30 days to obtain the compost.
Preferably, the effective viable count of the strain of claim 1 in the material obtained by mixing the fermentation inoculum and the fecal raw material is (1.5-26) x 10 9 CFU/kg。
Has the advantages that:
the invention provides a Brucella (Brucella sp.) WXX-3 strain, wherein the preservation number of the strain is CGMCC NO.22259. The WXX-3 strain provided by the invention can efficiently degrade fluoroquinolone antibiotics, and as can be seen from the examples, the strain is added into an LB liquid culture medium containing 20mg/L ciprofloxacin, and the highest degradation rate of the ciprofloxacin after light-shielding culture reaches 44.7%; in addition, the strain is inoculated into livestock and poultry manure for propagation for 8-10 days, and then conditioners such as sawdust, straws and the like are added for conventional composting for 30 days, so that the degradation rate of ciprofloxacin can reach 85.7%; meanwhile, the compost prepared by the strain can degrade antibiotic resistance genes, so that the safety of compost products is improved, and the strain is suitable for large-scale popularization and application.
Description of biological preservation
Brucella (Brucella sp.) WXX-3 strain is preserved in China general microbiological culture Collection center (CGMCC) at 29.04.29.2021, with the preservation address of No. 3 Xilu-Shih 1 of Beijing republic of the rising area and the preservation number of CGMCC NO.22259.
Drawings
FIG. 1 is the characterization and hemolytic assay results for the WXX-3 strain of example 1, wherein A is the macroscopic morphology of the strain; b is the microscopic morphology of the strain; c is a hemolytic reaction result, wherein 1 is a negative control bacterium: listeria lnnock (Listeria innocula); 2 is a positive control bacterium: staphylococcus aureus (Staphylococcus aureus); 3 is a sample: a WXX-3 strain; d is a bar graph of the effect of temperature on strain growth;
FIG. 2 is a phylogenetic tree of WXX-3 strain in example 1;
FIG. 3 shows the degradation of ciprofloxacin by WXX-3 strain in a liquid environment in example 2;
FIG. 4 is a graph showing the degradation of ciprofloxacin by the WXX-3 strain in pig manure compost in example 3;
FIG. 5 is a graph showing the abundance of fluoroquinolone antibiotic resistance genes in compost.
Detailed Description
The invention provides a Brucella (Brucella sp.) WXX-3 strain, wherein the preservation number of the strain is CGMCCNO.22259.
The WXX-3 strain of the invention preferably has the following properties:
(1) The nucleotide sequence of 16S rDNA of the strain is shown in SEQ ID NO:1, and the following steps: GAGCGCGTAGCAATACGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCATCACTAGGGAATAACTCAGGGAAACTTGTGCTAATACCCTATACGACCGAGAGGTGAAAGATTTATCGGTGATGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGATTTACTGGGCGTAAAGCGCACGTAGGCGGGCTAATAAGTCAGGGGTGAAATCCCGGGGCTCAACCCCGGAACTGCCTTTGATACTGTTAGTCTTGAGTATGGAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTTGGGGAGTTTACTCTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATCCCGATCGCGGTTAGTGGAGACACTTTCCTTCAGTTCGGCTGGATCGGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGCACGCGAGTGTGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGCGCTGTGCTAACCGCAA;
(2) After the bacterial strain is cultured on an LB solid culture medium for 24 hours, the bacterial colony is light yellow, the surface is wet, the bacterial colony is opaque, and the edge is neat;
(3) The thallus under the optical microscope is rod-shaped, the size is 0.4-0.6 Mum multiplied by 0.8-2.4 Mum, and the thallus is arranged singly or in pairs and is gram-negative;
(4) The hemolytic reaction of the strain is negative;
(5) The strain is suitable for growth at the temperature of 20-40 ℃ and can be inactivated when the temperature exceeds 50 ℃.
The WXX-3 strain provided by the invention can efficiently degrade fluoroquinolone antibiotics, and meanwhile, compost prepared by the strain can degrade antibiotic resistance genes, so that the safety of compost products is improved, and the strain is suitable for large-scale popularization and application.
The invention also provides a fermentation inoculant which comprises the strain.
In the present invention, the number of viable bacteria of the above-mentioned strain per g or per mL of the fermentation broth is preferably (1.5 to 2.6). Times.10 10 CFU, more preferably 2X 10 10 CFU。
The invention also provides a preparation method of the fermentation inoculant, which comprises the following steps:
and (3) inoculating the strain into an LB culture medium, and culturing for 24-40 h to obtain the fermentation inoculum.
In the present invention, the temperature of the culture is preferably 20 to 40 ℃, more preferably 20 to 25 ℃; the culturing mode preferably further comprises: activating in LB liquid culture medium for 12h, then transferring the bacteria liquid activated to logarithmic phase into fresh LB liquid culture medium again according to the inoculation amount of 1% (v/v), and carrying out amplification culture for 24h. The source of the LB medium is not particularly required in the present invention, and it is prepared using commercially available products well known to those skilled in the art or using preparation methods well known to those skilled in the art.
The invention also provides the application of the bacterial strain or the fermentation inoculant prepared by the preparation method in degrading antibiotics and/or antibiotic resistance genes.
In the present invention, the antibiotic preferably includes fluoroquinolone antibiotics; the fluoroquinolone antibiotics preferably include: one or more of ofloxacin, norfloxacin, ciprofloxacin and enrofloxacin, more preferably ciprofloxacin.
The strain and the fermentation inoculant containing the strain can efficiently degrade fluoroquinolone antibiotics, and the embodiment shows that the highest degradation rate of ciprofloxacin after light-shielding culture reaches 44.7 percent by adding the strain into an LB liquid culture medium containing 20mg/L ciprofloxacin; in addition, the strain is inoculated into livestock and poultry manure for propagation for 8-10 days, and then conditioners such as sawdust, straws and the like are added for conventional composting for 30 days, so that the degradation rate of ciprofloxacin can reach 85.7%; in addition, the compost prepared by the strain can degrade antibiotic resistance genes, thereby improving the safety of compost products and being suitable for large-scale popularization and application.
The invention also provides a composting method for degrading the fluoroquinolone antibiotics and/or the fluoroquinolone antibiotic resistance genes, which comprises the following steps:
mixing the fermentation inoculum with the fecal raw material, carrying out propagation at 20-40 ℃ for 8-10 days, adjusting the carbon-nitrogen ratio, and fermenting for 30 days to obtain the compost.
In the invention, the effective viable count of the bacterial strain in the material mixed by the zymophyte agent and the fecal raw material is preferably (1.5-26) multiplied by 10 9 CFU/kg, more preferably (7.5-13) prepared roll10 9 CFU/kg, more preferably 1X 10 10 CFU/kg。
In the present invention, the carbon-nitrogen ratio is preferably (23 to 27): 1, more preferably 25; the substance for adjusting the carbon-nitrogen ratio preferably comprises: wood chips and/or straw.
In the present invention, it is preferable to adjust the carbon-nitrogen ratio and also include: the water content of the compost mixture was adjusted to 55%. The fermentation mode is not particularly required, and the fermentation is preferably carried out in a manner of windrow compost fermentation.
The compost preparation method provided by the invention is simple to operate and convenient to control, the fermentation inoculum and the excrement are mixed at the early stage of compost fermentation, propagation culture is carried out for 8-10 days, and the potential of the strain for degrading the fluoroquinolone antibiotics can be effectively exerted; after propagation culture, conventional compost fermentation is carried out (the fermentation temperature is 60-70 ℃), and strains in the fermentation inoculum are killed at high temperature, so that the pollution of antibiotic resistance genes carried by the strains is avoided, the production process cannot cause secondary pollution, and the method is suitable for large-scale popularization and application.
In order to further illustrate the present invention, a fluoroquinolone antibiotic degrading bacterium and its application in compost provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Isolation, purification and characterization of the strains
The preparation of the culture medium used in the invention is as follows:
inorganic salt culture medium: k 2 HPO 4 5.8g，KH 2 PO 4 4.5g，(NH 4 ) 2 SO 4 2.0g，MgCl 2 0.16g，CaCl 2 0.02g，Na 2 MoO 4 ·2H 2 O 0.0024g，KNO 3 0.0012g，FeCl 3 0.0018g，MnCl 2 ·2H 2 0.0015g of O, adding ultrapure water to 1000mL, and adjusting the pH value to 7.0-7.5.
Separating a culture medium: 20g of agar powder is added to each liter of the inorganic salt culture medium.
LB liquid medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, adding ultrapure water to 1000mL, and adjusting the pH to 7.0-7.5.
LB solid medium: 20g of agar powder was added per liter of LB liquid medium.
Domestication, separation and purification of the strain:
first, about 5g of compost sample (organic fertilizer stacks from Jiangxi Zhenghe ecological agriculture Co., ltd.) was added to an inorganic salt medium containing 5mg/L ciprofloxacin, and the mixture was placed in a constant temperature shaker (30 ℃,180 rpm) and cultured for 7 days in the dark. After the culture is stood still, the supernatant is transferred to an inorganic salt culture medium containing 10mg/L ciprofloxacin, and after 7 days of light-shielding culture, the supernatant is transferred to an inorganic salt culture medium containing 20mg/L ciprofloxacin again for 7 days. 0.2ml of the culture was spread on a separation medium containing 20mg/L ciprofloxacin, and incubated at constant temperature for 48 hours. Selecting a single colony, inoculating the single colony into an inorganic salt culture medium test tube (containing ciprofloxacin and 20 mg/L), culturing for 7 days at constant temperature, measuring the antibiotic concentration by using a high performance liquid chromatography, and selecting a pure strain brucella WXX-3 with high antibiotic degradation capacity from the antibiotic concentration.
Screening for morphological and physiological-biochemical reaction characteristics of strains
After the strain WXX-3 is cultured on an LB solid medium for 24 hours, colonies are light yellow, the surface is wet, the colonies are not transparent, and the edges are neat (shown as A in figure 1); the thallus under the optical microscope is rod-shaped, the size is 0.4-0.6 μm multiplied by 0.8-2.4 μm, and the thallus is singly or pairwise arranged and gram-negative (see B in figure 1); the hemolytic reaction of the strain was negative (see C in fig. 1); the strain is suitably grown at a temperature of 20 to 40 ℃ and the temperature is above 50 ℃ to inactivate the strain (see D in FIG. 1). The physiological and biochemical characteristics of the strain are automatically identified by using a VITEK2 GN gram-negative bacteria identification card, and the specific identification result is shown in Table 1.
TABLE 1 physiological and biochemical characteristics of Strain WXX-3
Note: "+", positive; "-", negative.
Molecular biological identification of the screened strains:
an animal genome DNA extraction kit (TSP 201-200, beijing Optimalaceae, biotechnology Limited) is adopted to extract the total DNA of the strain WXX-3, and the specific steps are as follows: cell lysis, activation, elution and recovery. PCR amplification was then performed with 16S rDNA universal primer 27F/1492R. The amplification system comprises 1 mu L of upstream primer, 1 mu L of downstream primer, 1 mu L of DNA template, 27 mu L of gold medal mix, 30ul in total, pre-denaturation at 98 ℃ for 3min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s, extension at 72 ℃ for 10s, and 35 cycles. The PCR-amplified fragment was then subjected to 16S rDNA sequencing, and comparative analysis of the sequencing results (shown in SEQ ID NO: 1) in GenBank showed that strain WXX-3 was highly similar to Brucella sp. And belongs to Brucella in molecular phylogenetic taxonomy (FIG. 2).
Example 2
Research on degradation effect of Brucella WXX-3 on ciprofloxacin
Amplification culture of Brucella WXX-3
Firstly, the WXX-3 strain obtained by screening in example 1 is activated in LB liquid culture medium for 12h, then the bacterial liquid activated to logarithmic phase is transferred to fresh LB liquid culture medium again according to the inoculum size of 1% (volume concentration), the quantity of the bacterial body reaches 2X 10 after 24h of amplification culture 10 CFU/mL to obtain the fermentation inoculum.
Degradation effect of WXX-3 strain on ciprofloxacin in liquid environment
2 treatments are set: 1) CFC is LB liquid culture medium containing 20mg/L ciprofloxacin; 2) CFC + WXX-3, the strain after the expansion culture is inoculated into LB liquid culture medium containing 20mg/L ciprofloxacin according to the inoculation amount of 1 percent (volume concentration).
The reaction flask was covered with tinfoil paper to protect from light, and cultured in a constant temperature incubator at 30 ℃ and 180r/min for sampling times of 0, 2, 4, 6, 8, 10 and 14 days, respectively, to determine the ciprofloxacin concentration. The results are shown in Table 2 and FIG. 3.
TABLE 2 degradation Effect of ciprofloxacin for different treatment groups (unit: mg/L)
| Days/ |
0 | 2 | 4 | 6 | 8 | 10 | 14 |
| CFC | 19.6 | 19.2 | 19.8 | 19.8 | 19.8 | 19.3 | 19.2 |
| CFC+WXX-3 | 19.9 | 16.4 | 15.2 | 14.5 | 11.3 | 11.0 | 11.4 |
As can be seen from the table 2 and the figure 3, the WXX-3 strain can effectively degrade the ciprofloxacin under the liquid environment, the degradation rate reaches 43.2% -44.7% in 8 th to 10 th days, and then the ciprofloxacin concentration is basically not reduced any more.
Example 3
Application of WXX-3 strain in compost
The fermentation inoculum prepared in example 2 was selected
The experiment set up 3 treatments: 1) CK, adding no bacteria in the pig manure; 2) 0.5 per mill WXX-3, adding the WXX-3 microbial inoculum into pig manure according to the inoculation amount of 0.5 per mill (v/w, L/kg); 3) 10% WXX-3 by adding the WXX-3 inoculum to pig manure at an inoculum size of 10% (v/w, L/kg). The pig manure contains 20mg/L ciprofloxacin and the water content is 75 percent. The above process was set to 3 repetitions, respectively. The strain and the pig manure are evenly mixed and then are kept stand for 8 to 10 days at room temperature, and the concentration of effective viable bacteria after propagation reaches 5.0 multiplied by 10 8 CFU/g, then adding wood chips or straws, adjusting the C/N to be 25/1, adjusting the initial water content of the pile to be 55%, and performing strip-pile composting for 30 days, wherein the results are shown in a table 3 and a figure 4.
TABLE 3 degradation Effect of ciprofloxacin in composts of different treatment groups
As can be seen from Table 3 and FIG. 4, the degradation efficiency of ciprofloxacin was the highest in the 0.5% o WXX-3 treatment group, reaching 85.7%, which was increased by 23.0% compared to CK, and was not significantly different from the 10% WXX-3 treatment group.
Comprehensively considered, the composting method beneficial to the removal of antibiotics comprises the following steps: the microbial inoculum after the enlarged culture in the LB liquid culture medium is mixed into the excrement raw material according to the addition amount of 0.1-1 thousandth (v/w), the mixture is kept stand for 8-10 days to propagate the strains, and then conditioners such as sawdust, straw and the like are added to regulate C/N normal compost.
Example 4
Determination of resistance genes in Brucella (Brucella sp.) WXX-3 and compost
Reagent for extracting animal genome DNAExtracting DNA of bacterial liquid from kit (TSP 201-200, beijing Optimalaceae Biotechnology Co., ltd.), performing fluorescent quantitative PCR on 22 fluoroquinolone resistance genes, wherein the required primer sequence is shown in Table 4, and the detection instrument is StepOnePlus TM Real-time fluorescent quantitative PCR (Thermo). The qPCR reaction system comprises 5 mu L of TB Green chimeric fluorescent dye, 0.4 mu L of upstream primer, 0.4 mu L of downstream primer, 0.2 mu L of ROX reference dye, 1 mu L of DNA template and 3 mu L of sterile water, and the total amount is 10 mu L. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, extension at 60 ℃ for 30s, and 40 cycles. The results show that the strain mainly contains 4 fluoroquinolone resistance genes, namely acrB-01, mexA, qepA and qnrA.
TABLE 4 test primer sequences
0.1000g of each of the compost raw material and a sample after 30 days of compost fermentation was accurately weighed, and DNA of the sample was extracted with FastDNA Spin Kit for solids (MP Biomedicals), and the detailed procedures were referred to the Kit instructions. The extracted DNA is used for determining 4 fluoroquinolone resistance genes of acrB-01, mexA, qepA and qnrA by adopting the fluorescent quantitative PCR method. The results are shown in Table 5 and FIG. 5.
TABLE 5 Absolute abundance results (copies/g) for different resistance genes in different treatment groups
Note: # in table 5 indicates no detection.
As can be seen from Table 5 and FIG. 5, compost raw materials and compost were mainly storedThe resistance gene is qEPA, and the absolute abundance reaches 1.5 multiplied by 10 10 ～2.2×10 10 copies/g, three other resistance genes are less abundant. After composting, the abundance of the resistant genes in CK and 0.5 thousandth WXX-3 treatment is obviously reduced, and the abundance of the resistant genes in 0.5 thousandth WXX-3 treatment is obviously lower than that in CK treatment (p treatment)<0.05 Indicating that the WXX-3 strain can obviously reduce the pollution risk of resistance genes when being applied to compost.
In conclusion, the WXX-3 strain provided by the invention can efficiently degrade fluoroquinolone antibiotics, and meanwhile, the compost prepared by the strain can degrade antibiotic resistance genes, so that the safety of compost products is improved; in addition, the compost preparation method provided by the invention is simple to operate and convenient to control, does not cause secondary pollution in the production process, and is suitable for large-scale popularization and application.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> Nanjing soil institute of Chinese academy of sciences
<120> fluoroquinolone antibiotic degrading bacterium and application thereof in compost
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<210> 7
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cgtgcgcgaa cgaacat 17
<210> 8
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cgtgcgcgaa cgaaca 16
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
actttgcgcg ccatcttc 18
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
caaggaaacg aacgcaatac c 21
<210> 12
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gcggccaggc acaaaa 16
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tacgctcttc ccacggtttc 20
<210> 14
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aggacaacgc tatgcaacga a 21
<210> 15
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ccggaaaggg ccgaaat 17
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cactgcggag aactgtctgt aga 23
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggtcagcacc gacaaggtct ac 22
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
agctcgacgt acttgaggaa cac 23
<210> 20
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ccgcgagaag gccaaga 17
<210> 21
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ttgagttcgg cggtgatga 19
<210> 22
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
acgagagtgg cgtcgacaa 19
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ttctcccccg gcgggaagta c 21
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tttgcaggtt ttgttcctaa tgc 23
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gcagagcctg atttctcctt tg 22
<210> 28
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tggcaatagg agctatggtg ttt 23
<210> 29
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
aaggtaacac tattttcggt ccaaatc 27
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
<210> 31
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
atgctcgcct tccagaaaa 19
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
agaggatttc tcacgccagg 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gggttgtaca tttattgaat cg 22
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
<210> 36
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
cgagatcaat ttacggggaa ta 22
<210> 37
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
aacaagctga agcgcctg 18
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tctaaaccgt cgagttcggc g 21
<210> 40
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gcagttgcat atcgccttct c 21
<210> 41
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
cttcccggca aacagcat 18
<210> 42
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
tgctgatcgg gattctgatt g 21
<210> 43
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
caggcgcgac gaacataat 19
Claims (9)
1. Brucella (A. Bruxella)Brucellasp.) WXX-3 strain, wherein the preservation number of the strain is CGMCC NO.22259.
2. The strain of claim 1, wherein the nucleotide sequence of 16S rDNA of the strain is as set forth in SEQ ID NO:1 is shown.
3. A fermentation inoculum comprising the strain of claim 1 or 2.
4. The fermentation inoculant according to claim 3, wherein the number of viable bacteria per gram or per mL of the strain is (1.5 to 2.6). Times.10 10 CFU。
5. A method for preparing a fermentation inoculum according to claim 3 or 4, comprising the steps of:
the strain of claim 1 is inoculated in an LB culture medium and cultured for 24 to 40h to obtain the fermentation inoculant.
6. The method according to claim 5, wherein the temperature of the culture is 20 to 40 ℃.
7. The use of the strain of claim 1 or 2 or the fermentation inoculum of claim 3 or 4 or prepared by the preparation method of claim 5 or 6 for degrading antibiotics and/or antibiotic resistance genes;
the antibiotic is ciprofloxacin;
the antibiotic resistance gene is qepA and/or acrB-01.
8. A composting method for degrading fluoroquinolone antibiotics and/or degrading fluoroquinolone antibiotic resistance genes comprises the following steps:
mixing the fermentation inoculum according to claim 3 or 4 with a fecal raw material, expanding propagation at 20 to 40 ℃ for 8 to 10 days, adjusting the carbon-nitrogen ratio, and fermenting for 30 days to obtain the compost;
the antibiotic is ciprofloxacin;
the antibiotic resistance gene is qepA and/or acrB-01.
9. The method according to claim 8, wherein the effective viable count of the strain according to claim 1 in the material obtained by mixing the fermentation inoculum and the fecal material is (1.5 to 26). Times.10 9 CFU/kg。
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