CN114045237B - Aeromonas caviae W3 for degrading typical fluoroquinolone antibiotics and application thereof - Google Patents
Aeromonas caviae W3 for degrading typical fluoroquinolone antibiotics and application thereof Download PDFInfo
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Abstract
The invention relates to a guinea pig aeromonas W3 for degrading typical fluoroquinolone antibiotics and application thereof, wherein the guinea pig aeromonas has a preservation number of CCTCC M2021633 and is preserved in China center for type culture Collection (China) at 5 months and 31 days of 2021. The Aeromonas caviae (Aeromonas caviae) W3 is a gram-negative bacterium, colonies on an MSM flat plate are round, the surface is smooth, wetting is easy to pick up, and the color is transparent to white. The Aeromonas caviae (Aeromonas caviae) W3 provided by the invention has strong degradation capability on typical fluoroquinolone antibiotics under aerobic conditions, and the strain is suitable for treating pollution of the typical fluoroquinolone antibiotics in the environment and can achieve the purpose of environmental remediation.
Description
Technical Field
The invention belongs to the technical field of Aeromonas caviae. More particularly, it relates to a Aeromonas caviae W3 degrading a typical fluoroquinolone antibiotic and application thereof.
Background
Norfloxacin (NOR) and Ciprofloxacin (CIP) are third generation quinolone antibiotics commonly used in the clinical and livestock farming industries, and mainly enter the environment through domestic wastewater, hospital wastewater and farming wastewater, the quinolone antibiotics cannot be completely removed by municipal sewage treatment plants, and still exist in sewage and sludge of the sewage treatment plants, and can be transferred to surface water and groundwater, thereby negatively affecting non-target organisms, causing disappearance of certain microbial populations and ecological functions thereof, and even causing health hazards to human bodies through accumulation of food chains. In addition, antibiotic residues can promote the evolution and transfer of antibiotic resistant bacteria and antibiotic resistance genes in the environment, and further endanger the environmental health. No doubt, quinolone antibiotic pollution has become a great problem for social development and environmental protection.
Fluoroquinolones belong to the class of quinolones, also known as pyridonecids, and belong to the class of chemically synthesized antibacterial agents. The method for pollution control of fluoroquinolone antibiotics comprises a physical method, a chemical method, a biological method, a high-grade oxidation method and the like, and the microbial method has the advantages of economy, high efficiency and thorough degradation, so that the bacterial strain capable of efficiently degrading NOR is enriched and domesticated by aerobic sludge, purified and separated, the characteristics of the bacterial strain are researched, and the bacterial strain has important scientific significance and application value for environmental pollution treatment of fluoroquinolone antibiotics. However, the current high-efficiency strain for degrading fluoroquinolone antibiotics is very lacking, which definitely brings great obstacle to the treatment of NOR environmental pollution.
Strains reported in the prior art to have the ability to degrade fluoroquinolone antibiotics include E.coli, microbacterium, mycobacterium, bacillus and Labrys portucalensis. The patent CN110591948A adopts compound microorganism bacteria to degrade quinolone antibiotics in soil, the patent CN113023906A uses waste bacterial cells of escherichia coli to adsorb and reduce palladium ions and platinum ions in wastewater, and the prepared nano-scale bio-metal catalyst is used for degrading levofloxacin and norfloxacin. However, no reference has been made to Aeromonas caviae (Aeromonas caviae) for its ability to degrade fluoroquinolone antibiotics.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a guinea pig Aeromonas (Aeromonas caviae) W3 for degrading typical fluoroquinolone antibiotics and application thereof, wherein the guinea pig Aeromonas (Aeromonas caviae) W3 has stronger degradation capability on the typical fluoroquinolone antibiotics, namely norfloxacin and ciprofloxacin, under aerobic conditions, and the strain is suitable for treating pollution of the typical fluoroquinolone antibiotics in the environment and can achieve the aim of environmental remediation.
The first object of the present invention is to provide Aeromonas caviae (Aeromonas caviae) W3.
The second object of the present invention is to provide an application of Aeromonas caviae (Aeromonas caviae).
A third object of the present invention is to provide a formulation for degrading a typical fluoroquinolone antibiotic.
A fourth object of the present invention is to provide a method for degrading a typical fluoroquinolone antibiotic.
In order to achieve the above purpose, the invention adopts the following technical scheme:
aeromonas caviae (Aeromonas caviae) W3, wherein the Aeromonas caviae (Aeromonas caviae) W3 has a preservation number of CCTCC M2021633, and the Aeromonas caviae (Aeromonas caviae) W3 has been preserved in China center for type culture collection (China) for 5 months.
Use of Aeromonas caviae (Aeromonas caviae) for degrading and/or preparing a formulation which degrades a typical fluoroquinolone antibiotic which is one or both of norfloxacin and ciprofloxacin.
Preferably, the Aeromonas caviae (Aeromonas caviae) is Aeromonas caviae (Aeromonas caviae) W3, and the Aeromonas caviae (Aeromonas caviae) W3 has a preservation number of CCTCC M2021633 and is preserved in China center for type culture collection (China) for 5 th month 31 of 2021.
A formulation for degrading a typical fluoroquinolone antibiotic, wherein the formulation comprises Aeromonas caviae (Aeromonas caviae), and the typical fluoroquinolone antibiotic is one or both of norfloxacin and ciprofloxacin.
Preferably, the Aeromonas caviae (Aeromonas caviae) is Aeromonas caviae (Aeromonas caviae) W3, and the Aeromonas caviae (Aeromonas caviae) W3 has a preservation number of CCTCC M2021633 and is preserved in China center for type culture collection (China) for 5 th month 31 of 2021.
Preferably, the formulation is a formulation that degrades a typical fluoroquinolone antibiotic in a body of water.
A method for degrading a typical fluoroquinolone antibiotic, which is one or both of norfloxacin and ciprofloxacin, using Aeromonas caviae (Aeromonas caviae) for degradation.
Preferably, the Aeromonas caviae (Aeromonas caviae) is Aeromonas caviae (Aeromonas caviae) W3, and the Aeromonas caviae (Aeromonas caviae) W3 has a preservation number of CCTCC M2021633 and is preserved in China center for type culture collection (China) for 5 th month 31 of 2021.
Preferably, the degradation temperature is 25-35 ℃, the pH is 7.0-7.5, and the initial inoculation amount is OD600 = 0.5-0.8.
More preferably, the degradation temperature is 28 ℃, the pH is 7.3, and the initial inoculum size is od600=0.68.
Preferably, the method is a method of degrading a typical fluoroquinolone antibiotic in a body of water.
According to the invention, a strain of Aeromonas caviae (Aeromonas caviae) W3 with a typical norfloxacin degradation function under an aerobic condition is separated from an aerobic reactor added with Norfloxacin (NOR), and the strain can effectively degrade norfloxacin in a single carbon source matrix environment, and the degradation efficiency can reach more than 90%. The degradation efficiency of ciprofloxacin can reach 46 percent.
The Aeromonas caviae (Aeromonas caviae) W3 provided by the invention can effectively degrade typical fluoroquinolone antibiotics under a single carbon source.
Compared with the prior art, the invention has the following beneficial effects:
the invention determines that the Aeromonas caviae (Aeromonas caviae) W3 with the function of degrading typical fluoroquinolone antibiotics is grown on MSM flat plates for the first time, bacterial colonies are round, the surfaces are smooth, the colors are transparent to white, and gram staining is negative. The main physicochemical properties of Aeromonas caviae (Aeromonas caviae) W3 are: the strain grows at the temperature of 10-40 ℃ and the pH range of 6-8, and the strain grows aerobically and aerobically, and the oxidase is positive and the contact enzyme is positive. The Aeromonas caviae (Aeromonas caviae) W3 provided by the invention has strong capability of degrading typical fluoroquinolone antibiotics, especially norfloxacin, and the degradation rate can reach more than 90% after the norfloxacin is degraded for 8 days under aerobic conditions at the concentration of 75mg/L. The ciprofloxacin is degraded for 9 days under the aerobic condition with the concentration of 65mg/L and the degradation rate of 46 percent. The Aeromonas caviae (Aeromonas caviae) W3 provided by the invention is particularly suitable for treating pollution of typical fluoroquinolone antibiotics in the environment, and can achieve the purpose of environmental remediation.
Drawings
FIG. 1 is a phylogenetic tree of Aeromonas caviae (Aeromonas caviae) W3;
FIG. 2 is a graph showing the growth curve of Aeromonas caviae (Aeromonas caviae) W3;
FIG. 3 shows the effect of norfloxacin on Aeromonas caviae (Aeromonas caviae) W3 growth.
FIG. 4 shows the effect of ciprofloxacin on the growth of Aeromonas caviae (Aeromonas caviae) W3.
FIG. 5 is an effect of enrofloxacin on the growth of Aeromonas caviae (Aeromonas caviae) W3.
FIG. 6 shows the effect of ofloxacin on Aeromonas caviae (Aeromonas caviae) W3 growth.
Detailed Description
The invention will be further elaborated in connection with the drawings and the specific embodiments described below, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 isolation and characterization of Aeromonas caviae (Aeromonas caviae) W3
1. Enrichment and separation of strains
Taking aerobic sludge from a Guangzhou asphalt sewage treatment plant, and cleaning according to the following steps: (1) sieving out largeStanding and precipitating for 30min, and discarding supernatant; (2) the mixture was resuspended by addition of PBS (phosphate buffer saline) solution, vortexing for 10min, and the supernatant was discarded. 20mL of puree was added to 200mL of nutrient solution (the ingredients and contents are shown in Table 1), and Norfloxacin (NOR) was added to 10mg/L and sodium acetate 0.3g/L. 0.1mL of the bacterial suspension (dilution 10) was taken with uniform stirring of the rotor 1 ~10 4 Fold) was plated on MSM solid plate medium with norfloxacin as the sole carbon source and incubated at 37℃in the dark until single pure colonies appeared on the plates. And selecting colonies with good growth state and regular edges for purification for a plurality of times. The MSM culture medium contains 0.05g/L norfloxacin, 1.5g/L agar powder and other additive components and contents shown in Table 1.
Table 1: nutrient solution composition and content
2. Identification of strains
Molecular marker identification: extracting total DNA of bacteria by using a bacterial DNA extraction kit, amplifying a 16S rRNA sequence of the strain by using a bacterial 16S rRNA universal primer through PCR, performing BLAST comparison, and constructing a phylogenetic tree by using MEGA X software to obtain the phylogenetic relation tree of the strain.
The conditions for the PCR amplification were as follows:
universal primer:
27F:5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO:1)
1492R:5'-GGCTACCTTGTTACGACTT-3'(SEQ ID NO:2)
PCR amplification procedure:
sequencing the 16S rRNA, and BLAST comparison shows that the 16S rRNA sequence of the strain has up to 100% homology with Aeromonas caviae (Aeromonas caviae). Therefore, it can be judged that the strain belongs to Aeromonas caviae, is named as Aeromonas caviae W3, and is preserved in China Center for Type Culture Collection (CCTCC) at 5.31 of 2021, the preservation number is CCTCC M2021633, the preservation place is university of Wuhan in Hubei province of China, and the preservation name is Aeromonas caviae W3 (Aeromonas caviae W).
The domesticated strain is identified by 16S rRNA sequencing, BLAST comparison and MEGA X software is used for constructing a phylogenetic relationship tree, and can be referred to as figure 1, and is identified as Aeromonas caviae (Aeromonas caviae) and gram negative. The sequencing result is shown in SEQ ID NO. 3.
(3) Physiological and biochemical characteristics of the strain
Aeromonas caviae (Aeromonas caviae) W3 is gram-negative bacteria, and the colony on the MSM flat-plate culture medium is round, smooth in surface, wet and easy to pick up, slightly adhered, and transparent to white in color. The growth temperature of the strain is 10-40 ℃, the pH range is 6-8, the strain grows anaerobically and facultatively, the oxidase is positive, and the contact enzyme is positive. The growth curve of Aeromonas caviae (Aeromonas caviae) W3 is shown in FIG. 2.
3. Determination of Strain growth Curve
The optical density value of the culture solution under the wavelength of 600nm is measured by an ultraviolet-visible spectrophotometer, the OD600 value is shown in figure 2, after bacteria are inoculated to the culture medium, the curve is flat and stable for 0-4 h, the bacteria are less in propagation, and a transient adaptation process is provided for a new environment; after 4 hours, the bacteria enter a logarithmic phase, the number of viable bacteria on a growth curve rises linearly, and the bacteria grow extremely fast in a stable geometric progression for about 6 hours; after 10h, the bacterial growth speed slows down, and the bacterial growth enters a stable period around 22h, and the total number of growing bacterial flora is in a flat stage.
EXAMPLE 2 degradation test of Aeromonas caviae (Aeromonas caviae) W3 on Norfloxacin (NOR) under aerobic conditions
1. Conical flask experiments on isolated and purified Aeromonas caviae (Aeromonas caviae) W3 of example 1 were carried out under aerobic conditions to degrade norfloxacin, and the degradation rate of norfloxacin and the growth OD600 of the strain were detected. The temperature (10-40 ℃), pH (6-8) and initial OD600 (0.2-0.8) are taken as independent variables, the degradation rate is taken as a dependent variable, three-factor three-level response surface analysis is carried out, the design scheme is Box-Behnken, and the fitting model is quadric. Experimental data are shown in the following table:
TABLE 1 response surface data results
2. The OD600 of the growth condition of Aeromonas caviae (Aeromonas caviae) is generally reduced under the condition that norfloxacin is taken as a single carbon source. Corresponding surface analysis model Adjusted R 2 For 0.9558, the fit equation is as follows:
removal(%)=-861.89+1.06A+238.40B+121.20C+0.96A×B+0.57A×C+10.92B ×C-0.15A 2 -18.47B 2 -114.68C 2
where A is temperature (. Degree.C.), B is pH, and C is the initial OD600. Therefore, the optimal experimental condition is selected to be 27.493 ℃, pH is 7.336, initial OD600 is 0.681, and the fitting degradation rate can reach 100% through fitting by Design Expert 11 software. In actual operation, the temperature is controlled to be 28 ℃, the pH is 7.3, and the initial OD600 is 0.67-0.69.
EXAMPLE 3 Effect of different fluoroquinolone antibiotics on Aeromonas caviae (Aeromonas caviae) W3 growth
Four types of fluoroquinolone antibiotic drugs were added to a conical flask containing 100mL of the nutrient solution described in example 1, so that the initial concentration of Norfloxacin (NOR) was 75mg/L, the initial concentration of CIP (CIP) was 65mg/L, the initial concentration of Enrofloxacin (ENR) was 37.5mg/L, and the initial concentration of Ofloxacin (OFL) was 75mg/L, respectively. Centrifuging the bacterial liquid in logarithmic phase at 6000rpm at 4deg.C for 6min, discarding supernatant, washing with PBS solution, centrifuging, collecting precipitate, and adding small amount of PBS solution to obtain bacterial suspension. According to the experimental results of example 2, a certain amount of bacterial suspension was inoculated at the initial OD600 = 0.68, and the culture was performed at 28 ℃ at ph7.3 in the absence of light, and the effect of different fluoroquinolone drugs on the growth of strain W3 was examined.
The results show that the growth of Aeromonas caviae (Aeromonas caviae) W3 can be inhibited by fluoroquinolone drugs, and the Aeromonas caviae and ciprofloxacin have degradation effects, and the effect of the Aeromonas caviae (Aeromonas caviae) W3 on the growth of Aeromonas caviae (Aeromonas caviae) W3 is shown in the figure 3, and the degradation rate can reach more than 90% in the aerobic condition under the condition that the concentration of the Aeromonas caviae is 75mg/L. FIG. 4 shows the effect of ciprofloxacin on Aeromonas caviae W3 growth, with ciprofloxacin concentration of 65mg/L and ciprofloxacin degradation rate of 46% by day 9 of the experiment under aerobic conditions. The degradation rate is calculated as follows:
FIGS. 5 and 6 show that Aeromonas caviae (Aeromonas caviae) W3 has no degrading effect on enrofloxacin and ofloxacin. Therefore, the Aeromonas caviae (Aeromonas caviae) W3 has better degradation effect on norfloxacin and ciprofloxacin which are two fluoroquinolone antibiotics, and has the best degradation effect on norfloxacin.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Sequence listing
<120> Aeromonas caviae W3 degrading typical fluoroquinolone antibiotics and application thereof
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ggctaccttg ttacgactt 19
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tggctcagat tgaacgctgg cggcaggcct aacacatgca agtcgagcgg cagcgggaaa 60
gtagcttgct acttttgccg gcgagcggcg gacgggtgag taatgcctgg gaaattgccc 120
agtcgagggg gataacagtt ggaaacgact gctaataccg catacgccct acgggggaaa 180
gcaggggacc ttcgggcctt gcgcgattgg atatgcccag gtgggattag ctagttggtg 240
aggtaatggc tcaccaaggc gacgatccct agctggtctg agaggatgat cagccacact 300
ggaactgaga cacggtccag actcctacgg gaggcagcag tggggaatat tgcacaatgg 360
gggaaaccct gatgcagcca tgccgcgtgt gtgaagaagg ccttcgggtt gtaaagcact 420
ttcagcgagg aggaaaggtc agtagctaat acctgctggc tgtgacgtta ctcgcagaag 480
aagcaccggc taactccgtg ccagcagccg cggtaatacg gagggtgcaa gcgttaatcg 540
gaattactgg gcgtaaagcg cacgcaggcg gttggataag ttagatgtga aagccccggg 600
ctcaacctgg gaattgcatt taaaactgtc cagctagagt cttgtagagg ggggtagaat 660
tccaggtgta gcggtgaaat gcgtagagat ctggaggaat accggtggcg aaggcggccc 720
cctggacaaa gactgacgct caggtgcgaa agcgtgggga gcaaacagga ttagataccc 780
tggtagtcca cgccgtaaac gatgtcgatt tggaggctgt gtccttgaga cgtggcttcc 840
ggagctaacg cgttaaatcg accgcctggg gagtacggcc gcaaggttaa aactcaaatg 900
aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgatgc aacgcgaaga 960
accttacctg gccttgacat gtctggaatc ctgcagagat gcgggagtgc cttcgggaat 1020
cagaacacag gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080
cgcaacgagc gcaacccctg tcctttgttg ccagcacgta atggtgggaa ctcaagggag 1140
actgccggtg ataaaccgga ggaaggtggg gatgacgtca agtcatcatg gcccttacgg 1200
ccagggctac acacgtgcta caatggcgcg tacagagggc tgcaagctag cgatagtgag 1260
cgaatcccaa aaagcgcgtc gtagtccgga ttggagtctg caactcgact ccatgaagtc 1320
ggaatcgcta gtaatcgcaa atcagaatgt tgcggtgaat acgttcccgg gccttgtaca 1380
caccgcccgt cacaccatgg gagtgggttg caccagaagt agatagctta accttcggga 1440
gggcgtttac cacggtgtga ttcatgactg gggtga 1476
Claims (6)
1. Aeromonas caviae (guinea pig)Aeromonas caviae) W3 is characterized in that the Aeromonas caviae is [ ]Aeromonas caviae) W3 preservation number is CCTCC NO: m2021633, which was deposited with the China center for type culture Collection, was 5.31 in 2021.
2. The Aeromonas caviae strain according to claim 1Aeromonas caviae) The use of a typical fluoroquinolone antibiotic in a body of water or in the preparation of a formulation for degrading a typical fluoroquinolone antibiotic, wherein the typical fluoroquinolone antibiotic is one or both of norfloxacin and ciprofloxacin.
3. A preparation for degrading a typical fluoroquinolone antibiotic, wherein the preparation comprises the Aeromonas caviae strain according to claim 1Aeromonas caviae) The typical fluoroquinolone antibiotics are one or two of norfloxacin and ciprofloxacin.
4. A method for degrading a typical fluoroquinolone antibiotic in a water body, comprising the step of using the Aeromonas caviae strain according to claim 1Aeromonas caviae) Degradation is carried out, and the typical fluoroquinolone antibiotics are one or two of norfloxacin and ciprofloxacin.
5. The method for degrading a classical fluoroquinolone antibiotic in a water body according to claim 4, wherein the degradation temperature is 25-35 ℃, the pH is 7.0-7.5, and the initial inoculation amount is OD 600 =0.5~0.8。
6. The method for degrading a classical fluoroquinolone antibiotic in a water body according to claim 5, wherein the degradation temperature is 28 ℃, the pH is 7.3, and the initial inoculation amount is OD 600 =0.68。
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