A method of simultaneously degrading florfenicol and inhibiting biofilm tolerance induced by florfenicol

Abstract

The present application relates to the technical field of antibiotic pollution remediation, and particularly relates to a method for simultaneously degrading florfenicol and inhibiting drug resistance induced by florfenicol. On the basis of traditional microbial degradation, the method regulates the community structure of a biofilm by using nano zero-valent iron, and through detection of degradation products and microbial metagenome, the method achieves effective inhibition of the singleization of the biofilm community structure and the risk of drug resistance induced by florfenicol while maintaining the microbial degradation effect, overcomes the environmental risk and public health and safety problems caused by traditional biological degradation of antibiotics, and can achieve safe and efficient removal of antibiotic pollution in water bodies.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibiotic pollution remediation technology, and particularly relates to a method for simultaneously degrading florfenicol and inhibiting drug resistance induced by florfenicol. BACKGROUND

[0002] Antibiotics have been widely used in the treatment of infectious diseases in humans and livestock due to their excellent antibacterial and bacteriostatic effects, and the risk of microbial drug resistance caused by antibiotics has attracted widespread attention. Among many antibiotics, chloramphenicol antibiotics such as chloramphenicol, thiamphenicol and florfenicol are low in price, wide in antibacterial spectrum, and are used in large quantities in the livestock breeding industry. In 2013, the use of florfenicol in China reached 10,000 tons, ranking in the top three among all antibiotics. However, with the widespread use of florfenicol in the breeding industry, intensive farms, especially aquaculture ponds, have become heavily polluted areas of antibiotics. The absorption and transformation of organisms to antibiotics is very limited. Florfenicol is slightly soluble in water and has weak binding ability with soil organic matter, and has strong mobility. At present, such chloramphenicol antibiotics are commonly found in solid waste, water and soil in many places in China, and the concentration of some samples exceeds the environmental quality standard. More importantly, the long-term presence of antibiotics such as florfenicol in the environment can lead to changes in microbial community structure and induce antibiotic resistance of biofilms, ultimately threatening human health. It is urgent to develop safe and efficient control technologies for florfenicol pollution and its drug resistance.

[0003] Microbial degradation is one of the main mechanisms for the reduction of florfenicol in nature. A large number of studies have found many functional degrading bacteria through enrichment culture, isolation and screening techniques from biofilms in high-concentration antibiotic-polluted environments, which can convert florfenicol into degradation products such as florfenicol amine through biological effects such as hydrolysis. For example, in actual water environments, these microorganisms will attach to solid surfaces to form biofilms and respond to antibiotic pollution through community changes. It is worth noting that the ability of microorganisms to degrade antibiotics is a macroscopic manifestation of their drug resistance, and the stronger the antibiotic-degrading ability of biofilms, the higher the abundance of drug-resistant microorganisms, especially pathogenic drug-resistant bacteria. Therefore, single microbial degradation technology cannot balance the antibiotic degradation performance and the risk of drug resistance induced by it, and breakthroughs are needed.

[0004] Nano zero-valent iron (NZVI) is a new type of reductive adsorbent widely used in environmental pollution remediation in recent years. It has the characteristics of large specific surface area, small particle size and high reactivity. Iron is the fourth most abundant element in the earth's crust and the most abundant transition metal on earth. Therefore, NZVI is considered to be an environmentally friendly pollution remediation material. In recent years, the principles and effects of NZVI for antibiotic pollution remediation have been continuously revealed. Studies have shown that NZVI can degrade chloramphenicol antibiotics through reductive dechlorination. Some studies have also shown that NZVI has good antibacterial effect and can inhibit the growth of Escherichia coli and tetracycline-resistant bacteria. Therefore, NZVI is expected to be used to simultaneously degrade florfenicol and inhibit the biofilm community drug resistance induced by florfenicol. It is urgent to develop a technology to reduce the abundance of pathogenic bacteria and the risk of biofilm drug resistance in aquaculture wastewater based on the regulation of NZVI on the community structure of water biofilms. SUMMARY

[0005] The application provides a method for simultaneously degrading florfenicol and inhibiting the biofilm community drug resistance induced by florfenicol. The technology adds NZVI to the traditional biological degradation experiment, effectively inhibits the microbial community structure simplification and drug resistance risk induced by florfenicol, to some extent, overcomes the environmental risk and public health safety problems caused by traditional biological degradation of antibiotics, and realizes safe and efficient removal of antibiotic pollution in water.

[0006] The specific technical solutions are as follows:

[0007] A method for simultaneously degrading florfenicol and inhibiting the biofilm drug resistance induced by florfenicol, comprising the following steps:

[0008] (1) A simulated aquaculture wastewater microcosm system is constructed. The collected aquaculture water sample is resuspended and filtered through a microporous filter membrane with a pore size of 0.45 μm to remove impurities in the original water sample. The total viable bacterial count of the mixed microorganisms is determined by microscopic counting. The microorganisms obtained by sampling are configured into a certain concentration of bacterial solution in the culture medium, inoculated into the experimental container, and the container wall is used as the substrate of the biofilm. Circulating water is provided to maintain a room temperature of 20±1℃. The container inoculated with microorganisms is placed at room temperature of 20℃ for 14 days to form a preliminary biofilm;

[0009] (2) A certain amount of commercial NZVI (surface passivation) with a particle size of 80-100 nm is weighed and placed in a 50 mL centrifuge tube. Ultra-pure water is added to prepare a 10 g / L stock solution. The NZVI solution is uniformly dispersed by ultrasonic suspension for 30 minutes.

[0010] (3) In the container in which the biofilm has been formed, the reaction is carried out, the antibiotic-containing water body to be repaired and the nano zero-valent iron solution are added to the container, and mixing is carried out, and the degradation experiment is carried out at room temperature; during the reaction, sampling is carried out at a time, and the contents of florfenicol, florfenicol amine, de-chloro product and de-dichloro product in the water body are detected by using high performance liquid chromatography-tandem mass spectrometry.

[0011] (4) After the reaction is completed, a certain amount of biofilm is weighed and macro-genome sequencing is started, DNA extraction, sequencing, data quality control, splicing assembly and data analysis are carried out. According to the annotation and abundance value of NR, KEGG, ARDB and other databases, the changes of species and functions in different groups are analyzed by using clustering and alpha diversity index calculation methods, and are visualized in the form of column chart, box chart and Circos chart, and the significant difference between groups is tested by using statistical T test.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] In the present application, nano zero-valent iron is applied to the antibiotic-polluted water body, which can effectively inhibit antibiotic-induced pathogenic bacteria and drug resistance on the basis of maintaining the effect of microbial degradation of antibiotics, and realize safe and efficient removal of florfenicol in aquaculture wastewater. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The technical flowchart of the present application is shown in the figure;

[0015] Figure 2 The concentration changes of florfenicol and its degradation products in different treatment groups in Example 1 of the present application are shown in the figure;

[0016] Figure 3 The alpha diversity index difference test between groups of nano zero-valent iron, florfenicol and combined exposure in Example 1 of the present application is shown in the figure;

[0017] Figure 4 The drug resistance characteristic Circos chart of nano zero-valent iron, florfenicol and combined exposure in Example 1 of the present application is shown in the figure;

[0018] Figure 5 The difference of pathogenic bacteria genus abundance and average abundance in different treatment groups in Example 1 of the present application is shown in the figure;

[0019] Figure 6 The difference of pathogenic bacteria genus abundance under florfenicol single exposure and combined exposure with nano zero-valent iron in Example 1 of the present application is shown in the figure. DETAILED DESCRIPTION

[0020] The present application will be further described below in combination with specific examples, and the following examples are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto.

[0021] The method of the present application is specifically as follows:

[0022] (1) Preparation of florfenicol stock solution and nano zero-valent iron solution

[0023] A certain amount of florfenicol standard substance was accurately weighed and added to ultrapure water, and dissolved by ultrasonic at room temperature to prepare a 500 mg / L standard stock solution, which was sealed with Parafilm-M film and stored in a 4°C refrigerator. The 80-100 nm nano zero-valent iron was ultrasonically suspended in ultrapure water for 30 minutes to obtain a uniformly dispersed nano zero-valent iron solution.

[0024] (2) Biological membrane collection and culture before the experiment

[0025] The biological membrane used in the experiment was collected from the wastewater outlet of a certain aquaculture farm in Huzhou, Zhejiang. The collected sample was resuspended and filtered through a microporous filter membrane with a pore size of 0.45 μm to remove impurities in the original water sample. A portion of the sample was diluted, and the total viable bacterial count was determined by microscopic counting method. The microorganisms obtained by sampling were prepared into a bacterial solution with a concentration of 2×10 8 6 cfu / mL in the culture medium, and 10 mL was inoculated into a 2 L open glass water tank for the experiment, with the glass tank wall as the substrate for the biological membrane, and circulating water was provided to maintain the room temperature at 20±1°C. The open glass water tank inoculated with microorganisms was placed at room temperature of 20°C for 14 days to allow the biological membrane to form preliminarily. In the formal experiment, florfenicol stock solution and nano zero-valent iron solution were uniformly mixed in the glass water tank with formed biological membrane, and the treatment groups are shown in the following table, so that the final concentration of florfenicol is 3 mg / L and the concentration of nano zero-valent iron is 300 mg / L. During the exposure period, the circulating water pump was circulated for 24 hours, and water lost during the exposure process was supplemented every day, for a duration of 14 days.

[0026] Table 1 Treatment groups

[0027]

[0028] (3) Detection of florfenicol and its degradation products

[0029] During the experiment, samples were taken at 0, 1, 2, 4, 6, 8, 10, 12, 14 days, 1 mL each time, filtered through a 0.22 μm filter membrane, and then stored in a 2 mL liquid phase vial. Florfenicol and its degradation products were detected by high performance liquid chromatography-tandem mass spectrometry.

[0030] Chromatographic conditions: The chromatographic column was ACQUITY BEH C18 column (1.7 μm, 2.1 mm x 100 mm); mobile phase: 0.1% ammonia water (A) - acetonitrile (B); column temperature 313 K; injection volume 5 μL; flow rate 0.3 mL / min; mobile phase and gradient elution program were shown in Table 2.

[0031] Table 2 Gradient of mobile phase of high performance liquid chromatography-tandem mass spectrometry

[0032]

[0033] Mass spectrometry conditions: ion source temperature (TEM): 300℃; scanning mode: negative ion scanning; detection mode: MRM; electrospray voltage: 2.08 kV; desolvation gas temperature 350℃; desolvation gas flow rate: 800 L / Hr; collision gas: 0.25 mL / min; retention time and mass spectrometry parameters of target were shown in Table 3.

[0034] Table 3 Retention time and mass spectrometry parameters of target

[0035]

[0036] In the experiment, external standard method was used for quantification, the calibration range was 0.5-50 μg / L, the correlation coefficient R 2 of fitting was greater than 0.99. FF-2Cl was not detected in the experiment, which was lower than the detection limit of 0.5 μg / L.

[0037] (4) Macro-genomic sequencing of biofilm

[0038] After the experiment, the biofilm in the area of 5 cm x 5 cm on the same position of the exposed 14-day open glass tank wall in each group was scraped into a 2 mL centrifuge tube, weighed and subjected to macro-genomic sequencing.

[0039] In terms of DNA extraction and sequencing, E.Z.N.A.B Soil DNA Kit (Omega Bio-tek, USA) kit was used for DNA extraction of samples. Then TBS-380 and NanoDrop2000 were used to detect the concentration and purity of DNA, and 1% agarose gel electrophoresis was used to detect the integrity of DNA. DNA was fragmented by Covaris M220 (Gene Company, China), and fragments of about 400 bp were selected to construct PE library using NEXTFLEX Rapid DNA-Seq (Bioo Sientific, USA) library construction kit. After bridge PCR amplification, Illumina NovaSeg / Hiseq Xten (Illumina, USA) sequencing platform was used for macro-genomic sequencing.

[0040] In data quality control and assembly, BWA alignment software was used to align reads to the host, and Fastp software was used to control the DNA sequence of raw data, and remove high similarity of pollution reads. MEGAHIT was used to assemble the optimized sequences based on the principle of succinct de Bruijngraphs. In the assembly results, contigs greater than 300 bp were selected as the final assembly results. MetaGene was used to predict ORF of the contigs obtained by assembly, and CD-HIT was used to cluster all sample gene sequences predicted to construct a non-redundant gene set. Finally, SOAPaligner was used to align each sample's high-quality reads to the non-redundant gene set (95% identity), and obtain the abundance information of genes in the corresponding sample. When analyzing the data, BLASTP was used to align the amino acid sequences of the non-redundant gene set with the NR database, KEGG database, ARDB database and other databases for annotation, and obtain the related species and functional annotation, and then statistical analysis was performed according to the annotation results.

[0041] (5) Result analysis

[0042] Figure 2 The concentration changes of florfenicol and its degradation products in different treatment groups. The results showed that the addition of nano zero-valent iron promoted the dechlorination reaction of florfenicol, and the degradation rate of florfenicol reached 29.2% in 14 days, which had no significant difference with the single florfenicol treatment group. On this basis, the changes of microbial community composition caused by florfenicol or / and nano zero-valent iron were analyzed, and the results are shown in Figure 3 Single florfenicol stress significantly reduced the alpha diversity of water biofilm, and the addition of nano zero-valent iron made the biofilm community structure recover to a certain extent, and the alpha diversity had no significant difference with the control group. It is shown that nano zero-valent iron can ensure the richness and diversity of the community when used for antibiotic pollution remediation, and it is safe and reliable.

[0043] The results of the analysis of the risk of drug resistance in water are as follows Figure 4and the table below. Under single florfenicol exposure, florfenicol significantly increased chloramphenicol resistance (by 8%), sulfonamide resistance (by 9%), and streptomycin resistance (by 4%), while bacitracin resistance and tetracycline resistance decreased by 21% and 1%, respectively. There were significant differences in antibiotic species distribution and the dominant antibiotic species shifted from being dominated by sulfonamide and bacitracin resistance to being dominated by chloramphenicol resistance and tetracycline resistance. This resulted in a significant change in antibiotic species distribution and a significant shift in the dominant antibiotic species. Under complex exposure, compared with the florfenicol treatment group, in addition to a 6% increase in bacitracin resistance, there was no significant change in tetracycline resistance, and chloramphenicol resistance, sulfonamide resistance, and streptomycin resistance were significantly reduced.

[0044] Table 4. Resistance characteristics and proportion (%) of different treatment groups

[0045]

[0046] The abundance of pathogenic bacteria was analyzed. Among the 32 pathogenic microorganism genera that required high attention, the different bacterial genus abundances in each group were classified according to whether they were lower than the average abundance in the four groups, and the results are shown in Figure 5 In the control group, 16 pathogenic bacterial genera were higher than the average abundance, and 16 pathogenic bacterial genera were lower than the average abundance. In the nano zero-valent iron group, the number of pathogenic bacterial genera higher than the average abundance decreased to 6, and the remaining 26 were lower than the average abundance. In the florfenicol group and the complex exposure group, 20 pathogenic bacterial genera were higher than the average abundance, and 10 were lower than the average abundance. In order to distinguish the complex exposure group from the florfenicol single exposure group, a significant T test was performed on these two groups alone, and the results are shown in Figure 6 The results showed that there were 9 pathogenic bacterial genera in the complex exposure group that were significantly different from the single florfenicol exposure group, of which 5 pathogenic bacterial genera had significantly lower abundance, and Legionella, Mycobacterium, Pseudomonas, and Coxiella had significantly higher abundance. That is, under the same condition of being higher than the average abundance, the abundance of most pathogenic bacterial genera in the complex exposure group was lower than that in the florfenicol group. The above results show that nano zero-valent iron can effectively inhibit pathogenic bacteria in biofilm communities and reduce the risk of pathogenicity brought about by florfenicol.

Claims

1. A method for simultaneously degrading florfenicol and inhibiting its induced resistance in biofilm communities, characterized in that: Includes the following steps: (1) A micro-universe system for aquaculture wastewater was constructed by resuspending the collected aquaculture farm water samples and filtering them through a microporous membrane with a pore size of 0.45 μm to remove impurities from the original water samples; the total viable count of mixed microorganisms was determined by microscopic counting method; the microorganisms obtained from the sampling were prepared into a bacterial solution of a certain concentration in a culture medium and inoculated into an experimental container, with the container wall as the substrate of the biofilm, and circulating water was provided to maintain the room temperature at 20±1℃; the container inoculated with microorganisms was placed at room temperature at 20℃ for 14 days to allow the biofilm to initially form. (2) Weigh a certain amount of commercial nano zero-valent iron with a particle size of 80-100 nm and passivated surface, place it in a 50 mL centrifuge tube, add ultrapure water to prepare a 10 g / L mother liquor, and sonicate it for 30 minutes to obtain a uniformly dispersed nano zero-valent iron solution. (3) The reaction was carried out in a container in which a biofilm had been formed. The water to be remediated containing antibiotics and nano zero-valent iron solution were added to the container, mixed well, and the degradation experiment was carried out at room temperature. During the reaction, samples were taken regularly, and the contents of florfenicol, florfenicolamine, dechlorination products and dechlorination products in the water were detected by high performance liquid chromatography-tandem mass spectrometry. (4) After the reaction is complete, a certain amount of biofilm is weighed and metagenomic sequencing is started. DNA extraction, sequencing, data quality control, splicing and assembly and data analysis are performed.

2. The method for simultaneously degrading florfenicol and inhibiting its induced drug resistance in biofilm communities according to claim 1, characterized in that: The data analysis, based on the annotations and abundance values ​​of the NR, KEGG, and ARDB databases, used clustering and α diversity index calculation methods to analyze the changes in species and functions among different groups, and the statistical t-test was used to test the significance of differences between groups.

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