| Objective:In recent years,antibiotics are widely used in the fields of medical treatment and breeding.The residual antibiotics in water environment can cause aquatic bacteria to carry ARGs,form ARB,and even produce superbacteria.Exploring the mechanism of ARGs in water can solve the ecological problem fundamentally.At present,the traditional wastewater treatment method has the problems of high cost,unstable performance,large accumulation of ARGs and the like in the reduction effect of ARGs in a water environment.Therefore,a low-cost,high-efficiency and green ARGs reduction method has important scientific significance and application value for ecological restoration of ARGs polluted water body.In this study,a biological reduction method of ARGs based on Chlorella sp.was established to analyze the reduction effect of Chlorella on typical ARGs in water samples,and to provide some basis for exploring the mechanism of ARGs transmission in water.Methods:1.Detection of target genes in water samplesIn this study,water samples were collected from shallow water at the shore of a lake of Changchun City,and three sampling sites were selected.2 L of water samples were collected in sterile collection bags and brought back to the laboratory.Four typical ARGs(tetracyclines: tet G and tet W,sulfonamides: sul1 and sul2)with high absolute abundance reported in the aqueous environment,class I integrons(int I1)related to ARGs transmission and housekeeping genes(16S)were selected as target genes,and standard plasmids were synthesized by comparing universal primers to establish q PCR absolute quantification methods(including optimizing the annealing temperature and establishing the standard curve)The detection of target genes in water samples was carried out by and three technical replicates were performed for each sample.2.Correlation between dry weight of Chlorella cells and changes in target gene abundanceThe Chlorella concentrates were transferred into glass triangular flasks under aseptic conditions and sealed,and incubated in a light incubator for 3 days.The conditions were 25 ℃,2000 Lux light intensity,12 Hr Light / 12 Hr Dark.The flasks were rotated uniformly at 8:00,13:00 and 17:00 every day,and the position of the flasks was changed frequently so that Chlorella could receive uniform light.Subsequently,the expansion of Chlorella was carried out using BG11 medium at a volume ratio of 1:5.The expanded Chlorella was concentrated by centrifugation(8000 rpm,10 min)and the dry weight was measured: first,a 1.2 μm filter membrane was spread on the top layer of the filter core and the algal solution was poured into a vacuum filtration device for vacuum aspiration.The filter membranes were dried in a drying oven for 12 h(105 ℃)and the membranes were dried and weighed in the same way beforehand to calculate the dry weight of Chlorella cells using the difference method.The dry weight was measured and inoculated at a concentration of 90 mg/L into the water samples collected from South Lake.After inoculation with Chlorella,the experimental and control groups were placed in a light incubator and incubated under the same conditions as Chlorella expansion.Forty m L of water samples were collected on days 1,4,7 and 10,respectively,and DNA was extracted from the water samples in different phenotypes(plasmid,genomic and free),and the target genes of the water samples were detected using the established q PCR method.The dry weight of Chlorella cells was correlated with the change in target gene abundance to analyze the effect of Chlorella cell growth on target gene reduction.3.Reduction of target genes by Chlorella on water samples of different phenotypesThe absolute and relative abundance of target genes before and after Chlorella inoculation were used to analyze the reduction effect of Chlorella on different types of target genes in water samples,and the correlation between different types of ARGs and int I1 and 16 S.Results:1.Detection of target genes in water samplesThe results of target gene detection in this study showed that all six target genes were not detected in the free state in the water samples collected from South Lake,and only tetracycline ARGs(tet G and tet W)were not detected in the genome and plasmid,while sulfonamide ARGs(sul1 and sul2),int I1 and 16 S were detected in the genome and plasmid,and four target genes(sul1,sul2,int I1,16S)were detected at higher concentrations in the plasmid than in the genome(P < 0.05).The growth rate of dry weight of Chlorella cells and the reduction rate of target gene abundance showed that both the reduction rate of absolute abundance of ARGs and dry weight of Chlorella cells reached the highest growth rate on the 4th day after inoculation,and the growth rate decreased significantly on the 10 th day.The correlation analysis showed that the absolute abundance of target genes was negatively correlated with the dry weight of algal cells(P < 0.05),which indicated that the growth of algal cells had an effect on the reduction of target genes.3.The effect of Chlorella on the reduction of target genes in different types of water samplesThe results showed that Chlorella could achieve a good reduction effect on ARGs in the water samples.The absolute abundance of sul1,sul2,int I1 and 16 S in the genome could reach33.9 %,34.0 %,35.5 % and 24.3 %,respectively,and the relative abundance of sul1,sul2 and int I1 could reach 12.6 %,15.7 % and 35.5 %,respectively.Among the plasmids,the absolute abundance could reach 59.1 %,55.3 %,55.8 %,44.3 % for sul1,sul2,int I1 and 16 S,respectively,and the relative abundance could reach 25.3 %,22.0 %,50.0 % for sul1,sul2 and int I1,respectively.The absolute and relative abundance of these four target genes were significantly reduced by Chlorella,and the absolute abundance of ARGs on the plasmid decreased more significantly than that of the genome,and there was a positive correlation between the changes of ARGs of different phenotypes and the absolute abundance of int I1 and 16 S,and the association strength of ARGs with int I1 on the plasmid was stronger than that of the genome.The results showed that Chlorella vulgaris has a certain reduction effect on ARGs in water,and the biomass of algal cells is the influencing factor of the reduction effect,and the diffusion of these ARGs in the water environment is dominated by cellular plasmids,and there is an association with class I integrons(int I1)and microbial communities.Conclusion:1.In the water samples,none of the six target genes were detected in the free state.In the plasmids and genomes,tetracycline-like ARGs were not detected,while sulfa-like ARGs,int I1 and 16 S were detected.2.The absolute and relative abundance of ARGs in water samples inoculated with Chlorella showed a decreasing trend,indicating that Chlorella had a significant reduction effect on typical ARGs in water.3.After inoculation with Chlorella vulgaris in water samples,the sulforaphane ARGs(sul1 and sul2)in both plasmid water samples and genomic water samples were correlated with int I1 and 16 S,and the plasmid ARGs were more strongly correlated with transmission-related genes,which provided a basis for the transmission mechanism of ARGs integrated on the integrons of plasmid and transposon,with horizontal transfer as the main mechanism. |
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