The Regulation Of Acyl-homoserine Lactones Signal Molecules On The Response Of Sediment Nitrogen Cycle Under Florfenicol Stress

With the development of animal husbandry,the use of antibacterial drugs is increasing.After the antibacterial drugs are ingested by animals,only a small amount is absorbed by the animals,Most of them enter the environment with excreta such as feces and urine in the form of prototypes or metabolites.Remaining antibiotic in the environment will not only cause irreversible effects on the microorganisms,but also affect human health.Nitrogen cycle as one of the important components of the ecosystem cycle,the residues of antibiotics in the environment hinder its process to a certain extent,disrupt the metabolism of environmental microorganisms and affect the nitrogen balance.As a way of communication between bacteria,quorum sensing(QS)has a certain effect on itself and other bacteria when it communicates with other bacteria to adapt to the environment,and can regulate the metabolic pathways of microorganisms,thus affecting the process of nitrogen cycle.However,the effect of QS system on nitrogen cycling under antimicrobial exposure stress is still uncertain.In this paper,a common antimicrobial agent,florfenicol(FFC),was used as a stress factor to construct an indoor water sediment model by adding exogenous Acyl-homoserine Lactones(AHLs)signal molecules.Blank group(CK),florfenicol treatment group(FF 30μg/L),florfenicol plus AHLs treatment group(HF FFC: 30 μg/L,AHLs: 48 μg/L),were set up for three times,and samples were collected at 0 h,4 h,10 h,1 d,3 d,10 d and 20 d.The effects of FFC addition and AHLs addition on microbial community structure,functional diversity and nitrogen cycle in sediments were analyzed by metagenomics and q PCR.The results showed that the relative abundance of nir S was the highest in the treatment group,and the FF group could promote nir K.The relative abundance of nir K,nos Z and nir S genes in CK group and FF group was lower than that in HF group in the first 10 hours.The relative abundance of four denitrification genes in the first 10 hours in the FF group was about 5～10 times of that in the later sampling sites.However,at 10 hours,the relative abundance of four genes in the control group and the FF group was about 10 times higher than HF group.After 1 day,the change trend of relative gene abundance in each group was different.Compared with the FF group.The addition of AHLs increased the number of bacterial communities in the sediments.With the extension of the culture time,the detectable concentration of FFC in the samples decreased significantly(P<0.01).The content of FFC in the FF group was lower than that of the HF group 1d before incubation,and the FFC degradation in the HF group at 4 h and 1 d The rate was significantly higher than that in the FF group(P=0.046,P=0.014).The sediment samples in this experiment were analyzed by metagenome sequencing.The results of flora annotation showed that the dominant bacteria in the sediment were Proteobacteria,Chloroflexi and Euryarchaeota.The relative abundance of Proteobacteria and Euryarchaeota in both treatment groups was higher than that in CK group.The relative abundance of Chloroflexi in CK group was higher than that in HF group but lower than that in FF group.Among the 152 dominant genera in sediments,64 genera were significantly correlated with nitrogen levels(P<0.05),and most of them were negatively correlated with nitrate and nitrite.Functional annotation showed that FFC exposure stress significantly affected the total number of functional genes related to nitrogen cycling,and the addition of AHLs reduced the effect of FFC on the number of related functional gene annotation sequences.The abundance of "energy generation and transformation" and "amino acid transport and metabolism" were the highest in the treatment group,and the addition of AHLs reduced the response of some functions to FFC.Among the five modules of nitrogen metabolism,denitrification accounted for the highest proportion,followed by dissimilatory nitrate reduction.Among the enzymes related to nitrogen cycle,the abundance of nitrate reductase was the highest,followed by nitrite reductase,including nitrogenase,nitric oxide reductase,nitrous oxide reductase and ammonia monooxygenase,which responded to FFC and AHLs in different degrees.In addition,the correlation analysis between nitrogen cycle-related functional genes and dominant bacteria showed that many coding enzyme genes were significantly associated with more than one flora;correlation analysis between functional genes showed that nitrate reductase,nitrate/nitrite transport system and glutamase coding genes were the main participants in the correlation between genes.Most of the enzyme coding genes in the nitrogen cycle are affected by nitrogen levels,and most of them are negatively correlated with nitrate nitrogen and nitrite nitrogen,ammonia and total nitrogen contents are the main environmental parameters that determine the microbial structure.The above results show that florfenicol residues in water environment can rapidly change the microbial community structure in sediments,affect the functional diversity of microorganisms,and then hinder the process of nitrogen cycle.The response of microorganisms to florfenicol was enhanced or weakened by the addition of AHLs in florfenicol exposure.AHLs may affect the changes of functional microflora in sediments under FFC stress by regulating the expression of glutamate metabolic enzymes or nitrate /nitrite nitrogen transporters.