Mechanism Of Horizontal Transfer Of Antibiotic-Resistance Genes By Sublethal Photocatalysis In Typical Antibiotic Resistant Bacteria In Water Environment

The growing misuse of antibiotics,accompanied by the frequent occurrence of major public health events,has led to a growing awareness of the health risk issues posed by the medication crisis.Water is one of the most abundant resources on earth,and health risks caused by the spread of antibiotic resistant bacteria（ARB）and antibiotic resistance genes（ARGs）in the water bodies have become a research hotspot worldwide.Modes of transmission of bacterial antibiotic resistant include vertical gene transfer and horizontal gene transfer,which in turn include conjugative transfer,transformation,and transduction.Overall,however,most current studies of ARGs migratory diffusion by the academic community are limited to a certain diffusion pathway.The migration spreading law of ARGs has not been studied on the whole.Photocatalysis,as an emerging advanced oxidation process,can be used for terminal disinfection technology of water treatment,which may have an impact on ARG migration diffusion.Therefore,in this thesis,a set of methods for systematically evaluating the horizontal transfer mechanism of ARB as well as the migration of ARGs were established by setting up different kinds of E.coli bacterial strains,in UV_365nm+Ti O₂photocatalytic system.In which E.coli DH5α（CTX）,E.coli DH5α（MCR）,and E.coli C600 were used to investigate the conjugative transfer mechanism of ARGs;and E.coli DH5α,E.coli HB101were used to explore the transformation mechanism of ARGs.On this basis,the effects of sublethal photocatalysis on the horizontal transfer of ARGs were systematically evaluated by stud Ying the changes in bacterial activity,antioxidant system,cell membrane changes,ATP content,and the expression of related genes.The main findings of this thesis are shown as follows:（1）In the UV_365nm+nano-Ti O₂photocatalytic system,three kinds of bacteria（E.coli DH5α（CTX）,E.coli DH5α（MCR）,and E.coli C600）were set up,and the photocatalytic intensity was adjusted so that the bacteria were in a sublethal state.We observed the changes of ARG conjugative transfer under sublethal photocatalysis.The conjugative transfer mechanism of ARGs under sublethal photocatalysis was investigated according to the obtained changes from bacterial activity,antioxidant system,ATP system as well as the levels of relevant genes.The results showed that when only donor bacteria（E.coli DH5α（CTX）,E.coli DH5α（MCR））were induced with sublethal photocatalysis,the conjugative transfer frequency of ARGs increased 3-6-fold,and intracellular reactive oxygen species（ROS）level of the donor bacteria was also up-regulated up to 8-fold at the same time.In contrast,the conjugative transfer frequency of ARGs exhibited an inhibitory trend when only the recipient bacteria（E.coli C600）were induced by sublethal photocatalysis.However,when the donor or recipient bacteria were induced beforehand for a specific period,the conjugative transfer frequency could increase up to a 10–22-fold.In addition,the frequency of horizontal transfer and its mechanism were related to the oxidative stress system,ATP system as well as the expression of related genes.Moreover,the contribution of the transformability and horizontal transfer of ARB extracellular plasmids were investigated.The results showed that E.coli C600 transformants accounted for 50%of the total number of transconjugants,indicating that transformation may also be one of the main pathways for horizontal transfer of ARBs in water bodies by ARGs.The above results indicate that sublethal photocatalysis will increase the frequency of horizontal transfer of ARGs via conjugative transfer and transformation pathways and enhance the propagation risk of ARBs in water environments.（2）Through the UV_365nm+nano-Ti O₂photocatalytic system,two kinds of bacterial（E.coli DH5a,E.coli HB101）with a plasmid carr Ying the ampicillin resistance gene AMP（p UC19）to investigate the feasibility of ARGs transformation under sublethal photocatalytic conditions.And the transformation model of ARGs under sublethal photocatalysis was optimized by two aspects from recipient bacteria concentration to incubation temperature.On the optimized model,the ARGs transformation was observed under sublethal photocatalysis.Further,the transformation mechanism of ARGs under the sublethal photocatalysis was studied based on the results of bacterial activity,antioxidant system,cell membrane line,ATP system as well as the levels of relevant genes.The results showed that the sublethal photocatalysis would promote the transformation of ARGs in the water environment.Specifically,the transformation frequency of Ampicillin（AMP）resistant bacteria carried by p UC19 increased at most 3-5-fold after 50-60 min of sublethal photocatalytic induction by E.coli DH5α,E.coli HB101.The frequency of transformation of ARGs was also related to the concentration of recipient bacteria as well as the incubation temperature for transformation.It was found that after ARG transformation,the recipient bacteria were more able to adapt to the adverse effects brought about by sublethal photocatalysis.On mechanistic investigation,it was found that the elevation of ROS in bacterial could similarly promote ARGs transformation.We then further found that sublethal photocatalysis elevates the bacterial extracellular protein secretion as well as the expression of type IV pili,which is beneficial to elevate the size of the cell membrane transport channel of bacteria and thus facilitates the influx of free ARGs.The promotion of ROS with the promotion of cell membrane permeability,will promote the influx of Ca²⁺into the cell,Ca²⁺accumulation would not only activate the antioxidant system of bacteria,but also induce bacterial apoptosis,and then have an effect on the transformation of ARGs.In contrast,the binding transfer of ARGs,although there is no direct association between the bacterial ATP system and the transformation of ARGs,the inhibition of the bacterial ATP synthesis system may cause the blockage of the efflux function of Ca²⁺in the bacterial interior,and then lead to the accumulation of Ca²⁺in the bacteria,which may have an indirect effect on the transformation of ARGs.In summary,this paper found that if bacteria were induced to a sublethal state by photocatalysis,the spread of antibiotic resistance in water environment would be promoted.And the diffusion pathways of ARGs are diverse,including not only conjugative transfer,but also transformation.The above knowledge will provide some insights for comprehensively preventing the spread of drug resistance and eliminating ARGs as a emerging contaminant.