Inactivation Kinetics And Gene Transfer Mechanism Of Different Antibiotic Resistant Bacteria With Different Resistance Action Targets By Photocatalysis

The widespread spread of antibiotic resistance genes（ARGs）in the environment al waters caused the emergence of a large number of antibiotic resistant bacteria（ARB）,which posed a serious threat to human and environment.Therefore,developing an efficient method for inactivating ARB and ARGs from environmental water has become an urgent problem.Currently,commonly used disinfection methods such as UV,chlorination and ozonation cannot effectively inactivate ARB and ARGs.Therefore,the UVA-Ti O₂heterogenous photocatalytic oxidation,an advanced oxidative disinfection method,was used to systematically investigate the effectiveness of the inactivation of ARGs and ARB,whose respective resistance to six antibiotics with different modes of action targets（cell wall,cell membrane,protein,folate,DNA and RNA）.After that,the important physica l and chemical parameters of the sterilization process were screened and optimized.On this basis,the stress response of photocatalytic oxidation to ARB with different antibiotic resistance action targets under sub-lethal or lethal conditions and the effect of photocatalytic oxidation on the horizontal gene transfer（HGT）of ARGs were studied to reveal the underlying mechanisms.Based on the obtained results,more detailed information was provided for the practical application of photocatalytic oxidation t o inactivate ARB and hampering the transfer of ARGs in environmental water.The main results of this thesis are as follows:（1）UVA-TiO₂ heterogenous photocatalytic oxidation has efficient inactivation ability of ARB and ARGs in environmental water,and its inactivation efficiency is significantly higher than that of UV.First,based on the response surface methodology,the highest inactivation efficiency of ARB was obtained under the photocatalytic oxidation conditions of p H 7.0,25℃,dissolved oxygen concentration of 7.0 mg L^-1 and catalyst concentration of 1.5 g L^-1.The highest degradation efficiency of ARGs was obtained under the photocatalytic oxidation conditions of p H 7.5,30℃,dissolved oxygen concentration of 6.5 mg L^-1 and catalyst concentration of 1.5 g L^-1.Photocatalytic oxidation could accelerate the lysis of ARB,and induce DNA structural damage of plasmid carrying ARGs and change its conformation under the optimized and selected optimal conditions.（2）Antibiotic resistance action targets influence photocatalytic tolerance of ARB:The tolerance of ARB under the conditions of photocatalytic oxidation was significantly improved.Compared with ASB,it has a longer survival time and significantly improve d antibiotic resistance.The folds of increasing change of antibiotic-resistance level of different ARB were up to 16 for cefotaxime（CTX）,64 for polymyxin（PB）,32 for tetracycline（TET）,16 for sulfamethoxazole（SMZ）,32 for nalidixic acid（NAL）and 16for rifampicin（RA）at the process of photocatalytic oxidation The stress response mechanism of E.coli DH5α（TET）and E.coli DH5α（SMZ）targeting on the synthesis of protein and folate could rapidly induce global regulators by the overexpression of relat ive antibiotic resistance action target genes,mediating various stress response systems through a mechanism of cross-protection,which caused stronger tolerance to adverse environment than other ARB.Moreover,photocatalytic inactivation mechanism on bacterial cells and graded response of cellular stress mechanism caused the differences in the intensity of stress mechanism about antibiotic resistance action targets.The E.coli DH5α（CTX）and E.coli DH5α（PB）targeting on the synthesis of cell wall and cell membrane,could confer more advantages to bacterial survival in adverse environment than E.coli DH5α（NAL）and E.coli DH5α（RA）targeting on the synthesis of DNA and RNA.（3）UVA-TiO₂ heterogenous photocatalytic oxidation regulates HGT of ARGs:The concentration of ARGs decreased significantly under high-intensity,long-term photocatalytic oxidation induction,and some ARGs such as sul3 and mcr-1 could be completely inactivated under 24 h photocatalytic oxidation conditions.At the same time,it could be found that ARGs were hardly affected by photocatalytic oxidation in the lethal or sublethal state of ARB,and the concentration remained basically unchanged at the copy number level of 7.5 log.Moreover,20-min photocatalytic oxidation process promoted the horizontal gene transfer（HGT）of ARGs in environmental water under sub-lethal conditions of ARB.The frequency of conjugative transfer was related to the stress response mechanism of antibiotic resistance action targets.The stress response of high-intensity antibiotic resistance action targets could increase the frequency of conjugative transfer,which was the main way of HGT at the early stage of photocatalytic oxidation and the ARGs conjugative transfer is about 45%.In contrast,transduction is the main way of HGT in the middle to late stages of photocatalytic oxidation,which triggered the transduction of ARGs with a frequency of 58%.The natural transformation and transduction efficiency of ARGs were regulated by photocatalytic oxidation,and the recipient bacteria could overexpress related horizontal transfer genes to accelerate the spread of ARGs in environmental waters.The donor bacteria mediated the occurrence of natural transformation and transduction through the ctr A gene regulation.The overexpression of the Rc GTA gene increased the frequency of the transduction process so that the transduction was the main way in which HGT occurs in the middle and late stages of photocatalytic oxidation.Based on the obtained results,the mechanism about the changes in the tolerance of ARB at the process of photocatalytic oxidation disinfection were revealed in depth.At the same time,the main ways and reasons for the HGT occurrence of ARGs in the environmental waters were determined under exposure to the disinfection process of photocatalytic oxidation.The theoretical guidance for photocatalytic oxidation on inactivating ARB and preventing the spread of ARGs in the environmental waters were provided.