| As is known to all,the spread of antibiotic-resistant bacteria(ARB)and antibioticresistant genes(ARGs)and the formation of antibiotic-resistant biofilms have become two important issues affecting public health worldwide.As the default growth mode of many bacteria,biofilms exist widely in water supply systems,medical systems and natural water bodies.However,in many cases,the growth of biofilms seriously affects the sustainable development of human health and environmental systems.Biofilms can form on living and abiotic surfaces with adverse effects on the environment,industry and human health.Therefore,it is particularly important to inhibit and remove antibiotic-resistant biofilms.In addition,it has been reported that about 95 % of the biomass in the water environments is in the form of biofilms,which can also continuously release planktonic bacteria,causing persistent pollution in the water.Therefore,the removal of biofilm in water supply system is particularly important.Due to the mass production and application of nanomaterials,nanoparticles are released into the water system.Therefore,bacteria are easily exposed to sub-lethal photocatalysis in the process of ultraviolet disinfection in sewage treatment systems.It is well known that a certain degree of harsh environment can promote the growth of biofilms.However,it is not clear whether photocatalytic stimulation inhibits or accidentally promotes biofilm formation during wastewater treatment,or if more complex conditions occur.Moreover,in practice,when the concentration of reactive oxygen species(ROS)produced in the system is low,most bacteria usually escape lethal attack and only experience sublethal oxidative stress.This process can be defined as sublethal photocatalysis,and some bacteria will be subjected to sublethal attack by ROSs during this process,but a considerable number of bacteria can still grow into films,which may slow down the bacterial killing process during disinfection.Therefore,it is necessary to study the growth process of bacterial biofilm under sublethal photocatalytic stimulation and the killing mechanism of mature biofilm under photocatalytic stimulation in detail,so as to comprehensively understand its response mechanism to stress.The main results of this thesis are as follows:(1)Firstly,we explored the biofilm formation mechanism of resistant bacteria Pseudomonas aeruginosa(GEN)under the sub-lethal photocatalysis.We used enzyme standard instrument to detect the change of bacteria abundance in the biofilm and we found that sub-lethal photocatalytic stimulation reduced bacterial abundance in the biofilm.It was found that sub-lethal photocatalytic stimulation could reduce the thickness of biofilm growth by observing the change of thickness of biofilm by confocal laser fluorescence microscope.Scanning electron microscope was used to observe the morphology of biofilm,and it was found that the morphology of biofilm growing under the action of sub-lethal photocatalysis was significantly damaged.The changes of biofilm barrier function were observed by freezing slicing machine and laser confocal fluorescence microscope.It was found that the barrier ability of biofilm grown under sub-lethal photocatalysis was significantly damaged.The oxidative stress response and extracellular polymeric substance(EPS)secretion stress response of bacteria in the biofilm were detected by relevant biological kit.It was found that sub-lethal photocatalytic stimulation could cause strong oxidative stress response and EPS secretion stress response of bacteria in the biofilm.We found that the number of viable unculturable(VBNC)bacteria in the biofilm culturing under sub-lethal photocatalytic stimulation was less than that in the control group by using flow cytometry combined with live cell imager detection and enzyme standard instrument.Finally,we used real-time quantitative PCR to detect genes related to biofilm growth,antibiotic resistance,oxidative stress and EPS secretion,and found that the expression of related genes was up-regulated to varying degrees.We found that with the increase of EPS secretion,the barrier ability and thickness of biofilm also gradually increased,thus producing a defense against sub-lethal photocatalytic stimulation,and the expression of related genes would also be down-regulated,further leading to the reduction of oxidative stress response and EPS secretion response.Therefore,with the extension of culture time,biofilm growth and sub-lethal photocatalytic damage would reach a certain degree of dynamic balance,and finally the biofilm entered a slow growth state.This part revealed a series of stress response mechanisms of antibioticresistant biofilm under sub-lethal photocatalysis.In the meanwhile,this part also revealed a phenomenon that the sub-lethal photocatalysis could only inhibit rather than prevent the growth of biofilm and the cause of its occurrence.The conclusion of this thesis provides an effective strategy for the growth inhibition of biofilm in water supply circulation system,and has very important research significance for the control and elimination of biofilm pollution.(2)Further,we continued to explore the stress response mechanism of mature biofilm formed by conditioned bacteria(Pseudomonas aeruginosa)under the sub-lethal photocatalysis,further explored the killing mechanism of sub-lethal photocatalysis on mature biofilm and the comparative study of bacteria released from the biofilm and bacterioplankton.The destruction of sub-lethal photocatalysis on biofilm morphology was observed by scanning electron microscopy,the death of bacteria in biofilm was detected by flow cytometry combined with living cell imager,and the destruction of sub-lethal photocatalysis on biofilm barrier ability was observed by laser confocal fluorescence microscopy.The change of the pathogenicity of bacteria were detected by RTCA multi-functional real-time unlabeled cell analyzer,and the difference of the antibiotic-resistance of the bacteria dispersed from the biofilm was detected by minimum inhibitory concentration(MIC),and the change of the antibiotic-resistance mechanism was further detected.The results showed that under the sublethal photocatalysis,the mortality rate of bacteria within the biofilm was not significant.Within 4 h sub-lethal photocatalysis,the mortality of bacteria within biofilm reached only13.9 %.At the same time,the biofilm structure was collapsed,and the biofilm barrier ability was impaired.The biofilm gradually transformed from biofilm state to bacterioplankton state,but not eliminated the harm to the environment completely.The bacteria dispersed from the biofilm were more pathogenic and antibiotic resistant than bacterioplankton,and it was found that the pathogenicity and antibiotic resistance could not be inherited stably without stress stimulation.The increase in pathogenicity of the bacteria dispersed from the biofilm comparing with bacterioplankton was manifested as the increased in cytotoxicity and the promotion of apoptosis process,while the basic reason was caused by an increase in virulence factors of bacteria.The increased in antibiotic resistance was manifested as the change of MIC value,while the basic cause was the change of bacterial antibiotic resistance mechanism,including an increase of the efflux capacity of bacterial efflux pump and the permeability of bacterial cell membrane.This part revealed that sub-lethal photocatalysis could transform the biofilm from the biofilm state to the bacterioplankton state,but could not completely eliminated the harm to the environment and the reasons for their occurrence.Relevant conclusions obtained in this thesis can provide an effective strategy and theoretical support for the elimination of biofilm,which has very important research significance for the control and elimination of biofilm pollution.In summary,this study respectively explored the growth mechanism of biofilm formation by antibiotic-resistant bacteria under sub-lethal photocatalytic stimulation and the killing mechanism of mature biofilm by photocatalytic technology.It was found that sub-lethal photocatalysis could only inhibit the growth of biofilms,but could not completely prevent the growth of biofilms.With the extension of culture time,the growth of biofilms and the damage of sub-lethal photocatalysis would reach a certain degree of dynamic balance,and finally the biofilm would enter a slow growth state.The killing of biofilm by photocatalytic technology could transform the biofilm from biofilm state to plankton state,but could not completely inactivated the biofilm.Compared with original plankton bacteria,the free bacteria had been found to be stronger pathogenicity and stronger antibiotic resistance.Meanwhile,such pathogenicity and antibiotic resistance could not be inherited stably.On the whole,the research of this thesis can provide a new research idea for the further exploration of the formation of biofilm(attachment-growth-falling off-diffusion)and the corresponding stress strategy of biofilm to related stimuli,as well as a new method and idea for the elimination of biofilm.At the same time,this thesis reveals the sub-lethal condition,a series of stress response mechanism in the process of biofilm formation and mature biofilm diffusion mechanism,the relevant conclusions of biofilm growth inhibition in water circulation system provides an effective strategy,to control and eliminate the biofilm pollution has very important research significance. |
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