Bacterial Response Mechanism Of Typical Bacteria During Biofilm Growth On Different Metal Material Substrates And The Transfer Of Antibiotic-resistance Genes

Overwhelming bacterial biofilm grew onto different metal-based pipeline materials are intractable,which can pose a serious threat to public health when the tap water is running flow through these pipelines.However,many formation processes of microbial biofilm are still not elucidated,including growth mechanisms,stress strategies,and horizontal transfer of the genetic information.What drives the biofilm bacteria to evolve and adapt quickly?To fully understand and explain the mechanisms of biofilm formation and antibiotic-resistance gene diffusion,we need to better understand the biochemical and molecular biological mechanisms that control these processes.In this thesis,four kinds of metal materials were selected as the growth substrates of biofilm,and various Escherichia coli strains were selected as the research object.Biofilm was cultured in a home-made flow-cell biofilm reactor.With biofilm formation,the response mechanism of biofilm bacteria was investigated from the physiological and biochemical levels,protein levels to molecular levels.To investigate the stress strategies and changes of antibiotic resistance in the biofilms of antibiotic-resistant bacteria and compare them with those of sensitive bacteria.A simple hybrid biofilm system was established to investigate the changes of bacterial structure and horizontal transfer efficiency of antibiotic-resistance genes in the biofilm.Finally,the formation mechanism of biofilm growth on metal substrate and the spread behavior of antibiotic-resistance genes in biofilm were elucidated.The main results of this thesis are as follows:(1)First,a model bacterial strain,Escherichia coli K-12,was used for microbial growth and biofilm formation experiments.Four kinds of metals,stainless steel(SS),copper(Cu),titanium sheet(Ti)and nickel foam(Ni),were used for pipelines or are otherwise extensively used as materials were selected as substrates.Results revealed that bacteria proliferated when they grew on SS and Ti,quickly developing into bacterial biofilms.In contrast,the abundance of bacteria on Cu and Ni substates decreased sharply by 4.0-5.0 logs within 24 h.The morphological shrinkage and shortening of bacterial cells,as well as a sudden 64-fold increase of carbohydrate content in extracellular polymeric substances(EPS),were observed on Cu substrate.Furthermore,the generation of reactive oxygen species and fluctuation of enzymatic activities demonstrated the destruction of redox equilibrium in the bacterial cells.Bacteria cultured on Cu substrate showed the strongest response,followed by Ni,SS and Ti substrates.The oxidative stress increased quickly during the growth of bacterial biofilm,and almost all tested metals transporter-related genes were upregulated by 2-11 folds on Cu,which were higher than on other substrates(1-2 folds for SS and Ti,2-9 folds for Ni).Finally,these behaviors were compared under the biofilm regulatory molecular network.(2)Furthermore,the growth mechanisms of antibiotic--resistant bacteria(Escherichia coli DH5?(CTX))biofilm on SS and Cu substrates were further explored.The special mechanism of antibiotic-resistant bacteria biofilm in response to copper ion toxicity stress was compared with the stress strategy of sensitive bacteria biofilm.The results showed that the antibiotic-resistant biofilm grew faster than the antibiotic-sensitive biofilm.However,the bacterial abundance of the two strains on the Cu substrate decreased,indicating that the bacteria were partially killed by Cu poisoning regardless of whether the bacteria had antibiotic resistance or not.The difference is that the attenuation of the sensitive bacteria is relatively severe,which decreases by 4.5 logs within 3 days.EPS secreted by the sensitive bacteria on the Cu sheet increased to 940.0?g m L^-1 within 48 h.However,the content of EPS secreted by antibiotic-resistant bacteria increased slowly,and increased to 694.0?g m L^-1 within 48 h.This indicated that the antibiotic-resistant bacterial biofilm had better environmental adaptability.Based on the assessment of biofilm resistance,the biofilm resistance of antibiotic-resistant bacteria was greater than that of sensitive bacteria.Further investigation on the response of the antioxidant system of the resistant and sensitive bacteria on the Cu sheet revealed that the antioxidant enzyme activities of the antibiotic-resistant bacteria biofilm fluctuated less than that of the antibiotic-sensitive bacteria.Oxidative stress genes and metal transporter genes were also the most significant upregulation of the antibiotic-sensitive bacteria.At the mature stage of biofilm,the expression levels of biofilm genes of antibiotic-resistant bacteria were lower than that of antibiotic-sensitive bacteria.The ability of antibiotic-resistant bacteria to deal with the oxidative stress of Cu is obviously stronger than that of antibiotic-sensitive bacteria.(3)Finally,to explore the biofilm growth mechanism of mixed bacteria and the spread of antibiotic-resistance genes within biofilms,we selected two hybrid systems.One is of antibiotic-sensitive bacteria(Escherichia coli K-12)and bacteria carrying portable resistance gene plasmid(Escherichia coli RK2);another is of general antibiotic-resistant bacteria(Escherichia coli C600(Sm))and bacteria carrying portable resistance gene plasmid(Escherichia coli RK2).The growth activity,bacterial structure and gene transfer frequency of biofilm were compared in these two mixed systems.The relationship between the spread of antibiotic-resistance genes and the structure of biofilm was further explained by the changes in the expression levels of conjugation transfer related genes and biofilm related genes.The results showed that the growth activity of mixed bacterial biofilm was different from that of single bacterial biofilm.Bacterial structure and antibiotic-resistance gene conjugation in biofilm played an important role.The growth of the mixed biofilm composed of two kinds of antibiotic-resistant bacteria was more stable than that of the mixed biofilm composed of antibiotic-sensitive bacteria and antibiotic-resistant bacteria.Specifically,the maximum abundance of mixed bacteria of antibiotic-sensitive bacteria and antibiotic-resistant bacteria reached 4.170×10¹⁰CFU m L^-1 within 48 hours,and the maximum abundance of mixed antibiotic-resistant bacteria reached 7.760×10¹⁰CFU m L^-1 within 48 hours.Moreover,the biofilm of mixed antibiotic-resistant bacteria was abundant and thicker.The structural composition of bacteria was also changed.Specifically,antibiotic-sensitive bacteria accounted for 85%of the mixed bacteria and became the main species at 72 hours,while the proportion of bacteria in the mixed antibiotic-resistant bacteria system was relatively stable,between 40%and 50%.Clearly,both competition and cooperation were existed between different bacterial species.The results of the frequency of conjugation showed that the conjugation frequency of the bacteria in the biofilm increased significantly during the growth of biofilm,reached the highest in the maturity of biofilm,and the conjugation rate in the mixed antibiotic-resistant bacteria system increased by 100 times.The up-regulation of genes related to the conjugation and transfer mechanism and biofilm-regulated further indicate that these two processes are mutually reinforcing.Above all,this study explored the growth mechanism of single bacterial biofilm and mixed bacterial biofilm on different metal substrate,and the spread mechanism of antibiotic-resistance genes in during the formation of biofilm.It was found that bacterial biofilm had different stress strategies for different metal substrates.The stress response of antibiotic-resistant bacteria biofilm is different from that of common bacteria biofilm,and antibiotic-resistant bacteria adapt to the stimulation of environment more quickly.Antibiotic-resistant bacteria and antibiotic-sensitive bacteria can coexist in a mixed biofilm and have competitive and cooperative relationship.This thesis provides a research idea how to comprehensively explore the mechanism of biofilm growth and stress strategies.In addition,this thesis studied biofilm colonization on metal substrate and the spread of antibiotic-resistance genes in the membrane and proposed that the formation of bacteria biofilm and the spread of antibiotic-resistance genes in water distribution system threaten water environment and public health.