The Dissemination Of Antibiotic Resistance And Its Impact Factors In Atmospheric Environment And Artificial Systems In Vitro

Bacteria,as pathogens,spread diseases among humans or animals through physical contact,digestive tract,respiratory tract and other means.When antibiotics are used to treat bacterial infection-type diseases,bacteria can survive by developing or acquiring antimicrobial resistance(AMR).In recent years,antibiotics have been widely used in the medical and agricultural fields to prevent and treat bacterial infections.Antibiotics are the main antimicrobial drugs for clinical treatment of bacterial infections,which alleviate the problem of bacterial infections to a certain extent.However,long-term use of antibiotics will not only provide a selective advantage for the survival of antibiotic-resistant bacteria,but also promote the spread of AMR to pathogenic bacteria in the form of horizontal gene transfer(HGT),which also leads to the formation of antibiotic-resistant pathogens and even multi-drug-resistant(MDR)pathogens.Infections caused by MDR pathogens have doubled the difficulty of clinical treatment,and even faced the dilemma of no cure,which has posed a serious threat to human health.The production of MDR bacteria and the AMR dissemination in microbial communities have become research hotspots in recent years.The microbial communities commonly studied include intestinal tract,water environment,activated sludge and soil microbiome.However,as the air is an indispensable environmental factor in human life,there were relatively few studies on the microbiome and resistome on atmospheric particulate matter(APM).In recent years,with the spread of various respiratory infectious diseases,people's attention to the APM microbiome has increased.It is necessary and meaningful to explore and analyze the potential role of microorganisms on APM in the AMR dissemination.The AMR dissemination between microbial communities will produce MDR bacteria.Strictly exploring the AMR dissemination dynamics in various situations is the basis for understanding the AMR dissemination.Whether,how,and to what extent antibiotics affect the spread of AMR in different microbial communities was still unclear.Based on this,a self-made continuous culture device was used to simulate and explore the difference in AMR dissemination dynamics produced by antibiotic treatment at different concentrations and at different stages when MDR bacteria invaded different types of microbial communities,and initially explored the mechanism that caused these differences.The main content and results of the thesis are as follows:1.The biological and chemical components of APM and their potential role in the spread of AMR.From 2015.12.21 to 2016.01.20,114 APM samples were collected on the top floor of the teaching building of Shandong University in Jinan City,and the microbial structure,chemical element composition and prevalence of antibiotic resistant genes(ARGs)of the samples were investigated,and their correlation with the environment parameters was analyzed,and found that:(1)Two opportunistic pathogens,Klebsiella and Aeromonas,were significantly positively correlated with PM2.5 and temperature,indicating that they can proliferate on APM;(2)The chemical elements on PM2.5 and PM10 were different,but the microbiome structures were similar,indicating that although their sources were different,this will not affect the microbiome structure;(3)The changes of the microbiome structure in different sampling periods indicated that human activities will affect the composition of the microbial community in APM;(4)Rare metals including lanthanides have an inhibitory effect on pathogenic bacteria in APM;(5)Except for carbapenem,the resistance genes of the other antibiotics and the integrase genes of the genetic element"integrons" have been commonly detected,indicated that APM played a potential role in AMR dissemination.2.There was a significant correlation between the microbiome and the resistome in APM.A total of 112 APM samples were collected in different cities in the north and south of China.In addition to analyzing the microbiome of APM,investigating the prevalence of ARGs,and analyzing the impact of environmental parameters on the microbiome and resistome,bioinformatics analysis(Procrustes analysis and Mantel test)was used to further explore the correlation between the microbiome and the resistome in APM,and found that:(1)APM in northern and southern China have significantly different microbiome and resistome,but the distribution of them were similar from the perspective of urban clustering;(2)Environmental parameters have a significant impact on the microbiome and resistome in APM:the impact of particulate matters and chemical pollutants on them was opposite to the impact of temperature and wind speed on them;(3)There was a significant correlation between the microbiome and the resistome.The abundance of 40 microbial genera was significantly related to the resistome,including 39 bacterial genera and 1 archaeal genus Nitrosoarchaeum.(4)The correlation between the microbiome and the resistome may be related to the correlation between bacteria and some genes.For example,Proteobacteria has a significant positive correlation with intI1 and intI3,while Firmicutes was significantly negatively correlated with intI1,oqxA and blaVIM1.In addition to being significantly positively correlated with qnrB,Bacteroidetes was significantly negatively correlated with other quinolone resistance genes.It is worth noting that Latescibacteria were significantly positively correlated with the colistin resistance gene mcr-1 and vancomycin resistance gene vanA.3.The dynamics of antibiotics to promote the spread of MDR plasmids in E.coli.Based on the high prevalence of AMR in the atmospheric environment,a quadruple fermenter device was used to simulate the invasion of MDR bacteria in the environment,and to explore the AMR dissemination dynamics in E.coli species under the pressure of antibiotics.Found that:(1)AMR transmission events can still occur without antibiotic pressure;(2)Under the pressure of sublethal concentration of ceftazidime(CAZ),the rapid spread of AMR-carrying plasmids leads to a significant increase in the overall AMR in the flora;(3)The low concentration of CAZ at the ng/L level has no effect on the spread of AMR.4.Set up a continuous-culture device to simulate and analyze the AMR dissemination dynamics when MDR bacteria invade.A continuous-culture device was used to explore the AMR dissemination dynamics from E.coli to the pathogenic Salmonella enterica community under different tetracycline(TET)concentrations,and found that:(1)HGT and vertical gene transfer(VGT)both played an important role in promoting the spread of AMR,but the impact varies in different situations;(2)Although low-concentration TET cannot provide the selective advantage of the growth of resistant bacteria,it can promote the spread of AMR by enhancing HGT,which should be given enough attention;(3)The early invasion of MDR E.coli completely and rapidly destroys the effectiveness of subsequent antibiotic treatment;(4)After the occurrence of S.enterica infection,if antibiotics were used for treatment in time,even if the invasion of MDR E.coli occurs later,the antibiotic treatment still has a certain effectiveness;(5)TET at an environmental concentration will promote the conjugation frequency of MDR plasmids,which can cause the up-regulation of HGT-related genes and SOS response-related genes.5.In vitro simulation and evaluation of the AMR dissemination dynamics when S.enterica and MDR E.coli invade the Bifidobacterium infantis community early in life.The infant gut microbiome(IGM)dominated by Bifidobacterium is fragile and susceptible to bacterial infections.The spread of AMR between bacteria makes it more difficult to treat infant bacterial infections clinically.A self-built in vitro anaerobic continuous-culture device was used to simulate IGM bacterial infections of different severity,and the AMR dissemination dynamics under these events were analyzed,and the role of B.infantis was evaluated,and found that:(1)In the simulated event of mild bacterial infection,although the number of sensitive S.enterica will decrease significantly after antibiotic treatment,but the sensitive S.enterica will eventually colonize the B.infantis flora stably;(2)In the simulated event of moderate bacterial infection,B.infantis can effectively reduce the rapid spread of AMR by inhibiting the occurrence of VGT of drug-resistant bacteria;(3)In the severe bacterial infection simulation event,the ability of B.infantis to inhibit the spread of AMR was not observed;(4)In the microbial community,there was a sequence of conjugation frequency of plasmids.They will preferentially transfer to the same species,while the conjugation frequency to heterogeneous bacteria was lower;(5)The spread of AMR in the intestinal environment was low,which may be because B.infantis can inhibit the proliferation of MDR bacteria,and the presence of bile salts in the intestine can significantly reduce the conjugation frequency of AMR-carrying plasmid.In summary:The atmosphere was rich in AMR,and almost all types of ARGs are ubiquitous in APM;the phenomenon that pathogenic bacteria can proliferate on APM further revealed that APM,a widely accepted and ubiquitous pollutant,can be used as some pathogenic bacteria.(Such as Fusobacteria,Klebsiella and Aeromonas),and human activities will have a significant impact on changes in the microbiome structure;environmental parameters significantly affect the structure of the microbiome and resistome,among which the impact of environmental pollutants and particulate matter concentration were opposite to the effects of temperature and wind speed;there was a significant correlation between the microbiome and the resistome in APM,which was mainly caused by the abundance of 39 bacterial genera and 1 archaea(Nitrosoarchaeum),and the correlation between the microbiome and resistome may be related to the significant correlation between bacteria(such as Proteobacteria,Firmicutes,Bacteroidetes and Latescibacteria,etc.)and some genes(such as intI1,intI3,oqxA,blaVIM1,qnrB,mcr-1 and vanA).Based on the high prevalence of AMR in the atmospheric environment,the spread of AMR when a small amount of AMR-carrying bacteria in the environment invades different community systems has been further simulated and explored.The spread of AMR in bacterial communities was very common,even if there was no antibiotic pressure,AMR dissemination events still occur.The presence of antibiotics significantly promoted the AMR dissemination.However,the contribution of HGT and VGT in promoting the spread of AMR when treated with higher concentrations of antibiotics in different situations was different.Long-term exposure to low-concentration TET pollutants in the environment increased the conjugation frequency of plasmid between bacteria,which promoted the spread of AMR,and this process is not only related to SOS Response and HGT related pathways,but also exists unknown molecular mechanism.The bile salts in the intestinal environment can inhibit the conjugation frequency of plasmid and thus inhibit the spread of AMR.The effect of B.infantis in inhibiting the spread of AMR in bacterial infections can also be used in adjuvant treatment of bacterial infections,through reasonable use of B.infantis medicine ensures the therapeutic effect of antibiotics,while minimizing the spread of AMR and alleviating the current clinical situation of the rapid spread of AMR between bacteria.