Occurrence,Transcription,and Bacterial Hosts Of Antibiotic Resistome In Microplastic Biofilm

Microorganism is the vital component of structural and functional composition in freshwater environments,playing key roles in many important ecological processes,such as energy metabolism and elemental cycle.Lately,microplastic has been generally acknowledged as a widely distributed pollutant and has been detected in various freshwater environments on earth.The environmental behaviors and impacts of microplastics have aroused wide concern.Once entering the water,microplastics will be colonized by bacterial communities,which is the main response of microorganism in freshwater environments to the entering of microplastics.Most research focused on the distribution and biological toxicity of microplastics.However,relatively less attention was paid to the community composition and structural features of microplastic biofilm.The occurrence,expression and transfer of antibiotic resistome in plastisphere are also largely unknown.Hence,this study aimed to answer these questions and provided essential information for understanding the distinctiveness of microplastic when serving as a novel niche,and the function of microplastic during enriching and spreading antibiotic resistome.The research contents and main results of Chapter 2 to Chapter 6 in this study are listed as follows:1.Based on the combination of continuous culture by bioreactors and 16S r RNA gene sequencing,the study in Chapter 2 explored the differences of microbial community composition,structure,and function between microplastic biofilm and natural substrate（rock and leaf）biofilm.During the whole cultivation period（14 days）,the concentration of planktonic bacterial community in river water kept relatively stable at 2.7×10⁷ cells·m L^-1.The microplastic biofilm biomass increased from the first day to the seventh day and reached the peak(3.1×10⁸ cells·g^-1)on the eighth day.The biomass then became relatively stable.The biomass of microplastic biofilm was approximately 2.4 times of that of rock biofilm,but lower than leaf biofilm.By analyzing the 16S r RNA gene sequencing data,three bacteria phyla（Proteobacteria,Bacteroidetes,and Firmicute）were found to be the dominant groups in microplastic biofilm.The dominant groups in microplastic biofilm were similar to that in rock biofilm but were different compared with leaf biofilm.As forαdiversity,the Shannon-Wiener index displayed the trend of microplastic biofilm>rock biofilm>leaf biofilm,which showed that theα-diversity in microplastic biofilm was higher than biofilm on natural substrates and microplastic biofilm was more reluctant to disturbance of external environment.The PCo A analysis was conducted to illustrate theβ-diversity of all samples.The microplastic biofilm samples clustered in a group,separating from the group of rock and leaf biofilm samples.The differentiating factor of these three types of biofilms could be mainly attributed to the material features of the attached substrates.All biofilm samples showed significant differences compared with planktonic bacterial communities.Microplastic significantly enriched 296 OTUs from river water,higher than that in rock biofilm and leaf biofilm.The above results indicated that the bacteria groups attaching to solid surface were different compared with bacteria with planktonic lifestyle in river water.2.By metagenomic sequencing and alignment with database,the study in Chapter 3 investigated the resistance types,diversity,and abundance of antibiotic resistance genes（ARGs）in microplastic biofilm and natural substrates（rock and leaf）biofilm.Among all samples,a total of 267 ARGs belonged to 26 ARG types were detected.The resistome abundance in microplastic biofilm was approximately 3 times as much as that in river water,indicating that the biofilm structure has the ability of enriching ARGs.In microplastic biofilm,the ARGs with multidrug resistance type had the highest proportion.After annotating the taxonomy and calculating the proportions of ARG bacterial hosts,the resistance genes in microplastic biofilm were mainly carried by Pluralibacter,Pseudomonas,Leclercia,and Pantoe.On species level,the plant pathogen and human opportunistic pathogens were found in microplastic biofilm,including Pseudomonas monteilii,Pseudomonas mendocina,and Pseudomonas syringae.Specifically,these pathogens only existed in microplastic biofilm,but not in rock and leaf biofilm,which indicated that microplastic biofilm could selectively enrich specific bacterial pathogens.3.The studies in Chapter 4 and Chapter 5 characterized the microbial community composition,antibiotic resistome,and co-occurrence network structure of biofilm on two types of microplastic:biodegradable PLA and non-biodegradable PVC.According to the results,the PLA microplastic and PVC microplastic had significantly different biofilm community composition.A total of 150 ARGs were detected in PLA microplastic biofilm and 137 ARGs were detected in PVC microplastic biofilm.The abundance of ARGs in PLA biofilm was 1.29×10^-1,higher than that in PVC biofilm(1.26×10^-1),indicating that PLA microplastic could carry more ARGs.The co-occurrence network analysis revealed that Proteobacteria was the main bacterial phylum in both PLA and PVC biofilm.A number of human opportunistic pathogens were found in PLA biofilm,including Klebsiella oxytoca,Kluyvera intermedia,and Leclercia adecarboxylata.In PVC,the pathogen Klebsiella pneumoniae was detected.The bacterial pathogens mentioned above belonged to Enterobacterales.The phenomenon that pathogenic bacteria with antibiotic resistance colonized on microplastic surface was not a coincidence,which may be resulted from the physical and chemical properties of the microplastic material.4.By metatranscriptomic sequencing,the study in Chapter 6 detected,annotated,and calculated the abundance of transcripts of antibiotic resistance genes in PLA microplastic biofilm and PVC microplastic biofilm.Among the 173 ARGs in plastisphere,75 ARG transcripts with 18 ARG types were detected and 42%of the ARGs in PLA microplastic biofilm showed expressional activity.In PVC,the proportion was 46%.Although PLA microplastic biofilm loaded more ARGs than PVC,the proportion of activated ARGs was lower.By aligning with the protein database of plasmids,the plasmid associated ARGs were also studied.In plastisphere,the number of plasmids associated ARGs was 39.Plasmid p GMI1000MP carried gene acr B,acr F（acriflavine resistance type）,ade B,mex C（multidrug resistance type）simultaneously.The ARGs located in plasmids could complete gene replication independently and play vital role during the horizontal gene transfer of ARGs.The proportions of actively expressed plasmids associated ARGs in PLA microplastic biofilm and PVC microplastic biofilm were 6.45%and 5.59%,respectively.Only 2.54%of plasmid associated ARGs were observed with activity in river water.The results implied that plastisphere had an import effect on the process of horizontal gene transfer,posing potential ecological risks.This study compared the biofilms on artificial microplastic substrate and natural substrates,focusing on the responding process of microbial community to microplastic surface.This study provides an important step towards clarifying the environmental behavior and function of microplastic during antibiotic resistome dissemination in freshwater ecosystem.