| The provision of high-quality drinking water is one of the most important tasks in the modern city.Drinking water distribution system(DWDS)is the key step to ensure water quality at the end,and the regrowth of microorganisms becomes an important threat to tap water.Biofilms are the main habitat for the microorganisms in DWDS,and over 90% of microorganisms live in biofilms.Therefore,the problem related to the pipe wall biofilms needs attention.This study analyses the prevalence of antibiotic resistant genes(ARGs)and opportunistic pathogens(OPs)in drinking water biofilms,and investigated the microbial risk of drinking water.(1)The study on the prevalence of ARGs and OPs in drinking water and biofilms.The occurrence of the ARGs and OPs as well as the microbial community composition were investigated in a real drinking water supply system.The results showed that both ARGs including sul1,sul2,tet A,tet M,amp C,bla TEM,and OPs including Mycobacterium spp.,Legionella pneumophila,Pseudomonas aeruginosa,and Hartmannella vermiformis were detected in both biofilm and water samples.Tet M was observed to present in relatively high concentrations,with average relative concentrations of 1.18×10-2 and 5.19×10-3 in biofilm and water samples,respectively.;Mycobacterium spp.was observed to present in relatively high concentrations,with average relative concentrations of 2.23×10-1and 1.00×10-1 in biofilm and water samples,respectively.The relative abundances of ARGs and OPs in biofilm samples were significantly higher than those in water samples,which indicated that the biofilms are the important reservoir of ARGs and OPs and provide proper conditions for the spread of ARGs.The analysis of the microbial community revealed that the abundances of Deltaproteobacteria,Cyanobacteria,and Sphingobacteriia show a significant difference between biofilm and bulk water samples.The abundance of sulfate-reducing bacteria Desulfovibrio in biofilms was significantly higher than that in water samples.,which indicated the occurrence of sulfate-reducing reaction in DWDSs.The correlation analysis showed that the abundance of the OPs was significantly correlated to the relative abundance of ARGs and the mobile gene elements(MGE)int I1(p < 0.05),which indicated that the horizontal gene transfer(HGT)might be responsible for the spread of ARGs in DWDSs and the OPs might be the potential host of ARGs.(2)The study on the impact of environmental conditions on the conjugation transfer of ARGs: The types(chlorine or chloramine)and concentrations of disinfectant,as well as the concentration of nutrients(carbon source),were selected since they are common changing conditions in DWDSs.The results indicated that low concentrations of chlorine and chloramine could facilitate the conjugation of ARGs in biofilm and suspended bacteria,while the high concentration of disinfectant would suppress the processes,which indicated the low concentrations of disinfectants in DWDSs might improve the spread risk of ARGs.The average conjugation rates in biofilm and water were 6.76×10-4 and 2.53×10-5,respectively,which indicated that the biofilm had higher spread and health risks.Since the chlorine was more active but easier to decay while chloramine is harder to decay but less active,the improvement of chlorine in the water was higher than that in biofilm and the improvement of chloramine in biofilm was higher.The concentration of carbon source impacted the conjugation transfer in both biofilm and water,and the low concentration facilitated the processes.The impact of carbon source concentration on the conjugation rates in biofilms was less significant since the existence of extracellular polymeric substances(EPS)can protect the bacteria from starvation.(3)The study on the mechanism of improvement in the conjugation transfer of ARGs: Based on the results of(1)and(2),the investigation of cell membrane permeability,the formation of reactive oxygen species(ROS)as well as the expression of functional genes were conducted to reveal the related mechanisms.The results showed that chlorine/chloramine and starvation could increase the cell membrane permeability as well as the production of ROS,resulting in the improvement of conjugation transfer of ARGs.The results of reverse transcription-q PCR also showed that the low concentrations of chlorine/chloramine facilitate the expression of rpo S which was related to the formation of ROS;the down-regulated general functional genes including kor A,kor B,and trb A could activate the mating pair formation system,therefore resulting in the connection between the donor and receptor bacteria to facilitate the conjugation transfer.(4)The study on the safety risk and mechanism of biofilm detachment: Based on the above research on the spread mechanism of ARGs in biofilms,we further discussed the impact of biofilm detachment on the microbial community and ARGs in water.The results indicated that the detaching rates from biofilms were accelerated with increasing chloramine concentrations.The community diversity of detached biofilms was significantly higher than that of stubborn biofilms.The abundance of Nitrospira was significantly higher in stubborn biofilms than that in detached biofilms.The relative abundance of several chemoautotrophic bacteria,including Bryobacter,Hyphomicrobium,and Pedomicrobium,also showed a significant difference between stubborn and detached biofilms.The abundance of ARGs in detached biofilms was higher than that in stubborn biofilms.The total relative abundance of ARGs in biofilms after starvation treatment(0.022 and 0.055 copies/cell)was significantly higher than that without starvation treatment(0.016 and 0.0347 copies/cell),indicating that starvation treatment promoted the spread of resistance genes,leading to an increase in the relative abundance of ARGs.The hosts of ARGs detected in the different samples included Pseudomonas,Mycobacteriaceae,and Nitrospira,and the numbers of hosts detected in detached biofilms were significantly higher than those in stubborn biofilms,which indicated that the detached biofilms have higher risks on human health.In this study,the microbial community composition,as well as the prevalence of ARGs and OPs,were investigated.The results of the study confirmed that biofilms could be the reservoir of ARGs and OPs and the potential risk of the ARGs spread in drinking water need more attention.The analysis of conjugation transfer in suspended bacteria and biofilms with different concentrations and types of disinfectant and carbon sources revealed that low concentration disinfectant and starvation treatment would facilitate the conjugation transfer of ARGs.The detaching experiment studied the impact of chloramine concentration and starvation treatment on the detachment of biofilms and confirm the potential risk of ARGs spread through the biofilm detachment.The results of this research not only provide new thoughts for the evaluation of the biological safety about the spread of antibiotic resistance in drinking water and biofilms but also provide the theoretical basis and technical support for reducing the microbial risks and ensuring water quality safety. |
