Study On The Safety And Hazard Formation Mechanism Of Microbe In Mineral Water Treatment System

It has been a serious public health problem that pathogens cause large-scale outbreaks of infectious diseases via contaminated drinking water.Previous investigations showed that about 350 million people worldwide were infected with waterborne pathogens each year,and10 to 20 million of them die from severe infections.Although the mineral water for consumers to drink has strict microbial standards,the production of mineral water that meets quality standards does not necessarily guarantee microbial safety.Inparticular,recent studies showed that mineral water contained pathogens and opportunistic pathogens,which will have a bad impact on the health of consumers.Bacterial community was ubiquitous in the entire mineral water treatment system（MWTS）.Therefore,the comprehensive understanding of the composition and dynamics of the bacterial community in MWTS is the key to ensure the microbial safety of mineral water.In this paper,the bacterial diversity and dominant microbiota in different seasons and different treatment processes in MWTS were systematically studied by using high-throughput sequencing technology and pure culture method.The bacterial community composition and dynamic change in MWTS were understood,providing necessary data for the prevention and control of microbial contamination in mineral water.In addition,as Pseudomonas aeruginosa was serious pollution and difficult to control in MWTS,we determined the ability of biofilm formation,virulence genes,disinfectant-resistant genes and antimicrobial resistance of 132waterborne P.aeruginosa isolates,and further characterized the isolates using multilocus sequence typing（MLST）for the future molecular tracing sources library construction to provide basic data.The main conclusions of this paper are as follows:（1）The biomass and biological activity of bacterial were performed by measuring total heterotrophic bacterial counts（HPC）,and adenosine tri-phosphate（ATP）respectively in MWTS.The biomass and biological activity of bacteria gradually declined with the progress of each treatment process.The bacterial biomass decreased significantly after filtration by quartz sand and fine filter（p<0.01）,but increased slightly after filtration by activated carbon.The overall trend of bacterial biological activity decreased,especially the concentration of ATP decreased by 98%after the quartz sand filter（p<0.01）,while the concentration of ATP increased significantly after the activated carbon filter（p<0.01）.The Pearson correlation analysis showed that turbidity,TOC and COD_Mn were the key factors affecting bacterial biomass and biological activity in MWTS,and there was also a very significant correlation between bacterial bioactive and biomass（p<0.01）.（2）The bacterial diversity of MWTS was studied using high-throughput sequencing technology in different seasons.The bacterial diversity index of water samples in dry season and in wet season had no significantly different.On the whole,the bacterial diversity of the raw water was the highest,and gradually decreased along with treatment processes.There was little difference in bacterial community composition between wet season and dry season in MWTS.The total sequences of Proteobacteria accounted for more than 60%of all sequences,which was the dominant phylum.The relative abundance of Bacteroidetes,Actinobacteria and Firmicutes changed greatly along with treatment processes.The relative abundance of Firmicutes increased sharply after ozone sterilization,indicating that Firmicutes had disinfection resistance.In the level of genus classification,the relative abundance of 25bacterial genera in all water samples was>1%,and the bacterial diversity was very high.The relative abundance of unclassified bacteria was more than 60%,which indicates that MWTS contains a large number of unknown microorganisms.The dominant genera in final product water were Pseudomonas and Acinetobacter.In the present study,the core microbiome comprised 15 families,which contains a large number of pathogens and opportunistic pathogens.As the dominant genera in MWTS,the relative abundance of Pseudomonas was high in all water samples.（3）In total,300 strains were isolated from raw water samples of MWTS in wet and dry season.All 300 isolates were distributed in 5 phyla,7 classes,17 families,19 genera.In the level of phylum classification,Proteobacteria was the dominant phylum,with a relative abundance of 55.7%.Compared with the bacterial community composition of raw water samples in two seasons,the relative abundance of Proteobacteria and Bacteroidetes significant changed.The relative abundance of Proteobacteria was 60.4%in the dry season and 50.4%in the wet season.The relative abundance of Bacteroidetes was 3.1%in the dry season and 13.5%in the wet season.In the genus classification level,Pseudomonas was the dominant genus,with a relative abundance of 19.3%.Mycobacterium,Flavobacterium and Hydrogenophaga were not isolated in the dry season,and Porphyrobacter and Pimelobacter were not isolated in the wet season.In particular,some genera,such as Pseudomonas and Acinetobacter,which are highly likely to contain pathogens and opportunistic pathogens.（4）To identify the biofilm forming ability of 132 waterborne P.aeruginosa isolates,crystal violet semi-quantitative assay were conducted and found that more than 85%isolates could form biofilms after 24 hours of culture.Among all isolates,29.5%had strong biofilm forming ability,indicating that P.aeruginosa had a very high possibility of biofilm forming in the production and processing of mineral water.The results of static culture showed that the strong biofilm-forming isolates could form distinct biofilms at both gas-liquid and solid-liquid interfaces.According to the observation of scanning electron microscope（SEM）and laser scanning confocal microscope（LSCM）,the cells of the non-biofilm forming isolates were loosely distributed and did not release extracellular polymeric substance;and the cells of strong biofilm-forming isolates were closely distributed and can form more extracellular polymeric substance.Compare to non-biofilm isolates,as the rapid biofilm formation ability of the strong biofilm-forming isolates,cells of nutrient supply from the medium were restricted,resulting in more normal cells and dead cells in strong biofilm-forming isolates.（5）All 132 waterborne P.aeruginosa isolates were sensitive to 14 antibiotics of 7categories（fluoroquinolones,aminoglycosides,polymyxin,carbapenems,monocyclic,cephalosporins and penicillin）.Tox A,phz M,las B,Exo U and Exo S have very high detection rates in all isolates,and 98（74.2%）isolates were virulence profile B,which was the dominant virulence profile.Moreover,all the isolates contained at least three virulence genes,indicating that these isolates were potentially pathogenic.81.1%of isolates carried qac E△1,which is likely to cause the resistance of P.aeruginosa to disinfectant.All isolates were divided into 42ST types,among which ST235 contained 11（8.3%）isolates,which was the dominant ST type of waterborne p.aeruginosa.Further analysis showed that the ST type of P.aeruginosa had a good correlation with its virulence profile.Systematic study of bacterial diversity in MWTS has not been conducted.The aim of this paper was for the first time to investigate the composition and dynamics of the bacterial community in a full-scale MWTS,which is helpful for us to further understand the contamination prevalence of potential pathogens in MWTS.In addition,this paper focused on the biofilm formation ability and harmfulness of P.aeruginosa,providing basic data for the risk identification of p.aeruginosa.The data and information in this study will provide a theoretical basis for the optimization of disinfection process and the prevention of microbial safety in MWTS,which will better ensure the microbial safety of mineral water.