Diversity And Risks Of Human Bacterial Pathogens In The Urban Waters For Landscape And Recreation

The urban recreational water body is an important reservoir and transportation media of water-borne pathogenic microorganisms.The microbial quality of urban water bodies is closely related to public health.At present,there is limited knowledge on the diversity detection,distribution,risk levels and source tracking of human bacterial pathogens in urban waters.In this study,the recreational lake in Beijing Olympic Forest Park and fifteen urban rivers in Changzhou City were investigated.The purposes were to explore the bacterial pathogen diversity,temporal and spatial distribution characteristics,risk levels and potential sources in urban recreational waters.The 16S rRNA gene high-throughput sequencing method was first improved and combined with qPCR to detect bacterial pathogen diversity.Additionally,the potential effects of in-situ ecological purification/restoration measures in urban waters on the bacterial pathogen levels were evaluated.This study will provide the research foundation for microbial quality management in urban waters.The 16S rRNA gene next-generation sequencing（NGS）method for detecting pathogen diversity was improved to be more comprehensive and accurate by expanding the database and selecting optimal sequencing conditions.The new database included 145genera and 303 potential pathogen species,which largely broaden the previous databases.The read length and sequencing depth were increased,and the variable region was also optimized into V34,which elevated the resolution ratio of the species sequences in each genus by an average of 22.7%.In the urban recreational lake of Beijing Olympic Forest Park,the dominant pathogens detected by NGS included mainly environmental pathogens,like Acinetobacter johnsonii,Mycobacterium avium and Aeromonas spp.qPCR was further combined to quantify enteric pathogens/indicator,Escherichia coli and Salmonella,and the other three environmental pathogens like Aeromonas spp.It was found that the target genes of these pathogens were prevalent in the lake,and 34.8%of all the samples harbored all four pathogens（E.coli excluded）.The quantitative microbial risk assessment showed that the annual infection risks of Salmonella,M.avium and Pseudomonas aeruginosa in activities,like boating,fishing and playing with water,were mostly greater than the U.S.EPA risk limit（0.032）for recreational contacts,and children,who playing with water,may be exposed to the greatest infection risk.In the urban rivers of Changzhou,more diverse and abundant potential pathogens were detected by NGS than in the park lake,especially the enteric ones.In total,16potential enteric pathogen species were detected,mainly including Arcobacter spp.and Bacteroides spp.Further quantification of twelve concerned pathogens by qPCR showed that the enteric pathogens/indicators,Arcobacter cryaerophilus,Campylobacter jejuni and Enterococcus faecalis were ubiquitously detected,indicating enteric pathogen pollution were prevalent.The abundances of enteric pathogens were mostly higher than the levels in other surface waters previously reported.In addition,seven environmental pathogens were also found abundant,especially A.johnsonii、Acinetobacter lwoffii and Aeromonas spp.The kinds and abundances of the dominant environmental pathogen species were similar as in the Beijing lake.A variety of potential pathogens were detected by NGS in artificial floating island biofilms in the urban rivers.Most of the quantified pathogens（9/12）in the biofilms had higher or similar relative abundances than or as in the river water,indicating that the artificial floating island biofilms might be reservoirs of various pathogens in the rivers and possess high risks.The constructed wetland system of the park lake in Beijing significantly increased the concentrations of Aeromonas spp.in effluent,and had weak removal effects on the other pathogens/indicator(log₁₀ reduction rate of 95^thh concentrations was 0.30-1.44 for E.coli,Salmonella,M.avium and P.aeruginosa).These results indicated that the in-situ ecological purification/restoration measures in urban waters have potential microbial risks.Microbial source tracking of the enteric pathogens in the urban rivers showed that urban domestic sewage discharge carrying a lot of human feces would be the main pollution pathway.The levels of enteric pathogens had spatial differences across rivers.The abundances of most enteric pathogens were significantly correlated with the levels of the human-source microbial marker instead of nutrients.Therefore,it is suggested that the microbial pollution levels or risks should be determined by microbial source tracking or direct detection of pathogens in practical management.