| Research of microbial risk in urban recreational water is of great importance to the public health.First grasping the environmental fate of bacterial pathogens in water is conducive to the accurate risk evaluation and control.However,among the environmental factors affecting pathogen fate in water,knowledge about how suspended algae govern pathogen survival and distribution in recreational water is scarce.In this study,bacterial pathogens in three typical recreational lakes in Beijing were investigated.The purposes were to show the effects of suspended algae on the pathogen's persistence and distribution,as well as effects on the spread of antibiotic resistance in water.This study also made a supplement of an activity parameter?Pi?to Quantitative Microbial Risk Assessment?QMRA?in urban recreational water.This study will provide useful suggestions for microbial risk control in water management.Analyses of physicochemical parameters in three sampling lakes revealed that suspended particles in water were mainly composed of algae,on which bacterial biofilm were observed.Next-generation sequencing?NGS?was employed to determine the diversity of genera containing pathoge ns,and the results showed obvious composition differences between particle-attached?PA?and free-floating?FL?samples.It revealed stable distribution characteristics of each genus:some genera were primarily FL status?e.g.Leptospira?,some genera were preferentially PA status?e.g.Aeromonas and Mycobacterium?,and some genera might have no preference between two fractions.qPCR results of 5pathogen species presented that their absolute concentrations were consistently higher in PA fractions than in FL fractions?p<0.001?,and relative abundance data exhibited that the species maintained the same distribution pattern with their genus?e.g.Aeromonas spp.and Mycobacterium avium?.A combination of culturing and NGS method was employed to explore the diversity of potential antibiotic-resistant pathogens?ARPs?in recreational water,and results showed that most of the identified ARPs were environmental opportunistic pathogens with emerging clinical concerns,?e.g.Aeromonas junii?A.hydrophila?,showing multi-resistance to several antibiotics.Further comparision indicated that bacteria in PA fractions had a higher tolerance?ARI?to antibiotics than those in FL fractions?p<0.001?,and ARPs could persist for a longer time under the particles protection.However,qPCR results of 7antibiotic-resistant genes?ARGs?showed that their relative abundances in PA samples were not higher than those in FL samples,suggesting no clear effect from suspended particles on the spread of ARGs.Simulation study was further conducted to analyze how suspended algae govern pathogen survival and distribution in recreational water.Results in three sets of experiment all indicated that algae was a favorable factor for pathogen survival,but the effect varied under different conditions:with limited nutrients,algae with low biomass performed better than high biomass,presenting the decay rates order 15 mg/L group<30 mg/L group<0 mg/L group;whereas with sufficient nutrients,algae grew steadily and higher biomass enhanced more pathogens,presenting the decay rates order 4×group<2×group<1×group;additionally,decay rates of different particle types displayed that organic?algae<inorganic.Moreover,there were substantial differences among the 5target species.Escherichia coli and Enterococcus faecalis with no distinctions between PA/FL fractions showed higher decay rates(0.9-1.3 day-1;0.8-1.2day-1),while other three opportunistic species with higher abundances in PA fractions exhibited lower decay rates(0.6-0.9 day-1).It suggested that the decay rates of species were correlated with their ability attaching to algae.In order to accurately evaluate the pathogen risk in water,the activity parameter Pi,a critical factor for risk calculation,was primarily determined by a combination of PMA-qPCR,RT-qPCR and culture counting methods.The results all showed that pathogen activity was significantly higher in PA fractions than that of FL fractions?p<0.001?.Take Pseudomonas aeruginosa as an example,while incorporating different Pi values between PA and FL fractions,risks were 7.9 times higher than the original value according to the RT-qPCR results,and 15.4 times higher according to the culturing results.The supplement of parameter Pi improved the accuracy of microbial risk assessment. |
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