Mapping the Terrain for Pathogen Persistence and Proliferation in Non-potable Reuse Distribution Systems: Interactive Effects of Biofiltration, Disinfection, and Water Age

Abstract

Diverse pathogens can potentially persist and proliferate in reclaimed water distribution systems (RWDSs). The goal of this study was to evaluate interactive effects of reclaimed water treatments and water age on persistence and proliferation of multiple fecal (e.g., Klebsiella, Enterobacter) and non-fecal (e.g., Legionella, mycobacteria) gene markers in RWDSs. Six laboratory-scale RWDSs were operated in parallel receiving the influent with or without biologically active carbon (BAC) filtration + chlorination, chloramination, or no disinfectant residual. After 3 years of operation, the RWDSs were subject to sacrificial sampling and shotgun metagenomic sequencing. We developed an in-house metagenome-derived pathogen quantification pipeline, validated by quantitative polymerase chain reaction and mock community analysis, to estimate changes in abundance of ∼30 genera containing waterborne pathogens. Microbial community composition in the RWDS bulk water, biofilm, and sediments was clearly shaped by BAC filtration, disinfectant conditions, and water age. Key commonalities were noted in the ecological niches occupied by fecal pathogen markers in the RWDSs, while non-fecal pathogen markers were more varied in their distribution. BAC-filtration + chlorine was found to most effectively control the widest range of target genera. However, filtration alone or chlorine secondary disinfection alone resulted in proliferation of some of these genera containing waterborne pathogens.

Metagenomic Profiling of Pathogens in Simulated Reclaimed Water Distribution Systems Operated at Elevated Temperature Reveals Distinct Response of Mycobacteria to Filtration and Disinfection Conditions

“…However, without BAC filtration, disinfectants were actually detrimental to pathogen control, with the highest levels of FP markers observed in the chlorinated condition followed by the chloraminated condition in bulk water (Figure ). This was consistent with prior studies of these systems that noted elevated pathogens and ARGs, including clinically relevant and mobile ARGs, when unfiltered water was chlorinated. , …”

“…Detailed methods for quantification of FPs and non-FPs of interest (Supporting Information Table S2) are available in GitHub (Sudeshna-Ghosh/pathogen-fluctuations). The in-house metagenome-derived pathogen quantification pipeline based on UniRef90 gene-family annotation was described in detail by Ghosh et al 2021 . Here, we used the HUMAnN3 pipeline for UniRef90 gene-family annotation along with the “Unique to genus” database in the GitHub repository.…”

“…Here, we used the HUMAnN3 pipeline for UniRef90 gene-family annotation along with the “Unique to genus” database in the GitHub repository. Absolute abundances were quantified as described previously . Metagenome-derived Mycobacterium spp.…”

“…An extra biologically activated carbon (BAC) filtration step prior to disinfection marginally improved disinfectant stability. Microbial and chemical analysis indicated that loss of disinfectant was due to a combination of factors, including elevated nitrification rates (in the chloramine condition) as well as elevated rates of chlorine and chloramine decay stimulated by high organic matter concentrations and accumulated sediments. ,, Additionally, at 22 °C, it was noted that relative abundances of antibiotic resistance genes (ARGs) and pathogens of concern tended to be elevated in RWDSs receiving unfiltered water, especially when chlorinated. , While BAC filtration and disinfection on their own each elevated the levels of ARGs, filtration in combination with chlorination effectively mitigated ARG and pathogen levels.…”

“…The objectives of this study were to profile pathogen and ARG abundances in RWDSs when conditions have deteriorated due to elevated temperature and to assess the extent to which optimal treatment and disinfectant conditions identified at lower temperatures extend to higher temperatures. This was achieved through a head-to-head comparison of six continuous flow laboratory-scale simulated RWDSs operated at 30 °C, compared to prior studies when they were operated at 22 °C. , The RWDSs received reclaimed feed water (with or without BAC filtration), followed by chlorine or chloramine addition to an influent target of 4 mg/L. Two control RWDSs were fed with breakpoint chlorinated (no residual) feed water with and without BAC filtration.…”