Correlation of crAssphage qPCR Markers with Culturable and

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Ecotoxicology and Human Environmental Health

Correlation of crAssphage-based qPCR markers with culturable and molecular indicators of human fecal pollution in an impacted urban watershed Elyse Stachler, Benay Akyon, Nathalia Aquino de Carvalho, Christian Ference, and Kyle Bibby Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00638 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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Environmental Science & Technology

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Correlation of crAssphage qPCR markers with

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culturable and molecular indicators of human

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fecal pollution in an impacted urban watershed

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Elyse Stachler a, Benay Akyon a, Nathalia Aquino de Carvalho a, Christian Ference

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a

, Kyle Bibby a,b*

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a

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Pittsburgh, PA, 15261, USA;

Department of Civil and Environmental Engineering, University of Pittsburgh,

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b

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Notre Dame, South Bend, IN, 46556, USA

Department of Civil and Environmental Engineering and Earth Sciences, University of

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* Corresponding Author: Kyle Bibby, [email protected], +1-574-631-1130

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Keywords

crAssphage, qPCR, source tracking, virus, bacteriophage, water quality

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1

ACS Paragon Plus Environment


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ABSTRACT

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Environmental waters are monitored for fecal pollution to protect public health. Many

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previously developed human-specific fecal pollution indicators lack adequate sensitivity

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to be reliably detected in environmental waters or do not correlate well with viral

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pathogens. Recently, two novel human sewage-associated source tracking qPCR

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markers were developed based on the bacteriophage crAssphage, CPQ_056 and

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CPQ_064. These assays are highly human specific, abundant in sewage, and are viral-

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based, suggesting great promise for environmental application as human fecal pollution

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indicators. A 30-day sampling study was conducted in an urban stream impacted by

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combined sewer overflows to evaluate the crAssphage markers’ performance in an

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environmental system. The crAssphage markers were present at concentrations of 4.02-

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6.04 log10 copies/100 mL throughout the study period, indicating their high abundance

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and ease of detection in polluted environmental waters. In addition, the crAssphage

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assays were correlated with rain events, molecular markers for human polyomavirus and

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HF183, as well as culturable E. coli, enterococci, and somatic coliphage. The CPQ_064

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assay correlated strongly to a greater number of biological indicators than the CPQ_056

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assay. This study is the first to evaluate both crAssphage qPCR assays in an extended

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environmental application of crAssphage markers for monitoring of environmental

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waters. It is also the first study to compare crAssphage marker concentration with other

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viral-based indicators.

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TOC Art

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INTRODUCTION

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The release of inadequately or untreated wastewater into the environment is a major

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source of microbial impairment of environmental waters. Release of viral pathogens

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occurs from WWTP effluent, where viruses are not eliminated as successfully as

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bacteria through treatment processes and can often pass through the system.1-3 Another

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source of pollution comes from leaking septic tanks which have been shown to be the

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main source of fecal bacteria into the environment in some watersheds.4 In addition,

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approximately 40 million people in the United States in 772 communities are serviced by

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combined sewer systems (CSOs).5 CSOs are engineered to overflow untreated sewage

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into natural water bodies during wet weather events, releasing viruses and bacteria from

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fecal material and posing a potential health risk to the public.

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Fecal contamination in environmental waters historically has been monitored via

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quantification of culturable fecal indicator bacteria (FIB). Well-known limitations of

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monitoring environmental waters with FIB include the lack of differentiation between

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sources of fecal contamination and inadequately capturing viral risk to human health.

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The field of microbial source tracking (MST) has emerged to address the former

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problem, aiming to develop host-specific assays for water monitoring applications.

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Assays have been developed for the detection of numerous animal species such as 3



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cattle,6 dog,7 and gulls,8 as well as human-derived fecal matter.9-13 Human-specific

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markers are particularly important since this pollution has been shown to pose greater

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health risks to humans than pollution from other animal sources.14-16

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In addition to the inability to differentiate animal sources of fecal pollution, FIB

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have been shown to not correlate well with enteric viruses in environmental waters and

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through wastewater treatment processes.1, 2, 17 The public may be exposed to enteric

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viruses present in environmental waters through multiple routes, for example direct

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contact with impaired waters or through irrigating crops with contaminated water. Enteric

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viruses cause a significant amount of disease burden and cost society billions of dollars

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every year in health system costs. For example, the estimated annual cost of norovirus

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infections are $4.2 billion in direct health systems costs and $60.3 billion in societal costs

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worldwide.18 Approximately 16% of all norovirus gastroenteritis illness is caused by

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environmental sources, such as consuming contaminated water and shellfish.19 In

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addition, the occurrence of outbreaks caused by enteric viruses is rising.17, 18, 20

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Assays have been developed based on viruses such as adenovirus,11, 21 human

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polyomavirus (HPyV),12 and pepper mild mottle virus (PMMoV)9 and are used to monitor

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environmental waters contaminated with fecal pollution to capture viral health risk in

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these waters. Adenovirus and HPyV assays exhibit high specificity toward human fecal

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waste but are still detected at concentrations orders of magnitude lower than the HF183

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assay, a human-specific, bacteria-based assay commonly used for source tracking

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applications.12, 22-27 These lower concentrations in sewage may necessitate

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concentration of large volumes of environmental water for accurate detection.22, 24 Viral

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concentration methods often exhibit low recovery efficiencies and can co-concentrate

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inhibitors that interfere with downstream testing, such as PCR.28-30 PMMoV is present at 4


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higher concentrations in sewage, easing its detection; however, PMMoV cross-reacts

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with other animal sources.9 Effort has been expended to develop coliphage as a viral-

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based measure of health risk due to fecal pollution; however, coliphage repeatedly fails

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at specifically targeting human fecal pollution, even when genotyping is applied.22, 23

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Many researchers have emphasized the need for a human-associated viral marker that

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is abundant and reliably detected.1, 31, 32

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The bacteriophage crAssphage was discovered by metagenomic data mining

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and found to be highly human-associated and more abundant than other human gut

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phages and viruses.33 CrAssphage was subsequently evaluated for MST technology

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development and the crAssphage-based qPCR assays used in this manuscript, named

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CPQ_056 and CPQ_064, were recently published.34, 35 Other primers not evaluated in

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this manuscript have also been recently published.36, 37 Results in the initial publication

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indicated that the CPQ_056 and CPQ_064 targets were as abundant as current

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bacterial-based markers in sewage and had high specificity to sewage.35 The initial study

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also confirmed crAssphage presence in a sewage-impacted environmental sample.35 A

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recent study evaluating CPQ_056 also confirmed high abundance in sewage and

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successful detection in a storm sewer outfall.38 Despite prior successful short-term

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demonstrations, environmental assay testing over an extended study period is valuable

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to evaluate assay performance and determine the environmental correlation of

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crAssphage markers with existing markers and pollution events.

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The goals of the present study were to perform an environmental evaluation of

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crAssphage qPCR markers in an impacted urban stream. Nine Mile Run in Pittsburgh,

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PA was sampled daily for 30 days and analyzed for chemical and biological indicators of

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water quality. This test site was chosen due to known fecal contamination from CSO 5



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events and leaky sanitary sewers,39 facilitating detection and evaluation of fecal source

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tracking markers in a relevant environmental matrix. Spearman’s rank correlation was

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determined between all monitored parameters and assays to evaluate correlation

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between each biological and chemical indicator, including crAssphage. This is the first

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extended environmental validation study of both crAssphage-based assays for source

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tracking of human fecal pollution, correlating the assays with culturable indicators,

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molecular indicators, and pollution events. This is also the first study to correlate

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crAssphage indicators with other viral indicators, including both culture and molecular-

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based indicators.

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MATERIALS AND METHODS

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Study Site

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Sampling for the present study was conducted at Nine Mile Run in Pittsburgh, PA, USA

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daily from September 6, 2016 through October 5, 2016. The selected location has been

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previously used as a water quality study site.39 Nine Mile Run is a small urban stream

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located in Pittsburgh’s Frick Park, with a watershed of approximately 19.4 km2 and

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50,000 residents .40 Nine Mile Run has been shown to be chronically polluted, including

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high levels of fecal indicator bacteria (E. coli and fecal coliforms), despite undergoing a

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restoration project in 2006.40, 41 For example, approximately 90% of samples collected in

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2011 and 2012 exceeded the EPA limit of 235 cfu/100 mL for culturable E. coli.41 The

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small stream can become overwhelmed during large rain events, and two combined

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sewer outfalls discharge directly into the stream. The specific sampling location

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(40.426385, -79.905262) used in this study is a public access location point directly

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downstream of one combined sewer outfall and was investigated to capture changes in

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concentration to the stream from this point source (Figure 1).

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141 142 143 144 145 146

Figure 1: Study sampling site in Pittsburgh, Pennsylvania (Adapted from 2013 State of the Watershed Report, Nine Mile Run Association. Copyright 2013 Nine Mile Run Association. Used with Permission.). Google Map image (Map data © 2017 Google) shows the sampling location downstream of where a CSO outfall joins with Nine Mile Run (NMR).

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Sampling and Data Collection

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A grab sample was collected daily at 9:00 AM (EDT) for 30 days. Each day, three liters

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of stream water were collected in sterile containers and transported on ice to the

151

laboratory, where samples were processed within six hours (hrs). One additional sample

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was collected on September 30th during an active CSO event. The temperature of both 7



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the stream and the air were measured at the time of sampling. Rainfall data was

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collected in real-time and reported every 15 minutes from 3 Rivers Wet Weather

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(http://www.3riverswetweather.org/municipalities/calibrated-radar-rainfall-data), a

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nonprofit organization that operates and maintains rain gauges throughout the region.

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The data used for this project comes from rain gauge #11, which corresponds to Nine

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Mile Run. Combined sewer overflow (CSO) events were monitored from the local

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wastewater treatment and conveyance authority’s (Alcosan) online Sewer Overflow

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Advisory Key status changes

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(http://www.alcosan.org/SewerOverflowAdvisories/SOAKStatusChanges2017/tabid/115/

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Default.aspx). A CSO advisory is issued based on the wet well elevation at Alcosan’s

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Main Pump Station, which may or may not correspond to a CSO at Nine Mile Run due to

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physical distance and sensitivity of the NMR system.

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Chemical Parameter Characterization

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Chemical parameters were measured throughout the course of this study to monitor

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water quality. pH was measured using a FiveEasy Plus pH meter (Mettler Toledo,

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Columbus, OH, USA). Turbidity was measured using Program 745 on a Hach DR900

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meter (Loveland, CO, USA). Total Organic Carbon (TOC) was measured by filtering 40

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mL of sample through a 0.45-µm filter and analyzing filtrate on a TOC-L TOC Analyzer

172

(Shimadzu, Kyoto, Japan). Total dissolved solids (TDS) were determined by filtering the

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sample through a 0.45-µm filter and drying the filtrate in a lab oven at 180°C according

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to EPA Method 8163. Values were reported as non-detect (ND) if they were below the

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limit of detection (LOD) of the measuring system. The LOD for turbidity testing was 1

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FAU and the LOD for TDS testing was 0.001 mg. For statistical analysis for turbidity and 8


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TDS, ND data points were given a value of zero. All chemical parameters were tested in

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triplicate for each sample so individual readings could be averaged for subsequent data

179

analysis.

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Enumeration of Culturable Indicators

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Culturable bacteria and phage indicators were measured for each sample. E. coli were

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measured by membrane filtration and culturing on HiChrome m-TEC agar (Sigma-

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Aldrich, St. Louis, MO, USA) following the protocol of EPA Method 160342. Enterococci

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were measured by membrane filtration following EPA Method 1600 and culturing on mEI

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plates43, made with Difco mE agar (BD, Franklin Lakes, NJ, USA) with the addition of

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indoxyl β-D-glucoside (Chem-Impex International, Wood Dale, IL, USA). Somatic

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coliphages were enumerated via a single-agar layer plaque assay procedure according

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to EPA Method 1602.44

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qPCR Assays

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Stream water was simultaneously concentrated for viruses and bacteria as previously

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described.12, 30 Briefly, 500 mL of stream water was pH adjusted to pH=3.5 and filtered

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through a 47-mm 0.45-µm mixed cellulose HAWG filter (Millipore, Billerica, MA, USA).

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Duplicate filters for each sample were then placed in bead beating tubes and frozen at -

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80°C. For the simultaneous DNA extraction of all samples, filters were thawed to room

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temperature and DNA was extracted using a DNeasy PowerWater Kit (Qiagen, Valencia,

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CA, USA), following the manufacturer’s protocol. Extracted DNA was stored at -20°C.

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Assay primers and probes used in the current study are reported in Table 1. TaqMan

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hydrolysis probe qPCR assays targeting crAssphage (CPQ_056 and CPQ_064),35 a 9



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Bacteroides species (HF183/BacR287)13, and human polyomavirus (HPyV) 12 were

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performed as described previously. A patent application has been filed with the USPTO

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on the crAssphage assays described in this manuscript. Universities and non-profit

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researchers interested in using this technology must obtain a research license from the

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USEPA. To apply for a research license, please request additional information

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from [email protected]. Each 25 µL qPCR reaction for the crAssphage assays and

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HF183/BacR287 contained 1x TaqMan Environmental Master Mix 2.0 (Thermo Fisher

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Scientific), 5 µg Bovine Serum Albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA), 1 µM

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of each primer, 80 nM FAM-labeled probe, 80 nM VIC-labeled probe (HF183/BacR287

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only), molecular grade water, and 2 µL of a 10x dilution of extracted DNA. Each 25 µL

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reaction for HPyV contained 1x TaqMan Environmental Master Mix 2.0, 5 µg Bovine

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Serum Albumin, 0.5 µM of each primer, 0.4 µM FAM-labeled probe, molecular grade

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water, and 2 µL of extracted DNA. All qPCR tests were performed in triplicate on a CFX

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Connect Real-Time System (BioRad, Hercules, CA, USA). Each qPCR plate included a

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standard curve with 101-105 target copies per reaction that was prepared as previously

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described.35 Briefly, a customized gBlock gene fragment (Integrated DNA Technologies,

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Coralville, IA, USA) containing all target sequences used in this study was synthesized

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and was serially diluted to supply standard curve concentrations. Performance

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characteristics of standard curves are reported in Table S1. Thermocycler conditions for

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each assay were as previously reported.12, 35 Fluorescence drift correction was applied

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and the threshold for quantification cycle (Cq) determination was auto-calculated using a

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baseline determined from cycle numbers 10 to 20. Concentration was calculated as:

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logC=(Cq-Yint)/slope, where Yint and slope are the plate-specific y-intercept and slope

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generated from the standard curve. Averages and standard deviations (ntotal=6) of logC 10


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were then calculated and used for statistical analysis. If an assay did not detect any

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target molecules within the limit of detection of the assay for a sampling event, this

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sample was considered a non-detect and the data point was excluded from statistical

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analysis (n = 1, for HPyV).

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Table 1: Primer and probe information for qPCR assays in this study. qPCR assay CPQ_056

primer or probe 056F1 056R1 056P1

CPQ_064

HF183/BacR287

HPyV

064F1 064R1 064P1 HF183 BacR287 BacP234MGB SM2 P6 KGJ3

sequence 5' → 3'

Reference

CAGAAGTACAAACTCCTAAAAAACGTAGAG GATGACCAATAAACAAGCCATTAGC (FAM)-AATAACGATTTACGTGATGTAAC(MGB) TGTATAGATGCTGCTGCAACTGTACTC CGTTGTTTTCATCTTTATCTTGTCCAT (FAM)-CTGAAATTGTTCATAAGCAA-(MGB) ATCATGAGTTCACATGTCCG CTTCCTCTCAGAACCCCTATCC (FAM)-CTAATGGAACGCATCCC-(MGB) AGTCTTTAGGGTCTTCTACCTTT GGTGCCAACCTATGGAACAG (FAM)-TCATCACTGGCAAACAT-(MGBNFQ)

34

34

44

12

231 232

Controls

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Negative process controls for mTEC (ntotal = 31) and mEI (ntotal = 31) plates were

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performed each sampling day by filtering sterile buffer rinse water through a filter and

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plating. Negative process controls (ntotal = 31) for the somatic coliphage procedure were

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performed each sampling day by adding DI water to the test agar. Filter control DNA

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extractions (n = 3) were performed by placing a sterile filter in a bead mill tube, and

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extraction blanks (n = 3) were performed and tested by qPCR for all assays. Triplicate

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no template controls (NTCs) were included on each qPCR plate (ntotal = 36). All NTCs

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and process controls were negative throughout the course of the study. In addition, the 11



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HF183/BacR287 assay includes an internal amplification control (IAC) that allows for

242

evaluation of PCR inhibition. Potential inhibition introduced through the water

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environment or concentration procedures was determined as previously described.35

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Briefly, the Cq values of IAC assay in the NTCs were compared to the Cq values of the

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IAC assay in the samples. If the Cq value of a sample was more than the average Cq of

246

the NTCs plus 3 standard deviations, that test was determined to be inhibited. No

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samples exhibited PCR inhibition as determined through the evaluation of the IAC

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control; however, since the IAC was run on 1:10 dilution of DNA it is possible that some

249

inhibition effects may not have been captured within the HPyV data.

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Statistical Analyses

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Spearman’s rank correlation coefficients (r) were calculated on the means of log

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transformed data in GraphPad Prism 7 (La Jolla, CA, USA) between culturable

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indicators, qPCR indicators, and chemical parameters, using two-tailed 95% confidence

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intervals. Coefficients are characterized by the following scale for comparison purposes:

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0.2-0.39 (weak correlation), 0.4-0.59 (moderate correlation), 0.6-0.79 (strong

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correlation), and 0.8-1 (very strong correlation). Graphs were drawn in GraphPad Prism

258

7 using averages and standard deviations of data sets. Additionally, log10 transformed

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concentration data and chemical parameter data between dry weather days and wet

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weather days were fit to a general linear model ANOVA in Minitab 18 (State College, PA,

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USA) to determine if rain events significantly (p < 0.05) impacted water quality

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parameters. Multiple pairwise comparisons were corrected for using Bonferroni 95%

263

confidence intervals. Rainfall is reported as the total amount of precipitation recorded in

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the 24 hrs before sampling. A sampling day was considered a wet weather sampling

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event if it had rained at least 0.25 mm within the past 24 hrs.

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RESULTS & DISCUSSION

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Pollution events during study period

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The overarching goal of the current study was to demonstrate the use of crAssphage

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based qPCR assays to detect and quantify human fecal pollution in an impacted urban

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watershed and correlate these assays with other culturable and molecular indicators

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along with inputs of fecal pollution. During the 30-day study period, it rained a total of 12

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days, with 8 of those days reporting active CSOs. Total rainfall ranged from 0.51 mm to

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25.65 mm of rain in a 24-hr period.

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Chemical parameters during the study period and correlation with pollution events

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Chemical parameters were measured throughout the length of the study to monitor

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water quality. Results for TOC, pH, turbidity, and TDS can be seen in Figure S1 along

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with a plot of the rainfall throughout the study. Throughout the study period, TOC ranged

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from 1.6-6.3 mg/L (1.6-3.4 mg/L for dry weather days and 2.4-6.3 mg/L for wet weather

281

days). The pH remained stable throughout sampling, ranging from 7.62 to 8.05. Turbidity

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ranged from non-detect (ND) to 40.7 Formazin Attenuation Units (FAU) (ND-10 FAU for

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dry weather days and 2.7-40.7 FAU for wet weather days). TDS ranged from ND-947

284

mg/L (500-947 mg/L for dry weather days and ND-692 mg/L for wet weather days). The

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chemical values measured during the active CSO event fell within the range observed

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on study days for TOC and TDS but were observed outside of the wet weather range for

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pH (6.73) and turbidity (87.3 FAU). 13



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One-way ANOVA analysis was performed on observed chemical values to

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compare dry weather days versus wet weather days. The analysis demonstrated a

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statistically significant difference (p < 0.05) for all chemical parameters between dry and

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wet weather days. TOC and turbidity were both positively correlated with rain events, i.e.

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their values increased significantly with rainfall. TDS and pH were both negatively

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correlated with rainfall, i.e. these parameters were characterized by significantly lower

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values on wet weather sampling days. No significant differences in any chemical

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parameter values were observed between wet weather events that resulted in a reported

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CSO event versus no reported CSO event.

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Culturable and qPCR indicators during study period and correlation with pollution events

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Culture-based and qPCR assays were evaluated to compare the crAssphage qPCR

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assays to fecal pollution indicators (Figure 2). Ranges observed for culturable indicator

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concentrations (per 100 mL) were 2.38-4.49 log10 cfu E. coli, 2.06-4.31 log10 cfu

302

enterococci, and 2.02-3.74 log10 pfu somatic coliphage. Ranges observed for qPCR

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indicator concentrations (per 100 mL) were 4.02-6.04 log10 copies CPQ_056, 4.33-5.94

304

log10 copies CPQ_064, 2.80-5.64 log10 copies HF183/BacR287, and 1.50-4.21 log10

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copies HPyV. All indicators were detected each day of the study except for HPyV, which

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was not detected on one sampling day due to the limit of detection of the assay.

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Figure 2: Culturable and qPCR indicators along with daily rainfall during the Nine Mile Run sampling period. Rainfall is presented as the amount of rain that fell within the 24-hr period before that day’s sampling. A red bar on the rainfall graph indicates rainfall that corresponded to a reported CSO event. The individual data points outlined in black for the indicators represent an additional sampling time point (day 25) during an active CSO event. Data points represent averages of triplicate data points and error bars represent standard deviations. 15



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Similar to the chemical parameters, one-way ANOVA analysis was performed on

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all biological indicators to understand if rain and/or CSO events significantly affected

319

concentrations of the markers. The abundance of all markers was positively correlated

320

with rain events, i.e. in all cases the indicator increased after a rainfall event. In addition,

321

the abundance of all markers was found to be statistically significantly different (p < 0.05)

322

between wet and dry weather conditions. No statistically significant difference was found

323

between wet weather events that corresponded to a reported CSO event versus a rain

324

event without a reported CSO event.

325 326

Correlation of chemical and biological water quality parameters

327

Spearman’s rank correlation coefficients were calculated between each pair of culturable

328

indicators, molecular indicators, and chemical parameters (Figure 3, Table S1). The

329

analysis generally showed strong correlation between markers, with 91% (50 out of 55)

330

of p-values being statistically significant (p < 0.05, Table S2). Notably, pH did not

331

strongly correlate with any other biological or chemical parameter. Although the two

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crAssphage assays strongly correlated with each other, their correlation with other

333

indicators varied. The only chemical parameter that correlated strongly with crAssphage

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abundance was TOC with the CPQ_064 assay. For culturable markers, the CPQ_064

335

assay strongly correlated with culturable E. coli and enterococci, while the CPQ_056

336

assay correlated strongly with somatic coliphage. For molecular markers, the CPQ_056

337

assay was only strongly correlated with CPQ_064. In addition to CPQ_056, the

338

CPQ_064 assay was also strongly correlated with the HF183 and HPyV assays.

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In addition to the crAssphage correlations described above, there were several

340

other correlations between other parameters measured in this study. The strongest

341

correlations were observed between bacterial-based indicators: E. coli vs. enterococci

342

(0.93), E. coli vs. HF183 (0.88), and enterococci vs. HF183 (0.86). The bacterial-based

343

indicators (E. coli, enterococci, and HF183) also had stronger correlations with chemical

344

parameters than the viral indicators exhibited, with each being strongly correlated with

345

TOC, turbidity, and TDS.

346

347 348 349 350 351 352

Figure 3: Heat map of Spearman’s rank correlation coefficients matrix for culturable indicators, qPCR indicators, and chemical parameters. Coefficients are colored based on the following scale of the absolute value of the coefficients: weak correlation (0.2 to 0.39), moderate correlation (0.4 to 0.59), strong correlation (0.6 to 0.79), and very strong 17



353 354

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correlation (0.8 to 1). Values of correlation coefficients and p-values are provided in Tables S2 and S3.

355 356

Comparison of crAssphage qPCR assays

357

Overall, levels of both crAssphage markers were detected at similar concentrations to

358

HF183/BacR287 and approximately two to three orders of magnitude greater than

359

HPyV. Despite positive correlations between all microbial indicators, the two

360

crAssphage-based assays differed in the strength of their correlations with other

361

indicators. The most notable difference was the strong correlation between the

362

CPQ_064 assay and the HPyV assay, while the p-value between CPQ_056 and HPyV

363

was one of the few in the study to not show statistical significance. Differences in assay

364

performance highlighted in this study are notable and warrant further investigation, as

365

future investigations will likely choose a single crAssphage assay for application. For

366

example, a recent study only investigated the CPQ_056 assay.38 This study demonstrates the high abundance of crAssphage-based indicators

367 368

compared to other indicators of human fecal pollution in the environment and the ease at

369

which they can be detected along with other DNA-based assays. Due to the high levels

370

of fecal pollution at the selected study site, HPyV was detected in nearly all samples;

371

however, HPyV had the lowest quantities detected of all the qPCR assays. Based on the

372

results of this study, CPQ_064 correlates well with HPyV yet is orders of magnitude

373

more abundant, making crAssphage assays potentially useful in waters polluted at lower

374

levels.

375

Recently, a separate crAssphage-based qPCR assay not evaluated in the

376

current study was developed by targeting a short (78 bp) genetically conserved genome

377

fragment and evaluated.36 In the initial evaluation of this assay, 61% of fecal samples 18


Page 19 of 25


378

derived from cows, pigs, and poultry were positive for this crAssphage sequence.36 In

379

comparison, the assays evaluated in this study have been shown to exhibit specificities

380

of 98.6% (n = 222), cross-reacting with a single dog fecal sample and two gull

381

samples.35 A recent study also showed CPQ_056 cross-reaction with poultry litter but

382

still determined a specificity of 92.7%.38 This further demonstrates the differing suitability

383

of crAssphage genome regions for human-specific applications. Some genome regions

384

of crAssphage may have homology to other animal-associated phages, while some

385

regions may be human-specific. Hence, it is likely that crAssphage assays will perform

386

differently based on the genomic region on which they are designed.

387 388

Site-specific considerations

389

While the goal of this study was to demonstrate performance of crAssphage qPCR

390

assays in an impacted urban stream whose results can be applied widely, the specific

391

site chosen has some unique characteristics that should be considered. Nine Mile Run

392

was selected as a test site due to its well-known contamination from sewage inputs and

393

the well-characterized nature of the system. The present study detected peaks in

394

biological marker concentrations after rain events, which then decayed to a background

395

level of pollution. Ideally, the peak in concentration would decay until the biological

396

markers are no longer detectable to confirm that the marker signal is not present if fecal

397

pollution is no longer present. However, NMR has been shown to be impacted by

398

leaking sanitary sewers during baseflow conditions,39, 45 resulting in a signal decay to this

399

lower level of constant sewage input. Additional research should be conducted to

400

quantify the decay of the crAssphage markers. In addition, it has been shown that during

401

rain events, more pristine storm water dilutes the nitrate signal in the stream.39, 45 This 19



402

effect could also be present in this study, muting the peak concentrations of the

403

biological markers.

404

Page 20 of 25

The current study evaluated a single system and results may vary between

405

environmental systems. Previous studies have suggested the potential for geographic

406

variation of crAssphage abundance in sewage;34 the crAssphage indicators should be

407

studied in additional sites to verify findings. In addition, the study site demonstrated high

408

levels of microbial water quality impairment. The concentrations of E. coli and

409

enterococci were above the 2012 Recreational Water Quality Criteria recommendations

410

throughout the duration of our study;46 however, elevated levels of these indicator

411

bacteria due to rain events were readily observed which allowed observations above

412

background levels of pollution. Future studies should investigate both less chronically

413

polluted systems and systems polluted by non-point sources. Further, this study found

414

no significant differences from CSO events and rain events not resulting in a reported

415

CSO. This could be due to limitations in data resolution since the study system is located

416

away from where the CSO levels are determined. Overflow events may have been

417

occurring even when they were not officially reported. In addition, overflow events may

418

have impacted the study site faster than the reporting system due to the small volume of

419

the study stream.

420

Finally, samples were necessarily concentrated for qPCR analysis. The

421

concentration method used in this study conveniently allowed for simultaneous

422

concentration of both viral and bacterial indicators. This method has previously been

423

shown to perform well, with HPyV recovery efficiencies of 40 and 78% in tap and river

424

water, respectively;30 however, the use of any concentration method likely

425

underestimates the levels of both crAssphage and HPyV. 20


Page 21 of 25


426 427

Environmental implications

428

This study demonstrated the first extended environmental application of crAssphage-

429

based qPCR assays as indicators of human fecal pollution in an impacted urban stream.

430

The crAssphage assays correlated with both viral and bacterial culturable and molecular

431

fecal pollution indicators, as well as rain events. This demonstrates the fluctuation of the

432

crAssphage signal with fecal pollution presence, with the signal decaying after a large

433

input of pollution. The assays also detected targets in contaminated waters at high

434

abundance, orders of magnitude higher than the viral-based HPyV assay. This suggests

435

the ease of detection and utility of crAssphage as a fecal pollution indicator in less

436

chronically impaired waters where other viruses may be too dilute to detect. While

437

crAssphage-based assays remain a promising tool for water management applications,

438

more research is necessary to fully understand marker performance. Additional study

439

sites should be evaluated that are affected by non-point sources of pollution and that are

440

less chronically polluted than Nine Mile Run. In addition, studies should be conducted to

441

understand the persistence of crAssphage marker signals in different water matrices

442

compared to other pathogenic viruses to understand if crAssphage behaves in a similar

443

manner. Ultimately, these developments will improve our ability to assess viral presence

444

and health risk in environmental waters for public health protection.

445 446

SUPPORTING INFORMATION

447

Supporting Information. Standard curve performance metrics for qPCR assays, data

448

values and p-values of Spearman’s rank correlation coefficients, figure of chemical

449

parameters plotted against rainfall during the study period. 21



Page 22 of 25

450 451

ACKNOWLEDGEMENTS

452

This material is based upon work supported by the National Science Foundation

453

Graduate Research Fellowship Program under Grant No. 1747452 and NSF Grant No.

454

1510925. Any opinions, findings, and conclusions or recommendations expressed in this

455

material are those of the author(s) and do not necessarily reflect the views of the

456

National Science Foundation. The authors declare the following competing financial

457

interest(s): The primers reported in the manuscript are the subject of a patent application

458

entitled “Cross-Assembly Phage DNA Sequences, Primers and Probes for PCR-based

459

Identification of Human Fecal Pollution Sources” (Application Number: 62/386,532).

460 461

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Correlation of crAssphage qPCR Markers with Culturable and

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