Real-Time Quantitative PCR Measurements of Fecal Indicator Bacteria

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Real-time Quantitative PCR Measurements of Fecal Indicator Bacteria and Human-associated Source Tracking Markers in a Texas River following Hurricane Harvey Vikram Kapoor, Indrani Gupta, ABM Tanvir Pasha, and Duc Phan Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00237 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 11, 2018

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Environmental Science & Technology Letters

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Real-time Quantitative PCR Measurements of Fecal

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Indicator Bacteria and Human-associated Source

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Tracking Markers in a Texas River following Hurricane

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Harvey

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Vikram Kapoor*, Indrani Gupta, A.B.M. Tanvir Pasha, and Duc Phan

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Department of Civil and Environmental Engineering, University of Texas at San Antonio, San

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Antonio, TX 78249, USA

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Keywords. Fecal source tracking, human-associated Bacteroidales, quantitative PCR, genetic

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markers, water quality, Hurricane Harvey

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ABSTRACT

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Hurricane Harvey has caused unprecedented devastation to huge parts of southeastern Texas,

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particularly damaging the wastewater infrastructure resulting in release of sewage contamination

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into environmental waters. The purpose of this study was to conduct a preliminary assessment of

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fecal indicator bacteria (E. coli and enterococci) and human-associated fecal genetic markers

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(Human-associated Bacteroidales), measured using qPCR assays, across a Texas river impacted

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by Hurricane Harvey. Water samples were collected along the Guadalupe River during

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September – December 2017. The most heavily flooded sites showed highest abundance of fecal

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indicator bacteria and human-associated Bacteroidales markers indicating that a large number of

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sewage overflows and stormwater runoff occurred during Harvey flooding. These findings

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suggest that high levels of human fecal contamination were introduced into waterways draining

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into the Gulf of Mexico and impaired surface water quality. The human-associated Bacteroidales

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markers exhibited low to slightly strong correlation with conventional fecal indicators suggesting

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the variable occurrence of different markers and uncertainty of enterococci and E. coli for

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detection of human fecal pollution. In general, results of this initial microbiological contaminant

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assessment will serve as baseline information for follow-on studies to monitor existing and

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emerging public health risks to residents of Texas and potential long-term environmental impacts

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upon the water resources in the impacted regions.

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INTRODUCTION

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Hurricane Harvey originated as a tropical storm over the Atlantic Ocean, becoming a Category 4

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hurricane on August 25, 2017 as it approached the coast of Texas.1 After a week of storms and

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heavy flooding, Hurricane Harvey devastated huge parts of southeastern Texas, particularly

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damaging the wastewater infrastructure resulting in release of sewage contamination into 2 ACS Paragon Plus Environment

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environmental waters.2,3 Immediately following Harvey, both the Texas Commission on

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Environmental Quality (TCEQ) and the U.S. Environmental Protection Agency (EPA) reported

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the release of wastewater from sanitary sewers due to the historic flooding and more than 800

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wastewater treatment plants reported spills.4,5 The widespread flooding resulted in sewage

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overflows and contamination of floodwaters which eventually make their way into surface

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waters, before finally discharging into the Gulf of Mexico. These sewage overflows contain high

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levels of fecal bacteria and potentially pathogenic organisms posing a serious risk to human and

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environmental health via waterborne disease outbreaks, deterioration of recreational and drinking

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water quality, and degradation of aquatic ecology.6 Initial water samples collected from

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floodwaters in affected areas showed E. coli levels greater than 100 times the EPA criteria for

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recreation water quality standard,7 strongly suggesting mixture of sewage in water.2,8 Water

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quality impacts have been reported for previous hurricanes but have primarily been limited to

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chemical contaminants, and transient increases in nutrient loading.9-12 Moreover, these studies

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have focused on contamination of localized water bodies such as the contamination of Lake

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Pontchartrain (Louisiana) during Hurricane Katrina12,13 and limited data is available on the

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distribution of microbial contaminants in impacted rivers and streams.

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Hurricanes and large storms play a significant role in the transport of water contaminants across

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environmental waters.10,11 Among these contaminants, the mobilization and distribution of fecal

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indicator bacteria and human-associated fecal markers due to sewage overflows and stormwater

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runoff resulting from flooding is poorly understood. There is a critical need to understand the

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distribution and transport of fecal bacteria in contaminated surface waters due to ecological as

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well as human health concerns. Emerging real-time quantitative PCR (qPCR) methods designed

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to estimate the concentration of fecal pollution by targeting genomic DNA from fecal indicator



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bacteria such as E. coli, Enterococcus spp., and Bacteroidales are now available and can

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generate test results in just a few hours after sampling, which is critical during extreme events

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such as hurricanes. These genetic markers may also detect viable but nonculturable (VBNC)

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cells that are not detected by conventional cultivation approaches but may still pose a public

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health risk. In this study, we have documented the levels of fecal indicator bacteria (E. coli and

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enterococci) including human-associated fecal genetic markers (Human-associated

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Bacteroidales), measured using qPCR assays, across a Texas river impacted by Hurricane

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Harvey. In addition, we studied the correlation between human-associated Bacteroidales markers

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and traditional fecal indicators to better evaluate the efficacy of using these markers for the

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detection of human fecal pollution.

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The primary objective of this study was to investigate the extent to which the flooded

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waterways in Texas have been contaminated with microbial contaminants following Hurricane

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Harvey. To accomplish this, we have evaluated the presence and abundance of fecal indicator

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bacteria and selected human-associated markers in flood-impacted environmental waters.

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Surface water samples were collected along the Guadalupe River, ranging from sites in Victoria,

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TX (which recorded devastating flooding; see Figure S1)14 to its outfall in the Texas coast.

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Samples were also collected from the San Antonio River as a control site since it is in close

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proximity to Guadalupe River and was not severely impacted due to flooding.14 We applied a

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suite of qPCR assays targeting E. coli, enterococci, and human-associated Bacteroidales markers

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(HF183 and BacHum) to identify and quantify the fecal contamination in water. The results

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presented here provide the much needed and critical data on the effects of human fecal

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contamination sources on the surface water quality following Hurricane Harvey.

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MATERIALS AND METHODS

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Sampling sites and sample collection. The Guadalupe River runs from Kerr County, Texas to

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the San Antonio Bay in the Gulf of Mexico. The excessive rainfall from Hurricane Harvey

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resulted in major flooding over the Guadalupe River at Victoria, TX during which the river

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crested at about 32 feet (i.e. around 10 feet above the flood stage; see Figure S1). The water

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quality of the river is a major concern as it is an important drinking water source for several

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cities and some sections are used in recreational activities. Thus, fecal contamination of the river

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is a significant public health concern and has a negative economic impact.

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An a priori identification of representative sampling locations was not possible due to the

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considerable logistical complications involving access to the sites and safety concerns. An initial

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rapid assessment was performed on September 8, 2017 where multiple water samples were

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collected from the Guadalupe River in flooded regions in Victoria, TX, and potential sampling

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sites were identified. A more intensive sampling campaign was conducted two weeks later on

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September 22, 2017 followed by three more sampling events each in October, November and

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December 2017. During the intensive sampling campaign, water samples were collected from six

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different sites along the Guadalupe River (G1, G2, G3, G4, G5 and G6) and two sites on the San

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Antonio River (SA1 and SA2) over a period of 10 weeks from September 2017 to December

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2017 (Figure 1). The sampling locations were chosen and categorized into flooded or non-

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flooded sites based on the severity of impact due to rainfall and flooding (see Figure S2). The

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description and coordinates of the sites has been listed in Table 1. Additional description of the

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sampling sites has been given in Supporting Information.

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Water samples were collected in duplicate from each site using sterilized 1 L bottles (Nalgene, Rochester, NY) and transported on ice to the laboratory at the University of Texas at 5 ACS Paragon Plus Environment


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San Antonio (San Antonio, TX) within 6 h of collection. The water samples (200 to 1,000 mL)

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were filtered onto 0.45 μm pore-size, 47 mm diameter polycarbonate membranes (Pall Life

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Sciences, Ann Arbor, Michigan) and stored at −20 °C until DNA extraction.

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qPCR analyses. The occurrence and relative abundance of four different fecal bacterial markers

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in environmental water samples was measured using TaqMan qPCR assays (Table S1) and DNA

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extracts as the templates. DNA was extracted from filter samples using the DNeasy PowerLyzer

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PowerSoil Kit (Qiagen, Germantown, MD) according to the manufacturer’s protocol. The

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targeted fecal bacterial groups were E. coli (EC23S857 assay),15 Enterococcus spp. (Entero1

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assay),16 and human-associated Bacteroidales (HF183 and BacHum assays).17,18 The qPCR

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assays were performed as previously described.19 Additional details regarding DNA extraction

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and qPCR assays have been provided in Supporting Information. The genetic markers used in

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our study for the detection of E. coli and enterococci, have been correlated to public health risk20

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and Entero1 assay has been incorporated by the EPA into water quality standards in the United

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States.7 The two human-associated Bacteroidales qPCR markers have been evaluated in a

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number of studies and been shown to be highly specific to human feces, with little to no cross

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reactivity with other host feces.21,22 In addition, all the four qPCR markers have been validated

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and tested in several studies for measuring human fecal contamination in environmental

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waters.19,21-23

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Data analyses. The marker copy number per 100 mL of water was calculated for those samples

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with values above the limit of quantification for each assay, and all data were log10 transformed

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before statistical analysis. The qPCR results for initial grab samples were excluded from any

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subsequent analyses as these samples were obtained from slightly different locations than the

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sampling sites established in this study. Differences in marker concentrations were analyzed 6 ACS Paragon Plus Environment

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using non-parametric Kruskal-Wallis one-way analysis of variance. The correlation between

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human-associated Bacteroidales and conventional fecal indicators was analyzed using the

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logistic regression analysis. All analyses were performed using Microsoft Excel (2016) and

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correlation strength was interpreted according to an accepted scale for biological statistics.24 All

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statistical test outcomes were regarded as significant at p < 0.001.

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RESULTS AND DISCUSSION

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Performance of qPCR assays. Standard curves were generated using serial dilutions of known

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copy numbers to determine the amplification efficiencies and linear range of the qPCR assays.

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The qPCR amplification efficiencies for all the assays ranged from 92.5 to 105.6%, with r2

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values between 0.986 and 0.999. The linear range of quantification for qPCR assay of human-

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associated Bacteroidales (HF183 and BacHum) and E. coli markers were between 10 and 106

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copies, while the linear range for qPCR assay of Entero1 was 20 – 2 × 106 copies. PCR

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inhibition tests were performed with 10-fold dilutions of each DNA extract as described in

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previous study.23 In these tests, a Ct value proportional to a 10-fold dilution relative to the

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undiluted DNA templates resulted, suggesting that PCR inhibition did not interfere with the

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amplification efficiency. No template controls (two per PCR plate) and DNA extraction controls

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indicated the absence of contamination in the qPCR experiments.

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Distribution of fecal indicator bacteria and human-associated marker levels. Initial grab

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water samples (n = 4) were collected on September 8, 2017 near sites G4 and G5. The average

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concentration of fecal bacterial indicators in these samples were 1.31 × 102 copies and 5.05 × 105

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copies per 100 mL water for E. coli and enterococci, respectively. The average concentration of

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human-associated Bacteroidales in these samples were 6.74 × 101 copies and 5.67 × 101 copies



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per 100 mL water for BacHum and HF183, respectively. This was followed by a more intensive

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sampling campaign from September to December 2017.

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The targeted fecal bacterial groups were frequently detected in the surface water samples

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(n = 32) (40–100%). E. coli and enterococci were present in all the samples using the EC23S857

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and Entero1 assays, respectively while the human-associated Bacteroidales were detected less

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frequently. Enterococci exhibited the highest levels across all the sampling sites with mean

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marker abundance greater than 103 copies per 100 mL water. The spatial distribution of the

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levels of markers across the study sites is represented in Figure 2. The water samples yielded

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significantly different amplification results across study sites with both the EC23S857 and

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Entero1 assays, (Kruskal-Wallis test, p < 0.001). Site G4 had the highest concentrations of E.

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coli and enterococci among all the study sites. The two human-associated Bacteroidales markers,

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HF183 and BacHum, exhibited a similar spatial distribution pattern across the sampling sites,

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although the level of BacHum marker was slightly higher for all the samples. Both of the

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markers tested positive at most of the sites, with the exception of sites G1 and SA2 where HF183

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was not detected in any of the samples. The levels of the human-associated Bacteroidales

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markers were statistically different (Kruskal-Wallis test, p < 0.001) from each other among the

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study sites. Site G4 had the highest mean copy number for the human-associated Bacteroidales

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markers which is consistent with the excessive flooding at this site, and also because it was

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located right after a WWTP and the discharge from the WWTP was fed into this region. Site G5

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also had a relatively high concentration of human-associated Bacteroidales compared to other

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sites. However, for site G6, which is located right after the confluence of Guadalupe River and

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San Antonio River, the concentrations of both human-associated Bacteroidales markers

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decreased, indicating that San Antonio River was not severely impacted by human fecal


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contamination. This was also true for sites SA1 and SA2 (control sites on San Antonio River)

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which had considerably lower levels of human-associated Bacteroidales.

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The temporal distribution of the average levels of fecal indicator bacteria and human-

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associated markers is presented in Figure 3. Sites G4 and G5 were classified as flooded regions

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of the Guadalupe River, while sites G1, G2, G3 and G6 were located in the non-flooded areas.

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Sites SA1 and SA2 were located in the non-flooded segments of the San Antonio River. On

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average, all the markers had the highest concentrations for the flooded regions of the Guadalupe

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River. Although, the concentrations of human-associated Bacteroidales was significantly higher

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for the flooded sites than the non-flooded sites throughout the collection period, the observed

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levels of human-associated fecal markers were considered relatively low for an impacted site.25

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However, the concentrations for human-associated Bacteroidales remained elevated for flooded

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sites even 2-3 months after Harvey suggesting that human fecal pollution may be a chronic and

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relatively constant source of fecal contamination for the flood-impacted sites, both before and

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after the hurricane. Samples collected on September 22, 2017, ~3 weeks after flooding,

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demonstrated highest levels for E. coli, BacHum and HF183, both in the flooded and non-

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flooded segments; however, no significant variation occurred over time (Kruskal-Wallis test, p >

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0.5). Initial grab samples collected on September 8, 2017 (during recovery period from flooding)

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from the flooded segment of Guadalupe River (near sites G4 and G5) had higher concentrations

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of Entero1 marker than the samples collected on September 22, 2017 (3 weeks after flooding),

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however the concentrations of other markers was slightly lower. This may be due to the

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differences in the locations of the initial grab samples and the actual sampling sites, which were

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not accessible earlier due to safety concerns.



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The presence of high concentrations of bacterial indicators in surface water samples

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collected from flooded regions of the river were indicative of the fecal contamination in the

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environmental waters of the impacted areas. The most heavily flooded sites showed the highest

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abundance of fecal indicator bacteria and human-associated Bacteroidales indicating that the

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large number of sewage overflows and stormwater runoff which occurred during Harvey

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flooding introduced high levels of fecal bacteria into waterways draining into the Gulf of Mexico

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and thereby impairing surface and coastal water quality. The concentrations of fecal indicator

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bacteria, such as Enterococcus spp., in recreational waters impacted by sewage have been linked

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to adverse health outcomes in swimmers through epidemiology studies.20 The U.S. EPA recently

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published revised recreational water quality criteria for enterococci that aim to keep the risk of

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gastrointestinal illness (GI) in swimmers below approximately 30 illnesses per 1000 primary

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contact recreators.7 All initial water samples collected from the flooded regions of the Guadalupe

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River had E. coli and enterococci concentrations above the regulatory level for contact

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recreational waters,7 although it should be noted that our results were measured by qPCR

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methods while the Recreational Water Quality Criteria is based on culturable E. coli or

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enterococci. A recent study used quantitative microbial risk assessment (QMRA) to simulate the

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risk of GI illness associated with swimming in waters containing different concentrations of the

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HF183 qPCR marker, and estimated there was a linear relationship between HF183 median

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concentrations (in units of copies per 100 mL) and simulated GI illness rates [log10(GI risk) =

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−4.93 + 0.94 × log10HF183; RSQ = 0.99].25 On the basis of this regression relationship, the

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simulated GI illness rate per 1000 swimmers varied between 5.2 × 10-4 and 0.005 for the flooded

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sites in Guadalupe River. This level of predicted human health risk based on HF183 marker is

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relatively low as compared to the EPA-adopted health target of ∼30 GI illnesses per 1000


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swimmers. Although the QMRA model used here relies on a scenario in which raw sewage

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contaminates recreational waters, there are many other potential sources of human fecal pollution

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such as leaking septic systems, open defecation, and treated wastewater. Future studies to

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explore the relationships between human health and human-associated qPCR marker levels in

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recreational waters impacted by sewage, treated effluents, and human feces are needed.

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Stormwater runoff resulting from flooding can cause an influx of indicator bacteria to

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receiving waters. Previous studies have demonstrated increased indicator bacteria levels in

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coastal waters influenced by stormwater runoff.26-28 However, there are limited studies relating

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the impact of flooding caused by hurricanes to the sources of fecal pollution in inland waterways.

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Fecal bacteria may be introduced into surface waters during flooding through numerous sources

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such as municipal waste from household sewage treatment systems, combined sewer overflows

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(CSOs), sanitary sewer overflows (SSOs), damaged WWTPs, leaky septic tanks, and stormwater

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and urban runoff.6,19 In this study we quantified the extent of human fecal contamination in

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environmental waters by an integrated analysis of host-specific markers and fecal bacteria. The

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assessment of study sites for human-associated Bacteroidales demonstrates that sewage sources

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of fecal pollution are major contributors to water quality deterioration within our study area.

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Because the Guadalupe River does not encompass agriculture or farming runoff, and there are no

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CSOs in the sampling region, the primary source of human-associated Bacteroidales can be

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attributed to the waste influx from damaged wastewater infrastructure and stormwater runoff. As

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the river flow into the Gulf of Mexico, these sources appear to represent a chronic and relatively

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constant source of human contamination for inland and near shore communities.

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Correlation between human-associated Bacteroidales and fecal indicator bacteria. The

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numbers of markers per 100 mL water sample were used to study the correlation between 11 ACS Paragon Plus Environment


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human-associated Bacteroidales and conventional fecal indicators (enterococci and E. coli). The

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human-associated Bacteroidales markers showed low to slightly strong correlation with

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enterococci and E. coli markers, which are the most frequently used water quality indicators

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(Figure S3). There was a weak correlation between the BacHum and Entero1 markers in the

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samples (correlation coefficient, r2 = 0.24, p < 0.001), although E. coli exhibited slightly stronger

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correlation with the BacHum marker (r2 = 0.72, p < 0.001). HF183 also showed similar

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correlation as BacHum with both Entero1 (r2 = 0.32, p < 0.001) and E. coli (r2 = 0.75, p

Real-Time Quantitative PCR Measurements of Fecal Indicator Bacteria

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