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Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement Elyse Stachler, Catherine Kelty, Mano Sivaganesan, Xiang Li, Kyle Bibby, and Orin C. Shanks Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02703 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017
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Environmental Science & Technology
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Quantitative CrAssphage PCR Assays for Human Fecal
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Pollution Measurement
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Elyse Stachler a, Catherine Kelty b, Mano Sivaganesanb, Xiang Li b, Kyle Bibby a,c*,
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Orin C. Shanksb*
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a
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Pittsburgh, PA, 15260 USA;
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b
Department of Civil and Environmental Engineering, University of Pittsburgh,
U.S. Environmental Protection Agency, Office of Research and Development, National
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Risk Management Research Laboratory, Cincinnati, OH, 45268 USA;
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c
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Pittsburgh, PA, 15260 USA
Department of Computational and Systems Biology, University of Pittsburgh,
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* Corresponding Authors:
Orin C. Shanks,
[email protected] Kyle Bibby,
[email protected] 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 and water
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resources. Traditionally, general fecal indicator bacteria are used; however, they cannot
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distinguish human fecal waste from other animal pollution sources. Recently, a novel
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bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to
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be abundant in and closely associated with human fecal waste. To confirm bioinformatic
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predictions, 384 primer sets were designed along the length of the crAssphage genome.
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Based upon initial screening, two novel crAssphage qPCR assays (CPQ_056 and
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CPQ_064) were designed and evaluated in reference fecal samples and water matrices.
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The assays exhibited high specificities (98.6%) when tested against an animal fecal
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reference library and crAssphage genetic markers were highly abundant in raw sewage
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and sewage impacted water samples. In addition, CPQ_056 and CPQ_064 performance
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was compared to HF183/BacR287 and HumM2 assays in paired experiments. Findings
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confirm viral crAssphage qPCR assays perform at a similar level to well established
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bacterial human-associated fecal source identification approaches. These new viral based
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assays could become important water quality management and research tools.
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INTRODUCTION Many environmental waters are polluted with human fecal waste originating from
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numerous sources such as leaking sewer lines, faulty septic systems, improperly
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connected downspouts, and combined sewer overflows. Human fecal waste can harbor
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disease-causing pathogens that contribute to poor public health, reduced ecological
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outcomes, and economic burdens. Many public health managers rely on general fecal
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indicator methods (e.g. E. coli and enterococci) to monitor fecal pollution levels, which
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do not discriminate between human and other potential animal sources of fecal pollution.
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General indicators provide limited information which prevents focused remediation,
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because many areas are polluted by a combination of human, agricultural, and/or wildlife
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sources. Information on human waste (i.e. sewage) is particularly important because it
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may pose a greater risk to public health compared to fecal pollution from other animal
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sources1-3. To compliment general indicator measurements and better characterize human
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fecal pollution, many researchers and water quality managers use fecal source
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identification technologies.
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Most currently available human fecal source identification technologies target
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fecal bacteria, mainly Bacteroides species4. These fecal bacteria are abundant in the
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human gut and sewage, facilitating their detection in diluted environmental water
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samples. Bacterial human fecal genetic markers, such as HF183/BacR287 and HumM2
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are highly human-associated and reproducible in multiple laboratory validation studies4-6.
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Although bacterial methods exist for human fecal source characterization, technologies
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targeting viruses are also needed7-9.
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Enteric viruses, such as norovirus, adenovirus, and enterovirus are reported to be
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the dominant etiological agents of waterborne and shellfish-borne disease10, 11. Several
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studies suggest enteric viruses react to environmental and waste treatment conditions in
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markedly different ways compared to bacterial fecal indicators8, 12-15. In addition,
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waterborne viral outbreaks have occurred when general bacterial fecal indicators are not
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detected or are below regulated levels10. Reliance solely on bacterial indicators limits the
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ability of water quality managers to link measures of human fecal pollution with public
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health risk; thus, viral human-associated technologies offer an attractive alternative to
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bacterial fecal source identification methods. Researchers have previously recognized the
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potential value of viral human-associated methodologies, leading to the development of
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technologies targeting enterovirus16-20, adenovirus21, 22, norovirus23, 24, polyomavirus BK
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and JC25, somatic coliphage26, 27, Bacteroides phages28-30, and pepper mild mottle virus31,
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among others. However, a recent multiple laboratory study evaluated the performance of
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many of these virus methods and concluded that the technologies tested either lacked
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sensitivity or exhibited poor specificity, potentially limiting suitability for widespread
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water quality management applications32. A recent comparison of human polyomavirus
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levels to predicted public health risk also illustrates that more sensitive viral
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methodologies are needed33.
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An ideal viral human-associated method for environmental water quality testing
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would target a virus that is both highly human-associated and consistently abundant in
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human waste sources. Recently, a novel bacteriophage, “crAssphage”, was described via
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metagenome cross-assembly and was predicted to be a Bacteroides phage by co-
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occurrence profiling34. The double-stranded DNA crAssphage putative genome was
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assembled from shotgun metagenomic libraries isolated from an individual human fecal
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sample34. Further bioinformatic testing predicted that the crAssphage genome is highly
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abundant and was identified in 73% of human fecal metagenomes surveyed34. A
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subsequent metagenome survey detected crAssphage in sewage from the United States
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and Europe, while crAssphage was absent in other environments, such as non-human
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fecal samples and water environments35. In addition, the crAssphage genome is estimated
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to be up to 10 times more abundant in sewage than other known human-associated
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viruses, including norovirus and adenovirus35.
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Near-ubiquity across human fecal metagenomes and the high abundance
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compared to other sewage-derived viruses, combined with potential human specificity,
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motivates the development of crAssphage as a fecal source identification technology35.
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However, several unknown issues remain that must be addressed for the successful
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development of a crAssphage fecal source identification tool. For instance, the
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crAssphage genome likely represents a viral quasi-species consensus sequence compiled
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from a collection of DNA regions with unknown sequence variability. Furthermore, most
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information about the crAssphage genome has been generated from computer predictions
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with minimal laboratory testing to verify findings. Extensive laboratory testing of fecal
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samples gathered from a wide variety of animal species and sewage collected across a
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broad geographic range is necessary to evaluate the suitability of the crAssphage genome
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for human fecal source identification applications.
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The goals of the present study are to survey the crAssphage genome for human-
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associated genetic regions, develop qPCR methods as potential future environmental
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water quality monitoring tools, and compare their performance to top performing
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bacterial human-associated technologies. We employed a “biased genome shotgun
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strategy” where select genetic regions of the crAssphage genome were screened using
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end-point PCR for highly specific and abundant human-associated fecal pollution genetic
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regions. These genetic regions were subsequently utilized to develop two novel qPCR
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fecal source identification assays. Findings suggest that high throughput laboratory
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screening of novel virus genomes discovered through metagenomic DNA sequence
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mining is a successful strategy to develop host-associated qPCR methods that may be
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important for future research and water quality management activities.
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MATERIALS AND METHODS
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Sample Collection. Individual fecal samples (n=222) were collected from various
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locations across the continental United States as previously described6. Animal fecal
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samples represent ten species including Anser spp. (Canada goose; n=18), Canis
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familiaris (dog, n=41), Bos taurus (cow, n=61), Larus spp. (gull, n=25), Equus caballus
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(horse, n=20), Cervus canadensis (elk, n=20), Gallus gallus (chicken, n=11), Sus scrofa.
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(pig, n=9), Castor canadensis (beaver, n=8), and Odocoileus virginianus (deer, n=9) (see
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Table S1 for sample details). Each fecal sample was collected from a different individual
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as previously described6 and stored at -80°C until time of DNA extraction (
