Interlaboratory accuracy and precision among results of three sewage-associated marker genes in urban environmental estuarine waters and freshwater streams

doi:10.1016/j.scitotenv.2020.140071

Science of The Total Environment

Volume 741, 1 November 2020, 140071

https://doi.org/10.1016/j.scitotenv.2020.140071 Get rights and content

Highlights

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A good agreement was observed between two laboratories for the MST marker gene results.
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Standards, qPCR platforms, nucleic acid extraction efficiency contributed to the observed discrepancies
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A standardized protocol could greatly improve fecal contamination tracking in aquatic environments.

Abstract

The application of quantitative polymerase chain reaction (qPCR) based microbial source tracking (MST) marker genes are increasingly being used to identify contaminating sources and inform management decisions. In this study, we assessed interlaboratory agreement on duplicate environmental water samples collected from estuarine and freshwater locations, by comparing results of qPCR based testing for Bacteroides HF183, crAssphage CPQ_056, and pepper mild mottle virus (PMMoV). The overall agreements (co-detection and non-co-detection) between CSIRO Land and Water (CLW) laboratory and Sydney Water (SW) laboratory for the HF183, crAssphage CPQ_056 and PMMoV marker genes for duplicate water samples were 74, 75 and 74%, respectively. Cohene's kappa (k) revealed fair to moderate agreements and acceptable relative percent difference (RPD) values of <15% for duplicate samples. The pooled mean abundances of HF183, CPQ_056, and PMMoV in measurable samples at the CLW laboratory were 5.19 ± 0.93, 5.12 ± 0.82, and 4.42 ± 0.65 log₁₀ copies/L, respectively. However, the pooled mean abundances were significantly lower at the SW laboratory, HF183 (4.58 ± 0.84 log₁₀ copies/L), crAssphage CPQ_056 (4.20 ± 0.63 log₁₀ copies/L), and PMMoV (3.89 ± 0.41 log₁₀ copies/L). At individual sample level, most of the paired samples had <1 log₁₀ difference. Significant positive Spearman rank correlations were obtained between two laboratories for the HF183 (R_s = 0.65; p < 0.05), CPQ_056 (R_s = 0.79; p < 0.05), and PMMoV (R_s = 0.54; p < 0.05) marker genes. Several factors such as standards, qPCR platforms, PCR inhibitors, nucleic acid extraction efficiency and low levels of targets in some samples may have contributed to the observed discrepancies. Results presented in this study highlight the importance of standardized protocol, laboratory equipment (such as digital PCR), sample processing strategies and appropriate quality controls that may need implementation to further improve accuracy and precision of results between laboratories.

Graphical abstract

Introduction

The application of quantitative polymerase chain reaction (qPCR) based microbial source tracking (MST) tools offer several advantages over traditional fecal indicator bacteria (FIB) monitoring. These include the identification of contaminating sources (Harwood et al., 2014) to enable management of persistent contamination issues (Converse et al., 2012), and linking fecal contamination levels to human health risks using quantitative microbial risk assessment (QMRA) (Crank et al., 2019). qPCR based marker gene assays are generally developed and evaluated in research laboratories followed by evaluation of the performance characteristics of those particular assays/marker genes by other research laboratories within the same or different geographical location (Ahmed et al., 2012; Boehm et al., 2013). These assay performance characteristics are generally limited to host sensitivity and specificity of the marker gene by testing a collection of fecal and wastewater samples from various animal species (Green et al., 2014; Stachler et al., 2017; Ahmed et al., 2019a, Ahmed et al., 2019b).

The Source Identification Protocol Project (SIPP) in California, USA, attempted to determine the performance characteristics (i.e., most accurate and specific assays) of 41 qPCR based MST assays among 27 participating laboratories (Boehm et al., 2013). In this study, blind test samples were prepared by seeding a single or double source of human and animal fecal material in water samples. After the sample filtration, membranes were shipped to 27 participating laboratories specialized in MST analysis. The SIPP study revealed considerable differences in results among laboratories. These differences may be attributable to differences in qPCR platforms, reagents, standards, nucleic acid extraction protocols, and the proficiency of technicians using a particular assay (Ebentier et al., 2013).

Stewart et al. (2013) identified several limitations of the SIPP study design. In the SIPP study, reference fecal materials were seeded in an artificial freshwater matrix, which is different than actual environmental samples such as estuarine or marine waters that are prone to event based fecal contamination. This could affect PCR amplification due to the presence of different levels of inhibitors (Steele et al., 2018; Ahmed et al., 2020). Also, a maximum of two sources of contamination was seeded; therefore, determining the source in blind samples was relatively easy compared to real-world environmental samples, which may contain fecal contamination from multiple sources at low levels, making accurate identification challenging.

Sydney Water is a New South Wales Government-owned corporation providing potable drinking water and wastewater services to approximately five million people in Sydney and the surrounding regions of the Illawarra and Blue Mountains. The annual rainfall in Sydney ranges from 900 to 1440 mm, which primarily falls between October and May. During periods of excessive rain, the sewerage system can become overloaded, and wet weather overflows (WWOs) will discharge to environmental waters to alleviate sewer surcharge into households and business premises. Our recent study has established a link between WWOs and sewage contamination levels of estuarine waters in Australia (Ahmed et al., 2020). This study also demonstrated the capability of the MST monitoring approach to understanding sources (sewage or animal) of fecal contamination. This capability will significantly enhance management decisions assisting in the prioritization of remediation efforts of the sewerage system to improve estuarine bathing water quality and diminish human health risk. Sydney Water is investigating the application of MST in the long term WWO monitoring and solution planning. This will support Sydney Water in meeting regulatory measures.

Commonwealth Scientific Industrial Research Organization (CSIRO) has been maintaining its capabilities to remain at the forefront of dealing with fecal contamination tracking in Australia using MST tools. CSIRO is assisting Sydney Water to incorporate these tools into their existing water quality monitoring regimes. However, results obtained for environmental water samples must be reproducible in interlaboratory comparison for their wider acceptance by water quality managers and for regulatory uses (Ebentier et al., 2013). It is important to identify factors that may alter MST results. Such information is vital for the transfer of MST tools from research laboratories to major utilities and other water quality laboratories. Particularly given these laboratories can operate at regional scale in a production manner processing relatively large numbers of samples. The main aim of this study was to assess interlaboratory precision and accuracy between two laboratories on duplicate environmental water samples collected from three estuarine and one freshwater site by using Bacteroides HF183, crAssphage CPQ_056, and pepper mild mottle virus (PMMoV) assays with different instruments and analyst experience levels.

Section snippets

Participating laboratories

CSIRO Land and Water (research provider) and Sydney Water (collaborator) participated in this study. Both laboratories are not ISO 17025 certified for analysis of sewage-associated marker genes in water samples. CSIRO Land and Water (will be referred to as CLW laboratory hereafter) laboratory and the Sydney Water Monitoring Services Laboratory (will be referred to as SW laboratory hereafter) took part in this study. CLW laboratory is located at Ecosciences Precinct, Dutton Park, Brisbane. SW

qPCR performance characteristics and PCR inhibition

Six qPCR standard curves were analyzed to determine the qPCR performance characteristics for each qPCR assay at the CLW laboratory, while two to three standard curves were analyzed at the SW laboratory. Slopes ranged from −3.51 to −3.41 (for HF183), −3.41 to −3.15 (for CPQ_056) and −3.43 to −3.33 for PMMoV at the CLW laboratory (Table 3). Similarly, value of slopes ranged from −3.43 to −3.27 (for HF183), −3.44 to −3.33 (for CPQ_056) and −3.43 to −3.33 (for PMMoV) at the SW laboratory. Slope

Discussion

Microbial source tracking (MST) marker genes are mostly used by specialized research laboratories to determine the sources of fecal contamination in waterways. Routine monitoring utilizing MST analysis is still to become a reality for utilities and local governments in Australia. The analysis is limited to highly trained personnel due to the complex nature of qPCR analysis and data interpretation. This is further exacerbated by the use of standards, reagents, nucleic acid extraction kits, qPCR

Conclusions

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Despite several dissimilarities in laboratory testing protocols between two laboratories, >74% agreements (co-detection and non-co-detection) were found for the HF183, CPQ_056 and PMMoV marker genes for duplicate water samples collected from urban environmental estuarine waters and freshwater streams.
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Discrepancies were observed on the quantitative detection of sewage-associated marker genes between two laboratories. In general, the CLW laboratory reported greater concentrations (<1 log₁₀) of

CRediT authorship contribution statement

Warish Ahmed: Investigation, Resources, Writing - original draft, Writing - review & editing. Sudhi Payyappat: Investigation, Resources, Writing - original draft, Writing - review & editing. Michele Cassidy: Resources, Writing - original draft, Writing - review & editing. Nathan Harrison: Resources, Writing - original draft, Writing - review & editing. Colin Besley:Resources, Writing - original draft, Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Sydney Water for financial assistance and for support in collection of water samples and laboratory work. We also thank Merran Griffith and Kaye Power from Sydney Water for providing valuable feedback on the manuscript.

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Interlaboratory accuracy and precision among results of three sewage-associated marker genes in urban environmental estuarine waters and freshwater streams

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Participating laboratories

qPCR performance characteristics and PCR inhibition

Discussion

Conclusions

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgements

Environ. Int.

Sci. Total Environ.

Water Res.

Water Res.

Water Res.

Sci. Total Environ.

Water Res.

Water Res.

Biophys. J.

Anal. Biochem.

J. Microbiol. Methods

Water Res.

Water Res.

Trends Biotechnol.

Consistency in the host specificity and host sensitivity of the Bacteroides HF183 marker for sewage pollution tracking

Lett. Appl. Microbiol.

Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters

Appl. Environ. Microbiol.

Distributions of fecal markers in wastewater from different climatic zones for human fecal pollution tracking in Australian Surface Waters

Appl. Environ. Microbiol.