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Evaluation of molecular- and culture-dependent MST markers to detect fecal contamination and indicate viral presence in good quality groundwater David Diston, Michael Sinreich, Stephanie Zimmermann, Andreas Baumgartner, and Richard Felleisen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00515 • Publication Date (Web): 14 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015
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Evaluation of molecular- and culture-dependent MST markers to detect fecal contamination
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and indicate viral presence in good quality groundwater
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Diston, D. 1*, Sinreich, M.2, Zimmermann, S.2, Baumgartner, A.1, and Felleisen, R.1
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Federal Food Safety and Veterinary Office FSVO, Bern, Switzerland
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Federal Office for the Environment FOEN, Bern, Switzerland
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Corresponding author:
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Federal Food Safety and Veterinary Office, Sector Laboratories, Schwarzenburgstrasse 165, 3003
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Bern, Switzerland
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Tel.: +41 58 325 40 69
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Fax: +41 58 322 95 74
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Email: [email protected]
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ABSTRACT
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Microbial contamination of groundwater represents a significant health risk to resource users. Culture-
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dependent Bacteroides phage and molecular-dependent Bacteroidales 16S rRNA assays are employed
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in Microbial Source Tracking (MST) studies globally, however little is known regarding how these
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important groups relate to each other in the environment and which is more suitable to indicate the
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presence of waterborne fecal pollution and human enteric viruses. This study addresses this knowledge
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gap by examining 64 groundwater samples from sites with varying hydrogeological properties using a
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MST toolbox containing two bacteriophage groups (phage infecting GB-124 and ARABA-84), and
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two Bacteroidales 16S rRNA markers (Hf183 and BacR); those were compared to fecal indicator
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bacteria, somatic coliphage, Bacteroidales 16S rRNA marker AllBac, four human enteric viruses
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(norovirus G1 & 2, enterovirus and group A rotavirus) and supplementary hydrogeological/chemical
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data. Bacteroidales 16S rRNA indicators offered a more sensitive assessment of both human-specific
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and general fecal contamination than phage indicators, but may overestimate the risk from enteric viral
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pathogens. Comparison with hydrogeological and land use site characteristics as well as auxiliary
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microbiological and chemical data proved the plausibility of the MST findings. Sites representing
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karst aquifers were of significantly worse microbial quality than those with unconsolidated or fissured
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aquifers, highlighting the vulnerability of these hydrogeological settings.
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1.0 INTRODUCTION
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Enteric viruses are frequently detected in groundwater used as drinking water resources (1-3), with
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gastrointestinal illness caused by the consumption of microbially contaminated drinking water
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representing a significant health risk. Many countries rely on groundwater as the primary source of
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drinking water and contamination events represent a significant economic and health burden. Of
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particular importance, from a water resource management perspective, is the high vulnerability of
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karst aquifers, found in areas with limestone geologies, as well as highly heterogeneous fissured
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aquifers to contamination by rainfall-driven fecal pollution (4-6). 2
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Although animal fecal material may contain pathogens such as Cryptosporidium, Giardia,
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Campylobacter, Salmonella and enterohemorrhagic E. coli, amongst others, which are able to infect
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humans (7), viral pathogens from human feces are more likely to infect and cause illness in humans
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than viral pathogens present in animal feces. The health risk, therefore, posed to water consumers is
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primarily dependent on the source of the fecal contamination (7).
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Traditionally fecal coliforms and fecal enterococci (termed together as fecal indicator bacteria; FIB)
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have been used to identify water of poor microbial quality, but these parameters offer neither an
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indication of pollutant source nor of pathogen presence (8). Numerous culture- and molecular-
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dependent microbial source tracking (MST) techniques have been developed in order to identify
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sources of aquatic fecal pollution, offering varying degrees of sensitivity and specificity [see Harwood
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et al. 2013 and Roslev and Bukh, 2011 for reviews (8,9)]. MST methods are powerful tools in
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identifying fecal contributors to aquatic systems and allow informed risk assessments regarding water
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usage to be conducted. At present there is no “gold standard” MST marker for the identification of
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either human- or non-human fecal pollution in waterbodies, with studies benefiting from a “toolbox”
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approach (10-12). Two groups of MST tools, amongst the many proposed indicators, have shown
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promise: culture-dependent Bacteroides bacteriophage (13-17) and Bacteroidales 16S rRNA
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molecular-dependent assays (18-22). In addition to MST markers, chemical markers may also be used
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to indicate origins of waterborne pollution. Many markers have been proposed (including
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pharmaceuticals, optical brighteners or caffeine amongst others) and they are a useful addition to a
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source tracking ‘tool-box’(23).
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In order to be a useful water management and risk assessment tool, proposed indicators should fulfill a
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number of requirements; i) presence in host feces only, ii) inability to replicate in natural waters, iii)
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possession of a simple, reliable, rapid and inexpensive assay method, iv) not requiring the culturing of
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isolates, v) not requiring a large reference strain library, and vi) simple methods of sample collection
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and storage (24). In addition, if the MST indicators are to be used as surrogates for enteric viruses they
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should be present when the pathogen of concern is present (ideally in higher densities), low to absent
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when no pathogen is present, and have similar inactivation kinetics (24,25). For Bacteroidales 16S 3
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rRNA markers, some of these requirements have, in part, been satisfied. However, for phage infecting
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Bacteroides strains GB-124 and ARABA-84, little information regarding these requirements is
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available [inactivation ecology has, in part, been investigated for phage infecting GB-124 (26,27)].
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Investigation into the relationship between fecal indicators and pathogens is key research need,
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fundamental to the deployment of any proposed MST marker (9,11).
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At present, no studies have been identified assessing the relationship between both molecular-
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dependent Bacteroidales 16S rRNA assays and culture-dependent Bacteroides phage with enteric
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viruses in groundwater. This study aims to contribute to filling this knowledge gap by applying a
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toolbox of both culture-dependent and molecular-dependent source tracking methods to groundwater
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of different geologies and aquifer types, respectively, assessing which group(s) of indicators give the
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best prediction of enteric viral presence. This aim will be achieved by carrying out the following
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objectives:
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1. To assess the suitability of Bacteroidales 16S rRNA general fecal (AllBac) and source-specific
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markers, Hf183 and BacR) and Bacteroides phage (capable of infecting strains GB-124 and
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ARABA-84) to indicate the presence of norovirus genogroups I & II, human enterovirus, and
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human group A rotavirus in groundwater;
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2. To compare the ability of culture-dependant Bacteroides phage and molecular-dependent
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Bacteroidales 16S rRNA MST methods to detect fecal contamination in groundwater as
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indicated by human enteric viruses, general fecal indicators and chemical parameters.
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This will be the first time Bacteroides strains GB-124 and ARABA-84 have been directly compared
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with Bacteroidales 16S rRNA Hf183 (and other Bacteroidales 16S rRNA non-human markers) in an
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environmental MST study, and will give insights into the strengths and weaknesses of the cultivation-
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dependent and cultivation-independent methods when aiming to identify fecal material and enteric
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viral pathogens presence in groundwater from varying hydrogeological settings.
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2.0. MATERIALS AND METHODS
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2.1 Groundwater samples
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Previous data collected during the NAQUA National Groundwater Monitoring programme allowed
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the identification of groundwater sites in Switzerland that have experienced either high FIB counts or
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recorded incidences of human enteric viruses (6). Eight sites were chosen (figure S1), each of which
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was sampled eight times from January 2012 to September 2013 for a suite of 14 parameters. The
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hydrogeological characterization of these sites is varied, three are related to unconsolidated aquifers
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(NTG34, NTG44 and NTG51), three to karst aquifers (NTQ03, NTQ25 and NTQ27), and two to
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fissured aquifers (NTQ31 and NTQ32). For all sites, 30L grab samples were collected in sterile
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polypropylene carboys (Nalgene, USA) with sample containers being pre-rinsed in situ three times
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before sample collection. Water samples were then kept on ice blocks in a polystyrene cool box and
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transported back to the Federal Food Safety and Veterinary Office laboratory (Köniz, Bern,
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Switzerland) within three hours.
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2.2 Fecal indicators/MST indicators assayed
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Groundwater samples were assayed for three bacteriophage groups: human-specific host strains
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Bacteroides fragilis GB-124 (13,28) – selected as it appears to be both sensitive and highly specific –
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and B. thetaiotaomicron ARABA-84 (17) – selected as it is the ‘local’ bacteriophage host strain –, and
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also the general host strain Escherichia coli WG-5 [somatic coliphage (29)]. The optimized
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bacteriophage filtration method of Mendez et al. (30) was used. Briefly, MgCL2 was added to 1 L of
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groundwater to a final concentration of 0.05 M, and filtered through a 47 mm 0.22 µm nitrocellulose
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membrane (Millipore, Ireland) at a flow rate of 2 L per hour. Filters containing phage were then cut
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into 8 fragments, and transferred to a flask containing 5 mL of eluting solution [3 % (w/v) beef extract,
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3% (v/v) Tween 80, and 0.5 M NaCl, pH 9.0]. The flask was then placed in an ultrasonic bath for 4
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minutes, and the resulting 5 mL eluate was assayed according to the International Standard for the
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respective phage group (29,31). Fragments of the filtration membrane were placed face down on a 5
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host monolayer for assay and incubated at 37 °C for 18 hours. Positive and blank assays were also
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conducted.
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Both fecal coliforms (FC) and intestinal enterococci (ENT) are validated, non-specific indicators of
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fecal pollution, and are often employed as legislative tools for water quality throughout the world. FC
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and ENT were enumerated following International Standards (32,33) by membrane filtration on a 47
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mm 0.45 µm mixed cellulose ester membrane (Millipore, Ireland). Positive (sterile Elga Purelab Ultra
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water spiked with reference bacteria) and blank filtrations (sterile Elga Purelab Ultra water only) were
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conducted in parallel.
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Real-time PCR (qPCR) assays to target species- (or group-) specific Bacteroidales 16S rRNA gene
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sequences were used to indicate human, ruminant and general fecal contamination. These assays
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represent the “state of the art” and are an important research area. Three markers were assayed:
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human-specific Hf183 (18,34) – selected because of high specificity (35) and widespread use –,
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ruminant-specific BacR (19,36,37) – selected because it was developed in a karst region, i.e., similar
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to present study area and high specificity (35) –, and a general fecal marker AllBac (21) – selected
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because it has been widely used in other studies allowing comparison of the present data. The filtration
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method used was that of Gourmelon et al. (34). Briefly, 1 L of sample was filtered using a 47 mm 0.2
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µm cyclopore track etched membrane (Whatman, USA). The filter was then folded and placed into a
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15 mL DNA-free, screw-top test tube containing 0.5 mL of GITC buffer (5M Guanidine
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isothiocyanate, 100 mM EDTA (pH 8), 0.5 % Sarkosyl). The tube was inverted several times to ensure
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the entire filter was wetted with GITC buffer and stored at -20 °C until DNA extraction. DNA was
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extracted from thawed filters using QIAmp DNA mini kit (Qiagen, USA) employing a modified
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protocol (38). qPCR assays containing 3 µL DNA (and dilutions thereof) for the three Bacteroidales
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16S rRNA parameters were run on an ABI 7500 Real Time PCR System (Applied Biosystems) with
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either ABI TaqMan Universal PCR MasterMix (AllBac and BacR) or Roche SYBR green Fast Start
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Probe MasterMix (Hf183). Gene copy numbers were quantified using plasmids (TOPO TA Cloning®
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Kit, E. coli DH5α-T1R, Invitrogen, Germany) constructed from PCR amplification of DNA (extracted
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with Stool Mini kit, Qiagen, USA) from fecal samples containing appropriate target sequences. 6
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Standard curves from 1.0 x 105 to 1 copy/reaction were run in the qPCR assays. The concentrations of
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the primers and probes in the qPCR systems remained unchanged from the original papers and are
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detailed in table S1 (18,19,21). Blank filtrations (sterile Elga Purelab Ultra water) were conducted in
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parallel whilst inhibition of DNA during qPCR assay was tested using a TaqMan exogenous internal
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positive control (Applied Biosystems, USA).
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2.3 Human enteric viruses
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Three pathogenic human RNA viruses were selected for assay; norovirus (genogroups I and II),
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human enterovirus and human rotavirus (group A); the assay oligonucleotides are shown in table S1.
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The viral concentration method of Katayama et al. (39) was used. Briefly, 1 L groundwater samples
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were amended with MgCl2, achieving a final concentration of 25 mM, and spiked with 100 plaque
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forming units (PFU) of process control phage MS2. Samples were acidified to pH 3.75 using 10 %
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(v/v) C2H4O2 and filtered through a 47 mm 0.45 µm HA negatively charged membrane (Millipore,
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Ireland). 100 mL of 0.5 mM H2SO4 was passed through the membrane and 10 mL of 1 mM NaOH
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poured onto membrane to elute virions. Eluate was recovered in a tube containing 0.1 mL of 50 mM
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H2SO4 and 0.1 mL of 100X Tris-EDTA (TE) buffer and then further purified, concentrated, and
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desalted using Centriprep YM-50 concentrator columns (Millipore, Ireland) according to the
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manufacturer’s instructions.
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RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen, USA) and eluted into 60 µL AVE
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buffer. Reverse transcription was achieved using High Capacity cDNA Archive Kit (Applied
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Biosystems, USA) and cDNA was stored at -20 °C until needed.
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MS2 phage was a process control throughout the complete viral assay (allowing the filtration, elution,
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reverse transcription and qPCR assay to be monitored) and the qPCR system was based on the MS2
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phage lysis protein from O’Connell et al. (40).
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All assays containing 3 µL cDNA (and dilutions thereof), 25 mL equivalent volume of groundwater
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with a sensitivity of 40 virions per litre, were run on an ABI 7500 Real Time PCR System (Applied
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Biosystems) with ABI Taqman Universal PCR MasterMix throughout. Positive stool samples for the
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four viral types were obtained from Biolytix (Basel, Switzerland), Medica Medical laboratories
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(Zürich, Switzerland), and St. Gallen Cantonal Laboratory (St. Gallen, Switzerland). Standard curves
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from 1.0 x 105 to 1 copy/reaction were included in the qPCR assays. The concentrations of the primers
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and probes in the qPCR systems remained unchanged from the original papers (41-43) and blank
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filtrations (sterile Elga Ultralab Pure water), positive and negative controls were conducted in parallel.
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When positive results were returned (in any number of wells), cDNA aliquots were sent for
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subsequent assay in an external lab (Biosmart, Bern, Switzerland) using methods employed previously
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using water samples from these sites (4,44). Furthermore, in qPCR assays where either two or three
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wells were positive, these samples were retested to see if result was reproducible.
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2.4 Other parameters
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In conjunction with both general and source specific microbial markers, soluble reactive phosphorus
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(SRP; PO4) and boron (B) concentrations were assayed in order to determine if the parameters were
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suitable for source identification in groundwater. Strong correlation between SRP and B may indicate
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discharges from wastewater treatment works [WwTW(45)]. Both B and SRP were measured in
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triplicate using photometry (AQUANAL®-professional Spectro 1000, methods 85 and 320
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respectively).
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Auxiliary hydro-chemical and microbiological data were available from the sampling program of the
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NAQUA National Groundwater Monitoring. These data were site-specifically processed in
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conjunction with information on aquifer characteristics (including vulnerability) and catchment land
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use. Both land use and chemical data were grouped and classified according to their indication for
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human or ruminant sources and compared to the host-specific MST results obtained from the present
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study. Groundwater level and spring discharge data recorded in the framework of NAQUA were used 8
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to identify relationships between high water stages and microbiological parameters. Daily means at
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sampling dates were therefore normalized to long-term mean values obtained from continuous
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measurement.
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2.6 Statistical methods
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All data were non-normally distributed (log transformation did not produce normally distributed data),
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and therefore non-parametric statistical methods were used throughout. To allow comparison of results
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with previous/future studies, both mean and median statistics are provided. Furthermore, because the
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water was of “good” quality and as such the parameters were infrequently detected at certain
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monitoring stations, the median across all samples in conjunction with positive sample only medians
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are reported. Normality tests, descriptive statistics and Spearman rank correlations were computed
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using Microsoft Excel 2007 for Windows. The interpretation of Spearman Rank ρ was as follows: 0.9
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to 1 - very strong, 0.7 to 0.89 – strong, 0.5 to 0.69 – moderate, 0.3 to 0.49 - low, 0.16 to 0.29 – weak, and
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40
>300
and/or
40
>1.0
>5
> 0.1 (> 1 substance)
503
504 505
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* NAQUA National groundwater monitoring results from former microbiological pilot studies (6, 7) and regular chemical sampling 2012 and 2013 (maximum concentration, sampling frequency 2-4 per year) ** Group 1: Benzotriazole and pharmaceutical residues (Sulfamethoxazole, Carbamazepine, Diatrizoic acid) 25
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Jothikumar, N.; Kang, G.; Hill, V. R. Broadly reactive TaqMan assay for real-time RT-PCR detection of rotavirus in clinical and environmental samples. [email protected]. J. Virol. Methods. 2009, 155 (2), 126-131.
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Article
Evaluation of molecular- and culture-dependent MST markers to detect fecal contamination and indicate viral presence in good quality groundwater David Diston, Michael Sinreich, Stephanie Zimmermann, Andreas Baumgartner, and Richard Felleisen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00515 • Publication Date (Web): 14 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015
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1
Evaluation of molecular- and culture-dependent MST markers to detect fecal contamination
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and indicate viral presence in good quality groundwater
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Diston, D. 1*, Sinreich, M.2, Zimmermann, S.2, Baumgartner, A.1, and Felleisen, R.1
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1
Federal Food Safety and Veterinary Office FSVO, Bern, Switzerland
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2
Federal Office for the Environment FOEN, Bern, Switzerland
*
Corresponding author:
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9
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Federal Food Safety and Veterinary Office, Sector Laboratories, Schwarzenburgstrasse 165, 3003
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Bern, Switzerland
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Tel.: +41 58 325 40 69
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Fax: +41 58 322 95 74
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Email: [email protected]
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ABSTRACT
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Microbial contamination of groundwater represents a significant health risk to resource users. Culture-
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dependent Bacteroides phage and molecular-dependent Bacteroidales 16S rRNA assays are employed
24
in Microbial Source Tracking (MST) studies globally, however little is known regarding how these
25
important groups relate to each other in the environment and which is more suitable to indicate the
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presence of waterborne fecal pollution and human enteric viruses. This study addresses this knowledge
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gap by examining 64 groundwater samples from sites with varying hydrogeological properties using a
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MST toolbox containing two bacteriophage groups (phage infecting GB-124 and ARABA-84), and
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two Bacteroidales 16S rRNA markers (Hf183 and BacR); those were compared to fecal indicator
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bacteria, somatic coliphage, Bacteroidales 16S rRNA marker AllBac, four human enteric viruses
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(norovirus G1 & 2, enterovirus and group A rotavirus) and supplementary hydrogeological/chemical
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data. Bacteroidales 16S rRNA indicators offered a more sensitive assessment of both human-specific
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and general fecal contamination than phage indicators, but may overestimate the risk from enteric viral
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pathogens. Comparison with hydrogeological and land use site characteristics as well as auxiliary
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microbiological and chemical data proved the plausibility of the MST findings. Sites representing
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karst aquifers were of significantly worse microbial quality than those with unconsolidated or fissured
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aquifers, highlighting the vulnerability of these hydrogeological settings.
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1.0 INTRODUCTION
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Enteric viruses are frequently detected in groundwater used as drinking water resources (1-3), with
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gastrointestinal illness caused by the consumption of microbially contaminated drinking water
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representing a significant health risk. Many countries rely on groundwater as the primary source of
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drinking water and contamination events represent a significant economic and health burden. Of
44
particular importance, from a water resource management perspective, is the high vulnerability of
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karst aquifers, found in areas with limestone geologies, as well as highly heterogeneous fissured
46
aquifers to contamination by rainfall-driven fecal pollution (4-6). 2
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47
Although animal fecal material may contain pathogens such as Cryptosporidium, Giardia,
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Campylobacter, Salmonella and enterohemorrhagic E. coli, amongst others, which are able to infect
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humans (7), viral pathogens from human feces are more likely to infect and cause illness in humans
50
than viral pathogens present in animal feces. The health risk, therefore, posed to water consumers is
51
primarily dependent on the source of the fecal contamination (7).
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Traditionally fecal coliforms and fecal enterococci (termed together as fecal indicator bacteria; FIB)
53
have been used to identify water of poor microbial quality, but these parameters offer neither an
54
indication of pollutant source nor of pathogen presence (8). Numerous culture- and molecular-
55
dependent microbial source tracking (MST) techniques have been developed in order to identify
56
sources of aquatic fecal pollution, offering varying degrees of sensitivity and specificity [see Harwood
57
et al. 2013 and Roslev and Bukh, 2011 for reviews (8,9)]. MST methods are powerful tools in
58
identifying fecal contributors to aquatic systems and allow informed risk assessments regarding water
59
usage to be conducted. At present there is no “gold standard” MST marker for the identification of
60
either human- or non-human fecal pollution in waterbodies, with studies benefiting from a “toolbox”
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approach (10-12). Two groups of MST tools, amongst the many proposed indicators, have shown
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promise: culture-dependent Bacteroides bacteriophage (13-17) and Bacteroidales 16S rRNA
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molecular-dependent assays (18-22). In addition to MST markers, chemical markers may also be used
64
to indicate origins of waterborne pollution. Many markers have been proposed (including
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pharmaceuticals, optical brighteners or caffeine amongst others) and they are a useful addition to a
66
source tracking ‘tool-box’(23).
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In order to be a useful water management and risk assessment tool, proposed indicators should fulfill a
68
number of requirements; i) presence in host feces only, ii) inability to replicate in natural waters, iii)
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possession of a simple, reliable, rapid and inexpensive assay method, iv) not requiring the culturing of
70
isolates, v) not requiring a large reference strain library, and vi) simple methods of sample collection
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and storage (24). In addition, if the MST indicators are to be used as surrogates for enteric viruses they
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should be present when the pathogen of concern is present (ideally in higher densities), low to absent
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when no pathogen is present, and have similar inactivation kinetics (24,25). For Bacteroidales 16S 3
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rRNA markers, some of these requirements have, in part, been satisfied. However, for phage infecting
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Bacteroides strains GB-124 and ARABA-84, little information regarding these requirements is
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available [inactivation ecology has, in part, been investigated for phage infecting GB-124 (26,27)].
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Investigation into the relationship between fecal indicators and pathogens is key research need,
78
fundamental to the deployment of any proposed MST marker (9,11).
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At present, no studies have been identified assessing the relationship between both molecular-
80
dependent Bacteroidales 16S rRNA assays and culture-dependent Bacteroides phage with enteric
81
viruses in groundwater. This study aims to contribute to filling this knowledge gap by applying a
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toolbox of both culture-dependent and molecular-dependent source tracking methods to groundwater
83
of different geologies and aquifer types, respectively, assessing which group(s) of indicators give the
84
best prediction of enteric viral presence. This aim will be achieved by carrying out the following
85
objectives:
86
1. To assess the suitability of Bacteroidales 16S rRNA general fecal (AllBac) and source-specific
87
markers, Hf183 and BacR) and Bacteroides phage (capable of infecting strains GB-124 and
88
ARABA-84) to indicate the presence of norovirus genogroups I & II, human enterovirus, and
89
human group A rotavirus in groundwater;
90
2. To compare the ability of culture-dependant Bacteroides phage and molecular-dependent
91
Bacteroidales 16S rRNA MST methods to detect fecal contamination in groundwater as
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indicated by human enteric viruses, general fecal indicators and chemical parameters.
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This will be the first time Bacteroides strains GB-124 and ARABA-84 have been directly compared
94
with Bacteroidales 16S rRNA Hf183 (and other Bacteroidales 16S rRNA non-human markers) in an
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environmental MST study, and will give insights into the strengths and weaknesses of the cultivation-
96
dependent and cultivation-independent methods when aiming to identify fecal material and enteric
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viral pathogens presence in groundwater from varying hydrogeological settings.
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2.0. MATERIALS AND METHODS
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2.1 Groundwater samples
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Previous data collected during the NAQUA National Groundwater Monitoring programme allowed
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the identification of groundwater sites in Switzerland that have experienced either high FIB counts or
103
recorded incidences of human enteric viruses (6). Eight sites were chosen (figure S1), each of which
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was sampled eight times from January 2012 to September 2013 for a suite of 14 parameters. The
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hydrogeological characterization of these sites is varied, three are related to unconsolidated aquifers
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(NTG34, NTG44 and NTG51), three to karst aquifers (NTQ03, NTQ25 and NTQ27), and two to
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fissured aquifers (NTQ31 and NTQ32). For all sites, 30L grab samples were collected in sterile
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polypropylene carboys (Nalgene, USA) with sample containers being pre-rinsed in situ three times
109
before sample collection. Water samples were then kept on ice blocks in a polystyrene cool box and
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transported back to the Federal Food Safety and Veterinary Office laboratory (Köniz, Bern,
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Switzerland) within three hours.
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2.2 Fecal indicators/MST indicators assayed
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Groundwater samples were assayed for three bacteriophage groups: human-specific host strains
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Bacteroides fragilis GB-124 (13,28) – selected as it appears to be both sensitive and highly specific –
116
and B. thetaiotaomicron ARABA-84 (17) – selected as it is the ‘local’ bacteriophage host strain –, and
117
also the general host strain Escherichia coli WG-5 [somatic coliphage (29)]. The optimized
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bacteriophage filtration method of Mendez et al. (30) was used. Briefly, MgCL2 was added to 1 L of
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groundwater to a final concentration of 0.05 M, and filtered through a 47 mm 0.22 µm nitrocellulose
120
membrane (Millipore, Ireland) at a flow rate of 2 L per hour. Filters containing phage were then cut
121
into 8 fragments, and transferred to a flask containing 5 mL of eluting solution [3 % (w/v) beef extract,
122
3% (v/v) Tween 80, and 0.5 M NaCl, pH 9.0]. The flask was then placed in an ultrasonic bath for 4
123
minutes, and the resulting 5 mL eluate was assayed according to the International Standard for the
124
respective phage group (29,31). Fragments of the filtration membrane were placed face down on a 5
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host monolayer for assay and incubated at 37 °C for 18 hours. Positive and blank assays were also
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conducted.
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Both fecal coliforms (FC) and intestinal enterococci (ENT) are validated, non-specific indicators of
128
fecal pollution, and are often employed as legislative tools for water quality throughout the world. FC
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and ENT were enumerated following International Standards (32,33) by membrane filtration on a 47
130
mm 0.45 µm mixed cellulose ester membrane (Millipore, Ireland). Positive (sterile Elga Purelab Ultra
131
water spiked with reference bacteria) and blank filtrations (sterile Elga Purelab Ultra water only) were
132
conducted in parallel.
133
Real-time PCR (qPCR) assays to target species- (or group-) specific Bacteroidales 16S rRNA gene
134
sequences were used to indicate human, ruminant and general fecal contamination. These assays
135
represent the “state of the art” and are an important research area. Three markers were assayed:
136
human-specific Hf183 (18,34) – selected because of high specificity (35) and widespread use –,
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ruminant-specific BacR (19,36,37) – selected because it was developed in a karst region, i.e., similar
138
to present study area and high specificity (35) –, and a general fecal marker AllBac (21) – selected
139
because it has been widely used in other studies allowing comparison of the present data. The filtration
140
method used was that of Gourmelon et al. (34). Briefly, 1 L of sample was filtered using a 47 mm 0.2
141
µm cyclopore track etched membrane (Whatman, USA). The filter was then folded and placed into a
142
15 mL DNA-free, screw-top test tube containing 0.5 mL of GITC buffer (5M Guanidine
143
isothiocyanate, 100 mM EDTA (pH 8), 0.5 % Sarkosyl). The tube was inverted several times to ensure
144
the entire filter was wetted with GITC buffer and stored at -20 °C until DNA extraction. DNA was
145
extracted from thawed filters using QIAmp DNA mini kit (Qiagen, USA) employing a modified
146
protocol (38). qPCR assays containing 3 µL DNA (and dilutions thereof) for the three Bacteroidales
147
16S rRNA parameters were run on an ABI 7500 Real Time PCR System (Applied Biosystems) with
148
either ABI TaqMan Universal PCR MasterMix (AllBac and BacR) or Roche SYBR green Fast Start
149
Probe MasterMix (Hf183). Gene copy numbers were quantified using plasmids (TOPO TA Cloning®
150
Kit, E. coli DH5α-T1R, Invitrogen, Germany) constructed from PCR amplification of DNA (extracted
151
with Stool Mini kit, Qiagen, USA) from fecal samples containing appropriate target sequences. 6
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152
Standard curves from 1.0 x 105 to 1 copy/reaction were run in the qPCR assays. The concentrations of
153
the primers and probes in the qPCR systems remained unchanged from the original papers and are
154
detailed in table S1 (18,19,21). Blank filtrations (sterile Elga Purelab Ultra water) were conducted in
155
parallel whilst inhibition of DNA during qPCR assay was tested using a TaqMan exogenous internal
156
positive control (Applied Biosystems, USA).
157
158
2.3 Human enteric viruses
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Three pathogenic human RNA viruses were selected for assay; norovirus (genogroups I and II),
160
human enterovirus and human rotavirus (group A); the assay oligonucleotides are shown in table S1.
161
The viral concentration method of Katayama et al. (39) was used. Briefly, 1 L groundwater samples
162
were amended with MgCl2, achieving a final concentration of 25 mM, and spiked with 100 plaque
163
forming units (PFU) of process control phage MS2. Samples were acidified to pH 3.75 using 10 %
164
(v/v) C2H4O2 and filtered through a 47 mm 0.45 µm HA negatively charged membrane (Millipore,
165
Ireland). 100 mL of 0.5 mM H2SO4 was passed through the membrane and 10 mL of 1 mM NaOH
166
poured onto membrane to elute virions. Eluate was recovered in a tube containing 0.1 mL of 50 mM
167
H2SO4 and 0.1 mL of 100X Tris-EDTA (TE) buffer and then further purified, concentrated, and
168
desalted using Centriprep YM-50 concentrator columns (Millipore, Ireland) according to the
169
manufacturer’s instructions.
170
RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen, USA) and eluted into 60 µL AVE
171
buffer. Reverse transcription was achieved using High Capacity cDNA Archive Kit (Applied
172
Biosystems, USA) and cDNA was stored at -20 °C until needed.
173
MS2 phage was a process control throughout the complete viral assay (allowing the filtration, elution,
174
reverse transcription and qPCR assay to be monitored) and the qPCR system was based on the MS2
175
phage lysis protein from O’Connell et al. (40).
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176
All assays containing 3 µL cDNA (and dilutions thereof), 25 mL equivalent volume of groundwater
177
with a sensitivity of 40 virions per litre, were run on an ABI 7500 Real Time PCR System (Applied
178
Biosystems) with ABI Taqman Universal PCR MasterMix throughout. Positive stool samples for the
179
four viral types were obtained from Biolytix (Basel, Switzerland), Medica Medical laboratories
180
(Zürich, Switzerland), and St. Gallen Cantonal Laboratory (St. Gallen, Switzerland). Standard curves
181
from 1.0 x 105 to 1 copy/reaction were included in the qPCR assays. The concentrations of the primers
182
and probes in the qPCR systems remained unchanged from the original papers (41-43) and blank
183
filtrations (sterile Elga Ultralab Pure water), positive and negative controls were conducted in parallel.
184
When positive results were returned (in any number of wells), cDNA aliquots were sent for
185
subsequent assay in an external lab (Biosmart, Bern, Switzerland) using methods employed previously
186
using water samples from these sites (4,44). Furthermore, in qPCR assays where either two or three
187
wells were positive, these samples were retested to see if result was reproducible.
188
189
2.4 Other parameters
190
In conjunction with both general and source specific microbial markers, soluble reactive phosphorus
191
(SRP; PO4) and boron (B) concentrations were assayed in order to determine if the parameters were
192
suitable for source identification in groundwater. Strong correlation between SRP and B may indicate
193
discharges from wastewater treatment works [WwTW(45)]. Both B and SRP were measured in
194
triplicate using photometry (AQUANAL®-professional Spectro 1000, methods 85 and 320
195
respectively).
196
Auxiliary hydro-chemical and microbiological data were available from the sampling program of the
197
NAQUA National Groundwater Monitoring. These data were site-specifically processed in
198
conjunction with information on aquifer characteristics (including vulnerability) and catchment land
199
use. Both land use and chemical data were grouped and classified according to their indication for
200
human or ruminant sources and compared to the host-specific MST results obtained from the present
201
study. Groundwater level and spring discharge data recorded in the framework of NAQUA were used 8
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202
to identify relationships between high water stages and microbiological parameters. Daily means at
203
sampling dates were therefore normalized to long-term mean values obtained from continuous
204
measurement.
205
206
2.6 Statistical methods
207
All data were non-normally distributed (log transformation did not produce normally distributed data),
208
and therefore non-parametric statistical methods were used throughout. To allow comparison of results
209
with previous/future studies, both mean and median statistics are provided. Furthermore, because the
210
water was of “good” quality and as such the parameters were infrequently detected at certain
211
monitoring stations, the median across all samples in conjunction with positive sample only medians
212
are reported. Normality tests, descriptive statistics and Spearman rank correlations were computed
213
using Microsoft Excel 2007 for Windows. The interpretation of Spearman Rank ρ was as follows: 0.9
214
to 1 - very strong, 0.7 to 0.89 – strong, 0.5 to 0.69 – moderate, 0.3 to 0.49 - low, 0.16 to 0.29 – weak, and
215
40
>300
and/or
40
>1.0
>5
> 0.1 (> 1 substance)
503
504 505
506
* NAQUA National groundwater monitoring results from former microbiological pilot studies (6, 7) and regular chemical sampling 2012 and 2013 (maximum concentration, sampling frequency 2-4 per year) ** Group 1: Benzotriazole and pharmaceutical residues (Sulfamethoxazole, Carbamazepine, Diatrizoic acid) 25
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