1. Maija Jedynak1,2, Jonathan Choi1,2,Sophia Tracy1,2, Kelly McCullum1,2, Paul Tuffy2, and Paul Melchior1
1North Hennepin Community College, Brooklyn Park, Minnesota , USA
2Galway Mayo Institute of Technology, Galway, Republic of Ireland
RT-qPCR Detection and Quantification of Waterborne Pathogens in
Celtic-Christian Holy Wells of Western Ireland
Irish Holy Wells – An Introduction
• Holy wells are springs, pools or seeps venerated as sacred by pre-Christian
Celtic (pagan) and Christian cultures in Ireland beginning in the Iron Age;
• Approximately 600 holy wells, typically named for Catholic saints or parishes,
exist in Ireland. Many of remain in use;
• Important Irish cultural heritage sites. Wells are often in pastures, church
yards, or on hillsides;
• Because holy wells have purported healing qualities, they are frequently
visited by parishioners, pilgrims, and tourists;
• Visitors often drink, collect, or wash with the water while at holy wells.
Pathogen Surveillance Project
• Domestic livestock and poultry are the primary sources of food and
waterborne microbial infections in northern Europe. Other sources
include contaminated surface waters;
• Many holy wells are located on or near agricultural land grazed by sheep
and cattle. Others are closely associated with surface waters including
streams, rivers or the Atlantic Ocean;
• We hypothesized that holy wells near agricultural sites or surface waters
would frequently harbor the fecal indicator bacteria Enterococcus spp. and
Escherichia coli.
• Thirty-three holy wells in six counties in western Ireland were sampled
and analyzed for the presence and population density fecal indicator
bacteria and pathogens (Figure 1, Table 1).
Acknowledgements
• We are grateful for technical and logistical assistance at GMIT by Drs. Rick Officer and Seamus Lennon, and Ms. Angela Jacobsen
(Minnesota Dept. of Health). We thank the Minnesota Department of Health, Division of Molecular Epidemiology for providing
reference cultures. Funding for this project was provided by a grant from the NHCC Foundation, as well as the CCURI grant
Results
Our research team of four NHCC students spent fall semester 2013 at
the Galway-Mayo Institute of Technology, Galway, Ireland
St. Joseph’s Well: Leenaun, Co. Mayo, Ireland (rural)
Tubrid Well, Tubrid, Co. Cork, (rural)
Figure 1
Sampled Holy Well Locations, Republic of Ireland
Scale: 1 cm equals approx. 15 km
Tober Murray, Creegh, Co Clare
References
• Fukushima, H., Tsunomori, Y., and R. Seki. (2003). Duplex real-time SYBR green PCR assays for detection of 17 species of food-or
waterborne pathogens in stools. Journal of Clinical Microbiology 41.11, 5134-5146.
• Healy, E. 2001. Ireland’s Holy Wells. Wolfhound Press, Dublin, Ireland.
• Rinttilä, T., Lyra, A., Krogius-Kurikka, L., and Palva, A. (2011). Real-time PCR analysis of enteric pathogens from fecal samples of
irritable bowel syndrome subjects. Gut Pathogens, 3(1), 6-6.
Indicator Organism: Target Gene
Amplicon
( bp)
Enterococcus spp. 16S rDNA
Escherichia coli K12 β-D Glucuronidase (uidA) 61
Bactroides spp. 16S rDNA
Pathogens Target Gene
Amplicon
(bp)
Bacillus cereus group Flagella motor protein (motB) 285
Campylobacter jejuni Hipuricase (hipO) 244
Clostridium difficile Cd Toxin A (cdtA) 158
Cd Toxin B (cdtB) 101
Enterotoxigenic E. coli Heat-labile enterotoxin (LT) 190
Escherichia coli 0157:H7 Shiga toxin 1 (stx1) 76
Shiga toxin 2 (stx2) 78
Legionella pneumophila
Macrophage infectivity
potentiator (mips) 66
Listeria monocytogenes Listeriolysin O (hylA) 77
Salmonella enteritidis/enterica
Invasion-associated protein
(invA) 88
Shigella sp.
Invasion plasmid antigen H
(ipaH) 7.8 62
Vibrio parahemolyticus
Thermostabile direct
hemolysin (tdh) 251
Vibrio vulnificus Cytotoxin hemolysin (cth) 383
Yersinia enterocolitica Attachment-invasion locus (ail) 90
Giardia lamblia/muris β-Giardin (bg) 74
Cryposporidium parvum Oocyst wall protein (COWP) 150
Well Name
Well
Number Enterococcus spp.
Diseart/Tobar Nano 1
St. Brendan's 2
Our Lady 7
St. Senan's 13
Tobermurray 15
St. Michael 17
St. Brigid's 23
St. Joseph's 24
Ballyvaghn 31
St. Joseph's 32 +
Holy Mother of God 33 +
South (Pasture w/ Mary) 34 +
North (Next to cemetery) 35 +
Tober Macdaugh 36
TBD 37 +
Lady's Well 39
St Brendan's 40
St. Ciaran's 41 +
Blessed Well 42
Tobernault/Calvary 43
St. Patrick's 44
Tober na Molt (Wethers) 45 +
Tobar na Croiche Naofa 46
Tobarin 47
Tobar 48
Tobar Feasa? St. Michaels 49
St. Crohan 50
Shronebeg 51 +
Tubrid Millstreet 52
St John of Mushera 53
St. Augustine 54
St. John's Well A 27A
St. John's Well B 27B
Methods
Sampling
• Wells with evidence of significant visitor use were
chosen for sampling.
• Well water was collected with sterile syringes and
collection bottles and stored at 4°C for transport.
DNA Extraction
• Samples were filtered (0.45 μm) to collect microbes.
DNA extraction was performed using MoBio Labs
Power Water DNA Extraction kit ™. Processed DNA was
stored at -20°C until analysis.
Pathogen Detection and Quantification
• The presence of bacterial genomic DNA in well extracts
was confirmed by end-point 16S rDNA PCR and gel
electrophoresis.
• TaqMan qPCR amplification of species-specific target
genes (Table 1) was performed on a Thermo Scientific
PikoReal quantitative thermal cycler.
• Quantification of pathogen population density was
determined by comparison with control organism
standard curve data.
Table 1: Surveillance Pathogens and qPCR Gene Targets Table 2: qPCR Amplification Results
Figure 2: 16S rDNA
Amplification from Holy Well-
Derived Bacterial DNA (1.5%
Agarose TAE Gel)
Figure 3: qPCR Amplification Profile of 10X Serially
Diluted Enterococcus DNA
Figure 4: Standard Curve of 10X Serially Diluted
Enterococcus DNA
• End-point PCR and gel electrophoresis were used to
confirm bacterial DNA presence in each well water
sample (universal bacterial rDNA primers; ~1400 bp
amplicon) (Fig. 2).
• Ten-fold serial dilutions of Enterococcus faecalis DNA
were amplified using TaqMan qPCR to develop a
standard curve (e.g. Fig. 4) to correlate viable cell
density in well samples with Cq.
• Eight wells tested positive for the presence of
Enterococcus faecalis 16S rDNA gene using qPCR.