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

ResultsOur 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 GeneAmplicon

( bp)Enterococcus spp. 16S rDNA Escherichia coli K12 β-D Glucuronidase (uidA) 61Bactroides spp. 16S rDNA  

Pathogens Target GeneAmplicon

(bp)

Bacillus cereus group Flagella motor protein (motB) 285Campylobacter jejuni Hipuricase (hipO) 244Clostridium difficile Cd Toxin A (cdtA) 158 Cd Toxin B (cdtB) 101

Enterotoxigenic E. coli Heat-labile enterotoxin (LT) 190Escherichia coli 0157:H7 Shiga toxin 1 (stx1) 76 Shiga toxin 2 (stx2) 78

Legionella pneumophilaMacrophage infectivity potentiator (mips) 66

Listeria monocytogenes Listeriolysin O (hylA) 77

Salmonella enteritidis/entericaInvasion-associated protein (invA) 88

Shigella sp.Invasion plasmid antigen H (ipaH) 7.8 62

Vibrio parahemolyticusThermostabile 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 NameWell

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

MethodsSampling• 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.