AQUAMANCHE. Development of methods for discriminating sources of faecal pollution of waters: Application to the catchment Havre de l Ay

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1 Unité de Recherche Risques Microbiens Université de Caen Basse-Normandie QUMNCHE France (Channel) England INTERREG IV Program (FEDER) Development of methods for discriminating sources of faecal pollution of waters: pplication to the catchment Havre de l y

2 E 4655 Unité de Recherche Risques Microbiens (U2RM) (Pr.. Rincé) Enterococci Study of adaptation to changes in their environment Study of mechanisms of virulence Study of mechanisms of antibiotic resistance Typing of faecal pollution sources (Microbial Source Tracking)

3 E 4655 Unité de Recherche Risques Microbiens (U2RM) (Pr.. Rincé) Enterococci Study of adaptation to changes in their environment Study of mechanisms of virulence Study of mechanisms of antibiotic resistance Typing of faecal pollution sources (Microbial Source Tracking) - Faecal indicators of contamination (an approach that limits possible bias related to variations in the concentrations of MST markers and indicators of contamination). - Have the advantage of being easily enriched using a selective medium in order to lower the detection limit.

4 Contribution of the University of Caen to the quamanche project ction 2.1. Development and evaluation of novel MST methods The main objective was to develop a new approach to discriminate between Ovine and Bovine origins. Bovin Ovine e ction 2.2. Targeted river catchment and coastal case studies (site: y estuary). Monthly sampling over 2 years at 10 geographical points (240 samples) nalysis of a set of parameters for each sample - Enumeration of faecal indicators - Physico-chemical parameters - Typing of pollution source MST markerss ctions Data analyses, public participation

5 ction 2.1. Development and evaluation of novel MST methods. Work done as part of a previous study Typing of Enterococcus faecalis strains in order to discriminate the origins of faecal pollution in waters Study supported by: - Le Conseil Régional de Basse-Normandie - L'gence de l'eau Seine Normandie (ESN DEPEE/DLM-EU et SNTE) Samples Samples from STP (human origin) Faeces from cows Faeces from sheep Isolation of Enterococcus faecalis strains

6 Typing of Enterococcus faecalis strains in order to discriminate the origins of faecal pollution in waters Study supported by: - Le Conseil Régional de Basse-Normandie - L'gence de l'eau Seine Normandie (ESN DEPEE/DLM-EU et SNTE) Samples Samples from STP (human origin) Faeces from cows Faeces from sheep Isolation of Enterococcus faecalis strains sal gki mplification and sequencing of DN from 109 strains 11 genes: gdh, gyd, psts, gki, aroe, xpt, yqil, sal, efa, ace, ddle xpt Chromosome d E.faecalis ace ddle gdh nalyses of DN sequences to obtain a marker allowing discrimination of Ovine vs. Bovine. efa gyd psts aroe yqil

7 Enterococcus faecalis markers T C G Origin 1 G C T Origin 2 - Hu EF - Hu+Bo EF Faeces Cow (n=15) Sheep (n=15) Human (STP: n=10) Deliberately Contaminated Water with faeces (DCW) (E. coli = 500 (+/-100) CFU/100ml) Cow (n=10) Sheep (n=10) Human (n=10) Enterococci enrichment (Slanetz and Bartley) DN extraction qpcr Filtration - Enrichment (Slanetz and Bartley), DN extraction qpcr Marker Sensitivity Specificity Faeces DCW Faeces DCW Hu+Bo EF 96.0 % (n=25) 100 % (20) 100 % (15) 100 % (10) Hu EF 100 % (10) 100 % (10) 96.7 % (30) 100 % (20)

Bacteroidales markers Bacteroidales marker EF447F /EF990R from Elk Elk Bac

2005 We showed that this marker produced a signal from DN extracted from

Positive results with sheep French faeces samples Cow feces DCW Hu Elk Bac DCW

8 Bacteroidales markers Bacteroidales marker EF447F /EF990R from Elk Elk Bac marker (Dick et al. 2005, EM, 71, ) US faeces samples Dick et al We showed that this marker produced a signal from DN extracted from faeces of sheep, but not from faeces of cattle. Positive results with sheep French faeces samples Cow feces DCW Hu Elk Bac DCW Bo sheep faeces Ct > Ct neg > Ct neg DCW Ov Therefore, Elk Bac marker allows us to discriminate pollution from sheep

9 ction 2.2. Targeted river catchment and coastal case studies (site: Havre de l Y). Human Sheep St Germain sur y Plage F B C St Germain sur y G H l y I le Dun Créances J Lessay LESSY Cow Shellfish production E D Pirou plage

10 Monthly sampling for 2 years: July 2009-June sites tested every month (240 samples) nalysis of a set of parameters for each sample Water sample Q U N T I T T I V E Enumeration of faecal indicators -E. coli : MPN -Enterococci: MPN Discrimination of the origin of contamination using Bacteroidales markers -Extraction of DN from filters -qpcr Bacteroidales markers: marker) - ll Bac - HF183 (human marker) - Rum2Bac (ruminant - Elk Bac Filtrations of 200 ml (0,45µm) - Enrichment of enterococci (Slanetz and Bartley) - DN extraction - qpcr E. faecalis markers: - Hu+Bo EF - Hu EF Determination of physicochemical parameters -Temperature -ph -Salt -Disolved oxygen -Turbidity Discrimination of the origin of contamination using E. faecalis markers N O N Q U N T I T T I V E Diagram of the protocol used for the study of the site: Havre de l Y

11 F B G H le Dun I l y J C Points F, G, H and I pollution were systematically high (mean >4000 E. coli/100ml) Pollution relatively high at point J (mean = 1400 E. coli /100ml) E D, B and E: very high variation C et D : good quality water

12 F B G le Dun I H l y J C t points, B, but also C, D and F, pollution is higher during high tidal ranges. E D For some points, including G, pollution was higher in summer than in winter

F B G le Dun I H l y J Sheep C Frequency (%) 100 80 60 Origin of water pollution Frequency (%) of samples for which [E.

13 F B G le Dun I H l y J Sheep C Frequency (%) Origin of water pollution Frequency (%) of samples for which [E. coli >500 UFC/100ml] Frequency (%) of sample for which typing predicted a n origin: Série6 Not determined animal, Série5 usually sheep Human 40 animal, Série4 usually sheep and caw animal, Série3 usually caw Cow E D B2 C3 D4 E5 F6 G7 H8 I9 10 J mixed Série2 human Série1 Sampling site

14 Conclusions: During high tides (range> 90), pollution in the estuary and outside seems to be mostly related to sheep. However, at points C and D, seawater is still mostly good quality and occasionally medium quality. The markers used in this study are complementary to discriminate between three sources of pollution: Human, Sheep and Cow. We showed that identification of pollution from Sheep is possible. However, the detection threshold using the Elk Bac marker is quite high compared with Rum2Bac and HF183 markers.

15 Thanks:

16 cknowledgments: gence Régionale de Santé (St Lô) Syndicat Mixte Espace Littoraux de la Manche Determination of sampling sites Relations with farmers Farmers Faeces sampling SUR Sampling from STPs ll the quamanche partners (University of Brighton, Environment gency, Ifremer) ll staff at the laboratory S. La Carbona, N. Sauvageot,. Benachour, N. Verneuil, J.C. Giard, I. Rincé, E. Marchand,. Blandin, S. Mascré, Y. uffray

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