Unité de Recherche Risques Microbiens Université de Caen Basse-Normandie AQUAMANCHE France (Channel) – England INTERREG IVA Program (FEDER) Development of methods for discriminating sources of faecal pollution of waters: Application to the catchment Havre de l’Ay
EA 4655 Unité de Recherche Risques Microbiens (U2RM) (Pr. A. Rincé) Study of adaptation to changes in their environment Study of mechanisms of virulence Enterococci Study of mechanisms of antibiotic resistance Typing of faecal pollution sources (Microbial Source Tracking)
EA 4655 Unité de Recherche Risques Microbiens (U2RM) (Pr. A. Rincé) Study of adaptation to changes in their environment Study of mechanisms of virulence Enterococci 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.
Contribution of the University of Caen to the Aquamanche project Action 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 Action 2.2 . Targeted river catchment and coastal case studies (site: Ay estuary). Monthly sampling over 2 years at 10 geographical points (240 samples) Analysis of a set of parameters for each sample - Enumeration of faecal indicators - Physico-chemical parameters - Typing of pollution source – MST markerss Actions 2.3. - 2.4 . Data analyses, public participation
Action 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'Agence de l'Eau Seine Normandie (AESN DEPEE/DLM-EAU et SANTE) Samples Samples from STP Faeces from cows Faeces from sheep (human origin) Isolation of Enterococcus faecalis strains
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'Agence de l'Eau Seine Normandie (AESN DEPEE/DLM-EAU et SANTE) Samples Samples from STP Faeces from sheep Faeces from cows (human origin) Isolation of Enterococcus faecalis strains salA gki Chromosome ddlE d’ E.faecalis Amplification and sequencing of DNA from 109 strains xpt 11 genes: gdh , gyd , pstS , gki , aroE , xpt , yqiL, salA, efaA, ace, ddlE gdh ace efaA yqiL gyd aroE pstS Analyses of DNA sequences to obtain a marker allowing discrimination of Ovine vs. Bovine.
Enterococcus faecalis markers A C Origin 1 T G G T Origin 2 C A - Hu EF - Hu+Bo EF Faeces Deliberately Contaminated Water with faeces (DCW) ( E. coli = 500 (+/-100) CFU/100ml) Cow (n=15) Sheep (n=15) Cow (n=10) Human (STP: n=10) Sheep (n=10) Human (n=10) Filtration - Enrichment (Slanetz and Bartley), Enterococci enrichment (Slanetz and Bartley) DNA extraction DNA extraction qPCR qPCR Marker Sensitivity Specificity Faeces DCW Faeces DCW 96.0 % 100 % 100 % 100 % Hu+Bo EF (n=25) (20) (15) (10) 100 % 100 % 96.7 % 100 % Hu EF (10) (10) (30) (20)
Bacteroidales markers Bacteroidales marker EF447F /EF990R from Elk – Elk Bac marker (Dick et al. 2005, AEM, 71, 3179-3183) USA faeces samples Dick et al. 2005 We showed that this marker produced a signal from DNA extracted from Positive results faeces of sheep, but not from faeces of cattle. with sheep French faeces samples Cow feces sheep faeces Ct DCW Hu > Ct neg Elk Bac DCW Bo > Ct neg DCW Ov 26 - 34 Therefore, Elk Bac marker allows us to discriminate pollution from sheep
Action 2.2 . Targeted river catchment and coastal case studies (site: Havre de l’AY). St Germain sur Ay Plage St Germain sur Ay F A G H l’Ay J I B LESSAY le Dun Lessay Human C Cr é ances Sheep Cow E D Pirou plage Shellfish production
Monthly sampling for 2 years: July 2009-June 2011 10 sites tested every month (240 samples) Analysis of a set of parameters for each sample Water sample Enumeration of faecal Determination of indicators physicochemical parameters Filtrations of 200 ml -Temperature - E. coli : MPN -pH (0,45µm) -Enterococci: MPN -Salt -Disolved oxygen -Turbidity N O Discrimination of the origin of Discrimination of the origin of N Q contamination using contamination using E. faecalis U Bacteroidales markers markers Q A U N A -Extraction of DNA from filters - Enrichment of enterococci T N (Slanetz and Bartley) I T -qPCR Bacteroidale s markers: T I - DNA extraction A T - All Bac T A - HF183 (human marker) - qPCR E. faecalis markers : I T - Rum2Bac (ruminant V I - Hu+Bo EF marker) E V - Hu EF - Elk Bac E Diagram of the protocol used for the study of the site: Havre de l’AY
F A G l’Ay H J I B le Dun 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) A, B and E: very high variation E C et D : good quality water D
F G A l’Ay H J I B le Dun C At points A, B, but also C, D and F, pollution is higher during high tidal ranges. For some points, including G, pollution was higher in summer than in winter E D
F G A H l’Ay J I B le Dun Origin of water pollution Frequency (%) Frequency (%) of samples for C 100 which [ E. coli >500 UFC/100ml] Frequency (%) of sample for which 80 typingpredicted a n origin: Série6 Not determined Sheep 60 animal, usually sheep Série5 animal, usually sheep and caw Série4 40 Human animal, usually caw Série3 mixed Série2 20 E human Série1 Cow 0 D 1 2 3 4 5 6 7 8 9 10 A B C D E F G H I J Sampling site
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.
Thanks:
Acknowledgments: Agence Régionale de Santé (St Lô) Determination of sampling sites Syndicat Mixte Espace Littoraux de Relations with farmers la Manche Farmers Faeces sampling SAUR Sampling from STPs All the Aquamanche partners (University of Brighton, Environment Agency, Ifremer) All staff at the laboratory S. La Carbona, N. Sauvageot, A. Benachour, N. Verneuil, J.C. Giard, I. Rincé, E. Marchand, A. Blandin, S. Mascré, Y. Auffray
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