faecal pollution of waters: Application to the catchment Havre de lAy - - PowerPoint PPT Presentation

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Aug 18, 2022 1.32k likes •1.54k views

Unit de Recherche Risques Microbiens Universit 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

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AQUAMANCHE

Unité de Recherche Risques Microbiens Université de Caen Basse-Normandie

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

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EA 4655 Unité de Recherche Risques Microbiens (U2RM) (Pr. A. Rincé) 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) Enterococci

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EA 4655 Unité de Recherche Risques Microbiens (U2RM) (Pr. A. Rincé) 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) Enterococci

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.

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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.

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

Ovine Bovin e

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Action 2.1. Development and evaluation of novel MST methods.

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)

Work done as part of a previous study

Faeces from cows Faeces from sheep Samples from STP (human origin)

Samples Isolation of Enterococcus faecalis strains

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Amplification and sequencing of DNA from 109 strains 11 genes: gdh, gyd, pstS, gki, aroE, xpt, yqiL, salA, efaA, ace, ddlE Analyses of DNA sequences to obtain a marker allowing discrimination of Ovine vs. Bovine.

gdh gyd pstS gki aroE xpt yqiL salA ace efaA ddlE Chromosome d’E.faecalis

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)

Faeces from cows Faeces from sheep Samples from STP (human origin)

Samples Isolation of Enterococcus faecalis strains

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Hu EF
Hu+Bo EF

Origin 1

C T C A G A

Origin 2

T G 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)

Faeces Cow (n=15) Sheep (n=15) Human (STP: n=10)

Enterococci enrichment (Slanetz and Bartley) DNA extraction qPCR

Deliberately Contaminated Water with faeces (DCW) (E. coli = 500 (+/-100) CFU/100ml) Cow (n=10) Sheep (n=10) Human (n=10)

Filtration - Enrichment (Slanetz and Bartley), DNA extraction qPCR

Enterococcus faecalis markers

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Dick et al. 2005

We showed that this marker produced a signal from DNA extracted from faeces of sheep, but not from faeces of cattle. Bacteroidales marker EF447F /EF990R from Elk – Elk Bac marker (Dick et al. 2005, AEM, 71, 3179-3183) Therefore, Elk Bac marker allows us to discriminate pollution from sheep Ct DCW Hu > Ct neg DCW Bo > Ct neg DCW Ov 26 - 34 Elk Bac

Bacteroidales markers

USA faeces samples

Cow feces sheep faeces

French faeces samples

Positive results with sheep

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LESSAY Action 2.2. Targeted river catchment and coastal case studies (site: Havre de l’AY).

Shellfish production

Pirou plage Créances Lessay St Germain sur Ay St Germain sur Ay Plage

Sheep Human Cow

l’Ay le Dun A F B I H C D E J G

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Diagram of the protocol used for the study of the site: Havre de l’AY

Water sample Enumeration of faecal indicators

E. coli : MPN
Enterococci: MPN

Determination of physicochemical parameters

Temperature
pH
Salt
Disolved oxygen
Turbidity

Filtrations of 200 ml

(0,45µm)

Extraction of DNA from filters
qPCR Bacteroidales markers:
All Bac
HF183 (human marker)
Rum2Bac (ruminant

marker)

Elk Bac

Discrimination of the origin of contamination using Bacteroidales markers

Enrichment of enterococci

(Slanetz and Bartley)

DNA extraction
qPCR E. faecalis markers:
Hu+Bo EF
Hu EF

Discrimination of the origin of contamination using E. faecalis markers

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

Q U A N T I T A T I V E N O N Q U A N T I T A T I V E

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l’Ay le Dun

A F B I H C D E J G

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 C et D : good quality water

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l’Ay le Dun

A F B I H C D E J G

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

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l’Ay le Dun

F A B C D E H J

Sheep Human Cow

I

G

20 40 60 80 100 1 2 3 4 5 6 7 8 9 10 Série6 Série5 Série4 Série3 Série2 Série1 Frequency (%) of samples for which [E. coli >500 UFC/100ml]

Not determined animal, usually sheep animal, usually sheep and caw animal, usually caw mixed human

Frequency (%) of sample for which typingpredicted a n origin: Sampling site A B C D E F G H I J Frequency (%)

Origin of water pollution

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Conclusions:

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. 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

ccasionally medium quality.

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Thanks:

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Acknowledgments:

All the Aquamanche partners (University of Brighton, Environment Agency, Ifremer) Agence Régionale de Santé (St Lô) Determination of sampling sites Syndicat Mixte Espace Littoraux de la Manche Relations with farmers Farmers Faeces sampling SAUR Sampling from STPs 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