<table><caption>**Table 1** Prevalence of *stx1*, *stx2* and *eae* genes in enrichment broths</caption><thead><tr><th>Sample (n)</th><th>stx1* n (%)</th><th>stx2† n (%)</th><th>stx1 and stx2 n (%)</th><th>Total stx1 and/or stx2 n (%)</th><th>eae n (%)</th></tr></thead><tbody><tr><td>Cockles (24)</td><td>3 (12.5)</td><td>3 (12.5)</td><td>6 (25.0)</td><td>12 (50.0)</td><td>21 (87.5)</td></tr><tr><td>Mussels (52)</td><td>12 (23.1)</td><td>8 (15.4)</td><td>1 (1.9)</td><td>21 (40.4)</td><td>43 (82.7)</td></tr><tr><td>Oysters (50)</td><td>7 (14.0)</td><td>2 (4.0)</td><td>2 (4.0)</td><td>11 (22.0)‡</td><td>28 (56.0)§</td></tr><tr><td>Total Shellfish (126)</td><td>22 (17.5)</td><td>13 (10.3)</td><td>9 (7.1)</td><td>44 (34.9)</td><td>92 (73.0)</td></tr><tr><td>Waters (117)</td><td>10 (8.5)</td><td>21 (17.9)</td><td>76 (65.0)</td><td>107 (91.4)¶</td><td>116 (99.1)**</td></tr><tr><td>Sediment (39)</td><td>3 (7.7)</td><td>6 (13.4)</td><td>2 (5.1)</td><td>11 (28.2)</td><td>19 (48.7)</td></tr></tbody></table>

*stx1* was found but not *stx2*.

†*stx2* was found but not *stx1*.

‡*P* value (Chi-squared test) between oysters and other shellfish = 0.0136.

§*P* value (Chi-squared test) between oysters and other shellfish = 3.23 × 10⁻⁵.

¶*P* value (Chi-squared test) between waters and other samples = 2.33 × 10⁻²².

***P* value (Chi-squared test) between waters and other samples = 4.87 × 10⁻¹¹.

<table><caption>**Table 2** Prevalence of strains of the five highly pathogenic serogroups in the BPW broths and number of isolated strains belonging to the STEC or EPEC pathotype</caption><thead><tr><th>Sample (n)</th><th>stx<sup>+</sup> and eae<sup>+</sup></th><th>O157<sup>+</sup> (%)*</th><th>O26<sup>+</sup> (%)*</th><th>O103<sup>+</sup> (%)*</th><th>O111<sup>+</sup> (%)*</th><th>O145<sup>+</sup> (%)*</th><th>stx<sup>+</sup>, eae<sup>+</sup> and serotype<sup>+</sup>† (%)<sup>‡</sup></th><th>Isolated strains</th></tr></thead><tbody><tr><td>Shellfish (126)</td><td>37</td><td>7 (18.9)</td><td>13 (35.1)</td><td>20 (54.1)</td><td>3 (8.1)</td><td>15 (40.5)</td><td>28 (22.2)</td><td>5</td></tr><tr><td>Waters (117)</td><td>107</td><td>65 (60.7)</td><td>67 (62.6)</td><td>93 (86.9)</td><td>30 (28.0)</td><td>83 (77.6)</td><td>106 (90.6)§</td><td>5</td></tr><tr><td>Sediment (39)</td><td>8</td><td>1 (12.5)</td><td>1 (12.5)</td><td>5 (65.5)</td><td>0 (0.0)</td><td>4 (50.0)</td><td>7 (17.9)</td><td>0</td></tr></tbody></table>

*% Calculated based on the number of samples stx⁺ and eae⁺.

†*stx⁺*, *eae⁺* and at least one of the major serotypes.

‡% Calculated based on total number of samples.

§*P* value (Chi-squared test) between waters and other samples = 1.62 × 10⁻³⁰.

EPEC isolation. We have to consider the possibility that the targeted *stx*, *eae* and serotypes markers could also be detected in the absence of culturable STECs. Indeed, their detection can be due to the presence free bacteriophages, dead or viable but nonculturable bacteria or other bacteria such as *Shigella* and *Citrobacter*, which are also known to carry these genes (Muniesa *et al.* 1999; Martinez-Castillo *et al.* 2013). A poor isolation rate of highly pathogenic STEC was also previously described for other matrices, such as cattle faeces (Bibbal *et al.* 2014), retail-minced beef (Auvray *et al.* 2007) and raw-milk cheeses (Madic *et al.* 2011) from which six (n = 150), zero (n = 164) and zero (n = 400) strains were isolated respectively. In the present study, EPEC were more frequently isolated than STEC. This may be the consequence of the loss of *stx* genes by STEC discharged in the environment. Indeed, previous studies have demonstrated the capacity of STEC to lose *stx* genes in bovine, avian human and environmental sources (Feng *et al.* 2001; Wetzel and LeJeune 2007).

A low detection rate of STEC in shellfish is in agreement with previous studies. Indeed, three studies previously described STEC belonging to the O157-serogroup in shellfish using an immunomagnetic method. One,

conducted in France on 150 oyster samples, led to the isolation of only one O157-*E. coli* strain (O157:H- *stx1*⁺, *stx2c*⁺, *eae*⁺) (Guyon *et al.* 2000), a second failed to isolate O157:H7 strains from 192 batches of different shellfish species in the UK (MacRae *et al.* 2005) and the third described the isolation of one EPEC strain (O157:H7) from 72 shellfish batches collected in France (Gourmelon *et al.* 2006). Other approaches focused on the isolation of *E. coli* strains followed by investigation for the presence of *stx* genes, which demonstrated low percentages of STEC among the isolates (6.2% in shellfish from Morocco (Bennani *et al.* 2011) and 4.1% in clams from India (Sanath Kumar *et al.* 2001)). Furthermore, five STEC strains not belonging to the five major serogroups have previously been isolated in France from shellfish by analyzing STEC by hybridization, following culture enrichment (Gourmelon *et al.* 2006).

These results suggest that STEC and EPEC are occasionally present in the environment and can contaminate shellfish. According to our results, the risk of human infection by highly pathogenic STEC resulting from the consumption of shellfish from the investigated areas seems to be limited as only one STEC strain was isolated from 126 batches taken from the area and shellfish which
<table><caption>**Table 3** Characterization of STEC and EPEC strains isolated from shellfish and water</caption><thead><tr><th>Strain</th><th>Sample type</th><th>Escherichia coli count</th><th>Serotype</th><th>stx (type)</th><th>eae</th></tr></thead><tbody><tr><td>10</td><td>Mussel</td><td>15 000*</td><td>O26:H11</td><td>+(stx1)</td><td>+</td></tr><tr><td>9</td><td>Cockle</td><td>12 000*</td><td>O26:H11</td><td>—</td><td>+</td></tr><tr><td>7</td><td>Mussel</td><td>15 000*</td><td>O26:H11</td><td>—</td><td>+</td></tr><tr><td>4</td><td>Water</td><td>3570†</td><td>O26:H11</td><td>—</td><td>+</td></tr><tr><td>5</td><td>Water</td><td>5200†</td><td>O26:H11</td><td>—</td><td>+</td></tr><tr><td>8</td><td>Water</td><td>16 620†</td><td>O26:H11</td><td>—</td><td>+</td></tr><tr><td>2</td><td>Cockle</td><td>22 000*</td><td>O145:H28</td><td>—</td><td>+</td></tr><tr><td>3</td><td>Mussel</td><td>11 000*</td><td>O145:H28</td><td>—</td><td>+</td></tr><tr><td>1</td><td>Water</td><td>357†</td><td>O103:H2</td><td>—</td><td>+</td></tr><tr><td>6</td><td>Water</td><td>16 620†</td><td>O103:H2</td><td>—</td><td>+</td></tr></tbody></table>

are from category B or C areas, which are depurated or which undergo protracted relaying prior to sale. As with the *E. coli* indicators, we may expect STEC to be eliminated during the depuration or relaying step. However, further assays are currently being undertaken in our laboratory to compare STEC behaviour with that of *E. coli* indicators in shellfish.

## Conclusion

In conclusion, this study shows that the ISO/TS-13136 protocol effectively detects STEC or EPEC strains belonging to the five major serotypes in shellfish and environmental samples. Even though *stx* and *eae* genes were often detected, the presence of STEC in such environments appears to be limited, as only one STEC and nine EPEC were isolated from water or shellfish samples. Furthermore, the human health risk associated with consuming the shellfish collected from these sites should be reduced as they are depurated or relayed prior to consumption.

## Materials and methods

### Origin and collection of samples

Three French shellfish sites from the English Channel coastal area were studied. One is located in the 'Cotes d'Armor' department (Brittany region) while others are in the department 'La Manche' (Lower-Normandy region). All three sites are characterized as receiving inputs from agricultural catchments with high stocking densities (cattle, sheep, pigs, or poultry) but are also impacted by microbial pollution of human origin. The bays are the focus of extensive shellfish gathering

activities. According to the European classification of shellfish harvesting areas (Anonymous 2004), these shellfish sites are classified as category B (*E. coli* <4600 CFU per 100 g total flesh for 90% of the samples) for mussels and oysters and as category C (*E. coli* <46 000 CFU per 100 g) for cockles. Shellfish from category B areas are only suitable for human consumption following depuration, relaying or cooking by an approved method, whereas shellfish from category C areas can only be consumed after relaying for at least 2 months or cooking by an approved method. For each site, mussels, oysters and cockles were collected monthly, from February 2013 to February 2014. A total of 126 batches of shellfish (52 batches for mussels (n ≈ 70), 50 for oysters (n ≈ 14) and 24 for cockles (n ≈ 80)) were analysed. In parallel, 117 water samples from nine different points located upstream of the shellfish areas and 39 samples of sediment were collected. Samples, which were transported in insulated cooler boxes, were analysed within 24 h. Before analysis, shellfish were scrubbed under running tap water to remove debris and algae.

### Escherichia coli enumeration

Escherichia coli enumeration was performed using the Most Probable Number method in MUG-microtitre plates according to the EN/ISO-9308-3 method (Anon 1998) and using an impedance method according to NF/V-08-106 for shellfish (Anon 2000).

### Escherichia coli enrichment

After opening, shellfish were crushed in a Warring blender for 60 s. Twenty-five grammes of crushed flesh and intravalvular liquid were introduced into 225 ml of buffered peptone water (BPW). For sediment, 10 g were introduced into the same quantity of BPW while for water samples, 1 l was filtered using 0.45 µm membranes and the filter placed in 225 ml of BPW. Incubation was performed at 37°C, for 24 h.

### Detection of *stx* and *eae* genes and markers associated with the five major serotypes

DNA was extracted from 1 ml of enrichment cultures in BPW using an automatic method (EZ1-biorobot, and DNA tissue card kit (Qiagen®, Cournaboeuf, France)). *stx* and *eae* genes were detected by real-time PCR, according to ISO/TS-13136. For samples positive for *stx* and *eae*, genetic markers associated with the major serogroups O157, O26, O103, O111, and O145 (rfbEO157, wzxO26, wzxO103, wbd1O111 and ihp1O145 genes respectively) were detected by real-time PCR, also according to ISO/

<footnote>*E. coli count per 100 g.</footnote>

<footnote>†E. coli count per 100 ml.</footnote>
TS-13136 and using primers and probes published previ-
ously (Nielsen and Andersen 2003; Perelle *et al.* 2007).

Isolation of Shiga-toxin-producing *Escherichia coli*

The isolation of STEC strains was performed only for the
positive samples for which the targeted combination of
genetic marker was detected, i.e. stx, *eae* and at least one
O-group marker. From the positive BPW broths, direct
streaking and streaking after immuno-magnetic separation
(IMS), were performed on the following media. The
O157 serogroups were searched by direct streaking onto
chromID™ O157:H7 + cefixime-tellurite (bioMérieux®,
Marcy l'Etoile, France) and streaking after the IMS onto
cefixime-tellurite-Sorbitol-MacConkey agar (Biokar,
Beauvais, France) and chromID™ O157 (bioMérieux).
Streakings for O26 serogroups were performed onto Sor-
bitol-MacConkey agar (SMAC) (bioMérieux) and after
onto SMAC and chromID™ O26 (bioMérieux) after the
IMS, while serogroups O103, O145 and O111 were
directly detected using Posse media (Posse *et al.* 2008) or
using SMAC and chromID™ after the IMS. All media
were incubated for 18–24 h at 37°C. Confirmation of the
clones belonging to a particular serotype and presence of
stx and/or *eae* genes were then tested by PCR on pre-
sumptive colonies as described in ISO/TS-13136. Charac-
terization of the *fliCH* alleles (*fliCH2*, *fliCH7*, *fliCH8*,
*fliCH11* and *fliCH28*) was investigated by PCR, as
described by Madic *et al.* (2011).

Acknowledgements

This work was funded by the European Regional Devel-
opment Fund Interreg IVA Programme as part of the col-
laborative project RiskManche. Charlotte Balière was
supported by a grant from Ifremer and Agence de l’Eau
Loire-Bretagne. We thank Clémence Balière and Quentin
Bruey, for their participation in the sampling and techni-
cal assistance and Huw Taylor for his critical review of
the manuscript.

Conflict of Interest

No conflict of interest declared.

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# Chapitre 3 : PREVALENCE ET CARACTERISATION DES E. COLI PRODUCTEURS DE SHIGA-TOXINES ET DES E. COLI ENTEROPATHOGENES DANS DES ZONES CONCHYLICOLES FRANCAISES ET LEUR BASSINS VERSANTS

## A. Contexte de la deuxième partie de l'étude

Ce troisième chapitre décrit la recherche et la caractérisation des STEC et EPEC isolés au cours de la la totalité de l'étude (Février 2013 à Janvier 2015).

Si la méthode ISO-TS-13136 apparaissait pertinente pour rechercher les sérotypes majeurs dans les échantillons environnementaux, elle ne permettait toutefois pas de mettre en évidence les souches STEC ou EPEC appartenant aux autres sérotypes. Aussi, deux approches complémentaires à la norme ISO et assez similaires sur le plan cultural et moléculaire ont été appliquées en parallèle au cours de cette étude.

La première approche était basée sur l'utilisation de l'enrichissement effectué dans le cadre de la méthode ISO. La même méthodologie de détection par PCR en temps réel a été utilisée pour la détection des gènes stx1, stx2 et eae dans les bouillons d'enrichissement au laboratoire à Plouzané (LSEM- Ifremer) et au laboratoire de l'Université de Caen (U2RM EA4655 Stress/Virulence).

Dans le cas de la détection des gènes de virulence (stx1 et/ou stx2 et eae) une procédure d'isolement de souches et de confirmation de la présence des gènes caractéristiques a été mise en place.

La deuxième approche s'est basée sur l'isolement direct de souches de E. coli. Pour les coquillages, une portion des chairs et liquides intervalvaires broyés a été ensemencée en masse dans un milieu sélectif (10 g ; Trypton-Bile-X-glucuronidase agar ; TBX). Pour les eaux, des volumes de 10, 50 et 100 ml ont été filtrés et les filtres ont été déposés directement sur un milieu sélectif solide (TBX). Le sédiment n'a pas été traité avec cette approche car trop peu contaminé en E. coli. L'ensemble des milieux a été incubé à 44°C pendant 24 heures. Pour les échantillons qui présentaient un résultat positif pour la détection des gènes stx et eae par PCR en temps réel dans les enrichissements, une procédure de screening et de confirmation de la présence des gènes caractéristiques a été mise en place sur les colonies suspectes obtenues sur ces milieux de culture.

Ces deux méthodes complémentaires ont été appliquées sur les échantillons récoltés mensuellement sur la période de 13 mois (de Février 2013 à Janvier 2014) et poursuivies sur une période de 11 mois supplémentaires (de Février 2014 à Janvier 2015) avec le même plan d'échantillonnage. L'utilisation de la méthode ISO a été arrêtée à partir de Mars 2014.
Ce chapitre du manuscrit de thèse porte donc sur la prévalence et la caractérisation des souches STEC et EPEC, isolées à partir d'un total de 505 échantillons environnementaux soit 238 lots de coquillages, 216 eaux douces, 39 sédiments (prélevés entre Février 2013 et Janvier 2015) et 12 eaux de mer (prélevées entre Février 2014 et Janvier 2015).

La caractérisation des souches a été réalisée en collaboration avec plusieurs équipes.

J'ai tout d'abord rassemblé toutes les souches STEC et EPEC isolées au cours de cette étude, ce qui a permis d'obtenir au total une collection de 174 souches conservées à – 80°C au Laboratoire de l'Ifremer de Plouzané.

Toutes les souches STEC et EPEC isolées au cours de l'étude ont ensuite été caractérisées au laboratoire en utilisant la méthode de génotypage par séquençage multilocus MLST (Multilocus sequence typing).

Cette méthode MLST est une méthode standardisée et universelle, basée sur l'examen de séquences nucléotidiques de plusieurs locus correspondant à des gènes ou des fragments de gène de ménage. Elle a permis d'évaluer la relation génétique entre les souches. Actuellement, il existe trois régimes MLST pour *E. coli* dans des bases de données publiques : EcMLST (http://www.shigatox.net), Institut Pasteur Base de données *E. coli* MLST (http://www.pasteur.fr/mlst) et Bases de données MLST (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). Nous avons fait le choix de travailler sur le séquençage de sept gènes de ménage (*adk*, *fumc*, *gyrB*, *icd*, *recA*, *purA* et *mdh*) avec la base de données « mLst.warwick.ac.uk », la plus riche en données (5 844 souches disponibles).

Au cours de séjours au Service de Microbiologie, CNR Associé *Escherichia coli*, AP-HP, Hôpital Robert Debré, Paris, France (Octobre, Novembre 2013, Mars 2014), j'ai caractérisé les 103 premières souches isolées par la méthode PFGE (*Pulsed-Field Gel Electrophoresis*) et initié la recherche de gènes de virulence (pour les 174 souches) en recherchant les gènes de virulence *ehxA* (codant pour l'entérohémolysine A) et *saa* (STEC *autoagglutining adhesin*). Pour les souches restantes (n= 71), les analyses PFGE ont été poursuivies par le Service de Microbiologie, CNR Associé *Escherichia coli*.

La méthode d'empreinte génétique PFGE est une technique de génotypage reposant sur le polymorphisme de macro-restriction de l'ADN total. Cette méthode a permis d'établir la relation génétique entre les souches étudiées et de mettre en évidence des liens génétiques avérés ou possibles entre les souches (Tenover *et al.*, 1995). La PFGE est considérée comme la méthode de référence pour le typage de souches de *E. coli*.

Les souches ont également été caractérisées en collaboration avec plusieurs équipes :

* le Département de Microbiologie et Parasitologie, Faculté vétérinaire, Université de Santiago de Compostela, Lugo, Espagne : pour le sérotypage des souches (Expédition des souches)
* le laboratoire de l'Université de Caen, U2RM EA4655, Antibio-Résistance, France : pour la caractérisation des phylogroupes et l'évaluation des profils d'antibio-résistance des souches (Expédition des souches)

* le Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Centre de Recherche d'Infectiologie Porcine et Avicole, Université de Montréal, Saint-Hyacinthe, QC, Canada : pour l'étude de la capacité des souches à former des biofilms appliquée à une collection de 13 EPEC et 9 STEC (Expédition des souches)

Au final, l'ensemble de ces travaux ont permis de :

* isoler un total de 28 souches STEC et 89 souches EPEC différentes à partir des 505 échantillons analysés

* montrer que 87,2 % des souches isolées présentaient un seul des gènes de virulence étudiés (dont *eae* 70,1 %, *stx2* 11,1 % ou *stx1* 7,7 %) et d'identifier huit profils de virulence différents combinant les gènes *eae*, *stx1*, *stx2*, *ehxA* et *saa*

* d'identifier 75 sérotypes différents dont les sérotypes O26 :H11, O145 :H28 et O103 :H2

* d'identifier 79 profils PFGE différents et 46 profils ST (Sequence type)

* de mettre en évidence la présence de souches similiaires dans plusieurs échantillons environnementaux et à des périodes différentes : notion de persistance des souches dans la zone côtière ou les bassins versants en amont

* de montrer que les souches de *E. coli* isolées étaient majoritairement classées au sein des phylogroupes B1 et B2

* de mettre en évidence la capacité forte à modérée de certaines souches à former des biofilms à 18° C plutôt qu'à 30° C

* de montrer que plus de 79 % des souches analysées étaient sensibles aux 16 antibiotiques testés

* de montrer que les souches STEC semblent plus isolées en été et les souches EPEC en été et en automne et qu'aucune influence de la température et de la pluviométrie sur l'isolement des souches n'a été mise en évidence (dans les conditions de cette étude)

En conclusion, ce travail constitue la première étude de la prévalence et de la diversité des STEC et des EPEC au niveau de l'environnement littoral, à notre connaissance. Cette étude a permis d'apporter des éléments de réponse concernant la circulation des souches STEC et EPEC au niveau des deux environnements littoraux sélectionnés. Ces résultats confirment que l'environnement peut être le réservoir de souches STEC et EPEC. L'évaluation de la circulation des souches dans l'environnement peut permettre de mieux appréhender l'émergence de nouvelles souches STEC pathogènes pour l'Homme.
Au regard de la diversité des souches isolées (87,2 % des souches isolées présentaient un seul gène de virulence parmi les gènes recherchés *stx1*, *stx2*, *eae*, *ehxA* et *saa*) et la réglementation appliquée aux sites conchylicoles de catégorie B (purification et reparcage) ou C (reparcage longue durée ou non commercialisation), le risque sanitaire semble limité dans les sites étudiés.

Néanmoins, plusieurs souches STEC et EPEC appartenant aux sérotypes majeurs impliqués dans des cas d'infections humaines ont été isolées dans des coquillages, des eaux de rivières et du sédiment dans cette étude. Ces observations nous ont suggéré d'approfondir plus largement l'analyse du pouvoir pathogène de ces souches en recherchant la présence d'autres éléments qui pourraient permettre d'estimer le niveau de virulence des souches comme la détection d'autres gènes de virulence.
B. Publication N°2

Prevalence and characterization of Shiga Toxin-producing and enteropathogenic *Escherichia coli* in shellfish-harvesting areas and their watersheds

Charlotte Balière¹, Alain Rincé², Jorge Blanco³, Ghizlane Dahbi³, Josée Harel⁴, Philippe Vogeleer⁴, Jean-Christophe Giard⁵, Patricia Mariani-Kurkdjian⁶,⁷,⁸, Michèle Gourmelon¹*

1 Laboratoire Santé Environnement et Microbiologie, Unité Santé, Génétique et Microbiologie des Mollusques, Département Ressources Biologiques et Environnement, Ifremer, Plouzané, France

2 U2RM EA4655 Stress/Virulence, Normandie-Université, University of Caen Normandy, Caen, France

3 Departamento de Microbioloxía e Parasitoloxía, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain

4 Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Centre de Recherche d'Infectiologie Porcine et Avicole, Université de Montréal, Saint-Hyacinthe, QC, Canada

5 U2RM EA4655 Antibio-Résistance, Normandie-Université, University of Caen Normandy, Caen, France

6 Service de Microbiologie, CNR Associé Escherichia coli, AP-HP, Hôpital Robert-Debré, Paris, France

7 Infection, Antimicrobials, Modelling, Evolution, UMR 1137, INSERM, Paris, France

8 Infection, Antimicrobials, Modelling, Evolution, UMR 1137, Université Paris Diderot – Sorbonne Paris Cité, Paris, France
# Prevalence and Characterization of Shiga Toxin-Producing and Enteropathogenic *Escherichia coli* in Shellfish-Harvesting Areas and Their Watersheds

Charlotte Balière<fnref n="1" />, Alain Rincé², Jorge Blanco³, Ghizlane Dahbi³, Josée Harel⁴,
Philippe Vogeleer⁴, Jean-Christophe Giard⁵, Patricia Mariani-Kurkdjian⁶,⁷,⁸ and Michèle Gourmelon<fnref n="1" />*

## OPEN ACCESS

**Edited by:**
Anton F. Post,
University of Rhode Island, USA

**Reviewed by:**
Erin Katherine Lipp,
University of Georgia, USA
Dale Leavitt,
Roger Williams University, USA

*Correspondence:
Michèle Gourmelon
michele.gourmelon@ifremer.fr

**Specialty section:**
This article was submitted to
Aquatic Microbiology,
a section of the journal
Frontiers in Microbiology

Received: 14 August 2015
Accepted: 16 November 2015
Published: 01 December 2015

**Citation:**
Balière C, Rincé A, Blanco J, Dahbi G, Harel J, Vogeleer P, Giard J-C, Mariani-Kurkdjian P and Gourmelon M (2015) Prevalence and Characterization of Shiga Toxin-Producing and Enteropathogenic Escherichia coli in Shellfish-Harvesting Areas and Their Watersheds. *Front. Microbiol.* 6:1356.
doi: 10.3389/fmicb.2015.01356

During a 2-years study, the presence of Shiga-toxin producing *Escherichia coli* (STEC) and enteropathogenic *E. coli* (EPEC) was investigated in shellfish (n = 238), seawater (n = 12), and surface sediment (n = 39) collected from three French coastal shellfish-harvesting areas and freshwaters (n = 216) in their watersheds. PCR detection of Shiga toxin- (stx1/stx2) and intimin- (eae) genes following enrichment from these samples revealed the presence of least one of the stx genes in 30.3% of shellfish batches, 85.9% of freshwater, 41.7% of seawater, and 28.2% of sediment samples, while the eae gene was observed in 74.8, 100, 100, and 43.6% of shellfish batches, freshwater, seawater, and sediment samples, respectively. Twenty-eight STEC and 89 EPEC strains were isolated and analyzed in order to determine their serotype, phylogroup, and genetic relatedness and to evaluate the presence of the saa and ehxA genes encoding the STEC autoagglutinating adhesin and the enterohemolysin A, respectively. Finally, the ability to form biofilms and antimicrobial susceptibility were investigated for a selection of strains. Eighteen serotypes were identified among the STEC isolates and 57 among the EPEC isolates. A high diversity was observed within these strains, as 79 different PFGE patterns and 48 distinguishable sequence types were identified. Strains were found to belong mainly to phylogroups B1 and B2 and virulence was observed to be low as more than 85% of the strains possessed only stx1, stx2, or eae genes. One STEC and several EPEC strains belonged to three of the five highly pathogenic serogroups (i.e., O26, O103, and O145). The subset of strains tested for their capacity to form biofilms was mainly strongly to moderately adherent and

<footnote n="1">¹ Laboratoire Santé Environnement et Microbiologie, Unité Santé, Génétique et Microbiologie des Mollusques, Département Ressources Biologiques et Environnement, Ifremer, Plouzané, France, ² U2RM EA4655 Stress/Virulence, Normandie-Université, University of Caen Normandy, Caen, France, ³ Departamento de Microbioloxía e Parasitoloxía, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain, ⁴ Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Centre de Recherche d'Infectiologie Porcine et Avicole, Université de Montréal, Saint-Hyacinthe, QC, Canada, ⁵ U2RM EA4655 Antibio-Résistance, Normandie-Université, University of Caen Normandy, Caen, France, ⁶ Service de Microbiologie, CNR Associé Escherichia coli, AP-HP, Hôpital Robert-Debré, Paris, France, ⁷ Infection, Antimicrobials, Modelling, Evolution, UMR 1137, INSERM, Paris, France, ⁸ Infection, Antimicrobials, Modelling, Evolution, UMR 1137, Université Paris Diderot – Sorbonne Paris Cité, Paris, France</footnote>
more strains formed a strong biofilm at 18 than at 30°C. Finally, more than 85% of analyzed strains were found to be sensitive to the 16 tested antibiotics. These data suggest the low risk of human infection by STEC if shellfish from these shellfish-harvesting areas were consumed.

Keywords: STEC, EPEC, shellfish, water, MLST, PFGE, biofilms

INTRODUCTION

The microbiological quality of coastal environments can be impacted by urban and agricultural fecal wastes from watersheds. Moreover, shellfish can accumulate and concentrate pathogenic micro-organisms, such as Salmonella, pathogenic Escherichia coli (E. coli) and noroviruses present in surrounding waters by their filter-feeding activities (Potasman et al., 2002). This can lead to closures or downgrading of shellfish-harvesting areas and to outbreaks of food poisoning through consumption of contaminated shellfish (Iwamoto et al., 2010).

Enumeration of E. coli, a fecal bacterial indicator, is the standard way to assess the level of fecal microorganisms in water and shellfish and indirectly, to estimate the associated potential risk to human health from all waterborne enteric pathogens (e.g., through classification of bathing areas and shellfish-harvesting areas; Anonymous, 2004). However, in addition to being a fecal indicator and a commensal bacterium, E. coli includes strains that can be pathogenic to humans. These can cause diarrhea and extra-intestinal diseases after acquiring virulence genes by genetic mobile elements such as bacteriophages, pathogenicity islands, and plasmids (Touchon et al., 2009). Pathogenic E. coli are distributed into diarrheagenic E. coli pathotypes including enterotoxigenic E. coli (ETEC), Shigella/enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC; for review, Croxen et al., 2013) and into extra-intestinal E. coli pathotypes (Russo and Johnson, 2000).

*Escherichia coli* occurrence in seafood is considered a sanitary case and may represent a risk to the consumers if related to diarrheagenic *E. coli* (for review, Costa, 2013).

The study presented here focuses on EPEC (one of the main causes of diarrhea in infants) and STEC (an emerging zoonotic pathogen).

Enteropathogenic *E. coli* is an important cause of infantile watery diarrhea, which is more frequently encountered in low-income countries than in the industrialized world (Nataro and Kaper, 1998). They are known to create distinctive lesions on the surface of intestinal epithelial cells, called attaching and effacing (A/E) lesions. This property is encoded by genes, including *eae*, grouped together in a pathogenicity island referred to the 'locus of enterocyte effacement' (LEE; Paton and Paton, 1998). EPEC is transmitted from host to host via the fecal-oral route through contaminated surfaces, waters and food and human carriers. Humans, including symptomatic and asymptomatic children and asymptomatic adults, are the most likely source (Levine and Edelman, 1984). Animals, such as cattle and wildlife species, have been found to be additional sources (Singh et al., 2015). Twelve O serogroups have been recognized as EPEC by the World Health

Organization: O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158 (WHO, 1987).

Shiga-toxin producing *Escherichia coli* are responsible for the mucoid-bloody diarrhea that can progress to hemolytic uremic-syndrome (HUS), especially in children. One of the most important pathogenicity factors produced by STEC strains is the Shiga toxin (Stx), encoded by a lambdaoid bacteriophage (O'Brien et al., 1984). Shiga toxins can be divided into two types, Stx1 (almost identical to Shiga toxin produced by *Shigella dysenteriae* type 1) and Stx2, encoded by *stx1* and *stx2* genes, respectively (Scheutz et al., 2012). In addition, the STEC strains are often able to produce the A/E lesions as a result of the presence of the LEE pathogenicity island, as in EPEC. This subset of STEC strains is also known as enterohemorrhagic *E. coli* (EHEC; McDaniel et al., 1995). Instead of this LEE pathogenicity island, they can also possess the auto-agglutinating adhesin factor designated Saa (STEC autoagglutinating adhesin; Paton et al., 2001). Adhesion to the intestinal mucosa is an essential step in the infection cycle of *E. coli*, which contributes to pathogenesis in humans. Other factors are involved in the virulence of STEC but also of EPEC, such as enterohemolysin A, encoded by the *ehxA* gene and associated with cytotoxic effects on endothelial cells that may contribute to the development of HUS (Jiang et al., 2015). STEC infections have been reported following the ingestion of contaminated food or water, after bathing in contaminated waters or contact with animals (for review, Croxen et al., 2013). The principal reservoir of STEC is the digestive tract of animals, particularly of cattle that are healthy carriers (Bibbal et al., 2015). Other animals, such as sheep, goats, swine, birds, and other wild animals, as well as humans, can also harbor STEC (Mora et al., 2012; Chandran and Mazumder, 2013).

Most human illness is caused by the serotype STEC O157:H7 (Paton and Paton, 1998). However, it is becoming evident that non-O157 isolates belonging to the serogroups O26, O45, O91, O103, O111, O113, O121, O145 also cause significant human illness (Mellmann et al., 2009; USDA, 2011). In Europe, O157:H7 and the four serotypes: O26:H11, O103:H2, O111:H8, and O145:H28 are the most widely implicated in human STEC infections, constituting the five highly pathogenic serotypes (EFSA, 2013).

Shiga-toxin producing *Escherichia coli* and EPEC contamination of the environment may occur through the spreading of livestock manure, animal waste on pastures, via wastewaters from slaughterhouses or from treatment plant effluents and by wildlife (Muniesa et al., 2006; Singh et al., 2015).

In such environments, STEC and EPEC strains are exposed to various stresses, such as low temperature or nutrient depletion and the ability to form biofilm could be an advantage to increase persistence (Vogeleer et al., 2014).
To date, very few studies have focused on the detection and isolation of pathogenic *E. coli* belonging to the STEC and EPEC pathovars in coastal environments (Gourmelon et al., 2006; Bennani et al., 2011). The aim of the study presented here was to detect and characterize STEC and EPEC strains from French shellfish-harvesting areas and their upstream watershed in order to assess the diversity of these pathogenic *E. coli* potentially present in this type of hostile environment. For this purpose, during a 2-years study, shellfish batches, freshwater, seawater, and surface sediment samples from three selected shellfish-harvesting areas and their upstream watersheds, the location of intensive livestock activities (cattle, swine, poultry, and/or sheep), were analyzed monthly to evaluate the presence of STEC and EPEC strains.

# MATERIALS AND METHODS

## Sampling Locations and Sample Description

Shellfish, water, and surface sediment samples were collected from three shellfish-harvesting sites on the French coast of within the Eastern English Channel and their watersheds. One of these sites, located in Brittany (site 1), corresponded to a 121 km² watershed, characterized by intensive livestock farming (cattle, swine, and poultry), with a human population of about 9,000 inhabitants. The two others were situated in Normandy; site 2 was characterized by a 1,000 km² catchment, with large livestock farming (cattle, sheep, swine, and poultry) and about 40,000 inhabitants, while the second site (site 3) corresponded to a 50 km² watersheds with large livestock farming (cattle, sheep, and swine) and about 7,000 inhabitants. These two latter watersheds are geographically closer together than the Brittany site (location of the sites Supplementary Figure S1). The three shellfish-harvesting areas are classified as category B for oysters (*Crassostrea gigas*) and mussels (*Mytilus edulis*) and as category C for common cockles (*Cerastoderma edule*) according to European regulation (European Directive 91/492/EEC; Anonymous, 2004). Shellfish from category B shellfish-harvesting areas must be depurated before being sold and shellfish from category C areas must be relayed at least 2 months prior to sale for consumption. Shellfish [oyster, mussel, and common cockle batches (site 1, n = 120; site 2, n = 72; and site 3, n = 46)] and freshwater samples from nine sampling sites upstream of shellfish-harvesting areas (site 1, n = 96; site 2, n = 72; site 3, n = 48) were collected monthly from February 2013 to January 2015, whereas surface sediment samples (site 1, n = 13; site 2, n = 13; site 3, n = 13) were collected from February 2013 to January 2014 and seawater samples (site 1, n = 12) from February 2014 to January 2015.

## Isolation of STEC and EPEC Strains

Samples were transported in insulated cooler boxes to the laboratory and analyzed within 24 h. After opening, total shellfish flesh, including shellfish flesh and intravascular liquid, were homogenized in a commercial blender (Waring Products Division, Torrington, CT, USA) for 60 s at high speed.

Twenty-five grams of homogenized total shellfish flesh were inoculated into 225 ml of buffered peptone water (BPW). For surface sediments, 10 g were introduced into the same volume of BPW. For water samples, 1 L was filtered using 0.45 µm cellulose membranes (Pall Gelman GN-6 Metricel; Pall Corporation, St Germain-en-Laye, France) and the filter was placed in 225 ml of BPW. Incubation was performed at 37°C for 24 h.

Shiga-toxin producing *Escherichia coli* and EPEC strains were isolated from the environmental samples using three additional protocols. The first one, described by Balière et al. (2015), involves application of the ISO/TS-13136 method, which focuses on isolation of strains belonging to the five highly pathogenic serotypes and was applied to samples collected from February 2013 to February 2014. The two other protocols involve the isolation of STEC and EPEC with or without an enrichment step and independently from the serotype. These were applied to samples collected from February 2013 to January 2015.

For the protocol with an enrichment step (described in Balière et al., 2015), DNA was extracted from 500 µL of each BPW enrichment broth using NucliSENS Nucleic Acid Extraction Reagents for miniMAG (BioMérieux, Marcy l'Etoile, France), according to the manufacturer's instructions. The *stx* and *eae* genes were detected by real-time PCR (Agilent MX3000P, Waldbronn, Germany), using primers and probes published previously (Nielsen and Andersen, 2003; Perelle et al., 2007), according to the ISO/TS-13136: 2012 technical specification, with slight modifications concerning the PCR cycles [denaturation for 10 s at 95°C, primer annealing for 5 s at 55°C, and extension for 25 s at 60°C (45 cycles)]. BPW broths identified positive for *stx* and *eae* were screened for STEC and EPEC isolates by streaking 1 µL of these broths onto Tryptone-Bile-X-glucuronide agar (TBX; AES chemunex, Bruz, France) and onto chromIDTM agar (BioMérieux), followed by incubation at 44°C for 24 h.

The final protocol to be used involves the screening of *E. coli* isolated directly from the water and shellfish samples without an enrichment step. For this protocol, 1, 10, and 100 ml of water were filtered through 0.45 µm cellulose membranes and the filters were placed onto TBX agar. For shellfish, 10 g of blended total shellfish flesh were distributed onto five empty and sterile plates with overlay super-cooled TBX agar. All TBX plates were incubated at 44°C for 24 h. Presumptive STEC and EPEC isolates were confirmed by real-time PCRs targeting *stx1*, *stx2*, and *eae* genes, as described above after a DNA extraction of each isolate by boiling at 100°C, for 15 min.

The STEC and EPEC isolates were characterized using several protocols as described below.

## Serotyping

The serotypes of the STEC and EPEC strains were characterized using the serotyping method by agglutination, as described by Blanco et al. (2003).

More precisely, determination of O and H antigens was carried out by agglutination as previously described (Guinée et al., 1981), employing all available O (O1-O185) and
H (H1-H56) antisera. All antisera were absorbed with the corresponding cross-reacting antigens to remove the non-specific agglutinins. The O and H antisera were produced in the Laboratorio de Referencia de *E. coli* (USC, Lugo, Spain). Isolates that did not react with O antisera were considered as non-typeable (ONT) and those non-motile were HNM.

## Detection of Enterohemolysin and Adhesin

The presence of *ehxA* (encoding enterohemolysin A) and *saa* (encoding STEC autoagglutining adhesin) genes in these isolates was investigated by conventional PCR using primers previously described by Paton and Paton (2002).

## Phylogenetic Group

Isolates were classified into the four main *E. coli* phylogenetic groups (A, B1, B2, or D) using a conventional triplex PCR method based on the detection of two genes, *chuA*, required for heme transport in enterohemorrhagic O157:H7 *E. coli*, *yjaA*, initially identified in the recent complete genome sequence of *E. coli* K-12, for which the function is unknown, and of an anonymous DNA fragment designated TSPE4.C2 using primers described previously by Clermont et al. (2000).

## Pulsed-field Gel Electrophoresis

The genetic relatedness of the isolates was studied by the pulsed-field gel electrophoresis method (PFGE) according to Bidet et al. (2005). Isolated strains were inoculated in nutrient broth containing 1.3% NaCl (Bio-Rad, Marnes-la-coquette, France) and incubated at 37°C for 24 h. Bacterial DNA was extracted from 400 µl of the enrichment broth using the CHEF Bacterial Genomic DNA Plug Kit (Bio-Rad) according to the manufacturer's recommendations. Bacterial DNA was digested for between 16 and 20 h at 37°C with the restriction endonuclease *Xba*I (Roche Diagnostic, Meylan, France) according to the manufacturer's recommendations. Each electrophoresis was performed using a lambda ladder molecular mass marker (Bio-Rad) for the normalization of gel images. The migration was performed on a 1% agarose gel using the CHEF-DRIII apparatus (Bio-Rad) at 6 V cm⁻¹ for 27 h, with pulse times varying linearly between two and 49 s. The bacterial DNA restriction patterns were analyzed using the Bionumerics software 7.5 (Applied Maths, Kortrijk, Belgium). The similarity of PFGE profiles was compared and a dendrogram was created using the band-based Dice unweighted-pair group method, using average linkages (UPGMA), based on 1% position tolerance and 0% position optimization. Branch quality was evaluated using Cophenetic correlation. PFGE patterns were considered clonally related when they had a similarity coefficient higher than 80%.

## Multilocus Sequencing Typing

The genetic relatedness of the isolates was also studied using the multilocus sequencing typing method (MLST). Fragments of seven housekeeping genes, i.e., *adk* (adenylate

kinase), *fumC* (fumarate hydratase), *gyrB* (DNA gyrase), *icd* (isocitrate/isopropylmalate dehydrogenase), *mdh* (malate dehydrogenase), *purA* (adenylosuccinate dehydrogenase), and *recA* (ATP/GTP binding motif) were amplified and sequenced using suitable primers (Wirth et al., 2006) with minor modifications for the *recA* primers (*recAR* 5'-*TCG-TCG-AAA-TCT-ACG-GAC-CGG-A-3′; *recAF1* 5'-*ACC-TTT-GTA-GCT-GTA-CCA-CG-3′). The PCR cycle included denaturation for 60 s at 95°C, primer annealing for 60 s at 56°C (for *adk*, *purA*, *recA*, and *icd*), at 65°C (for *mdh* and *gyrB*), or at 68°C (for *fumC*), and extension for 60 s at 72°C (35 cycles) in MJ Research PTC-200 (DNA Engine, Waltham, MA, USA). Sequencing was performed in both directions with the fluorescent dye terminator Sanger method on ABI3730 (Applied Biosystem) by Eurofins Genomics (Ebersberg, Germany). The alleles and sequence types (ST) were assigned in accordance with the *E. coli* MLST database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli).

## Static Biofilm Formation Assay

A biofilm formation assay was performed as previously described by Tremblay et al. (2015). In addition to the incubation temperature of 30°C, the ability to form biofilms was also tested at 18°C, in order to reproduce marine temperate environmental conditions (Moldoveanu, 2012). Briefly, overnight cultures at 37°C in LB media were diluted (1:100) in 5 ml of M9 medium with glucose (0.4% wt/vol) and minerals (1.16 mM MgSO₄, 2 µM FeCl₃, 8 µM CaCl₂, and 16 µM MnCl₂) and incubated for 24 h at 37°C. These cultures were diluted (1:100) in M9 medium supplemented with glucose and minerals and were inoculated in triplicate into microtitre plates (Costar 3370; Corning, NY, USA). After 24 h of incubation at 18 or 30°C, unattached cells were removed by washing three times with phosphate-buffered saline (PBS). Plates were dried at 37°C for 15 min and biofilms were stained with crystal violet (0.1% wt/vol) for 2 min. After removal of crystal violet solution, the biofilms were washed three times with PBS and dried at 37°C for 15 min. The biofilm stain was dissolved with 150 µl of 80% (vol/vol) ethanol and 20% (vol/vol) acetone and biofilms were quantified by measuring the absorbance at 590 nm (OD₅₀₀) with a microplate reader (Powerwave; BioTek Instruments, Winooski, VT, USA). The results for the static biofilms formed at 18 and 30°C were compared using two-way analysis of variance (ANOVA) followed by a Bonferroni *post hoc* comparison using GraphPad Prism, version 4.02 (GraphPad Software, San Diego, CA, USA). Strains were divided into three groups based on the OD₅₀₀ of bacterial biofilm: strong (A₅₀₀ > 0.6), medium (0.6 ≥ A₅₀₀ ≥ 0.3) and weak or none (A₅₀₀ < 0.3).

## Antibiotic Resistance

Antimicrobial susceptibility testing based on the disk diffusion method was performed on a selection of STEC and EPEC isolates. Sixteen antibiotics were tested: Tobramycin (10 µg), Fosfomycin (50 µg), Cefalotin (30 µg), Imipenem (10 µg), Tigecyclin (15 µg), Gentamycin (15 µg), Cefotaxim (30 µg), Cefoxitin (30 µg), Doxycyclin (30 µg), Ciprofloxacin (5 µg), Augmentin (20 µg
amoxicillin; 10 µg clavulanic acid), Ticarcillin (75 µg), Bactrim (1.25 µg trimethoprim; 23.75 µg sulfamethoxazol), Nalidixic acid (30 µg), Amikacin (30 µg), Amoxicillin (25 µg) on Mueller-Hinton medium (AES chemunex, Bruz, France). Plates were incubated at 37°C for 24 h.

## Environmental Data and Statistical Analysis

Rainfall data (2-days cumulative rainfall before sampling date) were provided by the meteorological stations from Meteo France at Pleurtuit (site 1) and at Coutances (sites 2 and 3). The water temperature was measured manually at each sampling. The data on temperature and precipitation were categorized into three groups whose boundaries were defined so that they are likely to categorize the data significantly for the studied sites and they allow to have in each category a number of sample consistent with a reliable statistical analysis. Comparisons of STEC and EPEC prevalence between the type of samples, the site, the season, the temperature and the precipitation were analyzed by the chi-square test. A *p*-value of <0.05 was considered statistically significant.

## RESULTS

### Detection and Isolation of STEC and EPEC Strains

The *stx* gene was detected in 30.3, 85.9, 41.7, and 28.2% of shellfish, freshwater, seawater and surface sediment enrichment broths, respectively (Table 1). The *eae* gene was detected in 74.8, 100, 100, and 43.6% of shellfish, freshwater, seawater, and surface sediment enrichment broths, respectively (Table 1). STEC were isolated from 5.0% of the 238 shellfish, 5.6% of the 216 freshwater, 8.3% of the 12 seawater, and 2.6% of the 39 surface sediment samples analyzed, whereas EPEC were isolated from 8.0, 21.3, and 33.3% of the shellfish, freshwater, and seawater samples, respectively. No EPEC were isolated from surface sediments (Table 2). A total of 57 STEC and 117 EPEC isolates were obtained from these samples. However, as 29 STEC and 28 EPEC had identical serotypes, PFGE and MLST patterns, virulence gene profiles, and phylogroups to other isolates cultivated from the same samples, they were considered to be replicates and not retained. The remaining 28 STEC and 89 EPEC isolates represented 0.2 and 0.7% of the total *E. coli* (n = 12,016), respectively (Table 3).

<table><caption>TABLE 2 | Isolation of STEC and EPEC as regard to sample parameters.</caption><thead><tr><th>Sample parameter</th><th>No. of samples</th><th>No. of samples with at least one STEC isolate (%)<sup>a</sup></th><th>No. of samples with at least one EPEC isolate (%)<sup>a</sup></th></tr></thead><tbody><tr><td><strong>Type</strong></td><td></td><td></td><td></td></tr><tr><td>Shellfish</td><td>238</td><td>12 (5.0)</td><td>19 (8.0)</td></tr><tr><td>Freshwater</td><td>216</td><td>12 (5.6)</td><td>46 (21.3)</td></tr><tr><td>Seawater</td><td>12</td><td>1 (8.3)</td><td>4 (33.3)</td></tr><tr><td>Superficial sediment</td><td>39</td><td>1 (2.6)</td><td>0</td></tr><tr><td>χ<sup>2</sup> test</td><td></td><td><i>p</i> = 0.845</td><td><i>p</i> = 3.09 10<sup>-5</sup></td></tr><tr><td><strong>Site</strong></td><td></td><td></td><td></td></tr><tr><td>Site 1</td><td>241</td><td>10 (4.1)</td><td>34 (14.1)</td></tr><tr><td>Site 2</td><td>157</td><td>7 (4.5)</td><td>20 (12.7)</td></tr><tr><td>Site 3</td><td>107</td><td>9 (8.4)</td><td>15 (14.0)</td></tr><tr><td>χ<sup>2</sup> test</td><td></td><td><i>p</i> = 0.244</td><td><i>p</i> = 0.931</td></tr><tr><td><strong>Season</strong></td><td></td><td></td><td></td></tr><tr><td>Fall</td><td>146</td><td>4 (2.7)</td><td>11 (7.5)</td></tr><tr><td>Summer</td><td>126</td><td>17 (13.5)</td><td>22 (17.5)</td></tr><tr><td>Autumn</td><td>124</td><td>3 (2.4)</td><td>24 (19.4)</td></tr><tr><td>Winter</td><td>109</td><td>2 (1.8)</td><td>12 (11.0)</td></tr><tr><td>χ<sup>2</sup> test</td><td></td><td><i>p</i> = 4.44 10<sup>-5</sup></td><td><i>p</i> = 0.0314</td></tr><tr><td><strong>Temperature (°C)</strong></td><td></td><td></td><td></td></tr><tr><td>0-<10</td><td>172</td><td>5 (2.9)</td><td>15 (8.7)</td></tr><tr><td>10-<15</td><td>189</td><td>14 (7.4)</td><td>29 (15.3)</td></tr><tr><td>15-&gt;15</td><td>144</td><td>7 (4.9)</td><td>25 (17.4)</td></tr><tr><td>χ<sup>2</sup> test</td><td></td><td><i>p</i> = 0.167</td><td><i>p</i> = 0.086</td></tr><tr><td><strong>Precipitation (mm)<sup>b</sup></strong></td><td></td><td></td><td></td></tr><tr><td>0-<1</td><td>253</td><td>17 (6.7)</td><td>30 (11.9)</td></tr><tr><td>1-<10</td><td>144</td><td>5 (3.5)</td><td>21 (14.6)</td></tr><tr><td>10-&gt;10</td><td>108</td><td>4 (3.7)</td><td>18 (16.7)</td></tr><tr><td>χ<sup>2</sup> test</td><td></td><td><i>p</i> = 0.296</td><td><i>p</i> = 0.495</td></tr><tr><td><strong>Total</strong></td><td>505</td><td>26 (5.1)</td><td>69 (13.7)</td></tr></tbody></table>

Shiga-toxin producing *Escherichia coli* strains represented 0.2, 0.2, and 0.3%, of the isolated *E. coli* from sites 1–3, respectively. EPEC strains represented 0.8, 0.6, and 0.7% of the isolated *E. coli* from sites 1–3, respectively (Table 3).

For the three sites, the majority of STEC strains derived from freshwater samples (50, 57.1, and 44.4% of samples from sites 1–3, respectively) and from shellfish batches (41.7, 42.9, and 44.4% in the sites 1–3, respectively; Table 3). Only two STEC strains were isolated from seawater and surface sediment samples from sites

<table><caption>TABLE 1 | Prevalence of *stx* and *eae* genes in shellfish, freshwater, seawater, and superficial sediment enrichment broths.</caption><thead><tr><th>Type of sample</th><th>No. of samples</th><th>No. of stx-positive broth (%)<sup>a</sup></th><th>No. of eae-positive broth (%)<sup>a</sup></th><th>No. of stx- and eae-positive broth (%)<sup>a</sup></th></tr></thead><tbody><tr><td>Shellfish</td><td>238</td><td>72 (30.3)</td><td>178 (74.8)</td><td>64 (26.9)</td></tr><tr><td>Freshwater</td><td>216</td><td>196 (85.9)</td><td>216 (100)</td><td>196 (85.9)</td></tr><tr><td>Seawater</td><td>12</td><td>5 (41.7)</td><td>12 (100)</td><td>5 (41.7)</td></tr><tr><td>Superficial sediment</td><td>39</td><td>11 (28.2)</td><td>17 (43.6)</td><td>8 (20.5)</td></tr><tr><td><strong>Total</strong></td><td>505</td><td>284 (56.2)</td><td>423 (83.7)</td><td>273 (54.1)</td></tr></tbody></table>

<footnote><sup>a</sup>Percentage calculated based on the total of no. samples for each type of sample.</footnote>

<footnote><sup>b</sup>Precipitation: 2-days cumulative rainfall before sampling date.</footnote>

<footnote><sup>a</sup>Percentage calculated based on the total of no. samples for each type of sample.</footnote>
<table><caption>TABLE 3 | Number of STEC and EPEC strains isolated from shellfish, freshwater, seawater, and superficial sediment samples collected in the three shellfish-harvesting areas and their watersheds, as regard to the total number of *E. coli* isolates.</caption><thead><tr><th>Type of sample</th><th>Total (%)<sup>a</sup></th><th>Shellfish (%)</th><th>Freshwater (%)</th><th>Seawater (%)</th><th>Superficial sediment (%)</th></tr></thead><tbody><tr><td><strong>Site 1</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No. <em>E. coli</em> isolates</td><td>5,676</td><td>1,343</td><td>3,410</td><td>225</td><td>30</td></tr><tr><td>No. STEC strains</td><td>12 (0.2)</td><td>5 (41.7)<sup>b</sup></td><td>6 (50.0)<sup>b</sup></td><td>1 (8.3)<sup>b</sup></td><td>0</td></tr><tr><td>No. EPEC strains</td><td>47 (0.8)</td><td>8 (17.4)<sup>b</sup></td><td>35 (76.1)<sup>b</sup></td><td>4 (8.5)<sup>b</sup></td><td>0</td></tr><tr><td><strong>Site 2</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No. <em>E. coli</em> isolates</td><td>3,682</td><td>757</td><td>2,925</td><td>nd<sup>c</sup></td><td>4</td></tr><tr><td>No. STEC strains</td><td>7 (0.2)</td><td>3 (42.9)<sup>b</sup></td><td>4 (57.1)<sup>b</sup></td><td>nd</td><td>0</td></tr><tr><td>No. EPEC strains</td><td>23 (0.6)</td><td>10 (43.5)<sup>b</sup></td><td>13 (56.5)<sup>b</sup></td><td>nd</td><td>0</td></tr><tr><td><strong>Site 3</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No. <em>E. coli</em> isolates</td><td>2,658</td><td>678</td><td>2,036</td><td>nd</td><td>83</td></tr><tr><td>No. STEC strains</td><td>9 (0.3)</td><td>4 (44.4)<sup>b</sup></td><td>4 (44.4)<sup>b</sup></td><td>nd</td><td>1 (11.1)<sup>b</sup></td></tr><tr><td>No. EPEC strains</td><td>19 (0.7)</td><td>5 (26.3)<sup>b</sup></td><td>14 (73.7)<sup>b</sup></td><td>nd</td><td>0</td></tr><tr><td><strong>Total of the three sites</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No. <em>E. coli</em> isolates</td><td>12,016</td><td>2,778</td><td>8,371</td><td>225</td><td>117</td></tr><tr><td>No. STEC strains</td><td>28 (0.2)</td><td>12 (42.9)<sup>b</sup></td><td>14 (50.0)<sup>b</sup></td><td>1 (3.6)<sup>b</sup></td><td>1 (3.6)<sup>b</sup></td></tr><tr><td>No. EPEC strains</td><td>89 (0.7)</td><td>23 (25.8)<sup>b</sup></td><td>62 (69.7)<sup>b</sup></td><td>4 (4.5)<sup>b</sup></td><td>0</td></tr></tbody></table>

1 and 3, respectively. The majority of EPEC strains derived from the freshwater samples (76.1, 56.5, and 73.7% of samples from sites 1–3, respectively) and the remaining EPEC derived from shellfish batches (17.4, 43.5, and 26.3%, respectively) and seawater samples (8.5% only in the site 1; Table 3).

Nearly one third of the STEC strains were obtained from samples collected in May 2013 (32.1%, 9/28), whereas the EPEC strains were mostly obtained from samples collected in November 2013 (21.3%, 19/89) and August 2014 (12.4%, 11/89; Table 4). The entire sample set demonstrated a seasonal effect with potential pathogenic *E. coli* as STEC strains were significantly more frequently isolated in Summer and EPEC strains in Summer and Autumn ($p < 0.05$; Table 2). However, no correlation between the prevalence of both STEC and EPEC and pluviometry nor temperature was observed (Table 2).

## Virulence Gene Profiles

By considering the presence of a single virulence gene or a combination of the four virulence genes investigated (i.e., *stx*, *eae*, *ehxA*, and *saa*) in the 117 STEC or EPEC strains, eight virulence gene profiles were found. The most frequent profile presented the *eae* gene only (70.1% of the strains) followed by the profile presenting the *stx2* gene only (11.1%) and the profile presenting the *stx1* gene only (7.7%). Seven strains (6.0%) were shown to possess the *eae* and *ehxA* genes. The *stx1-stx2-ehxA-saa* profile was found in three strains and three other virulence gene profiles were observed only once, i.e., the *stx1-eae-ehxA*, the *stx1-stx2-saa*, and the *stx2-ehxA-saa* profiles (Table 4).

Seven STEC strains carrying *stx1*, three carrying *stx2*, and two carrying both *stx1* and *stx2* genes were isolated from the site 1 whereas three STEC strains harboring the *stx1* gene, 11 the *stx2* gene, and two presenting both *stx1* and *stx2* genes were recovered from sites 2 and 3.

## Phylogroups

The STEC strains ($n = 28$) were mainly distributed among the phylogroups A, B1, and D (39.3, 35.7, 21.4%, respectively). Only one strain belonged to phylogroup B2. The EPEC strains ($n = 89$) belonged to all the phylogroups; the strains from phylogroup B1 and B2 (38.2 and 38.2%, respectively) being more prevalent than those from phylogroups A and D (18.0 and 5.7%, respectively; Table 5). More precisely, at site 1, the STEC strains were mainly divided between phylogroups B1 and D. At sites 2 and 3, the STEC strains were divided between phylogroups A and B1. At sites 1–3, the EPEC strains belonged to all the investigated phylogroups, with a majority belonging to phylogroups B1 and B2.

## Serotyping

The 117 (STEC or EPEC) strains selected in this study belonged to 44 O antigens and 24 H antigens and presented 75 distinguishable serotypes (Table 4). Among all strains, 13 strains were non-typable (NT) for the O antigen [ONT:H2 ($n = 1$), ONT:H31 ($n = 1$), ONT:H34 ($n = 1$), ONT:H6 ($n = 6$), ONT:H8 ($n = 2$), ONT:H10 ($n = 1$), ONT:H11 ($n = 1$)] and 24 strains were uncharacterized for the H antigen (HNM: non-motile or HNT: non-typable). Two strains were non-typable for either antigens (ONT:HNT).

Eighteen different serotypes (O:H) were identified among the STEC strains. Only one STEC belonging to one of the five highly pathogenic serotypes was isolated: an O26:H11 *stx1*<sup>+</sup>, *eae*<sup>+</sup>, and *ehxA*<sup>+</sup> strain, from a mussel batch. One strain from serotype O91:H21 and carrying *stx1*, *stx2*, *ehxA*, and *saa* genes was also identified among the STEC strains. The most detected serotype among the STEC strains was the O100:HNM ($n = 9$). Fifteen additional serotypes (O149:H31/HNM, O154:H31/HNM, O130:H11, O15:H16, O185:H28, O2:H32, O28:H1, O63:H11, O76:H19, O8:H12, O88:H25, ONT:H10, and ONT:H11) were

<footnote><sup>a</sup>Percentage calculated based on the total of no. *E. coli* isolates from each sites. <sup>b</sup>Percentage calculated based on the total of no. STEC or EPEC strains isolated from the three sites. <sup>c</sup>nd: non-determined.</footnote>
<table><caption>TABLE 4 | Characteristics of STEC and EPEC strains isolated from the three French shellfish-harvesting sites from the Eastern English Channel coastal area and their watersheds.</caption><thead><tr><th>Serotype (no. of isolate)</th><th>Virulence gene (no. of isolate)</th><th>Sample origin (no. of isolate)</th><th>Sampling site (no. of isolate)</th><th>Sampling month-year</th><th>Precipitation (mm)</th></tr></thead><tbody><tr><td colspan="6"><strong>STEC</strong></td></tr><tr><td>O100:HNM (9)</td><td>stx2</td><td>SFm(1), SFc(1),<br/>FW(1), SFo(1), SFm(1)<br/>SFm(1), S(1)<br/>SFm<br/>SFo</td><td>2<br/>3<br/>3<br/>2<br/>3</td><td>May-13<br/>May-13<br/>June-13<br/>March-14<br/>June-14</td><td>0.1<br/>0.1<br/>6.1<br/>0.1<br/>0</td></tr><tr><td>O15:H16(1)</td><td>stx2</td><td>SFm</td><td>1</td><td>June-13</td><td>0.2</td></tr><tr><td>O2:H32(1)</td><td>stx2</td><td>SW</td><td>1</td><td>February-14</td><td>0</td></tr><tr><td>O8:H19(2)</td><td>stx2</td><td>FW<br/>FW</td><td>3<br/>3</td><td>June-14<br/>July-14</td><td>0<br/>1.4</td></tr><tr><td>O149:H1(1)</td><td>stx1</td><td>FW</td><td>1</td><td>April-13</td><td>1.6</td></tr><tr><td>O149:HNM(1)</td><td>stx1</td><td>FW</td><td>1</td><td>April-13</td><td>1.6</td></tr><tr><td>O154:H31(2)</td><td>stx1</td><td>SFo(1), FW(1)</td><td>1</td><td>May-13</td><td>0.1</td></tr><tr><td>O154:HNM(1)</td><td>stx1</td><td>SFm<br/>SFc</td><td>1<br/>1</td><td>November-14<br/>March-14</td><td>15.4<br/>0.1</td></tr><tr><td>O28: H1(1)</td><td>stx1</td><td>FW</td><td>2</td><td>August-14</td><td>26.0</td></tr><tr><td>O76:H19(1)</td><td>stx1</td><td>FW</td><td>2</td><td>September-14</td><td>0.4</td></tr><tr><td>O88:H25(1)</td><td>stx1</td><td>FW</td><td>2</td><td>April-14</td><td>0.3</td></tr><tr><td>ONT:H10(1)</td><td>stx1</td><td>FW</td><td>2</td><td>November-13</td><td>15.4</td></tr><tr><td>O26:H11(1)</td><td>stx1+eae+ehxA</td><td>SFm</td><td>1</td><td>November-14</td><td>24.0</td></tr><tr><td>O63:H6(1)</td><td>stx2+ehxA+saa</td><td>FW</td><td>1</td><td>May-13</td><td>1.8</td></tr><tr><td>ONT:H11(1)</td><td>stx1+stx2+saa</td><td>FW</td><td>1</td><td>Apr-13</td><td>1.6</td></tr><tr><td>O185:H28(1)</td><td>stx1+stx2+ehxA+saa</td><td>FW</td><td>2</td><td>December-13</td><td>0</td></tr><tr><td>O130:H11(1)</td><td>stx1+stx2+ehxA+saa</td><td>FW</td><td>1</td><td>May-13</td><td>1.8</td></tr><tr><td>O91:H21(1)</td><td>stx1+stx2+ehxA+saa</td><td>FW</td><td>3</td><td>March-14</td><td>0.1</td></tr><tr><td colspan="6"><strong>EPEC</strong></td></tr><tr><td>O103:H25(1)</td><td>eae</td><td>FW</td><td>3</td><td>February-14</td><td>0</td></tr><tr><td>O103:HNM(1)</td><td>eae</td><td>FW</td><td>1</td><td>November-13</td><td>0.1</td></tr><tr><td>O108:H21(3)</td><td>eae</td><td>FW<br/>SFc<br/>FW</td><td>3<br/>2<br/>3</td><td>January-14<br/>July-14</td><td>6.5<br/>1.4</td></tr><tr><td>O113:H6(4)</td><td>eae</td><td>SFm(1), SFc(1)<br/>FW<br/>SFo</td><td>1<br/>3<br/>3</td><td>November-13<br/>November-13<br/>August-14</td><td>15.4<br/>0.1<br/>26</td></tr><tr><td>O116:H20(1)</td><td>eae</td><td>FW</td><td>1</td><td>November-13</td><td>15.4</td></tr><tr><td>O125:H6(2)</td><td>eae</td><td>FW<br/>FW</td><td>1<br/>2</td><td>November-13<br/>September-14</td><td>15.4<br/>0.4</td></tr><tr><td>O128:H2(1)</td><td>eae</td><td>FW</td><td>2</td><td>November-13</td><td>0</td></tr><tr><td>O137:H6(2)</td><td>eae</td><td>FW<br/>FW</td><td>1<br/>3</td><td>August-13<br/>January-15</td><td>1.0<br/>2.3</td></tr><tr><td>O145:H34(1)</td><td>eae</td><td>FW</td><td>1</td><td>July-14</td><td>6.5</td></tr><tr><td>O146:H21(1)</td><td>eae</td><td>SFm</td><td>3</td><td>August-14</td><td>26.0</td></tr><tr><td>O146:H6(1)</td><td>eae</td><td>SFc</td><td>2</td><td>September-14</td><td>0.4</td></tr><tr><td>O15:H2(2)</td><td>eae</td><td>FW<br/>SFm</td><td>3<br/>2</td><td>August-14<br/>January-15</td><td>26.0<br/>2.3</td></tr><tr><td>O153:H21(1)</td><td>eae</td><td>FW</td><td>1</td><td>January-14</td><td>7.2</td></tr><tr><td>O157:H16(1)</td><td>eae</td><td>FW</td><td>1</td><td>July-14</td><td>6.5</td></tr><tr><td>O159:H7(1)</td><td>eae</td><td>FW</td><td>1</td><td>November-14</td><td>24.0</td></tr><tr><td>O167:H3(1)</td><td>eae</td><td>FW</td><td>1</td><td>August-13</td><td>2.8</td></tr><tr><td>O179:H31(2)</td><td>eae</td><td>FW<br/>FW</td><td>1<br/>3</td><td>January-14<br/>June-14</td><td>7.2<br/>0</td></tr><tr><td>O2:H45(1)</td><td>eae</td><td>FW</td><td>2</td><td>December-13</td><td>0</td></tr><tr><td>O20:HNT(1)</td><td>eae</td><td>SW</td><td>1</td><td>April-14</td><td>0</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 4 | Continued</caption><thead><tr><th>Serotype (no. of isolate)</th><th>Virulence gene (no. of isolate)</th><th>Sample origin (no. of isolate)</th><th>Sampling site (no. of isolate)</th><th>Sampling month-year</th><th>Precipitation (mm)</th></tr></thead><tbody><tr><td rowspan="2">O23:H8(2)</td><td rowspan="2">eae</td><td>FW</td><td>2</td><td>August-13</td><td>0.7</td></tr><tr><td>SFc</td><td>2</td><td>August-14</td><td>26.0</td></tr><tr><td>O25:H2(1)</td><td>eae</td><td>FW</td><td>1</td><td>September-14</td><td>0</td></tr><tr><td>O28:H16(1)</td><td>eae</td><td>SFm</td><td>1</td><td>November-13</td><td>15.4</td></tr><tr><td>O29:H19(1)</td><td>eae</td><td>FW</td><td>2</td><td>October-13</td><td>22.8</td></tr><tr><td>O33:H6(1)</td><td>eae</td><td>FW</td><td>1</td><td>February-14</td><td>0</td></tr><tr><td>O39:HNM(1)</td><td>eae</td><td>FW</td><td>1</td><td>January-14</td><td>7.2</td></tr><tr><td>O40:HNM(1)</td><td>eae</td><td>FW</td><td>1</td><td>February-14</td><td>0</td></tr><tr><td>O42:H37(1)</td><td>eae</td><td>FW</td><td>3</td><td>March-13</td><td>0</td></tr><tr><td>O5:H40(1)</td><td>eae</td><td>SFm</td><td>2</td><td>February-13</td><td>0.8</td></tr><tr><td>O51:HNM(1)</td><td>eae</td><td>FW</td><td>2</td><td>October-13</td><td>22.8</td></tr><tr><td rowspan="4">O63:H6(4)</td><td rowspan="4">eae</td><td>FW</td><td>1</td><td>November-13</td><td>15.4</td></tr><tr><td>FW</td><td>2</td><td>November-13</td><td>0.1</td></tr><tr><td>FW</td><td>1</td><td>January-14</td><td>7.2</td></tr><tr><td>FW</td><td>3</td><td>October-14</td><td>0.5</td></tr><tr><td>O63:HNM(1)</td><td>eae</td><td>FW</td><td>3</td><td>October-14</td><td>0.5</td></tr><tr><td>O71:H49(2)</td><td>eae</td><td>SFm(1), SFc(1)</td><td>1</td><td>October-13</td><td>0.4</td></tr><tr><td>O71:HNM(1)</td><td>eae</td><td>FW</td><td>1</td><td>August-14</td><td>1.0</td></tr><tr><td>O8:H14(1)</td><td>eae</td><td>FW</td><td>1</td><td>November-13</td><td>15.4</td></tr><tr><td>O85:H31(1)</td><td>eae</td><td>FW</td><td>1</td><td>October-14</td><td>8.3</td></tr><tr><td>O85:HNM(1)</td><td>eae</td><td>FW</td><td>1</td><td>August-14</td><td>1.0</td></tr><tr><td>O86:H31(1)</td><td>eae</td><td>SFc</td><td>2</td><td>August-14</td><td>26.0</td></tr><tr><td>O88:H25(1)</td><td>eae</td><td>SFm</td><td>3</td><td>September-14</td><td>0.4</td></tr><tr><td>O88:H8(1)</td><td>eae</td><td>SFo</td><td>3</td><td>August-14</td><td>26.0</td></tr><tr><td>O9:HNM(1)</td><td>eae</td><td>FW</td><td>2</td><td>November-13</td><td>0.1</td></tr><tr><td>O91:H10(1)</td><td>eae</td><td>SFc</td><td>1</td><td>July-14</td><td>6.5</td></tr><tr><td>O98:H56(1)</td><td>eae</td><td>FW</td><td>1</td><td>December-14</td><td>0</td></tr><tr><td>O98:H8(1)</td><td>eae</td><td>SFm</td><td>1</td><td>November-13</td><td>15.4</td></tr><tr><td rowspan="2">O98:HNM(2)</td><td rowspan="2">eae</td><td>SW</td><td>1</td><td>February-14</td><td>0</td></tr><tr><td>FW</td><td>1</td><td>March-14</td><td>0</td></tr><tr><td>O98:HNT(1)</td><td>eae</td><td>FW</td><td>1</td><td>December-14</td><td>0</td></tr><tr><td>ONT:H2(1)</td><td>eae</td><td>FW</td><td>1</td><td>August-13</td><td>2.8</td></tr><tr><td>ONT:H31(1)</td><td>eae</td><td>FW</td><td>1</td><td>October-14</td><td>8.3</td></tr><tr><td>ONT:H34(1)</td><td>eae</td><td>FW</td><td>3</td><td>December-14</td><td>10.6</td></tr><tr><td rowspan="6">ONT:H6(6)</td><td rowspan="6">eae</td><td>FW</td><td>1</td><td>September-13</td><td>1.0</td></tr><tr><td>FW</td><td>1</td><td>August-14</td><td>1.0</td></tr><tr><td>FW</td><td>3</td><td>September-14</td><td>0.4</td></tr><tr><td>FW</td><td>3</td><td>October-14</td><td>0.5</td></tr><tr><td>SFm</td><td>2</td><td>December-14</td><td>0</td></tr><tr><td>SW</td><td>1</td><td>December-14</td><td>10.6</td></tr><tr><td rowspan="2">ONT:H8(2)</td><td rowspan="2">eae</td><td>FW</td><td>1</td><td>April-14</td><td>0</td></tr><tr><td>FW</td><td>1</td><td>August-14</td><td>1</td></tr><tr><td rowspan="2">ONT:HNT(2)</td><td rowspan="2">eae</td><td>SW</td><td>1</td><td>November-14</td><td>24.0</td></tr><tr><td>FW</td><td>3</td><td>October-14</td><td>10.6</td></tr><tr><td>O28:HNM(1)</td><td>eae+ehxA</td><td>FW</td><td>1</td><td>June-14</td><td>0.2</td></tr><tr><td>O145:H28(2)</td><td>eae+ehxA</td><td>SFm(1), SFc(1)</td><td>2</td><td>June-13</td><td>6.1</td></tr><tr><td>O177:H11(1)</td><td>eae+ehxA</td><td>FW</td><td>2</td><td>July-14</td><td>1.4</td></tr><tr><td rowspan="2">O26:H11(6)</td><td rowspan="2">eae(5), eae+ehxA(1)</td><td>FW</td><td>2</td><td>August-13</td><td>0.7</td></tr><tr><td>FW(3), SFm(1), SFc(1)</td><td>1</td><td>November-13</td><td>15.4</td></tr><tr><td rowspan="3">O103:H2(3)</td><td rowspan="3">eae(2), eae+ehxA(1)</td><td>FW</td><td>2</td><td>February-13</td><td>0.8</td></tr><tr><td>FW</td><td>2</td><td>June-13</td><td>6.1</td></tr><tr><td>FW</td><td>1</td><td>November-13</td><td>15.4</td></tr><tr><td rowspan="2">O153:H2(3)</td><td rowspan="2">eae(2), eae+ehxA(2)</td><td>SFm(1), FW(1)</td><td>2 (1), 3 (1)</td><td>March-14</td><td>0.1</td></tr><tr><td>SFc</td><td>2</td><td>July-14</td><td>1.4</td></tr></tbody></table>

NT, non-typable; NM, non-motile. Sample origin: SF: shellfish, o: oyster, m: mussel, c: cockle, FW: freshwater, SW: seawater, S: superficial sediment. (:): number of strain and when it is not specified the no. of strain is one. (1) Brittany site, (2) first Normandy site, and (3) second Normandy site. Precipitation: 2-days cumulative rainfall before sampling date.
<table><caption>TABLE 5 | Distribution of phylogroup A, B1, B2, and D among STEC and EPEC strains isolated in the three French shellfish-harvesting sites from the Eastern English Channel coastal area and their watersheds.</caption><thead><tr><th>Phylogroup</th><th>Total</th><th>A (%)</th><th>B1 (%)</th><th>B2 (%)</th><th>D (%)</th></tr></thead><tbody><tr><td><strong>Site 1</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No. STEC strains (%)<sup>a</sup></td><td>12</td><td>2 (16.7)</td><td>4 (33.3)</td><td>0</td><td>6 (50.0)</td></tr><tr><td>No. EPEC strains (%)<sup>b</sup></td><td>47</td><td>13 (27.7)</td><td>13 (27.7)</td><td>19 (40.4)</td><td>2 (4.4)</td></tr><tr><td><strong>Site 2</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No. STEC strains (%)<sup>a</sup></td><td>7</td><td>3 (42.9)</td><td>3 (42.9)</td><td>1 (14.2)</td><td>0</td></tr><tr><td>No. EPEC strains (%)<sup>b</sup></td><td>23</td><td>2 (8.7)</td><td>13 (56.5)</td><td>5 (21.7)</td><td>3 (13.0)</td></tr><tr><td><strong>Site 3</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No. STEC strains (%)<sup>a</sup></td><td>9</td><td>6 (66.7)</td><td>3 (33.3)</td><td>0</td><td>0</td></tr><tr><td>No. EPEC strains (%)<sup>b</sup></td><td>19</td><td>1 (5.3)</td><td>8 (42.1)</td><td>10 (52.6)</td><td>0</td></tr><tr><td><strong>Total of the three sites</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No. STEC strains (%)<sup>c</sup></td><td>28</td><td>11 (39.3)</td><td>10 (35.7)</td><td>1 (3.6)</td><td>6 (21.4)</td></tr><tr><td>No. EPEC strains (%)<sup>c</sup></td><td>89</td><td>16 (18.0)</td><td>34 (38.2)</td><td>34 (38.2)</td><td>5 (5.7)</td></tr></tbody></table>

identified within the STEC strains and contained one or two individual isolates each.

Fifty-seven serotypes were identified among the EPEC strains. Eleven strains belonged to the highly pathogenic serotypes: O26:H11 (n = 6), O103:H2 (n = 3), and O145:H28 (n = 2). The remaining EPEC strains belong to a large diversity of serotypes listed in Table 4.

It should be noted that some serotypes were isolated at different months and in different types of samples. For example, the nine strains of serotype O100:HNM stx2⁺ were isolated from seven shellfish batches (n = 7; oyster, mussel, and common cockle batches), from one freshwater (n = 1) and from one surface sediment sample (n = 1). Three O154:H31 stx1⁺ and their immotile form were isolated from two shellfish batches (n = 2; oyster and mussel batches) and from one freshwater sample (n = 1). The EPEC serotypes, O108:H21 (n = 3), O113:H6 (n = 4), O15:H2 (n = 2), O153:H2 (n = 3), O23:H8 (n = 2), O26:H11 (n = 6), and O71:H49/HNM (n = 3) were all isolated from shellfish batches and also from freshwater samples (Table 4).

The same serotypes were sometimes isolated from geographically independent sites. For example, serotypes O103:H2, O125:H6, and O26:H11 were isolated from sites 1 and 2 and serotypes O113:H6, O137:H6, and O179:H31 from sites 1 and 3. Finally, the O63:H6 and ONT:H6 serotypes were isolated from all the three sites (Table 4).

## PFGE and MLST Profiles

The genetic relatedness of 26 STEC and 79 EPEC strains was investigated by PFGE and MLST analysis (Supplementary Figure S2). Seventy-nine distinguishable PFGE patterns (PT) and 46 distinguishable sequence types (STs) were obtained. Seven other STs (seven strains, one STEC and six EPEC) were obtained but have not as yet been described. These results demonstrate a high level of genetic diversity among the strains isolated. The highest diversity was observed for the EPEC strains, which represented 8.1% of the PTs identified (64/79) and 71.7% of the STs (33/46).

The STEC serotype O100:HNM (n = 9) presented identical PT (D) and ST (ST933; Figure 1A) despite of their three specificities:

isolated (a) from oyster, mussel, and common cockle batches, freshwater, and superficial sediment samples, (b) from the sites 2 and 3, (c) during the sampling campaigns of May 2013, June 2013, March 2014, and June 2014. With regards to the major serotypes (Figure 1B), the six EPEC O26:H11 strains presented three additional PTs (i.e., L, BV, and AL) and two STs (i.e., ST29 and ST48) isolated from mussel and common cockle batches and freshwater samples. One of those belonging to the ST29 was isolated from the same mussel batch from which the STEC O26:H11 belonging to the ST21 was isolated. A unique PT (I) was observed for the two EPEC O145:H28 (ST not yet described), isolated from mussel and common cockle batches and sampled during the same campaign (June 2013). Both O103:H2 isolated during two different months (i.e., June 2013 and November 2013) had distinguishable PTs (i.e., M and N) and STs (i.e., ST1146 and ST343). Additionally, identical PTs and STs were found among the other STEC and EPEC strains isolated from different types of sample (i.e., shellfish vs. freshwater) or between shellfish batches (i.e., mussel vs. common cockle) and between freshwater samples, often from samples taken from the same sites on the same date (e.g., O153:H2, O108:H21 serotypes; Supplementary Figure S2).

## Biofilm Formation

Biofilm formation by a subset of 13 EPEC and nine STEC strains was evaluated at 18 and 30°C on plastic surface. At both temperatures, strains varied in their ability to form biofilm (OD590 = 0.03 for the lowest, OD590 = 1.9 for the highest). In general, a large number of strains were strongly to moderately adherent and more strains formed a biofilm at 18 than at 30°C (Figure 2). Indeed, 11 of the 22 strains formed strong biofilms [serotypes O2:H32, O149:H1, ONT:H11, O91:H21, O185:H28, O26:H11 (n = 2), O145:H28 (n = 1), O103:H2 (n = 3)], six formed medium biofilms [serotypes O26:H11 (n = 3), O145:H28 (n = 2) and O125:H6] and five formed weak biofilms or no biofilm at all [serotypes O100:HNM, O154:H31, O15:H16, and O26:H11 (n = 2)]. At 30°C, 11 strains formed strong biofilms [serotypes O2:H32, O149:H1, ONT:H11, O91:H21, O185:H28, O145:H28 (n = 3), O103:H2 (n = 2) and O125:H6] but

<footnote><sup>a</sup>Percentage calculated based on the total no. of STEC strains isolated from each site. <sup>b</sup>Percentage calculated based on the total no. of EPEC strains isolated from each site. <sup>c</sup>Percentage calculated based on the total no. of STEC and EPEC strains isolated from the three sites.</footnote>
<figure><img src="image_27.png" /><figcaption>A</figcaption></figure>

<table><caption>B</caption><thead><tr><th>Samples</th><th>Site</th><th>Month-Year</th><th>virulence gene</th><th>Serotype</th><th>Phylogroup</th><th>PT</th><th>ST</th></tr></thead><tbody><tr><td>SFm</td><td>3</td><td>Jun-13</td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>S</td><td>3</td><td>Jun-13</td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFm</td><td>3</td><td>May-13</td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFc</td><td>2</td><td>May-13</td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFm</td><td>2</td><td>May-13</td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>FW</td><td>3</td><td>May-13</td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFo</td><td>3</td><td>May-13</td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFm</td><td>2</td><td>Mar-14</td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFo</td><td>3</td><td>Jun-14</td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>FW</td><td>1</td><td>Nov-13</td><td>eoe</td><td>O125:H6</td><td>B2</td><td>J</td><td>583</td></tr></tbody></table>

<figure><img src="image_28.png" /></figure>

<table><thead><tr><th>Samples</th><th>Site</th><th>Month-Year</th><th>virulence gene</th><th>Serotype</th><th>Phylogroup</th><th>PT</th><th>ST</th></tr></thead><tbody><tr><td>FW</td><td>1</td><td>Nov-13</td><td>eoe</td><td>O26:H11</td><td>B1</td><td>BV</td><td>29</td></tr><tr><td>FW</td><td>1</td><td>Nov-13</td><td>eoe</td><td>O103:H2</td><td>A</td><td>M</td><td>1146</td></tr><tr><td>FW</td><td>1</td><td>Nov-13</td><td>eoe-ehxA</td><td>O26:H11</td><td>A</td><td>AL</td><td>48</td></tr><tr><td>SFm</td><td>1</td><td>Nov-13</td><td>eoe</td><td>O26:H11</td><td>B1</td><td>L</td><td>29</td></tr><tr><td>FW</td><td>1</td><td>Nov-13</td><td>eoe</td><td>O26:H11</td><td>B1</td><td>L</td><td>29</td></tr><tr><td>FW</td><td>1</td><td>Nov-13</td><td>eoe</td><td>O26:H11</td><td>B1</td><td>L</td><td>29</td></tr><tr><td>FW</td><td>2</td><td>Aug-13</td><td>eoe</td><td>O26:H11</td><td>B1</td><td>L</td><td>29</td></tr><tr><td>FW</td><td>2</td><td>Jun-13</td><td>eoe-ehxA</td><td>O103:H2</td><td>B1</td><td>N</td><td>343</td></tr><tr><td>SFc</td><td>2</td><td>Jun-13</td><td>eoe-ehxA</td><td>O145:H28</td><td>D</td><td>I</td><td>nd</td></tr><tr><td>SFm</td><td>2</td><td>Jun-13</td><td>eoe-ehxA</td><td>O145:H28</td><td>D</td><td>I</td><td>nd</td></tr><tr><td>FW</td><td>3</td><td>May-13</td><td>stx1-stx2-ehxA-saa</td><td>O91:H21</td><td>B1</td><td>T</td><td>442</td></tr><tr><td>SFm</td><td>1</td><td>Nov-13</td><td>stx1-eoe-ehxA</td><td>O26:H11</td><td>B1</td><td>AK</td><td>21</td></tr><tr><td>FW</td><td>1</td><td>Feb-14</td><td>eoe</td><td>O103:HNM</td><td>A</td><td>AJ</td><td>3747</td></tr></tbody></table>

FIGURE 1 | Focused dendograms of XbaI PFGE patterns (PTs), characteristics and Sequence Types (STs) of 9 STEC O100:HNM (A) and the 13 major serotypes (B) isolated from freshwater (FW), shellfish (SF) [oyster (o), mussel (m), or cockle (c)] and superficial sediment (S), from the Brittany site (1) and the two Normandy sites (2 and 3). The similarity of PFGE profiles was compared and dendogram was created with the Bionumerics software 7.5 (Applied Maths, Belgium), using the band-based Dice unweighted-pair group method, using average linkages (UPGMA), based on 1% position tolerance. NT: non-typable, NM: non-motile, nd: not determined.

fewer (n = 2) were able to form a medium biofilm (O26:H11 and O145:H28) and nine formed a weak biofilm [serotypes O100:HNM, O15:H16, O154:H31, O26:H11 (n = 6); Figure 2]. Interestingly, all O26:H11 strains formed significantly (Mann-Whitney test) more biofilm (p < 0.05) at 18°C than at 30°C.

## Antibiotic Resistance

Antimicrobial susceptibility testing based on the disk diffusion method was performed on 38 EPEC strains and on all STEC strains (n = 28). Most of the analyzed STEC and EPEC strains [i.e., 85.7% (24/28) and 73.7% (28/38), respectively] were sensitive toward all 16 used antibiotics. Only four STEC strains were resistant to one antibiotic (i.e., Doxycyclin). With regard to EPEC, 7.9% (3/38) of strains [i.e., serotypes O63:H6, O71:H49, and O98:H8] were resistant to one antibiotic (i.e., Doxycyclin or Imipenem) and 13.2% (5/38) of strains were resistant to two antibiotics (i.e., Cefotaxim/Cefoxitin, O63:H6

or Doxycyclin/Bactrim, O26:H11 or Ticarcillin/Amoxicillin, O26:H11, n = 3). One of the O26:H11 serotype was resistant to three antibiotics (i.e., Gentamycin/Ticarcillin/Amoxicillin). Finally, the O2:H45 serotype (n = 1) was resistant to four antibiotics (i.e., Cefalotin/Imipenem/Cefotaxim/Cefoxitin).

## DISCUSSION

To our knowledge, this study is the first to focus on the detection and characterization of environmental STEC and EPEC strains from shellfish-harvesting areas and their upstream watershed. Overall, among the environmental samples analyzed (n = 505), very few STEC (0.2%, n = 28) or EPEC (0.7%, n = 89) strains were obtained from the E. coli that were isolated (n = 12,016) and in comparison with the number of shellfish, water or surface sediment samples that were found to be positive for stx and
<figure><img src="image_29.png" /></figure>

eae genes (54.1%, 273/505). The higher proportion of EPEC than STEC strains isolated from these environmental samples is in agreement with the results obtained in previous studies (Hamilton et al., 2010; Chandran and Mazumder, 2015). For example, only 3.6% EPEC and no STEC were detected among the 24,493 *E. coli* isolated from seawater collected in Santa Catalina Island, CA, USA (Hamilton et al., 2010). Conversely, more STEC strains (6.2%) than EPEC strains (1.8%) were isolated from water samples from the Yeongsan river basin in South Korea (n = 3,480 *E. coli*; Jang et al., 2014). The low level of isolation of STEC or EPEC vs. the high frequency of detection of genetic markers in the analyzed samples has also been observed in various studies focusing on cattle feces, food, and samples from the environment (Miyagi et al., 2001; Bai et al., 2015; Bibbal et al., 2015). The low level of isolation of STEC vs. the high detection of *stx* genes in the environmental samples could be explained by the presence of free *stx*-encoding bacteriophages in the environment (Martinez-Castillo et al., 2013) and the presence of viable but non-culturable or dead bacteria as a result of the stressful conditions (sunlight, salinity, oligotrophy, predation, etc.) in riverine and especially coastal environments (Gourmelon et al., 1997, for review Rozen and Belkin, 2001). The difficulties of isolating these bacteria from environmental samples containing a significant background flora could also contribute to this low recovery of strains (Pradel et al., 2000; Gourmelon et al., 2006).

The strains isolated in coastal areas of Brittany (site 1) and Normandy (sites 2 and 3) present a high diversity of serotypes

as has been previously reported for environmental samples in California or Spain (García-Aljaro et al., 2005; Cooley et al., 2014). A subset of the serotypes isolated in the present study was previously isolated from humans, animals, or the environment. Serotypes such as O8:H14, O26:H11, O76:H19, O91:H21, O103:H2/HNM, O145:H28, and O154:H31 have previously been associated with human infections (Beutin and Fach, 2014). In addition, STEC serotypes O130:H11 and O154:HNM were detected in healthy cattle and waters (Hornitzky et al., 2002), the serotype O157:H16 in dogs, humans, and in the environment (Feng et al., 2012), and the serotype O149:H1 in shellfish (Gourmelon et al., 2006). The STEC O100:HNM was previously detected in swine fecal samples, wild boar feces, and drinking water contaminated by waste water (García-Aljaro et al., 2005; Lienemann et al., 2011; Mora et al., 2012).

In addition, a high genetic diversity among the 105 genotyped strains was observed, with 79 PFGE patterns and 46 distinguishable sequence types in agreement with the high genetic diversity observed by PFGE in other studies for STEC and EPEC strains (Bibbal et al., 2015; Singh et al., 2015). In this study, PFGE was found to be more discriminatory than MLST as previously described for bacteria such as *Salmonella* isolated in Californian coastal waters (Walters et al., 2013). For example, seven strains belonging to ST10 (seven different serotypes) were further discriminated into seven distinct PTs (S, AG, AH, AW, X, G, and Y). The identification of numerous PTs and STs highlights the potential presence of different strains in a same sample and
the presence of genetic diversity between strains belonging to the same serotype (e.g., *E. coli* O26:H11 ST21 and ST29 in the same mussel batch).

The non-detection of *E. coli* from the O157:H7 serotype in the shellfish, water and surface sediment samples investigated (from February 2013 to February 2014 (n = 282, Balière et al., 2015) is in agreement with the low detection or absence of *E. coli* O157 in shellfish and environmental water previously observed (Miyagi et al., 2001; Manna et al., 2008).

Several EPEC strains belonging to the highly pathogenic serogroups (i.e., O26, O103, and O145) were also isolated from some of the shellfish batches or freshwater samples that were analyzed. The STEC O26:H11 ST21 found in a mussel batch was shown to be implicated in STEC infections and has been detected in cattle in Europe (Zweifel et al., 2013).

The EPEC O26:H11 ST29 isolated in this study can be strains with no previous contact with *stx*-bacteriophages or bacteria that have lost the *stx*-bacteriophage either during their passage from their original source to water or shellfish or during their isolation steps. The presence of these bacteria in coastal environments could present a risk to human health as these EPEC could be lysogenized by *stx1*- or *stx2*-converting bacteriophages, which are present in the same environment and could become STEC of the highly pathogenic serotypes. In fact, Bielaszewska et al. (2007) demonstrated that STEC O26 strains can lose their *stx*-bacteriophages and become EPEC O26, and conversely EPEC O26 can be lysogenized by *stx1*- or *stx2*-bacteriophages and become STEC O26. Even if the conversion of strains was found to occur in the digestive tract of different animals (Toth et al., 2003) and in various food matrices (Imamovic et al., 2009), the environment could also provide the conditions for conversion of strains. However, the potential conversion of *E. coli* strains in the environment still needs to be evaluated in more details (Dumke et al., 2006). Interestingly, Solheim et al. (2013) have demonstrated the conversion of an *E. coli* strain (serotype O103:H25) by bacteriophages in a biofilm at 37°C, but also at 20°C.

In addition, another STEC strain isolated in a freshwater sample could present a potential human risk. Indeed, an O91:H21 *E. coli* was found to belong to ST442, a sequence type that had previously been isolated from adult patients in Germany with symptoms that ranged from diarrhea to hemolytic uremic syndrome (Mellmann et al., 2009).

The majority of strains isolated in this study would present low virulence as most of the isolated strains (87.2%) possessed only one of the five virulence genes (*stx1*, *stx2*, *eae*, *ehxA*, or *saa*); i.e., 70.1% of the strains carried the *eae* gene, 7.7% *stx1* and 11.1% *stx2*. The STEC O26:H11 was the only STEC isolate to carry the *eae* gene. These results are in agreement with previous studies describing STEC strains isolated from the environment (García-Aljaro et al., 2005) with the exception of the analysis of water samples from a Californian central coast agricultural region where Cooley et al. (2014) showed that the majority of STEC strains isolated contained *stx1*, *stx2*, and *eae* genes. The low level of isolation of STEC strains carrying the *saa* gene encoding another adherence factor, the STEC autoagglutinating adhesion, or the gene *ehxA* encoding enterohemolysin A was in

agreement with the results obtained from water samples in Spain by García-Aljaro et al. (2005).

Most of the STEC strains in this study were classified into the A, B1, and D phylogroups. Phylogroups A and B1 were also the main phylogroups of environmental STEC strains isolated by García-Aljaro et al. (2005) in Spain. In the Yeongsan river basin of South Korea, STEC strains isolated belonged mainly to phylogroup D and to a lesser extent to phylogroups A, B1, and B2 (Jang et al., 2014). In Brittany and Normandy, overall, the EPEC strains belonged mainly to phylogroups B1 and B2 and to a lesser extent to A and D. EPEC strains isolated from water samples in South Korea belonged mainly to the B2 phylogroups (Jang et al., 2014). The frequent isolation of *E. coli* from B1 phylogroup in the present study is in agreement with recent data showing that environmentally persistent *E. coli* belong mainly to the B1 phylogroup (Berthe et al., 2013).

Phenotypic differences in the ability to form biofilms among tested strains underline the genetic diversity of STEC or EPEC strains. Our study demonstrates that more than half of the tested strains (17/22) were able to form biofilms on polystyrene at 18 or 30°C, and most of these strains were able to form strong biofilm at 18°C, a temperature close to marine environment condition. A similar result had been observed previously when *E. coli* K12 biofilm was grown at low temperature (White-Ziegler et al., 2008). It has been shown that low temperatures (<30°C) promote the expression of genes associated with biofilm development, including genes involved in curli (csgA and *mlrA*) or cellulose (*yaiC*) production (Olsen et al., 1989; Arnqvist et al., 1992; White-Ziegler et al., 2008). Interestingly, although O26:H11 strains formed weaker biofilms than other strains, they formed significantly stronger biofilms at 18°C than at 30°C. Also, all but one O26:H11 strains were negative for *pgaA*, a gene coding for the export of poly-N-acetyl-D-glucosamine (PGA) that promotes biofilm formation (Itoh et al., 2008; data not shown). In addition, *pgaA* sequence is also absent in the sequenced genomes of STEC O26 strains available in GenBank. In conclusion, the ability of strains to form a biofilm might contribute to their persistence in coastal environments.

This study highlights the presence of a specific geographic distribution of some of the STEC and EPEC serotypes and a persistence of some of these serotypes in the coastal environments from Brittany and Normandy investigated in this study. The isolation of the serotype O100:HNM positive for *stx2* (PT D and ST993), at different dates (over a period of 1 year), in shellfish, waters and surface sediments from both sites in Normandy (sites 2 and 3), but its absence in Brittany (site 1), highlights potential specific contamination sources in these region and the higher persistence of some of these specific strains. This had previously been shown in water samples from California for O157 strains isolated up to 19 months apart by Cooley et al. (2014). The various livestock breeding in the three watersheds, i.e., mainly swine, poultry and bovine in Brittany and sheep, bovine and swine in Normandy, could explain differences in strain detection at specific sites. A potential explanation of the frequent isolation of the STEC O100:HNM is the high carriage of this *E. coli* strain in the animals in the upstream watersheds. Bibbal et al. (2015) have identified farms harboring STEC bovine carriers, highlighting the
fact that STEC of a given serotype could be carried by several animals belonging to the same farm. A probable prediction of the presence of these STEC strains in the coastal environment is their re-introduction to the water and consequently to shellfish from animal reservoirs, which enables persistence at high titer for months (Cooley et al., 2014). Another explanation could be that they are present in surface sediments in which a better persistence could occur and then they are re-introduced to the water and then filtered and accumulated by shellfish. The evaluation of the persistence of STEC and EPEC (especially those isolated several times in this study, i.e., *E. coli* O100 and *E. coli* O26) in freshwaters and seawaters and in shellfish needs to be studied to better understand their frequent detection in such shellfish-harvesting areas. Several studies have been carried out to evaluate the persistence of STEC in water or superficial sediments and these have shown that some *E. coli* strains are able to persist in the environment for periods of a few days to several months (Fremaux et al., 2010) and that the persistence could be variable according to the serotypes (Ma et al., 2014).

This study critically evaluated the nature of STEC and EPEC strains present in coastal environments. Knowledge of strains circulating in the environment is crucial to the investigation of potential new STEC serotypes and their human health risk. These results confirm that the environment is a reservoir for these strains. The presence of both EPEC strains and *stx*-converting bacteriophages in the same samples could lead to new pathogenic *E. coli*.

The risk of a human infection by STEC caused by shellfish consumption seems to be limited for two reasons. First, a depuration step or relaying step has to be performed before

shellfish from category B and C areas, respectively, reach market. Secondly, STEC were present in only a few samples and the majority of STEC strains lacked genes associated with high human virulence, such as *eae*, and few of the STEC isolated in this study have previously been shown to be involved in human infections.

## ACKNOWLEDGMENTS

This work was funded by the European Regional Development Fund Interreg IVA Programme, as part of the collaborative project RiskManche. The thesis of CB was supported by a grant from Ifremer and the Agence de l'Éau Loire-Bretagne. We thank Clémence Balière from the University of Caen and Céline Courroux from Hôpital Robert-Debré, for their participation in the sampling and technical assistance and Dominique Hervio-Heath (Ifremer) and Huw Taylor (University of Brighton, UK) for their critical review of the manuscript. Work in the LREC-USC-laboratory was financed by the grant CN2012/303 from Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) and The European Regional Development Fund, ERDF).

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2015.01356

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Balière. Rincé, Blanco, Dahbi, Harel, Vogeleer, Giard, Mariani-Kurkdjian and Gourmelon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
<figure><img src="image_30.png" /></figure>

**Figure 1S:** Map of the three studied shellfish-harvesting sites on the French coast of within the Eastern English Channel. Localization of the Brittany site (site 1) and the two Normandy sites (site 2 and site 3).
<figure><img src="image_31.png" /></figure>

PFGE-Xbal
Dice (Opt 0%, Tol 1%)

<table><thead><tr><th>Samples</th><th>Site</th><th>Month</th><th>Year</th><th> virulence gene</th><th>Serotype</th><th>Phylogroup</th><th>PT</th><th>ST</th></tr></thead><tbody><tr><td>FW</td><td>1</td><td>Jul-14</td><td></td><td>eeae</td><td>O145:H34</td><td>B2</td><td>AV</td><td>526</td></tr><tr><td>SFm</td><td>1</td><td>Nov-13</td><td></td><td>eeae</td><td>O113:H6</td><td>B2</td><td>AC</td><td>121</td></tr><tr><td>FW</td><td>3</td><td>Sep-14</td><td></td><td>eeae</td><td>ONT:H6</td><td>B2</td><td>AT</td><td>28</td></tr><tr><td>FW</td><td>1</td><td>Aug-14</td><td></td><td>eeae</td><td>ONT:H6</td><td>B2</td><td>AT</td><td>28</td></tr><tr><td>SFc</td><td>1</td><td>Nov-13</td><td></td><td>eeae</td><td>O113:H6</td><td>B2</td><td>AD</td><td>1583</td></tr><tr><td>FW</td><td>1</td><td>Nov-13</td><td></td><td>eeae</td><td>O103:H2</td><td>A</td><td>M</td><td>1146</td></tr><tr><td>FW</td><td>1</td><td>Aug-14</td><td></td><td>eeae</td><td>O85:HNM</td><td>B2</td><td>AQ</td><td>803</td></tr><tr><td>FW</td><td>1</td><td>Oct-14</td><td></td><td>eeae</td><td>O85:H31</td><td>B2</td><td>AQ</td><td>803</td></tr><tr><td>SFm</td><td>3</td><td>May-13</td><td></td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFc</td><td>2</td><td>May-13</td><td></td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFo</td><td>3</td><td>May-13</td><td></td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFm</td><td>3</td><td>Jun-13</td><td></td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>S</td><td>3</td><td>Jun-13</td><td></td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFm</td><td>2</td><td>Mar-14</td><td></td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFo</td><td>3</td><td>Jun-14</td><td></td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>SFc</td><td>1</td><td>Jul-14</td><td></td><td>eeae</td><td>O91:H10</td><td>B2</td><td>BS</td><td>641</td></tr><tr><td>SFm</td><td>2</td><td>May-13</td><td></td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>FW</td><td>3</td><td>May-13</td><td></td><td>stx2</td><td>O100:HNM</td><td>A</td><td>D</td><td>993</td></tr><tr><td>FW</td><td>1</td><td>Nov-13</td><td></td><td>eeae</td><td>O125:H6</td><td>B2</td><td>J</td><td>583</td></tr><tr><td>FW</td><td>2</td><td>Nov-13</td><td></td><td>eeae</td><td>O63:H6</td><td>B2</td><td>V</td><td>122</td></tr><tr><td>FW</td><td>2</td><td>Sep-14</td><td></td><td>eeae</td><td>O125:H6</td><td>B2</td><td>BF</td><td>583</td></tr><tr><td>FW</td><td>1</td><td>Aug-14</td><td></td><td>eeae</td><td>ONT:H8</td><td>B1</td><td>BQ</td><td>327</td></tr><tr><td>FW</td><td>2</td><td>Dec-13</td><td></td><td>eeae</td><td>O2:H45</td><td>B2</td><td>P</td><td>1907</td></tr><tr><td>FW</td><td>1</td><td>May-13</td><td></td><td>stx1-stx2-saa</td><td>ONT:H11</td><td>B1</td><td>B</td><td>295</td></tr><tr><td>FW</td><td>2</td><td>Apr-14</td><td></td><td>stx1</td><td>ONT:H10</td><td>B1</td><td>CC</td><td>1258</td></tr><tr><td>SFc</td><td>1</td><td>Mar-14</td><td></td><td>stx1</td><td>O28:H1</td><td>D</td><td>AO</td><td>132</td></tr><tr><td>SW</td><td>1</td><td>Apr-14</td><td></td><td>eeae</td><td>O20:HNT</td><td>A</td><td>BR</td><td>3747</td></tr><tr><td>FW</td><td>2</td><td>Aug-14</td><td></td><td>stx1</td><td>O76:H19</td><td>B1</td><td>AZ</td><td>675</td></tr><tr><td>FW</td><td>1</td><td>Aug-13</td><td></td><td>eeae</td><td>O167:H3</td><td>D</td><td>E</td><td>2558</td></tr><tr><td>FW</td><td>3</td><td>Jul-14</td><td></td><td>stx2</td><td>O8:H19</td><td>B1</td><td>AY</td><td>162</td></tr><tr><td>SFc</td><td>2</td><td>Aug-14</td><td></td><td>eeae</td><td>O86:H31</td><td>D</td><td>CB</td><td>2569</td></tr><tr><td>FW</td><td>1</td><td>Nov-14</td><td></td><td>eeae</td><td>O159:H7</td><td>B1</td><td>BK</td><td>nd</td></tr><tr><td>FW</td><td>3</td><td>Nov-13</td><td></td><td>eeae</td><td>O108:H21</td><td>B1</td><td>AN</td><td>337</td></tr><tr><td>SFc</td><td>2</td><td>Jan-14</td><td></td><td>eeae</td><td>O108:H21</td><td>B1</td><td>AN</td><td>337</td></tr><tr><td>SFm</td><td>3</td><td>Aug-14</td><td></td><td>eeae</td><td>O146:H21</td><td>B1</td><td>BH</td><td>442</td></tr><tr><td>SFc</td><td>2</td><td>Sep-14</td><td></td><td>eeae</td><td>O146:H6</td><td>B1</td><td>BI</td><td>442</td></tr><tr><td>FW</td><td>1</td><td>Apr-13</td><td></td><td>stx1</td><td>O149:H1</td><td>D</td><td>A</td><td>132</td></tr><tr><td>FW</td><td>1</td><td>Apr-13</td><td></td><td>stx1</td><td>O149:HNM</td><td>D</td><td>A</td><td>132</td></tr><tr><td>SFc</td><td>2</td><td>Aug-14</td><td></td><td>eeae</td><td>O23:H8</td><td>B1</td><td>BD</td><td>327</td></tr><tr><td>SW</td><td>1</td><td>Dec-14</td><td></td><td>eeae</td><td>ONT:H6</td><td>B2</td><td>BY</td><td>28</td></tr><tr><td>FW</td><td>2</td><td>Oct-13</td><td></td><td>eeae</td><td>O51:HNM</td><td>B2</td><td>AB</td><td>589</td></tr><tr><td>FW</td><td>1</td><td>Jan-14</td><td></td><td>eeae</td><td>O39:HNM</td><td>B1</td><td>O</td><td>39</td></tr><tr><td>FW</td><td>1</td><td>Nov-13</td><td></td><td>eeae</td><td>O8:H14</td><td>B2</td><td>AA</td><td>4178</td></tr><tr><td>FW</td><td>1</td><td>Dec-13</td><td></td><td>stx1-stx2-ehxA-saa</td><td>O130:H11</td><td>B1</td><td>U</td><td>nd</td></tr><tr><td>SW</td><td>1</td><td>Feb-14</td><td></td><td>stx2</td><td>O2:H32</td><td>A</td><td>X</td><td>10</td></tr><tr><td>FW</td><td>3</td><td>Nov-13</td><td></td><td>eeae</td><td>O113:H6</td><td>B2</td><td>BC</td><td>121</td></tr><tr><td>SFo</td><td>3</td><td>Aug-14</td><td></td><td>eeae</td><td>O113:H6</td><td>B2</td><td>BE</td><td>121</td></tr><tr><td>FW</td><td>3</td><td>Mar-14</td><td></td><td>eeae</td><td>O103:H25</td><td>B1</td><td>N</td><td>343</td></tr><tr><td>FW</td><td>2</td><td>Jun-13</td><td></td><td>eeae-ehxA</td><td>O103:H2</td><td>B1</td><td>N</td><td>343</td></tr><tr><td>FW</td><td>1</td><td>Apr-14</td><td></td><td>eeae</td><td>ONT:H8</td><td>B1</td><td>BP</td><td>327</td></tr><tr><td>FW</td><td>2</td><td>Oct-13</td><td></td><td>eeae</td><td>O29:H19</td><td>B1</td><td>Z</td><td>517</td></tr><tr><td>FW</td><td>1</td><td>Nov-13</td><td></td><td>eeae</td><td>O26:H11</td><td>B1</td><td>BV</td><td>29</td></tr><tr><td>FW</td><td>1</td><td>Oct-14</td><td></td><td>eeae</td><td>ONT:H31</td><td>B1</td><td>AR</td><td>40</td></tr><tr><td>FW</td><td>1</td><td>Jan-14</td><td></td><td>eeae</td><td>O179:H31</td><td>B2</td><td>AE</td><td>1092</td></tr><tr><td>SFm</td><td>2</td><td>Feb-13</td><td></td><td>eeae</td><td>O5:H40</td><td>A</td><td>S</td><td>10</td></tr><tr><td>FW</td><td>1</td><td>Nov-13</td><td></td><td>eeae</td><td>O116:H20</td><td>A</td><td>AG</td><td>10</td></tr><tr><td>SFm</td><td>1</td><td>Nov-13</td><td></td><td>eeae</td><td>O28:H16</td><td>A</td><td>AH</td><td>10</td></tr><tr><td>FW</td><td>1</td><td>Jul-14</td><td></td><td>eeae</td><td>O157:H16</td><td>A</td><td>AW</td><td>10</td></tr><tr><td>FW</td><td>1</td><td>Nov-14</td><td></td><td>stx2-ehxA-saa</td><td>O63:H6</td><td>B1</td><td>BM</td><td>2105</td></tr><tr><td>FW</td><td>1</td><td>Jan-14</td><td></td><td>eeae</td><td>O153:H21</td><td>B1</td><td>AI</td><td>40</td></tr><tr><td>FW</td><td>2</td><td>Nov-13</td><td></td><td>eeae</td><td>O9:HNM</td><td>A</td><td>BB</td><td>nd</td></tr><tr><td>SFo</td><td>3</td><td>Aug-14</td><td></td><td>eeae</td><td>O88:H8</td><td>B1</td><td>BG</td><td>590</td></tr></tbody></table>
<figure><img src="image_32.png" /><figcaption>**Figure 2S:** Dendograms of *XbaI* PFGE patterns (PT), characteristics and Sequence Type (ST) of STEC and EPEC strains isolated from freshwater (FW), shellfish (SF) [oyster (o), mussel (m) or cockle (c)] and superficial sediment (S), from the Brittany site (1) and the two Normandy sites (2 and 3). The similarity of PFGE profiles was compared and dendogram was created with the Bionumerics software 7.5 (Applied Maths, Belgium), using the band-based Dice unweighted-pair group method, using average linkages (UPGMA), based on 1% position tolerance. NT: non-typable, NM: non-motile, nd: not determined.</figcaption></figure>
# Chapitre 4 : PROFIL DE VIRULENCE DES E. COLI PRODUCTEURS DE SHIGA-TOXINES ET DES E. COLI ENTEROPATHOGENES ISONES EN ZONES CONCHYLICOLES FRANCAISES

## A. Contexte de la troisième partie de l'étude

Le chapitre précédent a montré que des souches STEC et EPEC étaient présentes dans l'environnement littoral et qu'elles possédaient des gènes de virulence codant pour les Shiga-toxines, pour l'intimine, l'entérohémolysine A et l'alpha-hémolysine. Toutefois, dans des études récentes, d'autres facteurs de virulence impliqués dans l'adhésion, la production d'autres toxines et dans la virulence ont été mis en évidence dans des souches de E.coli pathogènes (Coombes et al., 2008, Bugarel et al., 2010, Delannoy et al., 2013, Chui et al., 2015). Aussi, pour avoir une meilleure évaluation de la virulence de ces souches environnementales, il nous est apparu important d'approfondir la recherche des gènes de virulence présents dans ces souches.

Au regard des progrès actuels, nous avons voulu poursuivre la caractérisation des souches STEC et EPEC isolées à partir des échantillons environnementaux présentés dans le chapitre N°3 (Publication N°2) en axant ce 4ème chapitre sur le potentiel de virulence des souches issues du littoral et des eaux en amont par une approche moléculaire.

Dans un premier temps, nous avons sélectionné dans la littérature des gènes codant pour des facteurs de virulence et des facteurs impliqués dans la formation de biofilms présents chez des souches de E. coli STEC et EPEC.

Ce travail a été réalisé en collaboration avec le laboratoire de sécurité alimentaire de l'ANSES, Plateforme IdentyPath, de l'Université Paris-Est, Maisons-Alfort, où ont été réalisées ensuite les PCR haut débit sur les souches environnementales.

Le choix des gènes codant des facteurs impliqués dans la formation de biofilms a été réalisé en collaboration avec le Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Centre de Recherche d'Infectiologie Porcine et Avicole, Université de Montréal, Saint-Hyacinthe, QC, Canada.

Soixante-quinze gènes cibles ont été sélectionnés en association avec leur rôle dans la virulence chez E. coli, leur implication dans des pathologies graves chez l'Homme et les animaux et leur capacité à être des marqueurs de différenciation des souches EHEC, des STEC et des EPEC. Dix gènes en association avec la capacité à former des biofilms ont également été sélectionnés. Ces gènes ont été recherchés par PCR haut débit au moyen de la technologie Fluidigm Puce 96.96 marqueur EvaGreen.
De plus, les variants des gènes *stx* et *eae* ont été caractérisés au laboratoire Ifremer de Plouzané et à l'Université de Caen par PCR et par séquençage pour les variants non identifiés par les systèmes PCR.

Le manuscrit présenté ci-après expose les résultats obtenus à partir d'une sélection de 28 souches STEC et de 75 souches EPEC jugées différentes après l'acquisition de l'ensemble des paramètres de caractérisation présentés dans le chapitre N°3 (Publication N°2) et d'une étude précédente concernant la détection et l'isolement de STEC dans d'autres coquillages du littoral français (n=4) (Gourmelon et al., 2006).

Ce travail a permis de mettre en évidence par exemple :

Pour les STEC :

* La présence de 17 profils de virulence différents, avec de 2 à 47 gènes détectés par souche en plus des gènes *eae* et/ou *stx1* et/ou *stx2*

* La souche EHEC O26:H11 *stx1*⁺ *eae*⁺, isolée dans un lot de moules par la méthode ISO-TS-13136 (Publication N°1), dispose de 45 gènes de virulence supplémentaires

* La souche STEC de sérotype O100:HNM *stx2*⁺, la plus fréquemment isolée dans les échantillons environnementaux, ne possède que 2 gènes de virulence supplémentaires

* Peu de gènes associés aux OI-122, OI-43-48, OI-57 et OI-71 sont détectés dans les souches STEC, hormis le O26:H11 *stx1*⁺ *eae*⁺, soit entre 1 et 2 gènes sur les 20 gènes recherchés

* La détection de plusieurs gènes codant des protéines d'adhésion pour les souches STEC LEE-négatives

Pour les EPEC :

* La présence de 56 profils de virulence différents, avec de 11 à 50 gènes détectés par souche en plus du gène *eae*

* Les souches appartenant aux sérotypes O26:H11, O157:H7, O103:H2, O145:H28 et O103:H25 possèdent entre 33 et 49 gènes de virulence supplémentaires

* Entre 1 et 19 gènes de virulence associés aux OI-122, OI-43-48, OI-57 et OI-71 (sur un total de 20 gènes) sont détectés chez 67 souches EPEC (dont les sérotypes O26:H11, O157:H7, O103:H2, O145:H28 et O103:H25 avec entre 13 et 19 gènes)

En conclusion, cette étude est, à notre connaissance, la première qui évalue la virulence de souches STEC et EPEC isolées d'échantillons de l'environnement littoral par une approche moléculaire par PCR haut débit. Au regard des 75 gènes de virulence sélectionnés dans cette étude et de la caractérisation des variants des gènes *stx* et *eae*, plusieurs souches EPEC et quelques souches STEC ont montré des combinaisons de gènes caractéristiques de souches associées à des pathologies humaines et animales. A ce jour, à notre connaissance, aucune toxi-infection alimentaire liée à la consommation de coquillages contaminés par des STEC ou des EPEC n'a été recensée dans le monde.
La réglementation sanitaire appliquée aux zones de production conchylicole (purification des coquillages en catégorie B avant commercialisation, par exemple) est surement un acteur important pour expliquer cette dernière observation. L'acquisition de ces données sur la virulence des souches STEC et EPEC circulant en zone littorale permet d'enrichir les connaissances sur la prévalence de ce type de *E. coli* pathogènes dans cet environnement.
# Molecular profiling of STEC and EPEC strains isolated from French coastal environments

Balière C.<fnref n="1" />, Rincé A.<fnref n="2" />, Delannoy S<fnref n="3" />, Fach P<fnref n="3" />, Gourmelon M.<fnref n="1" />

<footnote n="1">¹ Laboratoire Santé Environnement et Microbiologie, Unité Santé, Génétique et Microbiologie des mollusques, Département Ressources Biologiques et Environnement, IFREMER, Plouzané, France</footnote>

<footnote n="2">² U2RM EA4655 Stress/Virulence, Normandie-Université; UCBN, Caen, France</footnote>

<footnote n="3">³ Université Paris-Est, ANSES Food Safety Laboratory, Platform IdentyPath, Maisons-Alfor</footnote>
Molecular profiling of STEC and EPEC strains isolated from French
coastal environments

Balière C.¹, Rincé A.², Delannoy S.³, Fach P.³, Gourmelon M.¹*

¹ Laboratoire Santé Environnement et Microbiologie, Unité Santé, Génétique et Microbiologie des mollusques, Département Ressources Biologiques et Environnement, IFREMER, Plouzané, France

² U2RM EA4655 Stress/Virulence, Normandie-Université; UCBN, Caen, France

³ Université Paris-Est, ANSES Food Safety Laboratory, Platform IdentyPath, Maisons-Alfort

**Running title:** Molecular profiling of environmental *E. coli*

**Keywords:** *E. coli*, STEC; EPEC, environment, virulence genes, high-throughput real-time PCR method

*Corresponding author:

Michèle Gourmelon

Laboratoire Santé Environnement et Microbiologie LSEM, Ifremer, CS10070 Plouzané,
France

E-mail: Michèle.Gourmelon@ifremer.fr
ABSTRACT

Shiga toxin-producing *Escherichia coli* (STEC) and enteropathogenic *E. coli* (EPEC) strains
may be responsible for foodborne infections in humans. Twenty-eight STEC and 75 EPEC
previously isolated from French shellfish-harvesting areas and their watersheds and belonging
to 68 distinguishable serotypes were characterized in this study. High-throughput real-time
PCR was used to search for the presence of 75 *E. coli* virulence-associated gene targets and
genes encoding Shiga-toxin (*stx*) and intimin (*eae*) were subtyped using PCR tests and DNA
sequencing, respectively. Results showed a high diversity between strains with 17 unique
virulence gene profiles for STEC and 56 for EPEC. Six STEC and 15 EPEC strains were
found to display a large number or a particular combination of virulence genetic markers and
the presence of *stx* and/or *eae* variants suggesting their potential pathogenicity for humans.
From them, a O26:H11 *stx1a eaeβ1* strain was associated with a large number of virulence-
associated genes (i.e. 47) including genes carried on the locus of enterocyte effacement (LEE)
or other pathogenicity islands such as OI-122, OI-71, OI-43/48, OI-50, OI-57, and HPI. One
O91:H21 STEC containing 4 *stx* variants (*stx1a*, 2a, 2c, and 2d) was found to possess genes
associated with pathogenicity islands OI-122, OI-43/48, and OI-15. From EPEC strains
harboring a large number of virulence genes (between 34 and 50), eight belonged to the
serotypes O26:H11, O103:H2, O103:H25, O145:H28, O157:H7, and O153:H2. This study
clearly highlights that if the majority of strains isolated from coastal environment are
potentially weakly pathogenic, then some of these are likely to be more pathogenic.
INTRODUCTION

*Escherichia coli* is a commensal aerobic bacterium of the warm-blooded animal intestinal microbiota and is used as a fecal indicator in the environment to classify shellfish-harvesting and bathing areas (1). However, *E. coli* can become pathogenic through the acquisition of mobile genetic elements such as bacteriophages, pathogenicity islands, and plasmids. Among pathogenic *E. coli*, are the Shiga toxin-producing *Escherichia coli* (STEC) and enteropathogenic *E. coli* (EPEC).

STEC can cause infections ranging from uncomplicated diarrheas to hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Several STEC serotypes have been involved in numerous food-borne outbreaks worldwide (2, 3), and these strains have been identified as enterohemorrhagic *E. coli* (EHEC). Although *E. coli* O157:H7 has been the main serotype implicated in HC and HUS since the early 1980s, recent studies have shown that non-O157 serotypes are also responsible for numerous human STEC infections. The most commonly implicated serogroups associated with human STEC infections in the US are O26, O45, O103, O111, O121, O145, and O157 (4) whereas in Europe five major EHEC serogroups (Top 5; O157, O26, O103, O111, O145) dominate (5).

The primary virulence factor of STEC is the Shiga-toxin, encoded by a lambdoid bacteriophage, which inhibits host cell protein synthesis (6). Within the two major types of Shiga-toxin, namely Stx1 and Stx2, three subtypes of stx1 gene (stx1a, stx1c, and stx1d) and seven subtypes of stx2 gene (stx2a, 2b, 2c, 2d, 2e, 2f, and 2g) have been identified (7). Specific stx subtypes are involved in human infections (e.g. stx2a and stx2c are more often isolated from patients with HUS) (8) whereas others are related to non-human animal infections (e.g. stx2e causing edema disease in pigs; 9). Certain stx-subtypes present within the strain may indicate its source: stx2c is more prevalent in cattle (10) whereas stx2f is mainly associated with pigeon sources (11).

The STEC strains implicated in the major cases of human infection (also referred to as 'typical EHEC') possess the locus of enterocyte effacement (LEE) pathogenicity island, which is involved in attaching and effacing lesions on intestinal epithelial cells (12). Eighteen types and nine subtypes of the *eae* gene, namely α, α2, β1 to -3, γ1, γ2, δ, ε, ε2 to -4, ζ, η, η2, θ, ι, ι2, κ, λ, μ, ν, ξ, ο, π, ρ and σ
have been deposited in the Genbank database (13). The *eae* subtypes are responsible for some host
tissue cell tropisms (14) and are related to human infections; *eae* subtypes β, γ, ε and θ are those most
frequently associated with human infections (15, 16).

However, the emergence of human infections linked to LEE-negative STEC indicates that this
pathogenicity island is not the only factor responsible for the adherence of bacteria and suggests the
presence of other virulence factors carried by other pathogenicity islands or plasmids (17). Additional
proteins associated with attachment were proposed as adhesive factors. For example, Paa is involved
in intimate attachment of the bacteria to enterocytes and induced typical A/E (Attaching and Effacing)
lesions in the pig ileal (18). EhaA, an enterohemorrhagic *E. coli* autotransporter, is involved in
attachment to biotic and abiotic surfaces (19). Saa encodes an autoagglutinating adhesion unique to
LEE-negative STEC (20). Lpf, long polar fimbriae facilitates attachment of the bacteria to murine
Peyer's patches (21).

In addition to genes located on the LEE, a large number of non-LEE effector genes located on other
pathogenicity islands were identified in strains responsible for human infections (i.e. *nleA*, *nleB*, *nleC*,
*nleD*, *nleE*, *nleG*, etc.). These genes are involved in various functions, such as inhibiting phagocytosis,
disruption of host innate immune responses, blockage of cell division, etc. (22).

Besides Shiga-toxins, other hemolysins or toxins have been identified in the pathogenesis of STEC
strains. These include, for example, the enterohemolysin, encoded by the *ehxA* gene, linked to
cytotoxic effects on endothelial cells (23), the enteroaggregative *E. coli* (EAEC) heat-stable
enterotoxin encoded by the *astA* gene (24) or the alpha hemolysin encoded by the *hlyA* gene (25).

Among the virulence factors, the presence of various combinations of type III effector genes, toxin-
producing genes or adhesin-producing genes present in pathogenicity islands (OI-122, OI-43/48, OI-
57, OI-71, and HPI), plasmids or chromosome were used to discriminate EHEC from STEC strains
and to perform a molecular risk assessment based on the presence of several virulence genes in STEC
strains (26-30).
Among pathogenic *E. coli*, there are also EPECs. These are involved in the majority of infantile watery diarrheas in low-income countries but rarely in adult diarrhea (31). EPEC strains are characterized by the presence of the LEE, containing the *eae* gene, as described above. They are classified into typical EPEC and atypical EPEC, based on the presence of the EPEC adherence factor (EAF) plasmid. The plasmid harbors the *bfp* operon encoding the Bundle-Forming Pilus, involved in the initial adherence of strains to intestinal epithelial cells (31). Twelve serogroups have been recognized as EPEC by the World Health Organization: O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158 (32).

EPEC could possess the majority of the virulence genes described above other than *stx* genes. Furthermore, EPEC strains could be lysogenized by *stx*-converting bacteriophages and consequently could become EHEC. Conversely, STEC strains possessing the *eae* gene could lose the *stx*-converting bacteriophage and become EPEC or EHEC-like strains (33).

The main reservoir of STEC is cattle (10). However, other animals, such as sheep, goats, swine, birds, wild animals, and humans, can also harbor STEC strains in their digestive tract (34-36). For typical EPEC, the only reservoir is humans whereas for atypical EPEC, both humans and other animals can be reservoirs (36). Contamination of the environment by STEC and EPEC may occur through the spreading of livestock manure, animal wastes on pastures, via wastewaters from slaughterhouses or from treatment plant effluents, and by wildlife (37, 38). Only a few studies have focused on the prevalence and description of STEC and EPEC strains in the environment and particularly on the virulence gene profiles of such strains (39-42). Thus, the authors were prompted to characterize STEC and EPEC strains isolated from French shellfish-harvesting areas.

In the present study, high-throughput microfluidic real-time PCR methods, which had been previously developed and used to investigate the pathogenic potential of *E. coli* strains isolated mainly from animals feces (9), from carcasses of cattle (43), and from food and animals (44, 45), were used. During a two-year study, STEC and EPEC strains were isolated from three French shellfish-harvesting areas
(from shellfish, sediment, and seawater samples) and their watersheds (from river water samples),
(46). In addition to the stx and eae genes already investigated, 75 E. coli virulence-associated gene
targets were examined in these strains using high throughput microfluidic PCR.

MATERIALS AND METHODS

**Bacterial strains and DNA extraction.** Most of the strains used in this study were isolated between February 2013 and January 2015 from three French shellfish-harvesting areas and their watersheds located on the English Channel coast (46), except three strains, which were isolated from other French shellfish-harvesting areas during 2006 (47). A total of 28 STEC and 75 EPEC strains belonging to 68 distinguishable serotypes isolated from three types of shellfish (oysters, mussels, and cockles), from freshwater, seawater and surface sediment samples were investigated. The determination of their serotype, phylogroups and MLST sequence type has been previously described (46, 47). After cultivation of bacteria on Trypton-Bile-X-glucuronide agar (TBX) (AES chemunex, Bruz, France) at 37°C for 24 hours, DNA was extracted with the InstaGene™ Matrix kit (Bio-Rad, Nanterre, France) according to the manufacturer's instructions.

**Characterization of the stx subtypes.** STEC strains (n=28) were previously identified by Baliere *et al.* (46) by PCR using the stx primers and probes described by Perelle *et al.* (48) and the stx-subtyping was performed here using the PCR tests described by Scheutz *et al.* (7) for targeting the stx1a, stx1c and stx1d subtypes and stx2a, stx2b, stx2c, stx2d, stx2e, stx2f and stx2g subtypes.

**Characterization of the eae subtypes.** *E. coli* eae-positive strains (n=76) were previously identified by Baliere *et al.* (46) with the eae primers and probe described by Nielsen and Andersen (49). The eae-subtypes were determined here by sequencing 311 to 722bp amplicons of the 3' variable region of the eae gene. Amplicons were obtained with the universal EAE-F and EAE-RB primers, which target the 3' variable region of the *eae-α1*, -α2, -β1, -β2, -γ2, -θ, -δ, -κ, -ζ and -ι subtypes or with primers targeting the 3' variable region of the *eae-ε*, -η, -ξ, -μ and -ν subtypes (Table 1). The amplicons obtained following the PCR program described by Blanco *et al.* (50) were then subjected to gel
electrophoresis before being sequenced in both directions with the corresponding primers and using
the fluorescent dye terminator Sanger method ABI3730 (Applied Biosystem) by Eurofins Genomics
(Ebersberg, Germany). DNA sequences were edited using the BioEdit program (52) and were
compared with the GenBank database. The first valid published sequence with 100% similarity was
chosen to identify the *eae*-variant.

Nine *E. coli* strains, which were PCR-negative for these primer combinations, were chosen for sequencing the entire *eae* gene. Two overlapping DNA fragments of the *eae* gene were PCR amplified using primers eae-F1 and escD-R1 on the one hand and cesT-F3 and eae-R3 on the other hand (Table 1). The PCR program included a 10 min initial denaturing step at 94°C, followed by 35 cycles of amplification (94°C for 20 s, 56°C for 20 s, and 72°C for 90 s) and a final extension step at 72°C for 5 min. The PCR amplicons were then subjected to gel electrophoresis and purified using the Nucleospin Extract II Kit (Macherey-Nagel, Dünen, Germany) before being sequenced as described above.

**High-throughput real-time PCR system.** The BioMark real-time PCR system (Fluidigm, San Francisco, CA) was used for high-throughput microfluidic real-time PCR amplification using the 96.96 dynamic arrays (Fluidigm). Amplifications were performed in accordance with the recommendations of the manufacturer, using the EvaGreen DNA binding dye (Biotium Inc., Hayward, CA) followed by a melting curve analysis. The BioMark real-time PCR system was used with the following thermal profile: 95°C for 10 min (enzyme activation) followed by 35 cycles of 95°C for 15 s and 60°C for 1 min (annealing of primers and amplification step).

Seventy-five *E. coli* virulence-associated gene targets were selected according to their role in pathogenesis, their ability to be associated with human and non-human animal illness and because they had previously been shown to be useful for the characterization of STEC and EPEC strains, isolated mainly from finishing swine (9), human patients and animals feces, food and animals (44, 45), carcasses of cattle originating from different farms and food (43) and adult and children's feces (27, 29, 30, 53).
Virulence gene targets were classified in five groups according to their function; adhesion group
[eibG, iha, saa, toxB, orfA, orfB, paa, stcE, sab, efa1/LifA, bfpA, espP, F6/F987P (fasA), F18/F107
(fedA), F41 (fim41a), lpfAO157, lpfAO26, lpfAO113, ehaA, epeA; n=20], the type III secretion system
group (espB, espD, espA, tir, espZ, escC, espG, escD, escN, escV, escJ, nleB, nleE, nleF, nleG, nleH1-
2, nleA, espM1, nleC, nleD, nleH1-1, nleG2, nleG5-1, nleG5-2, nleG6-2, espV, espK, espN, espJ,
espM2; n= 30), toxin group (subA, ent, ehxA, cdt-I, cdt-III, cdt-V, STa, LT, hlyA, cnf1, cnf2, astA;
n=12) and other function group (katP, ecf1, pagC, terE, ureD, aggR, pic, irp2, fyuA, Z2098, ecs1763,
ecs1822, etpD; n=13). The wecA gene was used as an E. coli reference genetic marker.

RESULTS

**STEC and EHEC virulence gene profiles.** A collection of *E. coli* strains, comprising 27 STEC (*stx*-positive, *eae*-negative) and one EHEC (*stx*-positive, *eae*-positive) collected from French coastal areas, was investigated. Eleven STEC strains were positive for the *stx1* gene only, 13 were positive for the *stx2* gene only, while four strains harbored a combination of the *stx1* and *stx2* gene. The most commonly identified *stx1* subtype was *stx1d* (23% of detected *stx* genes), followed by *stx1a* (17%) (Figure 1, Table 2). The most common *stx2* subtype was *stx2a* (17%) (Figure 1). Five strains were identified with several *stx* subtypes; three possessed *stx1a* and *stx2a* and belonged to the serotypes O185:H28, ONT:H11 and O130:H11; one possessed *stx2a* and *stx2d* and one possessed *stx1a*, *stx2a*, *stx2c* and *stx2d* corresponding to the serotypes O8:H19 and O91:H21, respectively (Table 2). Three strains harbored either the *stx1c*, *stx2e* or the *stx2g* variant.

Among the STEC strains investigated, 17 unique virulence gene profiles were identified, based on
virulence gene detection (Table 2). The EHEC O26:H11 strain harbored the profile with the highest
number of *E. coli* virulence-associated gene targets, with 47 genes detected (profile N°1). In addition
to the genes carried on the LEE and linked to the type III secretion system and to the *eae* gene (and
precisely the *eae*β1 subtype; Table 2), this strain harbors the genes associated with pathogenicity
islands such as OI-122 (*nleB*, *nleE*, and *ent*), OI-71 (*espM1*), OI-43/48 (*ureD* and *terE*), OI-50 (*espK*
and espN), OI-57 (Z2098) and HPI (irp2 and fuyA). One strain of serotype O91:H21 (profile N°2) was isolated and found to possess genes associated with some pathogenicity islands, such as OI-122 (pagC), OI-43/48 (iha), OI-15 (ehaA) and with plasmids (pO113: sab and epeA and pO157: espP and ehxA). For the other STEC strains, between two and 12 genes were detected, corresponding to 15 additional virulence gene profiles. These strains did not possess genes from the LEE, nor genes associated with the pathogenicity islands OI-122, OI-71, OI-50, and HPI. The serotype O100:HNM, which was the most commonly represented serotype in isolated STECs (n=9), was only associated with two additional virulence genes.

In those STEC strains without the LEE pathogenicity island, other genes involved in adhesion were detected. Of these, the *paa* gene, which encodes the porcine A/E associated gene, was the most frequently detected (57% of STEC strains) (Figure 2). This was followed by the *ehaA* gene, which was detected in 54% of strains. The other genes involved in adhesion detected were mainly those encoding long polar fimbriae (*lpfAO113* (39%) and *lpfAO26* (18%)), genes encoding extracellular serine protease (*espP*) (32%), IrgA homologue adhesion *iha* (21%), autoagglutinating adhesion (*saa*) (18%), and autotransporter (*sab*) (18%).

In addition to the *stx* genes, genes that could lead to the production of other toxins or hemolysins were present in 11 different STEC strains (corresponding to 11 different virulence profiles). The most frequent toxin-associated gene detected was *ehxA* (25% of strains) (Figure 2). The *subA*, *cdt-V* and *astA* genes encoding the subtilase cytotoxin, a cytolethal distending toxin, and an EAEC heat-stable enterotoxin, respectively were each detected in 11% of the strains. The *STa* gene, encoding a heat-stable toxin and the *hlyA* gene, which encodes the alpha hemolysin, were both detected in 7% of strains.

**EPEC virulence gene profiles.** Seventy-five EPEC (i.e. *eae*-positive and *stx*-negative strains) from the French coastal areas were also investigated. The isolated EPEC strains were mainly atypical EPEC strains (93%) as the typical EPEC marker *bfpA* was detected only in 7% of EPEC strains (Table 2).
The most commonly identified *eae* subtype was *eaeθ* (26% of strains), followed by *eaeβ1* (17%), *eaeβ2* (16%) and *eaeα2* (11%) (Figure 1). All four O26:H11 EPEC isolated from shellfish and freshwater harbored the *eaeβ1* (Table 2). All the strains of serotypes O63:H6/HNM (n=5) and O125:H6 (n=2) harbored *eaeα2*, even though they belonged to four different profiles (Nº 30, 32, 37 and 43). The same observation was made for the strains from serotypes O33:H6 (n=2), and O113:H6 (n=4), which belonged to six different profiles but which all harbored the *eaeβ2* subtype and with strains from serotypes O23:H8 (n=2) and ONT:H8 (n=2), which belonged to four different profiles but which all harbored the *eaeθ* subtype. The 21 other strains harbored one of the following *eae* subtypes: ε, γ1, κ/δ, ι, ζ or η (Table 2).

Among the 75 EPEC strains, 56 unique virulence gene profiles were identified, based on the virulence genes detected, suggesting a high diversity of virulence genes in the EPEC isolated from the environment (Table 2). The number of virulence genes detected fluctuated between 11 and 50. Most of the profiles were represented by one serotype, except for seven EPEC profiles (nº 3, 13, 37, 42, 45, 52 and 53), which were represented by two to four different serotypes. The EPEC profile number 37, corresponding to the serotypes O63:H6/HNM and ONT:H6 with 17 virulence genes, was the most represented in EPEC (n=5). The profiles with the highest numbers of *E. coli* virulence-associated genes were represented by 10 strains for which the serotypes belonged to the Top 5 EHEC serotypes (i.e., O26:H11, O103:H2, O103:H25, O145:H28, and O157:H7) and to the serotype O153:H2, harboring between 34 and 50 virulence genes. These strains possess genes carried by some pathogenicity islands such as OI-122, OI-71, OI-43/48 and at least one of the following genes: *espM1*, *espK*, *espV*, *espN*, *ureD* and *Z2098* that are highly associated with typical EHEC. These latter genes were also detected in 13 additional EPEC strains corresponding to serotypes O125:H6, O128:H2, O159:H7, O167:H3, O23:H8, O28:HNM, O29:H19, O40:HNM, O71:HNM, O86:H31, O88:H8 and O98:H8.

Among EPECs, in addition to *eae*, seven genes (i.e. *espB*, *espD*, *escC*, *espG*, *escD*, *escV*, and *escJ*) were detected in 100% of strains, all of which carried by the LEE. The other most frequently detected genes (i.e. *espM2*, *escN*, *nleC*, *nleH1-2*, *espJ*, and *nleH1-1*) were observed in more than 60% of strains
and are also genes associated with the type III secretion system (Figure 2). Among the 20 genes of the adhesion function group investigated, *paa* was the most frequently detected gene (60% of strains) followed by *ehaA*, *lpfAO113* and *lpfAO26* genes, with 39%, 33% and 29% of strains, respectively, and by the *efa1/LifA* gene encoding EHEC factor for adherence (20%) (Figure 2). A number of genes that encode toxins and hemolysins were present in EPEC strains. For example, 33% of strains carried the *astA* gene, which encodes the EAEC heat-stable toxin. The *ent* gene, which encodes a Ankyrin repeat was also detected in 33% of the EPEC strains. The *ehxA* gene, which encodes an entero-haemolysin and the *cdt-I*, which encodes a cytolethal distending toxin, were detected in 11% and 7% of strains, respectively. Finally, the *hlyA* gene, which encodes for the alpha-haemolysin was present in 5% of EPECs.

**Genes not detected in STEC and EPEC.** Nine virulence genes were never detected in STEC and EPEC strains, four of which are linked to the adhesion function [*eibG*, *F6/987P (fasA)*, *F18,F107 (fedA)*, *F41 (fimF41a)*], four to the toxin production function [*cdt-III*, *LT (elt)*, *cnf1* and *cnf2*] and one, [*aggR*], to the entero-aggregative function. No obvious difference was observed between the virulence profiles and the type of sample from which the strains were isolated (shellfish, waters or sediments).

DISCUSSION

This study presents a molecular risk assessment of STEC and EPEC strains isolated in France from shellfish, seawater and sediment samples, collected from shellfish-harvesting areas and from freshwater samples in their upstream watersheds. These strains derived from a larger collection of *E. coli* strains (12,016 isolates) isolated during a recent study (46). The molecular risk assessment of 28 STEC and 75 EPEC strains was conducted by testing a large panel of virulence genetic markers (i.e. a total of 75 markers) associated with human and animal infections using a high-throughput real-time PCR approach and by identifying *stx* and *eae* subtypes.
Some STEC and EPEC strains characterized in this study were found to display a large number or a
particular combination of virulence genetic markers and the presence of *stx* and/or *eae* variants,
suggesting their potential pathogenicity for humans.

Among the *stx*-positive strains, only the EHEC O26:H11 *stx1a eaeβ1* was associated with a large number of virulence-associated gene targets (60% of genes). Furthermore, this strain, isolated from shellfish, was the only one to harbor the two EHEC gene markers *stx* and *eae*. The combined presence of *stx*, *eae* and the 45 supplementary virulence genes has been associated with enhanced virulence. Similar O26:H11 *stx1a eaeβ1* strains (sequence type 21; phylogroup B1) have previously been isolated from human patients with HUS or diarrhea in Europe (54, 55)

Although lacking the *eae* gene, most of the STEC strains isolated here have the genetic potential to adhere to host cells through other structures. For example, 18% of *eae*-negative STEC strains displayed the *saa* gene, encoding the STEC auto-agglutinating adhesion factor. This gene was only observed in LEE-negative STEC, in agreement with previous findings from strains of human and bovine origin (56). Other genes encoding proteins associated with attachment were detected in some strains. These include *paa*, detected here in 15 *eae*-negative STEC as well as *ehaA*, *lpfAO113*, *lpfAO26*, *espP*, *iha*, and *sab*. Many of these genes have previously been detected in *eae*-negative strains isolated from other studies and could play a role in adhesion to the host cells and consequently in the virulence potential of the isolated strains (18-21, 57).

Among the strains that were negative for the *eae* gene, STEC strain O91:H21, isolated from
freshwater, could potentially be pathogenic for humans as a result of: 1) the presence of seven
alternative adhesion factors (*saa*, *ehaA*, *lpfAO113*, *lpfAO26*, *espP*, *iha*, and *sab*), some of which are
included in pathogenicity islands such as OI-15 or OI-43/48; 2) the presence of three *stx2* variants,
including *stx2d*. This latter *stx2* variant has been found to be associated with the ability to cause severe
diseases, including bloody diarrhea and hemolytic uremic syndrome, in atypical EHEC (58).
Furthermore, this strain also possesses the *cdt-V* gene, which has been found in STEC strains involved
in serious diseases (59). Finally, this strain presented the sequence type ST442, which has been found
to be the unique ST associated with hemolytic uremic syndrome among the 10 ST identified in 100 STEC O91 isolated from different patients (60).

In addition to the O91:H21 strain, four other STEC strains (i.e. O8:H19, O185:H28, ONT:H11 and O130:H11) were found to combine several distinct *stx1* and/or *stx2* subtypes. The presence of a combination of *stx* genes has previously been observed among strains of similar serotypes isolated from humans (17). Furthermore, in the study of Bertin *et al.* (61), strains harboring two or three *stx* subtypes were found to be more frequently highly cytotoxic toward Vero cells than the other strains. We can hypothesize that strains with a combination of *stx1* and/or *stx2* subtypes are more virulent. Furthermore, strains of serotypes O8:H19 and O130:H11 were previously isolated from human patients (62). The latter strain, like the O91:H21 strain described above, harbor the *iha*, *lpfA*0113, *ehxA*, and *cdt-V* genes, suggesting a potential virulence trait for humans. Indeed, these genetic markers have already been identified in human LEE-negative STEC strains associated with diseases (17, 63).

The subtypes *stx1a* and *stx2a* were each found in 17% of the STEC strains (essentially from freshwater samples) and have been associated with only six to eight supplementary virulence genes. These subtypes have frequently been identified in STEC strains from human, animal, environmental and food samples (64-67). STEC strains with *stx2a* have been found to be associated with several clinical symptoms, such as HUS and HC, whereas STEC with *stx1a* have been mainly associated with diarrhea without HUS (8).

On the other hand, the simultaneous detection of the subtype *stx2e*, and of the *paa*, *orfA*, *hlyA* and *ecs1763* genes in the strain of O2:H32 serotype (from seawater sample) suggests that this strain could potentially be associated with swine edema disease (9) or from a pig source (66). In the same way, strain O15:H16 (from shellfish sample) associated with the *stx2g* subtype and four supplementary virulence genes (two toxins and two adhesion factors), could potentially be linked with cattle sources as it has been previously shown for similar strains (40, 68). We can hypothesize that this strain is associated with a low human risk. Similarly, the O38:H26 STEC strain isolated from shellfish was associated with a low human risk; indeed it was associated with the *stx1c* subtype and only one other
toxin gene (subA) and one adhesion gene (espP) and may potentially derive from a sheep source as previously shown (66, 69). Finally, the stx1d subtype dominates in STEC strains isolated (23% of detected stx genes) in our study (from shellfish and freshwater samples). This subtype does not appear to be common in STEC (64-66). However, a proportion of stx1d-variants similar to those observed here has been observed in STEC strains from ruminant stools in India (18.7%) (70). Little is known about the clinical significance of this subtype, but it seems to be associated with a low risk (71). In agreement with this suspected low virulence, STECs with stx1d were found to be associated with only one to four additional virulence genes from the panel of 75 genes investigated.

Among the EPEC strains isolated in this study, some appear potentially pathogenic for humans due to the high number and the selection of virulence genes they possess. First, the E. coli O157:H7 isolated from shellfish possessed the highest number of virulence genes (50 genes) and especially the following genes (ehxA, astA, lpfA, katP,etpD,espP, terE,and ureD) identified by using whole genome sequencing analyses in STEC O157:H7 and the EPEC O157:H7 isolated from patients with gastrointestinal complaints in the Netherlands (72). These EPEC strains were mostly related to the STEC group and might be referred to as EHEC strains that have lost the Shiga-toxin (EHEC-LST; 72). Another category of such strains could be “EHEC –like”, as previously proposed for strains of the O26:H11 serotype (73). In addition, non-O157 serotypes, such as O145:H28, O103:H25, O103:H2, O26:H11 isolated in this study, were also found to possess a high number of genes (from 27 to 39 virulence genes) and could also be regarded as EHEC strains that have lost the Shiga-toxin. The large number of virulence genes in these strains is in agreement with the fact that members of the Top 5 serotypes are the strains most frequently associated with human diseases (5). Among the strains with the highest number of virulence genes (> 25 virulence genes), there are also strains of serotypes O153:H2 and O23:H8. STEC of the O153:H2 serotype has already been isolated from human patients (62) whereas EPEC strains of serotype O23:H8 and with similarities to those of the present study (same ST: ST327, with genes encoding the same adhesins [lpfAO26, lpfAO113, and paa] and genes of the OI-122) were previously associated with non-bloody diarrhea (74).
In addition to the high number of virulence genes, several strains could be potentially pathogenic as specific virulence gene associations were found. For example, the detection of the four genetic markers, *espK*, *espV*, *ureD* and Z2098, in strains belonging to the O103:H2 and O103:H25 serotypes suggests that these strains, isolated from freshwater samples, could be highly virulent for humans, as previously proposed by studies into the detection of EHEC and their stx-negative derivative strains (29) and on prediction of virulence of strains (28).

Furthermore, ten strains displayed more than 60% of the genetic markers related to the pathogenicity islands OI-122 (*efa1*, *pagC*, *nleB*, *nleE* and *ent*), OI-57 (*nleG2*, *nleG5-1*, *nleG5-2*, *nleG6-2*, Z2098 and *ecs1763*), OI-71 (*espM1*, *nleA*, *nleF*, *nleG*, and *nleH1-2*) and OI-43-48 (*iha*, *terE* and *ureD*) and to the High Pathogenic Island (HPI) (*irp2* and *fuyA*), used to identify strains that are able to cause severe disease outbreaks (21, 42). These strains were isolated from shellfish and freshwater samples and belonged to the serotypes O23:H8, O26:H11, O103:H25, O103:H2, O145:H28 and O157:H7. They are among the strains described above as having the largest number of virulence genes and represent a group of strains with a high virulence potential for humans. This is also corroborated by their serotype, which is associated with the classical EHEC serotype.

The presence of *nle* (non-LEE effector) genes and the number of genes carried by an *E. coli* strain is an important criterion for estimating its virulence potential (26, 75). Eight strains carried at least 10 of the 13 *nle* genes analyzed. These strains belong to the O26:H11, O23:H8, O157:H7 serotypes described above and two strains of serotypes O157:H16, O39:HNM, isolated from shellfish and freshwater samples.

The majority of the EPEC strains isolated in this study was found to possess the four main intimin subtypes, which could be highly related to pathogenic serotypes (*eae* subtypes ß, γ, ε, and θ; 72% of the EPEC strains; 16). The *eaeγ1* subtype was found in the strain from serotype O157:H7 and the two O145:H28, while *eaeβ1* was found in the four strains from the O26:H11 serotype (isolated from shellfish and freshwater samples). These correlations are consistent with results previously shown for strains belonging to the Top 5 EHEC serotypes isolated from slaughtered adult cattle (10) and from
strains linked to human diseases (50). These data are also consistent with publications that showed that the *eaeβ* and *eaeγ* subtypes were the two most frequently detected subtypes in clinical isolates associated with human infections (14, 50, 76). Moreover, these serotypes were associated with the highest number of genes studied for virulence, with 26 to 49 supplementary virulence genes and the *E. coli* strains isolated here could be EHEC strains that have lost the *stx* gene as suggested above.

Some correlations were found between the *eae* subtype and specific virulence genes or phylogroups. Among EPECs, all strains with the *eaeγ1* subtype, and only these, harbored the *espA* and *espZ* genes. Although from different serotypes, the three *eaeγ1*-containing phylogrouped strains were from phylogroup D and harbored the *tir* gene that was also detected in seven of the nine *eaeα2* variants. Similarly, all *eae-β2* strains (n = 12) were from phylogroup B2 and contained the *fyuA* and *irp2* genes (HPI), while only 18% of other EPEC variant strains harbored these genes. This resulted in a greater proportion of strains carrying these *fyuA* and *irp2* genes among members from phylogroup B2. However, strains from phylogroup B1 preferentially carried the *ehaA* (89.2%), *lpfAO113* (83.8%), *lpfAO26* (70.3%), *nleE* (62.2%), *nleB* (59.5%) and *ent* (59.5%) genes compared with those of phylogroups A, B1 and D (less than 15%).

In conclusion, this is the first molecular risk assessment study on STEC and EPEC strains isolated from the coastal environment in which genetic markers associated with human or non-human animal diseases were used to evaluate the potential risk for shellfish consumers. Six STEC (corresponding to profiles number 1-4, 7 and 9) and 15 EPEC strains (corresponding to profiles number 1-9, 36), which could be 'EHEC-like' displayed a large number or particular combination of virulence genetic markers, suggesting their association with human or non-human animal infections. This study clearly highlights the ubiquitous presence of potentially pathogenic STEC and EPEC strains in coastal environment (shellfish, waters and sediment samples) even if these strains may be less prevalent in such environment than in upstream watersheds as a result of the distance from the source and the negative impact of a saline environment. To date, no shellfish outbreak involving STEC or EPEC
strains has been described. The sanitary classification of shellfish-harvesting areas in Europe is
probably an important feature that helps to prevent shellfish-foodborne outbreaks caused by these
bacteria. However, the absence of a case description could also be linked to an underestimation of the
potential hazard associated with potentially pathogenic STEC and EPEC. Therefore, acquisition of
data on circulating strains in the environment is crucial to prevent the risk of human infection and to
improve our understanding of STEC and EPEC.

ACKNOWLEDGMENTS

This work was funded by the European Regional Development Fund Interreg IVA
Programme, as part of the collaborative project RiskManche. The thesis of Charlotte Balière
was supported by a grant from Ifremer and the Agence de l'Eau Loire-Bretagne. We thank
Huw Taylor (University of Brighton, UK) for his critical review of the manuscript.

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**Table 1: PCR primers used in this study for stx and eae subtyping**

**Table 2: Distribution of STEC and EPEC strains by serotype and virulence gene profiles<sup>a, b</sup>**

<sup>a</sup> Serotype, Phylogroup and ST (MLST-Sequence Type) as previously determined by Balière *et al.* (46) and Gourmelon *et al.* (47).

<sup>b</sup> Virulence gene targets *eibG*, F6/987P (*fasA*), F18,F107 (*fedA*), F41 (*fimF41a*), *cdt-III*, LT (*elt*), *cnf1*, *cnf2* and *aggR* that were detected in any of the strains are not listed in this table.

<sup>c</sup> nd: not done

**Figure 1: Shiga toxin and intimin encoding genes subtyping.**

A: distribution of *stx* gene subtypes found in STEC strains. B: distribution of *eae* gene subtypes found in *E.coli* strains.

**Figure 2: Prevalence of virulence gene targets among STEC and EPEC strains.**

Results are expressed as a percentage of the strains bearing the corresponding gene.
<table><caption>Table 1: PCR primers used in this study for stx and eae subtyping.</caption><thead><tr><th>Gene</th><th>Primers</th><th>Sequence of primer (5'-3')</th><th>Fragment size (bp)</th><th>Reference</th></tr></thead><tbody><tr><td>stx2</td><td>F4<br>R1</td><td>GGCACTGTCTGAACTGCTCCTGT<br>ATTAAACTGCACTTCAGCAAATCC</td><td>627</td><td>7</td></tr><tr><td>stx2</td><td>F4-f<br>R1-e/f</td><td>CGCTGTCTGAGGCATCTCCGCT<br>TAAACTTCACCTGGGCAAAGCC</td><td>625</td><td>7</td></tr><tr><td>ea e<sup>a</sup></td><td>EAE-F<br>EAE-RB</td><td>ATTACTGAGATTAAGGCTGAT<br>ATTTATTTGCAGCCCCCAT</td><td>682</td><td>50</td></tr><tr><td>ea e-ε</td><td>EAE-F<br>LP5</td><td>ATTACTGAGATTAAGGCTGAT<br>AGCTCACTCGTAGATGACGGCAAGCG</td><td>722</td><td>50</td></tr><tr><td>ea e-η</td><td>EAE-F<br>LP8</td><td>ATTACTGAGATTAAGGCTGAT<br>TAGATGACGGTAAGCGAC</td><td>712</td><td>50</td></tr><tr><td>ea e-ξ</td><td>EAE-F<br>B49R</td><td>ATTACTGAGATTAAGGCTGAT<br>ACCACCTTTAGCAGTCAATTTG</td><td>468</td><td>50</td></tr><tr><td>ea e-μ</td><td>FV373F<br>FV373R</td><td>CAACGGTAAGTCTCAGACAC<br>CATAATAAGCTTTTTGGCCTACC</td><td>443</td><td>50</td></tr><tr><td>ea e-ν</td><td>IH1229aF<br>IH1229aR</td><td>CACAGCTTACAATTGATAACA<br>CTCACTATAAGTCATACGACT</td><td>311</td><td>50</td></tr><tr><td>ea e<sup>b</sup></td><td>ea e-F1<br>escD-R1</td><td>ACTCCGATTCCTCTGGTGAC<br>GTATCAACATCTCCCGCCCA</td><td>~1800-2100 depending<br>on the allele</td><td>51</td></tr><tr><td>ea e<sup>b</sup></td><td>cesT-F3<br>ea e-R3</td><td>CAGGAGCACAATCGCTGTTG<br>CAGACGATACGATCCAGACC</td><td>1727</td><td>This study</td></tr></tbody></table>

<footnote><sup>a</sup> universal primers targeting the 3' variable region of the *eae*-α1, -α2, -β1, -β2, -γ2, -θ, -δ, -κ, -ζ and -ι subtypes</footnote>

<footnote><sup>b</sup> primers targeting overlapping DNA fragments of the *eae* gene</footnote>
A

<figure><img src="image_33.png" /></figure>

B

<figure><img src="image_34.png" /><figcaption>**Figure 1:** Shiga toxin and intimin encoding genes subtyping.</figcaption></figure>

A: distribution of *stx* gene subtypes found in STEC strains. B: distribution of *eae* gene subtypes found in *E.coli* strains.
<figure><img src="image_35.png" /><figcaption>Figure 2: Prevalence of virulence gene targets among STEC and EPEC strains.</figcaption></figure>

Results are expressed as a percentage of the strains bearing the corresponding gene.
# Chapitre 5 : EVALUATION DE LA CINETIQUE DE CONTAMINATION ET DE DECONTAMINATION DE COQUILLAGES AU CONTACT DE SOUCHES STEC

L'objectif de ce chapitre était d'évaluer la cinétique de contamination et de décontanimation d'huîtres en balnéation dans de l'eau de mer naturelle au contact de souches STEC isolées au cours de cette étude, parallèlement à celle d'huîtres contaminées par des souches de *E. coli* non STEC.

## A. Contexte de la quatrième partie de l'étude

*E. coli* est utilisé comme indicateur de contamination fécale pour classer les eaux de baignade et les zones conchylicoles (Anonyme 2004, 2006). Les zones conchylicoles sont classées en trois principales catégories : A, B et C selon les concentrations en *E. coli* retrouvées dans les coquillages. Les coquillages en catégorie B sont placés dans des bassins de purification pendant un temps suffisant pour permettre la diminution des concentrations en *E. coli* des coquillages et obtenir des concentrations < 230 *E. coli* / 100 g de CLI (correspondant à des coquillages en catégorie A). Plusieurs études ont été réalisées pour évaluer la cinétique de contamination et de décontamination de coquillages au contact de souches de *E. coli* ou d'autres bactéries entériques (de Abreu Corrêaa et al., 2007, Love et al., 2010, Chinnadurai et al., 2014, Ottaviani et al., 2015). La phase de contamination des coquillages par les *E. coli* est le plus souvent très rapide de l'ordre d'une heure, jusqu'à une contamination maximale appelée « l'équilibre cinétique ». Les *E. coli* sont éliminés rapidement du coquillage entre deux et six heures (Marino et al., 2005). Un rapport entre la concentration en *E. coli* initiale présente dans l'eau de mer et la concentration mesurée dans les coquillages à l'équilibre cinétique permet de définir le facteur de bioaccumulation de chaque type de coquillage dans des conditions environnementales particulières. Ce facteur est extrêmement dépendant de l'état physiologique du coquillage, de sa taille, de l'espèce étudiée mais également des conditions environnementales (température, salinité, pH...) (Love et al., 2010).

Les *E. coli*, en plus d'être des bactéries commensales et des indicateurs de contamination fécale, peuvent aussi être des bactéries pathogènes telles que des STEC. A notre connaissance, une seule étude s'est intéressée à la cinétique de contamination et de décontamination de coquillages au contact d'une souche appartenant au sérotype O157:H7 mais *stx* négatif (Ottaviani et al., 2015).

L'objectif de notre étude était donc d'évaluer la cinétique de contamination et de décontamination de souches STEC isolées de l'environnement dans des lots d'huîtres en balnéation et de la comparer à celle de bactéries *E. coli* non STEC issues également de l'environnement.
# B. Matériels et méthodes

## Souches bactériennes, huîtres et eau de mer

Deux souches de *E. coli* STEC ont été sélectionnées parmi la collection de souches environnementales obtenue à partir de l'analyse précédente d'échantillons de coquillages, d'eaux et de sédiments (Publication N°2). La première souche STEC est une souche O100:HNM stx2+ (code : *E. coli* stx2) et la seconde une souche O154:H31 stx1+ (code : *E. coli* stx1), isolées à partir de lots de coquillages. Deux souches de *E. coli* non STEC (codes : *E. coli* 1 et *E. coli* 2) ont été sélectionnées parmi la collection de souches environnementales obtenue dans le cadre plus global du projet Riskmanche isolées à partir des mêmes lots de coquillages que les souches STEC sélectionnées pour cette étude.

Les huîtres (*Crassostrea gigas*) de calibre 3-4 provenaient d'une exploitation conchylicole finistérienne (COIC ; Pointe du Château) située en Rade de Brest. Les tests ont été réalisés en Octobre 2014 sur des huîtres maigres en fin de période de ponte.

L'eau de mer utilisée pour les expérimentations a été prélèvée directement à la mer. Les paramètres turbidité, concentrations en ammonium, nitrate, phosphate et chlorophylle A de cette eau de mer ont été mesurés avant son utilisation dans les microcosmes (données non présentées). Pendant l'expérience, les paramètres physico-chimiques de l'eau de mer : le taux d'oxygène, la température, la salinité ont été mesurés à l'aide d'une sonde PT100 et d'un analyseur multiparamétrique (Eutech Instrument PD650 ; ThermoScientific).

## Préparation des souches bactériennes et dénombrement des *E. coli*

Chaque souche a été mise en culture dans un bouillon d'eau peptonée tamponnée, à 37°C pendant 24 heures. Les suspensions bactériennes ont été titrées à l'aide de la méthode de dénombrement *E. coli* par étalement sur le milieu solide Tryptone-Bile-X-glucuronidase agar (TBX ; AES cheminex, Bruz, France), selon la norme ISO NF-EN-8199. Les suspensions bactériennes ont été préparées à une concentration d'environ 6 log₁₀ UFC/100 mL (10 mL de suspension bactérienne) pour avoir une contamination de l'eau mer d'environ 4 log₁₀ UFC/100 mL une fois la suspension bactérienne versée dans le bac.

La numération des *E. coli* dans l'eau de mer a été réalisée par filtration de 1, 10, 100 ou 500 mL d'eau sur membrane 0,45 µm (Pall Gelman GN-6 Metrocel : Pall Corporation, St Germin-en-Laye, France) et le dépôt du filtre sur le milieu TBX, incubé 24h à 44°C ; inspiré des normes ISO NF-EN-8199 et ISO TS-16649-3:2005. La concentration en *E. coli* réelle dans les bacs de contamination à T0 a été mesurée après quelques minutes d'homogénéisation de l'eau de mer avec la suspension bactérienne titrée versée. La numération des *E. coli* dans les huîtres a été réalisée par impédancemétrie selon la norme NF V 08-106.