# High carriage of adherent invasive E. coli in wildlife and healthy individuals

Oumaira Rahmouni, Cecile Vignal, Marie Titecat, Benoit Foligne, Benjamin

Pariente, Laurent Dubuquoy, Pierre Desreumaux, Christel Neut

► To cite this version:

Oumaira Rahmouni, Cecile Vignal, Marie Titecat, Benoit Foligne, Benjamin Pariente, et al. High carriage of adherent invasive E. coli in wildlife and healthy individuals. Gut Pathogens, 2018, Gut Pathogens, 10, pp.23. 10.1186/s13099-018-0248-7. hal-04429524

HAL Id: hal-04429524

https://hal.univ-lille.fr/hal-04429524

Submitted on 31 Jan 2024

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# High carriage of adherent invasive E. coli in wildlife and healthy individuals

Oumaira Rahmouni, Cecile Vignal, Marie Titecat, Benoit Foligne, Benjamin

Pariente, Laurent Dubuquoy, Pierre Desreumaux, Christel Neut

► To cite this version:

Oumaira Rahmouni, Cecile Vignal, Marie Titecat, Benoit Foligne, Benjamin Pariente, et al. High carriage of adherent invasive E. coli in wildlife and healthy individuals. Gut Pathogens, 2018, Gut Pathogens, 10, pp.23. 10.1186/s13099-018-0248-7. hal-04429524

HAL Id: hal-04429524

https://lilloa.hal.science/hal-04429524v1

Submitted on 31 Jan 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
SHORT REPORT

Open Access

<figure><img src="image_2.png" /></figure>

CrossMark

# High carriage of adherent invasive *E. coli* in wildlife and healthy individuals

Oumaïra Rahmouni¹, Cécile Vignal¹, Marie Titécat¹,², Benoît Foligné¹, Benjamin Pariente¹,³, Laurent Dubuquoy¹, Pierre Desreumeaux¹,³ and Christel Neut¹,<fnref n="4" />*

**Abstract**

**Background:** Adherent invasive *Escherichia coli* (AIEC) are suspected to be involved in the pathogenesis of inflammatory bowel diseases. Since AIEC was first described in 1999, despite important progress on its genomic and immune characterizations, some crucial questions remain unanswered, such as whether there exists a natural reservoir, or whether there is asymptomatic carriage. The ECOR collection, including *E. coli* strains isolated mainly from the gut of healthy humans and animals, constitutes an ideal tool to investigate AIEC prevalence in healthy condition. A total of 61 *E. coli* strains were examined for characteristics of AIEC.

**Methods:** The adhesion, invasion and intramacrophage replication capabilities (AIEC phenotype) of 61 intestinal *E. coli* strains were determined. The absence of virulence-associated diarrheagenic *E. coli* pathotypes (EPEC, ETEC, EIEC, EHEC, DAEC, EAEC), and uropathogenic *E. coli* was checked.

**Results:** Out of 61 intestinal strains, 13 (21%) exhibit the AIEC phenotype, 7 are from human origin and 6 are from animal origin. Prevalence of AIEC strains is about 24 and 19% in healthy humans and animals respectively. These strains are highly genetically diverse as they are distributed among the main described phylogroups. Among *E. coli* strains from the ECOR collection, we also detected strains able to detach I-407 cells.

**Conclusions:** Our study described for the first time AIEC strains isolated from the feces of healthy humans and animals.

**Keywords:** Adherent invasive *Escherichia coli* (AIEC), Healthy individuals, ECOR collection, Pathobiont

**Background**

Crohn's disease (CD), one of the clinical presentation of inflammatory bowel diseases (IBD), is characterized by chronic lesions of varying intensity along the gastrointestinal tract resulting from an exacerbated reaction of a defective immune system [1-3]. The etiology of CD is complex and is multifactorial, one of these factors is the state of the intestinal microbiota [4]. Over the last 20 years, *Escherichia coli* has attracted the most attention in respect to CD etiology. A high mucosal colonization level by *E. coli* was first demonstrated in CD [5]. A new pathogenic group of *E. coli*, called adherent invasive *E.*

* coli* (AIEC), harboring adhesive and invasive abilities to intestinal epithelial cells, was described [6-8]. The main characteristics of AIEC are (i) the ability to adhere to and invade intestinal epithelial cells, (ii) the ability to survive and replicate expansively within macrophages without triggering host cell death, and (iii) the lack of known invasive determinants [10].

The AIEC strains isolated to date are clonally diverse and belong to distinct serotypes [9-11]. The B2 phylogroup is most prevalent, but strains of A, B1, and D phylogroups have also been isolated [8, 10, 12, 13]. AIEC strains have virulence factors in common with other pathogenic *E. coli* and are closely related to extra-intestinal pathogenic *E. coli* (ExPEC), associated with urinary tract infections and neonatal meningitis [14, 15]. In absence of common and specific genetic signatures between AIEC strains, this pathotype can up to now

<footnote>*Correspondence: christelneut@nordnet.fr</footnote>

<footnote n="4">⁴ Laboratoire de Bactériologie, 3, Rue de Pr. Laguesse, B.P. 83, 59006 Lille Cedex, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
SHORT REPORT

Open Access

<figure><img src="image_2.png" /></figure>

CrossMark

# High carriage of adherent invasive *E. coli* in wildlife and healthy individuals

Oumaïra Rahmouni¹, Cécile Vignal¹, Marie Titécat¹,², Benoît Foligné¹, Benjamin Pariente¹,³, Laurent Dubuquoy¹, Pierre Desreumeaux¹,³ and Christel Neut¹,<fnref n="4" />*

**Abstract**

**Background:** Adherent invasive *Escherichia coli* (AIEC) are suspected to be involved in the pathogenesis of inflammatory bowel diseases. Since AIEC was first described in 1999, despite important progress on its genomic and immune characterizations, some crucial questions remain unanswered, such as whether there exists a natural reservoir, or whether there is asymptomatic carriage. The ECOR collection, including *E. coli* strains isolated mainly from the gut of healthy humans and animals, constitutes an ideal tool to investigate AIEC prevalence in healthy condition. A total of 61 *E. coli* strains were examined for characteristics of AIEC.

**Methods:** The adhesion, invasion and intramacrophage replication capabilities (AIEC phenotype) of 61 intestinal *E. coli* strains were determined. The absence of virulence-associated diarrheagenic *E. coli* pathotypes (EPEC, ETEC, EIEC, EHEC, DAEC, EAEC), and uropathogenic *E. coli* was checked.

**Results:** Out of 61 intestinal strains, 13 (21%) exhibit the AIEC phenotype, 7 are from human origin and 6 are from animal origin. Prevalence of AIEC strains is about 24 and 19% in healthy humans and animals respectively. These strains are highly genetically diverse as they are distributed among the main described phylogroups. Among *E. coli* strains from the ECOR collection, we also detected strains able to detach I-407 cells.

**Conclusions:** Our study described for the first time AIEC strains isolated from the feces of healthy humans and animals.

**Keywords:** Adherent invasive *Escherichia coli* (AIEC), Healthy individuals, ECOR collection, Pathobiont

**Background**

Crohn's disease (CD), one of the clinical presentation of inflammatory bowel diseases (IBD), is characterized by chronic lesions of varying intensity along the gastrointestinal tract resulting from an exacerbated reaction of a defective immune system [1-3]. The etiology of CD is complex and is multifactorial, one of these factors is the state of the intestinal microbiota [4]. Over the last 20 years, *Escherichia coli* has attracted the most attention in respect to CD etiology. A high mucosal colonization level by *E. coli* was first demonstrated in CD [5]. A new pathogenic group of *E. coli*, called adherent invasive *E.*

* coli* (AIEC), harboring adhesive and invasive abilities to intestinal epithelial cells, was described [6–8]. The main characteristics of AIEC are (i) the ability to adhere to and invade intestinal epithelial cells, (ii) the ability to survive and replicate expansively within macrophages without triggering host cell death, and (iii) the lack of known invasive determinants [10].

The AIEC strains isolated to date are clonally diverse and belong to distinct serotypes [9–11]. The B2 phylogroup is most prevalent, but strains of A, B1, and D phylogroups have also been isolated [8, 10, 12, 13]. AIEC strains have virulence factors in common with other pathogenic *E. coli* and are closely related to extra-intestinal pathogenic *E. coli* (ExPEC), associated with urinary tract infections and neonatal meningitis [14, 15]. In absence of common and specific genetic signatures between AIEC strains, this pathotype can up to now

<footnote>*Correspondence: christelneut@nordnet.fr</footnote>

<footnote n="4">⁴ Laboratoire de Bactériologie, 3, Rue de Pr. Laguesse, B.P. 83, 59006 Lille Cedex, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
only be identified by phenotypical traits. AIEC have been
described in other human intestinal disorders such as
ulcerative colitis (UC), another form of IBD, and colo-
rectal cancer but also in animals with intestinal diseases
[16–18]. However, AIEC carriage is not restricted to
intestinal inflammatory conditions and AIEC have also
been found in biopsies of patients with functional intes-
tinal disorders and in asymptomatic subjects undergoing
surveillance colonoscopy, subjects termed as “healthy” in
AIEC prevalence studies [6, 9, 12, 13, 16]. A better knowl-
edge of AIEC carriage in healthy condition whether in
human or animals will help in understanding more about
AIEC natural reservoir and transmission.

The aim of this work was to determine if AIEC is pre-
sent in the *E. coli* Reference (ECOR) Collection. The
ECOR collection is a set of *E. coli* strains of natural origin,
isolated from the gastrointestinal tract of healthy humans
and non-human mammalians from a variety of geo-
graphic locations, and designed to represent the variation
and genetic structure of *E. coli* in natural populations
[19]. 61 ECOR strains were isolated from feces of healthy
hosts (Table 1), making this collection an ideal tool to
study the prevalence of AIEC in healthy conditions.

Results

Adherent and invasive capacity of ECOR strains isolated
from the feces of healthy individuals

The ECOR collection contains 61 strains of fecal origin. Of these, 29 were from human and 32 from animals (Table 1). An invasion assay was performed on these strains using I-407 cells based on gentamicin protection, according to previously described methods [6, 15, 21, 22]. All of the strains were first tested for their susceptibility to gentamicin using disc diffusion method as only strains susceptible to gentamicin can be tested. All the strains were found to be susceptible to gentamicin, determined as a zone diameter superior to 17 mm, according to EUCAST recommendations.

The invasion levels of the noninvasive references *E. coli*
strain K-12 C600 and *E. coli* Nissle were 0.0561 ± 0.0373%
and 0.0254 ± 0.0265% respectively. Reference strain LF82,
included in all of the assays as a positive invasive control,
gave a mean invasion level of 1.0101 ± 0.9880%. Another
positive invasive control *E. coli* NRG857c showed a mean
invasion of 0.1683 ± 0.0368%. The invasion levels were
done in triplicate.

Among strains belonging to the ECOR collection, 18
strains (30%) were classified as invasive based on this
assay, with invasion rates ranging between 0.10 and
0.64%. Eight of these were of human origin (28%), four
belonged to the phylogroup A (ECOR 1, 2, 9 and 15),
one to phylogroup B2 (ECOR 63), two to phylogroup D

(ECOR 35 and ECOR 36) and one to the phylogroup E
(ECOR 43). Ten were of animal origin (31%), two belong-
ing to the phylogroup A (ECOR 7 and 23), four to the
phylogroup B1 (ECOR 45, 67, 69 and 70), two to the phy-
logroup B2 (ECOR 52 and 57) and two to the phylogroup
D (ECOR 44 and 46) (Fig. 1).

During the invasion assay, for five of the strains, an
unexpected pathogenic feature was discovered: the ability
to detach intestinal cells from the microplate. All of these
strains were of human origin, one belonging to the phy-
logroup A (ECOR 6) and the 4 others to the phylogroup
B2 (ECOR 51, 53, 54 and 59) (Table 3). Invasion scores
of these cell-detaching strains were established but were
not interpretable due to their capacity to detach adher-
ent cells from the microplate. The actual concentration of
the intracellular bacteria was therefore skewed. Further
investigations are necessary to define their propensity to
invade.

The strains revealed as invasive were tested for their
ability to adhere. All the invasive strains from the ECOR
collection were adherent, with an adhesion index ranging
between 67 and 334, all showed an adhesion index super-
rior to 1 bacteria per cell (Table 3).

Virulence genotyping of the invasive strains
and cell-detaching strains

The 18 fecal strains previously classified as adherent
and invasive were genotyped. The presence of 12 selected
virulence genes associated with diarrheagenic *E. coli*
pathotype was assessed. This included common virulence
factors present in diarrheagenic *E. coli* pathotypes EHEC
(stx-1, stx-2, eae and ehxA), EIEC (ipaC), ETEC (estA,
elt and tia), EPEC (eae, ehxA, bfpA), EAEC (aggR and
pCVD432) and DAEC (afaD) (Additional file 1: Table S1).
Three strains were positive for the tia gene (ECOR 57)
and tia and afaD genes (ECOR 63 and 70). These strains
are not affiliated as AIEC pathovars and belong to ETEC/
DAEC pathovars. The other 15 invasive strains (ECOR 1,
2, 9, 15, 35, 36, 43, 7, 23, 45, 67, 69, 52, 44, 46) were nega-
tive for the tested virulence genes excluding their affilia-
tion to the previously described pathotypes (Table 2).

The cell-detaching strains were also tested for their
expression of virulence genes associated of enteropatho-
genic strains. The ECOR 51 strain was shown to express
the *afaD* virulence gene, showing that this strain is clas-
sified as a DAEC. The other cell-detaching strains were
negative for the tested genes (Table 2).

Survival and replication ability of the invasive strains
from the ECOR collection

The 15 strains revealed as adherent and invasive and
without virulence genes associated with diarrheagenic *E.
coli* pathotype were tested for their ability to survive and
only be identified by phenotypical traits. AIEC have been
described in other human intestinal disorders such as
ulcerative colitis (UC), another form of IBD, and colo-
rectal cancer but also in animals with intestinal diseases
[16–18]. However, AIEC carriage is not restricted to
intestinal inflammatory conditions and AIEC have also
been found in biopsies of patients with functional intes-
tinal disorders and in asymptomatic subjects undergoing
surveillance colonoscopy, subjects termed as “healthy” in
AIEC prevalence studies [6, 9, 12, 13, 16]. A better knowl-
edge of AIEC carriage in healthy condition whether in
human or animals will help in understanding more about
AIEC natural reservoir and transmission.

The aim of this work was to determine if AIEC is pre-
sent in the *E. coli* Reference (ECOR) Collection. The
ECOR collection is a set of *E. coli* strains of natural origin,
isolated from the gastrointestinal tract of healthy humans
and non-human mammalians from a variety of geo-
graphic locations, and designed to represent the variation
and genetic structure of *E. coli* in natural populations
[19]. 61 ECOR strains were isolated from feces of healthy
hosts (Table 1), making this collection an ideal tool to
study the prevalence of AIEC in healthy conditions.

Results

Adherent and invasive capacity of ECOR strains isolated
from the feces of healthy individuals

The ECOR collection contains 61 strains of fecal origin. Of these, 29 were from human and 32 from animals (Table 1). An invasion assay was performed on these strains using I-407 cells based on gentamicin protection, according to previously described methods [6, 15, 21, 22]. All of the strains were first tested for their susceptibility to gentamicin using disc diffusion method as only strains susceptible to gentamicin can be tested. All the strains were found to be susceptible to gentamicin, determined as a zone diameter superior to 17 mm, according to EUCAST recommendations.

The invasion levels of the noninvasive references *E. coli*
strain K-12 C600 and *E. coli* Nissle were 0.0561 ± 0.0373%
and 0.0254 ± 0.0265% respectively. Reference strain LF82,
included in all of the assays as a positive invasive control,
gave a mean invasion level of 1.0101 ± 0.9880%. Another
positive invasive control *E. coli* NRG857c showed a mean
invasion of 0.1683 ± 0.0368%. The invasion levels were
done in triplicate.

Among strains belonging to the ECOR collection, 18
strains (30%) were classified as invasive based on this
assay, with invasion rates ranging between 0.10 and
0.64%. Eight of these were of human origin (28%), four
belonged to the phylogroup A (ECOR 1, 2, 9 and 15),
one to phylogroup B2 (ECOR 63), two to phylogroup D

(ECOR 35 and ECOR 36) and one to the phylogroup E
(ECOR 43). Ten were of animal origin (31%), two belong-
ing to the phylogroup A (ECOR 7 and 23), four to the
phylogroup B1 (ECOR 45, 67, 69 and 70), two to the phy-
logroup B2 (ECOR 52 and 57) and two to the phylogroup
D (ECOR 44 and 46) (Fig. 1).

During the invasion assay, for five of the strains, an
unexpected pathogenic feature was discovered: the ability
to detach intestinal cells from the microplate. All of these
strains were of human origin, one belonging to the phy-
logroup A (ECOR 6) and the 4 others to the phylogroup
B2 (ECOR 51, 53, 54 and 59) (Table 3). Invasion scores
of these cell-detaching strains were established but were
not interpretable due to their capacity to detach adher-
ent cells from the microplate. The actual concentration of
the intracellular bacteria was therefore skewed. Further
investigations are necessary to define their propensity to
invade.

The strains revealed as invasive were tested for their
ability to adhere. All the invasive strains from the ECOR
collection were adherent, with an adhesion index ranging
between 67 and 334, all showed an adhesion index super-
rior to 1 bacteria per cell (Table 3).

Virulence genotyping of the invasive strains
and cell-detaching strains

The 18 fecal strains previously classified as adherent
and invasive were genotyped. The presence of 12 selected
virulence genes associated with diarrheagenic *E. coli*
pathotype was assessed. This included common virulence
factors present in diarrheagenic *E. coli* pathotypes EHEC
(stx-1, stx-2, eae and ehxA), EIEC (ipaC), ETEC (estA,
elt and tia), EPEC (eae, ehxA, bfpA), EAEC (aggR and
pCVD432) and DAEC (afaD) (Additional file 1: Table S1).
Three strains were positive for the tia gene (ECOR 57)
and tia and afaD genes (ECOR 63 and 70). These strains
are not affiliated as AIEC pathovars and belong to ETEC/
DAEC pathovars. The other 15 invasive strains (ECOR 1,
2, 9, 15, 35, 36, 43, 7, 23, 45, 67, 69, 52, 44, 46) were nega-
tive for the tested virulence genes excluding their affilia-
tion to the previously described pathotypes (Table 2).

The cell-detaching strains were also tested for their
expression of virulence genes associated of enteropatho-
genic strains. The ECOR 51 strain was shown to express
the *afaD* virulence gene, showing that this strain is clas-
sified as a DAEC. The other cell-detaching strains were
negative for the tested genes (Table 2).

Survival and replication ability of the invasive strains
from the ECOR collection

The 15 strains revealed as adherent and invasive and
without virulence genes associated with diarrheagenic *E.
coli* pathotype were tested for their ability to survive and
<table><caption>Table 1 Fecal strains investigated in this study</caption><thead><tr><th rowspan="2">ID</th><th rowspan="2">Serotype</th><th rowspan="2">Phylogroup</th><th colspan="3">Source</th><th rowspan="2">ID</th><th rowspan="2">Serotype</th><th rowspan="2">Phylogroup</th><th colspan="3">Source</th></tr><tr><th>Host</th><th>Sex</th><th>Location</th><th>Host</th><th>Sex</th><th>Location</th></tr></thead><tbody><tr><td>ECOR1</td><td>ON:HN</td><td>A</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR34</td><td>O88:NM</td><td>B1</td><td>Dog</td><td></td><td>USA</td></tr><tr><td>ECOR2</td><td>ON:H32</td><td>A</td><td>Human</td><td>Man</td><td>USA</td><td>ECOR35</td><td>O1:NM</td><td>D</td><td>Human</td><td>Man</td><td>USA</td></tr><tr><td>ECOR3</td><td>O1:NM</td><td>A</td><td>Dog</td><td></td><td>USA</td><td>ECOR36</td><td>O79:H25</td><td>D</td><td>Human</td><td>Woman</td><td>USA</td></tr><tr><td>ECOR4</td><td>ON:HN</td><td>A</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR37</td><td>ON:HN</td><td>E</td><td>Marmoset</td><td></td><td>USA</td></tr><tr><td>ECOR5</td><td>O79:NM</td><td>A</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR38</td><td>O7:NM</td><td>D</td><td>Human</td><td>Woman</td><td>USA</td></tr><tr><td>ECOR6</td><td>ON:NM</td><td>A</td><td>Human</td><td>Man</td><td>USA</td><td>ECOR39</td><td>O7:NM</td><td>D</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR7</td><td>O85:HN</td><td>A</td><td>Orangutan</td><td></td><td>USA</td><td>ECOR41</td><td>O7:NM</td><td>D</td><td>Human</td><td>Man</td><td>Tonga</td></tr><tr><td>ECOR8</td><td>O86:HN</td><td>A</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR42</td><td>ON:H26</td><td>E</td><td>Human</td><td>Man</td><td>USA</td></tr><tr><td>ECOR9</td><td>ON:NM</td><td>A</td><td>Human</td><td>Woman</td><td>Sweden</td><td>ECOR43</td><td>ON:HN</td><td>E</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR10</td><td>O6:H10</td><td>A</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR44</td><td>ON:HN</td><td>D</td><td>Cougar</td><td></td><td>USA</td></tr><tr><td>ECOR12</td><td>O7:H32</td><td>A</td><td>Human</td><td>Woman</td><td>Sweden</td><td>ECOR45</td><td>ON:HM</td><td>B1</td><td>Pig</td><td></td><td>Indonesia</td></tr><tr><td>ECOR13</td><td>ON:HN</td><td>A</td><td>Human</td><td>Woman</td><td>Sweden</td><td>ECOR46</td><td>O1:H6</td><td>D</td><td>Celebese ape</td><td></td><td>USA</td></tr><tr><td>ECOR15</td><td>O25:NM</td><td>A</td><td>Human</td><td>Woman</td><td>Sweden</td><td>ECOR47</td><td>OM:H18</td><td>D</td><td>Sheep</td><td></td><td>New Guinea</td></tr><tr><td>ECOR16</td><td>ON:H10</td><td>A</td><td>Leopard</td><td></td><td>USA</td><td>ECOR49</td><td>O2:NM</td><td>D</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR17</td><td>O106:NM</td><td>A</td><td>Pig</td><td></td><td>Indonesia</td><td>ECOR51</td><td>O25:HN</td><td>B2</td><td>Human infant</td><td></td><td>USA</td></tr><tr><td>ECOR18</td><td>O5:NM</td><td>A</td><td>Celebese ape</td><td></td><td>USA</td><td>ECOR52</td><td>O25:H1</td><td>B2</td><td>Orangutan</td><td></td><td>USA</td></tr><tr><td>ECOR19</td><td>O5:HN</td><td>A</td><td>Celebese ape</td><td></td><td>USA</td><td>ECOR53</td><td>O4:HN</td><td>B2</td><td>Human</td><td>Woman</td><td>USA</td></tr><tr><td>ECOR20</td><td>O89:HN</td><td>A</td><td>Steer</td><td></td><td>Bali</td><td>ECOR54</td><td>O25:H1</td><td>B2</td><td>Human</td><td></td><td>USA</td></tr><tr><td>ECOR21</td><td>O121:HN</td><td>A</td><td>Steer</td><td></td><td>Bali</td><td>ECOR56</td><td>O6:H1</td><td>B2</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR22</td><td>ON:HN</td><td>A</td><td>Steer</td><td></td><td>Bali</td><td>ECOR57</td><td>ON:NM</td><td>B2</td><td>Gorilla</td><td></td><td>USA</td></tr><tr><td>ECOR23</td><td>O86:H43</td><td>A</td><td>Elephant</td><td></td><td>USA</td><td>ECOR58</td><td>O112:H8</td><td>B1</td><td>Lion</td><td></td><td>USA</td></tr><tr><td>ECOR24</td><td>O15:NM</td><td>A</td><td>Human</td><td>Woman</td><td>Sweden</td><td>ECOR59</td><td>O4:H40</td><td>B2</td><td>Human</td><td>Man</td><td>USA</td></tr><tr><td>ECOR25</td><td>ON:HN</td><td>A</td><td>Dog</td><td></td><td>USA</td><td>ECOR61</td><td>O2:NM</td><td>B2</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR26</td><td>O104:H21</td><td>B1</td><td>Human infant</td><td></td><td>USA</td><td>ECOR63</td><td>ON:NM</td><td>B2</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR27</td><td>O104:NM</td><td>B1</td><td>Giraffe</td><td></td><td>USA</td><td>ECOR65</td><td>ON:H10</td><td>B2</td><td>Celebese ape</td><td></td><td>USA</td></tr><tr><td>ECOR28</td><td>O104:NM</td><td>B1</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR66</td><td>O4:H40</td><td>B2</td><td>Celebese ape</td><td></td><td>USA</td></tr><tr><td>ECOR29</td><td>O150:H21</td><td>B1</td><td>Kangaroo rat</td><td></td><td>USA</td><td>ECOR67</td><td>O4:H43</td><td>B1</td><td>Goat</td><td></td><td>Indonesia</td></tr><tr><td>ECOR30</td><td>O113:H21</td><td>B1</td><td>Bison</td><td></td><td>Canada</td><td>ECOR68</td><td>ON:NM</td><td>B1</td><td>Giraffe</td><td></td><td>USA</td></tr><tr><td>ECOR31</td><td>O79:H43</td><td>E</td><td>Leopard</td><td></td><td>USA</td><td>ECOR69</td><td>ON:NM</td><td>B1</td><td>Celebese ape</td><td></td><td>USA</td></tr><tr><td>ECOR32</td><td>O7:H21</td><td>B1</td><td>Giraffe</td><td></td><td>USA</td><td>ECOR70</td><td>O78:NM</td><td>B1</td><td>Gorilla</td><td></td><td>USA</td></tr><tr><td>ECOR33</td><td>O7:H21</td><td>B1</td><td>Sheep</td><td></td><td>USA</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

This table was built according to data from previously published papers [19, 20].

replicate within macrophages. Among these strains, 13 were able to survive and replicate in macrophages. The percent intracellular bacteria at 1 and 24 h postinfection ranged between 114.06 and 881.36%. The ECOR 1, 2, 9, 15, 35, 36, 43, 23, 45, 67, 69, 52, 44 were thus considered as belonging to the AIEC pathotype.

The two other strains (ECOR 46 and 7) were not able to replicate inside macrophages with an index of 70.42 and 74.51% respectively (Table 3). These strains were thus not affiliated to the AIEC pathovar.

## Discussion

The ECOR collection is a collection of *E. coli* that reflects the genetic diversity of non-pathogenic *E. coli* isolated from humans and animals of diverse geographical origins [19] (Table 1). We studied the 61 strains of *E. coli* isolated from the feces of healthy individuals for an invasive phenotype. Our study allowed the demonstration of the presence of AIEC strains in feces of healthy individuals. On the other hand this also gave an insight into the prevalence of AIEC in diverse conditions. Our results showed
<table><caption>Table 1 Fecal strains investigated in this study</caption><thead><tr><th rowspan="2">ID</th><th rowspan="2">Serotype</th><th rowspan="2">Phylogroup</th><th colspan="3">Source</th><th rowspan="2">ID</th><th rowspan="2">Serotype</th><th rowspan="2">Phylogroup</th><th colspan="3">Source</th></tr><tr><th>Host</th><th>Sex</th><th>Location</th><th>Host</th><th>Sex</th><th>Location</th></tr></thead><tbody><tr><td>ECOR1</td><td>ON:HN</td><td>A</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR34</td><td>O88:NM</td><td>B1</td><td>Dog</td><td></td><td>USA</td></tr><tr><td>ECOR2</td><td>ON:H32</td><td>A</td><td>Human</td><td>Man</td><td>USA</td><td>ECOR35</td><td>O1:NM</td><td>D</td><td>Human</td><td>Man</td><td>USA</td></tr><tr><td>ECOR3</td><td>O1:NM</td><td>A</td><td>Dog</td><td></td><td>USA</td><td>ECOR36</td><td>O79:H25</td><td>D</td><td>Human</td><td>Woman</td><td>USA</td></tr><tr><td>ECOR4</td><td>ON:HN</td><td>A</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR37</td><td>ON:HN</td><td>E</td><td>Marmoset</td><td></td><td>USA</td></tr><tr><td>ECOR5</td><td>O79:NM</td><td>A</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR38</td><td>O7:NM</td><td>D</td><td>Human</td><td>Woman</td><td>USA</td></tr><tr><td>ECOR6</td><td>ON:NM</td><td>A</td><td>Human</td><td>Man</td><td>USA</td><td>ECOR39</td><td>O7:NM</td><td>D</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR7</td><td>O85:HN</td><td>A</td><td>Orangutan</td><td></td><td>USA</td><td>ECOR41</td><td>O7:NM</td><td>D</td><td>Human</td><td>Man</td><td>Tonga</td></tr><tr><td>ECOR8</td><td>O86:HN</td><td>A</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR42</td><td>ON:H26</td><td>E</td><td>Human</td><td>Man</td><td>USA</td></tr><tr><td>ECOR9</td><td>ON:NM</td><td>A</td><td>Human</td><td>Woman</td><td>Sweden</td><td>ECOR43</td><td>ON:HN</td><td>E</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR10</td><td>O6:H10</td><td>A</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR44</td><td>ON:HN</td><td>D</td><td>Cougar</td><td></td><td>USA</td></tr><tr><td>ECOR12</td><td>O7:H32</td><td>A</td><td>Human</td><td>Woman</td><td>Sweden</td><td>ECOR45</td><td>ON:HM</td><td>B1</td><td>Pig</td><td></td><td>Indonesia</td></tr><tr><td>ECOR13</td><td>ON:HN</td><td>A</td><td>Human</td><td>Woman</td><td>Sweden</td><td>ECOR46</td><td>O1:H6</td><td>D</td><td>Celebese ape</td><td></td><td>USA</td></tr><tr><td>ECOR15</td><td>O25:NM</td><td>A</td><td>Human</td><td>Woman</td><td>Sweden</td><td>ECOR47</td><td>OM:H18</td><td>D</td><td>Sheep</td><td></td><td>New Guinea</td></tr><tr><td>ECOR16</td><td>ON:H10</td><td>A</td><td>Leopard</td><td></td><td>USA</td><td>ECOR49</td><td>O2:NM</td><td>D</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR17</td><td>O106:NM</td><td>A</td><td>Pig</td><td></td><td>Indonesia</td><td>ECOR51</td><td>O25:HN</td><td>B2</td><td>Human infant</td><td></td><td>USA</td></tr><tr><td>ECOR18</td><td>O5:NM</td><td>A</td><td>Celebese ape</td><td></td><td>USA</td><td>ECOR52</td><td>O25:H1</td><td>B2</td><td>Orangutan</td><td></td><td>USA</td></tr><tr><td>ECOR19</td><td>O5:HN</td><td>A</td><td>Celebese ape</td><td></td><td>USA</td><td>ECOR53</td><td>O4:HN</td><td>B2</td><td>Human</td><td>Woman</td><td>USA</td></tr><tr><td>ECOR20</td><td>O89:HN</td><td>A</td><td>Steer</td><td></td><td>Bali</td><td>ECOR54</td><td>O25:H1</td><td>B2</td><td>Human</td><td></td><td>USA</td></tr><tr><td>ECOR21</td><td>O121:HN</td><td>A</td><td>Steer</td><td></td><td>Bali</td><td>ECOR56</td><td>O6:H1</td><td>B2</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR22</td><td>ON:HN</td><td>A</td><td>Steer</td><td></td><td>Bali</td><td>ECOR57</td><td>ON:NM</td><td>B2</td><td>Gorilla</td><td></td><td>USA</td></tr><tr><td>ECOR23</td><td>O86:H43</td><td>A</td><td>Elephant</td><td></td><td>USA</td><td>ECOR58</td><td>O112:H8</td><td>B1</td><td>Lion</td><td></td><td>USA</td></tr><tr><td>ECOR24</td><td>O15:NM</td><td>A</td><td>Human</td><td>Woman</td><td>Sweden</td><td>ECOR59</td><td>O4:H40</td><td>B2</td><td>Human</td><td>Man</td><td>USA</td></tr><tr><td>ECOR25</td><td>ON:HN</td><td>A</td><td>Dog</td><td></td><td>USA</td><td>ECOR61</td><td>O2:NM</td><td>B2</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR26</td><td>O104:H21</td><td>B1</td><td>Human infant</td><td></td><td>USA</td><td>ECOR63</td><td>ON:NM</td><td>B2</td><td>Human</td><td>Woman</td><td>Sweden</td></tr><tr><td>ECOR27</td><td>O104:NM</td><td>B1</td><td>Giraffe</td><td></td><td>USA</td><td>ECOR65</td><td>ON:H10</td><td>B2</td><td>Celebese ape</td><td></td><td>USA</td></tr><tr><td>ECOR28</td><td>O104:NM</td><td>B1</td><td>Human</td><td>Woman</td><td>USA</td><td>ECOR66</td><td>O4:H40</td><td>B2</td><td>Celebese ape</td><td></td><td>USA</td></tr><tr><td>ECOR29</td><td>O150:H21</td><td>B1</td><td>Kangaroo rat</td><td></td><td>USA</td><td>ECOR67</td><td>O4:H43</td><td>B1</td><td>Goat</td><td></td><td>Indonesia</td></tr><tr><td>ECOR30</td><td>O113:H21</td><td>B1</td><td>Bison</td><td></td><td>Canada</td><td>ECOR68</td><td>ON:NM</td><td>B1</td><td>Giraffe</td><td></td><td>USA</td></tr><tr><td>ECOR31</td><td>O79:H43</td><td>E</td><td>Leopard</td><td></td><td>USA</td><td>ECOR69</td><td>ON:NM</td><td>B1</td><td>Celebese ape</td><td></td><td>USA</td></tr><tr><td>ECOR32</td><td>O7:H21</td><td>B1</td><td>Giraffe</td><td></td><td>USA</td><td>ECOR70</td><td>O78:NM</td><td>B1</td><td>Gorilla</td><td></td><td>USA</td></tr><tr><td>ECOR33</td><td>O7:H21</td><td>B1</td><td>Sheep</td><td></td><td>USA</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

This table was built according to data from previously published papers [19, 20].

replicate within macrophages. Among these strains, 13 were able to survive and replicate in macrophages. The percent intracellular bacteria at 1 and 24 h postinfection ranged between 114.06 and 881.36%. The ECOR 1, 2, 9, 15, 35, 36, 43, 23, 45, 67, 69, 52, 44 were thus considered as belonging to the AIEC pathotype.

The two other strains (ECOR 46 and 7) were not able to replicate inside macrophages with an index of 70.42 and 74.51% respectively (Table 3). These strains were thus not affiliated to the AIEC pathovar.

Discussion

The ECOR collection is a collection of *E. coli* that reflects the genetic diversity of non-pathogenic *E. coli* isolated from humans and animals of diverse geographical origins [19] (Table 1). We studied the 61 strains of *E. coli* isolated from the feces of healthy individuals for an invasive phenotype. Our study allowed the demonstration of the presence of AIEC strains in feces of healthy individuals. On the other hand this also gave an insight into the prevalence of AIEC in diverse conditions. Our results showed
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Invasion score of ECOR strains of fecal origin according to their phylogroup (excluding the cell-detaching strains)</figcaption></figure>

that intestinal *E. coli* from the ECOR collection could be divided into three groups according to their phenotypes observed in vitro in I-407 cells: (i) non-invasive strains, (ii) invasive strains defined as AIEC and (iii) strains able to disrupt I-407 cell layer.

Of a total of 61 fecal *E. coli* strains, 48 were not invasive, 18 presented adherent and invasive properties and 5 were able to detach I-407 cells. Out of 18 adherent and invasive strains, 13 were able to survive and replicate inside macrophages, these strains were thus affiliated to the AIEC pathotype. Among all the fecal strains, the prevalence of AIEC was 21% (24% in the human set and 19% among animal strains). All cell-detaching strains were of human origin, which accounted for 8% of the strains among animal and human isolates (17% of all human strains tested).

The AIEC pathotype was first described in inflammatory bowel diseases patients. Several independent research groups have reported a higher prevalence of AIEC in biopsies from IBD patients compared to non-IBD controls. However, AIEC prevalence greatly varies between studies. Indeed, AIEC prevalence was shown to range between 10 and 52% in ileal biopsies of CD patients [9–12]. This decreases to 3.7% in colonic biopsies of CD patients, and AIEC has yet to be isolated from colonic biopsies of UC patients [6]. In non-IBD controls, AIEC prevalence was shown to be between 0 and 17% [9–12]. In our study, we showed for the first time that AIEC strains can also be detected in stools of healthy subjects, with a prevalence of 21%.

AIEC pathovars from the ECOR collection are genetically highly diverse as they belong to different phylogroups. Indeed, among the 13 intestinal adherent and
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Invasion score of ECOR strains of fecal origin according to their phylogroup (excluding the cell-detaching strains)</figcaption></figure>

that intestinal *E. coli* from the ECOR collection could be divided into three groups according to their phenotypes observed in vitro in I-407 cells: (i) non-invasive strains, (ii) invasive strains defined as AIEC and (iii) strains able to disrupt I-407 cell layer.

Of a total of 61 fecal *E. coli* strains, 48 were not invasive, 18 presented adherent and invasive properties and 5 were able to detach I-407 cells. Out of 18 adherent and invasive strains, 13 were able to survive and replicate inside macrophages, these strains were thus affiliated to the AIEC pathotype. Among all the fecal strains, the prevalence of AIEC was 21% (24% in the human set and 19% among animal strains). All cell-detaching strains were of human origin, which accounted for 8% of the strains among animal and human isolates (17% of all human strains tested).

The AIEC pathotype was first described in inflammatory bowel diseases patients. Several independent research groups have reported a higher prevalence of AIEC in biopsies from IBD patients compared to non-IBD controls. However, AIEC prevalence greatly varies between studies. Indeed, AIEC prevalence was shown to range between 10 and 52% in ileal biopsies of CD patients [9–12]. This decreases to 3.7% in colonic biopsies of CD patients, and AIEC has yet to be isolated from colonic biopsies of UC patients [6]. In non-IBD controls, AIEC prevalence was shown to be between 0 and 17% [9–12]. In our study, we showed for the first time that AIEC strains can also be detected in stools of healthy subjects, with a prevalence of 21%.

AIEC pathovars from the ECOR collection are genetically highly diverse as they belong to different phylogroups. Indeed, among the 13 intestinal adherent and
<table><caption>Table 2 Characteristics summary of invasive strains of fecal origin</caption><thead><tr><th>Origin</th><th>Type of strains</th><th>ID</th><th>Serotype</th><th>Phylogroup</th><th>Source</th><th colspan="10">Virulence genes</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th></th><th>stx-1</th><th>stx-2</th><th>ipaC</th><th>estA</th><th>elt</th><th>tia</th><th>bfpA</th><th>eae</th><th>exhA</th><th>aggR</th><th>pCVD432</th><th>afaD</th></tr></thead><tbody><tr><td rowspan="10">Humans</td><td rowspan="10">Invasive strains</td><td>ECOR1</td><td>ON:HIN</td><td>A</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR2</td><td>ON:H32</td><td>A</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR9</td><td>ON:NM</td><td>A</td><td>Human, Sweden</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR15</td><td>O25:NM</td><td>A</td><td>Human, Sweden</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR63</td><td>ON:NM</td><td>B2</td><td>Human, Sweden</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td></tr><tr><td>ECOR35</td><td>O1:NM</td><td>D</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR36</td><td>O79:H25</td><td>D</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR43</td><td>ON:HIN</td><td>E</td><td>Human, Sweden</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR7</td><td>O85:HIN</td><td>A</td><td>Orangutan, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td rowspan="10">Animals</td><td rowspan="10">Cell detaching strains</td><td>ECOR23</td><td>O86:H43</td><td>A</td><td>Elephant, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR45</td><td>ON:HIM</td><td>B1</td><td>Pig, Indonesia</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR67</td><td>O4:H43</td><td>B1</td><td>Goat, Indonesia</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR69</td><td>ON:NM</td><td>B1</td><td>Celebese ape, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR70</td><td>O78:NM</td><td>B1</td><td>Gorilla, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td></tr><tr><td>ECOR52</td><td>O25:H1</td><td>B2</td><td>Orangutan, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR57</td><td>ON:NM</td><td>B2</td><td>Gorilla, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR44</td><td>ON:HIN</td><td>D</td><td>Cougar, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR46</td><td>O1:H6</td><td>D</td><td>Celebese ape, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td rowspan="4">Humans</td><td rowspan="4">Cell detaching strains</td><td>ECOR6</td><td>ON:HIM</td><td>A</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR51</td><td>O25:HIN</td><td>B2</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td></tr><tr><td>ECOR53</td><td>O4:HIN</td><td>B2</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR54</td><td>O25:H1</td><td>B2</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td></td><td></td><td>ECOR59</td><td>O4:H40</td><td>B2</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr></tbody></table>
<table><caption>Table 2 Characteristics summary of invasive strains of fecal origin</caption><thead><tr><th>Origin</th><th>Type of strains</th><th>ID</th><th>Serotype</th><th>Phylogroup</th><th>Source</th><th colspan="10">Virulence genes</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th></th><th>stx-1</th><th>stx-2</th><th>ipaC</th><th>estA</th><th>elt</th><th>tia</th><th>bfpA</th><th>eae</th><th>exhA</th><th>aggR</th><th>pCVD432</th><th>afaD</th></tr></thead><tbody><tr><td rowspan="10">Humans</td><td rowspan="10">Invasive strains</td><td>ECOR1</td><td>ON:HIN</td><td>A</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR2</td><td>ON:H32</td><td>A</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR9</td><td>ON:NM</td><td>A</td><td>Human, Sweden</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR15</td><td>O25:NM</td><td>A</td><td>Human, Sweden</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR63</td><td>ON:NM</td><td>B2</td><td>Human, Sweden</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td></tr><tr><td>ECOR35</td><td>O1:NM</td><td>D</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR36</td><td>O79:H25</td><td>D</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR43</td><td>ON:HIN</td><td>E</td><td>Human, Sweden</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR7</td><td>O85:HIN</td><td>A</td><td>Orangutan, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td rowspan="10">Animals</td><td rowspan="10">Cell detaching strains</td><td>ECOR23</td><td>O86:H43</td><td>A</td><td>Elephant, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR45</td><td>ON:HIM</td><td>B1</td><td>Pig, Indonesia</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR67</td><td>O4:H43</td><td>B1</td><td>Goat, Indonesia</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR69</td><td>ON:NM</td><td>B1</td><td>Celebese ape, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR70</td><td>O78:NM</td><td>B1</td><td>Gorilla, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td></tr><tr><td>ECOR52</td><td>O25:H1</td><td>B2</td><td>Orangutan, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR57</td><td>ON:NM</td><td>B2</td><td>Gorilla, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR44</td><td>ON:HIN</td><td>D</td><td>Cougar, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR46</td><td>O1:H6</td><td>D</td><td>Celebese ape, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR6</td><td>ON:HIM</td><td>A</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td rowspan="4">Humans</td><td rowspan="4">Cell detaching strains</td><td>ECOR51</td><td>O25:HIN</td><td>B2</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td></tr><tr><td>ECOR53</td><td>O4:HIN</td><td>B2</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR54</td><td>O25:H1</td><td>B2</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>ECOR59</td><td>O4:H40</td><td>B2</td><td>Human, USA</td><td></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr></tbody></table>
<table><caption>Table 3 Capacity of adhesion in I-407 cells and survival and replication in macrophages among the 15 invasive strains form the ECOR collection</caption><thead><tr><th></th><th>ECOR strains</th><th>Mean adhesion index</th><th>Mean of bacteria recovered at 1 and 24 h postinfection (%)</th></tr></thead><tbody><tr><td rowspan="12">Invasive strains</td><td>ECOR1</td><td>197.89±29.77</td><td>387.47%±236.00%</td></tr><tr><td>ECOR35</td><td>210.53±208.41</td><td>881.36%±295.06%</td></tr><tr><td>ECOR52</td><td>73.26±2.38</td><td>133.33%</td></tr><tr><td>ECOR2</td><td>111.58±32.75</td><td>168.72%±5.67%</td></tr><tr><td>ECOR36</td><td>145.26±68.48</td><td>511.41%±70.89%</td></tr><tr><td>ECOR43</td><td>82.11±20.84</td><td>114.06%±142.24%</td></tr><tr><td>ECOR44</td><td>334.74±229.25</td><td>255.70%±35.56%</td></tr><tr><td>ECOR45</td><td>94.74±20.84</td><td>314.08%±268.26%</td></tr><tr><td>ECOR46</td><td>92.63±17.86</td><td>70.42%</td></tr><tr><td>ECOR7</td><td>170.53±74.43</td><td>74.51%</td></tr><tr><td>ECOR9</td><td>157.89±56.57</td><td>152.00%</td></tr><tr><td>ECOR15</td><td>153.68±44.66</td><td>279.07%</td></tr><tr><td>ECOR23</td><td>260.63±48.23</td><td>395.48%</td></tr><tr><td>ECOR67</td><td>67.37±23.82</td><td>361.90%</td></tr><tr><td>ECOR69</td><td>117.89±41.68</td><td>887.64%</td></tr><tr><td rowspan="2">Noninvasive strains</td><td><i>E. coli</i> K12</td><td>51.58±16.38</td><td>24.59%±2.84</td></tr><tr><td><i>E. coli</i> Nissle</td><td>32.21±8.30</td><td>87.78%±131.98%</td></tr><tr><td rowspan="2">Invasive strains</td><td>AIEC LF82</td><td>166.32±20.84</td><td>220.59%</td></tr><tr><td>AIEC NRG857c</td><td>54.32±4.17</td><td>194.90%±39.93</td></tr></tbody></table>

invasive strains identified in this study, 5 belong to the phylogroup A, 3 to the phylogroup B1, 1 to the phylogroup B2, 3 to the phylogroup D and 1 to the phylogroup E. It has been shown that *E. coli* belonging to B2 and D phylogroups possess higher numbers of virulence genes than do A and B1 phylogroups. However, recently, no association was found between the phylogenetic background and the occurrence of diarrhea in calves [23]. The majority of AIEC strains isolated to date, coming from diseased human or animals, belong to the B2 phylogroup [10, 11, 15, 17, 18]. AIEC strains isolated from non-IBD controls (mainly patients suffering from rectorrhagia, hemorrhoids, irritable bowel syndrome, or diverticulitis) also mainly belong to the B2 phylogroup [10]. We showed here that AIEC strains isolated from healthy individuals, either humans or animals, mainly belong to other phylogroups.

Some studies have reported virulence determinant genes and cytotoxic profiles in *E. coli* strains from the ECOR collection [24–27]. Strains revealed to be invasive and capable of detaching I-407 cells were found to express some virulent genes such as pyelonephritis associated pili (pap), S-family adhesions (sfa), hemolysin (hly), long polar fimbriae (lpf), and secreted autotransporter toxin (sat). Among the 13 adherent and invasive strains, 10 are known to express at least one of these

genes (ECOR 2, 35, 36, 43, 23, 45, 67, 69, 52 and 44) and among the five cell-detaching strains, three harbor at least one of these virulence determinants (ECOR 51, 53 and 54) (Table 4). Some of these genes were also found in AIEC strains isolated from biopsies of both IBD and non-IBD patients. Martinez et al. showed that of a total of 16 AIEC strains recovered from biopsies of IBD patients, 8 express *papC* gene, 2 express *sfa*, 4 express *hlya*, and all *fimH*. AIEC strains from non-IBD patients (n=6) also express these genes, 4 were positive for *papC*, 2 for *sfa*, 2 for *hlya* and all for *fimH* [10].

Our work showed for the first time the detection of AIEC strains in healthy animal hosts; one from a healthy orangutan, one from a healthy elephant in captivity, one from a healthy celebese ape in captivity, one from a healthy pig, one from a healthy cougar and one from a healthy goat. These results provide further support for the absence of host specificity of this pathotype [28]. Further studies analyzing the prevalence of AIEC in well-characterized diseased and healthy animals are needed in order to detect putative reservoirs of AIEC strains and to evaluate the dimension of the risk with respect to the implementation of prevention and control measures and to public health.

To conclude, this study showed a prevalence of asymptomatic intestinal carriage of invasive pathogenic *E. coli*,
<table><caption>Table 3 Capacity of adhesion in I-407 cells and survival and replication in macrophages among the 15 invasive strains form the ECOR collection</caption><thead><tr><th></th><th>ECOR strains</th><th>Mean adhesion index</th><th>Mean of bacteria recovered at 1 and 24 h postinfection (%)</th></tr></thead><tbody><tr><td rowspan="12">Invasive strains</td><td>ECOR1</td><td>197.89±29.77</td><td>387.47%±236.00%</td></tr><tr><td>ECOR35</td><td>210.53±208.41</td><td>881.36%±295.06%</td></tr><tr><td>ECOR52</td><td>73.26±2.38</td><td>133.33%</td></tr><tr><td>ECOR2</td><td>111.58±32.75</td><td>168.72%±5.67%</td></tr><tr><td>ECOR36</td><td>145.26±68.48</td><td>511.41%±70.89%</td></tr><tr><td>ECOR43</td><td>82.11±20.84</td><td>114.06%±142.24%</td></tr><tr><td>ECOR44</td><td>334.74±229.25</td><td>255.70%±35.56%</td></tr><tr><td>ECOR45</td><td>94.74±20.84</td><td>314.08%±268.26%</td></tr><tr><td>ECOR46</td><td>92.63±17.86</td><td>70.42%</td></tr><tr><td>ECOR7</td><td>170.53±74.43</td><td>74.51%</td></tr><tr><td>ECOR9</td><td>157.89±56.57</td><td>152.00%</td></tr><tr><td>ECOR15</td><td>153.68±44.66</td><td>279.07%</td></tr><tr><td>ECOR23</td><td>260.63±48.23</td><td>395.48%</td></tr><tr><td>ECOR67</td><td>67.37±23.82</td><td>361.90%</td></tr><tr><td>ECOR69</td><td>117.89±41.68</td><td>887.64%</td></tr><tr><td rowspan="2">Noninvasive strains</td><td><i>E. coli</i> K12</td><td>51.58±16.38</td><td>24.59%±2.84</td></tr><tr><td><i>E. coli</i> Nissle</td><td>32.21±8.30</td><td>87.78%±131.98%</td></tr><tr><td rowspan="2">Invasive strains</td><td>AIEC LF82</td><td>166.32±20.84</td><td>220.59%</td></tr><tr><td>AIEC NRG857c</td><td>54.32±4.17</td><td>194.90%±39.93</td></tr></tbody></table>

invasive strains identified in this study, 5 belong to the phylogroup A, 3 to the phylogroup B1, 1 to the phylogroup B2, 3 to the phylogroup D and 1 to the phylogroup E. It has been shown that *E. coli* belonging to B2 and D phylogroups possess higher numbers of virulence genes than do A and B1 phylogroups. However, recently, no association was found between the phylogenetic background and the occurrence of diarrhea in calves [23]. The majority of AIEC strains isolated to date, coming from diseased human or animals, belong to the B2 phylogroup [10, 11, 15, 17, 18]. AIEC strains isolated from non-IBD controls (mainly patients suffering from rectorrhagia, hemorrhoids, irritable bowel syndrome, or diverticulitis) also mainly belong to the B2 phylogroup [10]. We showed here that AIEC strains isolated from healthy individuals, either humans or animals, mainly belong to other phylogroups.

Some studies have reported virulence determinant genes and cytotoxic profiles in *E. coli* strains from the ECOR collection [24–27]. Strains revealed to be invasive and capable of detaching I-407 cells were found to express some virulent genes such as pyelonephritis associated pili (pap), S-family adhesions (sfa), hemolysin (hly), long polar fimbriae (lpf), and secreted autotransporter toxin (sat). Among the 13 adherent and invasive strains, 10 are known to express at least one of these

genes (ECOR 2, 35, 36, 43, 23, 45, 67, 69, 52 and 44) and among the five cell-detaching strains, three harbor at least one of these virulence determinants (ECOR 51, 53 and 54) (Table 4). Some of these genes were also found in AIEC strains isolated from biopsies of both IBD and non-IBD patients. Martinez et al. showed that of a total of 16 AIEC strains recovered from biopsies of IBD patients, 8 express *papC* gene, 2 express *sfa*, 4 express *hlya*, and all *fimH*. AIEC strains from non-IBD patients (n=6) also express these genes, 4 were positive for *papC*, 2 for *sfa*, 2 for *hlya* and all for *fimH* [10].

Our work showed for the first time the detection of AIEC strains in healthy animal hosts; one from a healthy orangutan, one from a healthy elephant in captivity, one from a healthy celebese ape in captivity, one from a healthy pig, one from a healthy cougar and one from a healthy goat. These results provide further support for the absence of host specificity of this pathotype [28]. Further studies analyzing the prevalence of AIEC in well-characterized diseased and healthy animals are needed in order to detect putative reservoirs of AIEC strains and to evaluate the dimension of the risk with respect to the implementation of prevention and control measures and to public health.

To conclude, this study showed a prevalence of asymptomatic intestinal carriage of invasive pathogenic *E. coli*,
<table><caption>Table 4 Virulence determinants in AIEC and cell-detaching strains from the ECOR collection</caption><thead><tr><th rowspan="2">ECOR strains</th><th colspan="4">Cell-detaching strains</th><th rowspan="2">References</th></tr><tr><th>ECOR53</th><th>ECOR51</th><th>ECOR6</th><th>ECOR59</th><th>ECOR54</th></tr></thead><tbody><tr><td colspan="6">Virulence determinants associated with pathogenicity islands</td></tr><tr><td>Two adhesin gene clusters sfa and pap</td><td>+ pap1.2.3<br>sfa1.2</td><td>+ pap1.2.3<br>sfa1.2</td><td>—</td><td>—</td><td>+sfa1.2</td><td>Boyd et al. [25]</td></tr><tr><td>The kps capsule operon</td><td>+kps1.2</td><td>+ kps1.2</td><td>—</td><td>—</td><td>+ kps1.2</td><td></td></tr><tr><td>The alpha-hemolysin loci hlyI and hlyII</td><td>+hly2</td><td>+hly2</td><td>—</td><td>—</td><td>+ hly2</td><td></td></tr><tr><td>Haemolytic activity</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PCR detection of the hlyA gene</td><td>+ Strong haemolysis</td><td>+ Strong haemolysis</td><td>ND</td><td>ND</td><td>+ Strong haemolysis</td><td>Lai et al. [24]</td></tr><tr><td>Distribution of STEC-lpfAs</td><td></td><td></td><td>ND</td><td>ND</td><td></td><td></td></tr><tr><td>Four lpfA genetic variants designated lpfAO157/OI-141, lpfAO157/OI-154, lpfAO26 and lpfAO113</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Toma et al. [26]</td></tr><tr><td colspan="6">Antibiotic resistance in the ECOR collection</td></tr><tr><td>Streptomycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Spectinomycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Sulfonamide</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Tetracycline</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Ampicillin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Kanamycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Chloramphenicol</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Mercuric chloride</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Ethidium bromide</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><th colspan="6">ECOR strains</th><th colspan="4">AIEC phenotype</th><th colspan="4">References</th></tr><tr><th>ECOR43</th><th>ECOR23</th><th>ECOR36</th><th>ECOR69</th><th>ECOR52</th><th>ECOR1</th><th>ECOR2</th><th>ECOR9</th><th>ECOR15</th><th>ECOR35</th><th>ECOR45</th><th>ECOR67</th><th>ECOR44</th></tr><tr><td colspan="6">Virulence determinants associated with pathogenicity islands</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Two adhesin gene clusters sfa and pap</td><td>—</td><td>+ pap1.2.3</td><td>—</td><td>+ pap1.2.3<br>sfa1.2</td><td>—</td><td>+ pap1.2.3</td><td>—</td><td>—</td><td>+ pap1</td><td>—</td><td>—</td><td>+ pap1.2.3</td><td>Boyd et al. [25]</td></tr><tr><td>The kps capsule operon</td><td>—</td><td>+ kps1.2</td><td>—</td><td>+ kps1.2</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+ kps1.2</td><td>—</td><td>—</td><td>+ kps1.2</td><td></td></tr><tr><td>The alpha-hemolysin loci hlyI and hlyII</td><td>+ hly2</td><td>—</td><td>—</td><td>+ hly2</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Haemolytic activity</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PCR detection of the hlyA gene</td><td>No haemoly-<br>sis</td><td>ND</td><td>ND</td><td>ND</td><td>+ Strong<br>haemolysis</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>Lai et al. [24]</td></tr><tr><td>Distribution of STEC-lpfAs</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Four lpfA genetic variants<br>designated lpfAO157/<br>OI-141, lpfAO157/OI-154,<br>lpfAO26 and lpfAO113</td><td>lpfAO26+<br>lpfAO157/<br>OI-141+</td><td>lpfAO26+<br>lpfAO113+</td><td>lpfAO113+</td><td>lpfAO113+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>lpfAO113+</td><td>lpfAO26+<br>lpfAO113+</td><td>lpfAO26+<br>lpfAO113+</td><td>lpfAO113+</td><td>Toma et al. [26]</td></tr></tbody></table>
<table><caption>Table 4 Virulence determinants in AIEC and cell-detaching strains from the ECOR collection</caption><thead><tr><th rowspan="2">ECOR strains</th><th colspan="4">Cell-detaching strains</th><th rowspan="2">References</th></tr><tr><th>ECOR53</th><th>ECOR51</th><th>ECOR6</th><th>ECOR59</th><th>ECOR54</th></tr></thead><tbody><tr><td colspan="6">Virulence determinants associated with pathogenicity islands</td></tr><tr><td>Two adhesin gene clusters sfa and pap</td><td>+ pap1.2.3<br>sfa1.2</td><td>+ pap1.2.3<br>sfa1.2</td><td>—</td><td>—</td><td>+sfa1.2</td><td>Boyd et al. [25]</td></tr><tr><td>The kps capsule operon</td><td>+kps1.2</td><td>+ kps1.2</td><td>—</td><td>—</td><td>+ kps1.2</td><td></td></tr><tr><td>The alpha-hemolysin loci hlyI and hlyII</td><td>+hly2</td><td>+hly2</td><td>—</td><td>—</td><td>+ hly2</td><td></td></tr><tr><td>Haemolytic activity</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PCR detection of the hlyA gene</td><td>+ Strong haemolysis</td><td>+ Strong haemolysis</td><td>ND</td><td>ND</td><td>+ Strong haemolysis</td><td>Lai et al. [24]</td></tr><tr><td>Distribution of STEC-lpfAs</td><td></td><td></td><td>ND</td><td>ND</td><td></td><td></td></tr><tr><td>Four lpfA genetic variants designated lpfAO157/OI-141, lpfAO157/OI-154, lpfAO26 and lpfAO113</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Toma et al. [26]</td></tr><tr><td colspan="6">Antibiotic resistance in the ECOR collection</td></tr><tr><td>Streptomycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Spectinomycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Sulfonamide</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Tetracycline</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Ampicillin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Kanamycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Chloramphenicol</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Mercuric chloride</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td>Ethidium bromide</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><th colspan="6">ECOR strains</th><th colspan="4">AIEC phenotype</th><th rowspan="2">References</th></tr><tr><th>ECOR43</th><th>ECOR23</th><th>ECOR36</th><th>ECOR69</th><th>ECOR52</th><th>ECOR1</th><th>ECOR2</th><th>ECOR9</th><th>ECOR15</th><th>ECOR35</th><th>ECOR45</th><th>ECOR67</th><th>ECOR44</th></tr><tr><td colspan="6">Virulence determinants associated with pathogenicity islands</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Two adhesin gene clusters sfa and pap</td><td>—</td><td>+ pap1.2.3</td><td>—</td><td>+ pap1.2.3<br>sfa1.2</td><td>—</td><td>+ pap1.2.3</td><td>—</td><td>—</td><td>+ pap1</td><td>—</td><td>—</td><td>+ pap1.2.3</td><td>Boyd et al. [25]</td></tr><tr><td>The kps capsule operon</td><td>—</td><td>+ kps1.2</td><td>—</td><td>+ kps1.2</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+ kps1.2</td><td>—</td><td>—</td><td>+ kps1.2</td><td></td></tr><tr><td>The alpha-hemolysin loci hlyI and hlyII</td><td>+ hly2</td><td>—</td><td>—</td><td>+ hly2</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td></td></tr><tr><td colspan="6">Haemolytic activity</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PCR detection of the hlyA gene</td><td>No haemoly-<br>sis</td><td>ND</td><td>ND</td><td>ND</td><td>+ Strong<br>haemolysis</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>Lai et al. [24]</td></tr><tr><td>Distribution of STEC-lpfAs</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Four lpfA genetic variants<br>designated lpfAO157/<br>OI-141, lpfAO157/OI-154,<br>lpfAO26 and lpfAO113</td><td>lpfAO26+<br>lpfAO157/<br>OI-141+</td><td>lpfAO26+<br>lpfAO113+</td><td>lpfAO113+</td><td>lpfAO113+</td><td>—</td><td>—</td><td>—</td><td>—</td><td>lpfAO113+</td><td>lpfAO26+<br>lpfAO113+</td><td>lpfAO26+<br>lpfAO113+</td><td>lpfAO113+</td><td>Toma et al. [26]</td></tr></tbody></table>
<table><caption>**Table 4 (continued)**</caption><thead><tr><th rowspan="2">ECOR strains</th><th colspan="10">AIEC phenotype</th><th rowspan="2">References</th></tr><tr><th>ECOR43</th><th>ECOR23</th><th>ECOR36</th><th>ECOR69</th><th>ECOR52</th><th>ECOR1</th><th>ECOR2</th><th>ECOR9</th><th>ECOR15</th><th>ECOR35</th><th>ECOR45</th><th>ECOR67</th><th>ECOR44</th></tr></thead><tbody><tr><td colspan="14">Antibiotic resistance in the ECOR collection</td><td></td></tr><tr><td>Streptomycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Mazel et al. [32]</td></tr><tr><td>Spectinomycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Sulfonamide</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Tetracyclin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Ampicillin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Kanamycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Chloramphenicol</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Mercuric chloride</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Ethidium bromide</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr></tbody></table>
<table><caption>Table 4 (continued)</caption><thead><tr><th rowspan="2">ECOR strains</th><th colspan="10">AIEC phenotype</th><th colspan="4">References</th></tr><tr><th>ECOR43</th><th>ECOR23</th><th>ECOR36</th><th>ECOR69</th><th>ECOR52</th><th>ECOR1</th><th>ECOR2</th><th>ECOR9</th><th>ECOR15</th><th>ECOR35</th><th>ECOR45</th><th>ECOR67</th><th>ECOR44</th></tr></thead><tbody><tr><td colspan="14">Antibiotic resistance in the ECOR collection</td></tr><tr><td>Streptomycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Mazel et al. [32]</td></tr><tr><td>Spectinomycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Sulfonamide</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Tetracyclin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Ampicillin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Kanamycin</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Chloramphenicol</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Mercuric chloride</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Ethidium bromide</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr></tbody></table>
supporting the argument that AIEC may be considered
as pathobiont [9, 16, 29]. Large-scale studies in humans
(both CD and UC patients, irritable bowel disease
patients and healthy individuals) are necessary for a bet-
ter knowledge of AIEC distribution and are currently in
progress in our laboratory. Our clinical study is consist-
ent with these data, showing notably a high prevalence
of AIEC in feces of healthy individuals (manuscript in
preparation).

Conclusions

Our study has evaluated the prevalence of AIEC in the
standard reference *E. coli* collection (ECOR). Among
all the strains from the ECOR collection, 21% exhibited
the AIEC phenotype, 24% were from healthy human ori-
gin and 19% from different healthy animal hosts. This is
of major interest from an epidemiological point of view,
highlighting potential environmental niches and possible
human transmission of AIEC and susceptibility in further
inflammatory events in a healthy population.

Methods

Bacterial strains

Bacteria from the ECOR collection were kindly provided
by Laurent Debarbieux (Institut Pasteur Paris) (Table 1).
*E. coli* referent strains belonging to different pathogenic
groups respectively ETEC (ATCC 35401), EHEC (ATCC
43895), EPEC (ATCC 43887), EAEC (ATCC 33780),
EIEC (ATCC 43893), DAEC (AfaA30, kindly provided
by Pr Eric Oswald, IRSD, INSERM UMR 1220, Toulouse,
France) were used as positive controls in PCR genotyp-
ing; while *E. coli* K12 was used as a negative control. *E.
coli* ATCC 25922 was used as a positive control for the
gentamicin susceptibility assay. Bacteria were routinely
grown on Luria-Bertani (LB) agar plates or in LB broth
at 37 °C.

Gentamicin susceptibility assay

All the strains were assessed for their susceptibility to
gentamicin by applying the in vitro diffusion method.
Agar plates were inoculated with a 0.5 McFarland bac-
terial suspension. Then, gentamicin discs (Oxoid, 10 µg)
were placed in the inoculated media and incubated at
37 °C for 24 h. The susceptibility of the bacteria to gen-
tamicin was determined by measurement of the diam-
eters of the inhibition zones surrounding the discs in
millimeters according EUCAST values. A control stand-
ard strain (E. coli ATCC 25922) for which the suscepti-
bility to antimicrobial agents is known is included in the
test to validate the assay [30].

Cells

Intestine-407 cell lines (I-407, ATCC accession num-
ber CCL-6™) were used to determine bacterial invasive-
ness. I-407 cells were maintained in Basal medium Eagle
(BME, Thermofisher, France) with 10% heat-inactivated
fetal calf serum (Euroclone Italy) supplemented with 1%
Glutamax and 1% penicillin/streptomycin (Thermofisher,
France), in 5% CO₂ atmosphere at 37 °C.

Adhesion and invasion assays in epithelial cells

Intestine-407

For AIEC identification, adhesion and invasion assays,
based on gentamicin protection, were performed accord-
ing to a previously described method [6, 15, 21, 22].
24-well plates containing 4 × 10⁵ cells/well incubated for
20 h without antibiotics were infected with the *E. coli*
strains at a multiplicity of infection of 10 during 3 h of
incubation time. For the bacterial adhesion assays, the
cell monolayers were washed with phosphate-buffered
saline and then lysed with 1% Triton X-100. Adherent
bacteria were quantified by plating them on LB agar. Plat-
ing was performed over a maximum period of 30 min
in order to avoid bacterial lysis by Triton X-100. Adher-
ence ability (I_ADH) was determined by calculating
the mean number of bacteria per cell. For the bacterial
invasion assays, cells were washed twice with phosphate
buffer saline (PBS), and fresh cell culture medium con-
taining 100 µg ml⁻¹ of gentamicin was added for 1 h to
kill extracellular bacteria. Cells were then washed three
times with PBS and 1 ml of 1% Triton X-100 was added in
each well for 5 min to lyse the eukaryotic cells. After cell
lysis, the number of intracellular bacteria was determined
by plating. The invasive ability was expressed as the per-
centage of intracellular *E. coli* recovered from the ini-
tial inoculum, taken as 100%. An isolate was considered
invasive when invasion was ≥ 0.1%. Each experiment was
done in triplicate. The non-invasive strains of *E. coli* K12
and *E. coli* Nissle and the invasive AIEC LF82 and AIEC
NRG857c strains were used as negative and positive con-
trol in each experiment.

Survival and replication in J774 macrophages

The macrophage-like J774A.1 cell line (ATCC TIB-67)
was used as a model in *E. coli* survival and replication
assays. *E. coli* isolates with known adherence and inva-
sion properties were checked for their ability to survive
and replicate inside macrophages. Briefly, macrophages
were seeded at 4 × 10⁵ cells per well in two 24-well plates
and incubated for 20 h. After incubation, the medium
was replaced with fresh medium and bacteria were
supporting the argument that AIEC may be considered
as pathobiont [9, 16, 29]. Large-scale studies in humans
(both CD and UC patients, irritable bowel disease
patients and healthy individuals) are necessary for a bet-
ter knowledge of AIEC distribution and are currently in
progress in our laboratory. Our clinical study is consist-
ent with these data, showing notably a high prevalence
of AIEC in feces of healthy individuals (manuscript in
preparation).

Conclusions

Our study has evaluated the prevalence of AIEC in the
standard reference *E. coli* collection (ECOR). Among
all the strains from the ECOR collection, 21% exhibited
the AIEC phenotype, 24% were from healthy human ori-
gin and 19% from different healthy animal hosts. This is
of major interest from an epidemiological point of view,
highlighting potential environmental niches and possible
human transmission of AIEC and susceptibility in further
inflammatory events in a healthy population.

**Methods**

Bacterial strains

Bacteria from the ECOR collection were kindly provided
by Laurent Debarbieux (Institut Pasteur Paris) (Table 1).
*E. coli* referent strains belonging to different pathogenic
groups respectively ETEC (ATCC 35401), EHEC (ATCC
43895), EPEC (ATCC 43887), EAEC (ATCC 33780),
EIEC (ATCC 43893), DAEC (AfaA30, kindly provided
by Pr Eric Oswald, IRSD, INSERM UMR 1220, Toulouse,
France) were used as positive controls in PCR genotyp-
ing; while *E. coli* K12 was used as a negative control. *E.
coli* ATCC 25922 was used as a positive control for the
gentamicin susceptibility assay. Bacteria were routinely
grown on Luria–Bertani (LB) agar plates or in LB broth
at 37 °C.

Gentamicin susceptibility assay

All the strains were assessed for their susceptibility to
gentamicin by applying the in vitro diffusion method.
Agar plates were inoculated with a 0.5 McFarland bac-
terial suspension. Then, gentamicin discs (Oxoid, 10 µg)
were placed in the inoculated media and incubated at
37 °C for 24 h. The susceptibility of the bacteria to gen-
tamicin was determined by measurement of the diam-
eters of the inhibition zones surrounding the discs in
millimeters according EUCAST values. A control stand-
ard strain (E. coli ATCC 25922) for which the suscepti-
bility to antimicrobial agents is known is included in the
test to validate the assay [30].

Cells

Intestine-407 cell lines (I-407, ATCC accession num-
ber CCL-6™) were used to determine bacterial invasive-
ness. I-407 cells were maintained in Basal medium Eagle
(BME, Thermofisher, France) with 10% heat-inactivated
fetal calf serum (Euroclone Italy) supplemented with 1%
Glutamax and 1% penicillin/streptomycin (Thermofisher,
France), in 5% CO₂ atmosphere at 37 °C.

Adhesion and invasion assays in epithelial cells

Intestine-407

For AIEC identification, adhesion and invasion assays,
based on gentamicin protection, were performed accord-
ing to a previously described method [6, 15, 21, 22].
24-well plates containing 4 × 10⁵ cells/well incubated for
20 h without antibiotics were infected with the *E. coli*
strains at a multiplicity of infection of 10 during 3 h of
incubation time. For the bacterial adhesion assays, the
cell monolayers were washed with phosphate-buffered
saline and then lysed with 1% Triton X-100. Adherent
bacteria were quantified by plating them on LB agar. Plat-
ing was performed over a maximum period of 30 min
in order to avoid bacterial lysis by Triton X-100. Adher-
ence ability (I_ADH) was determined by calculating
the mean number of bacteria per cell. For the bacterial
invasion assays, cells were washed twice with phosphate
buffer saline (PBS), and fresh cell culture medium con-
taining 100 µg ml⁻¹ of gentamicin was added for 1 h to
kill extracellular bacteria. Cells were then washed three
times with PBS and 1 ml of 1% Triton X-100 was added in
each well for 5 min to lyse the eukaryotic cells. After cell
lysis, the number of intracellular bacteria was determined
by plating. The invasive ability was expressed as the per-
centage of intracellular *E. coli* recovered from the ini-
tial inoculum, taken as 100%. An isolate was considered
invasive when invasion was ≥ 0.1%. Each experiment was
done in triplicate. The non-invasive strains of *E. coli* K12
and *E. coli* Nissle and the invasive AIEC LF82 and AIEC
NRG857c strains were used as negative and positive con-
trol in each experiment.

Survival and replication in J774 macrophages

The macrophage-like J774A.1 cell line (ATCC TIB-67)
was used as a model in *E. coli* survival and replication
assays. *E. coli* isolates with known adherence and inva-
sion properties were checked for their ability to survive
and replicate inside macrophages. Briefly, macrophages
were seeded at 4 × 10⁵ cells per well in two 24-well plates
and incubated for 20 h. After incubation, the medium
was replaced with fresh medium and bacteria were
seeded at a multiplicity of infection of 100. To promote
internalization of bacteria by the macrophages, the sam-
ples were centrifuged at 1000 rpm for 10 min and incu-
bated for an additional 10 min at 37 °C with 5% CO2.
Nonphagocytosed bacteria were killed with gentamicin.
Intracellular bacteria were quantified in the same manner
as described for the invasion assays after 1 and 24 h of
infection. The results are expressed as the mean percent-
ages of bacteria recovered at 1 and 24 h postinfection:
I_REPL (%) = (CFU ml⁻<fnref n="1" /> at 24 h/CFU ml⁻<fnref n="1" /> at 1 h) × 100.
Those strains with an I_INV of > 0.1 and an I_REPL of
> 100% were classified as AIEC strains in the present
study. All assays were performed in duplicate.

Virulence genotyping by polymerase chain reaction (PCR)

DNA was extracted from bacteria by suspending one
bacterial colony in 50 µl of deionized water, boiling the
suspension for 10 min, and centrifuging it at 10,000×g
for 1 min. The supernatant was then used as the DNA
template for PCR.

The presence of 12 virulence genes specifically associ-
ated with diarrheagenic *E. coli* pathotypes (EPEC, ETEC,
EIEC, EHEC, DAEC, EAEC), and uropathogenic *E. coli*
(UPEC) was analyzed (Additional file 1: Table S1) [8, 31].
*E. coli* reference strains were used as positive controls.

Adherent/invasive bacteria able to survive and to mul-
tiply within macrophage, without expressing virulence
genes associated with diarrheagenic *E. coli* pathotypes
and uropathogenic *E. coli* were considered as AIEC
strains.

**Additional file**

**Additional file 1: Table S1.** Primers used for *E. coli* virulence genes detection.

Abbreviations

AIEC: adherent invasive *Escherichia coli*; BME: Basal medium eagle; CD: Crohn's disease; ECOR: *Escherichia coli* Reference Collection; FCS: Fetal calf serum; I-407: Intestine-407 cell lines; IBD: inflammatory bowel disease; IEC: intestinal epithelial cells; LB: Luria Bertani; MLEE: multilocus enzyme electrophoresis; PBS: phosphate buffer saline; PCR: polymerase chain reaction; S: susceptible; TNF: tumor necrosis factor; UC: ulcerative colitis; USA: United States of America.

Authors' contributions

OR, MT and CV designed, performed and analyzed experiments. PD and LD conceived and designed the project. OR, MT, CN interpreted data. OR and CN wrote the manuscript. PD, LD, CN and BP supervised the study. BF, CN and CV critically revised the manuscript. PD and CN obtained funding. All authors read and approved the final manuscript.

Author details

Acknowledgements

We are grateful to Dr. Laurent Debarbieux (Institut Pasteur Paris) for the gift of the ECOR collection strains. We also thank Pr Eric Oswald for the *E. coli* referent strains belonging to different pathogenic groups.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

Not applicable.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Funding

We would like to thank DigestScience foundation for their financial support.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Received: 16 March 2018 Accepted: 23 May 2018

Published online: 14 June 2018

References

1. Kaplan GG, Ng SC. Understanding and preventing the global increase of inflammatory bowel disease. Gastroenterology. 2017;152:313-21.

2. de Souza HSP, Fiocchi C. Immunopathogenesis of IBD: current state of the art. Nat Rev Gastroenterol Hepatol. 2016;13:13-27.

3. Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease. Nature. 2007;448:427-34.

4. Sartor RB, Wu GD. Roles for intestinal bacteria, viruses, and fungi in patho-
genesis of inflammatory bowel diseases and therapeutic approaches.
Gastroenterology. 2017;152:327-39.

5. Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening-Baucke V, Ortner M, Weber J, Hoffmann U, Schreiber S, Dietel M, Lochs H. Mucosal flora in inflammatory bowel disease. Gastroenterology. 2002;122:44-54.

6. Darfeuille-Michaud A, Bouneau J, Bulois P, Neut C, Glasser A-L, Barnich N, Bringer M-A, Swidsinski A, Beaugerie L, Colombel J-F. High prevalence of adherent-invasive *Escherichia coli* associated with ileal mucosa in Crohn's disease. Gastroenterology. 2004;127:412-21.

7. Palmela C, Chevarin C, Xu Z, Torres J, Sevrin G, Hirten R, Barnich N, Ng SC, Colombel J-F. Adherent-invasive *Escherichia coli* in inflammatory bowel disease. Gut. 2017;67:574-87.

8. Martin HM, Campbell BJ, Hart CA, Mpofu C, Nayar M, Singh R, Eng-
lyst H, Williams HF, Rhodes JM. Enhanced *Escherichia coli* adherence
and invasion in Crohn's disease and colon cancer. Gastroenterology.
2004;127:80-93.

9. Conte M, Longhi C, Marazzato M, Conte A, Aleandri M, Lepanto M, Zagaglia C, Nicoletti M, Aloi M, Totino V, Palamara A, Schippa S. Adherent-invasive *Escherichia coli* (AIEC) in pediatric Crohn's disease patients: phenotypic and genetic pathogenic features. BMC Res Notes. 2014;7:748.

10. Martinez-Medina M, Aldeguer X, Lopez-Siles M, González-Huix F, López-Oliu C, Dahbi G, Blanco JE, Blanco J, Garcia-Gil LJ, Darfeuille-Michaud A. Molecular diversity of *Escherichia coli* in the human gut: new ecological evidence supporting the role of adherent-invasive *E. coli* (AIEC) in Crohn's disease. Inflamm Bowel Dis. 2009;15:872-82.

11. Céspedes S, Saitz W, Del Canto F, De la Fuente M, Quera R, Hermoso M, Muñoz R, Ginard D, Khorrami S, Girón J, Assar R, Rosselló-Mora R, Vidal RM. Genetic diversity and virulence determinants of *Escherichia coli* strains isolated from patients with Crohn's disease in Spain and Chile. Front Microbiol. 2017;8:639.

12. Baumgart M, Dogan B, Rishniw M, Weitzman G, Bosworth B, Yantiss R, Orsi RH, Wiedmann M, McDonough P, Kim SG, Berg D, Shukken Y, Scherl E, Simpson KW. Culture independent analysis of ileal mucosa reveals a

<footnote n="1">¹ Lille Inflammation Research International Center, UMR 995 Inserm, Lille University, CHRU Lille, Lille, France. ² Centre de Biologie Pathologie Génétique, CHU Lille, Lille, France. ³ Service des Maladies de l'Appareil Digestif et de la Nutrition, Hôpital Claude Huriez, CHU Lille, 59037 Lille, France. ⁴ Laboratoire de Bactériologie, 3, Rue de Pr. Laguesse, B.P. 83, 59006 Lille Cedex, France.</footnote>
seeded at a multiplicity of infection of 100. To promote
internalization of bacteria by the macrophages, the sam-
ples were centrifuged at 1000 rpm for 10 min and incu-
bated for an additional 10 min at 37 °C with 5% CO2.
Nonphagocytosed bacteria were killed with gentamicin.
Intracellular bacteria were quantified in the same manner
as described for the invasion assays after 1 and 24 h of
infection. The results are expressed as the mean percent-
ages of bacteria recovered at 1 and 24 h postinfection:
I_REPL (%) = (CFU ml⁻<fnref n="1" /> at 24 h/CFU ml⁻<fnref n="1" /> at 1 h) × 100.
Those strains with an I_INV of > 0.1 and an I_REPL of
> 100% were classified as AIEC strains in the present
study. All assays were performed in duplicate.

Virulence genotyping by polymerase chain reaction (PCR)

DNA was extracted from bacteria by suspending one
bacterial colony in 50 µl of deionized water, boiling the
suspension for 10 min, and centrifuging it at 10,000×g
for 1 min. The supernatant was then used as the DNA
template for PCR.

The presence of 12 virulence genes specifically associ-
ated with diarrheagenic *E. coli* pathotypes (EPEC, ETEC,
EIEC, EHEC, DAEC, EAEC), and uropathogenic *E. coli*
(UPEC) was analyzed (Additional file 1: Table S1) [8, 31].
*E. coli* reference strains were used as positive controls.

Adherent/invasive bacteria able to survive and to mul-
tiply within macrophage, without expressing virulence
genes associated with diarrheagenic *E. coli* pathotypes
and uropathogenic *E. coli* were considered as AIEC
strains.

**Additional file**

**Additional file 1: Table S1.** Primers used for *E. coli* virulence genes detection.

**Abbreviations**

AIEC: adherent invasive *Escherichia coli*; BME: Basal medium eagle; CD: Crohn's disease; ECOR: *Escherichia coli* Reference Collection; FCS: Fetal calf serum; I-407: Intestine-407 cell lines; IBD: inflammatory bowel disease; IEC: intestinal epithelial cells; LB: Luria Bertani; MLEE: multilocus enzyme electrophoresis; PBS: phosphate buffer saline; PCR: polymerase chain reaction; S: susceptible; TNF: tumor necrosis factor; UC: ulcerative colitis; USA: United States of America.

**Authors' contributions**

OR, MT and CV designed, performed and analyzed experiments. PD and LD conceived and designed the project. OR, MT, CN interpreted data. OR and CN wrote the manuscript. PD, LD, CN and BP supervised the study. BF, CN and CV critically revised the manuscript. PD and CN obtained funding. All authors read and approved the final manuscript.

**Author details**

**Acknowledgements**

We are grateful to Dr. Laurent Debarbieux (Institut Pasteur Paris) for the gift of the ECOR collection strains. We also thank Pr Eric Oswald for the *E. coli* referent strains belonging to different pathogenic groups.

**Competing interests**

The authors declare that they have no competing interests.

**Availability of data and materials**

Not applicable.

**Consent for publication**

Not applicable.

**Ethics approval and consent to participate**

Not applicable.

**Funding**

We would like to thank DigestScience foundation for their financial support.

**Publisher's Note**

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Received: 16 March 2018 Accepted: 23 May 2018

Published online: 14 June 2018

**References**

1. Kaplan GG, Ng SC. Understanding and preventing the global increase of inflammatory bowel disease. Gastroenterology. 2017;152:313-21.

2. de Souza HSP, Fiocchi C. Immunopathogenesis of IBD: current state of the art. Nat Rev Gastroenterol Hepatol. 2016;13:13-27.

3. Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease. Nature. 2007;448:427-34.

4. Sartor RB, Wu GD. Roles for intestinal bacteria, viruses, and fungi in pathogenesis of inflammatory bowel diseases and therapeutic approaches. Gastroenterology. 2017;152:327-39.

5. Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening-Baucke V, Ortner M, Weber J, Hoffmann U, Schreiber S, Dietel M, Lochs H. Mucosal flora in inflammatory bowel disease. Gastroenterology. 2002;122:44-54.

6. Darfeuille-Michaud A, Boudeau J, Bulois P, Neut C, Glasser A-L, Barnich N, Bringer M-A, Swidsinski A, Beaugerie L, Colombel J-F. High prevalence of adherent-invasive *Escherichia coli* associated with ileal mucosa in Crohn's disease. Gastroenterology. 2004;127:412-21.

7. Palmela C, Chevarin C, Xu Z, Torres J, Sevrin G, Hirten R, Barnich N, Ng SC, Colombel J-F. Adherent-invasive *Escherichia coli* in inflammatory bowel disease. Gut. 2017;67:574-87.

8. Martin HM, Campbell BJ, Hart CA, Mpofu C, Nayar M, Singh R, Englyst H, Williams HF, Rhodes JM. Enhanced *Escherichia coli* adherence and invasion in Crohn's disease and colon cancer. Gastroenterology. 2004;127:80-93.

9. Conte M, Longhi C, Marazzato M, Conte A, Aleandri M, Lepanto M, Zagaglia C, Nicoletti M, Aloi M, Totino V, Palamara A, Schippa S. Adherent-invasive *Escherichia coli* (AIEC) in pediatric Crohn's disease patients: phenotypic and genetic pathogenic features. BMC Res Notes. 2014;7:748.

10. Martinez-Medina M, Aldeguer X, Lopez-Siles M, González-Huix F, López-Oliu C, Dahbi G, Blanco JE, Blanco J, Garcia-Gil LJ, Darfeuille-Michaud A. Molecular diversity of *Escherichia coli* in the human gut: new ecological evidence supporting the role of adherent-invasive *E. coli* (AIEC) in Crohn's disease. Inflamm Bowel Dis. 2009;15:872-82.

11. Céspedes S, Saitz W, Del Canto F, De la Fuente M, Quera R, Hermoso M, Muñoz R, Ginard D, Khorrami S, Girón J, Assar R, Rosselló-Mora R, Vidal RM. Genetic diversity and virulence determinants of *Escherichia coli* strains isolated from patients with Crohn's disease in Spain and Chile. Front Microbiol. 2017;8:639.

12. Baumgart M, Dogan B, Rishniw M, Weitzman G, Bosworth B, Yantiss R, Orsi RH, Wiedmann M, McDonough P, Kim SG, Berg D, Shukken Y, Scherl E, Simpson KW. Culture independent analysis of ileal mucosa reveals a

<footnote n="1">¹ Lille Inflammation Research International Center, UMR 995 Inserm, Lille University, CHRU Lille, Lille, France. ² Centre de Biologie Pathologie Génétique, CHU Lille, Lille, France. ³ Service des Maladies de l'Appareil Digestif et de la Nutrition, Hôpital Claude Huriez, CHU Lille, 59037 Lille, France. ⁴ Laboratoire de Bactériologie, 3, Rue de Pr. Laguesse, B.P. 83, 59006 Lille Cedex, France.</footnote>
selective increase in invasive *Escherichia coli* of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum. ISME J. 2007;1:403-18.

13. Sasaki M, Sitaraman SV, Babbin BA, Gerner-Smidt P, Ribot EM, Garrett N, Alpern JA, Akyildiz A, Theiss AL, Nusrat A, Klapproth JMA. Invasive *Escherichia coli* are a feature of Crohn's disease. Lab Invest. 2007;87:1042-54.

14. Nash JH, Villegas A, Kropinski AM, Aguilar-Valenzuela R, Konczy P, Mascarenhas M, Ziebell K, Torres AG, Karmali MA, Coombes BK. Genome sequence of adherent-invasive *Escherichia coli* and comparative genomic analysis with other *E. coli* pathotypes. BMC Genomics. 2010;11:667.

15. Martinez-Medina M, Mora A, Blanco M, López C, Alonso MP, Bonacorsi S, Nicolas-Chanoine M-H, Darfeuille-Michaud A, Garcia-Gil J, Blanco J. Similarity and divergence among adherent-invasive *Escherichia coli* and extraintestinal pathogenic *E. coli* strains. J Clin Microbiol. 2009;47:3968-79.

16. Martinez-Medina M, Garcia-Gil LJ. *Escherichia coli* in chronic inflammatory bowel diseases: an update on adherent invasive *Escherichia coli* pathogenicity. World J Gastrointest Pathophysiol. 2014;3:213-27.

17. Simpson KW, Dogan B, Rishniw M, Goldstein RE, Klaessig S, McDonough PL, German AJ, Yates RM, Russell DG, Johnson SE, Berg DE, Harel J, Bruant G, McDonough SP, Schukken YH. Adherent and invasive *Escherichia coli* is associated with granulomatous colitis in boxer dogs. Infect Immun. 2006;74:4778-92.

18. Martinez-Medina M, Garcia-Gil J, Barnich N, Wieler LH, Ewers C. Adherent-invasive *Escherichia coli* phenotype displayed by intestinal pathogenic *E. coli* strains from cats, dogs, and swine. Appl Environ Microbiol. 2011;77:5813-7.

19. Ochman H, Selander RK. Standard reference strains of *Escherichia coli* from natural populations. J Bacteriol. 1984;157:690-3.

20. Clermont O, Cordevant C, Bonacorsi S, Marecat A, Lange M, Bingen E. Automated ribotyping provides rapid phylogenetic subgroup affiliation of clinical extraintestinal pathogenic *Escherichia coli* strains. J Clin Microbiol. 2001;39:4549-53.

21. Drouet M, Vignal C, Singer E, Djouina M, Dubreuil L, Cortot A, Desruemaux P, Neut C. AIEC colonization and pathogenicity: influence of previous antibiotic treatment and preexisting inflammation. Inflamm Bowel Dis. 2012;18:1923-31.

22. Boudeau J, Glasser AL, Masseret E, Joly B, Darfeuille-Michaud A. Invasive ability of an *Escherichia coli* strain isolated from the ileal mucosa of a patient with Crohn's disease. Infect Immun. 1999;67:4499-509.

23. Coura FM, de Araújo Diniz S, Mussi JMS, Silva MX, Lage AP, Heinemann MB. Characterization of virulence factors and phylogenetic group determination of *Escherichia coli* isolated from diarrheic and non-diarrheic calves from Brazil. Folia Microbiol. 2017;62:139-44.

24. Lai XH, Wang SY, Uhlin BE. Expression of cytotoxicity by potential pathogens in the standard *Escherichia coli* collection of reference (ECOR) strains. Microbiology. 1999;145:3295-303.

25. Boyd EF, Hartl DL. Chromosomal regions specific to pathogenic isolates of *Escherichia coli* have a phylogenetically clustered distribution. J Bacteriol. 1998;180:1159-65.

26. Toma C, Higa N, Iyoda S, Rivas M, Iwanaga M. The long polar fimbriae genes identified in Shiga toxin-producing *Escherichia coli* are present in other diarrheagenic *E. coli* and in the standard *E. coli* collection of reference (ECOR) strains. Res Microbiol. 2006;157:153-61.

27. Restieri C, Garriss G, Locas M-C, Dozois CM. Autotransporter-encoding sequences are phylogenetically distributed among *Escherichia coli* clinical isolates and reference strains. Appl Environ Microbiol. 2007;73:1553-62.

28. Dogan B, Suzuki H, Herlekar D, Sartor RB, Campbell BJ, Roberts CL, Stewart K, Scherl EJ, Araz Y, Bitar PP, Lefébure T, Chandler B, Schukken YH, Stanhope MJ, Simpson KW. Inflammation-associated adherent-invasive *Escherichia coli* are enriched in pathways for use of propanediol and iron and M-cell translocation. Inflamm Bowel Dis. 2014;20:1919-32.

29. Chassaing B, Koren O, Carvalho FA, Ley RE, Gewirtz AT. AIEC pathobiont instigates chronic colitis in susceptible hosts by altering microbiota composition. Gut. 2014;63:1069-80.

30. European Committee on Antimicrobial Susceptibility Testing. http://www.eucast.org/clinical_breakpoints/.

31. Cabal A, Gómez-Barrero S, Porrero C, Bárcena C, López G, Cantón R, Gortázar C, Domínguez L, Álvarez J. Assessment of virulence factors characteristic of human *Escherichia coli* pathotypes and antimicrobial resistance in O157:H7 and non-O157:H7 isolates from livestock in Spain. Appl Environ Microbiol. 2013;79:4170-2.

32. Mazel D, Dychinco B, Webb V-A, Davies J. Antibiotic resistance in the ECOR collection: integrons and identification of a novel aad gene. Antimicrob. Agents Chemother. 2000;44:1568-74.

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* thorough peer review by experienced researchers in your field

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selective increase in invasive *Escherichia coli* of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum. ISME J. 2007;1:403-18.

13. Sasaki M, Sitaraman SV, Babbin BA, Gerner-Smidt P, Ribot EM, Garrett N, Alpern JA, Akyildiz A, Theiss AL, Nusrat A, Klapproth JMA. Invasive *Escherichia coli* are a feature of Crohn's disease. Lab Invest. 2007;87:1042-54.

14. Nash JH, Villegas A, Kropinski AM, Aguilar-Valenzuela R, Konczy P, Mascarenhas M, Ziebell K, Torres AG, Karmali MA, Coombes BK. Genome sequence of adherent-invasive *Escherichia coli* and comparative genomic analysis with other *E. coli* pathotypes. BMC Genomics. 2010;11:667.

15. Martinez-Medina M, Mora A, Blanco M, López C, Alonso MP, Bonacorsi S, Nicolas-Chanoine M-H, Darfeuille-Michaud A, Garcia-Gil J, Blanco J. Similarity and divergence among adherent-invasive *Escherichia coli* and extraintestinal pathogenic *E. coli* strains. J Clin Microbiol. 2009;47:3968-79.

16. Martinez-Medina M, Garcia-Gil LJ. *Escherichia coli* in chronic inflammatory bowel diseases: an update on adherent invasive *Escherichia coli* pathogenicity. World J Gastrointest Pathophysiol. 2014;3:213-27.

17. Simpson KW, Dogan B, Rishniw M, Goldstein RE, Klaessig S, McDonough PL, German AJ, Yates RM, Russell DG, Johnson SE, Berg DE, Harel J, Bruant G, McDonough SP, Schukken YH. Adherent and invasive *Escherichia coli* is associated with granulomatous colitis in boxer dogs. Infect Immun. 2006;74:4778-92.

18. Martinez-Medina M, Garcia-Gil J, Barnich N, Wieler LH, Ewers C. Adherent-invasive *Escherichia coli* phenotype displayed by intestinal pathogenic *E. coli* strains from cats, dogs, and swine. Appl Environ Microbiol. 2011;77:5813-7.

19. Ochman H, Selander RK. Standard reference strains of *Escherichia coli* from natural populations. J Bacteriol. 1984;157:690-3.

20. Clermont O, Cordevant C, Bonacorsi S, Marecat A, Lange M, Bingen E. Automated ribotyping provides rapid phylogenetic subgroup affiliation of clinical extraintestinal pathogenic *Escherichia coli* strains. J Clin Microbiol. 2001;39:4549-53.

21. Drouet M, Vignal C, Singer E, Djouina M, Dubreuil L, Cortot A, Desru Maux P, Neut C. AIEC colonization and pathogenicity: influence of previous antibiotic treatment and preexisting inflammation. Inflamm Bowel Dis. 2012;18:1923-31.

22. Boudeau J, Glasser AL, Masseret E, Joly B, Darfeuille-Michaud A. Invasive ability of an *Escherichia coli* strain isolated from the ileal mucosa of a patient with Crohn's disease. Infect Immun. 1999;67:4499-509.

23. Coura FM, de Araújo Diniz S, Mussi JMS, Silva MX, Lage AP, Heinemann MB. Characterization of virulence factors and phylogenetic group determination of *Escherichia coli* isolated from diarrheic and non-diarrheic calves from Brazil. Folia Microbiol. 2017;62:139-44.

24. Lai XH, Wang SY, Uhlin BE. Expression of cytotoxicity by potential pathogens in the standard *Escherichia coli* collection of reference (ECOR) strains. Microbiology. 1999;145:3295-303.

25. Boyd EF, Hartl DL. Chromosomal regions specific to pathogenic isolates of *Escherichia coli* have a phylogenetically clustered distribution. J Bacteriol. 1998;180:1159-65.

26. Toma C, Higa N, Iyoda S, Rivas M, Iwanaga M. The long polar fimbriae genes identified in Shiga toxin-producing *Escherichia coli* are present in other diarrheagenic *E. coli* and in the standard *E. coli* collection of reference (ECOR) strains. Res Microbiol. 2006;157:153-61.

27. Restieri C, Garriss G, Locas M-C, Dozois CM. Autotransporter-encoding sequences are phylogenetically distributed among *Escherichia coli* clinical isolates and reference strains. Appl Environ Microbiol. 2007;73:1553-62.

28. Dogan B, Suzuki H, Herlekar D, Sartor RB, Campbell BJ, Roberts CL, Stewart K, Scherl EJ, Araz Y, Bitar PP, Lefébure T, Chandler B, Schukken YH, Stanhope MJ, Simpson KW. Inflammation-associated adherent-invasive *Escherichia coli* are enriched in pathways for use of propanediol and iron and M-cell translocation. Inflamm Bowel Dis. 2014;20:1919-32.

29. Chassaing B, Koren O, Carvalho FA, Ley RE, Gewirtz AT. AIEC pathobiont instigates chronic colitis in susceptible hosts by altering microbiota composition. Gut. 2014;63:1069-80.

30. European Committee on Antimicrobial Susceptibility Testing. http://www.eucast.org/clinical_breakpoints/.

31. Cabal A, Gómez-Barrero S, Porrero C, Bárcena C, López G, Cantón R, Gortázar C, Domínguez L, Álvarez J. Assessment of virulence factors characteristic of human *Escherichia coli* pathotypes and antimicrobial resistance in O157:H7 and non-O157:H7 isolates from livestock in Spain. Appl Environ Microbiol. 2013;79:4170-2.

32. Mazel D, Dychinco B, Webb V-A, Davies J. Antibiotic resistance in the ECOR collection: integrons and identification of a novel aad gene. Antimicrob. Agents Chemother. 2000;44:1568-74.

Ready to submit your research? Choose BMC and benefit from:

* fast, convenient online submission

* thorough peer review by experienced researchers in your field

* rapid publication on acceptance

* support for research data, including large and complex data types

* gold Open Access which fosters wider collaboration and increased citations

* maximum visibility for your research: over 100M website views per year

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions

BMC