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Supplementary online material
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TEXT
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Experimental procedures
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Sampling procedures. In October 2006, and in June 2008, we conducted two sampling
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campaigns in Trois-Sauts, an isolated French Guiana village. Characteristics of the village,
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the humans and the human sampling are described elsewhere (Ruimy et al., 2010; Woerther et
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al., 2010). Briefly, the village is located in the Amazonian pristine forest, in the south of
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French Guiana, nearby the Oyapock river source and in a protected area, where access is
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administratively controlled (Fig. S1). The village is divided in 4 hamlets, Zidock, Roger, Pina
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and Yawapa from where human samples came from (Fig. S1). The villagers are native
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Amerindian Wayampis. Their way of life is comparable to traditional societies as they share
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large huts with no access to hygienic facilities, and eat local food (traditional agriculture,
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fishing and hunting). However they receive education and medical care from metropolitan
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French teachers and nurses living in the village. Human associated animals were sampled in
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Zidock, Roger and Pina (Fig. S1), they mostly belong to dog and chicken populations, living
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free in the hamlets, hence not as closed to villagers as could be pets from industrialised
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country inhabitants. Wild animals were sampled by trapping along two transects (one
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connecting Zidock and Roger and the other issued from Zidock and ending 20 km away in
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North-West direction in the Amazonian forest) and then released alive in the forest or killed
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and kept as representative specimens of rare species. Wild animals were also sampled thanks
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to hunting by the villagers for food purpose. In this case, they were brought immediately to
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the village and sampled as fast as possible. Main characteristics of individuals including
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Linnean denomination, order, class, body mass, diet and sampling localisation [obtained by
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the Global Positionning System (GPS)] were collected (Table S1). The study was approved
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by ad hoc Guadeloupe Ethics Committee (Comité de protection des personnes de
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Guadeloupe, France; no 06-05).
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We sampled 393 individuals comprising 162 adult Wayampi Amerindians, 33 human
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associated animals living in the village and 198 wild animals. Among the 162 human
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samples, 94 (58 %), 37 (23%), 15 (9%) and 16 (10%) were sampled in the hamlets Zidock,
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Roger, Pina and Yawapa, respectively (Fig. S1). Among the 33 human associated animal
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samples, 22 (67%) came from Zidock, 2 (6%) from Roger and 9 (27%) from Pina (Fig. S1).
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Among the 198 wild animal sampled during the two collects, 122 in 2006 and 76 in 2008, 117
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(59%) were collected in or close to the hamlets Zidock, Roger, Pina and Yawapa [69 (57%) in
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2006 and 48 (63%) in 2008] and 81 (41%) were collected outside the hamlets along two
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transects or captured by the villagers around the village [53 (43%) in 2006 and 28 (37%) in
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2008] (Fig. S1).
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Strain isolation. Fresh faecal samples and rectal swabs (from humans and animals,
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respectively) were inoculated extemporaneously onto Drigalski agar slants in screw-cup
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tubes, stored at room temperature and sent to metropolitan France two weeks later. There, the
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whole culture from each tube was suspended in 1.5 mL of brain-heart infusion broth with
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10% glycerol and stored at −80°C. 100µL aliquots of each stored broth were cultured on
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chromogenic plates (Uriselect®; BioRad). Pink colonies were tested for their indole
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production and identified as E. coli if positive, according to the manufacturer’s
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recommendations. The first strain identified as E. coli was chosen to be representative of each
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sample and considered as randomly selected.
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Antibiotic resistance pattern. The antimicrobial susceptibility of the strains to 32
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antibiotics (see list in Table S3) was determined using the disk-diffusion method, as described
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elsewhere (http://www.sfm.asso.fr). For each strain, phenotype of resistance was classified
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into 2 categories: S (sensitivity to all antibiotics tested) or R (resistance to at least one
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antibiotic). Antibiotic resistance scores were also determined as the relative proportion of the
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number of identified resistances on the total number of tested resistances *100.
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The presence of penicillinase TEM and CMY-type ß-lactamases were detected by
PCR as in (Branger et al., 2005; Courpon-Claudinon et al., 2011).
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Strain genotyping. Phylogenetic groups were firstly assigned to one of the seven groups A0,
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A1, B1, B22, B23, D1 and D2 using the triplex PCR method (Clermont et al., 2000; Escobar-
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Paramo et al., 2004). MLST was then performed using the Pasteur Institute scheme
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(http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi.html) (Jaureguy et al., 2008) on 96
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B22, B23, D1 and D2 strains allowing the assignation of the strains to the B2, D, E and F
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phylogroups (Tenaillon et al., 2010). C group strains (Moissenet et al., 2010) were determined
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among A1 strains using an allele-specific C group PCR on the trpA gene developed in this
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study. The PCR steps were as follows: denaturation for 4 min at 94°C, 30 cycles of 5 s at
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94°C and 10 s at 59°C, and a final extension step of 5 min at 72°C using the primers
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trpAgpC1
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TCTGCGCCGGTCACGCCC-3’) (product size: 219 bp). An allele-specific E group PCR was
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also developed to verify that no strain typed as A0, A1, and B1 by the triplex PCR method was
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belonging to this group. The chosen target was the arpA gene using the ArpAgpE.f 5’-
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GATTCCATCTTGTCAAAATATGCC-3’
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GAAAAGAAAAAGAATTCCCAAGAG-3’ primers amplifying a 301 bp fragment. The
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PCR conditions were as above except that the annealing temperature was of 57°C. Among
(5’-AGTTTTATGCCCAGTGCGAG-3’)
and
and
trpAgpC2
ArpAgpE.r
(5’-
5’-
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the 176 A0, A1, and B1 strains, no one belongs to the E group. At the opposite, all strains
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classified as E by the MLST were positive with our E group PCR assay. A maximum-
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likelihood phylogenetic tree was reconstructed with the PHYML program (Guindon et al.,
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2005) using the concatenated MLST sequences from the 96 E. coli strains included in this
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study, 35 strains of the ECOR collection (including 15, 6, 3, 6, 2, 2, and 1 strains belonging to
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the B2, D, E/UG, F, A, B1 and C phylogenetic groups, respectively) (Ochman and Selander,
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1984; Lescat et al., 2009), 13 representative B2 phylogenetic subgroup strains previously
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analysed by Le Gall et al. (Le Gall et al., 2007), 5 representative D and F group strains
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(Touchon et al., 2011) and the reference strains ED1a, E2348/69, 536, TN03, 042 and
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EDL933. The tree was rooted on E. fergusonii ATCC 35469T. Detection of clade strains was
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performed by PCR as described by Clermont et al. (Clermont et al., 2011a).
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Extraintestinal virulence genes (hly, cnf, aer, papC, iroN, traT, fyuA, sfa, and kpsE)
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(Bingen-Bidois et al., 2002; Johnson et al., 2006) and intraintestinal virulence genes (afaD,
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stx1, stx2, ipaH, eae, bfpA, ST and LT coding genes, aaiC) (Clermont et al., 2011b) were
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detected by PCR. Bacteriocins, including colicins and microcins, were detected as follow.
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Colicins have firstly been detected by a slightly modified phenotypic method (Schamberger
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and Diez-Gonzalez, 2005). At first a suspension in phosphate buffered saline of E. coli K-12
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(at 0.5 Mc Farland) as a sensitive strain has been plated on a Luria Bertani (LB) agar medium
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containing mitomycin (concentration at 0.25 mg/L). Then, 10 μl of an over night (O/N)
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culture in LB medium of each strain were spotted on the mitomycin-LB agar plate. After an
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O/N culture at 37°C, the presence of colicin (or phage) has been detected for the strains
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surrounded by a halo traducing an inhibition of the culture of the E. coli K-12 strain. Strains
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positive for the phenotypic test have then been tested by PCR for the presence of the most
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frequent colicin genes (colIa/Ib, colE1, colB) (Gordon and O'Brien, 2006). The detection of
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the main microcins genes (micH47 and micV), which production is related to the iron
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concentration in the medium and known to be associated to the iroN gene (Waters and Crosa,
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1991), have been detected among the iroN positive strains (Gordon and O'Brien, 2006). For
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each strain, genotypes of extraintestinal virulence, intraintestinal virulence and bacteriocin
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were separately classified in 2 categories: absence or presence of at least one character.
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Relevant scores for each type of characters were then determined by the relative proportion of
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the number of identified extraintestinal, intraintestinal virulence factors and bacteriocins on
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the total number of tested characters of each type *100.
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Septicemia mouse model. A mouse model of systemic infection was used to assess the
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intrinsic virulence of 8 B2 strains chosen to be representative of the B2 phylogenetic
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subgroups, according to the tree reconstructed from the MLST data obtained in this study
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(Picard et al., 1999). Ten outbreed female OF1 mice (6-week-old, 14-16 g) were challenged
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subcutaneously in the neck with a standardized bacterial inoculum for each strain (0.2 ml of a
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Ringer solution containing 109 cfu/ml of log-phase bacteria). Mortality was assessed over 7
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days post-challenge. Each experimental series included a positive control (urosepsis strain
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CFT073) and a negative control (commensal derived strain K-12 MG1655). In this model,
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lethality is a rather clear-cut parameter and strains are usually classified either as non-killer
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(strains killing none or one mouse out of 10) or killer (strains killing 9 or 10 mice out of 10)
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(Johnson et al., 2006). Strains that did not fall in these two categories were considered as
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being intermediate killer. Animal experimentations were done according to the authorization
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n° 6665 given by the Ministère de l’Agriculture, France.
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Factorial analysis of correspondence (FAC). A FAC was used to describe associations
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among the data (Greenacre, 1992). FAC was conducted with SPAD.N software (Cisia, Saint
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Mandé, France) from a two-way table. This table had 272 rows, one for each studied E. coli
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strains and 6 columns corresponding to the 6 variables: the origins of the strains [human
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strains (HS), human associated animal strains (HAAS) and wild animal strains (WAS)], the
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phylogenetic group (A, B1, B2, C, D, E and F) according to the phylogrouping method and
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the MLST data, the presence of extra-intestinal virulence determinants, of intra-intestinal
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virulence determinants and of bacteriocins, the presence of resistance to antibiotics. For each
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column, the character of each strain was coded as a binary code: present =1, absent = 0.
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Comparison of nucleotide diversity of the MLST data. Nucleotide diversity per site (Pi) of
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the strains obtained from Trois Sauts village and belonging to the B2, D, E and F
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phylogenetic groups (96 strains) was compared to the nucleotide diversity per site of strains
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belonging to the same phylogroups but obtained from (i) the ECOR collection representative
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of the E. coli species genetic diversity (Ochman and Selander, 1984) (31 strains), (ii) the
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Broad
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(http://www.broadinstitute.org/annotation/genome/escherichia_antibiotic_resistance/MultiHo
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me.html) from which strains have been chosen for their origin from various species and
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completely sequenced (40 strains) and (iii) a personal collection of 137 strains (ROAR
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collection) composed of 38, 36 and 63 strains from human, domestic and wild animals,
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respectively, sampled in metropolitan France in the 2000’s (Skurnik, Clermont, Brisse and
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Denamur, personal data). Nucleotide diversities per site of the 4 collections were estimated
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using DnaSP (Rozas, 2009) and then compared with a t-test.
collection
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FIGURE LEGENDS
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Figure S1: Location of study site and sample collection points in Trois-Sauts, French Guiana.
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The study was located in a protected area where human access is restricted. Locations of
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human, human associated animal and wild animal samples are indicated in white, grey and
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black circles, according to the Global Positioning System (GPS) collected for each sample.
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Circles are proportional to the numbers of sampled individuals.
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TABLE TITLES
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Table S1. Main characteristics of individuals sampled during the 1st collect in October 2006
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[humans (H), human associated animals (HAA) and wild animals (S1-WA)] and the 2nd
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collect in June 2008 [wild animals (S2-WA)] as well as the E. coli presence in the faeces.
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Table S2. Presence of extraintestinal virulence factors, intraintestinal virulence factors,
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bacteriocins and corresponding scores of strains of E. coli issued from humans, human
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associated animals and wild animals from both collects (2006-2008).
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Table S3. Presence of resistance to antibiotics and resistance score of E. coli issued from
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humans, human associated animals and wild animals from both collects (2006-2008).
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Table S4. Character mapping of the 96 E. coli strains belonging to the B2, D, E and F
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phylogroups and ordered as in the Fig. 1.
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