Diversity of Shiga toxin-producing Escherichia coli in sheep flocks of Paraná State, southern Brazil

Veterinary Microbiology 175 (2015) 150–156

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Short Communication

Diversity of Shiga toxin-producing Escherichia coli in sheep flocks of Parana´ State, southern Brazil Fernando Henrique Martins a,1, Beatriz Ernestina Cabilio Guth b,*, Roxane Maria Piazza c, Sylvia Cardoso Lea˜o b, Agostinho Ludovico d, Marilu´cia Santos Ludovico a, Ghizlane Dahbi e, Juan Marzoa e, Azucena Mora e, Jorge Blanco e, Jacinta Sanchez Pelayo a a

Departamento de Microbiologia, Universidade Estadual de Londrina, Londrina, PR, Brazil Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil c Laborato´rio de Bacteriologia, Instituto Butantan, Sa˜o Paulo, SP, Brazil d Faculdade de Medicina Veterina´ria, Universidade Norte do Parana´, Arapongas, PR, Brazil e Laboratorio de Referencia de E. coli (LREC), Departamento de Microbioloxı´a e Parasitoloxı´a, Facultade de Veterinaria, Universidade de Santiago de Compostela (USC), Lugo, Spain b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 28 August 2014 Received in revised form 31 October 2014 Accepted 2 November 2014

Sheep constitute an important source of zoonotic pathogens as Shiga toxin-producing Escherichia coli (STEC). In this study, the prevalence, serotypes and virulence profiles of STEC were investigated among 130 healthy sheep from small and medium farms in southern Brazil. STEC was isolated from 65 (50%) of the tested animals and detected in all flocks. A total of 70 STEC isolates were characterized, and belonged to 23 different O:H serotypes, many of which associated with human disease, including hemolytic-uremic syndrome (HUS). Among the serotypes identified, O76:H19 and O65:H– were the most common, and O75:H14 and O169:H7 have not been previously reported in STEC strains. Most of the STEC isolates harbored only stx1, whereas the Stx2b subtype was the most common among those carrying stx2. Enterohemolysin (ehxA) and intimin (eae) genes were detected in 61 (87.1%) and four (5.7%) isolates, respectively. Genes encoding putative adhesins (saa, iha, lpfO113) and toxins (subAB and cdtV) were also observed. The majority of the isolates displayed virulence features related to pathogenesis of STEC, such as adherence to epithelial cells, high cytotoxicity and enterohemolytic activity. Ovine STEC isolates belonged mostly to phylogenetic group B1. PFGE revealed particular clones distributed in some farms, as well as variations in the degree of genetic similarity within serotypes examined. In conclusion, STEC are widely distributed in southern Brazilian sheep, and belonged mainly to serotypes that are not commonly reported in other regions, such as O76:H19 and O65:H–. A geographical variation in the distribution of STEC serotypes seems to occur in sheep. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Sheep Shiga toxin-producing E. coli Serotypes Virulence factors Genetic diversity

* Corresponding author at: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo – Rua Botucatu, 862, 04023-062 Sa˜o Paulo, SP, Brazil. Tel.: +55 11 55764201. E-mail address: [email protected] (B.E.C. Guth). 1 Present address: Laborato´rio de Bacteriologia, Instituto Butantan, Sa˜o Paulo, SP, Brazil. http://dx.doi.org/10.1016/j.vetmic.2014.11.003 0378-1135/ß 2014 Elsevier B.V. All rights reserved.

F.H. Martins et al. / Veterinary Microbiology 175 (2015) 150–156

1. Introduction Sheep farming is an important activity in the animal production sector of Brazil. Currently, this country has both the largest flock (approximately 17 million head) and sheep meat production (85,000 tonnes) of the American continent (FAO, 2012). The increasing consumption of ovine meat has boosted the expansion of Brazilian sheep industry. Consequently, there is an increased risk of exposure to zoonotic pathogens as Shiga toxin-producing Escherichia coli (STEC), which can be transmitted by food production chain (Schimmer et al., 2008), as well as contact with these animals and their environments (Ogden et al., 2002). STEC infection can lead to life-threatening complications such as hemorrhagic colitis (HC) and hemolyticuremic syndrome (HUS). Although cattle are considered to be the major reservoir of STEC strains, sheep also represent important carriers of these pathogens, and higher prevalence of STEC have been detected in ovine feces (Mora et al., 2011; Ame´zquitaLo´pez et al., 2012) and meat (Momtaz et al., 2013) when compared to samples of bovine origin. Nevertheless, only a few studies have reported the occurrence of STEC in Brazilian sheep populations (Vettorato et al., 2009; Maluta et al., 2014; Martins et al., 2014) and, particularly in the South region where approximately 30% of the national flock is found, these data are unknown so far. Thus, the aim of this study was to investigate the occurrence of STEC in sheep flocks from Parana´ State, southern Brazil, to better understand the role played by these animals as reservoir of potentially pathogenic strains. 2. Methods

151

GeneAmp1 PCR System 9700 thermal cycler (Applied Biosystems, USA), with mixtures of 25 mL containing 2 mL of bacterial lysates, 200 mM dNTPs, 1X PCR buffer, 2 mM MgCl2, 25 pmol of each primer and 1 U of Taq DNA Polymerase (Invitrogen, USA). PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized under ultraviolet light (Vilbert Loumart, France) after staining with ethidium bromide. 2.3. Serotyping Determination of O and H antigens was carried out as previously described (Guine´e et al., 1981), employing all available O (O1–O185) and H (H1–H56) antisera. All antisera were absorbed with the corresponding crossreacting antigens to remove the nonspecific agglutinins. The O and H antisera were produced in the Laboratorio de Referencia de E. coli (USC, Lugo, Spain). Isolates that did not react with O antisera were considered as non-typeable (ONT) and those nonmotile were H–. 2.4. Genotypic characterization of STEC STEC isolates were investigated by PCR for the presence of genes encoding enterohemolysin (ehxA), STEC autoagglutinating adhesin (saa), Irg homologous adhesin (iha), long polar fimbriae (lpfO113), subtilase cytotoxin (subAB) and cytolethal distending toxin (cdtV). The stx2 subtyping was carried out by PCR using primers for stx2a, stx2b, stx2c and stx2d genes. Typing of the eae gene was performed by PCR and sequencing. All primers used in this study and corresponding references are listed in Table S1 (Supplementary information). 2.5. Phenotypic assays

2.1. Sample collection and isolation of E. coli A single rectal swab was obtained from 130 randomly selected sheep without diarrhea (one week to eight years old) between April and September 2010. The animals were originated from ten small and medium farms located in Parana´ State, southern Brazil, and each locality was visited once. Samples were transported to the laboratory in CaryBlair medium (Difco, USA), streaked onto MacConkey Agar (Difco, USA) and incubated overnight at 37 8C. At least three lactose-fermenting colonies were selected from each bacterial growth, confirmed as E. coli by standard biochemical tests, and then stored in Brain Heart Infusion broth (Difco, USA) with 20% glycerin at 20 8C until use. 2.2. Detection of STEC by multiplex polymerase chain reaction (mPCR) Bacterial DNA was obtained by boiling method. Briefly, the isolates were cultivated onto Luria-Bertani (LB) Agar (Difco, USA) overnight at 37 8C. A loopful for each bacterial growth was resuspended in 300 mL of sterile ultrapure water, boiled at 100 8C for 10 min and centrifuged to 10,000  g for 5 min. The supernatants were utilized as templates in mPCR for detection of the Shiga toxin (stx1 and stx2) and intimin (eae) genes, according to Paton and Paton (1998). Amplification reactions were performed on

The cytotoxicity assay on Vero (African green monkey kidney) cells was carried out according to Beutin et al. (2002) with some modifications. STEC supernatants were prepared as described previously (Rocha and Piazza, 2007), and 1:10 dilutions were tested in duplicates on Vero cells. Supernatants of E. coli O157:H7 (EDL933) and K-12 (DH5a) strains were used as positive and negative controls, respectively. After 72 h-exposure to STEC supernatants, cellular metabolic activity was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma–Aldrich, USA) assay, according to manufacturer’s instructions. The level of cytotoxicity was calculated using the following formula: (absorbance of the sample absorbance of the negative control)/(absorbance of the positive control absorbance of the negative control)  100. Three independent experiments were performed. Adherence capacity of STEC isolates to HEp-2 (human laryngeal epithelial carcinoma) cells was tested according to Cravioto et al. (1979) after 6 h of bacteria–cell interaction. Production of hemolysin was determined by the method described by Beutin et al. (1989). 2.6. Phylogenetic grouping The phylogenetic group (A, B1, B2 and D) was determined using a triplex PCR for chuA, yjaA genes and

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the DNA fragment TspE4.C2, according to Clermont et al. (2000).

Table 1 Prevalence of STEC in sheep farms of Parana´ State, southern Brazil. Farm

Na

2.7. Pulsed-field gel electrophoresis (PFGE) A total of 32 STEC isolates representing the six most frequent serotypes found in the study was processed according to the protocol described by Gautom (1997), with slight modifications. Briefly, the chromosomal DNA was digested using 45 U of XbaI (Invitrogen, USA), and electrophoresis was performed on a CHEF-DRIII PFGE apparatus with the following conditions: initial time of 2.2 s, final time of 35 s, run time of 16 h, angle of 1208, gradient of 6.0 V cm 1, temperature of 14 8C. When required, 100 mM thiourea (Sigma–Aldrich, USA) was added to the running buffer to prevent DNA degradation (Liesegang and Tschape, 2002). The band patterns were analyzed by using the BioNumerics (version 5.1) program (Applied Maths, Belgium), and the similarity of PFGE profiles was compared and dendograms were prepared using the band-based Dice unweighted-pair group method using average linkages (UPGMA), based on 1.5% position tolerance and optimization.

A B C D E F G H I J Total a b c

Prevalence nb

% (CI)c

24 12 8 5 17 23 9 18 7 7

11 7 4 5 9 14 6 3 6 2

45.8 58.3 50.0 60.0 52.9 60.8 66.6 16.6 85.7 28.5

130

65

50.0 (41.5–58.4)

(27.9–64.9) (31.9–80.6) (21.5–78.5) (23.0–88.2) (30.9–73.8) (40.8–77.8) (35.4–87.9) (5.8–39.2) (48.7–97.4) (8.2–64.1)

Number of animals sampled. Number of animals positive for STEC. Confidence intervals of 95%.

O76:H19, O65:H–, and O75:H14 also showed a significant association (p < 0.05) with farms A, B, D, F and H, respectively. In contrast, other serotypes were restricted to a single locality, such as O171:H2 (farm A) and ONT:H28 (farm F) (p < 0.05).

2.8. Statistical analysis 3.3. Virulence genes The prevalence rate of STEC was calculated as the quotient of the number of animals with stx-positive E. coli and the total number of animals sampled, using exact 95% confidence intervals (CIs). Associations between serotypes, the presence of virulence factors, phylogroups and farms were evaluated using Chi-square (x2) test or Fisher exact test, and p values < 0.05 were considered to be significant. 3. Results 3.1. Prevalence of STEC in sheep All sheep flocks studied were found to be positive for STEC, and the prevalence on farms ranged from 16.6% (95% CI: 5.8–39.2) to 85.7% (95% CI: 48.7–97.4) (Table 1). Overall, STEC was detected in 65 (50.0%) of 130 fecal samples. Only one isolate per stx-genotype was selected from each positive sample. In animals harboring both stxpositive/eae-negative and stx-positive/eae-positive isolates, one of each was selected. Thus, a total of 70 STEC isolates were recovered for further characterization. 3.2. Serotypes of ovine STEC isolates STEC isolates belonged to 17 O serogroups, 10 H types and 23 different O:H serotypes (Table 2). The most frequent serotype observed was O76:H19, followed by O65:H–, O75:H–, O91:H–, O75:H14 and O128:H2. These six serotypes represented 65.7% of the recovered STEC isolates. Two new serotypes not previously reported in STEC strains (O75:H14 and O169:H7) were found in this study. None of the STEC isolates belonged to O157 serogroup. Nine serotypes were found to be distributed on more than one flock (Table 2), but serotypes O128:H2, ONT:H16,

Most of the STEC isolates (52.8%) possessed the stx1 gene alone, whereas 32.9% carried both stx1 and stx2, and 14.3% harbored only stx2. Isolates carrying only stx1 gene were associated to the serotypes O65:H– and O76:H19, while the ones harboring stx1 and stx2 showed a strong association with the serotypes O75:H14 and O91:H– (p < 0.05). Among the 33 STEC isolates carrying stx2, 28 (84.8%) presented the stx2b subtype, and most of them belonged to serotypes O75:H14, O91:H– O128:H2, O171:H2 and ONT:H–. Eight isolates possessed the stx2c gene, seven of which concomitantly carried stx2b. The stx2d and stx2a genes were detected in two and one isolates, respectively. The stx2 subtyping was not possible in one isolate belonging to O169:H7 serotype (Table 2). Four of the STEC isolates (5.7%) presented the eae gene. Two of them showed the eae-e1 subtype and belonged to O103:H2 and O172:H– serotypes. The eae-g1 subtype was identified in an ONT:H7 isolate, whereas O169:H7 isolate was untypeable for the eae gene. The ehxA, cdtV and subAB genes were detected in 87.1%, 2.8% and 1.4% of the STEC isolates, respectively. All but one isolate possessed at least one of the adhesin-encoding genes investigated. Specifically, 68 (97.1%) isolates carried the lpfAO113 gene, whereas saa and iha were found in 54.3% and 10% of the isolates, respectively. The saa and iha genes presented a strong association (p < 0.05) with serotypes O76:H19 and O91:H–, respectively. A total of 25 different combinations of virulence genes were observed (Table 2). A significant association (p < 0.05) was found between harboring saa and ehxA or saa and stx1, as also for cdtV and stx2. The most common genetic profile was stx1 ehxA saa lpfO113 (32.8%), followed by stx1 ehxA lpfO113 (11.4%) and stx1 stx2b ehxA saa

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153

Table 2 Characteristics of STEC isolated from sheep in Parana´ State, southern Brazil. Serotype (no. isolates)

Farm

O5:H–a O65:H–a (4) O65:H–a (3) O65:H–a O75:H–a (3) O75:H–a (2) O75:H–a O75:H–a O75:H14b (2) O75:H14b O75:H14b O76:H19a (13)

A F,G B,F F G B,I I G E,H H E A,C,D,E, F,H,I E,F I C F A A E B G E B J C A B A A C E E A A A E F I F F B J F F F

O76:H19a (2) O76:H19a O76:H19a O76:H19a O79:H14a O91:H–a (2) O91:H–a O91:H–a O91:H–a O102:H6a O102:H6a O103:H2a,* O106/0185:H7 O116:H21a O123:H–a O128:H2a (2) O128:H2a O128:H2a O169:H7b O170:H7a O171:H2a O171:H2a O171:H2a O172:H–a O174:H8a ONT:H–a ONT:H–a ONT:H7a ONT:H16a (2) ONT:H16a ONT:H28a ONT:H28a ONT:H42a

Virulence genes stx1 + + + + + + + + + + + + + + + + + + + + +

**

stx2

+ +

+ +

eae

+(b) +(b,c) +(c) +(b) +(b,c) +(b,c)

+(b) +(b) +(b) +(b) +(b,c) +(b)

e1

+

e1

g1

+ +

Ehly

HEp-2

+ + + + + + + + + + + +

+ + + + + + + + + + + +

+ + + + + + + + + + + +(12)

+ +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + +

AA UND (3), AA AA (2), UND AA UND AA AA AA UND AA UND UND (7), AA (3), DA (3) AA, UND DA UND UND UND DA, UND UND UND UND UND AA LAL DA UND UND UND UND AA LAL AA AA UND AA LAL UND AA DA LAL AA UND UND UND UND

+ + + + + + + + + + + +

+ + + +

+(b) + + +

+ +(b)

+

+ + + + + + +

+(d) +(d) +(b) +(b) +(b) + +(b) +(b) +(b) +(b,c) +(a) +(b,c) +(b,c) +(b,c)

lpfAO113

+

+ + + +

+

+ + + +

+

+

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + +

cdtV

+ + +

+ +

+

+

+

+

Stx

Phylogroup e

saa

+

subAB

d

+ + + + + + + + + + + +

+

iha

c

ehxA

+ + + + + + +

+(b)

+

+ + + +

Phenotypic assays

+ + +

+ + + + + + + + + +

+ +

+ +

B1 B1 B1 D B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 D B1 B1 B1 B1 B1 A A B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 A B1 B1 A D B1 B1 B1 B1 B1

a

Serotypes previously reported in human STEC strains – in bold: serotypes associated with HUS. New serotypes not previously described in STEC strains. Cytotoxic effect on Vero cells. d Production of enterohemolysin on washed sheep blood agar. e Adherence patterns to HEp-2 cells. AA (aggregative adherence); DA (diffuse adherence); LAL (localized adherence-like); UND (undefined pattern). * Previously described by Martins et al. (2014). ** stx2 subtypes are shown in parenthesis. b c

lpfO113 (10%). All eae-positive isolates also possessed ehxA and lpfO113, and were negative for the saa gene. 3.4. Phenotypic features All STEC supernatants produced cytotoxic effect on Vero cells, and 90% (63/70) of the isolates showed a high cytotoxicity (above 50%) as determined by MTT assay (Fig. S1 Supplementary information). All isolates adhered to HEp-2 cells after 6 h of interaction, and distinct

adherence phenotypes were identified. Specifically, STEC isolates carrying eae showed localized-like adhesion (LAL), and among those lacking eae, 31.4% (22/70) displayed aggregative adherence (AA), 10% (7/70) expressed diffuse adherence (DA), and the remaining exhibited an undefined adherence pattern (UND). Enterohemolytic activity was observed in 55 (78.5%) of the 70 STEC isolates, whereas production of a-hemolysin was not detected. Table 2 summarizes the phenotypic features of ovine STEC isolates.

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3.5. Phylogenetic typing Most of the STEC isolates fell into phylogroup B1 (90.0%), followed by group A (5.7%) and D (4.3%). STEC isolates from the flock F were the most heterogeneous, and were grouped into the three phylogroups identified in this study (Table 2). A significant association (p < 0.05) was found between isolates belonging to phylogroup D and farm F. All STEC isolates belonging to phylogroup A were found to be positive for stx2 and negative for stx1 and saa (p < 0.05). 3.6. PFGE PFGE analysis was performed separately for each serotype. Initially, all STEC O76:H19 isolates were untypeable as no banding patterns were defined. However, after addition of 100 mM thiourea 10 distinct pulsetypes were identified among these isolates (Fig. 1). A high genetic similarity (above 90%) was observed among 9 of 11 STEC O76:H19 isolates, indicating a clonal relationship within this serotype, even among isolates originated from distinct farms. STEC O65:H– isolates were also sensitive to DNA degradation, and could be only typed after addition of 100 mM thiourea. Three out of 5 STEC O65:H– isolates showed identical pulsetypes, and were isolated from distinct animals of the same farm (F). The other serotypes could be analyzed without addition of thiourea and in general, high diversity of genetic profiles was observed (Fig. 1). As an exception, two O75:H– isolates showed the same pulsetype, and other two O75:H14 isolates were closely related (above 95% similarity). STEC isolates belonging to O91:H– and O128:H2 serotypes showed the highest genetic diversity (below 80% similarity). 4. Discussion To our knowledge, this is the first report on STEC occurrence in sheep populations from southern Brazil, being isolated from 50% (65/130) of the tested animals and detected in all farms. High prevalence of STEC have also been reported in sheep farms from different countries (Mora et al., 2011; Schilling et al., 2012), emphasizing the importance of these animals as STEC reservoir. Interestingly, the most common STEC serotypes O76:H19 and O65:H– identified in this study were not frequently recovered from sheep in other geographical areas (Vettorato et al., 2009; Mora et al., 2011; Ame´zquitaLo´pez et al., 2012; Schilling et al., 2012; Maluta et al., 2014). Serotype O65:H– is rarely reported in STEC strains from different sources, whereas O76:H19 is often associated with STEC strains of caprine origin (Martin and Beutin, 2011; Mora et al., 2011). Despite this fact, ovine STEC O76:H19 have been recently implicated in human illness (Sa´nchez et al., 2014). Other two serotypes found herein (O75:H14 and O169:H7) have not been previously described in STEC strains. Among the remaining serogroups/serotypes identified in this study, only eight (O5:H–, O75, O91, O128:H2, O172:H–, O174:H8, ONT:H16 and ONT:H–) were previously reported in Brazilian sheep

(Vettorato et al., 2009; Maluta et al., 2014). Taken together, these data suggest a geographical variation in the distribution of STEC serotypes in sheep. STEC O157 was not found in this population studied, which is in agreement with the previously reported data in Brazil (Vettorato et al., 2009; Maluta et al., 2014). However, it is noteworthy that none sensitive technique for STEC O157 isolation as immunomagnetic separation (IMS) was employed herein. Thus, the true carriage rate of STEC O157 in Brazilian sheep remains unclear, and further studies are necessary to elucidate that. On the other hand, 23 serotypes were identified in this study, most of which associated with human illness, including HUS, such as O5:H–, O75:H–, O76:H19, O91:H–, O103:H2 and O128:H2 (Beutin et al., 2004; EFSA, 2013), what reinforces the role played by sheep as potential source of STEC infection. Of particular interest was the detection of STEC O103:H2, a serotype that has been associated with human illness in Brazil (Guth et al., 2005), but was only recently isolated from animals in this country (Martins et al., 2014), suggesting a possible link between sheep and O103:H2 infections in our settings. The Stx2b subtype, which was predominant among the isolates studied herein, is epidemiologically linked to ovine STEC, and occurs in the great majority of stx2-carrying strains isolated from feces, carcass and feed of sheep (Ramachandran et al., 2001; Vettorato et al., 2009; Martin and Beutin, 2011; Maluta et al., 2014). Although the Stx2b subtype is associated with lower virulence, stx2b-carrying STEC has been implicated with sporadic cases of HUS (Stritt et al., 2012), indicating that these strains can also cause serious infections in humans. It is notable that other Stx subtypes associated with severe human illness as Stx2a, Stx2c and Stx2d (Beutin et al., 2004), were also presently identified. A low frequency of eae was observed among the STEC isolates, which is in accordance with previously reported data (Mora et al., 2011; Ame´zquita-Lo´pez et al., 2012; Schilling et al., 2012). In contrast, ehxA gene was often detected, confirming that the combination stx/ehxA occurs more commonly among ovine STEC strains than stx/eae (Ramachandran et al., 2001; Vettorato et al., 2009; Maluta et al., 2014). Although the presence of the eae gene is associated with increased pathogenicity, ovine STEC strains lacking eae have also been implicated with severe human illness (Bekal et al., 2014). PFGE results showed isolates showing indistinguishable profiles (100% similarity) were restricted to the same farm. These specific clones were recovered from distinct animals, indicating a cross-contamination within these flocks. Variations in the degree of genetic similarity were observed among STEC isolates belonging to the same serotype. Specifically, most of the STEC O76:H19 isolates were closely related independently of the sampling site, suggesting the dissemination of specific clones. On the other hand, a higher genetic diversity was observed within O91:H– and O128:H2 serotypes, even among isolates from the same farm. These results demonstrate a high heterogeneity within some serotypes of ovine STEC strains, corroborating with previous findings (Vettorato et al., 2009; Ame´zquita-Lo´pez et al., 2012; Maluta et al., 2014).

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Fig. 1. Pulsed-field gel electrophoresis (PFGE) patterns of 32 representative STEC strains belonging to the six most frequent serotypes found in this study. A dendogram was constructed separately for each serotype.

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5. Conclusion In conclusion, STEC are widely distributed in southern Brazilian sheep, and these animals carry some serotypes which are not commonly reported in other regions, such as O76:H19 and O65:H–, as well as new serotypes that had not been previously described in STEC strains. Moreover, attention should be given to ovine-human contact as feces derived from these animals contain potentially pathogenic STEC. Acknowledgments We thank Evanilde Maria Gonc¸alves for technical assistance, and Laborato´rio de Virologia (Universidade Estadual de Londrina, Parana´, Brazil) for providing the HEp-2 cells used in the adherence tests. This work was supported by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) (Brazil); Consellerı´a de Cultura, Educacio´n e Ordenacio´n Universitaria, Xunta de Galicia, the European Regional Development Fund (ERDF) (CN2012/303) and the Ministerio de ˜a Economı´a y Competitividad, Gobierno de Espan (AGL2013-47852-R). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2014.11.003. References ˜ ones, B., Cooley, M.B., Leo´n-Fe´lix, J., CastroAme´zquita-Lo´pez, B.A., Quin del-Campo, N., Mandrell, R.E., Jime´nez, M., Chaidez, C., 2012. Genotypic analyses of shiga toxin-producing Escherichia coli O157 and nonO157 recovered from feces of domestic animals on rural farms in Mexico. PLoS ONE 7, e51565. Bekal, S., Ramsay, D., Rallu, F., Pilon, P., Gilmour, M., Johnson, R., Tremblay, C., 2014. First documented case of human infection with ovine Shigatoxin-producing Escherichia coli serotype O52:H45. Can. J. Microbiol. 60, 417–418. Beutin, L., Montenegro, M.A., Ørskov, I., Ørskov, F., Prada, J., Zimmermann, S., Stephan, R., 1989. Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli. J. Clin. Microbiol. 27, 2559–2564. Beutin, L., Zimmermann, S., Gleier, K., 2002. Evaluation of the VTEC-Screen Seiken test for detection of different types of Shiga toxin (Verotoxin)producing Escherichia coli (STEC) in human samples. Diagn. Microbiol. Infect. Dis. 42, 1–8. Beutin, L., Krause, G., Zimmermann, S., Kaulfuss, S., Gleier, K., 2004. Characterization of Shiga toxin-producing Escherichia coli strains isolated from human patients in Germany over a 3-year period. J. Clin. Microbiol. 42, 1099–1108. Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 4555–4558. Cravioto, A., Gross, R.J., Scotland, S., Rowe, B., 1979. An adhesive factor found in strains of Escherichia coli belonging to the traditional enteropathogenic serotypes. Curr. Microbiol. 3, 95–99. European Food Safety Authority (EFSA), 2013. Scientific opinion on VTECseropathotype and scientific criteria regarding pathogenicity assessment. EFSA J. 11, 3138.

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