Clonal Diversity of ESBL-Producing Escherichia coli in Pigs at Slaughter Level in Portugal

Abstract

We aimed to determine the prevalence of extended-spectrum beta-lactamase (ESBL)–producing Escherichia coli in fecal samples of healthy pigs, and to evaluate their clonality and associated resistance. Forty-nine percent of pigs sampled (n=35/71) in a slaughterhouse in Portugal revealed ESBL-producing E. coli isolates. Most isolates produced CTX-M-1 enzyme (71.4%; n=25/35), followed by CTX-M-9 (11.4%; n=4/35), CTX-M-14 (5.7%; n=2/35), SHV-12 (5.7%; n=2/35), and CTX-M-32 (5.7%; n=2/35). Ninety-four percent of the isolates presented a phenotype of multi-resistance. Most isolates belonged to phylogroups B1 (42.8%; n=15/35) and A (40%; n=14/35). Multilocus sequence typing (MLST) analysis revealed nine sequence types (STs) under six clonal complexes (CCs) and nine singletons, including overrepresentation of CC10 and three new STs (ST2524, ST2525, ST2528). We observed the frequent presence of CTX-M–producing E. coli in pigs at slaughter level, most of them belonging to CC10, commonly recovered from clinical samples.

Introduction

E scherichia coli is a common inhabitant of the gastrointestinal tract of human and animals, with the capability of acquiring and preserving antibiotic resistance genes. (Murray, 1997). In addition, E. coli is an important human pathogen; about 80% of the urinary tract infections (UTIs) that occur globally are associated with this bacterium (Russo and Johnson, 2003; Vincent et al., 2010), and β-lactams are frequently used for treatment (Paterson et al., 2001).

Extended-spectrum beta-lactamases (ESBLs) are bacterial enzymes that confer resistance to a broad range of commonly used β-lactams, including cephalosporins (ceftriaxone, cefotaxime, and ceftazidime), as well as to aztreonam and related oxyimino-β-lactams (Bradford, 2001). Moreover, resistance caused by ESBLs is often associated with resistance to other classes of antibiotics such as aminoglycosides, fluoroquinolones, and trimethoprim-sulfamethoxazole. In addition, most ESBLs genes are plasmid-borne and are often located within transposons and integrons, which facilitates transfer between and within bacterial species (Eckert et al., 2006).

ESBL-producing E. coli are increasingly reported in healthy food-producing animals in several countries in Europe, Asia, and North Africa, and the genotypes sometimes correspond to locally dominant human types (Agersø et al., 2012; Bortolaia et al., 2010; Costa et al., 2009; Escudero et al., 2010; Hiroi et al., 2012; Randall et al., 2011).

Since healthy food-producing animals slaughtered for human consumption can be reservoirs for ESBL strains, the aim of this study was to evaluate the carriage level and type of ESBLs in E. coli obtained from fecal samples in pigs slaughtered in Portugal. In addition, multilocus sequence typing (MLST) was performed based on a greater discriminatory ability and the capacity to define genetically related ESBL-producing E. coli isolates.

Materials and Methods

Samples and bacterial isolates

Seventy-one fecal samples from pigs were collected from September 2008 to March 2009 in a slaughterhouse located in the center of Portugal, where animals from different geographical regions of Portugal are slaughtered. Each fecal sample corresponded to a different flock of animals, ranging from 30 to 50 animals, and were collected directly from the rectum during animal slaughter with the aid of sterile gloves, and transported to the laboratory in a Cary-Blair medium before being processed. The samples were seeded onto Levine agar plates (Levine-CTX plates) supplemented with 2 mg/L cefotaxime (BD product no. 236950, BD Difco™ Tryptic Soy Agar; Oxoid Limited, Basingstoke, UK). Plates were incubated at 37°C for 24 h. Three colonies per sample, with typical E. coli morphology, were selected and identified by classical biochemical methods (Gram-staining, catalase, oxidase, indol, Methyl-Red, Voges-Proskauer, citrate, and urease), and confirmed by the API 20E system (BioMérieux, La Balme-Les Grottes, France).

Antimicrobial susceptibility testing

Susceptibility to 16 antimicrobial agents (ampicillin, amoxicillin+clavulanic acid, cefoxitin, cefotaxime, ceftazidime, aztreonam, imipenem, gentamicin, amikacin, tobramycin, streptomycin, nalidixic acid, ciprofloxacin, sulfamethoxazole/trimethoprim, tetracycline, and chloramphenicol) was tested by the disk-diffusion method in all isolates, and ESBL-phenotypic detection was carried out by double-disk synergy test (CLSI, 2011). E. coli American Type Culture Collection 25922 was used as a quality control strain.

Characterization of antibiotic resistance mechanisms

The presence of genes encoding TEM, OXA, SHV, and CTX-M β-lactamases was studied by polymerase chain reaction (PCR) in all the E. coli isolates using primers and conditions previously reported (Briñas et al., 2003; Jouini et al., 2007). The obtained DNA amplicons were sequenced on both strands, and sequences were compared with those included in the GeneBank database, as well as with those deposited at a website (www.lahey.org/Studies/), in order to determine the specific type of β-lactamase gene (Machado et al., 2005). Additionally, the genetic environment of bla _CTX-M genes (ISEcp1, IS903, IS26, and orf477) was studied by PCR (Eckert et al., 2006). The following genes were also studied by PCR: tet(A), tet(B), tet(C), tet(D), and tet(E) (in tetracycline-resistant isolates); aadA and strA/B (in streptomycin-resistant isolates); aac(3)-II and aac(3)-IV (in gentamicin-resistant isolates); cmlA and floR (in chloramphenicol-resistant isolates); and sul1, sul2, and sul3 (in trimethoprim-sulfamethoxazole-resistant isolates) (Sáenz et al., 2004). In addition, the presence of the intI1 and intI2 genes, encoding class 1 and 2 integrases, respectively, and their variable region was also analyzed by PCR and sequencing (Machado et al., 2005). The quinolone resistance–determining region of gyrA and parC genes was amplified by PCR, sequenced, and examined in all quinolone-resistant isolates (Sáenz et al., 2004). Positive and negative PCR controls from the bacterial collection of the University of Trás-os-Montes and Alto Douro (Vila Real, Portugal) were used in all the assays.

MLST and phylogenetic groups analysis

ESBL-containing E. coli isolates were characterized by MLST. The sequences obtained were analyzed using Bionumerics version 3.5 software (Applied Maths, Sint-Martens-Latem, Belgium) and compared against the http://mlst.ucc.ie/ database. The combination of the seven obtained alleles for each isolate allows us to determine the corresponding sequence type (ST) and clonal complex (CC) (Tartof et al., 2005).

The isolates were also classified into one of the four main phylogenetic groups A, B1, B2, and D, following the PCR strategy described previously (Clermont et al., 2000).

Results

β-Lactamases detected in CTX-resistant E. coli isolates

CTX-resistant E. coli isolates were detected in 35 of 71 fecal samples of slaughtered pigs, representing 49.3% of the analyzed samples, and three isolates per sample were identified and their antimicrobial susceptibility profiles determined. All three isolates exhibited the same antimicrobial resistance profile, and for this reason, only one isolate per positive sample was maintained for further studies. All 35 CTX-resistant E. coli isolates exhibited a positive ESBL production test, and the characteristics of the isolates are shown in Table 1. β-Lactamase genes found in the E. coli isolates were as follows: bla _TEM-1+bla _CTX-M-1 (n=18), bla _CTX-M-1 (n=7), bla _CTX-M-9 (n=4), bla _CTX-M-14 (n=2), bla _SHV-12 (n=2), bla _TEM-1+bla _CTX-M-32 (n=1), and bla _CTX-M-32 (n=1). The insertion sequences ISEcp1, IS903, and orf477, surrounding the CTX-M genes, were detected in 26 ESBL-producing E. coli isolates (Table 1). The ISEcp1 sequence was found upstream of the bla _CTX-M genes in two of 33 CTX-M E. coli isolates. In these CTX-M isolates, an 80-bp region between the β-lactamase–encoding region and ISEcp1 sequence was detected. In addition, 24 isolates harbored the orf477 or IS903 sequence downstream of the bla _CTX-M-1/bla _CTX-M-32 or bla _CTX-M-9/bla _CTX-M-14 genes, respectively.

Table 1.

Characteristics of Cefotaxime-Resistant Escherichia coli Isolates Recovered from Pigs

					Amino acid changes ^c		MLST ^d
E. coli isolate	β-Lactamases	Environment of bla _CTX-M genes	Phenotype of resistance to non-β-lactams ^b	Resistance genes	GyrA	ParC	ST	Clonal complex	Phylogenetic group
SU01	CTX-M-1	ISEcp1-bla _CTX-M-1 -orf477	TET-STR	strA; strB; tet(B)	—	—	ST56	CC155	B1
SU65	CTX-M-1	—	TET	tet(B)	—	—	ST218	CC10	A
SU66	CTX-M-1	—	TET-STR-NA	aadA	Ser83Leu	wild	ST1397	Singleton	B1
SU31	CTX-M-1	bla _CTX-M-1 -orf477	TET-STR	aadA; tet(A)	—	—	ST877	Singleton	B1
SU46	CTX-M-1	bla _CTX-M-1 -orf477	CN-TET-NA-STR-SXT-CHL	sul1; sul2; aadA; tet(B); aac(3)-II-cmlA	Ser83Leu	wild	ST10	CC10	A
SU53	CTX-M-1	bla _CTX-M-1 -orf477	TET-STR-SXT	sul2; aadA; tet(A); tet(B)	—	—	ST10	CC10	A
SU63	CTX-M-1	bla _CTX-M-1 -orf477	TET-STR-SXT	sul3; aadA; tet(B)	—	—	ST10	CC10	A
SU02	CTX-M-1^a	bla _CTX-M-1 -orf477	TET	tet(A); tet(B)	—	—	ST540	Singleton	B1
SU03	CTX-M-1^a	bla _CTX-M-1 -orf477	CIP-CN-TET-TOB-NA-SXT-CHL	sul1; sul3; tet(A); aac(3)-II; aac(3)-IV; cmlA	Ser83Leu+Asp87Asn	Ser80Ile	ST2528	Singleton	B1
SU05	CTX-M-1^a	bla _CTX-M-1 -orf477	CIP-TET-NA-TOB-SXT-CHL	sul1; sul3; tet(A); cmlA	Ser83Leu+Asp87Asn	Ser80Ile	ST2528	Singleton	B1
SU10	CTX-M-1^a	bla _CTX-M-1 -orf477	CIP-TET-NA-SXT-CHL	sul1; sul3; tet(A)	Ser83Leu+Asp87Asn	Ser80Ile	ST2528	Singleton	B1
SU14	CTX-M-1^a	bla _CTX-M-1 -orf477	CIP-TET-STR-NA-SXT-CHL	sul1; sul2; aadA; tet(A); floR	Ser83Leu+Asp87Asn	Ser80Ile	ST398	CC398	A
SU16	CTX-M-1^a	ISEcp1-bla _CTX-M-1 -orf477	TET-STR-SXT	sul2; aadA; tet(A)	—	—	ST58	CC155	B1
SU32	CTX-M-1^a	bla _CTX-M-1 -orf477	STR	aadA	—	—	ST1508	Singleton	D
SU29	CTX-M-1^a	bla _CTX-M-1 -orf477	TET-SXT-STR	sul1; sul3; aadA; tet(A); tet(B)	—	—	ST10	CC10	A
SU39	CTX-M-1^a	bla _CTX-M-1 -orf477	TET-STR-SXT	sul3; aadA; tet(A)	—	—	ST10	CC10	A
SU47	CTX-M-1^a	bla _CTX-M-1 -orf477	TET-STR-NA-SXT-CHL-TOB	sul3; aadA; tet(A); tet(B); cmlA	Ser83Leu	wild	ST1832	Singleton	D
SU50	CTX-M-1^a	bla _CTX-M-1 -orf477	TET-CHL	tet(A); cmlA	—	—	ST48	CC10	A
SU51	CTX-M-1^a	bla _CTX-M-1 -orf477	TET-STR-SXT-CHL	sul3; aadA; tet(B); cmlA	—	—	ST10	CC10	A
SU54	CTX-M-1^a	bla _CTX-M-1 -orf477	TET-SXT-CHL	sul3; tet(A); tet(B); cmlA	—	—	ST10	CC10	A
SU68	CTX-M-1^a	—	TET-STR-SXT-CHL	sul3; aadA; cmlA	—	—	ST10	CC10	A
SU69	CTX-M-1^a		TET-STR-NA-SXT-CHL	sul1; sul2; aadA; tet(A)	Ser83Leu	wild	ST10	CC10	A
SU70	CTX-M-1^a	bla _CTX-M-1 -orf477	CN-TET-TOB-STR-SXT	sul1; sul2; sul3; aadA; tet(B); aac(3)-IV	—	—	ST2525	Singleton	B1
SU77	CTX-M-1^a	bla _CTX-M-1 -orf477	TET-STR-NA-SXT-CHL	sul1; sul2; aadA; tet(A)	Ser83Leu	wild	ST23	CC23	A
SU80	CTX-M-1^a	—	TET-SXT	sul1; sul2; sul3; tet(A)	—	—	ST398	CC398	A
SU26	CTX-M-9	bla _CTX-M-9 - IS903	TET-STR-SXT	sul1; sul2; aadA; tet(A)	—	—	ST58	CC155	B1
SU28	CTX-M-9	bla _CTX-M-9- IS903	TET-STR-SXT	sul1; sul2; aadA; tet(A)	—	—	ST58	CC155	B1
SU30	CTX-M-9	bla _CTX-M-9 - IS903	TET-STR-SXT	sul1; aadA	—	—	ST1790	Singleton	B1
SU55	CTX-M-9	—	CIP-CN-TET-TOB-NA-SXT-STR	aadA; tet(B); aac(3)-II	Ser83Leu+Asp87Asn	Ser80Ile+Glu84Gly	ST354	CC354	D
SU23	CTX-M-14	—	CN-CIP-TET-TOB-NA-STR-SXT	strA; tet(B); aac(3)-II	Ser83Leu+Asp87Asn	Ser80Ile	ST354	CC354	D
SU42	CTX-M-14	bla _CTX-M-14 - IS903	TET-STR-SXT-CHL	sul3; aadA; tet(A); cmlA	—	—	ST101	CC101	B1
SU25	CTX-M-32	bla _CTX-M-32 -orf477	TET-STR-SXT	sul2; aadA; tet(B)	—	—	ST1832	Singleton	D
SU21	CTX-M-32^a	bla _CTX-M-32 -orf477	TET-STR-SXT-CHL	sul3; aadA; tet(B); cmlA	—	—	ST1832	Singleton	D
SU62	SHV-12	—	TET	tet(A)	—	—	ST2524	Singleton	B1
SU64	SHV-12	—	TET	tet(A); tet(B)	—	—	ST101	CC101	B1

Additionally bla _TEM-1 was detected in these E. coli isolates.

CN, gentamicin; TOB, tobramycin; STR, streptomycin; TET, tetracycline; SXT, sulfamethoxazole-trimethoprim; NA, nalidixic acid; CIP, ciprofloxacin; CHL, chloramphenicol.

Sequences were compared with gyrA and parC genes included in the GenBank database with the accession numbers X06373 for gyrA and M58408 with the modification in L22025 for parC.

MLST, multilocus sequence typing: ST, sequence type; CC, clonal complex. New STs are indicated in boldface.

Antimicrobial resistance

With respect to the pattern of resistance of the ESBL-producing E. coli isolates, most of them (94.2%) presented a phenotype of multidrug-resistance that included antimicrobial agents belonging to at least three different categories, and 22.8% showed resistance to seven different antimicrobial categories (Magiorakos et al., 2012) Table 1.

A wide variety of resistance genes [tet(A), tet(B), aadA, strA, strB, cmlA, floR, aac(3)-II, aac(3)-IV, sul1, sul2, sul3] were detected among our ESBL-producing E. coli isolates (Table 1). Two amino acid changes were identified in GyrA protein (Ser83Leu+Asp87Asn) in six quinolone-resistant isolates, and in ParC protein (Ser80Ile+Glu84Gly) in one isolate. In addition, one amino acid changes was identified in GyrA protein (Ser83Leu) in five nalidixic acid-resistant isolates, and in ParC protein (Ser80Ile) in five nalidixic acid and ciprofloxacin-resistant isolates (Table 1). Twelve isolates harbored class 1 integrons and the gene cassette arrangement dfrA1+aadA1 was identified in all of them. Seven isolates harbored class 2 integrons with the following gene cassettes in their variable regions: aadA1+dfrA1+sat2 (six isolates) and sat1+aadA1 (one isolate). Two E. coli isolates (SU14 and SU80) contained simultaneously class 1 and 2 integrons.

MLST typing

Among the 35 ESBL-producing E. coli isolates, MLST analysis revealed nine different STs under six CCs and nine singletons ST, including overrepresentation of CC10 (31%; n=11/35). Three of the isolates were assigned in a new allele number for the fumC gene, which were included in the MLST database (www.mlst.net) and registered as ST2528, and two isolates were registered as ST2524 and ST2525 through new combination of alleles (Table 1).

The CTX-M-1–producers were distributed into four different CCs (16 isolates) and seven singletons (nine isolates). The CTX-M-9–producers were distributed among two CCs (three isolates) and one singleton (one isolate). The CTX-M-32–producers were distributed among one singleton (two isolates). The CTX-M-14–producers were distributed among two CCs (two isolates). On the other hand, E. coli isolates with different variants of CTX-M, grouped into the same CC, were observed. Three isolates, CTX-M-9– and CTX-M-1–producing E. coli, are associated with the ST58 (CC58); two isolates, CTX-M-14– and CTX-M-9–producing E. coli, are associated with the ST354 (CC354); and three isolates, CTX-M-32– and CTX-M-1–producing E. coli, are associated with the singleton ST1832. The SHV-12–producers were distributed among one CC (one isolate) and one singleton (one isolate; Table 1). Concerning the phylogenetic groups, 42.8% of the 35 ESBL-producing E. coli isolates belonged to the B1 phylogenetic group, 40.0% ofthe isolates to A, and 17.1% of the isolates to D.

Discussion

The detection of ESBL-producing E. coli in 49.3% of the analyzed samples highlights the wide dissemination of these resistant bacteria among healthy pigs for human consumption in Portugal. Similar studies on the prevalence of ESBL-producing E. coli in pigs have been performed at the national level in Portugal and in other European countries. However, significantly lower prevalence rates of ESBL-positive E. coli isolates were described in these reports when compared with our study (Agersø et al., 2012; Escudero et al., 2010; Geser et al., 2011; Gonçalves et al., 2010). Thus, 25% was found in fecal samples of intensive pig farm in Portugal (Gonçalves et al., 2010), while non-ESBL-producing E. coli were found in fecal samples recovered from healthy swine in the north and central region of the country (Machado et al., 2008). Moreover, percentages of 15–26% were described in recent studies in slaughter pigs in Switzerland and Spain, respectively (Escudero et al., 2010; Geser et al., 2011). Some of the variability between our data and previous studies can be related to differences in geographical conditions or even differences in methodologies. However, the wide dissemination of ESBL-producing E. coli among fecal isolates of healthy food-producing animals found in our study is a problem of food safety, and it is important to analyze the factors that could contribute to this situation.

Our results confirm that CTX-M-1 enzyme was the most dominant ESBL, followed by CTX-M-9. In previous studies, performed in Spain and Denmark, a high prevalence of CTX-M-1 was also reported in E. coli isolates recovered from healthy swine (Agersø et al., 2012; Blanc et al., 2006). Moreover, also in Spain, Briñas et al. (2005) described E. coli isolates recovered from sick pigs that were found to harbor CTX-M-14–, CTX-M-32–, and SHV-12–encoding genes (Briñas et al., 2005). In our study, isolates from healthy pigs also showed CTX-M-14, CTX-M-32, and SHV-12 β-lactamases. The occurrence of ESBL-producing E. coli in Portugal was reported in 2010 in a study conducted in an intensive pig farm in the north of the country by Gonçalves et al. (2010). In that study, the only ESBL-encoding genes detected were the CTX-M-1. In addition, ESBL-producing E. coli, particularly those producing CTX-M type ESBLs, are commonly associated with human healthcare and community (Guimarães et al., 2009; Mendonça et al., 2007).

The plasmid-located bla _CTX-M genes have been previously found associated to the ISEcp1 or the IS903 insertion sequences (Lartigue et al., 2007). In our study, the bla _CTX-M-1 gene has been identified as associated with the ISEcp1 sequence in two E. coli isolates, and the IS903 element was found downstream of bla _CTX-M-9 (three isolates) and bla _CTX-M-14 (one isolate). The presence of these insertion sequences upstream and downstream of the bla _CTX-M genes may play an important role in the mobilization and expression of different β-lactamase genes (Eckert et al., 2006).

Resistance to quinolones in E. coli is primarily related to mutations in GyrA, and additional mutations in ParC also produce higher levels of resistance. The substitutions Ser83Leu and Asp87Asn (GyrA) and Ser80Ile (ParC) were the most frequently detected in our quinolones-resistance E. coli isolates and similar findings were reported by others (Drago et al., 2010; Jouini et al., 2007; Leflon-Guibout et al., 2004).

Class 1 and/or class 2 integrons carrying resistance gene cassettes were detected in 48.6% of E. coli isolates. Similar to our results, class 1 integrons carrying dfrA1 and aadA1 cassettes were frequently observed in E. coli isolates from pigs in Europe (Guerra et al., 2003; Kadlec and Schwarz, 2008; Sunde, 2005). In addition, in class 2 integrons, the aadA1 cassette gene was found in association with the arrangements dfrA1-sat2 or sat1. These gene combinations have also been frequently detected among resistant E. coli isolates from pigs (Kadlec and Schwarz, 2008; Sunde, 2005). The increased prevalence of integrons observed in bacteria isolated from healthy swine for human consumption is a cause for concern, and even if they do not have the capacity for genetic mobilization, integrons can be disseminated through plasmids or transposons to other bacteria.

A great heterogeneity of MLST types was observed among our ESBL-producing E. coli isolates. CC10 and CC155 were the most common CCs. Some ESBL-producing E. coli clones of phylogroups A and B1 associated with CC10 and CC155, respectively, have been increasingly found in human clinical samples in Spain (Valverde et al., 2009). CC10 is overrepresented among isolates of phylogroup A, and is frequently identified among ESBL- and non-ESBL-producing E. coli isolates from fecal samples of healthy people (Leflon-Guibout et al; Valverde et al., 2009), enterotoxigenic E. coli isolates from community-based populations (Turner et al., 2006), and ESBL-producers E. coli causing UTIs (Oteo et al., 2009; Valverde et al., 2009). It is also important to underline the incidence of two ST101 E. coli isolates in our study, which had recently been associated with clinical isolates with New Delhi metallo-β-lactamase (NDM) enzymes in the United Kingdom, Pakistan, and Canada (Nordmann et al., 2011).

Moreover, six of our ESBL-producing E. coli isolates were included in three new sequences type, and all of them were assigned to phylogroup B1. Three of these isolates were identified in a new allele number for the fumC gene that originated a new ST (ST2528). Moreover, a new combination of alleles was obtained for two isolates and designated as ST2524 and ST2525. Through the MLST database, we found that ST656 (CC10) and ST8 (CC165) have a higher homology to ST2524 and ST2525, respectively. The ST2524 is a single locus variant of ST656 which belongs to CC10, while ST2525 is a double locus variant of ST8 included in CC165. CCs are defined as a group of multi-locus genotypes in which every genotype shares at least five loci in common with at least one other member of the group (Feil et al., 2001); the new STs, ST2524 and ST2525, most likely belong to CC10 and CC165, respectively.

E. coli isolates belonging to the most common ST or CC could carry almost all of the ESBL types known. For example, in a study performed in Spain, the ST10 complex (the most frequent in this study and also in the MLST database) carried five different ESBLs (CTX-M-14, SHV-12, CTX-M-9, CTX-M-15, and CTX-M-32) (Oteo et al., 2009). In a French study, ST10 complex isolates (associated with ST131) were found to be the most prevalent among fecal samples from healthy carriers of nalidixic acid–resistant (but ESBL-negative) E. coli (Leflon-Guibout et al., 2008).

Conclusion

This study demonstrated a high prevalence of ESBL-producing E. coli recovered in pigs slaughtered for human consumption in Portugal. Our findings raise important questions for human health due to the possible spreading of ESBL-producing E. coli via foodborne transmission or even by environmental pathways, such as farm waste. From a food safety perspective, the presence of ESBL-producing E. coli in fecal samples of pigs represents a risk for carcass contamination at slaughter and therefore also the potential for contamination of retail meat products. Although the bacteria can be easily killed during the cooking process, if the meat is not thoroughly cooked E. coli might survive and may lead to colonization of humans with ESBL-producing E. coli. Further investigation is required to understand the magnitude of this selection and to assess the risk of zoonotic transmission via the food chain and by contact with animals.

Footnotes

Acknowledgments

S.R. and D.D. were supported by national funds of the Fundação para a Ciência e Tecnologia (FCT) (SFRH/BD/47706/2008 and SFRH/ BD/80001/2011, respectively), co-financed by POPH QREN Type 4.1–Advanced Training, and subsidized by the European Social Fund and National Funds of Ministry of Science and Technology for High Education (MCTES). N.S. was supported by Programa Ciência (2008), co-financed by POPH QREN Type 4.2–Employment Promotion Scientific, and subsidized by the MCTES.

Disclosure Statement

No competing financial interests exist.

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