Clonal Diversity of ESBL-Producing Escherichia coli in Pigs at Slaughter Level in Portugal So ´ nia Ramos, 1–4, * Nuno Silva, 1, * Daniela Dias, 5 Margarida Sousa, 1–4 Jose ´ Luis Capelo-Martinez, 6,7 Francisco Brito, 1,2 Manuela Canic ¸ a, 5 Gilberto Igrejas, 3,4 and Patrı ´cia Poeta 1,2 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 pro- duced 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 pheno- type 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 gas- trointestinal tract of human and animals, with the capa- bility 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 b-lactams are fre- quently 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 b-lactams, including cephalosporins (ceftriaxone, cefo- taxime, and ceftazidime), as well as to aztreonam and related oxyimino-b-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 trans- posons 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; Borto- laia 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 hu- man 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 slaugh- tered 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 1 Centre of Studies of Animal and Veterinary Sciences, Vila Real, Portugal. 2 Veterinary Sciences Department, 3 Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, and 4 Department of Genetics and Biotechnology, University of Tra ´s-os-Montes and Alto Douro, Vila Real, Portugal. 5 National Reference Laboratory of Antimicrobial Resistances, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal. 6 REQUIMTE, Chemistry Department, Science and Technology Faculty (FCT), New University of Lisbon, Caparica, Portugal. 7 BIOSCOPE Group, Physical Chemistry Department, Science Faculty, University of Vigo at Ourense Campus, Ourense, Spain. *These two authors contributed equally to this work. FOODBORNE PATHOGENS AND DISEASE Volume 10, Number 1, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2012.1173 74