The aim of the present study was to prospectively evaluate the prevalence of intestinal carriage by ESBL-producing Enterobacteriaceae among Portuguese students attending healthcare bachelors, and to determine the molecular features of ESBL-producing isolates. One-hundred and eleven fecal samples recovered from Portuguese healthcare students were screened for either ESBL-producing, carbapenem-resistant, colistin-resistant, or pan-aminoglycoside-resistant Enterobacteriaceae, using respective

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... ning media. All recovered isolates were tested for antimicrobial susceptibility and characterized by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). A total of 17 ESBL-producing Enterobacteriaceae (16 Escherichia coli and a single Klebsiella pneumoniae) were recovered from 16 students, corresponding to a prevalence of 14.5%. The E. coli isolates were distributed into three sequence types (ST) and seven PFGE types. The most common ESBL identified was CTX-M-1 (n = 13; 76%), followed by CTX-M-15 (n = 3; 18%), and CTX-M-8 (n = 1; 6%). The majority of the strains were resistant to sulfonamides (88%) and fosfomycin (71%). Resistance to aminoglycosides was observed at low rate, i.e. 12% for both tobramycin and kanamycin. No colistin-, carbapenem, or pan-aminoglycoside-resistant isolates were recovered. A major clone, ST10-blaCTX-M-1, included 12 E. coli isolates. The blaCTX-M-1 gene was always located onto an IncFIA/FIB plasmid type, co-harboring genes encoding resistance to tetracycline, sulfonamides, trimethoprim-sulfamethoxazole, and fosfomycin. The most commonly identified ESBL gene in E. coli was blaCTX-M-1, usually identified among ESBL-producing isolates recovered from animals. A high prevalence of fecal carriage of ESBL-producing E. coli was found among healthy healthcare students, underlying this population as an important reservoir.