| Summary: | We aimed to investigate the occurrence of acquired AmpC β-lactamases (qAmpC), and characterize qAmpC-producing <i>Enterobacteriaceae</i> from different non-clinical environments in Portugal. We analysed 880 <i>Enterobacteriaceae</i> resistant to third-generation cephalosporins recovered from 632 non-clinical samples [healthy human and healthy animal (swine, chickens) faeces; uncooked chicken carcasses; aquatic and trout aquaculture samples]. Bacterial and qAmpC identification, antibiotic susceptibility, clonal (PFGE, MLST) and plasmid (S1-/I-<i>Ceu</i>I-PFGE, replicon typing, hybridization) analysis were performed using standard methods. The occurrence of qAmpC among <i>Enterobacteriaceae</i> from non-clinical origins was low (0.6%; n = 4/628 samples), corresponding to CMY-2-producing <i>Escherichia coli</i> from three healthy humans (HH) and one uncooked chicken carcass (UCC). We highlight a slight increase in CMY-2 human faecal carriage in the two periods sampled [1.0% in 2013–2014 versus 0% in 2001–2004], which is in accordance with the trend observed in other European countries. CMY-2-producing <i>E. coli</i> belonged to B2<sub>2</sub>-ST4953 (n = 2, HH), A<sub>0</sub>-ST665 (n = 1, HH) or A<sub>1</sub>-ST48 (n = 1, UCC) clones. <i>bla</i><sub>CMY-2</sub> was identified in non-typeable and IncA/C<sub>2</sub> plasmids. This study is one of the few providing an integrated evaluation of the qAmpC-producing <i>Enterobacteriaceae</i> occurrence, which was low, from a very large collection of different non-clinical origins. Further surveillance in contemporary collections can provide an integrated epidemiological information of potential shifts in reservoirs, transmission routes and mechanisms of dissemination of <i>bla</i><sub>qAmpC</sub> in non-clinical settings.
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