| Summary: | Background: <i>Enterobacteriaceae</i> are major players in the spread of resistance to β-lactam antibiotics through the action of CTX-M β-lactamases. We aimed to analyze the diversity and genetic characteristics of ESBL-producing <i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> isolates from patients in a Northern Portuguese hospital. Methods: A total of 62 cefotaxime/ceftazidime-resistant <i>E. coli</i> (<i>n</i> = 38) and <i>K. pneumoniae</i> (<i>n</i> = 24) clinical isolates were studied. Identification was performed by MALDI-TOF MS. Antimicrobial susceptibility testing against 13 antibiotics was performed. Detection of ESBL-encoding genes and other resistance genes, phylogenetic grouping, and molecular typing (for selected isolates) was carried out by PCR/sequencing. Results: ESBL activity was detected in all 62 <i>E. coli</i> and <i>K. pneumoniae</i> isolates. Most of the ESBL-producing <i>E. coli</i> isolates carried a <i>bla</i><sub>CTX-M</sub> gene (37/38 isolates), being <i>bla</i><sub>CTX-M-15</sub> predominant (<i>n</i> = 32), although <i>bla</i><sub>CTX-M-27</sub> (<i>n</i> = 1) and <i>bla</i><sub>CTX-M-1</sub> (<i>n</i> = 1) were also detected. Two <i>E. coli</i> isolates carried the <i>bla</i><sub>KPC2/3</sub> gene. The lineages ST131-B2 and ST410-A were detected among the ESBL-producing blood <i>E. coli</i> isolates. Regarding the 24 ESBL-producing <i>K. pneumoniae</i> isolates, 18 carried a <i>bla</i><sub>CTX-M</sub> gene (<i>bla</i><sub>CTX-M-15</sub>, 16 isolates; <i>bla</i><sub>CTX-M-55</sub>, 2 isolates). All <i>K. pneumoniae</i> isolates carried <i>bla</i><sub>SHV</sub> genes, including ESBL-variants (<i>bla</i><sub>SHV-12</sub> and <i>bla</i><sub>SHV-27</sub>, 14 isolates) or non-ESBL-variants (<i>bla</i><sub>SHV-11</sub> and <i>bla</i><sub>SHV-28</sub>, 10 isolates); ten <i>K. pneumoniae</i> isolates also carried the <i>bla</i><sub>KPC2/3</sub> gene and showed imipenem-resistance. ESBL-positive <i>E. coli</i> isolates were ascribed to the B<sub>2</sub> phylogenetic group (82%), mostly associated with ST131 lineage and, at a lower rate, to ST410/A. Regarding <i>K. pneumoniae</i>, the three international lineages ST15, ST147, and ST280 were detected among selected isolates. Conclusions: Different ESBL variants of CTX-M (especially CTX-M-15) and SHV-type (specially SHV-12) were detected among CTX/CAZ<sup>R</sup><i>E. coli</i> and <i>K. pneumoniae</i> isolates, in occasions associated with carbapenemase genes (<i>bla</i><sub>KPC2/3</sub> gene).
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