Background: Enterobacteriaceae 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 Escherichia coli and Klebsiella pneumoniae isolates from patients in a Northern Portuguese hospital. Methods: A total of 62 cefotaxime/ceftazidime-resistant E. coli (n = 38) and K. pneumoniae (n = 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 E. coli and K. pneumoniae isolates. Most of the ESBL-producing E. coli isolates carried a bla_CTX-M gene (37/38 isolates), being bla_CTX-M-15 predominant (n = 32), although bla_CTX-M-27 (n = 1) and bla_CTX-M-1 (n = 1) were also detected. Two E. coli isolates carried the bla_KPC2/3 gene. The lineages ST131-B2 and ST410-A were detected among the ESBL-producing blood E. coli isolates. Regarding the 24 ESBL-producing K. pneumoniae isolates, 18 carried a bla_CTX-M gene (bla_CTX-M-15, 16 isolates; bla_CTX-M-55, 2 isolates). All K. pneumoniae isolates carried bla_SHV genes, including ESBL-variants (bla_SHV-12 and bla_SHV-27, 14 isolates) or non-ESBL-variants (bla_SHV-11 and bla_SHV-28, 10 isolates); ten K. pneumoniae isolates also carried the bla_KPC2/3 gene and showed imipenem-resistance. ESBL-positive E. coli isolates were ascribed to the B₂ phylogenetic group (82%), mostly associated with ST131 lineage and, at a lower rate, to ST410/A. Regarding K. pneumoniae, 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^RE. coli and K. pneumoniae isolates, in occasions associated with carbapenemase genes (bla_KPC2/3 gene).

The purpose of this study was to analyse the prevalence and genetic characteristics of ESBL and acquired-AmpC (qAmpC)-producing Escherichia coli isolates from healthy and sick dogs in Portugal. Three hundred and sixty-one faecal samples from sick and healthy dogs were seeded on MacConkey agar supplemented with cefotaxime (2 µg/mL) for cefotaxime-resistant (CTX^R) E. coli recovery. Antimicrobial susceptibility testing for 15 antibiotics was performed and the ESBL-phenotype of the E. coli isolates was screened. Detection of antimicrobial resistance and virulence genes, and molecular typing of the isolates (phylogroups, multilocus-sequence-typing, and specific-ST131) were performed by PCR (and sequencing when required). CTX^RE. coli isolates were obtained in 51/361 faecal samples analysed (14.1%), originating from 36/234 sick dogs and 15/127 healthy dogs. Forty-seven ESBL-producing E. coli isolates were recovered from 32 sick (13.7%) and 15 healthy animals (11.8%). Different variants of bla_CTX-M genes were detected among 45/47 ESBL-producers: bla_CTX-M-15 (n = 26), bla_CTX-M-1 (n = 10), bla_CTX-M-32 (n = 3), bla_CTX-M-55 (n = 3), bla_CTX-M-14 (n = 2), and bla_CTX-M-variant (n = 1); one ESBL-positive isolate co-produced CTX-M-15 and CMY-2 enzymes. Moreover, two additional CTX^R ESBL-negative E. coli isolates were CMY-2-producers (qAmpC). Ten different sequence types were identified (ST/phylogenetic-group/β-lactamase): ST131/B2/CTX-M-15, ST617/A/CTX-M-55, ST3078/B1/CTX-M-32, ST542/A/CTX-M-14, ST57/D/CTX-M-1, ST12/B2/CTX-M-15, ST6448/B1/CTX-M-15 + CMY-2, ST5766/A/CTX-M-32, ST115/D/CMY-2 and a new-ST/D/CMY-2. Five variants of CTX-M enzymes (CTX-M-15 and CTX-M-1 predominant) and eight different clonal complexes were detected from canine ESBL-producing E. coli isolates. Although at a lower rate, CMY-2 β-lactamase was also found. Dogs remain frequent carriers of ESBL and/or qAmpC-producing E. coli with a potential zoonotic role.