| Summary: | While antibiotic resistance is rising to dangerously high levels, resistance mechanisms are spreading globally among diverse bacterial species. The emergence of antibiotic-resistant <i>Klebsiella pneumoniae</i>, mainly due to the production of antibiotic-inactivating enzymes, is currently responsible for most treatment failures, threatening the effectiveness of classes of antibiotics used for decades. This study assessed the presence of genetic determinants of β-lactam resistance in 102 multi-drug resistant (MDR) <i>K. pneumoniae</i> isolates from patients admitted to two central hospitals in northern Portugal from 2010 to 2020. Antimicrobial susceptibility testing revealed a high rate (>90%) of resistance to most β-lactam antibiotics, except for carbapenems and cephamycins, which showed antimicrobial susceptibility rates in the range of 23.5–34.3% and 40.2–68.6%, respectively. A diverse pool of β-lactam resistance genetic determinants, including carbapenemases- (i.e., <i>bla</i><sub>KPC-like</sub> and <i>bla</i><sub>OXA-48-like</sub>), extended-spectrum β-lactamases (ESBL; i.e., <i>bla</i><sub>TEM-like</sub>, <i>bla</i><sub>CTX-M-like</sub> and <i>bla</i><sub>SHV-like</sub>), and AmpC β-lactamases-coding genes (i.e., <i>bla</i><sub>CMY-2-like</sub> and <i>bla</i><sub>DHA-like</sub>) were found in most <i>K. pneumoniae</i> isolates. <i>bla</i><sub>KPC-like</sub> (72.5%) and ESBL genes (37.3–74.5%) were the most detected, with approximately 80% of <i>K. pneumoniae</i> isolates presenting two or more resistance genes. As the optimal treatment of β-lactamase-producing <i>K. pneumoniae</i> infections remains problematic, the high co-occurrence of multiple β-lactam resistance genes must be seen as a serious warning of the problem of antimicrobial resistance.
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