| Summary: | Carbapenemase-producing <i>Enterobacterales</i> (CPE) are a growing concern, representing a major public health threat to humans, especially in healthcare settings. In the present study, we evaluated the persistent contamination by carbapenem-resistant <i>Enterobacterales</i> in water from Douro River, Portugal. KPC-producing <i>Enterobacterales</i> were detected in five water samples separated chronologically by 15 days each. Susceptibility testing was performed by disk-diffusion-method according to Clinical and Laboratory Standards Institute (CLSI), phenotypic carbapenemase activity was evaluated by carbapenem inactivation method, presumptive identification of the isolates was performed by CHROMagar orientation and confirmed by API-20E. Carbapenemase genes were screened by PCR and the clonality of all isolates was assessed by <i>XbaI</i>-Pulsed Field Gel Electrophoresis (PFGE). Fifteen KPC-producing <i>Enterobacterales</i> isolates were selected, identified as multidrug-resistant and showed a resistance profile to non-beta-lactam antibiotics: sulfamethoxazole + trimethoprim (7/15), ciprofloxacin (3/15), fosfomycin (3/15) and chloramphenicol (2/15). Isolates were identified as (6) <i>Escherichia coli</i> and (9) <i>Klebsiella pneumoniae</i>. Our results suggest a punctual contamination with KPC-producing <i>Enterobacterales</i> continued through the time. The absence of clonality between the isolates suggests a circulation of mobile genetic element harbouring KPC gene in the origin of contamination. This work provides a better understanding on the impacts of water pollution resulting from human activities on aquatic environments.
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