| Summary: | Wastewater treatment plants (WWTPs) are significant reservoirs of bacterial resistance. This work aims to identify the determinants of resistance produced by Gram-negative bacteria in the influent and effluent of two WWTPs in Portugal. A total of 96 wastewater samples were obtained between 2016 and 2019. The numbers of total aerobic and fecal contamination bacteria were evaluated, and genomic features were searched by polymerase chain reaction (PCR) and Next-Generation Sequencing (NGS). <i>Enterobacteriaceae</i> corresponded to 78.6% (<i>n</i> = 161) of the 205 isolates identified by 16sRNA. The most frequent isolates were <i>Escherichia</i> spp. (57.1%, <i>n</i> = 117), followed by <i>Aeromonas</i> spp. (16.1%, <i>n</i> = 33) and <i>Klebsiella</i> spp. (12.7%, <i>n</i> = 26). The remaining 29 isolates (14.1%) were distributed across 10 different genera. Among the 183 resistant genes detected, 54 isolates produced extended spectrum β-lactamases (ESBL), of which <i>bla</i><sub>CTX-M-15</sub> was predominant (37 isolates; 68.5%). A KPC-3 carbapenemase-producing <i>K. oxytoca</i> was identified (<i>n</i> = 1), with <i>bla</i><sub>KPC-3</sub> included in a transposon <i>Tn4401</i> isoform b. A higher number of virulence genes (VG) (19 genes) was found in the <i>E. coli</i> 5301 (O25b-ST131-B2) isolate compared with a commensal <i>E. coli</i> 5281 (O25b-ST410-A) (six genes). Both shared five VG [Enterobactin; Aerobactin, CFA/1 (clade α); Type1 (clade γ1); Type IV]. In conclusion, this work highlights the role of relevant clinical bacteria in WWTPs, such as KPC-3-producing <i>K. oxytoca,</i> and, for the first time, a CTX-M-15-producing <i>Ochromobactrum intermedium</i>, a human opportunistic pathogen, and a SED-1-producing <i>Citrobacter farmeri</i>, an uncommon CTX-M-type extended-spectrum beta-lactamase.
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