Klebsiella spp. are opportunistic pathogens capable of acquiring antimicrobial resistance genes, posing a public health concern. Surface water can act as a reservoir for resistant bacteria, facilitating their dissemination in the environment. This study aimed to identify Klebsiella species in surface water and characterize their antimicrobial resistance and virulence profiles through whole-genome sequencing.
Water samples were collected from 77 sites in the Douro River Basin, Portugal, including rivers, streams, wells, and fountains. Water was filtered through a 0.45 μm membrane and incubated in BHI broth at 37°C for 24 h. Cultures were plated on Chromogenic Coliform Agar and subcultured on HiChrome Klebsiella Selective Agar. Purple colonies were subcultured on BHI agar and identified by MALDI-TOF MS. Antimicrobial susceptibility was evaluated using the Kirby–Bauer disk diffusion method, while whole-genome sequencing was conducted to identify resistance and virulence genes, along with clonal lineages.
Out of the 77 samples, 21 (27.3%) Klebsiella spp. were isolated. Among the isolates, thirteen K. pneumonia, five K. aerogenes, two K. michiganensis, and one K. oxytoca were identified. Among K. pneumoniae, blaCTX-M-15, blaTEM-1, blaSHVvariants, and blaKPC-3 (3/13) were detected. Aminoglycoside resistance genes (aph(3'')-Ib, aph(6)-Id) and fluoroquinolone resistance genes (qnrB1, qnrS1) were present. All isolates carried fosA and heavy-metal resistance genes (sil, pco). Regarding the molecular typing, K. pneumoniae isolates belonged to a wide ranges of sequence types, including ST34, ST716, ST45, ST198, ST39, ST234, ST276, ST422, ST6, and ST37, while K. oxytoca was ascribed to ST155. K. aerogenes harbored ampC, fosA9, and efflux pump regulators (ramA, marA, baeR). K. michiganensis carried blaOXY-1, fosA, and qnrS1 (1/2), while K. oxytoca had blaOXY-2-8, fosA, and oqxA/B efflux genes.
These findings highlight the presence of multidrug-resistant Klebsiella species in surface water, emphasizing the potential role of aquatic environments in the dissemination of antimicrobial resistance genes.
