Rafael da Silva Rosa, Micaela Santana Ramos, Letícia Franco Gervasoni, Lucas David Rodrigues dos Santos, Eduardo Angelino Savazzi, João Pedro Rueda Furlan, Eliana Guedes Stehling
Abstract Groundwater is a critical freshwater resource, but its quality can be compromised by anthropogenic contamination. Metals may exert selective pressure on microbial communities, promoting metal-tolerant bacteria carrying metal tolerance genes (MTGs), which are often associated with antimicrobial resistance genes (ARGs) and virulence determinants. Therefore, this study aimed to investigate Gram-negative opportunistic bacteria from groundwater, focusing on antimicrobial resistance (AMR), metal tolerance, and virulence profiles. A total of 101 groundwater samples were collected from 58 cities in São Paulo State, Brazil. Physicochemical analyses revealed that some samples exceeded recommended limits for metals, dissolved solids, fluoride, total coliforms, or E. coli . For these samples, 66 bacterial isolates were recovered, including Pseudomonas aeruginosa , Acinetobacter baumannii , Stenotrophomonas maltophilia , Burkholderia cepacia complex, Klebsiella pneumoniae species complex, and Enterobacter hormaechei . Antimicrobial susceptibility testing revealed diverse AMR profiles, with five isolates classified as multidrug-resistant. The ARGs qnrS , bla TEM , aac(6')-Ib , aadA , and tetB were detected. Metal tolerance assays showed tolerance mainly to silver, arsenic, and mercury, with multimetal tolerance detected in several isolates. A wide diversity of MTGs was identified, including silA , pcoA , merA , znuA , and rcnA . Virulence genes related to biofilm formation, immune evasion, and invasion were detected in clinically relevant species. Molecular typing revealed two novel sequence types, P. aeruginosa ST6404 and A. baumannii ST3355, along with clinically associated lineages such as A. baumannii ST744 and K. pneumoniae ST1536. These findings highlight groundwater as a potential environmental reservoir of opportunistic pathogens carrying ARGs, MTGs, and virulence determinants, reinforcing the importance of monitoring these ecosystems to mitigate AMR.