Insights of bacterial community structure and occurrence of antibiotic resistance and antimicrobial compounds in an urban stream in the megacity of São Paulo, Brazil

• Urban streams are heavily impacted by slum untreated sewage. • Polluted urban waterways accelerate the dispersion of antibiotic resistance. • Co-occurrence of antimicrobials, resistant bacteria and resistance genes is alarming. • Klebsiella pneumoniae and Serratia marcescens showed high chlorhexidine MICs. • Stream bacterial community shows fecal and antibiotic bacterial family’s indicator. This study aimed to investigate the microbial community structure and the presence of Antibiotic-Resistant Bacteria (ARB), Antibiotic Resistance Genes (ARGs), and antimicrobial residues in an urban stream adjacent to an informal settlement in Brazil. Water samples collected during the winter of 2021 (n = 7) were analysed using culture-dependent and -independent methods. Microbial community structure was assessed by 16S rRNA sequencing, while bacterial isolates were characterized by Chlorhexidine Minimum Inhibitory Concentration (CHX-MIC) and whole-genome sequencing. Free carbapenemase ARGs were screened by conventional PCR and selected antimicrobial residues were quantified by UHPLC-MS/MS. The bacterial community was dominated by phyla Firmicutes, Bacteroidota, Proteobacteria, Campylobacterota, Actinobacteriot a, and Patescibacteria . Abundant (> 5%) families included Ruminococcaceae, Prevotellaceae, Moraxellaceae , and Arcobacteraceae , associated with faecal contamination and antimicrobial resistance. Urban stream bacterial isolates (31/66) belonged mainly to the genera Aeromonas, Citrobacter, Escherichia, Serratia, Chryseobacterium, Elizabethkingia with a high percentage of phenotypic resistance to third-generation cephalosporin and carbapenems. WGS of some isolates (16/31) revealed the co-production of ESBL and carbapenemase ARGs as well as Inc -type plasmids, efflux pumps and virulence genes. CHX-MICs ranged from 0.5 to 128 mg/L, with Klebsiella pneumoniae and Serratia marcescens showing the highest values and harboring qac genes and Inc HI2 plasmids, respectively. Azithromycin, ceftriaxone, levofloxacin, and chlorhexidine were detected at up to 2.39, 1.18, 0.14, and 0.43 mg/L, respectively. Notably, ceftriaxone-positive water samples also contained ARB and free ARGs ( bla NDM , bla KPC , bla VIM ). These findings highlight urban streams as reservoirs of ARB, ARGs, and antimicrobial residues, stressing the urgent need for local water treatment to mitigate antimicrobial resistance and public health risks.