Culture, qPCR, and genome-based surveillance of,, andin school wastewater from Santiago, Chile.

BACKGROUND: Antimicrobial resistance (AMR) surveillance remains predominantly centered on clinical settings, potentially underestimating the community circulation of high-priority carbapenemase determinants. METHODS: We integrated culture-based isolation, absolute qPCR, and whole-genome sequencing (WGS) to monitor bla KPC , bla NDM , and bla VIM in school wastewater from Santiago, Chile. Four 8-h composite influent samples (one per season) were collected in 2024 from an educational establishment comprising primary and secondary levels. Wastewater was screened on MacConkey agar supplemented with ceftazidime or ciprofloxacin (2 μg/mL), followed by disk diffusion testing. Carbapenem-non-susceptible isolates were assessed by Blue-Carba and PCR targeting bla KPC , bla NDM , and bla VIM ; carbapenemase-confirmed isolates were further characterized by WGS and resistome analysis. In parallel, gene copy numbers were quantified directly from wastewater DNA using plasmid-based standard curves. RESULTS: Total bacterial recovery on antibiotic-free control plates was comparable across seasons, supporting consistent sampling performance. Resistance rates peaked in winter for both ceftazidime (3.7% ± 0.87) and ciprofloxacin (11.1% ± 1.72). Across seasons, 92 morphotypes were recovered, dominated by the genera Aeromonas (n = 43) and Pseudomonas (n = 17), with Enterobacterales most frequent in winter (10/18). Twenty-one isolates were non-susceptible to at least one carbapenem; 12 were Blue-Carba positive (winter, n = 9; fall, n = 3), while no carbapenem-resistant isolates were recovered in spring and no carbapenemase producers were detected in summer. Multi-carbapenemase genotypes were detected, with bla NDM restricted to winter. WGS confirmed bla KPC -bla NDM -bla VIM co-carriage in five winter isolates, including a critical-priority Escherichia coli ST1193. Genomes also revealed complex Aeromonas resistomes and the presence of mcr-1 and mcr-3.17 in Aeromonas allosaccharophila isolates. Absolute qPCR showed a pronounced winter peak (up to 107 copies for bla KPC ; >106 for bla NDM and bla VIM ) and low loads in spring-summer, mirroring isolate-based findings. CONCLUSIONS: School wastewater provided a sensitive community sentinel matrix for targeted carbapenemase surveillance, and bla KPC emerged as a robust biomarker capturing seasonal AMR dynamics.

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Antimicrobial resistance (AMR) surveillance remains predominantly centered on clinical settings, potentially underestimating the community circulation of high-priority carbapenemase determinants.

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