Free-living amoebae as vectors of antimicrobial resistance in regenerated sludge for agricultural systems.

The dissemination of antimicrobial resistance (AMR) in the environment plays a relevant role in the clinical area, since agriculture can act as a bypass that connects the environment with the population. In recent years, it is being studied the potential role of Free-Living Amoebae (FLA) as reservoirs and vectors of AMR. This study aims to analyse the role of FLA as a reservoir of AMR, focusing specifically on beta-lactam and, concretely, carbapenem resistance, in regenerated sludge for agricultural purposes. Forty sludge samples, 20 of dehydrated sludge and 20 of anaerobically digested sludge, were collected and analysed using a multidisciplinary approach combining molecular and culture-based techniques. FLA populations and their associated microbiome were characterized using 18S and 16S rRNA amplicon sequencing. Vermamoeba vermiformis (97.5%) and Naegleria spp. (95.0%) were the dominant FLA among the samples. The FLA-associated microbiome was dominated by Stenotrophomonas and Aeromonas, while dehydrated sludge exhibited significantly higher bacterial diversity. HT-qPCR detected clinically relevant AMR determinants with significant differences between sludge types. A total of 63 viable beta-lactam-resistant bacterial isolates, predominantly Pseudomonas and Klebsiella, were recovered from the FLA microbiome. Most isolates were resistant to third-generation cephalosporins, whereas approximately half were resistant to carbapenems. These findings demonstrate the role of FLA as AMR reservoirs and the importance of including FLA in sludge safety monitoring for agricultural reuse.