Abordagem metagenômica para análise do microbioma e resistoma em rios do bioma amazônico

Abstract

Although Brazil has a rich biodiversity, the Amazon biome and its aquatic matrices have been undergoing intense degradation, being exposed to pollutants, including antimicrobials. Environments contaminated by these compounds may become sites for the selection of antimicrobial resistance (AMR) genes, which can be transferred to environmental or pathogenic bacteria. AMR already constitutes a serious public health problem, being associated with more than 4.95 million deaths annually, reinforcing the need for interdisciplinary techniques for rapid detection, such as metagenomic analyses combined with bioinformatics for the identification of resistance genes. This study aimed to identify and analyze the microbiome and the resistome in Amazonian aquatic matrices, as well as to develop pipelines for the detection of natural AMR reservoirs. DNA from samples originating from effluents of rivers in the state of Amapá was isolated using the commercial DNeasy® PowerSoil® kit and then sequenced using Nanopore technology. Subsequently, standardized pipelines were applied for quality assessment, filtering, and assembly. For pipeline validation, pilot samples from the Jaguaribe River (João Pessoa, PB) were used, due to the good contiguity of the assemblies and excellent N50 and L50 values. Taxonomic analyses were performed using Kraken2, and the resistome was characterized with different databases, such as CARD, ResFinder, and NCBI. Visual materials addressing AMR and One Health were developed. The application to Amazonian data was efficient in both assembly strategies. In the Flye approach, dominant taxa such as the phylum Pseudomonadota prevailed, with emphasis on Alphaproteobacteria and Gammaproteobacteria, in addition to Actinomycetota and Thermoproteota at several sites. The MEGAHIT assembly showed greater fragmentation but enabled the detection of abundant and low- coverage taxa, as well as possible pathogens such as Pseudomonas and Enterobacterales. From the MEGAHIT assemblies at sites AP2 and AP9, the resistance genes blaOXA-60 and RbpA were identified, associated with the genera Ralstonia and Mycolicibacterium, respectively. The developed pipelines proved to be effective for the analysis of the microbiome and resistome of environmental metagenomic DNA from aquatic matrices, and the detection of these genes reinforces the importance of surveillance in biomes under degradation.