21The vast biochemical repertoire found in microbial communities from a wide-range of environments allows screening 22 and isolation of novel enzymes with improved catalytic features. In this sense, metagenomics approaches have been 23 of high relevance for providing enzymes used in diverse industrial applications. For instance, glycosyl hydrolases, 24 which catalyze the hydrolysis of carbohydrates to sugars, are essential for bioethanol production from renewable 25resources. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities 26 from microbial communities inhabiting a soil sample by using the lacZα-based plasmid pSEVA232 in the generation 27 of a screenable metagenomic library. For this, we used a functional screen based on skimmed milk agar and a pH 28 indicator dye as previously reported in literature. Although we effectively identified nine positive clones in the 29 screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded 30 in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within 31 the coding region of the lacZα gene present in the original vector. We concluded that the current method has a higher 32 tendency for false positive clones' recovery, when used in combination with a lacZα-based vector. Finally, we discuss 33 the molecular explanation for positive phenotype recovering and highlight the importance of reporting boundaries in 34 metagenomic screenings methodologies. 35

The rise of multi-antibiotics resistant bacteria represents an emergent threat to human health. Here, we investigate antibiotic resistance mechanisms in bacteria of several species isolated from an intensive care unit in Brazil. We used whole-genome analysis to identify antibiotic resistance genes (ARGs) and plasmids in 35 strains of Gram-negative and Gram-positive bacteria, including the first genomic description of Morganella morganii and Ralstonia mannitolilytica clinical isolates from South America. We identify a high abundance of beta-lactamase genes in highly resistant organisms, including seven extended-spectrum β-lactamases shared between organisms from different species. Additionally, we identify several ARGs-carrying plasmids indicating the potential for fast transmission of resistance mechanism between bacterial strains, comprising a novel IncFII plasmid recently introduced in Brazil from Asia. Through comparative genomic analysis, we demonstrate that some pathogens identified here are very distantly related to other bacteria isolated worldwide, demonstrating the potential existence of endemic bacterial pathogens in Brazil. Also, we uncovered at least two couples of (near)-identical plasmids exhibiting multi-drug resistance, suggesting that plasmids were transmitted between bacteria of the same or different species in the hospital studied. Finally, since many highly resistant strains carry several different ARGs, we used functional genomics to investigate which of them were indeed functional. In this sense, for three bacterial strains (Escherichia coli, Klebsiella pneumoniae, and M. morganii), we identify six beta-lactamase genes out of 15 predicted in silico as the main responsible for the resistance mechanisms observed, corroborating the existence of redundant resistance mechanisms in these organisms.