**1. Introduction**

Drug-resistant bacteria kill an estimated 700,000 people worldwide each year, and the discovery of new antimicrobial drugs is urgently needed [1–3]. This is motivating the search for new ecologies for novel natural products of potential therapeutic value. Human-proximal terrestrial life has been screened for diverse natural products to a much greater extent than larger but less accessible marine ecosystems. Blue biotechnology (or marine biotechnology) is an emerging field that investigates the rich diversity of bioactive molecules produced by marine organisms with potential industrial and therapeutic

**Citation:** Prichula, J.; Primon-Barros, M.; Luz, R.C.Z.; Castro, Í.M.S.; Paim, T.G.S.; Tavares, M.; Ligabue-Braun, R.; d'Azevedo, P.A.; Frazzon, J.; Frazzon, A.P.G.; et al. Genome Mining for Antimicrobial Compounds in Wild Marine Animals-Associated Enterococci. *Mar. Drugs* **2021**, *19*, 328. https://doi.org/10.3390/md19060328

Academic Editor: Max Crüsemann

Received: 1 May 2021 Accepted: 3 June 2021 Published: 6 June 2021

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applications [4–9]. Early successes include compounds derived from a gastropod (e.g., ziconotide, commercial name Prialt [10]), sponge (e.g., eribulin mesylate, commercial name Halaven [11]), cyanobacteria (e.g., dolastatin 10 [12], apratoxin A [13], and barbamide [14]), fungi (e.g., penicillipyrone A and B [15], and aszonapyrone A [16]), algae (e.g., neolaurene [17] and diphlorethohydroxycarmalol (DPHC) [18]), and bacteria (e.g., salinosporamide A [19], abyssomicin C [20], forazoline A [21], and farnesylquinone [22].

Recently, host-associated microbes also have drawn attention as a potential source for low toxicity agents compatible with host health but active against pathogenic microbes [23,24]. It was, therefore, of interest to us to explore marine animals from remote habitats for hostassociated microbes that encode novel natural product biosynthetic pathways. Further, we focused on host-associated enterococci, a genus of gut microbes associated with all classes of land animals studied [25], and with animals that have returned to the marine environment [26]. Although most enterococci exist as harmless commensals, some lineages of the species *Enterococcus faecalis* and *Enterococcus faecium* have emerged as leading causes of multidrug-resistant hospital infection [25,27–30].

Enterococci are known to produce bacteriocins with narrow to broad antimicrobial activity [31–33]. Bacteriocins have found use as natural antimicrobial agents so far, mainly in the food industry but could complement traditional antibiotics in controlling important human and animal pathogens [34,35]. Different classification schemes have been proposed for bacteriocins produced by Lactic Acid Bacteria (LAB), although still a subject of debate [33,36,37]. Class I bacteriocins are posttranslationally modified peptides with less than 10 kDa that require enzymatic modification during biosynthesis, and thereby, the molecules have uncommon amino acids and structures that impact their properties [36]. Class II bacteriocins are also less than 10 kDa, although they are heat stable and unmodified peptides [36] with the exception of disulfide bridging, circularization, and methionine formylation [33]. This class has been subclassified: IIa—pediocin-like bacteriocins; IIb two-peptide bacteriocins; circular bacteriocins; leaderless; and other bacteriocins that do not fall into any of the recognized subclasses [33]. On the other hand, Class III bacteriocins are large-molecular-weight (more than 10 kDa) and heat-labile antimicrobial proteins usually composed of different domains [36]. Divergently, some authors have been classified circular bacteriocins as class IV [38] or as Class Ib [36] since these head-to-tail cyclized peptides whose N- and C-termini are linked by a peptide bond, thereby rendering a circular molecule [36].

The bacteriocins synthetized by enterococci, enterocins, are generally small molecular weight (20–60 amino acids), often post-translationally modified peptides with cationic, hydrophobic, and heat-stable properties [32,33,36]. They vary in their mode of action, activity spectrum (restricted or broad), molecular mass, biochemical properties, and genetic origin [33,39,40]. Most known enterocins are produced by *E. faecium* and *E. faecalis*, but a few peptides have also been isolated from *Enterococcus mundtii*, *Enterococcus avium*, *Enterococcus durans*, *Enterococcus hirae*, and *Enterococcus lactis* [33,38]. Most characterized enterocins derive from enterococci associated with food, waste, feces, and gastrointestinal tract of humans and other animals [32,33,41]. Few have been described from enterococci from wild ecologies [8,26,42–44].

Traditionally, new bioactive compounds have been identified by screening microorganism extracts for biological activity or by amplification of new genes using polymerase chain reaction (PCR) [45–48]. These screening strategies are limited by time-consuming and laborious test methods [24,49]. Advances in molecular biology, bioinformatics, and genomics have been providing important new tools for exploration and development [50–52]. Genome screening has identified a large pool of potential compounds encoded by biosynthetic gene clusters (BGCs) in DNA databases [1,53–56]. The identification of new BGCs may be performed by applying algorithms based on indicators (e.g., evolutionary hallmarks, signature protein domains, and distant paralogs of primary metabolic enzymes) and using bioinformatic tools, such as antiSMASH5 [57] and BAGEL4 [58]. High throughput

computational technologies are being used for screening, presumptive chemical elucidation, and understanding of activities and biological aspects of new compounds [7,24].

Therefore, genome mining may represent a fertile strategy for identifying new biomolecules for future therapeutic and industrial applications. In this sense, the aim of the present study was to examine 22 genomes of *Enterococcus* species isolated from fecal samples of 17 wild marine animals from remote ecologies for potential antimicrobial compounds and/or probiotics strains.
