*2.4. Phylogenetic Relationship among Class II and III Bacteriocins Predicted from Wild Marine Animal-Associated Enterococcal Genomes*

To gain insights into the phylogeny of the 30 class II and 19 class III bacteriocins genes identified, phylogenetic analysis was performed (Figure 2) to determine their relationship (Supplementary Table S5) to 16 reference sequences in Bagel4 and Uniprot databases (Supplementary Table S6). This identified two groups with significant branch support (Figure 2). Group 1 included bacteriocins of both classes II and III. Class II bacteriocin gene clusters in Group 1 could be divided into subclasses a, b, and others. Included within each are: *IIa*) mundticin AT06, enterocin P, bacteriocin T8, bacteriocin 31, and enterocin SE-K4; *IIb*) enterocin X chain alpha, enterocin X chain beta; *II leaderless*) enterocin EJ97; *II circular bacteriocin*) carnocyclin A; *II other subclasses*) sakacin Q, enterocin 96, uviB, and enterocin NKR-5-3D; and unknown bacteriocins I, II, III, IV, and V. Class III bacteriocins in Group 1 included: enterolysin A, propionicin SM1, and unknown bacteriocin VI. In contrast, phylogenetic Group 2 included only the class II bacteriocin, lactococcin 972.

Interestingly, the 17 Class III enterolysin A-related sequences occurring in Group 1 could be grouped into three subclades. The first and second branches included sequences derived from *E. hirae* strains C7, MP1-1, MP1-2, MP1-4, MP1-5, DMW1-1, while the third branch included enterolysins A from *E. faecalis* strains GT3-2, ST1-20, MP8-1, RD1-1, MP5-1, MP9-10, and B9. The alignment of enterolysin A sequences within each branch (Supplementary Figures S1–S3) shows high similarity among them, although they have few conserved amino acids compared to the enterolysin A reference sequences (Supplementary Figure S4).

The alignment of the other bacteriocin sequences with reference sequences was performed (Supplementary Figures S5–S10). Among identities found were conserved motifs such as YGN and cysteine residues (all class IIa bacteriocins can be found in Supplementary Figure S6), and GxxxG or AxxxA motifs among class IIb and circular bacteriocin members (Supplementary Figures S7 and S8).

**Figure 1.** Biosynthetic bacteriocins genes were found within 22 *Enterococcus* spp. genomes from wild marine animals. The *Enterococcus* genomes are represented in the external circle (grey). Diversity of bacteriocin genes within 22 *Enterococcus* spp. genomes are represented by color gradients: Class I (green gradient) and Class II (blue gradient), and Class III (purple). \* Genomes showing duplicated bacteriocin genes (rectangles indicate the number of these genes). The illustration was designed using a D3 and Adobe Illustrator.

**Figure 2.** The phylogenetic relationships among bacteriocins (Class II and III) predicted for wild marine animals-associated enterococci genomes. The different groups are represented by grey colors (light grey: Group 1 and dark grey: Group 2). Class II is represented in blue and class III in purple (bold purple are enterolysins A from *E. hirae*, and regular purple are enterolysins A from *E. faecalis*). Unknown bacteriocins are highlighted in bold blue (I, II, III, IV, and V) and bold purple (VI).

> New putative bacteriocins I, II, and VI showed greater similarity to carnocyclin A, while the unknown bacteriocins III, IV and V were more closely related to enterocin X chain alpha (Xα) (Figure 2). Alignment of unknown bacteriocins with carnocyclin A and Enterocin Xα reference sequences allowed detection of conserved amino acid residues and motifs such as GxxxG or AxxxA (Figure 3). Putative novel bacteriocins I, II, VI and carnocyclin A showed only 1.3% overall amino acid sequence identity (Figure 3A), whereas bacteriocins I and II share 55.22% identity between them (Figure 3B). Putative bacteriocins III, IV, and V, which were closely related to enterocin Xα, have 9.2% overall amino acid sequence identity (Figure 3C); and bacteriocins III and V share 43.4% identity between them (Figure 3D). Structural modeling of these putative class II and III bacteriocins using the I-TASSER [62] package to build models using a combination of fragment and ab initio model building [63] is shown in Figure 4. Insights into structural features are important for the biosynthesis, mode of action, and biological activity of bacteriocins. The molecular models are in agreement with the expected protein folds (mostly alpha-helices with coil regions). Likewise, the most divergent model (Bacteriocin VI) is also isolated in its group in the phylogenetic reconstruction, supporting its uniqueness among other unknown bacteriocins.


**Figure 3.** *Cont*.

*Mar. Drugs* **2021**, *19*, 328


**Figure 3.** The alignment of putative unknown Class II bacteriocins and reference sequences using Clustal Omega software. (**A**) Alignment among I, II, VI, and carnocyclin A (reference) (Identity (\*): 1.3%; Strongly similar (:): 2.2%; Weakly similar (.): 4.4%]. (**B**) The alignment between I and II [Identity (\*): 55.22%; Strongly similar (:): 11.94%; Weakly similar (.) 10.45%]. (**C**) Alignment among III, VI, V, and enterocin Xα (reference) [Identity (\*): 9.2%; Strongly similar (:): 11.8%; Weakly similar (.): 9.2%]. (**D**) Alignment between I and II [Identity (\*): 43.4%; Strongly similar (:): 14.5%; Weakly similar (.) 11.8%). Identical residues are shaded in grey, and GxxxG or AxxxA motives are represented in red color. (-) Gaps introduced to optimize alignments. (\*) Positions with a single conserved residue. (:) Conservation between groups with strongly similar properties. (.) Conservation between groups with weakly similar properties.

**Figure 4.** The structural modeling of unknown Class II enterococcal bacteriocins from wild marine animals.

#### *2.5. Detection of Genes Associated with Enhanced Enterococcal Virulence*

Among the 22 genomes evaluated, *E. avium* (L8) and *E. mundtii* (MP7-18) were found to be devoid of determinants that have mainly been identified in *E. faecalis* and *E. faecium* strains associated with enhanced virulence (Figure 5A,B). All other enterococci strains possessed at least one potential virulence-associated trait (Figure 5B). As expected, these were most common in *E. faecalis*, where they have been most thoroughly studied. Some of these traits are encoded within the core genomes [25,26]. The unique *E. lactis* harbored *efa*Afm and *acm* genes, while all *E. faecalis* contained several genes associated with adhesion (*ace*, *efa*Afs), biofilm production (*ebp*A, *ebp*B and *ebp*C), proteases (*gel*E and *srt*A), protection against oxidative stress (*tpx*), and quorum sensing and sex pheromone (*cad*, *cam*E, *c*CF10, *c*OB1, and *fsr*B). *Enterococcus faecalis* genomes varied in the presence of hyaluronidase genes (*hyl*A and *hyl*B) and adhesion-associated gene (*Elr*A).

(**A**) **Figure 5.** *Cont*. 112

**Figure 5.** Wild marine animals-associated enterococci might represent a potentially valuable source of new compounds for biotechnological application and generation of new drug leads and potential probiotic application. (**A**) Scheme showing the main marine enterococci biotechnological applications suggested in this study. (**B**) Virulence markers analysis revealed potential probiotic enterococci from wild marine animals. Determinants of resistance (light yellow) and virulence (dark yellow) were associated with the results of in silico screening by bacteriocins (green, blue, and purple colors). \* Genomes showing duplicated bacteriocin genes (rectangles are representing the number of these genes). Blue dash representing the potential probiotic candidate strains (L8 and MP7-18). The illustration was designed using D3, R software, and Adobe Illustrator.

Resistome analysis (Figure 5B) revealed that all *E. casseliflavus* genomes (*n* = 3) possessed genes related to low-level vancomycin resistance (*van*RC and *van*XYC), as expected since these are part of the core genome for that species [64]. All *E. faecalis* genomes (*n* = 10) contained genes within the core genomes [26] conferring resistance to trimethoprim (*dfr*E); to macrolide, fluoroquinolone, and rifamycin (*efr*A and *efr*B); to pleuromutilin, lincosamide, and streptogramin (*lsa*A); and have a multidrug and toxic compound extrusion transporter (*eme*A). On the other hand, the unique *E. lactis* genome possessed genes related to the resistance to aminoglycosides (*aac*(6 )-Ii); to macrolide, lincosamide, streptogramin, tetracycline, oxazolidinone, phenicol, pleuromutilin (*eat*Av); and to macrolide, lincosamide, streptogramin (*msr*C). In addition, *E*. *hirae* genomes harbored genes related to aminoglycoside (*acc*(6 )-Iid; *n* = 6), and tetracycline [*tet*(W/N/M), *n* = 2; *tet*(L); *n* = 1] resistance.

#### **3. Discussion**

Microbes associated with marine animals from remote ecologies may be important sources for new tools to manage human and/or microbial interactions. In this study, we explored *Enterococcus* strains from the microbiota of wild sea birds, sea turtles, and marine mammals that range from the Antarctic to the coast of Brazil to identify potentially novel BGCs. These prospective BCGs were found in generalist species *E. faecalis*, as well as less common and less studied species, including *E. avium*, *E. casseliflavus*, *E. hirae*, *E. lactis*, and *E. mundtii*.

Putative bacteriocin genes were present in all enterococcal strains investigated, highlighting the competitive nature of the gut niche. Bacteriocin-encoding genes are known to be widely disseminated among enterococci species of different origins [33,54,55]. However, likely because of the novel environmental source of these strains, we found considerable diversity and novelty (Figure 1), with eight genomes possessing four or more bacteriocin gene clusters. This may be driven by variation in wild marine animal diets along migratory routes, combined with selection pressure for factors to control population structure and niche control in the host gut.

Enterococcal bacteriocins are of interest because of their antimicrobial activities, with activity against different Gram-positive and Gram-negative bacteria, including species of *Listeria*, *Clostridium*, *Staphylococcus*, *Streptococcus*, *Cutibacterium*, *Pseudomonas*, and *Salmonella* [6,33,34,65]. Enterocins have also been described as effective agents against antibiotic-resistant bacteria such as vancomycin-resistant enterococci (VRE) and methicillinresistant *Staphylococcus aureus* (MRSA) [35,46]. Furthermore, antiviral activities have been reported against herpes simplex viruses (HSV-1 and HSV-2), polio virus (PV3), measles virus, and influenza virus [41,66]. Immunomodulatory and anticancer properties of enterocins have not been widely explored but may also be of potential interest [67–69].

In this study, we identified known bacteriocins, natural variants of known bacteriocins, and potentially new bacteriocins distributed among different enterococcal species. The potency and spectrum of bacteriocins against important pathogens vary according to the peptide subclass [34,35,66,70]. Class I bacteriocins were identified in our in silico screening, with sactipeptides, new lanthipeptides I, lasso peptides, and thiopeptides being found in high numbers (Figure 1). Sactipeptides are produced mainly by Gram-positive organisms, and according to previous studies, the sactipeptides from *Bacillus subtilis* (subtilisin A) and *Bacillus thuringiensis* (Thuricin CD) have broad and narrow antimicrobial activity spectra, respectively [34,71]. A previous study also identified sactipeptide BGC in *Enterococcus mudtii* QU25 [36], similar to one found in this study. Lantibiotics and thiopeptides are most active against Gram-positive pathogens, including MRSA, VRE, and *Clostridium difficile* [23,34]. In contrast, most lasso peptides show activity against Gram-negative pathogens, e.g., bacteriocin MccJ25, which is active against some strains of *Escherichia coli* and *Salmonella* spp. [34].

The present study provides further evidence of the significant biodiversity of BGCs for class II, 19 bacteriocins, including five new putative bacteriocins (Figures 1 and 2; Supplementary Table S4). Class II bacteriocins are of special interest as potential therapeutic

agents and have been proposed on a larger scale production, whether in the food industry or in human health and veterinary medicine [72–74]. Because they consist of unmodified peptides, they do not require enzymes for their maturation and are small structures, less than 10 kDa [36,73], that may subject to low-cost production than other classes by chemical synthesis [73]. Complementing the recombinant technologies, chemical synthesis of bacteriocins may allow further molecular engineering for enhanced potency, improved pharmacological properties, increased stability and modified spectra of activity [73]. Class II bacteriocins and analogs thereof have been successfully prepared by chemical syntheses, such as aureocin A53 (AucA), durancin A5-11, enterocin CRL35, lactococcin MMFII, leucocin A, pediocin PA-1, curvacin A, lacticin Q (LnqQ), mesentericin Y105, and sakacin P [72–74].

In general, the class II bacteriocins are most active against Gram-positive pathogens, especially the class IIa bacteriocins, which are active against *L. monocytogenes* and other Gram-positive pathogens [33,34,72,75]. Enterocin SE-K4 and enterocin P were the most frequent class II bacteriocins in this study (Figure 1). Enterocin SE-K4 has been reported to exhibit antimicrobial activity against Gram-positive bacteria, *B. subtilis*, *Clostridium beijerinckii*, *E. faecium*, *E. faecalis*, and *L. monocytogenes* [40]. In contrast, enterocin P has a broad antimicrobial spectrum that includes activity against food-borne pathogens, *C. botulinum*, *C. perfringens*, *L. monocytogenes*, and *S. aureus* [64], as well as clinical strains, *L. monocytogenes*, *Salmonella* (S.) *typhi*, *Salmonella paratyphi* C, *Shigella dysenteriae*, vancomycin-resistant enterococci (VRE), and carbapenem-resistant *Pseudomonas aeruginosa* [75,76].

It is also important to highlight that class III bacteriocins were most common and widely distributed from wild marine animals and also included the unknown bacteriocin VI (Figure 1). Furthermore, three different enterolysin A sequences were verified among enterococci species, with two of them from *E. hirae* genomes that are reported for the first time in this species. Enterolysin A is a cell wall-degrading bacteriocin first reported to be produced by *E. faecalis* isolated from fish in Iceland [77]. Despite class III bacteriocins are large proteins (more than 10 kDa) and complex produced by chemical approaches [61], enterolysin A have been reported as broad-spectrum activity against pathogenic and nonpathogenic bacteria; acting on cleave the peptide bonds within the stem peptide as well as in the interpeptide bridge of Gram-positive bacterial cell walls [33,78].

In addition to bacteriocins, a wide variety of novel gene clusters encoding putative terpenes, NRPs, polyketides, and other active compounds have been uncovered by in silico analysis, creating new opportunities for drug development [23,24,49,79]. NRPs and terpenes have been reported with activity against several antibiotic-resistant strains [80–85]. A small library of predicted NRP peptides was chemically synthesized, based on the primary sequence of NRP clusters in the human microbiome, and a potent anti-MRSA (methicillin-resistant *Staphylococcus aureus*) peptide with a new mechanism of action, named humimycin, was identified [80]. The antitubercular agent levesquamide is a new polyketidenonribosomal peptide (PK-NRP) hybrid of a marine natural product (BGC) identified and isolated from *Streptomyces* sp. [84]. Furthermore, the antibacterial activity of 33 free terpenes commonly found in essential oils was evaluated, with 16 compounds showing antimicrobial activity, including eugenol, which exhibited rapid bactericidal action against *Salmonella enterica* serovar *Typhimurium*. Further, terpineol showed excellent bactericidal activity against *S. aureus* strains, and carveol, citronellol, and geraniol were rapidly bactericidal for *E. coli* [81]. In this study, we also found terpene biosynthesis-related clusters in *E. casseliflavus*, *E. hirae*, and *E. mundtii* species. Terpenes are secondary metabolites found in plants, bacteria, and fungi and have been shown to act as antibiotics, hormones, flavor or odor constituents, and pigments [86–88]. Beukers and collaborators [89] also identified putative genes or operons involved in terpene synthesis in *E. hirae*, *E. villorum*, *E. gallinarum*, *E. durans*, and *E. casseliflavus* strains isolated from bovine feces. The role of terpenes in enterococcal biology, including their possible involvement as bacteriocins, remains unclear [89].

Previous studies have examined the probiotic potential of enterococci from the marine environment [43,90,91]. Marine probiont strains have been used in finfish aquaculture due to their health beneficial effect and low potential to transfer antibiotic resistance genes to pathogens through horizontal gene transfer [92]. The potential of 13 enterococci isolated from wild seals was evaluated in a previous study from our group, and five (36.46%) showed activity against *L. monocytogenes* ATCC 35152 in the double-agar layer test, and one of them should be a good candidate for probiotic application [43]. In the present study, genome screening for bacteriocins highlighted potential probiotic enterococcal strains lacking known virulence or resistance traits (Figure 5A, B). In particular, the *E. avium* (L8) genome contained gene clusters for bicereucin BsjA1 and BsjA2, enterocin NKR-5-3D, mundticin AT06, and unknown bacteriocin I; and the *E. mundtii* genome (MP7-18) encoded sacpeptide and mundticin AT06 variants. Members of the genus *Enterococcus* have not yet obtained the status of generally recognized as safe (GRAS), although some are already being used as probiotics and in the production of animal food additives to prevent diseases or to improve growth [93,94]. New regulations for probiotics that distinguish between safe and potentially harmful strains are needed [35]. The application of genomic approaches in probiotic research would improve the understanding of the molecular mechanisms that endow the genera with safe and favorable traits [95].

Host-associated microbes are a rich source of factors that regulate community structure in a manner compatible with host health [96,97]. Our findings show a considerable novelty of biosynthetic pathways to be found by exploring the genomes of wild marine-animalsassociated microbes in remote ecologies with the potential to shape host-associated microbial population structures. The novel compounds and natural bacteriocin variants were discovered to provide the first leads for deriving new approaches for managing humanmicrobe interactions in health and disease. Besides, this data will inform and broaden the limits of known structural variation, knowledge of how structure relates to activity, and synthetic biology. In this context, as a perspective for further studies, the data generated here may be associated with recombinant technologies, chemical synthesis, molecular engineering, and other strategies to increase the biological potency, stability, and pharmacological properties in order to guarantee or modify the antimicrobial activity. Therefore, our results may contribute to promote the future development of bacteriocin-based drugs for potential use in managing animal and human health and as food preservatives.
