1. Introduction
Multidrug-resistant bacteria cause persistent hospital infections that increase morbidity and mortality, especially in developing countries [
1,
2]. Their impact on health care systems is mostly due to the unavailability of effective antibiotics [
1]. The main nosocomial antibiotic-resistant pathogens are
Acinetobacter baumannii,
Pseudomonas aeruginosa, extended-spectrum beta-lactamase-producing
Escherichia coli, methicillin-resistant
Staphylococcus aureus (MRSA),
Klebsiella pneumoniae, carbapenem-resistant
Enterobacterales (CRE), and vancomycin-resistant
Enterococci (VRE) [
1,
3,
4]. Antimicrobial resistance is the ability of microorganisms to inactivate or decrease the effectiveness of antibiotics. Resistance can occur spontaneously due to genetic modifications; nonetheless, this process can be accelerated by the inappropriate use of antibiotics, resulting in evolutionary pressures for genetic mutations and the exchange of genetic material between bacteria and phages [
5]. Since the discovery of antibiotics, between 1930–1962, more than 20 new classes have been described. However, resistance continues to evolve, and the search for new antimicrobial compounds is an urgent challenge [
5]. Only three new classes of antibiotics against gram-positive bacteria have been described recently: the Oxazolidinones class with Linezolid (2001) and tedizolid (2014); the daptomycin class, consisting of cyclic lipopeptides, discovered in 2006; and the fidaxomicin class, a macrocycle drug, discovered in 2011 [
5].
Bacteria are a source of many antimicrobial compounds. They produce lipopeptide, comprising non-ribosomal peptides synthetases (NRPSs), such as circular lipopeptides (surfactin, iturine, and phengycine families), polyketide (PKS) compounds, and siderophores [
6]. Some of these compounds are products of secondary metabolism, such as antibiotics, while others are bioactive molecules ribosomally synthesized, such as antimicrobial peptides and bacteriocins. Bacteriocins are viable alternatives to antibiotics that are no longer effective due to antimicrobial resistance [
7,
8]. Traditional bioprospecting strategies for new antibiotics are not efficient in finding new substances [
9]. Since traditional methods for screening antimicrobial substances can last a long time and have high costs, genomic analyses provide a new opportunity to search these substances in a more practical and less expensive way. Genome sequencing, gene annotation, and the activation of silent gene clusters constitute the basis of new methods for massive screening and new antibiotics discovery and yet success is limited [
9].
Aeromonas strains are known to produce several antimicrobial substances with the potential to become new antibiotics and therefore are worthy of detailed genomic investigations. The
Aeromonas genus comprises gram-negative, facultative anaerobic bacteria often found in aquatic environments [
10], the human gastrointestinal tract, and other animals, including fish, reptiles, and amphibians [
10,
11,
12].
Aeromonas species can cause several animal diseases. Furunculosis, for example, is a condition observed in fish [
13], which is associated with significant economic losses in pisciculture [
11,
12,
14]. Many
Aeromonas’ virulence genes have already been reported:
vapA (layer A);
act,
alt, and
ast (cytotonic enterotoxins);
ahyB (elastase);
exu (DNases) [
15,
16].
Aeromonas strains are considered opportunistic pathogens, infecting mainly immunosuppressed patients [
11,
12,
17]. Although there are reports correlating
Aeromonas sp. to gastroenteritis and few cases of more severe infections, the etiological role of the genus in this pathogenicity remains controversial [
17,
18]. The World Health Organization’s “One-World-One Health” concept highlights that healthiness is based on a balance of human, animal, microbe and environmental interactions [
19]. In this manner, solutions to these problems are likely to be found in nature. Antagonistic interactions are continually observed and are a part of nature, and are in natural environments. This concept was a guide for the research presented in this article. Bacteriocins receive special focus because they possess great potential in preventing the spread of infectious bacteria, controlling spoilage of industrialized products, and mitigating the indiscriminate and excessive use of other antibiotics [
20].
There are numerous reports on
Aeromonas strains producing bacteriocin-like substances (BLS) [
21,
22]. However, to date, the activity and presence of BLS has not been linked to its genetic origin. Antimicrobial peptides are important compounds for microorganisms, which grant competitiveness in different environments [
23]. These molecules are synthesized by several organisms for their defense. Amongst these are peptides called bacteriocins, which can kill or inhibit the growth of other microorganisms [
7]. Bacteriocins from gram-positive bacteria are frequently described as inhibiting other gram-positive strains [
24]. However, important gram-negative pathogens, such as
Salmonella and
Escherichia coli, have not yet been targeted by bacteriocins [
25,
26]. Thus, there is a need to discover and report on bacteriocins that target gram-negative disease-causing bacteria.
Biotechnological applications of bacteriocins include their use as antibiotics, food preservatives and bacteriocin, such as Nisin, used by food industries [
8,
23]; and as probiotics [
27]. Bacteriocins may also have applications as anticancer agents [
7,
24]. There are new assays that use bacteriocins in agriculture for the biocontrol of phytopathogens [
8]. Colicin is used for the biocontrol of pests in tobacco plants and considered an efficient strategy that meets GRAS (FDA) safety protocols for controlling bacteria [
28]. Bacteria from aquatic environments have already been described as great candidates for the production of antimicrobial substances. The
Aeromonas genus has been reported as capable of producing bacteriocins. This is an interesting genus, since it is found either in animal, water, or human, which requires a certain level of adaptation to different niches, where bacteriocins and toxins may play a role in the competition and maintenance of those species in respective niches [
29]. Therefore, our work aimed to isolate
Aeromonas bacteria from fish to investigate the antimicrobial substances’ production and to perform the genomic characterization of the producing strain. Here are described the bioprospecting and screening of a wide range of wild
Aeromonas strains looking for novel antagonistic behavior, followed by genomic mining to search for genes related to bacteriocins and antimicrobial activity.
4. Discussion
Messi et al., 2003 [
22], had previously reported on the potential of
Aeromonas strains to produce antimicrobial substances. Following their rationale, a screening for antimicrobial activity was performed and confirmed that strains of the
Aeromonas genus are widely antagonistic, as observed in
Figure 1. Thirty-eight of the 57 strains tested demonstrated some type of antagonistic activity towards at least one highly pathogenic bacterial strain. Screening analysis detected a group of seven strains, which can inhibit both gram-positive and gram-negative bacteria, with a different profile from others described in the literature, this is unusual and important for bacteriocin research. A lack of bacteriocin patents suggests they have perhaps been neglected and are an opportunity for novel discoveries. A glance further back in the literature reveals that bacteriocin-producing strains have been described to inhibit
Yersinia ruckeri,
Listonella anguillarum, and
Photobacterium damselae [
64]; fish pathogens, such as
Vibrio tubiashii [
65]; and strains associated with food contamination, such as
Staphylococcus sp. and
Lactobacillus sp. [
21,
22].
Strain AE59-TE2 stands out for being able to inhibit 14 of the 16 indicator strains tested. This strain exhibited antagonistic activity towards
K.
pneumoniae KPC (
Klebsiella pneumoniae carbapenemase). KPC-producing bacteria are a group of microorganisms with elevated resistance to various antibiotics, which causes infections that commonly available antibiotics can no longer effectively treat [
66]. This result is highlighted, since the majority of bacteriocins come from gram-positive bacteria and are not reported as being antagonistic towards gram-negative pathogenic microorganisms. There is a need for bacteriocins that target gram-negative food-spoilage strains, such as those from the genera
Salmonella and
Escherichia [
25,
26]. These findings shine a light on possible new solutions for medical, pharmaceutical, and food sectors.
In this work, the characterization of the
Aeromonas AE59-TE2 strain was proposed. Species identification and taxonomy within the
Aeromonas genus is controversial, even with the contribution of genomic analyses. Based on our data and as described in the literature,
16S rRNA gene sequences are highly conserved and do not contain enough genetic signal to separate
A. veronii from
A. allosaccharophila [
12]. Separating these taxons requires more than
16S rRNA sequence and biochemical tests [
67]. DDH analysis and MLSA [
45] phylogenetic inference were used with multiple housekeeping genes, including
rpoD, for taxonomic identification, and the strain was classified as
Aeromonas allosaccharophila AE59-TE2.
Since
Aeromonas strains are described as opportunistic pathogens, an equally important factor was assessing the strain’s virulence potential, which could impair its biotechnological applications in the future [
11,
12]. Aerolysin (
aerA) [
68], toxin A(
rtxA) [
69], layer A (
vapA) and secretion systems types II (T2SS) (
exeAB and exeC-N operons), T3SS (
ascV,
aopP,
aopH,
ascC and
aexT genes), T4SS (
traB,
traC,
traD,
traE,
trbJ,
traA,
traF,
traG,
traH,
traI,
traJ and
traK genes, with
traA,
traF-
traI as core components) and T6SS (
hcp (haemolysin), vgrG2 (valine), vgrG (glycine), vgrG1 (ADP-ribosyltransferase activity),
vasH (transcription regulator) and the
vasK (unknown function genes) altogether make up for the major virulence factors identified in the
Aeromonas genus [
70,
71,
72,
73].
The virulence factors database (VFDB), from the ABRicate tool, identified the
ascV and
ascC genes, which are type III secretion system (T3SS) structural genes, and
aopH, one of the main effector genes. Vanden Bergh and Frey (2014) [
74] demonstrated that, due to several mutations and genetic rearrangements, changes may occur in the type III secretion system. Thus, to affirm its integrity, one must analyze whether the structural genes (
ascV and
ascC) are intact and whether the main effector genes (
aopH,
aexT,
ati2,
aopO,
aopP and
aopS) are present [
74]. The T3SS is a complex structure used by gram-negative bacteria, which is capable of injecting effector proteins directly into the host cell cytoplasm. Only one effector gene was found in the AE59-TE2 genome. Furthermore, a progressive loss of virulence potential in
A. salmonicida is observed as constant genetic deletions and additions occur due to horizontal gene transfer with environmental bacteria. This is especially observed in strains grown in laboratories that do not undergo the selective pressures of natural environments [
74]. It is worth mentioning that some genes found in the AE59-TE2 genome may not be functional because they are truncated, as has already been described for
Aeromonas virulence mechanisms [
75]. Further analyses found the genes
blaOXA (a beta-lactamase),
arsD (an arsenite efflux transporter metallochaperone),
arsC (glutaredoxin-dependent arsenate reductase),
rsmA and
adeF (antibiotic efflux pump),
OXA-726 (beta-lactamase/antibiotic inactivation), and
EF-Tu (resistance to Pulvomycin). These genes are mostly related to antibiotic resistance. Concerns about virulence with this strain are founded but can be circumvented by using bacteriocins in a purified form. There is increasing interest in the pharmaceutical industry for the use of purified bacteriocins [
76].
The antiSMASH tool identified a homoserine lactone cluster in the AE59-TE2 genome. The N-acyl homoserine-lactone (AHL) is a “signal” molecule in gram-negative bacteria and is responsible for the regulation of several biological processes, such as biofilm formation, antibiotic production, and motility [
10]. Thus, this is a vital cluster that may be related to the antimicrobial activity observed in our analyses.
RAST server annotation uncovered a CvpA protein in the AE59-TE2 genome. This protein is required for colicin V production and was originally identified in plasmid pColV-K30 from
Escherichia coli. Nonetheless, this is not the structural gene for the colicin V bacteriocin [
77]. The
cvpA gene is chromosomal and is required for colicin V production and secretion 77]. It encodes an inner membrane protein that is involved in the colicin V export machinery [
77]. The colicin V structural
cvaC gene and the
cvaA and
cvaB genes are required for toxin processing and export. The protein that confers immunity on the host cell is encoded by the
cvi gene [
78]. Gene clusters similar to known bacteriocins have been described in other
Aeromonas genomes [
79], and the receptor for ferrienterochelin and colicins was identified in
A. salmonicida subsp.
pectinolytica 34melT genome [
80]. However, no correlation between the presence of these clusters and bacteriocin activity has been reported until now. BLAST analysis between a CvpA protein identified in the AE59-TE2 genome and a CvpA colicin V production protein from
Escherichia coli str. K-12 substr. MG1655 revealed a 64.59% identity. This result suggests that the gene could be associated with the production and secretion of colicin V peptide,
cvaC gene, or a similar structural peptide gene [
78]. However, no significant homology to the
E. coli cvaC gene was found.
Blast analyses identified four sequences with similarities to the
zooA gene. This gene encodes a Zn-metalloprotease called zoocin A, belonging to the M23/M37 family, and isolated initially from
Streptococcus equi subsp.
zooepidemicus. This protein functions as an enzybiotic that is active against gram-positive bacteria, cleaving peptides from their cell wall [
81]. The AE59-TE2 strain was able to inhibit the growth of gram-positive bacteria, namely
Enterococcus sp. and
Staphylococcus sp., which is not a common feature for gram-negative bacteriocin-producing strains. These data suggest that the AE59-TE2 strain might use different mechanisms to inhibit gram-negative and gram-positive bacteria. Multiple alignments between the four sequences found in the AE59-TE2 genome and other zooA sequences from the UniProt database demonstrated a highly conserved region for a peptidase M23 domain, suggesting that they may be new sequences related to the production of bacteriocins similar to Zoocin A.
The BLAST against DoBiscuit Database resulted in six sequences with more than 60% identity with sequences related to antibiotics. These sequences were annotated in the PROKKA software as RpoC, InfA (translation initiation factor IF-1), ThiC (phosphomethylpyrimidine synthase), FadH (2,4-dienoyl-CoA reductase), and MetK (S-adenosylmethionine synthase). Proteins RpoC and InfA could be related with resistance to Ansamycin and Rubradirin, respectively. The ThiC protein is associated with thiamine biosynthesis. The FadH protein is a NADPH-dependent 2,4-dienoyl-CoA reductase, and MetK protein catalyzes the formation of S-adenosylmethionine (AdoMet) from methionine and ATP and is associated with tylosin production [
82]. Tylosin is a macrolide antibiotic that is used as a feed additive in veterinary medicine.
KEGG analysis provided a general characterization of the genome and highlighted several important pathways to be studied. These results were further explored by investigating and comparing sequences with genes of known important antimicrobial compounds. For instance, the peptidase family C39 contains bacteriocin processing endopeptidases. In this genome, the
mepM gene was identified and is related to peptidoglycan synthesis [
14]. Also identified was the
nlpC gene, which is related to cell wall remodeling, cell separation during division, and cleaving non-canonical peptide bonds [
83]. Zoocin A is a D-alanyl-L-alanyl endopeptidase, which hydrolyses cross bridges in the peptidoglycan structure of susceptible
streptococci [
84]. The PROKKA software identified two sequences as D-alanyl-D-alanine endopeptidases (GAJHKBHP_00392 and GAJHKBHP_03927), corroborating with previous results of Zoocin A sequences. One protein containing an HNH endonuclease domain (Peg.1792) was annotated by the PATRIC server. HNH-type endonucleases are known as Nuclease Bacteriocins (NB) [
85]. Polyketides (PKS) were also pursued due to their antimicrobial activity, as described in the literature. Kegg analysis identified the rfb operon, which comprises four genes (
rfbABCD) and is involved in dTDP-rhamnose biosynthesis. Genes
rfbAB transform D-glucose-1-phosphate into dTDP-4-oxo-6-deoxy-D-glucose, an essential substance in polyketide sugar unit biosynthesis. This substance is further processed by genes
rfbCD, resulting in dTDP-
l-rhamnose. This latter substance can be involved in the biosynthesis of enediyne antibiotics and streptomycin. Streptomycin, for instance, is an aminoglycoside that possesses antimicrobial activity towards many bacteria, such as
Bacillus subtilis,
E. coli, certain strains of
Salmonella,
B. mycoides,
B. cereus, and
P. aeruginosa [
86]. Several enzyme complexes can be produced by the secondary metabolism of bacteria. Type I PKSs, known as modular/iterative, are multicatalytic enzymes, which give rise to known natural products, such as macrolides (erythromycin) and polyenes (nystatin). On the other hand, type II aromatic PKSs are mono and bifunctional enzymes that interact during the synthesis of polycyclic aromatic compounds, such as tetracycline or doxorubicin [
87]. Polyketide synthase modules and other related proteins were annotated in the PATRIC server (peg.1909). Antibiotic biosynthesis monooxygenase (ABM) is a protein superfamily that is involved in the production of several antibiotics, playing an important role in the biosynthesis of aromatic polyketides. ABM leads to a significant increase in antibiotic production [
88]. The PATRIC server identified an antibiotic biosynthesis monooxygenase (peg.705), demonstrating another important sequence related to antimicrobial activity and how rich is the genome. The PATRIC server also demonstrated several important pathways related to antimicrobial activity to be further explored in the future.
These genomic analyses of an A. allosaccharophila strain fill in a knowledge gap for this species, which has not been studied in such detail before. Furthermore, the A. allosaccharophila AE59-TE2 genome has similarities with the enzybiotic zoocin A endopeptidase sequences from streptococci bacteria. AE59-TE2 possesses a broad spectrum of inhibitory activity, targeting gram-positive and gram-negative multidrug resistant pathogens. Genomic analyses revealed important sequences associated with antimicrobial activity. Further analyses are required to better elucidate this antimicrobial substance, since it holds promising biotechnological use for the health and food sectors.