open access

Abstract

As the search for new antibiotics continues, the resistance to known antimicrobial compounds continues to increase. Many researchers around the world, in response to antibiotics resistance, have continued to search for new antimicrobial compounds in different ecological niches such as the marine environment. Marine habitats are one of the known and promising sources for bioactive compounds with antimicrobial potentials against currently drug-resistant strains of pathogenic microorganisms. For more than a decade, numerous antimicrobial compounds have been discovered from marine environments, with many more antimicrobials still being discovered every year. So far, only very few compounds are in preclinical and clinical trials. Research in marine natural products has resulted in the isolation and identification of numerous diverse and novel chemical compounds with potency against even drug-resistant pathogens. This review is an attempt to explore marine antimicrobial peptides (AMPs) as a rich source of molecules with antimicrobial activity. In fact, the sea is poorly explored in terms of AMPs, but it represents a resource with plentiful antibacterial agents performing their role in a harsh environment. For the application of AMPs in the medical field limitations correlated to their peptide nature, their inactivation by environmental pH, presence of salts, proteases, or other components have to be solved. Thus, these peptides may act as templates for the design of more potent and less toxic compounds.

Keywords: Antibiotics, Antimicrobial peptides (AMPs), Marine AMPs, Microorganisms, Multidrug resistance

Introduction

The indiscriminate worldwide overuse and misuse of antibiotics has led to high rates of microbial resistance (Aiello and Larson, 2003; Arnold, 2007), posing new challenges to human health. Hence, we are facing a worldwide re-emergence of infectious diseases and a rapid increase in pathogenic multidrug-resistant (MDR) bacteria, resistant to commercially available antibiotics, threatening the world with a return to the pre-antibiotic era. The search of novel molecules with antibacterial activity that could overcome the resistance phenomenon is a priority. Therefore, naturally-occurring, cationic antimicrobial peptides (AMPs) are considered the new hope and attract attention of scientists as suitable templates for the development of alternatives to conventional antibiotics. AMPs have been isolated from a variety of organisms (Yeaman and Yount 2003) and have been shown to play a key role in building their defense strategies, being part of the humoral natural defense against infections (Galdiero et al., 2015). The number of AMPs effective against pathogenic bacteria continues to grow. They are relatively small peptides (<60 amino acids) with a broad-spectrum of activity against microorganisms (Gram-positive and Gram-negative bacteria, fungi, viruses, parasites) (Cruz et al., 2014) and a low likelihood of developing resistance (Hancock and Sahl, 2006; Marr et al., 2006). Moreover, AMPs may play multifunctional roles which extend far beyond their ability to function as antibiotics. As a matter of fact, some of these peptides have anticancer activity, stimulate the immune system by favoring cytokine release, chemotaxis, antigen presentation, angiogenesis, inflammatory responses, and adaptive immune induction (Gaspar et al., 2013).

Much effort is devoted to attaining novel antimicrobial compounds with broad spectrum activity against a wide spectrum of pathogenic microorganisms and recent research is focused on the development of modified compounds to obtain more selective and efficient drugs. In contrast to most antibiotics, which usually target specific steps in bacterial growth and replication, AMPs’ antimicrobial activity is correlated to membrane disruption and osmotic lysis of microbes and/or to acting on intracellular targets of microbes inhibiting the synthesis of proteins, nucleic acids, and the cell wall. The mechanism used by AMPs to kill specific bacteria exhibits notable differences from peptide to peptide and specificity for particular AMP-bacteria coupling; moreover, a clear relationship between AMP structures and killing mechanisms has not yet arisen. It has also been proposed that molecules able to disrupt the membrane bilayer are a promising alternative to antibiotics since they present the additional advantage of being active against dormant bacteria with slow or no growth. In fact, dormant bacteria can survive high concentrations of antibiotics and need extensive treatment for efficacy (Hurdle et al., 2011; Lai and Gallo, 2009).

As for the mechanism inducing the damage of the membrane and/or internalization, AMPs essentially exploit major differences in the composition of bacterial versus eukaryotic membranes. Selectivity is in fact correlated to the difference between membrane compositions and characteristics of host and pathogen cells. Differences, such as the absence of cholesterol, greater presence of anionic lipids, and a stronger inward directed electric field are key for the specificity. Moreover, it is more difficult for bacteria to alter these features compared to the more circumscribed molecular targets of conventional antibiotics and development of resistance is unlikely because it would require a change in the bacterial membrane. Biophysical studies have provided models for the mechanism of membrane damage; the main proposed mode of action are the carpet model, barrel stave model, and toroidal-pore model (Shai, 2002). In all proposed models, the initial interaction between the peptide and the bacterial membrane is electrostatic and involves the positively-charged residues in the peptides and the negatively-charged moieties on the surface of the bacterial membrane. The main features of the three models are reported below. According to the carpet model, peptides accumulate in a parallel fashion on the lipid membrane surface forming a carpet-like structure. Following the initial electrostatic interaction between AMPs and the phospholipids, the peptide reaches a threshold concentration and inserts into the membrane, breaking the lipid structure and causing cell lysis in a detergent-like manner involving a large-scale micellization of the bilayer. This mode of action has been proposed for peptides, like dermaseptin and cecropins, with positively-charged amino acids distributed along the peptide sequence which do not cause hemolysis because of their weak interaction with zwitterionic membranes (Brogden, 2005). The barrel-stave model is typical of helical peptides with spatially-separated, and distinctly-hydrophobic and -hydrophilic regions; moreover, the net charge of these amphipathic peptides is close to neutral. Amphipathic helices insert into the hydrophobic core of the membrane establishing interactions with lipid polar head groups using their hydrophilic portion and interactions with the hydrophobic chains using its hydrophobic portion. As a result, transmembrane pores are formed. According to this model, peptides, such as alamethicin, bind to the membrane, recognize each other and oligomerize, the oligomer inserts into the hydrophobic core of the membrane, forming a transmembrane pore inserted perpendicular to the bilayer surface (Brogden, 2005). The toroidal-pore model is typical of AMPs, such as magainins, protegrins, and melittin. This model differs from the barrel-stave model in a way that peptides are always associated with lipid head groups even when they are perpendicularly inserted into the highly-curved lipid surfaces (Brogden, 2005).

Recently, Marrink et al. (2009), using molecular dynamic simulations, proposed a “chaotic pore” model, involving a continuously changing situation characterized by a localized permeabilization caused by a time varying number of peptide and lipid molecules. In conclusion, both cationic amino acid residues and hydrophobic residues play key roles in the interaction of peptides with phospholipid bilayer and subsequent membrane perturbation. The absence of a clear correlation between structure and function, further supports the idea that interfacial activity determines the ability of a peptide to permeabilize membranes (Wimley, 2010). This is key, also, to distinguish between antibacterial peptides able to damage the membrane bilayer and cell-penetrating peptides able to cross the bilayer without damages (Falanga et al., 2015; Galdiero et al., 2012; Galdiero et al., 2015). In fact, some cell-penetrating peptides are also antibacterial and the switch is correlated to their concentration (Galdiero et al., 2015). Alternatively, some AMPs act on intracellular targets inhibiting cell-wall synthesis, nucleic acid binding and synthesis, protein production, and enzyme activity.

As part of the ongoing global effort to discover novel antimicrobials to treat infections caused by resistant pathogenic organisms, many laboratories are now devoted to the discovery of novel antimicrobial compounds against antibiotic resistant bacteria and putative antibacterial compounds are directly derived from the plant and animal kingdoms. The marine environment is extremely hydrophilic and contains not only a wide range of microorganisms but also a high salt content. In this review, we reviewed the very recent research results in the field of marine AMPs, which were characterized and identified. We have classified marine AMPs according to their biological effects into antibacterial, antimycobacterial, antifungal, antiviral, and antiprotozoal.

Antibacterial marine peptide 

Almost there is no marine organism that does not produce natural antibacterial compounds as an essential line of defense to survive, hence marine antibacterial peptides is a rich class of antimicrobials with new discoveries (Table 1).

Table 1.Selection of some peptide from different classes

Aurelin is an AMP derived from the mesoglea of a scyphoid jellyfish called Aurelia aurita. It is composed of 40 amino acid residues and described to be cationic in nature with a molecular weight of 4296.4 Da. It has demonstrated modest antibacterial activity against both Gram-positive and Gram-negative bacteria with MIC of 10 µM against the Bacillus megaterium, strain B-392, and 40 µM against Micrococcus luteus, strain Ac-2229 which were determined to be the most sensitive species to Aurelin. shows that Aurlein is the first AMP to acquire Shk fold that enables Aurelin to have a diversity in its mechanism of action which can be attributed to two different mechanisms; (i) acting as a peptide toxin bocking K+ channels, (ii) acting as a membrane active antimicrobial peptide (Shenkarev et al., 2012).

Mytimacin-AF is an AMP derived from marine mollusks, particularly from the mucus of Achatina fulica snail. It is characterized to be rich in cysteine as it is composed of 80 amino acid residues 10 of which are cysteines, and its molecular weight is 9711.41 Da. Mytimacin-AF showed activity against both Gram-positive and Gram-negative bacteria, however it was most potent against Staphylococcus aureus with minimum inhibitory concentration (MIC) value of 1.9 µg/ml. In addition, the most promising feature about mytimacin-AF is its activity against the human Klebseilla pneumonia, one of the most common hospital-acquired bacterial infections, what makes mytimacin-AF a very promising antibacterial agent (Zhong et al., 2013).

Myticusin TDHQMAQSACIGVSQDNAYASAIPRDCHGGKTCEGICAD ATATMDRYSDTGGPLSIARCVNAFHFYKRRGEENVSYKPFVVSW KYGVAGCFYTHCGPNFCCCIS is another cysteine-rich peptide derived from mussels. It was characterized and identified from the hemolymph of Mytilus coruscus. Its involvement in the host immune response has been proven, hence it plays a role in bacterial infection eradication. The cysteine amino acid comprises 10 out of 104 amino acid residues which makes myticusin-1 a long polypeptide chain with a molecular weight of 11,279.63 Da. This molecule has demonstrated greater potency against Gram positive compared to Gram-negative bacteria, as it has recorded an MIC < 5 mM against a variety of tested Gram-positive strains including S. aureus compared to an MIC >10 mM against a variety of Gram-negative bacteria including E. coli (Liao et al., 2013).

EC-hepcidin3 (APAKCTPYCYPTHDGVFCGVRCDFQ), is a novel isoform that belongs to the hepcidin class of AMPs. This class is also known to be rich in cysteine, but this isoform is rather a four cysteinehepcidine unlike the typical eight cysteinehepcidin. It was cloned from the marine fish, the orange spotted grouper Epinephelus coioides. The purified Ec–hepcidin 3 has a theoretical molecular weight of 2798.2 Da. The kinetic studies proved that this molecule has a rapid and strong antibacterial activity against Staphylococcus aureus (MIC 1.5–3 µM, MBC 1.5–3 µM) and Pseudomonas stutzeri (MIC < 1.5 µM and minimum bactericidal concentration (MBC) < 1.5 µM) (Qu et al., 2013).

Marine’s bacteria are also important source for AMP, Gageostatins A-C. They are examples of three new nonribosomal lipopeptide molecules that were derived from the broth of the bacterium Bacillus subtilis. These three molecules are consisting of heptapeptides and new fatty acid which is 3 beta hydroxy fatty acid and are anionic in nature with a uniform net charge of 3. Those molecules were tested as anticancer agents against a panel of six cancer cell lines; MDA-MB-231 breast cancer cell line, HCT-15 colon cancer cell line, PC-3 prostate cancer cell line, NCI-H23 lung cancer cell line, NUGC-3 stomach cancer cell line, and ACHN renal cancer cell line, and their GI50 values ranged from 4.6 to 19.6 mg/ml. They were also tested for antifungal and bacterial activities against R. solani, C. acutatum, and B. cinera fungi, in addition to S. aureus, B. subtilis, S. typhi, and P. aeruginosa with MIC ranging from 4 to 64 mg/ml. It was reported that a combination between Gageostatins A and B showed greater antibacterial activity than individuals. The three compounds differ in molecular weight and structure, but they all possess the same amino acid sequence (ELLVDLL) (Tareq et al., 2014a).

Acosta et al. (2013) were capable of identifying three AMPs from piscidin that were isolated from the Teloest fish, tilapia gills called (Oreochromis niloticus). These peptides had the names and molecular weights of oreochromicins (Oreoch-1, 2524.40 Da; Oreoch-2, 2981.60 Da; and Oreoch-3, 3654.19 Da), the three AMPs are composed of a signal peptide which is highly cationic in nature with uniform net charges of 5, 7, and 10, and constituted of 23, 25, and 32 amino acid residues, respectively. The three peptides displayed activity against a wide range of bacteria and fungi, specifically Oreoch-1 (FIHHIIGGLFSVGKHIHGLIHGH) that showed activity against Gram-positive bacteria like S. aureus (MIC = 5 mM) and B. subtilis (MIC = 3 mM), and Gram-negative bacteria like P. aeruginosa (MIC = 35 mM) and E. coli (MIC = 6.7 mM), aswell as antifungal activity against C. albicans (MIC = 20 mM) (Mygind et al., 2005). While Oreoch-2 (FIHHIIGGLFSAGKAIHRLIRRRRR) showed similar potency to Oreoch-1 against S. aureus (MIC = 5 mM), B. subtilis (MIC = 1.7 mM), P. aeruginosa (MIC = 6.7 mM), E. coli (MIC = 5 mM), E. tarda (MIC = 20 m M), and C. albicans (MIC = 26.7 mM). Lastly, the third AMPs Oreoch-3 (IWDAIFHGAKHFLHRLVNPGGKDAVKDVQQKQ) showed greater antifungal activity compared to antibacterial, where it was active mainly against C. albicans (MIC = 40 mM) but very weak against B. subtilis (MIC = 106 mM), E. tarda (MIC = 160 mM), and Vibrio sp. (MIC = 106 mM). The ability of oreoch compounds to induce an immune response and serve as adjuvant candidates for vaccine development was investigated by their co-administration to fish and mice infected with inactivated bacteria and virus that served as the subunit antigen. The results of this in vivo study indicated that the three oreoch compounds were capable of inducing a hormonal and cellular responses in the infected species as well. The study described the powerful impact of the combination of Oreoch-2 alongwith Oreoch-3 as this combination resulted in a significant increase in the produced antibodyamounts. Hence, according to the study results, oreoch compounds are to be considered as strong and successful candidates for molecular adjuvants in vaccination (Acosta et al., 2014).

ß-defensins are a large family of broad-spectrum AMPs. Cod ß-defensin [defb] [WSCPTLSGVCRKVCLPTEMFFGPLGCG KEFQCCVSHFF] is a novel antimicrobial peptide that belongs to the Defenisins family, this family of compounds has a diversity of functions and protective roles. Defb was identified from the Atlantic cod Gadusmorhua. The structure is enriched with six cysteine residues that make three disulfide bridges, which are accommodated in the compound’s tertiary structure composed of one a helix and three ß sheets. The compound has a molecular weight of 4490 Da, and a uniform net charge of +1. Defb shows activity against Gram-positive M. lutes ATCC 4698 (MIC = 25–50 mM) and P. citreus NCIMB 1493 (MIC = 0.4–0.8 mM). However, the in vivo study suggested potential activity for this compound against Gram-negative bacteria as this gene expression was highly upregulated in the head kidney of the challenged atlantic cod with Vibrio anguillarum. In addition, the Defb showed phagocytic activity on the prepared head kidney leucocytes obtained from five Atlantic cods (Ruangsri et al., 2013).

The Clam antimicrobial peptide V. philippinarum defensing (VpDef) (GFGCPEDEYECHNHCKNSVGCRGGYCDAGTLRQRCTCYGCN QKGRSIQE) possesses combined helix and beta structure, and four potential disulfide bonds. The recombinant VpDef (rVpDef) was expressed in E. coli Origami (DE3) as a C-terminal His6-tagged fusion protein. VpDef transcripts were significantly induced in the hemocytes post Vibrio anguillarum infection. rVpDef showed high activity against Gram-positive bacteria Micrococcus luteus (MIC = 6.25–12.5 mM) and less activity against Gram-negative bacteria like Proteus mirabilis (MIC = 50–100 mM) (Zhang et al., 2015).

CATH-BRALE is a salmonoid cathelicidin that is expressed from an ancient fish called Bracgymysttazlenok. This AMP belongs to the Cathelicidins family which are considered to be an essential component of the innate immune system that is capable of playing antibacterial and immunomodulatory role. This compound is composed of 199 amino acids, rich in arginine and glycine giving the compound an intense positive charge [uniform net charge +13]. CATH-BRALE is described to be composed of antiparallel b-sheets protruded by a-helices. This compound exhibited greater potency against Gram-negative bacteria where it has shown to effectively inhibit Gram negative fish bacterial pathogens, Aeromonas salmonicida and Aeromonas hydrophila with a low MIC value against both pathogens (9.38 lM). It has also displayed some antifungal activity against C. albicans ATCC 2002 (MIC = 2.3 mM) (Li et al., 2013).

As-CATH4 (amino acid sequence: RRGLFKKLRRKIKKGFK KIFKRLPPVGVGVSIPLAGRR) and As-CATH5 (amino acid sequence: TRRKFWKKVLNGALKIAPFLLG) are two novel compounds that belong to the cathelicidins family of AMPs. These compounds impact on the immune system was tested by their administration to the Chinese mitten crab. The injection of both compounds in the crab induced an increase in the activity of two enzymes, acid phosphatase and alkaline phosphatase. Hence, this in vivo study demonstrates the role of these compounds as immunostimulants. Additionally, this immunostimulatory effect had a positive impact on improving the anti-infective capacity of the drugs, as the bacterial load in the two groups of infected crabs was significantly reduced upon As-CATH4 and As-CATH5 administration. The bactericidal activity was further supported by the in vitro results where both compounds were effective against the tested ten different bacterial strains. Among these ten strains, eight strains were known to be ampicillin-resistant pathogens, and yet the compounds proved efficacy in low concentrations against them. This highlights the potential unique application of As-CATH4 and As-CATH 5 against drug resistant pathogens. The killing kinetics assay revealed the feature of the rapid killing effect where it took less than 20 min to have the colony-forming unit of the treated bacteria with As-CATH5 to be reduced and remain zero. When the bactericidal mechanism was investigated for these compounds, it showed their ability of targeting the cell membrane and increasing the permeability and cell disruption. These results provide evidence of the potent anti-infective ability of both As-CATH4 and As-CATH5 in crab’s aquatic organisms (Guoa et al., 2017).

A cathelicidin from the sea snake Hydrophis cyanocinctus was identified as Hc-CATH having the amino acid sequence KFFKRLLK SVRRAVKKFRKKPRLIGLSTLL. It demonstrated potent and broad spectrum antimicrobial activity through disruption of the cell membrane and lysis of the bacterial cell. Hc-CATH displayed powerful activity against human pathogenic microorganisms, with MICs ranging from 0.16 mM (toward Shigella dysenteriae) to 20.67 mM (toward Klebsiella 8). Keeping into consideration that the a helix conformation as well as the positive charge of the peptide are essential for the antimicrobial activity. It is unlikely that the bacteria would develop resistance to this peptide because of its rapid bactericidal effect. In addition to that, Hc-CATH is reported to possess anti-inflammatory effects, extreme stability in aqueous solutions and very low cytotoxicity toward tested mammalian cells. It was also shown that human serum proteins can degrade and bind to Hc-CATH. On the other hand, unlike many antimicrobial peptides, Hc-CATH’s antimicrobial activity is highly salt resistant implying great potency for application (Wei et al., 2015). Li et al. (2014) were capable of identifying, characterizing, and purifying a new AMP from the hemolymph of Mytilud coruscus a mollusks species called mytichitin-CB (TVKCGMNGKMPCKHGAFYTDTCDKNVFYRCVWGRPVKKHCGRGLVWNPRGFCDYA) with a molecular weight of 6621.55 Da. This molecule has some special features that include the chitin-binding domain for the drug, where preformed studies have shown C-terminal chitin-binding domain of human ChT allowed the drug from binding to chitin. It is constituted of 55 amino acid 6 of which are cysteines forming 3 disulfide linkages. This drug is specific against Gram-positive bacteria, and some fungi where it showed activity against B. subtilis, S. aureus, S. luteus; and B. megaterium (MIC < 5 mM), fungi C. albicans and M. albicans (MIC 6.2–25 mM). SM HEP1p and SM HEP2p are two hepcidin AMPs that are rich in cysteines which make 4 disulfide bond needed for the molecule’s tertiary structure. They are isolated from turbot called Scophthalmus maximus. SMHEP1 which is composed of 26 amino acids has a uniform net charge of 4 and its sequence is (QSHISLCRWCCNCCKANKGCGFCCKF) with a molecular weight of 2940 Da while SMHEP2 which is made up of 22 amino acid residues has a uniform net charge of 3 and the following sequence (GMKCKFCCNCCNLNGCGVCCRF) and its molecular weight is 2410 Da. When it comes to their antimicrobial activity, it was noted that against Gram-positive bacteria SEM HEP1p showed greater potency, where it was active against both S. aureus (MIC= 2 mM) and M. luteus (MIC = 1 mM). Unlike SEM HEP2p that showed much greater activity against Gram-negative bacteria, where it was potent against the following Gram-negative E. tarda (MIC = 1 mM) and V. anguillarum (MIC = 2 mM), and both compounds displayed antiviral activity. As for their mechanism of action, it is most likely that they act as bactericidal by inducing cell membrane destruction and causing severe permeability disruption. Despite the described activity for both compounds, the preformed in vivo and in vitro studies confirm that SmHep2p was superior to SmHep1p as antimicrobial agents. The preformed in vitro studies showed that both compounds were successful in inhibiting bacterial growth in fish cells. However, the in vivo study done on the turbot given the two compounds showed an increase in its bacterial and viral resistance (Zhang et al., 2014).

SA-hepcidin1 (QSHLSMCRYCCNCCRNNKGCGFCCKF) and SAhepcidin2 (NPAGCRFCCGCCPNMIGCGVCCRF) are cationic hepcidins from the spotted scat fish (Scatophagus argus). The antibacterial activity of both SA-hepcidin1 and SA-hepcidin2 were similar against Gram-positive bacteria Staphylococcus aureus (MIC = 50 mM) and Gram-negative bacteria Vibrio anguillarum (MIC = 50 mM) and Vibrio alginolyticus (MIC = 50 mM). Moreover SA-hepcidin1 showed strong antibacterial activity against Gram negative bacteria Vibrio fluvialis (MIC = 25 mM) and Escherichia coli (MIC = 50 mM), whereas SAhepcidin2 did not. In addition to the antibacterial effect, SA-hepcidin2 showed antiviral activity against enveloped and non-enveloped viruses, being most effective against MsReV. One possible mechanism of action as antiviral is causing virion collapse, thereby preventing viral attachment to host cells (Gui et al., 2016).

YFGPA(VKVGINGFGRIGRLVTRAAFHGKKVEVVAIND) AMP is considered as a GAPDH related compound. GAPDH is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase that has multiple essential intracellular functions, in addition to the newly discovered antimicrobial properties. YFGPA was obtained from the yellowfin tuna, Thunnus albacares. Its molecular weight is 3400 Da. The antimicrobial spectrum was proven to cover Gram positive bacteria such as Bacillus subtilis, Micrococcus luteus, and Streptococcus iniae with minimal effective concentrations (MECs) of 1.2–17.0 mg/mL, as well it showed activity against Gram negative bacteria like Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs = 3.1–12.0 mg/mL). The preformed killing kinetic study against B. subtilis, and E. coli D31 showed that YFGPA has a bacteriostatic activity rather than bactericidal activity, as it acts through non-lytic pathway. The low cytotoxicity of YFGPA makes it a potential alternative therapeutic agent for humans or a substitute to conventional antibiotics for managing fish diseases (Seo et al., 2014a).

Piscidins are known to have good antimicrobial activity and are important for innate host defense. Rock bream piscidin (Rbpisc) (GEGFLGMLLHGVGHAIHGLIHGK) has a tertiary structure which includes amphipathic helix-loop-helix structure. This unique structure of the peptide enables it to interact electrostatically with the negatively-charged bacterial membrane inserting the hydrophobic region into the lipid bilayer and puncturing the bacteria membrane, which is a crucial step to the antimicrobial activity. In vitro, it showed very strong antibacterial activity against S. iniae, V. harveyi and V. ordalii with MICs lower than 0.9 mM, and against V. ordalii (7.8–15.6 mM). Rbpisc also showed weak antimicrobial activity against E. coli, Vibrio alginolyticus and V. campbellii, as indicated by the MIC and IC50 values (1.9–3.9 and 125–250 mM, respectively). It is known that in order to apply any antimicrobial peptide as an antibiotic, it should have low toxicity or hemolytic activity, unfortunately the analysis done by Bae et al. (2016) revealed that the peptide exhibited significant hemolytic effect against fish erythrocytes at concentrations higher than 500 µM which is the concentration that effectively inhibited E. tarda and V. vulnificus. However, Rbpisc did not exhibit cytotoxicity at the effective concentration against S. iniae, V. alginlyticus, V. harveyi, and V. ordalii. Peng et al. (2012) discovered five piscidin-like AMPs, two of which are having potent antimicrobial activity which are named TP3 (FIHHIIGGLFSVGKHIHSLIHGH) and TP4 (FIHHIIGGLFSAGKAIHRLIRRRRR). These compounds were derived from Nile tilapia, Oreochromis niloticus. They share similar amphipathic a-helical structure and cationic nature that attribute in part to their activity. The selective toxicity and hemolytic activity of these two compounds were examined by comparing the toxicity against human mammalian normal cells and Hela cells of cervical tumors. The preformed in vitro study conveyed that TP3 was selective to tumor cells over normal cells. In contrast, TP4 showed no selectivity between both the normal and tumor cells. In addition, the hemolytic studies showed that TP4 is capable of inducing hemolysis to human erythrocytes while TP3 exerted less capability of inducing it. Both compounds showed antibacterial activity against both Gram-positive and Gram-negative bacteria where TP3 which is O. niloticus dicentracin-like peptide had a minimal inhibitory concentration range of 0.6–20 mg/mL against Gram-positive S. agalactiae 819, E. faecalis BCRC 10066, and S. agalactiae BCRC 10787 and Gram-negative V. vulnificus 204, V. alginolyticus. TP4 which is O. niloticus moronecidin-like peptide had a minimal inhibitory concentration range of 0.03–2 µg/ml against a variety of Gram positive including Acteria S. agalactiae 819, E. faecalis BCRC 10066, and S. agalactiae BCRC 10787 and Gram-negative including V. vulnificus 204, A. hydrophila BCRC 13018, V. alginolyticus, and P. aeruginosa ATCC 19660. Also TP4 showed promising activity against H. pylori, and triple-negative breast cancer (Peng et al., 2012). In addition, the activities of TP3 and TP4 were examined in vivo, where the antimicrobial potencies of TP3 and TP4 were investigated by the eradication of acute bacterial infection from hybrid tilapia fish. Upon infection of the Tilapia fish with V. vulnificus, the co-administration of TP3 and TP4 at a dose of 20 mg/fish for each agent, resulted in a survival rate of 95.3% and 88.9%, respectively, seven days following the treatment. Moreover, this study demonstrated that the pretreatment of the fish with TP3 and TP4 prior to infection would result in a survival rates of 28.9% and 37.8%, respectively, while injecting the fish with TP3 and TP4 30 min after infection gave 33.3% and 48.9% survival rates, respectively (Pan et al., 2017).

SpHyastatin protein from the Crab Scylla paramamosain displays a broad antimicrobial spectrum. Potent activities against Gram positive bacteria (Micrococcus luteus, Staphylococcus aureus, Corynebacterium glutamicum, Micrococcusluteus) and Gram negative bacteria (Pseudomonas stutzeri, Pseudomonas fluorescens, Aeromonashydrophila) with MIC values of 0.63–2.5 mM, as well as values of MBC lower than 10 mM were demonstrated. In an in vivo study, Scylla paramamosain crab was infected with V. parahaemolyticus to compare whether suppression of SpHyastatin would affect the survival rates following infection with V. parahaemolyticus. The results revealed that at 60 h, 13% of the control group survived while none of the SpHyastatin-suppressed crabs survived. It was proposed that this compound was capable of eradicating the bacterial infection via an immune reaction. Furthermore, this in vivo study revealed a unique property about SpHyastatin, it has the highest transcription level among six other tested AMPs on the infected crab, indicating the high potential of pathogen resistance to this compound (Shan et al., 2016). Sphistin is a cationic, amphiphilic a-helical antimicrobial peptide with a uniform net charge of +9. The 38 amino acid sequence of Sphistin peptide (MAGGKAGKDSGKAKAKAVSRSARAGLQFPV GRIHRHLK) was derived from the N terminus of the crab (Scylla paramamosain) histone H2A. It exerts its antimicrobial function through electrostatic attraction because of its cationic characteristics. Sphistin was effective against the growth of Gram-positive bacteria (S. aureus, C. glutamicum, Bacillus subtilis, Micrococcus lysodeikticus, and Micrococcus luteus) and Gram-negative bacteria (Shigella flexneri, Pseudomonas stutzeri, and Pseudomonas fluorescens) with MIC values lower than 1.5 mMas well MBC lower than 12 mM. It was demonstrated that sphistin exerted its antimicrobial activity via adsorption, followed by permeabilization and finally damaging the bacterial cell membranes. Fortunately, sphistin did not display toxicity towards either mammal or crab normal cells (Chen et al., 2015).

Pt5e is a peptide derived from fish egg yolk protein phosvitin. Its recombinant analogue (rPt5e) was expressed in E. coli Transetta (DE3) strain. It was purified by inverse transition cycling (ITC) technique, and the yield was 2.39 mg from 100 ml culture. It has shown the ability to kill clinical multidrug resistant bacteria; E. coli 577 (MIC = 2.1 mM), E. coli 4457 (MIC = 2.1 mM), Klebsiella pneumoniae 2182 (MIC = 2.1 mM), E. coli 140237 (MIC = 2.1 mM), and Acinetobacter baumannii7225 (MIC = 1.4 mM) in a dose dependent manner. To confirm the interaction between Pt5e and the bacteria, the membrane proteins were extracted from the multidrug resistant (MDR) bacteria and treated with Pt5e, then they were subjected to Western blotting analysis. The extracted membrane proteins of the MDR bacteria that were treated with Pt5e reacted with anti-His tag mouse monoclonal antibody, on the other hand, the membrane proteins of the same bacteria treated with phosphate buffered saline (PBS) did not react, indicating that the extracted proteins of the MDR bacteria treated with Pt5e contained recombinant Pt5e. These findings suggested that Pt5e interacted with the membranes of the MDR bacteria causing membrane permeabilization, depolarization, and increased the intracellular reactive oxygen species, leading to bacterial cell death (Li et al., 2016).

From the fish orange-spotted grouper (Epinephelu scoioides), ecPis-4 (FFRHIKSFWKGAKAIFRGARQG) was identified as having strong antibacterial activities, in addition to the two others; ecPis-2 and ecPis-3. The three piscidins were synthesized in order to determine their biological activity; they demonstrated antibacterial activities against fish pathogen V. parahaemolyticus, and human pathogen E. coli and S. aureus. MBCs were lower than 5.0 mmol/l for EcPis-2 and ecpis-4. However ecPis-3 MBCs were 5.0 mmol/l against V. parahaemolyticus, 10 mmol/l against E. coli, and 20 mmol/l against S. aureus (Zhuang et al., 2017).

MoroPC-NH2 (FFGHLFRGIINVGKHIHGLLSG-NH2) is synthetic, amidated moronecidin-like peptides from the Antarctic Fish Parachaenichthys charcoti. It showed strong activity against Gram negative Shigella sonnei, Psychrobacter sp., and E. coli DH5a (MICs < 12.5 µM). In addition, Gram-positive bacteria; Enterococcus faecalis, Streptococcus pyogenes, Staphylococcus aureus, and Listeria monocytogenes are sensitive to the peptide below 25 µM. On the other hand, moroNC-NH2 (FFWHHIGHALDAAKRVHGMLSG-NH2) is another synthetic, amidated moronecidin-like peptides obtained from the Antarctic Fish Notothenia coriiceps; having lower antibacterial activity and a narrower spectrum. This might be a result from its lower hydrophobicity. Salts can decrease the activity of antimicrobial peptides since they disrupt the electrostatic interactions needed for forming pores in the microbial membrane. The activity of moro-NH2 was affected by salt concentration as evidenced by the increase in the MIC in the presence of 500 mM NaCl or 5 mM CaCl2. Besides, it was concluded that it is difficult to consider moroNC-NH2 for clinical use since it has a narrow spectrum of antibacterial activity and high salt sensitivity. Upon examining the effect of the temperature on the antimicrobial peptides (moro-NH2, moroNC-NH2, and moroPC-NH2), none of the temperatures tested (15 °C, 20 °C, 30°C, and 37°C) affected the activities of the peptides (Shin et al., 2017).

PaLEAP-2 (MTPLWRVMGNKPFGAYCQDHVECSTGICKGGHCITSQPIKS) is an antimicrobial peptide from the teleost fish Plecoglossus altivelis, after being chemically synthesized; it exhibited selective antimicrobial activity in vitro against various bacteria. Its potencies towards E. tarda and V. anguillarum are the highest (6.25 mg/ml). While the MIC value against Escherichia coli DH5a is 50 mg/ml. Also, P. putida, V. alginolyticus, and V. vulnificus were sensitive to PaLEAP-2 (MIC = 100 mg/ml). The mode of action of LEAP-2 was suggested as hydrolyzing the DNA as a means to kill the bacteria. In addition, in vivo studies revealed that at high concentrations of PaLEAP-2 the mortality caused by V. anguillarum infection was significantly decreased; this is promising since the main causative agent for Vibriosis, which is a prevalent disease in ayu culture in China, is V. anguillarum (Li et al., 2015).

EeCentrocin 1 (GWWRRTVDKVRNAGRKVAGFASKACGALGH), EeCentrocin 2 (WGHKLRSSWNKVKHAVKKGAGYASGACRVLGH) and EeStrongylocin 2 (WNPFKKIAHRHCYPKNECITTNGKKTCKDYSCCQIVLFGKKTRSACTVVAQ) are peptides isolated from the edible sea urchin Echinus esculentus. EeCentrocin 1 is potent against the Gram-positive bacteria; C. glutamicum (MIC = 0.78 µM) and S. aureus (MIC = 0.78 µM), and against the Gram-negative bacteria, E. coli (MIC = 0.1 µM) and P. aeruginosa (MIC = 0.78 µM), whereas the MIC of EeStrongylocin 2 was found to range from 0.78 to 3.13 µM (Solstad et al., 2016).

From the large yellow croaker (Larimichthys crocea) which is a marine fish in China, Lc-NK-lysin was extracted with the amino acid sequence: MNSSSVLFVCILGACSVWTVHGRNLKVNDDDQEG AELDISVEARKLPGLCWV CKWSLNKVKKLLGRNTTAESVKEKLMRVCNEI GLLKSLCKKFVKGHLGELIEELTTSDDVRTICVN LKACKPKELSELDFESDEDAHTEMNDLLFE. Lc-NK-lysin mature peptide was divided into Lc-NKlysin-1 and Lc-NK-lysin-2, and both of these two peptides showed antibacterial activities. The Lc-NK-lysin-1 peptide exerted activity that could inhibit and kill S. aureus (MIC = 12–24 mM) and V. harveyi (MIC = 12–24 mM). In addition, it could inhibit but could not kill B. subtilis (MIC = 24–48 mM) and E. coli (MIC = 12–24 mM). In contrast, even the highest concentration of Lc-NK-lysin-1 peptide could not affect V. parahaemolyticus, A. hydrophila, and P. damselae. For the Lc-NK-lysin-2 peptide, it exerted activities against S. aureus (MIC = 12–24 mM) and E. coli (MIC = 24–48 mM) (Zhou et al., 2016).

WB Piscidin 5 (LIGSLFRGAKAIFRGARQGWRSHKAVSRYRARYVRRPVIYYHRVYP) is a host defense peptide found in the white bass Morone chrysops. It was chemically synthesized and its antibacterial activity was tested. WB Piscidin 5 exhibited activity against E. faecalis (MIC = 4.52 mM), S. aureus (MIC = 4.52 mM), Shigella flexneri (MIC = 1.13–2.26 mM), and E. coli (MIC = 1.13 mM) (Salger et al., 2016).

Lysozyme from the seahorse Hippocampus abdominalis can catalyze the hydrolysis of the bacterial cell wall, making it a good antibacterial agent in the innate immune system. ShLysG is a polypeptide obtained from H. abdominalis containing 184 amino acids (MGYGDIMKVDTSGASMKTAGQDRLT YAGVAASNTMAQTD LGRMNNYKAIIQRVGGKKD VDPAIIAGIISRESRAGNVLVNGWGDNGNAWG LMQVDKRYHTPQGGWNSEEHLSQGTDII SFIKQVQGKFPSWTAEQQLKGGIAAYNIGLGGV QTYERMDVGTTGDDYSSDVVARAQWYKSQGGF). It has a molecular mass of 20,000 Da. The conserved catalytic bacterial soluble lytic transglycosylase domain in ShLysG contains three catalytic residues and seven N-acetyl-D-glucosamine binding sites. This domain is crucial for the hydrolysis of the b-1,4-glycosidic bonds between the N-acetylmuramic acid andN-acetylglucosamine of peptidoglycan in the bacterial cell wall. X-ray crystal structure reported that Glu73 and two aspartic acid residues (Asp90 and Asp101) are also directly associated with the catalytic reaction. rShLysG presented antimicrobial activity towards the five bacterial strains; V. salmonicida, V. parahemolyticus, L. monocytogenes, S. iniae, and V. anguillarum (Ko et al., 2016).

A set of Histone H2A fragments were isolated from the Russian sturgeon (Acipenser gueldenstaedtii). They were called acipensins1, 2, 6 (Ac1, Ac2, Ac6). The peptides Ac1 and Ac2 displayed potent antimicrobial activity towards Gram-negative and Gram-positive bacteria, while Ac6 was only effective against Gram-negative bacteria. Ac1 (SGRGKTGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAQRVGAGAPVY) presented MIC = 0.7 mM against E. coli ML35p, MIC = 1.1 mM against Listeria monocytogenes EGD, and MIC = 0.9 mM against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591. Meanwhile, Ac2 (SGRGKTGGKARAKAKTRSSRAGLQFPVGRV HRLLR) presented MIC of 0.3 mM against E. coli, MIC = 1 mM against L. monocytogenes, and MIC = 0.6 Mm against MRSA. Finally, Ac6 (ILELAGNAARDNKKTRIIPRHLQL) had activity only against E. coli with MIC = 2.5 mM. All three peptides were devoid of hemolytic activity against human erythrocytes at concentrations of 1–40 lM and at concentrations 1–20 mM they did not exert toxic effects on the cells (Shamova et al., 2014).

SJGAP is another AMP (VKVGINGFGRIGRLVTRAAFHGKKVEIVAIND) obtained from skin extract of skipjack tuna (Katsuwonus pelamis). It exhibited potent antimicrobial activity against Gram-positive bacteria, such as B. subtilis, M. luteus, S. aureus, and S. iniae (MEC = 1.2–17.0 mg/mL), and Gram-negative bacteria, such as A. hydrophila, E. coli D31, and V. parahaemolyticus (MEC = 3.1–1 2.0 mg/mL). SJGAP consists of one a helix and two parallel bstrands, and it forms an amphipathic structure. Also, it acts through bacteriostatic process rather than bactericidal one since it displays a small degree of leakage ability (Seo et al., 2014b).

LEAP-2 is an important molecule of miiuy croaker’s innate immune system as it provides resistance against bacterial infections. It was found that LEAP-2 (MTPLWRIMNSKPFGAYCQNNYECSTGLCRAGHCSTSHRATSETVNY) has a molecular mass of 5000 Da and its instability index is 34.74, which means that the polypeptide is a stable protein. It was effective in controlling A. hydrophila showing bacteriostatic diameter of 10 mm (Liu et al., 2014).

Hemocyanins are multifunctional proteins that can be found in invertebrates including molluscs. Zhuang et al. (2015) identified haliotisin, a hemocyanin-derived antimicrobial peptide from the mollusk Haliotis tuberculate. Haliotisin peptide 3-4-5 (DTFDYKKFGYRYDSLELEGRSISR IDELIQQRQEKDRTFAGFLLKGFGTSAS) exhibited antibacterial activities. Peptide 3 displayed MIC = 0.3–1 mM against B. subtilis, while peptide 4 displayed MIC = 1.6–2.6 mM against E. carotovor, in addition to peptide 5 which showed 1.2–1.5 mM against E. carotovar. The novel 55 amino acid peptide cgMolluscidin (AATAKKGAKKADAPAKPKKATKPKSPK KAAKKAGAKKGVKRAGKKGAKKTTKAKK) with a molecular weight of 5500 Da, extracted from the pacific oyster Crassostrea giga, exerted potent antimicrobial activity against both Gram-positive bacteria including B. subtilis, M. luteus, and S. aureus (MEC = 1.3–31.3 mg/mL), and Gram-negative bacteria including E. coli, S. enterica, and V. parahaemolyticus (MEC = 0.4–2.3 mg/mL) (Seo et al., 2013a,b).

PdBD-2 (YDTGIQGWTCGSRGLCRKHCYAQEHTVGYHGCPRRYRC CALRF) was identified from the Chinese loach fish, Paramisgurnus dabryanus and it significantly inhibited the growth of the Gram negative bacteria A. hydrophila and the Gram-positive B. subtilis (Chen et al., 2013a).

From the pacific oyster Crassostrea gigas, the 74 amino acid cgUbiquitin (MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIP PDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLR) was identified, having a molecular weight of 8471 Da. It is composed of three a-helices and four b-strands separated by 7 loop regions. It is active against both Gram-positive and negative bacteria including S. iniae (MEC = 7.8 mg/mL) and V. parahemolyticus (MEC = 9.8 mg/mL) without causing hemolysis to human red blood cells up to 100 mg/mL. Moreover, according to the kinetics of killing and membrane permeabilization studies, it was illustrated that cgUbiquitin was not membrane permeable and acted through a bacteriostatic process (Seo et al.,2013a,b).

Chionodracine (FFGHLYRGITSVVKHVHGLLSG), isolated from the icefish species Chionodracohamatus, exhibited significant effect against Antarctic psychrophilic bacteria strains Psychrobacter sp. TAD1 (MIC = 10 mM) and TA144 (MIC = 15 mM at 15°C), the Gram positive B. cereus (MIC = 5 mM at 25_C), and the Gram-negative E. coli (MIC = 5 mM at 25_C). However, when the activity of the peptide was tested at 37 _C against E. coli and B. cereus the MIC levels increased to 20 mM and 10 mM, respectively. Buonocore et al. suggested that this could be an indication that chionodracine is adapted to low temperatures or that there was a conformational change in the bacterial membrane due to the lower temperature, leading to higher susceptibility to the peptide. A test was done in vitro on human erythrocytes and no significant lysis occurred with the use of Chionodracine until the concentration reached 50 mM (Buonocore et al., 2012).

The NZ17074 (N1) is a marine peptides derived from the marine invertebrate lugworm Arenicola marina. It is characterized by disulfide bonds. It was proven to show a potent antibacterial activity against Gram-negative bacteria, antifungal and cytotoxicity. However, to reduce its cytotoxicity and to improve its physicochemical properties, varieties of analogues were prepared including, N2, N3, N4, N5, N6 and N7 by structural modification of the natural peptide N1 through changing the number and locations of the disulfide bonds. The NMR spectral analysis identified four different classes of peptides designed based on structural determinants: (I) Rocket analogues with two disulfide bonds (N2: Gly1, 12Ala; N3: Trp4, Asn5Ala; N4: Trp0). (II) Kite analogue with a Cys7-Cys16 disulfide bond (N6: Cys3,20Ala). (III) Bullet analogue with a Cys3-Cys20 disulfide bond (N7: Cys7,16Ala). (IV) Other disulfide bond-free linear analogues (N5: DCys3,7,16,20; N8: Cys3,7,16,20 Ala) (Harwing et al., 1996; Yang et al., 2017). The four classes displayed in vitro activity against a wide range of microorganisms including Gram-negative and Gram-positive bacteria, and fungi. The Rocket N2 analogue, with two disulfide bonds, showed the strongest antimicrobial activity against Gram-negative bacteria with MIC values ranging from 0.25 to 1 mg/ml against E. coli, Salmonella, and Pseudomonas strains and lower cytotoxicity compared to other analogues (Wang et al., 2017). N2 and N6 analogues were less cytotoxic against RAW 264.7 macrophages, compared with N1. Both N2 and N6 analogues induced cell cycle arrest in I- and Rphases, respectively, in E. coli and S. enteritidis. In E. coli and in S. enteritidis, both peptides inhibited DNA/RNA/cell wall synthesis by 18.7–43.8% and 5.7–61.8%, respectively. They also modified the morphology of E. coli cells to become collapsed and filamentous after treatment with the two peptides. The survival rate of peritonitisand endotoxemia-induced mice was prolonged, the serum levels of interleukin-6 (IL-6), IL-1b, and tumor necrosis factor a (TNF-a) decreased following doses ranging from 2.5 to 7.5 mg/kg body weight. These peptides prevented lipopolysaccharide (LPS)-induced lung injury in mice (Harwing et al., 1996; Yang et al., 2017).

Several bacterial flora were isolated from marine ecosystem (Bacillus subtilis, B. amyloliquifaciens, Pseudomonas putida and P. aeroginosa) with potential activities (> 20 mm inhibition zone) against pathogenic Vibrios (Chakraborty et al., 2010). The antibacterial component in the CHCl3 fraction of P. aerogenosa was found to be N-substituted methyl-octahydro-1-phenazinecarboxylate. The other important antibacterial molecules were found to be propyl 2-oxoacetate and phenethyl 2-oxoacetate. About 4530 bacterial isolates were purified from seaweeds and sediments, and 23 isolates (B. subtilis MTCC 10402, 10403 and 10407, B. amyloliquifaciens 10456, P. putida MTCC 10458, P. aeroginosa MTCC 10610) were found to be potential against pathogenic Vibrios. N-substituted phenazinecarboxylate, propyl/phenethyl 2-oxoacetates were the major antibacterial molecules in bacteria.

Screening and development of aquaculture-grade chemicals from bacterial flora could be a highly promising approach to produce these bioactive molecules. Members of the genus Pseudomonas and Bacillus either free living or associated with marine flora are common beneficial bacterial candidates, and are known to produce a wide range of secondary metabolites (Raaijmakers et al., 1997) inhibiting a wide range of pathogenic bacteria (Rengpipat et al., 998). The metabolites 6-oxo-de-O-methyllasiodiplodin, (E)-9-ethenolasiodiplodin, lasiodiplodin, de-O-methyllasiodiplodin, and 5-hydroxy-de-Omethyllasiodiplodin, were isolated from the mycelium extracts of a microbe obtained from South China Sea (Yang et al., 2006). Bioactive compounds were isolated from a marine bacterium Bacillus circulans (Chakraborty et al., 2010). Labda-14-ene-3a,8a-diol and labda-14-ene-8a-hydroxy-3-one were found to be inhibitory to the growth of Vibrio parahaemolyticus with minimum inhibitory concentrations of 30-40 μg/mL (Chakraborty et al., 2010), and their structures have been elucidated by 1H NMR and 13C NMR spectra, including 2D NMR. Several bacterial flora were isolated from marine ecosystem (Bacillus subtilis, B. amyloliquifaciens, Pseudomonas putida and P. aeroginosa) with potential activities (> 20 mm inhibition zone) against pathogenic Vibrios (Chakraborty et al., 2010). The antibacterial component in the CHCl3 fraction of P. aerogenosa was found to be N-substituted methyl-octahydro-1-phenazinecarboxylate. The other important antibacterial molecules were found to be propyl 2-oxoacetate and phenethyl 2-oxoacetate. About 4530 bacterial isolates were purified from seaweeds and sediments, and 23 isolates (B. subtilis MTCC 10402, 10403 and 10407, B. amyloliquifaciens 10456, P. putida MTCC 10458, P. aeroginosa MTCC 10610) were found to be potential against pathogenic Vibrios. Nsubstituted phenazinecarboxylate, propyl/phenethyl 2-oxoacetates were the major antibacterial molecules in bacteria.

The ability of marine microorganisms to produce novel antimicrobial compounds has been well demonstrated, and clearly they have a future role in the fight against antibiotic resistant pathogens. Ongoing research efforts to isolate and screen new marine microorganism species should be accompanied by efforts to understand their ecology. Extensive culture-dependent and -independent surveys of marine microorganisms should be prioritized to determine the extent to which marine diversity differs, e.g. is the isolation of rare microorganisms’ genera from the sea merely due to the fact that terrestrial-to-sea input skews the species distribution. The isolation of seawater-obligate microorganisms has proved that marine adaptation has occurred in this lineage, but so far this property has only been identified at the genus and species level, an indication that marine adaptation is a comparatively recent evolutionary event. If such adaptation is rare within the microorganisms, it is reasonable to expect that seawater-obligate strains will represent species that have no terrestrial counterparts, and thus they are unlikely to have been previously screened for antimicrobial compounds. This raises the intriguing possibility that there are antimicrobial compounds unique to marine species. Whole-genome analysis of the genus Salinispora indicates hat differences in secondary metabolite biosynthetic genes may be a driver of speciation, supporting the hypothesis that new species will produce new compounds. Further analysis is needed to determine whether this property will hold as more species are described. Finally, if antimicrobial compounds are to make it from the ocean to the clinic, big pharma must re-engage in drug discovery from microbes. Currently, small pharmaceutical and biotechnology companies have been, or are currently engaged in antimicrobial discovery from marine microorganisms.

The study conducted by Phan et al., (2018) has revealed the presence of high numbers of marine fungi from Nha Trang waters having antimicrobial activity against the human microbial pathogens. Among the 100 isolates, 59 strains exhibited antimicrobial activity against at least two tested pathogens, that 57% against S. aureus, 50% against L. monocytogenes, 49% against B. cereus, 45% against S. faecalis, 7% against E. coli, 5% against C. albicans,and only 2% against P. aeruginosa.

The review conducted by Youssef et al. (2019) was summarize the chemistry and the biological activities of peptides that were isolated and structurally elucidated from marine fungi. Relevant fungal genera including Acremonium, Ascotricha, Aspergillus, Asteromyces, Ceratodictyon, Clonostachys, Emericella, Exserohilum, Microsporum, Metarrhizium, Penicillium, Scytalidium, Simplicillium, Stachylidium, Talaromyces, Trichoderma, as well as Zygosporium were extensively reviewed. About 131 peptides were reported from these 17 genera and their structures were unambiguously determined using 1D and 2D NMR (one and two dimensional nuclear magnetic resonance) techniques in addition to HRMS (high resolution mass spectrometry). Marfey and Mosher reactions were used to confirm the identity of these compounds. About 53% of the isolated peptides exhibited cytotoxic, antimicrobial, and antiviral activity, meanwhile, few of them showed antidiabetic, lipid lowering, and anti-inflammatory activity. However 47% of the isolated peptides showed no activity with respect to the examined biological activity and thus required further in depth biological assessment. In conclusion, when searching for bioactive natural products, it is worth exploring more peptides of fungal origin and assessing their biological activities (Youssef et al., 2019).

One domain of organisms, the Archaea, containing hyperthermophiles, extreme halophiles and the methanogens, is just beginning to be scrutinized for the production of peptide antibiotics. Production of archaeal proteinaceous antimicrobials (archaeocins) from extreme halophiles (halocins) is a nearly universal feature of the rod-shaped haloarchaea. Halocin activity is first detectable in culture supernatants at the beginning of the transition into stationary phase, concomitant with an induction of transcription of the structural gene. Halocins are diverse in size, consisting of proteins as large as 35 kDa and peptide ‘‘microhalocins’’ as small as 3.6 kDa. The 36 amino acids of microhalocin HalS8 are located in the interior of a 311-residue pro-protein from which they are liberated by an unknown mechanism (O’Connor and Shand, 2002).

In the study conducted by Villegas et al. (2020), they assayed the antimicrobial activity of two new haloarchaeal strains against a collection of typical fish (Streptococcus parauberis DSM 6631T, Lactococcus garvieae CECT 4531T, Tenacibaculum maritimum CECT 4276, Tenacibaculum soleae CECT 7292T, Pseudomonas anguilliseptica CECT 899T, Pseudomonas moraviensis DSM 16007T, Pseudomonas plecoglossicida DSM 15088T, Edwardsiella tarda CECT 849T, Edwardsiella tarda CECT 849T, Vibrio anguillarum CECT 522T, Vibrio harveyi CECT 525T, Vibrio tapetis CECT 4600T, Aeromonas salmonicida salmonicida CECT 894T, Pseudomonas baetica a390T, Mycobacterium marinum CECT 7091T, Yersinia ruckeri CECT 4319T, Photobacterium damsela damselae CECT 626T) and human pathogenic bacteria (Micrococcus luteus CECT 245, Bacillus cereus CECT 40 and Staphylococcus aureus), against representative microalgae and yeast species and over other haloarchaea typically found in hypersaline environments, by using the agar diffusion method. This screening revealed that only the acetone extracts showed considerable antimicrobial activity. Acetone extracts from the two tested haloarchaeal strains, H. hispanica HM1 and H. salinarum HM2, showed to be active against bacteria, microalgae, and archaea, but not on yeasts. Zones of inhibition, with diameters which varied according to the susceptibility of the target microorganism from 0.5 to 6 cm, were used as indicators of antimicrobial activity. Both acetone extracts inhibited the growth of all the human pathogenic bacteria assayed and of most fish pathogenic bacteria studied, with inhibition halos ranging from 0.5 to 2 cm. In addition, both extracts exhibited important antimicrobial activity against microalgae, which was especially pronounced over halophilic microalgae from the Dunaliella genus, with inhibition halos sized from 2 to 6 cm. Moreover, high activity was found against all the tested extreme halophilic archaea and bacteria, with inhibition zones from 0.5 to 4 cm (Villegas et al., 2020).

There is a lot of information about antimicrobial activity of the total extractive substances of marine seaweeds. In the study conducted by Chingizova et al., 2017, they tested three type of algal extracts (total extracts and their hydrophilic and lipophilic fraction) against gram-positive bacteria S. aureus, gram-negative bacteria Escherichia fungi Candida albicans. The results of this survey did not show clear taxonomic trends in the activities of the crude extracts as well as their hydrophilic and lipophilic fractions against the tested microorganisms, although Rhodophyta extracts showed highest inhibitory effect against fungi among all algae tested. Antimicrobial activities of extracts varied considerably among all studied algal species, and between assayed microorganisms, suggesting that microbial growth inhibition is mediated by a variety of antimicrobial metabolites. Overall, fewer extracts were active against fungi than gram-positive and gram-negative bacteria. In addition, larger more lipophilic than hydrophilic extracts inhibited the growth of all studied microbes and fungi indicating that liphophilic secondary metabolites may play a critical role in determination of antimicrobial properties of the algal extracts. Neorhodomela larixsubsp. aculeata, Fucus evanescens, Costaria costata, Saccharina cichorioides, S. japonica,Sargassum pallidumand Scytosiphon lomentaria appear to be the most promising species for further investigation, as they have a broad spectrum of biological activity and so is likely to yield more than one active compound. These bioactive compounds will need further studies to identify the chemical structure of these active compounds and to examine their beneficial effect for inhibition of some pathogenic bacteria and fungi.

In addition, the natural antimicrobials derived from marine algae offer shelf-life extension and increased safety from bacteria that cause food poisoning, without the side effects associated with many synthetic preservatives or high salt intake. Gupta et al. (2010) evaluated the antibacterial activity of three edible Irish brown seaweeds, Himanthalia elongata, Saccharina latissima, and Laminaria digitata in raw and heat processed (95°C) form. Their activity was tested against pathogens which commonly cause problems in the food industry, Listeria monocytogenes, Salmonella abony, Enterococcus faecalis, and Pseudomonas aeruginosa. In microtiter assays, methanol extracts of raw Himanthalia elongata (60 mg/mL) inhibited Listeria monocytogenes by 98.7%, compared to 96.5% inhibition by the synthetic preservative standard sodium benzoate, and sodium nitrite (96.2%). Raw Himanthalia elongata was also more potent than the standards against Enterococcus faecalis and Pseudomonas aeruginosa. Raw Saccharina latissima extracts were almost as potent against all four bacteria, followed by Laminaria digitata. Heat treatment significantly reduced the antibacterial activity of all seaweeds. These raw seaweed extracts may be useful for incorporation into products such as raw meats and fish, as they would exert their bacterial inhibition during the uncooked, cold storage phase, before inactivation by heat, at which stage the product would be consumed. A recent study by Dussault et al. (2016) explored the potential of developing several commonly consumed Pacific Island seaweeds into food preservation agents. In broth dilution assays, methanol extracts of the brown species, Padina and Dictyota, were found to inhibit the growth of Gram-positive foodborne pathogens Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus at low concentrations (≼500 µg/mL). However, the extracts had no activity against Gram-negative species, possibly due to the inability of the moderate to low polarity, hydrophobic extracts to breach the hydrophilic lipopolysaccharide, Gram-negative bacterial membrane. The extracts are not known to have any toxicity at the concentrations used, making them good candidates for incorporation into foods prone to Gram-positive bacterial growth.

The ability of marine microorganisms to produce novel antimicrobial compounds has been well demonstrated, and clearly they have a future role in the fight against antibiotic resistant pathogens. Ongoing research efforts to isolate and screen new marine microorganism species should be accompanied by efforts to understand their ecology. Extensive culture-dependent and -independent surveys of marine microorganisms should be prioritized to determine the extent to which marine diversity differs, e.g. is the isolation of rare microorganisms’ genera from the sea merely due to the fact that terrestrial-to-sea input skews the species distribution.

Challenges in the use of AMPs as drugs

Many reports describe the potential role of AMPs as antimicrobial agents and the possibility to exploit them to solve the problem of resistance. In particular, although there is a wealth of information on their activities in vitro, there are considerable challenges for their clinical application. These include doubts on the ability to achieve high antimicrobial activities under physiological salt, pH, and serum conditions; the rapid degradation by proteases; the poor oral availability; the difficult transportation across cell membranes; the non-selective receptor binding; the lack of information about potential toxicities in vivo; and the challenging multistep preparations and consequently high costs associated with their production (Avan et al., 2014).

Moreover, AMPs often lack efficacy compared to conventional antibiotics. One possible strategy that has been exploited to improve the antimicrobial efficacy and reduce the toxicity is the combined use of two or more molecules. For example, colistin and bacteriocin have been used together to attain a synergistic effect and overcome some defects. Colistin is a polypeptide antibiotic which was withdrawn because of its toxicity, but when used in combination with bacteriocidin it was effective at lower concentrations. Rational design has gained great importance and represents a major revolution in the area of development of AMPs which are more active, less cytotoxic and possible to produce on an industrial scale. Bioinformatic and biophysical studies aimed at identification of physicochemical features such as appropriate hydrophobicity, charge, amphipathic structural arrangement, and amphipathicity have acquired a key role. Natural AMPs may serve as templates for the design of new antibacterial agents.

Thus, peptidomimetics, which structurally mimic the key binding elements of the native peptide and retain the ability to interact with the biological target and produce the same biological effect, offer a strategy to overcome the issues correlated to the use of peptides in clinical therapeutics (Avan et al., 2014). In particular, to develop improved AMPs by structural modification, it is essential to understand the structure of native AMPs and to focus on regions responsible for activity. AMPs can be optimized to enhance their effectiveness and stability through modification of their primary sequences in order to obtain good templates for the development of therapeutic agents. Studies of AMP activity have often included the systematic change of amino acids or other chemical modifications which allow the obtainment of higher activities such as: chemical modification of terminal ends of peptides (Danial et al., 2012), development of analogues containing unnatural amino acids (Papo et al., 2002), shortening of the native sequence, modifications of their amphipathic character, cyclization. The appropriate balance of hydrophobicity, amphipathicity, and positive charge plays a pivotal role for the enhancement of their therapeutic potential. The reduction in the hydrophobicity determines a reduction of mammalian cell interactions while favoring the targeting of bacterial cell membranes, as long as the peptide has sufficient positive charge. Surface immobilization of AMPs also represents an attractive contact-killing technique which could further enhance AMP stability, broad range of action, and the low likelihood for the development of microbial resistance, reducing leaching, proteolysis, and cytotoxicity (Costa et al., 2002). Several authors have described modification of native sequences to dissect the mechanism of antibacterial activity modulating changes in hydrophobicity, length, and net charge of the peptides.

Chemical modification of marine AMPs 

Synthetic and modified AMPs derived from marine peptides can sustain physiological salt concentration and protease activity. The collection of post-translational modifications exploited by marine AMPs may help in the design of AMPs with enhanced stability and efficacy, for therapeutic applications in humans. In particular, the high salinity (up to 600 mM) of the marine environment makes it likely that marine AMPs naturally possess greater salt resistance than those derived from other sources, which may allow them to keep their biological activities in relatively high-salt environments, such as in saliva, gastrointestinal fluid, serum, or other body fluids. Marine AMPs undergo various post-translational modifications, which play a key role in the survival of marine organisms; most of them are required to induce proper folding into structural scaffolds that are necessary for the interactions with target bacterial surfaces and their membranes and to provide the necessary stability (Wang, 2012).

Post-translational modifications include: disulfide bonds, bromination, chlorination, C-terminal amidation, high content of specific amino acids (such as phenylalanine, arginines), modification of single amino acids (such as 3-methylisoleucine), presence of fatty acid linked to the peptide sequence, and presence of D-amino acids. Some are specific to marine AMPs and other modifications are shared with terrestrial AMPs. Bromination is observed in cathelicidins and protects the peptide from proteases in marine environment, as also evidenced by the absence of bromination of cathelicidins derived from terrestrial mammals (Shinnar et al., 2003).

Callinectin occurs in three different form which vary according to the modification present on the tryptophan residue (Noga et al., 2011). Modified tryptophans have been found in AMPs from a number of aquatic animals. The most frequent modification is bromination (bromotryptophan). Strongylocins contain a bromotryptophan residue in their sequence which makes them less susceptible to protease digestion, which is a key feature of AMPs with biotechnological potential (Li et al., 2008). The presence of bromotryptophan is associated with increased resistance to proteolysis. A hydroxylated tryptophan is present in MGD-2, an arthropod defensin from Mytilus galloprovincialis (Yang et al., 2000) and in piscidin 4 (Noga et al., 2009). The presence of the modified tryptophan is in some cases needed for full expression of antimicrobial activity; in fact, MGD-2 without tryptophan hydroxylation presents reduced activity against certain Gram-negative bacteria (Li et al., 2008).

Styelin contains unusual amino acids such as dihydroxyarginine, dihydroxylysine, 6-bromotryptophan, and 3,4-dihydroxyphenylalanine which are important for the antimicrobial activity at high salt concentrations (Taylor et al., 2008). Polydiscamide A contains a 3-methylisoleucine. Halicylindronides show a N-terminus blocked by a formyl group and a further lactonized C-terminal threonine. This modification provides proteolytic resistance and is sometimes found as an alternative to C-terminal amidation. As stated, the interaction of cationic peptides with anionic membranes represent the initial step of the antibacterial mechanism, thus AMP activity may be compromised by the presence of high salt concentrations. Pleurocidin can actively kill bacteria up to 625 mM NaCl (Cole et al., 1997); another AMP derived from the marine invertebrate Ciona intestinalis can resist salt concentrations up to 450 mM (Fedders et al., 2008).

Marine AMPs have evolved to adapt to the high salt concentration in sea water and this has probably been achieved by the substitution of lysines with arginines. Understanding the chemical reason that supports this salt independent activity could aid in the design of novel AMPs that could address pathogens under a wide range of normal and abnormal salt concentrations. In fact, human beta defensin-1 (hBD-1) is unable to inhibit P. aeruginosa due to a 120 mM concentration of NaCl in the lungs of cystic fibrosis patients (Goldman et al., 1997); while defensin-3 (hBD-3) shows antibacterial activity also at high salt concentrations (Scudiero et al., 2010; Scudiero et al., 2013). As a matter of fact, hBD-3 contains a C-terminal domain rich in arginine residues which has been demonstrated to be involved in the activity at high ionic strength through the synthesis of chimeric peptides obtained from hBD-1 and hBD-3 (Scudiero et al., 2010; Scudiero et al., 2013; Scudiero et al., 2015). Therefore, the understanding of the general rules underlining the ability of an AMP to inhibit microorganisms under physiological salt concentrations (120–150 mM) is a significant aspect in the success of an AMP under in vivo studies.

Rational modification of the amino acid sequence of mixinidin further supported the key role played by an appropriate balance of hydrophobicity, amphipathicity, and positive charge in helical peptides for the enhancement of their therapeutic potential. The highest activity was obtained for the WMR peptide, which contains an extra tryptophan residue and three arginines (His3, Asp4, and Pro11 were substituted with arginines). The simultaneous substitutions of residues present in positions 3, 4, and 11 with Arg and the addition of the Trp at the N-terminus result in a significant increase in activity (Cantisani et al., 2013; Cantisani et al., 2014).

Mai et al. (Mai et al., 2011) designed a small, target-specific, salt-resistant AMP that selectively killed S. mutans, combining the targeting domain of the S. mutans (ComC signaling peptide) to an active portion of pleurocidin which was highly effective against all S. mutans strains. The obtained molecule was capable of retaining antibacterial activity in physiological or even higher concentrations of salt compared to its native peptide. Interestingly, the molecule was also able to exert its antibacterial activity at low pH, (pH 5.5 to 6.0) though with a 20% to 25% lower activity than that at pH 7.5.

Cardoso et al. (Cardoso et al., 2016) designed a novel polyalanine-rich cationic peptide, named Pa-MAP 1.9, which was rationally designed based on the HPLC-8 peptide derived from the polar fish Pleuronectes americanus. They proved that the peptide is a valuable candidate for the treatment of gram-negative bacterial infections especially in their biofilm state; moreover, it is not chemolytic and cytotoxic against mammalian cells. The interaction with the bacterial membrane is favored by the adoption of an -helical conformation which helps the orientation and insertion into lipid bilayers mimicking the bacterial membrane.

Conclusion

Nature is a rich and still-undisclosed source of bioactive molecules for developing novel drugs against serious bacterial pathogens and to contrast the rapid emergence of multi drug resistance. This review article provides a brief description of marine AMP scaffolds amenable to the development of therapeutic candidates. In particular, marine microorganisms, which usually experience extreme and stressful environments, are becoming a rich source of templates for the design of novel AMPs which could be developed into effective drugs for human and veterinary medicine. The structure of marine AMPs may be exploited to achieve novel sequences with greater activity and stability in high ionic strength environments because marine organisms are constantly under an enormous microbial challenge from the ocean environment, which is also continuously altered by industrialization and transportation.

The need to produce novel molecules able to reduce the possibility of developing resistance has enhanced the interest toward molecules which target the cell membrane for which no resistance has been reported (Galdiero et al., 2015; Cantisani et al., 2012; Franci et al., 2015; Galdiero et al., 2003; Notomista et al., 2015; Boman Boman, 2003; Bradshaw, 2003). Thus, the difficulties in accomplishing resistance against AMPs by the pathogens make them potential substitutes for antibiotics and marine AMPs present us with a vast resource of biomolecules for assessing novel therapeutics as antimicrobials. Moreover, use of combinatorial therapeutics should be promising in the future. Marine peptides have also a high potential for the nutraceutical and medicinal industry and some of them are already on the market or in different phases of the clinical and preclinical pipeline (Cheung et al., 2015).

Treatment of MRSA-infected mice with epenecidin-1(Pan et al., 2007) allowed mice to survive by considerably decreasing the bacterial counts with further evidence of wound closure and angiogenesis enhancement (Huang et al., 2013). Epinecidin-1 is a promising candidate for topical application against vaginal or skin infections and to have a synergistic effect with commercial cleaning solutions. Moreover, its activity was not influenced by low pH or storing at room temperature and at 4°C for up to 14 days (Pan et al., 2010). Interestingly, chrysophsin-1 has been used to create antimicrobial surfaces capable of killing around 82% of E. coli bacteria (Ivanov et al., 2012).

Moreover, bacterial infections are rarely unique and occur as polymicrobial communities (biofilms). The anti-biofilm activity against Candida albicans, P. aeruginosa, and Bacillus pumilus of a small peptide isolated from the marine Bacillus liqueniformis D1 has been reported (Dusane et al., 2011). This peptide is also able to inhibit preformed biofilms at concentrations equivalent to MIC for planktonic cells, a feature which plays essential key role in fighting chronic established infections.

Bioactive compounds from the marine environment possess properties of biomedical importance, which make them attractive templates for rational design strategies of new drugs and pharmaceuticals with huge biotechnological and pharmaceutical potential. New technologies, close collaboration between researchers of various fields and increasing economic support will be key to the development of marine peptides as novel therapeutics.

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