Mammalian skin, organ epithelium, blood, and saliva store different cellular and molecular components, including AMPs, which provide a defense mechanism against potential pathogens [82]. Mammalian-derived peptides have great potential to combat bacterial infections caused by resistant strains of S. aureus, including VRSA and VISA strains (Table 2). Cathelicidins are among the best-known mammalian-derived AMPs and have strong antibacterial activity [83]. There are different cathelicidins identified in many mammalian species, among which the LL-37 peptide stands out [41]. This AMP is a human cathelicidin identified in neutrophils that has shown broad-spectrum in vitro activity against virus and Gram-negative and Gram-positive bacteria, including VRSA and VISA strains (MIC = 64 μg/mL), as well as a low cytotoxic effect [41][84][85]. LL-13 and LL-17 are shorter peptides, derived from fragments of the canonical sequence of LL-37, which showed activity against VRSA strains [41]. Both LL-13 and LL-17 showed high inhibitory concentrations against VRSA and VISA strains compared to the canonical LL-37 peptide (Table 2) [41].
Table 2. Animal-derived AMPs with antibacterial activity against VRSA and VISA strains.
Source |
AMP Name |
Strain ID |
MIC Value |
Reference |
Toxicity/Properties |
Apis mellifera |
Melittin |
VISA-9 |
2 μM |
[50] |
High toxicity to erythrocytes and other human cells |
Mellitin analog |
Hec |
VRSA-4 |
80 μM |
[35] |
Moderate toxic effect at high concentrations |
Musca domestica |
Formicin C |
VRSA-27 |
[ |
41 |
] |
– |
Mammalian skin, organ epithelium, blood, and saliva store different cellular and molecular components, including AMPs, which provide a defense mechanism against potential pathogens [101]. Mammalian-derived peptides have great potential to combat bacterial infections caused by resistant strains of S. aureus, including VRSA and VISA strains (Table 2). Cathelicidins are among the best-known mammalian-derived AMPs and have strong antibacterial activity [102]. There are different cathelicidins identified in many mammalian species, among which the LL-37 peptide stands out [41]. This AMP is a human cathelicidin identified in neutrophils that has shown broad-spectrum in vitro activity against virus and Gram-negative and Gram-positive bacteria, including VRSA and VISA strains (MIC = 64 μg/mL), as well as a low cytotoxic effect [41,103,104]. LL-13 and LL-17 are shorter peptides, derived from fragments of the canonical sequence of LL-37, which showed activity against VRSA strains [41]. Both LL-13 and LL-17 showed high inhibitory concentrations against VRSA and VISA strains compared to the canonical LL-37 peptide (Table 2) [41].
3.1.2. Bacteria-Derived AMPs
Diverse interactions occur naturally between bacterial species sharing the same habitat, which are determined by the nutritional resources available [86]. Through various mechanisms, such as the production of toxic molecules or compounds, many bacterial species can favor their own survival and evolution, affecting other bacterial species they live with [86]. One of these mechanisms involves peptides that may be naturally produced by some bacterial species to control the survival of other bacteria [86]. Due to their strong effect, some bacterial-derived AMPs have been evaluated as alternatives to control Gram-positive bacteria resistant to conventional antibiotics [87]. In this regard, numerous AMPs identified in bacteria have shown promising characteristics against resistant VRSA and VISA strains (Table 3). Depending on the biosynthetic route they use, bacterial-derived peptides can be classified into two groups: (1) ribosomally synthesized peptides such as bacteriocins and (2) non-ribosomal peptides, such as bacitracins and glycopeptides [88]. Bacteriocins are a group of AMPs with a wide variety in size, structure, and mode of action [89]. Bacteriocins derived from Gram-positive bacteria can be grouped into four different classes: (I) lantibiotics, (II) non-lantibiotics, (III) large peptides, and (IV) bacteriocins containing lipids or carbohydrates [89]. Within the lantibiotics, two subclasses are identified: subclass Ia, which includes AMPs such as nisin, hominicin, and mutancin 1140, and subclass Ib, which includes mersacidin [89]. One of the best known bacteriocins is nisin derived from Lactococcus lactis [88]. This AMP has a strong antimicrobial effect, and according to in vitro assays, it showed activity against VISA strains, with MIC between 4.1 and 8.3 μg/mL, and a slight hemolytic effect against sheep erythrocytes [53][90]. Similarly, hominicin produced by Staphylococcus hominis has shown activity against Gram-positive bacteria [52]. This AMP showed a strong antibacterial effect against VISA strains (MIC = 3.82 μg/mL) in antimicrobial assays [52]. The mutancin 1140 AMP derived from Streptococcus mutans has been widely studied and showed strong activity against Gram-positive-resistant strains [91][92]. In particular, this peptide showed activity against VRSA and VISA strains, with MIC ranging from 4 to 8 μg/mL [44]. In addition, mutancin 1140 sensitization tests have shown that no BR to this AMP has been generated [44]. On the other hand, mersacidin is an anionic AMP that has successfully inhibited the in vitro growth of S. aureus; more specifically, it showed antimicrobial activity against resistant VISA-type strains (MIC = 35 μg/mL) [55][93]. Non-lantibiotic AMPs are classified into four subclasses: IIa, IIb, IIc, and IId [89]. Within subclass IId, we recognize bactofencin A, which is a short AMP derived from Lactobacillus salivarius isolated from the pig intestine. This AMP inhibits the growth of clinically significant pathogens [28]. Bactofencin A showed very strong activity against Gram-positive bacteria; specifically, analog 5 showed an antibacterial effect against VRSA strains isolated from bovine mastitis (MIC between 4.3 μM and 100 μM) but did not show activity against Enterococcus fecalis and Streptococcus pyogenes [28]. Additionally, non-ribosomally synthesized peptides from bacteria have also shown activity against susceptible and resistant strains of wide range of Gram-positive bacteria [58]. In particular, the human commensal Staphylococcus lugdunensis produces lugdunin, which is a thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus [58]. Lugdunin showed a potent antimicrobial activity against VISA strains (MIC = 3 μg/mL) and did not show lysis of human neutrophils and erythrocytes [58].
Table 3. Bacteria-derived AMPs with antibacterial activity against VRSA and VISA strains.
Source |
AMP Name |
Strain ID |
MIC Value |
Reference |
Toxicity/Properties |
Lactococcus lactis |
Nisin |
Table 4. Artificial AMPs with antibacterial activity against VRSA and VISA strain.
AMP Name |
Strain ID |
MIC Value |
Reference |
Toxicity/Properties |
VISA-19 |
4.1 mg/L |
LTX-109 |
VRSA-5 |
2–4 μg/mL[53] |
Hemolytic effect on sheep erythrocytes |
[ | 36 | ] |
Phase III of a clinical trial |
VISA-20 |
8.3 mg/L |
[53] |
VISA-3 |
2–4 μg/mL |
[36] |
32 μg/mL |
[46] |
Non toxic to the intradermal model of the larva Hermetia illucens |
VISA-21 |
4.1 mg/L |
[ |
Omiganan (Indolicidin analog) | 53] |
VRSA-19 |
16 μg/mL |
Protonectin |
| [42] |
Topical antimicrobial agent in phase III of a clinical trial |
ILGTILGLLKGL |
12 |
+1 |
10.1 |
47.67 |
58.33 |
[48 |
Hyalophora cecropia |
Cecropin A |
VISA-8 |
64 μg/mL |
[49] |
VISA-10 |
16 μg/mL |
[42 | Low cytotoxic effect on human lung carcinoma |
Parachartergus fraternus and Agelaia pallipes pallipes |
VISA-22 |
8.3 mg/L |
[53] |
] |
Agelaia-MPI |
VRSA-33 |
VISA-23 |
8.3 mg/L | 4–8 μg/mL |
VISA-11 |
16 μg/mL |
[42][48] |
Strong hemolytic effect on human erythrocytes |
[ | 53] |
Protonectin |
VRSA-33 |
16 μg/mL |
VISA-24 |
4.1 mg/L |
[ |
] |
Protonectin-F |
|
IFGTILGFLKGL |
12 |
+1 |
10.1 |
50.16 |
58.33 |
WR12 | 53] |
VRSA-6 | [48] |
4 μM | Toxic to cancerous and non-cancerous cell lines, but moderated hemolytic effect against human erythrocytes |
[ | 37] |
– |
Agelaia-MPI analog |
NeuroVAL |
VRSA-33 |
Staphylococcus hominis |
Hominicin>128 μg/mL |
[48] |
Non toxic to human erythrocytes, and cancerous and non-cancerous cells lines. |
VISA-18 |
3.82 μg/mL |
VRSA-7 | [ | 52 | ] |
– |
8 μM |
[37] |
– |
Protonectin analog |
Protonectin-F |
VRSA-33 |
16 μg/mL |
[ |
Streptococcus mutans |
Mutacin 1140 |
VRSA-23 | 48 |
4–8 μg/mL | ] |
[44 | Toxic to cancerous and non-cancerous cell lines, but moderated hemolytic effect against human erythrocytes |
[ | 48 | ] |
– |
] |
VRSA-8 |
8 μM |
[37] |
– |
Chaerilus tricostatus |
Ctriporin |
VRSA-1 |
10 μg/mL |
VISA-15 |
4 μg/mL |
[44] |
VRSA-9 | [ | 29 | ] |
Histological results showed recovery of the skin |
4 μM | – |
[37] |
– |
VRSA-2 |
10 μg/mL |
Bacillus sp. | [29] |
Mersacidin |
VISA-27 |
35 μg/mL |
[55] |
– |
VRSA-10 |
4 μM |
[37] |
– |
VISA-1 |
10 μg/mL |
[29] |
Lactobacillus salivarius |
Bactofencin A (analog 5) |
VRSA-25 |
][12378]. Five AMPs with antibacterial activity against VRSA and VISA combine α-helix and β-folded sheet structures, with net charges ranging from +1.9 to +6 (
Table 2). In this regard, different members of defensins are characterized by preserving α-helix and β-folded sheet structural motifs, which are stabilized through disulfide bridges
[12479]. These motifs are highly preserved and have been observed in some species of insects, mussels, plants, and fungi
[12479]. Formin C is a defensin synthesized by the common housefly M. domestica, composed of 40 amino acids, 40% of which are hydrophobic amino acids
[6180]. This AMP has a net charge of +3, an isoelectric point of 8.3, and a structure composed of an α-helix and two anti-parallel β-folded sheets
[6180] (
Table 2). Likewise, tick-derived AMPs, such as IP, IR, HAE, and OMBAC, are defensins whose structure is also composed of an α-helix and two anti-parallel β-folded sheets, similar to formicin C (
Table 2). Despite this, these AMPs possess different physicochemical properties, in terms of net charge at physiological pH, isoelectric point, hydrophobicity, and percentage of hydrophobic residues (
Table 2).
3.2.4. AMPs of Atypical Structure: Cyclic, Complex, and with Unusual Amino Acids
Another classification that includes AMPs with structural characteristics different from the conventional ones is peptides that have unusual amino acids or cyclic structures (
Table 3 and
Table 4). In general, these AMPs are cationic, with 9 to 60 residues and low molecular weight. They have different action mechanisms, which are mainly based on the permeabilization of the bacterial cell membrane
[6281]. This category includes peptides, such as bacteriocins, which are characterized by cyclic structures of polypeptide chains, where the amino acid residues are covalently linked to form a ring that is favored by the interaction between chemical bonds, such as amide, lactone, ether, thioether, or disulfide
[6382]. Lantibiotic bacteriocins of subclass Ia (such as nisin, hominicin, and mutancin 1140) and of subclass Ib (such as mersacidin), are small AMPs with molecular weights less than 5 kDa possessing between 19 and 38 amino acids with post-translational modifications
[6281]. Subclass Ia AMPs are elongated peptides with positive charges, whereas those of subclass Ib are globular and rigid with negative charges
[6281]. Nisin is an AMP containing 34 amino acids with five rings based on lanthionine or methyllanthionine from the N-terminal to the C-terminal end
[6483] (
Table 3). This peptide is formed from the post-translational modification of an inactive 21 amino acid precursor synthesized by the precursors NisinA, NisinB, and NisinC, which catalyze the dehydration of serine and threonine residues and participate in the cyclization of cysteine
[6584]. Mutancin 1140 is characterized by having four thioether rings in its chemical structure and a molecular weight of 2.26 kDa, while mersacidin consists of 20 amino acids and forms four intramolecular thioether bridges that form a compact globular structure. It is characterized by a net charge of −1.2, an isoelectric point of 3.3, and a high percentage of hydrophobic amino acids
[6685][6786] (
Table 3). Homicin is a bacteriocin with a molecular weight of 2.03 kDa that does not have a specific tertiary structure and possesses thermotolerant properties and high stability
[6887]. In contrast, bactophencin A, a non-antibiotic bacteriocin, is a cationic AMP consisting of 22 amino acids linked in a loop through a disulfide bond between cysteine residue 7 and 22
[6988]. Especially in analog 5, the methionine residues of the original peptide were replaced by the amino acid leucine at positions 14 and 18, and therefore its physicochemical properties show that it is an AMP with a net charge of +7 at physiological pH and with a hydrophobicity of 27%
[28]. AMP BCP61 is another bacterial peptide with an atypical structure consisting of nine amino acids, a low percentage of which are hydrophobic, and which has a net charge equal to −1 and an isoelectric point of 3.1
[7089]. Lugdunin is a small cyclic bacterial peptide of 0.78 kDa, comprising an unusual thiazolidine heterocycle and five amino acids
[7190]. Finally, fusaricidin analogs were prepared through modification of the lipid tail, substitution of amino acid, and ester-to-amide substitution
[7291]. In this respect, LI-F04a analogs 5, 6, 8, and 11 comprising a lipid tail of 12-guanidinododecanoic acid and macrocyclic ring consisting of six amino acids, four of which, Thr1, D-Val2, D-Asn5, and D-Ala6, are conserved throughout all peptides
[7190] (
Table 3).
Table 3. AMPs of atypical structure derived from bacteria that showed antibacterial activity against VRSA and VISA strains.
AMP Name |
Aminoacid Sequences and Structures |
Molecular Weight (KDa) |
Reference |
Table 4. Artificial AMPs of atypical structure that showed antibacterial activity against VRSA and VISA strains.
Peptide Name |
Chemical Structure |
Molecular Weight (KDa) |
Reference |
Nisin |
|
3.35 |
[53] |
Hominicin |
|
2.03 |
[52] |
– |
[ | 40 | ] |
Mutacin 1140 (MU1140) |
Lipopeptide 2 * | |
2.26 |
[44] |
– |
[ | 40 | ] |
Mersacidin |
|
1.82 |
[55][ |
Lipopeptide 3 * |
|
– |
[407392] |
] |
LL-37 |
|
LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES |
37 |
+6 |
Bactofencin A (analog 5) |
|
2.77 | 11.1 |
35.14 |
34.62 |
[ |
Lipopeptide 4 * |
| [ |
–28] | 41 | ] |
[ | 40 |
LL-13 |
|
IGKEFKRIVQRIKDFLRNLVPRTES |
25 |
+4 |
11.4 |
39.37 |
BCP61 | 36.00 |
|
9.50 |
[45[41] |
] |
LL-17 |
|
LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES |
13 |
+4 |
12.2 |
35.69 |
46.15 |
[41] |
4.3 μM |
[ |
Lugdunin |
|
0.78 |
Melittin |
|
GIGAVLKVLTTGLPALISWIKRKRQQ |
26 |
+5 |
12.5 |
49.39 |
46.15 |
[50] |
Hec |
|
FALALKALKKALKKLKKALKKAL |
23 |
+9 |
11.4 |
39.47 |
60.87 |
LI-F04a analog 6 |
| 28] |
[ |
– |
[ | 58] | 35 | ] |
Smp24 |
|
IWSFLIKAATKLLPSLFGGG-KKDS |
24 |
+4 |
10.6 |
50.39 |
45.83 |
[54] |
VRSA-11 |
8 μM |
[37– |
] |
– |
Scorpio maurus palmatus |
Smp24 |
VISA-25 |
32 μg/mL |
[54 |
LI-F04a analog 8 |
Ctriporin |
|
FLWGLIPGAVTSLIAISKK |
19 |
+2 |
10.6 |
55.47 |
57.89 |
] |
Toxic to sheep erythrocytes |
[ | 29 | ] |
VRSA-26 |
|
– |
[ | 58 |
VRSA-12 | 100 μM |
4 μM | [28] |
[37] | – |
– |
Magainin-2 |
|
GIGKFLHSAKKFGKAFVGEIMNS |
23 |
+3 |
10.6 |
40.19 |
43.48 |
[49] |
VISA-26 |
64 μg/mL |
[54] |
Staphylococcus lugdunensis |
Lugdunin |
VISA-30 |
3 μg/mL |
[ |
VISA-4 | 58 | ] |
No lysis of primary human erythrocytes or neutrophils. |
1 μM |
Temporin-CPa |
|
Omiganan |
|
1.96 |
[ |
37 |
] |
– |
IPPFIKKVLTTVF |
13 |
+2 |
10.6 |
41.02 |
53 |
Ixodes persulcatus |
Persulcatusin (IP) |
VRSA-3 |
2 μg/mL |
[34] |
Bacillus sp. |
BCP61 |
VRSA-24 |
10 μg/mL |
[ |
VISA-5 | Non toxic to fibroblasts, colon epithelial cells, and erythrocytes |
56 | ] |
[45] |
– |
1 μM |
VISA-2 |
8 μg/mL |
Bacillus subtilis subsp. inaquosorum |
P138-C[34] |
VRSA-14 |
20 μg/mL |
[39] |
– |
Ixodes ricinus |
IR |
VRSA-3 |
32 μg/mL |
[34 |
Temporin-CPb |
|
FLPIVGRLISGIL |
13 |
+1 |
11.1 |
46.35 |
61 |
[56] |
Temporin-1Ga |
|
SILPTIVSFLSKVF |
14 |
+1 |
10.1 |
52.43 |
57 |
[56Bacillus amyloliquefaciens | ] |
CSPK14 |
VRSA-13 |
64 μg/mL |
[ | – |
] |
38 | ] |
– |
Temporin-1OLa |
|
FLPFLKSILGKIL |
13 |
+2 |
10.6 |
48.08 |
61 |
[56] |
VISA-2 |
>32 μg/mL |
[34 |
Fusaricidin analogs |
LI-F04a analog 5 | ] |
– |
VISA-29 |
16 μg/mL |
Temporin-1Spa |
| [57] |
Hemolysis on human erythrocytes |
Haemaphysalis longicornis |
HAE |
VRSA-3 |
>32 μg/mL |
[34] |
– |
LI-F04a analog 6 |
VISA-29 |
16 μg/mL |
[57] |
Hemolysis on human erythrocytes |
VISA-2 |
>32 μg/mL |
[34] |
– |
[ | 37] |
– |
VISA-6 |
1 μM |
[37] |
– |
DIK-8 |
VRSA-6 |
8 μM |
[37] |
– |
VRSA-7 |
16 μM |
[37] |
– |
FLSAITSILGKFF |
13 |
+1 |
10.1 |
47.05 |
61 |
[56] |
VRSA-8 |
16 μM |
[37] |
– |
Temporin-1Oc |
|
FLPLLASLFSRLF |
13 |
+1 |
11.1 |
59.16 |
69 |
[56] |
VRSA-9 |
Ornithodoros moubata |
LI-F04a analog 8 |
VISA-29 |
16 μg/mL |
[57] |
16 μM |
[37]Hemolysis on human erythrocytes |
– |
OMBAC |
FL9 |
|
GVVDILKGLAKDIAGHLASKVMNKL |
25 |
+2 |
10.2 |
41.54 |
VRSA-3 |
8 μg/mL |
[34] |
– |
52 |
[ | 60 | ] |
LI-F04a analog 11 |
VISA-29 |
VRSA-10 |
16 μM | 16 μg/mL |
[37][57] |
Hemolysis on human erythrocytes |
FL10 |
| – |
GVVDILKGALKDIAGHLASKVMNKL |
25 |
+2 |
10.2 |
41.31 |
52 |
[60] |
VISA-2 |
>32 μg/mL |
[34] |
– |
Xenopus laevis |
VRSA-11 |
16 μM |
[37] |
– |
Magainin-2 |
VISA-8 |
16 μg/mL |
FA-12 |
|
GVVDILKGAAKAIAGHLASKVMNKL |
[49] |
25 |
+3 |
10.6 |
37.87 |
56 |
VRSA-12 | – |
[ | 60 | ] |
16 μM |
FL-14 |
| [37] |
– |
GVVDILKGAAKDILGHLASKVMNKL |
25 |
+2 |
10.2 |
41.54 |
52 |
[60] |
Lithobates capito |
Temporin-CPa |
VISA-28 |
>25 μM |
[56] |
Hemolysis of human erythrocytes at high concentrations |
VISA-4 |
8 μM |
[37] |
– |
CSPK-14 |
|
HYDPGDDSGNTG |
12 |
−2.9 |
3.6 |
5.66 |
0 |
[38] |
Temporin-CPb |
VISA-28 |
12.5 μM |
[56] |
Hemolysis of human erythrocytes at high concentrations |
VISA-5 |
8 μM |
WR12 |
| [37] |
– |
RWWRWWRRWWRR |
12 |
+6 |
13.2 |
50.42 |
50.00 |
[37] |
Rana grylio |
Temporin-1Ga |
VISA-28 |
6.2 μM |
[56] |
Strong hemolytic effect on human erythrocytes |
VISA-6 |
8 μM |
[37] |
– |
RR |
|
WLRRIKAWLRR |
11 |
+5 |
13.0 |
33.04 |
Rana okaloosae |
Temporin-1OLa |
VISA-28 |
3.1 μM |
[56] |
Strong hemolytic effect on human erythrocytes |
Rana septentrionalis |
Temporin-1 SPa |
54 |
[ | 47 | ] |
MP196 |
VISA-16 |
16 μg/mL |
[51] |
Light hemolytic effect on cell lines of breast cancer. Acute toxicity in mice cells. |
RRIKA |
|
WLRRIKAWLRRIKA |
14 |
+6 |
13.0 |
39.90 |
57 |
[47] |
VISA-28 |
12.5 μM |
[56] |
VISA-17 |
64 μg/mL |
[51Moderate hemolytic effect on human erythrocytes |
] |
Rana ornativentris |
Temporin-1Oc |
VISA-28 |
1.6 μM |
[56] |
Strong hemolytic effect on human erythrocytes |
Fallaxin analogs |
FL10 |
VISA-32 |
50 μM |
[60] |
High hemolytic effect on human erythrocytes |
FL9 |
VISA-32 |
50 μM |
[60] |
Moderate hemolytic effect on human erythrocytes |
FA12 |
VISA-32 |
50 μM |
[60] |
High hemolytic effect on human erythrocytes |
FL14 |
VISA-32 |
50 μM |
[60] |
High hemolytic effect on human erythrocytes |
Homo sapiens |
LL-37 |
VRSA-18 |
64 μg/mL |
[41] |
Low cytotoxic effect |
VISA-7 |
64 μg/mL |
[41] |
Derived from LL-37 |
LL-13 |
VRSA-18 |
512 μg/mL |
[41] |
– |
VISA-7 |
1024 μg/mL |
[41] |
– |
Derived from LL-37 |
LL-17 |
VRSA-18 |
512 μg/mL |
[41] |
– |
VISA-7 |
1024 μg/mL |
On the other hand, a great variety of AMPs derived from bacteria of the Bacillus genus with different biological functions have been identified. In particular, the BCP61 peptide produced by bacteria of the Bacillus genus was isolated from a fermented food of Asian origin called “kimchi” [94][95]. This AMP has shown activity against different Gram-positive bacteria, such as S. aureus and E. fecalis. More specifically, it showed potent antibacterial activity against resistant VRSA strains (MIC = 10 μg/mL) [45]. The AMP P138-C—derived from Bacillus subtilis, subsp. inaquosorum, strain KCTC 13429 and present in a fermented food product—showed activity against a wide diversity of Gram-positive bacteria [39]. This peptide showed MIC of 20 μg/mL and MBC of 640 μg/mL against VRSA strains, and its activity was enhanced when combined with antibiotics, such as oxacillin, ampicillin, and penicillin [39]. Additionally, the peptide CSPK14 derived from Bacillus amyloliquefaciens showed activity against VRSA strains with an MIC of 64 μg/mL [38]. The effect of this peptide against these strains was enhanced when tested in synergy with the antibiotics ciprofloxacin and ampicillin [38]. On the other hand, from bacteria of the Paenibacillus genus, some naturally occurring peptides with antimicrobial potential have been identified [57]. In particular, fusaricidins (LI-F) are a family of cyclic lipodepsipeptide with antimicrobial activity against a variety of fungi and Gram-positive bacteria [57]. A total of 18 fusaricidin A analogs were designed and synthesized, and then evaluated against ATCC strains of S. aureus [57]. In this respect, the analogs 5, 6, 8, 11, and 14 showed the lowest MIC values against VISA strain Mu50 (MIC = 16 μg/mL) and considerable hemolysis [57].
3.1.2. Bacteria-Derived AMPs
3.1.3. Artificial AMPs
Testing of artificial AMPs and their ability to control pathogenic bacteria has gained momentum in recent years because they offer numerous comparative advantages over many natural peptides [96]. For example, many artificial peptides have an enhanced antibacterial effect and fewer adverse effects [61][97]. Thus, de novo design of more stable and effective artificial AMPs and their evaluation is a strategy against infections caused by resistant bacteria, which could be of great clinical importance. Artificial AMPs that demonstrated antimicrobial activity against VRSA and VISA strains are summarized in Table 4. An example of the application of de novo peptide design with activity against Gram-positive bacteria is the LTX-109 peptide designed by Lytix Biopharma [98]. This AMP is emerging as a topical therapeutic alternative against diabetic foot bacterial infections caused by S. aureus, as it has shown to be highly effective against resistant clinical isolates [36][98]. This AMP in particular has shown a strong bactericidal effect against VISA and VRSA clinical isolates (MIC = 2–4 μg/mL), demonstrating that the LTX-109 peptide has an antibacterial effect regardless of the resistance patterns of the strains [36][98] (Table 4). Omiganan, an analog peptide of indolicidin, has demonstrated broad-spectrum activity against Gram-positive and Gram-negative bacteria and fungi [99]. This AMP has shown strong activity against VRSA strains, showing a MIC of 16 μg/mL against VRSA and VISA strains [42]. In this regard, omiganan is emerging as a topical treatment used primarily against catheter-related local and bloodstream infections caused by resistant S. aureus strains [42][100]. In addition to de novo design, many researchers are using other strategies to enhance the antimicrobial activity and decrease the hemolytic or cytotoxic effects of AMPs [61][97]. Among the strategies that have shown promising results in the design of artificial AMPs we can highlight the following: addition of amino acids to AMPs canonical sequences, synthesis of hybrid peptides by combining sections of different peptides, synthesis of shorter peptides derived from canonical sequences of longer AMPs or proteins, and rational substitution of amino acids in the canonical sequences of AMPs [61][97]. With these strategies, it is possible to manage and modify physicochemical properties of AMPs, such as net charge, hydrophobicity, and amphipathicity [61][97]. In this regard, AMPs, such as MP196, WR12, and DIK-8, designed exclusively with highly specific amino acids, showed antibacterial activity against S. aureus strains resistant to conventional antibiotics [37]. The hexapeptide MP196 is a short sequence rich in tryptophan (W) and arginine (R) residues with chemical modifications, such as organoleptic derivatization, fatty acyl, and multivalent studies with promising antimicrobial characteristics [51]. This peptide showed antibacterial activity against VISA strains, with MIC between 16 and 64 μg/mL, and had no significant hemolytic or cytotoxic effects when evaluated against erythrocytes, rat kidney epithelial cells, and human T-cell lymphoblasts [51]. Likewise, the WR12 peptide, also composed exclusively of W and R residues, exhibited broad-spectrum antimicrobial activity, showing very strong activity against VRSA and VISA strains (MIC = 1–8 μM) [37]. DIK-8 is a short AMP composed exclusively of the amino acids isoleucine (I) and lysine (K), which showed antibacterial activity against VRSA (MIC = 8–16 μM) and VISA (MIC = 8 μM) strains, and low toxicity against mammalian cells [37]. Additionally, the design of AMPs by substituting and adding special amino acids has been used to improve antimicrobial activity and reduce the detrimental impact on host cells [101]. For example, the peptide P-113 derived from the human salivary protein histatin 5, which showed antibacterial activity against VRSA and VISA strains (MIC > 64 μg/mL), had its histidine (H) residues replaced by bulky unnatural amino acids [43]. This way the Phe-P-113, Bip-P-113, Dip-P-113, and Nal-P-113 peptides were obtained, which showed an enhanced antibacterial effect against VRSA and VISA strains (Table 4). On the other hand, AMPs with added lipoamino acids have been designed, namely, lipopeptides (lipopeptide-1 to -6). These molecules have shown broad antimicrobial activity against Gram-positive bacteria, including VRSA and VISA strains. However, they have shown toxicity against embryonic and renal cells [40][43] (Table 4). Additionally, other family of artificial small lipopeptides was designed and constructed with a combination of two or three basic, cationic, and/or anionic amino acids attached to an acyl chain of 14 carbons [59]. Seven peptides of this family (C14-KK, C14-RRR, C14-LK, C14-RW, C14-WR, C14-KWI, and C14-LKK) showed antibacterial activity against VISA strain Mu50 with MIC values between 1.56 and >12.5 μM, and strong hemolytic activity against human red blood cells [59]. Finally, two short artificial peptides (RRIKA and RR) exhibited potent and rapid antimicrobial effect against VRSA and VISA clinical isolates with MIC between 2 and 32 μM [47]
Formicin C |
|
|
ATCDLLSGTGVGHSACAAHCLLRGNRGGYCNGKGVCVCRN |
40 |
+3 |
8.3 |
30.58 |
42.50 |
[ |
46 |
] |
P-113 |
VRSA-20 |
>64 μg/mL |
[ | 43] |
IP |
| – |
|
GFGCPFNQGACHRHCRSIGRRGGYCAGLFKQTCTCYSR |
38 |
+6 |
9.3 |
29.58 |
34.21 |
[34] |
VRSA-21 |
>64 μg/mL |
[43] |
– |
IR |
|
GGYYCPFFQDKCHRHCRSFGRKAGYCGGFLKKTCICV |
37 |
+6 |
9.2 |
36.11 |
37.84 |
[34] |
VRSA-22 |
>64 μg/mL |
HAE |
| [43] |
– |
GCPLNQGACHNHCRSIGRRGGYCAGIIKQTCTCYRK |
36 |
+6 |
9.3 |
23.43 |
33.33 |
[41] |
VISA-12 |
>64 μg/mL |
[43] |
– |
OMBAC |
|
GFGCPFNQYECHAHCSGVPGYKGGYCKGLFKQTCNCY |
37 |
+2 |
8.0 |
32.12 |
32.43 |
[41] |
VISA-13 |
>64 μg/mL |
[43] |
– |
VISA-14 |
>64 μg/mL |
[43] |
– |
Phe-P-113 |
VRSA-20 |
>64 μg/mL |
[43] |
– |
VRSA-21 |
>64 μg/mL |
[43] |
– |
VRSA-22 |
>64 μg/mL |
[43] |
– |
VISA-12 |
>64 μg/mL |
[43] |
– |
VISA-13 |
>64 μg/mL |
[43] |
– |
VISA-14 |
>64 μg/mL |
[43] |
– |
Bip-P-113 |
VRSA-20 |
16 μg/mL |
[43] |
– |
VRSA-21 |
16 μg/mL |
[43] |
– |
VRSA-22 |
8 μg/mL |
[43] |
– |
VISA-12 |
16 μg/mL |
[43] |
– |
VISA-13 |
16 μg/mL |
[43] |
– |
VISA-14 |
8 μg/mL |
[43] |
– |
Dip-P-113 |
VRSA-20 |
32 μg/mL |
[43] |
– |
VRSA-21 |
32 μg/mL |
[43] |
– |
VRSA-22 |
32 μg/mL |
[43] |
– |
VISA-12 |
16 μg/mL |
[43] |
– |
VISA-13 |
16 μg/mL |
[43] |
– |
VISA-14 |
16 μg/mL |
[43] |
– |
Nal-P-113 |
VRSA-20 |
8 μg/mL |
[43] |
– |
VRSA-21 |
8 μg/mL |
[43] |
– |
VRSA-22 |
16 μg/mL |
[43] |
– |
VISA-12 |
8 μg/mL |
[43] |
– |
VISA-13 |
8 μg/mL |
[43] |
– |
VISA-14 |
8 μg/mL |
[43] |
– |
Lipopeptide 1 |
VRSA-15 |
0.5 μM |
[40] |
Low toxicity in human embryonic and kidney cells |
VRSA-16 |
0.7 μM |
[40] |
VRSA-17 |
0.9 μM |
[40] |
Lipopeptide 2 |
VRSA-15 |
2.8 μM |
[40] |
Low toxicity in human embryonic and kidney cells |
VRSA-16 |
1.9 μM |
[40] |
VRSA-17 |
2.8 μM |
[40] |
Lipopeptide 3 |
VRSA-15 |
>30 μM |
[40] |
Low toxicity in human embryonic and kidney cells |
VRSA-16 |
>30 μM |
[40] |
VRSA-17 |
>30 μM |
[40] |
Lipopeptide 4 |
VRSA-15 |
>30 μM |
[40] |
Low toxicity in human embryonic and kidney cells |
VRSA-16 |
>30 μM |
[40] |
VRSA-17 |
>30 μM |
[40] |
Lipopeptide 5 |
VRSA-15 |
0.2 μM |
[40] |
Low toxicity in human embryonic and kidney cells |
VRSA-16 |
0.1 μM |
[40] |
VRSA-17 |
0.1 μM |
[40] |
Lipopeptide 6 |
VRSA-15 |
2.8 μM |
[40] |
Low toxicity in human embryonic and kidney cells |
VRSA-16 |
1.9 μM |
[40] |
VRSA-17 |
1.9 μM |
[40] |
C14-KK |
VISA-31 |
12.5 μM |
[59] |
Strong hemolytic effect on human erythrocytes |
C14-RRR |
VISA-31 |
3.1 μM |
[59] |
Strong hemolytic effect on human erythrocytes |
C14-LK |
VISA-31 |
1.56 μM |
[59] |
Strong hemolytic effect on human erythrocytes |
C14-RW |
VISA-31 |
>12.5 μM |
[59] |
Strong hemolytic effect on human erythrocytes |
C14-WR |
VISA-31 |
3.1 μM |
[59] |
Strong hemolytic effect on human erythrocytes |
C14-KWI |
VISA-31 |
12.5 μM |
[59] |
Strong hemolytic effect on human erythrocytes |
C14-LKK |
VISA-31 |
3.1 μM |
[59] |
Strong hemolytic effect on human erythrocytes |
RRIKA |
VISA-33 |
2 μM |
[47] |
Low hemolytic activity, but show toxicity in mammalian cell lines |
VRSA-29 |
4 μM |
[47] |
VRSA-30 |
4 μM |
[47] |
VRSA-31 |
4 μM |
[47] |
VRSA-32 |
4 μM |
[47] |
Antimicrobial peptides have various physicochemical and structural properties that play a key role in regulating their antimicrobial activity, their mechanism of action, and their specificity towards molecular targets [121–124]. In this sense, AMPs with antibacterial activity against VRSA and VISA strains have different physicochemical properties in terms of amino acid sequence, charge, hydrophobicity, and isoelectric point, which determine their activity against these resistant strains (Tables 5–7). Likewise, these peptides have different structures, which allow them to be grouped into four categories: α-helical peptides, β-pleated sheet peptides, mixed-structure peptides (α-helix and β-pleated sheet) (Table 2), and peptides with atypical structure, which include cyclic and complex AMPs, as well as AMPs with unusual amino acids (Tables 6 and 7). The physicochemical structures and properties of some peptides were reported in some of the papers included in this review. However, when a paper did not report these characteristics for any AMP, its respective prediction was made from the amino acid sequences using the servers I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER/, accessed on 1 June 2021), ThermoFisher (https://www.thermofisher.com/co/en/home/life-science/protein-biology/peptides-proteins/custom-peptide-synthesis-services/peptide-analyzing-tool.html, accessed on 1 June 2021), and CALCAMPI (https://ciencias.medellin.unal.edu.co/gruposdeinvestigacion/prospeccionydisenobiomoleculas/InverPep/public/herramientas, accessed on 1 June 2021).
3.2.1. α-helix AMPs
AMPs with α-helix structure are the most widely spread in nature
[54]. This conformation is important for interaction with bacterial membranes, especially because of the arrangement of amino acids in the helix conformation, where polar residues are segregated on the polar side of the helix and hydrophobic residues on the apolar side of the helix
[54][55]. This results in the production of an amphipathic α-helical structure that provides the ability to insert into the hydrophobic sector of bacterial lipid bilayers and cause lethal damage
[54][55]. In general, AMPs that adopt this conformation tend to be short and easy to synthesize, as well as having a wide range of antibacterial mechanisms of action
[56]. A total of 26 AMPs with antibacterial activity against VRSA and VISA form α-helical structure, with net charges ranging from −2.9 to +9 (
Table 2). Cecropins are characterized by their tendency to form α-helical structures
[57]. For example, cecropin A, composed of 37 amino acids in length, charge of +6, and an isoelectric point of 10.6, exhibits an α-helical pattern with hydrophobic charged surfaces that make it a highly amphipathic AMP
[58]. Likewise, other insect-derived AMPs, such as agelaia-MPI, protonectin, and protonectin-F, also exhibit a α-helical structure, charge of +1, and high incidence of hydrophobic amino acids
[59]; a conserved glycine (G) residue gives the flexibility to all these peptides
[59]. In contrast, both cathelicidin LL-37 and the derived peptides LL-13 and LL-17 form α-helical structure (
Table 2). LL-37 is an AMP consisting of 37 amino acids, 34.6% of which are hydrophobic residues, has a net charge of +6, and has an isoelectric point of 11.1
[60] (
Table 2). AMPs synthesized by A. mellifera with activity against VRSA and VISA strains, such as melittin and Hec, also form α-helical structure (
Table 2). Melittin is an AMP that is synthesized in the bee venom gland as a 70-amino acid propeptide, which is subsequently cleaved to its compact form consisting of 26 residues
[10661]. The first 20 amino acids of this AMP have polar properties, while the remaining six are hydrophobic, and therefore their net charge of +6 at physiological pH is distributed as +4 in the N-terminal region and +2 in the C-terminal region
[10661][10762] (
Table 2). The Hec peptide is characterized by a high incidence of positively charged amino acids, an α-helix structure, high cationic charge (+9), and a high percentage of hydrophobic amino acids
[10863]. Peptides, such as smp24 and ctriporin, synthesized by different scorpion species, are structurally composed of a single α-helix (
Table 2). The AMP smp24, synthesized by the scorpion S. maurus, is composed of 24 amino acids and has a +4 net charge, high hydrophobicity, and a helical structure extending from the N-terminal residue to residue 18
[10964] (
Table 2). This peptide features an alteration in the central proline residue to enhance antibacterial activity; specifically, a kink in the middle of the α-helix structure provides the AMP potent pore formation and selective antimicrobial activity by prokaryotic membranes
[10964]. Ctriporin forms an α-helix structure, comprising mainly a hydrophobic face and a hydrophilic face
[29]. This AMP, with a net charge of +2 due to two positively charged lysine residues, contains more than 50% of hydrophobic amino acids
[29]. Magainins produced by amphibians are a family of AMPs that has been structurally well characterized, and many of its members form α-helical structures
[11065]. One of them is magainin-2, which has a net charge of +3 and is composed mainly of L-amino acids forming a sequence of 23 residues, 43% of which are hydrophobic
[11065]. Temporins, composed of 13 and 14 amino acids, exhibit an α-helical pattern and net charges ranging from +1 to +2. All temporins with antibacterial activity against VISA strains have more than 50% of hydrophobic residues
[11166]. Additionally, fallaxin analogs exhibit a α-helical structure; these peptides consisting of 25 amino acids, with more than 50% of hydrophobic residues, have a net charge between +2 and +3
[11267]. Several bacteriocins form α-helix structure, such as AMP CSPK-14, which has a low molecular weight (10 kDa), a net charge of −2.9, and no hydrophobic amino acids
[11368][11469]. The antibacterial activity of AMPs with anionic charges are enhanced due to the presence of divalent cations, such as Ca
+2, Mg
+2, and Mn
+2, which allow for the formation of an ion–peptide complex that reduces the overall negative charge of the AMP and favors the affinity for bacterial membranes
[11570]. In this regard, CSPK-14 improved its bactericidal activity when synergistically evaluated with metal ions, such as Ca
+2 [11368]. Finally, three artificial AMPs form amphipathic α-helix: WR12, RR, and RRIKA
[11671][11772]. WR12, composed of 12 very particular amino acids, contains six arginines and six tryptophans, and therefore it has 50% hydrophobicity and a net charge of +6 helix
[11671]. RR, composed of 11 amino acids, 54% of which are hydrophobic amino acids, has a net charge of +5, while RRIKA, with 14 amino acids and 57% of hydrophobicity, has a net charge of +6
[11772] (
Table 2).
Table 2. Structural and physicochemical properties of AMPs that showed antibacterial activity against VRSA and VISA strains.
[ |
42 | ] | [ | 7493] |
] |
Lipopeptide 5 * |
|
– |
[ |
[ | 58 | ] |
40 |
LI-F04a analog 5 |
|
– |
[ |
] |
VRSA-33 |
4 μM |
[ |
47 |
] |
RR |
Lipopeptide 1 * |
] |
Lipopeptide 6 * |
|
– |
[40] |
58] |
VISA-33 |
16 μM |
[ |
47 |
] |
Low hemolytic activity, but show toxicity in mammalian cell lines |
VRSA-29 |
32 μM |
[ |
47 |
] |
VRSA-30 |
16 μM |
[ |
47 |
] |
VRSA-31 |
16 μM |
[ |
47 |
] |
VRSA-32 |
32 μM |
[ |
47 |
] |
VRSA-33 |
32 μM |
[ |
47 |
] |
LI-F04a analog 11 |
|
– |
[ |
58 |
] |
Strain ID * |
Interpretive Categories for Conventional Antibiotics |
Method |
Genotype |
Reference |
PEN 1 |
AMX 1 |
OXA 1 |
ERY 2 |
VAN 3 |
TET 4 |
DAP 5 |
LZD 6 |
CLI 7 |
VRSA-1 |
R |
– |
– |
– |
R |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[29] |
VRSA-2 |
R |
– |
– |
– |
R |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[29] |
VRSA-3 |
– |
– |
– |
– |
R |
– |
– |
– |
– |
– |
– |
[34] |
VRSA-4 |
R |
R |
– |
R |
R |
S |
– |
– |
– |
Disc diffusion |
VanA |
[35] |
VRSA-5 |
– |
– |
– |
– |
R |
– |
S |
S |
R |
MIC (μg/mL) |
SCCmec II |
[36] |
VRSA-6 |
– |
– |
R |
– |
R |
– |
– |
– |
– |
MIC (μM) |
– |
[37] |
VRSA-7 |
– |
– |
R |
– |
R |
– |
– |
– |
– |
MIC (μM) |
– |
[37] |
VRSA-8 |
– |
– |
R |
– |
R |
– |
– |
– |
– |
MIC (μM) |
– |
[37] |
VRSA-9 |
– |
– |
R |
– |
R |
– |
– |
– |
– |
MIC (μM) |
– |
[37] |
VRSA-10 |
– |
– |
R |
– |
R |
– |
– |
– |
– |
MIC (μM) |
– |
[37] |
VRSA-11 |
– |
– |
R |
– |
R |
– |
– |
– |
– |
MIC (μM) |
– |
[37] |
VRSA-12 |
– |
– |
R |
– |
R |
– |
– |
– |
– |
MIC (μM) |
– |
[37] |
VRSA-13 |
R |
– |
R |
– |
R |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[38] |
VRSA-14 |
– |
– |
– |
– |
R |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[39] |
VRSA-15 |
– |
– |
– |
– |
R |
– |
– |
– |
– |
MIC (μM) |
– |
[40] |
VRSA-16 |
– |
– |
– |
– |
R |
– |
– |
– |
– |
MIC (μM) |
– |
[40] |
VRSA-17 |
– |
– |
– |
– |
R |
– |
– |
– |
– |
MIC (μM) |
– |
[40] |
VRSA-18 |
– |
– |
– |
– |
R |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[41] |
VRSA-19 |
– |
– |
S |
R |
R |
R |
– |
– |
R |
MIC (μg/mL) |
– |
[42] |
VRSA-20 |
– |
– |
– |
- |
R |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[43] |
VRSA-21 |
– |
– |
– |
- |
R |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[43] |
VRSA-22 |
– |
– |
– |
- |
R |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[43] |
VRSA-23 |
– |
– |
– |
- |
R |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[44] |
VRSA-24 |
– |
– |
– |
- |
R |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[45] |
VRSA-25 |
– |
– |
– |
- |
R |
– |
– |
– |
– |
– |
– |
[28] |
VRSA-26 |
– |
– |
– |
- |
R |
– |
– |
– |
– |
– |
– |
[28] |
VRSA-27 |
– |
– |
– |
- |
R |
– |
– |
– |
– |
MIC (μg/mL) |
MecA |
[46] |
VRSA-28 |
– |
– |
– |
- |
R |
– |
– |
S |
– |
– |
– |
[47] |
VRSA-29 |
– |
– |
– |
- |
R |
– |
– |
S |
– |
– |
– |
[47] |
VRSA-30 |
– |
– |
– |
- |
R |
– |
– |
S |
– |
– |
– |
[47] |
VRSA-31 |
– |
– |
– |
- |
R |
– |
– |
S |
– |
– |
– |
[47] |
VRSA-32 |
– |
– |
– |
- |
R |
– |
– |
R |
– |
– |
– |
[47] |
VRSA-33 |
– |
– |
– |
- |
R |
– |
– |
– |
– |
– |
– |
[48] |
VISA-1 |
R |
– |
– |
- |
I |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[29] |
VISA-2 |
– |
– |
– |
- |
I |
– |
– |
– |
– |
MIC (μg/mL) |
– |
[34] |
VISA-3 |
– |
– |
– |
- |
I |
– |
R |
S |
R |
MIC (μg/mL) |
SCCmec II |
[36] |
VISA-4 |
– |
– |
– |
R |
I |
– |
– |
S |
– |
MIC (μM) |
– |
[37] |
VISA-5 |
– |
– |
– |
R |
I |
– |
– |
S |
– |
MIC (μM) |
– |
[37] |
VISA-6 |
– |
– |
– |
R |
I |
– |
– |
S |
– |
MIC (μM) |
– |
[37] |
VISA-7 |
– |
– |
– |
Diverse interactions occur naturally between bacterial species sharing the same habi- tat, which are determined by the nutritional resources available [105]. Through various mechanisms, such as the production of toxic molecules or compounds, many bacterial species can favor their own survival and evolution, affecting other bacterial species they live with [105]. One of these mechanisms involves peptides that may be naturally pro- duced by some bacterial species to control the survival of other bacteria [105]. Due to their strong effect, some bacterial-derived AMPs have been evaluated as alternatives to control Gram-positive bacteria resistant to conventional antibiotics [106]. In this regard, numerous AMPs identified in bacteria have shown promising characteristics against re- sistant VRSA and VISA strains (Table 3). Depending on the biosynthetic route they use, bacterial-derived peptides can be classified into two groups: (1) ribosomally synthesized peptides such as bacteriocins and (2) non-ribosomal peptides, such as bacitracins and glycopeptides [107]. Bacteriocins are a group of AMPs with a wide variety in size, struc- ture, and mode of action [108]. Bacteriocins derived from Gram-positive bacteria can be grouped into four different classes: (I) lantibiotics, (II) non-lantibiotics, (III) large peptides, and (IV) bacteriocins containing lipids or carbohydrates [108]. Within the lantibiotics, two subclasses are identified: subclass Ia, which includes AMPs such as nisin, hominicin, and mutancin 1140, and subclass Ib, which includes mersacidin [108]. One of the best known bacteriocins is nisin derived from Lactococcus lactis [107]. This AMP has a strong antimicrobial effect, and according to in vitro assays, it showed activity against VISA strains, with MIC between 4.1 and 8.3 μg/mL, and a slight hemolytic effect against sheep erythrocytes [53,109]. Similarly, hominicin produced by Staphylococcus hominis has shown activity against Gram-positive bacteria [52]. This AMP showed a strong antibacterial effect against VISA strains (MIC = 3.82 μg/mL) in antimicrobial assays [52]. The mutancin 1140 AMP derived from Streptococcus mutans has been widely studied and showed strong activity against Gram-positive-resistant strains [110,111]. In particular, this peptide showed activity against VRSA and VISA strains, with MIC ranging from 4 to 8 μg/mL [44]. In addition, mutancin 1140 sensitization tests have shown that no BR to this AMP has been generated [44]. On the other hand, mersacidin is an anionic AMP that has successfully inhibited the in vitro growth of S. aureus; more specifically, it showed antimicrobial activity against resistant VISA-type strains (MIC = 35 μg/mL) [55,112]. Non-lantibiotic AMPs are classified into four subclasses: IIa, IIb, IIc, and IId [108]. Within subclass IId, we recognize bactofencin A, which is a short AMP derived from Lactobacillus salivarius isolated from the pig intestine. This AMP inhibits the growth of clinically significant pathogens [28]. Bactofencin A showed very strong activity against Gram-positive bacteria; specifically, analog 5 showed an antibacterial effect against VRSA strains isolated from bovine mastitis (MIC between 4.3 μM and 100 μM) but did not show activity against Enterococcus fecalis and Streptococcus pyogenes [28]. Additionally, non-ribosomally synthesized peptides from bacteria have also shown activity against susceptible and resistant strains of wide range of Gram-positive bacteria [58]. In particular, the human commensal Staphylococcus lugdunen- sis produces lugdunin, which is a thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus [58]. Lugdunin showed a potent antimicrobial activity against VISA strains (MIC = 3 μg/mL) and did not show lysis of human neutrophils and erythrocytes [58].
On the other hand, a great variety of AMPs derived from bacteria of the Bacillus genus with different biological functions have been identified. In particular, the BCP61 peptide produced by bacteria of the Bacillus genus was isolated from a fermented food of Asian ori- gin called “kimchi” [113,114]. This AMP has shown activity against different Gram-positive bacteria, such as S. aureus and E. fecalis. More specifically, it showed potent antibacterial activity against resistant VRSA strains (MIC = 10 μg/mL) [45]. The AMP P138-C—derived from Bacillus subtilis, subsp. inaquosorum, strain KCTC 13429 and present in a fermented food product—showed activity against a wide diversity of Gram-positive bacteria [39]. This peptide showed MIC of 20 μg/mL and MBC of 640 μg/mL against VRSA strains, and its activity was enhanced when combined with antibiotics, such as oxacillin, ampicillin, and penicillin [39]. Additionally, the peptide CSPK14 derived from Bacillus amyloliquefaciens showed activity against VRSA strains with an MIC of 64 μg/mL [38]. The effect of this peptide against these strains was enhanced when tested in synergy with the antibiotics ciprofloxacin and ampicillin [38]. On the other hand, from bacteria of the Paenibacillus genus, some naturally occurring peptides with antimicrobial potential have been iden- tified [57]. In particular, fusaricidins (LI-F) are a family of cyclic lipodepsipeptide with antimicrobial activity against a variety of fungi and Gram-positive bacteria [57]. A total of 18 fusaricidin A analogs were designed and synthesized, and then evaluated against ATCC strains of S. aureus [57]. In this respect, the analogs 5, 6, 8, 11, and 14 showed the lowest MIC values against VISA strain Mu50 (MIC = 16 μg/mL) and considerable hemolysis [57].
3.1.3. Artificial AMPs
Testing of artificial AMPs and their ability to control pathogenic bacteria has gained momentum in recent years because they offer numerous comparative advantages over many natural peptides [115]. For example, many artificial peptides have an enhanced antibacterial effect and fewer adverse effects [80,116]. Thus, de novo design of more stable and effective artificial AMPs and their evaluation is a strategy against infections caused by resistant bacteria, which could be of great clinical importance. Artificial AMPs that demonstrated antimicrobial activity against VRSA and VISA strains are summarized in Table 4. An example of the application of de novo peptide design with activity against Gram-positive bacteria is the LTX-109 peptide designed by Lytix Biopharma [117]. This AMP is emerging as a topical therapeutic alternative against diabetic foot bacterial infec- tions caused by S. aureus, as it has shown to be highly effective against resistant clinical isolates [36,117]. This AMP in particular has shown a strong bactericidal effect against VISA and VRSA clinical isolates (MIC = 2–4 μg/mL), demonstrating that the LTX-109 peptide has an antibacterial effect regardless of the resistance patterns of the strains [36,117] (Table 4).
3.2. AMPs Classification Based on Their Physicochemical and Structural Properties
Omiganan, an analog peptide of indolicidin, has demonstrated broad-spectrum activity against Gram-positive and Gram-negative bacteria and fungi [118]. This AMP has shown strong activity against VRSA strains, showing a MIC of 16 μg/mL against VRSA and VISA strains [42]. In this regard, omiganan is emerging as a topical treatment used primarily against catheter-related local and bloodstream infections caused by resistant S. aureus strains [42,119]. In addition to de novo design, many researchers are using other strategies to enhance the antimicrobial activity and decrease the hemolytic or cytotoxic effects of AMPs [80,116]. Among the strategies that have shown promising results in the design of artificial AMPs we can highlight the following: addition of amino acids to AMPs canonical sequences, synthesis of hybrid peptides by combining sections of different peptides, synthe- sis of shorter peptides derived from canonical sequences of longer AMPs or proteins, and rational substitution of amino acids in the canonical sequences of AMPs [80,116]. With these strategies, it is possible to manage and modify physicochemical properties of AMPs, such as net charge, hydrophobicity, and amphipathicity [80,116]. In this regard, AMPs, such as MP196, WR12, and DIK-8, designed exclusively with highly specific amino acids, showed antibacterial activity against S. aureus strains resistant to conventional antibiotics [37]. The hexapeptide MP196 is a short sequence rich in tryptophan (W) and arginine (R) residues with chemical modifications, such as organoleptic derivatization, fatty acyl, and multivalent studies with promising antimicrobial characteristics [51]. This peptide showed antibacterial activity against VISA strains, with MIC between 16 and 64 μg/mL, and had no significant hemolytic or cytotoxic effects when evaluated against erythrocytes, rat kidney epithelial cells, and human T-cell lymphoblasts [51]. Likewise, the WR12 peptide, also composed exclusively of W and R residues, exhibited broad-spectrum antimicrobial activity, showing very strong activity against VRSA and VISA strains (MIC = 1–8 μM) [37]. DIK-8 is a short AMP composed exclusively of the amino acids isoleucine (I) and lysine (K), which showed antibacterial activity against VRSA (MIC = 8–16 μM) and VISA (MIC = 8 μM) strains, and low toxicity against mammalian cells [37]. Additionally, the design of AMPs by substitut- ing and adding special amino acids has been used to improve antimicrobial activity and reduce the detrimental impact on host cells [120]. For example, the peptide P-113 derived from the human salivary protein histatin 5, which showed antibacterial activity against VRSA and VISA strains (MIC > 64 μg/mL), had its histidine (H) residues replaced by bulky unnatural amino acids [43]. This way the Phe-P-113, Bip-P-113, Dip-P-113, and Nal-P-113 peptides were obtained, which showed an enhanced antibacterial effect against VRSA and VISA strains (Table 4). On the other hand, AMPs with added lipoamino acids have been designed, namely, lipopeptides (lipopeptide-1 to -6). These molecules have shown broad antimicrobial activity against Gram-positive bacteria, including VRSA and VISA strains. However, they have shown toxicity against embryonic and renal cells [40,43] (Table 4). Additionally, other family of artificial small lipopeptides was designed and constructed with a combination of two or three basic, cationic, and/or anionic amino acids attached to an acyl chain of 14 carbons [59]. Seven peptides of this family (C14-KK, C14-RRR, C14-LK, C14-RW, C14-WR, C14-KWI, and C14-LKK) showed antibacterial activity against VISA strain Mu50 with MIC values between 1.56 and >12.5 μM, and strong hemolytic activity against human red blood cells [59]. Finally, two short artificial peptides (RRIKA and RR) exhibited potent and rapid antimicrobial effect against VRSA and VISA clinical isolates with MIC between 2 and 32 μM [47]
Antimicrobial peptides have various physicochemical and structural properties that play a key role in regulating their antimicrobial activity, their mechanism of action, and their specificity towards molecular targets [102][103][104][105]. In this sense, AMPs with antibacterial activity against VRSA and VISA strains have different physicochemical properties in terms of amino acid sequence, charge, hydrophobicity, and isoelectric point, which determine their activity against these resistant strains (Table 5, Table 6 and Table 7). Likewise, these peptides have different structures, which allow them to be grouped into four categories: α-helical peptides, β-pleated sheet peptides, mixed-structure peptides (α-helix and β-pleated sheet) (Table 5), and peptides with atypical structure, which include cyclic and complex AMPs, as well as AMPs with unusual amino acids (Table 6 and Table 7). The physicochemical structures and properties of some peptides were reported in some of the papers included in this review. However, when a paper did not report these characteristics for any AMP, its respective prediction was made from the amino acid sequences using the servers I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER/, accessed on 1 June 2021), ThermoFisher (https://www.thermofisher.com/co/en/home/life-science/protein-biology/peptides-proteins/custom-peptide-synthesis-services/peptide-analyzing-tool.html, accessed on 1 June 2021), and CALCAMPI (https://ciencias.medellin.unal.edu.co/gruposdeinvestigacion/prospeccionydisenobiomoleculas/InverPep/public/herramientas, accessed on 1 June 2021). 3.2. AMPs Classification Based on Their Physicochemical and Structural Properties
Table 5. Structural and physicochemical properties of AMPs that showed antibacterial activity against VRSA and VISA strains.
AMP Name |
3D Structure |
Sequence |
L |
C |
IP |
H |
%H |
Reference |
Cecropin A |
|
GIGKFLHSAKKFGKAFVGEIMNS |
23 |
+6 |
10.6 |
40.19 |
43.48 |
[37] |
Agelaia-MPI |
|
INWLKLGKAIIDAL |
14 |
+1 |
9.9 |
45.73 |
64.29 |
[48] |