Antimicrobial Peptides Eradicate Bacterial Biofilms of Multi-Drug Resistant: History
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Bacterial resistance is an emergency public health problem worldwide, compounded by the ability of bacteria to form biofilms, mainly in seriously ill hospitalized patients. The World Health Organization has published a list of priority bacteria that should be studied and, in turn, has encouraged the development of new drugs. Herein, we explain the importance of studying new molecules such as antimicrobial peptides (AMPs) with potential against multi-drug resistant (MDR) and extensively drug-resistant (XDR) bacteria and focus on the inhibition of biofilm formation. This review describes the main causes of antimicrobial resistance and biofilm formation, as well as the main and potential AMP applications against these bacteria. Our results suggest that the new biomacromolecules to be discovered and studied should focus on this group of dangerous and highly infectious bacteria. Alternative molecules such as AMPs could contribute to eradicating biofilm proliferation by MDR/XDR bacteria; this is a challenging undertaking with promising prospects. 

  • Antimicrobial Peptides
  • Multi-Drug Resistant Bacteria
  • Biofilms

1. Introduction

Bacterial resistance is a current emergency problem that has claimed millions of lives in recent years [1]. The hospital environment is the main place in which these deaths occur and, normally, the cause of this lethal bacterial resistance is the non-elimination of all petrogenic microorganisms in the treatment of patients. Thus, the microorganisms remaining after this insufficient treatment proliferate despite the use of antibiotics [2]. The recurrence of cases such as this leads to the resistance of several bacteria to different drugs, allowing their massive proliferation, which, compounded with the ability to form biofilm, makes them more resistant and more difficult to fight [3]. Bacterial resistance can be innate or acquired since many bacteria have resistance genes that are expressed only when they feel threatened; and they can easily transfer these genes when found in microecosystems to other species for community survival [4].
Naturally, bacteria capable of forming biofilms are practically everywhere. To form biofilms, these microorganisms adhere to surfaces, which can be biotic (composed of living beings or parts of them) or abiotic (composed of non-living substances). The biofilms cover the surfaces with an extracellular polymeric substance (EPS) [3]. This substance has conglomerates of proteins, polysaccharides, and exogenous DNA [5]. Diseases related to these bacteria develop slowly, allowing for greater adaptation and production of biofilm and subsequently leading to severe local inflammation [6].

2. Antimicrobial Peptides and Applications

AMPs are biomolecules formed by amino acids that vary in length, usually composed of 12–50 amino acids [60]. They are known to have great antifungal, antiviral, and antibacterial properties and are capable of reducing the bacterial load and avoiding resistance due to their ability to associate rapidly with the membrane [7]. AMPs are also small protein fractions with biological activity and are part of the body’s first line of defense for pathogen inactivation [61]. The first AMPs discovered and studied were based on structures related to defensins since these molecules were produced innately when some pathogenic agent came into contact with organisms [4]. AMPs are capable of modulating the immune system and generating a better response to defend the host since previous studies would indicate this potential as a single molecule [62,63,64], or in combination with another drugs, causing an even more beneficial and less toxic synergistic effect [65,66].
AMPs have an amphipathic nature because they are composed of hydrophilic and hydrophobic regions, although they are mostly hydrophobic. This allows them to interact with biological membranes due to van der Waals interactions with the membrane lipid tails, which are natural in cell membranes [67]. Most AMPs have a cationic behavior that promotes the interaction with membrane headgroup components [68,69]. They can adopt different secondary structures that influence their mechanism according to their physicochemical characteristics (Figure 1); the mechanism of action is also influenced by the net charge, amphipathicity, and number of amino acids [70]. AMPs act against bacteria due to membrane disruption and/or pore formations. Other actions consist of the inhibition of proteins, enzymes, and cell wall synthesis when they are present in the cytoplasm [67]. Due to this ability, shown in many studies, AMPs are considered effective against MDR bacteria and fungi cells [71].
The neutralization or disassembly of lipopolysaccharides in these strategies uses AMPs, which can penetrate through the lipid bilayer, since they have a hydrophobic side and a hydrophilic side, allowing their solubilization in an aquatic environment [63]. AMPs are able to infiltrate the biofilm and cause bacterial death [72] due to their ability to electrostatically bind to lipopolysaccharides (LPS), involving interaction between two cationic amino acids (lysine and arginine) and their respective heads of groups, forming a complex. This complex destabilizes lipid groups due to the formation of multiple pores, impairing the integrity of the bacterial cell membrane [73]. This is due to the fact that the complex is stabilized through hydrophobic interactions between the hydrophobic amino acids of the peptide and the fatty acyl chains of LPS [63].
AMPs are considered an excellent alternative against resistant bacteria, in comparison with conventional antibiotics. This is due to their non-specific mechanism (ability to reach a variety of sites), which reduces the chances of resistance development. In addition, some AMPs have great anti-multi-resistant biofilm activity, interfering with the initiation of biofilm formation (preventing bacteria from adhering to surfaces) or destroying mature biofilms (killing the bacteria present or causing them to detach) [74]. 
According to Wang et al. [99], AMPs may have the ability to inhibit the expansion of biofilms, and not always eliminating all microorganisms such as Nal-P-113 against Porphyromonas gingivalis W83 biofilms formation; therefore, the authors suggest its application with other drugs currently used for the oral treatment of this potentially virulent bacterium. Likewise, some studies report that their synergistic or combined effect could improve with the inclusion or structural modification of AMP; for example, chimeric peptide-Titanium conjugate (TiBP1-spacer-AMP y TiBP2-spacer-AMP) against Streptococcus mutans, Staphylococcus epidermidis, and Escherichia coli [100], A3-APO (proline-rich AMP) combined with imipenem against ESKAPE pathogens, biofilm-forming bacteria and in vivo murine model [101,102]. In addition, it was reported that modifications in the C terminal with fatty acids could further improve the specificity and activity of AMPs against superbugs and their respective biofilms [103,104]. Another study revealed that the addition of a hydrazide and using perfluoroaromatic (tetrafluorobenzene and octofluorobiphenyl) linkers enhance the antibacterial and antibiofilm activity demonstrated against MDR and XDR A. baumannii [105]. Table 3 shows promising applications of AMPs against biofilm formation.
Table 3. Promising AMPs against biofilm formation, potential antibacterial properties, and highlights of the promising results.
Peptide Sequence and Properties Antimicrobial Activity Highlights Reference
Myxinidin2 Myxinidin3 KIKWILKYWKWS RIRWILRYWRWS P. aeruginosa, S. aureus, and L. monocytogenes Effects against a wide range of bacteria, with its mechanism of action based on its ability to insert into bacterial membranes to produce an ion channel or pore that disrupts membrane function. [106]
Colistin (colistin–imipenem and colistin–ciprofloxacin) ALYKKLLKKLLKSAKKLG Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae Bactericidal mechanism by a detergent-like effect. Recommended as a last choice in the treatment of infections caused by MDR Gram-negative bacteria because it rarely causes bacterial resistance. [107]
S4(1–16)M4Ka ALWKTLLKKVLKAAAK-NH2 P. fluorescens Greater antimicrobial effect and less toxicity than its parent peptide (dermaseptin S4) [108]
Pexiganan GIGKFLKKAKKFGKAFVKILKK-NH2 S. aureus, S. epidermidis, S. pyogenes, S. pneumoniae, E. coli and P. aeruginosa Weak anti-biofilm agent against structures formed on CL. [109]
Citropin 1.1 GLFDVIKKVASVIGGL-NH2 Potent anti-biofilm agent against S. aureus strains.
Temporin A: FLPLIGRVLSGIL-NH2 Strong activity against vancomycin-resistant strains.
Palm-KK-NH2 Palm-KK-NH2 (Palm–hexadecanoic acid residue) Effective against most strains in the form of a biofilm. Activity potentiated when combined with standard antibiotics.
Palm-RR-NH2 Palm-RR-NH2 (Palm–hexadecanoic acid residue) Efficiency potentiated when combined with standard antibiotics.
HB AMP KKVVFWVKFK + HAp-binding heptapeptide (HBP7) S. mutans, L. acidophilus and A. viscosus Adsorption capacity on the dental surface. [110]
KSLW KKVVFWVKFK Promising peptide for oral use as it is resistant to the gastrointestinal tract and stable in human saliva.
TiBP1-GGG-AMP RPRENRGRERGKGGGLKLLKKLLKLLKKL S. mutans, S. epidermidis, and E. coli. Bifunctional peptide capable of binding to titanium materials, enabling its use in biomaterials. Antibacterial functionality. [100]
BA250-C10 RWRWRWK(C10) P. aeruginosa Great activity when used in synergism with two conventional anti-pseudomonas antibiotics to inhibit the planktonic growth of four strains of P. aeruginosa. [111]
D-HB43 FAKLLAKLAKKLL Methicillin-resistant S. aureus strains High cytotoxic and hemolytic effect. [112]
D-Ranalexin FLGGLIKIVPAMICAVTKKC Methicillin-resistant S. aureus strains Effective in dose-dependent biofilm killing, but high cytotoxic and hemolytic effect.
FK13-a1 WKRIVRRIKRWLR-NH2 Methicillin-resistant S. aureus, MDR P. aeruginosa and vancomycin-resistant E. faecium Mechanism of action based on the induction of cytoplasmic membrane potential loss, permeabilization, and rupture. [113]
FK13-a7 WKRWVRRWKRWLR-NH2 Methicillin-resistant S. aureus, MDR P. aeruginosa and vancomycin-resistant E. faecium Mechanism of action based on the induction of cytoplasmic membrane potential loss, permeabilization, and rupture.
KR-12-a5 KRIVKLILKWLR-NH2 E. coli, P. aeruginosa, S. typhimurium, S. aureus, B. subtilis, S. epidermidis This peptide and its analogs kill microbial cells by inducing loss of cytoplasmic membrane potential, permeabilization, and disruption. [114]
AMP2 KRRWRIWLV E. coli, P. aeruginosa, S. aureus, E. faecalis, S. epidermidis 76% reduction of the biofilm area. [115]
GH12 GLLWHLLHHLLH-NH2 S. mutans Antimicrobial activity against cariogenic bacteria and its biofilms in vitro. [116]
TP4 FIHHIIGGLFSAGKAIHRLIRRRRR P. aeruginosa, K. pneumoniae, S. aureus Peptide driven into helix shape by an LPS-like surfactant before binding to the target. [117]
LyeTxI IWLTALKFLGKNLGKHLALKQQLAKL F. nucleatum, P. gingivalis, A. actinomycetemcomitans Active against periodontopathic bacteria. Rapid bactericidal effect, prevention of biofilm development. Can be used in the dental field. [118]
Esc(1–21) GIFSKLAGKKIKNLLISGLKG-NH2 P. aeruginosa Mechanism of action causes membrane thinning. [119]
L12 LKKLLKKLLKKL-NH2 P. aeruginosa, K. pneumoniae, S. aureus, E. coli Mechanism of action based on pore formation, inducing rapid permeabilization of bacterial membranes, inhibition of biofilm formation, disruption of drug-resistant biofilms, and suppression of LPS-induced pro-inflammatory mediators, even at low peptide concentrations. [120]
W12 WKKWWKKWWKKW-NH2 Suppression of LPS-induced pro-inflammatory mediators, even at low peptide concentrations.
WLBU2 RRWVRRVRRVWRRVVRVVRRWVRR E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species Mechanism of action based on preventing bacterial adhesion and interfering with gene expression. [121,122]
LL37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species One of the most important human AMPs that play roles in the defense against local and systemic infections. Bactericidal mechanism against Gram-positive and Gram-negative bacteria based on phospholipid-dependent bacterial membrane disruption. [121,123]
SAAP-148 LKRVWKRVFKLLKRYWRQLKKPVR E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species Promising peptide fights difficult-to-treat infections due to its broad antimicrobial activity against MDR, biofilm, and persistent bacteria. [124]
WAM-1 KRGFGKKLRKRLKKFRNSIKKRLKNFNVVIPIPLPG A. baumannii This peptide originates from LL37 AMPs and is more effective in inhibiting biofilm dispersion than its parent peptide. [125]
H4 KFKKLFKKLSPVIGKEFKRIVERIKRFLR S. aureus, S. epidermidis, S. pneumoniae, E. coli, E. faecium, K. pneumoniae, and P. aeruginosa Insignificant rates of toxicity to eukaryotic cells. [126]
RWRWRWA-(Bpa) RWRWRWA-(4-benzophenylalanine) P. aeruginosa It targets the bacterial lipid membrane, but there is no specific receptor. It only affects a range of cellular processes. [127]
Pse-T2 LNALKKVFQKIHEAIKLI-NH2 P. aeruginosa, S. aureus, E. coli Mechanism of action based on the ability to disrupt the outer and inner membrane of Gram-negative bacteria and to bind DNA. [128]
Magainin 2 GIGKFLHSAKKFGKAFVGEIMNS-NH2 A. baumannii strains Strong antibacterial activity against A. baumannii, including MDR strains. Non-toxic to mammalian cells. [129]
Magainin I GIGKFLHSAGKFGKAFVGEIMKS E. coli strains Demands more energy metabolism, translational processes, and bacterial defense in E. coli strains when present. [130]
TC19 LRCMCIKWWSGKHPK B. subtilis strains Promising peptide against Gram-positive bacteria, as its activity on the membrane interferes with several essential cellular processes, leading to bacterial death. [131]
TC84 LRAMCIKWWSGKHPK Promising peptide against Gram-positive bacteria, as its activity on the membrane interferes with several essential cellular processes, leading to bacterial death.
BP2 GKWKLFKKAFKKFLKILAC B. subtilis strains Promising peptide against Gram-positive bacteria, as its activity by perturbation of the membrane interferes with several essential cellular processes, leading to bacterial death. [132]
Nisin A MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCHCSIHVSK B. subtilis spores Application as an adjuvant to antibiotic peptides in providing a bactericidal coating for the spores. [131,133]

This entry is adapted from the peer-reviewed paper 10.3390/pharmaceutics14030642

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