Antimicrobial Peptides Eradicate Bacterial Biofilms of Multi-Drug Resistant

Antimicrobial Peptides Eradicate Bacterial Biofilms of Multi-Drug Resistant: History

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Subjects: Biotechnology & Applied Microbiology

Contributor:

Cesar Roque-Borda

Janaína Pontes

Anna Beatriz Borges

Fernando Rogério Pavan

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	S. mutans, L. acidophilus and A. viscosus	Promising peptide for oral use as it is resistant to the gastrointestinal tract and stable in human saliva.	[110]
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.	[112]
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.	[113]
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	P. aeruginosa, K. pneumoniae, S. aureus, E. coli	Suppression of LPS-induced pro-inflammatory mediators, even at low peptide concentrations.	[120]
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	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]
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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