**4. Therapeutic Potential of Antimicrobial Peptides**

Human infections are typically polymicrobial and stem mainly from oral infections, surgical wounds, diabetic foot ulcers, cystic-fibrosis-related lung infections, urinary tract infections, and otitis media infections [69–71]. Therefore, the treatment of polymicrobial infection is more challenging when compared with monomicrobial infections. In contrast to traditional antibiotics, AMPs are characterized by their ability to target both monomicrobial and polymicrobial infections without the development of cross-resistance [72]. Thus, the advantage of AMPs over traditional antibiotics is their ability to act directly on the bacterial membrane when compared to their indirect action on the intracellular targets. Other advantages of AMPs over conventional antibiotics involve the actions mediated by their different characteristics, including their ability to function against both antibiotic-resistant and sensitive microbial pathogens and their ability to target monomicrobial and polymicrobial infections without the development of cross-resistance [64,73]. However, the therapeutic success of AMPs in the treatment and prevention of bacterial infection may result from their ability to act directly on the bacterial membrane, rather than their indirect action on intracellular targets [74]. Furthermore, the ability of a single AMP to exert its antimicrobial activity via multiple mechanisms, and through different pathways [75], suggests the clinical relevance of AMPs in the treatment and prevention of microbial pathogens. However, the establishment of novel, clinically relevant therapeutic approaches that target multiple pathogens in mixed populations, thereby replacing traditional antibiotics, is tangible.

The production of endogenous AMPs by multicellular organisms constitutes a host defense mechanism against pathogenic microbes. Based on the broad spectrum of their antimicrobial activity, AMPs are promising therapeutic agents for infection control [25]. In addition to their antimicrobial activities against various pathogens, including bacteria (Gram-positive and Gram-negative bacteria), fungi, and viruses [76], many AMPs are effective against multi-drug resistant (MDR) bacteria and have low propensity for the development of resistance [77]. AMPs are also involved in the promotion and regulation of the innate immunity system [78]. Finally, the use of AMPs against biofilm formation has been widely reported over the last few decades [79]. Many AMPs kill cells in biofilms and inhibit biofilm formation via the interference with the abundant extracellular polymeric

substances (EPSs) of microbial cells. These EPSs are known to be functionally responsible for the protection of microbial cells from the surrounding environment [80].

Despite the potential therapeutic benefits of AMPs when compared with existing antibiotics, AMPs have some limitations that hinder their development for clinical use [81]. Most natural AMPs are characterized by poor absorption, distribution, metabolism, and excretion, in addition to their short half-life and low permeability and solubility [82]. Moreover, AMPs have a high production cost and a degree of toxicity, particularly in oral administration. All these properties are considered as major hindrances for the development of novel AMP-based treatments. To overcome these AMP limitations that hamper clinical application, several studies are urgently needed to improve the functional properties of AMPs, such as their absorption, distribution, metabolism, excretion, cytotoxicity, and proteolytic stability. An improvement in the functional properties of AMPs may involve the alteration of the peptide composition and the modification of their post translation of AMPs.

To that end, several technical procedures have been proposed to improve the functional properties of AMPs. These include the modification of the chemical structure of AMPs via the introduction of unusual amino acids, such as D-form amino acids, or by the acetylation or amidation of the terminal regions of AMPs. As widely reported, the modification of the chemical structure of AMPs was found to improve the stability of their peptides and prevent their proteolytic degradation [83]. Similarly, the delivery of AMPs using liposome encapsulation was found to preserve the stability of AMPs and to reduce their toxicity [84].

AMPs are an essential component of the innate skin defense mechanisms and are considered to be a first-line barrier providing protection against microbial pathogens [85]. AMPs are closely associated the with innate skin immunity and are known to regulate immunity by interacting with various immune cells and linking innate and adaptive immune responses during infection. These AMPs include, β-defensins (BD) [86], cathelicidins (human hCAP18/LL37) [87], RNase 7 [88], and secretory leukocyte protease inhibitor (SLPI) [89]. Apart from their significant role in the regulation of innate skin immunity, AMPs such as defensins and cathelicidins have also been reported to play a key role in the regulation of the innate immunity of the lung [86,90]. To that end, both defensins and cathelicidins belong to a family of AMPs, which are mostly detected in the secretion of airways [87]. The exogenous administration of defensins and cathelicidins has been reported as an effective strategy in the prevention and treatment of infection. In particular, tachyplesin III, a β-sheet peptide isolated from the hemocytes of the horseshoe crab, has been evaluated for antimicrobial activity in lung polymicrobial co-infection pneumonia [89,91].

Naturally produced AMPs in the oral cavity play key roles in the maintenance of microbial homeostasis and oral cavity health stasis [91,92]. These AMPs are characterized by their antimicrobial activity against oral bacteria, which has been evaluated against oral infections, as widely reported in several studies [91,92]. D-Cateslytin (D-Ctl), an AMP derived from L-Cateslytin, has been observed to have therapeutic potential against bacterial infection in combination with several antimicrobials [92,93] and has been reported to be an antifungal agent in the treatment of oral infections associated with Candida albicans [94].

The most important advantages of AMPs over conventional therapeutics are attributed to the potential of AMPs to offer innovative and effective solutions to the treatment of mixed populations with polyinfections and to differentiate between pathogenic bacteria and protective normal flora. Therefore, the development and evaluation of AMPs with the ability to target multiple pathogens in mixed populations without the destruction of the protective normal flora represents an important public health issue.

#### **5. Conclusions**

AMPs are characterized by their broad spectrum of antimicrobial activities and are powerful regulators of innate immunity. AMPs have a strong cell-killing efficiency on

microbial pathogens, particularly MDR bacteria. In addition, AMPs offer an alternative approach to overcome the antibiotic resistance of most microbial pathogens. Although AMPs may be able to overcome the limitations of current antibiotics due to their antimicrobial activity, their shortcomings include poor stability, toxicity, and unexplored adverse effects, which limit their clinical application. However, continued development and evaluation of functional AMPs may allow for the modification of natural AMPs, thereby facilitating the production of new AMPs with clinically desirable characteristics. Some AMPs have been approved for clinical application, while others remain under investigation in clinical trials. Therefore, the development and evaluation of AMPs with the ability to target multiple pathogens in mixed populations without the destruction of the protective normal flora represents an exciting antimicrobial therapeutic strategy.

**Author Contributions:** Conceptualization, M.H. and Y.H.; methodology, M.H.; software, M.H.; validation, M.H. and C.B.; resources, D.M.; data curation, T.W.F.; writing—original draft preparation, M.H.; writing—review and editing, N.K.; visualization, N.K. and C.B.; supervision, M.H.; project administration, M.H.; funding acquisition, Y.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.
