The Demand for New Antibiotics: Antimicrobial Peptides, Nanoparticles, and Combinatorial Therapies as Future Strategies in Antibacterial Agent Design

1Development of antibacterial resistance is considered one of the leading public health problems, since it has a significant impact on the economy worldwide. Since therapeutic options to treat infections are increasingly being limited due to antibacterial resistance, this escalates the morbidity and mortality associated with infectious diseases caused by bacteria. ESKAPE pathogens are responsible for the majority of life-threatening nosocomial infections and are capable of “escaping” the biocidal action of antimicrobial agents. The term “ESKAPE” is an acronym for six bacterial pathogens associated with multidrug resistance: Enterococcus faecium (E. faecium), Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), and Enterobacter spp.. Multidrug-resistant (MDR) bacteria are resistant to more than one antimicrobial drug, and extensively drug-resistant (XDR) bacteria are types of drug-resistant organisms that are resistant to all, or almost all, approved antimicrobial agents. For these reasons, it is essential to design and engineer new promising classes of antibiotics.

2As we described above, the development of antimicrobial resistance represents a major threat to public health, and this has been echoed by different health organizations around the globe. Antimicrobial peptides (AMPs) and nanoparticles (NPs) and the design of novel combinatorial therapies are among the new promising alternatives to fight infections caused by MDR- and XDR-resistant bacteria.

3Antimicrobial peptides are a highly diverse family of small proteins with a varying number of amino acids; they have also been referred to as cationic host defense peptides. A variety of synthetic AMPs have been synthesized in the laboratories, but there are also a wide diversity of AMPs produced by bacteria and yeast, in addition to those found naturally in animals and plants. AMPs have demonstrated to participate in a variety of biological activities, including as antimicrobial antiviral, antifungal, and anti-mitogenic agents, in addition to their antitumor and anti-inflammatory properties and their ability to act as immune modulators. Therefore, AMPs represent a potential alternative to replace a wide variety of commonly used drugs. Moreover, most of the available studies demonstrate that AMPs exhibit therapeutic activity in in vitro and in vivo models.

4The use of AMPs alone or in combination with conventional drugs has proven effective in combating different infectious agents, mainly MDR bacteria. AMPs are promising potential candidates to counteract multiresistant pathogens since they possess many advantages: they display potent microbicidal activity in the micromolar range, they have demonstrated a rapid bacterial death action, and they have low resistance selection. Their mechanism of antibacterial action is multifunctional because it alters the cell membrane and also attacks specific targets that take part in the development of different intracellular processes, such as inhibition of transcription, translation, protein synthesis, and bacterial cell wall formation. These general mechanisms of action of AMPs are displayed in Figure 1A.

5One AMP of particular interest is human cathelicidin peptide (LL-37), which has been reported to have wound-healing effects on the host in addition to exhibiting antimicrobial and anti-biofilm activity against a variety of Gram-positive and Gram-negative human pathogens. LL-37 and its derivatives are considered excellent candidates as antimicrobial therapeutic agents and have been the subject of many studies. Especially, in 2018, De Breij et al. synthesized an LL-37 derivative (SAAP-148), with potent antimicrobial activities, by replacing an amino acid from the terminal carbon of the LL-37 chain. This LL-37 derivative exhibited a minimum inhibitory concentration [MIC] between 0.4 and 12.8 μM against various ESKAPE pathogenic bacteria (e.g., E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species) without selection of resistance. Furthermore, this AMP derivative showed anti-biofilm activity against S. aureus, A. baumannii, and P. aeruginosa.

6Colistin is another important peptide antibiotic (produced Bacillus polymyxa var. colistinus) used as a last-resort drug to treat MDR infections. It has emerged as an important agent in the treatment of Gram-negative bacterial infections, especially those caused by MDR pathogens in hospitalized patients. Notably, two new colistin-derived AMPs (AA139 and SET-M33), with a mechanism similar to colistin, are in development and have shown excellent therapeutic potential both in vitro against MDR bacteria and in in vivo infection models.

7The main limiting factor for the systemic use of AMPs is their sensitivity to proteolytic digestion in different body fluids (e.g., intestinal mucosa, gastrointestinal tract, and blood plasma), which directly affect both their in vivo stability and their pharmacokinetic profile. Therefore, the search for new AMPs continues, particularly in a new class of peptides with high specificity and potency, known as “selectively targeted AMPs” (STAMPs), which show increased sensitivity to specific pathogens, demonstrating a significant increase in their bactericidal capacity without direct effects on the microbiota. The STAMP technology requires two functionally independent peptide domains integrated through a small linker. One peptide domain serves as the killing AMP moiety and the other peptide domain consisting of a high-affinity binding peptide which functions as a targeting moiety. These properties increase the binding to the surface of a targeted pathogen by enhancing the local concentration of the AMP and thus lead to improve bactericidal efficiency. In recent years, several new and promising STEMs have been developed against Streptococcus mutans, Pseudomonas aeruginosa, and Streptococcus mutants together, methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, and clinical isolates (Pseudomonas aeruginosa; ). Nonetheless, more preclinical and clinical research is needed in the development of targeted antimicrobial therapy.

8An additional alternative to fighting infections caused by antibiotic-resistant bacteria is the development of NPs since it has been amply reported that metal nanoparticles (MNPs) have antibacterial activity against ESKAPE pathogens. Some of the mechanisms of the antimicrobial mode of action of MNPs are summarized in Figure 1B. In the search for new antimicrobials to treat the ESKAPE pathogens, silver has been highlighted as a potential candidate to treat infectious diseases. Silver nanoparticles (AgNPs) possess antimicrobial activity, and they act by disturbing cell membrane permeability, interacting with sulfur- and phosphorous-containing compounds including DNA, in addition to their ability to release silver ions, contributing to the antibacterial effect. Gold (Au) nanoparticles have also been reported as effective antibacterial agents for antibiotic-resistant bacterial strains such as S. aureus, E. faecium, Enterococcus faecalis (E. faecium), Escherichia coli (E. coli), Vibrio cholerae (V. cholerae), Salmonella typhimurium (S. typhimurium), and Salmonella dysenteriae (S. dysenteriae; ).

9Among metal oxide nanoparticles, zinc oxide (ZnO) nanoparticles have shown antimicrobial activity against both Gram-negative and Gram-positive bacteria, including Bacillus subtilis (B. subtilis), S. aureus, E. coli, P. aeruginosa, and A. baumannii. On the other hand, among photocatalytic nanoparticles, titanium dioxide (TiO2) NPs have been extensively studied due to their antimicrobial activity. Several studies have reported the antimicrobial activity of TiO2 NPs against methicillin-resistant S. aureus and MDR E. coli.

10Despite the advantages that nanoparticles offer, such as a broad therapeutic index, controlled drug release, less prone to bacterial resistance, and fewer side effects than chemical antimicrobials, to treat infections caused by the ESKAPE pathogens, there are still challenges remaining to be tackled such as improvement of physicochemical properties, better pharmacokinetic profiles, and comprehensive studies on long-term exposure to humans. In terms of design and application, there is a particular interest in the generation of nanohybrids combining different metals with different antimicrobial and sensitizer agents. Metal nanoparticle-based compounds (alone or in combination with other antimicrobial agents) provide promising alternatives to combat the development of antibacterial resistance. Therefore, it is imperative to develop a comprehensive understanding of the mechanisms of action responsible for the bactericidal properties as well as the identification of the most promising antimicrobial agents for future clinical translation.

11The strategies to reduce antibiotic resistance include the limited use of antibiotics and the application of more effective antibacterial therapies. Because the time of exposure to antibiotics correlates with the development of resistance, it is necessary to use drugs with a broad spectrum of action and pharmacokinetic properties that facilitate their rapid access to the target site. However, most of the available treatments do not have all these characteristics, so an alternative option is the use of combination therapies, which can lead to a synergistic and more effective response. It has been shown that the combination of drugs leads to a considerably more potent effect, compared to the individual drug. Figure 2 displays the disadvantages of using single drugs (Figure 2A) and the advantages of using combinatorial treatments (Figure 2B).

12Combinations of AMPs with antibiotics have been reported to show synergistic effects in the treatment of bacterial infections. The mechanism of antibacterial action in these combinations involves the disruption of the outer membrane. Moreover, the use of AMPs in combinatorial treatments has certain advantages over their use as a single treatment since it has been observed that in combinatorial treatments, AMPs work as enhancers of the antimicrobial effects. This characteristic allows the reduction of their dose, and it also unlocks the bactericidal application of molecules with low molecular weight, which typically do not exhibit antimicrobial properties.

13Recent studies have demonstrated the synergistic activity of antibiotics combined with AMPs. reported the synergism and other drug interactions between melittin, a cationic amphipathic peptide, and antibiotics such as doripenem, doxycycline, colistin, and ceftazidime, against MDR isolates of A. baumannii and P. aeruginosa. Likewise, combinatorial treatments of conventional antibiotics with new synthetic peptides inspired by human cationic peptides LL-37 and thrombocidin-1 (TC-1) have shown synergistic activity against S. aureus (antibacterial and anti-biofilm activity; ). In addition, the synergistic activity of 30 short AMPs combined with several conventional antibiotics such as beta-lactam antibiotics, cephalosporins, aminoglycosides, and quinolones was tested against an MDR P. aeruginosa isolate (PA910; ). Several combinations between peptides, polymyxin B, erythromycin, and tetracycline, as well as novel variants of indolicidin were found to be synergistic. Furthermore, the results showed that a single amino acid substitution within the peptides can have a powerful effect on the ability to synergize, which represents an opportunity to design treatment strategies based on synergistic interactions.

14Metal nanoparticles should be considered as an attractive alternative to potentiate the antimicrobial effect of old and current antibiotics, since they have a high tendency to act synergistically when combined with a wide variety of antibiotics. This, in addition to the increased biocompatibility achieved by synthesizing them through green chemistry, allows considering the use of MNPs as adjuvant agents for the treatment of infectious diseases.

15In the past years, there has been a marked increase in the use of biopolymers (e.g., proteins, nucleic acids, and polysaccharide) as capping agents to functionalize and stabilize MNPs. Exopolysaccharides are biocompatible and eco-friendly biomolecules; therefore, they can be used in the synthesis of MNPs. Recently, a silver-based nanobiocomposite was synthesized using an exopolysaccharide produced by Rhodotorula mucilaginosa UANL-001L (EPS). The results showed an increased antibacterial and anti-biofilm activity of this nanobiocomposite against pathogens of clinical relevance. Moreover, nanocomposites have been synthesized through green chemistry, such as zinc (Zn) and nickel (Ni) MNPs capped with EPS as capping agents, and they have displayed interesting antimicrobial properties as well. Ni-EPS nanoparticles exhibited both antimicrobial and anti-biofilm activity against resistant MDR strains of S. aureus and P. aeruginosa. Furthermore, Zn-EPS nanoparticles showed antimicrobial activity for treatments against MDR S. aureus and P. aeruginosa.

16Among the most studied nanomaterials are silver nanoparticles due to their antimicrobial activity against Gram-positive and Gram-negative bacteria. They can be used in combinatorial treatments with currently used antibiotics for enhanced antimicrobial activity. Nonetheless, some other MNPs such as gold, copper, and zinc have been used in combination with a variety of antibiotic families to enhance bactericidal efficacy.

17Some other interesting studies of silver-based nanomaterials have been reported. A novel silver-microfibrillated cellulose biocomposite has been synthesized, and its antimicrobial activity was determined against relevant clinical strains. The results showed that this biocomposite has antimicrobial activity against Gram-negative and Gram-positive bacteria so that it could be applied in the development of biocompatible biomedical devices. Another promising approach toward the development of new antimicrobial combinatorial treatments is the use of transition metals, since they exhibit rapid and significant toxicity, at low concentrations, in different bacterial strains. Garza-Cervantes et al. evaluated the synergistic antimicrobial effects of silver/transition-metal combinatorial treatments. Their results showed combinatorial treatments that exhibited synergism (Ag–Zn, Ag–Co, Ag–Cd, Ag–Ni, and Ag–Cu) since their antimicrobial effects are increased up to 8-fold, compared to the effects observed for the treatments with the individual metals. Furthermore, Montelongo-Peralta et al. reported synergism between transition metals and antibiotics used to treat first-line drug-resistant strains of Mycobacterium tuberculosis (M. tuberculosis). Combinatorial treatments composed of isoniazid/silver exhibited a synergistic bactericidal effect in an isoniazid-resistant clinical strain of M. tuberculosis.

18Moreover, a previous study showed the ability of silver to potentiate the activity of a broad range of antibiotics against Gram-negative bacteria, as well as to restore antibiotic susceptibility (re-sensitizing) to a resistant bacterial strain. Recently, a group of researchers achieved to re-sensitize antibiotic-resistant E. coli using transition-metal micronutrients (Cu2+, Zn2+, Co2+, Cd2+, and Ni2+) combined with antibiotics (ampicillin and kanamycin). These combinatorial treatments showed a therapeutic activity and no toxicological effects in a murine topical infection model caused by antibiotic-resistant strains. The above data therefore strongly suggest that combination therapies are a potential strategy in the development of new treatments against infectious diseases.

19The search for a new generation of antimicrobials to mitigate the spread of antibiotic resistance is urgent. Current research and technology developments have promoted the improvement of antimicrobial agents that selectively target a target site (e.g., a gene, a cellular process, or a specific pathogen;; ). AMPs and MNPs exemplify a novel approach for treating infectious diseases. Nonetheless, the combinatorial treatments are considered as an excellent option for designing and developing next-generation antibacterial agents. As summary, Table 1 describes the mechanism of action, tested bacterial strains, advantages and disadvantages of AMPs, MNPs, and combinatorial treatments.

20The selection of appropriate combinatorial treatment is critical for the successful prevention of infections. The most important challenges include (i) selection of agents with ideal physical–chemical properties (hydrosolubility and chemical stability in biological fluids; ), (ii) selection of antimicrobials that display appropriate pharmacokinetics and pharmacodynamics properties, (iii) selection of biocompatible capping agents or biopolymer-based materials that enable drug release, and (iv) development of a process that ensures the stability and does not compromise the performance of the combination therapy formulation as a whole. Therefore, the design of combinatorial treatment provides a pathway to develop antimicrobial therapeutics with broad-spectrum antimicrobial activity, bactericidal instead of bacteriostatic mechanism of action, and better efficacy against MDR bacteria.

21Conceptualization, AL-B and JM-R. Writing-original draft preparation AL-B, CG-C, JG-C, JL-E, and JM-R. Graphic design, JL-E. Writing-review and editing, AL-B, CG-C, JG-C, and JM-R. Supervision, AL-B and JM-R. All authors contributed to the article and approved the submitted version.

22The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.