Antibiotic Resistance among Escherichia coli Isolates, Antimicrobial Peptides and Cell Membrane Disruption to the Control of E. coli Infections

Abstract

The treatment of Escherichia coli infections has been seriously complicated due to the appearance of multidrug-resistant isolates and the rapid distribution of extended-spectrum β-lactamase-producing species. In recent years there has been considerable effort to develop alternative therapies to traditional antibiotics for infection diseases caused by antimicrobial agents. The mechanisms by which antimicrobial compounds induce bacterial damage have been suggested to be interaction with membranes, formation of pores lined by both lipids and peptides, or by a more general “Anionic lipid clustering,” and other specific mechanisms. The major constituents of the lipid bilayer on the outer membrane of E. coli as a Gram-negative bacteria are lipopolysaccharide, zwitterionic core oligosaccharides, saturated fatty acid chains with zwitterionic phospholipid head groups, and lipid A functionalized with anionic phosphate groups. Research findings emphasize the importance of the membrane composition of E. coli in determining the susceptibility to certain antimicrobial agents, such as antimicrobial peptides (AMPs) and successful treatment.

1. Introduction

By the discovery of penicillin in 1928, the twentieth century was the golden age of antibiotics based on small molecule natural products, for instance, tetracyclines, β-lactams, and aminoglycosides [1]. These products were successful in the treatment of infectious diseases, and they saved the lives of many human beings from different types of bacterial infections. However, common antibiotics have become ineffective due to the constant evolution of most bacterial strains against them [2]. These bacterial strains can spread all around the world and lead to fatal infectious diseases because of antimicrobial resistance (AMR), which is currently one of the most crucial global health concerns [3]. Not only the antibiotics misuse and overuse have an important role in increasing AMR, but also the relatively slow pace of the development of novel antibiotics has aggravated this problem [4]. The latter reason, the so-called “discovery void,” occurred because no major class of antibiotics has been introduced since the introduction of lipopeptide antibiotics (e.g., daptomycin) in the mid-1980s. While in over 50 years no new class of antibiotics has been approved, the antibacterial treatments for Gram-negative bacteria become more difficult [2, 3, 4, 5]. In 2016, multidrug resistance (MDR) was announced as one of the major health challenges of that time by the World Economic Forum (WEF). The foundation believed without urgent action, the estimated global death because of MDR could reach 10 million by 2050 [6]. Hence, the design and synthesis of new antibiotics with new antimicrobial mechanisms is evident [7]. According to the literature, bacterial cell membranes have a critical role in modulating antibiotic resistance, during recent years, studies on bacterial cell membranes perturbed by new compounds to overcome antibiotic resistance have been developed [8].

In comparison to Gram-positive bacteria, all Gram-negative bacteria have an extra membrane that surrounds them and is called the outer membrane (OM) (Figure 1) [8]. Unlike the cytoplasmic membrane (CM), the OM is very asymmetric, containing phospholipids on the inner leaflet, and lipopolysaccharides (LPSs) on the outer leaflet [9]. LPSs are the major constituents on the OM of Gram-negative bacteria which have zwitterionic oligosaccharides as core [10], zwitterionic phospholipid head groups on saturated fatty acid chains [11], and lipid A with anionic phosphate groups [12].

Figure 1.

Comparing gram-positive and gram-negative bacterial cell membranes.

The OM is essential for cell viability and prevents the entry of harmful toxic substances by blocking permeability. LPSs play a central role in the selective permeability and integrity of OM. While many hydrophobic molecules are able to limit diffusion [13], LPSs play an important factor in providing selectivity to them. Because of the anionic phosphate groups, LPS molecules are able to form intermolecular electrostatic bonds with neighbors. The cross-bridging of neighboring LPS molecules significantly contributes to the resistance against hydrophobic antimicrobial agents. The anionic nature of lipid A seems to be the Achilles heel for OM integrity. The OM of E. coli is composed not only of LPS but also outer membrane proteins (OMP), lipoproteins (LPP), and porins [14, 15, 16]. Porins are charged proteins that allow the penetration of drugs, nutrients, and small molecules inside bacterial cells [17].

So far, intracellular processes are the target of many antibiotics to create holes in the bacterial cell envelope. In particular, there is a formidable barrier on the outer membrane (OM) of Gram-negative bacteria that must be overcome by antibiotics. There are two different pathways that help antibiotics to take through the OM, which are general diffusion porins for hydrophilic antibiotics and a lipid-mediated pathway for hydrophobic antibiotics. Some outer membrane structures such as protein and lipid, and their modifications have a striking influence on the bacterial antibiotics sensitivity and resistance [18]. The ability of OM disruption to change the rules of Gram-negative entry, overcome pre-existing and spontaneous resistance. Disruption of the OM expands the threshold of hydrophobicity compatible with Gram-negative activity to include hydrophobic molecules. Together, OM disruption overcomes many of the traditional hurdles encountered during antibiotic treatment and is a high-priority approach for further development [19].

2. Factors associated with antibiotic resistance in Escherichia coli

It is absolutely clear to us today that the antibiotic resistance of E. coli and some other bacteria involves a combination of different factors [20]. Drug-resistant E. coli can be transmitted to human beings from the environment through direct or indirect contact (e.g., consumption of contaminated food and water) [21]. The uncontrolled use of antibiotics in domestic animals, as well as dietary supplements, could be one of the main reasons for high antimicrobial resistance [22]. In addition, colonization of healthy adult workers with extended-spectrum β-lactamases (ESBLs) producing E. coli may be related to consumption of food and water contaminated with ESBL-producing bacteria [23].

The main causes of antibiotic resistance may involve aimless antibiotic use, deficiencies in health centers and infection-control programs in hospitals, insufficient staff training, poor hygiene and other preventive measures in veterinary medicine, and lack of right management steps in animal farms, that may cause a high frequency of ESBL producing E. coli isolates in human (42%) and animal sample (63%) [24].

According to the worldwide antibiotic sales database, comparing antibiotic use for the years 2000 to 2015, an evident rise from about 11 doses per 1000 inhabitants per day to almost 16 is noticed [25]. Analyzing research findings with the statistics demonstrates that the mean value of antibiotic consumption was largely impacted by mid-income and low-income countries [26]. The highest number of MDR bacterial infections were observed in these countries [20]. In the past 10 years, a growing number of resistance genes have been identified in E. coli isolates, and many of these resistance genes were received by horizontal gene transfer. E. coli acts as a donor and a recipient of resistance genes in the enterobacterial gene pool, and as a result can acquire resistance genes from other bacteria but can also pass on its resistance genes to other bacteria. AMR in E. coli is considered one of the most important disputes in both animals and humans on a global scale as a real public health concern [20].

According to research studies, proper monitoring of disposal processes in hospitals, systematic surveillance of hospital-associated infections, monitoring the consumption of antibiotics in animals, evaluation and monitoring of antibiotic-sensitivity patterns, and preparation of safe antibiotic strategies may ease more corrective steps for the control and inhibition of E. coli infections in all around the world [24].

The three most common manners by which Gram-negative bacteria develop antibiotic resistance are: (i) cleaving the antibiotic drugs such as β-lactam in the periplasmic space by secretion of enzymes like β-lactamase [27]; (ii) decreasing the size and number of the porins that facilitate drug transport [12]; and, (iii) changing the selective permeation and electrostatic field within the constriction zones of porins where the antibiotic docks first and then translocate inside the cells [28]. In attention to these facts, it seems the nature charge of the OM plays definitive roles in the interactions of electrostatic binding, charged molecules transportation, and drugs killing/inhibitory actions of wild-type and antibiotic-resistant species [29].

3. Antimicrobial peptides and alternative antimicrobial agents

The two major classes of alternate antimicrobial candidates are cationic, gene-encoded antimicrobial peptides (AMPs) [30], and, π-conjugated oligo/polyelectrolytes [29]. Initial studies suggested that bacteria still find it difficult to show resistance against the second class of antimicrobial molecules [31].

The bounded ability to cross the bacterial cell membrane is the major limitation in the subsequent development of peptides as antimicrobial agents. To overcome this problem and achieve a more efficient cellular uptake, peptides and a delivery vector were combined in a single molecule. For this purpose, proline-rich antimicrobial peptides (PrAMPs), as a part of the innate immune response [32] via the inner membrane transporters SbmA and MdtM are transported into a large panel of Gram-negative bacterial cells. Results showed that PrAMPs could be suitable carriers to transfer the other non-penetrating AMPs into the bacterial cells [33, 34]. According to this, PrAMPS are considered as a novel class of antibiotics [32, 33, 34, 35].

Antimicrobial peptides (AMPs) are one of the most promising candidates for a novel class of antibiotics [36]. Antimicrobial peptides (also called host-defense peptides) occur in nature as an ancient class of polypeptides [37]. AMPs are part of the innate immune system and exhibit antibacterial activity against Gram-negative and Gram-positive bacteria. According to this, AMPs serve as templates for the design of new antibacterial agents against multidrug resistance. Gramicidin and defense are natural AMPs that were discovered at the beginning of the twentieth century [38]. Today, lots of cationic AMPs are known to permeabilize real bacterial membranes [39]. After the emergence of antimicrobial-resistant bacteria, AMPs were considered as potential antibiotic drugs. The advantages of AMPs over conventional antibiotics and exigent need for the development of novel antibiotics lead to the upsurge of AMP research and their clinical trials activity in recent years [36]. In addition, synthetic AMPs or a variety of peptidomimetic antimicrobials have been very investigated to overcome the inherent drawbacks (e.g., stability) of peptides in physiological conditions [7].

Antimicrobial peptides have terrific chemical diversity and are based on some common structural characteristics set apart from traditional antibiotics. AMPs generally contain less than 100 amino acids, most of them including positively charged residues, such as lysine, arginine, and histidine, and more than 50% of them have a large portion of hydrophobic. In addition to the structural differences, AMPs directly target the bacterial cell membrane in most cases. Antimicrobial peptides based on their structure are classified into four different groups—α-helical, β-sheet, extended, and cyclic. For example, while some AMPs consist of a single helix or sheet entirely, others have a more complicated structure. The extended peptides are characterized by non-recognizable structural motifs and consist of specific amino acids, such as arginine, tryptophan, glycine, and histidine [40].

Natural antimicrobial peptides isolated are effective against Gram-positive and Gram-negative bacteria, enveloped and non-enveloped viruses, yeasts, fungi, molds, and parasites [41]. A single AMP may not be effective against all pathogens, however, may exhibit the same antimicrobial activity between different germs with anionic membranes. In addition, due to their mechanism of action, some isolated AMPs from natural sources can display species-specific antimicrobial activity [42]. This may be an outcome of a highly specialized environmental niche and evolutionary advantage that specific antimicrobial peptides present for survival [41]. As many antimicrobial peptides act on lipid components of the bacterial cell membrane, they often demonstrate broad-spectrum antimicrobial activity [43].

4. Molecular mechanisms of antimicrobial peptides action

Antimicrobial peptides can alter bacterial membrane properties by different mechanisms. Alteration of the bulk physical properties of the membrane is one way (Figure 2) [8]. AMPs can modify bulk properties while not having a specific target on the membrane. Changes in the spatial distribution of cell membrane molecules within or modification of a bulk physical property as intrinsic curvature or fluidity are examples of these alterations. Contrary to this, altering the bulk biophysical properties by AMPs can occur by targeting a class of particular lipids. Specific phospholipids are the potential targets which AMPs being effective against both Gram-negative and Gram-positive bacteria. These mechanisms are not completely independent of each other. For example, membrane clustering will lead to packing defects at the boundary between domains and physical curvature can drive clustering. In addition, directly targeting lipids can lead to any of the three phenomena that are mentioned.

Figure 2.

Different outcomes of AMPs on properties of the bacterial cell membrane. AMPs can affect the physical properties of the cellular membrane, such as (A) induction of membrane physical curvature [44], (B) lipid clustering, (C) prompting packing defects resulting in complete or partial loss of the permeability barrier, and (D) directly targeting components of a membrane such as lipids leading to a variety of consequences.

Antimicrobial agents also can target membrane phospholipids. Cardiolipin; CL, phosphatidylglycerol; PG, and phosphatidylethanolamine; PE, are the three main phospholipids in most bacteria. PG is the most abundant of them. It is an anionic lipid, and therefore, attracts cationic antimicrobial peptides. Modifying the PG head group by adding lysine and or alanine and reducing the negative charge on the membrane is one of the ways that bacteria use to protect themselves from these peptides and thus will be more resistant to the cationic antimicrobial peptides. In Gram-negative bacteria, PE is generally abundant. Several cyclic peptides are able to specifically bind to PE, and therefore, can be used to target these bacteria. In addition, PE and anionic lipid mixtures can create segregated clusters when the anionic lipid is bound to an AMP. The activity of antimicrobial agents that bind to cardiolipin is also based on a clustering mechanism [9]. Some AMPs can bind to either CL or PE and target specific bacteria. So, the cell membrane is a multipurpose target for AMPs that serves as targets or wards them off in resistance and provides a crucial site for toxic activities [8].

However, the head group structure of phospholipids is the same in bacteria and eukaryotes, the acyl chains in bacteria are shorter and more saturated [45]. In addition, while anionic lipids and PE are sequestered to the cytoplasmic surface of eukaryotic membranes, they are exposed to the external surface of bacterial membranes. These differences provide the feasibility of designing antibacterial agents that target specific bacterial lipids [8].

The antimicrobial activities of AMPs against various types of pathogens, including Gram-positive and Gram-negative bacteria, viruses, and fungi occur through a wide range of mechanisms, for example, membrane disruption, intracellular penetration, and immunomodulation [46]. Although AMPs may have different mechanisms of action, it is thought that their ability to act against such diverse cellular organisms is related to membrane activity [7]. The positive charge of cationic amino acid AMPs enables electrostatic interaction to the negatively charged microbial membranes [47, 48] and that the hydrophobic region is involved in the penetration of the cells [49]. The nature of the cell surface, in particular, the composition of the OM of Gram-negative bacteria has a major impact on the antimicrobial activity and efficacy of antimicrobial agents including cationic AMPs [50]. Antimicrobial peptides penetrate the bacterial membranes through several different mechanisms [51]. Briefly, AMPs binding to cell membrane break down the membrane potential, lead to alteration membrane permeability and metabolite leakage, and finally cause bacterial cell death [41].

Structural antimicrobial peptide studies have strongly suggested that the physicochemical properties of AMPs are responsible for their microbiological activities, rather than any specific amino acid residues. Since the amphiphilic topology is fundamental for insertion into the cytoplasmic membrane and disruption of cells, AMPs and their mimics have been considered as attractive targets for drug development. In particular, they are able to kill bacteria quickly and development of the bacterial antibiotic resistance is relatively difficult [7].

There is a clear phenomenological link between anionic lipid clustering and the bacterial species specificity of a number of antimicrobial agents. Direct activity on the bacterial cell membrane is the most prevalent mechanism of antimicrobial peptides [52]. Antimicrobial peptides can interact with the bacterial membranes due to their amphipathic nature. Most AMPs have a net positive charge, and therefore, are named cationic antimicrobial peptides. The binding of cationic antimicrobial peptides to the bacterial membranes is stabilized through electrostatic interactions between the cationic parts of AMPs and anionic compounds on bacterial membranes. Consequently, the bacterial membrane integrity is disrupted, causing antimicrobial peptides penetration into the membranes, and in most cases, finally forming the pores [53].

The clustering of anionic lipids to a region of the bacterial membrane would concentrate negative charge in a domain to which cationic peptides would congregate, possibly leading to the formation of a pore. After increasing the concentration of cationic antimicrobial agents on the anionic surface of the membrane, the rest of the membrane will surround the domain of anionic lipids and lead to less membrane stability under line tension. It seems that there are always domains with phase boundary defects in bacterial membranes [54] and those that would form in the presence of lipid clustering AMPs would appear suddenly [55]. Under these conditions, bacteria would not have enough time to repair this rearrangement and would be damaged as a result of the redistribution of membrane’s lipids. Consequently, disruption of functional natural domains or decreasing the availability of anionic lipids that may be necessary for the specific protein function in the cytoplasmic membrane would happen [8].

5. Antimicrobial peptides in Escherichia coli clinical trials

Naturally occurring cationic antimicrobial peptides (AMPs) and their mimics form a diverse class of antibacterial agents currently validated in preclinical and clinical settings for the treatment of infections caused by antimicrobial-resistant bacteria [56]. Clinical trials have been cautious of toxicity at these doses, as other peptide-based antibiotics (such as colistin) are toxic in high concentrations.

AMPs can be classified into three distinct approaches based on their clinical development: (i) direct antimicrobial effect on the cell membrane, (ii) indirect antimicrobial activity through immune regulation, and (iii) blocking the intracellular functions. Among forty-four peptides that have been undergoing clinical and preclinical trials, 35 target the bacterial cell membrane directly, eight affect the immune system to regulate the response of the body to infection, and three act on intracellular targets. Sixteen of these that show broad-spectrum activity, have been considered for treatment of Gram-negative infections [50].

Until 2020, FDA approves seven AMPs for clinical usages. Vancomycin and dalbavancin (vancomycin derivative) block bacterial wall synthesis, while oritavancin and telavancin (other derivatives) have both membranolytic and cell wall synthesis inhibition actions. Gramicidin D is a linear peptide that forms in the membrane. Daptomycin, colistin (polymyxin E), and cyclic lipopeptide lysis the membrane [7]. Among the AMPs that FDA approves, Buforin II that binds to nucleic acid, Colicin E1 and Bac8c that disrupt the bacterial membrane, specifically are used for E. coli treatment [41]. LPS in the outer membrane of Gram-negative bacteria, act as a protective shield and prevent from transporting the large glycopeptide antibiotics, such as vancomycin to intracellular targets. Recent studies have shown that vancomycin, when given together with other AMPs acts against vancomycin-resistant Gram-positive bacteria [57]. Corbett et al. reported SPR741 (polymyxin B derivative) that potentiates the efficacy of conventional antibiotics on Gram-negative bacteria whose spectrum of activity is limited because of bacterial outer membrane permeability obstacles [58]. Studies show the MICs in eight out of 35 antibiotics while combined with SPR741 were reduced 32 to 8000-fold against E. coli and Klebsiella pneumoniae. Interestingly, based on research E. coli becomes susceptible to vancomycin under cold stress. Moreover, the mechanism of vancomycin action to eradicate E. coli is similar to the Gram-positive bacteria, which is through inhibition of peptidoglycan biosynthesis [59]. It was also shown that silver ions can increase membrane permeability of Gram-negative bacteria and can potentiate the Gram-positive-specific antibiotic vancomycin against Gram-negative bacteria [60].

The permeability barrier of the outer membrane of Gram-negative bacteria limits the efficacy of vancomycin. So, a synergistic mechanism of action for P-113 derivatives (e.g., Bip-P-113, Dip-P-113, and Nal-P-113) and vancomycin was proposed. Study results showed, however, P-113 derivatives could perturb the outer membrane of Gram-negative bacteria and increase vancomycin entry into the resistant species. In addition, P-113 derivatives bind to the extra hydrophobic motif of lipid A and neutralize LPS protective actions [61].

Employing long-chain amino acid sequences increases the output cost of peptides and thereby the cost of research; hence, synthetic short-chain cationic peptides with potential antimicrobial activity have been attempted [62]. In particular, Indolicidin, a tridecapeptide isolated from the cytoplasmic granules of bovine neutrophils, was reported to exhibit membrane permeabilization effects and antimicrobial activity against Gram-negative and Gram-positive bacteria, fungi, HIV-1 virus, and protozoa [63, 64].

Enteroaggregative Escherichia coli (EAEC), an emerging foodborne pathogen, is implicated in endemic and epidemic diarrheal episodes. Multidrug resistance toward the antibiotics of first-line empirical therapy (fluoroquinolones and b-lactams) has been evident globally among the EAEC isolates [65, 66]. Indolicidin as an antimicrobial peptide exhibited a complete elimination of multidrug-resistant EAEC isolates in the time-kill kinetic assay by 2 h pi, while meropenem represented a similar effect after 60 min. These results indicate a unique advantage of AMPs over conventional antibiotics for better treatment of resistant antibacterial species. Studies about the antimicrobial effect of Indolicidin against MDR-EAEC strains in the G. mellonella larval model reported that Indolicidin is stable at high temperatures, in the presence of proteinase K and at physiological concentration of cationic salts. In addition, results demonstrated that while Indolicidin could eliminate MDR-EAEC completely, to be safe for commensal gut flora and eukaryotic cells [67].

Peptide 35,409 contains 20 amino acid residues and has been exhibited antibacterial activity against Escherichia coli ML35 at 22 ìM minimum inhibitory concentration (MIC). In spite, this peptide did not have cytotoxic activity against human cell lines such as HeLa and HepG2, showing hemolytic effects on human red blood cells at 1.5 μM minimum concentration. According to the low selectivity of peptide 35,409 at the therapeutic index for E. coli ML35 (calculated equal 0.045), its therapeutic use is restricted [7]. However, considering the essential need for developing new compounds with activity against microorganisms, 17-residue-long peptide 35,409-1 was obtained from peptide 35,409. This shorter peptide synthesized chemically with less charge but had greater hydrophobicity and amphipathicity properties than the original sequence. Peptide 35,409-1 sequence could inhibit E. coli multiresistant isolates and seemed to be highly selective for Gram-negative E. coli bacteria because it does not act against Gram-positive bacteria or human red blood cells. Peptide 35,409-1 permeabilizes into the bacterial membrane and leads to E. coli cytoplasmatic content leakage [7]. The interactions of AMPs with membranes have been very considered due to serious implications regarding AMPs therapeutic advantages [68, 69]. Five of the seven AMPs that are approved by the FDA are active on the membrane [70], so this mechanism must be surveyed for 35,409-1 profoundly. In comparison to conventional antibiotics, peptide 35,409-1 exhibited a lower potential for inducing resistance significantly. Therefore, it seems that peptide 35,409-1 could be a potential candidate for clinical therapy usages or developing highly selective new AMPs against Gram-negative E. coli. The stability in the presence of sera, efficacy against MRD- E. coli, and low inducing resistance of peptide 35,409-1 propose its significant clinical advantages for overcoming recent antibacterial E. coli resistance [71].

Some substitutes of histidine-rich antimicrobial peptide P-113 were developed recently [72]. Among them, Bip-P-113 showed serum proteolytic stability, enhanced salt resistance, peptide-induced permeabilization, zeta potential measurements, LPS condensed, and in vitro and in vivo neutralizing activities against LPS [70].

Polymyxin B and its derivatives are able to interact with anionic LPS in the outer membrane (OM) of Gram-negative bacteria. The derivatives of polymyxin B act as “permeabilizers” or “potentiators” and sensitize bacteria to antibiotics, Moreover, reinforce the action of other antibiotics [58]. Studies showed synergistic effects between colistin and bacteriocins that led to inhibit Gram-negative bacteria and reduction of antibiotic toxicity [73]. Ionic silver (Ag+) in silver nitrate salt (AgNO3) was found to increase the permeability of the bacterial outer membrane and sensitize Gram-negative bacteria to vancomycin [60]. Synergistic effects also have been proved between highly membrane-active AMPs and intracellular targeting antibiotics [61].

Stationary phase bacteria are much more resistant than exponentially growing cells to killing by conventional antibiotics, such as ampicillin, tetracycline, ciprofloxacin, and streptomycin [74]. The susceptibility of E. coli to human α-defensin 5 (HD5ox) was shown to be lower in the stationary phase compared to mid-log phase cells [75]. The authors suspected a correlation between bacterial susceptibility and altered cellular morphology [39]. Treated β-lactam resistant E. coli with ampicillin displayed changes in cell elasticity, membrane permeability, nanoscale morphology, and hydrophilic/hydrophobic interactions. Moreover, different ampicillin-resistant E. coli strains exhibited different traits phenotypically [76]. Therefore, exploring the interactions of conjugated molecules with wild-type and ampicillin-resistant bacterial strains is crucial since the cell drug interaction is highly dependent on the type of strains and the drug molecules applied [29].

6. Future perspectives

The development of novel antimicrobial compounds is critical for averting multidrug-resistant bacterial strains. Clinical trials showed that a large number of antimicrobial peptides have clinical potential. While AMPs have antimicrobial activity, in many cases, their clinical use has not been yet fully confirmed. Because of improper trial study design or lack of enough efficacy, many of the AMPs in clinical trials failed to progress to market. Thus, more research into the interaction between antimicrobial peptides and the human host would help to assess the true potential of these compounds.

Indeed, many of the antimicrobial compounds in clinical trials have some sort of chemical modification to improve their drug ability. The sophisticated digital libraries and modeling software would be useful for further optimization of the development of these compounds. In the future, we must try seriously to reduce the resistance to novel antimicrobial compounds. While AMPs have shown a lower tendency for resistance, this is an inevitable phenomenon due to evolutionary consequences. In fact, following the development of diverse antimicrobial agents and their mechanisms of antimicrobial action will impact antimicrobial resistance. In conclusion, it seems that through detailed monitoring and analysis of new antimicrobial drugs, limiting the use of antimicrobials in nonessential cases, and coadministration with antibiotics, the risk of appearance resistant bacterial strains will decrease in the future.

7. Conclusion

Outer membrane targeting is a revolutionary strategy for antibiotic discovery. Gram-negative pathogens will become sensitive to the range of clinically approved Gram-positive active antibiotics when their OM is perturbed. In this way, chemical space compatible with novel antimicrobial peptides would be expanded. However, there are many obstacles before performing this approach in the clinic successfully. The success or failure of this approach depends on the correct selection or development of the outer membrane perturbant and antibiotic adjutant combination. In comparison to monotherapy approaches as other combination therapies, dosage optimizing for adequate overlap in bioavailability approves difficulty and needs more complicated clinical trials [77]. Spontaneous resistance development, horizontally acquired resistance genes, and biofilm formations are all significant barriers to successful antibiotic treatment. The capacity for OM disruption to overcome many of these challenges, uniquely positioning this approach among discovery efforts in the Gram-negative resistance crisis [19].