**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 Grampositive 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) *Antibiotic Resistance among* Escherichia coli *Isolates, Antimicrobial Peptides and Cell Membrane… DOI: http://dx.doi.org/10.5772/intechopen.101936*

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].
