Synergistic Combinations between Human-Derived Antimicrobial Peptides and Traditional Antibiotics against Escherichia coli

Watkins, Nicholas

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Synergistic Combinations between Human-Derived Antimicrobial Peptides and Traditional Antibiotics against Escherichia coli

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The growing prevalence of antimicrobial resistance to clinical antibiotic treatments has been declared a healthcare crisis by several national and international public health agencies. The emergence of broad-range drug resistance in Escherichia coli (E. coli), a bacterium associated with severe infections of the gastrointestinal system, urinary tract, and bloodstream, has been identified as a serious threat by the Centers of Disease Control and Prevention (CDC). A limited pipeline of drug development and discovery demands that novel and creative antibiotic solutions be explored to combat multi-drug resistant bacterial infections. The human-derived antimicrobial peptide, LL37, and its synthetic short-chain analogues, FK16 and FK13, have been proposed as novel therapeutic treatments in combination with traditional antibiotics. Antimicrobial peptides (AMPs) are being widely explored as a new class of antibiotics with a wide range of efficacy against gram-positive and gram-negative bacteria. Their proposed mechanism of action, permeabilization of bacterial membranes, is particularly well-suited for combinations with antibiotics that are rendered ineffective by the outer membrane barrier of gram-negative bacteria. In addition, drug-combinations have lower risk of generating antimicrobial resistance than monotherapies. The combinatory benefits may be amplified when synergistic combinations are utilized. A checkerboard assay was used to explore antimicrobial combinations, and synergism was analyzed using the fractional inhibitory combination index (FICI). Synergistic combinations of LL37, FK16, and FK13 have been identified with the traditional antibiotics, vancomycin, polymyxin B, and colistin, against three strains of clinically isolated E. coli. These synergistic combinations were further investigated in medium with cationic concentrations resembling human blood. Vancomycin was shown to be an ineffective treatment for E. coli due to its inability to diffuse through the outer membrane. However, pre-treatment of E. coli with sub-inhibitory concentrations of LL37, FK16, or FK13 was shown to significantly decrease the minimum inhibitory concentration of vancomycin, demonstrating their membrane-permeabilizing capabilities. Synergistic combinations were particularly apparent between the antimicrobial peptides and two other membrane-targeting antibiotics, polymyxin B and colistin. The interaction of LL37, FK16, and FK13 with the outer membrane of bacteria was investigated by measuring the zeta potential of bacterial suspensions. The cationic peptides were shown to neutralize the membrane potential of bacteria at much lower concentrations than an intracellular-targeting antibiotic (vancomycin) with lower net charge. Neutralization efficiency was directly correlated to the net charge of the AMP, with LL37 having a +6 net charge and both FK16 and FK13 having a +4 net charge. The neutralization of E. coli liposaccharide molecules, the endotoxin primarily responsible for septic shock, was also investigated via zeta potential. This thesis explores and introduces information about antimicrobial resistance mechanisms, E. coli anatomy and infections, existing treatments, combinatory antibiotic methods, and antimicrobial peptides that was used to guide the research performed. The outcomes of this research suggest further investigation of LL37, FK16, and FK13 in synergistic combination with traditional antibiotics as a promising therapeutic method. Results from this research are compared to related studies in the field. Current hurdles facing the implementation of antimicrobial peptides in clinical settings are discussed. Finally, intentions for future research are presented.

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