Combination treatment can hinder the evolution of resistance to antimicrobial peptides

Abstract

Antibiotic resistant microbial pathogens are becoming a major threat to human health. Therefore, there is an urgent need to develop new alternatives to conventional antibiotics. One such promising alternative is antimicrobial peptides (AMPs), which are produced by virtually all organisms and typically inhibit bacteria via membrane disruption. However, previous studies demonstrated that bacteria can rapidly develop AMP resistance. Here, we study whether combination therapy, known to be able to inhibit the evolution of resistance to conventional antibiotics, can also hinder the evolution of AMP resistance. To do so, we evolved the opportunistic pathogen S. aureus in the presence of individual AMP, AMP pairs, and a combinatorial antimicrobial peptide library. Treatment with some AMP pair indeed hindered the evolution of resistance compared with individual AMPs. In particular, resistance to pairs was delayed when resistance to the individual AMPs came at a cost of impaired bacterial growth, and did not confer cross-resistance to other tested AMPs. The lowest level of resistance evolved during treatment with the combinatorial antimicrobial peptide library termed random antimicrobial peptide mixture, which contains more than a million different peptides. A better understanding of how AMP combinations affect the evolution of resistance is a crucial step in order to design 'resistant proof' AMPs cocktails that will offer a sustainable treatment option for antibiotic resistant pathogens.

Differences in Relevant Physicochemical Properties Correlate with Synergistic Activity of Antimicrobial Peptides

“…Bacterial resistance to individual AMPs is rare yet possible, 31−34 while bacterial resistance is less probable to AMP−AMP combinations. 23,35 This is one of the reasons why AMP−AMP combinations are more likely to not only be more effective than individual AMPs 36−38 but also serve as long-term antimicrobials in contrast to traditional antibiotics. 39−41 Several possible sources of cooperativity have been proposed.…”

“…This is currently accomplished by using several classes of specific small organic molecules that are generally called antibiotics . However, in the last 30 years, we have witnessed an increasing resistance to antibiotics in bacteria, which threatens to severely decrease our ability to protect human health. − These alarming trends stimulated a broad search for novel antibacterial agents and techniques. , Antimicrobial peptides (AMPs), which are produced by multicellular organisms as part of their immune responses to external infections, came out as promising alternatives to antibiotics. − Some organisms, e.g., frogs, produce a wide variety of AMPs in their skin secretions. , AMPs are relatively short peptide-chain molecules with typically large fractions of separated positively charged and hydrophobic residues that exhibit activities against multiple classes of bacteria, fungi, viruses, and even cancer. − It was also observed that combinations of some specific types of AMPs frequently work much more efficiently than single-type peptides. − Although it is known that, in contrast to single AMPs, AMP combinations are less toxic, can better hinder the ability of bacteria to develop resistance, and can associate to bacterial cells faster, the microscopic mechanisms of AMP–AMP cooperativity remain not well understood. − …”

“…The microscopic origin of antibacterial cooperativity between different types of AMPs is unclear. Bacterial resistance to individual AMPs is rare yet possible, − while bacterial resistance is less probable to AMP–AMP combinations. , This is one of the reasons why AMP–AMP combinations are more likely to not only be more effective than individual AMPs − but also serve as long-term antimicrobials in contrast to traditional antibiotics. − Several possible sources of cooperativity have been proposed. It was suggested, based on experimental findings, that two AMPs might be synergistic because they individually target different types of bacteria [e.g., Gram-positive and Gram-negative.…”

Differences in Relevant Physicochemical Properties Correlate with Synergistic Activity of Antimicrobial Peptides

“…AMPs are relatively short peptide-chain molecules with large fractions of separated positively charged and hydrophobic residues that exhibit activities against multiple classes of bacteria, fungi, viruses, and even cancer [10][11][12][13][14]. It was also observed that combinations of some specific types of AMPs frequently work much more efficiently than single-type peptides [15][16][17][18][19]. Although it is known that AMPs combinations are less toxic, can hinder the ability of bacteria to develop resistance, and can associate to bacterial cells faster, the mechanisms of AMPs cooperativity remain not well understood [18][19][20][21].…”

“…It was also observed that combinations of some specific types of AMPs frequently work much more efficiently than single-type peptides [15][16][17][18][19]. Although it is known that AMPs combinations are less toxic, can hinder the ability of bacteria to develop resistance, and can associate to bacterial cells faster, the mechanisms of AMPs cooperativity remain not well understood [18][19][20][21].…”