Clinical potential of engineered cationic antimicrobial peptides against drug resistant biofilms

Biofilms are involved in the pathogenesis of 80% of microbial infections in humans [1]. This prevalence is of major clinical importance as microbial populations growing in biofilms are far more tolerant to traditional antibiotics than when growing planktonically due to altered metabolic programs and increased expression of antimicrobial defenses [2]. Biofilms are a major source of recurrent and chronic infections, leading to tens of millions of infections and hundreds of thousands of deaths in the USA each year [1]. The majority of nosocomial infections are caused by biofilm colonization of medical implants, for which removal of the colonized implant is often the only effective treatment [3]. Additionally, biofilms are often polymicrobial [4], making treatment more complex as well as possibly further modulating antimicrobial susceptibility [5]. Many strategies to combat biofilm-associated infections have been proposed [6], focusing on preventing biofilm biogenesis as well as disrupting established biofilms. Towards biofilm prevention, compounds capable of blocking microbial adhesion to host or medical implant structures or inhibiting production of extracellular matrix (ECM) are ideal. For biofilm disruption, compounds capable of destabilizing ECM, interfering with microbial community communication to initiate dispersal, or killing slow-growing/persister cells are desirable. Antimicrobial peptides (AMPs), especially engineered cationic antimicrobial peptides (eCAPs), are able to perform many of these functions, including killing cells in biofilms, disrupting ECM production and stability, and interfering with microbial communication and as such have begun to be more recognized for their potential as antibiofilm antimicrobial agents [7]. Cationic AMPs, or cationic host defense peptides, are an incredibly diverse family of antimicrobial compounds, with approximately 4000 discovered to date [8], and are widely distributed in nature, constituting a major aspect of the immune defense system [9]. While some of these peptides display effective killing in vitro, efficacy in vivo is often limited. This loss of activity is largely due to the fact that many physiological conditions deactivate or inhibit natural AMPs [10]. Thus, the reason for the diversity of AMPs in nature is perhaps that they have evolved for activity in particular biological environments or for activity against specific microbial species common to those sites. Additionally, many natural AMPs have immunomodulatory functions [9], and this requirement for dual functionality may constrain their ability to maintain microbial killing in diverse physiological environments. eCAPs are synthetic peptides designed based on natural AMP sequences and structures that can overcome many of these limitations, as rational design has yielded peptides capable of maintaining killing activity in a wide range of environments and against a breadth of microbial species and strains [6]. Initial design of an eCAP should include consideration of the potential treatment environment; for instance, an eCAP produced for treatment of biofilms in the lungs of cystic fibrosis patients should maintain activity in low pH, high sodium chloride concentrations, and the presence of mucus that inactivate many natural AMPs [11]. Some eCAPs have also been shown to modulate inflammation to increase clearance of microbial pathogens, which can contribute to their antimicrobial efficacy [9]. Most AMPs, including eCAPs, are generally thought to kill microbial pathogens via membrane disruption. This mechanism of action is a key component of the potential efficacy of eCAPs for treatment of biofilm infections. Traditional antibiotics require bacteria to be actively growing in order to kill, making them ineffective against slow-growing or persister cells that are common in mature biofilms [2]. Membrane disruption kills largely regardless of metabolic status, and accordingly eCAPs have been shown to efficiently kill persisters [10]. An additional detrimental effect of the inability of traditional antibiotics to kill slow-growing cells is the potential for highly tolerant microbes to develop resistance to antibiotics that they are exposed to for long durations. eCAPs kill rapidly, on the seconds to minutes timescale [12,13]. The rates of resistance to eCAPs are generally low, while the mutants that do arise display variable crossresistance development rates for natural AMPs [14], which is a topic under intense investigation [15,16]. To attempt to limit cross-resistance, an intriguing strategy is to utilize eCAPs in conjunction with traditional antibiotics, as the distinct mechanisms of action often result in additive or synergistic activity [11,17]. One major benefit of the dual treatment approach is that traditional antibiotics may be able to kill dispersed or planktonic cells, which are more metabolically active, while eCAPs can kill the remaining slow-growing or persister cells [6].