Abstract
Antimicrobial peptides (AMPs) represent a potential new class of antimicrobial drugs with potent and broad-spectrum activities. However, knowledge about the mechanisms and rates of resistance development to AMPs and the resulting effects on fitness and cross-resistance is limited. We isolated antimicrobial peptide (AMP) resistant Salmonella typhimurium LT2 mutants by serially passaging several independent bacterial lineages in progressively increasing concentrations of LL-37, CNY100HL and Wheat Germ Histones. Significant AMP resistance developed in 15/18 independent bacterial lineages. Resistance mutations were identified by whole genome sequencing in two-component signal transduction systems (pmrB and phoP) as well as in the LPS core biosynthesis pathway (waaY, also designated rfaY). In most cases, resistance was associated with a reduced fitness, observed as a decreased growth rate, which was dependent on growth conditions and mutation type. Importantly, mutations in waaY decreased bacterial susceptibility to all tested AMPs and the mutant outcompeted the wild type parental strain at AMP concentrations below the MIC for the wild type. Our data suggests that resistance to antimicrobial peptides can develop rapidly through mechanisms that confer cross-resistance to several AMPs. Importantly, AMP-resistant mutants can have a competitive advantage over the wild type strain at AMP concentrations similar to those found near human epithelial cells. These results suggest that resistant mutants could both be selected de novo and maintained by exposure to our own natural repertoire of defence molecules.
Highlights
Antibiotic resistance in pathogenic bacteria is spreading at an alarming rate and we are in urgent need of new antimicrobial drugs
Minimum inhibitory concentration (MIC)-assays in refined LB were performed to confirm differences in antimicrobial peptides (AMPs) susceptibility between AMP-cycled lineages and lineages that had only been cycled in media without AMPs
Given that the initial serial passage experiment only ran for roughly 500 generations, it is alarming that several mutations connected with AMP resistance were obtained so readily
Summary
Antibiotic resistance in pathogenic bacteria is spreading at an alarming rate and we are in urgent need of new antimicrobial drugs. Interesting new candidates for future antimicrobial drugs are antimicrobial peptides (AMPs) and since their discovery in the cecropia larvae in 1980 [1], there has been major interest in developing antimicrobial drugs from peptide scaffolds. These small molecules are part of the innate immune system and found in abundance in nature, in organisms ranging from bacteria to humans. AMPs protect the producing organisms by directly killing invading pathogens or by functioning as signalling molecules and immune system modulators They are often referred to as host defence peptides and are often broad-spectrum and potent in their antimicrobial activities [2]. It has been suggested that resistance development is unlikely given that AMPs often act non- on conserved targets like the bacterial membrane, resulting in a high cost of resistance [3]
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