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Mechanisms and Fitness Costs of Resistance to Antimicrobial Peptides LL-37, CNY100HL and Wheat Germ Histones
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2013 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 7, e68875- p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2013. Vol. 8, no 7, e68875- p.
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-210752DOI: 10.1371/journal.pone.0068875ISI: 000325211000049OAI: oai:DiVA.org:uu-210752DiVA: diva2:664231
Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2016-06-15Bibliographically approved
In thesis
1. Mechanisms and Biological Costs of Bacterial Resistance to Antimicrobial Peptides
Open this publication in new window or tab >>Mechanisms and Biological Costs of Bacterial Resistance to Antimicrobial Peptides
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development.

In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4.

The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways.

Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host.

In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 61 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1226
antimicrobial peptides, antibiotic resistance, fitness cost, Salmonella Typhimurium, Staphylococcus aureus, bile, serum, pH response, growth rate, mice, phoP, pmrB, waaY, LPS, LL-37, defensins, membrane potential
National Category
Microbiology in the medical area
Research subject
urn:nbn:se:uu:diva-284119 (URN)978-91-554-9579-4 (ISBN)
Public defence
2016-06-14, C8:305, BMC, Husargatan 3, Uppsala, 13:00 (English)
Available from: 2016-05-20 Created: 2016-04-15 Last updated: 2016-06-15

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