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Mechanisms and Biological Costs of Bacterial Resistance to Antimicrobial Peptides
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Dan I Andersson)
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
Keyword [en]
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: urn:nbn:se:uu:diva-284119ISBN: 978-91-554-9579-4 (print)OAI: diva2:920021
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
List of papers
1. Mechanisms and Fitness Costs of Resistance to Antimicrobial Peptides LL-37, CNY100HL and Wheat Germ Histones
Open this publication in new window or tab >>Mechanisms and Fitness Costs of Resistance to Antimicrobial Peptides LL-37, CNY100HL and Wheat Germ Histones
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-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.

National Category
Natural Sciences
urn:nbn:se:uu:diva-210752 (URN)10.1371/journal.pone.0068875 (DOI)000325211000049 ()
Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2017-12-06Bibliographically approved
2. Fitness of Salmonella mutants resistant to antimicrobial peptides
Open this publication in new window or tab >>Fitness of Salmonella mutants resistant to antimicrobial peptides
2015 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 70, no 2, 432-440 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: To examine the effects of mutations in the waaY, phoP and pmrB genes, which confer resistance to antimicrobial peptides (AMPs), on fitness of Salmonella Typhimurium. Methods: Survival during low pH, oxidative stress, stationary-phase incubation, exposure to serum and bile and growth in mice and laboratory media were determined by time-kills, disc inhibition assays, competition experiments and optical density measurements. Results: Individual mutations in the waaY gene (involved in LPS core biosynthesis) and in the phoP and pmrB genes (part of two different two-component regulatory systems, phoPQ and pmrAB) conferred no or minor effects on bacterial survival during stressful in vitro conditions or in mice. In contrast, a waaY-phoP-pmrB triple mutant was compromised under most assay conditions. Conclusions: Results from this study show that AMP resistance can be cost-free, as assessed by several assays that attempt to mimic the conditions a bacterium might encounter within a host. Our findings imply that future therapeutic use of AMPs could select for fit mutants with cross-resistance to human defence peptides and that potential resistance development in response to therapeutic use of AMPs needs to be carefully monitored.

Salmonella Typhimurium, resistance, bile, serum, mice, pH response, oxidative stress, growth rate
National Category
Pharmacology and Toxicology
urn:nbn:se:uu:diva-249022 (URN)10.1093/jac/dku423 (DOI)000350211600011 ()25362575 (PubMedID)
Available from: 2015-04-24 Created: 2015-04-10 Last updated: 2017-12-04
3. Prolonged antimicrobial peptide exposure selects for Staphylococcus aureus resistance to human defense peptides
Open this publication in new window or tab >>Prolonged antimicrobial peptide exposure selects for Staphylococcus aureus resistance to human defense peptides
Show others...
2016 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723Article in journal (Other academic) Submitted
Abstract [en]

To combat the rapid increase in multi-drug resistant bacterial pathogens, the clinical development of antimicrobial peptides (AMPs) with broad-spectrum bactericidal activity is currently under evaluation. However, many of these closely resemble products of the host innate immune system, and the ramifications of prolonged bacterial exposure to host-derived AMPs are not fully understood. Here we show that in vitro serial passage of a clinical USA300 methicillin-resistant Staphylococcus aureus strain in a hostmimicking environment containing host-derived AMPs results in the selection of stable AMP-resistance. This phenotype coincided with diminished susceptibility to clinically prescribed antibiotics and occurred in the absence of a fitness cost to the bacterium. Further, AMP-resistant S. aureus were capable of causing invasive disease in mice and were less susceptible to human defensins. These findings suggest that therapeutic use of AMPs could select for virulent mutants with cross-resistance to both human defensins and antibiotics, ultimately rendering us more susceptible to infection. Thus, therapeutic use of AMPs and the implications of cross-resistance need to be carefully monitored and evaluated.

antimicrobial peptides, host defense peptides, antibiotic resistance
National Category
Pharmacology and Toxicology
urn:nbn:se:uu:diva-281683 (URN)
Available from: 2016-04-12 Created: 2016-03-29 Last updated: 2017-11-30Bibliographically approved

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