Differential Adaptive Responses of Staphylococcus aureus to In Vitro Selection with Different Antimicrobial Peptides

Abstract

We subjected Staphylococcus aureus ATCC 29213 to serial passage in the presence of subinhibitory concentrations of magainin 2 and gramicidin D for several hundred generations. We obtained S. aureus strains with induced resistance to magainin 2 (strain 55MG) and gramicidin D (strain 55GR) that showed different phenotypic changes in membrane properties. Both exhibited a change in membrane phospholipid content and an increase in membrane rigidity, while an alteration in net charge compared to that of the control occurred only in the case of 55MG.T he growing problem of resistance to conventional antibiotics in pathogens like Staphylococcus aureus and the need for new alternatives has stimulated interest in the development of antimicrobial peptides (AMPs) as human therapeutics (1). This has led to many AMPs, such as pexiganan (analogue of magainin), gramicidins, polymyxin, nisin, daptomycin, and defensin-mimetic molecule, being entered into clinical trials (2-6). However, it has been argued in several in vitro studies and recent reports that the use of AMPs in therapeutic amounts over an extended period of time might lead to reduced susceptibility due to adaptive changes in phenotypic and genotypic characteristics of the organism (7-13). Therefore, the aim of our study was to examine whether S. aureus bacteria, on continuous exposure to sublethal concentrations of certain well-studied AMPs (magainin 2 and gramicidin D), alter their susceptibility and whether resistant strains are selected. Since the putative mechanism of action of the AMPs studied involves targeting of the bacterial membrane, analyses of various cell membrane (CM) parameters were performed to understand their role in the in vitro-selected strains. (This study was presented in part at the 113th General Meeting, American Society for Microbiology, Denver, CO, 18 to 21 May 2013.) To generate AMP-resistant strains, S. aureus ATCC 29213 was chosen and two cell lines were maintained for each peptide in Mueller-Hinton broth (MHB). Serial passage was done in two cell lines for each AMP, one in the presence of noninhibitory and increasing AMP concentrations (positive-selection line) and the other in the absence of peptide (control selection line), as described elsewhere (10). The experiment was conducted for 55 serial passages, constituting 600 to 700 bacterial generations. Each transfer was given a strain designation to indicate the serial passage number. After every 5 transfers, evolution of resistance in the positive-selection line against magainin 2 and gramicidin D was identified by determining the MIC in MHB, following NCCLS guidelines (14). Further confirmation of resistance was done by performing a bactericidal assay as described before (15,16).The development of resistance occurred for both of the AMPs studied, as indicated by a gradual rise in MIC values with increasing passage number in the positive-selection lines compared to the MICs in the respective control selection lines. At the 55th passage, the positive-selection strains showed an Ն8-and ...

Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance

“…They analyzed the effects of sub-lethal concentrations of two AMP, magainin 2 and gramicidin D, on the rise of resistant strains of S. aureus , and found that the two peptides induced different resistance strategies that result in an increase of membrane rigidity (Shireen et al, 2013). …”

Bacterial Evasion of Host Antimicrobial Peptide Defenses

“…Human β-defensin (HBD-3) triggers the upregulation of the cell wall stress response pathway in S. aureus to counteract HBD-3-induced perturbation of peptidoglycan synthesis (13). Exposure of S. aureus to sub-lethal concentrations of magainin 2 and gramicidin D promotes to resistance to these AMPs through the enhancement of membrane rigidity (218). Changes in membrane fluidity induced by incorporation of longer chain unsaturated fatty acids into the lipid bilayer (resulting in increased membrane fluidity), or carotenoid staphyloxanthin pigment (resulting in increased membrane rigidity), promotes S. aureus resistance to platelet-derived AMPs (tPMPs), or polymyxin B and human neutrophil defensin 1, respectively (219, 220).…”

Bacterial resistance to antimicrobial peptides an evolving phenomenon

“…Similarly, a previous study showed that a less fluid membrane could mediate the resistance to daptomycin in E. faecalis and E. faecium (111). It has been documented that in Listeria monocytogenes the resistance to nisin is accompanied by changes in the constituent fatty acids of the membrane, which increase its rigidity (112). Moreover, an increase in the S. aureus membrane rigidity was a common characteristic in a strain with induced resistance to magainin 2 and another to gramicidin (113).…”