Substantiation in Enterococcus faecalis of Dose-Dependent Resistance and Cross-Resistance to Pore-Forming Antimicrobial Peptides by Use of a Polydiacetylene-Based Colorimetric Assay

Abstract

A better understanding of the antimicrobial peptide (AMP) resistance mechanisms of bacteria will facilitate the design of effective and potent AMPs. Therefore, to understand resistance mechanisms and for in vitro assessment, variants of Enterococcus faecalis that are resistant to different doses of the fungal AMP alamethicin (Alm r ) were selected and characterized. The resistance developed was dose dependent, as both doses of alamethicin and degrees of resistance were colinear. The formation of bacterial cell aggregates observed in resistant cells may be the prime mechanism of resistance because overall, a smaller cell surface in aggregated cells is exposed to AMPs. Increased rigidity of the membranes of Alm r variants, because of their altered fatty acids, was correlated with limited membrane penetration by alamethicin. Thus, resistance developed against alamethicin was an adaptation of the bacterial cells through changes in their morphological features and physiological activity and the composition of membrane phospholipids. The Alm r variants showed crossresistance to pediocin, which indicated that resistance developed against both AMPs may share a mechanism, i.e., an alteration in the cell membrane. High percentages of colorimetric response by both AMPs against polydiacetylene/lipid biomimetic membranes of Alm r variants confirmed that altered phospholipid and fatty acid compositions were responsible for acquisition of resistance. So far, this is the only report of quantification of resistance and cross-resistance using an in vitro colorimetric approach. Our results imply that a single AMP or AMP analog may be effective against bacterial strains having a common mechanism of resistance. Therefore, an understanding of resistance would contribute to the development of a single efficient, potent AMP against resistant strains that share a mechanism of resistance.

Contribution of Cell Surface Hydrophobicity in the Resistance ofStaphylococcus aureusagainst Antimicrobial Agents

“…The emergence and spread of resistance against known bacteriocins in food spoilage and pathogenic bacteria would threaten the safety of using bacteriocins as food preservatives [ 19 ]. The target bacteria adopt various strategies to overcome the effect of antimicrobial agents by either altering the cell envelope composition which no more remains a suitable target for AMPs or forming bacterial cell clumps by aggregation of large number of bacteria [ 15 , 22 , 23 ]. S. aureus is highly resistant to antimicrobial factors of the innate immune system such as cationic antimicrobial peptides (CAMPs) [ 14 ], which are produced by epithelial cells and neutrophils [ 24 , 25 ].…”

“…The pedA gene encoding pediocin was detected using PCR and sequenced [ 17 ]. Pediocin produced was purified by three-step purification procedure, which included ammonium sulfate precipitation, cation-exchange chromatography, and reverse-phase high-performance liquid chromatography [ 15 ]. The purity and antimicrobial activity of the pediocin fraction were checked using SDS-PAGE [ 18 ].…”

“…The development of resistance against bacteriocins threatens their safe use as biopreservatives. The available reports suggest that there are changes in the membrane phospholipid composition from sensitive to resistant varieties against barrel-stave pore-forming AMPs [ 12 – 15 ]. The understanding of mechanism of resistance development will help better understanding of structure of the target bacterial cell envelope and activity of bacteriocin.…”

Evidence for a Molecular Diode-based Mechanism in a Multispecific ATP-binding cassette (ABC) Exporter

“…After culturing for 24 h, we measured absorbance at 600 nm (A 600 ). We used this value to calculate the percentage of inhibition (%I) as follows: %I ϭ 1 Ϫ (Ac/Ao) ϫ 100, where Ac is the A 600 of the culture at a given concentration of drugs, and Ao is the A 600 of the positive control (19). We then determined the IC 50 as the concentration of drugs resulting in 50% inhibition.…”

Defensive remodeling: How bacterial surface properties and biofilm formation promote resistance to antimicrobial peptides

“…So far, it is suggested that peptides show cross resistance when they share common mechanisms of action [191], but this assumption should be further evaluated. In addition, in some cases the resistance to the AMPs was abolished after removing the AMP [129,190,192].…”