Efficacy of Antibacterial Peptides Against Peptide-Resistant MRSA Is Restored by Permeabilization of Bacteria Membranes

Abstract

Clinical application of antimicrobial peptides (AMPs), as with conventional antibiotics, may be compromised by the development of bacterial resistance. This study investigated AMP resistance in methicillin resistant Staphylococcus aureus, including aspects related to the resilience of the resistant bacteria toward the peptides, the stability of resistance when selection pressures are removed, and whether resistance can be overcome by using the peptides with other membrane-permeabilising agents. Genotypically variant strains of S. aureus became equally resistant to the antibacterial peptides melittin and bac8c when grown in sub-lethal concentrations. Subculture of a melittin-resistant strain without melittin for 8 days lowered the minimal lethal concentration of the peptide from 170 μg ml-1 to 30 μg ml-1. Growth for 24 h in 12 μg ml-1 melittin restored the MLC to 100 μg ml-1. Flow cytometry analysis of cationic fluorophore binding to melittin-naïve and melittin-resistant bacteria revealed that resistance coincided with decreased binding of cationic molecules, suggesting a reduction in nett negative charge on the membrane. Melittin was haemolytic at low concentrations but the truncated analog of melittin, mel12-26, was confirmed to lack haemolytic activity. Although a previous report found that mel12-26 retained full bactericidal activity, we found it to lack significant activity when added to culture medium. However, electroporation in the presence of 50 μg ml-1 of mel12-26, killed 99.3% of the bacteria. Similarly, using a low concentration of the non-ionic detergent Triton X-100 to permeabilize bacteria to mel12-26 markedly increased its bactericidal activity. The observation that bactericidal activity of the non-membranolytic peptide mel12-26 was enhanced when the bacterial membrane was permeablized by detergents or electroporation, suggests that its principal mechanism in reducing bacterial survival may be through interaction with intracellular organelles or processes. Additionally, our results showed that the haemolytic peptide bac8c, had increased antibacterial activity at non-haemolytic concentrations when used with membrane-permeabilizing surfactants.

Methods Used for the Eradication of Staphylococcal Biofilms

“…For this reason, several approaches (such as ionic liquid coatings) were introduced to overcome those limitations [105,106]. Furthermore, SEM imaging was found to be useful in research on novel antimicrobials as it can directly indicate how they affect the cell morphology [107,108]. On the other hand, SEM is also used to confirm the presence of a biofilm.…”

“…On the other hand, SEM is also used to confirm the presence of a biofilm. In the study by Nishitani et al, SEM and bioluminescent imaging allowed them to investigate the mechanism of S. aureus proliferation and stasis during implant-associated osteomyelitis [108]. Furthermore, SEM also proved to be a useful tool in studies on S. aureus implant-associated infections [109][110][111].…”

Overcoming Antimicrobial Resistance in Bacteria Using Bioactive Magnetic Nanoparticles and Pulsed Electromagnetic Fields

“…The method is non-thermal and already made its way to food science and food processing as a sterilization or extraction method ( Bobinaite et al, 2014 ; Golberg et al, 2016 ; Pal, 2017 ). However, electroporation is an emerging technique for sensitization of bacteria to antimicrobials ( Khan et al, 2016 ; Novickij et al, 2016b ; Ravensdale et al, 2016 ). Recently, a proof of concept that it is possible to overcome the nisin-resistance of Gram-negative bacteria using electroporation and nisin-loaded NPs was presented ( Novickij et al, 2016b ).…”

Selectivity Modulation and Structure of α/aza‐β3 Cyclic Antimicrobial Peptides

“…Previous studies have explored the use of host‐defense antimicrobial peptides (AMPs) that primarily target the bacterial membrane as novel antibiotic agents . AMPs have been studied as potential therapeutic agents owing to the low frequency of bacterial resistance observed for this class of compound . Most AMPs interact with and disrupt the bacterial cell membrane, triggering cell death .…”

“…[6][7][8] AMPs have been studiedas potential therapeutica gents owingt ot he low frequency of bacterial resistanceo bserved for this class of compound. [5,[9][10][11] Most AMPs interact with and disrupt the bacterial cell membrane, triggering cell death. [12] Accordingly,acommon feature among AMPs is their amphiphilic structure, wherein residues are segregated into hydrophobic and cationic regions.…”