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Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
Published on: May 8, 2013
Mechanistic Study of Antimicrobial Effectiveness of Cyclic Amphipathic Peptide [R4W4] against Methicillin-Resistant
Ajayi David Akinwale1,2, Keykavous Parang1, Rakesh Kumar Tiwari3
1Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences, Harry and Diane Rinker Health Science Campus, Chapman University School of Pharmacy, Irvine, CA 92618, USA.
Abstract:
Antimicrobial peptides (AMPs) are being explored as a potential strategy to combat antibiotic resistance due to their ability to reduce susceptibility to antibiotics. This study explored whether the [R4W4] peptide mode of action is bacteriostatic or bactericidal using modified two-fold serial dilution and evaluating the synergism between gentamicin and [R4W4] against Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) by a checkered board assay. [R4W4] exhibited bactericidal activity against bacterial isolates (MBC/MIC ≤ 4), with a synergistic effect with gentamicin against E. coli (FICI = 0.3) but not against MRSA (FICI = 0.75). Moreover, we investigated the mechanism of action of [R4W4] against MRSA by applying biophysical assays to evaluate zeta potential, cytoplasmic membrane depolarization, and lipoteichoic acid (LTA) binding affinity. [R4W4] at a 16 mg/mL concentration stabilized the zeta potential of MRSA -31 ± 0.88 mV to -8.37 mV. Also, [R4W4] at 2 × MIC and 16 × MIC revealed a membrane perturbation process associated with concentration-dependent effects. Lastly, in the presence of BODIPY-TR-cadaverine (BC) fluorescence dyes, [R4W4] exhibited binding affinity to LTA comparable with melittin, the positive control. In addition, the antibacterial activity of [R4W4] against MRSA remained unchanged in the absence and presence of LTA, with an MIC of 8 µg/mL. Therefore, the [R4W4] mechanism of action is deemed bactericidal, involving interaction with bacterial cell membranes, causing concentration-dependent membrane perturbation. Additionally, after 30 serial passages, there was a modest increment of MRSA strains resistant to [R4W4] and a change in antibacterial effectiveness MIC [R4W4] and vancomycin by 8 and 4 folds with a slight change in Levofloxacin MIC 1 to 2 µg/mL. These data suggest that [R4W4] warrants further consideration as a potential AMP.
Insights
The antimicrobial peptide [R4W4] demonstrates bactericidal action against bacteria, showing synergy with gentamicin against E. coli. Its mechanism involves membrane perturbation, with modest resistance developing in MRSA after serial passages.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Antimicrobial peptides (AMPs) are crucial in combating antibiotic resistance.
- The [R4W4] peptide's efficacy and mechanism against resistant bacteria require elucidation.
Purpose of the Study:
- To determine if the [R4W4] peptide is bacteriostatic or bactericidal.
- To evaluate the synergistic potential of [R4W4] with gentamicin against *E. coli* and MRSA.
- To investigate the mechanism of action of [R4W4] against MRSA.
Main Methods:
- Modified two-fold serial dilution assays to determine Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
- Checkerboard assays to assess synergistic effects between [R4W4] and gentamicin.
- Biophysical assays including zeta potential measurements, membrane depolarization, and lipoteichoic acid (LTA) binding affinity studies.
Main Results:
- [R4W4] exhibited bactericidal activity (MBC/MIC ≤ 4) against tested bacterial isolates.
- Synergistic effect observed between [R4W4] and gentamicin against *E. coli* (FICI = 0.3), but not MRSA (FICI = 0.75).
- [R4W4] demonstrated concentration-dependent membrane perturbation in MRSA and binding affinity to LTA, stabilizing zeta potential.
Conclusions:
- The [R4W4] peptide acts via a bactericidal mechanism involving bacterial membrane interaction and perturbation.
- While [R4W4] shows promise, modest resistance emerged in MRSA after serial passages, necessitating further investigation.
- The findings support [R4W4] as a potential candidate for further development as an antimicrobial agent.
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