Synergistic Effect of Synthetic Cationic Anitimicrobial Peptides And Antibiotics Against Mycobacteria: An In Vitro Study
Ankur Sharma1, Rahul kumar Verma1, Shripad A. Patil2, and Amit Kumar Singh2*
1 Institute of Nano Science and Technology (INST), Phase-10, Sector-64, Mohali, Punjab 160062, India
2 National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Tajganj, Agra 282004, India
Cationic anitimicrobial peptides (CAMPs) have drawn the attention of researchers due to their broad range of activity and multiple mechanism of action.finding a peptide sequences with antimycobacterial activity toward antibiotic resistant pathogen will be helpful as new treatment strategy against tuberculosis. The HHC-8 and MM-10 CAMPs have demonstrated activity against pathogenic bacteria including mycobacteria, however, the reported MICs were high and peptides were unstable in the solution. To overcome this limitation, nanoparticles based approach was adopted in present work for sustained release, protection from proteases and other chelating agents. We studied the combined effect and try to identify the additive efficacy between nanoencapsulated CAMPs with a suboptimal concentration of anti-TB drug (rifampicin).
Synthesis of HHC-8 and MM-10 CAMPs was carried-out by Solid Phase Peptide Synthesis. HHC8 and MM-10-loaded poly(ε-caprolactone) (PCL) nanoparticles were prepared using water-in-oil-in-water (W/O/W) double-emulsion solvent evaporation method and characterized for size, morphology, encapsulation efficiency, in vitro drug release profile. The MIC determination and determination of antimicrobial synergism was performed against M. tuberculosis and M. segmatis. M. smegmatis was chosen as surrogate model for a primary screen to shortlist the cAMP for advanced screening against MDR M. tuberculosis.
The PCL-nanoparticles having hydrodynamic diameter of 319.8 ± 16.5 nm (mean ± SD, n = 3) were synthesized for the work. The Entrapment Efficiency of HHC-8 and MM-10 (ɑ-helical peptide) in optimized PCL nanoparticles was found to be 18.9±9.32% and 21.1±6.85% respectively. MIC values of HHC-8 and MM-10 were initially determined against M. smegmatis MTCC994 and M. tuberculosis H37Rv respectively. The non-encapsulated HHC-8 and MM-10 AMPs showed MIC values of >75 µg/ml and 75 µg/ml respectively. In contrast, during the same incubation time the HHC-8 and MM-10-loaded nanocarriers respectively exerted a strong bactericidal activity against M. tuberculosis and M. smegmatis. The observed MIC against M. smegmatis was 18.75 µg/ml for both HHC-8 and MM-10-loaded nanocarrier. Interestingly, the MIC values were lower against M. tuberculosis (9µg/ml) than against the M. smegmatis strain (18.75 µg/ml). The combinatorial MIC assays with rifampicin antibiotics against M. smegmatis via the checkerboard assay containing 2-fold dilutions for each compound demonstrated synergism between HHC-8 -PCL-NPs and rifampicin with an FICI value of 0.09. Notably, a low peptide concentration equivalent to 1/18th its MIC, reduced the amount of rifampicin required to inhibit M. smegmatis growth by 1/20th of its MIC. It is likely that peptide-mediated destruction of membrane integrity facilitated the entry of rifampicin to cytoplasmic targets and was responsible for the synergism observed.
These results support the published report that encapsulation of AMPs could protect peptides from proteolytic cleavage while retaining its activity at physiological salt concentration which would otherwise hindered its action. The above findings can guide the preclinical development of AMP toward becoming a new generation of urgently needed antimicrobials.