Antimicrobial efficacy of Cecropin A (1-7)-Melittin and Lactoferricin (17- 30) against multi-drug resistant Salmonella Enteritidis

Abstract

The present study evaluated intracellular antibacterial efficacy of two short-chain cationic antimicrobial peptides (AMPs) namely, Cecropin A (1–7)-Melittin and lactoferricin (17–30) against three field strains of multi-drug resistant Salmonella Enteritidis. Initially, antimicrobial ability of both the AMPs was evaluated for their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against multi-drug resistant S. Enteritidis strains. Besides, the AMPs were evaluated for its in vitro stability (high-end temperatures, proteases, physiological concentrations of cationic salts and pH) and safety (haemolytic assay in sheep erythrocytes; cytotoxicity assay in murine macrophage RAW 264.7 cell line and human epithelioma HEp-2 cell line and beneficial gut lactobacilli). Later, a time-kill assay was performed to assess the intracellular antibacterial activity of Cecropin A (1–7)-Melittin and lactoferricin (17–30) against multi-drug resistant S. Enteritidis in RAW 264.7 and HEp-2 cells. The observed MBC values of Cecropin A (1–7)-Melittin and lactoferricin (17–30) against multi-drug resistant S. Enteritidis (128 μM; 256 μM) were generally twice or four-fold greater than the MIC values (64 μM). Further, both the AMPs were found variably stable after exposure at high-end temperatures (70 °C and 90 °C), protease treatment (trypsin, proteinase K, lysozyme), higher concentration of physiological salts (150 mM NaCl and 2 mM MgCl2) and hydrogen ion concentrations (pH 4.0 to 8.0). Both the AMPs were found non-haemolytic on sheep erythrocytes, revealed minimal cytotoxicity in RAW 264.7 and HEp-2 cells, and was tested safe against beneficial gut lactobacilli (L. acidophilus and L. rhamnosus). Intracellular bacteriostatic effect with both cationic AMPs against multi-drug resistant S. Enteritidis was evident in RAW 264.7 cells; however, in both the cell lines, the significant bactericidal effect was not observed (P > 0.05) with both cationic AMPs understudy against multi-drug resistant S. Enteritidis. Based on the results of the present study, both the cationic AMPs understudy may not be useful for the intracellular elimination of multi-drug resistant S. Enteritidis; hence, further studies such as conjugation of these AMPs with either cell-penetrating peptides (CPP) and/or nanoparticles (NPs) are warranted.