Two-Phase Bactericidal Mechanism of Silver Nanoparticles against Burkholderia pseudomallei.

Silver nanoparticles (AgNPs) have a strong antimicrobial activity against a variety of pathogenic bacteria. The killing mechanism of AgNPs involves direct physical membrane destruction and subsequent molecular damage from both AgNPs and released Ag+. Burkholderia pseudomallei is the causative agent of melioidosis, an endemic infectious disease primarily found in northern Australia and Southeast Asia. B. pseudomallei is intrinsically resistant to most common antibiotics. In this study, the antimicrobial activity and mechanism of AgNPs (10-20 nm) against B. pseudomallei were investigated. The MIC and MBC for nine B. pseudomallei strains ranged from 32-48 µg/mL and 96-128 µg/mL, respectively. Concentrations of AgNPs less than 256 µg/mL were not toxic to human red blood cells. AgNPs exhibited a two-phase mechanism: cell death induction and ROS induction. The first phase was a rapid killing step within 5 min, causing the direct damage of the cytoplasmic membrane of the bacterial cells, as observed by a time-kill assay and fluorescence microscopy. During the period of 5-30 min, the cell surface charge was rapidly neutralized from -8.73 and -7.74 to 2.85 and 2.94 mV in two isolates of B. pseudomallei, as revealed by zeta potential measurement. Energy-dispersive X-ray (EDX) spectroscopy showed the silver element deposited on the bacterial membrane, and TEM micrographs of the AgNP-treated B. pseudomallei cells showed severe membrane damage and cytosolic leakage at 1/5 MIC and cell bursting at MBC. During the killing effect the released Ag+ from AgNPs was only 3.9% from the starting AgNPs concentration as observed with ICP-OES experiment. In the second phase, the ROS induction occurred 1-4 hr after the AgNP treatment. Altogether, we provide direct kinetic evidence of the AgNPs killing mechanism, by which cell death is separable from the ROS induction and AgNPs mainly contributes in the killing action. AgNPs may be considered a potential candidate to develop a novel alternative agent for melioidosis treatment with fast action.

Effect of acyl chain length on therapeutic activity and mode of action of the CX-KYR-NH2 antimicrobial lipopeptide.

Peptide lipidation has proven to be an inexpensive and effective strategy for designing next-generation peptide-based drug compounds. In this study, the effect of the acyl chain length of ultrashort LiPs (CX-KYR-NH2; X=10, 12, 14 and 16) on their bacterial killing and membrane disruption kinetics was investigated. The geometric mean of the minimum inhibitory concentration (MIC) values for 4 pathogenic bacterial strains was 25 µM, with a selectivity index of 10.24 for C14-KYR-NH2. LiPs at all concentrations exhibited no cytotoxicity towards human erythrocytes, but towards Vero cells at 80 µM. All the LiPs adopted secondary structure in a membrane mimicking environment. C14-KYR-NH2 aggregated above 256 µM, while C16-KYR-NH2 did above 80 µM. All LiPs showed outer membrane permeabilization within 3 min after treatment, yet the extent and kinetics of inner membrane penetration and depolarization were dependent on the acyl chain length. Cell death subsequently occurred within 10 min, and killing activity appeared to correlate most with depolarization activity but not with outer or inner membrane permeability. AFM imaging of cells treated with C14-KYR-NH2 revealed rupture of the cell surface and cytosolic leakage depending on the length of incubation. This study highlights and follows the progression of events that occur during the membrane disintegration process over time, and determines the optimal amphipathicity of ultrashort LiPs with 12-14 carbon atoms for this membrane disrupting activity. The fast acting bactericidal properties of ultrashort LiPs with optimal chain lengths make them promising candidates for drug lead compounds.