title: Two-Phase Bactericidal Mechanism of Silver Nanoparticles against Burkholderia pseudomallei.
abstract: 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.
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.|Protein and Proteomics Research Center for Commercial and Industrial Purposes, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Pawinee
authors.identifier: http://orcid.org/0000-0003-1465-4972
authors.initials: P
authors.lastname: Siritongsuk
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.|Protein and Proteomics Research Center for Commercial and Industrial Purposes, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Nuttaya
authors.initials: N
authors.lastname: Hongsing
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Saengrawee
authors.initials: S
authors.lastname: Thammawithan
authors.affiliation: Protein and Proteomics Research Center for Commercial and Industrial Purposes, Khon Kaen University, Khon Kaen, Thailand.|Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Sakda
authors.initials: S
authors.lastname: Daduang
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.|Protein and Proteomics Research Center for Commercial and Industrial Purposes, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Sompong
authors.initials: S
authors.lastname: Klaynongsruang
authors.affiliation: College of Veterinary Medicine and Emerging Pathogens Institute, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Apichai
authors.initials: A
authors.lastname: Tuanyok
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.|Protein and Proteomics Research Center for Commercial and Industrial Purposes, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Rina
authors.initials: R
authors.lastname: Patramanon
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology*; D016957:Burkholderia pseudomallei / Q000187:drug effects*; D053768:Metal Nanoparticles / Q000737:chemistry*; D008826:Microbial Sensitivity Tests; D008856:Microscopy, Fluorescence; D012834:Silver / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D012834:Silver