The Influence of Osmolytes on the Antimicrobial Activity of Silver Nanoparticles

Silver nanoparticles (Ag-NPs) are gaining popularity as antimicrobial agents due to their broad- spectrum activity and lower propensity to develop resistance in bacteria. However, with increased frequency of use, there remains a possibility for bacteria to develop resistance or adaptive mechanisms against Ag-NPs overtime. Mechanisms used by bacteria to resist antimicrobial agents include efflux pumps, heat shock proteins (Hsp) and accumulation of compatible solutes, generally referred to as osmolytes. The latter mechanism is typically employed when bacteria are under osmotic stress they accumulate osmolytes either through de novo synthesis or exogenously. Osmolytes act to stabilize the bacterial cell membrane by maintaining the native protein structure, while at the same time, ensuring compatibility with other cellular structures and functions. The most common osmolytes accumulated by bacteria are glycine betaine, proline, carnitine, choline, trehalose and glutamate. Since Ag-NPs target the cell membrane it is conceivable that osmolytes may suppress its bactericidal activity. In the present study, we assess the antimicrobial efficacy of Ag-NPs in the presence of glycine betaine and proline. Exponential phase cultures (106 cfu/ml) of Escherichia coli O157:H7, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus were exposed to a predetermined the minimum inhibitory concentration (MIC) of 0.4 mM of citrate-stabilized Ag-NPs and incubated at 37°C for 48 h. This was replicated with the addition of 1 mM of either glycine betaine or proline. Growth was monitored by optical density, standard plate count, resazurin assay and LIVE/DEAD analyses. The result showed that Ag-NPs had no detectable effect on osmolyte treated cells. The average plate count of cultures supplemented with either glycine betaine or proline ranged from 108 to 109 cfu/ml after 48 h. Resazurin assay also showed that there was a significant increase in the cells supplemented with glycine betaine, 90% for E.coli O157:H7 and 44.3% for L. monocytogenes. No detectable metabolic activity was observed in the cells exposed to Ag-NPs alone. These results were corroborated by LIVE/DEAD analyses, which revealed over 86% cell viability in L. monocytogenes and E.coli O157:H7 treated with the osmolytes compared to 0.3% viability in those treated with Ag-NPs alone. SDS-PAGE analysis of the periplasmic proteins of the cells treated with Ag-NP and glycine betaine showed no qualitative difference in the protein profiles compared to the control cells grown in Trypticase Soy Broth alone. However, protein bands of cells treated with proline showed detectable difference relative to the control. The present findings suggest that glycine betaine and proline can suppress the antimicrobial activity of Ag-NPs.