Title: Antimicrobial activity of spherical silver nanoparticles prepared using a biocompatible macromolecular capping agent: evidence for induction of a greatly prolonged bacterial lag phase Authors
Submitted to: Journal of Nanobiotechnology (Biomed Central Open Access)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 6, 2010
Publication Date: December 21, 2010
Citation: Irwin, P.L., Martin, J., Nguyen, L.T., He, Y., Gehring, A.G., Chen, C. 2010. Antimicrobial activity of spherical silver nanoparticles prepared using a biocompatible macromolecular capping agent: evidence for induction of a greatly prolonged bacterial lag phase. Journal of Nanobiotechnology (Biomed Central Open Access). 34:1-12. Interpretive Summary: Recently, silver (Ag) nanoparticles (Np = uncharged 3-5 nm particles) have been shown to be more effective antimicrobial agents than their ionic Ag counterparts. This enhancement in relative antimicrobial activity has led researchers to consider their use in conjunction with medical products such as wound dressings by means of their fixation on textile surfaces and other materials to inhibit microbial growth such as flesh-consuming bacterial skin infections. The greatest challenge in integrating silver Nps with commercial products is attaining proper adhesion yet retaining functionality throughout the lifetime of the product. In the past we have found the adhesion of the citrate-stabilized silver Nps to textiles is poor. Furthermore, many of the options available for functionalizing the surfaces of textiles either degrade the material or affect some of the fabric’s desirable intrinsic properties. Toward achieving the goal of creating Nps with biomolecular stabilizers which better adhere to textile surfaces we have been exploring the use of biocompatible protein stabilizers such as keratin to allow for facile attachment of the nanomaterial to textile surfaces. This process produced discrete spherical silver Nps with a diameter of 3.4+/-0.74 nm that could be freeze dried and easily re-suspended in water without without additional treatment. Unfortunately, very little is known about the effect of such macromolecular stabilizers on antimicrobial properties when encapsulating silver Nps of similar size and shape. In this study, we investigate the growth kinetics and inhibition of one Gram-positive (Staphylococcus aureus) and two Gram-negative bacteria (Escherishia coli O157:H7 and Salmonella Typhimurium) in the presence of both a citrate-stabilized (traditional stabilizing agent) and keratin-capped Nps at various concentrations using a real-time spectrophotometric assay. We have shown that either keratin- or citrate-stabilized Ag Nps act by greatly increasing the bacterial lag time (T) seven- to twenty-fold. However, particle for particle, the citrus-based Ag Nps were about 5-fold more effective than the keratin-based Nps. This information will be useful to microbiologists, chemists and engineers interested in using Ag Nps for medical purposes whereupon an increase in T is significant. This work could also be used by microbiologists to induce predictable lag time delays in order to study the genetic regulation of T.
Technical Abstract: We have evaluated the antimicrobial properties of Ag-based nanoparticles (Np) using two solid platform-based bioassays and found that 10-20 uL of 0.3-3 uM keratin-based Nps (depending on the starting bacteria concentration = CI) completely inhibited the growth of an equivalent volume of ca. 1,000 to 10,000 CFU/mL Staphylococcus aureus, Salmonella Typhimurium, or Escherishia coli O157:H7 on solid surfaces. Even after one week at 37 deg C on such solid media, no growth was observed. At lower [Np], visible colonies were observed but eventually they ceased growing. To further study the physiology of this growth inhibition, we repeated these experiments in the liquid phase by observing microbial growth via optical density at 590 nm (OD) at 37 deg C in the presence [Np] = 0 to 1000 nM. To extract various growth parameters we fit all OD[t] data to a common sigmoidal function which provides measures of the beginning and final OD values, a first-order rate constant (k), as well as the time to calculated ½-maximal OD (tm) which is a function of CI, k, as well as the true microbiological lag time (T). Performing such experiments using a 96-well microtitre plate reader, we found that growth always occurred in solution but tm varied between 7 (controls) and > 20 hrs using either the citrate- ([Np] ~ 300 nM) or keratin-based ([Np] ~ 1000 nM) Nps whereupon (alpha-tm/alpha[Np])citrate ~ 50,000,000 and (alpha-tm/alpha[Np])keratin ~ 10,000,000 hoL/mole. We also found that there was little effect of Nps on S. aureus growth rates which varied only between k = 1.0/hr and 1.2/hr. To test the idea that the Nps were changing the initial concentration (CI) of bacteria (i.e., cell death), we performed probabilistic calculations assuming that the perturbations in tm were completely due to CI alone. We found that such large perturbations in tms could only come about at CIs where the probability of any growth at all was small. This result indicates that much of the Np-induced change in tm was due to a greatly increased value for T (from ca. 1 to 15-20 hrs). In the solid state assays the bacteria eventually expired probably since they were inhibited from undergoing further cell division. We propose that the difference between the solid and liquid system relates to obvious difference in the residence time of the Nps with respect to the bacterial cell membrane inasmuch as when the Np-inhibited, small colonies were selected and streaked on fresh (i.e., no Nps present) media, growth proceeded normally: e.g., one, small growth-inhibited colony resulted in a plateful of typical S. aureus colonies.