SINGLE-WALLED CARBON NANOTUBES ARE NOT BACTERICIDE FOR STAPHYLOCOCCUS AUREUS JÁNOS FENT AND SUSAN LAKATOS DEPARTMENT OF PATHOPHYSIOLOGY, LABORATORY INSTITUTE FOR HEALTH PROTECTION, MEDICAL CENTRE OF HUNGARIAN DEFENSE FORCES H-1134, RÓBERT KÁROLY KRT. 44, BUDAPEST, HUNGARY E-MAIL: LATYAKOS24@GMAIL.COM
INTRODUCTION
METHODS
Vast amount of literature data (cf. references) have shown that carbon nanotubes possess strong antimicrobial activity. These kinds of conclusions were mostly derived from findings that the colony forming units decreased when various bacterial species were incubated with carbon nanotubes. At the same time microscopy revealed that bacteria can form heteroaggregates with the nanotubes. Yang and his coworkers used not only the plating method but the optical density growth curve measurement. This latter method still being time consuming gives access to the time dependence of bacterial growth. In our study we combined the optical density growth curve measurements with flow cytometry in order to detect any physical changes in the bacteria due to the presence of carbon nanotubes.
Bacterium culture S.aureus was cultivated on blood agar, 37°C, overnight, and suspended in physiological saline. Initial bacterium counts were adjusted with flow cytometry (5000/µl in 20-fold dilution)
Treatment of bacteria Bacteria were incubated with 0,1 mg/ml (final concentration) of respective nanoparticle at room temperature parallel with respective controls, agitated (500 rpm)
Flow cytometry Samples were stained with Syto-9 (Live/Dead BacLight Bacterial Viability Kit, Invitrogen – Molecular Probes)
Growth curves Samples were diluted into nutrient broth medium and kinetics of absorption (OD 600 nm) changes were detected at 25°C in an ELISA reader (Multiskan Spectrum, Thermo Labsystems).
RESULTS Formation of aggregates upon nanoparticle treatment
Control
Light microscopic photograph - 600x
Light microscopic photograph - 600x Flow cytometric - SS vs. Syto dot plot
Flow cytometric - SS vs.Syto dot plot
Time dependence of bacterium growths (OD600nm) can be fitted with 5 parameter sigmoid curve and characterized by crossing points (CP)
Shifts in CPs due to the presence of nanoparticles do not indicate substantial decrease in bacteria counts relative to control
In the presence of various nanoparticles different amounts of heterocomplexes are formed
CONCLUSION Formation of heterocomplexes between S. aureus and nanoparticles tested does not inhibit growth of bacteria. Our findings do not correlate with many literature data pointing to that antimicrobial effect of the single-walled carbon nanotubes is a complex phenomenon depending not only on the physico-chemical characters of the nanoparticle but on the test circumstances as well. Therefore if the antibacterial effect of a given product is in question it needs to be tested against bacteria in all possible test setups .
MATERIALS NANOPARTICLES: CB SW CO-SW NH-SW PEG-SW
carbon black (Printex 90) - Evonik-Degussa (Essen, Germany). S-purified single-walled carbon nanotubes - SES Research (Houston, TX, USA) Carboxylated single-walled carbon nanotubes – NanoAmor (LosAlamos, USA) - Nanoshel (Wilmington, USA) Amidated single-walled carbon nanotubes – Sigma–Aldrich Pegylated single-walled carbon nanotubes – Sigma-Aldrich
Nanoparticles were dispersed in physiological saline by sonication
BACTERIUM: S. Aureus
Hungarian National Collection of Medical Bacteria (HNCMB 113003)
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