NOVEL ASPECTS OF THE INTERACTION BETWEEN STAPHYLOCOCCUS AUREUS BACTERIUM AND SINGLE-WALLED CARBON NANOTUBES JÁNOS FENT AND SUSAN LAKATOS DEPARTMENT OF PATHOPHYSIOLOGY, LABORATORY INSTITUTE FOR HEALTH PROTECTION, MEDICAL CENTRE OF HDF H-1134, RÓBERT KÁROLY KRT. 44, BUDAPEST, HUNGARY E-MAIL: LATYAKOS24@GMAIL.COM
Introduction
Discussion
To reveal the effect of single-walled carbon
Some SWNTs counteract to the shaking caused bacterial count decrease.
nanotubes (SWNTs) exerted on bacteria
The protecting effect is in accordance with the affinity of bacteria towards the given
close contacts are needed between them. Shaking
the
sample
ensures
probability of close contacts.
higher
nanotube, as reflected in the amount of heterocomplexes. The few amount of heterocomplex between carboxyl modified nanotube and S. aureus can be
Changing of
bacterial counts were studied as a function of
explained by the repulsive effect between their negative charges. Changing in bacterial counts upon shaking can be explained by cell adhesion to the wall of the
shaking speed.
sample container and/or by mechanical cell destruction.
Effect of 1 hour shaking on the bacterial counts
S. aureus counts after 700 RPM shaking and percent of heterocomplexes in the presence of SWNTs
PEG- and amide-modified SWNTs • practically completely counteract to the decrease of bacterial counts. • draw almost all the bacteria into heterocomplexes
carboxylated SWNT • Do not counteract to the decrease of bacterial counts. • Shaking S. aureus bacteria samples in
• Do
physiological salt solution at room
not
draw
bacteria
into
heterocomplexes
temperature for one hour results in bacterial count decrease.
Pristine SWNT • Partly counteract to the decrease of
• The
bacterial
shaking
count
speed
decrease
dependent
and
is
bacterial counts.
it
• Partly
becomes significant at 700 RPM
draw
bacteria
into
heterocomplexes
relative to that of at 0 RPM.
Materials and Methods Bacterium culture Staphylococcus aureus - Hungarian National Collection of Medical Bacteria (HNCMB 113003) S.aureus was cultivated on blood agar, 37°C, overnight, and suspended in physiological saline. Initial bacterium counts were estimated with optical density measurement (OD600=0.025)
Nanoparticles SWNT- S-purified single-walled carbon nanotubes - SES Research (Houston, TX, USA)* - Sigma–Aldrich* COOH-SWNT - Carboxylated single-walled carbon nanotubes - NanoAmor (LosAlamos, USA)* - Nanoshel (Wilmington, USA)* NH-SW - Amidated single-walled carbon nanotubes – Sigma–Aldrich PEG-SW - Pegylated single-walled carbon nanotubes – Sigma-Aldrich Nanoparticles were dispersed in physiological saline by sonication -------------------------------------------------------------------------------------------------------------------------------* The effects were independent of suppliers
Treatment of bacteria Bacteria were incubated with 0.1 mg/ml (final concentration) of respective nanoparticle at room temperature parallel with respective controls, agitated on a table-top orbital shaker (Labinco) at 500 or 700 rpm
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). Changes in bacterial counts were characterized by crossing point analysis cf: http://www.nanopaprika.eu/group/nanoposter/page/p16-28
Flow cytometry Samples were stained with Syto-9 (Live/Dead BacLight Bacterial Viability Kit, Invitrogen – Molecular Probes). Heteroaggregates were identified based on increased side-scatter of the Syto-9 positive events. cf: http://www.nanopaprika.eu/group/nanoposter/page/p16-28