Biofilm formation by pathogen bacteria is an important host defense mechanism especially against oxidative stress, which can be fatal for the organism. This work showed that 50Hz, 1 mT ELF-EMF affected biofilm formation, surface charge (as it is directly related to electrophoretic mobility), and hydrophobic character of the cell surface. The increase in biofilm formation upon exposure to ELF-EMF implies a field induced increase in oxidative stress that the organism responded by an increase in biofilm mass. The increase in hydrophobicity and the decrease in zeta potential correlate with the probable increase in adhesion of S. epidermidis, the first step of biofilm formation. A decrease of about 25% in zeta potential indicates a reduction of electrostatic repulsive forces and a 20% increase in hydrophobicity indicates an increase in attractive forces. However the increase in biofilm mass was found to be 70% which implies that ELF-EMF might have affected the second stage of biofilm formation that is the aggregation stage where intercellular adhesins are involved. This work clearly shows that ELF-EMF is a stress factor for prokaryotic organisms and further studies are needed to elucidate the mechanism of the effect of ELF-EMF on biofilm formation.

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measuring cell-surface hydrophobicity. FEMS Microbiol. Lett. 9:29-33.6. Deighton MA, Capstick J, Domalewski E. (2001) Methods for studying biofilms produced by Staphylococcus

epidermidis. Enzymology. 336:177–95.7. Burton E, Yakandawala N, LoVetri K, Madhyastha MS. (2007) A microplate spectrofluorometric assay for

bacterial biofilms. J Ind Microbiol Biotechnol. 34: 1-4.

Effect of extremely low frequency electromagnetic fields on biofilm

formation of S.epidermidis; correlation to surface charge and

hydrophobicityBurak Aksu1, M. Esra Cüce2, G.Ayşe İnhan Garip2

1 Marmara Univ. School of Medicine, Dept. of Microbiology, Haydarpasa, Istanbul2 Marmara Univ. School of Medicine, Dept. of Biophysics, Haydarpasa, Istanbul

Cell Surface HydrofobicityThe hydrophobicity of the bacteria was expressed as the percentage of bacteria that adhered to hydrocarbon. The results of microbial adhesion to hydrocarbon are summarized in Table 1. ELF-EMF exposed bacterial cells showed an increase about 21% in cell surface hydrophobicity (Fig. 2).

Zeta Potential MeasurementZeta potential of ELF-EMF exposed S. epidermidis was seen to decrease by about 25% toward positive values with respect to control (Table 1). This result implies a reduction in electrostatic repulsion between bacterial cell and substratum.

* Results represent the mean of three independent experiments

Exposure systemELF-EMF was produced with Helmholtz coils (20 cm inner diameter, 320 turns copper wire each) by a generator that delivered 50 Hz electric current. 1 mT field intensity was adjusted relative to local field intensity (i.e. after zeroing the local magnetic field). The exposure system was placed in a room and kept at ambient (25C) temperature. The exposure system was turned on at least 1 h before experiment to achieve field stability and thermal equilibrium. Field intensity varied by 0.05 mT during exposure. For the measurement a gaussmeter ( F.W. Bell Sypris 5100 series,U.S.A ) was used. Bacterial strainS. epidermidis RP62A (ATCC 35984) was used in all experimental settings. The strain known as a strong biofilm producer.Cell Surface Hydrofobicity AssayBacterial cell-surface hydrophobicity was determined by measuring the bacterial adhesion to a hydrocarbon in a hydrocarbon-water system according to Rosenberg et al.(5). Briefly, S. epidermidis RP62A cells were exposed to1 mT ELF-EMF for 18 hours. ELF-EMF exposed and control bacterial suspensions with optical density 0.4-0.6 at 600 nm (A0) were prepared and mixed with toluene (Merck, Germany). After 15 min rest period, second O.D. measurement (A1)was obtained from the aqueous phase. Cell-surface hydrofobicity was calculated by the following formula: [1-(A1/A0)]x100.Biofilm Production AssayOvernight culture of S. epidermidis RP62A was diluted 1/100 in fresh medium and dispensed on two of sterile 96-well microtiter plates. One plate used as control, the other plate was exposed to 1 mT ELF-EMF and both were incubated for 18 hours.After incubation, wells were washed three times with PBS (pH 7.4) and allowed to dry. Wells were stained with 200 ul of 0.4% crystal violet for 15 min and then washed with PBS for three times. 33% acetic acid added to each well and the plates were read at 620 nm using a microtiter plate reader (Multiskan Ascent, Labsystems, Finland) (6,7).Zeta Potential MeasurementZeta potential of S.epidermidis RP62A was obtained from the electrophoretic mobility measurements taken with Malvern NanoZS Measurement of ELF-EMF applied and control samples were made at 25C in Tryptic Soy Broth. Measurement was made in triplicate for ecery independent experiment.Zeta potential was calculated from the Helmholtz-Smoluchowski equation δ=μ 4πη/E, where μ is the electrophoretic mobility or velocity at unit potential gradient, ηis the viscosity of the liquid, and E is the dielectric constant.

Bacterial biofilms are sessile microbial

communities formed on solid surfaces in nature.

They have important implications in medicine

and engineering. It is found that an ever

increasing number of infections such as lung

infections, dental disease, and urinary tract

infections arise from biofilm producing

microorganisms (1,2).

Biofilm formation is a step by step dynamic

process including attachment of the cells to the

surface (adhesion), increase in cell population,

and maturation of the biofilm. Once the

organism reaches critical proximity to a surface

(usually ~1 nm), the final determination of

adhesion depends on the net sum of attractive

or repulsive forces generated between the two

surfaces. These forces include electrostatic and

hydrophobic interactions, steric hindrance, van

der Waals forces, temperature, and

hydrodynamic forces. Electrostatic interactions

tend to favor repulsion, because most bacteria

and inert surfaces are negatively charged while

hydrophobicity of the strain and hydrophobic

forces favor adhesion to substratum. After

adhesion, cellular aggregation is mediated by

polysaccharide intercellular adhesin (3).

Extremely Low Frequency Electromagnetic

Fields (ELF-EMF, 300Hz) have been shown to

affect living systems. For prokaryotic systems,

the exposure to electromagnetic fields produces

oxidative stress effects causing phenotypic and

transcriptional changes and decrease in growth

rate on free cells.

Staphylococcus epidermidis is a gram-positive

bacteria commonly found in normal individuals’

skin, eye and mucosal surfaces and is one of

the leading pathogens of hospital related

infections for its propensity to infect biomedical

implants and transcutaneous devices.

Since the process of bacterial attachment to an

available surface (living or abiotic) is

determined by the nonspecific repulsive

electrostatic and attractive hydrophobic

interactions and since it was previously shown

that (4) ELF-EMF changed the surface physical

properties, this work aimed to determine the

effect of Extremely Low Frequency

Electromagnetic Fields (ELF-EMF) on the biofilm

mass, the surface charge and hydrophobicity of

S.epidermidis.

INTRODUCTION METHODS and MATERIALS

CONCLUSIONS

RESULTS (cont’d)

REFERENCES

Control

Fig. 1

ELF-EMF exposed

RESULTS

Biofilm ProductionIntensity of biofilm produced by S.epidermidis was significantly increased with ELF-EMF exposure (Fig.1). The results of biofilm production are also showed in Table 1. Under the ELF-EMF exposure, bacterial biofilm formation was increased about 68% compared to control.

Assay Control* ELF-EMF expsd*

%difference

Biofilm production (O.D. at 620 nm)

1.213 2.036 +67.9 8.8

Cell surface hydrophobicity

45.26 54.62 +20.7 5.1

Zeta potential (mV) - 10.1 - 7.6 +24.7 1.0

Table 1.Effect of ELF-EMF on biofilm production and

cell surface characteristics

T0 T1

Fig.2