Abstract

The mechanisms by which various biological effects are triggered by exposure to an electromagnetic field are not fully understood and have been the subject of debate. Here, the effects of exposing typical representatives of the major microbial taxa to an 18 GHz microwave electromagnetic field (EMF) were studied. It appeared that the EMF exposure induced cell permeabilisation in all of the bacteria and yeast studied, while the cells remained viable (94% throughout the exposure), independent of the differences in cell membrane fatty acid and phospholipid composition. The resulting cell permeabilisation was confirmed by detection of the uptake of propidium iodine and 23 nm fluorescent silica nanospheres using transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Upon EMF exposure, the bacterial cell membranes are believed to become permeable through quasi-endocytosis processes. The dosimetry analysis revealed that the EMF threshold level required to induce the uptake of the large (46 nm) nanopsheres was between three and six EMF doses, with a specific absorption rate (SAR) of 3 kW/kg and 5 kW/kg per exposure, respectively, depending on the bacterial taxa being studied. It is suggested that the taxonomic affiliation and lipid composition (e.g. the presence of phosphatidyl-glycerol and/or pentadecanoic fatty acid) may affect the extent of uptake of the large nanospheres (46 nm). Multiple 18 GHz EMF exposures over a one-hour period induced periodic anomalous increases in the cell growth behavior of two Staphylococcus aureus strains, namely ATCC 25923 and CIP 65.8T.

Introduction

An electromagnetic field (EMF) is capable of triggering a variety of biological effects [1–4] upon genes [5–9], proteins and enzyme kinetics [10–14], depending on the EMF strength, frequency, and time of interaction [15, 16]. Despite many studies having been undertaken, the mechanisms responsible for the EMF effects are not fully understood and have been the subject of debate [1–4, 8, 10, 12, 16].

Whilst the bulk temperature rises that occur during EMF exposure may impact the cells, several studies have reported specific effects taking place that cannot be explained solely by this increase in bulk temperature. These effects may be a result of microthermal temperature increases that are not detectable at the macro level [4, 15, 17–20], strong polarization effects or subsequent changes in the dielectric constants being induced by the EMF. Other reports, however, suggested that exposure to EMF energy can influence the enzyme kinetics within the cells [15, 17, 21, 22]. Recently, it was reported that exposing bacterial cells to an 18 GHz EMF with a specific energy absorption rate (SAR) of approximately 5.0 kW kg-1 at temperature of 40°C induced permeability in the cell walls of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Planococcus maritimus cells without undermining the viability of the cells [20]. It is thought that the membrane permeation is dependent on the membrane fluidity, which in turn is dependent on the membrane lipid composition, cell microenvironment and the presence of charged phospholipid head groups [23, 24]. Modulation of the membrane fluidity may arise due to the ease of movement of water molecules, and the dielectric constant of water, which is affected by the EMF [25]. It has been reported that a temperature increase would cause an increase in the membrane fluidity, as confirmed by the diffusion of calcein molecules throughout the phosphatidylcholine bilayer membrane [25]. Lande et al. (1995) investigated the permeability of various artificial large unilamellar vesicles (LUVs) containing different fatty acid compositions that mimic that of biological membranes [24]. The authors concluded that membrane fluidity was affected by the presence of ionic substances [24]. Bacteria maintained their membrane fluidity by modulating the fatty acid composition in their cell walls [26]. It was also reported that charged phospholipid head groups developed a substantial potential at the lipid ˗ solution interface, influencing the concentration of ions at the interface and hence the permeability properties of the cell membrane [23]. Significant change in the dielectric constant of water due to exposure to the EMF results in the modification of the Debye length, which determines the interaction range between charged groups and thus, can destabilize the lipid bilayer.

Since the fatty acid and phospholipid compositions vary between different bacterial cell types [27, 28], it is of considerable interest to understand whether exposure to 18 GHz EMF will induce cell permeability in typical representatives of the major microbial taxa possessing different compositions of membrane fatty acids and phospholipids (S1 Table). Therefore, the aim of this study was to investigate the effects and dosage requirements of 18 GHz EMF on several prokaryotic organisms that had not been the subject of previous EMF exposure studies, such as Branhamella catarrhalis ATCC 23246 (Gram negative bacillus), Kocuria rosea CIP 71.15T (Gram positive coccus), Streptomyces griseus ATCC 23915 (Gram positive actinobacterium) [29], and the eukaryotic unicellular organism, yeast Saccharomyces cerevisiae ATCC 287. The effects resulting from prolonged multiple EMF exposures using two strains of Staphylococcus aureus bacteria as model organisms were also studied for the first time.

Materials and Methods

Cells, growth conditions and sample preparation

Branhamella catarrhalis ATCC 23246, Kocuria rosea CIP 71.15T, Staphylococcus aureus CIP 65.8, ATCC 25923, Streptomyces griseus ATCC 23915 bacterial strains, and the yeast Saccharomyces cerevisiae ATCC 287 were obtained from the American Type Culture Collection (ATCC, USA), and the Culture Collection of the Pasteur Institute (CIP, France). These cells were selected due to their distinct taxonomic affiliation and differences in their membrane lipid composition and structure. B. catarrhalis is an aerobic, non-motile Gram-negative diplococcus, opportunistic human pathogen, which is often found in the upper respiratory tract of humans. B. catarrhalis can cause respiratory infections, acute otitis media, sinusitis and infections such as endocarditis, meningitis and bacterial tracheitis [30]. The bacterium can grow well at temperatures as low as 22°C. Cells are kidney bean shaped, with a diameter of 0.6 to 1.0 μm, often appearing in pairs or as tetrads [31]. B. catarrhalis is saccharolytic, DNase, oxidase and catalase-positive with butyrate esterase activity [30]. K. rosea is an aerobic, non-motile, non-encapsulated, non-sporulated Gram-positive coccus, opportunistic human pathogen, which is commonly found on the surface of human skin, mucous membranes, in the oral cavity, and the outer ear canal [29, 32]. The bacterium can also be found in freshwater, saltwater, and soil environments [29, 32]. Cells have a diameter of approximately 1.0 to 1.8 μm and occur in pairs, tetrads or clusters [29]. Streptomyces griseus is non-motile, aerobic, Gram-positive filamentous, spore forming rod-shaped bacterium, which is a typical inhabitant of soil [33]. Streptomyces griseus is alkaliphilic, produces an aerial mycelium, which has modes of branching that eventually leads the hyphae to form chains of spores called arthospores [33, 34]. The optimum temperature for cell growth is in the range 25 to 35°C [33]. Considered relatively harmless to humans, Streptomyces griseus produces many useful secondary metabolites such as enzyme inhibitors, and they comprise 70% of naturally-occurring antibiotics [34]. Saccharomyces cerevisiae is considered to be a “model organism” for scientists because it has a fast rate of growth, being both a unicellular and eukaryotic organism [35]. This yeast has been used since ancient times in fermentation processes that convert sugar into alcohol, and baking processes as a leavening agent [35]. Yeast strains can be isolated from the surfaces of plants, surfaces of insects and warm-blooded animals, soils from all regions of the world and even in aquatic environments [35]. The optimum growth temperature is in the range 30 to 35°C [36]. Saccharomyces cerevisiae cells are round to ovoid, 5 to 10 μm in diameter, grow in either the haploid or diploid form [36]. Pure cultures were stored at -80°C in nutrient broth (NB, Oxoid Ltd., Basingstoke, Hampshire, England) with the addition of 20% (v/v) glycerol. All strains were grown on nutrient agar (NA, Oxoid Ltd., Basingstoke, Hampshire, England), brain-heart infused agar (BHIA, Becton Dickinson, Sparks, NV, USA) or potato dextrose agar (PDA, Becton Dickinson), depending on the requirements of a particular strain. Prior to each experiment, each strain was grown to the stationary phase of growth as confirmed by growth curves (data not shown) at 25°C (B. catarrhalis, K. rosea, Streptomyces griseus), 30°C (Saccharomyces cerevisiae), or 37°C (Staphylococcus aureus). Freshly prepared working suspensions were used for each independent experiment. The bacterial cell density was adjusted to OD600 0.1 in 10 mM phosphate buffered saline (PBS) at pH 7.4, using a spectrophotometer (Dynamica Halo RB-10 UV-Vis, Precisa Gravimetrics AG, Dietikon, Switzerland). The Streptomyces cerevisiae yeast cell density was adjusted to 7 × 105 cells mL-1 using a Neubauer-improved haemocytometer (Paul Marienfeld, Lauda-Königshofen, Germany).

EMF exposure

Exposure of the samples to the EMF was carried out as described elsewhere [4, 20]. In brief, 2 mL of the working suspension was transferred into a micro Petri dish (35 mm diameter, Griener Bio One, Frickenhausen, Germany). The EMF apparatus used for all experiments was a Vari-Wave Model LT 1500 (Lambda Technologies, Morrisville, NC, USA) instrument with a fixed frequency of 18 GHz. The samples were placed onto a ceramic pedestal PD160 (Pacific Ceramics, Sunnyvale, CA, USA, ε’ = 160, loss tangent < 10−3) at a position that had been identified, using electric field modelling of CST Microwave Studio 3D Electromagnetic Simulation Software (CST MWS) (CST of America, Framingham, MA, USA), to be the position that provided the most consistent heating environment (Fig 1). The calculated wavelength of the EMF in water was determined to be 2.34 mm, which is greater than the linear dimensions of each bacterial cell. The depth of penetration was calculated to be 1.04 mm, which was greater than the thickness of the bacterial suspension in the Petri dish. Hence, the possibility of subjecting the samples to non-even heating due to the presence of a non-uniform field distribution was considered to be negligible. The temperature of the suspension was constantly monitored during EMF exposures via a built-in temperature probe, a Luxtron Fiber Optic Temperature Unit (LFOTU) (LumaSense Technologies, Santa Clara, CA, USA), and a portable Cyclopes 330S infrared/thermal monitoring camera (Minolta, Osaka, Japan).

 

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