Bacterial growth rates are influenced by cellular characteristics of individual species when immersed in electromagnetic fields
Lucas W.E. Tessaro, Nirosha J. Murugan. Michael A. Persinger. Bacterial growth rates are influenced by cellular characteristics of individual species when immersed in electromagnetic fields. Microbiological Research. 172:26-33. March 2015,
Abstract
Previous studies have shown that exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) have negative effects on the rate of growth of bacteria.In the present study, two Gram-positive and two Gram-negative species were exposed to six magnetic field conditions in broth cultures. Three variations of the ‘Thomas’ pulsed frequency-modulated pattern; a strong-static “puck” magnet upwards of 5000 G in intensity; a pair of these magnets rotating opposite one another at ∼30 rpm; and finally a strong dynamic magnetic field generator termed the ‘Resonator’ with an average intensity of 250 μT were used. Growth rate was discerned by optical density (OD) measurements every hour at 600 nm.
ELF-EMF conditions significantly affected the rates of growth of the bacterial cultures, while the two static magnetic field conditions were not statistically significant. Most interestingly, the ‘Resonator’ dynamic magnetic field increased the rates of growth of three species (Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli), while slowing the growth of one (Serratia marcescens).
We suggest that these effects are due to individual biophysical characteristics of the bacterial species.
The behavioural effects of electromagnetic fields (EMFs) have been reported since the 1970s (Persinger, 1970 and Persinger and Pear, 1972). Recent developments in the field have shown that exposure to non-ionizing, electromagnetic radiation can induce numerous and quite varied biological effects (Adey, 1981, Santini et al., 2009 and Cifra et al., 2010). For example, exposure to extremely low frequency (ELF)-EMFs, ranging from 0 to 100 Hz, are capable of activating cellular immune responses (Simkò and Mattson, 2004 and Akan et al., 2010). ELF-EMFs are known to produce voltage gradients in tissues in the range of 10–10−1 V/cm. These gradients are essential to various physiological functions at the level of the cell (Adey 1993).
The majority of research regarding non-ionizing radiation has fixated on the probability of analogous effects. Despite this, there have been an increasing number of studies suggesting that exposure to certain patterned ELF-EMFs can inhibit the growth of cancer cells (Hu et al., 2010 and Karbowski et al., 2012). Other beneficial effects have been reported in studies where bisected planarian flatworms exposed to various ELF-EMFs have increased the rate of regeneration of the worms (Novikov et al., 2008, Goodman et al., 2009 and Tessaro and Persinger, 2013).
One field in which the application of EMFs has not been well-defined is the influence of EMF on microorganisms. Goodman et al. (1994) conducted a series of studies on the bacterium Escherichia coli which demonstrated that 60-min exposure to weak, frequency-pulsed magnetic fields (1.5 mT peak) increased the intracellular levels of a number of proteins by at least a factor of 2. A subsequent study revealed that as little as 15-min of exposure to a 60 Hz, 1.1 mT sine-wave magnetic field can enhance the intracellular level of σ32 mRNA ( Cairo et al. 1998). Gross colony morphology effects and slowed growth after exposure to ELF-EMFs were reported in a study by Strašák et al. (2002).
Nawrotek et al. (2014) have noted that the application of 50 Hz rotating magnetic fields (RMF) significantly increased the rate of growth in two species, E. coli and S. aureus, with the largest effect seen within the exponential phase, or approximately 150 min into their exposure. It was also found that 50 Hz RMF applications can modulate a variety of bacterial functions in various species, such as metabolic activity, biofilm formation and overall growth dynamics ( Fijalkowski et al. 2014). These studies taken together demonstrate the variety of biological processes susceptible to electromagnetic stimuli, in particular growth dynamics, and emphasize the importance of the spatial–temporal component of the applied field.
In the present study, we exposed four bacterial strains for a total of 12 h to one of six magnetic field conditions using nutrient broth as a growth medium. The purpose of the present study was to investigate what effects the different magnetic field conditions may have upon the rate of bacterial growth. This is pertinent given the prevalence of electromagnetic fields in our everyday environments. Here we demonstrate a differential, although transient, effect of specific field strengths and patterns upon specific strains of bacteria and their observed rates of growth as inferred by optical density measurements.
The prepared strain samples were exposed to one of six magnetic field conditions. Three were variations of a complex, pulsed frequency-modulated field (“Thomas pulse”) that has been shown to affect analgesic thresholds in mice or cell-relevant reactions in variety of invertebrates and vertebrates (Thomas et al., 1997, Martin et al., 2004a and Martin et al., 2004b). We also utilized strong, static magnets with an intensity of 5000 G, a set of these magnets rotated counter-clockwise, and a magnetic field generator termed the ‘Resonator’ with an intensity of 250 μT.
The Thomas field is a frequency-modulated pattern that begins with an initial presentation of 25 Hz (Fig. 1). Thereafter, the frequency slows to 6 Hz. At the centre of the coil, bacteria were subjected to an average field strength of 3.8 μT (ranging from 2.1 to 5.5 μT). Bacteria not receiving the magnetic field exposure (i.e. shams) were placed in an identical non-active Helmholtz coil for 12 h housed within a separate identical incubator. The sham cultures were started at the same time as the experimental conditions to ensure all other experimental variables were controlled for excluding the magnetic field presentation.
The results of this experiment indicate that exposures to ELF-EMFs are capable of differentially affecting the growth rates of the bacterial species utilized in this experiment. The fact that the strong static magnetic fields showed no effects is perhaps unsurprising. The Earth has an average magnetic field strength of 45 μT. The static magnet condition would thus increase this substantially, but the intensity would be constant. Bacteria growing within static fields are able to adapt to these stable environmental conditions (Cellini et al. 2008). These results stress the importance of the temporal component of the magnetic field pattern.
To test the effects of pre-exposure, an additional condition utilized nutrient broth exposed to the various Thomas-fields for 48 h prior to inoculation with bacteria and subsequently continuously exposed for a further 12 h. Buckner (2011) also found that changing point durations did elicited differential effects on cancer cells, with (3,3) being most optimal. Thus, we tested three variations of point durations under the hypothesis that the cell membrane was the locus of any possible effects. However, we found no significant differences when broth was pre-exposed to the Thomas field.
But the question still remains as to how a patterned field can affect cellular processes? Previous work conducted by Buckner (2011) indicated that the frequency modulated Thomas pulse at 3 ms point duration induced a massive influx of Ca2+ into B16-melanoma cells by acting upon T-type calcium channels. It is possible that this same mechanism is at work in bacterial cells, as bacterial cells also possess an equivalent T-type channel that displays similar properties and functions (Matasushita et al. 1989). This is also supported by researched conducted using a Ca2+ chelator, where when it was added to growing cultures of E. coli the effects of the EMF were significantly decreased ( Belyaev et al., 1995 and Belyaev et al., 1999).
Furthermore, the effect of ELF-EMF on bacteria is cell density dependent, with a critical mass plateau which act directly upon DNA, specifically chromatin conformation (Belyaev et al., 1998 and Belyaev and Alipov, 2001). Our findings are in agreement with this previous study in that the critical window for EMF effects (4–6 h) coincides with the critical mass plateau reported by Belyaev et al. The timing also coincides with the expression of cytoplasmic calcium channels in that they are dependent on the growth phase of bacteria. The complexes are present in the cytoplasmic membrane at low concentrations during exponential growth, higher during stationary phase, and particularly high during suspension in ice-cold calcium buffers (Reusch et al. 1995). That the final optical density measures for some conditions were no different from controls merely reflects the transient nature of the effects of EMF on bacterial growth rates. If EMFs are considered to be an environmental stressor, then a return to normal growth activity reflects an inherent capability for bacteria to adjust to that stressor.
Norris et al. (1996) explored calcium signalling in bacteria which has been difficult to demonstrate. Calcium influx in E. coli and Streptococcus sp. is associated with transport membrane complexes which are found in higher levels during the stationary phase. Therefore a Ca2+ influx due to the ELF-EMF exposure could lead to higher expression of these membrane complexes, leading the cells to metabolise at a state associated with stationary phase, delaying the exponential phase. Within the cell itself Ca2+ has been shown to interact directly with bacterial DNA effecting the expression of genes (Norris et al. 1996).
Earlier studies were in agreement with our results (Strašák et al., 2002 and Inhan-Garip et al., 2011). It has been demonstrated that exposure to ELF-EMFs may induce heat-shock proteins as a protective response against environmental stimuli (Del Re et al. 2003). Moreover, Norris et al. (1996) confirmed the induction of DnaK as one of three 70 kDa heat-shock proteins (HSPs) expressed in E. coli after stress exposures. Furthermore, stress stimuli may also increase reactive oxygen species (ROSs) and decrease oxidoreductase activity, which leads to oxidative damage to microbial cell proteins and reduction in growth rates (Mega-Tiber et al., 2008 and Strašák et al., 2002).
DnaK also has an autophosphorylation activity stimulated by calcium (Norris et al. 1996). Given the known effects of Thomas to induce calcium influx (Buckner 2011) and the effects of ELF-EMF on hsp70 (Goodman et al. 2009) a plausible mechanism for the effect of ‘Thomas’ is a combination of the two: Ca2+ influx induced by ‘Thomas’ interacts with cellular DNA inhibiting DnaK that is involved in chromosomal DNA replication, preventing reproduction of microbial cells (Sakakibara, 1998 and Shobin et al., 2012). Living cells are composed of charges and dipoles that can interact with electric and magnetic fields by various mechanisms. Blank and Goodman (2008) demonstrated that EMFs accelerated the rate of chemical reactions and activated Na,K-ATPase (0.2–0.3 μT), cytochrome oxidase (0.5–0.6 μT), and the Belousov–Zhabotinsky reaction (<0 .5="" across="" applications="" are="" discussed="" field="" magnetic="" mechanisms="" of="" present="" range="" similar="" span="" studies.="" study="" suggesting="" the="" these="" thresholds="" within="">
The ‘Resonator’ effects were quite unique; three of four species (S. epidermidis, S. aureus, E. coli) had their growth rates increased, while one (S. marcescens) had its rate decreased. This suggests a fundamental difference between these species which contributes to their specific responses to the magnetic fields. Indeed, both S. epidermidis and S. aureus are Gram-positive species, whereas E. coli and S. marcescens are Gram-negative – yet E. coli and S. marcescens reacted differently to the ‘Resonator’ as well, suggesting membrane morphology and composition is not the sole factor. It is far more likely that these fields interact with biological systems on multiple levels simultaneously ...
... Yet the question remains as to why E. coli had an increased rate of growth whereas S. marcescens had a reduced rate. If the above calculation is repeated, only using the DNA as the mass, and the length of each genome as the displacement, the values for S. marcescens in ‘swarmer cells’ once again reaches 10−20 J. S. marcescens is known to form aggregates of 10–30 cells that function as a single unit; if the mechanical energies act to disrupt this self-organizing phenomenon, then reduced efficiency of replication would be expected ( Alberti and Harshey, 1990 and Harshey, 1994).
It is most probable that an aggregate of the presented mechanisms are responsible for the phenomena observed in this experiment. Given the persistent occurrence of 10 −20 J as a fundamental quanta of energy across various levels of discourse, there is cause to suggest a physical interaction within cells subjected concurrently to magnetic and mechanical stressors. The solutions presented here utilize quantitative values, and suggest testable hypotheses for further study and verification.
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Joel M. Moskowitz, Ph.D., Director
Center for Family and Community Health
School of Public Health
University of California, Berkeley
Electromagnetic Radiation Safety
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