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Frequency Electromagnetic field effects on Gram-Positive and Gram-Negative Bacteria

Abstract Objective: The effects of electromagnetic waves on the growth of living organisms and the determination of the threshold of these radiations have remained elusive. Therefore, in this research, we have investigated the growth rate of gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria that had been exposed to the different frequencies of electromagnetic fields. Results: The more frequency increased the slower bacteria grew; however, in gram-positive bacteria such as S. aureus, this effect was seen less. The effect of the 1mT electromagnetic field in the growth of S. aureus was significant between the two groups, nonetheless, in the 2mT electromagnetic field, the effect was not significant between the two groups at different frequencies. Noteworthy, no significant change was observed by increasing the frequency in S. aureus exposed bacteria in comparison to the control group. The study of bacterial growth in terms of frequency in both case and control groups showed an increasing trend. Increasing the frequency from 50 Hz to 150Hz, significantly, enhanced the rate of bacterial growth. On the whole, the magnetic field had an increment effect on the growth of bacteria; in fact, this effect was greater on the gram-negative than on the gram-positive bacteria.


Introduction
Electrical devices, today, have had a great impact on human life. Electromagnetic fields, created by overhead power lines and other electrical devices may lead to any biological changes in living organisms. Being widely exposed to the electromagnetic fields in the last two decades, people have concerned the Low-Frequency Magnetic Fields (ELF-EMF) as the most common magnetic fields in the human environment with the frequencies below 300 Hz, as a great threat to their health quality. (1,2). Lee HC et al. and Ansari RM et al have investigated the effects of ELF-EMF on the cell functions (1,2). Other studies, also, have evaluated the effects of ELF-EMF on bacteria (3)(4)(5). The results of these studies sometimes contradict each other. For example Inhan-Garip A et al. and Segatore B et al. have shown in their studies that ELF-EMF can negatively affect the living system function (6,7). On the other hand, some studies reported the positive effects of ELF-EMF on the living organism function, and the bacteria sensitivity to the antibiotics (8,9). Although the previous studies have shown that the effects of ELF-EMF are depending on some parameters such as frequency and intensity of the field, time of exposure and type of bacteria cells, due to the complexity of biological systems, there is not a final agreement on the effect of ELF on bacteria as a living system (10). Therefore, more investigations are needed to shed light on this controversial subject. In the present study, the effect of ELF-EMF exposure on bacteria growth was investigated in the respect of changing the frequency (50, 75, 100 and 150 Hz), magnetic field intensity (1 and 2 mT) and type of bacteria (gram-positive and gram-negative).

Electromagnetic generation system:
Electromagnetic exposures were produced by a homemade electromagnetic generator (previous study) in the Department of Medical Physics at Arak University of Medical Sciences. The system, basically, was consisted of a pair of Helmholtz coils, a power supply and a signal generator (Fig 1).
The developed electromagnetic generator has equipped with buttons that enable the user to select the desired frequency between 0 (as a static electromagnetic field) to 300 Hz (as a pulsating electromagnetic field), and it can produce electromagnetic fields with intensities of 0 to 8 mT. The efficiency and precision of the system were checked using a gauss-meter. The uniformity of the electromagnetic field between the coils provided a 4 simultaneous exposure for the cultures. The yield of the produced electromagnetic field was in agreement with the computation of the field distribution and homogeneity of the electromagnetic field which was calculated by a Laplace equation simulation program. The program takes the finite dimensions of coils into account. In this study, electromagnetic fields with different frequencies of 50, 75, 100 and 150 Hz and intensities of 1 and 2 mT were applied to the two types of gram-positive and gram-negative bacteria.  (11). In order to identify the E. coli strain, the samples were cultured on the Simmons citrate agar medium and gram staining procedure was applied to the bacteria.
Also, the following tests were performed to determine the E. coli strain including Lysine Decarboxylase, Motility, Methyl Red, Indole, Voges-Proskauer, Ornithine Decarboxylase, and Triple Sugar Iron test (12).

Counting the number of colony-forming units
The pour plate method, in a fixed amount of inoculum (generally 1 ml), was used for counting colony-forming units. In the present study, we used a dilution of 0.5 McFarland 5 standard. One milliliter dilution of bacterial strain was poured into an 8 centimeter sterile Petri dish, then Muller Hinton agar (approx. 20mL) was added to the Petri dish and was mixed well. After incubating at 37 °C, and using the following formula: (CFU/mL= CFU * dilution factor * 1/aliquot), the number of colony-forming units in one milliliter of dilution of bacterial strain (CFU/mL) was calculated.

Bacterial culture treatment
A serial dilution of bacteria was provided in the glass tubes, and the tubes were exposed to the electromagnetic fields with different frequencies of 50, 75, 100 and 150 Hz. Then, the exposed bacteria were cultured in the Muller Hinton agar and the number of colonyforming bacteria was counted using the Pour plate method. The dilution was prepared in 1000 μl volume, then 900 μl of each tube was poured into 7 tubes. The media cultures

Statistical Analysis:
The electromagnetic fields' effect on the bacterium growth was demonstrated by counting colony-forming units on the Muller Hinton agar medium. The CFU/mL for the control group, the bacteria with the control dilution of 10 -5 -10 -7 , 0.5 McFarland standards, was more than that of bacterium exposed to the electromagnetic fields. The CFU/mL for the 10 -5 and 10 -6 of 0.5 McFarland standards dilution of E.coli in the 50 Hz electromagnetic field was 27 and 12, respectively, while this amount for the control group was 720 and 480. To show the CFU/ml difference between the two groups, the logarithm on 10 bases was used to demonstrate the minor changes between the two groups more obvious. Also, the difference between 10 -5 and 10 -7 of 0.5 McFarland standard dilution was big and the logarithm has made this difference small. On the other hand, the effect of the electromagnetic field on the bacterium growth was so small and the logarithm has made this difference big. The data analysis was performed and the results are shown in figure 2.
The one way ANOVA ststistical test showed this difference (P-value < 0.05) in the electromagnetic field's effect on E-coli bacterium growth. In other hand this effect is not significant in S. aureus bacterium growth (P-value > 0.05).

Discussion 7
Electrical devices have a major role in human life and their effects on different biological structures have been discussed in recent years. In the current study, we investigated the effect of electromagnetic field intensity with different frequencies. E. coli and S. aureus bacteria, gram-negative and gram-positive bacteria, respectively, were selected for this study. These two bacteria can be found in abundance in nature; in addition, they cause various infections including diarrhea, wound infection and septicemia in humans (13,14).
After placing E. coli in an electromagnetic field of 1mT, the number of colonies was increased in comparison to the control group that had not been exposed to the waves showed a significant increase in cell proliferation and survival in comparison to the control group. Therefore, it can be implied that the presence of these bacteria in the vicinity of the electromagnetic field can make it difficult to treat the diseases caused by these pathogens.

Limitations
The Electrical devices that were used in the present study were homemade although they had a major effect on the study. This step can be considered as the main limitation of the study design.

Abbreviations
Exposure to Low-Frequency Electromagnetic Fields (ELF-EMF)

Ethics approval and consent to participate
The study was approved in the Ethical Committee of Arak University of Medical Sciences and the ethic approval code is IR.ARAKMU.REC.1397.846. All of the participants in the study gave informed consent prior to enrolment in the study.