Evaluation of Short-Term Exposure to 2.4 Ghz Radiofrequency Radiation

GMJ.2020;9:e1580 www.gmj.ir

Received 2019-04-24 Revised 2019-07-14 Accepted 2019-08-06

Evaluation of Short-Term Exposure to 2.4 GHz Radiofrequency Radiation Emitted from Wi-Fi Routers on the Antimicrobial Susceptibility of Pseudomonas aeruginosa and Staphylococcus aureus

Samad Amani 1, Mohammad Taheri 2, Mohammad Mehdi Movahedi 3, 4, Mohammad Mohebi 5, Fatemeh Nouri 6 , Alireza Mehdizadeh3 

1 Shiraz University of Medical Sciences, Shiraz, Iran 2 Department of Medical Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran 3 Department of Medical Physics and Medical Engineering, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 4 Ionizing and Non-ionizing Radiation Protection Research Center (INIRPRC), Shiraz University of Medical Sciences, Shiraz, Iran 5 School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 6 Department of Pharmaceutical Biotechnology, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran

Abstract

Background: Overuse of antibiotics is a cause of bacterial resistance. It is known that electromagnetic waves emitted from electrical devices can cause changes in biological systems. This study aimed at evaluating the effects of short-term exposure to electromagnetic fields emitted from common Wi-Fi routers on changes in antibiotic sensitivity to opportunistic pathogenic bacteria. Materials and Methods: Standard strains of bacteria were prepared in this study. An- tibiotic susceptibility test, based on the Kirby-Bauer disk diffusion method, was carried out in Mueller-Hinton agar plates. Two different antibiotic susceptibility tests for Staphylococcus aureus and Pseudomonas aeruginosa were conducted after exposure to 2.4-GHz radiofrequency radiation. The control group was not exposed to radiation. Results: Our findings revealed that by increasing the duration of exposure to electromagnetic waves at a frequency of 2.4 GHz, bacterial resistance increased against S. aureus and P. aeruginosa, especially after 24 hours (P<0.05). Conclusion: The use of electromagnetic waves with a frequency of 2.4 GHz can be a suitable method for infection control and treatment. [GMJ.2020;9:e1580] DOI:10.31661/gmj.v9i0.1580

Keywords: Pseudomonas aeruginosa; Staphylococcus aureus; Radiofrequency; Drug Resis- tance

Introduction communication (GSM), several changes have occurred in the natural environment. Wi-Fi uring the last decades, due to the increase radiation, as a part of non-ionizing radiation Din the telecommunication systems uses, in the electromagnetic spectrum, usually pro- such as wireless fidelity (Wi-Fi) and mobile duce radiofrequency waves with a frequency

GMJ  Correspondence to: Dr. Alireza Mehdizadeh, Ph.D. in Medical Physics, Copyright© 2020, Galen Medical Journal. This is School of Medicine, Shiraz University of Medical Sci- an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International ences, Shiraz, Iran License (http://creativecommons.org/licenses/by/4.0/) Telephone Number: 0711-2349332 Email:[email protected] Email Address: [email protected] Amani S, et al. Antimicrobial Susceptibility after Wi-Fi Exposure Antimicrobial Susceptibility after Wi-Fi Exposure Amani S, et al.

of 2.4 GHz [1]. It is evident that electromag- posure of Wi-Fi emitted from routers, on the netic radiation either ionizing or non-ionizing susceptibility of S. aureus and P. aeruginosa, can affect biological systems via thermal or as opportunistic microorganisms, to various non-thermal effects [2]. As a result, the elec- antibiotics. tromagnetic effects on the biological functions of living microorganisms represent an inter- Materials and Methods esting area of research regarding the environ- mental impacts on human health. It has been Antimicrobial Agents reported that electromagnetic field (EMFs) In this study, the following antibiotics were can either negatively [3-6] or positively [5, 7, evaluated against P. aeruginosa; levofloxacin 8] affect cell growth and viability, as well as (5 μg), piperacillin (100 μg), aztreonam (30 bacterial sensitivity to antibiotics, depending μg), ciprofloxacin (5 μg), imipenem (10 μg), on its physical parameters, such as frequen- amikacin (30 μg), cefotaxime (30 μg), and cy, exposure time, intensity and magnetic flux ceftriaxone (30 μg). Also, ampicillin (10 μg), density and bacterial strain. The non-thermal vancomycin (30 μg), cotrimoxazole (30 μg), effects of Wi-Fi exposure on different strains tetracycline (30 μg), amikacin (30 μg), levo- of bacteria have been studied [5–11]. Overall, floxacin (5 μg), piperacillin (100 μg), and cef- the effects of EMFs on the biological systems triaxone (30 μg) were used for S. aureus. The have been investigated in several studies [1, antibiotic susceptibility pattern was performed 9]. However, the results have been controver- according to the Clinical and Laboratory Stan- sial, and different cell responses have been dards Institute guidelines. The Kirby-Bauer reported. In this regard, our previous study disc diffusion method was applied in Muel- reported that Wi-Fi exposure could not only ler-Hinton Agar (MHA; Biolife, Italy). The change the antibacterial susceptibility but also antibiotic disks were purchased by ROSCO cause changes in the growth rate of pathogenic Diagnostica (DK-2630 Taastrup, Denmark). bacteria such as Listeria monocytogenes and Escherichia coli [10]. Another study revealed Antimicrobial Susceptibility Testing that Wi-Fi radiation significantly enhanced The two bacterial strains, including P. aeru- the susceptibility of Klebsiella pneumoniae ginosa ATCC 27853, and S. aureus ATCC to various antibiotics [11]. Accordingly, it 25923 were obtained. Fresh bacterial cultures is necessary to evaluate the EMF effects on (0.5 McFarland turbidity, 1.5×108 CFU) were the bacteria and also find strategies to - con prepared separately. Each bacterial suspension trol antibacterial susceptibility in the clinical was poured on the plate. Afterward, different laboratories or even in the environment [12, types of antibiotic disks were placed on the 13]. Pseudomonas aeruginosa is a Gram-neg- plates for each bacterium and kept in incuba- ative, oxidase-positive, non-stimulating, aer- tors at 37°C. The test group was exposed to a obic bacterium. It accounts for 11% to 23% Wi-Fi router at 2.4 GHz in a 14.5 cm distance of hospital infections, especially in patients (far field) 18[ ], while the control group had no with cystic fibrosis, burn or immune deficien- exposure to such radiation. In the test group, cy, and dependence on respiratory equipment Wi-Fi exposure was achieved at intervals of 2, [14]. The development of multi-drug resistant 4, 6, 8, 10, and 24 hours. The antibiotic sus- (MDR) strains of P. aeruginosa is critical in ceptibility results were measured and record- the acquisition and development of resis- ed before and after exposure to waves emitted tance in Gram-negative bacteria [15, 16]. On from the Wi-Fi router. The inhibition zone di- the other hand, Staphylococcus aureus is a ameters were also recorded (mm). Gram-positive bacterium and also one of the most common pathogens in human communi- Statistical Analysis ties and hospitals. Despite the availability of The inhibition zone diameters in the exposed effective antibiotics, this pathogen considered and non-exposed groups were analyzed using the most common cause of skin and surgi- IBM SPSS Inc. version 22. The tests were car- cal wound infections [17]. The present study ried out in triplicate, and the non-parametric aimed to evaluate the effects of short-term ex- Mann-Whitney U test was performed for data

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analysis. P-value<0.05 was considered as sig- cin, aztreonam; and finally after 24 hours of nificant differences. exposure with all antibiotics. All of the observed changes were statistically significant Results (P<0.05). The mean inhibition zone diameters of P. aeruginosa are presented in Figure-1. Effects of Radiation Emitted from Wi-Fi Rout- According to this Figure, bacteria, which were ers on P. aeruginosa exposed to radiofrequency radiation, showed In P. aeruginosa bacteria, the inhibition zone remarkable changes in susceptibility to anti- diameters decreased significantly compared to biotics in comparison with the control group with the control group at the following times: at different exposure intervals. As shown in after two hours of Wi-Fi exposure emitted Figure-1, reduction in the inhibition zone di- from the Wi-Fi router for ceftriaxone; after ameter was related to increased resistance to four hours of treatment with ceftriaxone and antibiotics; therefore, after 24 hours of expo- cefotaxime; after six hours of treatment with sure to 2.4-GHz radiation, resistance patterns ceftriaxone, cefotaxime, and piperacillin; af- were determined for all antibiotics; changes ter eight and ten hours of treatment with cef- in antibacterial susceptibility to ciprofloxacin triaxone, cefotaxime, piperacillin, ciprofloxa- were evident (Figure-2).

Figure 1. The mean inhibition zone diameter of P. aeruginosa bacteria before and after exposure to Wi-Fi radiation.

Figure 2. The mean inhibition zone diameter of P. aeruginosa after 24 hours of exposure to Wi-Fi radiation.

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Effects of Wi-Fi Router Radiation on S. aureus changes in susceptibility, compared with anti- Although the inhibition zone diameter of S. biotics used in the control group. Consequent- aureus decreased after two hours of exposure ly, S. aureus bacteria, which were exposed to to Wi-Fi router radiation at a frequency of 2.4 radiofrequency radiation at 2.4 GHz, showed GH, this change was not statistically signif- resistance to antibiotics, including ceftriax- icant for vancomycin. Our findings showed one, amikacin, vancomycin, ampicillin, and that after four hours of exposure to radiation, piperacillin, compared with the control group; no significant difference was observed in sus- these changes were more significant for tetra- ceptibility to the studied antibiotics in com- cycline and vancomycin. parison with the control group. Also, after six hours of exposure to levofloxacin, the mean Discussion inhibition zone diameter decreased, whereas it increased for tetracycline in comparison with The aim of the present study was to examine the control group. These changes were signif- the short-term exposure effect to radiation icant for tetracycline and levofloxacin after emitted from a Wi-Fi router on the sensitivity four hours of exposure (P<0.05). Based on of pathogenic microorganisms to various anti- the findings, after eight hours of exposure, the biotics. The results of this study showed that mean inhibition zone of levofloxacin and tet- there are significant changes in the resistance racycline decreased and increased, respective- of S. aureus and P. aeruginosa to different an- ly; however, it remained unchanged for cotri- tibiotics due to exposure to 2.4 GHz radiation moxazole, compared with the control group. after 24 hours. P. aeruginosa was resistant to According to Figure-3, after 10 hours of ex- all antibiotics after 24 hours of exposure to posure to radiation, the mean inhibition zone radiofrequency waves. Moreover, S. aureus diameter of tetracycline was significantly dif- was resistant to some antibiotics, such as am- ferent from the non-exposed group (P<0.05). picillin, amikacin, ceftriaxone, vancomycin, After 24 hours of Wi-Fi exposure, the inhibi- and piperacillin. These results are in line with tion zone diameters for ceftriaxone, amikacin, the findings reported by Stansell et al. [19]. ampicillin, and piperacillin were decreased They showed that static magnetic fields with (Figure-4). On the other hand, it increased sig- moderate intensity can reduce the susceptibil- nificantly for tetracycline in comparison with ity of E. coli to piperacillin after 45 minutes the control group (P<0.05). The results of an- of exposure and make it more resistant to antimicrobial susceptibility tests for S. aureus tibiotics. Although the duration and frequency are presented in Figure-3. S. aureus bacteria, of exposure and type of microorganism and which were exposed to radiofrequency radia- antibiotic are different in these studies, the tion for different periods, showed remarkable non-ionizing radiation source was similar;

Figure 3. The mean inhibition zone diameter of S. aureus bacteria before and after exposure to Wi-Fi radiation.

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Figure 4. The mean inhibition zone diameter of S. aureus after 24 hours of exposure to Wi-Fi radiation.

accordingly, susceptibility changes were re- resistance of E. coli to all antibiotics after six markable. In the present study, by increasing hours of exposure to 900-MHz waves from a the exposure time to 24 hours for 2.4-GHz radiofrequency simulator; However, L. mono- waves emitted from the Wi-Fi router, the sen- cytogenes remained resistant to ciprofloxacin sitivity of P. aeruginosa to different antibiot- and cefotaxime after nine hours of exposure ics decreased in comparison with the control to 2.4-GHz waves from a Wi-Fi router; this group. In our previous study [11], the effect of finding is similar to the present study. On the exposure to 2.4-GHz electromagnetic waves other hand, Salmen [21]ÿÿÿÿ‑ reported that emitted from Wi-Fi routers on K. pneumoni- high EMFs, at frequencies of 900 and 1800 ae was evaluated for 4.5 hours. The results MHz, had no remarkable effects on S. aureus showed the increased sensitivity of this bacte- bacteria, treated with 30 µg of amoxicillin in rium to ceftriaxone, cefotaxime, piperacillin, the exposed group. Moreover, Belyaev [7] imipenem, and aztreonam. The discrepancy showed that, under particular conditions, ex- between the results of antibacterial suscepti- tremely-low frequency (ELF)-EMF can be bility tests may be attributed to the nature of considered a non-toxic tool stimulating the bacteria (Gram-positive or Gram-negative) growth of E. coli GE499. On the other hand, and duration of exposure. In contrast with Aslanimehr et al. [6] evaluated the effect of our findings, Segatore et al. [20], in a study ELF-EMF on the growth rate and viability on the effects of low-frequency (2 mT, 50 Hz) of S. aureus and E. coli. A reduction was ob- EMFs on the antibacterial susceptibility of E. served in the growth rate of bacteria; there- coli and P. aeruginosa, reported no remark- fore, it was suggested that ELF-EMF may be able differences in the antibiotic sensitivity a therapeutic approach for controlling acute of these bacteria in the exposed groups in and chronic infections. Two factors, which comparison with the control group. Also, in- can affect antimicrobial susceptibility, are the consistent findings have been reported for S. structure and function of antibiotics. Antibiot- aureus. In the present study, after exposure ics show antibacterial functions through dif- to Wi-Fi radiation, these bacteria were more ferent mechanisms, such as inhibition of DNA susceptible to certain antibiotics, such as tet- and cell-wall synthesis and prevention of di- racycline and levofloxacin, compared with hydrofolate production and protein synthesis. the control group, although they were more In this regard, Taheri et al. showed that elec- resistant to ceftriaxone and amikacin. The tromagnetic waves can cause some changes in mentioned finding is in line with the results of the structure of efflux pumps and ion channels our previous study [10] on E. coli and Listeria in the cell wall and alter their permeability to monocytogenes, which showed the increased molecules entering the bacterial cell; there-

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fore, antibacterial susceptibility of bacteria at the frequency of the 2.4 GHz radiofrequen- to different antibiotics changes [22]. Overall, cy radiation. To extrapolate the results of the the present study showed that 2.4-GHz waves current study, further studies with different emitted from Wi-Fi routers can change the an- microorganisms and different exposure condi- tibacterial susceptibility of microorganisms. It tions are suggested. seems that these waves can change the function of ion channels and efflux pumps of bac- Conclusion terial cell walls upon the entry of molecules into the cell; however, there are still many It is believed that EMF, relative to its frequen- other unknown mechanisms that can influence cy, can change antibacterial susceptibility. In bacterial responses. Several researchers have this regard, it is suggested that future stud- examined different effects of EMF on bac- ies investigate the effects of electromagnetic teria; these effects (e.g., bactericidal effects) waves emitted from different devices, exam- vary depending on the frequency and inten- ine the use of different antimicrobial agents, sity of EMF, coherence, and exposure time and evaluate the effect of longer exposure [23-25]. Furthermore, the phase of bacterial time. Also, the use of electromagnetic waves growth, medium ingredients, genetic proper- with a frequency of 2.4 GHz can be consid- ties, growth in the presence or absence of oxy- ered as a suitable method for infection control gen, and membrane features in bacteria should and treatment. be considered [25]. It is assumed that EMF, relative to its frequency, can change antibac- Acknowledgment terial susceptibility. In this regard, a previous study reported an increase in the antibacterial This study was supported by Shiraz Univer- effects with 51.8 GHz radiation [26]. More- sity of Medical Sciences, Iran. This research over, disruption of cation transport reduced was a part of Mohammad Mohebi thesis adenosine triphosphate content [27], and dif- (thesis No. 94.1013; ethical approval No.IR. ferent proteome contents in the membrane can SUMS.REC.1395.245). enhance antibiotic susceptibility [28]. Study Limitations Conflict of Interest This research study was carried out only on the two kinds of pathogenic bacteria, and only None declared.

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