The Distinguishing Effects of Low‐Intensity Electromagnetic Radiation

Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE The distinguishing effects of low-intensity electromagnetic radiation of different extremely high frequencies on Enterococcus hirae: growth rate inhibition and scanning electron microscopy analysis K. Hovnanyan1, V. Kalantaryan2 and A. Trchounian3

1 Institute of Molecular Biology of National Academy of Sciences of Armenia, Yerevan, Armenia 2 Department of Radiophysics of High Frequences and Telecommunications, Faculty of Radiophysics, Yerevan State University, Yerevan, Armenia 3 Department of Biochemistry, Microbiology and Biotechnology, Faculty of Biology, Yerevan State University, Yerevan, Armenia

Significance and Impact of the Study: A stronger inhibitory effect of low-intensity electromagnetic field on Enterococcus hirae ATCC 9790 bacterial growth rate was observed with 53 GHz vs 51Á8 GHz, regardless of exposure duration. Scanning electron microscopy analysis showed that almost all irradiated cells in the population have spherical shapes similar to nonirradiated ones, but they have increased diame- ters in case of irradiated cells at 53 GHz, but not 51Á8 GHz. The results are novel, showing distinguishing effects of low-intensity electromagnetic field of different frequencies. They could be applied in treatment of food and different products in medicine and veterinary, where E. hirae plays an important role.

Keywords Abstract action mechanisms, bacterial growth, electromagnetic radiation, electron A low-intensity electromagnetic field of extremely high frequency has microscopy, Enterococcus hirae. inhibitory and stimulatory effects on bacteria, including Enterococcus hirae.It was shown that the low-intensity (the incident power density of À Correspondence 0Á06 mW cm 2) electromagnetic field at the frequencies of 51Á8 GHz and Armen Trchounian, Department of Biochem- 53 GHz inhibited E. hirae ATCC 9790 bacterial growth rate; a stronger effect istry, Microbiology and Biotechnology, Faculty was observed with 53 GHz, regardless of exposure duration (0Á5h,1h or of Biology, Yerevan State University, 1 Manoukian Str., 0025 Yerevan, Armenia. 2 h). Scanning electron microscopy analysis of these effects has been done; the E-mail: [email protected] cells were of spherical shape. Electromagnetic field at 53 GHz, but not 51Á8 GHz, changed the cell size—the diameter was enlarged 1Á3 fold at 2017/0191: received 30 January 2017, revised 53 GHz. These results suggest the difference in mechanisms of action on 5 May 2017 and accepted 6 June 2017 bacteria for electromagnetic fields at 51Á8 GHz and 53 GHz. doi:10.1111/lam.12764

It has been previously shown that the low-intensity Introduction EMF at the frequencies of 51Á8 GHz and 53 GHz inhib- Electromagnetic field (EMF) of extremely high frequencies ited Enterococcus hirae growth (Ohanyan et al. 2008), pro- is an environmental stress factor, which has different stimu- longing lag growth phase duration and decreasing specific lative and depressive effects on living organisms, including growth rate during the log growth phase. Interestingly, bacteria (Soghomonyan et al. 2016). The interest to study the effects have increased with the EMF frequency from EMF effects is of significance because of developing electro- 45 GHz to 53 GHz, and 53 GHz had stronger effects magnetic technologies and widening of their applications (Ohanyan et al. 2008). Similar inhibitory effects have in telecommunications, environment control, as well as been reported with Escherichia coli (Trchounian et al. treatment of food and different medical and veterinary 2001; Torgomyan et al. 2013) and others, especially lactic products (Soghomonyan et al. 2016; Pan et al. 2017). acid bacteria (Soghomonyan and Trchounian 2013) at

220 Letters in Applied Microbiology 65, 220--225 © 2017 The Society for Applied Microbiology K. Hovnanyan et al. Electromagnetic field effects on E. hirae different frequencies (Soghomonyan et al. 2016). Note dependent on the exposure duration of EMF: the inhibi- that Enterococci belong to lactic acid bacteria (Fisher and tory effect was observed at 0Á5 h of exposure, the stron- Phillips 2009). In addition, the changes in E. coli cell gest effect was at 1 h of exposure and the decrease at 2 h morphology, showing increased cellular sizes, have been was only a little lower one than that at 1 h of exposure determined (Torgomyan et al. 2013). However, in con- (see Fig. 1). This confirms the results on inhibition of trast to E. coli, antibacterial effects with E. hirae were not growth rate and colony-forming activity reported before dependent on pH, EMF exposure duration and other for E. hirae for different growth conditions (Ohanyan factors (Ohanyan et al. 2008). et al. 2008). Moreover, there are probably some compen- The changes in water structure and properties (Golovl- satory cellular mechanisms which might prevent the eva et al. 1997; Torgomyan et al. 2011, 2013) and alter- increase in the EMF stress effects on bacteria (Soghomon- ations in E. hirae and E. coli cell plasma membrane yan et al. 2016). bioenergetic characteristics, ion transport and enzymatic Electron microscopy study of E. hirae irradiated by the properties (Trchounian et al. 2001; Torgomyan et al. EMF at 51Á8 GHz (Fig. 2) and 53 GHz (Fig. 3) showed 2012; Pham et al. 2016) can be considered as cellular that almost all cells in the population have spherical shapes mechanisms for the influence of the EMF on bacteria. similar to nonirradiated (control) bacteria; only several The change in sensitivity to different chemicals, including enterococci of oblong shape were seen. Changes in the aver- various antibiotics, is among the EMF effects (Dardalhon age diameter were observed during cell size measurements et al. 1981; Tadevosyan et al. 2008; Torgomyan et al. 2012, (Table 1). Such changes in cell size by irradiation of EMF 2013). But these effects were more strongly expressed in could be due to the intracellular hydration. Interestingly, E. hirae, than in E. coli (Torgomyan et al. 2012). It is destabilization of hydrogen bond structure in water near clear that cellular mechanisms of EMF effects should be the surface of the cell membrane and within the cell has studied further. These effects are of significance, since been suggested (Sinitsyn et al. 2000; Kuznetsov et al. 2006), E. hirae can be considered as a model organism for this can create conditions for cellular response. Gram-positive bacteria (Gaechter et al. 2012; Torgomyan Moreover, the marked increase in cell sizes for E. hirae et al. 2012); in addition, they are among commensal and grown till stationary growth phase (see Materials and pathogenic bacteria in nature (Vela et al. 2015), they can Methods) was observed by EMF at 53 GHz, but not be used as probiotics, applied in dairy production or, on 51Á8 GHz (see Table 1). This could be due to temporary the other hand, can be linked to different diseases in alterations in cell morphology by the EMF at 51Á8 GHz, human organism (Gilad et al. 1998; Fisher and Phillips as shown with E. coli (Torgomyan et al. 2013). It is likely 2009; Chan et al. 2012; El-Ghaish et al. 2015; Garcıa- to the mediated effects of EMF at these frequencies on Hernandez et al. 2016; Jain et al. 2016). Thus, structural E. coli, when the difference in the effects between and functional peculiarities of E. hirae, especially mem- 51Á8 GHz and 53 GHz is observed (Torgomyan et al. brane composition and properties, are the focus of this 2012, 2013). These effects could be due to the partial study. Moreover, the understanding of specific effects of absorbance of EMF energy of different value by the liquid EMF at 53 GHz, but not 51Á8 GHz is required; appropriate mechanisms would be suggested. This will allow the 0·9 clarification of the applications of EMF at different frequencies in food industry, biotechnology, medicine and ) 0·85 –1 veterinary fields, where E. hirae plays an important role. 0·8 The aim of this study was to provide scanning electron 0·75 microscopy analysis of E. hirae irradiated by EMF at 0·7 51Á8 GHz and 53 GHz, in addition to the effects on bacterial cell growth. Special attention would be paid to dis- 0·65 tinguish between the effects of EMF at these two 0·6 frequencies. Spesific growth rate (h 0·55 0·5 Control 0·5 1·0 2·0 Results and discussion Exposure to EMF irradiation (h) Low-intensity EMF at the frequency of 51Á8 GHz and Figure 1 The changes in specific growth rate of Enterococcus hirae 53 GHz had inhibitory effects on E. hirae ATCC 9790 ATCC 9790 after irradiation by electromagnetic field at the frequen- growth. Indeed, specific growth rate during the log cies of 51Á8 GHz (■) and 53 GHz ( ) with different exposure dura- growth phase was decreased, and these effects were more tion. Control is nonirradiated cells. For details, see Materials and strongly expressed at 53 GHz (Fig. 1). The effects were methods.

(a) (b)

Figure 2 Scanning electron microscopy images of Enterococcus hirae ATCC 9790 cells in control (a) and after bacterial suspension irradiation by an electromagnetic ﬁeld at the frequency of 51Á8 GHz with exposure of 0Á5 h (b), 1 h (c) and 2 h (d). Scale bar is shown. For details, see Materials (c) (d) and methods. [Colour ﬁgure can be viewed at wileyonlinelibrary.com]

(water) medium, when 51Á8 GHz, but not 53 GHz is a 2015). This can trigger structural changes in the cell and resonant frequency for the water molecules (Sinitsyn et al. activity inducing different deformations. However, in 2000; Kuznetsov et al. 2006). Indeed, the absorption of spite of remarkable alterations in cell membrane-asso- water molecules at near ultraviolet region was shown to ciated activity, a resonant frequency for E. hirae, as well be more increased by the EMF at 51Á8 GHz, than at as for other bacteria, has not been yet established. Fur- 53 GHz and other frequencies (Torgomyan et al. 2013). thermore, the alterations in cell plasma membrane struc- Therefore, changes in other targets of the EMF in bacte- ture and properties and cell wall (Torgomyan et al. 2012) ria, particularly cell plasma membrane, are probable could result in morphological changes in the cell (see (Trchounian et al. 2001; Torgomyan et al. 2012; Pham Table 1, Fig. 3). The targets for EMF influence on bacte- et al. 2016): marked decrease in energy-dependent ion ria seem to be well established, but the mechanisms are transport across the plasma membrane and the ATPase very complex: further studies are required. activity of E. hirae by a low-intensity EMF have been Thus, low-intensity EMFs at the frequencies of 51Á8 GHz determined (Torgomyan et al. 2012); changes in mem- and 53 GHz both inhibited E. hirae ATCC 9790 bacterial brane permeability are suggested. The stronger effects of growth rate; a stronger effect was observed with 53 GHz vs 53 GHz vs 51Á8 GHz were revealed for these changes 51Á8 GHz, regardless of exposure duration. Scanning elec- (Torgomyan et al. 2012). These might be due to the tron microscopy (SEM) analysis of these effects showed absorbance of EMF energy of a different value (even if that both nonirradiated and irradiated cells were of spheri- the difference in energy value is small) by cell membrane, cal shape. But electromagnetic irradiation at 53 GHz, but especially by membrane-associated transport and enzy- not 51Á8 GHz, increased the cell diameter. These results matic proteins, causing different conformational and suggest the difference in mechanisms for EMF effects on other type of changes in membrane structure and proper- bacteria at 51Á8 GHz and 53 GHz; further studies are ties (Betskii et al. 2000). It is also possible that cell mem- required. The results pointed out the frequency of the EMF brane or other structures are being excited by EMF of a with the strongest inhibitory effect and could be applied in specific frequency; this depends on their electric dipoles; treatment of food and different products, in biotechnology, Bose-condensed phonons can be formed (Anton et al. medicine and veterinary fields.

(a) (b)

Figure 3 Scanning electron microscopy images of Enterococcus hirae ATCC 9790 cells in control (a) and after bacterial suspension irradiation by an electromagnetic ﬁeld at the frequency of 53 GHz with exposure duration of 0Á5 h (b), 1 h (c) and 2 h (d). Scale bar is shown. For details, see materials and Methods. [Colour ﬁgure can be (c) (d) viewed at wileyonlinelibrary.com]

Table 1 The changes in cell diameter of Enterococcus hirae ATCC filled completely with growth medium) at 37°C till sta- 9790 cells after irradiation by a low-intensity electromagnetic field at tionary growth phase (12–16 h), as described (Torgomyan Á 51 8 GHz and 53 GHz et al. 2012). Note that this bacterium is a facultative Diameter (lm) anaerobic one (Fisher and Phillips 2009), and the conditions used were appropriate for anaerobic growth. Á Conditions* 51 8 GHz 53 GHz Bacterial growth was monitored with a Spectro UV– Control (nonirradiated) 0Á72 Æ 0Á01 0Á73 Æ 0Á01 VIS Auto spectrophotometer (Labomed, Los Angeles, CA, Irradiated cells during 0Á5h 0Á60 Æ 0Á01 0Á91 Æ 0Á02 USA) at a wavelength of 600 nm by obtaining absorbance P < 0Á05† P < 0Á01 changes. The specific growth rate (ln2/d, where d is bacte- Á Æ Á Á Æ Á Irradiated cells during 1 h 0 70 0 01 0 86 0 01 rial suspension absorbance doubling time within an inter- P > 0Á05 P < 0Á05 val, when the alteration of absorbance logarithm to the Irradiated cells during 2 h 0Á75 Æ 0Á02 0Á90 Æ 0Á01 P > 0Á05 P < 0Á01 time was linear (log growth phase)) was determined, as described elsewhere (Ohanyan et al. 2008; Tadevosyan *For details, see Materials and Methods. et al. 2008; Torgomyan et al. 2011, 2012, 2013). Grown † For the difference between the irradiated (for the irradiation duration cells were harvested, washed twice and diluted in bidis- mentioned) and nonirradiated (control) cells for the same series tilled water providing an osmotic shock. Thereafter, the (columns). concentrated bacterial suspension (with titre of ~1010 cells per mL) was divided into two parts and transferred into a Materials and methods plastic plate (Petri dish): the first part was the control without irradiation (nonirradiated) and the second one Bacteria, bacterial growth and preparation to assays was for subsequent irradiation. Note that E. hirae as Enterococcus hirae ATCC 9790 wild-type strain was used Gram-positive bacteria are tolerant to osmotic shock throughout. Bacteria were grown in a glucose (0Á4%) con- (Fisher and Phillips 2009), and no changes in bacterial taining medium (1% tryptone, 0Á5% yeast extract, 1% count were determined during the osmotic shocks used

K2HPO4;pH8Á0) under anaerobic conditions (in ﬂasks and subsequent irradiation (Torgomyan et al. 2012).

bacteria and the nonirradiated cells (control) in the same The electromagnetic irradiation of bacteria series) were determined using SigmaPlot software (San Bacterial suspension with thickness of ~1 mm (the cell Jose, CA, USA); the difference was estimated to be valid density of ~5Á109 cells per mL) was exposed to irradiation if P < 0Á05 (Ohanyan et al. 2008; Tadevosyan et al. 2008; by EMF at the frequencies of 51Á8 GHz and 53 GHz Torgomyan et al. 2012). using an EMF generator; model G4-141 with conical antenna (State Scientific-Production Enterprise “Istok”, Acknowledgements Fryazino, Moscow Region, Russia), as described in details elsewhere (Ohanyan et al. 2008; Tadevosyan et al. 2008; We thank Dr. Vahe Ohanyan (Yerevan State University) Torgomyan et al. 2011, 2012; Torgomyan and Trchounian for participation in some experiments, Dr. Lilit Hakobyan 2015). The frequency stability of generator in continuous (Yerevan State University) for improving English. This wave mode was up to 20 MHz; the amplitude-modula- study was done within the basic support framework of tion frequency was 1 Hz. The distance from the radiating State Committee of Science, Ministry of Education and end of the conical antenna to the object of irradiation Science of Armenia. was ~20 cm (the far zone) which provided equal distribu- tion of power to the exposed sample. For this distance, the intensity of EMF was measured to be low (the inci- Conflict of Interest À2 dent power density of 0Á06 mW cm ) with no heat The authors declare that they have no conflict of effects (the temperature change was <0Á1°C), as shown interests. (Torgomyan et al. 2013). The exposure duration was 0Á5 h, 1 h and 2 h. In the control (nonirradiated cells), bacteria were held during References the same period of time as the cells for irradiation and Anton, E., Rotaru, A., Covatariu, D., Giobica, A., Timofte, D., then subjected to appropriate growth (dilution of 1 : 100) Popescu, R. and Anton, C. (2015) Links between extremely and to assays, without exposure to EMF (Torgomyan high frequency electromagnetic waves and their biological et al. 2012). Note that the effects of low-intensity EMF on manifestations. Arch Biol Sci 67, 895–897. bacterial growth were the same for different numbers of Betskii, O., Devyatkov, N. and Kislov, V. (2000) Low intensity bacteria exposed to irradiation (Dardalhon et al. 1981; millimeter waves in medicine and biology. Crit Rev Biomed Torgomyan et al. 2012). Eng 28, 247–268. Chan, T., Wu, M.S., Suk, F.M., Chenm, C.N., Chenm, Y.F. and Houm, Y.H. (2012) Enterococcus hirae-related acute Electron microscopy assays pyelonephritis and cholangitis with bacteremia: an Enterococcus hirae cell sizes and shapes were visualized by unusual infection in humans. Kaohsiung J Med Sci 28, SEM, as described previously in details (Torgomyan et al. 111–114. 2013; Hovnanyan et al. 2015); the electron microscope type Dardalhon, M., Averbeck, D. and Berteaud, A. (1981) Studies VEGA TS 5130MM (TESCAN, Brno, Czech Republic) was on possible genetic effects of microwaves in procaryotic 20 – used; the number of cells for scoring was >20. Electron and eucaryotic cells. Radiat Environ Biophys ,37 51. microscopy imaging analysis was performed using the soft- El-Ghaish, S., El-Baz, A., Hwanhlem, N., Zommara, M., Ayad, ware “Video-Test Structure-5, Nanotechnology” (St. Peter- E., Choiset, Y., Haertle, T. and Chobert, J.M. (2015) Bacteriocin production and safety evaluation of non-starter burg, Russia). Enterococcus faecium IM1 and Enterococcus hirae IM1 strains isolated from homemade Egyptian dairy products. Reagents and data processing Eur Food Res Technol 240, 1211–1223. Fisher, C. and Phillips, C. (2009) The ecology, epidemiology Agar, tryptone and yeast extract were from Sigma-Ardrich and virulence of Enterococcus. Microbiology 155, 1749– (St. Louis, MO), glucose was from Borisov Medical 1757. Preparations Plant (Borisov City, Belarus). The other Gaechter, T., Wunderlin, C., Schmidheini, T. and Solioz, M. reagents of analytical grade (Carl Roths GmbH, Karlsruhe, (2012) Genome sequence of Enterococcus hirae Germany; Unichem, Hangshou, China) were used. (Streptococcus faecalis) ATCC 9790, a model organism for The average data processed were the mean from three the study of ion transport, bioenergetics, and copper replicates of independent measurements. The standard homeostasis. J Bacteriol 194, 5126–5127. errors (<2%) and the Student’s t test calculation with the Garcıa-Hernandez, Y., Perez-Sanchez, T., Boucourt, R., validity criteria (P) (validity for the difference between Balcazar, J.L., Nicoli, J.R., Moreira-Silva, J., Rodrıguez, Z., the changed values for each average data with irradiated Fuertes, H. et al. (2016) Isolation, characterization and

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