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Bactericidal effects of low-intensity extremely high frequency electromagnetic field: An overview with phenomenon, mechanisms, targets and consequences

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The user has requested enhancement of the downloaded file. Bactericidal effects of low-intensity extremely high frequency EMF Critical Reviews in Microbiology, 2013; 39(1): 102–111 © 2013 Informa Healthcare USA, Inc. H. Torgomyan and A. Trchounian ISSN 1040-841X print/ISSN 1549-7828 online DOI: 10.3109/1040841X.2012.691461 Critical Reviews in Microbiology 2013 REVIEW ARTICLE

39 1 Bactericidal effects of low-intensity extremely high 102 frequency electromagnetic field: an overview with phenomenon, mechanisms, targets and consequences 111

15March2012 Heghine Torgomyan1 and Armen Trchounian2

18April2012 1Yerevan State University, , Yerevan, and 2Yerevan State University, Microbiology and Biotechnology, Yerevan, Armenia 03May2012

Abstract 1040-841X Low-intensity electromagnetic field (EMF) of extremely high frequencies is a widespread environmental factor. This field is used in telecommunication systems, therapeutic practices and food protection. Particularly, in medicine 1549-7828 and food industries EMF is used for its bactericidal effects. The significant targets of cellular mechanisms for EMF effects at resonant frequencies in bacteria could be water (H2O), cell membrane and genome. The changes in H2O cluster structure and properties might be leading to increase of chemical activity or hydration of proteins and other © 2013 Informa Healthcare USA, Inc. cellular structures. These effects are likely to be specific and long-term. Moreover, cell membrane with its surface characteristics, substance transport and energy-conversing processes is also altered. Then, the genome is affected 10.3109/1040841X.2012.691461 because the conformational changes in DNA and the transition of bacterial pro-phages from lysogenic to lytic state have been detected. The consequences for EMF interaction with bacteria are the changes in their sensitivity to different chemicals, including antibiotics. These effects are important to understand distinguishing role of bacteria in environment, leading to changed metabolic pathways in bacteria and their antibiotic resistance. This EMF may also affect the cell-to-cell interactions in bacterial populations, since bacteria might interact with each other through EMF

For personal use only. of sub-extremely high frequency range. Keywords: Bactericidal effects, non-thermal processes by extremely high frequency electromagnetic field, microbial physiology and ecology, cell membrane, antibiotics

Extremely high frequency electromagnetic field in the environment EMF discernment by different organisms (Hyland, 2008). Furthermore, it has been established that small and very Electromagnetic field (EMF) of extremely high small doses of EMF (the power flux density is of 5 µW/ frequencies (of 30–300 GHz) or millimeter waves (the cm2) are affecting living cells and organisms, including wave length of 0.1–10 mm) are relatively novel and bacteria. Regarding to this property, EMF is valuable for widespread environmental factor. The dramatic changes using in therapeutic practices, in soil and agricultural in environment are significant for the variety of reasons. wastewater disinfection, and in food (fresh products, EMF of low-energetic and non-thermal intensity is used rice, raw meat and juices) protection technologies

Critical Reviews in Microbiology Downloaded from informahealthcare.com by IBI Circulation - Ashley Publications Ltd on 01/10/13 in satellite communication, radiometry, radar and/or (Rojavin and Ziskin, 1998; Banik et al., 2000; Betskii et al., remote sensing technologies. But the question exists: 2000; Usichenko et al., 2006; Geveke et al., 2007; Shamis whether such frequencies are potentially hazardeus for et al., 2008; Ukuku et al., 2008; Geveke et al., 2009; Zand living organisms (Pakhomov and Murphy, 2000; Silva, et al., 2010). The EMF use in food industry gives a benefit 2001; Belyaev, 2005). It was revealed that these devices to inactivate pathogenic at moderately produce EMF of much higher degree of coherence than low temperatures to maintain the food nutritional do the sources of natural origin. This feature facilitates attributes and its safety after packaging.

Address for Correspondence: Armen Trchounian PhD., DSc., Yerevan State University, Microbiology and Biotechnology, 1 A. Manoukian Street, Yerevan, 0025 Armenia. E-mail: [email protected] (Received 15 March 2012; revised 18 April 2012; accepted 03 May 2012)

102 Bactericidal effects of low-intensity extremely high frequency EMF 103 Moreover, the recent finding is in that bacteria and minutes following EMF; the optimal exposure time is 1 h. the other cells might interact with each other through In contrast, the other EMF frequencies such as of 41–43 ultrasonic irradiation by remising secondary photons GHz, 61 GHz and 99 GHz (Bulgakova et al., 1996; Cohen of sub-extremely high frequency range (Nikolaev, 2000; et al., 2010) have no sufficient effects on E. coli. On the Trushin, 2003; Belyaev, 2005; Reguera, 2011). This is other hand, EMF effect can be intensified with longer interesting because of bacteria, in this case, can be easily exposure period. As it was shown for 99 GHz, EMF effect affected by external extremely high frequency EMF. So, accumulated in cell and revealed at higher exposure the external EMF of extremely high frequency can imitate times (Cohen et al., 2010). cellular control signals and induce the cells to vibrate at specific frequencies. This can alter membrane processes The EMF bactericidal effects and change the metabolic state of bacteria, especially their reaction to other factors (chemicals, antibiotics) Different research groups during long-term study have (Bulgakova et al., 1996; Caubet et al., 2004; Tadevosyan prescribed that EMF causes different effects, including et al., 2008; Torgomyan et al., 2011b; Torgomyan and bactericidal ones. These are depending on EMF wave- Trchounian, 2012). length or frequency, intensity, coherence, exposure Based on all statements mentioned above about duration, post-exposure time, mediated and repeated extremely high frequencies EMF and its bacterial effects irradiation (Belyaev et al., 1992, 1993, 1996; Bulgakova further clarifications are necessary in order to under- et al., 1996; Scheglov et al., 2002; Alipov et al., 2003; stand the mechanisms of affection to propose further Isakhanyan and Trchounian, 2005; Tadevosyan et applications and directions of development. al., 2006, 2007, 2008; Torgomyan et al., 2011a, 2012). Additionally, bacterial growth phase, anaerobic or Extremely high frequency EMF aerobic conditions, composition of growth media, cell characteristics and studied bacteria density, cell-to-cell interaction, genetic features, pecu- liarities of metabolism and membrane properties in bac- Two types of low-intensity (the power flux density of terial species or strains and others should be mentioned 0.06 mW/cm2) EMF of extremely high frequencies have (Torgomyan and Trchounian, 2012). been used in the study: “noise” EMF (of broadband There were presented the data of EMF bacterial effects frequencies and randomly changing phases) usually for different environmental conditions – (i) bacteria in of 45–53 GHz (Trchounian et al., 2001; Isakhanyan water suspension, (ii) bacteria on a surface (agar) and and Trchounian, 2005), and coherent EMF (in time) of (iii) bacteria in the form of a biofilm. This is important 42, 54, 66 and 78 GHz (Bulgakova et al., 1996), 51.8, 53 when studying the propagation of this EMF in various (Tadevosyan et al., 2006, 2007, 2008; Tadevosyan and media, because such results can have different applica- For personal use only. Trchounian, 2009), 70.6 and 73 GHz (Torgomyan et al., tions for the future. 2011a; Torgomyan and Trchounian, 2011, 2012) frequen- Water is the major constituent of medium and of bio- cies, using the respective generators (Russian made logical systems as well. The low intensity extremely high

or others). Moreover, low-intensity EMF of extremely frequencies EMF effects on H2O molecules may be the high frequencies has been applied to different bacte- first mechanism in creation of conditions for biological ria such as Escherichia coli (Trchounian et al., 2001; response. That is why in this revew the main attention Isakhanyan and Trchounian, 2005; Tadevosyan et al., was given to the EMF effects on bacterial suspension. 2006, 2007, 2008; Tadevosyan and Trchounian, 2009; Different depressive effects on the growth and viability Torgomyan et al., 2011a,b; Torgomyan and Trchounian, of E. coli after bacterial susupension exposure to EMF of 2011, 2012), Enterococcus hirae (Ohanyan et al., 2008; 51.8, 53, 70.6 and 73 GHz frequencies have been shown Torgomyan et al., 2012), Bacillus firmus, B. mucilaginous, (Tadevosyan et al., 2007, 2008; Torgomyan et al., 2011a,b; Staphylococcus aureus, Methanosarcina barkeri and oth- Torgomyan and Trchounian, 2011, 2012). EMF effects ers (Bulgakova et al., 1996; Pakhomov et al., 1998; Banik on E. coli suspension can have a strong evidences at the et al., 2003; Belyaev, 2005; Zand et al., 2010). frequencies (50.3, 51.8, 64.5, 65.5, 95, 105 GHz), which But, the coherent EMF had more interest (Hyland, are resonant for H O molecules (effects are mediated by Critical Reviews in Microbiology Downloaded from informahealthcare.com by IBI Circulation - Ashley Publications Ltd on 01/10/13 2 2008). The effect is revealed when a change can be mea- water) (Fesenko et al., 1995; Betskii et al., 2000; Sinitsyn et sured in a biological system after introduction to EMF al., 2000; Isakhanyan and Trchounian, 2005; Tadevosyan stimuli. The clear effects of EMF at mentioned frequencies et al., 2006, 2007). It was shown, that E. coli growth after on E. coli, which is considered as the best characterized irradiation of bacterial suspension in bi-distilled water bacteria and a model organism (Belyaev, 2005; Cohen was more visible with 53 GHz, 51.8 GHz than with 73 et al., 2010; Torgomyan et al., 2011a; Torgomyan and GHz and 70.6 GHz (Figure 1). The effects are not only Trchounian, 2012), have been shown well (Trchounian linked with the energy of EMF, because the higher is the et al., 2001; Belyaev, 2005; Isakhanyan and Trchounian, frequency of EMF the greater is the energy of a photon 2005; Tadevosyan et al., 2006, 2007, 2008; Tadevosyan and associated with it. Differences also are attached with the Trchounian, 2009; Torgomyan et al., 2011a,b; Torgomyan different EMF effects on the structure of surrounding

and Trchounian, 2012). The effects arise within tenths of H2O molecules and creation of conditions for biological

© 2013 Informa Healthcare USA, Inc. 104 H. Torgomyan and A. Trchounian 51.8 GHz and 53 GHz the growth decrease after E. coli suspension irradiation was less compared with the direct irradiation of bacteria on solid medium. Especially that was also more in the case of 53 GHz. Probably, EMF

impinging with H2O probably loses its energy, but the transferred amount depends on a specific frequency (Fesenko et al., 1995; Golovleva et al., 1997; Sinitsyn et al., 2000; Torgomyan et al., 2011a; Torgomyan and Trchounian, 2012). The EMF power reflection from the exposed samples as was revealed, by the use of waveguide system, was insignificant (Tadevosyan et al., 2008). Moreover, difference between direct bactericidal effects of EMI of 51.8, 53, 70.6 and 73 GHz frequencies might also be associated with their primary resonant interaction with bacteria. EMI, therefore, can cause Figure 1. The effects of low-intensity EMF at the frequencies bacteria to vibrate and change metabolic activity, espe- of 51.8, 53, 70.6 and 73 GHz in combination with antibiotics – chloramphenicol (Chl), ceftriaxone (Cef), kanamycin (Kan) and cially connected with their membranes (Ruediger, 2009). tetracycline (Tet) on E. coli K12(λ) specific growth rate. Bacteria Also, it is probable that EMF interacts with the free and were grown in peptone medium. The control was without EMF and bounded water molecules of bacterial membranes and antibiotics. Antibiotics at their minimal inhibitory concentrations changes their structure. This by its turn can change (4, 0.4, 15 and 4 µM, respectively) with glucose (0.2%) were the membrane ion conductance, the processes con- added into the growth medium immediately before inoculation (Tadevosyan et al., 2008; Torgomyan et al., 2011a; Torgomyan and nected with membrane and afterwards – the cell activity Trchounian, 2012). For the others, see the legends to Figure 2. (Torgomyan and Trchounian, 2012). The direct irradiation of bacteria on solid growth medium with 51.8, 53, 70.6 and 73 GHz frequencies decreased the colonies numbers compared with non- irradiated control. The effects were more with 51.8 GHz and 53 GHz (Figure 2). Interestingly, the EMF influenced not only on decreases of the colonies numbers, but also on their dimensions (Torgomyan et al., 2011a). It is known well, that EMF of 41–43 GHz, 53 GHz, 70.6 GHz and 73 GHz frequencies can have resonant-like bacte- For personal use only. ricidal effects on E. coli, which probably occur by their direct interaction with bacteria (Guofen et al., 2002; Torgomyan et al., 2011a). It is known well that bacteria in biofilm are markedly different, environmentally and physiologically, from planktonic bacteria of the same species (Kolter, 2010). Those extracellular polysaccharide products are able to block antibacterial effect and penetration of antibiotics. Figure 2. The changes in E. coli K12(λ) colony-forming units For instance, bacteria in a biofilm environment can be number spread on solid growth medium irradiated by low-intensity 1000 times (or more) resistant to a specific antibiotic EMF at the frequencies of 51.8, 53, 70.6 and 73 GHz. Irradiation time than the same bacteria in a planktonic environment 2 was 1 h, the power flux density was of 0.06 mW/cm . In the control, (Caubet et al., 2004; Zieli ski et al., 2007). The bacteria were without irradiation (100%). The presented data of ń 70.6 and 73 GHz were adapted from Torgomyan et al. (2011a). influence of non-thermal intensity microwave EMF (2.45 GHz) on bacterial biofilm structure and function was demonstrated (Zelinski et al., 2007). Also it was

Critical Reviews in Microbiology Downloaded from informahealthcare.com by IBI Circulation - Ashley Publications Ltd on 01/10/13 response to EMF. Besides, the mediated effects of 70.6 GHz and 73 GHz by irradiated medium used (water, assay reported earlier, that radio frequency EMF (10 MHz) buffer or growth liquid medium) on E. coli growth were (Caubet et al., 2004) and electric field (Blenkinsopp insignificant (Torgomyan et al., 2011a). But the results et al., 1992) can increase of the antibiotic efficacy on were not the same for 51.8 GHz and 53 GHz, because E. coli biofilms. Thus, the nature of the mechanisms has clear EMI mediated effects on bacterial growth were yet to be confirmed, but if the effects hold true for a wide demonstrated (Tadevosyan et al., 2006, 2007). That was variety of biofilms and for extremely high frequencies more revealed with 53 GHz. Moreover, it was shown that EMF this technology will have great promise in with 70.6 GHz and 73 GHz the cell growth was similarly circumstances where sterilization and removal of suppressed after direct irradiation of E. coli in water biofilms for medical devices and biomaterials (for (liquid medium) or on solid medium. Interestingly, for example, food) are needed.

Critical Reviews in Microbiology Bactericidal effects of low-intensity extremely high frequency EMF 105

Bacterial cellular mechanisms and changes of H2O some physical and chemical properties targets of EMF effects (absorbance, electroconductivity and pH) have been dem- onstrated well (Tadevosyan et al., 2007; Torgomyan et al., The general mechanisms of EMF bactericidal and other 2011a). More visible evidences have been brought up with effects are not clear. Low-intensity (<10 mW/cm2) EMF at the increase of optical density of H2O in near ultraviolet extremely high frequency does not cause direct physical region (200–340 nm) exposed to 51.8 GHz and 70.6 GHz. damage, ionization or heating (>0.5°C) of exposed sur- The effect has been less by EMF of 53 GHz and no change face (Betskii et al., 2000; Belyaev, 2005; Hyland 2008). And has been detected by 73 GHz (Figure 4A). Interestingly, the most significant targets responsible for the cellular 1 h irradiation also changed the pH of bi-distilled water effects of EMF at resonant frequencies are H2O (Fesenko (Figure 4B) (Tadevosyan et al., 2007; Torgomyan et al., et al,. 1995; Golovleva et al., 1997; Sinitsyn et al., 2000; 2011a). In case of 51.8 GHz the acidification occurred for Isakhanyan and Trchounian, 2005; Novoselova et al., 2005; water at two pH values. But in cases of 70.6 GHz and 73 Tadevosyan et al., 2006, 2007; Torgomyan et al., 2011a), GHz water pH at 6.0 was increased, and water pH at 8.0 bacterial membrane with its surface characteristics, sub- was decreased. The explanation of these detected effects stance transport and energy-conversing processes (Neshev is in that EMF energy could be accumulated into the and Kirilova, 1994; Bulgakova et al., 1996; Kandashev and + structures of H2O till critical values and increase of free Н Savin, 1997; Trchounian et al., 2001; Tadevosyan et al., and ОН– dispersions by modification of molecular cluster 2008; Tadevosyan and Trchounian, 2009; Torgomyan et structuring (Fesenko et al., 1995; Sinitsyn et al., 2000). al., 2011a; Torgomyan and Trchounian, 2011, 2012), and Besides, the mechanism with EMF transformation genome (Belyaev et al., 1992, 1993, 1996; Scheglov et al., into informative biological signals at specific resonant 2002; Alipov et al., 2003; Ruediger, 2009) (Figure 3). frequencies is also proposed (Betskii et al., 2000; Guofen

Thus, the first target is medium, especially2 H O. EMI et al., 2002; Pakhomov and Murphy, 2000; Belyaev, 2005).

at resonant frequencies changes H2O cluster structuring EMF might imitate control signals produced by cells and properties and leads to increase of chemical activity and drive a cellular response by correction of metabolic or hydration of proteins and other cellular structures. It processes, especially which are taking part in cell has been determined that these effects might be specific membranes. However, bacterial cell response to EMF can and long-term (Fesenko et al., 1995; Golovleva et al., 1997; be integrative of different mechanisms. But experimental Sinitsyn et al., 2000; Belyaev, 2005; Novoselova et al., 2005; results are not enough to give an overall picture of EMF Tadevosyan et al., 2007; Torgomyan et al,. 2011a). The action on bacteria and cells. It was shown that EMF of 51.8 For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by IBI Circulation - Ashley Publications Ltd on 01/10/13

Figure 3. Probable ways and three main primary cellular targets for extremely high frequency EMF in bacteria – membrane proteins (the + proton F0F1-ATPase (F0F1), K transporter (TrkA), water molecules (H2O) and genome. F0F1 generates proton electrochemical gradients across + the membrane (ΔµH ). For the others see the text.

© 2013 Informa Healthcare USA, Inc. 106 H. Torgomyan and A. Trchounian GHz, 53 GHz, 70.6 GHz and 73 GHz frequencies changes as a result of EMF impact also occurred (Belyaev et al., 1992, ion (of H+ and K+) transport processes and enzymatic 1993, 1996; Scheglov et al., 2002; Alipov et al., 2003; Ruediger,

activities (the proton F0F1-ATPase and hydrogenase) of 2009). But all these effects on bacterial DNA or genome bacterial cell membrane. Also, the changed bacterial were strongly dependent on EMF frequency (Scheglov et al., sensitivity to chemical reagents, especially to inhibitors 2002). Also, it is more probable that EMF genotoxic effects of membrane proteins and antibiotics with the were mediated in other ways including membranous effects membranotropic action by these frequencies evidenced and disturbance of DNA-repair processes (Ruediger, 2009). about the main role of membrane in the cellular response to EMF action. The appropriate changes in The EMF effects on E. coli membrane- the membrane properties mentioned have been clearly associated energy-dependent processes revealed (Trchounian et al., 2001; Tadevosyan et al., 2008; Tadevosyan and Trchounian, 2009; Torgomyan et The bactericidal effects of 51.8, 53, 70.6 and 73 GHz al., 2011b; Torgomyan and Trchounian, 2011, 2012). frequencies EMF seem to be a result of complex effects The oscillations in the regulatory DNA sequences and connected with alterations in membrane structures conformational changes in bacterial genome are probable as conformations and functions. Bacterial membrane well (Belyaev, 2005). The effective rearrangement of electron structure and dynamics depends on its composition, subsystem levels in DNA might be induced (Belyaev et al., specific interactions between components, functional 1996; Ruediger, 2009). Moreover, it was considered that the state of proteins and electrochemical gradients across transition of bacterial pro-phages from lysogenic to lytic state the membrane (Tadevosyan and Trchounian, 2009; Torgomyan and Trchounian, 2011, 2012). The role of membranes in EMF primary targeting was suggested by its effects on energy-dependent H+/K+ exchange processes across the membrane of whole cells (Figure 5A and 5B) and membrane-associated ATPase For personal use only. Critical Reviews in Microbiology Downloaded from informahealthcare.com by IBI Circulation - Ashley Publications Ltd on 01/10/13

Figure 5. The low-intensity EMF effects on energy (glucose, 22 Figure 4. Bi-distilled water absorbance spectra (A) in near mM) depended H+ efflux (A) and+ K influx (B) through the E. coli ultraviolet region (200–340 nm of wavelength) and pH value K-12(λ) whole cells membrane. The data (K+ influx data after 70.6 (B) after by low-intensity EMF irradiation. In the control non- and 73 GHz irradiations were unpublished) were combined from

irradiated H2O was used. The data were adapted from Tadevosyan Tadevosyan et al. (2008), Tadevosyan and Trchounian (2009), et al. (2007) and Torgomyan et al. (2011b). For the others, see the Torgomyan and Trchounian (2012). For the others, see the legends legends to Figure 1. to Figure 1.

Critical Reviews in Microbiology Bactericidal effects of low-intensity extremely high frequency EMF 107 activity changes (Figure 6). Actually, bacteria using ATPase activities were changed negligibly by 70.6 GHz energy equivalents (glucose) to extrude H+ by the work and 73 GHz; a little one occurred only with 70.6 GHz + of F0F1 and to uptake K by their secondary transport irradiation (Figure 6). So, probably the conformational

system (TrkA) (Trchounian et al., 2001; Mnatsakanyan or other changes in F0F1 suggested for 51.8 and 53 GHz et al., 2002; Trchounian, 2004; Tadevosyan et al., 2008; did not occur with 70.6 and 73 GHz frequencies. The Tadevosyan and Trchounian, 2009; Torgomyan et al., lowered H+ efflux could be a result of changed interac-

2011b; Torgomyan and Trchounian, 2011, 2012). The tions between membrane proteins, especially of F0F1 and rates of H+/K+ fluxes in irradiated bacteria were depressed TrkA, and alterations in their activities. Also, the decrease compared with non-irradiated bacteria. Such as, H+ of organic acids secretions, which are the end products secretion from cells after glucose addition was more of processes, might affect on +H flux rate depressed with 53 GHz, than with 51.8 GHz and less with (Trchounian, 2004; Torgomyan and Trchounian, 2011, 70.6 GHz and 73 GHz EMF (Figure 5A). But K+ influx was 2012). a little more depressed in the case of 73 GHz compared Such changes in membrane-connected processes

with the other frequencies (Figure 5B). probably follow the changes of H2O molecules structur- The changed overall andN,N’ -dicyclohexyl ing and properties, which, in turn, can change the prop- carbodiimide (DCCD) sensitive H+-transporting erties of membrane proteins. So, frequency dependent ATPase activity has confirmed (Tadevosyan et al., 2008; EMF bacterial primary effects, especially membranous Tadevosyan and Trchounian, 2009) the low-intensity ones, require further more detailed study. Interestingly,

EMF possible action on F0F1. The influence of 51.8 and EMF at the frequencies used had no effect on bacterial 53 GHz frequencies EMF on these ATPase activities were membrane potential. crucial. Especially, the loss of DCCD-sensitive ATPase activity indicated about the probable conformational Changing bacterial sensitivity to changes in F F with these frequencies (Trchounian, 2004; 0 1 antibiotics by EMF Tadevosyan et al., 2008; Tadevosyan and Trchounian,

2009). The role of F0F1 as EMF target is likely since this is Different antibiotics as tetracycline, chloramphenicol, the main protein, which is presented in a higher amount kanamycin and ceftriaxone at their minimal inhibitory

in E. coli membrane (Mnatsakanyan et al., 2002). F0F1 concentrations affect E. coli growth and survival and these also is associated with secondary solute transport sys- effects could be reinforced by extremely high frequency tems and/or enzymes of anaerobic oxidation-reduction EMF (Tadevosyan et al., 2008; Torgomyan et al., 2011b; processes (Golovleva et al., 1997; Trchounian, 2004) and, Torgomyan and Trchounian, 2012). Similar effects have therefore, has a significant role in habitability of ferment- been obtained with E. hirae (Torgomyan et al., 2012). ing bacteria. Interestingly, overall and DCCD-sensitive The other antibiotics mainly having membranotropic For personal use only. properties are in study. EMF is assumed to change antibiotic sensitivity or resistance, which depends on frequency. The raised effects have been revealed by 51.8 GHz and more by 53 GHz (Figure 1) (Tadevosyan et al., 2008; Torgomyan et al., 2011a; Torgomyan and Trchounian, 2012). Membrane permeabilization;

discharging proton-motive force, generated by the F0F1- ATPase under fermentation conditions; disturbances in H+, K+ and Na+ transport, alterations in appropriate transport systems and lowering ATP level (Trchounian, 2004) as well as differences in membrane proteome (Xu et al., 2006) can increase sensitivity to antibiotics. In addition, alteration in DNA gyrase and other changes in gene expression or protein synthesis are also important for sensitivity to antibiotics (Cambau and Gutmann, Critical Reviews in Microbiology Downloaded from informahealthcare.com by IBI Circulation - Ashley Publications Ltd on 01/10/13 1993). Besides, this seems to be in accordance with the results of changed sensitivity of S. aureus to various antibiotics by extremely high frequency EMF with non- Figure 6. Overall, DCCD-treated and DCCD-sensitive ATPase thermal intensity (Bulgakova et al., 1996). Moreover, that activity of the membrane vesicles for EMF exposed E. coli K12(λ). is likely to the bioelectric effect with radio frequency In the control, bacteria were without irradiation DCCD-treated alternating electric current (10 MHz) for E. coli biofilms activity was determined after 10 min pre-incubation with 0.2 mM treated with the other antibiotics (Blenkinsopp et al., DCCD. DCCD-sensitive value was determined as a difference 1992). Dramatic increase of the efficacy of the antibiotic between the values in the presence and absence of DCCD in parallel measurements. The data were adapted from Tadevosyan et in biofilms was explained due to a specific action of al. (2008) and Torgomyan and Trchounian (2012). For the others, the radio frequency EMF on H2O molecules in liquid see the legends to Figure 1. surrounding or in biofilms matrix (Caubet et al., 2004).

© 2013 Informa Healthcare USA, Inc. 108 H. Torgomyan and A. Trchounian However role of membrane properties changes is not conditions biological systems or some of biological struc- ruled out. tures can be excited by interaction with coherent excita- Antibiotics are known to cause modifications and dis- tion of a specific frequency and precise value of which ruptions of bacterial cell cytoplasmic membrane those depends on particular electric dipolar unit characteriz- reflect in changes of cell membrane properties, their ing a given material (Betski et al., 2000; Belyaev, 2005). morphology and biochemical processes (Nakae and The provided EMF wavelength is largely compared with Nakae, 1982; Chung et al., 2006; Kohanski et al., 2007; the spatial dimension of the system, which coherent exci- Guliy et al., 2008). This might be occured in combined tation requires a supply of energy from irradiation. The action with EMF as well. As it was shown 51.8 GHz and 53 system becomes strong excited triggering fundamental GHz EMF with antibiotics depressed energy-dependent biological processes by excitation stimulation and then H+ and K+ transport, in which the main role belonged system acts to stabilize this mode and induces deforma-

to F0F1 (Torgomyan et al., 2011b). So, the enhanced cell tion of whole system (Hyland, 2008). death could be resulted (Figure 7) and detected. The probable targets which can be involved in a range Enhanced bacterial biofilm control by EMF in combi- of different processes and create bacterial responses to nation with different antibiotics is a phenomenon estab- extremely high frequency EMF are discussed above (see lished with a relatively weak continuous electric current Figure 3). The alterations in bacterial cell membrane and (McLeod et al., 1999) and a pulsed EMF (Pickering et al., in other macromolecular conformational components 2003). This EMF has been recently shown to increase containing charged particle or polar subsystems by low- antimicrobial activity against Staphilococcus epidermitis intensity EMF at specific frequencies could cause changes of the other substance, for instance, lime oil (Matewele, in cell function (Neshev and Kirilova, 1994; Fesenko 2010). However, the changing antibiotic activity for dif- et al., 1995; Kandashev and Savin, 1997; Pakhomov and ferent bacteria by low-intensity EMF of extremely high Murphy, 2000; Trchounian et al., 2001; Caubet et al., frequencies can be considered as the new phenom- 2004; Belyaev, 2005; Isakhanyan and Trchounian, 2005; enon which may have a specific explanation and a wide Tadevosyan et al., 2006, 2007, 2008; Ohanyan et al., application. 2008; Tadevosyan and Trchounian, 2009; Reguera, 2011; Torgomyan and Trchounian, 2011, 2012; Torgomyan et al., Model for EMF interaction with bacteria 2011a,b, 2012). Such as, EMF energy can be accumulated in H2O molecules till critical values by changing cluster To explain the mechanisms of the extremely high fre- structuring and triggering oscillations in them (Fesenko quency EMF bactericidal effects, quantum-mechanical et al., 1995; Guofen et al., 2002; Belyaev, 2005; Novoselova approach and physics of non-equilibrium and nonlinear et al., 2005; Cohen et al., 2010). This might cause changes systems are considered and a model is suggested for EMF in H O properties (Tadevosyan et al., 2007; Torgomyan

For personal use only. 2 interaction (Sinitsyn et al., 2000). Under appropriate et al., 2011b). The latter by its turn can alter membrane Critical Reviews in Microbiology Downloaded from informahealthcare.com by IBI Circulation - Ashley Publications Ltd on 01/10/13

Figure 7. Combined action of extremely high frequency EMF and antibiotics on bacteria. EMF signals are primary transformed to change

water cluster structuring, membrane proteins properties, DNA or prophage conformations and other cellular structures. H2O by its turn can change membrane-associated metabolic processes by altering membrane protein conformation, their hydration degree, electrochemical gradients across the membrane and other properties; a metabolic energy is required. These all create conditions, when the action of antibiotics is facilitated to bring enhanced cell damage and subsequently cell death (Torgomyan et al., 2011b). For the others, see the legends to Figure 3 and the text.

Critical Reviews in Microbiology Bactericidal effects of low-intensity extremely high frequency EMF 109 proteins conformation, their hydration degree and obtain of biomass and end-products; in medicine – to other properties (Neshev and Kirilova, 1994; Kandashev treate the broad range of bacterial diseases. The problem and Savin, 1997; Trchounian et al., 2001; Belyaev, 2005; also is to define the EMF exposure conditions depending Isakhanyan and Trchounian, 2005; Tadevosyan et al., on specific goals. 2008; Ukuku et al., 2008; Tadevosyan and Trchounian, 2009; Torgomyan et al., 2011b; Torgomyan and Acknowledgements Trchounian, 2011, 2012). Moreover, difference between bactericidal effects of EMF of different frequencies might The authors thank to Dr. V. Kalantaryan (Department of also be associated with their primary resonant interaction Telecommunication and High Frequency Radiophysics, with bacteria. EMF, therefore, can cause bacteria to Yerevan State University, Armenia), all researchers in vibrate and change metabolic activity. But the energy of Prof. Trchounian’s lab taking part in the study. This study weak EMF is not sufficient to destabilize of the H-bonds was done within the framework supported by Ministry of cellular macromolecules or to break the chemical of Education and Science of the Republic of Armenia bonds in DNA (Ruediger, 2009). EMF is able to primary (Grants # 0167–2005, # 1012–2008, # 11F-202–2011) and induce DNA damage but this should depend on EMF by US Armenian National Science & Education Fund frequency and intensity (Ruediger, 2009) so it requires (Grant # NS-Microbiol-1635). a further study. Elastic forces in the cell membranes or walls help to dampen those oscillatory forces by taking Declaration of interest part in coherent self-sustained oscillations, resulting in possible macromolecular conformational transitions The authors have no declarations of interest. which are fed with metabolic energy. Moreover, the straightened effects of antibiotics on References EMF exposed bacteria evidenced their common and sup- plementary action mechanism on cells, in which the cell Alipov ED, Shcheglov VS, Sarimov RM, Belyaev IY. (2003). Cell-density membrane probably played the main role (Trchounian dependent effects of low-dose ionizing radiation on E. coli cells. Radiats Biol Radioecol, 43, 167–171. et al., 2001; Tadevosyan et al., 2008; Tadevosyan and Banik S, Bandyopadhyay S, Ganguly S. (2003). Bioeffects of microwave–a Trchounian, 2009; Torgomyan et al., 2011a; Torgomyan brief review. Bioresour Technol, 87, 155–159. and Trchounian, 2011, 2012; Torgomyan et al., 2012). Belyaev I. (2005). Non-thermal biological effects of microwaves. Furthermore, the combined effects of EMF and anti- Microwave Rev, 11, 13–29. biotics are connected with the cell energy status. For Belyaev IY, Scheglov VS, Alipov YD, Radko SP. (1993). Regularities of separate and combined effects of circularly polarized millimeter example, in case of de-energization of cell by depression waves on E. coli cells at different phases of culture growth. of proton motive force across membrane (James et al., Bioelectrochem Bioenerg, 31, 49–63. For personal use only. 2009), antibiotics act with enhanced efficacy (McMurry Belyaev IY, Shcheglov VS, Alipov YD, Polunin VA. (1996). Resonance et al., 1986) (see Figure 7). However, mechanisms for effect of millimeter waves in the power range from 10(-19) to 3 x combined action of EMF and antibiotics are complex and 10(-3) W/cm2 on Escherichia coli cells at different concentrations. Bioelectromagnetics, 17, 312–321. not clear yet. Belyaev IYa, Alipov YD, Scheglov VS, Lystsov VN. (1992). Resonance effect of microwaves on the genome conformational state of E. coli cells. Z Naturforsch, 47, 621–627. Concluding remarks Betskii OV, Devyatkov ND, Kislov VV. (2000). Low intensity millimeter Over the past century and nowadays the environment waves in medicine and . Crit Rev Biomed Eng, 28, 247–268. Blenkinsopp SA, Khoury AE, Costerton JW. (1992). Electrical has been drastically changed due to increased amount enhancement of biocide efficacy against Pseudomonas aeruginosa of emissions, man-made EMF of extremely high frequen- biofilms. Appl Environ Microbiol, 58, 3770–3773. cies, etc. The latter even with low-intensity becomes a Bulgakova VG, Grushina VA, Orlova TI, Petrykina ZM, Polin AN, Noks PP, novel factor affecting different bacteria and their popu- Kononenko AA, Rubin AB. (1996). [The effect of millimeter-band lations. This is of significance for microbial physiology. radiation of nonthermal intensity on sensitivity of Staphylococcus to various antibiotics]. Biofizika, 41, 1289–1293. Probably, EMF has bactericide action, its effects lead to Cambau E, Gutmann L. (1993). Mechanisms of resistance to quinolones. the altered metabolic pathways in bacteria and to the

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