Extremely low frequency electromagnetic field affects virulence and antibiotic susceptibility of multidrug resistant Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus

Extremely low frequency electromagnetic field affects virulence and antibiotic susceptibility of multidrug resistant Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Extremely low frequency electromagnetic field affects virulence and antibiotic susceptibility of multidrug resistant Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus Mohamed Hosny, Mostafa Elnakib, Ahmed Gad This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6791184/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: The extremely low frequency electromagnetic field (ELF-EMF) effect on microorganisms has attracted attention due to its potential for industrial and medical applications as a promising candidate to combat multi-drug resistant pathogens. This study aimed to assess the effect of ELF-EMF on Gram-negative ( P. aeruginosa ) and Gram-positive ( S. aureus ) bacterial isolates. This effect was determined phenotypically (bacterial virulence and antibiotics susceptibility) and genotypically (mutations induced in whole genome). The test organisms were exposed to ELF-EMF with frequency of 0.7 Hz and 0.8 Hz for P. aeruginosa and S. aureus , respectively for different exposure times (6 and 12 hrs). Results: By comparing the results of exposed bacterial cultures with their counterparts non-exposed controls; remarkable differences were found in virulence, antibiotics susceptibility and genome structure. Whole genome sequencing revealed missense mutated genes that were associated directly/indirectly with the observed inhibition in protease and oxidase production, biofilm formation (in case of P. aeruginos a), coagulase and catalase production and biofilm formation (in case of S. aureus ). Also, the antibiotic susceptibility tests of both bacterial species indicated enhancement in the sensitivity. Conclusions: Therefore, it was concluded that each organism responds differently to ELF-EMF and exposure of P. aeruginosa and S. aureus test isolates to ELF-EMF at the stated frequencies affects the cellular activity as well as the structure and that effect depends on the duration of exposure. This study provides an evidence for the use of ELF-EMF as an efficient technique against skin bacterial infections especially those that are caused by pathogens with multiple drug resistance to different antimicrobial agents. Extremely low electromagnetic field Bacterial resistance S. aureus P. aeruginosa Virulence Mutation biofilm Multiple Drug resistance Growth Genome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Nowadays, the current environment is filled with electromagnetic fields (EMFs) which become as one of the most prevalent and quickly expanding factors that are affecting the environment 1 . The electromagnetic field spectrum includes radiofrequency, infrared, and X-rays, as well as static electric and magnetic fields. In various publications on radiation and field hazards, the extremely low frequency electromagnetic field (ELF-EMF) was given a range of 3 Hz to 3,000 Hz 2 . Later on, entities such as World Health Organization (WHO) and Occupational Safety and Health Administration (OSHA) referred the term extremely low frequency (ELF) to the frequency range of 0-300 Hz 3 . Currently, the ELF range is defined by the International Telecommunication Union (ITU) as the frequency range between 3 and 30 Hz only 4 . The ELF-EMFs are emitted from power lines, electric devices, electric appliances, communication systems and electric transmission lines 5 . Several studies were conducted to confirm the direct influence that extremely low frequencies electromagnetic fields (ELF-EMFs) could have on cell functions. Various responses of cells have been studied involving the effect on gene expression 6 , regulators of cell membrane signal transduction 7 , premature cell differentiation and proliferation 8 , ion channel expression and function 9 , cell apoptosis 10 , modification of the cellular redox state and modulation of the cellular metabolic activity 11 . Many studies in last decades have examined the impact of ELF-EMF on prokaryotes which appears promising in terms of medical or industrial uses 5 . The studies (that investigated the influence of ELF-EMF on Gram-positive or Gram-negative bacteria) dealt with this impact in terms of its effect on bacterial growth 12 , 13 morphology 14 , virulence 15 , and especially antimicrobial resistance (as it is one of the most bothering crisis that humanity is currently experiencing) 12 , 16 , 17 . Most of these studies have been recruited to find innovative method for the treatment of bacterial infections especially those that are caused by pathogens with multiple drug resistance to different antimicrobial agents. Several other studies have also worked on the mechanism and how this ELF-EMF introduces its effect on the bacteria or other cells. Some suggested that the active physiological mechanisms and biological systems produce electric currents and magnetic fields in vivo 18 and all bioelectric signals that produced by ionic movements, are involved in various activities. The ions' flow rate and direction produce an electric impulse with a certain frequency, and amplitude 18 . In order to control physiological functions in biological systems, it is necessary to apply electric fields with a frequency corresponding to the bioelectric impulses produced during metabolic processes. Ion motion in the biological system will be impacted by this interference and depending on the mechanism of interference, the ion motion may be promoted or inhibited. Considering that the bioelectric signals produced by metabolic processes in cells are known to be in the extremely low frequency range, the applied electromagnetic wave should have the same frequency as the bioelectric signal 19 . This study aimed to assess the effect of extremely low frequency electromagnetic field (ELF-EMF) on bacteria, both, phenotypically and genotypically by measuring the effect on virulence factors, antibiotics susceptibility and genome structure. Materials and Methods 1. Collection of some clinical isolates of certain bacterial species including S. aureus and P. aeruginosa. A number of 38 isolates from wound swabs were collected from surgical ICU of a tertiary hospital, Cairo, Egypt, in the period from June 2020 to August 2021. The isolates were identified first with the Vitek 2 system (AES software, bioMérieux, Marcy l'Étoile, France) according to the manufacturer's instructions. These isolates included, Methicillin-resistant S. aureus (MRSA), Acinetobacter, Proteus, E. coli, Pseudomonas and Klebsiella. The distribution of these isolates is shown in Table 1 ; these isolates included 6 Gram-positive isolates and 32 Gram-negative isolates. The Gram-positive isolates were, Methicillin-resistant S. aureus (MRSA) while the Gram-negative isolates included Proteus , Acinetobacter, E. coli, Pseudomonas and Klebsiella. Table 1 Distribution of isolates recovered from wound swabs Isolates Genus/Type Number Pseudomonas 12 MRSA 6 Klebsiella 15 Proteus 2 E. coli 2 Acinetobacter 1 Total 38 A number of 5 out of 12 P. aeruginosa isolates were multidrug resistant. These 5 isolates together with 6 Methicillin-Resistant S. aureus (MRSA) were selected to complete this study. The five multidrug resistant P. aeruginosa isolates were given the codes of P1, P2, P3, P4 and P5, while the six Methicillin-Resistant S. aureus (MRSA) were given the codes of S1, S2, S3, S4, S5 and S6. Different virulence factors of the collected isolates were studied before and after exposure to extremely low electromagnetic field; these factors included protease production, oxidase production, and biofilm formation for P. aeruginosa whereas coagulase production, catalase production, and biofilm formation were the virulence factors studied for MRSA isolates. 2. Preparation of the bacterial cultures for exposure to extremely low frequency electromagnetic field The bacterial culture used for subsequent experiments was prepared as follows: from the stock culture (nutrient agar plate culture) of each bacterial isolate, a loopful was transferred to 5 ml double strength nutrient broth contained in a test tube. The test tube was incubated overnight at 37 o C. From the obtained tube culture, a loopful was streaked for isolation on selective media of cetrimide agar (in case of P. aeruginosa ) and mannitol salt agar (in case of S. aureus ) followed by incubation at 37 o C for 24 h. Each isolate was cultured on the selective medium in triplicates, one to be used as a control while the second and the third ones were exposed to ELF-EMF for two separate and different durations of 6 and 12 hrs, respectively (three culture plates were used for each exposure duration period). The control culture was kept in the same conditions as the exposed ones but without exposure. 3. Exposure to extremely low frequency electromagnetic field Figure 1. shows the prototype apparatus for generation of the applied extremely low frequency electromagnetic waves of different frequencies (0.7 Hz and 0.8 Hz) and its mechanistic use. The isolate cultures' plates were placed between two parallel copper disk plates that emitted the ELF-EMF. Each copper plate was of 3 mm thickness and the distance between the two plates 30 cm. The cultures of bacteria were exposed to ELF-EMF with frequency of 0.7 Hz and 0.8 Hz for P. aeruginosa and S. aureus , respectively for different exposure durations (6 and 12 hrs for each bacterial species). 4. Effect of ELF-EMF on some virulence factors of P. aeruginosa 4.1. Effect on protease production A colony from the control culture and ELF-EMF exposed culture of the test isolate were line streaked separately on plates containing 15 ml of gelatin agar medium. The plates were incubated at 37 o C for 24 h. After the incubation period, the plates were flooded with mercuric chloride solution which causes opacity in the medium with clear zone around streaked growth. The clear zone width in mm was taken as a measure for the amount of the produced protease 20 . 4.2. Effect on oxidase production Ready-made oxidase discs were used; the disc was applied to an isolated colony of either the control culture or ELF-EMF exposed culture of the test isolate after had been cultured on nutrient agar and incubated at 37 o C for 24 h. A reaction was observed within 10–30 sec 21 . The results were recorded as follows: (++); microorganism that is oxidase positive with oxidase disc color changed to deep purple color within 10 sec, (+); microorganism that is delayed oxidase positive with disc color changed to deep purple color in more than 10 sec up to 30 sec, (-); microorganism that is oxidase negative and the disc color did not turn into deep purple in 30 sec. 4.3. Effect on biofilm formation An isolated colony from both the control culture and ELF-EMF exposed culture of the test isolate were separately streaked on nutrient agar plate. The plates were incubated at 37°C for 24 h. From the obtained culture an isolated colony was transferred into 5 ml of trypticase soy broth contained in a test tube followed by incubation at 37°C for 24 h. The cultures were diluted 1:100 with fresh trypticase soy broth. Each individual well of 96 U-shaped bottom polystyrene microtitre plate were filled with 0.2 ml aliquots of diluted culture. Control wells containing fresh trypticase soy broth were included to check sterility and nonspecific binding of the media. The plates were then incubated at 37°C for 24 h, after incubation the plates were inverted for discarding their contents with gentle tapping of the plates. The Plates were washed three times, each time with 0.2 ml of phosphate buffer saline (pH 7.2). The plates were then incubated at 37°C for an hour while inverted to get dried followed by transferring 0.2 ml aliquots of 0.1% crystal violet and leaving at room temperature for 10 min. Excess stain was removed by inverting the plates followed by washing three times with phosphate buffer saline and the plates were left at room temperature while inverted for drying. Aliquots of 200 µl of 95% ethanol were added to the wells. Optical densities (OD s ) of the stained plates were determined using micro ELISA auto reader (BIORAD 680) at a wavelength of 570 nm (OD 570 nm ) 22 . Interpretation of biofilm production based on optical density values was carried out as described in reference 25 where OD value of 0.12 was considered positive biofilm production. 5. Effect of ELF-EMF on some virulence factors of S. aureus (MRSA) 5.1. Effect on coagulase production Tube coagulase test was used, the following procedure 23 was followed for each isolate. An isolated colony from either the control culture or ELF-EMF exposed culture of the test isolate (retrieved from 24 h nutrient agar subculture) was placed in a test tube containing 0.5 ml of human plasma (from Medical Research Laboratories and Blood Bank of Armed Forces, Egypt), then the tube was incubated at 37°C and examined at 30 minutes intervals for up to 4 h. Clot formation was observed and the results were interpreted according to Sperber & Tatini (1975) as follows: (-); no evidence of fibrin formation, (+); have small unorganized clots, (++); feature of a large clot formation; and (+++); when the entire contents of the tube are coagulated and unable to be displaced upon tube inversion. 5.2. Effect on catalase production On a slide, 1 or 2 drops of 3% H 2 O 2 were added. An isolated colony from either the control culture or ELF-EMF exposed culture of the test isolate (retrieved from 24 h subculture nutrient agar) was picked up and mixed with the H 2 O 2 drops on a slide using a sterile applicator stick. The slide was placed over a dark surface and observed for immediate bubble formation 24 . Positive catalase production is evident by immediate effervescence (bubble formation), while no bubble formation (no catalase enzyme to hydrolyze the hydrogen peroxide) represents a negative catalase production. 5.3. Effect on biofilm formation This was carried out as mentioned earlier in section 4.3 . 6. Antimicrobial sensitivity test Kirby-Bauer method was used for determining the sensitivity of the test isolates to different antibiotics after exposure to ELF-EMF against their corresponding controls as follows. An isolated colony from either the control culture or ELF-EMF exposed culture of the test isolate was streaked on nutrient agar plate. The plates were incubated at 37°C for 24 h. From the obtained culture, an isolated colony was transferred and mixed with about 2 ml saline and adjusted to 0.5 McFarland standard, then a sterile swab was dipped in the isolate suspension obtained. After removal of the excess suspension by pressing the swab against the inner tube surface, it was used for entire streaking the surface of a Müeller–Hinton agar plate. The antibiotic discs were applied onto the surface of the inoculated plates and gently pressed. The plates were then incubated at 37°C for 24 h. The diameter of inhibition zones were measured in millimeters and recorded. The results were interpreted according to Clinical and Laboratory Standards Institute, 2021 (CLSI 2021). 7. Next-generation sequencing (NGS) using Illumina MiSeq machine (Whole Genome Sequencing). This was carried on one P. aeruginosa isolate (P4) and one S. aureus isolate (S3) 7.1. DNA Extraction The genomics DNA was extracted using the QIAamp® DNA Minikit (QIAGEN, Germany) following the manufacturer’s instructions. 7.2. Library Preparation and Next Generation Sequencing The preparation of the library was carried out utilizing the Nextera XT DNA Library preparation kit. 7.2.1. Tagment Genomic DNA In this step the Nexteratransposome was used to tagment DNA where the DNA was fragmented and then tagged with adapter sequences in a single step. 7.2.2. Amplify Library In this step the tagmented DNA was amplified using a limited-cycle PCR program where the Index 1 (i7), Index 2 (i5), and full adapter sequences were added to the tagmented DNA obtained in the previous step (7.2.1). The index primers and Nextera PCR Master Mix were added directly to the 25 µl of tagmented DNA obtained. 7.2.3. Clean Up Libraries In these step AMPure XP beads was used to purify the library DNA and remove short library fragments. 7.2.4. Check Libraries In this step 1 µl of library was run on an Agilent Technology 2100 Bio-analyzer using Agilent DNA 1000 chip. Typical libraries show a broad size distribution of ~ 250–1000 bp. Various libraries can be sequenced with average fragment sizes as small as 250 bp or as large as 1500 bp. 7.2.5. Normalize Libraries In this step the quantity of each library was normalized to ensure more equal library representation in the pooled library. 7.2.6. Pooling Library Equal volumes of normalized libraries were combined in a single tube to get the pooled library. The pooled library was diluted and heat-denatured before its loading for the sequencing run which was conducted according to Illumina manufacturing instructions. 7.3. Quality control, genome assembly, and alignment Reads were filtered, inspected and adaptors were removed using fastp. After that, the filtered reads were de novo assembled using Unicycler and annotated using Prokka. The assessment of the assembled files was carried out with QUAST. Previously filtered reads were mapped to the reference using Burrows-Wheeler Aligner (BWA) followed by variant calling using GATK and Picard. After that, most similar sequences were identified and phylogenetic tree was constructed using PATRIC. Finally, taxonomy profiling was performed using kraken2 and visualized using Pavian. 7.4. Variant calling and variant effect prediction. Previously merged reads in case of P. aeruginosa and S. aureus were mapped to the P. aeruginosa and S. aureus references (GenBank accession number NC_007795.1 and ASM676v1, respectively) using BWA. Variant identification and filtration were performed using GATK and Picard. The resulting variants were used to predict the variant effect using SnpEff, as well as consensus sequence generation using BCFtools. After that, the multiple sequence alignment was done. 7.5. Workflow diagram Statistical Analysis: The collected data was revised, coded, tabulated and introduced to a PC using Statistical package for Social Science (SPSS 27). Data was presented and suitable analysis was done according to the type of data obtained for each parameter. Descriptive statistics: Median and Interquartile range (IQR) for non-parametric numerical data. Frequency and percentage of non-numerical data. Analytical statistics: Wilcoxon signed rank test was used assess the statistical significance of the difference of an ordinal variable (score) measured twice for the same study group. McNemar test was used assess the statistical significant of the difference between a qualitative variable measured twice for the same study group. Marginal homogeneity test assesses the statistical significance of the difference of a variable with multiple categories measured twice for the same study group. p -value : level of significance: -P > 0.05: Non- significant (NS), P < 0.05: Significant (S). Results 1. Effect of ELF-EMF on some virulence factors of P. aeruginosa Table 2 . Presents the relation between different virulence factors of P. aeruginosa across different time points after exposure and the table shows that there was a significant decrease of the diameter of clearance zone (mm) between before radiations Vs. after 6 and 12 hrs of exposure as p -value was (0.042), but there was no statistically difference between after 6 hrs Vs. 12 hrs of exposure as p -value was (0.068). Regarding oxidase production there was a decrease in number of isolates that produce an oxidase across different time points of exposure without statistically significance difference between them. Regarding biofilm formation there was statistically significant decrease in absorbances readings that obtained at (OD 570nm ) as p -value was (0.043) between all different time points. Table 2 Effect of ELF-EMF* on some virulence factors of P. aeruginosa isolates Pseudomonas (N = 5) Before After 6 hrs After 12 hrs Test of significance (a) Median (IQR) N (%) Median (IQR) N (%) Median (IQR) N (%) p 1 p 2 p 3 Diameter of clearance zone (mm) (b) 20 (19–20) 10 (9–10) 7 (7–9) 0.042 0.042 0.068 Oxidase production (c) (+) 0 (0%) 2 (40%) 4 (80%) 0.5 0.125 0.5 (++) 5 (100%) 3 (60%) 1 (20%) Biofilm formation (b) 0.14 (0.14–0.24) 0.07 (0.06–0.15) 0.05 (0.05–0.06) 0.043 0.043 0.043 (a) p 1: p -value between Reading before Vs. Reading after 6 hrs. p 2: p -value between Reading before Vs. Reading after 12 hrs. p 3: p -value between Reading after 6 hrs Vs. Reading after 12 hrs. (b) Wilcoxon sign rank test was used to assess the significance. (c) McNemar test was used to assess the significance. * ELF-EMF was used at 0.7 Hz frequency 2. Effect of ELF-EMF on some virulence factors of S. aureus (MRSA) Table 3 . Presents the relation between different virulence factors of S. aureus (MRSA) across different time points after exposure and the table shows that there was no statistically significant difference in number of isolates in catalase production as all isolates were (+). Regarding biofilm formation there was statistically significant decrease in absorbances that obtained at (OD 570nm ) as p -value was (0.028) between all different time points. Table 3 Effect of ELF-EMF* on some virulence factors by S. aureus (MRSA) isolates MRSA (N = 6) Before After 6 hrs After 12 hrs Test of significance (a) Median (IQR) N (%) Median (IQR) N (%) Median (IQR) N (%) p 1 p 2 p 3 Catalase production (b) (+) 6 (100%) 6 (100%) 6 (100%) ---- ---- ---- Biofilm formation (c) 0.16 (0.06–0.19) 0.06 (0.05–0.08) 0.03 (0.02–0.03) 0.028 0.028 0.028 (a) p 1: p -value between Reading before Vs. Reading after 6 hrs. p 2: p -value between Reading before Vs. Reading after 12 hrs. p 3: p -value between Reading after 6 hrs Vs. Reading after 12 hrs. (b) Wilcoxon sign rank test was used to assess the significance. (c) McNemar test was used to assess the significance. * ELF-EMF was used at 0.8 Hz frequency Table 4 . illustrates the relation between coagulase production by S. aureus (MRSA) at different time intervals across different time points of exposure and there was no difference in number of isolates before and after 6 hrs of exposure. Regarding the relation between before and after 12 hrs of exposure at reading time interval of two hours, there was no statistically significance difference in number of isolates that produced coagulase as p -value was (0.083), while there were statistically significance decrease in number of isolates that showed entire contents are complete coagulated and are not displaced when the tube inverted at reading time interval of three hours and four hours as p -value were (0.046 & 0.031), respectively. Table 4 Effect of ELF-EMF* on coagulase produced by S. aureus (MRSA) isolates Time interval After 2 hours After 3 hours After 4 hours N (%) N (%) N (%) Before (+) 6 (100%) 0 (0%) 0 (0%) (++) 0 (0%) 6 (100%) 0 (0%) (+++) 0 (0%) 0 (0%) 6 (100%) After 6 hrs (+) 6 (100%) 0 (0%) 0 (0%) (++) 0 (0%) 6 (100%) 0 (0%) (+++) 0 (0%) 0 (0%) 6 (100%) After 12 hrs Negative 3 (50%) 0 (0%) 0 (0%) (+) 3 (50%) 4 (66.67%) 0 (0%) (++) 0 (0%) 2 (33.33%) 6 (100%) Test of significance (Before or After 6 hrs) Vs. After 12 hrs 0.083 (a) 0.046 (a) 0.031 (b) (a) Marginal Homogeneity test was used to assess the significance. (b) McNemar test was used to assess the significance. 3. Effect of exposure to ELF-EMF on the susceptibility of P. aeruginosa and S. aureus test isolates to antibiotics Table 5 . demonstrates antimicrobial susceptibility testing of P. aeruginosa across different time points exposure, there were no statistically significance difference in zone diameter of antimicrobial panel except TZP there were statistically significant increase in zone diameter between after 12 hrs exposure Vs. (before and after 6 hrs exposure) as p -value were (0.041 & 0.034), respectively. Table 5 Antibiotic susceptibility of P. aeruginosa isolates after exposure to ELF-EMF* as compared with unexposed control cultures Pseudomonas (N = 5) Before After 6 hrs After 12 hrs Wilcoxon Signed Ranks Test (a) Median (IQR) Median (IQR) Median (IQR) P1 P2 P3 Piperacillin-tazobactam (TZP) 8 (5–8) 9 (9–9) 12 (11–12) 0.066 0.041 0.034 Meropenem (MEM) 5 (5–5) 5 (5–5) 5 (5–8) 0.317 0.180 0.317 Ciprofloxacin (CIP) 5 (5–6) 5 (5–6) 5 (5–6) 1.000 0.317 0.317 Amikacin (AK) 5 (5–5) 5 (5–5) 5 (5–5) 1.000 1.000 1.000 Ceftazidime (CAZ) 5 (5–5) 5 (5–5) 5 (5–5) 1.000 1.000 1.000 Cefepime (FEP) 5 (5–5) 5 (5–5) 5 (5–5) 0.317 0.317 1.000 Levofloxacin (LEV) 5 (5–5) 5 (5–5) 5 (5–5) 1.000 1.000 1.000 Nitrofurantoin (NI) 5 (5–5) 5 (5–5) 5 (5–5) 1.000 1.000 1.000 Gentamicin (CN) 5 (5–5) 5 (5–5) 5 (5–5) 0.317 0.317 1.000 (a) p 1: p -value between Reading before Vs. Reading after 6 hrs. p 2: p -value between Reading before Vs. Reading after 12 hrs. p 3: p -value between Reading after 6 hrs Vs. Reading after 12 hrs. *ELF-EMF was used at 0.7 Hz frequency The method used to measure the sensitivity of the bacterial cells toward different antibiotics was disc method by Bauru-Kirby technique (Baker et al ., 1980) The method used to measure the sensitivity of the bacterial cells toward different antibiotics was disc method by Bauru-Kirby technique (Baker et al ., 1980) The method used to measure the sensitivity of the bacterial cells toward different antibiotics was disc method by Bauru-Kirby technique (Baker et al ., 1980) Table 6 . shows antimicrobial susceptibility testing of S. aureus (MRSA) across different time points exposure, there were no statistically significance difference in zone diameter of antimicrobial panel except (LEV, LZD & CLR) there were statistically significanct increase in zone diameter between after 12 hrs exposure Vs. (before and after 6 hrs exposure) as p -value were (0.042 & 0.043), (0.027 & 0.026) and (0.024 & 0.041), respectively. While there was statistically significant increase in zone diameter between before Vs. after 12 hrs exposure as p -value was (0.042) Table 6 Antibiotic susceptibility of S. aureus isolates after exposure to ELF-EMF* as compared with unexposed control cultures MRSA (N = 6) Before After 6 hrs After 12 hrs Wilcoxon Signed Ranks Test (a) Median (IQR) Median (IQR) Median (IQR) p 1 p 2 p 3 Amoxicillin/clavulanic acid (AMC) 5 (5–8) 8.5 (5–10) 11 (11–12) 0.109 0.042 0.068 Levofloxacin (LEV) 18 (5–20) 21 (5–24) 25 (14–25) 0.102 0.042 0.043 Linezolid (LZD) 23 (22–25) 25.5 (24–27) 26.5 (26–30) 0.102 0.027 0.026 Clindamycin (CD) 8.5 (5–14) 10 (5–16) 11 (5–19) 0.109 0.102 0.109 Cefoxitin (FOX) 8.5 (5–12) 8.5 (5–12) 12 (5–13) 1.000 0.109 0.109 Ciprofloxacin (CIP) 19.5 (14–21) 21 (14–25) 22.5 (14–25) 0.109 0.066 0.317 Gentamicin (CN) 5 (5–5) 5 (5–5) 5 (5–5) 1.000 1.000 1.000 Clarithromycin (CLR) 13 (5–20) 14.5 (8–22) 17 (12–24) 0.102 0.024 0.041 Ampicillin (Amp) 5 (5–5) 5 (5–5) 5 (5–5) 1.000 1.000 1.000 Oxacillin (Ox) 5 (5–5) 5 (5–5) 7.5 (5–10) 1.000 0.102 0.102 Penecillin G (P) 5 (5–5) 5 (5–5) 6.5 (5–10) 1.000 0.102 0.102 Methicillin (Met) 5 (5–5) 5 (5–5) 5 (5–8) 1.000 0.180 0.180 (a) p 1: p -value between Reading before Vs. Reading after 6 hrs. p 2: p -value between Reading before Vs. Reading after 12 hrs. p 3: p -value between Reading after 6 hrs Vs. Reading after 12 hrs. *ELF-EMF was used at 0.8 Hz frequency 4. Whole genome sequencing of P. aeruginosa isolate P4 and S. aureus isolate S3 before and after exposure to ELF-EMF 4.1. Gene Ontology for genes affected by mutations Gene Ontology (GO) of identified sequences of the two test isolates, P. aeruginosa P4 and S. aureus S3 before and after exposure to ELF-EMF was determined using Uniprot website ( https://www.uniprot.org/ ) accessed on 19July, 2022. The results are shown in Figs. 2&3 ( P. aeruginosa P4 ) and Figs. 4&5 ( S. aureus S3). 4.2. Variant calling (missense mutations) of affected genes in whole genome sequences of P. aeruginosa P4 and S. aureus S3 upon exposure to ELF-EMF Filtered reads of the two test isolates, P. aeruginosa P4 and S. aureus S3 after exposure to ELF-EMF for 6 and 12 hrs in comparison to the parent isolate before exposure were mapped using BWA software followed by variant calling using GATK and Picard software. The results are shown in Table 7 ( P. aeruginosa P4 ) and Table 8 ( S. aureus S3). Table 7 Intersect of missense mutation of affected genes due to exposure of P. aeruginosa P4 to ELF-EMF for 6 and 12 hrs in comparison to the parent isolate before exposure. Gene name Protein name Length (amino acids) Genomic change Protein change PA0259 DUF2875 domain-containing protein 480 290406A > T 200V > 200E PA0690 Haemagg_act domain-containing protein 4180 756738A > T 2261E > 2261V PA0982 Thioredoxin-like_fold domain-containing protein 182 1064704C > T 134D > 134N fliC PA1092 B-type flagellin 488 1183439G > A + 1183441T > C 128D > 128N cobTcobU PA1279 Nicotinate-nucleotide–dimethylbenzimidazolephosphoribosyltransferase (NN: DBI PRT) (EC 2.4.2.21) (N(1)-alpha-phosphoribosyltransferase) 351 1390304G > T 27R > 27L PA1416 FAD-binding PCMH-type domain-containing protein 460 1540435T > C 233T > 233A PA1874 BapA prefix-like domain-containing protein 2468 2041147C > G 1569I > 1569M pvdP PA2392 PvdP 544 2648261A > C 50D > 50E pvdF PA2396 Pyoverdine synthetase F 275 2652921T > C 46Q > 46R pvdD PA2399 Pyoverdine synthetase D 2448 2660959G > A 1396L > 1396F PA2402 Probable non-ribosomal peptide synthetase 5149 2678603G > A 2859A > 2859V pvdL PA2424 PvdL 4342 2713781G > A 2305A > 2305V PA2431 FUSC family protein 724 2726207T > G + 2726209C > T 645G > 645S PA2650 Methyltransf_11 domain-containing protein 269 2998633G > A 265R > 265K PA2760 Probable outer membrane protein 425 3120238A > C + 3120240G > A 56K > 56Q PA4503 Probable permease of ABC transporter 336 5042647G > T 194A > 194S Table 8 Intersect of missense mutations of affected genes due to exposure of S. aureus S3 to ELF-EMF for 6 and 12 hrs in comparison to the parent isolate before exposure. Gene name Protein name Length (amino acids) Genomic change Protein change SAOUHSC_00036 Rhodanese domain-containing protein 444 40440G > C 325E > 325Q SAOUHSC_00052 Uncharacterized lipoprotein SAOUHSC_00052 255 55881A > T 140E > 140D SAOUHSC_00235 PTS glucose transporter subunit IIABC 263 256400A > G 61T > 61A SAOUHSC_00245 Truncated transposase 134 264761T > A + 264763G > A 128W > 128R SAOUHSC_00255 DUF5080 family protein 195 274510G > A + 274512T > A 151V > 151I SAOUHSC_00276 TIGR01741 family protein 166 293353T > A 94L > 94H ebh SAOUHSC_01447 Extracellular matrix-binding protein ebh (ECM-binding protein homolog) 9535 1381611G > A + 1381613T > G 7696N > 7696H SAOUHSC_01557 Conserved hypothetical phage protein 68 1496261C > A 40C > 40F SAOUHSC_01558 PVL orf 51-like protein 80 1496625C > T 4S > 4N SAOUHSC_01582 Bacteriophage integrase 401 1508260T > A 323N > 323K SAOUHSC_02205 Conserved hypothetical phage protein 178 2060410T > G 64E > 64D SAOUHSC_02215 Conserved hypothetical phage protein 52 2063075T > G 51E > 51A SAOUHSC_02228 Conserved hypothetical phage protein 65 2069287A > T 51N > 51K SAOUHSC_02788 Uncharacterized lipoprotein SAOUHSC_02788 261 2560676C > A 21G > 21V SAOUHSC_A02795 Uncharacterized protein 56 2714202T > A 12L > 12H SAOUHSC_02991 Flavin_Reduct domain-containing protein 230 2767298T > G + 2767300C > T 228E > 228N Discussion The spreading and aggressiveness of infections which are caused by antimicrobial resistance and the lack of progress towards novel antibiotic discovery over the past decade were the reason behind investigating ELF-EMF as a novel technique against multidrug resistant infections. The difficulty in finding new antibiotic compounds has provoked the developing and utilizing of novel and complex techniques, such as electromagnetic technique. In order to assess its effect, in vitro , we employed extremely low frequency electromagnetic waves on bacteria, which retrieved from surgical site infections, and evaluated the effect of extremely low frequency electromagnetic field (ELF-EMF) on the bacterial genome, some virulence factors and antibiotics susceptibilities of both multiple drug resistant P. aeruginosa and S. aureus MRSA test isolates. The results have driven the study to significant conclusions and recommendations regarding using and application of ELF-EMF. Many studies have been carried out to verify the direct impact of extremely low frequency electromagnetic fields (ELF-EMFs) on the functioning of bacterial cells. Most of the research examined the effects on bacterial DNA, growth, morphology, biofilm, and antimicrobial susceptibility. Most of these studies revealed that ELF-EMF has a direct effect on bacteria. According to Inhan-Garip et al ., 5 who applied ELF-EMF on Gram-negative P. aeruginosa and Gram-positive S. aureus for six hrs, it was observed that (in comparison to control), all exposed strains to ELF-EMF were seen to have a significant decrease in growth rate suggesting that a mutation in bacterial genome was induced and that mutation became irreversible. Additionally, the investigations of Fojt et al. , Strasak et al ., El-Sayed et al ., Aslanimehr et al ., Segatore et al ., Martirosyan, Chen et al ., Oncul et al ., 25 , 26 , 27 , 16 , 28 , 29 , 30 , 14 showed that, ELF-EMF exposure has an impairing effect on different bacterial species in time-dependent manner. In addition the proper selection of frequency and exposure duration play a significant role in the effect of extremely low frequency electromagnetic field on bacteria. After reviewing literature, most of the research indicate that the bacterial cell wall and membrane are both responsible for the effects of extremely low frequency electromagnetic fields on bacteria, as it was confirmed by Fang et al ., 31 (after using the transmission electron microscopes), the cell wall of the exposed bacteria was broken forming irreversible perforations on the cell membrane; the cell inclusions and cell components were leaked and the cytoplasm and nucleolus substances flowed away leading to cell death. As well as based on several other studies of Garip et al ., Fadel et al ., Volpe et al ., Cellini et al ., Del Re et al ., 5 , 32 , 33 , 34 , 35 which documented that after exposure to ELF-EMF, the bacterial membrane hyperpolarization was induced causing changes in bacterial surface charge that affects the membrane potential and changes ions conduction which affect directly on bacterial viability and the electron transport system that lead to cell damage. Strahl and Hamoen 36 , showed that the presence of membrane potential is required for bacterial growth and survival. It was also determined that exposure to electromagnetic fields would change the physicochemical characteristics and activity of bacteria. Cell surface charge is essential for bacterial adhesion and defense mechanisms in host and have an impact on virulence, viability, and survival. We tested in our study the hypothesis (ELF-EMF induce its effect on bacteria through altering morphology of both cell wall and cell membrane) by first studying the effect of ELF-EMF on production of extracellular virulence factors such as protease, oxidase and biofilm (in case of Gram negative P . aeruginosa ) as well as coagulase, catalase and biofilm (in case of S . aureus MRSA). In addition, we studied the effect on bacterial antimicrobial susceptibility then we took an advanced step and investigated more by using whole genome sequencing and bioinformatics analysis of two bacteria isolates to find whether ELF-EMF has an effect at molecular level or not and which genes are mutated and responsible for the effect of ELF-EMF. Effect of extremely low frequency electromagnetic field (ELF-EMF) on some virulence factors and antibiotics susceptibilities of P. aeruginosa and S. aureus test isolates Exposure of P. aeruginosa test isolates to ELF-EMF of 0.7 Hz for 6 and 12 hrs affected extracellular protease production. The exposure caused significant decrease in protease production. As shown in Table 2 there was a significant decrease of the diameter of clearance zone (mm) produced in gelatin agar plates after 6 and 12 hrs of exposure to ELF-EMF compared to that obtained before exposure as p -value was (0.042), but there was no statistically difference after 6 hrs Vs. 12 hrs of exposure as p -value was (0.068). On other hand, regarding oxidase production by P. aeruginosa test isolates, exposure to ELF-EMF produced unexpected effects as the production of extracellular oxidase enzyme was not altered significantly. Although, there was a decrease in number of isolates that produce oxidase across different time points of exposure, but without statistically significant difference before exposure and after 6 hrs or 12 hrs exposure as p -value was (0.5 & 0.125), respectively. It was found that studies that investigated the effect of ELF-EMF on protease or oxidase production and the reason behind that are lacking and this could be because of the complexity of P. aeruginosa which makes studying the effect of ELF-EMF on extracellular protein (protease & oxidase) and corelating exposure with enzymes productions is difficult. Many genes entangled in their functions and take part directly or indirectly in the production cascade of protease and oxidase. Protease is extracellular enzyme that require cellular channel to be excreted. We detected a significant reduction in protease production as a results of cell wall distortion as was also confirmed by Fang et al ., 31 . It was found that as shown in Table 2 , ELF-EMF produces its effect in time dependent manner on bacterial cell wall. Our results revealed that, protease production was decreased gradually after 6 hrs &12 hrs., this reduction in protease productivity gives a chance to use ELF-EMF as a method against P. aeruginosa protease which plays a significant role in infections. In contrast to protease results, oxidase production (as shown in Table 2 ) showed non-significant change pheno-typically in 6 hrs & 12 hrs VS before exposure, although it is an extracellular enzyme. In addition, the number of isolates that produce oxidase across different time points of exposure in time dependent manner was decreased. According to Arai et al , the opportunistic pathogen P. aeruginosa encodes large number and diverse oxidases, and oxidase gene expression can be compensatory, such that loss of one or more oxidases leads to induction of others 37 , that's why we were unable to find any variations in oxidase production after exposure. In case of S. aureus test isolates, exposure to 0.8 Hz ELF-EMF caused inhibition in coagulase production in 5 out of 6 isolates and this effect was only observed after 12 hrs exposure. As shown in Table 4 , the relation between before and after 12 hrs of exposure at reading time interval of two hours, there was no statistically significant difference in number of isolates that produced coagulase p -value was (0.083), while there was statistically significant decrease in number of isolates that showed complete coagulation with no displacement when the tube inverted at reading time interval of three hours and four hours as p -value were (0.046 & 0.031), respectively. Interestingly exposure of the six S. aureus test isolates to ELF-EMF showed no effect on catalase production either after 6 or 12 hrs exposure. After statistical analysis of results of S. aureus (MRSA) across different time points after exposure, there was no statistically significant difference in number of isolates regarding catalase production as all isolates were (+) as shown in Table 3 . To provide an interpretation of the obtained findings that are demonstrated by inhibition of protease extracellular production while oxidase production showed non-significant changes (in case of P. aeruginosa ) and inhibition of coagulase extracellular production while catalase was not (in case of S. aureus ), we explained these results due to the channels which present in bacterial cell and responsible for excretion of these extracellular enzymes, these channels were impaired in case of protease and coagulase while the channels which are responsible for excretion of oxidase and catalase were not impaired at used frequency and exposure duration of ELF-EMF. This raise new inquiries about the appropriate choice of ELF-EMF frequency and the relationship between ELF-EMF frequency and/or duration with the precise area of the bacterial cell wall and membrane that ELF-EMF affects. It is suggested that longer exposure duration than 12 hrs is needed (based on our results) in case of Gram positive to obtain a complete distortion of cell wall and that because of the nature composition of Gram positive cell wall is thicker than Gram negative. Regarding biofilm formation, exposure of P. aeruginosa test isolates to ELF-EMF caused significant reductions in biofilm formation in all test isolates. As shown in Table 2 biofilm formation was statistically significant decrease in the absorbances readings that obtained at (OD 570nm ) as p -value was (0.043) between all different time points; between before exposure Vs. after 6 and 12 hrs of exposure as p -value was (0.043), and also between reading after 6 hrs Vs. reading after 12 hrs. In case of the effect of ELF-EMF on S. aureus test isolates biofilm formation, the results were similar to that exhibited by P. aeruginosa test isolates, where S. aureus test isolates showed a significant decrease in biofilm formation upon exposure to ELF-EMF. As shown in Table 3 there was statistically significant decrease in absorbances readings obtained at (OD 570nm ) as p -value was (0.028) between all different time points; between before exposure Vs. after 6 and 12 hrs of exposure as p -value was (0.028), as well as between reading after 6 hrs Vs. reading after 12 hrs. These results were aligned with Karaguler et al ., and Haagensen et al ., 38 , 39 which indicated that ELF-EMF has direct effects on biofilm formation and this effect is caused by their impact on the electrical charges embedded in the cell membrane, which alters the behavior of the cells that’s in turn affecting the exopolymeric matrix structure formation. Considering how the electromagnetic field affects bacterial resistance to antibiotics, there are plenty of opposing reports. It was established that exposure to electromagnetic field increases the permeability of ion channels in the cytoplasmic membrane, breaking down the cell wall and the cytoplasmic membrane which in turn influence the permeation and movement of antibiotics molecules across bacterial cell 40 . In addition, Segatore et al ., 16 who used P. aeruginosa to evaluate the effect of ELF-EMF on antibiotics susceptibility and concluded that by interfering with the surface charges on bacterial membrane, the rate at which antimicrobials penetrate is increased which in turn affected on bacterial susceptibility. On the contrary, Kamel et al. , 41 showed that the exposure to ELF-EMF could induce a reversible defensive mechanism to repair the provoked damage in bacterial membrane, allowing an adaptive response with no remarkable change or even cause decrease in the susceptibility. This study demonstrated that while exposure to ELF-EMF caused significant inhibitory effect on the tested virulence factors of both P. aeruginosa and S. aureus test isolates, that significant effect was not generally observed in case of susceptibility of both bacterial species to antibiotics. The susceptibility of both P. aeruginosa and S. aureus test isolates to antibiotics was not affected significantly by exposure to ELF-EMF in nearly all cases. In few cases the exposure to ELF-EMF increases the susceptibility to some antibiotics (Piperacillin/tazobactam, Meropenem and Cefepime) in case of P. aeruginosa and (Amoxicillin/clavulanic acid, Levofloxacin, linezolid, Clarithromycin) in case of S. aureus test isolates. After statistical analysis of antimicrobial susceptibility testing of P. aeruginosa across the two time points of exposure, there were no statistically significant difference in zone diameter of antimicrobial panel except TZP which showed statistically significant increase in zone diameter between after 12 hrs exposure Vs. (before and after 6 hrs exposure) as p -value were (0.041 & 0.034), respectively (Table 5 ). Antimicrobial susceptibility testing of S. aureus (MRSA) across the two time points of exposure, showed no statistically significance difference in zone diameter of antimicrobial panel except LEV, LZD & CLR which showed statistically significant increase in zone diameter between after 12 hrs exposure Vs. before and after 6 hrs exposure as p -value were (0.042 & 0.043), (0.027 & 0.026) and (0.024 & 0.041), respectively. There was statistically significant increase in zone diameter between before Vs. after 12 hrs exposure as p -value was (0.042) (Table 6 ). The results showed that, bacterial antibiotics susceptibility after exposure varies; depending on their mode of action. However, the apparent increase in susceptibility in certain antibiotics explains how membrane proteins and cell wall structure are crucial as they are the first barriers to ELF-EMF and play a role in transporting antibiotics and extracellular virulence factors. All of that has driven our future perspectives to study the relation between ELF-EMF and different antibiotics classes. Effect of exposure to extremely low frequency electromagnetic field (ELF-EMF) on whole genome sequences of P. aeruginosa and S. aureus test isolates. The major goal of this research was to determine the impact of extremely low frequency electromagnetic field (ELF-EMF) exposure on P. aeruginosa or S. aureus and to investigate the potential alterations at the genetic level. Therefore, a device that emits extremely low frequency electromagnetic field of 0.7 Hz in case of P. aeruginosa 18 and 0.8 Hz in case of S. aureus 32 was applied. Sequencing allowed quick answers to the change in genetic features (variations) of bacteria in response to exposure to externally ELF-EMF. The changes at the genetic level of the whole genome were scored and analyzed by bioinformatics tools to estimate the presence of noticeable variations/mutations induced in bacterial genome structure. The resulted effect was compared to the parent control isolate before exposure to the magnetic field. The results obtained showed differences in genome sequences between the non-exposed versus exposed bacterial isolates for both test bacterial species. Also, the results showed that both bacterial species, P. aeruginosa and S. aureus (Gram-negative and Gram-positive, respectively), have diverse responses to ELF-EMF and exposure duration times. The obtained bacterial genome mutations (as shown in Tables s9 & s10) after exposure in case of P. aeruginosa showed 27 and 30 missense mutated genes (involved in molecular function, cellular component and biological processes) after 6 and 12 h exposure times, respectively. While in S. aureus , the obtained bacterial genome mutations (as shown in Tables s11 & s12) after exposure showed 42 and 26 missense mutated genes (involved in molecular function, cellular component and biological processes) after 6 and 12 h exposure times, respectively. It is important to realize that these results were obtained after single subculture of the exposed cells in both test isolates. The results demonstrated that: (i) the Gram-positive S. aureus isolate S3 was more sensitive to ELF-EMF than the Gram-negative P. aeruginosa isolate P4, since more mutated genes were detected after 6 h exposure in case of S. aureus isolate as compared to P. aeruginosa ; (ii) although the number of mutated genes in case of P. aeruginosa increased from 27 to 30 by the extension of exposure to ELF-EMF from 6 to 12 hrs, this increase was insignificant only (10%), despite of doubling the exposure period; (iii) in case of S. aureus and in contrast to P. aeruginosa , doubling the exposure time to ELF-EMF showed un-expected significant decrease in the number of mutated genes by (38%); (iv) We suggest that the reason behind that obtained effect with the Gram-positive S. aureus isolate is, in this bacterial species, longer exposure apparently induces not only tolerance to ELF-EMF but also the repair system that recover the harmful effect induced by this magnetic field, this was demonstrated by the reduction in the number of mutated genes appeared after 12 hrs exposure time which resulted from a single subculture only; (v) the repair system detected in P. aeruginosa test isolate against the damaged induced by ELF-EMF was not so efficient as that demonstrated in case of S. aureus (zero percentage versus 38% reduction in the number of mutated genes in case of P. aeruginosa and S. aureus , respectively ) ; (vi) the high sensitivity of S. aureus to ELF-EMF corresponds to high efficient repair system while the resistance of P. aeruginosa to ELF-EMF corresponds to less efficient repair system, a physiological balance against the environmental harmful effects. The whole genome sequencing represents an important approach in understanding the effect of ELF-EMF. As shown in Figs. 2 and 4, the membrane proteins and cell wall structure genes are the most affected by ELF-EMF, and this effect could be expected since both the cell wall and cell membrane are the first cell components in direct contact with the applied EMF. As a result, the role of aforementioned structures in exporting/displaying extracellular virulence factors and transporting antibiotics, as well as their role in forming the structures that regulate adhesion and motility could be first and adversely affected by exposure to ELF-EMF. In case of P. aeruginosa several genes exhibited the same mutation at the two exposure times, 6 and 12 hrs, (Table 7 ). The direct/indirect effect of ELF-EMF on virulence and antibiotics susceptibility could be demonstrated by the missense mutation occurred in permease of ABC transporter gene which encodes for permease of ABC transporter protein 42 , 43 , pvdD gene that encodes for Pyoverdine synthetase D 44 and lptD gene which encodes for LPS-assembly protein LptD 45 , 46 , 47 . Missense mutations in those genes by ELF-EMF might enhance the efficacy of cell wall acting antibiotics and that was reflected phenotypically in this study as shown by the increase in sensitivity of P. aeruginosa to piperacillin-tazobactam (Table 5 ). While in case of S. aureus, ebh gene, which encodes for extracellular matrix-binding protein and the SAOUHSC_00192 gene which encodes for coagulase enzyme, both exhibited missense mutation. Both genes are responsible for the virulence character exhibited by coagulase enzyme of S. aureus . Interestingly, the mutation occurred in these two genes was reflected phenotypically on coagulase production (Table 4 ). Many other genes which encode for bifunctional autolysin protein, autolysin LytO 48 , 49 , Serine-aspartate repeat-containing protein D 50 , 51 , 52 and neutral metalloproteinase 53 , 54 showed missense mutation by exposure to ELF-EMF. These genes have a role in biofilm formation of S. aureus and The mutations occurred in these genes were also reflected phenotypically on the biofilm formation by this organism (Table 3 ). While genes encode for Bcr/CflA family efflux transporter protein 55 , 56 , 57 Lipid II glycine glycyltransferase 58 , 59 Lysine–tRNA ligase protein 60 and for ribosome biogenesis GTPase A protein 61 , 62 , 63 all these genes have a role in antibiotics susceptibility of S. aureus. The mutations occurred in these genes were also reflected phenotypically on the antibiotics susceptibility by the test isolate (Table 6 ). Conclusions From this study, it can be concluded that: (i) exposure to ELF-EMF induces harmful effects on microbial population, this effect is both bacterial species and exposure time dependent; (ii) although no killing effect could be exhibited by ELF-EMF on microbial cells, this magnetic field significantly affect the physiology of microbial cells that reflected on their virulence and pathogenicity; (iii) bacterial antimicrobial susceptibility is less affected by exposure to ELF-EMF as compared to the bacterial virulence; (iv) the harmful effect on bacterial cell induced by ELF-EMF is mediated by missense mutation in the genes controlling various physiological functions (transport of elements into and out of the cell, synthesis of structural components, metabolic enzymes, etc.); (v) this study provides a good evidence for the use of ELF-EMF as an efficient approach against bacterial pathogens especially those exhibiting multiple drug resistance to antimicrobial agents; (vi) the use of ELF-EMF as therapeutic approach against infectious diseases could efficiently contribute against the development of multiple drug resistance which occurs as a result of miss- and extensive use of antibiotics. Abbreviations ELF-EMF Extremely low frequency electromagnetic field MRSA Methicillin-resistant S. aureus OD s Optical densities BWA Burrows-Wheeler Aligner Software GATK Genome Analysis ToolKit QUAST Quality Assessment Tool PATRIC The Patho Systems Resource Integration Center Declarations Ethics approval and consent to participate This study was approved by the Institutional Review Board (IRB) of the Military Medical Academy under approval number [08 -2025], dated [12/06/2025]. Although the experimental work was using bacterial isolates collected as part of routine clinical care, the IRB reviewed the study retrospectively and confirmed that it met ethical standards. The isolates were anonymized, and no additional samples were collected beyond those required for routine clinical care. Consent for publication Not applicable. Availability of data and materials The datasets generated and analyzed during the current study are available in the NCBI SRA repository, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA999246 /accession number PRJNA999246 Competing interest: Not applicable. Funding: Not applicable. Authors' contributions: M. Elnakib: conceptualization, supervision, data interpretations, manuscript writing and revision. M. Hosny: methodology, data collection, writing original draft and the revised form. Acknowledgments: Genomics Program, Department of Basic Research, Children’s Cancer Hospital Egypt 57357, Cairo, Egypt for the contribution in methodology, data analysis and discussion of whole genome sequencing. References Ahlbom A, Feychting M. Electromagnetic radiation. Br Med Bull. 2003;68(1):157–65. Feychting M, Ahlbom A, Kheifets L. EMF AND HEALTH. Annu Rev Public Health. 2005;26(1):165–89. Extremely Low Frequency (ELF). 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J Bacteriol. 2014;196:4206–15. Karaguler T, Kahraman H, Tuter M. Analyzing effects of ELF electromagnetic fields on removing bacterial biofilm. Biocybernetics Biomedical Eng. 2017;37(2):336–40. Haagensen JAJ, Bache M, Giuliani L, Blom NS. Effects of Resonant Electromagnetic Fields on Biofilm Formation in Pseudomonas aeruginosa. Appl Sci. 2021;11(16):7760. Mousavian-Roshanzamir S, Makhdoumi-Kakhki A. The Inhibitory Effects of Static Magnetic Field on Escherichia coli from two Different Sources at Short Exposure Time. Kamel FH, Saeed CH, Qader SS. THE STATIC MAGNETIC FIELD EFFECT ON PSEUDOMONAS AERUGINOSA. Akhtar AA, Turner DPJ. The role of bacterial ATP-binding cassette (ABC) transporters in pathogenesis and virulence: Therapeutic and vaccine potential. Microb Pathog. 2022;171:105734. Podbielski A, Pohl B, Woischnik M, Körner C, Schmidt KH, Rozdzinski E, et al. Molecular characterization of group A streptococcal (GAS) oligopeptide permease (opp) and its effect on cysteine protease production. Mol Microbiol. 1996;21(5):1087–99. Kang D, Revtovich AV, Chen Q, Shah KN, Cannon CL, Kirienko NV. Pyoverdine-Dependent Virulence of Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients. Front Microbiol. 2019;10:2048. Pandey S, Delgado C, Kumari H, Florez L, Mathee K. Outer-membrane protein LptD (PA0595) plays a role in the regulation of alginate synthesis in Pseudomonas aeruginosa. J Med Microbiol. 2018;67(8):1139–56. Andolina G, Bencze LC, Zerbe K, Müller M, Steinmann J, Kocherla H, et al. A Peptidomimetic Antibiotic Interacts with the Periplasmic Domain of LptD from Pseudomonas aeruginosa. ACS Chem Biol. 2018;13(3):666–75. Lehman KM, Grabowicz M. Countering Gram-Negative Antibiotic Resistance: Recent Progress in Disrupting the Outer Membrane with Novel Therapeutics. Antibiotics. 2019;8(4):163. Osipovitch DC, Therrien S, Griswold KE. Discovery of novel S. aureus autolysins and molecular engineering to enhance bacteriolytic activity. Appl Microbiol Biotechnol. 2015;99(15):6315–26. Oshida T, Sugai M, Komatsuzawa H, Hong YM, Suginaka H, Tomasz A. A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization. Proc Natl Acad Sci USA. 1995;92(1):285–9. Askarian F, Ajayi C, Hanssen AM, van Sorge NM, Pettersen I, Diep DB, et al. The interaction between Staphylococcus aureus SdrD and desmoglein 1 is important for adhesion to host cells. Sci Rep. 2016;6(1):22134. Paharik AE, Horswill AR. The Staphylococcal Biofilm: Adhesins, Regulation, and Host Response. Kudva IT, Nicholson TL, editors. Microbiol Spectr. 2016;4(2):4.2.06. Schilcher K, Horswill AR. Staphylococcal Biofilm Development: Structure, Regulation, and Treatment Strategies. Microbiol Mol Biol Rev. 2020;84(3):e00026–19. Wu JW, Chen XL. Extracellular metalloproteases from bacteria. Appl Microbiol Biotechnol. 2011;92(2):253–62. Elhakim YA, Ali AE, Hosny AEDMS, Abdeltawab NF. Zinc Deprivation as a Promising Approach for Combating Methicillin-Resistant Staphylococcus aureus: A Pilot Study. Pathogens. 2021;10(10):1228. Kumar S, Varela MF. Biochemistry of Bacterial Multidrug Efflux Pumps. IJMS. 2012;13(4):4484–95. Marklevitz J, Harris LK. Prediction driven functional annotation of hypothetical proteins in the major facilitator superfamily of S. aureus NCTC 8325. Bioinformation. 2016;12(4):254–62. Seukep AJ, Mbuntcha HG, Kuete V, Chu Y, Fan E, Guo MQ. What Approaches to Thwart Bacterial Efflux Pumps-Mediated Resistance? Antibiotics. 2022;11(10):1287. Rohrer S, Ehlert K, Tschierske M, Labischinski H, Berger-Bächi B. The essential Staphylococcus aureus gene fmhB is involved in the first step of peptidoglycan pentaglycine interpeptide formation. Proc Natl Acad Sci USA. 1999;96(16):9351–6. Punekar AS, Samsudin F, Lloyd AJ, Dowson CG, Scott DJ, Khalid S, et al. The role of the jaw subdomain of peptidoglycan glycosyltransferases for lipid II polymerization. Cell Surf. 2018;2:54–66. Roy H, Ibba M. RNA-dependent lipid remodeling by bacterial multiple peptide resistance factors. Proc Natl Acad Sci USA. 2008;105(12):4667–72. Bennison DJ, Nakamoto JA, Craggs TD, Milón P, Rafferty JB, Corrigan RM. The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions. mBio. 2021;12(6):e0267921. Hwang J, Inouye M. The tandem GTPase, Der, is essential for the biogenesis of 50S ribosomal subunits in Escherichia coli. Mol Microbiol. 2006;61(6):1660–72. Champney WS. Antibiotics targeting bacterial ribosomal subunit biogenesis. J Antimicrob Chemother. 2020;75(4):787–806. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6791184","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476831871,"identity":"6074db89-a21f-430f-87b2-e0d4c882cadf","order_by":0,"name":"Mohamed Hosny","email":"","orcid":"","institution":"Ain Shams University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"Hosny","suffix":""},{"id":476831872,"identity":"3e3c9700-c856-473e-a6fc-7835264cb61e","order_by":1,"name":"Mostafa Elnakib","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYLCCBwwMBmwSzAcYGBuI1ZIA1sKWQKIWBgkeA+K06La3P3yQuMPOmE+655vEzx02cgzsh49uwKfF7MwZY4PEM8lmbDJnt0n2nkkzZuBJS7uBV8uNHDaJxDZmGzaJ3G0SvG2HExskeMwIaEl//iOxrR6oJeeZ5F/itCSYMSS2HTYDamGTJs4WoF+ADjtuzCZzzNhati3NmI2gX463P/zwsa3acP7s5oc337bZyPGzHz6GVwsyYJEAkWzEKgcB5g+kqB4Fo2AUjIKRAwC710nRck/L1gAAAABJRU5ErkJggg==","orcid":"","institution":"Military Medical Academy","correspondingAuthor":true,"prefix":"","firstName":"Mostafa","middleName":"","lastName":"Elnakib","suffix":""},{"id":476831873,"identity":"071cdfed-9a7c-4bc7-a40c-95b577c30fab","order_by":2,"name":"Ahmed Gad","email":"","orcid":"","institution":"Military Medical Academy","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"","lastName":"Gad","suffix":""}],"badges":[],"createdAt":"2025-05-31 13:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6791184/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6791184/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85744983,"identity":"247f9cd4-d668-46ea-8f43-c7d5f59e3f23","added_by":"auto","created_at":"2025-07-01 09:20:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":246248,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectromagnetic wave prototype apparatus (A) and a sketch diagram showing bacterial exposure method to ELF-EMF (B)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Electromagnetic wave prototype apparatus\u003c/p\u003e\n\u003cp\u003e(B) sketch diagram showing bacterial exposure to ELF-EMF\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6791184/v1/6c52fb505923e4345a497278.png"},{"id":85744979,"identity":"5983c47f-6d62-4b55-aa0e-0571ca7a0139","added_by":"auto","created_at":"2025-07-01 09:20:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":188738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe intersect of missense mutations and their counts occurred in biological processes (BP), cellular components (CC), and molecular functions (MF) genes due to exposure of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eP4 to ELF-EMF for 6 and 12 hrs relative to the parent isolate before exposure.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6791184/v1/dfe6ae440fb43e127b306f4c.png"},{"id":85746923,"identity":"bdebbea0-c2ed-4f1e-bb5c-79c18e3ce796","added_by":"auto","created_at":"2025-07-01 09:36:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":322872,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGO annotations and counts of missense mutations occurred in biological processes (BP), cellular components (CC), and molecular functions (MF) genes due to exposure of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eP4 to ELF-EMF relative to the parent isolate before exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) GO annotations and counts of missense mutations occurred in \u003cem\u003eP. aeruginosa \u003c/em\u003eP4 due to exposure to ELF-EMF for 6 h\u003c/p\u003e\n\u003cp\u003e(B) GO annotations and counts of missense mutations occurred in \u003cem\u003eP. aeruginosa \u003c/em\u003eP4 due to exposure to ELF-EMF for 12 h\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6791184/v1/eb72aa44de42e349bc5e225f.png"},{"id":85744980,"identity":"9ab5b50c-6ef5-454a-bfe2-2e726e80337d","added_by":"auto","created_at":"2025-07-01 09:20:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":104471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe intersect of missense mutations and their counts occurred in biological processes (BP), cellular components (CC), and molecular functions (MF) genes due to exposure of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eS3 to ELF-EMF for 6 and 12 hrs relative to the parent isolate before exposure.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6791184/v1/ed0ec4671dc7c5fd7bf56daf.png"},{"id":85746240,"identity":"b94e58b8-7864-4ff3-b2d8-7551a2fce858","added_by":"auto","created_at":"2025-07-01 09:28:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":231824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGO annotations and counts of missense mutations occurred in biological processes (BP), cellular components (CC), and molecular functions (MF) genes due to exposure of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eS3 to ELF-EMF relative to the parent isolate before exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) GO annotations and counts of missense mutations occurred in \u003cem\u003eS. aureus \u003c/em\u003eS3 due to exposure to ELF-EMF for 6 h\u003c/p\u003e\n\u003cp\u003e(b) GO annotations and counts of missense mutations occurred in \u003cem\u003eS. aureus \u003c/em\u003eS3 due to exposure to ELF-EMF for 12 h\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6791184/v1/59423edd4c4d6ef6ceff9250.png"},{"id":85746237,"identity":"6067a4be-ad91-4a80-b84c-5430500fd731","added_by":"auto","created_at":"2025-07-01 09:28:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":60238,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Materials \u0026amp; Methods section.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6791184/v1/da6b3249d95ba5a349e0e9d0.png"},{"id":85749036,"identity":"29c7cc30-acfa-4402-a13a-dff506845abb","added_by":"auto","created_at":"2025-07-01 09:52:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4108497,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6791184/v1/8862dfa0-9eff-46f1-ab5c-7f73a9eaa5cb.pdf"},{"id":85746922,"identity":"d36c8172-d9f1-43b8-bf11-3626ceba9051","added_by":"auto","created_at":"2025-07-01 09:36:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":60157,"visible":true,"origin":"","legend":"","description":"","filename":"TablesSupplementryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6791184/v1/6c62a5c67a3bc5f2105975d0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eExtremely low frequency electromagnetic field affects virulence and antibiotic susceptibility of multidrug resistant \u003cem\u003ePseudomonas aeruginosa \u003c/em\u003eand methicillin resistant Staphylococcus aureus\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eNowadays, the current environment is filled with electromagnetic fields (EMFs) which become as one of the most prevalent and quickly expanding factors that are affecting the environment\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The electromagnetic field spectrum includes radiofrequency, infrared, and X-rays, as well as static electric and magnetic fields. In various publications on radiation and field hazards, the extremely low frequency electromagnetic field (ELF-EMF) was given a range of 3 Hz to 3,000 Hz \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Later on, entities such as World Health Organization (WHO) and Occupational Safety and Health Administration (OSHA) referred the term extremely low frequency (ELF) to the frequency range of 0-300 Hz \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Currently, the ELF range is defined by the International Telecommunication Union (ITU) as the frequency range between 3 and 30 Hz only \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The ELF-EMFs are emitted from power lines, electric devices, electric appliances, communication systems and electric transmission lines \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Several studies were conducted to confirm the direct influence that extremely low frequencies electromagnetic fields (ELF-EMFs) could have on cell functions. Various responses of cells have been studied involving the effect on gene expression \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, regulators of cell membrane signal transduction \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, premature cell differentiation and proliferation \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, ion channel expression and function \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, cell apoptosis \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, modification of the cellular redox state and modulation of the cellular metabolic activity \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Many studies in last decades have examined the impact of ELF-EMF on prokaryotes which appears promising in terms of medical or industrial uses \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The studies (that investigated the influence of ELF-EMF on Gram-positive or Gram-negative bacteria) dealt with this impact in terms of its effect on bacterial growth \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e morphology \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, virulence \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and especially antimicrobial resistance (as it is one of the most bothering crisis that humanity is currently experiencing) \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Most of these studies have been recruited to find innovative method for the treatment of bacterial infections especially those that are caused by pathogens with multiple drug resistance to different antimicrobial agents. Several other studies have also worked on the mechanism and how this ELF-EMF introduces its effect on the bacteria or other cells. Some suggested that the active physiological mechanisms and biological systems produce electric currents and magnetic fields in vivo \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and all bioelectric signals that produced by ionic movements, are involved in various activities. The ions' flow rate and direction produce an electric impulse with a certain frequency, and amplitude \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In order to control physiological functions in biological systems, it is necessary to apply electric fields with a frequency corresponding to the bioelectric impulses produced during metabolic processes. Ion motion in the biological system will be impacted by this interference and depending on the mechanism of interference, the ion motion may be promoted or inhibited. Considering that the bioelectric signals produced by metabolic processes in cells are known to be in the extremely low frequency range, the applied electromagnetic wave should have the same frequency as the bioelectric signal \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This study aimed to assess the effect of extremely low frequency electromagnetic field (ELF-EMF) on bacteria, both, phenotypically and genotypically by measuring the effect on virulence factors, antibiotics susceptibility and genome structure.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003e1. Collection of some clinical isolates of certain bacterial species including\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eP. aeruginosa.\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eA number of 38 isolates from wound swabs were collected from surgical ICU of a tertiary hospital, Cairo, Egypt, in the period from June 2020 to August 2021. The isolates were identified first with the Vitek 2 system (AES software, bioM\u0026eacute;rieux, Marcy l\u0026apos;\u0026Eacute;toile, France) according to the manufacturer\u0026apos;s instructions. These isolates included, Methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA), \u003cem\u003eAcinetobacter, Proteus, E. coli, Pseudomonas\u003c/em\u003e and \u003cem\u003eKlebsiella.\u003c/em\u003e The distribution of these isolates is shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; these isolates included 6 Gram-positive isolates and 32 Gram-negative isolates. The Gram-positive isolates were, Methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) while the Gram-negative isolates included \u003cem\u003eProteus\u003c/em\u003e, \u003cem\u003eAcinetobacter, E. coli, Pseudomonas\u003c/em\u003e and \u003cem\u003eKlebsiella.\u003c/em\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDistribution of isolates recovered from wound swabs\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolates Genus/Type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMRSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eProteus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eA number of 5 out of 12 \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates were multidrug resistant. These 5 isolates together with 6 Methicillin-Resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) were selected to complete this study. The five multidrug resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates were given the codes of P1, P2, P3, P4 and P5, while the six Methicillin-Resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) were given the codes of S1, S2, S3, S4, S5 and S6.\u003c/p\u003e\n\u003cp\u003eDifferent virulence factors of the collected isolates were studied before and after exposure to extremely low electromagnetic field; these factors included protease production, oxidase production, and biofilm formation for \u003cem\u003eP. aeruginosa\u003c/em\u003e whereas coagulase production, catalase production, and biofilm formation were the virulence factors studied for MRSA isolates.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2. Preparation of the bacterial cultures for exposure to extremely low frequency electromagnetic field\u003c/h2\u003e\n \u003cp\u003eThe bacterial culture used for subsequent experiments was prepared as follows: from the stock culture (nutrient agar plate culture) of each bacterial isolate, a loopful was transferred to 5 ml double strength nutrient broth contained in a test tube. The test tube was incubated overnight at 37\u003csup\u003eo\u003c/sup\u003eC. From the obtained tube culture, a loopful was streaked for isolation on selective media of cetrimide agar (in case of \u003cem\u003eP. aeruginosa\u003c/em\u003e) and mannitol salt agar (in case of \u003cem\u003eS. aureus\u003c/em\u003e) followed by incubation at 37\u003csup\u003eo\u003c/sup\u003eC for 24 h. Each isolate was cultured on the selective medium in triplicates, one to be used as a control while the second and the third ones were exposed to ELF-EMF for two separate and different durations of 6 and 12 hrs, respectively (three culture plates were used for each exposure duration period). The control culture was kept in the same conditions as the exposed ones but without exposure.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e3. Exposure to extremely low frequency electromagnetic field\u003c/h3\u003e\n\u003cp\u003eFigure 1. shows the prototype apparatus for generation of the applied extremely low frequency electromagnetic waves of different frequencies (0.7 Hz and 0.8 Hz) and its mechanistic use. The isolate cultures\u0026apos; plates were placed between two parallel copper disk plates that emitted the ELF-EMF. Each copper plate was of 3 mm thickness and the distance between the two plates 30 cm. The cultures of bacteria were exposed to ELF-EMF with frequency of 0.7 Hz and 0.8 Hz for \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, respectively for different exposure durations (6 and 12 hrs for each bacterial species).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Effect of ELF-EMF on some virulence factors of\u003c/strong\u003e \u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/p\u003e\n\u003ch3\u003e4.1. Effect on protease production\u003c/h3\u003e\n\u003cp\u003eA colony from the control culture and ELF-EMF exposed culture of the test isolate were line streaked separately on plates containing 15 ml of gelatin agar medium. The plates were incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 24 h. After the incubation period, the plates were flooded with mercuric chloride solution which causes opacity in the medium with clear zone around streaked growth. The clear zone width in mm was taken as a measure for the amount of the produced protease \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003e4.2. Effect on oxidase production\u003c/h3\u003e\n\u003cp\u003eReady-made oxidase discs were used; the disc was applied to an isolated colony of either the control culture or ELF-EMF exposed culture of the test isolate after had been cultured on nutrient agar and incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 24 h. A reaction was observed within 10\u0026ndash;30 sec \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The results were recorded as follows: (++); microorganism that is oxidase positive with oxidase disc color changed to deep purple color within 10 sec, (+); microorganism that is delayed oxidase positive with disc color changed to deep purple color in more than 10 sec up to 30 sec, (-); microorganism that is oxidase negative and the disc color did not turn into deep purple in 30 sec.\u003c/p\u003e\n\u003ch3\u003e4.3. Effect on biofilm formation\u003c/h3\u003e\n\u003cp\u003eAn isolated colony from both the control culture and ELF-EMF exposed culture of the test isolate were separately streaked on nutrient agar plate. The plates were incubated at 37\u0026deg;C for 24 h. From the obtained culture an isolated colony was transferred into 5 ml of trypticase soy broth contained in a test tube followed by incubation at 37\u0026deg;C for 24 h. The cultures were diluted 1:100 with fresh trypticase soy broth. Each individual well of 96 U-shaped bottom polystyrene microtitre plate were filled with 0.2 ml aliquots of diluted culture. Control wells containing fresh trypticase soy broth were included to check sterility and nonspecific binding of the media. The plates were then incubated at 37\u0026deg;C for 24 h, after incubation the plates were inverted for discarding their contents with gentle tapping of the plates. The Plates were washed three times, each time with 0.2 ml of phosphate buffer saline (pH 7.2). The plates were then incubated at 37\u0026deg;C for an hour while inverted to get dried followed by transferring 0.2 ml aliquots of 0.1% crystal violet and leaving at room temperature for 10 min. Excess stain was removed by inverting the plates followed by washing three times with phosphate buffer saline and the plates were left at room temperature while inverted for drying. Aliquots of 200 \u0026micro;l of 95% ethanol were added to the wells. Optical densities (OD\u003csub\u003es\u003c/sub\u003e) of the stained plates were determined using micro ELISA auto reader (BIORAD 680) at a wavelength of 570 nm (OD \u003csub\u003e570 nm\u003c/sub\u003e) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Interpretation of biofilm production based on optical density values was carried out as described in reference 25 where OD value of \u0026lt;\u0026thinsp;0.12 was considered negative biofilm production and values\u0026thinsp;\u0026gt;\u0026thinsp;0.12 was considered positive biofilm production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Effect of ELF-EMF on some virulence factors of\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003e(MRSA)\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e5.1. Effect on coagulase production\u003c/h2\u003e\n \u003cp\u003eTube coagulase test was used, the following procedure \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e was followed for each isolate. An isolated colony from either the control culture or ELF-EMF exposed culture of the test isolate (retrieved from 24 h nutrient agar subculture) was placed in a test tube containing 0.5 ml of human plasma (from Medical Research Laboratories and Blood Bank of Armed Forces, Egypt), then the tube was incubated at 37\u0026deg;C and examined at 30 minutes intervals for up to 4 h. Clot formation was observed and the results were interpreted according to Sperber \u0026amp; Tatini (1975) as follows: (-); no evidence of fibrin formation, (+); have small unorganized clots, (++); feature of a large clot formation; and (+++); when the entire contents of the tube are coagulated and unable to be displaced upon tube inversion.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e5.2. Effect on catalase production\u003c/h3\u003e\n\u003cp\u003eOn a slide, 1 or 2 drops of 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were added. An isolated colony from either the control culture or ELF-EMF exposed culture of the test isolate (retrieved from 24 h subculture nutrient agar) was picked up and mixed with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e drops on a slide using a sterile applicator stick. The slide was placed over a dark surface and observed for immediate bubble formation \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Positive catalase production is evident by immediate effervescence (bubble formation), while no bubble formation (no catalase enzyme to hydrolyze the hydrogen peroxide) represents a negative catalase production.\u003c/p\u003e\n\u003ch3\u003e5.3. Effect on biofilm formation\u003c/h3\u003e\n\u003cp\u003eThis was carried out as mentioned earlier in section \u003cspan class=\"InternalRef\"\u003e4.3\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e6. Antimicrobial sensitivity test\u003c/h2\u003e\n \u003cp\u003eKirby-Bauer method was used for determining the sensitivity of the test isolates to different antibiotics after exposure to ELF-EMF against their corresponding controls as follows. An isolated colony from either the control culture or ELF-EMF exposed culture of the test isolate was streaked on nutrient agar plate. The plates were incubated at 37\u0026deg;C for 24 h. From the obtained culture, an isolated colony was transferred and mixed with about 2 ml saline and adjusted to 0.5 McFarland standard, then a sterile swab was dipped in the isolate suspension obtained. After removal of the excess suspension by pressing the swab against the inner tube surface, it was used for entire streaking the surface of a M\u0026uuml;eller\u0026ndash;Hinton agar plate. The antibiotic discs were applied onto the surface of the inoculated plates and gently pressed. The plates were then incubated at 37\u0026deg;C for 24 h. The diameter of inhibition zones were measured in millimeters and recorded. The results were interpreted according to Clinical and Laboratory Standards Institute, 2021 (CLSI 2021).\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003e7. Next-generation sequencing (NGS) using Illumina MiSeq machine (Whole Genome Sequencing).\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eThis was carried on one \u003cem\u003eP. aeruginosa\u003c/em\u003e isolate (P4) and one \u003cem\u003eS. aureus\u003c/em\u003e isolate (S3)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e7.1. DNA Extraction\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe genomics DNA was extracted using the QIAamp\u0026reg; DNA Minikit (QIAGEN, Germany) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e7.2. Library Preparation and Next Generation Sequencing\u003c/h2\u003e\n \u003cp\u003eThe preparation of the library was carried out utilizing the Nextera XT DNA Library preparation kit.\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.2.1.\u003c/strong\u003e Tagment Genomic DNA\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eIn this step the Nexteratransposome was used to tagment DNA where the DNA was fragmented and then tagged with adapter sequences in a single step.\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.2.2.\u003c/strong\u003e Amplify Library\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eIn this step the tagmented DNA was amplified using a limited-cycle PCR program where the Index 1 (i7), Index 2 (i5), and full adapter sequences were added to the tagmented DNA obtained in the previous step (7.2.1). The index primers and Nextera PCR Master Mix were added directly to the 25 \u0026micro;l of tagmented DNA obtained.\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.2.3.\u003c/strong\u003e Clean Up Libraries\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eIn these step AMPure XP beads was used to purify the library DNA and remove short library fragments.\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.2.4.\u003c/strong\u003e Check Libraries\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eIn this step 1 \u0026micro;l of library was run on an Agilent Technology 2100 Bio-analyzer using Agilent DNA 1000 chip. Typical libraries show a broad size distribution of ~\u0026thinsp;250\u0026ndash;1000 bp. Various libraries can be sequenced with average fragment sizes as small as 250 bp or as large as 1500 bp.\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.2.5.\u003c/strong\u003e Normalize Libraries\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eIn this step the quantity of each library was normalized to ensure more equal library representation in the pooled library.\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.2.6.\u003c/strong\u003e Pooling Library\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eEqual volumes of normalized libraries were combined in a single tube to get the pooled library. The pooled library was diluted and heat-denatured before its loading for the sequencing run which was conducted according to Illumina manufacturing instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e7.3. Quality control, genome assembly, and alignment\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eReads were filtered, inspected and adaptors were removed using fastp. After that, the filtered reads were de novo assembled using Unicycler and annotated using Prokka. The assessment of the assembled files was carried out with QUAST. Previously filtered reads were mapped to the reference using Burrows-Wheeler Aligner (BWA) followed by variant calling using GATK and Picard. After that, most similar sequences were identified and phylogenetic tree was constructed using PATRIC. Finally, taxonomy profiling was performed using kraken2 and visualized using Pavian.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e7.4. Variant calling and variant effect prediction.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePreviously merged reads in case of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e were mapped to the \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e references (GenBank accession number NC_007795.1 and ASM676v1, respectively) using BWA. Variant identification and filtration were performed using GATK and Picard. The resulting variants were used to predict the variant effect using SnpEff, as well as consensus sequence generation using BCFtools. After that, the multiple sequence alignment was done.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003e7.5. Workflow diagram\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe collected data was revised, coded, tabulated and introduced to a PC using Statistical package for Social Science (SPSS 27). Data was presented and suitable analysis was done according to the type of data obtained for each parameter.\u003c/p\u003e\n \u003cp\u003eDescriptive statistics: Median and Interquartile range (IQR) for non-parametric numerical data. Frequency and percentage of non-numerical data.\u003c/p\u003e\n \u003cp\u003eAnalytical statistics: Wilcoxon signed rank test was used assess the statistical significance of the difference of an ordinal variable (score) measured twice for the same study group. McNemar test was used assess the statistical significant of the difference between a qualitative variable measured twice for the same study group. Marginal homogeneity test assesses the statistical significance of the difference of a variable with multiple categories measured twice for the same study group.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e \u003cstrong\u003e-value\u003c/strong\u003e: level of significance: -P\u0026thinsp;\u0026gt;\u0026thinsp;0.05: Non- significant (NS), P\u0026thinsp;\u0026lt;\u0026thinsp;0.05: Significant (S).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Effect of ELF-EMF on some virulence factors of\u003c/strong\u003e \u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Presents the relation between different virulence factors of \u003cem\u003eP. aeruginosa\u003c/em\u003e across different time points after exposure and the table shows that there was a significant decrease of the diameter of clearance zone (mm) between before radiations Vs. after 6 and 12 hrs of exposure as \u003cem\u003ep\u003c/em\u003e-value was (0.042), but there was no statistically difference between after 6 hrs Vs. 12 hrs of exposure as \u003cem\u003ep\u003c/em\u003e-value was (0.068).\u003c/p\u003e\n \u003cp\u003eRegarding oxidase production there was a decrease in number of isolates that produce an oxidase across different time points of exposure without statistically significance difference between them.\u003c/p\u003e\n \u003cp\u003eRegarding biofilm formation there was statistically significant decrease in absorbances readings that obtained at (OD\u003csub\u003e570nm\u003c/sub\u003e) as \u003cem\u003ep\u003c/em\u003e-value was (0.043) between all different time points.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of ELF-EMF* on some virulence factors of \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth colspan=\"2\" rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBefore\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 6 hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 12 hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eTest of significance\u003csup\u003e(a)\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e3\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eDiameter of clearance zone (mm) \u003csup\u003e(b)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 (19\u0026ndash;20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (9\u0026ndash;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 (7\u0026ndash;9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.042\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.042\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.068\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eOxidase production\u003csup\u003e(c)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (40%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (60%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eBiofilm formation\u003csup\u003e(b)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.14 (0.14\u0026ndash;0.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07 (0.06\u0026ndash;0.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05 (0.05\u0026ndash;0.06)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.043\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.043\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.043\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003e(a)\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e1: \u003cem\u003ep\u003c/em\u003e-value between Reading before Vs. Reading after 6 hrs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e2: \u003cem\u003ep\u003c/em\u003e-value between Reading before Vs. Reading after 12 hrs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e3: \u003cem\u003ep\u003c/em\u003e-value between Reading after 6 hrs Vs. Reading after 12 hrs.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e(b)\u003c/sup\u003e Wilcoxon sign rank test was used to assess the significance.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e(c)\u003c/sup\u003e McNemar test was used to assess the significance.\u003c/p\u003e\n\u003cp\u003e* ELF-EMF was used at 0.7 Hz frequency\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Effect of ELF-EMF on some virulence factors of\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003e(MRSA)\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Presents the relation between different virulence factors of \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) across different time points after exposure and the table shows that there was no statistically significant difference in number of isolates in catalase production as all isolates were (+).\u003c/p\u003e\n \u003cp\u003eRegarding biofilm formation there was statistically significant decrease in absorbances that obtained at (OD\u003csub\u003e570nm\u003c/sub\u003e) as \u003cem\u003ep\u003c/em\u003e-value was (0.028) between all different time points.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of ELF-EMF* on some virulence factors by \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) isolates\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth colspan=\"2\" rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eMRSA\u003c/p\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBefore\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 6 hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 12 hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eTest of significance\u003csup\u003e(a)\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e3\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCatalase production\u003csup\u003e(b)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e----\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eBiofilm formation\u003csup\u003e(c)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16 (0.06\u0026ndash;0.19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06 (0.05\u0026ndash;0.08)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03 (0.02\u0026ndash;0.03)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.028\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.028\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.028\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003e(a)\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e1: \u003cem\u003ep\u003c/em\u003e-value between Reading before Vs. Reading after 6 hrs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e2: \u003cem\u003ep\u003c/em\u003e-value between Reading before Vs. Reading after 12 hrs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e3: \u003cem\u003ep\u003c/em\u003e-value between Reading after 6 hrs Vs. Reading after 12 hrs.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e(b)\u003c/sup\u003e Wilcoxon sign rank test was used to assess the significance.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e(c)\u003c/sup\u003e McNemar test was used to assess the significance.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e*\u003c/sup\u003e ELF-EMF was used at 0.8 Hz frequency\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. illustrates the relation between coagulase production by \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) at different time intervals across different time points of exposure and there was no difference in number of isolates before and after 6 hrs of exposure.\u003c/p\u003e\n\u003cp\u003eRegarding the relation between before and after 12 hrs of exposure at reading time interval of two hours, there was no statistically significance difference in number of isolates that produced coagulase as \u003cem\u003ep\u003c/em\u003e-value was (0.083), while there were statistically significance decrease in number of isolates that showed entire contents are complete coagulated and are not displaced when the tube inverted at reading time interval of three hours and four hours as \u003cem\u003ep\u003c/em\u003e-value were (0.046 \u0026amp; 0.031), respectively.\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of ELF-EMF* on coagulase produced by \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) isolates\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth colspan=\"2\" rowspan=\"3\" align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eTime interval\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 2 hours\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 3 hours\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 4 hours\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eBefore\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(+++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eAfter 6 hrs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(+++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eAfter 12 hrs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (66.67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (33.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTest of significance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(Before or After 6 hrs) Vs. After 12 hrs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.083\u003csup\u003e(a)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.046\u003c/strong\u003e\u003csup\u003e(a)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.031\u003c/strong\u003e\u003csup\u003e(b)\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\u003csup\u003e(a)\u003c/sup\u003e Marginal Homogeneity test was used to assess the significance.\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\u003csup\u003e(b)\u003c/sup\u003e McNemar test was used to assess the significance.\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\u003cstrong\u003e3. Effect of exposure to ELF-EMF on the susceptibility of\u003c/strong\u003e \u003cstrong\u003eP. aeruginosa\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003etest isolates to antibiotics\u003c/strong\u003e\u003c/div\u003e\n\u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. demonstrates antimicrobial susceptibility testing of \u003cem\u003eP. aeruginosa\u003c/em\u003e across different time points exposure, there were no statistically significance difference in zone diameter of antimicrobial panel \u003cstrong\u003eexcept\u003c/strong\u003e TZP there were statistically significant increase in zone diameter between after 12 hrs exposure Vs. (before and after 6 hrs exposure) as \u003cem\u003ep\u003c/em\u003e-value were (0.041 \u0026amp; 0.034), respectively. \u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAntibiotic susceptibility of \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates after exposure to ELF-EMF* as compared with unexposed control cultures\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBefore\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 6 hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 12 hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eWilcoxon Signed Ranks Test\u003csup\u003e(a)\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP3\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePiperacillin-tazobactam\u003c/p\u003e\n \u003cp\u003e(TZP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (5\u0026ndash;8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (9\u0026ndash;9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12 (11\u0026ndash;12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.041\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.034\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMeropenem\u003c/p\u003e\n \u003cp\u003e(MEM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCiprofloxacin\u003c/p\u003e\n \u003cp\u003e(CIP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmikacin\u003c/p\u003e\n \u003cp\u003e(AK)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCeftazidime\u003c/p\u003e\n \u003cp\u003e(CAZ)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCefepime\u003c/p\u003e\n \u003cp\u003e(FEP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevofloxacin\u003c/p\u003e\n \u003cp\u003e(LEV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNitrofurantoin\u003c/p\u003e\n \u003cp\u003e(NI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGentamicin\u003c/p\u003e\n \u003cp\u003e(CN)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003e(a)\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e1: \u003cem\u003ep\u003c/em\u003e-value between Reading before Vs. Reading after 6 hrs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e2: \u003cem\u003ep\u003c/em\u003e-value between Reading before Vs. Reading after 12 hrs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e3: \u003cem\u003ep\u003c/em\u003e-value between Reading after 6 hrs Vs. Reading after 12 hrs.\u003c/p\u003e\n\u003cp\u003e*ELF-EMF was used at 0.7 Hz frequency\u003c/p\u003e\n\u003cp\u003eThe method used to measure the sensitivity of the bacterial cells toward different antibiotics was disc\u003c/p\u003e\n\u003cp\u003emethod by Bauru-Kirby technique (Baker \u003cem\u003eet al\u003c/em\u003e., 1980)\u003c/p\u003e\n\u003cp\u003eThe method used to measure the sensitivity of the bacterial cells toward different antibiotics was disc\u003c/p\u003e\n\u003cp\u003emethod by Bauru-Kirby technique (Baker \u003cem\u003eet al\u003c/em\u003e., 1980)\u003c/p\u003e\n\u003cp\u003eThe method used to measure the sensitivity of the bacterial cells toward different antibiotics was disc\u003c/p\u003e\n\u003cp\u003emethod by Bauru-Kirby technique (Baker \u003cem\u003eet al\u003c/em\u003e., 1980)\u003c/p\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. shows antimicrobial susceptibility testing of \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) across different time points exposure, there were no statistically significance difference in zone diameter of antimicrobial panel \u003cstrong\u003eexcept\u003c/strong\u003e (LEV, LZD \u0026amp; CLR) there were statistically significanct increase in zone diameter between after 12 hrs exposure Vs. (before and after 6 hrs exposure) as \u003cem\u003ep\u003c/em\u003e-value were (0.042 \u0026amp; 0.043), (0.027 \u0026amp; 0.026) and (0.024 \u0026amp; 0.041), respectively. While there was statistically significant increase in zone diameter between before Vs. after 12 hrs exposure as \u003cem\u003ep\u003c/em\u003e-value was (0.042)\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAntibiotic susceptibility of \u003cem\u003eS. aureus\u003c/em\u003e isolates after exposure to ELF-EMF* as compared with unexposed control cultures\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eMRSA\u003c/p\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBefore\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 6 hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter 12 hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eWilcoxon Signed Ranks Test\u003csup\u003e(a)\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (IQR)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e3\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmoxicillin/clavulanic acid\u003c/p\u003e\n \u003cp\u003e(AMC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.5 (5\u0026ndash;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11 (11\u0026ndash;12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.042\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.068\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLevofloxacin\u003c/p\u003e\n \u003cp\u003e(LEV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18 (5\u0026ndash;20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21 (5\u0026ndash;24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25 (14\u0026ndash;25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.042\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.043\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLinezolid\u003c/p\u003e\n \u003cp\u003e(LZD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23 (22\u0026ndash;25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.5 (24\u0026ndash;27)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.5 (26\u0026ndash;30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.027\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.026\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClindamycin\u003c/p\u003e\n \u003cp\u003e(CD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.5 (5\u0026ndash;14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (5\u0026ndash;16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11 (5\u0026ndash;19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCefoxitin\u003c/p\u003e\n \u003cp\u003e(FOX)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.5 (5\u0026ndash;12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.5 (5\u0026ndash;12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12 (5\u0026ndash;13)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCiprofloxacin\u003c/p\u003e\n \u003cp\u003e(CIP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.5 (14\u0026ndash;21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21 (14\u0026ndash;25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.5 (14\u0026ndash;25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGentamicin\u003c/p\u003e\n \u003cp\u003e(CN)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClarithromycin\u003c/p\u003e\n \u003cp\u003e(CLR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13 (5\u0026ndash;20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.5 (8\u0026ndash;22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17 (12\u0026ndash;24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.024\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.041\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmpicillin\u003c/p\u003e\n \u003cp\u003e(Amp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOxacillin\u003c/p\u003e\n \u003cp\u003e(Ox)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.5 (5\u0026ndash;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePenecillin G\u003c/p\u003e\n \u003cp\u003e(P)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.5 (5\u0026ndash;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethicillin\u003c/p\u003e\n \u003cp\u003e(Met)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (5\u0026ndash;8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.180\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003csup\u003e(a)\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e1: \u003cem\u003ep\u003c/em\u003e-value between Reading before Vs. Reading after 6 hrs.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e2: \u003cem\u003ep\u003c/em\u003e-value between Reading before Vs. Reading after 12 hrs.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e3: \u003cem\u003ep\u003c/em\u003e-value between Reading after 6 hrs Vs. Reading after 12 hrs.\u003c/p\u003e\n \u003cp\u003e*ELF-EMF was used at 0.8 Hz frequency\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003e4. Whole genome sequencing of\u003c/strong\u003e \u003cstrong\u003eP. aeruginosa\u003c/strong\u003e \u003cstrong\u003eisolate P4 and\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003eisolate S3 before and after exposure to ELF-EMF\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1. Gene Ontology for genes affected by mutations\u003c/h2\u003e\n \u003cp\u003eGene Ontology (GO) of identified sequences of the two test isolates, \u003cem\u003eP. aeruginosa\u003c/em\u003e P4 and \u003cem\u003eS. aureus\u003c/em\u003e S3 before and after exposure to ELF-EMF was determined using Uniprot website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003c/span\u003e) accessed on 19July, 2022. The results are shown in Figs.\u0026nbsp;2\u0026amp;3 (\u003cem\u003eP. aeruginosa\u003c/em\u003e P4\u003cem\u003e)\u003c/em\u003e and Figs.\u0026nbsp;4\u0026amp;5 (\u003cem\u003eS. aureus\u003c/em\u003e S3).\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.2. Variant calling (missense mutations) of affected genes in whole genome sequences of\u003c/strong\u003e \u003cstrong\u003eP. aeruginosa\u003c/strong\u003e \u003cstrong\u003eP4 and\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003eS3 upon exposure to ELF-EMF\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eFiltered reads of the two test isolates, \u003cem\u003eP. aeruginosa\u003c/em\u003e P4 and \u003cem\u003eS. aureus\u003c/em\u003e S3 after exposure to ELF-EMF for 6 and 12 hrs in comparison to the parent isolate before exposure were mapped using BWA software followed by variant calling using GATK and Picard software. The results are shown in Table \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e (\u003cem\u003eP. aeruginosa\u003c/em\u003e P4\u003cem\u003e)\u003c/em\u003e and Table \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e (\u003cem\u003eS. aureus\u003c/em\u003e S3).\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eIntersect of missense mutation of affected genes due to exposure of \u003cem\u003eP. aeruginosa\u003c/em\u003e P4 to ELF-EMF for 6 and 12 hrs in comparison to the parent isolate before exposure.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGene name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLength\u003c/p\u003e\n \u003cp\u003e(amino acids)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenomic change\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein change\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA0259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF2875 domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e480\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e290406A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200V\u0026thinsp;\u0026gt;\u0026thinsp;200E\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA0690\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHaemagg_act domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e756738A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2261E\u0026thinsp;\u0026gt;\u0026thinsp;2261V\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA0982\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThioredoxin-like_fold domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1064704C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e134D\u0026thinsp;\u0026gt;\u0026thinsp;134N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efliC PA1092\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB-type flagellin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e488\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1183439G\u0026thinsp;\u0026gt;\u0026thinsp;A\u0026thinsp;+\u0026thinsp;1183441T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128D\u0026thinsp;\u0026gt;\u0026thinsp;128N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecobTcobU PA1279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNicotinate-nucleotide\u0026ndash;dimethylbenzimidazolephosphoribosyltransferase (NN: DBI PRT) (EC 2.4.2.21) (N(1)-alpha-phosphoribosyltransferase)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e351\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1390304G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27R\u0026thinsp;\u0026gt;\u0026thinsp;27L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA1416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFAD-binding PCMH-type domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e460\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1540435T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e233T\u0026thinsp;\u0026gt;\u0026thinsp;233A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA1874\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBapA prefix-like domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2041147C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1569I\u0026thinsp;\u0026gt;\u0026thinsp;1569M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epvdP PA2392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePvdP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e544\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2648261A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50D\u0026thinsp;\u0026gt;\u0026thinsp;50E\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epvdF PA2396\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyoverdine synthetase F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2652921T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46Q\u0026thinsp;\u0026gt;\u0026thinsp;46R\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epvdD PA2399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyoverdine synthetase D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2660959G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1396L\u0026thinsp;\u0026gt;\u0026thinsp;1396F\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA2402\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProbable non-ribosomal peptide synthetase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2678603G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2859A\u0026thinsp;\u0026gt;\u0026thinsp;2859V\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epvdL PA2424\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePvdL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2713781G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2305A\u0026thinsp;\u0026gt;\u0026thinsp;2305V\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA2431\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFUSC family protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e724\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2726207T\u0026thinsp;\u0026gt;\u0026thinsp;G\u0026thinsp;+\u0026thinsp;2726209C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e645G\u0026thinsp;\u0026gt;\u0026thinsp;645S\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA2650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethyltransf_11 domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2998633G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e265R\u0026thinsp;\u0026gt;\u0026thinsp;265K\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA2760\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProbable outer membrane protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3120238A\u0026thinsp;\u0026gt;\u0026thinsp;C\u0026thinsp;+\u0026thinsp;3120240G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56K\u0026thinsp;\u0026gt;\u0026thinsp;56Q\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA4503\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProbable permease of ABC transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e336\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5042647G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e194A\u0026thinsp;\u0026gt;\u0026thinsp;194S\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab8\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eIntersect of missense mutations of affected genes due to exposure of \u003cem\u003eS. aureus\u003c/em\u003e S3 to ELF-EMF for 6 and 12 hrs in comparison to the parent isolate before exposure.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGene name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLength\u003c/p\u003e\n \u003cp\u003e(amino acids)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenomic change\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein change\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_00036\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRhodanese domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40440G\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e325E\u0026thinsp;\u0026gt;\u0026thinsp;325Q\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_00052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUncharacterized lipoprotein SAOUHSC_00052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55881A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e140E\u0026thinsp;\u0026gt;\u0026thinsp;140D\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_00235\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS glucose transporter subunit IIABC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e263\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e256400A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61T\u0026thinsp;\u0026gt;\u0026thinsp;61A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_00245\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTruncated transposase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e264761T\u0026thinsp;\u0026gt;\u0026thinsp;A\u0026thinsp;+\u0026thinsp;264763G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128W\u0026thinsp;\u0026gt;\u0026thinsp;128R\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_00255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF5080 family protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e274510G\u0026thinsp;\u0026gt;\u0026thinsp;A\u0026thinsp;+\u0026thinsp;274512T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e151V\u0026thinsp;\u0026gt;\u0026thinsp;151I\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_00276\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTIGR01741 family protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e293353T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94L\u0026thinsp;\u0026gt;\u0026thinsp;94H\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eebh SAOUHSC_01447\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExtracellular matrix-binding protein ebh (ECM-binding protein homolog)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9535\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1381611G\u0026thinsp;\u0026gt;\u0026thinsp;A\u0026thinsp;+\u0026thinsp;1381613T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7696N\u0026thinsp;\u0026gt;\u0026thinsp;7696H\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_01557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConserved hypothetical phage protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1496261C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40C\u0026thinsp;\u0026gt;\u0026thinsp;40F\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_01558\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVL orf 51-like protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1496625C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4S\u0026thinsp;\u0026gt;\u0026thinsp;4N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_01582\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBacteriophage integrase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e401\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1508260T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e323N\u0026thinsp;\u0026gt;\u0026thinsp;323K\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_02205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConserved hypothetical phage protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2060410T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64E\u0026thinsp;\u0026gt;\u0026thinsp;64D\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_02215\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConserved hypothetical phage protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2063075T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51E\u0026thinsp;\u0026gt;\u0026thinsp;51A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_02228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConserved hypothetical phage protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2069287A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51N\u0026thinsp;\u0026gt;\u0026thinsp;51K\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_02788\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUncharacterized lipoprotein SAOUHSC_02788\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e261\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2560676C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21G\u0026thinsp;\u0026gt;\u0026thinsp;21V\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_A02795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUncharacterized protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2714202T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12L\u0026thinsp;\u0026gt;\u0026thinsp;12H\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAOUHSC_02991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavin_Reduct domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2767298T\u0026thinsp;\u0026gt;\u0026thinsp;G\u0026thinsp;+\u0026thinsp;2767300C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e228E\u0026thinsp;\u0026gt;\u0026thinsp;228N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe spreading and aggressiveness of infections which are caused by antimicrobial resistance and the lack of progress towards novel antibiotic discovery over the past decade were the reason behind investigating ELF-EMF as a novel technique against multidrug resistant infections.\u003c/p\u003e \u003cp\u003eThe difficulty in finding new antibiotic compounds has provoked the developing and utilizing of novel and complex techniques, such as electromagnetic technique. In order to assess its effect, \u003cem\u003ein vitro\u003c/em\u003e, we employed extremely low frequency electromagnetic waves on bacteria, which retrieved from surgical site infections, and evaluated the effect of extremely low frequency electromagnetic field (ELF-EMF) on the bacterial genome, some virulence factors and antibiotics susceptibilities of both multiple drug resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e MRSA test isolates. The results have driven the study to significant conclusions and recommendations regarding using and application of ELF-EMF.\u003c/p\u003e \u003cp\u003eMany studies have been carried out to verify the direct impact of extremely low frequency electromagnetic fields (ELF-EMFs) on the functioning of bacterial cells. Most of the research examined the effects on bacterial DNA, growth, morphology, biofilm, and antimicrobial susceptibility. Most of these studies revealed that ELF-EMF has a direct effect on bacteria. According to Inhan-Garip \u003cem\u003eet al\u003c/em\u003e., \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e who applied ELF-EMF on Gram-negative \u003cem\u003eP. aeruginosa\u003c/em\u003e and Gram-positive \u003cem\u003eS. aureus\u003c/em\u003e for six hrs, it was observed that (in comparison to control), all exposed strains to ELF-EMF were seen to have a significant decrease in growth rate suggesting that a mutation in bacterial genome was induced and that mutation became irreversible. Additionally, the investigations of Fojt \u003cem\u003eet al.\u003c/em\u003e, Strasak \u003cem\u003eet al\u003c/em\u003e., El-Sayed \u003cem\u003eet al\u003c/em\u003e., Aslanimehr \u003cem\u003eet al\u003c/em\u003e., Segatore \u003cem\u003eet al\u003c/em\u003e., Martirosyan, Chen \u003cem\u003eet al\u003c/em\u003e., Oncul \u003cem\u003eet al\u003c/em\u003e., \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e showed that, ELF-EMF exposure has an impairing effect on different bacterial species in time-dependent manner. In addition the proper selection of frequency and exposure duration play a significant role in the effect of extremely low frequency electromagnetic field on bacteria.\u003c/p\u003e \u003cp\u003eAfter reviewing literature, most of the research indicate that the bacterial cell wall and membrane are both responsible for the effects of extremely low frequency electromagnetic fields on bacteria, as it was confirmed by Fang \u003cem\u003eet al\u003c/em\u003e., \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e (after using the transmission electron microscopes), the cell wall of the exposed bacteria was broken forming irreversible perforations on the cell membrane; the cell inclusions and cell components were leaked and the cytoplasm and nucleolus substances flowed away leading to cell death.\u003c/p\u003e \u003cp\u003eAs well as based on several other studies of Garip \u003cem\u003eet al\u003c/em\u003e., Fadel \u003cem\u003eet al\u003c/em\u003e., Volpe \u003cem\u003eet al\u003c/em\u003e., Cellini \u003cem\u003eet al\u003c/em\u003e., Del Re \u003cem\u003eet al\u003c/em\u003e., \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e which documented that after exposure to ELF-EMF, the bacterial membrane hyperpolarization was induced causing changes in bacterial surface charge that affects the membrane potential and changes ions conduction which affect directly on bacterial viability and the electron transport system that lead to cell damage.\u003c/p\u003e \u003cp\u003eStrahl and Hamoen \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, showed that the presence of membrane potential is required for bacterial growth and survival. It was also determined that exposure to electromagnetic fields would change the physicochemical characteristics and activity of bacteria. Cell surface charge is essential for bacterial adhesion and defense mechanisms in host and have an impact on virulence, viability, and survival.\u003c/p\u003e \u003cp\u003eWe tested in our study the hypothesis (ELF-EMF induce its effect on bacteria through altering morphology of both cell wall and cell membrane) by first studying the effect of ELF-EMF on production of extracellular virulence factors such as protease, oxidase and biofilm (in case of Gram negative \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eaeruginosa\u003c/em\u003e) as well as coagulase, catalase and biofilm (in case of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eaureus\u003c/em\u003e MRSA). In addition, we studied the effect on bacterial antimicrobial susceptibility then we took an advanced step and investigated more by using whole genome sequencing and bioinformatics analysis of two bacteria isolates to find whether ELF-EMF has an effect at molecular level or not and which genes are mutated and responsible for the effect of ELF-EMF.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of extremely low frequency electromagnetic field (ELF-EMF) on some virulence factors and antibiotics susceptibilities of\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e \u003cb\u003etest isolates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExposure of \u003cem\u003eP. aeruginosa\u003c/em\u003e test isolates to ELF-EMF of 0.7 Hz for 6 and 12 hrs affected extracellular protease production. The exposure caused significant decrease in protease production. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e there was a significant decrease of the diameter of clearance zone (mm) produced in gelatin agar plates after 6 and 12 hrs of exposure to ELF-EMF compared to that obtained before exposure as \u003cem\u003ep\u003c/em\u003e-value was (0.042), but there was no statistically difference after 6 hrs Vs. 12 hrs of exposure as \u003cem\u003ep\u003c/em\u003e-value was (0.068). On other hand, regarding oxidase production by \u003cem\u003eP. aeruginosa\u003c/em\u003e test isolates, exposure to ELF-EMF produced unexpected effects as the production of extracellular oxidase enzyme was not altered significantly.\u003c/p\u003e \u003cp\u003eAlthough, there was a decrease in number of isolates that produce oxidase across different time points of exposure, but without statistically significant difference before exposure and after 6 hrs or 12 hrs exposure as \u003cem\u003ep\u003c/em\u003e-value was (0.5 \u0026amp; 0.125), respectively. It was found that studies that investigated the effect of ELF-EMF on protease or oxidase production and the reason behind that are lacking and this could be because of the complexity of \u003cem\u003eP. aeruginosa\u003c/em\u003e which makes studying the effect of ELF-EMF on extracellular protein (protease \u0026amp; oxidase) and corelating exposure with enzymes productions is difficult. Many genes entangled in their functions and take part directly or indirectly in the production cascade of protease and oxidase.\u003c/p\u003e \u003cp\u003eProtease is extracellular enzyme that require cellular channel to be excreted. We detected a significant reduction in protease production as a results of cell wall distortion as was also confirmed by Fang \u003cem\u003eet al\u003c/em\u003e., \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It was found that as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, ELF-EMF produces its effect in time dependent manner on bacterial cell wall. Our results revealed that, protease production was decreased gradually after 6 hrs \u0026amp;12 hrs., this reduction in protease productivity gives a chance to use ELF-EMF as a method against \u003cem\u003eP. aeruginosa\u003c/em\u003e protease which plays a significant role in infections.\u003c/p\u003e \u003cp\u003eIn contrast to protease results, oxidase production (as shown in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) showed non-significant change pheno-typically in 6 hrs \u0026amp; 12 hrs VS before exposure, although it is an extracellular enzyme. In addition, the number of isolates that produce oxidase across different time points of exposure in time dependent manner was decreased. According to Arai \u003cem\u003eet al\u003c/em\u003e, the opportunistic pathogen \u003cem\u003eP. aeruginosa\u003c/em\u003e encodes large number and diverse oxidases, and oxidase gene expression can be compensatory, such that loss of one or more oxidases leads to induction of others\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, that's why we were unable to find any variations in oxidase production after exposure.\u003c/p\u003e \u003cp\u003eIn case of \u003cem\u003eS. aureus\u003c/em\u003e test isolates, exposure to 0.8 Hz ELF-EMF caused inhibition in coagulase production in 5 out of 6 isolates and this effect was only observed after 12 hrs exposure. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the relation between before and after 12 hrs of exposure at reading time interval of two hours, there was no statistically significant difference in number of isolates that produced coagulase \u003cem\u003ep\u003c/em\u003e-value was (0.083), while there was statistically significant decrease in number of isolates that showed complete coagulation with no displacement when the tube inverted at reading time interval of three hours and four hours as \u003cem\u003ep\u003c/em\u003e-value were (0.046 \u0026amp; 0.031), respectively.\u003c/p\u003e \u003cp\u003eInterestingly exposure of the six \u003cem\u003eS. aureus\u003c/em\u003e test isolates to ELF-EMF showed no effect on catalase production either after 6 or 12 hrs exposure. After statistical analysis of results of \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) across different time points after exposure, there was no statistically significant difference in number of isolates regarding catalase production as all isolates were (+) as shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eTo provide an interpretation of the obtained findings that are demonstrated by inhibition of protease extracellular production while oxidase production showed non-significant changes (in case of \u003cem\u003eP. aeruginosa\u003c/em\u003e) and inhibition of coagulase extracellular production while catalase was not (in case of \u003cem\u003eS. aureus\u003c/em\u003e), we explained these results due to the channels which present in bacterial cell and responsible for excretion of these extracellular enzymes, these channels were impaired in case of protease and coagulase while the channels which are responsible for excretion of oxidase and catalase were not impaired at used frequency and exposure duration of ELF-EMF.\u003c/p\u003e \u003cp\u003eThis raise new inquiries about the appropriate choice of ELF-EMF frequency and the relationship between ELF-EMF frequency and/or duration with the precise area of the bacterial cell wall and membrane that ELF-EMF affects. It is suggested that longer exposure duration than 12 hrs is needed (based on our results) in case of Gram positive to obtain a complete distortion of cell wall and that because of the nature composition of Gram positive cell wall is thicker than Gram negative.\u003c/p\u003e \u003cp\u003eRegarding biofilm formation, exposure of \u003cem\u003eP. aeruginosa\u003c/em\u003e test isolates to ELF-EMF caused significant reductions in biofilm formation in all test isolates. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e biofilm formation was statistically significant decrease in the absorbances readings that obtained at (OD\u003csub\u003e570nm\u003c/sub\u003e) as \u003cem\u003ep\u003c/em\u003e-value was (0.043) between all different time points; between before exposure Vs. after 6 and 12 hrs of exposure as \u003cem\u003ep\u003c/em\u003e-value was (0.043), and also between reading after 6 hrs Vs. reading after 12 hrs.\u003c/p\u003e \u003cp\u003eIn case of the effect of ELF-EMF on \u003cem\u003eS. aureus\u003c/em\u003e test isolates biofilm formation, the results were similar to that exhibited by \u003cem\u003eP. aeruginosa\u003c/em\u003e test isolates, where \u003cem\u003eS. aureus\u003c/em\u003e test isolates showed a significant decrease in biofilm formation upon exposure to ELF-EMF. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e there was statistically significant decrease in absorbances readings obtained at (OD\u003csub\u003e570nm\u003c/sub\u003e) as \u003cem\u003ep\u003c/em\u003e-value was (0.028) between all different time points; between before exposure Vs. after 6 and 12 hrs of exposure as \u003cem\u003ep\u003c/em\u003e-value was (0.028), as well as between reading after 6 hrs Vs. reading after 12 hrs. These results were aligned with Karaguler \u003cem\u003eet al\u003c/em\u003e., and Haagensen \u003cem\u003eet al\u003c/em\u003e., \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e which indicated that ELF-EMF has direct effects on biofilm formation and this effect is caused by their impact on the electrical charges embedded in the cell membrane, which alters the behavior of the cells that\u0026rsquo;s in turn affecting the exopolymeric matrix structure formation.\u003c/p\u003e \u003cp\u003eConsidering how the electromagnetic field affects bacterial resistance to antibiotics, there are plenty of opposing reports. It was established that exposure to electromagnetic field increases the permeability of ion channels in the cytoplasmic membrane, breaking down the cell wall and the cytoplasmic membrane which in turn influence the permeation and movement of antibiotics molecules across bacterial cell \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In addition, Segatore \u003cem\u003eet al\u003c/em\u003e., \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e who used \u003cem\u003eP. aeruginosa\u003c/em\u003e to evaluate the effect of ELF-EMF on antibiotics susceptibility and concluded that by interfering with the surface charges on bacterial membrane, the rate at which antimicrobials penetrate is increased which in turn affected on bacterial susceptibility. On the contrary, Kamel \u003cem\u003eet al.\u003c/em\u003e, \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e showed that the exposure to ELF-EMF could induce a reversible defensive mechanism to repair the provoked damage in bacterial membrane, allowing an adaptive response with no remarkable change or even cause decrease in the susceptibility.\u003c/p\u003e \u003cp\u003eThis study demonstrated that while exposure to ELF-EMF caused significant inhibitory effect on the tested virulence factors of both \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e test isolates, that significant effect was not generally observed in case of susceptibility of both bacterial species to antibiotics.\u003c/p\u003e \u003cp\u003eThe susceptibility of both \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e test isolates to antibiotics was not affected significantly by exposure to ELF-EMF in nearly all cases. In few cases the exposure to ELF-EMF increases the susceptibility to some antibiotics (Piperacillin/tazobactam, Meropenem and Cefepime) in case of \u003cem\u003eP. aeruginosa\u003c/em\u003e and (Amoxicillin/clavulanic acid, Levofloxacin, linezolid, Clarithromycin) in case of \u003cem\u003eS. aureus\u003c/em\u003e test isolates.\u003c/p\u003e \u003cp\u003eAfter statistical analysis of antimicrobial susceptibility testing of \u003cem\u003eP. aeruginosa\u003c/em\u003e across the two time points of exposure, there were no statistically significant difference in zone diameter of antimicrobial panel except TZP which showed statistically significant increase in zone diameter between after 12 hrs exposure Vs. (before and after 6 hrs exposure) as \u003cem\u003ep\u003c/em\u003e-value were (0.041 \u0026amp; 0.034), respectively (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Antimicrobial susceptibility testing of \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) across the two time points of exposure, showed no statistically significance difference in zone diameter of antimicrobial panel except LEV, LZD \u0026amp; CLR which showed statistically significant increase in zone diameter between after 12 hrs exposure Vs. before and after 6 hrs exposure as \u003cem\u003ep\u003c/em\u003e-value were (0.042 \u0026amp; 0.043), (0.027 \u0026amp; 0.026) and (0.024 \u0026amp; 0.041), respectively.\u003c/p\u003e \u003cp\u003eThere was statistically significant increase in zone diameter between before Vs. after 12 hrs exposure as \u003cem\u003ep\u003c/em\u003e-value was (0.042) (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results showed that, bacterial antibiotics susceptibility after exposure varies; depending on their mode of action. However, the apparent increase in susceptibility in certain antibiotics explains how membrane proteins and cell wall structure are crucial as they are the first barriers to ELF-EMF and play a role in transporting antibiotics and extracellular virulence factors. All of that has driven our future perspectives to study the relation between ELF-EMF and different antibiotics classes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of exposure to extremely low frequency electromagnetic field (ELF-EMF) on whole genome sequences of\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e \u003cb\u003etest isolates.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe major goal of this research was to determine the impact of extremely low frequency electromagnetic field (ELF-EMF) exposure on \u003cem\u003eP. aeruginosa\u003c/em\u003e or \u003cem\u003eS. aureus\u003c/em\u003e and to investigate the potential alterations at the genetic level. Therefore, a device that emits extremely low frequency electromagnetic field of 0.7 Hz in case of \u003cem\u003eP. aeruginosa\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and 0.8 Hz in case of \u003cem\u003eS. aureus\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e was applied. Sequencing allowed quick answers to the change in genetic features (variations) of bacteria in response to exposure to externally ELF-EMF. The changes at the genetic level of the whole genome were scored and analyzed by bioinformatics tools to estimate the presence of noticeable variations/mutations induced in bacterial genome structure. The resulted effect was compared to the parent control isolate before exposure to the magnetic field. The results obtained showed differences in genome sequences between the non-exposed versus exposed bacterial isolates for both test bacterial species. Also, the results showed that both bacterial species, \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e (Gram-negative and Gram-positive, respectively), have diverse responses to ELF-EMF and exposure duration times. The obtained bacterial genome mutations (as shown in Tables s9 \u0026amp; s10) after exposure in case of \u003cem\u003eP. aeruginosa\u003c/em\u003e showed 27 and 30 missense mutated genes (involved in molecular function, cellular component and biological processes) after 6 and 12 h exposure times, respectively. While in \u003cem\u003eS. aureus\u003c/em\u003e, the obtained bacterial genome mutations (as shown in Tables s11 \u0026amp; s12) after exposure showed 42 and 26 missense mutated genes (involved in molecular function, cellular component and biological processes) after 6 and 12 h exposure times, respectively. It is important to realize that these results were obtained after single subculture of the exposed cells in both test isolates. The results demonstrated that: (i) the Gram-positive \u003cem\u003eS. aureus\u003c/em\u003e isolate S3 was more sensitive to ELF-EMF than the Gram-negative \u003cem\u003eP. aeruginosa\u003c/em\u003e isolate P4, since more mutated genes were detected after 6 h exposure in case of \u003cem\u003eS. aureus\u003c/em\u003e isolate as compared to \u003cem\u003eP. aeruginosa\u003c/em\u003e; (ii) although the number of mutated genes in case of \u003cem\u003eP. aeruginosa\u003c/em\u003e increased from 27 to 30 by the extension of exposure to ELF-EMF from 6 to 12 hrs, this increase was insignificant only (10%), despite of doubling the exposure period; (iii) in case of \u003cem\u003eS. aureus\u003c/em\u003e and in contrast to \u003cem\u003eP. aeruginosa\u003c/em\u003e, doubling the exposure time to ELF-EMF showed un-expected significant decrease in the number of mutated genes by (38%); (iv) We suggest that the reason behind that obtained effect with the Gram-positive \u003cem\u003eS. aureus\u003c/em\u003e isolate is, in this bacterial species, longer exposure apparently induces not only tolerance to ELF-EMF but also the repair system that recover the harmful effect induced by this magnetic field, this was demonstrated by the reduction in the number of mutated genes appeared after 12 hrs exposure time which resulted from a single subculture only; (v) the repair system detected in \u003cem\u003eP. aeruginosa\u003c/em\u003e test isolate against the damaged induced by ELF-EMF was not so efficient as that demonstrated in case of \u003cem\u003eS. aureus\u003c/em\u003e (zero percentage versus 38% reduction in the number of mutated genes in case of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, respectively\u003cem\u003e)\u003c/em\u003e; (vi) the high sensitivity of \u003cem\u003eS. aureus\u003c/em\u003e to ELF-EMF corresponds to high efficient repair system while the resistance of \u003cem\u003eP. aeruginosa\u003c/em\u003e to ELF-EMF corresponds to less efficient repair system, a physiological balance against the environmental harmful effects. The whole genome sequencing represents an important approach in understanding the effect of ELF-EMF. As shown in Figs.\u0026nbsp;2 and 4, the membrane proteins and cell wall structure genes are the most affected by ELF-EMF, and this effect could be expected since both the cell wall and cell membrane are the first cell components in direct contact with the applied EMF. As a result, the role of aforementioned structures in exporting/displaying extracellular virulence factors and transporting antibiotics, as well as their role in forming the structures that regulate adhesion and motility could be first and adversely affected by exposure to ELF-EMF. In case of \u003cem\u003eP. aeruginosa\u003c/em\u003e several genes exhibited the same mutation at the two exposure times, 6 and 12 hrs, (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The direct/indirect effect of ELF-EMF on virulence and antibiotics susceptibility could be demonstrated by the missense mutation occurred in permease of ABC transporter gene which encodes for permease of ABC transporter protein \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, pvdD gene that encodes for Pyoverdine synthetase D \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and lptD gene which encodes for LPS-assembly protein LptD \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Missense mutations in those genes by ELF-EMF might enhance the efficacy of cell wall acting antibiotics and that was reflected phenotypically in this study as shown by the increase in sensitivity of \u003cem\u003eP. aeruginosa\u003c/em\u003e to piperacillin-tazobactam (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile in case of \u003cem\u003eS. aureus, ebh\u003c/em\u003e gene, which encodes for extracellular matrix-binding protein and the SAOUHSC_00192 gene which encodes for coagulase enzyme, both exhibited missense mutation. Both genes are responsible for the virulence character exhibited by coagulase enzyme of \u003cem\u003eS. aureus\u003c/em\u003e. Interestingly, the mutation occurred in these two genes was reflected phenotypically on coagulase production (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Many other genes which encode for bifunctional autolysin protein, autolysin LytO \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, Serine-aspartate repeat-containing protein D \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e and neutral metalloproteinase \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e showed missense mutation by exposure to ELF-EMF. These genes have a role in biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e and The mutations occurred in these genes were also reflected phenotypically on the biofilm formation by this organism (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). While genes encode for Bcr/CflA family efflux transporter protein \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e Lipid II glycine glycyltransferase \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e Lysine\u0026ndash;tRNA ligase protein \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e and for ribosome biogenesis GTPase A protein \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e all these genes have a role in antibiotics susceptibility of \u003cem\u003eS. aureus.\u003c/em\u003e The mutations occurred in these genes were also reflected phenotypically on the antibiotics susceptibility by the test isolate (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eFrom this study, it can be concluded that: (i) exposure to ELF-EMF induces harmful effects on microbial population, this effect is both bacterial species and exposure time dependent; (ii) although no killing effect could be exhibited by ELF-EMF on microbial cells, this magnetic field significantly affect the physiology of microbial cells that reflected on their virulence and pathogenicity; (iii) bacterial antimicrobial susceptibility is less affected by exposure to ELF-EMF as compared to the bacterial virulence; (iv) the harmful effect on bacterial cell induced by ELF-EMF is mediated by missense mutation in the genes controlling various physiological functions (transport of elements into and out of the cell, synthesis of structural components, metabolic enzymes, etc.); (v) this study provides a good evidence for the use of ELF-EMF as an efficient approach against bacterial pathogens especially those exhibiting multiple drug resistance to antimicrobial agents; (vi) the use of ELF-EMF as therapeutic approach against infectious diseases could efficiently contribute against the development of multiple drug resistance which occurs as a result of miss- and extensive use of antibiotics.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELF-EMF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtremely low frequency electromagnetic field\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMethicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOD\u003csub\u003es\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptical densities\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBWA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBurrows-Wheeler Aligner Software\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGATK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGenome Analysis ToolKit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQUAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuality Assessment Tool\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePATRIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe Patho Systems Resource Integration Center\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board (IRB) of the Military Medical Academy under approval number [08 -2025], dated [12/06/2025]. Although the experimental work was using bacterial isolates collected as part of routine clinical care, the IRB reviewed the study retrospectively and confirmed that it met ethical standards. The isolates were anonymized, and no additional samples were collected beyond those required for routine clinical care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available in the NCBI SRA repository, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA999246 /accession number PRJNA999246\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. Elnakib: conceptualization, supervision, data interpretations, manuscript writing and revision. M. Hosny: methodology, data collection, writing original draft and the revised form.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomics Program, Department of Basic Research, Children\u0026rsquo;s Cancer Hospital Egypt 57357, Cairo, Egypt for the contribution in methodology, data analysis and discussion of whole genome sequencing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhlbom A, Feychting M. Electromagnetic radiation. Br Med Bull. 2003;68(1):157\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeychting M, Ahlbom A, Kheifets L. EMF AND HEALTH. Annu Rev Public Health. 2005;26(1):165\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eExtremely Low Frequency (ELF). 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Analyzing effects of ELF electromagnetic fields on removing bacterial biofilm. Biocybernetics Biomedical Eng. 2017;37(2):336\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaagensen JAJ, Bache M, Giuliani L, Blom NS. Effects of Resonant Electromagnetic Fields on Biofilm Formation in Pseudomonas aeruginosa. Appl Sci. 2021;11(16):7760.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMousavian-Roshanzamir S, Makhdoumi-Kakhki A. The Inhibitory Effects of Static Magnetic Field on Escherichia coli from two Different Sources at Short Exposure Time.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamel FH, Saeed CH, Qader SS. THE STATIC MAGNETIC FIELD EFFECT ON PSEUDOMONAS AERUGINOSA.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkhtar AA, Turner DPJ. The role of bacterial ATP-binding cassette (ABC) transporters in pathogenesis and virulence: Therapeutic and vaccine potential. Microb Pathog. 2022;171:105734.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePodbielski A, Pohl B, Woischnik M, K\u0026ouml;rner C, Schmidt KH, Rozdzinski E, et al. Molecular characterization of group A streptococcal (GAS) oligopeptide permease (opp) and its effect on cysteine protease production. Mol Microbiol. 1996;21(5):1087\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang D, Revtovich AV, Chen Q, Shah KN, Cannon CL, Kirienko NV. Pyoverdine-Dependent Virulence of Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients. Front Microbiol. 2019;10:2048.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePandey S, Delgado C, Kumari H, Florez L, Mathee K. Outer-membrane protein LptD (PA0595) plays a role in the regulation of alginate synthesis in Pseudomonas aeruginosa. 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RNA-dependent lipid remodeling by bacterial multiple peptide resistance factors. Proc Natl Acad Sci USA. 2008;105(12):4667\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBennison DJ, Nakamoto JA, Craggs TD, Mil\u0026oacute;n P, Rafferty JB, Corrigan RM. The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions. mBio. 2021;12(6):e0267921.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHwang J, Inouye M. The tandem GTPase, Der, is essential for the biogenesis of 50S ribosomal subunits in Escherichia coli. Mol Microbiol. 2006;61(6):1660\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChampney WS. Antibiotics targeting bacterial ribosomal subunit biogenesis. J Antimicrob Chemother. 2020;75(4):787\u0026ndash;806.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Extremely low electromagnetic field, Bacterial resistance, S. aureus, P. aeruginosa, Virulence, Mutation, biofilm, Multiple Drug resistance, Growth, Genome","lastPublishedDoi":"10.21203/rs.3.rs-6791184/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6791184/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The extremely low frequency electromagnetic field (ELF-EMF) effect on microorganisms has attracted attention due to its potential for industrial and medical applications as a promising candidate to combat multi-drug resistant pathogens. This study aimed to assess the effect of ELF-EMF on Gram-negative (\u003cem\u003eP. aeruginosa\u003c/em\u003e) and Gram-positive (\u003cem\u003eS. aureus\u003c/em\u003e) bacterial isolates. This effect was determined phenotypically (bacterial virulence and antibiotics susceptibility) and genotypically (mutations induced in whole genome). The test organisms were exposed to ELF-EMF with frequency of 0.7 Hz and 0.8 Hz for \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, respectively for different exposure times (6 and 12 hrs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e By comparing the results of exposed bacterial cultures with their counterparts non-exposed controls; remarkable differences were found in virulence, antibiotics susceptibility and genome structure. Whole genome sequencing revealed missense mutated genes that were associated directly/indirectly with the observed inhibition in protease and oxidase production, biofilm formation (in case of \u003cem\u003eP. aeruginos\u003c/em\u003ea), coagulase and catalase production and biofilm formation (in case of \u003cem\u003eS. aureus\u003c/em\u003e). Also, the antibiotic susceptibility tests of both bacterial species indicated enhancement in the sensitivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Therefore, it was concluded that each organism responds differently to ELF-EMF and exposure of \u003cem\u003eP. aeruginosa \u003c/em\u003eand\u003cem\u003e S. aureus \u003c/em\u003etest isolates to ELF-EMF at the stated frequencies affects the cellular activity as well as the structure and that effect depends on the duration of exposure. This study provides an evidence for the use of ELF-EMF as an efficient technique against skin bacterial infections especially those that are caused by pathogens with multiple drug resistance to different antimicrobial agents.\u003c/p\u003e","manuscriptTitle":"Extremely low frequency electromagnetic field affects virulence and antibiotic susceptibility of multidrug resistant Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-01 09:20:35","doi":"10.21203/rs.3.rs-6791184/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"133071830515858753990361546740941264822","date":"2025-12-16T07:54:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-04T13:02:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334590709877761713500235531599614013066","date":"2025-07-01T06:22:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29604657927128874051673851702002256333","date":"2025-06-26T11:11:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118280631474048470324835582706701756253","date":"2025-06-26T09:46:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-26T02:08:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-26T02:00:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-24T21:56:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-21T11:20:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-06-21T11:16:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"805a8d0e-e380-44cd-a789-a003f6037990","owner":[],"postedDate":"July 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-01T09:20:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-01 09:20:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6791184","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6791184","identity":"rs-6791184","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}