Electron-Beam Irradiation for inactivation of airborne Multidrug-Resistant Bacteria: Single-Pass Efficacy and morphological evidence of destructive mechanisms

Electron-Beam Irradiation for inactivation of airborne Multidrug-Resistant Bacteria: Single-Pass Efficacy and morphological evidence of destructive mechanisms | 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 Article Electron-Beam Irradiation for inactivation of airborne Multidrug-Resistant Bacteria: Single-Pass Efficacy and morphological evidence of destructive mechanisms Ashkan Seza, Kimia Mozahheb Yousefi, Sara Saeedifar, Sara Minaeian This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9234245/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Airborne and aerosol transmission of multidrug-resistant (MDR) bacteria pose a major challenge in healthcare environments. This study evaluated the performance of an electron-beam irradiation (EBI) system for inactivating airborne MDR bacteria under controlled laboratory conditions. To perform this assessment, clinical isolates of MDR Pseudomonas aeruginosa , Acinetobacter sp., Klebsiella sp., and methicillin-resistant Staphylococcus aureus (MRSA) were aerosolized to simulate realistic airborne transmission and subsequently introduced into the EBI system. Downstream bacterial aerosols were collected using liquid impingers and quantified by culture-based enumeration. CFU data were log₁₀-transformed prior to analysis. The EBI system reduced airborne bacterial loads by 4.01 log₁₀ for P. aeruginosa , 3.64 log₁₀ for Acinetobacter sp., 3.64 log₁₀ for Klebsiella sp., and 5.37 log₁₀ for MRSA, corresponding to disinfection efficiencies of 99.98–99.999%. Scanning electron microscopy (SEM) and elemental mechanistic study revealed morphological damage, including membrane rupture, surface collapse, and loss of structural integrity, indicating that electron impact and electroporation are the predominant mechanisms of inactivation. These results demonstrate that EBI is a rapid and effective method for mitigating airborne MDR bacterial threats, with potential for deployment in healthcare, laboratory, and other high-risk indoor environments. Health sciences/Diseases Health sciences/Medical research Biological sciences/Microbiology Electron-beam irradiation EBI Airborne transmission Aerosolized bacteria Electron-induced membrane disruption Electroporation Multidrug-resistant bacteria MDR Bioaerosol inactivation Bacteria elemental analysis Scanning electron microscopy SEM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Healthcare-associated infections (HAIs) remain a critical public health issue, representing one of the most significant contributors to morbidity and mortality in modern healthcare [ 1 ]. Airborne transmission of multidrug-resistant (MDR) bacteria amplifies this challenge by undermining traditional infection control measures within healthcare environments [ 2 , 3 ]. These pathogens account for a significant proportion of nosocomial infections, leading to prolonged hospital stays, elevated treatment costs, and increased mortality rates, particularly among immune-compromised patients [ 4 – 6 ]. Common MDR pathogens such as Pseudomonas aeruginosa , Acinetobacter species, Klebsiella species, and methicillin-resistant Staphylococcus aureus (MRSA) can persist in hospital air and on environmental surfaces, spreading via aerosolization and contact with inadequately disinfected areas [ 7 – 9 ]. In enclosed healthcare settings with poor ventilation, these microorganisms can form stable airborne reservoirs, substantially heightening the risk of hospital-acquired infections and localized outbreaks [ 10 , 11 ]. Conventional air purification technologies, such as High-Efficiency Particulate Air (HEPA) filtration, capture airborne particles as small as 0.3 µm with 99.97% efficiency; however, their effectiveness is limited to mechanical trapping rather than microbial inactivation [ 12 , 13 ]. Pathogens retained on filter surfaces can remain viable, creating a potential source for re-aerosolization or cross-contamination during filter maintenance and replacement [ 14 , 15 ]. Moreover, HEPA systems require high installation and operational costs, frequent maintenance, and exhibit reduced performance in humid environments [ 16 , 17 ]. They also show limited efficiency against smaller viral particles and lack real-time pathogen neutralization capability [ 18 , 19 ]. These drawbacks underscore the need for innovative air disinfection technologies capable of both capturing and actively inactivating microorganisms in situ [ 20 , 21 ]. To address these limitations, several advanced air purification strategies have been explored, including ultraviolet germicidal irradiation (UVGI), electrostatic precipitation, photocatalytic oxidation, and non-thermal plasma systems [ 22 – 25 ]. Among these emerging technologies, electrically driven systems such as non-thermal plasma and electron-based disinfection methods offer distinct advantages by producing physical and electrical effects capable of directly disrupting microbial structures [ 26 , 27 ]. The EBI system evaluated in this study generates high-energy electrons that can directly damage microbial cell envelopes through electron impact and can also induce electroporation via intense localized electric fields, leading to irreversible membrane destabilization and cell death [ 28 ]. The single-pass air disinfection efficacy of the EBI system was tested using a controlled aerosolization laboratory model, to quantify the single-pass inactivation efficiency and log₁₀ reductions of aerosolized MDR bacteria of multidrug-resistant (MDR) bacteria, including P. aeruginosa, Acinetobacter species, Klebsiella species, and methicillin-resistant S. aureus (MRSA), isolated from clinical samples obtained at Hazrat Rasoul Hospital, Tehran, Iran. The findings from this study will inform the future application and use of electron-beam irradiation as an air-disinfection technology for enhancing indoor microbial air quality and strengthening infection-control strategies in healthcare and other critical indoor environments, addressing the urgent need for alternative and novel disinfection approaches to address the escalating threat of antimicrobial resistance. 2. Materials and methods 2.1 Bacterial isolates and Antibiogram Profiles Clinical isolates of Acinetobacter sp., S. aureus , Klebsiella and P. aeruginosa were obtained from Hazrat-e Rasoul Hospital in Tehran, Iran. These isolates were selected based on their demonstrated resistance to multiple antibiotic classes (Table 1 ). Antibiogram testing was performed using the Kirby-Bauer disk diffusion method in accordance with CLSI M100 guidelines (2023). All experiments and methods were performed in accordance with relevant guidelines and regulations. The study protocols and the use of clinical isolates were reviewed and approved by the Ethics Committee of Iran University of Medical Sciences (Approval ID: IR.IUMS.REC.1403.958). Informed consent was obtained from all subjects and/or their legal guardian(s). All isolates were completely anonymized to protect patient privacy. Table 1 Antibiogram profiles of multidrug-resistant (MDR) clinical isolates used in the study. Microorganism Sensitive (S) Resistant (R) Acinetobacter sp. — Ampicillin – Sulbactam (10/10 µg(, Ciprofloxacin (5 µg(, Cefepime (30 µg(, Ceftriaxone (30 µg (, Amikacin (30 µg(, Gentamicin (10 µg (, Trimethoprim –Sulfamethoxazole (1.25/23.75µg), Piperacillin – Tazobactam (100/10 µg(, Doxycycline (30 µg(, Staphylococcus aureus (MRSA) Linezolid (30 µg(, Doxycycline (30 µg(, Trimethoprim –Sulfamethoxazole (1.25/23.75µg) Ciprofloxacin (5 µg(, Clindamycin (2 µg(, Erythromycin (15 µg(, Cefoxitin (30µg(, Penicillin G (10 U(, Vancomycin (30 µg(, Klebsiella sp. — Doxycycline (30 µg(, Gentamicin (10 µg(, Amikacin (30 µg(, Ciprofloxacin (5 µg(, Imipenem (10 µg(, Meropenem (10 µg(, ,Piperacillin – Tazobactam(100/10 µg(, Ampicillin–Sulbactam (10/10 µg(, Pseudomonas aeruginosa Gentamicin (10 µg(, Amikacin (30 µg ( Piperacillin – Tazobactam (100/10 µg(, Cefazolin (30 µg(, Cefepime (30 µg(, Ciprofloxacin (5 µg(, Imipenem (10 µg(, Meropenem (10 µg), 2.2 Bacterial Culture Preparation and Inoculum Standardization Acinetobacter sp., S. aureus, Klebsiella sp., and P. aeruginosa cultures were grown on nutrient agar at 37°C for 24 h, then transferred to Mueller–Hinton (MH) broth and incubated in a shaker incubator at 37°C for 2 h to obtain a viable, standardized inoculum. Bacterial concentrations were adjusted to approximately 10⁹CFU/mL based on optical density at 600 nm (OD 600 ) using a spectrophotometer. The cultures were then centrifuged and resuspended in 0.9% physiological saline (Merck, Germany). Cell density was further standardized using McFarland standards, and serial dilutions were prepared. To confirm the final cell concentration, serial dilutions were spread-plated on nutrient agar and colony-forming units (CFU) were enumerated. 2.3 Experimental Setup The device used in this study is an air-cleaning system with multiple layers of electron beam emitters within a stainless-steel reactor (Model MD250, PlasmaShield Ltd, Australia). The device was incorporated into the experimental setup shown in Fig. 1 . Microorganisms were aerosolized using a 3-jet Collison nebulizer (CH Technologies, Westwood, NJ, USA) at an inoculum suspension concentration of 10⁹ CFU/mL in physiological serum. The nebulizer was positioned at the start of a detachable stainless-steel pipeline (25 cm diameter, 70 cm length) before entering the device. Airflow within the duct was maintained at 1 m/s, and nebulization was achieved using compressed air at a flow rate of 14 L/min. Microorganisms downstream of the EBI unit were collected 120 cm beyond the reactor outlet using three glass impingers (Unilab, 500 mL, borosilicate glass 3.3). The sampling points located inside the stainless-steel pipe, oriented perpendicular to the airflow, and spaced 120° apart were each connected to an individual impinger via silicone tubing. Each impinger operated at 26.67 L/min for 30 minutes per run using a dry vacuum pump with 0.9% saline as the collection medium. Among the three sampling points, the impinger with the highest collection efficiency was selected for subsequent serial dilutions, while the remaining two were monitored to confirm uniformity of aerosol distribution in the airstream. All experiments were performed under controlled laboratory conditions with an average temperature of 28 ± 0.8°C measured continuously at the chamber inlet. Collected samples were serially diluted in physiological saline, spread-plated onto nutrient agar in triplicate, and incubated at 37°C for 24 h prior to CFU enumeration. Each testing condition was conducted in triplicate. Conditions included the EBI turned on and also a negative control where the device was replaced with an empty casing. This was repeated for each microorganism concentration. 2.4. Statistical analysis Statistical analysis was performed using SPSS (version 27). Colony-forming unit (CFU) data were log₁₀-transformed prior to analysis to account for the log-normal distribution typically observed in microbial count data. Results are reported as mean log₁₀ (CFU) values with corresponding standard deviations (SD) for both control and EBI-treated conditions. Log₁₀ reductions were calculated as the difference between mean log₁₀ (CFU) values of control and treated samples. Standard deviations for log₁₀ reductions were calculated using error propagation to reflect the combined variability of both control and treated measurements. Disinfection efficacy (%) was calculated based on the corresponding CFU values derived from the log₁₀-transformed data. Samples with no detectable colony growth were assigned a value of 1 CFU prior to log₁₀ transformation. 2.5. Scanning Electron Microscopy (SEM) SEM analysis was performed to visualize the morphological alterations of multi-drug resistant (MDR) bacteria following exposure to electron beam irradiation. The experiments were conducted using the setup described in Section 2.3 (Fig. 1 ). Microorganisms captured in the liquid impinger were carefully deposited onto pre-cleaned glass slides and immediately fixed in a 2.5% glutaraldehyde solution prepared in physiological saline at 25°C for 24 hours to preserve cellular structures. After fixation, the glutaraldehyde solution was discarded, and the samples were sequentially dehydrated in graded ethanol solutions (70%, 90%, and 100%), each for 15 minutes, to ensure complete removal of water. The dehydrated slides were subsequently mounted on aluminum SEM stubs using conductive carbon adhesive tape and sputter-coated with a 5 nm gold layer using an NSC DSR1 sputter coater to enhance surface conductivity and imaging quality. Imaging was performed using a Mira 3 (Tescan, Czech Republic) scanning electron microscope operated at an accelerating voltage of 15 kV under high vacuum conditions. Elemental characterization of the same samples was carried out by energy dispersive X-ray spectroscopy (EDS) using Aztec software (Oxford Instruments, UK) to evaluate surface elemental composition and detect chemical alterations associated with bacterial planktonic structures after EBI treatment. 3. Results The single-pass disinfection performance of the EBI system against aerosolized multidrug-resistant (MDR) bacteria is summarized in Table 2 . Across all tested microorganisms, the EBI system demonstrated consistent reductions in airborne bacterial load under controlled laboratory conditions (n = 3). The highest inactivation performance was observed for S. aureus (MRSA), with a mean log₁₀ reduction of 5.37, corresponding to a 99.999% disinfection efficacy. The EBI system achieved a mean log₁₀ reduction of 4.01 for P. aeruginosa , corresponding to a 99.99% disinfection efficacy, while Acinetobacter sp. and Klebsiella sp. exhibited mean log₁₀ reductions of 3.64, corresponding to 99.98% disinfection efficacy. Table 2 Quantitative single-pass disinfection performance of the electron-beam irradiation (EBI) system against aerosolized multidrug-resistant (MDR) bacteria. Results are reported as mean log₁₀ (CFU) ± SD for control (device off) and EBI-treated (device on) conditions obtained from triplicate experiments (n = 3 per condition). Log₁₀ reductions were calculated as the difference between mean control and treated log₁₀ (CFU) values. Disinfection efficacy (%) was calculated from CFU values derived from log₁₀-transformed data. Samples with no detectable colony growth were assigned a value of 1 CFU prior to log₁₀ transformation. MDR pathogen tested Control log₁₀(CFU) ± SD EBI log₁₀(CFU) ± SD Disinfection efficacy (%) Mean Log₁₀ reduction Pseudomonas aeruginosa 4.65 ± 0.43 0.64 ± 1.11 99.99 4.01 Acinetobacter 5.64 ± 0.08 2.01 ± 1.75 99.98 3.64 Klebsiella 5.39 ± 0.14 1.75 ± 1.55 99.98 3.64 Staphylococcus aureus (MRSA) 6.22 ± 0.28 0.84 ± 1.46 99.999 5.37 To complement these quantitative findings, scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were performed to examine electron beam induced morphological and compositional alterations in representative bacterial species, as described in the following sections. Figure 2 presents scanning electron micrographs (SEM) of untreated (control) and EBI-treated multidrug-resistant (MDR) bacterial species, including P. aeruginosa (A–B), S. aureus (MRSA). (C–D), Klebsiella sp. (E–F), and Acinetobacter sp. (G–H). The control panels (A, C, E, G) show the characteristic morphology of viable cells: P. aeruginosa and Klebsiella sp. display smooth rod-shaped structures with intact outer membranes; S. aureus appears as spherical cocci with well-defined, continuous surfaces; and Acinetobacter sp. exhibits compact, oval-shaped cells with uniform texture, features typical of healthy, actively growing bacteria. In contrast, the EBI-treated panels (B, D, F, H) reveal extensive structural collapse and severe morphological distortion across all species. The treated P. aeruginosa and Klebsiella Spp. cells exhibit extensive surface rupture and elongation collapse, indicating envelope disintegration and cytoplasmic leakage. S. aureus cells lose their spherical integrity and transform into fragmented, porous remnants, while Acinetobacter sp. appears as completely disintegrated aggregates with loss of cellular definition. These profound deformations confirm that electron beam exposure leads to irreversible physical damage of bacterial envelopes [ 29 – 31 ]. The observed destruction pattern is consistent with electron induced damage, a primary mechanism in the EBI’s technology. The electrons generated by the electron-beam emitters collide directly with microbial cell walls, causing localized bond scission, surface sputtering, and nano-scale ablation of membrane biomolecules. This exposure disrupts lipid bilayers, peptidoglycan structures, and protein matrices, resulting in envelope perforation, membrane collapse, and eventual cellular implosion. The electron impact is further intensified by transient electroporation which accelerates structural breakdown. Together, the SEM evidence in Fig. 2 demonstrates that the EBI system exerts a direct electron-mediated physical destruction effect on both Gram-positive and Gram-negative MDR bacteria. This supports the quantitative results presented in Table 2 and confirms that the predominant inactivation mechanism is physical ablation through electron impact rather than other mechanisms [ 32 – 35 ]. Following the extensive evidence of electron impact–driven physical ablation presented in Fig. 2 , further analysis was conducted to explore additional EBI-induced damage pathways contributing to microbial inactivation. While collision with high-speed electrons primarily causes surface erosion and molecular ablation, the intense electric fields generated within the EBI’s discharge zone are also capable of inducing electroporation, a complementary mechanism that destabilizes bacterial membranes at the nanostructural level [ 36 ]. To visualize this process, the morphological effects of electron beam exposure on P. aeruginosa and S. aureus (MRSA) were examined by SEM, as shown in Fig. 3 . Figure 3 presents scanning electron micrographs (SEMs) depicting the morphological alterations of P. aeruginosa (A–B) and S. aureus (MRSA) (C–D) following exposure to high-speed electrons. The control panels (A, C) show typical intact morphology: P. aeruginosa cells exhibit smooth, rod-shaped surfaces with clearly defined envelopes, while MRSA appears as regular spherical cocci with continuous cell walls and well-preserved contours. In contrast, the treated cells (B, D) display distinct electroporation-induced structural damage. P. aeruginosa demonstrates localized membrane perforations, shallow depressions, and early signs of cell wall rupture, suggesting the formation of pores caused by the intense electric field generated within the electron discharge zone. Similarly, MRSA cells reveal partial collapse and the appearance of nanoscale pits and ruptures on the surface morphological evidence of electrical membrane destabilization and pore coalescence [ 37 – 39 ]. These deformations are characteristic of irreversible electroporation, a key antimicrobial mechanism of the EBI system. The device generates an intense electric field that induces transmembrane potential differences exceeding the dielectric threshold of bacterial membranes (typically > 1 V), resulting in the formation of transient nanopores. Under sustained exposure, these pores expand irreversibly, causing leakage of intracellular components, disruption of osmotic balance, and eventual cell death. The electroporation effect is further amplified by simultaneous high-energy electron collisions within the device reactor, which together accelerate the breakdown of the phospholipid bilayer and peptidoglycan structures [ 31 , 34 , 40 , 41 ]. Collectively, the SEM findings in Fig. 3 confirm that electroporation-driven membrane perforation is a primary mechanism of inactivation in both Gram-negative ( Pseudomona s) and Gram-positive ( S. aureus ) bacteria. This supports the earlier electron-impact mechanism and highlights the dual-action behavior of the EBI technology, where electrical and physical destructive processes work together to produce rapid, irreversible microbial inactivation. To further support the morphological observations and elucidate the physicochemical processes underlying bacterial destruction, scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were conducted on multidrug-resistant (MDR) Acinetobacter sp. cells exposed to EBI (Fig. 4 ). In Fig. 4 a, the untreated control cells exhibits a smooth, intact morphology with an elemental composition of 60.57 wt% C, 13.39 wt% O, 9.52 wt% Si, 0.59 wt% Mg, and 0.41 wt% Al, indicative of a carbon-rich, protein–lipid envelope with negligible substrate exposure [ 42 ]. After electron beam exposure, Fig. 4 b shows localized membrane deformation and the formation of a central crater. Corresponding EDS spectra display 53.27 wt% C (− 7.30 wt%), 22.60 wt% O (+ 9.21 wt%), 11.62 wt% Si (+ 2.10 wt%), 0.81 wt% Mg, and 0.46 wt% Al, suggesting partial oxidation, focal organic loss, and incipient exposure of the underlying glass substrate. These features are consistent with electroporation-driven membrane disruption, where transient electric fields induce localized pore formation and oxidative stress [ 43 – 45 ]. In contrast, Fig. 4 c reveals severe surface erosion and fragmentation. EDS analysis shows 23.06 wt% C (− 37.51 wt% vs. control), 34.49 wt% O (+ 21.10 wt%), 21.81 wt% Si (+ 12.29 wt%), 1.58 wt% Mg, and 0.78 wt% Al, signifying extensive ablation of organic components and exposure of the inorganic substrate. This quantitative shift—from ~ 12% relative carbon loss during electroporation to ~ 62% during ablation highlights a dual-mechanism pathway for bacterial inactivation: (1) pulsed electric field–induced membrane perforation, initiating early oxidative disruption, followed by (2) reactive species and electron bombardment driven etching, culminating in complete structural collapse [ 38 ][ 46 ]. 4. Discussion The EBI air-cleaning system demonstrated high efficacy in inactivating aerosolized multidrug-resistant (MDR) bacteria, including Staphylococcus aureus (MRSA), Pseudomonas aeruginosa , Klebsiella sp., and Acinetobacter sp., under controlled laboratory conditions. The system achieved robust single-pass disinfection performance, with average removal efficiencies ranging from 99.98% to 99.999%. The highest inactivation was observed for MRSA and P. aeruginosa , with mean log₁₀ reductions of 5.37 and 4.01, respectively, while Klebsiella sp. and Acinetobacter sp. exhibited mean log₁₀ reductions of 3.64. SEM and EDS analyses provided direct morphological and compositional evidence of the inactivation mechanism, revealing severe structural deformation, membrane rupture, and elemental redistribution characterized by carbon depletion and oxygen enrichment. These findings are further clarified by the schematic in Fig. 5 , which illustrates the proposed dual-action antimicrobial mechanism of the EBI system: high-energy electron impact causes direct ablation of the bacterial envelope, whereas electroporation creates nanoscale membrane pores that destabilize cell structure. In combination, these mechanisms operate synergistically, accelerating cytoplasmic leakage, structural collapse, and eventual cell death. When integrated with the quantitative reductions observed in aerosolized MDR bacteria, the morphological and elemental evidence underscores that EBI inactivates microbes predominantly through physical ablation and electrical destabilization, not chemical oxidation. This dual-action mechanism effectively accounts for the rapid and consistent log 10 reductions demonstrated across Gram-positive and Gram-negative organisms. This study has certain limitations that should be acknowledged. The bacterial reduction levels observed may have been negatively influenced by the high humidity produced by the nebulizer during aerosolization. Elevated moisture can temporarily reduce electrostatic activity and charge-transfer interactions within both the electron discharge field and filter matrix, which may have decreased the disinfection efficacy. This humidity effect reflects the experimental setup and is not necessarily representative of typical indoor environments, where lower relative humidity is more common [ 42 , 43 ]. In addition, the maximum log 10 reduction achieved may have been limited by the maximum concentration tested in the inoculum suspension. Previous research has suggested that EBI efficiency may increase with higher microbial loads; however, that was not feasible in this experiment with the MDR strains [ 28 ]. Future experiments under stricter containment conditions are needed to explore the maximum disinfection efficacy achieved when challenged with higher concentrations of MDR microorganisms. 5. Conclusions In summary, the electron-beam irradiation (EBI) system represents a promising technology for mitigating airborne infections and indoor air decontamination. Its chemical-free operation enables continuous inactivation of airborne microorganisms and offers a practical strategy for reducing exposure to multidrug-resistant bacteria aerosols. Therefore, in healthcare, laboratory, and other high-risk environments, EBI-based air treatment has the potential to contribute meaningfully to the prevention of hospital-acquired infections and ultimately save lives, especially among vulnerable patient populations. Future research should also investigate the molecular and genetic consequences of EBI exposure. Studies on bacterial gene expression will clarify how electron‑beam irradiation influences biofilm formation, antibiotic resistance behavior, susceptibility to other disinfection methods, and overall pathogenicity. In parallel, analyses of intracellular components including DNA integrity and chromosomal organization are needed to determine how electron beam‑induced damage mechanisms affect genetic material and internal structures. These investigations will extend evaluation from surface‑level morphological disruption to fundamental cellular and molecular pathways, providing deeper insight into the long‑term biological impact of EBI treatment. In addition, further evaluation under a range of environmental conditions, together with expanded testing against viral and fungal aerosols, will help define the full antimicrobial spectrum of EBI and enhance its suitability for real-world infection-control applications. Declarations Ethics approval and consent to participate: All experiments and methods were carried out in accordance with relevant guidelines and regulations. The experimental protocols were approved by the Ethics Committee of Iran University of Medical Sciences (Approval ID: IR.IUMS.REC.1403.958). Informed consent was obtained from all subjects and/or their legal guardian(s). Conflicts of Interest Both funders had no role in the conduct of the experimental work including selection of test organisms, data collection or analysis, interpretation of the results, decision to publish, or preparation of the manuscript. The authors declare that they have no conflicts of interest. Funding This study was funded by the Antimicrobial Resistance Research Center of the Institute of Immunology and Infectious Diseases at Hazrat Rasoul Hospital, Iran University of Medical Sciences (Grant number REC.1403.958). Plasma Shield Ltd (Australia) provided additional support limited to publication-related costs and providing funding to researchers at Flinders University, Australia to provide an external and independent review of the study design. Author Contribution A.S. and S.M. conceptualized the study and designed the methodology. A.S., K.M.Y., and S.S. conducted the investigations. Data curation and formal analysis were performed by K.M.Y., S.S., and S.M. K.M.Y. contributed to software application and data validation, while A.S. and S.S. prepared the visual representations of the data. S.M. provided resources, supervised the research, and managed project administration. A.S. was responsible for funding acquisition. The original draft of the manuscript was written by A.S. and K.M.Y., with S.M. providing critical review and editing. All authors reviewed the manuscript. Acknowledgement The authors would like to acknowledge the facilities, as well as the scientific and technical assistance, provided by the Antimicrobial Research Center under the Institute of Immunology and Infectious Diseases at Hazrat-e Rasoul Hospital, Iran University of Medical Sciences. We also appreciate Rahavard Fanavari Company for all helps and its technical support during the project. 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PLoS One . 12 , e0173618 (2017). Du, C. et al. Qualitation and quantitation on microplasma jet for bacteria inactivation. Sci. Rep. 6 , 18838 (2016). Huo, Z. Y. et al. Triboelectrification induced self-powered microbial disinfection using nanowire-enhanced localized electric field. Nat. Commun. 12 , 3693 (2021). Estifaee, P., Su, X., Yannam, S. K., Rogers, S. & Thagard, S. M. Mechanism of E. coli inactivation by direct-in-liquid electrical discharge plasma in low conductivity solutions. Sci. Rep. 9 , 2326 (2019). Barkhade, T., Nigam, K., Ravi, G., Rawat, S. & Nema, S. K. Investigating the effects of microwave plasma on bacterial cell structures, viability, and membrane integrity. Sci. Rep. 15 , 18052 (2025). Zhao, J. et al. Effect of plasma-activated solution treatment on cell biology of Staphylococcus aureus and quality of fresh lettuces. Foods 10 , 2976 (2021). Yeo, S. K. & Liong, M. T. Effects and applications of sub-lethal ultrasound, electroporation and UV radiations in bioprocessing. Ann. Microbiol. 63 , 813–824 (2013). Ma, Y. et al. Efficient robust yield method for preparing bacterial ghosts by Escherichia coli phage ID52 lysis protein E. Bioengineering 9 , 300 (2022). Park, J. T. & Uehara, T. How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan). Microbiol. Mol. Biol. Rev. 72 , 211–227 (2008). Sreedevi, P. R. & Suresh, K. Cold atmospheric plasma mediated cell membrane permeation and gene delivery-empirical interventions and pertinence. Adv. Colloid Interface Sci. 320 , 102989 (2023). Huo, Z. Y. et al. Cell transport prompts the performance of low-voltage electroporation for cell inactivation. Sci. Rep. 8 , 15832 (2018). Han, L. et al. Mechanisms of inactivation by high-voltage atmospheric cold plasma differ for Escherichia coli and Staphylococcus aureus. Appl. Environ. Microbiol. 82 , 450–458 (2016). Patinglag, L. et al. Non-thermal plasma-based inactivation of bacteria in water using a microfluidic reactor. Water Res. 201 , 117321 (2021). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 16 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviews received at journal 04 May, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers invited by journal 08 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Editor invited by journal 02 Apr, 2026 Submission checks completed at journal 31 Mar, 2026 First submitted to journal 31 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-9234245","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":622258145,"identity":"ad98c3fe-951d-4917-8003-51fdde01289d","order_by":0,"name":"Ashkan Seza","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ashkan","middleName":"","lastName":"Seza","suffix":""},{"id":622258146,"identity":"6c088f04-9c08-45f9-bb0b-735f3c56713f","order_by":1,"name":"Kimia Mozahheb Yousefi","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kimia","middleName":"Mozahheb","lastName":"Yousefi","suffix":""},{"id":622258147,"identity":"1d1da350-1712-479b-9617-ee83e4cb7752","order_by":2,"name":"Sara Saeedifar","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Saeedifar","suffix":""},{"id":622258148,"identity":"2bd1c1c5-653d-46da-95b4-141ab0ba4fa5","order_by":3,"name":"Sara Minaeian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYPACCSBmbGBgqADSzMwNpGg5A9LCSJQWKGBsY4DqxQPk288+/MCYY5HH397c+LlwXm00fztQy4+KbTi1GJxJN5Zg3CZRLHHmYLP0zG3Hc2ccZmxg7DlzG7cWhjQGkJbEhhuJDdK8247lNgC1MDO24dYi3/+M+QdIy/z7D5t/8845ljufkBaGG2lsYFs23GBsk+ZtqMndQEiLwY1nbBaJQC0bzyS2WfMcO5C7EajlID6/yPenMd/4uK0ucd7x449v89TU5c47f/jggx8VeBwGAgkI5mEweQC/elRQR4riUTAKRsEoGCEAAFGfW39GLhyHAAAAAElFTkSuQmCC","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Sara","middleName":"","lastName":"Minaeian","suffix":""}],"badges":[],"createdAt":"2026-03-26 12:40:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9234245/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9234245/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106986298,"identity":"b0db21c6-a0e3-4917-8616-6f2778a9e6b7","added_by":"auto","created_at":"2026-04-15 13:06:23","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112286,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of experimental setup.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9234245/v1/99cea2b4166bd7bd74401593.jpeg"},{"id":106994248,"identity":"bf8be702-1b08-4c78-aa53-c4c31191137d","added_by":"auto","created_at":"2026-04-15 15:06:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1254756,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscope images of baseline/negative control (A) Pseudomonas aeruginosa (C) Staphylococcus aureus (E) Klebsiella sp and (G) Acinetobacter sp. and EBI destroyed microorganisms (B) P. aeruginosa (D) S. aureus (F) Klebsiella sp. and (H) Acinetobacter sp.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9234245/v1/eb12b4d2e7a69dfd2a7b9ce0.png"},{"id":106986299,"identity":"73ec71e4-336b-49e8-9cc2-653cc966c66f","added_by":"auto","created_at":"2026-04-15 13:06:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":670165,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of P. aeruginosa (A–B) and S aureus (MRSA) (C–D) before and after EBI exposure. Electron beam-treated cells show nano-scale perforations and deformation caused by electroporation and membrane destabilization.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9234245/v1/d6f11e60112eefc702641e7f.png"},{"id":106986302,"identity":"e741e5ce-6f96-49ad-95f7-6d81bba4f7a6","added_by":"auto","created_at":"2026-04-15 13:06:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":775683,"visible":true,"origin":"","legend":"\u003cp\u003eSEM and EDS analyses of MDR Acinetobacter sp. after exposure to EBI. (a) Untreated control showing intact morphology and high carbon content. (b) Moderate electron beam exposure showing crater formation and partial carbon loss, indicative of electroporation-type damage. (c) Severe electron beam exposure showing extensive erosion and carbon depletion, consistent with electron impact–driven ablation. Yellow boxes indicate regions selected for EDS analysis.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9234245/v1/561b56de613e36c8ae35b941.png"},{"id":106986301,"identity":"43b4748e-5d8b-48ea-84d5-73638d204cb9","added_by":"auto","created_at":"2026-04-15 13:06:23","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":141038,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of EBI’s proposed dual‑action antimicrobial mechanism. Figure 5.A. electron bombardment causing particle ablation. Figure 5.B: electroporation generating nanoscale membrane pores. Figure 5.C. combined effect showing synergistic membrane rupture and collapse.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9234245/v1/a877e8eb1580af15688ed607.jpeg"},{"id":107480290,"identity":"8d9057b7-7214-4450-90a7-02c59e60fccb","added_by":"auto","created_at":"2026-04-22 02:07:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3343640,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9234245/v1/4a9c994e-f335-4e1c-9081-47ab101dea6e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Electron-Beam Irradiation for inactivation of airborne Multidrug-Resistant Bacteria: Single-Pass Efficacy and morphological evidence of destructive mechanisms","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHealthcare-associated infections (HAIs) remain a critical public health issue, representing one of the most significant contributors to morbidity and mortality in modern healthcare [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Airborne transmission of multidrug-resistant (MDR) bacteria amplifies this challenge by undermining traditional infection control measures within healthcare environments [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These pathogens account for a significant proportion of nosocomial infections, leading to prolonged hospital stays, elevated treatment costs, and increased mortality rates, particularly among immune-compromised patients [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Common MDR pathogens such as \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eAcinetobacter\u003c/em\u003e species, \u003cem\u003eKlebsiella\u003c/em\u003e species, and methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) can persist in hospital air and on environmental surfaces, spreading via aerosolization and contact with inadequately disinfected areas [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In enclosed healthcare settings with poor ventilation, these microorganisms can form stable airborne reservoirs, substantially heightening the risk of hospital-acquired infections and localized outbreaks [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Conventional air purification technologies, such as High-Efficiency Particulate Air (HEPA) filtration, capture airborne particles as small as 0.3 \u0026micro;m with 99.97% efficiency; however, their effectiveness is limited to mechanical trapping rather than microbial inactivation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Pathogens retained on filter surfaces can remain viable, creating a potential source for re-aerosolization or cross-contamination during filter maintenance and replacement [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, HEPA systems require high installation and operational costs, frequent maintenance, and exhibit reduced performance in humid environments [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. They also show limited efficiency against smaller viral particles and lack real-time pathogen neutralization capability [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These drawbacks underscore the need for innovative air disinfection technologies capable of both capturing and actively inactivating microorganisms \u003cem\u003ein situ\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To address these limitations, several advanced air purification strategies have been explored, including ultraviolet germicidal irradiation (UVGI), electrostatic precipitation, photocatalytic oxidation, and non-thermal plasma systems [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Among these emerging technologies, electrically driven systems such as non-thermal plasma and electron-based disinfection methods offer distinct advantages by producing physical and electrical effects capable of directly disrupting microbial structures [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe EBI system evaluated in this study generates high-energy electrons that can directly damage microbial cell envelopes through electron impact and can also induce electroporation via intense localized electric fields, leading to irreversible membrane destabilization and cell death [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The single-pass air disinfection efficacy of the EBI system was tested using a controlled aerosolization laboratory model, to quantify the single-pass inactivation efficiency and log₁₀ reductions of aerosolized MDR bacteria of multidrug-resistant (MDR) bacteria, including \u003cem\u003eP. aeruginosa, Acinetobacter\u003c/em\u003e species, \u003cem\u003eKlebsiella\u003c/em\u003e species, and methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA), isolated from clinical samples obtained at Hazrat Rasoul Hospital, Tehran, Iran. The findings from this study will inform the future application and use of electron-beam irradiation as an air-disinfection technology for enhancing indoor microbial air quality and strengthening infection-control strategies in healthcare and other critical indoor environments, addressing the urgent need for alternative and novel disinfection approaches to address the escalating threat of antimicrobial resistance.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bacterial isolates and Antibiogram Profiles\u003c/h2\u003e \u003cp\u003eClinical isolates of \u003cem\u003eAcinetobacter\u003c/em\u003e sp., \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eKlebsiella\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e were obtained from Hazrat-e Rasoul Hospital in Tehran, Iran. These isolates were selected based on their demonstrated resistance to multiple antibiotic classes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Antibiogram testing was performed using the Kirby-Bauer disk diffusion method in accordance with CLSI M100 guidelines (2023). All experiments and methods were performed in accordance with relevant guidelines and regulations. The study protocols and the use of clinical isolates were reviewed and approved by the Ethics Committee of Iran University of Medical Sciences (Approval ID: IR.IUMS.REC.1403.958). Informed consent was obtained from all subjects and/or their legal guardian(s). All isolates were completely anonymized to protect patient privacy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibiogram profiles of multidrug-resistant (MDR) clinical isolates used in the study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicroorganism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSensitive (S)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResistant (R)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcinetobacter\u003c/b\u003e \u003cb\u003esp.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmpicillin \u0026ndash; Sulbactam (10/10 \u0026micro;g(, Ciprofloxacin (5 \u0026micro;g(, Cefepime (30 \u0026micro;g(, Ceftriaxone (30 \u0026micro;g (, Amikacin (30 \u0026micro;g(, Gentamicin (10 \u0026micro;g (, Trimethoprim \u0026ndash;Sulfamethoxazole (1.25/23.75\u0026micro;g), Piperacillin \u0026ndash; Tazobactam (100/10 \u0026micro;g(, Doxycycline (30 \u0026micro;g(,\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStaphylococcus aureus\u003c/b\u003e \u003cb\u003e(MRSA)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLinezolid (30 \u0026micro;g(, Doxycycline (30 \u0026micro;g(, Trimethoprim \u0026ndash;Sulfamethoxazole (1.25/23.75\u0026micro;g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCiprofloxacin (5 \u0026micro;g(, Clindamycin (2 \u0026micro;g(, Erythromycin (15 \u0026micro;g(, Cefoxitin (30\u0026micro;g(, Penicillin G (10 U(, Vancomycin (30 \u0026micro;g(,\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKlebsiella\u003c/b\u003e \u003cb\u003esp.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDoxycycline (30 \u0026micro;g(, Gentamicin (10 \u0026micro;g(, Amikacin (30 \u0026micro;g(, Ciprofloxacin (5 \u0026micro;g(, Imipenem (10 \u0026micro;g(, Meropenem (10 \u0026micro;g(,\u003c/p\u003e \u003cp\u003e,Piperacillin \u0026ndash; Tazobactam(100/10 \u0026micro;g(, Ampicillin\u0026ndash;Sulbactam (10/10 \u0026micro;g(,\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePseudomonas aeruginosa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGentamicin (10 \u0026micro;g(, Amikacin (30 \u0026micro;g (\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePiperacillin \u0026ndash; Tazobactam (100/10 \u0026micro;g(, Cefazolin (30 \u0026micro;g(, Cefepime (30 \u0026micro;g(, Ciprofloxacin (5 \u0026micro;g(, Imipenem (10 \u0026micro;g(, Meropenem (10 \u0026micro;g),\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Bacterial Culture Preparation and Inoculum Standardization\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAcinetobacter\u003c/em\u003e sp., \u003cem\u003eS. aureus, Klebsiella\u003c/em\u003e sp., and \u003cem\u003eP. aeruginosa\u003c/em\u003e cultures were grown on nutrient agar at 37\u0026deg;C for 24 h, then transferred to Mueller\u0026ndash;Hinton (MH) broth and incubated in a shaker incubator at 37\u0026deg;C for 2 h to obtain a viable, standardized inoculum. Bacterial concentrations were adjusted to approximately 10⁹CFU/mL based on optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) using a spectrophotometer. The cultures were then centrifuged and resuspended in 0.9% physiological saline (Merck, Germany). Cell density was further standardized using McFarland standards, and serial dilutions were prepared. To confirm the final cell concentration, serial dilutions were spread-plated on nutrient agar and colony-forming units (CFU) were enumerated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experimental Setup\u003c/h2\u003e \u003cp\u003eThe device used in this study is an air-cleaning system with multiple layers of electron beam emitters within a stainless-steel reactor (Model MD250, PlasmaShield Ltd, Australia). The device was incorporated into the experimental setup shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Microorganisms were aerosolized using a 3-jet Collison nebulizer (CH Technologies, Westwood, NJ, USA) at an inoculum suspension concentration of 10⁹ CFU/mL in physiological serum. The nebulizer was positioned at the start of a detachable stainless-steel pipeline (25 cm diameter, 70 cm length) before entering the device. Airflow within the duct was maintained at 1 m/s, and nebulization was achieved using compressed air at a flow rate of 14 L/min.\u003c/p\u003e \u003cp\u003eMicroorganisms downstream of the EBI unit were collected 120 cm beyond the reactor outlet using three glass impingers (Unilab, 500 mL, borosilicate glass 3.3). The sampling points located inside the stainless-steel pipe, oriented perpendicular to the airflow, and spaced 120\u0026deg; apart were each connected to an individual impinger via silicone tubing. Each impinger operated at 26.67 L/min for 30 minutes per run using a dry vacuum pump with 0.9% saline as the collection medium. Among the three sampling points, the impinger with the highest collection efficiency was selected for subsequent serial dilutions, while the remaining two were monitored to confirm uniformity of aerosol distribution in the airstream.\u003c/p\u003e \u003cp\u003eAll experiments were performed under controlled laboratory conditions with an average temperature of 28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u0026deg;C measured continuously at the chamber inlet. Collected samples were serially diluted in physiological saline, spread-plated onto nutrient agar in triplicate, and incubated at 37\u0026deg;C for 24 h prior to CFU enumeration. Each testing condition was conducted in triplicate. Conditions included the EBI turned on and also a negative control where the device was replaced with an empty casing. This was repeated for each microorganism concentration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS (version 27). Colony-forming unit (CFU) data were log₁₀-transformed prior to analysis to account for the log-normal distribution typically observed in microbial count data. Results are reported as mean log₁₀ (CFU) values with corresponding standard deviations (SD) for both control and EBI-treated conditions. Log₁₀ reductions were calculated as the difference between mean log₁₀ (CFU) values of control and treated samples. Standard deviations for log₁₀ reductions were calculated using error propagation to reflect the combined variability of both control and treated measurements. Disinfection efficacy (%) was calculated based on the corresponding CFU values derived from the log₁₀-transformed data. Samples with no detectable colony growth were assigned a value of 1 CFU prior to log₁₀ transformation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Scanning Electron Microscopy (SEM)\u003c/h2\u003e \u003cp\u003eSEM analysis was performed to visualize the morphological alterations of multi-drug resistant (MDR) bacteria following exposure to electron beam irradiation. The experiments were conducted using the setup described in Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Microorganisms captured in the liquid impinger were carefully deposited onto pre-cleaned glass slides and immediately fixed in a 2.5% glutaraldehyde solution prepared in physiological saline at 25\u0026deg;C for 24 hours to preserve cellular structures. After fixation, the glutaraldehyde solution was discarded, and the samples were sequentially dehydrated in graded ethanol solutions (70%, 90%, and 100%), each for 15 minutes, to ensure complete removal of water. The dehydrated slides were subsequently mounted on aluminum SEM stubs using conductive carbon adhesive tape and sputter-coated with a 5 nm gold layer using an NSC DSR1 sputter coater to enhance surface conductivity and imaging quality. Imaging was performed using a Mira 3 (Tescan, Czech Republic) scanning electron microscope operated at an accelerating voltage of 15 kV under high vacuum conditions. Elemental characterization of the same samples was carried out by energy dispersive X-ray spectroscopy (EDS) using Aztec software (Oxford Instruments, UK) to evaluate surface elemental composition and detect chemical alterations associated with bacterial planktonic structures after EBI treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe single-pass disinfection performance of the EBI system against aerosolized multidrug-resistant (MDR) bacteria is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Across all tested microorganisms, the EBI system demonstrated consistent reductions in airborne bacterial load under controlled laboratory conditions (n\u0026thinsp;=\u0026thinsp;3). The highest inactivation performance was observed for \u003cem\u003eS. aureus\u003c/em\u003e (MRSA), with a mean log₁₀ reduction of 5.37, corresponding to a 99.999% disinfection efficacy. The EBI system achieved a mean log₁₀ reduction of 4.01 for \u003cem\u003eP. aeruginosa\u003c/em\u003e, corresponding to a 99.99% disinfection efficacy, while \u003cem\u003eAcinetobacter\u003c/em\u003e sp. and \u003cem\u003eKlebsiella\u003c/em\u003e sp. exhibited mean log₁₀ reductions of 3.64, corresponding to 99.98% disinfection efficacy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantitative single-pass disinfection performance of the electron-beam irradiation (EBI) system against aerosolized multidrug-resistant (MDR) bacteria. Results are reported as mean log₁₀ (CFU)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for control (device off) and EBI-treated (device on) conditions obtained from triplicate experiments (n\u0026thinsp;=\u0026thinsp;3 per condition). Log₁₀ reductions were calculated as the difference between mean control and treated log₁₀ (CFU) values. Disinfection efficacy (%) was calculated from CFU values derived from log₁₀-transformed data. Samples with no detectable colony growth were assigned a value of 1 CFU prior to log₁₀ transformation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDR pathogen tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl log₁₀(CFU)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEBI log₁₀(CFU)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDisinfection efficacy (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean Log₁₀ reduction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAcinetobacter\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo complement these quantitative findings, scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were performed to examine electron beam induced morphological and compositional alterations in representative bacterial species, as described in the following sections.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents scanning electron micrographs (SEM) of untreated (control) and EBI-treated multidrug-resistant (MDR) bacterial species, including \u003cem\u003eP. aeruginosa\u003c/em\u003e (A\u0026ndash;B), \u003cem\u003eS. aureus\u003c/em\u003e (MRSA). (C\u0026ndash;D), \u003cem\u003eKlebsiella\u003c/em\u003e sp. (E\u0026ndash;F), and \u003cem\u003eAcinetobacter\u003c/em\u003e sp. (G\u0026ndash;H). The control panels (A, C, E, G) show the characteristic morphology of viable cells: \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e sp. display smooth rod-shaped structures with intact outer membranes; \u003cem\u003eS. aureus\u003c/em\u003e appears as spherical cocci with well-defined, continuous surfaces; and \u003cem\u003eAcinetobacter\u003c/em\u003e sp. exhibits compact, oval-shaped cells with uniform texture, features typical of healthy, actively growing bacteria. In contrast, the EBI-treated panels (B, D, F, H) reveal extensive structural collapse and severe morphological distortion across all species. The treated \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eKlebsiella Spp.\u003c/em\u003e cells exhibit extensive surface rupture and elongation collapse, indicating envelope disintegration and cytoplasmic leakage. \u003cem\u003eS. aureus\u003c/em\u003e cells lose their spherical integrity and transform into fragmented, porous remnants, while \u003cem\u003eAcinetobacter\u003c/em\u003e sp. appears as completely disintegrated aggregates with loss of cellular definition. These profound deformations confirm that electron beam exposure leads to irreversible physical damage of bacterial envelopes [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe observed destruction pattern is consistent with electron induced damage, a primary mechanism in the EBI\u0026rsquo;s technology. The electrons generated by the electron-beam emitters collide directly with microbial cell walls, causing localized bond scission, surface sputtering, and nano-scale ablation of membrane biomolecules. This exposure disrupts lipid bilayers, peptidoglycan structures, and protein matrices, resulting in envelope perforation, membrane collapse, and eventual cellular implosion. The electron impact is further intensified by transient electroporation which accelerates structural breakdown. Together, the SEM evidence in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e demonstrates that the EBI system exerts a direct electron-mediated physical destruction effect on both Gram-positive and Gram-negative MDR bacteria. This supports the quantitative results presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and confirms that the predominant inactivation mechanism is physical ablation through electron impact rather than other mechanisms [\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing the extensive evidence of electron impact\u0026ndash;driven physical ablation presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, further analysis was conducted to explore additional EBI-induced damage pathways contributing to microbial inactivation. While collision with high-speed electrons primarily causes surface erosion and molecular ablation, the intense electric fields generated within the EBI\u0026rsquo;s discharge zone are also capable of inducing electroporation, a complementary mechanism that destabilizes bacterial membranes at the nanostructural level [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. To visualize this process, the morphological effects of electron beam exposure on \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) were examined by SEM, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents scanning electron micrographs (SEMs) depicting the morphological alterations of \u003cem\u003eP. aeruginosa\u003c/em\u003e (A\u0026ndash;B) and \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) (C\u0026ndash;D) following exposure to high-speed electrons. The control panels (A, C) show typical intact morphology: \u003cem\u003eP. aeruginosa\u003c/em\u003e cells exhibit smooth, rod-shaped surfaces with clearly defined envelopes, while MRSA appears as regular spherical cocci with continuous cell walls and well-preserved contours. In contrast, the treated cells (B, D) display distinct electroporation-induced structural damage. \u003cem\u003eP. aeruginosa\u003c/em\u003e demonstrates localized membrane perforations, shallow depressions, and early signs of cell wall rupture, suggesting the formation of pores caused by the intense electric field generated within the electron discharge zone. Similarly, MRSA cells reveal partial collapse and the appearance of nanoscale pits and ruptures on the surface morphological evidence of electrical membrane destabilization and pore coalescence [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese deformations are characteristic of irreversible electroporation, a key antimicrobial mechanism of the EBI system. The device generates an intense electric field that induces transmembrane potential differences exceeding the dielectric threshold of bacterial membranes (typically\u0026thinsp;\u0026gt;\u0026thinsp;1 V), resulting in the formation of transient nanopores. Under sustained exposure, these pores expand irreversibly, causing leakage of intracellular components, disruption of osmotic balance, and eventual cell death. The electroporation effect is further amplified by simultaneous high-energy electron collisions within the device reactor, which together accelerate the breakdown of the phospholipid bilayer and peptidoglycan structures [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCollectively, the SEM findings in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e confirm that electroporation-driven membrane perforation is a primary mechanism of inactivation in both Gram-negative (\u003cem\u003ePseudomona\u003c/em\u003es) and Gram-positive (\u003cem\u003eS. aureus\u003c/em\u003e) bacteria. This supports the earlier electron-impact mechanism and highlights the dual-action behavior of the EBI technology, where electrical and physical destructive processes work together to produce rapid, irreversible microbial inactivation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further support the morphological observations and elucidate the physicochemical processes underlying bacterial destruction, scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were conducted on multidrug-resistant (MDR) \u003cem\u003eAcinetobacter\u003c/em\u003e sp. cells exposed to EBI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the untreated control cells exhibits a smooth, intact morphology with an elemental composition of 60.57 wt% C, 13.39 wt% O, 9.52 wt% Si, 0.59 wt% Mg, and 0.41 wt% Al, indicative of a carbon-rich, protein\u0026ndash;lipid envelope with negligible substrate exposure [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter electron beam exposure, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb shows localized membrane deformation and the formation of a central crater. Corresponding EDS spectra display 53.27 wt% C (\u0026minus;\u0026thinsp;7.30 wt%), 22.60 wt% O (+\u0026thinsp;9.21 wt%), 11.62 wt% Si (+\u0026thinsp;2.10 wt%), 0.81 wt% Mg, and 0.46 wt% Al, suggesting partial oxidation, focal organic loss, and incipient exposure of the underlying glass substrate. These features are consistent with electroporation-driven membrane disruption, where transient electric fields induce localized pore formation and oxidative stress [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec reveals severe surface erosion and fragmentation. EDS analysis shows 23.06 wt% C (\u0026minus;\u0026thinsp;37.51 wt% vs. control), 34.49 wt% O (+\u0026thinsp;21.10 wt%), 21.81 wt% Si (+\u0026thinsp;12.29 wt%), 1.58 wt% Mg, and 0.78 wt% Al, signifying extensive ablation of organic components and exposure of the inorganic substrate. This quantitative shift\u0026mdash;from ~\u0026thinsp;12% relative carbon loss during electroporation to ~\u0026thinsp;62% during ablation highlights a dual-mechanism pathway for bacterial inactivation: (1) pulsed electric field\u0026ndash;induced membrane perforation, initiating early oxidative disruption, followed by (2) reactive species and electron bombardment driven etching, culminating in complete structural collapse [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e][\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe EBI air-cleaning system demonstrated high efficacy in inactivating aerosolized multidrug-resistant (MDR) bacteria, including \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eKlebsiella\u003c/em\u003e sp., and \u003cem\u003eAcinetobacter\u003c/em\u003e sp., under controlled laboratory conditions. The system achieved robust single-pass disinfection performance, with average removal efficiencies ranging from 99.98% to 99.999%. The highest inactivation was observed for MRSA and \u003cem\u003eP. aeruginosa\u003c/em\u003e, with mean log₁₀ reductions of 5.37 and 4.01, respectively, while \u003cem\u003eKlebsiella\u003c/em\u003e sp. and \u003cem\u003eAcinetobacter\u003c/em\u003e sp. exhibited mean log₁₀ reductions of 3.64.\u003c/p\u003e \u003cp\u003eSEM and EDS analyses provided direct morphological and compositional evidence of the inactivation mechanism, revealing severe structural deformation, membrane rupture, and elemental redistribution characterized by carbon depletion and oxygen enrichment. These findings are further clarified by the schematic in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, which illustrates the proposed dual-action antimicrobial mechanism of the EBI system: high-energy electron impact causes direct ablation of the bacterial envelope, whereas electroporation creates nanoscale membrane pores that destabilize cell structure. In combination, these mechanisms operate synergistically, accelerating cytoplasmic leakage, structural collapse, and eventual cell death. When integrated with the quantitative reductions observed in aerosolized MDR bacteria, the morphological and elemental evidence underscores that EBI inactivates microbes predominantly through physical ablation and electrical destabilization, not chemical oxidation. This dual-action mechanism effectively accounts for the rapid and consistent log\u003csub\u003e10\u003c/sub\u003e reductions demonstrated across Gram-positive and Gram-negative organisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis study has certain limitations that should be acknowledged. The bacterial reduction levels observed may have been negatively influenced by the high humidity produced by the nebulizer during aerosolization. Elevated moisture can temporarily reduce electrostatic activity and charge-transfer interactions within both the electron discharge field and filter matrix, which may have decreased the disinfection efficacy. This humidity effect reflects the experimental setup and is not necessarily representative of typical indoor environments, where lower relative humidity is more common [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In addition, the maximum log\u003csub\u003e10\u003c/sub\u003e reduction achieved may have been limited by the maximum concentration tested in the inoculum suspension. Previous research has suggested that EBI efficiency may increase with higher microbial loads; however, that was not feasible in this experiment with the MDR strains [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Future experiments under stricter containment conditions are needed to explore the maximum disinfection efficacy achieved when challenged with higher concentrations of MDR microorganisms.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, the electron-beam irradiation (EBI) system represents a promising technology for mitigating airborne infections and indoor air decontamination. Its chemical-free operation enables continuous inactivation of airborne microorganisms and offers a practical strategy for reducing exposure to multidrug-resistant bacteria aerosols. Therefore, in healthcare, laboratory, and other high-risk environments, EBI-based air treatment has the potential to contribute meaningfully to the prevention of hospital-acquired infections and ultimately save lives, especially among vulnerable patient populations.\u003c/p\u003e \u003cp\u003eFuture research should also investigate the molecular and genetic consequences of EBI exposure. Studies on bacterial gene expression will clarify how electron‑beam irradiation influences biofilm formation, antibiotic resistance behavior, susceptibility to other disinfection methods, and overall pathogenicity. In parallel, analyses of intracellular components including DNA integrity and chromosomal organization are needed to determine how electron beam‑induced damage mechanisms affect genetic material and internal structures. These investigations will extend evaluation from surface‑level morphological disruption to fundamental cellular and molecular pathways, providing deeper insight into the long‑term biological impact of EBI treatment. In addition, further evaluation under a range of environmental conditions, together with expanded testing against viral and fungal aerosols, will help define the full antimicrobial spectrum of EBI and enhance its suitability for real-world infection-control applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate:\u003c/h2\u003e\n\u003cp\u003eAll experiments and methods were carried out in accordance with relevant guidelines and regulations. The experimental protocols were approved by the Ethics Committee of Iran University of Medical Sciences (Approval ID: IR.IUMS.REC.1403.958). Informed consent was obtained from all subjects and/or their legal guardian(s).\u003c/p\u003e\n\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\n\u003cp\u003eBoth funders had no role in the conduct of the experimental work including selection of test organisms, data collection or analysis, interpretation of the results, decision to publish, or preparation of the manuscript. The authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was funded by the Antimicrobial Resistance Research Center of the Institute of Immunology and Infectious Diseases at Hazrat Rasoul Hospital, Iran University of Medical Sciences (Grant number REC.1403.958). Plasma Shield Ltd (Australia) provided additional support limited to publication-related costs and providing funding to researchers at Flinders University, Australia to provide an external and independent review of the study design.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eA.S. and S.M. conceptualized the study and designed the methodology. A.S., K.M.Y., and S.S. conducted the investigations. Data curation and formal analysis were performed by K.M.Y., S.S., and S.M. K.M.Y. contributed to software application and data validation, while A.S. and S.S. prepared the visual representations of the data. S.M. provided resources, supervised the research, and managed project administration. A.S. was responsible for funding acquisition. The original draft of the manuscript was written by A.S. and K.M.Y., with S.M. providing critical review and editing. All authors reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors would like to acknowledge the facilities, as well as the scientific and technical assistance, provided by the Antimicrobial Research Center under the Institute of Immunology and Infectious Diseases at Hazrat-e Rasoul Hospital, Iran University of Medical Sciences. We also appreciate Rahavard Fanavari Company for all helps and its technical support during the project. Authors also acknowledge Professor Kirstin Ross and Professor Harriet Whiley from Flinders University and their role in providing guidance on experimental design and constructive feedback during manuscript editing.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCDC Winnable Battles Final Report: Healthcare-associated infections (HAIs)., Centers Dis. Control Prev. 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Non-thermal plasma-based inactivation of bacteria in water using a microfluidic reactor. \u003cem\u003eWater Res.\u003c/em\u003e \u003cb\u003e201\u003c/b\u003e, 117321 (2021).\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Electron-beam irradiation, EBI, Airborne transmission, Aerosolized bacteria, Electron-induced membrane disruption, Electroporation, Multidrug-resistant bacteria, MDR, Bioaerosol inactivation, Bacteria elemental analysis, Scanning electron microscopy, SEM","lastPublishedDoi":"10.21203/rs.3.rs-9234245/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9234245/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAirborne and aerosol transmission of multidrug-resistant (MDR) bacteria pose a major challenge in healthcare environments. This study evaluated the performance of an electron-beam irradiation (EBI) system for inactivating airborne MDR bacteria under controlled laboratory conditions. To perform this assessment, clinical isolates of MDR \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eAcinetobacter\u003c/em\u003e sp., \u003cem\u003eKlebsiella\u003c/em\u003e sp., and methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) were aerosolized to simulate realistic airborne transmission and subsequently introduced into the EBI system. Downstream bacterial aerosols were collected using liquid impingers and quantified by culture-based enumeration. CFU data were log₁₀-transformed prior to analysis. The EBI system reduced airborne bacterial loads by 4.01 log₁₀ for \u003cem\u003eP. aeruginosa\u003c/em\u003e, 3.64 log₁₀ for \u003cem\u003eAcinetobacter\u003c/em\u003e sp., 3.64 log₁₀ for \u003cem\u003eKlebsiella\u003c/em\u003e sp., and 5.37 log₁₀ for MRSA, corresponding to disinfection efficiencies of 99.98\u0026ndash;99.999%. Scanning electron microscopy (SEM) and elemental mechanistic study revealed morphological damage, including membrane rupture, surface collapse, and loss of structural integrity, indicating that electron impact and electroporation are the predominant mechanisms of inactivation. These results demonstrate that EBI is a rapid and effective method for mitigating airborne MDR bacterial threats, with potential for deployment in healthcare, laboratory, and other high-risk indoor environments.\u003c/p\u003e","manuscriptTitle":"Electron-Beam Irradiation for inactivation of airborne Multidrug-Resistant Bacteria: Single-Pass Efficacy and morphological evidence of destructive mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-15 13:06:19","doi":"10.21203/rs.3.rs-9234245/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-16T20:54:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133177511039580115945400886718737909661","date":"2026-05-07T00:13:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T06:27:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4972205634439698011286399200413058492","date":"2026-04-23T06:08:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-08T06:27:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T06:55:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-02T17:18:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-31T08:16:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-31T06:34:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53b7e1d7-87bb-4dcf-87ed-6bc5bbe5c99f","owner":[],"postedDate":"April 15th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-16T20:54:37+00:00","index":63,"fulltext":""},{"type":"reviewerAgreed","content":"133177511039580115945400886718737909661","date":"2026-05-07T00:13:46+00:00","index":62,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T06:27:23+00:00","index":60,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66217750,"name":"Health sciences/Diseases"},{"id":66217751,"name":"Health sciences/Medical research"},{"id":66217752,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-15T13:06:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-15 13:06:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9234245","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9234245","identity":"rs-9234245","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}