Phage Inactivation Research Articles

Field Study and Evaluation of KrCl* Far UV-C Device Capability for Inactivation of Phi6 Bacteriophage.

"Far UV-C" is an effective disinfection method that can be deployed in occupied areas. Commercially available Krypton Chloride (KrCl*) excimer lamps filtered to emit at 222 nm are effective in disinfecting pathogens and safe for human exposure up to an allowable threshold exposure, which is much longer than for conventional UV lamps emitting at 254 nm. Laboratory and controlled field testing of a filtered KrCl* excimer lamp for disinfection of a virus suspended in a thin film aqueous solution in an occupied office setting was conducted. Complete inactivation of almost 6 log (99.9999%) of Phi6 bacteriophage virus was achieved in ~20 min of exposure time in a field setting, equivalent to a dose of about 10 mJ cm-2 . The Phi6 inactivation rate constant for the field test results were not statistically different from laboratory values (P > 0.05, paired t-test). When positioned at 1 m distance from possible human exposure, this device can be used safely for almost 4.5 h of continuous direct exposure without any acute or long-term adverse health effects. This study illustrates the applicability and deployment of Far UV-C for pathogen reduction and can help in decision making for implementation of Far UV-C for disinfection in human-occupied environments.

Chlorine Inactivation of PhiX174 Bacteriophage in Treated Sewage and Drinking Water Samples

Chlorine Inactivation of PhiX174 Bacteriophage in Treated Sewage and Drinking Water Samples

Shelf-life prediction and storage stability of Aeromonas bacteriophage vB_AsM_ZHF

Phage therapy is a potential alternative to antibiotics for the treatment of bacterial infections. Due to the good antibacterial and therapeutic effects of phages, phage therapy has received attention and the demand for clinical applications has gradually increased. Phage storage stability and shelf life are key aspects of biopharmaceutical development and registration. In this study, Aeromonas salmonicida phage was stored at different temperatures for 12 months. We found that 4 °C was the optimal storage temperature. In the case of cryopreservation, 10% dimethyl sulfoxide (DMSO) was more effective at protecting the phage at -20 °C and -80 °C than 30% glycerin. Indeed, the phage titer decreased by only one order of magnitude within one year when DMSO was added. Hydroxyapatite (HAP) reduced the inactivation of phages by six orders of magnitude during storage at 28ºC for 1 year, significantly lower than that of in SM buffer. In addition, for excipients in lyophilization, tryptic soy broth (TSB) and tryptone or skim milk powder (SMP) in combination with trehalose alleviated phage inactivation during lyophilization and subsequent storage at 28 °C. Furthermore, a model for predicting the phage shelf-life was established with the Accelerated Stability Assessment Program (ASAP) based on the Arrhenius equation. The error of the model was less than 15% by comparing the predicted value with the actual value at 28 °C, indicating high accuracy. The study demonstrated the storage stability and shelf-life model of phage for the first time, which provided a theoretical basis for the development and application of phage products.

Anti-Viral Photodynamic Inactivation of T4-like Bacteriophage as a Mammalian Virus Model in Blood.

The laboratorial available methods applied in plasma disinfection can induce damage in other blood components. Antimicrobial photodynamic therapy (aPDT) represents a promising approach and is approved for plasma and platelet disinfection using non-porphyrinic photosensitizers (PSs), such as methylene blue (MB). In this study, the photodynamic action of three cationic porphyrins (Tri-Py(+)-Me, Tetra-Py(+)-Me and Tetra-S-Py(+)-Me) towards viruses was evaluated under white light irradiation at an irradiance of 25 and 150 mW·cm−2, and the results were compared with the efficacy of the approved MB. None of the PSs caused hemolysis at the isotonic conditions, using a T4-like phage as a model of mammalian viruses. All porphyrins were more effective than MB in the photoinactivation of the T4-like phage in plasma. Moreover, the most efficient PS promoted a moderate inactivation rate of the T4-like phage in whole blood. Nevertheless, these porphyrins, such as MB, can be considered promising and safe PSs to photoinactivate viruses in blood plasma.

Container material dictates stability of bacteriophage suspensions: Light scattering and infectivity measurements reveal mechanisms of infectious titre decay.

To measure the infectious titre (IT) decay rate for various bacteriophages as a function of storage container material. Additionally, parallel light scattering and infectious titre measurements reveal distinct mechanisms for IT loss, depending on phage. Suspensions of bacteriophages 44AHJD, P68 and gh-1 were stored in various labware. IT of each suspension was repeatedly measured over the course of 2 weeks. Large variability in IT decay was observed, with >4 log10 loss in glass and low-binding polypropylene. Incubation of polymer containers with Bovine Serum Albumin (BSA) resulted in a consistent reduction in IT decay. Aggregation state of phage suspensions was studied by nanoparticle tracking analysis (NTA), revealing highest aggregation in glass-stored suspensions and lowest after storage in BSA-treated containers. Glass and 'low-binding' containers may aggravate IT decay while BSA treatment may present an easy mitigation strategy. IT versus NTA titre diagrams highlight the importance of phage inactivation in combination with aggregation. Container material is a significant determinant of bacteriophage IT decay. It is therefore essential to confirm IT following storage and tailor choice of phage storage containers accordingly. Aggregation of phages and adsorption onto labware surfaces are not only the mechanisms accounting for IT loss but also biological instability.

Experimental evidence for the impact of phages on mineralization of soil-derived dissolved organic matter under different temperature regimes

Microbial mineralization of dissolved organic matter (DOM) plays an important role in regulating C and nutrient cycling. Viruses are the most abundant biological agents on Earth, but their effect on the density and activity of soil microorganisms and, consequently, on mineralization of DOM under different temperatures remains poorly understood. To assess the impact of viruses on DOM mineralization, we added soil phage concentrate (active vs. inactive phage control) to four DOM extracts containing inoculated microbial communities and incubated them at 18 °C and 23 °C for 32 days. Infection with active phages generally decreased DOM mineralization at day one and showed accelerated DOM mineralization later (especially from day 5 to 15) compared to that with the inactivated phages. Overall, phage infection increased the microbially driven CO2 release. Notably, while higher temperature increased the total CO2 release, the cumulative CO2 release induced by phage infection (difference between active phages and inactivated control) was not affected. However, higher temperatures advanced the response time of the phages but shortening its active period. Our findings suggest that bacterial predation by phages can significantly affect soil DOM mineralization. Therefore, higher temperatures may accelerate host-phage interactions and thus, the duration of C recycling.

Assessment of Surface Disinfection Effectiveness of Decontamination System COUNTERFOG® SDR-F05A+ Against Bacteriophage ɸ29

The experience of COVID19 pandemic has demonstrated the real concern of biological agents dispersed in the air and surfaces environments. Therefore, the need of a fast and large-scale disinfection method has arisen for prevention of contagion. COUNTERFOG® is an innovative technology developed for large-scale decontamination of air and surfaces. The objective of this study is to assess experimentally the effectiveness of COUNTERFOG® in disinfecting viral-contaminated surfaces. We also aim to measure the necessary time to disinfect said surfaces. Stainless steel surfaces were contaminated with bacteriophage φ29 and disinfected using COUNTERFOG® SDR-F05A+, which uses a sodium hypochlorite solution at different concentrations and for different exposure times. A log reduction over 6 logs of virus titer is obtained in 1 min with 1.2% sodium hypochlorite when the application is direct; while at a radial distance of 5 cm from the point of application the disinfection reaches a reduction of 5.5 logs in 8 min. In the same way, a higher dilution of the sodium hypochlorite concentration (0.7% NaOCl) requires more exposure time (16 min) to obtain the same log reduction (> 6 logs). COUNTERFOG® creates, in a short time and at a distance of 2 m from the point of application, a thin layer of disinfectant that covers the surfaces. The selection of the concentration and exposure time is critical for the efficacy of disinfection. These tests demonstrate that a concentration between 0.7- 1.2% sodium hypochlorite is enough for a fast and efficient ɸ29 phage inactivation. The fact that ɸ29 phage is more resistant to disinfection than SARS-CoV-2 sustains this disinfection procedure.

UV tolerance of Lactococcus lactis 936-type phages: Impact of wavelength, matrix, and pH

Ultraviolet C (UVC) radiation is a widely used technology for the disinfection of surfaces, air flows, water and other liquids. Although extensive research has been conducted on the UV tolerance of bacteriophages used as surrogates for waterborne viruses, limited information is available on phages relevant to food processing. Phages of dairy starters may reach high numbers in dairy facilities and cause fermentation failure with great economic losses for the dairy industry. Here, the UV tolerance of virulent phages, belonging to the 936-group (Skunavirus) of Lactococcus lactis subsp. diacetylactis F7/2, was assessed, employing both host infectivity loss and qPCR assays. A highly heat-tolerant phage (P680) and a less heat-tolerant phage (P008) were exposed to UV radiation at 265 nm (UVC), 285 nm (UVB) and 365 nm (UVA), respectively, in an aqueous suspension, using UV Light-Emitting-Diodes (LEDs) in a static set-up. UVC at 265 nm achieved the highest total inactivation, leading to a 4 log10 reduction of the phage titer at a UV dose of 327 and 164 mJ/cm2 for P680 and P008, respectively. UVB at 285 nm achieved similar inactivation levels, while UVA at 365 nm did not cause major reductions. Phages were also suspended in yoghurt serum of pH 5.5 and pH 7.0 and exposed to UVC radiation at 265 nm. The heat-tolerant phage P680 was more UV tolerant for all wavelengths, matrices and pH values tested. A higher aggregation degree together with less DNA damage was observed for both phages at pH 5.5, especially for phage P680, indicating a UV light-shielding effect. Interestingly, there were indications of some phage survivors exhibiting higher UV tolerance on re-exposure, pointing out a need for further investigation. Our results show that UV LEDs emitting at 265 nm and 285 nm are efficient in reducing the phage population significantly, but also underline that 936-type phages are relatively UV resistant. A further understanding of the main factors influencing UV efficiency could enable future use of the UV technology as an alternative or complement to thermal treatment for phage inactivation.

Hydrogen peroxide-assisted pasteurization: An alternative for household water disinfection

Decentralized treatments applied at the household level may be alternatives to positively impact global health. Pasteurization is a conventional disinfection method and efforts have been made to increase its efficiency and productivity in point-of-entry (POE) applications. To overcome limitations associated to this practice, we hypothesized that including oxidation prior to pasteurization would improve the treatment. This research aimed to evaluate the performance of hydrogen peroxide-assisted pasteurization and effects of H2O2 concentration and temperature organized by a full factorial experimental design. At optimal conditions, >9.3 log10 inactivation of Escherichia coli and >5.8 log10 Phi X174 bacteriophage were obtained. Observed values were modeled as empirical equations for E. coli and phage inactivation (R2 = 0.76 and 0.72, respectively). Temperature did not lead to significant differences in H2O2 residual, which is favorable for practical implementation. Synergistic effects were found for E. coli, and inactivation of Phi X174 tops results obtained by individual disinfection by pasteurization and H2O2 oxidation. Our study also suggests H2O2-assisted pasteurization increases oxidation potential, inferred by cell lysis and protein removal. Additionally, analyzing disinfection and H2O2 residuals during temperature ramp time endorsed that inactivation might happen at lower temperatures, and stability of H2O2 may provide a safer setup when heat sources cannot guarantee pasteurization to occur. Broadly, our research underscores potentials of H2O2-assisted pasteurization and, although we recommend further assessments considering pathogens, as well as case studies for specific scenarios, we believe H2O2 may improve performance and resilience of classic disinfection by pasteurization as a POE solution.

The Effect of Zero-Valent Iron Nanoparticles (nZVI) on Bacteriophages.

Bacteriophages are viruses that attack and usually kill bacteria. Their appearance in the industrial facilities using bacteria to produce active compounds (e.g., drugs, food, cosmetics, etc.) causes considerable financial losses. Instances of bacteriophage resistance towards disinfectants and decontamination procedures (such as thermal inactivation and photocatalysis) have been reported. There is a pressing need to explore new ways of phage inactivation that are environmentally neutral, inexpensive, and more efficient. Here, we study the effect of zero-valent iron nanoparticles (nZVI) on four different bacteriophages (T4, T7, MS2, M13). The reduction of plaque-forming units (PFU) per mL varies from greater than 7log to around 0.5log depending on bacteriophages (M13 and T7, respectively). A comparison of the importance of oxidation of nZVI versus the release of Fe2+/Fe3+ ions is shown. The mechanism of action is proposed in connection to redox reactions, adsorption of virions on nZVI, and the effect of released iron ions. The nZVI constitutes a critical addition to available antiphagents (i.e., anti-bacteriophage agents).

Use of a Hydrogen Peroxide Nebulizer for Viral Disinfection of Emergency Ambulance and Hospital Waiting Room.

Disinfection of hospital facilities and ambulances is an important issue for breaking the chain of transmission of viral pathogens. Hydrogen peroxide has provided promising results in laboratory assays. Here, we evaluate the efficacy of a hydrogen peroxide nebulizer for the inactivation of surrogate MS2 bacteriophage and murine norovirus (MNV) in a patient waiting room and the fully equipped cabin of a medical ambulance. We observed an average 3 log10 titer reduction in both settings, which represents the destruction of over 106 and 109 infectious particles of MNV and MS2 per cm2, respectively. The potential for viral exposure is high for health workers when disinfecting confined and cluttered spaces, so the use of a hydrogen peroxide mist might offer an affordable and efficient solution to minimize the risk of viral contaminations.

Inactivation of MS2 bacteriophage on copper film deployed in high touch areas of a public transport system.

Although SARS-CoV-2 is primarily an airborne risk, the COVID-19 pandemic also highlighted the need for self-disinfection surfaces that could withstand the demand of high occupant densities characteristic of public transportation systems. The aim of this study was to evaluate the durability and antiviral activity of a copper film deployed for 90 days in two high touch locations within an active metropolitan bus and railcar. The antiviral efficacy of this copper film after being deployed in transit vehicles for 90 days (deployed copper film) was then compared to new (unused) copper film to determine if frequent touches and cleaning protocols could decrease the efficacy of the copper films. Deployed copper film, new copper film, and aluminium foil (positive control) coupons were inoculated with ~1 × 106 MS2 virus particles, allowed a contact time of either 5- or 10-min, and analysed for residual viral infectiousness. On both new and deployed copper films, MS2 was completely inactivated (≥5 log reduction) at both time points. These results suggest that the copper film may provide the durability demanded by high touch public spaces while maintaining the antiviral activity necessary to reduce exposure risk and viral transmission via surfaces in public transportation settings.

Combined Effect of Phage phT4A and Pressure-Based Strategies in the Inhibition of Escherichia coli.

The major concern regarding the bacteriophage (or phage) therapy approach is the regrowth of bacteria after treatment, a consequence of the emergence of phage-resistant mutants. However, this limitation can be overcome by combining different therapies. In this study, the potential of combining phage phT4A with pressure storage (HS) to enhance the control of Escherichia coli and bacterial regrowth after treatment was evaluated. For that, the combining effect of phage phT4A and HS was studied and compared with storage at atmospheric pressure (AP) under refrigeration (4 °C, RF) and room temperature (RT). Initially, the effect of high hydrostatic pressure (200, 300 and 400 MPa) and HS (75 MPa), as well as refrigeration in phage phT4A viability, was determined. However, a considerable phage inactivation was verified at 200 MPa and so only HS at 75 MPa was further studied for combined treatment. The combined treatment with phage phT4A and HS was more efficient (reduction of 2.5 log CFU/mL after 7 days of storage) than phage phT4A (E. coli concentration was similar to that of the bacterial control after 7 days of storage) and HS (reduction of 1.8 log CFU/mL after 7 days of storage) applied individually. The combination of phage phT4A with refrigerated storage did not decrease E. coli levels. However, both the combination of phage with HS and the treatment with HS at 75 MPa effectively reduced E. coli concentration and prevented its regrowth. Phage phT4A viability was slightly affected during HS; however, the efficiency of the combined treatment phage-HS was not compromised. Further studies are needed to validate these findings in food products.

Phi 6 Bacteriophage Inactivation by Metal Salts, Metal Powders, and Metal Surfaces.

The interaction of phages with abiotic environmental surfaces is usually an understudied field of phage ecology. In this study, we investigated the virucidal potential of different metal salts, metal and ceramic powders doped with Ag and Cu ions, and newly fabricated ceramic and metal surfaces against Phi6 bacteriophage. The new materials were fabricated by spark plasma sintering (SPS) and/or selective laser melting (SLM) techniques and had different surface free energies and infiltration features. We show that inactivation of Phi6 in solutions with Ag and Cu ions can be as effective as inactivation by pH, temperature, or UV. Adding powder to Ag and Cu ion solutions decreased their virucidal effect. The newly fabricated ceramic and metal surfaces showed very good virucidal activity. In particular, 45%TiO2 + 5%Ag + 45%ZrO2 + 5%Cu, in addition to virus adhesion, showed virucidal and infiltration properties. The results indicate that more than 99.99% of viruses deposited on the new ceramic surface were inactivated or irreversibly attached to it.

On thermal inactivation of pathogens in aerosolized droplets through electromagnetic heating

A simple model describing the most significant impact of electromagnetic heating on pathogen-containing aerosols is presented. While the physics of ohmic heating are well understood, the connection between the unsteady temperature increase with net pathogen inactivation in an aerosolized electrolyte solution over a range of frequencies is not clear. The model is composed of two parts: a thermal model of electromagnetic heating of the droplets and a survival model describing the active pathogen population as a function of time. The droplets of saline solution, whose electrical conductivity depends on salt concentration and carrier frequency, are assumed to be small enough so that ambient air flows are sufficient to counter gravity, and the droplets are assumed to be equally spaced. As the droplets move with the ambient air, energy transport is limited to conduction, and within an adiabatic system, the mean spatial mode dominates the thermal transients of the air-droplet system. The kill rate of the pathogen depends on temperature, and the result of our thermal model informs the pathogen population through the Arrhenius kill rate. The model shows strong qualitative agreement with microwave inactivation of MS2 bacteriophages in aerosolized droplets. Inactivation is improved with larger electric field amplitudes, even for small duty cycles. We show that for a range of viruses with known activation energy, the thermal inactivation mechanism is more reliable for viruses with larger activation energy.

A model to predict the kinetics of direct (endogenous) virus inactivation by sunlight at different latitudes and seasons, based on the equivalent monochromatic wavelength approach

Sunlight plays an important role in the inactivation of pathogenic microorganisms such as bacteria and viruses in water. Here we present a model that is able to predict the kinetics of direct virus inactivation (i.e. inactivation triggered by sunlight absorption by the virion, without the role played by photochemically produced reactive intermediates generated by water-dissolved photosensitizers) on a global scale (from 60 °S to 60 °N latitude) and for the different months of the year. The model is based on the equivalent monochromatic wavelength (EMW) approach that was introduced recently, and which largely simplifies complex polychromatic calculations by approximating them with a monochromatic equation at the proper wavelength, the EMW. The EMW equation was initially established for mid-July conditions at a mid-latitude, and was then extended to different seasons and to the latitude belt where the day-night cycle is always observed throughout the year. By so doing, the first-order rate constant of direct virus photoinactivation can be predicted on a global scale, with the use of a relatively simple equation plus tables of pre-calculated input data, as a function of latitude, month, and key water parameters. The model was here applied to the virus organism phiX174, a somatic phage that is often used as proxy for pathogenic viruses undergoing fast direct inactivation, and for which a wide array of published inactivation data is available. Model predictions are validated by comparison with field data of inactivation of somatic phages by sunlight.

Characteristics and Mechanisms of Bacteriophage MS2 Inactivation in Water by UV Activated Sodium Persulfate

Viruses in the aquatic environment have strong resistance to common disinfection techniques. To contribute to the development of efficient virus inactivation technologies, the characteristics and mechanisms of virus inactivation in a UV activated sodium persulfate(UV/PS) system were studied. The inactivation rate and kinetic characteristics of bacteriophage MS2 in water samples by the UV/PS were studied. The effects of PS dosage, pH, and the initial concentration of bacteriophages on the inactivation effect were also investigated. Furthermore, the morphologies of phages before and after UV/PS treatment were observed by transmission scanning electron microscope, and the free radicals in the reaction system were identified by electron paramagnetic resonance spectroscopy. By means of a free radical quenching experiment, the contribution rate of various factors in the UV/PS system to phage inactivation was also analyzed and calculated. The results showed that when the UV irradiation intensity was 160 μW·cm-2, the phage MS2 of 4.39 lg could be removed after UV/PS treatment for 4 min, which was 1.44 lg higher than that of the same UV dose alone. The inactivation of phage MS2 by the UV/PS system was in accordance with the first-order kinetic model. Increasing the initial concentration of PS in the system significantly improved the inactivation rate of phages, while pH and the initial concentration of phages had little effect on the inactivation rate. UV/PS treatment damages the capsid of phages and promotes the aggregation of phage particles. The presence of SO4-· and·OH in the UV/PS system was an important factor for the inactivation of MS2 phages. Finally, ·OH contributed more to MS2 phage inactivation than SO4-·.

UVC inactivation of MS2-phage in drinking water – Modelling and field testing

UVC disinfection has been recognised by the WHO as an effective disinfection treatment to provide decentralized potable water. Under real conditions there are still unknowns that limit this application including the influence of suspended solids and natural organic matter. This work aims to investigate the influence of two key parameters, suspended solids and natural organic matter, on the efficiency of UVC disinfection of surface water to achieve the drinking water quality requirements established by the WHO for point of use (POU) technologies. Kaolinite (turbidity agent) and humic acids (HA, model of organic matter) were used in a factorial design of experiments (Turbidity from 0 to 5 NTU, and HA from 0 to 3.5 mg/L) to investigate their effect on UVC inactivation of MS2 phage in surface water. A collimated beam (12 W) and a commercial UVC disinfection flow system (16 W) designed to provide drinking water at households were used. The UVC flow system both in the laboratory and in the field was able to achieve the reduction requirements established by WHO (LRV >3.5 for all tested conditions), confirming the good performance of the studied UVC disinfection system. The results found in the lab were used to establish a numerical model that predicts the disinfection rate constant as a function of water turbidity and transmittance at 254 nm (confidence level>95%). The model permitted to elucidate the critical effect of low concentrations of HA in reducing the inactivation rate by 40% for 3.5 mg/L-HA compared with 0, the non-significant detrimental effect of turbidity lower than 5 NTU, and the lack of synergistic effects between both parameters at these levels. The UVC flow system was also tested in the field, in Tzabalho, Chiapas (Mexico), and Antioquia (Colombia), with spiked MS2 into natural surface water. This investigation opens a potential application to monitor the performance of UVC systems with surface water by monitoring transmittance at 254 nm as a tool to control UVC domestic systems to deliver safe drinking water in a household without the need of expensive and laborious biological monitoring tools.

Visible-light photocatalysis over MIL-53(Fe) for VOC removal and viral inactivation in air

MIL-53(Fe), synthesized by a one-step hydrothermal method, was investigated for the removal of toluene and inactivation of bacteriophage ⲪX 174 in air under visible light illumination. MIL-53(Fe) exhibited superior photochemical activity to other metal organic frameworks synthesized by the same method with different metal precursors. Analytical methods of diffuse reflectance spectroscopy, BET specific surface area analysis, SEM-EDS, FT-IR analysis, XRD, Mott-Schottky analysis, and XPS were used to characterize MIL-53(Fe). The illuminated MIL-53(Fe) removed input toluene (C0 = 3.59 g/m3) by 66% in 6 h by adsorption and subsequent photocatalytic oxidation. High humidity and temperature, and the anoxic condition inhibited the toluene removal. MIL-53(Fe) showed sustainable toluene removal for five consecutive runs, even though the photocatalytic activity slightly decreased. Meanwhile, the illuminated MIL-53(Fe) also resulted in the inactivation of ΦX 174 suspended in air, achieving approximately 3 log inactivation in 60 min (N0 = ~106 PFU/mL air). Similar to toluene removal, the presence of oxygen and low humidity were beneficial for viral inactivation. The photo-generated holes are believed to be responsible for the organic degradation and viral inactivation by the illuminated MIL-53(Fe).

Validation of a Low-Cost on-Demand Compact Ozone Covid-19 Sterilization Chamber

The COVID-19 pandemic has resulted in limited availability of N95 masks and other personal protective equipment (PPE). The sterilization and re-use of PPE can be of immediate benefit to help alleviate shortages and offer peace of mind to healthcare workers. Also, an inexpensive, precise, and portable sterilizer will have many applications beyond the current health crisis in a variety of field disaster relief operations. The sterilization of PPE and medical or other instruments will reduce costs, improve safety, and reduce waste. The portable sterilizer can also be utilized by individuals and businesses outside of the health care sector with similar benefits.Ozone sterilization does not require chemical consumables, uses ambient oxygen to generate ozone on site, and results in no toxic residuals. KWJ Engineering led the development of a small benchtop sterilizer with on board ozone generation controlled by feedback from an electrochemical sensor module and complete with an ozone destruct cycle for safety. Relative humidity as well as temperature and O3 concentration are also continuously recorded and can be maintained at the most effective levels for sterilization.Validation of the ozone sterilization protocol for deactivation of surrogate viruses on N95 masks has been demonstrated using TLS, an E. coli bacteriophage (bacteria-infecting virus), to provide a measure of effectiveness for variables such as time, ozone and viral concentrations, temperature, relative humidity, and virus-contaminated substrate. Additional organisms and conditions of sterilization are reported in this study to provide further analysis of efficacy of equipment and procedures as well as deleterious effects, if any, on PPE.The experiments with bacteriophage indicate that there is 99.99% inactivation of the robust bacteriophage when placed on N95 mask material with a one-hour treatment at 200 ppm ozone as compared to a 0-ppm control. The inactivation was shown to be 99.8% with only 50 ppm ozone. These ozone levels are easily achieved with a low power source and the entire system is operated using 12 VDC from a charger or battery. This sterilization effect is highly dependent on humidity, and the higher inactivation rates require higher relative humidity. The inactivation was demonstrated on multiple types of substrates with and without prior drying of the deposited phage. We expect the covid19 to be easier to inactivate than the bacteriophage and will test the system on SARS-CoV2 in a separate series of experiments. The sterilizer apparatus is simple and low cost and can be constructed from readily available parts and software for automated control is open sourced by KWJ.