Inactivation of Airborne Bacteria by Direct Interaction with Non-Thermal Dielectric Barrier Discharge Plasma: The Involvement of Reactive Oxygen Species

The present study examined the effect of Dielectric Barrier Discharge (DBD) plasma on bioaerosol particles. Different DBD plasma devices were designed and tested for their efficacy in inactivation of airborne bacteria. Bacterial aerosols were injected in / through the plasma stream and the treated bioaerosols were analyzed. The results indicated a complete inactivation of bioaerosol upon a very short exposure in the range of milliseconds to plasma discharge. A large system was designed to evaluate its efficacy to inactivate bacterial spores. After preliminary studies, to study the underlying mechanisms of inactivation, a single filament DBD plasma generating probe was developed and used for subsequent studies. In parallel, a near uniform aerosol generator (nebulizer) was optimized, and aerosol particle size characterized. The kinetics of bacterial inactivation produced by this system was investigated, and sub-lethal dose determined. We hypothesized that the prototype bacteria, Escherichia coli when present in aerosols and exposed to single filament DBD plasma system, activates intracellular reactive oxygen species (ROS). The predetermined sub-lethal dose of DBD plasma was used to study the cellular responses of Escherichia coli during its inactivation. Cell membrane is more vulnerable when bacteria are present in aerosols, and hence the changes in features, such as cellular respiration and growth, permeation, and depolarization were investigated following exposure to single filament DBD plasma system. During studies, the catalase mediated defense system was found to be involved predominantly in the management of intracellular ROS pool. Through the use of E. coli derivatives of specific gene mutation, we analyzed the involvement of heat stress-responsive genes. Although the plasma is considered non-thermal, localized heating and the generated interactive stress is likely involved in the inactivation of E. coli bioaerosol. These findings provide a new dimension in underlying mechanisms of E. coli inactivation during DBD plasma exposure.Ph.D., Biomedical Engineering -- Drexel University, 201

Rapid inactivation of airborne bacteria using atmospheric pressure dielectric barrier grating discharge

IEEE Transactions on Plasma Science, 35(5): pp. 1501-1510.Dielectric barrier discharge plasma has been known to inactivate many different microorganisms on surfaces when treatment times are on the order of seconds or minutes in duration. In this paper, a unique plasma air cleaning facility was created which combines a dielectric barrier grating discharge (DBGD) with a filterless laboratory-scale ventilation system and is used to treat concentrated bacterial bioaerosol in a moving air stream at air flow rates of 25 L/s. Results indicate that plasma treatment times on the order of milliseconds corresponding to one pass through the DBGD device can achieve 1.5-log reduction in culturable E. coli immediately after contact with plasma and 5-log reduction totally following in the minutes after the plasma treatment. A numerical characterization study was performed to help predict and understand the mechanism of bacteria inactivation in the DBD plasma from a variety of plasma factors

Inactivating SARS-CoV-2 Surrogates on Surfaces Using Engineered Water Nanostructures Incorporated with Nature Derived Antimicrobials

The continuing cases of COVID-19 due to emerging strains of the SARS-CoV-2 virus underscore the urgent need to develop effective antiviral technologies. A crucial aspect of reducing transmission of the virus is through environmental disinfection. To this end, a nanotechnology-based antimicrobial platform utilizing engineered water nanostructures (EWNS) was utilized to challenge the human coronavirus 229E (HCoV-229E), a surrogate of SARS-CoV-2, on surfaces. The EWNS were synthesized using electrospray and ionization of aqueous solutions of antimicrobials, had a size in the nanoscale, and contained both antimicrobial agents and reactive oxygen species (ROS). Various EWNS were synthesized using single active ingredients (AI) as well as their combinations. The results of EWNS treatment indicate that EWNS produced with a cocktail of hydrogen peroxide, citric acid, lysozyme, nisin, and triethylene glycol was able to inactivate 3.8 logs of HCoV-229E, in 30 s of treatment. The delivered dose of antimicrobials to the surface was measured to be in pico to nanograms. These results indicate the efficacy of EWNS technology as a nano-carrier for delivering a minuscule dose while inactivating HCoV-229E, making this an attractive technology against SARS-CoV-2

Inactivating SARS-CoV-2 Surrogates on Surfaces Using Engineered Water Nanostructures Incorporated with Nature Derived Antimicrobials

The continuing cases of COVID-19 due to emerging strains of the SARS-CoV-2 virus underscore the urgent need to develop effective antiviral technologies. A crucial aspect of reducing transmission of the virus is through environmental disinfection. To this end, a nanotechnology-based antimicrobial platform utilizing engineered water nanostructures (EWNS) was utilized to challenge the human coronavirus 229E (HCoV-229E), a surrogate of SARS-CoV-2, on surfaces. The EWNS were synthesized using electrospray and ionization of aqueous solutions of antimicrobials, had a size in the nanoscale, and contained both antimicrobial agents and reactive oxygen species (ROS). Various EWNS were synthesized using single active ingredients (AI) as well as their combinations. The results of EWNS treatment indicate that EWNS produced with a cocktail of hydrogen peroxide, citric acid, lysozyme, nisin, and triethylene glycol was able to inactivate 3.8 logs of HCoV-229E, in 30 s of treatment. The delivered dose of antimicrobials to the surface was measured to be in pico to nanograms. These results indicate the efficacy of EWNS technology as a nano-carrier for delivering a minuscule dose while inactivating HCoV-229E, making this an attractive technology against SARS-CoV-2

Involvement of multiple stressors induced by non-thermal plasma-charged aerosols during inactivation of airborne bacteria.

A lab-scale, tunable, single-filament, point-to-point nonthermal dieletric-barrier discharge (DBD) plasma device was built to study the mechanisms of inactivation of aerosolized bacterial pathogens. The system inactivates airborne antibiotic-resistant pathogens efficiently. Nebulization mediated pre-optimized (4 log and 7 log) bacterial loads were challenged to plasma-charged aerosols, and lethal and sublethal doses determined using colony assay, and cell viability assay; and the loss of membrane potential and cellular respiration were determined using cell membrane potential assay and XTT assay. Using the strategies of Escherichia coli wildtype, over-expression mutant, deletion mutants, and peroxide and heat stress scavenging, we analyzed activation of intracellular reactive oxygen species (ROS) and heat shock protein (hsp) chaperons. Superoxide dismutase deletion mutants (ΔsodA, ΔsodB, ΔsodAΔsodB) and catalase mutants ΔkatG and ΔkatEΔkatG did not show significant difference from wildtype strain, and ΔkatE and ΔahpC was found significantly more susceptible to cell death than wildtype. The oxyR regulon was found to mediate plasma-charged aerosol-induced oxidative stress in bacteria. Hsp deficient E. coli (ΔhtpG, ΔgroEL, ΔclpX, ΔgrpE) showed complete inactivation of cells at ambient temperature, and the treatment at cold temperature (4°C) significantly protected hsp deletion mutants and wildtype cells, and indicate a direct involvement of hsp in plasma-charged aerosol mediated E. coli cell death

Colony assays demonstrating the relative susceptibilities of <i>E</i>. <i>coli</i> wildtype and its various derivatives to plasma charged aerosols.

<p><b>(A)</b> Superoxide dismutase single deletion (<i>ΔsodA</i>, <i>ΔsodB</i>) and double deletion (<i>ΔsodAΔsodB</i>) mutants do not show significant difference in susceptibilities from wildtype cells. <b>(B)</b> Catalase/ hydroperoxidase deletion mutants demonstrates that <i>katE</i> and <i>ahpC</i> are significantly more susceptible to plasma charged aerosols as compared to wildtype (*), whereas <i>katG</i> and double mutant (<i>katEkatG</i>) behaved like wildtype and do not show significantly different susceptibility from wildtype. <b>(C)</b> The responses of <i>oxyR</i> regulon demonstrate that its deletion mutant is significantly more susceptible (**), and overexpression (*) mutant is more resistant to plasma charged mediated inactivation as compared to wildtype, indicating direct involvement of <i>oxyR</i> in inactivation mechanism.</p

A customized miniature device of bioaerosol plasma treatment system.

<p>This system is tunable for rate of flow of bioaerosols, amount of bacterial load, plasma power and frequency, and thus for plasma energy dose. <b>(A)</b> The device picture showing parts of it. <b>(B)</b> A lateral view showing ignited plasma point to point discharge.</p

The colony assays showing effect of scavengers on rescue of plasma charged aerosol post-treated <i>E</i>. <i>coli</i> wildtype and mutants.

<p><b>(A)</b> Catalase, the specific scavenger of H₂O₂ was able to significantly protect mutant (<i>ΔkatE</i>) from inactivation. <b>(B)</b> A non-enzymatic ROS scavenger, D-mannitol was significantly able to rescue wildtype and <i>ΔkatE</i> cells from inactivation as compared to their corresponding non-scavenged conditions. <b>(C)</b> Thiourea, a wildly used ^.OH scavenger was able provide significant protection to deletion mutant (<i>ΔkatE</i>) but not the wildtype <i>E</i>. <i>coli</i>, suggesting that intracellular ^.OH pool created in hydroperoxidase deficient cells during post-exposure to plasma charged aerosols are effectively scavenged, and possibly inhibit further Fenton chemistry generating hydroxyl radicals. This also demonstrates that <i>katE</i> is an important mediator of survival in bioaerosolized <i>E</i>. <i>coli</i> under such condition. (* indicates <i>p</i> <0.05 against their corresponding conditions without scavenger. ** indicates <i>p</i> <0.05 against remaining concentrations of catalase).</p

The colony assays demonstrating the effect of plasma charged aerosols on heat shock protein (hsp) deficient <i>E</i>. <i>coli</i> derivatives.

<p><b>(A)</b> All <i>hsp</i> deficient <i>E</i>. <i>coli</i> (<i>ΔhtpG</i>, <i>ΔgroEL</i>, <i>ΔclpX</i>) showed complete inactivation of cells at ambient temperature. (*, p <0.05 against corresponding untreated bioaerosols) <b>(B)</b> When the treatments with plasma charged aerosols were carried out at cold temperature (4°C), all <i>hsp</i> deletion mutants and wildtype <i>E</i>. <i>coli</i> cells significantly survived. (*, p <0.05 against corresponding ‘no scavenging’ conditions). Together, these findings demonstrate that <i>hsp</i> activation occurs in <i>E</i>. <i>coli</i> cells in response to DBD charged aerosol exposure at room temperature, <i>htpG</i>, <i>groEL</i>, <i>clpX</i> and <i>grpE</i> are directly involved cell survival, and cold temperature scavenge intracellular localized heating effect and protect these specific mutants.</p