The antimicrobial efficacy of plasma-activated water against Listeria and E. coli is modulated by reactor design and water composition.

AIMS: This study aimed to compare the efficacy of plasma-activated water (PAW) generated by two novel plasma reactors against pathogenic foodborne illness organisms. METHODS AND RESULTS: The antimicrobial efficacy of PAW produced by a bubble spark discharge (BSD) reactor and a dielectric barrier discharge-diffuser (DBDD) reactor operating at atmospheric conditions with air, multiple discharge frequencies and Milli-Q and tap water, was investigated with model organisms Listeria innocua and Escherichia coli in situ. Optimal conditions were subsequently employed for pathogenic bacteria Listeria monocytogenes, E. coli and Salmonella enterica. DBDD-PAW reduced more than 6-log of bacteria within 1 min. The BSD-PAW, while attaining high log reduction, was less effective. Analysis of physicochemical properties revealed that BSD-PAW had a greater variety of reactive species than DBDD-PAW. Scavenger assays designed to specifically sequester reactive species demonstrated a critical role of superoxide, particularly in DBDD-PAW. CONCLUSIONS: DBDD-PAW demonstrated rapid antimicrobial activity against pathogenic bacteria, with superoxide the critical reactive species. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrates the potential of DBDD-PAW produced using tap water and air as a feasible and cost-effective option for antimicrobial applications, including food safety.

Mixing postharvest fungicides and sanitizers results in unpredictable survival of microbes that affect cantaloupes.

Postharvest treatments with sanitizers and fungicides are applied to increase the quality, safety and shelf life of fresh produce including cantaloupes (also known as rockmelons). The primary role of sanitizers during cantaloupe washing is to prevent cross contamination of potentially pathogenic bacteria in washwater. Postharvest fungicide sprays or dips are employed to inhibit spoilage-causing fungi. While assessing the compatibility of these antimicrobials based on the measurement of active ingredients levels provides some indication of antimicrobial capacity, there is limited data on whether the interaction between these chemicals in wash water modifies their overall efficacy against relevant microorganisms. The aim of this research was to determine how chlorine- and peroxyacetic acid-based sanitizers interact with commercial guazatine- and imazalil-based fungicide formulations used on cantaloupes, and whether mixing these augments or suppresses anti-microbial activity against relevant human pathogens and spoilage fungi in wash water. The results were unpredictable: while most combinations were antimicrobial, the chlorine-based sanitizer when mixed with the guazatine-based fungicide had significantly reduced efficacy against pathogenic Salmonella spp. (~2.7 log) and the fungal spoilage organisms, Trichothecium roseum and Rhizopus stolonifera. Mixing the chlorine-based sanitizer with an imazalil-based fungicide produced a range of outcomes with antagonistic, indifferent and synergistic interactions observed for the fungal species tested. The peroxyacetic acid-based sanitizer led to indifferent interactions with the guazatine-based fungicide, while antagonism and synergy were observed when mixed with the imazalil-based fungicide. This study demonstrates that mixing postharvest agrichemicals used in the cantaloupe industry may increase the risk of microbial contamination and thereby potentially compromise food safety and quality.

Bisphosphonates synergistically enhance the antifungal activity of azoles in dermatophytes and other pathogenic molds.

Superficial infections of the skin, hair, and nails by fungal dermatophytes are the most prevalent of human mycoses, and many infections are refractory to treatment. As current treatment options are limited, recent research has explored drug synergy with azoles for dermatophytoses. Bisphosphonates, which are approved to treat osteoporosis, can synergistically enhance the activity of azoles in diverse yeast pathogens but their activity has not been explored in dermatophytes or other molds. Market bisphosphonates risedronate, alendronate, and zoledronate (ZOL) were evaluated for antifungal efficacy and synergy with three azole antifungals: fluconazole (FLC), itraconazole (ITR), and ketoconazole (KET). ZOL was the most active bisphosphonate tested, displaying moderate activity against nine dermatophyte species (MIC range 64-256 µg/mL), and was synergistic with KET in eight of these species. ZOL was also able to synergistically improve the anti-biofilm activity of KET and combining KET and ZOL prevented the development of antifungal resistance. Rescue assays in Trichophyton rubrum revealed that the inhibitory effects of ZOL alone and in combination with KET were due to the inhibition of squalene synthesis. Fluorescence microscopy using membrane- and ROS-sensitive probes demonstrated that ZOL and KET:ZOL compromised membrane structure and induced oxidative stress. Antifungal activity and synergy between bisphosphonates and azoles were also observed in other clinically relevant molds, including species of Aspergillus and Mucor. These findings indicate that repurposing bisphosphonates as antifungals is a promising strategy for revitalising certain azoles as topical antifungals, and that this combination could be fast-tracked for investigation in clinical trials. IMPORTANCE: Fungal infections of the skin, hair, and nails, generally grouped together as "tineas" are the most prevalent infectious diseases globally. These infections, caused by fungal species known as dermatophytes, are generally superficial, but can in some cases become aggressive. They are also notoriously difficult to resolve, with few effective treatments and rising levels of drug resistance. Here, we report a potential new treatment that combines azole antifungals with bisphosphonates. Bisphosphonates are approved for the treatment of low bone density diseases, and in fungi they inhibit the biosynthesis of the cell membrane, which is also the target of azoles. Combinations were synergistic across the dermatophyte species and prevented the development of resistance. We extended the study to molds that cause invasive disease, finding synergy in some problematic species. We suggest bisphosphonates could be repurposed as synergents for tinea treatment, and that this combination could be fast-tracked for use in clinical therapy.

An Effective Sanitizer for Fresh Produce Production: In Situ Plasma-Activated Water Treatment Inactivates Pathogenic Bacteria and Maintains the Quality of Cucurbit Fruit.

The effect of plasma-activated water (PAW) generated with a dielectric barrier discharge diffusor (DBDD) system on microbial load and organoleptic quality of cucamelons was investigated and compared to the established sanitizer, sodium hypochlorite (NaOCl). Pathogenic serotypes of Escherichia coli, Salmonella enterica, and Listeria monocytogenes were inoculated onto the surface of cucamelons (6.5 log CFU g-1) and into the wash water (6 log CFU mL-1). PAW treatment involved 2 min in situ with water activated at 1,500 Hz and 120 V and air as the feed gas; NaOCl treatment was a wash with 100 ppm total chlorine; control treatment was a wash with tap water. PAW treatment produced a 3-log CFU g-1 reduction of pathogens on the cucamelon surface without negatively impacting quality or shelf life. NaOCl treatment reduced the pathogenic bacteria on the cucamelon surface by 3 to 4 log CFU g-1; however, this treatment also reduced fruit shelf life and quality. Both systems reduced 6-log CFU mL-1 pathogens in the wash water to below detectable limits. The critical role of superoxide anion radical (·O2-) in the antimicrobial power of DBDD-PAW was demonstrated through a Tiron scavenger assay, and chemistry modeling confirmed that ·O2- generation readily occurs in DBDD-PAW generated with the employed settings. Modeling of the physical forces produced during plasma treatment showed that bacteria likely experience strong local electric fields and polarization. We hypothesize that these physical effects synergize with reactive chemical species to produce the acute antimicrobial activity seen with the in situ PAW system. IMPORTANCE Plasma-activated water (PAW) is an emerging sanitizer in the fresh food industry, where food safety must be achieved without a thermal kill step. Here, we demonstrate PAW generated in situ to be a competitive sanitizer technology, providing a significant reduction of pathogenic and spoilage microorganisms while maintaining the quality and shelf life of the produce item. Our experimental results are supported by modeling of the plasma chemistry and applied physical forces, which show that the system can generate highly reactive ·O2- and strong electric fields that combine to produce potent antimicrobial power. In situ PAW has promise in industrial applications as it requires only low power (12 W), tap water, and air. Moreover, it does not produce toxic by-products or hazardous effluent waste, making it a sustainable solution for fresh food safety.

Plasma activated water as a pre-treatment strategy in the context of biofilm-infected chronic wounds.

Healing and treatment of chronic wounds are often complicated due to biofilm formation by pathogens. Here, the efficacy of plasma activated water (PAW) as a pre-treatment strategy has been investigated prior to the application of topical antiseptics polyhexamethylene biguanide, povidone iodine, and MediHoney, which are routinely used to treat chronic wounds. The efficacy of this treatment strategy was determined against biofilms of Escherichia coli formed on a plastic substratum and on a human keratinocyte monolayer substratum used as an in vitro biofilm-skin epithelial cell model. PAW pre-treatment greatly increased the killing efficacy of all the three antiseptics to eradicate the E. coli biofilms formed on the plastic and keratinocyte substrates. However, the efficacy of the combined PAW-antiseptic treatment and single treatments using PAW or antiseptic alone was lower for biofilms formed in the in vitro biofilm-skin epithelial cell model compared to the plastic substratum. Scavenging assays demonstrated that reactive species present within the PAW were largely responsible for its anti-biofilm activity. PAW treatment resulted in significant intracellular reactive oxygen and nitrogen species accumulation within the E. coli biofilms, while also rapidly acting on the microbial membrane leading to outer membrane permeabilisation and depolarisation. Together, these factors contribute to significant cell death, potentiating the antibacterial effect of the assessed antiseptics.