Biology subjects

Vyas, H. K. N.

Publications and source records attributed to Vyas, H. K. N..

6 recordsLinked to original sources

bioRxiv2026

Plasma-Activated Water: An Emerging Technology for the Control of Eggshell-Associated Biofilms

Biofilm formation on eggshell surfaces poses a significant food safety concern by promoting bacterial persistence and facilitating cross-contamination, contributing to foodborne illness outbreaks worldwide. Beyond public health impacts, bacterial contamination also drives spoilage, food loss, and waste, together placing substantial strain on healthcare systems and economic infrastructures. This study evaluated the anti-biofilm efficacy of plasma-activated water (PAW), a reactive oxygen and nitrogen species (RONS)-rich solution generated through the interaction of cold atmospheric plasma with water. 50 bacterial isolates were recovered from chicken eggshells, and the biofilm-forming capacities of eight representative species (Staphylococcus arlettae, Lysinibacillus fusiformis, Priestia megaterium, Peribacillus frigoritolerans, Acinetobacter lwoffii, Escherichia coli, Pseudomonas fulva, and Pseudomonas stutzeri) were characterised in vitro. All isolates formed biofilms, exhibiting variable culturable cell populations, biomass, and extracellular polymeric substance (EPS) matrix presence and composition. EPS analyses identified polysaccharides, extracellular DNA, and proteins as key matrix components, with relative abundance varying with biofilm maturity. PAW efficacy was assessed under short exposure times (1, 5, and 15 minutes) using freshly generated, 1-week-old, and 4-week-old PAW. Fresh PAW demonstrated potent anti-biofilm activity, achieving complete inactivation of E. coli and P. stutzeri biofilms below the limit of detection within 1 min, while biofilms of Gram-positive bacteria, L. fusiformis and P. megaterium, were generally more tolerant. Reduced efficacy of aged PAW was likely associated with RONS decay. Mechanistic analyses, including scanning electron microscopy and intracellular reactive oxygen species assays, linked PAW treatment to biofilm disruption and intracellular oxidative stress. This study provides the first detailed evaluation of biofilm formation by under-investigated eggshell-associated bacteria and elucidates mechanisms underlying PAW-mediated biofilm inactivation. Overall, PAW shows strong potential as a sanitising technology for the egg industry, supporting safer food production and reduced reliance on conventional antimicrobial agents amid rising global antimicrobial resistance rates.

microbiology↗

bioRxiv2024

Antimicrobial mechanism of in-situ plasma activated water treatment of pathogenic Escherichia coli and Staphylococcus aureus biofilms

AimsThis study investigated the efficacy and mechanisms of inactivation of against Escherichia coli UTI89 and Staphylococcus aureus NCTC8325 through an in-situ plasma-activated water (PAW) treatment. Methods and ResultsPAW was prepared by discharging atmospheric pressure cold plasma beneath the surface of sterile distilled water. The study investigated the inactivation of biofilm cells and biofilm matrix. A complete killing of biofilm cells was achieved on both of E. coli (6.76 {+/-} 0.01 log CFU/mL) and S. aureus (6.82 {+/-} 0.02 log CFU/mL). This process happened earlier in S. aureus. Simultaneously, PAW treatment disrupted the biofilm structure, inducing a significant reduction in general biofilm biomass and extracellular polymer substances (EPS) matrix. With the disruption of EPS, PAW was enabled to further interact with the bacterial membrane, causing a significant increase in membrane permeability and disrupted membrane structure. Finally, PAW treatment led to a significant accumulation of intracellular reactive oxygen and nitrogen species within the biofilm cells. ConclusionsCollectively, these findings indicate that PAW effectively inactivates biofilms by mechanically targeting the biofilm EPS matrix and biofilm cells in both gram-negative and gram-positive bacteria. Impact statementThis study contributes novel insights into plasma-activated waters mechanisms of action, particularly its impact on the biofilm extracellular polymeric substances matrix (exopolysaccharides, extracellular DNA, and protein), cell membrane permeability, depolarization, and intracellular ROS and RNS accumulation in both of Gram-positive and Gram-negative species. These findings highlight PAW-based treatments against biofilm-related challenges in antimicrobial development and water system decontamination.

microbiology↗

bioRxiv2024

Transcriptional Signatures Associated with the Survival of Escherichia coli Biofilm During Treatment with Plasma-Activated Water

Biofilm formation on surfaces, tools and equipment can damage their quality and lead to high repair or replacement costs. Plasma-activated water (PAW), a new technology, has shown promise in killing biofilm and non-biofilm bacteria due to its mix of reactive oxygen and nitrogen species (RONS), and in particular superoxide. However, the specific genetic mechanisms behind PAWs effectiveness, especially against biofilms, are not yet fully understood. Here, we examined the stress responses of Escherichia coli biofilms when exposed to sub-lethal PAW treatment with and without superoxide (by adding the scavenger Tiron to remove it). A 40% variation in gene expression was observed for PAW treated biofilms when compared to PAW-Tiron and controls. Specifically, PAW treatment resulted in 478 upregulated genes (> 1.5 log2FC) and 186 downregulated genes (< -1.5 log2FC) compared to the control. Pathway enrichment and biological process enrichment analysis revealed significant upregulation of sulfur metabolism, ATP-binding cassette transporter genes, amino acid metabolic/biosynthesis pathways, hypochlorite response systems and oxidative phosphorylation for biofilms treated with PAW compared to control. Knockout mutants of significantly upregulated genes associated with these pathways trxC (4.23-fold), cysP (1.58-fold) and nuoM (1.74-fold) were compared to the wild-type (WT) for their biofilm viability and intracellular RONS accumulation. Relative to PAW-treated WT,{Delta} trxC and{Delta} nuoM knockout mutants displayed significantly reduced biofilm viability (P [≤] 0.05) confirming their role in PAW-mediated response. Interestingly,{Delta} trxC biofilms had the highest intracellular ROS accumulation, as revealed by DCFDA staining after PAW treatment. This study gives a detailed insight into how E. coli biofilms respond to oxidative stress induced by PAW. It highlights the significance of superoxide in PAWs bactericidal effects. Overall, our findings shed light on the specific genes and pathways that help E. coli biofilms survive and respond to PAW treatment, offering a new understanding of plasma technology and its anti-biofilm mechanisms.

microbiology↗

bioRxiv2023

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 RONS 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.

microbiology↗

bioRxiv2023

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 minutes in situ with water activated at 1500 Hz and 120 V, and air as the feed gas; NaOCl treatment was a wash with 100 ppm total chlorine; and the 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 3-4 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 ({middle dot}O2-) in the antimicrobial power of DBDD-PAW was demonstrated through a scavenger assay, and chemistry modelling confirmed that {middle dot}O2- generation readily occurs in DBDD-PAW generated with the employed settings. Modelling 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 synergise with reactive chemical species to produce the acute antimicrobial activity seen with the in situ PAW system. ImportancePlasma 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 micro-organisms while maintaining the quality and shelf life of the produce item. Our experimental results are supported by modelling of the plasma chemistry and applied physical forces, which show that the system can generate highly reactive superoxide radicals and strong electric fields that combine to produce potent antimicrobial power. In situ PAW has promise in industrial applications as it only requires 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.

microbiology↗

bioRxiv2020

An Optimised GAS-pharyngeal cell biofilm model

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Biofilm formation has been implicated in both pharyngeal and dermal GAS infections. In vitro, plate-based assays have shown that several GAS M-types form biofilms, and multiple GAS virulence factors have been linked to biofilm formation. Although the contributions of these plate-based studies have been valuable, most have failed to mimic the host environment, with many studies utilising abiotic surfaces. GAS is a human specific pathogen, and colonisation and subsequent biofilm formation is likely facilitated by distinct interactions with host tissue surfaces. As such, a host cell-GAS model has been optimised to support and grow GAS biofilms of a variety of GAS M-types. Improvements and adjustments to the crystal violet biofilm biomass assay have also been tailored to reproducibly detect delicate GAS biofilms. We propose 72 h as an optimal growth period for yielding detectable biofilm biomass. GAS biofilms formed are robust and durable, and can be reproducibly assessed via staining/washing intensive assays such as crystal violet with the aid of methanol fixation prior to staining. Lastly, SEM imaging of GAS biofilms formed by this model are resemblant of those previously found on excised tonsils of patients suffering chronic pharyngo-tonsillitis. Taken together, we outline an efficacious GAS biofilm pharyngeal cell model that can support long-term GAS biofilm formation, with biofilms formed closely resembling those seen in vivo.

microbiology↗