Objectives:Chemical disinfectants are critical for infection control in healthcare environments and beyond, as exemplified by their vital role during the COVID-19 pandemic. Despite research repeatedly demonstrating that bacteria can develop adaptations that mitigate the efficacy of chemical disinfectants, the underlying molecular mechanisms remain poorly characterized. This study investigates the mechanisms that underpin resistance demonstrated by disinfectant-adapted Klebsiella pneumoniae NCTC 13443 samples. Methods:Resistant samples have previously undergone long-term in vitro adaptation via serial passage in increasing concentrations of common disinfectants benzalkonium chloride (BAC), didecydimethylammonium chloride (DDAC), polyhexamethylene biguanide (PHMB), chlorocresol or bronopol. A multi-omics approach was used to conduct in-depth molecular analyses of the adaptations that contribute to resistance. Results:K. pneumoniae adaptation to BAC, DDAC and PHMB was associated with the modification of lipid A causing the reduction of the net-negative charge of the outer surface, lowering the affinity of cationic disinfectants. This mechanism is also used for polymyxin and colistin resistance, highlighting a potential cross-resistance risk. Chlorocresol-adapted K. pneumoniae samples demonstrated increased expression of efflux pumps and expression changes linked to biofilm formation. Bronopol resistance was associated with promoting biofilm formation and increased thioredoxin expression to alleviate oxidative stress. Results indicate the potential role of N-ethylmaleimide reductase NemA in bronopol resistance via enzymatic degradation. Conclusions:These findings provide novel insights into how causative pathogens of healthcare-associated infections can adapt to and mitigate the effectiveness of common chemical disinfectants that are relied on globally every day as a critical infection control measure.

Background: Disinfectants are a critical infection control measure that are relied upon globally in a range of settings including healthcare, food production, and domestic environments. However, bacteria have been shown to survive disinfectant treatments when harboured in dry surface biofilms or when disinfectants are used ineffectively. This provides an opportunity for organisms to develop low-level tolerance to various disinfectants. The capability of bacteria to develop adaptations to non-antibiotic antimicrobial agents is often overlooked. Aim: To report on the capability and readiness of clinically relevant K. pneumoniae to adapt to common disinfectants that are relied upon every day across the world, delivering much-needed insights into an often-overlooked aspect of antimicrobial resistance. Methods: This study investigated the ability of Klebsiella pneumoniae NDM-1 strain NCTC 13443 to adapt to a range of common chemical disinfectants (benzalkonium chloride, didecyldimethylammonium chloride, polyhexamethylene biguanide, chlorocresol and bronopol) via serial passage exposure method. Findings: After long-term adaptation, K. pneumoniae developed tolerance to all tested disinfectants, exhibiting a minimum inhibitory concentration increase of between 30 and 413% compared with the untreated parent samples. Characterization of disinfectant cross- tolerance showed that while cross-tolerance can occur, most adapted samples became more susceptible to the second disinfectant treatment, probably because of the fitness cost of adaptation. Observed cross-tolerance/collateral sensitivity was not always reciprocated between disinfectant-tolerant samples. Conclusions: Results suggest the order of disinfectant exposure is important during tolerance development. This has significant implications for disinfectant cleaning routines, and is probably due to variations in the underpinning tolerance mechanisms, even when the disinfectants display similar mechanisms of action. (c) 2024 The Authors. Published by Elsevier Ltd on behalf of The Healthcare Infection Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Fresh produce-associated outbreaks of the foodborne pathogen Escherichia coli O157 are responsible for a number of disease cases, hospitalisations, and deaths. In many cases, the source of contamination can be linked to the growing media of the food, although pathogen detection is problematic in these complex soil ecosystems. In this study, direct qPCR without pre-enrichment was used to detect 310 copies of the translocated intimin receptor gene, using a primer sequence specific to E. coli O157, in horticultural compost purchased from a commercial supplier. The pathogen could not be cultured on selective media but was visualised using peptide nucleic acid fluorescence in situ hybridisation and cell elongation viability assay, confirming the viability. Enumeration of elongated E. coli O157 determined that there were 205 live cells per gram compost. The nonculturability and confirmation of viability of the pathogen indicates its viable but nonculturable (VBNC) status. The detection of VBNC foodborne pathogens in environmental samples challenges current understanding of the nature of foodborne pathogen contamination.

Preventing legionellosis in water systems demands effective hydrodynamic management and biofilm mitigation. This study investigates the complex relationship between hydrodynamics (80 RPM and stagnation), biofilm mesoscale structure and Legionella pneumophila colonization, by addressing three key questions: (1) How do low flow vs stagnation conditions affect biofilm response to L. pneumophila colonization?, (2) How do biofilm structural variations mediate L. pneumophila migration across the biofilm?, and (3) Can specific hydrodynamic conditions trigger L. pneumophila entrance in a viable but nonculturable (VBNC) state? It was found that Pseudomonas fluorescens biofilms exhibit different responses to L. pneumophila based on the prevailing hydrodynamic conditions. While biofilm thickness and porosity decreased under shear (80 RPM), thickness tends to significantly increase when pre-established 80 RPM-grown biofilms are set to stagnation upon L. pneumophila spiking. Imposing stagnation after the spiking also seemed to accelerate Legionella migration towards the bottom of the biofilm. Water structures in the biofilm seem to be key to Legionella migration across the biofilm. Finally, shear conditions favoured the transition of L. pneumophila to VBNC states (∼94 %), despite the high viable cell counts (∼8 log10 CFU/cm2) found throughout the experiments. This research highlights the increased risk posed by biofilms and stagnation, emphasizing the importance of understanding the mechanisms that govern Legionella behaviour in diverse biofilm environments. These insights are crucial for developing more effective monitoring and prevention strategies in water systems.

Bacteria can resist antibiotics and toxic substances within demanding ecological settings, such as low oxygen, extreme acid, and during nutrient starvation. MdtEF, a proton motive force-driven efflux pump from the resistance-nodulation-cell division (RND) superfamily, is upregulated in these conditions but its molecular mechanism is unknown. Here, we report cryo-electron microscopy structures of Escherichia coli multidrug transporter MdtF within native-lipid nanodiscs, including a single-point mutant with an altered multidrug phenotype and associated substrate-bound form. Drug binding domain and channel conformational plasticity likely governs substrate polyspecificity, analogous to closely related, constitutively expressed counterpart, AcrB. Whereas we discover distinct transmembrane state transitions within MdtF, which create a more engaged proton relay network, altered drug transport allostery and an acid-responsive increase in efflux efficiency. Our findings provide mechanistic insights necessary to understand bacterial xenobiotic and toxin removal by MdtF and its role within nutrient-depleted and acid stress settings, as endured in the gastrointestinal tract.

Poor hydraulic management at water systems is associated with an increased risk of Legionnaires' disease caused by Legionella. Stagnation periods, followed by sudden water flow, can promote biofilm detachment and the release of Legionella into the bulk water. Regardless of its importance, the simultaneous effects of shear stress on biofilm detachment and Legionella release into the bulk water remain poorly understood. This study investigates how shear stress affects biofilms containing Legionella pneumophila in terms of: a) biofilm detachment, b) release of L. pneumophila into the bulk phase, and c) shifting of L. pneumophila into the viable but nonculturable (VBNC) state. Pseudomonas fluorescens biofilms were formed in a Center for Disease Control (CDC) biofilm reactor at 125 RPM and spiked with L. pneumophila. After 6 days, the system was set for 48 h to stagnation before flow was resumed at rotational velocities of 125, 225, and 400 RPM, corresponding to turbulent regimes with Reynolds numbers of 1552, 2794 and 4966, respectively. Biofilm properties, L. pneumophila viability, culturability, and spatial distribution were monitored. Results show that biofilms containing L. pneumophila maintained a similar basal thickness (12 μm) despite the detachment of the upper layers under different shear stresses. L. pneumophila, located at the bottom of the biofilm, remains surface-attached after biofilm detachment and seems to enhance the cohesiveness of these layers compared to P. fluorescens biofilms. On the contrary, when Legionella is not present, biofilm detachment increases with the increase of applied shear forces. All tested rotational velocities triggered L. pneumophila to enter the VBNC state in the bulk phase, while biofilm-associated VBNC cells were only observed at 400 RPM. Finally, the contribution of the present work to Legionella control practices in water systems is discussed, highlighting the important insights that biofilms can provide in this context.

Biofilms are known to be critical for Legionella settlement in engineered water systems and are often associated with Legionnaire's Disease events. One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of Legionella pneumophila colonization of a Pseudomonas fluorescens biofilm, as information about the interactions between Legionella and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled L. pneumophila to tackle the following questions: (a) does the biofilm structure change upon L. pneumophila biofilm colonization?; (b) what happens to L. pneumophila within the biofilm over time and (c) where is L. pneumophila preferentially located within the biofilm? Results showed that P. fluorescens structure did not significantly change upon L. pneumophila colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled L. pneumophila showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old P. fluorescens biofilms, presumably entering in VBNC state by the end of the experiment. L. pneumophila was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced Legionella protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between L. pneumophila and biofilm structure that can provide, in the future, insights for public health Legionella management of water systems.

Flagellum-mediated motility has been suggested to contribute to virulence by allowing bacteria to colonize and spread to new surfaces. In Salmonella enterica and Escherichia coli species, mutants affected by their flagellar motility have shown a reduced ability to form biofilms. While it is known that some species might act as co-aggregation factors for bacterial adhesion, studies of food-related biofilms have been limited to single-species biofilms and short biofilm formation periods. To assess the contribution of flagella and flagellum-based motility to adhesion and biofilm formation, two Salmonella and E. coli mutants with different flagellar phenotypes were produced: the fliC mutants, which do not produce flagella, and the motAB mutants, which are non-motile. The ability of wild-type and mutant strains to form biofilms was compared, and their relative fitness was determined in two-species biofilms with other foodborne pathogens. Our results showed a defective and significant behavior of E. coli in initial surface colonization (p < 0.05), which delayed single-species biofilm formation. Salmonella mutants were not affected by the ability to form biofilm (p > 0.05). Regarding the effect of motility/flagellum absence on bacterial fitness, none of the mutant strains seems to have their relative fitness affected in the presence of a competing species. Although the absence of motility may eventually delay initial colonization, this study suggests that motility is not essential for biofilm formation and does not have a strong impact on bacteria’s fitness when a competing species is present.

Background: In recent years, hand drying has been highlighted as a key step in appropriate hand hygiene, as moisture on hands can increase the transfer of micro-organisms from hands to surfaces and vice versa. Aim: To understand bacterial and viral aerosolization following hand drying, and study the transfer of micro-organisms from hands to surfaces after drying using different methods. Methods: Groups of five volunteers had their hands pre-washed with soap, rinsed and dried, then inoculated with a concentrated mixture of Pseudomonas fluorescens and MS2 bacteriophage. Volunteers entered an empty washroom, one at a time, and rinsed their hands with water or washed their hands with soap prior to drying with a jet dryer or paper towels. Each volunteer applied one hand successively to various surfaces, while their other hand was sampled using the glove juice method. Both residual bacteria and viruses were quantified from the washroom air, surface swabs and hand samples. Findings: P. fluorescens and MS2 bacteriophages were rarely aerosolized while drying hands for any of the drying methods studied. Results also showed limited, and similar, transfer of both micro-organisms studied on to surfaces for all drying methods. Conclusion: The use of jet dryers or paper towels produces low levels of aerosolization when drying hands in a washroom. Similarly, all drying methods result in low transfer to surfaces. While the coronavirus disease 2019 pandemic raised concerns regarding public washrooms, this study shows that all methods tested are hygienic solutions for dry washed hands.

Background: Dry surface biofilms (DSBs) have been found abundantly across hospital surfaces within intensive care units and may explain how nosocomial pathogens can remain virulent and persist on surfaces for extended periods. Testing standards governing the performance of disinfectant products employ planktonic models under routine growth conditions, which are known to be less tolerant than their biofilm counterpart.Aim: To evaluate biofilm models cultured under artificial human sweat (AHS), a source of nutrient expected on touch surfaces, to assess the antimicrobial performance of common cleaning agents, including a quaternary ammonium, hydrogen peroxide and active chlorine.Methods: Five single-species biofilms, using pathogenic bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis, were generated on stainless-steel substrates using a sedimentation protocol under both AHS and nutrient-rich conditions for a direct comparison of phenotypic tolerance. The biofilm models were grown over five days followed by desiccation cycles, before being submerged into the disinfectant solutions for up to 25 min. Epifluorescence (EF) micro-scopy using LIVE/DEADTM stain was used to visualize microcolony viability. Findings: The results revealed biofilms cultured under AHS exhibited a greater anti-microbial tolerance and reduced speed of kill for all cleaning agents compared with the routine media; an average reduction of 72.4% vs 96.9%, respectively. EF microscopy revealed traces of viable bacteria across all coupons after disinfection indicating a potential opportunity for regrowth and recontamination. Conclusion: The notable difference in biocidal performance between the two growth conditions highlights potential pitfalls within current antimicrobial test standards, and the importance of accurate representation of the microbial challenge.(c) 2023 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Background: Pathogenic prions (PrPSc) are amyloid-rich hydrophobic proteins which bind avidly to surgical surfaces and represent some of the most difficult targets during the reprocessing of reusable surgical instruments. In-vitro methods to amplify and detect the presence of otherwise undetectable prion contamination are available, but they do not measure associated infectivity. Most of these methods rely on the use of proteinase K, however this can lead to the loss of a substantial portion of PrPSc, potentially producing false negatives.Aim: To develop a sensitive in-situ method without proteinase treatment for the dynamic quantification of amyloid accumulation in N2a #58 cells following 22L-prion infection from infected tissues and spiked stainless-steel surfaces. Methods: We spiked cultures of N2a #58 cells with the 22L prion strain in solution or dried on stainless-steel wires and directly measured the accumulation of prion amyloid aggre-gates over several passages using highly sensitive fluorescence microscopy.Findings: We demonstrated a 10-log dynamic range using our method to test residual prion infectivity, that was validated to show variable decontamination efficacy against prions from commercially available cleaning chemistries.Conclusions: The new cell-based infectivity method presented here avoids partial or possibly total proteinase K digestion of PrPSc in samples for greater sensitivity, in addition to low cost, no ethical concerns, and adaptability to detect different prion strains. This method can be used to test cleaning chemistries' efficacy with greater sensitivity than measuring total residual proteins, which may not correlate with residual prion infectivity.(c) 2022 The Authors. Published by Elsevier Ltd on behalf of The Healthcare Infection Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Background In this study, we aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil, by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18 O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled “heavy” and the unlabelled “light” SIP fractions were sequenced. Results After incubations, an increase of the 16S rRNA copy numbers in the “heavy” fractions of the treatments with 18 O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) are highly abundant after two days of incubation with antibiotics. Several Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. On the other hand, only two ARGs from the unbinned-assembled light fractions were identified. Conclusions The results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil, and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur.

Hospital surfaces contaminated with microbial soiling, such as dry surface biofilms (DSBs), can act as a reservoir for pathogenic micro-organisms, and inhibit their detection and removal during routine cleaning. Studies have recognized that such increases in bioburden can hinder the impact of disinfectants and mask the detection of potential pathogens. Cleanliness within healthcare settings is often determined through routine culture-based analysis, whereby surfaces that exhibit >2.5 colony-forming units (CFU) per cm2 pose a risk to patient health; therefore, any underestimation could have detrimental effects. This study quantified microbial growth on high-touch surfaces in four hospitals in England over 19 months. This was achieved using environmental swabs to sample a variety of surfaces within close proximity of the patient, and plating these on to non-specific low nutrient detection agar. The presence of DSBs on surfaces physically removed from the environment was confirmed using real-time imaging through episcopic differential interference contrast microscopy combined with epifluorescence. Approximately two-thirds of surfaces tested exceeded the limit for cleanliness (median 2230 CFU/cm2), whilst 83% of surfaces imaged with BacLight LIVE/DEAD staining confirmed traces of biofilm. Differences in infection control methods, such as choice of surface disinfectants and cleaning personnel, were not reflected in the microbial variation observed and resulting risk to patients. This highlights a potential limitation in the effectiveness of the current standards for all hospital cleaning, and further development using representative clinical data is required to overcome this limitation.

Background: Despite adherence to standard protocols, residues including live micro-organisms may remain on the various surfaces of reprocessed flexible endoscopes. Prions are infectious proteins that are notoriously difficult to eliminate.Aim: To test the potential of cold atmospheric plasma (CAP) for the decontamination of various surfaces of flexible endoscopes, measuring total proteins and prion residual infectivity as indicators of efficacy.Methods: New PTFE endoscope channels and metal test surfaces spiked with test soil or prion-infected tissues were treated using different CAP-generating prototypes. Surfaces were examined for the presence of residues using very sensitive fluorescence epimicro-scopy. Prion residual infectivity was determined using the wire implant animal model and a more sensitive cell infectivity assay. Findings: A CAP jet applied perpendicularly at close range on flat test surfaces removed soil within 3 min, but left microscopic residues and failed to eliminate prion infectivity according to the wire implant animal assay. The longitudinal gas flow from CAP prototypes developed for the treatment of long channels led to the displacement and sedimentation of residual soil towards the distal end, when applied alone. Observations of the plasma inside glass tubes showed temporal and spatial heterogeneity within a limited range. After the standard enzymatic manual pre-wash, 'CAP-activated' gas effluents prevented prion transmission from treated endoscope channels according to the prion infectivity cell assay. Conclusion: CAP shows promising results as a final step for decontamination of surgical surfaces. Optimizing CAP delivery could further enhance CAP efficacy, offering a safe, chemical-free alternative for the reprocessing of all luminal flexible endoscope surfaces.(c) 2023 The Author(s). Published by Elsevier Ltd on behalf of The Healthcare Infection Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Background We aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18 O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled “heavy” and the unlabelled “light” SIP fractions were sequenced. Results An increase of the 16S rRNA copy numbers in the “heavy” fractions of the treatments with 18 O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) were highly abundant after two days of incubation with antibiotics. Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. In comparison, only two ARGs from the unbinned-assembled light fractions were identified. Conclusions The results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur.

The emergence of biofilms on dry hospital surfaces has led to the development of numerous models designed to challenge the efficacious properties of common antimicrobial agents used in cleaning. This is in spite of limited research defining how dry surfaces are able to facilitate biofilm growth and formation in such desiccating and nutrient-deprived environments. While it is well established that the phenotypical response of biofilms is dependent on the conditions in which they are formed, most models incorporate a nutrient-enriched, hydrated environment dissimilar to the clinical setting. In this study, we piloted a novel culture medium, artificial human sweat (AHS), which is perceived to be more indicative of the nutrient sources available on hospital surfaces, particularly those in close proximity to patients. AHS was capable of sustaining the proliferation of four clinically relevant multidrug-resistant pathogens (Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa) and achieved biofilm formation at concentration levels equivalent to those found in situ (average, 6.00 log10 CFU/cm2) with similar visual characteristics upon microscopy. The AHS model presented here could be used for downstream applications, including efficacy testing of hospital cleaning products, due to its resemblance to clinical biofilms on dry surfaces. This may contribute to a better understanding of the true impact these products have on surface hygiene. IMPORTANCE Precise modeling of dry surface biofilms in hospitals is critical for understanding their role in hospital-acquired infection transmission and surface contamination. Using a representative culture condition which includes a nutrient source is key to developing a phenotypically accurate biofilm community. This will enable accurate laboratory testing of cleaning products and their efficacy against dry surface biofilms.

Chronic wounds fail to progress through the normal stages of healing, with the largest remediable cause of chronicity being presence of a multi-species biofilm. Removal of biofilm from the wound environment is central to wound care. A device for mechanically removing biofilms from wounds has been devised. The removal is caused by small-scale liquid currents and shear, generated by acoustically activated microscopic air bubbles. These bubbles and acoustic waves are delivered onto the wound by a gentle liquid stream, allowing cleaning in situ and removal of debris in the run-off liquid. We have investigated if this liquid acoustic wound stream (LAWS) can remove bacterial biofilm from soft biological wound models and studied the effect of LAWS on the cellular tissues of the substrate. LAWS will efficiently remove early Pseudomonas aeruginosa biofilm from an artificial wound in a pig's trotter, 24 hours-mature biofilm of P. aeruginosa from a pre-wounded human full thickness skin model (EpiDerm FT), and 3-day mature biofilm of P. aeruginosa or Staphylococcus aureus from a porcine skin explant. Histological examinations of uninfected EpiDerm models that had been treated by LAWS and then stained with Haematoxylin and Eosin, demonstrated no damage to the human tissue, and wound diameter was smaller in the treated skin models compared with untreated samples. Immunofluorescence staining for cytokeratin 14 showed that keratinocytes had migrated further across the wound in the uninfected samples treated by LAWS. We discuss the implications for wound healing and propose further laboratory and clinical studies to demonstrate the removal of biofilm from patients with chronic leg ulcers and the impact on healing.

The COVID-19 pandemic has caused a high demand for filtering facepiece respirators (FFRs), which has brought global challenges in sustaining the supply chain for FFRs. Because respirators are basic personal protective equipment to protect frontline healthcare workers against COVID-19, the chronic, global shortage of N95/N99 masks is one of the most urgent threats to our collective ability to save lives from the coronavirus. The reuse of masks may need to be considered as a crisis capacity strategy to ensure continued availability even though most of the masks are considered one-time use. Moreover, environmentalists warn that single-use masks add to the glut of plastic pollution, threatening the health of oceans and marine life. In this study, we develop a method to decontaminate respirators to reuse filtering facepiece respirators. Samples of SARS-CoV-2 are applied to the 4 × 4 cm2 samples of FFP2 and FFP3 respirator materials. The filtration efficiency of plasma treated samples is measured using a planar particle image velocimetry technique with a neutrally charged polydisperse aerosol particle of NaCl. The measured viral decontamination and filtration efficiencies show that the developed plasma decontamination system can achieve a 4-log reduction for the coronavirus without reducing the filtration efficiency of masks after 5-min plasma exposure. The developed plasma decontamination system demonstrates the feasibility to tackle the acute shortages of FFRs in many countries and their environmental and economic burdens against discarding reusable masks.

In this study, we investigated the effects of an ultrasonically activated stream (UAS) on the removal of microbial contaminants from spinach leaves. The microbial loads on samples cleaned with and without UAS were enumerated using the cell culture method and compared against unwashed samples on day 0 and day 6 after cleaning. The effects of UAS cleaning on leaf quality were also examined through both macroscopic and microscopic inspection, as well as measurement of the electrolyte leakage rate. Results showed that the microbial load on samples cleaned with UAS for 2 min was significantly lower on day 6 after cleaning than on those treated without ultrasound. Comparison between the cleaning effects of UAS for 40 s versus 2 min indicated that a cleaning duration of 2 min allowed sufficient time for UAS to disaggregate and detach the microbial contamination more effectively. In this case, the induction of bacteria into a viable but non-culturable state does not affect the shelf-life test results as much as it does with a 40 s clean. UAS cleaning for 2 min did not produce significant surface damage, which can affect overall leaf quality. These findings highlight the potential of UAS systems in the salad industry to improve the microbiological quality and shelf life of salads.