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.

Lactoferrin Is Broadly Active against Yeasts and Highly Synergistic with Amphotericin B.

Lactoferrin (LF) is a multifunctional milk protein with antimicrobial activity against a range of pathogens. While numerous studies report that LF is active against fungi, there are considerable differences in the level of antifungal activity and the capacity of LF to interact with other drugs. Here we undertook a comprehensive evaluation of the antifungal spectrum of activity of three defined sources of LF across 22 yeast and 24 mold species and assessed its interactions with six widely used antifungal drugs. LF was broadly and consistently active against all yeast species tested (MICs, 8 to 64 µg/ml), with the extent of activity being strongly affected by iron saturation. LF was synergistic with amphotericin B (AMB) against 19 out of 22 yeast species tested, and synergy was unaffected by iron saturation but was affected by the extent of LF digestion. LF-AMB combination therapy significantly prolonged the survival of Galleria mellonella wax moth larvae infected with Candida albicans or Cryptococcus neoformans and decreased the fungal burden 12- to 25-fold. Evidence that LF directly interacts with the fungal cell surface was seen via scanning electron microscopy, which showed pore formation, hyphal thinning, and major cell collapse in response to LF-AMB synergy. Important virulence mechanisms were disrupted by LF-AMB treatment, which significantly prevented biofilms in C. albicans and C. glabrata, inhibited hyphal development in C. albicans, and reduced cell and capsule size and phenotypic diversity in Cryptococcus Our results demonstrate the potential of LF-AMB as an antifungal treatment that is broadly synergistic against important yeast pathogens, with the synergy being attributed to the presence of one or more LF peptides.

Lactoferrin-Derived Peptide Lactofungin Is Potently Synergistic with Amphotericin B.

Lactoferrin (LF) is an iron-binding glycoprotein with broad-spectrum antimicrobial activity. Previously, we discovered that LF synergistically enhanced the antifungal efficacy of amphotericin B (AMB) across a variety of yeast species and subsequently hypothesized that this synergy was enhanced by the presence of small peptides derived from the whole LF molecule. In this study, LF was digested with pepsin under a range of conditions. The resulting hydrolysates exhibited enhanced synergy with AMB compared to its synergy with undigested LF. Samples were analyzed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and 14 peptides were identified. The sequences of these peptides were predicted by matching their molecular weights to those of a virtual digest with pepsin. The relative intensities of predicted peptides in each hydrolysate were compared with the activity of the hydrolysate, and the structural and physicochemical properties of the peptides were assessed. From this, a 30-residue peptide was selected for synthesis and dubbed lactofungin (LFG). Pure LFG was highly synergistic with AMB, outperforming native LF in all fungal species tested. With potential for further structural and chemical improvements, LFG is an excellent lead for development as an antifungal adjuvant.

Cryptococcus strains with different pathogenic potentials have diverse protein secretomes.

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.

Multilocus variable-number tandem-repeat analysis of clinical isolates of Aspergillus flavus from Iran reveals the first cases of Aspergillus minisclerotigenes associated with human infection.

BACKGROUND: Aspergillus flavus is intensively studied for its role in infecting crop plants and contaminating produce with aflatoxin, but its role as a human pathogen is less well understood. In parts of the Middle East and India, A. flavus surpasses A. fumigatus as a cause of invasive aspergillosis and is a significant cause of cutaneous, sinus, nasal and nail infections. METHODS: A collection of 45 clinical and 10 environmental A. flavus isolates from Iran were analysed using Variable-Number Tandem-Repeat (VNTR) markers with MICROSAT and goeBURST to determine their genetic diversity and their relatedness to clinical and environmental A. flavus isolates from Australia. Phylogeny was assessed using partial ß-tubulin and calmodulin gene sequencing, and mating type was determined by PCR. Antifungal susceptibility testing was performed on selected isolates using a reference microbroth dilution method. RESULTS: There was considerable diversity in the A. flavus collection, with no segregation on goeBURST networks according to source or geographic location. Three Iranian isolates, two from sinus infections and one from a paranasal infection grouped with Aspergillus minisclerotigenes, and all produced B and G aflatoxin. Phylogenic analysis using partial ß-tubulin and calmodulin sequencing confirmed two of these as A. minisclerotigenes, while the third could not be differentiated from A. flavus and related species within Aspergillus section flavi. Based on epidemiological cut-off values, the A. minisclerotigens and A. flavus isolates tested were susceptible to commonly used antifungal drugs. CONCLUSIONS: This is the first report of human infection due to A. minisclerotigenes, and it raises the possiblity that other species within Aspergillus section flavi may also cause clinical disease. Clinical isolates of A. flavus from Iran are not distinct from Australian isolates, indicating local environmental, climatic or host features, rather than fungal features, govern the high incidence of A. flavus infection in this region. The results of this study have important implications for biological control strategies that aim to reduce aflatoxin by the introduction of non-toxigenic strains, as potentially any strain of A. flavus, and closely related species like A. minisclerotigenes, might be capable of human infection.

A photosynthetic alveolate closely related to apicomplexan parasites.

Many parasitic Apicomplexa, such as Plasmodium falciparum, contain an unpigmented chloroplast remnant termed the apicoplast, which is a target for malaria treatment. However, no close relative of apicomplexans with a functional photosynthetic plastid has yet been described. Here we describe a newly cultured organism that has ultrastructural features typical for alveolates, is phylogenetically related to apicomplexans, and contains a photosynthetic plastid. The plastid is surrounded by four membranes, is pigmented by chlorophyll a, and uses the codon UGA to encode tryptophan in the psbA gene. This genetic feature has been found only in coccidian apicoplasts and various mitochondria. The UGA-Trp codon and phylogenies of plastid and nuclear ribosomal RNA genes indicate that the organism is the closest known photosynthetic relative to apicomplexan parasites and that its plastid shares an origin with the apicoplasts. The discovery of this organism provides a powerful model with which to study the evolution of parasitism in Apicomplexa.

Epidemiologic analysis of change in eyelash characteristics with increasing age in a population of healthy women.

BACKGROUND: Observations that eyelashes become thinner, shorter, and lighter, as women age has not been previously quantified. OBJECTIVE: This study was conducted to investigate associations between eyelash characteristics and age. MATERIALS AND METHODS: The upper natural eyelashes of 179 subjects were photographed and analyzed (digital image analysis); length, thickness, and darkness (intensity: 0 = white and 255 = black) were calculated. Linear regression, including race as a potentially confounding factor, was used to assess the association between age and mean eyelash characteristics. RESULTS: Subjects' mean age was 40.3 (±10.3) years; 46.1% were white, 36.5% Asian, 9.0% Hispanic, 5.1% East Indian, and 3.4% black. Mean eyelash length ranged from 6.39 (±1.02) to 7.98 (±1.15) mm (subjects aged 50-65 years and 22-29 years, respectively). Mean thickness ranged from 1.17 (±0.42) to 1.62 (±0.56) mm (subjects aged 50-65 years and 20-29 years, respectively). Mean intensity ranged from 118.2 (±19.8) to 129.4 (±17.3) (subjects aged 30-39 years and 50-65 years, respectively). Adjusted for race, eyelash length, thickness, and darkness decreased significantly with increasing age (p < .000, p = .0090, and p < .05, respectively). CONCLUSION: Advancing age among an ethnically diverse population of healthy women is associated with significant decreases in eyelash length, thickness, and darkness.

Pollen products collected from honey bee hives experiencing minor stress have altered fungal communities and reduced antimicrobial properties.

Fungi are increasingly recognized to play diverse roles within honey bee hives, acting as pathogens, mutualists, and commensals. Pollen products, essential for hive nutrition, host significant fungal communities with potential protective and nutritional benefits. In this study, we profile the fungal communities and antifungal properties of three pollen products from healthy and stressed hives: fresh pollen collected by forager bees from local plants; stored pollen packed into the comb inside the hive; and bee bread, which is stored pollen following anaerobic fermentation used for bee and larval nutrition. Using amplicon sequencing, we found significant differences in fungal community composition, with hive health and sample type accounting for 8.8% and 19.3% of variation in beta diversity, respectively. Pollen and bee bread extracts had species-specific antimicrobial activity and inhibited the fungal hive pathogens Ascosphaera apis, Aspergillus flavus, and Aspergillus fumigatus, and the bacterial hive pathogen Paenibacillus larvae. Activity was positively correlated with phenolic and antioxidant content and was diminished in stressed hives. The plant source of pollen determined by amplicon sequencing differed in stressed hives, suggesting altered foraging behaviour. These findings illustrate the complex interplay between honey bees, fungal communities, and hive products, which should be considered in hive management and conservation.

Long-term stability and the physical and chemical factors predictive for antimicrobial activity in Australian honey.

The growing burden of expired medicines contributes to environmental contamination and landfill waste accumulation. Medicinal honey, with its non-toxic nature and potentially long shelf-life, represents a promising and underutilised therapeutic that avoids some of these issues. However, limited knowledge on how its antimicrobial properties change over time combined with a lack of reliable processes in the honey industry for measuring antimicrobial potential, hinder its clinical adoption. Using a diverse selection of 30 Australian honey samples collected between 2005 and 2007, we comprehensively evaluated their antibacterial and antifungal activity and pertinent physical and chemical properties with the aims of assessing the effect of long-term storage on activity, pinpointing factors associated with antimicrobial efficacy, and establishing robust assessment methods. Minimum inhibitory concentration (MIC) assays proved superior to the standard phenol equivalence assay in capturing the full range of antimicrobial activity present in honey. Correlations between activity and a range of physical and chemical properties uncovered significant associations, with hydrogen peroxide, antioxidant content, and water activity emerging as key indicators in non-Leptospermum honey. However, the complex nature and the diverse composition of honey samples precludes the use of high-throughput chemical tests for accurately assessing this activity, and direct assessment using live microorganisms remains the most economical and reliable method. We provide recommendations for different methods of assaying various honey properties, taking into account their accuracy along with technical difficulty and safety considerations. All Leptospermum and fourteen of seventeen non-Leptospermum honey samples retained at least some antimicrobial properties after 15-17 years of storage, suggesting that honey can remain active for extended periods. Overall, the results of this study will help industry meet the growing demand for high-quality, medicinally active honey while ensuring accurate assessment of its antimicrobial potential.

The effects of season and soil type on microbial degradation of gasoline residues from incendiary devices.

The primary task of a fire debris chemist is to determine if there is an ignitable liquid present in a fire debris sample and, if so, to classify it according to its boiling point and carbon number range. However, in organic-rich substrates such as soil, the ignitable liquid residue is subject to microbial degradation due to the ease with which bacteria can metabolize the various hydrocarbons present. This is a rapid process which is problematic in many forensic laboratories as fire debris is often stored for extended periods of time due to case backlog. Although microbial degradation has been studied in laboratory samples, it has not been well-studied in "real-world" samples, which have not only been exposed to microbial degradation but have also suffered the effects of weathering due to the intense heat of the fire. In this work, the effects of microbial degradation of gasoline from an incendiary device have been evaluated over time. In addition to visually monitoring chromatographic changes, this work also utilizes multivariate statistical techniques to simplify the complex data set and elucidate trends that might not otherwise be observed. Results indicate a clear difference between glass samples, which suffered the loss of low boiling compounds, and soil, which suffered the loss of the normal alkanes and lesser substituted aromatics. Also, devices deployed on lawn soil and in the winter season appear to show the most extensive degradation of gasoline. Finally, while the ratio of the C(3)-alkylbenzenes is significantly altered in soil samples recovered from large devices, the overall chromatographic profile of gasoline recovered from smaller incendiary devices is significantly lower.

Unique antimicrobial activity in honey from the Australian honeypot ant (Camponotus inflatus).

Honey produced by the Australian honeypot ant (Camponotus inflatus) is valued nutritionally and medicinally by Indigenous peoples, but its antimicrobial activity has never been formally studied. Here, we determine the activity of honeypot ant honey (HPAH) against a panel of bacterial and fungal pathogens, investigate its chemical properties, and profile the bacterial and fungal microbiome of the honeypot ant for the first time. We found HPAH to have strong total activity against Staphylococcus aureus but not against other bacteria, and strong non-peroxide activity against Cryptococcus and Aspergillus sp. When compared with therapeutic-grade jarrah and manuka honey produced by honey bees, we found HPAH to have a markedly different antimicrobial activity and chemical properties, suggesting HPAH has a unique mode of antimicrobial action. We found the bacterial microbiome of honeypot ants to be dominated by the known endosymbiont genus Candidatus Blochmannia (99.75%), and the fungal microbiome to be dominated by the plant-associated genus Neocelosporium (92.77%). This study demonstrates that HPAH has unique antimicrobial characteristics that validate its therapeutic use by Indigenous peoples and may provide a lead for the discovery of novel antimicrobial compounds.

Low Levels of Hive Stress Are Associated with Decreased Honey Activity and Changes to the Gut Microbiome of Resident Honey Bees.

Honey bees (Apis mellifera) face increasing threats to their health, particularly from the degradation of floral resources and chronic pesticide exposure. The properties of honey and the bee gut microbiome are known to both affect and be affected by bee health. Using samples from healthy hives and hives showing signs of stress from a single apiary with access to the same floral resources, we profiled the antimicrobial activity and chemical properties of honey and determined the bacterial and fungal microbiome of the bee gut and the hive environment. We found honey from healthy hives was significantly more active than honey from stressed hives, with increased phenolics and antioxidant content linked to higher antimicrobial activity. The bacterial microbiome was more diverse in stressed hives, suggesting they may have less capacity to exclude potential pathogens. Finally, bees from healthy and stressed hives had significant differences in core and opportunistically pathogenic taxa in gut samples. Our results emphasize the need for understanding and proactively managing bee health. IMPORTANCE Honey bees serve as pollinators for many plants and crops worldwide and produce valuable hive products such as honey and wax. Various sources of stress can disrupt honey bee colonies, affecting their health and productivity. Growing evidence suggests that honey is vitally important to hive functioning and overall health. In this study, we determined the antimicrobial activity and chemical properties of honey from healthy hives and hives showing signs of stress, finding that honey from healthy hives was significantly more antimicrobial, with increased phenolics and antioxidant content. We next profiled the bacterial and fungal microbiome of the bee gut and the hive environment, finding significant differences between healthy and stressed hives. Our results underscore the need for greater understanding in this area, as we found even apparently minor stress can have implications for overall hive fitness as well as the economic potential of hive products.

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.

A randomized clinical trial of on-demand oral pre-exposure prophylaxis does not modulate lymphoid/myeloid HIV target cell density in the foreskin.

OBJECTIVES: As topical pre-exposure prophylaxis (PrEP) has been shown to cause immune modulation in rectal or cervical tissue, our aim was to examine the impact of oral PrEP on lymphoid and myeloid changes in the foreskin in response to dosing and timing of drug administration. DESIGN: HIV-negative male individuals ( n  = 144) were recruited in South Africa and Uganda into an open-label randomized controlled trial in a 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 ratio to control arm (with no PrEP) or one of eight arms receiving emtricitabine-tenofovir disoproxil fumarate (F/TDF) or emtricitabine-tenofovir alafenamide (F/TAF) at one of two different doses, 5 or 21 h before undergoing voluntary medical male circumcision (VMMC). METHODS: After dorsal-slit circumcision, foreskin tissue sections were embedded into Optimal Cutting Temperature media and analysed, blinded to trial allocation, to determine numbers of CD4 + CCR5 + , CD1a + cells and claudin-1 expression. Cell densities were correlated with tissue-bound drug metabolites and p24 production after ex-vivo foreskin challenge with HIV-1 bal . RESULTS: There was no significant difference in CD4 + CCR5 + or CD1a + cell numbers in foreskins between treatment arms compared with the control arm. Claudin-1 expression was 34% higher ( P  = 0.003) in foreskin tissue from participants receiving PrEP relative to controls, but was no longer statistically significant after controlling for multiple comparisons. There was neither correlation of CD4 + CCR5 + , CD1a + cell numbers, or claudin-1 expression with tissue-bound drug metabolites, nor with p24 production after ex-vivo viral challenge. CONCLUSION: Oral doses and timing of on-demand PrEP and in-situ drug metabolite levels in tissue have no effect on numbers or anatomical location of lymphoid or myeloid HIV target cells in foreskin tissue.

Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.