Broad-Spectrum Supramolecularly Reloadable Antimicrobial Coatings.

Antimicrobial surfaces limit the spread of infectious diseases. To date, there is no antimicrobial coating that has widespread use because of short-lived and limited spectrum efficacy, poor resistance to organic material, and/or cost. Here, we present a paint based on waterborne latex particles that is supramolecularly associated with quaternary ammonium compounds (QACs). The optimal supramolecular pairing was first determined by immobilizing selected ions on self-assembled monolayers exposing different groups. The QAC surface loading density was then increased by using polymer brushes. These concepts were adopted to develop inexpensive paints to be applied on many different surfaces. The paint could be employed for healthcare and food production applications. Its slow release of QAC allows for long-lasting antimicrobial action, even in the presence of organic material. Its efficacy lasts for more than 90 washes, and importantly, once lost, it can readily be restored by spraying an aqueous solution of the QAC. We mainly tested cetyltrimethylammonium as QAC as it is already used in consumer care products. Our antimicrobial paint is broad spectrum as it showed excellent antimicrobial efficiency against four bacteria and four viruses.

Non-thermal plasma inactivation of Salmonella Typhimurium on different matrices and the effect of selected food components on its bactericidal efficacy.

Non-thermal plasma (NTP) is known as an effective source of a variety of reactive species generated in the gas phase. Nowadays, NTP is gaining increasing interest from the food industry as a microbial inactivation technique. In the present study the effect of inoculation method and matrix on inactivation of Salmonella Typhimurium was examined by treating spread plated agar (2.2 log CFU/sample inactivation by NTP), spot inoculated agar (1.9 log CFU inactivation), glass beads (1.3 log CFU inactivation) and peppercorn (0.2 log CFU inactivation). Furthermore, multiple agar matrices supplemented with low and high concentrations of a certain food component (casein, starch, sunflower oil, vitamin C, sodium pyruvate or grinded peppercorns) were inoculated and treated to determine the effect of those components on NTP efficiency. Although starch, vitamin C and sodium pyruvate had no significant influence on the inactivation degree, the presence of 10% casein (2.1 log CFU/sample less inactivation compared to tryptone soy agar (TSA)), 10% pepper (2.1 log CFU less inactivation) or 1% and 10% sunflower oil (1.6 and 2.1 log CFU less inactivation, respectively) in TSA demonstrated the protective effect of these substances for NTP treatment. These experiments led to the conclusion that low inactivation on produce seemed not to arise from the inoculation method nor from the shape of the produce, but is the result of the food matrix.

Can a combination of vaccination and face mask wearing contain the COVID-19 pandemic?

The COVID-19 pandemic is going into its third year with Europe again being the focus of major epidemic activity. The present review tries to answer the question whether one can come to grip with the pandemic by a combination of vaccinations and non-pharmaceutical interventions (NPIs). Several COVID-19 vaccines are of remarkable efficacy and achieve high protection rates against symptomatic disease, especially severe disease, but mathematical models suggest that the current vaccination coverage in many countries is insufficient to achieve pandemic control. NPIs are needed as complementary measures because recent research has also revealed the limits of vaccination alone. Here, we review the evidence for efficacy of face mask wearing in various settings. Overall pooled analysis showed significant reduction in COVID-19 incidence with mask wearing, although heterogeneity between studies was substantial. Controlled trials of mask wearing are difficult to conduct, separating mask wearing effects in population studies from the impact of other NPIs is challenging and the efficacy of masks depend on mask material and mask fit. The combination of vaccination and mask wearing is potentially synergistic since vaccination protects so far well from disease development (the omicron variant is currently an unknown) but immunity from infection wanes over few months after vaccination. In comparison, masks interfere with the virus transmission process at a level of a physical barrier independent of coronavirus variant. Vaccination and masks are much less costly to apply than other NPI measures which are associated with high economic and social costs, but paradoxically both measures are the target of a vocal opposition by a sizable minority of the society. In parallel with biomedical research, we need more social science research into this opposition to guide political decisions on how to end the pandemic.

Electron Beam Susceptibility of Enteric Viruses and Surrogate Organisms on Fruit, Seed and Spice Matrices.

The objective of this study was to use high-energy electron beam (HEEB) treatments to find surrogate microorganisms for enteric viruses and to use the selected surrogates as proof of concept to investigate low-energy electron beam (LEEB) treatments for enteric virus inactivation at industrial scale on frozen blueberries. Six food matrices inoculated with HAV (hepatitis A virus), MNV S99 (murine norovirus), bacteriophages MS2 and Qß, and Geobacillus stearothermophilus spores were treated with HEEB at 10 MeV using 4, 8 and 16 kGy doses. G. stearothermophilus spores showed the highest inactivation on all matrices except on raisins, with a dose-dependent effect. HAV reached the maximum measurable log10 reduction (> 3.2 log10) when treated at 16 kGy on raisins. MNV showed the highest resistance of all tested microorganisms, independent of the dose, except on frozen blueberries. On frozen blueberries, freeze-dried raspberries, sesame seeds and black peppercorns, HAV showed a mean inactivation level in between those of MS2 and G. stearothermophilus. Based on this, we selected both surrogate organisms as first approximation to estimate HAV inactivation on frozen blueberries during LEEB treatment at 250 keV using 16 kGy. Reductions of 3.1 and 1.3 log10 were measured for G. stearothermophilus spores and MS2, respectively, suggesting that a minimum reduction of 1.4 log10 can be expected for HAV under the same conditions.

COVID 19: challenges for virologists in the food industry.

The COVID-19 pandemic is not only a challenge for public health and hospitals, but affects many aspects of our societies. This Lilliput minireview deals with problems that the pandemic causes for the food industry, addressing the presence and persistence of SARS-CoV-2 in the food environment, methods of virus inactivation and the protection of the food worker and the consumer. So far food has not been implicated in the transmission of the infection, but social disruptions caused by the pandemic could cause problems with food security.

Inactivation by osmotic dehydration and air drying of Salmonella, Shiga toxin-producing Escherichia coli, Listeria monocytogenes, hepatitis A virus and selected surrogates on blueberries.

Osmotically dehydrated and air dried berry fruits are used as ingredients for the production of yoghurts, chocolates, cereal bars and mixes, ice creams and cakes and these fruits are often subjected to mild thermal treatments only, posing questions around their microbiological safety. As osmotic dehydration methods and parameters vary considerably within the industry and minimally processed high quality fruits are increasingly sought, the scope of this study was to determine which temperatures are required for the inactivation of relevant bacteria and viruses during osmotic dehydration of berries, using blueberries as a model berry in a thawed state to mimic common industrial practices. Additionally, we studied the inactivation of osmotic dehydration at 23 °C, sometimes referred to "cold infusion" followed by air drying at 100 °C to determine the microbiological safety achieved by this combined treatment. Four pathogens (Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes and hepatitis A virus (HAV)) and five surrogates (Enterococcus faecium, Escherichia coli P1, Listeria innocua, murine Norovirus (MNV) and bacteriophage MS2) were inoculated on blueberries and reductions were measured after different treatment combinations. After osmotic dehydration of bacterial strains at 40 °C no survivors were detected on blueberries, with the exception of E. faecium. Inactivation of the viruses at 45 °C showed no survivors for MS2 and mean reductions of 1.5 and 3.4 log10 median tissue culture infectious dose (TCID50)/g for HAV and MNV, respectively. Similarly, in the sugar solution at 40 °C, no survivors were observed, with the exception of E. faecium and the three viruses. The combined process (osmotic dehydration at 23 °C followed by air-drying at 100 °C) achieved an >6 log reduction of all tested bacterial strains and MS2. For HAV and MNV, 2.6 and >3.4 log10 TCID50/g were measured. In summary, the present study shows that osmotic dehydration appears an efficient control measure for the control of L. monocytogenes, S. enterica and E. coli O157:H7 if carried out at 40 °C or at 23 °C and followed by air-drying at 100 °C. Based on the results generated with MNV, the combined treatment is also expected to reduce human Norovirus (NoV) but does not appear to be sufficient to fully control HAV. The results contribute to a better management of the microbial safety of osmotically dehydrated and dried berries and especially the results generated for the viruses emphasize that within a robust food safety management system, safety must be assured through the entire food supply chain and therefore must start at primary production with the implementation of Good Agricultural Practices (GAP).

Foodborne viruses: Detection, risk assessment, and control options in food processing.

In a recent report by risk assessment experts on the identification of food safety priorities using the Delphi technique, foodborne viruses were recognized among the top rated food safety priorities and have become a greater concern to the food industry over the past few years. Food safety experts agreed that control measures for viruses throughout the food chain are required. However, much still needs to be understood with regard to the effectiveness of these controls and how to properly validate their performance, whether it is personal hygiene of food handlers or the effects of processing of at risk foods or the interpretation and action required on positive virus test result. This manuscript provides a description of foodborne viruses and their characteristics, their responses to stress and technologies developed for viral detection and control. In addition, the gaps in knowledge and understanding, and future perspectives on the application of viral detection and control strategies for the food industry, along with suggestions on how the food industry could implement effective control strategies for viruses in foods. The current state of the science on epidemiology, public health burden, risk assessment and management options for viruses in food processing environments will be highlighted in this review.

UV-C inactivation of foodborne bacterial and viral pathogens and surrogates on fresh and frozen berries.

Outbreaks of foodborne illness associated with berries often involve contamination with hepatitis A virus (HAV) and norovirus but also bacteria such as Escherichia coli O157:H7 and parasites such as Cyclospora caytanensis. We evaluated the applicability of UV-C to the inactivation of pathogens on strawberries, raspberries and blueberries. Our three-step approach consisted of assessing the chemical safety of UV-C-irradiated berries, evaluating the sensory quality after UV-C treatment and finally studying the inactivation of the target microorganisms. Treatments lasting up to 9 min (4000 mJ cm-2) did not produce detectable levels of furan (<5 µg/kg), a known photolysis product of fructose with genotoxic activity and thus were assessed to be toxicologically safe. No effect on taste or appearance was observed, unless treatment was excessively long. 20 s of treatment (an average fluence of ~ 212 mJ cm-2) reduced active HAV titer by >1 log10 unit in 95% of cases except on frozen raspberries, while 120 s were required to inactivate murine norovirus to this extent on fresh blueberries. The mean inactivation of HAV and MNV was greater on blueberries (2-3 log10) than on strawberries and raspberries (<2 log10). MNV was more sensitive on fresh than on frozen berries, unlike HAV. Inactivation of Salmonella, E. coli O157:H7 and Listeria monocytogenes was poor on all three berries, no treatment reducing viable counts by >1 log10 unit. In most matrices, prolonging the treatment did not improve the result to any significant degree. The effect was near its plateau after 20 s of treatment. These results provide insight into the effectiveness of UV-C irradiation for inactivating bacterial and viral pathogens and surrogates on fresh and frozen berries having different surface types, under different physical conditions and at different levels of contamination. Overall they show that UV-C as single processing step is unsuitable to inactivate significant numbers of foodborne pathogens on berries.

Inactivation of viruses and bacteria on strawberries using a levulinic acid plus sodium dodecyl sulfate based sanitizer, taking sensorial and chemical food safety aspects into account.

The efficacy of levulinic acid (LVA) in combination with sodium dodecyl sulfate (SDS) in removal of foodborne viruses, enteric bacterial pathogens and their surrogates on fresh strawberries was investigated. Inoculated strawberries were treated with potable water, sodium hypochlorite solution (50ppm), 0.5% LVA plus 0.5% SDS solution, and 5% LVA plus 2% SDS solution respectively for 2min, followed by spray-rinsing with potable water. Water washing removed at least 1.0-log of the tested viral and bacterial strains from the strawberries' surfaces. The 50ppm chlorine wash induced 3.4, 1.5 and 2.1-log reductions for hepatitis A virus (HAV), murine norovirus-1 (MNV-1) and MS2 bacteriophage, respectively. In comparison, the tested bacterial strains showed uniform reductions around 1.6-log CFU/ml. The 0.5% LVA plus 0.5% SDS wash induced 2.7, 1.4 and 2.4-log reductions for HAV, MNV-1 and MS2, which were comparable with the reductions induced by chlorine (P>0.05). For bacteria, over 2.0-log reductions were obtained for Enterococcus faecium, Listeria monocytogenes and Salmonella, while Escherichia coli O157:H7 and Escherichia coli P1 showed reductions of 1.9 and 1.8-log CFU/ml. Higher concentration of LVA plus SDS showed no significantly higher reductions (P>0.05). Sensory tests of washed strawberries and chemical residue analysis of LVA on strawberries after washing were also performed. In conclusion, this study demonstrates good performance of 0.5% LVA plus 0.5% SDS to reduce the levels of enteric pathogens if present on strawberries without altering taste and introducing chemical safety issues.

Sample preparation prior to molecular amplification: complexities and opportunities.

Molecular amplification using Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) is currently considered as the gold standard to detect enteric human pathogenic viruses such as norovirus and hepatitis A virus in food and water. However, the molecular-based detection requires an adequate sampling strategy and a sample preparation specific for viruses. Sampling for enteric human viruses in water and food should not necessarily follow bacterial sampling plans. The development of a reference detection method including sample preparation as proposed in ISO/TS 15216 represents a milestone to facilitate the evaluation of the performance and eventually validation of future virus detection methods. The potential viral infectivity linked to a positive PCR result is a remaining issue and pretreatments allowing the differentiation of infectious viruses would be useful for future risk assessments.

Amplification and purification of T4-like escherichia coli phages for phage therapy: from laboratory to pilot scale.

We investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 10(9) to 10(10) PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-µm membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C.

T4 phages against Escherichia coli diarrhea: potential and problems.

A combination of in vitro and in vivo experiments with comparative phage genomics was used for the rational design of a phage cocktail against E. coli diarrhea. Orally applied T4 coliphages representing three different subgroups (T4-, RB49- and JS98-like phages) had no negative impact on the murine gut microbiota. T4 phages were found with high titers in the cecum and colon and lower titers in the small intestine, but were not detected in the blood, liver or spleen. No adverse effects were observed after one-month exposure to phage nor were serum anti-T4 antibodies detected. T4 phages belonging to the same subgroup showed closely related genomes that differed by 12 (phage JS10 vs. JS98 reference) to 17 (phage JSE vs. RB49 reference) insertion/deletions mostly representing single small ORFs. Bioinformatic analysis did not reveal undesired genes in the T4 genomes. Sequence variability was seen over the tail fibre genes, but the variability did not correlate with phage host range. The investigated T4 phages were not only species- but also strain-specific, necessitating the use of phage cocktails consisting of 10 and 16 T4 phage isolates to cover half to two thirds of E. coli strains representing the five main pathotypes isolated from diarrhea patients.

Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems.

Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life-threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 10(8) pfu ml(-1) of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (10(6) and 10(2) cfu ml(-1)). In contrast, broth inoculated with 10(4) phage and 10(2) bacteria per ml first showed normal bacterial growth until reaching a cell titre of 10(5) cfu ml(-1). Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml(-1). Phages could be produced with titres of 10(10) pfu ml(-1) in broth culture, but they were not stable upon freeze-drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature.

Genome analysis of phage JS98 defines a fourth major subgroup of T4-like phages in Escherichia coli.

Numerous T4-like Escherichia coli phages were isolated from human stool and environmental wastewater samples in Bangladesh and Switzerland. The sequences of the major head gene (g23) revealed that these coliphages could be placed into four subgroups, represented by the phages T4, RB69, RB49, and JS98. Thus, JS98 defines a new major subgroup of E. coli T4-like phages. We conducted an analysis of the 169-kb JS98 genome sequence. Overall, 198 of the 266 JS98 open reading frames (ORFs) shared amino acid sequence identity with the reference T4 phage, 41 shared identity with other T4-like phages, and 27 ORFs lacked any database matches. Genes on the plus strand encoded virion proteins, which showed moderate to high sequence identity with T4 proteins. The right genome half of JS98 showed a higher degree of sequence conservation with T4 and RB69, even for the nonstructural genes, than did the left genome half, containing exclusively nonstructural genes. Most of the JS98-specific genes were found in the left genome half. Two came as a hypervariability cluster, but most represented isolated genes, suggesting that they were acquired separately in multiple acquisition events. No evidence for DNA exchange between JS98 phage and the E. coli host genome or coliphages other than T4 was observed. No undesired genes which could compromise its medical use were detected in the JS98 genome sequence.

The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids.

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0.

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.

The G-protein alpha-subunit GasC plays a major role in germination in the dimorphic fungus Penicillium marneffei.

The opportunistic human pathogen Penicillium marneffei exhibits a temperature-dependent dimorphic switch. At 25 degrees, multinucleate, septate hyphae that can undergo differentiation to produce asexual spores (conidia) are produced. At 37 degrees hyphae undergo arthroconidiation to produce uninucleate yeast cells that divide by fission. This work describes the cloning of the P. marneffei gasC gene encoding a G-protein alpha-subunit that shows high homology to members of the class III fungal Galpha-subunits. Characterization of a DeltagasC mutant and strains carrying a dominant-activating gasC(G45R) or a dominant-interfering gasC(G207R) allele show that GasC is a crucial regulator of germination. A DeltagasC mutant is severely delayed in germination, whereas strains carrying a dominant-activating gasC(G45R) allele show a significantly accelerated germination rate. Additionally, GasC signaling positively affects the production of the red pigment by P. marneffei at 25 degrees and negatively affects the onset of conidiation and the conidial yield, showing that GasC function overlaps with functions of the previously described Galpha-subunit GasA. In contrast to the S. cerevisiae ortholog Gpa2, our data indicate that GasC is not involved in carbon or nitrogen source sensing and plays no major role in either hyphal or yeast growth or in the switch between these two forms.

G-protein signaling mediates asexual development at 25 degrees C but has no effect on yeast-like growth at 37 degrees C in the dimorphic fungus Penicillium mameffei.

The ascomycete Penicillium marneffei is an opportunistic human pathogen exhibiting a temperature-dependent dimorphic switch. At 25 degrees C, P. marneffei grows as filamentous multinucleate hyphae and undergoes asexual development, producing uninucleate spores. At 37 degrees C, it forms uninucleate yeast cells which divide by fission. We have cloned a gene encoding a G alpha subunit of a heterotrimeric G protein from P. marneffei named gasA with high similarity to fadA in Aspergillus nidulans. Through the characterization of a delta gasA strain and mutants carrying a dominant activating or a dominant interfering gasA allele, we show that GasA is a key regulator of asexual development but seems to play no role in the regulation of growth. A dominant activating gasA mutant whose mutation results in a G42-to-R change (gasA(G42R)) does not express brlA, the conidiation-specific regulatory gene, and is locked in vegetative growth, while a dominant interfering gasA(G203R) mutant shows inappropriate brlA expression and conidiation. Interestingly, the gasA mutants have no apparent defect in dimorphic switching or yeast-like growth at 37 degrees C. Growth tests on dibutyryl cyclic AMP (dbcAMP) and theophylline suggest that a cAMP-protein kinase A cascade may be involved in the GasA signaling pathway.