Effectiveness of a spontaneous carvacrol nanoemulsion against Salmonella enterica Enteritidis and Escherichia coli O157:H7 on contaminated broccoli and radish seeds.

The incidence of foodborne illness associated with the consumption of fresh produce has continued to increase over the past decade. Sprouts, such as mung bean, alfalfa, radish, and broccoli, are minimally processed and have been sources for foodborne illness. Currently, a 20,000 ppm calcium hypochlorite soak is recommended for the treatment of sprouting seeds. In this study, the efficacy of an antimicrobial carvacrol nanoemulsion was tested against Salmonella enterica subspecies enterica serovar Enteritidis (ATCC BAA-1045) or EGFP expressing Escherichia coli O157:H7 (ATCC 42895) contaminated sprouting seeds. Antimicrobial treatments were performed by soaking inoculated seeds in nanoemulsions (4000 or 8000 ppm) for 30 or 60 min. Following treatment, surviving cells were determined by performing plate counts and/or Most Probable Number (MPN) enumeration. Treated seeds were sprouted and tested for the presence of pathogens. Treatment successfully inactivated low levels (2 and 3 log CFU/g) of S. Enteritidis and E. coli on radish seeds when soaked for 60 min at concentrations ≥4000 (0.4%) ppm carvacrol. This treatment method was not affective on contaminated broccoli seeds. Total sprout yield was not influenced by any treatments. These results show that carvacrol nanoemulsions may be an alternative treatment method for contaminated radish seeds.

Purification and characterization of iso-ribonucleases from a novel thermophilic fungus.

A thermophilic fungus previously isolated from composted horse manure was found to produce extracellular iso-RNases that were purified 127.6-fold using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined electrophoretically using 4%-15% gradient polyacrylamide gels. RNase activity was dependent on the presence of a metal co-factor with significantly more activity with Zn2+ or Mn2+ than Mg2+. The RNases exhibited maximum activity at both pH 3.0 and pH 7.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the iso-RNase was 70 °C. The molecular weight of the iso-RNase was determined to be 69 kDa using a Sephadex G-75 column.

Purification of an inducible DNase from a thermophilic fungus.

The ability to induce an extracellular DNase from a novel thermophilic fungus was studied and the DNAse purified using both traditional and innovative purification techniques. The isolate produced sterile hyphae under all attempted growing conditions, with an average diameter of 2 µm and was found to have an optimal temperature of 45 °C and a maximum of 65 °C. Sequencing of the internal transcribed region resulted in a 91% match with Chaetomium sp., suggesting a new species, but further clarification on this point is needed. The optimal temperature for DNase production was found to be 55 °C and was induced by the presence of DNA and/or deoxyribose. Static growth of the organism resulted in significantly higher DNase production than agitated growth. The DNase was purified 145-fold using a novel affinity membrane purification system with 25% of the initial enzyme activity remaining. Electrophoresis of the purified enzyme resulted in a single protein band, indicating DNase homogeneity.

Oral Dysbiosis and Neurodegenerative Diseases: Correlations and Potential Causations.

Biofilms are a heterogenous complex community of vegetative cells and extracellular polymeric substances that can adhere to various surfaces and are responsible for a variety of chronic and acute diseases. The impact of bacterial biofilms on oral and intestinal health is well studied, but the correlation and causations of biofilms and neurodegenerative diseases are still in their infancy. However, the correlations between biofilms and diseases such as Alzheimer's Disease, Multiple Sclerosis, and even Parkinson's Disease are starting to demonstrate the role bacterial biofilms have in promoting and exasperating various illnesses. The review article provides insight into the role bacterial biofilms may have on the development and progression of various neurodegenerative diseases and hopefully shine a light on this very important area of research.

Draft Genome Sequences of Six Strains Isolated from the InSight Spacecraft and Associated Surfaces Using Oxford Nanopore- and Illumina-Based Sequencing.

Whole-genome sequencing and annotation have allowed planetary protection engineers to assess the functional capabilities of microorganisms isolated from spacecraft hardware and associated surfaces. Here, we report draft genomes of six strains isolated from the InSight mission, determined using Oxford Nanopore- and Illumina-based sequencing.

Biofilms-Impacts on Human Health and Its Relevance to Space Travel.

As the world looks towards the stars, the impacts of endogenous and exogenous microorganisms on human health during long-duration space flight are subjects of increased interest within the space community. The presence and continued growth of bacterial biofilms about spacecraft has been documented for decades; however, the impact on crew health is in its infancy. The impacts of biofilms are well known in the medical, agricultural, commercial, and industrial spaces. It less known that biofilms are undermining many facets of space travel and that their effects need to be understood and addressed for future space missions. Biofilms can damage space crew health and spoil limited food supply. Yet, at the same time, they can benefit plant systems for food growth, nutrient development, and other biological systems that are being explored for use in space travel. Various biofilm removal techniques have been studied to mitigate the hazards posed by biofilm persistence during space travel. Because the presence of biofilms can advance or hinder humanity's space exploration efforts, an understanding of their impacts over the duration of space flights is of paramount importance.

Inactivation of Salmonella on Sprouting Seeds Using a Spontaneous Carvacrol Nanoemulsion Acidified with Organic Acids.

Over the past decade, demand has increased for natural, minimally processed produce, including sprout-based products. Sanitization with 20,000 ppm of calcium hypochlorite is currently recommended for all sprouting seeds before germination to limit sprout-related foodborne outbreaks. A potentially promising disinfectant as an alternative to calcium hypochlorite is acidified spontaneous essential oil nanoemulsions. In this study, the efficacy of an acidified carvacrol nanoemulsion was tested against mung beans and broccoli seeds artificially contaminated with a Salmonella enterica Enteritidis cocktail (ATCC BAA-709, ATCC BAA-711, and ATCC BAA-1045). Treatments were performed by soaking inoculated seeds in acidified (50 mM acetic or levulinic acid) carvacrol nanoemulsions (4,000 or 8,000 ppm) for 30 or 60 min. After treatment, the number of surviving cells was determined via plate counts and/or the most probable number (MPN) approach. Treatment for 30 min successfully reduced Salmonella Enteritidis by 4 log CFU/g on mung beans (from an initial contamination level of 4.2 to 4.6 log CFU/g) and by 2 log CFU/g on broccoli seeds (from an initial contamination level of 2.4 to 2.6 log CFU/g) to below our detection limit (≤3 MPN/g). Treated seeds were sprouted and tested for the presence of pathogens and sprout yield. The final sprout product had no detectable pathogens, and total sprout yield was not influenced by any treatment.

Development of a novel affinity membrane purification system for deoxyribonuclease.

A membrane based affinity purification system was developed for the purification of the DNA specific nuclease, DNase I. Single stranded DNA was bound to unmodified polyvinylidene fluoride (PVDF) membranes which were used to purify DNase I from a solution of bovine serum albumin. Using coated membranes, a 6-fold increase in specific activity was achieved with 80 % enzyme recovery. This method provides a simple yet effective way to purify DNase I and can be very useful for the purification of other DNA specific enzymes.

Characterization of a recently purified thermophilic DNase from a novel thermophilic fungus.

A newly isolated thermophilic fungus was found to produce a partially inducible extracellular DNase. This manuscript focuses on the characterization of this novel thermophilic DNase in terms of optimal enzyme conditions, molecular weight, and certain kinetic properties. The DNase was found to be inactivated by the presence of EDTA demonstrating its dependence on metal cofactors for activity. Maximum activity occurred at pH 6.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the purified DNase was 65 °C. The thermophilic DNase was found to be an exonuclease with an estimated molecular weight of 56 kDa.

Effectiveness of a novel spontaneous carvacrol nanoemulsion against Salmonella enterica Enteritidis and Escherichia coli O157:H7 on contaminated mung bean and alfalfa seeds.

Outbreaks of foodborne illness from consumption of sprouts have been linked to contaminated seeds prior to germination. Due to the long sprouting period at ambient temperatures and high humidity, germinating seeds contaminated with low pathogen levels (0.1logCFU/g) can result in sprouts with high numbers (≥10(8)CFU/g) of pathogens. Currently, the recommended treatment method involves soaking seeds in 20,000ppm (2%) calcium hypochlorite prior to germination. In this study, an alternative treatment involving soaking seeds in a carvacrol nanoemulsion was tested for its efficacy against Salmonella enterica subspecies enterica serovar Enteritidis (ATCC BAA-1045) or EGFP expressing E. coli O157:H7 (ATCC 42895) contaminated mung bean and alfalfa seeds. The antimicrobial treatment was performed by soaking inoculated seed batches in the spontaneous nanoemulsion (4000 or 8000ppm) for 30 or 60min. The spontaneous nanoemulsion was formed by titrating the oil phase (carvacrol and medium chain triglycerides) and water-soluble surfactant (Tween 80®) into sodium citrate buffer. Following treatment, the numbers of surviving cells were determined by suspending the seeds in TSB and performing plate counts and/or Most Probable Number (MPN) enumeration. Treated seeds were sprouted and tested for the presence of the appropriate pathogen. This treatment successfully inactivated low levels (2 and 3logCFU/g) of S. Enteritidis and E. coli on either seed types when soaked for either 30 or 60min at nanoemulsion concentrations corresponding to 4000 (0.4%) or 8000 (0.8%) ppm carvacrol. Inoculated alfalfa seeds treated with 4000ppm nanoemulsion, required a 60min treatment time to show a similar 2-3 log reduction. Complete inactivation was confirmed by germinating treated seeds and performing microbiological testing. Total sprout yield was not compromised by any of the tested treatments. These results show that carvacrol nanoemulsions may be an alternative antimicrobial treatment method for mung bean and alfalfa seeds.