Salmonella enterica colonization and fitness in pre-harvest cantaloupe production.

Cantaloupes have emerged as significant vehicles of widespread foodborne illness outbreaks caused by bacterial pathogens, including Salmonella. The purpose of this study was to investigate the efficiency of Salmonella colonization and internalization in cantaloupes by relevant routes of contamination. Cantaloupe plants (Cucumis melo 'reticulatus') from two cultivars 'Athena' (Eastern) and 'Primo' (Western) were grown from commercial seed. Plants were maintained in the NCSU BSL-3P phytotron greenhouse. Salmonella enterica (a cocktail of cantaloupe-associated outbreak serovars Javiana, Newport, Panama, Poona and Typhimurium) contamination was introduced via blossoms or soil at ca. 4.4 log10 CFU/blossom or 8.4 log10 CFU/root zone, respectively. Cantaloupes were analyzed for Salmonella by enrichment in accordance with modified FDA-BAM methods. Five randomly chosen colonies from each Salmonella-positive sample were typed using the Agilent 2100 bioanalyzer following multiplex PCR. Data were analyzed for prevalence of contamination and serovar predominance in fruit, stems and soil. Of the total cantaloupe fruit harvested from Salmonella-inoculated blossoms (n = 63), 89% (56/63) were externally contaminated and 73% (46/63) had Salmonella internalized into the fruit. Serovar Panama was the most commonly isolated from the surface of fruit while S. Panama and S. Poona were the most prevalent inside the fruit. When soil was inoculated with Salmonella at one day post-transplant, 13% (8/60) of the plants were shown to translocate the organism to the lower stem (ca. 4 cm) by 7 days post-inoculation (dpi). We observed Salmonella persistence in the soil up to 60 dpi with S. Newport being the predominant serovar at 10 and 20 dpi. These data demonstrate that contaminated soil and blossoms can lead to Salmonella internalization into the plant or fruit at a relatively high frequency.

Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.

KEY MESSAGE: A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 'Bright Yellow-2' (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.).

KEY MESSAGE: A switchgrass protoplast system was developed, achieving a cost reduction of ~1000-fold, a threefold increase in transformation efficiency, and a fourfold reduction in required DNA quantity compared to previous methods. In recent years, there has been a resurgence in the use of protoplast systems for rapid screening of gene silencing and genome-editing targets for siRNA, miRNA, and CRISPR technologies. In the case of switchgrass (Panicum virgatum L.), to achieve economic feasibility for biofuel production, it is necessary to develop plants with decreased cell wall recalcitrance to reduce processing costs. To achieve this goal, transgenic plants have been generated with altered cell wall chemistry; however, with limited success owing to the complexity of cell walls. Because of the considerable cost, time, and effort required to screen transgenic plants, a protoplast system that can provide data at an early stage has potential to eliminate low performing candidate genes/targets prior to the creation of transgenic plants. Despite the advantages of protoplast systems, protoplast isolation in switchgrass has proven costly, requiring expensive lab-grade enzymes and high DNA quantities. In this paper, we describe a low-cost protoplast isolation system using a mesophyll culture approach and a cell suspension culture. Results from this work show a cost reduction of ~1000-fold compared to previous methods of protoplast isolation in switchgrass, with a cost of $0.003 (USD) per reaction for mesophyll protoplasts and $0.018 for axenic cell culture-derived protoplasts. Further, the efficiency of protoplast transformation was optimized threefold over previous methods, despite a fourfold reduction in DNA quantity. The methods developed in this work remove the cost barrier previously limiting high-throughput screening of genome-editing and gene silencing targets in switchgrass, paving the way for more efficient development of transgenic plants.

Origin of a novel regulatory module by duplication and degeneration of an ancient plant transcription factor.

It is commonly believed that gene duplications provide the raw material for morphological evolution. Both the number of genes and size of gene families have increased during the diversification of land plants. Several small proteins that regulate transcription factors have recently been identified in plants, including the LITTLE ZIPPER (ZPR) proteins. ZPRs are post-translational negative regulators, via heterodimerization, of class III Homeodomain Leucine Zipper (C3HDZ) proteins that play a key role in directing plant form and growth. We show that ZPR genes originated as a duplication of a C3HDZ transcription factor paralog in the common ancestor of euphyllophytes (ferns and seed plants). The ZPRs evolved by degenerative mutations resulting in loss all of the C3HDZ functional domains, except the leucine zipper that modulates dimerization. ZPRs represent a novel regulatory module of the C3HDZ network unique to the euphyllophyte lineage, and their origin correlates to a period of rapid morphological changes and increased complexity in land plants. The origin of the ZPRs illustrates the significance of gene duplications in creating developmental complexity during land plant evolution that likely led to morphological evolution.

Colonization and Internalization of Salmonella enterica and Its Prevalence in Cucumber Plants.

Consumption of cucumbers (Cucumis sativus var. sativus) has been linked to several foodborne outbreaks involving Salmonella enterica. The purpose of this work was to investigate the efficiency of colonization and internalization of S. enterica into cucumber plants by various routes of contamination. Produce-associated outbreak strains of Salmonella (a cocktail of serovars Javiana, Montevideo, Newport, Poona, and Typhimurium) were introduced to three cultivars of cucumber plants (two slicing cultivars and one pickling) via blossoms (ca. 6.4 log10 CFU/blossom, 4.5 log10 CFU/blossom, or 2.5 log10 CFU/blossom) or soil (ca. 8.3 log10 CFU/root zone) and were analyzed for prevalence of Salmonella contamination (internal and external) and serovar predominance in fruit and stems. Of the total slicing fruit harvested from Salmonella-inoculated blossoms (ca. 6.4, 4.5, or 2.5 log10 CFU/blossom), 83.9% (47/56), 81.4% (48/59) or 71.2% (84/118) were found colonized and 67.9% (38/56), 35.6% (21/59) or 22.0% (26/118) had Salmonella internalized into the fruit, respectively. S. Poona was the most prevalent serovar isolated on or in cucumber fruits at all inoculation levels. When soil was inoculated at 1 day post-transplant (dpt), 8% (10/120) of the plants were shown to translocate Salmonella to the lower stem 7 days post-inoculation (dpi). Results identified blossoms as an important route by which Salmonella internalized at a high percentage into cucumbers, and S. Poona, the same strain isolated from the 2015 outbreak of cucumbers imported from Mexico, was shown to be well-adapted to the blossom niche.

The Potential of Systems Biology to Discover Antibacterial Mechanisms of Plant Phenolics.

Drug resistance of bacterial pathogens is a growing problem that can be addressed through the discovery of compounds with novel mechanisms of antibacterial activity. Natural products, including plant phenolic compounds, are one source of diverse chemical structures that could inhibit bacteria through novel mechanisms. However, evaluating novel antibacterial mechanisms of action can be difficult and is uncommon in assessments of plant phenolic compounds. With systems biology approaches, though, antibacterial mechanisms can be assessed without the bias of target-directed bioassays to enable the discovery of novel mechanism(s) of action against drug resistant microorganisms. This review article summarizes the current knowledge of antibacterial mechanisms of action of plant phenolic compounds and discusses relevant methodology.

Computational Ranking of Yerba Mate Small Molecules Based on Their Predicted Contribution to Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus.

The aqueous extract of yerba mate, a South American tea beverage made from Ilex paraguariensis leaves, has demonstrated bactericidal and inhibitory activity against bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). The gas chromatography-mass spectrometry (GC-MS) analysis of two unique fractions of yerba mate aqueous extract revealed 8 identifiable small molecules in those fractions with antimicrobial activity. For a more comprehensive analysis, a data analysis pipeline was assembled to prioritize compounds for antimicrobial testing against both MRSA and methicillin-sensitive S. aureus using forty-two unique fractions of the tea extract that were generated in duplicate, assayed for activity, and analyzed with GC-MS. As validation of our automated analysis, we checked our predicted active compounds for activity in literature references and used authentic standards to test for antimicrobial activity. 3,4-dihydroxybenzaldehyde showed the most antibacterial activity against MRSA at low concentrations in our bioassays. In addition, quinic acid and quercetin were identified using random forests analysis and 5-hydroxy pipecolic acid was identified using linear discriminant analysis. We also generated a ranked list of unidentified compounds that may contribute to the antimicrobial activity of yerba mate against MRSA. Here we utilized GC-MS data to implement an automated analysis that resulted in a ranked list of compounds that likely contribute to the antimicrobial activity of aqueous yerba mate extract against MRSA.

Narrow terahertz attenuation signatures in Bacillus thuringiensis.

Terahertz absorption signatures from culture-cultivated Bacillus thuringiensis were measured with a THz photomixing spectrometer operating from 400 to 1200 GHz. We observe two distinct signatures centered at ∼955 and 1015 GHz, and attribute them to the optically coupled particle vibrational resonance (surface phonon-polariton) of Bacillus spores. This demonstrates the potential of the THz attenuation signatures as "fingerprints" for label-free biomolecular detection.

Antimicrobial activity of Hibiscus sabdariffa aqueous extracts against Escherichia coli O157:H7 and Staphylococcus aureus in a microbiological medium and milk of various fat concentrations.

Hibiscus sabdariffa L. calyces are widely used in the preparation of beverages. The calyces contain compounds that exhibit antimicrobial activity, yet little research has been conducted on their possible use in food systems as antimicrobials. Aqueous extracts prepared from the brand "Mi Costenita" were sterilized by membrane filtration (0.22-µm pore size) or autoclaving (121 °C, 30 min) and tested for antimicrobial activity against the foodborne pathogens Escherichia coli O157:H7 strains ATCC 43894 and Cider and Staphylococcus aureus strains SA113 and ATCC 27708 in a microbiological medium and ultrahigh-temperature-processed milk with various fat percentages. Extracts heated by autoclaving exhibited greater activity than did filtered extracts in a microbiological medium. Against E. coli, results of 20 mg/ml filtered extract were not different from those of the control, whereas autoclaved extracts reduced viable cells ca. 3 to 4 log CFU/ml. At 60 mg/ml, both extracts inactivated cells after 24 h. There were reduced populations of both strains of S. aureus (ca. 2.7 and 3 log CFU/ml, respectively) after 24 h of incubation in 40 mg/ml filtered extracts. When grown in autoclaved extracts at 40 mg/ml, both strains of S. aureus were inactivated after 9 h. Autoclaved extracts had decreased anthocyanin content (2.63 mg/liter) compared with filtered extracts (14.27 mg/liter), whereas the phenolic content (48.7 and 53.8 mg/g) remained similar for both treatments. Autoclaved extracts were then tested for activity in milk at various fat concentrations (skim [<0.5%], 1%, 2%, and whole [>3.25%]) against a 1:1 mixture of the two strains of E. coli O157:H7 and a 1:1 mixture of the two strains of S. aureus. Extracts at 40 mg/ml inactivated S. aureus after 168 h in skim and whole milk, and E. coli was inactivated after 96 h in 60 mg/ml extract in all fat levels. These findings show the potential use of Hibiscus extracts to prevent the growth of pathogens in foods and beverages.

Mega-nano detection of foodborne pathogens and transgenes using molecular beacon and semiconductor quantum dot technologies.

Signature molecules derived from Listeria monocytogenes, Bacillus thuringiensis, and Salmonella Typhimurium were detected directly on food substrates (mega) by coupling molecular beacon technology utilizing fluorescent resonance energy transfer (FRET), luminescent nanoscale semiconductor quantum dots, and nanoscale quenchers. We designed target DNA sequences for detecting hlyA, Bt cry1Ac, and invA genes from L. monocytogenes, B. thuringiensis and Salmonella Typhimurium, respectively, and prepared molecular beacons for specific targets for use in real-time monitoring. We successfully detected increased fluorescence in the presence of signature molecules at molecular beacon (MB) concentrations from 1.17 nM to 40 nM, depending upon system tested in (water, milk or plant leaves), respective target (hlyA, Bt cry1Ac, or invA) and genomic DNA target concentration (50-800 ng). We were able to detect bacterial genomic DNA derived from L. monocytogenes and Salmonella sp. in a food system, 2% milk ( > 20% of total volume). Furthermore, we infiltrated the Bt cry1Ac beacon in the presence of genomic DNA extracted from B. thuringiensis into Arabidopsis thaliana leaves and observed increased fluorescence in the presence of the target, indicating the ability to use these beacons in a plant system.

Aqueous extracts of yerba mate (Ilex paraguariensis) as a natural antimicrobial against Escherichia coli O157:H7 in a microbiological medium and pH 6.0 apple juice.

Ilex paraguariensis is popularly used in the preparation of a tea infusion (yerba mate), most commonly produced and consumed in the South American countries of Uruguay, Paraguay, Argentina, and Brazil. In this study, aqueous extracts of commercial tea, derived from the holly plant species I. paraguariensis were evaluated for their ability to inhibit or inactivate Escherichia coli O157:H7 in a microbiological medium and modified apple juice. Dialyzed, lyophilized aqueous extracts were screened for antimicrobial activity against E. coli O157:H7 strains ATCC 43894 and 'Cider' in tryptic soy broth (TSB) and apple juice (adjusted to pH 6.0 to allow for growth of the bacterium). A mixture of the two strains was used as the inoculum when apple juice was used as the medium. MBCs were determined to be ca. 5 and 10 mg/ml for ATCC 43894 and 'Cider', respectively, in TSB. Higher concentrations of the extract were required to inactivate E. coli O157:H7 in pH-adjusted apple juice. An approximate 4.5-log reduction was observed for E. coli O157:H7 treated with 40 mg/ml extract. It was concluded that aqueous extracts from commercial yerba mate have potential to be used as antimicrobials in foods and beverages against pathogenic E. coli O157:H7.

Antimicrobial activity of Yerba Mate (Ilex paraguariensis) aqueous extracts against Escherichia coli O157:H7 and Staphylococcus aureus.

UNLABELLED: Bioactive compounds from natural plant sources are becoming increasingly important to the food industry. Ilex paraguariensis is used in the preparation of a widely popular tea beverage (Yerba Mate) in the countries of Uruguay, Paraguay, Argentina, and Brazil. In this study, extracts of 4 brands of commercial tea, derived from the holly plant species, Ilex paraguariensis, were evaluated for their ability to inhibit or inactivate bacterial foodborne pathogens. The ultimate goal was to evaluate potential use of the extracts in commercial applications. Dialyzed aqueous extracts were screened for antimicrobial activity against Escherichia coli O157:H7 and Staphylococcus aureus. S. aureus was found to be the more sensitive to extracts than E. coli O157:H7. Minimum bactericidal concentrations (MBCs) were determined to be approximately 150 to 800 µg/mL and 25 to 50 µg/mL against E. coli O157:H7 and S. aureus, respectively. A Uruguayan brand had reduced activity against E. coli O157:H7 compared to the Argentinean brands tested. It was concluded that Yerba Mate could be used as a potential antimicrobial in foods and beverages against these pathogenic bacteria. PRACTICAL APPLICATION: Soluble extracts from Yerba Mate are natural antimicrobials that can be incorporated into food products to achieve longer shelf life.