Bacteriophages against Vibrio coralliilyticus and Vibrio tubiashii: Isolation, Characterization, and Remediation of Larval Oyster Mortalities.

Vibrio coralliilyticus and Vibrio tubiashii are pathogens responsible for high larval oyster mortality rates in shellfish hatcheries. Bacteriophage therapy was evaluated to determine its potential to remediate these mortalities. Sixteen phages against V. coralliilyticus and V. tubiashii were isolated and characterized from Hawaiian seawater. Fourteen isolates were members of the Myoviridae family, and two were members of the Siphoviridae In proof-of-principle trials, a cocktail of five phages reduced mortalities of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) by up to 91% 6 days after challenge with lethal doses of V. coralliilyticus Larval survival depended on the oyster species, the quantities of phages and vibrios applied, and the species and strain of Vibrio A later-generation cocktail, designated VCP300, was formulated with three lytic phages subsequently named Vibrio phages vB_VcorM-GR7B, vB_VcorM-GR11A, and vB_VcorM-GR28A (abbreviated 7B, 11A, and 28A, respectively). Together, these three phages displayed host specificity toward eight V. coralliilyticus strains and a V. tubiashii strain. Larval C. gigas mortalities from V. coralliilyticus strains RE98 and OCN008 were significantly reduced by >90% (P < 0.0001) over 6 days with phage treatment compared to those of untreated controls. Genomic sequencing of phages 7B, 11A, and 28A revealed 207,758-, 194,800-, and 154,046-bp linear DNA genomes, respectively, with the latter showing 92% similarity to V. coralliilyticus phage YC, a strain from the Great Barrier Reef, Australia. Phage 7B and 11A genomes showed little similarity to phages in the NCBI database. This study demonstrates the promising potential for phage therapy to reduce larval oyster mortalities in oyster hatcheries.IMPORTANCE Shellfish hatcheries encounter episodic outbreaks of larval oyster mortalities, jeopardizing the economic stability of hatcheries and the commercial shellfish industry. Shellfish pathogens like Vibrio coralliilyticus and Vibrio tubiashii have been recognized as major contributors of larval oyster mortalities in U.S. East and West Coast hatcheries for many years. This study isolated, identified, and characterized bacteriophages against these Vibrio species and demonstrated their ability to reduce mortalities from V. coralliilyticus in larval Pacific oysters and from both V. coralliilyticus and V. tubiashii in larval Eastern oysters. Phage therapy offers a promising approach for stimulating hatchery production to ensure the well-being of hatcheries and the commercial oyster trade.

Mechanisms for Pseudoalteromonas piscicida-Induced Killing of Vibrios and Other Bacterial Pathogens.

Pseudoalteromonas piscicida is a Gram-negative gammaproteobacterium found in the marine environment. Three strains of pigmented P. piscicida were isolated from seawater and partially characterized by inhibition studies, electron microscopy, and analysis for proteolytic enzymes. Growth inhibition and death occurred around colonies of P. piscicida on lawns of the naturally occurring marine pathogens Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Photobacterium damselae, and Shewanella algae Inhibition also occurred on lawns of Staphylococcus aureus but not on Escherichia coli O157:H7 or Salmonella enterica serovar Typhimurium. Inhibition was not pH associated, but it may have been related to the secretion of a cysteine protease with strong activity, as detected with a synthetic fluorogenic substrate. This diffusible enzyme was secreted from all three P. piscicida strains. Direct overlay of the Pseudoalteromonas colonies with synthetic fluorogenic substrates demonstrated the activity of two aminopeptidase Bs, a trypsin-like serine protease, and enzymes reactive against substrates for cathepsin G-like and caspase 1-like proteases. In seawater cultures, scanning electron microscopy revealed numerous vesicles tethered to the outer surface of P. piscicida and a novel mechanism of direct transfer of these vesicles to V. parahaemolyticus Vesicles digested holes in V. parahaemolyticus cells, while the P. piscicida congregated around the vibrios in a predatory fashion. This transfer of vesicles and vesicle-associated digestion of holes were not observed in other bacteria, suggesting that vesicle binding may be mediated by host-specific receptors. In conclusion, we show two mechanisms by which P. piscicida inhibits and/or kills competing bacteria, involving the secretion of antimicrobial substances and the direct transfer of digestive vesicles to competing bacteria.IMPORTANCEPseudoalteromonas species are widespread in nature and reduce competing microflora by the production of antimicrobial compounds. We isolated three strains of P. piscicida and characterized secreted and cell-associated proteolytic enzymes, which may have antimicrobial properties. We identified a second method by which P. piscicida kills V. parahaemolyticus It involves the direct transfer of apparently lytic vesicles from the surface of the Pseudoalteromonas strains to the surface of Vibrio cells, with subsequent digestion of holes in the Vibrio cell walls. Enzymes associated with these vesicles are likely responsible for the digestion of holes in the cell walls. Pseudoalteromonas piscicida has potential applications in aquaculture and food safety, in control of the formation of biofilms in the environment, and in food processing. These findings may facilitate the probiotic use of P. piscicida to inactivate pathogens and may lead to the isolation of enzymes and other antimicrobial compounds of pharmacological value.

In-package inhibition of E. coli O157:H7 on bulk Romaine lettuce using cold plasma.

Dielectric barrier discharge atmospheric cold plasma (DACP) treatment was evaluated for the inactivation of Escherichia coli O157:H7, surface morphology, color, carbon dioxide generation, and weight loss of bulk Romaine lettuce in a commercial plastic clamshell container. The lettuce samples were packed in a model bulk packaging configuration (three rows with either 1, 3, 5, or 7 layers) in the container and treated by DACP (42.6 kV, 10 min). DACP treatment reduced the number of E. coli O157:H7 in the leaf samples in the 1-, 3-, and 5-layer configurations by 0.4-0.8 log CFU/g lettuce, with no significant correlation to the sample location (P > 0.05). In the largest bulk stacking with 7 layers, a greater degree of reduction (1.1 log CFU/g lettuce) was observed at the top layer, but shaking the container increased the uniformity of the inhibition. DACP did not significantly change the surface morphology, color, respiration rate, or weight loss of the samples, nor did these properties differ significantly according to their location in the bulk stack. DACP treatment inhibited E. coli O157:H7 on bulk lettuce in clamshell containers in a uniform manner, without affecting the physical and biological properties and thus holds promise as a post-packaging process for fresh and fresh-cut fruits and vegetables.

Purification and Host Specificity of Predatory Halobacteriovorax Isolates from Seawater.

Halobacteriovorax (formerly Bacteriovorax) is a small predatory bacterium found in the marine environment and modulates bacterial pathogens in shellfish. Four strains of Halobacteriovorax originally isolated in Vibrio parahaemolyticus O3:K6 host cells were separated from their prey by an enrichment-filtration-dilution technique for specificity testing in other bacteria. This technique was essential, since 0.45-µm filtration alone was unable to remove infectious Vibrio minicells, as determined by scanning electron microscopy and cultural methods. Purified Halobacteriovorax strains were screened for predation against other V. parahaemolyticus strains and against Vibrio vulnificus, Vibrio alginolyticus, Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium DT104, all potential threats to seafood safety. They showed high host specificity and were predatory only against strains of V. parahaemolyticus. In addition, strains of Halobacteriovorax that were predatory for E. coli O157:H7 and S. Typhimurium DT104 were isolated from a tidal river at 5 ppt salinity. In a modified plaque assay agar, they killed their respective prey over a broad range of salinities (5 to 30 ppt). Plaques became smaller as the salinity levels rose, suggesting that the lower salinities were optimal for the predators' replication. These species also showed broader host specificity, infectious against each other's original hosts as well as against V. parahaemolyticus strains. In summary, this study characterized strains of Halobacteriovorax which may be considered for use in the development of broad-based biocontrol technologies to enhance the safety of commercially marketed shellfish and other foods.

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 105 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

Bio-based phenolic branched-chain fatty acid in wash water reduced populations of Listeria innocua on apple fruit.

Phenolic branched-chain fatty acid (PBC-FA) emulsion was produced by dissolving it in ethanol and mixing with water (pH 7). The resulting monodispersed emulsion droplets were approximately 200 nm in diameter. The stability of the emulsion was evaluated by storing it at 4 and 20 °C for 30 days. The antimicrobial activity of the PBC-FA emulsion was tested against Escherichia coli and Listeria innocua (8 log CFU/mL) by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a microdilution method. The PBC-FA was effective against L. innocua with MIC and MBC of 14.1 µg/mL and caused membrane permeation as determined with SEM and Live/Dead cell assay, but was not effective against E. coli O157:H7 at the tested concentrations (5-250 µg/mL). We also evaluated PBC-FA emulsion's potential to be used as a wash against L. innocua inoculated on apples. The results showed that the 500 µg/mL PBC-FA emulsion with 5 % ethanol had equivalent antimicrobial activity (2-3 logs reductions) against L. innocua as the 20 µg/mL chlorine solution, a commonly used sanitizer. 500 µg/mL PBC-FA emulsion had better antimicrobial efficacy when organic matter (chemical oxygen demand: 9.0 g/L) was present compared to 20 µg/mL of chlorine. The effect of PBC-FA on the quality of the apples, was determined by measuring changes in color, firmness, and soluble solids content over a 14-day storage period at 20 °C. The quality of the apples was not affected by PBC-FA over the 14-day storage period, suggesting that PBC-FA emulsion can be used as a wash for apples without affecting their quality.

Coating with phenolic branched-chain fatty acid reduces Listeria innocua populations on apple fruit.

An antimicrobial coating was produced by mixing phenolic branched-chain fatty acid (PBC-FA) with glycerol and a carboxymethyl cellulose solution (CMC) at pH 7. The resulting PBC-FA-CMC solution formed an emulsion with an average droplet size of 77 nm. The emulsion in the coating solution was stable for at least 30 days at 20 °C. The in vitro antimicrobial activity of the film formed from the PBC-FA emulsion was tested against a mixture of 3 strains of Listeria innocua (7 log CFU/mL). Film with a concentration of 1000 µg/mL of PBC-FA effectively reduced the population of L. innocua below the limit of detection (<1.48 log CFU/mL) in vitro. The effect of the 1000 µg/mL PBC-FA-CMC coating formulation was then evaluated against L. innocua inoculated on "Gala" apples. Results showed that compared with the non-coated control, the coating reduced L. innocua populations by ~2 log CFU/fruit and ~6 log CFU/fruit on the apple when enumerated on tryptic soy agar and selective media (PALCAM), respectively, indicating that PBC-FA applied as a coating on apples resulted in the sub-lethal injury of bacterial cells. When L. innocua was inoculated onto PBC-FA-coated apples, the L. innocua population decreased by ~4 log CFU/fruit during 14 days of shelf-life at 20 °C. The PBC-FA coating lowered the moisture loss but did not affect the color, firmness, or soluble solids content of apples during the 14-day at 20 °C. Overall, this study revealed that there is a potential that PBC-FA can be used as an antimicrobial coating to inactivate Listeria and preserve the quality of apples.

A high-throughput antibody-based microarray typing platform.

Many rapid methods have been developed for screening foods for the presence of pathogenic microorganisms. Rapid methods that have the additional ability to identify microorganisms via multiplexed immunological recognition have the potential for classification or typing of microbial contaminants thus facilitating epidemiological investigations that aim to identify outbreaks and trace back the contamination to its source. This manuscript introduces a novel, high throughput typing platform that employs microarrayed multiwell plate substrates and laser-induced fluorescence of the nucleic acid intercalating dye/stain SYBR Gold for detection of antibody-captured bacteria. The aim of this study was to use this platform for comparison of different sets of antibodies raised against the same pathogens as well as demonstrate its potential effectiveness for serotyping. To that end, two sets of antibodies raised against each of the "Big Six" non-O157 Shiga toxin-producing E. coli (STEC) as well as E. coli O157:H7 were array-printed into microtiter plates, and serial dilutions of the bacteria were added and subsequently detected. Though antibody specificity was not sufficient for the development of an STEC serotyping method, the STEC antibody sets performed reasonably well exhibiting that specificity increased at lower capture antibody concentrations or, conversely, at lower bacterial target concentrations. The favorable results indicated that with sufficiently selective and ideally concentrated sets of biorecognition elements (e.g., antibodies or aptamers), this high-throughput platform can be used to rapidly type microbial isolates derived from food samples within ca. 80 min of total assay time. It can also potentially be used to detect the pathogens from food enrichments and at least serve as a platform for testing antibodies.

Mechanism of Synergistic Photoinactivation Utilizing Curcumin and Lauric Arginate Ethyl Ester against Escherichia coli and Listeria innocua.

This study investigated the mechanism of how lauric arginate ethyl ester (LAE) improves the photoinactivation of bacteria by curcumin after diluting the 100 µmol/L stock curcumin-LAE micelle solution to the concentration used during the treatment based on the curcumin concentration. The photoinactivation of bacteria was conducted by irradiating the 1 µmol/L curcumin-LAE solution containing cocktails of Escherichia coli and Listeria innocua strains (7 log CFU/mL) for 5 min with UV-A light (λ = 365 nm). The changes in solution turbidity, curcumin stability, and bacterial morphology, viability, and recovery were observed using SEM, TEM, and live/dead cell assays. The study found that LAE enhances the photoinactivation of bacteria by increasing the permeability of cell membranes which could promote the interaction of reactive oxygen species produced by photosensitized curcumin with the cell components. The combination of curcumin and LAE was demonstrated to be more effective in inhibiting bacterial recovery at pH 3.5 for E. coli, while LAE alone was more effective at pH 7.0 for L. innocua.

The Effect of Previous Life Cycle Phase on the Growth Kinetics, Morphology, and Antibiotic Resistance of Salmonella Typhimurium DT104 in Brain Heart Infusion and Ground Chicken Extract.

Growth models are predominately used in the food industry to estimate the potential growth of selected microorganisms under environmental conditions. The growth kinetics, cellular morphology, and antibiotic resistance were studied throughout the life cycle of Salmonella Typhimurium. The effect of the previous life cycle phase [late log phase (LLP), early stationary phase (ESP), late stationary phase (LSP), and early death phase (EDP)] of Salmonella after reinoculation in brain heart infusion broth (BHI), ground chicken extract (GCE), and BHI at pH 5, 7, and 9 and salt concentrations 2, 3, and 4% was investigated. The growth media and previous life cycle phase had significant effects on the lag time (λ), specific growth rate (µ max), and maximum population density (Y max). At 2 and 4% salt concentration, the LLP had the significantly (p < 0.05) fastest µ max (1.07 and 0.69 log CFU/ml/h, respectively). As the cells transitioned from the late log phase (LLP) to the early death phase (EDP), the λ significantly (p < 0.05) increased. At pH 5 and 9, the EDP had a significantly (p < 0.05) lower Y max than the LLP, ESP, and LSP. As the cells transitioned from a rod shape to a coccoid shape in the EDP, the cells were more susceptible to antibiotics. The cells regained their resistance as they transitioned back to a rod shape from the EDP to the log and stationary phase. Our results revealed that growth kinetics, cell's length, shape, and antibiotic resistance were significantly affected by the previous life cycle phase. The results of this study also demonstrate that the previous life cycle should be considered when developing growth models of foodborne pathogens to better ensure the safety of poultry and poultry products.

Bacteriophages Against Pathogenic Vibrios in Delaware Bay Oysters (Crassostrea virginica) During a Period of High Levels of Pathogenic Vibrio parahaemolyticus.

Eastern oysters (Crassostrea virginica) from three locations along the Delaware Bay were surveyed monthly from May to October 2017 for levels of total Vibrio parahaemolyticus, pathogenic strains of V. parahaemolyticus and Vibrio vulnificus, and for strain-specific bacteriophages against vibrios (vibriophages). The objectives were to determine (a) whether vibriophages against known strains or serotypes of clinical and environmental vibrios were detectable in oysters from the Delaware Bay and (b) whether vibriophage presence or absence corresponded with Vibrio abundances in oysters. Host cells for phage assays included pathogenic V. parahaemolyticus serotypes O3:K6, O1:KUT (untypable) and O1:K1, as well as clinical and environmental strains of V. vulnificus. Vibriophages against some, but not all, pathogenic V. parahaemolyticus serotypes were readily detected in Delaware Bay oysters. In July, abundances of total and pathogenic V. parahaemolyticus at one site spiked to levels exceeding regulatory guidelines. Phages against three V. parahaemolyticus host serotypes were detected in these same oysters, but also in oysters with low V. parahaemolyticus levels. Serotype-specific vibriophage presence or absence did not correspond with abundances of total or pathogenic V. parahaemolyticus. Vibriophages were not detected against three V. vulnificus host strains, even though V. vulnificus were readily detectable in oyster tissues. Selected phage isolates against V. parahaemolyticus showed high host specificity. Transmission electron micrographs revealed that most isolates were ~ 60-nm diameter, non-tailed phages. In conclusion, vibriophages were detected against pandemic V. parahaemolyticus O3:K6 and O1:KUT, suggesting that phage monitoring in specific host cells may be a useful technique to assess public health risks from oyster consumption.

Characterization of growth inhibition of oral bacteria by sophorolipid using a microplate-format assay.

Sophorolipid (SL) is a class of glycolipid biosurfactant produced by yeast and has potent antimicrobial activity against many microorganisms. In this paper, a microplate-based method was developed to characterize the growth inhibition by SL on five representative species of caries-causing oral bacteria. Bacterial growth on microplate in the absence and presence of varying concentrations of SL was continuously monitored by recording the absorbance at 600nm of the cultures using a microplate reader. The results showed that SL completely inhibited the growth of the Lactobacilli at ≥1mg/ml and the Streptococci at much lower concentrations of ≥50µg/ml. More importantly, we further defined the mechanism of antimicrobial activity of SL by analyzing the pattern of the cell growth curves. SL at sublethal concentrations (<1mg/ml) is bactericidal towards the Lactobacilli; it lengthens the apparent cell-doubling time (Td) and decreases the final cell density (as indicated by A600nm) in a concentration-dependent manner. Against the oral Streptococci, on the other hand, SL at sublethal concentrations (<50µg/ml) is bacteriostatic; it delays the onset of cell growth in a concentration-dependent fashion, but once the cell growth is commenced there is no noticeable adverse effect on Td and the final A600nm. Scanning electron microscopic (SEM) study of L. acidophilus grown in sublethal concentration of SL reveals extensive structural damage to the cells. S. mutans grown in sublethal level of SL did not show morphological damage to the cells, but numerous protruding structures could be seen on the cell surface. At the respective lethal levels of SL, L. acidophilus cells were lysed (at 1mg/ml SL) and the cell surface structure of S. mutans (at 130µg/ml SL) was extensively deformed. In summary, this paper presents the first report on a detailed analysis of the effects of SL on Lactobacilli and Streptococci important to oral health and hygiene.

Effects of Particle Size on the Morphology and Water- and Thermo-Resistance of Washed Cottonseed Meal-Based Wood Adhesives.

Water washing of cottonseed meal is more cost-efficient and environmentally friendly than protein isolation by means of alkaline extraction and acidic precipitation. Thus, water-washed cottonseed meal (WCSM) is more promising as biobased wood adhesives. In this work, we examined the effects of the particle size on the morphology and adhesive performance of WCSM. Pilot-scale produced and dried WCSM was treated by three grinding methods: (1) ground by a hammer mill and passed through a 0.5-mm screen, (2) further ground by a cyclone mill and passed through a 0.5-mm screen, or (3) further ground by a ball mill and passed through a 0.18-mm screen. Micro-morphological examination revealed two types of particles. The filament-like particles were mainly fibrous materials from residual linters. Chunk-like particles were more like aggregates or accumulations of small particles, with proteins as the major component. Further grinding of the 0.5-mm Hammer product with the Cyclone and Ball mill led to more fine (smaller) particles in the WCSM products. The impact of further grinding on the dry and soaked adhesive strengths was minimal. However, the decrease of the hot and wet strengths of WCSM products by the additional grinding was significant (p ≤ 0.05). Data presented in this work is useful in developing the industrial standards of WCSM products used in wood bonding.

Inactivation of Escherichia coli O157:H7 and Aerobic Microorganisms in Romaine Lettuce Packaged in a Commercial Polyethylene Terephthalate Container Using Atmospheric Cold Plasma.

The effects of dielectric barrier discharge atmospheric cold plasma (DACP) treatment on the inactivation of Escherichia coli O157:H7 and aerobic microorganisms in romaine lettuce packaged in a conventional commercial plastic container were evaluated during storage at 4°C for 7 days. Effects investigated included the color, carbon dioxide (CO2) generation, weight loss, and surface morphology of the lettuce during storage. Romaine lettuce pieces, with or without inoculation with a cocktail of three strains of E. coli O157:H7 (~6 log CFU/g of lettuce), were packaged in a polyethylene terephthalate commercial clamshell container and treated at 34.8 kV at 1.1 kHz for 5 min by using a DACP treatment system equipped with a pin-type high-voltage electrode. Romaine lettuce samples were analyzed for inactivation of E. coli O157:H7, total mesophilic aerobes, and yeasts and molds, color, CO2 generation, weight loss, and surface morphology during storage at 4°C for 7 days. The DACP treatment reduced the initial counts of E. coli O157:H7 and total aerobic microorganisms by ~1 log CFU/g, with negligible temperature change from 24.5 ± 1.4°C to 26.6 ± 1.7°C. The reductions in the numbers of E. coli O157:H7, total mesophilic aerobes, and yeasts and molds during storage were 0.8 to 1.5, 0.7 to 1.9, and 0.9 to 1.7 log CFU/g, respectively. DACP treatment, however, did not significantly affect the color, CO2 generation, weight, and surface morphology of lettuce during storage (P > 0.05). Some mesophilic aerobic bacteria were sublethally injured by DACP treatment. The results from this study demonstrate the potential of applying DACP as a postpackaging treatment to decontaminate lettuce contained in conventional plastic packages without altering color and leaf respiration during posttreatment cold storage.

Inactivation of Salmonella spp. and Listeria spp. by Palmitic, Stearic, and Oleic Acid Sophorolipids and Thiamine Dilauryl Sulfate.

Food contaminated with human pathogens, such as Salmonella spp. and Listeria monocytogenes, frequently causes outbreaks of foodborne illness. Consumer concern over the use of synthesized antimicrobials to enhance microbial food safety has led to a search of natural alternatives. The objectives of this study were to evaluate the antimicrobial activity of various types of sophorolipids (SLs) and thiamine dilauryl sulfate (TDS) against pathogenic Salmonella spp. and Listeria spp. Both free and lactonic forms of SLs were synthesized from Candida bombicola using palmitic, stearic, and oleic acids as co-feedstocks. TDS and purified SLs were used to treat cocktails of Salmonella spp. and Listeria spp. Results showed that lactonic SLs had higher antimicrobial activity than the free-acid form, and Gram-positive Listeria spp. were more susceptible to SLs and TDS than Gram-negative Salmonella spp. Listeria populations were reduced from an initial concentration of 7.2 log CFU/mL to a non-detectible level within a 1 min treatment of 0.1% (w/v) lactonic SLs and TDS in the presence of 20% ethanol, which itself did not significantly reduce the populations. There were no significant differences in the antimicrobial efficacy among palmitic, stearic, and oleic acid-based SLs against Salmonella or Listeria spp. Ethanol was utilized to improve the antimicrobial activity of free-acid SLs against Gram-negative bacteria. In general, TDS was more effective than the SLs against Salmonella and Listeria spp. scanning electron microscopy and transmission electron microscopy images showed that SLs and TDS damaged Listeria cell membranes and resulted in cell lysis. Overall, our results demonstrated that SLs and TDS in the presence of ethanol can be used to inactivate foodborne pathogens, especially Gram-positive bacteria.

Electrospinning of agar/PVA aqueous solutions and its relation with rheological properties.

In this work, we report the successful fabrication of agar-based nanofibers by electrospinning technique, using water as solvent media. A tubeless spinneret was attached inside the electrospinning chamber, operating at 50°C, to avoid agar gelation. Agar pure solution (1 wt%) showed inadequate spinnability regardless of the used electrospinning conditions. The addition of a co-blending polymer such as PVA (10 wt% starting solution) improved the solutions viscoelasticity and hence, the solutions spinnability. Agar/PVA solutions were prepared with different mass ratios (100/0, 50/50, 40/60, 30/70, 20/80 and 0/100) and electrospun at various sets of electrospinning conditions. Best nanofibers were obtained with 30/70 and 20/80 agar/PVA blends while samples with higher agar contents (50/50 and 40/60 agar/PVA) were harder to process and led to discontinuous fibrous mats. This first set of encouraging results can open a new window of opportunities for agar-based biomaterials in the form of nanofibers.

Improving agar electrospinnability with choline-based deep eutectic solvents.

Very recently our group has produced novel agar-based fibers by an electrospinning technique using water as solvent and polyvinyl alcohol (PVA) as co-blending polymer. Here, we tested the deep eutectic solvent (DES), (2-hydroxyethyl)trimethylammonium chloride/urea prepared at 1:2 molar ratio, as an alternative solvent medium for agar electrospinning. The electrospun materials were collected with an ethanol bath adapted to a previous electrospinning set-up. One weight percent agar-in-DES showed improved viscoelasticity and hence, spinnability, when compared to 1 wt% agar-in-water and pure agar nanofibers were successfully electrospun if working above the temperature of sol-gel transition (∼80 °C). By changing the solvent medium we decreased the PVA concentration (5 wt% starting solution) and successfully produced composite fibers with high agar contents (50/50 agar/PVA). Best composite fibers were formed with the 50/50 and 30/70 agar/PVA solutions. These fibers were mechanically resistant, showed tailorable surface roughness and diverse size distributions, with most of the diameters falling in the sub-micron range. Both nano and micro forms of agar fibers (used separately or combined) may have potential for the design of new and highly functional agar-based materials.

Seasonal levels of the Vibrio predator bacteriovorax in atlantic, pacific, and gulf coast seawater.

Bacteriovorax were quantified in US Atlantic, Gulf, and Pacific seawater to determine baseline levels of these predatory bacteria and possible seasonal fluctuations in levels. Surface seawater was analyzed monthly for 1 year from Kailua-Kona, Hawaii; the Gulf Coast of Alabama; and four sites along the Delaware Bay. Screening for Bacteriovorax was performed on lawns of V. parahaemolyticus host cells. Direct testing of 7.5 mL portions of seawater from the Atlantic, Pacific, and Gulf coasts gave mean annual counts ≤12.2 PFU. Spikes in counts were observed at 3 out of 4 sites along the Delaware Bay 1 week after Hurricane Sandy. A comparison of summer versus winter counts showed significantly more Bacteriovorax (P ≤ 0.0001) in the Delaware Bay during the summer and significantly more (P ≤ 0.0001) in the Gulf during the winter, but no significant seasonal differences (P > 0.05) for Hawaiian seawater. Bacteriovorax counts only correlated with seawater salinity and temperature at one Delaware site (r = 0.79 and r = 0.65, resp.). There was a relatively strong negative correlation between temperature and Bacteriovorax levels (r = -0.585) for Gulf seawater. Selected isolates were sequenced and identified by phylogenetic analysis as Bacteriovorax clusters IX, X, XI, and XII.

Antibody microarray detection of Escherichia coli O157:H7: Quantification, assay limitations, and capture efficiency.

A sandwich fluorescent immunoassay in a microarray format was used to capture and detect E. coli O157:H7. Here, we explored quantitative aspects, limitations, and capture efficiency of the assay. When biotinylated capture antibodies were used, the signal generated was higher (over 5-fold higher with some cell concentrations) compared to biotinylated protein G-bound capture antibodies. By adjusting the concentration of reporter antibody, a linear fluorescent response was observed from approximately 3.0 x 10(6) to approximately 9.0 x 10(7) cells/mL, and this was in agreement with the number of captured bacteria as determined by fluorescence microscopy. Capture efficiency calculations revealed that, as the number of bacteria presented for capture decreased, capture efficiency increased to near 35%. Optimization experiments, with several combinations of capture and reporter antibodies, demonstrated that the amount of bacteria available for capture (10(6) versus 10(8) cells/mL) affected the optimal combination. The findings presented here indicate that antibody microarrays, when used in sandwich assay format, may be effectively used to capture and detect E. coli O157:H7.