Differentiating Live Versus Dead Gram-Positive and Gram-Negative Bacteria With and Without Oxidative Stress Using Buoyant Mass Measurements.

Expeditious and accurate determination of pathogenic bacteria cell viability is of great importance to public health for numerous areas including medical diagnostics, food safety, and environmental monitoring. In this work a cell buoyant mass classifier approach is presented to assess bacteria cell viability in real time. Buoyant mass measurements for live and dead Gram-positive and Gram-negative bacteria populations were acquired with a commercial suspended microchannel resonator, Archimedes, to generate receiver operating characteristic (ROC) curves. To quantitatively assess the difference in buoyant mass for live and dead bacteria populations, ROC curves were generated to demonstrate cell viability determination. The results are presented as a binary classifier with a decision boundary, above which cells are considered live and below which cells are considered dead. A decision threshold value is evaluated with consideration that a certain true positive rate (correct classification of a live cell) is maintained with an acceptable false positive rate. The potential for this approach to monitor cell viability in real time is significant, especially when considering multiple classifier dimensions such as buoyant mass and density. This classifier approach represents a next generation technique for rapid and label-free diagnostics based on cell feature measurements.

Survival of Salmonella enterica in Military Low-Moisture Food Products during Long-Term Storage at 4, 25, and 40°C.

ABSTRACT: Salmonella enterica has been increasingly implicated in foodborne outbreaks involving low-moisture foods (LMF) during the recent decade. This study aimed to investigate the potential for persistence of S. enterica in a range of LMF during storage at three temperatures. LMF products, boil-in-bag eggs (freeze-dried product), chocolate protein drink, cran-raspberry First Strike bars, mocha dessert bar, and peanut butter, were inoculated with a five-strain cocktail of S. enterica and stored at 4, 25, or 40°C for 36 months. Salmonella populations remained above 7 log CFU/g in all products stored at 4°C and above 6 log CFU/g in products stored at 25°C, excluding the cran-raspberry First Strike bars. Storage at 40°C resulted in Salmonella populations above 5.5 log CFU/g in boil-in-bag eggs after 36 months and demonstrated survivability for 12 months or less in the other five products. Additionally, a mocha bar production temperature profile study identified rapid cooling of bars in which the temperatures reached would have no measurable impact on Salmonella populations. The results indicate the ability of Salmonella to survive in a variety of LMF category foods, even under adverse storage conditions and identifies how the food matrix may affect Salmonella survivability. The data indicate the importance of establishing food processing procedures that adequately mitigate the presence of Salmonella throughout food processing systems, while also increasing comprehensive understanding of Salmonella survivability mechanisms.

Voltammetric detection of arsenic (III) using gold nanoparticles modified carbon screen printed electrodes: Application for facile and rapid analysis in commercial apple juice.

This paper describes a voltammetric method and data analysis program developed for the detection of arsenic(III) in commercial apple juice. Arsenic(III) was detected using square wave stripping voltammetry with gold nanoparticle modified screen printed electrodes. The only sample pretreatment performed was the addition of a 100 mM phosphate buffer with a pH of 7. To compensate for interference from high ascorbic acid concentrations, a data analysis program was developed in MATLAB to fit a non-linear baseline, allowing for accurate peak height measurement. With this data analysis program, the developed methodology had a sensitivity of 0.1007 µA (µg L-1)-1 and a limit of detection of 16.73 µg L-1. A comparison between the voltammetric method and graphite furnace atomic absorption spectroscopy showed no bias in the voltammetric results and a good correlation between the two sets of predicted concentrations, with an R2 of 0.939.

Dehydration of bacteriophages in electrospun nanofibers: effect of excipients in polymeric solutions.

Bacteriophages are viruses capable of infecting and lysing target bacterial cells; as such they have potential applications in agriculture for decontamination of foods, food contact surfaces and food rinse water. Although bacteriophages can retain infectivity long-term using lyophilized storage, the process of freeze-drying can be time consuming and expensive. In this study, electrospinning was used for dehydrating bacteriophages in polyvinylpyrrolidone polymer solutions with addition of excipients (sodium chloride, magnesium sulfate, Tris-HCl, sucrose) in deionized water. The high voltage dehydration reduced the infectivity of bacteriophages following electrospinning, with the damaging effect abated with addition of storage media (SM) buffer and sucrose. SM buffer and sucrose also provided the most protection over extended storage (8 weeks; 20 °C; 1% relative humidity) by mitigating environmental effects on the dried bacteriophages. Magnesium sulfate however provided the least protection due to coagulation effects of the ion, which can disrupt the native conformation of the bacteriophage protein coat. Storage temperatures (20 °C, 4 °C and -20 °C; 1% relative humidity) had a minimal effect while relative humidity had substantial effect on the infectivity of bacteriophages. Nanofibers stored in higher relative humidity (33% and 75%) underwent considerable damage due to extensive water absorption and disruption of the fibers. Overall, following storage of nanofiber mats for eight weeks at ambient temperatures, high infective phage concentrations (106-107 PFU ml-1) were retained. Therefore, this study provided valuable insights on preservation and dehydration of bacteriophages by electrospinning in comparison to freeze drying and liquid storage, and the influence of excipients on the viability of bacteriophages.

Electrospun water-soluble polymer nanofibers for the dehydration and storage of sensitive reagents.

The ability to preserve and deliver reagents remains an obstacle for the successful deployment of self-contained diagnostic microdevices. In this study we investigated the ability of bacteriophage T7 to be encapsulated and preserved in water soluble nanofibers. The bacteriophage T7 was added to mixtures of polyvinylpyrrolidone and water and electrospun onto a grounded plate. Trehalose and magnesium salts were added to the mixtures to determine their effect on the infectivity of the bacteriophage following electrospinning and during storage. The loss of T7 infectivity was determined immediately following electrospinning and during storage using agar overlay plating and plaque counting. The results indicate that the addition of magnesium salts protects the bacteriophage during the relatively violent and high voltage electrospinning process, but is not as effective as a protectant during storage of the dried T7. Conversely, the addition of trehalose into the electrospinning mix has little effect on the electrospinning, but a more significant role as a protectant during storage.

Mass and density measurements of live and dead Gram-negative and Gram-positive bacterial populations.

Monitoring cell growth and measuring physical features of food-borne pathogenic bacteria are important for better understanding the conditions under which these organisms survive and proliferate. To address this challenge, buoyant masses of live and dead Escherichia coli O157:H7 and Listeria innocua were measured using Archimedes, a commercially available suspended microchannel resonator (SMR). Cell growth was monitored with Archimedes by observing increased cell concentration and buoyant mass values of live growing bacteria. These growth data were compared to optical density measurements obtained with a Bioscreen system. We observed buoyant mass measurements with Archimedes at cell concentrations between 10(5) and 10(8) cells/ml, while growth was not observed with optical density measurements until the concentration was 10(7) cells/ml. Buoyant mass measurements of live and dead cells with and without exposure to hydrogen peroxide stress were also compared; live cells generally had a larger buoyant mass than dead cells. Additionally, buoyant mass measurements were used to determine cell density and total mass for both live and dead cells. Dead E. coli cells were found to have a larger density and smaller total mass than live E. coli cells. In contrast, density was the same for both live and dead L. innocua cells, while the total mass was greater for live than for dead cells. These results contribute to the ongoing challenge to further develop existing technologies used to observe cell populations at low concentrations and to measure unique physical features of cells that may be useful for developing future diagnostics.

Additive approach for inactivation of Escherichia coli O157:H7, Salmonella, and Shigella spp. on contaminated fresh fruits and vegetables using bacteriophage cocktail and produce wash.

The incidence of foodborne outbreaks involving fresh produce is of worldwide concern. Lytic bacteriophage cocktails and a levulinic acid produce wash were investigated for their effectiveness against the foodborne pathogens Escherichia coli O157:H7, Shigella spp., and Salmonella on broccoli, cantaloupe, and strawberries. Inoculated samples were treated with bacteriophage cocktails (BC) before storage at 10°C for 24 h, a levulinic acid produce wash (PW) after storage at 10°C for 24 h, or a combination of the washes (BCPW) before and after storage. All three treatments were compared against a 200-ppm free available chlorine wash. Wash solutions were prepared using potable water and water with an increased organic content of 2.5 g/liter total dissolved solids and total organic carbon. BCPW was the most effective treatment, producing the highest log reductions in the pathogens. Produce treated with BCPW in potable water with a PW exposure time of 5 min resulted in the highest reduction of each pathogen for all samples tested. The type of produce and wash solution had significant effects on the efficacy of the individual treatments. The chlorine wash in water with higher organic content was the least effective treatment tested. An additive effect of BCPW was seen in water with higher organic content, resulting in greater than 4.0-log reductions in pathogens. Our findings indicate that the combination of antimicrobial BC with a commercial produce wash is a very effective method for treating produce contaminated with E. coli O157:H7, Shigella spp., and Salmonella even in the presence of high loads of organic matter.

The effect of 3-thiopheneacetic Acid in the polymerization of a conductive electrotextile for use in biosensor development.

Investigations were conducted to develop an electrotextile using a nonwoven polypropylene fiber platform conformally coated in a conductive, functionalized copolymer of polypyrrole and 3-thiopheneacetic acid (3TAA). The objectives of this study were to determine: (1) if the inclusion of 3TAA in the polymerization process would have an effect on the availability of binding sites in the high-surface area electrotextile for biorecognition elements and (2) how the increase in the concentration of 3TAA would affect the physical characteristics of the coating, resistivity of the sample and availability of binding sites. It was found that the addition of 3TAA to the polymerization process resulted in an increase in the size of the polypyrrole coating, as well as the material resistivity and available binding sites for biorecognition elements. These factors were used to determine which of the tested concentrations was best for biosensor development. A polymer coated membrane sample containing a concentration within the range of 10-50 mg/mL of 3TAA was selected as the best for future biosensor work.

Synthesis of a functionalized polypyrrole coated electrotextile for use in biosensors.

An electrotextile with a biosensing focus composed of conductive polymer coated microfibers that contain functional attachment sites for biorecognition elements was developed. Experiments were conducted to select a compound with a pendant functional group for inclusion in the polymer, a fiber platform, and polymerization solvent. The effects of dopant inclusion and post-polymerization wash steps were also analyzed. Finally, the successful attachment of avidin, which was then used to capture biotin, to the electrotextile was achieved. The initial results show a nonwoven fiber matrix can be successfully coated in a conductive, functionalized polymer while still maintaining surface area and fiber durability. A polypropylene fiber platform with a conductive polypyrrole coating using iron (III) chloride as an oxidant, water as a solvent, and 5-sulfosalicylic acid as a dopant exhibited the best coating consistency, material durability, and lowest resistance. Biological attachment of avidin was achieved on the fibers through the inclusion of a carboxyl functional group via 3-thiopheneacetic acid in the monomer. The immobilized avidin was then successfully used to capture biotin. This was confirmed through the use of fluorescent quantum dots and confocal microscopy. A preliminary electrochemical experiment using avidin for biotin detection was conducted. This technology will be extremely useful in the formation of electrotextiles for use in biosensor systems.

Bacteriophage cocktail significantly reduces Escherichia coli O157: H7 contamination of lettuce and beef, but does not protect against recontamination.

Foods contaminated with Escherichia coli O157:H7 cause more than 63,000 foodborne illnesses in the United States every year, resulting in a significant economic impact on medical costs and product liabilities. Efforts to reduce contamination with E. coli O157:H7 have largely focused on washing, application of various antibacterial chemicals, and gamma-irradiation, each of which has practical and environmental drawbacks. A relatively recent, environmentally-friendly approach proposed for eliminating or significantly reducing E. coli O157:H7 contamination of foods is the use of lytic bacteriophages as biocontrol agents. We found that EcoShield™, a commercially available preparation composed of three lytic bacteriophages specific for E. coli O157:H7, significantly (p < 0.05) reduced the levels of the bacterium in experimentally contaminated beef by ≥ 94% and in lettuce by 87% after a five minute contact time. The reduced levels of bacteria were maintained for at least one week at refrigerated temperatures. However, the one-time application of EcoShield™ did not protect the foods from recontamination with E. coli O157:H7. Our results demonstrate that EcoShield™ is effective in significantly reducing contamination of beef and lettuce with E. coli O157:H7, but does not protect against potential later contamination due to, for example, unsanitary handling of the foods post processing.

Application of a biotin functionalized QD assay for determining available binding sites on electrospun nanofiber membrane.

BACKGROUND: The quantification of surface groups attached to non-woven fibers is an important step in developing nanofiber biosensing detection technologies. A method utilizing biotin functionalized quantum dots (QDs) 655 for quantitative analysis of available biotin binding sites within avidin immobilized on electrospun nanofiber membranes was developed. RESULTS: A method for quantifying nanofiber bound avidin using biotin functionalized QDs is presented. Avidin was covalently bound to electrospun fibrous polyvinyl chloride (PVC 1.8% COOH w/w containing 10% w/w carbon black) membranes using primary amine reactive EDC-Sulfo NHS linkage chemistry. After a 12 h exposure of the avidin coated membranes to the biotin-QD complex, fluorescence intensity was measured and the total amount of attached QDs was determined from a standard curve of QD in solution (total fluorescence vs. femtomole of QD 655). Additionally, fluorescence confocal microscopy verified the labeling of avidin coated nanofibers with QDs. The developed method was tested against 2.4, 5.2, 7.3 and 13.7 mg spray weights of electrospun nanofiber mats. Of the spray weight samples tested, maximum fluorescence was measured for a weight of 7.3 mg, not at the highest weight of 13.7 mg. The data of total fluorescence from QDs bound to immobilized avidin on increasing weights of nanofiber membrane was best fit with a second order polynomial equation (R(2) = .9973) while the standard curve of total fluorescence vs. femtomole QDs in solution had a linear response (R(2) = .999). CONCLUSION: A QD assay was developed in this study that provides a direct method for quantifying ligand attachment sites of avidin covalently bound to surfaces. The strong fluorescence signal that is a fundamental characteristic of QDs allows for the measurement of small changes in the amount of these particles in solution or attached to surfaces.

Enumeration of bacteriophage particles: Comparative analysis of the traditional plaque assay and real-time QPCR- and nanosight-based assays.

Bacteriophages are increasingly being utilized and considered for various practical applications, ranging from decontaminating foods and inanimate surfaces to human therapy; therefore, it is important to determine their concentrations quickly and reliably. Traditional plaque assay (PA) is the current "gold standard" for quantitating phage titers. However, it requires at least 18 h before results are obtained, and they may be significantly influenced by various factors. Therefore, two alternative assays based on the quantitative real-time polymerase chain reaction (QPCR) and NanoSight Limited (NS) technologies were recently proposed for enumerating phage particles. The present study compared the three approaches' abilities to quantitate Listeria monocytogenes-, Escherichia coli O157:H7- and Yersinia pestis-specific lytic phages quickly and reproducibly. The average coefficient of variation (CVS) of the PA method including all three phages was 0.15. The reproducibility of the PA method decreased dramatically when multiple investigators performed the assays, and mean differences of as much as 0.33 log were observed. The QPC R method required costly equipment and the synthesis of phage-specific oligonucleotide primers, but it determined phage concentrations faster (within about 4 h) and more precisely than did PA (CVS = 0.13). NS technology required costly equipment, was less precise (CVS = 0.28) than the PA and QPCR methods, and only worked when the phages were suspended in clear medium. However, it provided results within 5 min. After the overall correlation is established with the PA method, either of the two assays may be useful for quickly and reproducibly determining phage concentrations.

Predicting behavior of Staphylococcus aureus, salmonella serovars, and Escherichia coli O157: H7 in pork products during single and repeated temperature abuse periods.

Tools for predicting growth of Staphylococcus aureus, Salmonella, and Escherichia coli O157:H7 (THERM; temperature history evaluation for raw meats) have been developed using ground pork and sausage. THERM tools have been tested with three types of pork sausage but not with other pork products or during sequential temperature abuse periods. We conducted inoculation studies (five strains each of S. aureus and/or Salmonella plus E. coli O157:H7) with simulated cooling of warm sausages, inprocess warming of bratwurst, isothermal temperature abuse of pork frankfurter batter, and two sequential periods of 13, 15.6, or 21.1 degrees C temperature abuse of breakfast sausage, natural (additive-free) chops, and enhanced (phosphate solution-injected) loins. In sequential temperature abuse studies, a temperature abuse period (> or =24 h) occurred before and after either refrigeration (5 degrees C for 24 h), or freezing (-20 degrees C for 24 h) and thawing (24 h at 5 degrees C). Pathogen growth predictions from THERM developed using ground pork and sausage were compared with experimental results of 0 to 3.0 log CFU of growth. Across all temperature abuse conditions, qualitative predictions (growth versus no growth) made using the pork tool (n = 133) and the sausage tool (n = 115) were accurate (51 and 50%, respectively), fail-safe (44 and 50%), or fail-dangerous (5 and 0%). Quantitative predictions from the two tools were accurate (29 and 22% , respectively), fail-safe (59 and 73%), or fail-dangerous (12 and 5%). Pathogen growth was greater during the second sequential temperature abuse period but not significantly so (P > 0.05). Both THERM tools provide useful qualitative predictions of pathogen growth in pork products during isolated or sequential temperature abuse events.

Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7.

A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (10(10), 10(9), and 10(8) PFU/ml) resulted in statistically significant reductions (P = <0.05) of 99.99%, 98%, and 94%, respectively, in the number of E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 10(9) PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 +/- 4 h posttreatment of tomato samples) to 100% (at 24 +/- 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.

Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7.

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.

Survival of Staphylococcus aureus ATCC 13565 in intermediate moisture foods is highly variable.

Staphylococcus aureus is a gram-positive, enterotoxin-producing coccus. It is a hardy organism and known to survive over a wide range of water activities, pH values, and temperatures. The objective of this study was to model the survival or gradual inactivation of S. aureus ATCC 13565 in intermediate moisture foods (IMFs). Various initial concentrations (approximately 10(1), 10(2), 10(3), and 10(4) CFU/g) were used to inoculate three different IMFs (beefsteak, bread, and chicken pockets). Viable counts were determined up to 60 days using tryptic soy agar. Inoculum size did not influence the survival or gradual inactivation of S. aureus in these foods. The rate of change (increase or decrease) in log CFU/day was calculated for every consecutive pair of data points and by linear regression for each inactivation curve. Both consecutive pair and linear regression rates of change were fit to logistic distributions (with parameters alpha and beta) for each food. Based on the distribution parameters, survival or gradual inactivation of S. aureus was predicted by computer simulation. The simulations indicated an overall decline in S. aureus population over time, although a small fraction of samples in the consecutive pair simulation showed a slight population increase even after 60 days, consistent with the observed data. Simulation results were compared to predictions from other computer models. The models of Stewart et al., were fail-safe, predicting the possibility of significant growth only after > 3,000 days. The USDA pathogen modeling program predictions were found to be fail-dangerous, predicting declines at least four times faster than observed.