Advances in Foodborne Pathogen Analysis.

As the world population has grown, new demands on the production of foods have been met by increased efficiencies in production, from planting and harvesting to processing, packaging and distribution to retail locations. These efficiencies enable rapid intranational and global dissemination of foods, providing longer "face time" for products on retail shelves and allowing consumers to make healthy dietary choices year-round. However, our food production capabilities have outpaced the capacity of traditional detection methods to ensure our foods are safe. Traditional methods for culture-based detection and characterization of microorganisms are time-, labor- and, in some instances, space- and infrastructure-intensive, and are therefore not compatible with current (or future) production and processing realities. New and versatile detection methods requiring fewer overall resources (time, labor, space, equipment, cost, etc.) are needed to transform the throughput and safety dimensions of the food industry. Access to new, user-friendly, and point-of-care testing technologies may help expand the use and ease of testing, allowing stakeholders to leverage the data obtained to reduce their operating risk and health risks to the public. The papers in this Special Issue on "Advances in Foodborne Pathogen Analysis" address critical issues in rapid pathogen analysis, including preanalytical sample preparation, portable and field-capable test methods, the prevalence of antibiotic resistance in zoonotic pathogens and non-bacterial pathogens, such as viruses and protozoa.

Magnetic ionic liquids: interactions with bacterial cells, behavior in aqueous suspension, and broader applications.

Previously, we demonstrated capture and concentration of Salmonella enterica subspecies enterica ser. Typhimurium using magnetic ionic liquids (MILs), followed by rapid isothermal detection of captured cells via recombinase polymerase amplification (RPA). Here, we report work intended to explore the broader potential of MILs as novel pre-analytical capture reagents in food safety and related applications. Specifically, we evaluated the capacity of the ([P66614+][Ni(hfacac)3-]) ("Ni(II)") MIL to bind a wider range of human pathogens using a panel of Salmonella and Escherichia coli O157:H7 isolates, including a "deep rough" strain of S. Minnesota. We extended this exploration further to include other members of the family Enterobacteriaceae of food safety and clinical or agricultural significance. Both the Ni(II) MIL and the ([P66614+][Dy(hfacac)4-]) ("Dy(III)") MIL were evaluated for their effects on cell viability and structure-function relationships behind observed antimicrobial activities of the Dy(III) MIL were determined. Next, we used flow imaging microscopy (FIM) of Ni(II) MIL dispersions made in model liquid media to examine the impact of increasing ionic complexity on MIL droplet properties as a first step towards understanding the impact of suspension medium properties on MIL dispersion behavior. Finally, we used FIM to examine interactions between the Ni(II) MIL and Serratia marcescens, providing insights into how the MIL may act to capture and concentrate Gram-negative bacteria in aqueous samples, including food suspensions. Together, our results provide further characterization of bacteria-MIL interactions and support the broader utility of the Ni(II) MIL as a cell-friendly capture reagent for sample preparation prior to cultural or molecular analyses. Graphical abstract.

Capture, Concentration, and Detection of Salmonella in Foods Using Magnetic Ionic Liquids and Recombinase Polymerase Amplification.

We previously investigated the extraction and concentration of bacteria from model systems using magnetic ionic liquid (MIL) solvents while retaining their viability. Here, we combine MIL-based sample preparation with isothermal amplification and detection of Salmonella-specific DNA using recombinase polymerase amplification (RPA). After initial developmental work with Serratia marcescens in water, Salmonella Typhimurium ATCC 14028 was inoculated in water, 2% milk, almond milk, or liquid egg samples and extracted using one of two MILs, including trihexyl(tetradecyl)phosphonium cobalt(II) hexafluoroacetylacetonate ([P66614+][Co(hfacac)3-]) and trihexyl(tetradecyl)phosphonium nickel(II) hexafluoroacetylacetonate ([P66614+][Ni(hfacac)3-]). Viable cells were recovered from the MIL extraction phase after the addition of modified LB broth, followed by a 20 min isothermal RPA assay. Amplification was carried out using supersaturated sodium acetate heat packs and results compared to those using a conventional laboratory thermocycler set to a single temperature. Results were visualized using either gel electrophoresis or nucleic acid lateral flow immunoassay (NALFIA). The combined MIL-RPA approach enabled detection of Salmonella at levels as low as 103 CFU mL-1. MIL-based sample preparation required less than 5 min to capture and concentrate sufficient cells for detection using RPA, which (including NALFIA or gel-based analysis) required approximately 30-45 min. Our results suggest the utility of MILs for the rapid extraction and concentration of pathogenic microorganisms in food samples, providing a means for physical enrichment that is compatible with downstream analysis using RPA.

Long-Term Survival Phase Cells of Salmonella Typhimurium ATCC 14028 Have Significantly Greater Resistance to Ultraviolet Radiation in 0.85% Saline and Apple Juice.

Nonendospore-forming pathogenic bacteria in the long-term survival (LTS) phase can remain viable for months or years and may show reduced susceptibility to various antimicrobial interventions. In the present study, we investigated the response of LTS phase Salmonella enterica serovar Typhimurium (ATCC 14028) to ultraviolet (UV) radiation in 0.85% (w/v) saline and apple juice and the extent of sublethal injury in LTS phase survivors. The LTS-phase Salmonella Typhimurium cells were cultured at 35°C for 14 days in tryptic soy broth with 0.6% (w/v) yeast extract (TSBYE). Exponential- and stationary-phase cells, cultured in TSBYE (35°C) for 2.5 and 18 h, respectively, served as control samples. Cells (107 CFU [colony-forming unit]/mL) from each physiological state were exposed to UV light in saline (80 µW/cm2) and apple juice (1500 µW/cm2). The Salmonella Typhimurium survivors were plated for enumeration on either tryptic soy agar with 0.6% yeast extract or xylose-lysine-tergitol 4 (XLT4) agar and colonies counted after incubation (35°C, 24 h). Of all the growth phases tested, LTS phase cells were consistently impacted the least by UV treatment (p < 0.05). In saline, D-values of exponential, stationary, and LTS Salmonella Typhimurium were 0.35, 0.38, and 0.49 min, respectively. D-values in apple juice at pH 3.63 and pH 5.65 were 2.52, 3.19, and 3.57 min and 3.24, 3.50, and 4.18 min, respectively. UV radiation (80 µW/cm2) of Salmonella Typhimurium in saline for 2.5 min reduced the number of exponential- and stationary-phase cells by ∼7.19 and 6.30 log10 CFU/mL, respectively. In contrast, LTS cells were only reduced by 5.08 log10 CFU/mL. Among the three physiological states, LTS phase cells had the least sublethal injury in the surviving population (p < 0.05). These results indicate that the LTS state cross-protects Salmonella Typhimurium against UV radiation and should be considered in determination of the UV radiation D-value for this pathogen.

Design and evaluation of peptide nucleic acid probes for specific identification of Candida albicans.

Candida albicans is an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiating C. albicans from other Candida species are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel of C. albicans and various nontarget Candida spp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power for C. albicans than P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification of C. albicans in clinical and related applications, especially when combined with FCM.

Effects of different nitrite concentrations from a vegetable source with and without high hydrostatic pressure on the recovery of Listeria monocytogenes on ready-to-eat restructured ham.

Sodium nitrite exerts an inhibitory effect on the growth of Listeria monocytogenes. The objective of this study was to investigate the effects of various nitrite concentrations from a vegetable source with and without high hydrostatic pressure (HHP) on the recovery and growth of L. monocytogenes on ready-to-eat restructured ham. A preconverted celery powder was used as the vegetable source of nitrite. Targeted concentrations of natural nitrite investigated were 0, 50, and 100 mg/kg. HHP treatments evaluated were 400 MPa for 4 min and 600 MPa for 1 or 4 min at 12 ± 2 °C (initial temperature of the pressurization fluid). Viable L. monocytogenes populations were monitored on modified Oxford medium and thin agar layer medium through 98 days of storage at 4 ± 1 °C. Populations on both media did not differ. The HHP treatment at 600 MPa for 4 min resulted in L. monocytogenes populations below the detection limit of our sampling protocols throughout the storage period regardless of the natural nitrite concentration. The combination of HHP at 400 MPa for 4 min or 600 MPa for 1 min with natural nitrite resulted in initial inhibition of viable L. monocytogenes. Ham formulations that did not contain natural nitrite allowed faster growth of L. monocytogenes than did those with nitrite, regardless of whether they were treated with HHP. The results indicate that nitrite from a vegetable source at the concentrations used in this study resulted in slower growth of this microorganism. HHP treatments enhanced the inhibitory effects of natural nitrite on L. monocytogenes growth. Thus, the combination of natural nitrite plus HHP appears to have a synergistic inhibitory effect on L. monocytogenes growth.

Evaluation of the thin agar layer method for the recovery of pressure-injured and heat-injured Listeria monocytogenes.

A sublethally injured bacterial cell has been defined as a cell that survives a stress such as heating, freezing, acid treatment, or other antimicrobial intervention but can repair the cellular damage exerted by the stressor and later regain its original ability to grow. Consequently, sublethally injured cells are not likely to be included in conventional enumeration procedures, which could result in unrealistically low counts unless efforts are made to encourage recovery of the injured cells before enumeration. The objective of this study was to evaluate the use of the thin agar layer (TAL) method for the recovery of pressure-injured and heat-injured Listeria monocytogenes in a tryptic soy broth with 0.6% yeast extract system. Pressure injury consisted of treatment of a culture of mixed L. monocytogenes strains with high hydrostatic pressure at 400 or 600 MPa for 1 s, 2 min, 4 min, or 6 min at a process temperature of 12±2 °C. Heat injury consisted of treatment of a culture of mixed L. monocytogenes strains at 60±1 °C for 3, 6, or 9 min. Growth media were tryptic soy agar (TSA) with 0.6% yeast extract, modified Oxford medium (MOX), and TAL, which consisted of a 7-ml layer of TSA overlaid onto solidified MOX. Counts of viable L. monocytogenes on TAL were higher than those on MOX in the heat-injury experiment but not in the pressure-injury experiment. Therefore, the effectiveness of the TAL method may be specific to the type of injury applied to the microorganism and should be investigated in a variety of cellular injury scenarios.

Investigating the control of Listeria monocytogenes on a ready-to-eat ham product using natural antimicrobial ingredients and postlethality interventions.

Ready-to-eat (RTE) meat and poultry products manufactured with natural or organic methods are at greater risk for Listeria monocytogenes growth, if contaminated, than their conventional counterparts due to the required absence of preservatives and antimicrobials. Thus, the objective of this study was to investigate the use of commercially available natural antimicrobials and postlethality interventions in the control of L. monocytogenes growth and recovery on a RTE ham product. Antimicrobials evaluated were cranberry powder (90MX), vinegar (DV), and vinegar/lemon juice concentrate (LV1X). Postlethality interventions studied were high hydrostatic pressure at 400 (HHP400) or 600 (HHP600) MPa, lauric arginate (LAE), octanoic acid (OA), and postpackaging thermal treatment (PPTT). Parameters evaluated through 98 days of storage at 4±1°C were residual nitrite concentrations, pH, a(w), and viable L. monocytogenes on modified Oxford (MOX) media. On day 1, OA, 90MX, DV, and LV1X yielded lower residual nitrite concentrations than the control, whereas HHP400, HHP600, and LAE did not. LAE, HHP400, and OA reduced L. monocytogenes population compared to the control after 1 day of storage by 2.38, 2.21, and 1.73 log10 colony-forming units per gram, respectively. PPTT did not achieve a significant reduction in L. monocytogenes populations. L. monocytogenes recovered and grew in all postlethality intervention treatments except HHP600. 90MX did not inhibit the growth of L. monocytogenes, while DV and LV1X did. Results of this study demonstrate the bactericidal properties of HHP, OA, and LAE and the bacteriostatic potential of natural antimicrobial ingredients such as DV and LV1X against L. monocytogenes.

Investigating the control of Listeria monocytogenes on alternatively-cured frankfurters using natural antimicrobial ingredients or post-lethality interventions.

The objective of this study was to investigate natural antimicrobials including cranberry powder, dried vinegar and lemon juice/vinegar concentrate, and post-lethality interventions (lauric arginate, octanoic acid, thermal treatment and high hydrostatic pressure) for the control of Listeria monocytogenes on alternatively-cured frankfurters. Lauric arginate, octanoic acid, and high hydrostatic pressure (400 MPa) reduced L. monocytogenes populations by 2.28, 2.03, and 1.88 log 10 CFU per g compared to the control. L. monocytogenes grew in all post-lethality intervention treatments, except after a 600 MPa high hydrostatic pressure treatment for 4 min. Cranberry powder did not inhibit the growth of L. monocytogenes, while a dried vinegar and a vinegar/lemon juice concentrate did. This study demonstrated the bactericidal properties of high hydrostatic pressure, octanoic acid and lauric arginate, and the bacteriostatic potential of natural antimicrobial ingredients such as dried vinegar and vinegar/lemon juice concentrate against L. monocytogenes.

Ozonation-based decolorization of food dyes for recovery of fruit leather wastes.

Commercial manufacture of fruit leathers (FL) usually results in a portion of the product that is out of specification. The disposition of this material poses special challenges in the food industry. Because the material remains edible and contains valuable ingredients (fruit pulp, sugars, acidulates, etc.), an ideal solution would be to recover this material for product rework. A key practical obstacle to such recovery is that compositing of differently colored wastes results in an unsalable gray product. Therefore, a safe and scalable method for decolorization of FL prior to product rework is needed. This research introduces a novel approach utilizing ozonation for color removal. To explore the use of ozonation as a decolorization step, we first applied it to simple solutions of the commonly used food colorants 2-naphthalenesulfonic acid (Red 40), tartrazine (Yellow 5), and erioglaucine (Blue 1). Decolorization was measured by UV/vis spectrometry at visible wavelengths and with a Hunter colorimeter. Volatile and semivolatile byproducts from ozone-based colorant decomposition were identified and quantified with solid phase microextraction coupled with gas chromatography-mass spectrometry (SPME-GC-MS). Removal of Yellow 5, Red 40 and Blue 1 of about 65%, 80%, and 90%, respectively, was accomplished with 70 g of ozone applied per 1 kg of redissolved and resuspended FL. Carbonyl compounds were identified as major byproducts from ozone-induced decomposition of the food colorants. A conservative risk assessment based on quantification results and published toxicity information of potentially toxic byproducts, determined that ozone-based decolorization of FL before recycling is acceptable from a safety standpoint. A preliminary cost estimate based on recycling of 1000 tons of FL annually suggests a potential of $275,000 annual profit from this practice at one production facility alone.

Isolation of carotenoid hyperproducing mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by flow cytometry and cell sorting.

Approaches for improving astaxanthin yields in Xanthophyllomyces dendrorhous include optimization of fermentation conditions and generation of hyperproducing mutants through random mutagenesis using chemical or physical means. A key limitation of classical mutagenesis is the labor-intensive nature of the screening processes required to find relatively rare mutants having increased carotenoid content, as these are present against a high background of low-interest cells. Here, flow cytometry is described as a high-throughput, single-cell method for primary enrichment of mutagenized cells expressing high levels of astaxanthin. This approach improves the speed and productivity of classical strain selection, enhancing the chances for isolating the carotenoid hyperproducing mutants (CHMs) needed to enable high-titer, economical production of natural astaxanthin.

Combination of adhesive-tape-based sampling and fluorescence in situ hybridization for rapid detection of Salmonella on fresh produce.

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55°C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (≤ 500 µL) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37°C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalapeño peppers.

Value-added production of nisin from soy whey.

The objective of this study was to evaluate the potential of low/negative value soy whey (SW) as an alternative, inexpensive fermentation substrate to culture Lactococcus lactis subsp. lactis for nisin production. Initially, a microtiter plate assay using a Bioscreen C Microbiology Plate Reader was used for rapid optimization of culture conditions. Various treatments were examined in efforts to optimize nisin production from SW, including different methods for SW sterilization, ultrasonication of soy flake slurries for possible nutrient release, comparison of diluted and undiluted SW, and supplementation of SW with nutrients. In subsequent flask-based experiments, dry bacterial mass and nisin yields obtained from SW were 2.18 g/L and 619 mg/L, respectively, as compared to 2.17 g/L and 672 mg/L from a complex medium, de Man-Rogosa-Sharpe broth. Ultrasonication of soybean flake slurries (10% solid content) in water prior to production of SW resulted in ∼2% increase in biomass yields and ∼1% decrease in nisin yields. Nutrient supplementation to SW resulted in ∼3% and ∼7% increase in cell and nisin yields, respectively. This proof-of-concept study demonstrates the potential for use of a low/negative value liquid waste stream from soybean processing for production of a high-value fermentation end product.

Rapid identification of Candida albicans in blood by combined capillary electrophoresis and fluorescence in situ hybridization.

A CE method based on whole-cell molecular labeling via fluorescence in situ hybridization was developed for the detection of Candida albicans in whole blood. Removal of potentially interfering red blood cells (RBC) with a simple hypotonic/detergent lysis step enabled us to detect and quantitate contaminating C. albicans cells at concentrations that were orders of magnitude lower than background RBC counts ( approximately 7.0 x 10(9) RBC/mL). In the presence of the lysed blood matrix, yeast cells aggregated without the use of a blocking plug to stack the cells. Short (15 min) hybridizations yielded bright Candida-specific fluorescence in situ hybridization signals, enabling us to detect as few as a single injected cell. The peak area response of the stacked Candida cells showed a strong linear correlation with cell concentrations determined by plate counts, up to approximately 10(7) CFU/mL (or approximately 1 x 10(4) injected cells). This rapid and quantitative method for detecting Candida in blood may have advantageous applications in both human and veterinary diagnostics.

Antilisterial effects of gravinol-s grape seed extract at low levels in aqueous media and its potential application as a produce wash.

Grape seed extract (GSE) is a rich source of proanthocyanidins, a class of natural antioxidants reported to have wide-ranging bioactivity as anti-inflammatory, anticarcinogenic, and antimicrobial agents. The ability of GSE to rapidly inactivate Listeria monocytogenes in vitro and the generally recognized as safe status of GSE make this extract an attractive candidate for control of Listeria in or on foods. Previously, GSE has been used at relatively high concentrations (1%) in complex food matrices and in combination with other antimicrobials. We sought to characterize the antilisterial effects of a commercial GSE preparation (Gravinol-S) alone at much lower concentrations (0.00015 to 0.125%) in aqueous solution and to test its possible use as an antimicrobial wash for fresh produce surfaces. Based on broth microdilution tests, the MICs of GSE against L. monocytogenes Scott A and Listeria innocua ATCC 33090 were as low as 50 and 78 mug ml(-1), respectively. GSE was evaluated in 0.85% saline against live cells of L. innocua via flow cytometry, using propidium iodide as a probe for membrane integrity. At sub-MICs and after only 2 min of exposure, treatment with GSE caused rapid permeabilization and clumping of L. innocua, results that we confirmed for L. monocytogenes using fluorescence microscopy and Live/Dead staining. At higher concentrations (0.125%), GSE reduced viable cell counts for L. monocytogenes by approximately 2 log units within 2 min on tomato surfaces. These results suggest the potential for GSE as a natural control of Listeria spp. on low-complexity foods such as tomatoes.

Enhanced dark field microscopy for rapid artifact-free detection of nanoparticle binding to Candida albicans cells and hyphae.

We surveyed a panel of 13 metal nanoparticle (NP) catalysts for their antifungal activities against Candida albicans ATCC 90028. Initial characterization using scanning electron microscopy (SEM) suggested that our ability to detect NP binding to Candida surfaces with this method was impeded by preparation artifacts. As an alternative method for visualizing NP binding, we used an enhanced dark field illumination system (CytoViva) attached to a standard light microscope. When viewed using this system, all NP produced intense optical signals due to resonant light scattering. To assay binding, NP were allowed to interact with C. albicans hyphae and cells in spent RPMI broth for 15 min with gentle inversion, followed by viewing with the CytoViva system. The antifungal efficacy of NP preparations was determined separately using a 24-h broth microdilution test. For single-metal NP, observations of binding at 15 min made via CytoViva corresponded to antifungal efficacy at 24 h, with the most antifungal NP yielding complete coverage of hyphal surfaces. Our work suggests the utility of visual screening using the CytoViva system for rapid, simple and artifact-free viewing of NP-cell interactions in support of antimicrobial screening efforts. This approach provides a quick and accessible alternative to SEM for imaging of NP-cell interactions.

Flow-through imaging cytometry for characterization of Salmonella subpopulations in alfalfa sprouts, a complex food system.

We recently developed an approach combining fluorescence in situ hybridization (FISH) and flow cytometry for detecting low levels of Salmonella spp. (approximately 10(3) cells/mL sprout wash) against high levels of naturally occurring sprout flora (approximately 10(7)-10(8) CFU/g sprouts). Although this "FISH and flow" approach provided rapid presence/absence testing for Salmonella in this complex food system, it was not capable of more nuanced tasks, such as probing the phenotypic complexity of the microbes present in sprouts or determining the physical interactions of Salmonella with these microbes, or with sprout debris. In the present study, we have combined rapid FISH-based labeling of Salmonella spp. in sprout washes with flow-through imaging cytometry (FT-IC), using the ImageStream 100, a commercial FT-IC instrument. This approach enables image-based characterization of various subpopulations of interest occurring within these samples. Here, we demonstrate the ability of FT-IC to unambiguously identify cells, cell aggregates and other events within these subpopulations based on both cell morphology and hybridization status after reaction with a Salmonella-targeted probe cocktail. Our ability to directly explore the nature of these events expands the layers of information possible from cytometric analyses of these complex samples and clearly demonstrates that "a picture is worth a thousand dots".

Simple adhesive-tape-based sampling of tomato surfaces combined with rapid fluorescence in situ hybridization for Salmonella detection.

A simple adhesive-tape-based method for sampling of tomato surfaces was combined with fluorescence in situ hybridization for rapid culture-independent detection of Salmonella strains. Tapes could also be placed face-down on selective agar for on-tape enrichment of captured Salmonella cells. Overlay of cell-charged tapes with small volumes of liquid enrichment media enabled subsequent detection of tape-captured Salmonella via flow cytometry.

Combined capillary electrophoresis and DNA-fluorescence in situ hybridization for rapid molecular identification of Salmonella Typhimurium in mixed culture.

CE, long a staple in analytical chemistry for molecular separations, has recently been adapted for separating heterogeneous mixtures of microbial cells based on intrinsic differences in cell morphology and surface charge. In this application, CE enables effective separations of both relatively broad categories of cells, as well as of more similar cell types. As a phenotypic approach, CE may be less applicable to certain populations, including those comprised of pleiomorphic cells or chain-forming cells, where differences in cell size, shape, or chain length may lead to broad, "unfocusable" distributions in cell surface charge. At the other end of the spectrum, closely related species having similar surface charge profiles may not be separable via CE alone. Successful combination of microbial CE with a compatible method for generating cell-specific signals could address these limitations, increasing the diagnostic power of this approach. Fluorescence in situ hybridization (FISH) is a rapid molecular technique for fluorescence-based labeling of whole target cells. In this work, we combined a simple CE-based presence/absence test with FISH to develop a bacterial detection assay having an additional "layer" of molecular specificity. Using this approach, we were able to differentiate Salmonella Typhimurium from Escherichia coli in mixed populations via CE. Both hybridizations and CE run times were short (10-15 min), bacterial populations were highly focused ( approximately 2-3 s peak width) and there was no need for a posthybridization wash step. As few as three injected cells of S. Typhimurium were detected against a background of approximately 300 injected E. coli cells, suggesting the possibility for single-cell detection of pathogens using this technique. This proof of concept study highlights the potential of CE-FISH as a promising new tool for molecular detection of specific bacterial cells within mixtures of closely related, physiologically inseparable populations.

Design and evaluation of 16S rRNA-targeted peptide nucleic acid probes for whole-cell detection of members of the genus Listeria.

Six fluorescein-labeled peptide nucleic acid oligomers targeting Listeria-specific sequences on the 16S ribosomal subunit were evaluated for their abilities to hybridize to whole cells by fluorescence in situ hybridization (FISH). Four of these probes yielded weak or no fluorescent signals after hybridization and were not investigated further. The remaining two FISH-compatible probes, LisUn-3 and LisUn-11, were evaluated for their reactivities against 22 Listeria strains and 17 other bacterial strains belonging to 10 closely related genera. Hybridization with BacUni-1, a domain-specific eubacterial probe, was used as a positive control for target accessibility in both Listeria spp. and nontarget cells. RNase T1 treatment of select cell types was used to confirm that positive fluorescence responses were rRNA dependent and to examine the extent of nonspecific staining of nontarget cells. Both LisUn-3 and LisUn-11 yielded rapid, bright, and genus-specific hybridizations at probe concentrations of approximately 100 pmol ml(-1). LisUn-11 was the brightest probe and stained all six Listeria species. LisUn-3 hybridized with all Listeria spp. except for L. grayi, for which it had two mismatched bases. A simple ethanolic fixation yielded superior results with Listeria spp. compared to fixation in 10% buffered formalin and was applicable to all cell types studied. This study highlights the advantages of peptide nucleic acid probes for FISH-based detection of gram-positive bacteria and provides new tools for the rapid detection of Listeria spp. These probes may be useful for the routine monitoring of food production environments in support of efforts to control L. monocytogenes.