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Fluorescence <i>in situ</i> hybridization-based detection of <i>Salmonella</i> spp. and <i>Listeria monocytogenes</i> in complex food matrices
No full textCurrent methods for detection of Salmonella spp. and Listeria monocytogenes in food are culture-based methods and require performing numerous steps, between preenrichment, enrichment, selective plating, identification, and confirmation. Conducting these procedures can take several days; they require extensive manual labor and large amounts of media and reagents which can increase the cost of the testing. Molecular-based rapid high throughput methods can present a valid alternative to these methods, allowing for timely and sensitive detection of these bacterial pathogens before the contaminated products can reach the consumer, helping to prevent the occurrence of foodborne listeriosis and salmonellosis. Fluorescence in situ hybridization (FISH) is a sensitive and robust molecular method that uses sequence-specific rRNA-targeted fluorescently-labeled oligonucleotide probes to specifically label whole, permeabilized bacterial cells. When coupled with fluorescence microscopy or flow cytometry for analysis, FISH can be a powerful tool for detection of bacterial pathogens in food.
My hypothesis was that we could develop rapid and sensitive FISH-based methods for detection of these two pathogens in complex food matrices. My research concentrated on four objectives: 1. Optimize use of existing FISH probes and hybridization conditions for detection of Salmonella spp. and Listeria monocytogenes; 2. Develop pre-anayltical food sample preparation methods compatible with downstream approaches for whole-cell detection; 3. Utilize the results of objectives 1 and 2 to develop FISH-based assays for detection of Salmonella spp. and L. monocytogenes in foods; and 4. Establish the ultimate detection sensitivity of the developed methods.
Specifically, optimal combinations of existing Salmonella-specific probes were developed and applied for rapid (15 min) hybridizations of target cells. Use of these probe cocktails was integrated with pre-analytical sample preparation steps, including tangential flow filtration, adhesive tape sampling and immunomagnetic separation to enable sensitive detection of Salmonella spp. in complex food systems via flow cytometry or fluorescence microscopy. The food systems studied included alfalfa sprouts, fresh produce (tomatoes, jalapeyo peppers, spinach, cilantro), and peanut butter. Pre-analytical sample preparation using pulsification also improved the signal-to-noise ratio for cytometric detection of Listeria monocytogenes in pork frankfurters via flow cytometry following FISH. In addition, use of the PulsifierTM enabled detachment of surface-bound L. monocytogenes cells into minimal volumes of diluent, obviating the need for the subsequent cell concentration steps typically required prior to detection.
Results from this work suggest that, when paired with effective methods for upstream food sample preparation and with downstream analytical methods such as flow cytometry and fluorescence microscopy, FISH-based methods have great potential for rapid molecular detection of Salmonella spp. and L. monocytogenes in foods.</p
Control of Listeria monocytogenes in ready-to-eat (RTE) vacuum packaged turkey roll via single or combined use of organic acid salts, ALTA 2341, and electron-beam irradiation
Get PDFThe effectiveness of electron beam irradiation for controlling LIsteria monocytogenes in ready-to-eat (RTE) turkey roll, formulated with ALTA 2341 (6,000 or 12,000 AU) alone or combined with sodium lactate (SL, 2%) + sodium diacetate (SDA, 0.25%), was investigated. Slices of turkey roll were inoculated with a five-strain mixture of L. monocytogenes to give [Difference]106 CFU/cm2. Inoculated product without ALTA 2341 or SL + SDA served as controls. Meat samples were vacuum-packaged, then irradiated at 0, 1.0, 1.5, 2.0, and 2.5 kGy. Samples irradiated at 0, 1.5, and 2.5 kGy were stored at 40C (42 days) or 100C (30 days). L. monocytogenes were enumerated by plating serial dilutions of meat homogenate on Modified Oxford (MOX) agar and counting bacterial colonies on agar plates after incubation (350C, 48h). Irradiation at 1.5 and 2.5 kGy reduced initial populations of L. monocytogenes by [Difference]2.4 and [Difference]4.4 log, respectively, irrespective of product formulation. Irradiation D-values were not significantly affected by differences in formulation (p>0.05). Growth of survivors in RTE turkey roll with ALTA (6,000 or 12,000 AU) alone was not inhibited (p>0.05). Growth of survivors was completely inhibited in samples with SL + SDA (P<0.05). For example, L. monocytogenes in irradiated (2.5 kGy) turkey roll with SL +SDA + ALTA 2341 were less than 102 CFU/cm2 throughout storage at 40C or 100C. In contrast, survivors in 2.5 kGy-treated samples without SL + SDA reached [Difference]108 CFU/cm2 at 20 days (100C) and 35 days (40C). Irradiation (2.5 kGy) combined with SL (2.0%) + SDA (0.25%) with or without ALTA (6,000 or 12,000 AU) is effective in reducing initial populations of L. monocytogenes in RTE turkey roll and preventing growth of survivors during refrigeration (40C) and temperature abuse (100C).</p
Advances in Prevention of Foodborne Pathogens of Public Health Concern during Manufacturing
Get PDFAccording to a report from the U.S. Centers for Disease Control and Prevention (CDC), achieving safe and healthier foods was one of the top ten achievements of public health in the 20th century. However, considerable persisting challenges currently exist in developed nations and developing economies for further assuring the safety and security of the food supplies. According to CDC estimates, as many as 3000 American adults, as an example, and based on a recent epidemiological estimate of the World Health Organization, around 420,000 individuals around the globe, lose their lives annually due to foodborne diseases. This emphasizes the need for innovative and emerging interventions, for further prevention or mitigation of the risk of foodborne microbial pathogens during food processing and manufacturing. The current publication discusses recent advancements and progress in the elimination and decontamination of microbial pathogens during various stages of manufacturing and production. Special emphasis is placed on hurdle validation studies, investigating decontamination of non-typhoidal Salmonella enterica serovars, various serogroups of Shiga toxin-producing Escherichia coli, public health-significant serotypes of Listeria monocytogenes, and pathogenic species of Cronobacter
Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce
Get PDFThis 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 facedown 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.This article is from Journal of Visualized Experiments 44 (2010), doi:10.3791/2308.
This article includes a video component, which may be found at http://www.jove.com/video/2308/.</p
Simple Adhesive-Tape-Based Sampling of Tomato Surfaces Combined with Rapid Fluorescence In Situ Hybridization for Salmonella Detection
Get PDFA 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.This article is from Applied and Environmental Microbiology 75, no. 5 (March 2009): 1450–1455, doi:10.1128/AEM.01944-08.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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