combination-adhesive-tape-based-sampling-fluorescence-situ

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 ...

colorimetric-paper-based-detection-escherichia-coli-salmonella-spp

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

detection-of-viruses-from-bioaerosols-using-anion-exchange-resin

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

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.

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

Eine einfache Klebstoff-Bandger&auml;t f&uuml;r Probenahme von Tomaten Fl&auml;chen zusammen mit der Fluoreszenz in Situ Hybridisierung f&uuml;r kulturunabh&auml;ngig Schnellerkennung von Salmonella-St&auml;mme. B&auml;nder k&ouml;nnte auch verdeckte auf selektiv-Agar f&uuml;r die Anreicherung der auf Band aufgenommenen Salmonellen-Zellen platziert werden. &Uuml;berlagerung der Zelle aufgeladenen B&auml;nder mit kleinen Mengen an fl&uuml;ssigen Bereicherung Medien aktiviert nachfolgende Erkennung von Salmonellen &uuml;ber Durchflusszytometrie Band gefangen genommen.

Einfachen Klebstoff-bandbasierte Probenahme von Tomaten-Oberfl&auml;chen kombiniert mit schnellen Fluoreszenz in Situ Hybridisierung zum Nachweis von Salmonellen.

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".

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

Wir k&uuml;rzlich einen Ansatz, bei dem Fluoreszenz in situ Hybridisierung (FISH) und Durchflusszytometrie zum Erkennen von niedrigem Niveau Salmonella spp. (ca. 10 Absatz 3 Zellen/mL spriessen W&auml;sche) gegen hohe Niveaus von nat&uuml;rlich vorkommenden sprie&szlig;en Pflanzen (etwa 10(7)-10(8) KBE/g Sprossen). Obwohl dieser Ansatz &quot;Fisch und Flow&quot; Schnelle Anwesenheit/Abwesenheit Tests f&uuml;r Salmonellen in diesem komplexen Lebensmittel-System bereitgestellt, es war nicht mehr f&auml;hig nuanciert Aufgaben wie Sondierung der ph&auml;notypischen Komplexit&auml;t der die Mikroben in Sprossen vorhanden oder bestimmen die physikalischen Interaktionen von Salmonellen mit diesen Mikroben oder spriessen Tr&uuml;mmer. In der vorliegenden Studie haben wir kombiniert schnelle Fischgerichte Kennzeichnung von Salmonella spp. in spriessen Waschprogramme mit Passagen-bildgebenden Cytometry (FT-IC), mit dem ImageStream-100, einem kommerziellen FT-IC-Instrument. Dieser Ansatz erm&ouml;glicht bildbasierte Charakterisierung von verschiedenen Subpopulationen von Interesse, die innerhalb dieser Proben. Hier zeigen wir die F&auml;higkeit des FT-IC Zellen, Zelle Aggregate und andere Veranstaltungen innerhalb diese Subpopulationen basierend auf beide Zelle Morphologie und Hybridisierung-Status nach Reaktion mit einer Salmonellen-bezogene Pr&uuml;fspitze cocktail eindeutig zu bestimmen. Unsere F&auml;higkeit, die Natur dieser Ereignisse direkt zu erkunden erweitert die Schichten von Informationen, die dieser komplexen Proben von Cytometric Analysen m&ouml;glich und zeigt deutlich, dass sich &quot;ein Bild tausend Punkte lohnt&quot;.

Passagen-bildgebenden Cytometry zur Charakterisierung von Salmonellen Subpopulationen in Alfalfa Sprossen, eine komplexe Lebensmittel-System.

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.

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

Traubenkernextrakt (GSE) ist eine reiche Quelle von Proanthocyanidins, eine Klasse von nat&uuml;rlichen Antioxidantien berichtet weitreichende Bioaktivit&auml;t als entz&uuml;ndungshemmend, antikanzerogen und antimikrobielle Agenzien zu haben. Die F&auml;higkeit der GSE, rasch inaktivieren von Listeria Monocytogenes in-vitro- und die allgemein anerkannten als sicheren Status von GSE machen dadurch ein attraktives Kandidat f&uuml;r die Kontrolle der Listerien in oder auf Lebensmitteln extrahiert. Zuvor wurde GSE in relativ hohen Konzentrationen (1 %) in komplexe Lebensmittelmatrizes und in Kombination mit anderen Antibiotika eingesetzt. Wir versuchten, die Antilisterial Auswirkungen einer kommerziellen GSE Zubereitung (Gravinol-S) allein bei viel niedrigeren Konzentrationen (0.00015 0,125 %) in w&auml;ssriger L&ouml;sung zu charakterisieren und seine m&ouml;gliche Verwendung als eine antimikrobielle Waschanlagen f&uuml;r Frischwaren Oberfl&auml;chen zu testen. Basierend auf Br&uuml;he Microdilution Tests, waren die Mikrofone der GSE gegen L. Monocytogenes Scott A und Listeria PAM ATCC 33090 so niedrig wie 50 und 78 Becher ml(-1), beziehungsweise. GSE wurde in 0,85 % Kochsalzl&ouml;sung gegen lebenden Zellen von L. PAM &uuml;ber Durchflusszytometrie, bewertet mit Propidium Jodid als Pr&uuml;fspitze f&uuml;r Membran-Integrit&auml;t. Bei Sub-MICs und nach nur 2 min Exposition, Behandlung mit GSE verursacht schnelle Permeabilization und Horstig von L. PAM, ergibt sich, dass wir f&uuml;r L. Monocytogenes mit Fluoreszenzmikroskopie und Live/Dead F&auml;rbung best&auml;tigt. Bei h&ouml;heren Konzentrationen (0,125 %) reduziert die GSE lebensf&auml;higen Zellzahlen f&uuml;r L. Monocytogenes von etwa 2 Protokoll Einheiten innerhalb von 2 Minuten auf Tomaten-Oberfl&auml;chen. Diese Resultate schlagen das Potenzial f&uuml;r GSE als nat&uuml;rliche Steuerelement Listeria spp. auf Low-Complexity-Lebensmittel wie Tomaten.

Antilisterial Wirkungen der Gravinol-s-Traubenkernextrakt auf niedrigem Niveau in w&auml;ssrigen Medien und seine m&ouml;gliche Anwendung als ein Produkt-waschen.

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.

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

Eine CE-Methode basiert auf ganze Zellen Molekulare Kennzeichnung per Fluoreszenz in Situ Hybridisierung entwickelte sich zum Nachweis von Candida Albicans im Vollblut. Entfernung von potentiell st&ouml;rende rote Blutk&ouml;rperchen (RBC) mit einen einfachen hypoton/Waschmittel Lyse konnten wir zur Erkennung und Quantifizierung verunreinigen C. Albicans Zellen bei Konzentrationen, die waren Gr&ouml;&szlig;enordnungen niedriger als Hintergrund RBC (ca. 7,0 X Neubewerber RBC/mL) z&auml;hlt. In Anwesenheit der aufgel&ouml;ste Blut-Matrix aggregiert Hefezellen ohne den Einsatz von einem blockierenden Stecker, die Zellen zu stapeln. Kurzfilm (15 min) Hybridisierung ergab hell Candida-spezifische Fluoreszenz in Situ Hybridisierung Signale, die es uns erm&ouml;glicht, als auch nur eine einzelne injizierte Zelle erkennen. Die Peak-Fl&auml;che-ansprechen der gestapelten Candida Zellen zeigten eine starke lineare Korrelation mit Zelle Konzentrationen von Platte z&auml;hlt, bis zu ca. 7 KBE/mL (oder ca. 1 x 10 gespritzt 4-Zellen) bestimmt. Diese schnelle und quantitative Methode zum Nachweis von Candida im Blut vorteilhafte Anwendungen m&ouml;glicherweise in der Human- und veterin&auml;rmedizinische Diagnostik.

Schnelle Identifizierung von Candida Albicans im Blut kombiniert durch Kapillarelektrophorese und Fluoreszenz in Situ Hybridisierung.

Modified Moore swabs (MMS; consisting of a polyvinyl chloride cartridge filled with gauze) capture microorganisms within the packed gauze as water flows through the cartridge, while continuous flow centrifugation (CFC) uses centrifugation to sediment the microorganisms while water continuously flows in the system. This study evaluated and compared the efficacy of MMS and CFC for concentration and subsequent detection of Escherichia coli O157:H7 and Salmonella from large volumes of water (10 liters). Water samples were spiked at levels of 10(1), 10(2), 10(3), and 10(4) CFU/100 ml with three-strain cocktails of either E. coli O157:H7 or Salmonella serovars, which had been previously transformed with a plasmid to express resistance to ampicillin as well as green, red, or cyan fluorescent proteins. Plating was performed before and after concentration on tryptic soy agar supplemented with ampicillin in order to quantitate the concentration efficiencies of each method. The two lowest spiking levels were also enriched in low volumes of tryptic soy broth supplemented with ampicillin followed by testing via lateral flow devices. Significant (P < 0.05) concentrations of initial levels of E. coli O157:H7 in the range of 0.7 to 1.0 and 1.2 to 1.4 log were achieved within approximately 35 min of processing time via MMS and CFC, respectively. Similarly, significant (P < 0.05) concentrations were also achieved for Salmonella with 0.9 to 1.2 and 1.2 to 1.4 log concentration for MMS and CFC, respectively. There were no statistical differences (P > 0.05) between the two concentration methods in their ability to concentrate either of the two target bacteria. Significantly (P > 0.05) more spiked samples were detected by lateral flow devices following concentration and enrichment than for nonconcentrated, enriched samples. It is concluded that both MMS and CFC have potential to be used to enhance the sensitivity of downstream bacterial detection methods used to test irrigation water for the presence of foodborne pathogens.

Evaluation of modified moore swabs and continuous flow centrifugation for concentration of Salmonella and Escherichia coli O157:H7 from large volumes of water.

Ge&auml;ndert von Moore Tupfer (MMS; bestehend aus einer PVC-Patrone gef&uuml;llt mit Gaze) Mikroorganismen in die gepackte Gaze zu erfassen, wie Wasser in der Patrone, w&auml;hrend kontinuierlicher Fluss Zentrifugation (CFC) Verwendungen Zentrifugation auf Sediment Mikroorganismen flie&szlig;t, w&auml;hrend Wasser kontinuierlich in das System flie&szlig;t. Diese Studie ausgewertet und verglichen die Wirksamkeit von MMS und CFC f&uuml;r Konzentration und anschlie&szlig;enden Nachweis von Escherichia coli O157: H7 und Salmonellen aus gro&szlig;e Mengen an Wasser (10 Liter). Wasserproben waren Spike-Ebene 10 1 10 2, 10 Absatz 3 und 10 Absatz 4 CFU/100 ml mit drei-Dehnungs-Cocktails von E. Coli O157: H7 oder Salmonella-Serovare, die zuvor mit einem Plasmid, Resistenz gegen Ampicillin als auch gr&uuml;nen, roten oder Cyan fluoreszierende Proteine auszudr&uuml;cken umgewandelt wurde. Der &Uuml;berzug erfolgte vor und nach der Konzentration auf tryptic Soja-Agar mit Ampicillin erg&auml;nzen, um die Konzentration Wirkungsgrade der einzelnen Methoden Quantifizierung. Die beiden niedrigsten Blindkontrollen Ebenen wurden auch geringe Mengen an tryptic Soy Bouillon mit Ampicillin gefolgt von Tests &uuml;ber lateral-Flow-Ger&auml;te erg&auml;nzt bereichert. Bedeutende (P &lt; 0,05) konzentrationen des ausgangsniveaus von E. Coli o157: h7 im bereich von 0,7 bis 1.0 und 1.2 bis 1.4 Log erzielten innerhalb von etwa 35 min verarbeitungszeit per MMS und CFC, bzw.. Ebenso bedeutende (P &lt; 0,05) konzentrationen wurden auch f&uuml;r Salmonellen mit erreicht 0,9 bis 1,2 und 1.2 bis 1.4 protokoll-konzentration f&uuml;r MMS und CFC, bzw.. gab es keine statistischen unterschiede (P &gt; 0,05) zwischen den zwei Konzentration Methoden in ihrer F&auml;higkeit, entweder der Bakterien zwei Ziel konzentrieren. Signifikant (P &gt; 0,05) mehr Spike Proben von lateral-Flow-Ger&auml;te nach Konzentration und f&uuml;r nonconcentrated, angereicherten Proben als Bereicherung erkannt wurden. Wird der Schluss gezogen, dass MMS und CFC Potenzial, verbessert die Empfindlichkeit des nachgeschalteten bakterielle Nachweismethoden Bew&auml;sserung-Wasser auf das Vorhandensein von lebensmittelbedingten Krankheitserregern getestet haben.

Bewertung der modifizierten Moore-Tupfer und kontinuierlichen Fluss Zentrifugation f&uuml;r Konzentration von Salmonellen und Escherichia coli O157 aus gro&szlig;e Mengen an Wasser.

There has been much recent interest in the use of phages as biocontrol agents of foodborne pathogens in animals used for food production, and in the food products themselves. This interest seems to be driven by consumers' request for more natural foods, as well as the fact that foodborne outbreaks continue to occur, globally, in many foods, some of which (such as fresh produce), lack adequate methods to control any pathogenic contamination present. Also, the many successes with respect to regulatory approval of phage based products destined for use in foods is leading to an increase in the number of phage products that are commercially available. At present, these products are directed against three main foodborne pathogens including Escherichia coli O157:H7, Salmonella spp and Listeria monocytogenes. In the future, it is likely that new phage products will be targeted against emerging foodborne pathogens. Here, we review the current literature and status of phage based strategies aimed at reducing the presence of foodborne pathogenic bacteria in food and the food production environment.

Phage-based biocontrol strategies to reduce foodborne pathogens in foods.

Als Biocontrol Bevollm&auml;chtigte von lebensmittelbedingten Krankheitserregern bei Tieren, die f&uuml;r die Lebensmittelproduktion verwendet und in die Nahrungsmittel selbst gab es den letzten Interesse daran, die Verwendung von Phagen. Dieses Interesse scheint Verbraucher auf Wunsch angetrieben werden, f&uuml;r weitere Naturkostprodukte, sowie die Tatsache, dass lebensmittelbedingter Krankheitsausbr&uuml;che weltweit in vielen Lebensmitteln, einige davon (wie frische Produkte), auftreten, weiterhin angemessene Methoden zur Steuerung von pathogenen Verunreinigungen vorhanden fehlen. Auch die vielen Erfolge in Bezug auf die Zulassung der Phage basierte Produkte zur Verwendung in Lebensmitteln bestimmt f&uuml;hrt zu einer Zunahme der Zahl der Phagen-Produkte, die im Handel erh&auml;ltlich sind. Diese Produkte richten sich derzeit gegen drei wichtigsten Foodborne pathogene Escherichia coli O157: H7, Salmonella Spp und Listeria Monocytogenes. In Zukunft ist es wahrscheinlich, dass neue Phagen-Produkte betroffen sein werden, gegen emerging lebensmittelbedingten Krankheitserregern. Hier schauen wir uns die aktuelle Literatur und den Status der Phage basierte Strategien zur Verringerung der des Vorhandensein von Foodborne pathogene Bakterien in Lebensmitteln und der Lebensmittel-Produktionsumgebung.

Biocontrol Phage-basierte Strategien zur Verringerung der Foodborne Krankheitserreger in Lebensmitteln.

Foodborne pathogens are a major public health threat and financial burden for the food industry, individuals, and society, with an estimated 76 million cases of food related illness occurring in the United States alone each year. Three of the most important causative bacterial agents of foodborne diseases are pathogenic strains of Escherichia coli, Salmonella spp., and Listeria monocytogenes, due to the severity and frequency of illness, and disproportionally high number of fatalities. Their continued persistence in food has dictated the ongoing need for faster, simpler, and less expensive analytical systems capable of live pathogen detection in complex samples. Culture techniques for detection and identification of foodborne pathogens require 5-7 days to complete. Major improvements to molecular detection techniques have been introduced recently, including polymerase chain reaction (PCR). These methods can be tedious; require complex, expensive instrumentation; necessitate highly trained personnel; and are not easily amenable to routine screening. Here, a microfluidic paper-based analytical device (Î¼PAD) has been developed for the detection of E. coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in food samples as a screening system. In this work, a paper-based microspot assay was created using wax printing on filter paper. Detection is achieved by measuring the color change when an enzyme associated with the pathogen of interest reacts with a chromogenic substrate. When combined with enrichment procedures, the method allows for a enrichment time of 12 hours or less and is capable of detecting bacteria in concentrations in inoculated ready-to-eat (RTE) meat as low as 101 CFU/cm2.

Development of a Paper-Based Analytical Device for Colorimetric Detection of Select Foodborne Pathogens.

Foodborne Krankheitserreger sind einer gr&ouml;&szlig;ten Gesundheitsbedrohung und finanzielle Belastung f&uuml;r die Lebensmittelindustrie, Individuen und Gesellschaft mit etwa 76 Millionen F&auml;lle von Lebensmittel &auml;hnliche Krankheit, die in den Vereinigten Staaten alleine jedes Jahr auftreten. Drei der wichtigsten bakteriellen Erreger von lebensmittelbedingten Krankheiten sind pathogene St&auml;mme von Escherichia coli, Salmonella spp. und Listeria Monocytogenes, aufgrund der schwere und H&auml;ufigkeit der Erkrankung und unverh&auml;ltnism&auml;&szlig;ig hohe Zahl von Todesopfern. Ihre fortgesetzte Persistenz in Lebensmitteln hat laufend den Bedarf an schneller, einfacher und kosteng&uuml;nstiger Analysensysteme Lage Leben Erregernachweis in komplexen Proben diktiert. Kultur Techniken zum Nachweis und zur Identifizierung von lebensmittelbedingten Krankheitserregern erfordern 5-7 Tage in Anspruch. Hauptverbesserungen zu Molekulare Erkennungstechniken wurden vor kurzem einschlie&szlig;lich Polymerase-Kettenreaktion (PCR) eingef&uuml;hrt. Diese Methoden k&ouml;nnen langwierig sein; erfordern Sie komplexe, teure Instrumentierung; erfordern Sie gut ausgebildete Personal. und sind nicht leicht zug&auml;nglich, routinem&auml;&szlig;ige Pr&uuml;fung unterzogen wurden. Hier wurde eine mikrofluidische Papier-analytische Ger&auml;t (&mu;PAD) zum Nachweis von E. Coli O157: H7, Salmonella Typhimurium und Listeria Monocytogenes in Lebensmittelproben als ein Screening-Systems entwickelt. In dieser Arbeit wurde eine papiergebundene MicroSpot-Assay, mit Wachs Drucken auf Filterpapier. Erkennung erfolgt durch Messung der Farb&auml;nderung, wenn ein Enzym, das mit der Erreger von Interesse mit einem chromogenen reagiert. In Kombination mit Anreicherung Verfahren die Methode erm&ouml;glicht eine Bereicherung-Zeit von 12 Stunden oder weniger und ist Aufsp&uuml;ren von Bakterien in Konzentrationen in beimpften Ready-to-eat (RTE) Fleisch so niedrig wie 10&lt;/sup&gt; 1 KBE/cm2.

Entwicklung einer Papier-analytische Ger&auml;t f&uuml;r kolorimetrische Erkennung von ausgew&auml;hlten lebensmittelbedingten Krankheitserregern.

Most diagnostic approaches for Shiga toxin producing Escherichia coli (STEC) have been designed to detect only serogroup O157 that causes a majority, but not all STEC related outbreaks in the United States. Therefore, there is a need to develop methodology that would enable the detection of other STEC serogroups that cause disease. Three bacteriophages (phages) that infect STEC serogroups O26, O103, O111, O145 and O157 were chemically labeled with horseradish peroxidase (HRP). The enzyme-labeled phages (Phazymes) were individually combined with a sampling device (a swab), STEC serogroup-specific immunomagnetic separation (IMS) beads, bacterial enrichment broth and luminescent HRP substrate, in a self-contained test device, while luminescence was measured in a hand-held luminometer.The O26 and O157 Phazyme assays correctly identified more than 93% of the bacteria tested during this study, the O123 Phazyme assay identified 89.6%, while the O111 and O145 Phazyme assays correctly detected 82.4% and 75.9%, respectively. The decreased specificity of the O111 and O145 assays was related to the broad host ranges of the phages used in both assays. The Phazyme assays were capable of directly detecting between 10(5) and 10(6) CFU/ml in pure culture, depending on the serogroup. In food trials, the O157 Phazyme assay was able to detect E. coli O157:H7 in spinach consistently at levels of 1 CFU/g and occasionally at levels of 0.1 CFU/g. The assay detected 10(0) CFU/100 cm(2) on swabbed meat samples and 10(2) CFU/100 ml in water samples. The Phazyme assay effectively detects most STEC in a simple and rapid manner, with minimal need for instrumentation to interpret the test result.

Luminescence based enzyme-labeled phage (Phazyme) assays for rapid detection of Shiga toxin producing Escherichia coli serogroups.

Die meisten diagnostische Ans&auml;tze f&uuml;r Shiga-Toxin-produzierenden Escherichia coli (STEC) wurden entwickelt, um nur Serogruppe O157 zu erkennen, die bewirkt, eine Mehrheit dass, aber nicht alle STEC &auml;hnliche Ausbr&uuml;che in den Vereinigten Staaten. Es gibt deshalb m&uuml;ssen Methodik entwickelt, die die Erkennung von anderen STEC-Serogruppen erm&ouml;glichen w&uuml;rde, die Krankheiten hervorrufen. Drei Bakteriophagen (Phagen), die STEC Serogruppen O157, O26, O103, O111 und O145 infizieren wurden chemisch mit Meerrettich-Peroxidase (HRP) bezeichnet. Die Enzym-markierten Phagen (Phazymes) wurden individuell kombiniert mit ein Probenahmeger&auml;t (einen Tupfer), STEC-Serogruppe-spezifische Immunomagnetic Trennung (IMS) Perlen, bakterielle Anreicherung Br&uuml;he und leuchtende HRP-Substrat, in einem eigenst&auml;ndigen Testger&auml;t, w&auml;hrend Lumineszenz in einem handgef&uuml;hrten Luminometer gemessen wurde.Die O26 und O157 Phazyme Assays korrekt identifiziert mehr als 93 % der Bakterien, die bei dieser Studie getestet, identifiziert der O123 Phazyme-Assay 89,6 %, w&auml;hrend die O111 und O145 Phazyme Assays 82,4 % und 75,9 %, bzw. erkannt. Die verminderte Spezifit&auml;t der O111 und O145 Tests bezog sich die breiten Host-Reihen von den Phagen in beiden Proben verwendet. Die Phazyme-Assays wurden direkt zwischen 10 Absatz 5 und 10 6 KBE/ml in Reinkultur, abh&auml;ngig von der Serogruppe feststellen kann. In Essen-Studien konnte O157 Phazyme Assays E. Coli O157: H7 in Spinat konsequent auf Ebenen 1 KBE/g und gelegentlich in Stufen von 0,1 CFU/g zu erkennen. Der Assay 10(0) CFU/100 cm(2) tupfte Fleisch Proben und 10 Absatz 2 CFU/100 ml in Wasserproben erkannt. Im Phazyme-Test erkennt die meisten STEC effektiv auf einfache und schnelle Weise mit minimaler Instrumentierung, das Testergebnis zu interpretieren.

Lumineszenz basierte Enzym-markierten Phage (Phazyme)-Assays f&uuml;r schnelle Erkennung von Shiga-Toxin-produzierenden Escherichia coli Serogruppen.

Escherichia coli O157:H7 and Salmonella spp. are bacterial pathogens often associated with beef, and cause many cases of foodborne illness each year in the United States. During beef slaughter and processing, these bacteria may spread from the hide or intestines to the carcass. The objective of this research was to investigate the use of naturally occurring compounds citrus essential oils (CEOs) extracted from orange peel to reduce or eliminate these pathogens at the chilling stage of processing, or during fabrication. Brisket flats (used to simulate beef subprimals) were spot inoculated with approximately 6 log of surrogate generic E. coli cocktail (previously shown to be identical in growth and survival parameters to E. coli O157:H7 and Salmonella spp.). Following drying, CEOs were applied by spraying at concentrations of 3% and 6% to the surface of different pieces of meat. Treatments were applied using a custom built spray cabinet at 2.07 bar and applied at a rate of 3.79 L/min to replicate commercial practices. The CEOs significantly reduced (P < 0.05) the concentration of E. coli on the brisket flats in comparison to inoculated no spray or water sprayed controls over a period of 90 d, while causing an initial reduction of approximately 1.4 log units. Total aerobic bacteria and psychrotrophic counts were also reduced on uninoculated briskets following treatment. These results indicate that 3% cold-pressed terpeneless Valencia orange oil could be used as an additional intervention against E. coli O157:H7 and Salmonella spp. at the refrigerated storage stage of processing.

Activity of citrus essential oils against Escherichia coli O157:H7 and Salmonella spp. and effects on beef subprimal cuts under refrigeration.

Detecting low concentrations of enteric viruses in water is needed for public health-related monitoring and control purposes. Thus, there is a need for sensitive, rapid and cost effective enteric viral concentration methods compatible with downstream molecular detection. Here, a virus concentration method based on adsorption of the virus to an anion exchange resin and direct isolation of nucleic acids is presented. Ten liter samples of tap water spiked with different concentrations (10-10,000 TCID50/10 L) of human adenovirus 40 (HAdV-40), hepatitis A virus (HAV) or rotavirus (RV) were concentrated and detected by real time PCR or real time RT-PCR. This method improved viral detection compared to direct testing of spiked water samples where the ΔCt was 12.1 for AdV-40 and 4.3 for HAV. Direct detection of RV in water was only possible for one of the three replicates tested (Ct of 37), but RV detection was improved using the resin method (all replicates tested positive with an average Ct of 30, n=3). The limit of detection of the method was 10 TCID50/10 L for HAdV-40 and HAV, and 100 TCID50/10 L of water for RV. These results compare favorably with detection limits reported for more expensive and laborious methods.

Concentration of enteric viruses from tap water using an anion exchange resin-based method.

After harvest, pistachios are hulled with mechanical abrasion and then separated in a float tank containing water; the nuts that float (∼15%; floaters) and those that sink (∼85%; sinkers) are dried and stored separately. To determine the prevalence of Salmonella in pistachios, a total of 3,966 samples (1,032 floaters and 2,934 sinkers) were collected within 4 months of the 2010, 2011, and 2012 harvests from storage silos (12 samples from each silo, in most cases) and were stored at 4°C; 100-g subsamples were enriched for the presence of Salmonella. Twenty-one of the floater samples and 11 of the sinker samples were positive for Salmonella: 2.0% prevalence (95% confidence interval [CI], 1.3 to 3.1%) and 0.37% prevalence (95% CI, 0.21 to 0.67%), respectively, for a weighted average prevalence of 0.61%. Levels of Salmonella were determined for positive samples using a most-probable-number (MPN) method with multiple 50-g, three 5.6-g, and three 0.56-g subsamples. Geometric mean levels of Salmonella in floaters and sinkers were 0.66 MPN/100 g (0.14 to 5.3 MPN/100 g) and 0.18 MPN/100 g (0.10 to 0.62 MPN/100 g), respectively. Seven different serovars were identified among the isolates, with nine pulsed-field gel electrophoresis fingerprints; as many as four serovars were isolated from some samples. Salmonella serovars Montevideo (44%), Enteritidis (19%), Senftenberg (16%), Worthington (12%), and Liverpool (9.4%) were most commonly isolated from the initial 100-g samples. The prevalence and levels of Salmonella in pistachios are within those observed for other tree nuts, but the limited number of serovars isolated suggests a narrow and persistent contamination source.

Prevalence and Amounts of Salmonella Found on Raw California Inshell Pistachios.

The development of transparency-based electrochemical and paper-based colorimetric analytic detection platforms is presented as complementary methods for food and waterborne bacteria detection from a single assay. Escherichia coli and Enterococcus species, both indicators of fecal contamination, were detected using substrates specific to enzymes produced by each species. β-galactosidase (β-gal) and β-glucuronidase (β-glucur) are both produced by E. coli, while β-glucosidase (β-gluco) is produced by Enterococcus spp. Substrates used produced either p-nitrophenol (PNP), o-nitrophenol (ONP), or p-aminophenol (PAP) as products. Electrochemical detection using stencil-printed carbon electrodes (SPCEs) was found to provide optimal performance on inexpensive and disposable transparency film platforms. Using SPCEs, detection limits for electrochemically active substrates, PNP, ONP, and PAP were determined to be 1.1, 2.8, and 0.5 μM, respectively. A colorimetric paper-based well plate system was developed from a simple cardboard box and smart phone for the detection of PNP and ONP. Colorimetric detection limits were determined to be 81 μM and 119 μM for ONP and PNP respectively. While colorimetric detection methods gave higher detection limits than electrochemical detection, both methods provided similar times to positive bacteria detection. Low concentrations (10 CFU/mL) of pathogenic and nonpathogenic E. coli isolates and (10 CFU/mL) E. faecalis and E. faecium strains were detected within 4 and 8 h of pre-enrichment. Alfalfa sprout and lagoon water samples served as model food and water samples, and while water samples did not test positive, sprout samples did test positive within 4 h of pre-enrichment. Positive detection of inoculated (2.3 × 10 and 3.1 × 10 CFU/mL or g of E. coli and E. faecium, respectively) sprout and water samples tested positive within 4 and 12 h of pre-enrichment, respectively.

Colorimetric and Electrochemical Bacteria Detection Using Printed Paper- and Transparency-Based Analytic Devices.

The detection of aerosolized viruses can serve as an important surveillance and control tool in agriculture, human health, and environmental settings. Here, we adapted an anion exchange resin-based method, initially developed to concentrate negatively charged viruses from water, to liquid impingement-based bioaerosol sampling. In this method, aerosolized viruses are collected in a 20ml liquid sample contained within widely used impingers, BioSamplers (SKC Inc., Eighty Four, PA), and further concentrated via adsorption to an anion exchange resin that is suspended within this liquid. Viral nucleic acids are then extracted from the resin to facilitate molecular analyses through a reduction in the effective sample volume. For this study, various quantities of two negatively charged viruses, type A and type B influenza viruses (FluMist Quadrivalent vaccine) and the male-specific (F+) RNA coliphage MS2 (MS2), were nebulized into a custom-built bioaerosolization chamber, and sampled using BioSamplers with and without anion exchange resin. Compared to direct testing of the BioSampler liquid, detection was improved by 6.77× and 3.33× for type A and type B influenza viruses, respectively, by using the anion exchange resin. For MS2, the anion exchange resin method allowed for an average improvement in detection of 8.26×.

A method for the improved detection of aerosolized influenza viruses and the male-specific (F+) RNA coliphage MS2.

The World Health Organization recently recognized the Republic of Kosovo as one of the highest consumers per capita of antibiotics for human use among non-European Union Eastern European countries; however, data are limited regarding antimicrobial usage and antimicrobial resistance in the livestock sector for this recently formed country. The objective of this study was to conduct the first nationwide survey of antimicrobial resistance phenotypes in indicator bacteria collected from dairy farms in Kosovo. Composite fecal samples were collected from 52 farms located within all 7 administrative districts of Kosovo in the summer of 2014. Isolation and characterization of the indicator bacteria Escherichia coli (n = 165) and Enterococcus spp. (n = 153) from these samples was achieved by culturing on selective/differential media with and without select antibiotics, followed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry-based identification and antimicrobial susceptibility testing using the disk diffusion method. When no selective pressure was applied in culture-based isolation, the majority of E. coli and Enterococcus spp. collected were resistant to ≤1 of 16 and ≤2 of 12 antibiotics tested, respectively. In contrast, E. coli and Enterococcus spp. isolated using sub-minimum inhibitory concentrations of cefoxitin, ciprofloxacin, or erythromycin were typically resistant to at least one and often multiple antibiotic types, which primarily consisted of certain β-lactams, quinolones, sulfonamides, phenicols, and tetracyclines for E. coli isolates and macrolides, tetracyclines, and rifamycins for enterococci isolates.

ArticleTotal : 3	Year
Publication title Cited by 17	2010
Publication title Cited by 24	2014
Publication title	2018

Article	Year
Simple adhesive-tape-based sampling of tomato surfaces combined with rapid fluorescence in situ hybridization for Salmonella detection. Applied and environmental microbiology\| PubMed ID: 19124588	2009
Flow-through imaging cytometry for characterization of Salmonella subpopulations in alfalfa sprouts, a complex food system. Biotechnology journal\| PubMed ID: 19492327	2009
Antilisterial effects of gravinol-s grape seed extract at low levels in aqueous media and its potential application as a produce wash. Journal of food protection\| PubMed ID: 20132671	2010
Rapid identification of Candida albicans in blood by combined capillary electrophoresis and fluorescence in situ hybridization. Electrophoresis\| PubMed ID: 20665522	2010
Evaluation of modified moore swabs and continuous flow centrifugation for concentration of Salmonella and Escherichia coli O157:H7 from large volumes of water. Journal of food protection\| PubMed ID: 22054197	2011
Phage-based biocontrol strategies to reduce foodborne pathogens in foods. Bacteriophage\| PubMed ID: 22164346	2011
Development of a Paper-Based Analytical Device for Colorimetric Detection of Select Foodborne Pathogens. Analytical chemistry\| PubMed ID: 22320200	2012
Luminescence based enzyme-labeled phage (Phazyme) assays for rapid detection of Shiga toxin producing Escherichia coli serogroups. Bacteriophage\| PubMed ID: 22334866	2011
Activity of citrus essential oils against Escherichia coli O157:H7 and Salmonella spp. and effects on beef subprimal cuts under refrigeration. Journal of food science\| PubMed ID: 22417514	2011
Concentration of enteric viruses from tap water using an anion exchange resin-based method. Journal of virological methods\| PubMed ID: 24911889	2014
Prevalence and Amounts of Salmonella Found on Raw California Inshell Pistachios. Journal of food protection\| PubMed ID: 27497117	2016
Colorimetric and Electrochemical Bacteria Detection Using Printed Paper- and Transparency-Based Analytic Devices. Analytical chemistry\| PubMed ID: 28225595	2017
A method for the improved detection of aerosolized influenza viruses and the male-specific (F+) RNA coliphage MS2. Journal of virological methods\| PubMed ID: 28450173	2017
Short communication: A countrywide survey of antimicrobial-resistant indicator bacteria in Kosovo's dairy farms. Journal of dairy science\| PubMed ID: 29859693	2018

Bledar Bisha

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