Cesar Nadala

Draft Genome Sequence of blaNDM-1-Positive Escherichia coli O25b-ST131 Clone Isolated from an Environmental Sample

ABSTRACT A multidrug-resistant NDM-1 carbapenamase-producing Escherichia coli sequence type 131 (ST131) organism was obtained from vacuum cleaner dust collected from the home of a case patient. Here, we report the assembly and annotation of its genome.

Development and validation of a rapid test system for detection of pork meat and collagen residues.

Mislabeling, contamination, and economic adulteration of meat products with undeclared pork tissues are illegal under regulations promulgated by numerous regulatory agencies. Nonetheless, analysis of the European meat industry has revealed pervasive meat adulteration, necessitating more extensive application of meat authentication testing. As existing methods for meat speciation require specialized equipment and/or training, we developed a detection system based on a lateral flow device (LFD) assay format capable of rapidly (~35min) identifying porcine residues derived from raw meat, cooked meat, and gelatin down to 0.01%, 1.0%, and 2.5% contamination, respectively. Specificity analysis revealed no cross-reactivity with meat derived from chicken, turkey, horse, beef, lamb, or goat. Comparison with a commercial ELISA kit and PCR method revealed similar if not improved sensitivity, with the added feature that the LFD-based system required considerably less time to perform. Accordingly, this test system should aid the food industry and food control authorities in monitoring for adulteration with pork.

Development and Validation of a Lateral Flow Immunoassay Test Kit for Dual Detection of Casein and β-Lactoglobulin Residues.

Allergies to cows milk are very common and can present as life-threatening anaphylaxis. Consequently, food labeling legislation mandates that foods containing milk residues, including casein and/or β-lactoglobulin, provide an indication of such on the product label. Because contamination with either component independent of the other can occur during food manufacturing, effective allergen management measures for containment of milk residues necessitates the use of dual screening methods. To assist the food industry in improving food safety practices, we have developed a rapid lateral flow immunoassay test kit that reliably reports both residues down to 0.01 μg per swab and 0.1 ppm of protein for foods. The assay utilizes both sandwich and competitive format test lines and is specific for bovine milk residues. Selectivity testing using a panel of matrices with potentially interfering substances, including commonly used sanitizing agents, indicated reduction in the limit of detection by one-to fourfold. With food, residues were easily detected in all cows milk-based foods tested, but goat and sheep milk residues were not detected. Specificity analysis revealed no cross-reactivity with common commodities, with the exception of kidney beans when present at high concentrations (> 1%). The development of a highly sensitive and rapid test method capable of detecting trace amounts of casein and/or β-lactoglobulin should aid food manufacturers and regulatory agencies in monitoring for milk allergens in environmental and food samples.

Draft Genome Sequence of the Beer Spoilage Bacterium Megasphaera cerevisiae Strain PAT 1T.

ABSTRACT The genus Megasphaera harbors important spoilage organisms that cause beer spoilage by producing off flavors, undesirable aroma, and turbidity. Megasphaera cerevisiae is mainly found in nonpasteurized low-alcohol beer. In this study, we report the draft genome of the type strain of the genus, M. cerevisiae strain PAT 1T.

Quantitative Detection of Horse Contamination in Cooked Meat Products by ELISA

Recent news of many cases of adulteration of meats with pork has bolstered the need for a way to detect and quantify the unwanted contamination of pork in other meats. To address this need, Microbiologique, Inc. has produced a sandwich ELISA assay that can rapidly quantify the presence of pork in cooked horse, beef, chicken, goat, and lamb meats. We carried out a validation study and showed that this assay has an analytical sensitivity of 0.00014 and 0.00040% (w/v) for cooked and autoclaved pork, respectively, and an analytical range of quantitation of 0.05-3.2% (w/v) in the absence of other meats. The assay can measure pork contamination down to 0.1% (w/w) in the presence of cooked horse, beef, chicken, goat, and lamb meats. The assay is quick and can be completed in 1 h and 10 min.

An Economical and Scalable Preparation of Poly(Ethylene Glycol) Methyl Ether Amine, M.W. 5,000

Polyethylene glycols (PEGs) are polyether chemical polymers of ethylene oxide that have many applications in industry, cosmetics, medicine and chemical research. Polymerizations of PEGs are categorized statistically by molecular weight ranging from small (300 g/mol) to large (10,000,000 g/mol) with many different derivatives, branching patterns and functional groups. Recently, PEGs have been popularized for their use in drug targeting effects and as drug-polymer conjugate linkers to increase bio-availability through slow release. One decidedly important PEG derivation is poly(ethylene glycol) methyl ether amine (mPEG-amine). This is comprised of a methoxy ether initiator, polymer body and is functionalized with a terminal primary amine, with an average molecular weight of 5000 g/mol. This polymer has been attached to various drugs (e. g. atropine and procaine) and enzymes (e. g. insulin). In our laboratory, mPEG5000-amine is used to prepare immunomagnetic particles, where it was found that antibodies conjugated to carboxy-magnetic beads were significantly more efficient once rigidified with mPEG-amine polymer. The present discovery stems from our need to prepare PEG derivatives with suitable functionalities for conjugation, bulk quantities (>50 grams/month), and at a reduced cost (commercial mPEG-5000-amine,

Simultaneous Detection of Multiple Wine-Spoilage Organisms Using a PCR-Based DNA Dipstick Assay

104 USD/g, Sigma Aldrich Catalogue, 2017). The ease with which this compound can be synthesized is an important requirement. The reaction sequence we now describe requires no column chromatography and no costly reagents or overly complex reaction set-ups. Accordingly, we selected poly(ethylene glycol) methyl ether, M.W. 5000 (1) as an economical starting material (mPEG-5000-OH,

Rapid Detection of Listeria in Ice Cream in 13 Hours Using the Roka Listeria Detection Assay

0.18 USD/g, Sigma Aldrich Catalogue, 2017). In this simple reaction sequence (Scheme 1), chlorination of 1 (50 g) with SOCl2 and pyridine refluxed in toluene and precipitation with ether produced the corresponding alkyl chloride 2 in 95% yield. Chloride

Comparison of ELISA and DNA Lateral Flow Assays for Detection of Pork, Horse, Beef, Chicken, Turkey, and Goat Contamination in Meat Products

Background: The presence of microbial contaminants such as Brettanomyces in wine can lead to undesirable wine. Therefore, monitoring for the presence of these spoilage organisms is critical for winemakers to ensure the quality of their end product. Objective: To address this problem, Molecular Epidemiology, Inc. (MEI, Seattle, WA) has developed a wine-spoilage organism detection kit consisting of a multiplex PCR DNA dipstick that simultaneously detects these organisms. Methods: Wine samples obtained from local wineries that tested negative by routine microbiological culture were spiked with the target microorganisms, while samples that were designated as spoiled by the wineries were used as-is without spiking for assessing the performance characteristics of the DNA dipstick assay. Microbial enumeration was performed following standard microbiological plating methods. Samples spiked with low cell numbers (<5 cells per 100 mL) were enriched using wine enrichment media (WSE; optional component of the kit) prior to analysis using the DNA dipstick assay. Suitability of WSE medium to support the growth of wine-spoilage microorganisms was compared with standard microbiological media. Results: Testing of 92 diverse bacterial and yeast strains commonly found in winery and food operations and 50 various strains of spoilage organisms isolated from wineries indicated that the dipstick assay can exclusively detect the target wine-spoilage microorganisms. All target spoilage organisms in samples containing low cell numbers (<5 cells per 100 mL) were detected by dipstick assay 48 h postenrichment in WSE, except for a few strains of Brettanomyces bruxellensis that required longer incubation times. Conclusions: The wine-spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the wine-making process to detect multiple spoilage-causing microorganisms in a single assay, thus offering a convenient test system for winemakers interested in monitoring the quality of their product.

Quantitative Detection of Chicken and Turkey Contamination in Cooked Meat Products by ELISA

Background: Listeria contamination is a major concern in the ice cream industry; therefore, early and accurate detection is vital. Current detection methods require about a 24 h enrichment period for detection. Objective: Enhance the early detection of Listeria in ice cream using the highly sensitive isothermal ribosomal RNA-based Roka/Atlas Listeria Detection Assay. Methods: The R2 Medium was developed for Listeria enrichment by Molecular Epidemiology, Inc. (Seattle, WA). Comparative growth curve studies were performed on the new R2 Medium for Listeria and the currently validated media for the Roka Listeria Detection Assay. Subsequently, a method comparison between the Roka Listeria Detection Assay and the U.S. Food and Drug Administrations (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 reference method on ice cream was carried out. Results: The R2 Medium supports the growth of L. monocytogenes better than Buffered Listeria Enrichment Broth, Demi-Fraser broth, and Modified University of Vermont Broth, as indicated by the faster growth rate of the organism. When used as an enrichment medium in a method comparison study of ice cream, the results showed that R2 Medium-enriched samples tested with the Roka Listeria Detection Assay gave an equivalent performance compared with the 24 h FDA-BAM reference method at 10 and 18 h post-enrichment for Listeria. Conclusions: The results from this study indicate that the new R2 Medium and the highly sensitive Roka Listeria Detection Assay allowed for the rapid detection of Listeria species in ice cream in 13 h. Highlights: The Roka Listeria Detection Assay, in conjunction with a new media formulation (R2 Medium), allowed for the early detection of Listeria in ice cream and may be applied in other food matrixes and environmental samples.