Microbiological survey and genomic analysis of Cronobacter sakazakii strains isolated from US households and retail foods.

Cronobacter species are opportunistic pathogens that are capable of causing morbidity and mortality, particularly in infants. Although the transmission dynamics involved in Cronobacter infections remain largely unknown, contaminated powdered infant formula (PIF) has been linked to 30% of Cronobacter sakazakii cases involving invasive illness in infants. As several lines of evidence have implicated the domestic environment in PIF contamination, we undertook a microbiological survey of homes (N = 263) across the US. Cronobacter spp. and C. sakazakii were isolated from 36.1% and 24.7% of US homes, respectively, with higher recovery rates observed for floor and kitchen surfaces. Multi-locus sequence typing indicated that the dominant strain was C. sakazakii ST4, the sequence type most commonly associated with neonatal meningitis. For comparison purposes, retail foods (N = 4,009) were also surveyed, with the highest contamination frequencies (10.1%-26.3%) seen for nut products, seeds, and grains/baked goods/flours. The sequence type profile of isolates recovered from homes mirrored that of isolates recovered from retail foods, with increased representation of ST1, ST4, ST13, ST17, and ST40. Analysis of 386 whole genomic sequences revealed significant diversity. Redundancies were only observed for isolates recovered from within the same domicile, and there were no identical matches with sequences archived at the NCBI pathogen database. Genes coding for putative virulence and antibiotic resistance factors did not segregate with clinically significant sequence types. Collectively, these findings support the possibility that contamination events occurring within the home should not be overlooked as a contributor to community-onset Cronobacter infections. IMPORTANCE: Cronobacter sakazakii is an opportunistic pathogen that can cause significant morbidity and mortality in neonates. Its transmission dynamics are poorly understood, though powered infant formula (PIF) is thought to be the major transmission vehicle. How the PIF becomes contaminated remains unknown. Our survey shows that roughly 1/4 of US homes are contaminated with Cronobacter sakazakii, particularly in the kitchen setting. Our analyses suggest that the domestic environment may contribute to contamination of PIF and provides insights into mitigating the risk of transmission.

Performance Evaluation of the SAMBA II SARS-CoV-2 Test for Point-of-Care Detection of SARS-CoV-2.

Nucleic acid amplification for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory samples is the standard method for diagnosis. The majority of this testing is centralized and therefore has turnaround times of several days. Point-of-care (POC) testing with rapid turnaround times would allow more effective triage in settings where patient management and infection control decisions need to be made rapidly. The inclusivity and specificity of the Simple AMplification-Based Assay (SAMBA) II SARS-CoV-2 test were determined by both in silico analyses of the primers and probes and wet testing. The SAMBA II SARS-CoV-2 test was evaluated for performance characteristics. Clinical performance was evaluated in residual combined throat/nose swabs and compared to that of the Public Health England real-time PCR assay targeting the RdRp gene. The SAMBA II SARS-CoV-2 test has an analytical sensitivity of 250 copies/ml for detecting two regions of the genome (open reading frame 1ab [ORF1ab] and nucleocapsid protein [N]). The clinical performance was evaluated in 172 residual combined nose/throat swabs provided by the Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge (CMPHL), which showed an estimated positive percent agreement of 98.9% (95% confidence interval [CI], 93.83 to 99.97) and negative percent agreement of 96.4% (95% CI, 89.92 to 99.26) compared to testing by the CMPHL. The data show that the SAMBA II SARS-CoV-2 test performs equivalently to the centralized testing methods, but with a shorter turnaround time of 86 to 101 min. Point-of-care tests such as SAMBA should enable rapid patient management and effective implementation of infection control measures.

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

Background: Concerns about the contamination of meat products with undeclared meats and new regulations for the declaration of meat adulterants have established the need for a rapid test to detect chicken and turkey adulteration. Objective: To address this need, Microbiologique, Inc. has developed an ELISA that can quantify the presence of chicken and turkey down to 0.1% (w/w) in cooked pork, horse, beef, goat, and lamb meats. Results: This chicken/turkey authentication ELISA has an analytical sensitivity of 0.000037% and 0.000048% (w/v) for cooked and autoclaved chicken, respectively, and an analytical range of quantitation of 0.025-2% (w/v), in the absence of other meats. The assay cross-reacts with cooked duck and pheasant but does not demonstrate any cross-reactivity with cooked pork, horse, beef, goat, and lamb meats, egg, or common food matrixes. Conclusions: The assay is rapid, can be completed in 70 min, and can detect a 0.1% level of meat adulteration. Highlights: The Microbiologique Cooked Chicken/Turkey ELISA can quantitate cooked chicken/turkey in the presence of pork, horse, chicken, goat, or sheep meat to 0.1% (w/w) and is not affected by common food matrixes.

Quantitative Detection of Beef Contamination in Cooked Meat Products by ELISA.

Background: Concerns about the contamination of meat products with undeclared meats, and new regulations for the declaration of meat adulterants have established the need for a rapid test to detect beef adulteration to 0.1% sensitivity. Objective: To address this need, Microbiologique, Inc. has developed an ELISA that can quantify the presence of beef down to 0.1% (w/w) in cooked pork, horse, chicken, goat, and sheep meat. Results: The beef-authentication ELISA has an analytical sensitivity of 0.00022 and 0.00012% (w/v) for cooked and autoclaved beef, respectively, and an analytical range of quantitation of 0.025 to 2% (w/v), in the absence of other meats. Moreover, the assay is specific for cooked beef and does not cross react with common food matrixes. Conclusions: The assay is rapid, can be completed in 70 min, and can detect a 0.1% level of meat adulteration. The assay is an improvement over a previous U.S. Department of Agriculture's tested assay, which is sensitive to 1% adulteration and takes 2.5-3 h to complete. Highlights: The Microbiologique Cooked Beef ELISA can quantitate cooked beef in the presence of pork, horse, chicken, goat, and sheep meat to 0.1% (w/w) and is not affected by common food matrixes.

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

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.

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

Background: Concerns about the contamination of meat products with undeclared meats and new regulations for the declaration of meat adulterants have established the need for a sensitive test to detect meat adulteration. To address this need, Microbiologique, Inc. has developed ELISA assays that can detect the presence of pork, horse, beef, chicken, turkey, and goat meat adulterants to 0.1% (w/w) and a deoxyribonucleic acid (DNA) lateral flow assay for pork, horse, beef, chicken, turkey, goat, and lamb adulterants to 0.1% (w/w). Objective: We compared the results of the DNA lateral flow assay to the ELISA assays. Methods: ELISA and DNA lateral flow assays were performed on the same spiked meat samples, prepared meats, and pet foods. Results: Both the DNA lateral flow and the ELISA assays were sensitive to 0.1% meat adulterant, and the agreement between the DNA lateral flow and ELISA assays for spiked samples, prepared meat, and pet foods was 100%. Conclusions: Based on the 100% concordance between the two assay formats, the choice between the two is dependent on whether quantitation is desired, which assay is more familiar to the particular laboratory, availability of the required equipment, and time restrictions. Highlights: The ELISA assays are less time consuming, taking about 1.5 h, compared with about 2.5 h for the DNA lateral flow assay. Because the DNA lateral flow test detects seven species in one test, it can be more cost effective when the potential adulterant is not known, while the ELISA may be better for quantification.

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

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 Administration's (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.

Dipstick Assay for Rapid Detection of Beer Spoilage Organisms.

Background: Beer spoilage caused by wild yeast and bacteria is a major concern to both commercial and home brewers. Objective: To address this problem, Molecular Epidemiology Inc. (MEI, Seattle, WA) has developed a beer spoilage organism detection kit consisting of an enrichment media (BSE) and a multiplex PCR DNA dipstick that simultaneously detects these organisms within 2 h following enrichment. Methods: The kit was tested by using samples obtained from breweries located in the Greater Seattle area. Samples were spiked with the target microbes, when necessary, and used for assessing the performance characteristics of the DNA dipstick assay. Microbial enumerations were performed as per the standard microbiological plating methods. The suitability of the BSE medium to support the growth of beer spoilage microbes was compared with the industry-approved NBB-C medium (Dohler, Darmstadt, Germany). Results: Inclusivity (a panel of 50 isolates) and Exclusivity (a panel of 92 isolates) testing indicated that the dipstick assay can exclusively detect the indicated target beer spoilage microbes. When compared with the NBB-C medium (Dohler, Darmstadt, Germany) approved by the European Brewers Convention for beer spoilage organisms, the BSE medium supported faster growth of critical spoilage lactic acid bacteria such as Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Conclusions: The beer spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the brewing process, thus offering a convenient, cost effective, and faster test system for brewers interested in monitoring the quality of their product.

Quantitative Detection of Horse Contamination in Cooked Meat Products by ELISA.

Concerns about the contamination of meat products with horse meat and new regulations for the declaration of meat adulterants have highlighted the need for a rapid test to detect horse meat adulteration. To address this need, Microbiologique, Inc., has developed a sandwich ELISA that can quantify the presence of horse meat down to 0.1% (w/w) in cooked pork, beef, chicken, goat, and lamb meats. This horse meat authentication ELISA has an analytical sensitivity of 0.000030 and 0.000046% (w/v) for cooked and autoclaved horse meat, respectively, and an analytical range of quantitation of 0.05-0.8% (w/v) in the absence of other meats. The assay is rapid and can be completed in 1 h and 10 min. Moreover, the assay is specific for cooked horse meat and does not demonstrate any cross-reactivity with xenogeneic cooked meat sources.

Quantitative Detection of Pork 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.

New Delhi metallo-ß-lactamase-1 (NDM-1) Escherichia coli isolated from household vacuum cleaner-Oregon, 2013.

The first Oregon case of New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Escherichia coli was reported during November 2013. Epidemiologic investigation revealed only local outpatient medical care and no travel outside Oregon for both the patient and his household contact. Environmental sampling discovered a matching isolate from the patient's household vacuum cleaner, suggesting environmental persistence.

Novel Monoclonal Antibody-Based Immunodiagnostic Assay for Rapid Detection of Deamidated Gluten Residues.

Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food-processing measures can affect epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, and rye and deamidated gluten down to 2 ppm in food as well as its performance in food testing.

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

Allergies to cow's 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 ß-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 cow's 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 ß-lactoglobulin should aid food manufacturers and regulatory agencies in monitoring for milk allergens in environmental and food samples.

A Novel Immunoassay Test System for Detection of Modified Allergen Residues Present in Almond-, Cashew-, Coconut-, Hazelnut-, and Soy-Based Nondairy Beverages.

A growing number of plant-based milk substitutes have become commercially available, providing an array of options for consumers with dietary restrictions. Though several of these products rival cow's milk in terms of their nutritional profiles, beverages prepared with soy and tree nuts can be a significant concern to consumers because of potential contamination with food allergens. Adding to this concern is the fact that allergen residues from plant-based beverages are modified during manufacturing, thereby decreasing the sensitivity of antibody-based detection methods. Consequently, many commercially available allergen detection kits are less effective for allergens derived from nondairy milk substitutes. To address this limitation, we developed a panel of polyclonal antibodies directed against the modified proteins present in almond, cashew, coconut, hazelnut, and soy milks and incorporated them into rapid lateral flow immunoassay tests configured in both sandwich and competitive format. The tests had robust detection capabilities when used with a panel of various brand-name products, with a sensitivity of 1 ppm and selectivity values of 3 to 5 ppm in nondairy beverages. Minimal cross-reactivity to extracts prepared from common commodities was observed. The development of a highly sensitive and rapid test specifically designed to detect trace quantities of highly modified allergen residues in plant-based, dairy-free beverages will aid food manufacturers and regulatory agencies in monitoring products for these modified allergens when testing environmental and food samples.

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

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 1(T).

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

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.