Development of a Multiplex Polymerase Chain Reaction (PCR) Assay for the Potential Detection of Insect Contaminants in Food.

Molecular methods can potentially be used to detect insect contaminants of food products. In this study, we used three sets of group-specific primers, two of them targeting the amplification of two regions of the insect's mitochondrial cytochrome c oxidase subunit I (COI-Fa and COI-Fb) and the other targeting a region of the nuclear protein-coding wingless (wg) gene. Using singleplex and multiplex polymerase chain reaction (PCR), we evaluated the three sets of primers using genomic DNA (gDNA) from 48 insect species including food storage insect pests and known vectors of foodborne pathogens. Seven plant-based food matrices were also evaluated for exclusivity testing. Additionally, we spiked fragments from five insect species in a selected food matrix (whole wheat flour). Singleplex and multiplex PCR amplified single specific bands (401-449 bp), corresponding to the wg gene, from insect species belonging to families Blattidae and Formicidae, and in Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). The COI-Fa primers amplified specific bands (171-188 bp) in all Dipteran species and the COI-Fb primers amplified a specific band (∼140 bp) in DNA from Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and P. interpunctella. However, the presence of specific bands in most Coleopterans was not consistent. No amplicon bands were observed in any of the food matrixes tested and the expected pattern of amplicon bands was seen in multiplex reactions using gDNA from spiked food samples. Our multiplex PCR assay targeted specific groups of insects that commonly contaminate foods without amplifying bands from the food matrixes tested; thus, molecular methods may be suitable for detecting insects or their fragments in foods.

Prevalence of Salmonella in 11 Spices Offered for Sale from Retail Establishments and in Imported Shipments Offered for Entry to the United States.

The U.S. Food and Drug Administration conducted a survey to evaluate Salmonella prevalence and aerobic plate counts in packaged (dried) spices offered for sale at retail establishments in the United States. The study included 7,250 retail samples of 11 spice types that were collected during November 2013 to September 2014 and October 2014 to March 2015. No Salmonella-positive samples (based on analysis of 125 g) were found among retail samples of cumin seed (whole or ground), sesame seed (whole, not roasted or toasted, and not black), and white pepper (ground or cracked), for prevalence estimates of 0.00% with 95% Clopper and Pearson's confidence intervals of 0.00 to 0.67%, 0.00 to 0.70%, and 0.00 to 0.63%, respectively. Salmonella prevalence estimates (confidence intervals) for the other eight spice types were 0.19% (0.0048 to 1.1%) for basil leaf (whole, ground, crushed, or flakes), 0.24% (0.049 to 0.69%) for black pepper (whole, ground, or cracked), 0.56% (0.11 to 1.6%) for coriander seed (ground), 0.19% (0.0049 to 1.1%) for curry powder (ground mixture of spices), 0.49% (0.10 to 1.4%) for dehydrated garlic (powder, granules, or flakes), 0.15% (0.0038 to 0.83%) for oregano leaf (whole, ground, crushed, or flakes), 0.25% (0.03 to 0.88%) for paprika (ground or cracked), and 0.64% (0.17 to 1.6%) for red pepper (hot red pepper, e.g., chili, cayenne; ground, cracked, crushed, or flakes). Salmonella isolates were serotyped, and genomes were sequenced. Samples of these same 11 spice types were also examined from shipments of imported spices offered for entry to the United States from 1 October 2011 to 30 September 2015. Salmonella prevalence estimates (based on analysis of two 375-g composite samples) for shipments of imported spices were 1.7 to 18%. The Salmonella prevalence estimates for spices offered for sale at retail establishments for all of the spice types except dehydrated garlic and basil were significantly lower than estimates for shipments of imported spice offered for entry.

Presence of Undeclared Food Allergens in Cumin: The Need for Multiplex Methods.

Beginning in the autumn of 2014, millions of dollars of food and over 675 products were recalled in the United States due to the presence of undeclared peanut, attributed to cumin used in the manufacture of the products. Initial analyses also indicated the presence of almond. Subsequent research showed that the presence of peanut and almond did not fully explain the analytical results for the cumin samples. Using a combination of mass spectrometry, DNA-based methods (i.e., PCR and Sanger DNA Sequencing), microscopy, and antibody-based technologies (i.e., ELISA, Western blot analysis, and a novel xMAP multiplex assay) the presence of peanut was confirmed. Screening for secondary sources of adulteration (e.g., tree nuts, mahleb, peach, and cherry) supported the assessment that the cumin contained multiple contaminants. These results demonstrate the limitations of single analyte-specific assays and the need for orthogonal multiplex methods to detect food allergens irrespective of varietal or other differences.

Ingested Salmonella enterica, Cronobacter sakazakii, Escherichia coli O157:H7, and Listeria monocytogenes: transmission dynamics from adult house flies to their eggs and first filial (F1) generation adults.

BACKGROUND: The mechanical transmission of pathogenic bacteria by synanthropic filth flies is widely recognized. While many studies report the fate and the temporospatial distribution of ingested foodborne bacteria by filth flies, there is little evidence about the transmission dynamics of ingested foodborne bacteria by adult house flies (Musca domestica) to their progeny. In this study, we fed parental house fly adults with food contaminated with low, medium, and high concentrations of Salmonella enterica, Cronobacter sakazakii, Escherichia coli O157:H7, and Listeria monocytogenes and evaluated the probability of transmission of these pathogens to house fly eggs and the surface and the alimentary canal of their first filial (F1) generation adults. RESULTS: All foodborne pathogens were present in samples containing pooled house fly eggs. The probability of transmission was higher after parental house flies ingested food containing medium bacterial loads. Cronobacter sakazakii was 16, 6, and 3 times more likely to be transmitted to house fly eggs than S. enterica, E. coli O157:H7, and L. monocytogenes, respectively. Only S. enterica and C. sakazakii were transmitted to F1 generation adults and their presence was 2.4 times more likely on their body surfaces than in their alimentary canals. The highest probabilities of finding S. enterica (60 %) and C. sakazakii (28 %) on newly emerged F1 adults were observed after parental house flies ingested food containing medium and high levels of these pathogens, respectively. CONCLUSION: Our study demonstrates that adult house flies that fed from food contaminated with various levels of foodborne bacteria were able to transmit those pathogens to their eggs and some were further transmitted to newly emerged F1 generation adults, enhancing the vector potential of these insects. Understanding the type of associations that synanthropic filth flies establish with foodborne pathogens will help to elucidate transmission mechanisms and possible ways to mitigate the spread of foodborne pathogens.

Detection of foodborne bacterial pathogens from individual filth flies.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

Prevalence, level and distribution of Salmonella in shipments of imported capsicum and sesame seed spice offered for entry to the United States: observations and modeling results.

In response to increased concerns about spice safety, the United States Food and Drug Administration (FDA) initiated research to characterize the prevalence and levels of Salmonella in imported spices. 299 imported dried capsicum shipments and 233 imported sesame seed shipments offered for entry to the United States were sampled. Observed Salmonella shipment prevalence was 3.3% (1500 g examined; 95% CI 1.6-6.1%) for capsicum and 9.9% (1500 g; 95% Confidence Interval (CI) 6.3-14%) for sesame seed. Within shipment contamination was not inconsistent with a Poisson distribution. Shipment mean Salmonella level estimates among contaminated shipments ranged from 6 × 10(-4) to 0.09 (capsicum) or 6 × 10(-4) to 0.04 (sesame seed) MPN/g. A gamma-Poisson model provided the best fit to observed data for both imported shipments of capsicum and imported shipments of sesame seed sampled in this study among the six parametric models considered. Shipment mean levels of Salmonella vary widely between shipments; many contaminated shipments contain low levels of contamination. Examination of sampling plan efficacy for identifying contaminated spice shipments from these distributions indicates that sample size of spice examined is critical. Sampling protocols examining 25 g samples are predicted to be able to identify a small fraction of contaminated shipments of imported capsicum or sesame seeds.

Fluorometric detection of active alkaline phosphatase and gamma-glutamyl transferase in fluid dairy products from multiple species.

Over the past 80 years, a variety of methods have been developed to detect underpasteurized or improperly pasteurized milks used in dairy products. Existing methods are hampered by duration of analysis, poor reproducibility, and in some cases the use of hazardous chemicals. To overcome these issues, two new methods have been developed using fluorogenic substrates for two marker enzymes, alkaline phosphatase and gamma-glutamyl transferase. In 30 min, up to 18 samples can be analyzed in triplicate by both methods on two separate 96-well plates. Sample preparation is not necessary for liquid milks when using these methods. The relative standard deviation for each assay is less than 9%, and the correlation coefficient for results of the two methods is greater than 0.98. Using the new methods, milks from four species and nine commercially available liquid milk products were tested. The new methods were also tested directly against an existing phosphatase method (Fluorophos) in spiked whole milk samples.

Prevalence and relative risk of Cronobacter spp., Salmonella spp., and Listeria monocytogenes associated with the body surfaces and guts of individual filth flies.

Although flies are important vectors of food-borne pathogens, there is little information to accurately assess the food-related health risk of the presence of individual flies, especially in urban areas. This study quantifies the prevalence and the relative risk of food-borne pathogens associated with the body surfaces and guts of individual wild flies. One hundred flies were collected from the dumpsters of 10 randomly selected urban restaurants. Flies were identified using taxonomic keys before being individually dissected. Cronobacter spp., Salmonella spp., and Listeria monocytogenes were detected using the PCR-based BAX system Q7. Positive samples were confirmed by culture on specific media and through PCR amplification and sequencing or ribotyping. Among collected flies were the housefly, Musca domestica (47%), the blowflies, Lucilia cuprina (33%) and Lucilia sericata (14%), and others (6%). Cronobacter species were detected in 14% of flies, including C. sakazakii, C. turicensis, and C. universalis, leading to the proposal of flies as a natural reservoir of this food-borne pathogen. Six percent of flies carried Salmonella enterica, including the serovars Poona, Hadar, Schwarzengrund, Senftenberg, and Brackenridge. L. monocytogenes was detected in 3% of flies. Overall, the prevalence of food-borne pathogens was three times greater in the guts than on the body surfaces of the flies. The relative risk of flies carrying any of the three pathogens was associated with the type of pathogen, the body part of the fly, and the ambient temperature. These data enhance the ability to predict the microbiological risk associated with the presence of individual flies in food and food facilities.

Preparation and characterization of a suite of ephedra-containing standard reference materials.

The National Institute of Standards and Technology, the U.S. Food and Drug Administration, Center for Drug Evaluation and Research and Center for Food Safety and Applied Nutrition, and the National Institutes of Health, Office of Dietary Supplements, are collaborating to produce a series of Standard Reference Materials (SRMs) for dietary supplements. A suite of ephedra materials is the first in the series, and this paper describes the acquisition, preparation, and value assignment of these materials: SRMs 3240 Ephedra sinica Stapf Aerial Parts, 3241 E. sinica Stapf Native Extract, 3242 E. sinica Stapf Commercial Extract, 3243 Ephedra-Containing Solid Oral Dosage Form, and 3244 Ephedra-Containing Protein Powder. Values are assigned for ephedrine alkaloids and toxic elements in all 5 materials. Values are assigned for other analytes (e.g., caffeine, nutrient elements, proximates, etc.) in some of the materials, as appropriate. Materials in this suite of SRMs are intended for use as primary control materials when values are assigned to in-house (secondary) control materials and for validation of analytical methods for the measurement of alkaloids, toxic elements, and, in the case of SRM 3244, nutrients in similar materials.