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Whole genome sequencing (WGS) was performed on 201 Listeria monocytogenes isolates recovered from 102 of 27,389 refrigerated ready-to-eat (RTE) food samples purchased at retail in U.S. FoodNet sites as part of the 2010-2013 interagency L. monocytogenes Market Basket Survey (Lm MBS). Core genome multi-locus sequence typing (cgMLST) and in-silico analyses were conducted, and these data were analyzed with metadata for isolates from five food groups: produce, seafood, dairy, meat, and combination foods. Six of 201 isolates, from 3 samples, were subsequently confirmed as L. welshimeri. Three samples contained one isolate per sample; mmong the 96 samples that contained two isolates per sample, 3 samples each contained two different strains and 93 samples each contained duplicate isolates. After 93 duplicate isolates were removed, the remaining 102 isolates were delineated into 29 clonal complexes (CCs) or singletons based on their sequence type. The five most prevalent CCs were CC155, CC1, CC5, CC87, and CC321. The Shannon's diversity index for clones per food group ranged from 1.49 for dairy to 2.32 for produce isolates, which were not significantly different in pairwise comparisons. The most common molecular serogroup as determined by in-silico analysis was IIa (45.6%), followed by IIb (27.2%), IVb (20.4%), and IIc (4.9%). The proportions of isolates within lineages I, II, and III were 48.0%, 50.0% and 2.0%, respectively. Full-length inlA was present in 89.3% of isolates. Listeria pathogenicity island 3 (LIPI-3) and LIPI-4 were found in 51% and 30.6% of lineage I isolates, respectively. Stress survival islet 1 (SSI-1) was present in 34.7% of lineage I isolates, 80.4% of lineage II isolates and the 2 lineage III isolates; SSI-2 was present only in the CC121 isolate. Plasmids were found in 48% of isolates, including 24.5% of lineage I isolates and 72.5% of lineage II isolates. Among the plasmid-carrying isolates, 100% contained at least one cadmium resistance cassette and 89.8% contained bcrABC, involved in quaternary ammonium compound tolerance. Multiple clusters of isolates from different food samples were identified by cgMLST which, along with available metadata, could aid in the investigation of possible cross-contamination and persistence events.

A listeriosis outbreak in the United States implicated contaminated ice cream produced by one company, which operated 3 facilities. We performed single nucleotide polymorphism (SNP)-based whole genome sequencing (WGS) analysis on Listeria monocytogenes from food, environmental and clinical sources, identifying two clusters and a single branch, belonging to PCR serogroup IIb and genetic lineage I. WGS Cluster I, representing one outbreak strain, contained 82 food and environmental isolates from Facility I and 4 clinical isolates. These isolates differed by up to 29 SNPs, exhibited 9 pulsed-field gel electrophoresis (PFGE) profiles and multilocus sequence typing (MLST) sequence type (ST) 5 of clonal complex 5 (CC5). WGS Cluster II contained 51 food and environmental isolates from Facility II, 4 food isolates from Facility I and 5 clinical isolates. Among them the isolates from Facility II and clinical isolates formed a clade and represented another outbreak strain. Isolates in this clade differed by up to 29 SNPs, exhibited 3 PFGE profiles and ST5. The only isolate collected from Facility III belonged to singleton ST489, which was in a single branch separate from Clusters I and II, and was not associated with the outbreak. WGS analyses clustered together outbreak-associated isolates exhibiting multiple PFGE profiles, while differentiating them from epidemiologically unrelated isolates that exhibited outbreak PFGE profiles. The complete genome of a Cluster I isolate allowed the identification and analyses of putative prophages, revealing that Cluster I isolates differed by the gain or loss of three putative prophages, causing the banding pattern differences among all 3 AscI-PFGE profiles observed in Cluster I isolates. WGS data suggested that certain ice cream varieties and/or production lines might have contamination sources unique to them. The SNP-based analysis was able to distinguish CC5 as a group from non-CC5 isolates and differentiate among CC5 isolates from different outbreaks/incidents.

Background An effective environmental sampling method involves the use of a transport/neutralizing broth with the ability to neutralize sanitizer residues that are collected during sampling and to maintain viability of stressed Listeria monocytogenes ( Lm ) cells. Results We applied Lm onto stainless steel surfaces and then subjected Lm to desiccation stress for 16–18 h at room temperature (RT, 21–24 °C). This was followed by the subsequent application of Whisper™ V, a quaternary ammonium compound (QAC)-based sanitizer, diluted to 400 ppm and 8000 ppm of active quat, for 6 h. We then sampled Lm with sponges pre-moistened in three transport broths, Dey/Engley (D/E) broth, Letheen broth and HiCap™ broth, to generate environmental samples that contained sanitizer residues and low levels of stressed Lm , which were subsequently analyzed by an enrichment-based method. This scheme conformed with validation guidelines of AOAC International by using 20 environmental test portions per broth that contained low levels of Lm such that not all test portions were positive (i.e., fractional positive). We showed that D/E broth, Letheen broth and HiCap™ broth performed similarly when no quat or 400 ppm of quat was applied to the Lm contaminating stainless steel surfaces. However, when 8000 ppm of quat was applied, Letheen broth did not effectively neutralize the QAC in the samples. These comparisons were performed on samples stored under three conditions after collection to replicate scenarios of sample transport, RT for 2 h, 4 °C for 24 h and 4 °C for 72 h. Comparisons under the three different scenarios generally reached the same conclusions. In addition, we further demonstrated that storing Letheen and HiCap™ broths at RT for two months before sampling did not reduce their capacity to neutralize sanitizers. Conclusions We developed a scheme to evaluate the ability of transport broths to neutralize QAC sanitizers. The three transport broths performed similarly with a commonly used concentration of quat, but Letheen broth could not effectively neutralize a very high concentration of QAC. The performance of transport broths was not significantly affected under the assessed pre-sampling and post-sampling storage conditions.

Background. Using a novel combination of whole-genome sequencing (WGS) analysis and geographic metadata, we traced the origins of Salmonella Bareilly isolates collected in 2012 during a widespread food-borne outbreak in the United States associated with scraped tuna imported from India. Methods. Using next-generation sequencing, we sequenced the complete genome of 100 Salmonella Bareilly isolates obtained from patients who consumed contaminated product, from natural sources, and from unrelated historically and geographically disparate foods. Pathogen genomes were linked to geography by projecting the phylogeny on a virtual globe and produced a transmission network. Results. Phylogenetic analysis of WGS data revealed a common origin for outbreak strains, indicating that patients in Maryland and New York were infected from sources originating at a facility in India. Conclusions. These data represent the first report fully integrating WGS analysis with geographic mapping and a novel use of transmission networks. Results showed that WGS vastly improves our ability to delimit the scope and source of bacterial food-borne contamination events. Furthermore, these findings reinforce the extraordinary utility that WGS brings to global outbreak investigation as a greatly enhanced approach to protecting the human food supply chain as well as public health in general.

The objective of this research was to assess the microbiological status of leafy greens, sprouts, and melons from U.S. markets. A total of 14,183 samples of leafy greens, 2,652 samples of sprouts, and 3,411 samples of melons were collected throughout the United States from 2009 to 2014. The samples were analyzed for aerobic plate counts, total coliform counts, Escherichia coli counts, and the presence and levels of Salmonella, Shigella, Listeria monocytogenes, and Shiga toxin-producing E. coli (STEC), depending on the year and type of produce. Among the leafy greens, no E. coli O157:H7 or non-O157 STEC were detected from iceberg lettuce samples. The overall prevalences of Salmonella, E. coli O157:H7, non-O157 STEC, and L. monocytogenes in the 14,183 samples of leafy greens were 0.05, 0.01, 0.07, and 0.11%, respectively. Among sprout samples, no Salmonella or E. coli O157:H7 was detected, and the overall prevalences of non-O157 STEC and L. monocytogenes were 0.04 and 0.11%, respectively. Among melon samples, no Salmonella was detected from cucumbers, no L. monocytogenes was detected from cantaloupes, and the overall prevalences of Salmonella and L. monocytogenes were 0.12 and 0.23%, respectively. L. monocytogenes levels were 0.4 to 1,470 most probable number (MPN)/g in leafy greens, 0.36 to 1,100 MPN/g in sprouts, and <0.03 to 150 MPN/g in melons, and most positive samples had low levels of these pathogens. The isolates from these foods were very diverse genetically. Foodborne pathogens, including Salmonella, STEC, and L. monocytogenes, had relatively low prevalences in the produce surveyed. Because these foods are usually consumed raw, measures should be taken to significantly minimize the presence and levels of human pathogens.

In recent years, tree fruits have emerged as a new concern for Listeria monocytogenes contamination. The objective of the current study was to evaluate the potential internalization of L. monocytogenes from the surface of avocados into the edible portions of the fruit during certain postharvest practices simulated in a laboratory setting. One set of intact avocados was spot inoculated with L. monocytogenes on the stem scar, and the second set was hydrocooled in water contaminated with L. monocytogenes. Under these experimental conditions, L. monocytogenes internalized into the avocado pulp through the stem or stem scar after both spot inoculation and hydrocooling. In avocados spot inoculated with 50, 130, 500, and 1,300 CFU per fruit, bacteria were detected in the edible portion adjacent to the stem scar within 15 days postinoculation during storage at 4°C. In avocados hydrocooled in water containing L. monocytogenes at 10(6) and 10(8) CFU/ml, bacteria reached the bottom end of the fruit, and the populations in the edible portion adjacent to the stem scar reached up to 5.90 to 7.19 log CFU/g within 10 to 15 days during storage at 4°C. Dye mixed with inoculum was useful for guiding subsequent sampling, but dye penetration patterns were not always consistent with bacterial penetration.

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.

The complexity of the modern food supply makes identifying foodborne outbreaks difficult. In this report, FDA investigators identify a 44-state outbreak of Salmonella enterica serotype Montevideo, using molecular sequencing. To the Editor: We used next-generation sequencing (NGS) of microbiologic isolates in the molecular tracking of an outbreak source. Our investigation focused on isolates of Salmonella enterica serotype Montevideo that were associated with red and black pepper used in the production of Italian-style spiced meats in a New England processing facility. This manufacturer was implicated in a major salmonellosis outbreak that reportedly affected nearly 300 people in 44 states and the District of Columbia in 2009 and 2010. These clonal isolates confounded conventional epidemiologic traceback. For instance, pulsed-field gel electrophoresis (PFGE) was unable to distinguish spiced-meat food and clinical samples . . .

Nuts have been identified as a vector for salmonellosis. The objective of this project was to estimate the prevalence and contamination level of Salmonella in raw tree nuts (cashews, pecans, hazelnuts, macadamia nuts, pine nuts, and walnuts) at retail markets in the United States. A total of 3,656 samples of six types of tree nuts were collected from different types of retail stores and markets nationwide between October 2014 and October 2015. These samples were analyzed using a modified version of the Salmonella culture method from the U.S. Food and Drug Administration's Bacteriological Analytical Manual. Of the 3,656 samples collected and tested, 32 were culturally confirmed as containing Salmonella. These isolates represented 25 serotypes. Salmonella was not detected in pecans and in-shell hazelnuts. Salmonella prevalence estimates (and 95% confidence intervals) in cashews, shelled hazelnuts, pine nuts, walnuts, and macadamia nuts were 0.55% [0.15, 1.40], 0.35% [0.04, 1.20], 0.48% [0.10, 1.40], 1.20% [0.53, 2.40], and 4.20% [2.40, 6.90], respectively. The rates of Salmonella isolation from major or big chain supermarkets, small chain supermarkets, discount, variety, or drug stores, and online were 0.64% [0.38, 1.00], 1.60% [0.80, 2.90], 0.00% [0.00, 2.40], and 13.64% [2.90, 35.00], respectively (Cochran-Mantel-Haenszel test: P = 0.02). The rates of Salmonella isolation for conventional and organic nuts were not significantly different. Of the samples containing Salmonella, 60.7% had levels less than 0.003 most probable number (MPN)/g. The highest contamination level observed was 0.092 MPN/g. The prevalence and levels of Salmonella in these tree nut samples were comparable to those previously reported for similar foods.

A most-probable-number (MPN) method was used to enumerate Listeria monocytogenes in 2,320 commercial ice cream scoops manufactured on a production line that was implicated in a 2015 listeriosis outbreak in the United States. The analyzed samples were collected from seven lots produced in November 2014, December 2014, January 2015, and March 2015. L. monocytogenes was detected in 99% (2,307 of 2,320) of the tested samples (lower limit of detection, 0.03 MPN/g), 92% of which were contaminated at <20 MPN/g. The levels of L. monocytogenes in these samples had a geometric mean per lot of 0.15 to 7.1 MPN/g. The prevalence and enumeration data from an unprecedented large number of naturally contaminated ice cream products linked to a listeriosis outbreak provided a unique data set for further understanding the risk associated with L. monocytogenes contamination for highly susceptible populations.